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Digital polymerase chain reaction strategies for accurate and precise detection of vector copy number in chimeric antigen receptor T-cell products

  • Lindsey A. Murphy
    Affiliations
    Center for Cancer and Blood Disorders, Children's Hospital Colorado and Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA
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  • Russell C. Marians
    Affiliations
    Charles C. Gates Biomanufacturing Facility, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA
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  • Kristen Miller
    Affiliations
    Center for Cancer and Blood Disorders, Children's Hospital Colorado and Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA
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  • Matthew D. Brenton
    Affiliations
    Charles C. Gates Biomanufacturing Facility, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA
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  • Rebecca L.V. Mallo
    Affiliations
    Charles C. Gates Biomanufacturing Facility, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA
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  • M. Eric Kohler
    Affiliations
    Center for Cancer and Blood Disorders, Children's Hospital Colorado and Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA
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  • Terry J. Fry
    Affiliations
    Center for Cancer and Blood Disorders, Children's Hospital Colorado and Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA
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  • Amanda C. Winters
    Correspondence
    Correspondence: Amanda C. Winters, MD, PhD, Center for Cancer and Blood Disorders, Children's Hospital Colorado and Department of Pediatrics, University of Colorado Anschutz Medical Campus, 13123 E 16th Ave, B-115, Aurora, Colorado 80045, USA.
    Affiliations
    Center for Cancer and Blood Disorders, Children's Hospital Colorado and Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA
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Published:October 14, 2022DOI:https://doi.org/10.1016/j.jcyt.2022.09.004

      Abstract

      Background aims

      Vector copy number (VCN), an average quantification of transgene copies unique to a chimeric antigen receptor (CAR) T-cell product, is a characteristic that must be reported prior to patient administration, as high VCN increases the risk of insertional mutagenesis. Historically, VCN assessment in CAR T-cell products has been performed via quantitative polymerase chain reaction (qPCR). qPCR is reliable along a broad range of concentrations, but quantification requires use of a standard curve and precision is limited. Digital PCR (dPCR) methods were developed for absolute quantification of target sequences by counting nucleic acid molecules encapsulated in discrete, volumetrically defined partitions. Advantages of dPCR compared with qPCR include simplicity, reproducibility, sensitivity and lack of dependency on a standard curve for definitive quantification. In the present study, the authors describe a dPCR assay developed for analysis of the novel bicistronic CD19 × CD22 CAR T-cell construct.

      Methods

      The authors compared the performance of the dPCR assay with qPCR on both the QX200 droplet dPCR (ddPCR) system (Bio-Rad Laboratories, Inc, Hercules, CA, USA) and the QIAcuity nanoplate-based dPCR (ndPCR) system (QIAGEN Sciences, Inc, Germantown, MD, USA). The primer–probe assay was validated with qPCR, ndPCR and ddPCR using patient samples from pre-clinical CAR T-cell manufacturing production runs as well as Jurkat cell subclones, which stably express this bicistronic CAR construct.

      Results

      ddPCR confirmed the specificity of this assay to detect only the bicistronic CAR product. Additionally, the authors’ assay gave accurate, precise and reproducible CAR T-cell VCN measurements across qPCR, ndPCR and ddPCR modalities.

      Conclusions

      The authors demonstrate that dPCR strategies can be utilized for absolute quantification of CAR transgenes and VCN measurements, with improved test–retest reliability, and that specific assays can be developed for detection of unique constructs.

      Key Words

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      References

        • Brentjens RJ
        • Davila ML
        • Riviere I
        • Park J
        • Wang X
        • Cowell LG
        • et al.
        CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia.
        Sci Transl Med. 2013; 5: 177ra38
        • Davila ML
        • Brentjens RJ.
        CD19-Targeted CAR T cells as novel cancer immunotherapy for relapsed or refractory B-cell acute lymphoblastic leukemia.
        Clin Adv Hematol Oncol. 2016; 14: 802-808
        • Park JH
        • Geyer MB
        • Brentjens RJ.
        CD19-targeted CAR T-cell therapeutics for hematologic malignancies: interpreting clinical outcomes to date.
        Blood. 2016; 127: 3312-3320
        • Maude SL
        • Frey N
        • Shaw PA
        • Aplenc R
        • Barrett DM
        • Bunin NJ
        • et al.
        Chimeric antigen receptor T cells for sustained remissions in leukemia.
        N Engl J Med. 2014; 371: 1507-1517
        • Neelapu SS
        • Locke FL
        • Bartlett NL
        • Lekakis LJ
        • Miklos DB
        • Jacobson CA
        • et al.
        Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma.
        N Engl J Med. 2017; 377: 2531-2544
        • Leahy AB
        • Elgarten CW
        • Grupp SA
        • Maude SL
        • Teachey DT
        Tisagenlecleucel for the treatment of B-cell acute lymphoblastic leukemia.
        Expert Rev Anticancer Ther. 2018; 18: 959-971
        • Abramson JS
        • Palomba ML
        • Gordon LI
        • Lunning MA
        • Wang M
        • Arnason J
        • et al.
        Lisocabtagene maraleucel for patients with relapsed or refractory large B-cell lymphomas (TRANSCEND NHL 001): a multicentre seamless design study.
        Lancet. 2020; 396: 839-852
        • Mian A
        • Hill BT
        Brexucabtagene autoleucel for the treatment of relapsed/refractory mantle cell lymphoma.
        Expert Opin Biol Ther. 2021; 21: 435-441
        • Shen-Ong GL
        • Potter M
        • Mushinski JF
        • Lavu S
        • Reddy EP.
        Activation of the c-myb locus by viral insertional mutagenesis in plasmacytoid lymphosarcomas.
        Science. 1984; 226: 1077-1080
        • Varmus HE
        • Quintrell N
        • Ortiz S.
        Retroviruses as mutagens: insertion and excision of a nontransforming provirus alter expression of a resident transforming provirus.
        Cell. 1981; 25: 23-36
        • Vannucci L
        • Lai M
        • Chiuppesi F
        • Ceccherini-Nelli L
        • Pistello M.
        Viral vectors: a look back and ahead on gene transfer technology.
        New Microbiol. 2013; 36: 1-22
        • Schroder AR
        • Shinn P
        • Chen H
        • Berry C
        • Ecker JR
        • Bushman F.
        HIV-1 integration in the human genome favors active genes and local hotspots.
        Cell. 2002; 110: 521-529
        • Wu X
        • Li Y
        • Crise B
        • Burgess SM.
        Transcription start regions in the human genome are favored targets for MLV integration.
        Science. 2003; 300: 1749-1751
        • Cavazzana-Calvo M
        • Payen E
        • Negre O
        • Wang G
        • Hehir K
        • Fusil F
        • et al.
        Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassaemia.
        Nature. 2010; 467: 318-322
        • Scholler J
        • Brady TL
        • Binder-Scholl G
        • Hwang WT
        • Plesa G
        • Hege KM
        • et al.
        Decade-long safety and function of retroviral-modified chimeric antigen receptor T cells.
        Sci Transl Med. 2012; 4: 132ra53
        • Fraietta JA
        • Nobles CL
        • Sammons MA
        • Lundh S
        • Carty SA
        • Reich TJ
        • et al.
        Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells.
        Nature. 2018; 558: 307-312
        • Shah NN
        • Qin H
        • Yates B
        • Su L
        • Shalabi H
        • Raffeld M
        • et al.
        Clonal expansion of CAR T cells harboring lentivector integration in the CBL gene following anti-CD22 CAR T-cell therapy.
        Blood Adv. 2019; 3: 2317-2322
        • Lu A
        • Liu H
        • Shi R
        • Cai Y
        • Ma J
        • Shao L
        • et al.
        Application of droplet digital PCR for the detection of vector copy number in clinical CAR/TCR T cell products.
        J Transl Med. 2020; 18: 191
      1. Food and Drug Administration. Long Term Follow-Up After Administration of Human Gene Therapy Products: Guidance for Industry; 2020. https://www.fda.gov/media/113768/download. Accessed January 14, 2022.

        • Hollyman D
        • Stefanski J
        • Przybylowski M
        • Bartido S
        • Borquez-Ojeda O
        • Taylor C
        • et al.
        Manufacturing validation of biologically functional T cells targeted to CD19 antigen for autologous adoptive cell therapy.
        J Immunother. 2009; 32: 169-180
        • Singh H
        • Figliola MJ
        • Dawson MJ
        • Olivares S
        • Zhang L
        • Yang G
        • 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: e64138
        • Kunz A
        • Gern U
        • Schmitt A
        • Neuber B
        • Wang L
        • Huckelhoven-Krauss A
        • et al.
        Optimized Assessment of qPCR-Based Vector Copy Numbers as a Safety Parameter for GMP-Grade CAR T Cells and Monitoring of Frequency in Patients.
        Mol Ther Methods Clin Dev. 2020; 17: 448-454
        • Jennings LJ
        • George D
        • Czech J
        • Yu M
        • Joseph L.
        Detection and quantification of BCR-ABL1 fusion transcripts by droplet digital PCR.
        J Mol Diagn. 2014; 16: 174-179
        • Huggett JF
        • Foy CA
        • Benes V
        • Emslie K
        • Garson JA
        • Haynes R
        • et al.
        The digital MIQE guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments.
        Clin Chem. 2013; 59: 892-902
        • Hindson CM
        • Chevillet JR
        • Briggs HA
        • Gallichotte EN
        • Ruf IK
        • Hindson BJ
        • et al.
        Absolute quantification by droplet digital PCR versus analog real-time PCR.
        Nat Methods. 2013; 10: 1003-1005
        • Whale AS
        • Huggett JF
        • Cowen S
        • Speirs V
        • Shaw J
        • Ellison S
        • et al.
        Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation.
        Nucleic Acids Res. 2012; 40: e82
        • Zhang BO
        • Xu CW
        • Shao Y
        • Wang HT
        • Wu YF
        • Song YY
        • et al.
        Comparison of droplet digital PCR and conventional quantitative PCR for measuring EGFR gene mutation.
        Exp Ther Med. 2015; 9: 1383-1388
      2. Bio-Rad Laboratories, Inc. Droplet Digital PCR Applications Guide. 2022.http://www.bio-radcom/webroot/web/pdf/lsr/literature/Bulletin_6407.pdf. Accessed January 1, 2022.

        • Pabst T
        • Joncourt R
        • Shumilov E
        • Heini A
        • Wiedemann G
        • Legros M
        • et al.
        Analysis of IL-6 serum levels and CAR T cell-specific digital PCR in the context of cytokine release syndrome.
        Exp Hematol. 2020; 88: 7-14.e3
        • Mika T
        • Maghnouj A
        • Klein-Scory S
        • Ladigan-Badura S
        • Baraniskin A
        • Thomson J
        • et al.
        Digital-Droplet PCR for Quantification of CD19-Directed CAR T-Cells.
        Front Mol Biosci. 2020; 7: 84
        • Fehse B
        • Badbaran A
        • Berger C
        • Sonntag T
        • Riecken K
        • Geffken M
        • et al.
        Digital PCR Assays for Precise Quantification of CD19-CAR-T Cells after Treatment with Axicabtagene Ciloleucel.
        Mol Ther Methods Clin Dev. 2020; 16: 172-178
        • Badbaran A
        • Berger C
        • Riecken K
        • Kruchen A
        • Geffken M
        • Muller I
        • et al.
        Accurate In-Vivo Quantification of CD19 CAR-T Cells after Treatment with Axicabtagene Ciloleucel (Axi-Cel) and Tisagenlecleucel (Tisa-Cel) Using Digital PCR.
        Cancers (Basel). 2020; 12: 1-12
        • Haderbache R
        • Warda W
        • Hervouet E
        • da Rocha MN
        • Trad R
        • Allain V
        • et al.
        Droplet digital PCR allows vector copy number assessment and monitoring of experimental CAR T cells in murine xenograft models or approved CD19 CAR T cell-treated patients.
        J Transl Med. 2021; 19: 265
        • Lou Y
        • Chen C
        • Long X
        • Gu J
        • Xiao M
        • Wang D
        • et al.
        Detection and Quantification of Chimeric Antigen Receptor Transgene Copy Number by Droplet Digital PCR versus Real-Time PCR.
        J Mol Diagn. 2020; 22: 699-707
        • Qin H
        • Nguyen SM
        • Ramakrishna S
        • Tarun S
        • Yang L
        • Verdini NP
        • et al.
        Novel CD19/CD22 Bicistronic Chimeric Antigen Receptors Outperform Single or Bivalent Cars in Eradicating CD19+CD22+, CD19- and CD22- Pre-B Leukemia.
        Blood. 2017; 130 (810LP–810)
        • Koo TK
        • Li MY.
        A Guideline of Selecting and Reporting Intraclass Correlation Coefficients for Reliability Research.
        J Chiropr Med. 2016; 15: 155-163
        • Bell AD
        • Usher CL
        • McCarroll SA.
        Analyzing Copy Number Variation with Droplet Digital PCR.
        Methods Mol Biol. 2018; 1768: 143-160
        • Miotke L
        • Lau BT
        • Rumma RT
        • Ji HP.
        High sensitivity detection and quantitation of DNA copy number and single nucleotide variants with single color droplet digital PCR.
        Anal Chem. 2014; 86: 2618-2624
        • Lock M
        • Alvira MR
        • Chen SJ
        • Wilson JM
        Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR.
        Hum Gene Ther Methods. 2014; 25: 115-125
        • Milone MC
        • Bhoj VG.
        The Pharmacology of T Cell Therapies.
        Mol Ther Methods Clin Dev. 2018; 8: 210-221
        • Porter DL
        • Hwang WT
        • Frey NV
        • Lacey SF
        • Shaw PA
        • Loren AW
        • et al.
        Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia.
        Sci Transl Med. 2015; 7: 303ra139
        • Turtle CJ
        • Hay KA
        • Hanafi LA
        • Li D
        • Cherian S
        • Chen X
        • et al.
        Durable Molecular Remissions in Chronic Lymphocytic Leukemia Treated With CD19-Specific Chimeric Antigen Receptor-Modified T Cells After Failure of Ibrutinib.
        J Clin Oncol. 2017; 35: 3010-3020
        • Mueller KT
        • Maude SL
        • Porter DL
        • Frey N
        • Wood P
        • Han X
        • et al.
        Cellular kinetics of CTL019 in relapsed/refractory B-cell acute lymphoblastic leukemia and chronic lymphocytic leukemia.
        Blood. 2017; 130: 2317-2325
        • Sugimoto H
        • Chen S
        • Minembe JP
        • Chouitar J
        • He X
        • Wang H
        • et al.
        Insights on Droplet Digital PCR-Based Cellular Kinetics and Biodistribution Assay Support for CAR-T Cell Therapy.
        AAPS J. 2021; 23: 36
        • Wiltshire TD
        • Milosevic D
        • Jacob EK
        • Grebe SK
        • Dietz AB
        Sensitive detection of integrated and free transcripts in chimeric antigen receptor T-cell manufactured cell products using droplet digital polymerase chain reaction.
        Cytotherapy. 2021; 23: 452-458