Differences in Expansion Potential of Naive Chimeric Antigen Receptor T Cells from Healthy Donors and Untreated Chronic Lymphocytic Leukemia Patients

Jean-Marc Hoffmann; Maria-Luisa Schubert; Lei Wang; Angela Hückelhoven; Leopold Sellner; Sophia Stock; Anita Schmitt; Christian Kleist; Ulrike Gern; Angelica Loskog; Patrick Wuchter; Susanne Hofmann; Anthony D Ho; Carsten Müller-Tidow; Peter Dreger; Michael Schmitt

doi:10.3389/fimmu.2017.01956

Differences in Expansion Potential of Naive Chimeric Antigen Receptor T Cells from Healthy Donors and Untreated Chronic Lymphocytic Leukemia Patients

Front Immunol. 2018 Jan 10:8:1956. doi: 10.3389/fimmu.2017.01956. eCollection 2017.

Authors

Jean-Marc Hoffmann¹, Maria-Luisa Schubert¹, Lei Wang¹, Angela Hückelhoven¹, Leopold Sellner^{1

2}, Sophia Stock¹, Anita Schmitt¹, Christian Kleist³, Ulrike Gern¹, Angelica Loskog⁴, Patrick Wuchter^{1

5}, Susanne Hofmann¹, Anthony D Ho^{1

2}, Carsten Müller-Tidow^{1

2}, Peter Dreger^{1

2}, Michael Schmitt^{1

2}

Affiliations

¹ Cellular Immunotherapy, GMP Core Facility, Department of Internal Medicine V, Heidelberg University Hospital, Heidelberg, Germany.
² National Center for Tumor Diseases (NCT), Heidelberg, Germany.
³ Department of Nuclear Medicine, Heidelberg University Hospital, Heidelberg, Germany.
⁴ Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
⁵ Medical Faculty Mannheim, Institute of Transfusion Medicine and Immunology, Heidelberg University, German Red Cross Blood Service Baden-Württemberg - Hessen, Mannheim, Germany.

Abstract

Introduction: Therapy with chimeric antigen receptor T (CART) cells for hematological malignancies has shown promising results. Effectiveness of CART cells may depend on the ratio of naive (T_N) vs. effector (T_E) T cells, T_N cells being responsible for an enduring antitumor activity through maturation. Therefore, we investigated factors influencing the T_N/T_E ratio of CART cells.

Materials and methods: CART cells were generated upon transduction of peripheral blood mononuclear cells with a CD19.CAR-CD28-CD137zeta third generation retroviral vector under two different stimulating culture conditions: anti-CD3/anti-CD28 antibodies adding either interleukin (IL)-7/IL-15 or IL-2. CART cells were maintained in culture for 20 days. We evaluated 24 healthy donors (HDs) and 11 patients with chronic lymphocytic leukemia (CLL) for the composition of cell subsets and produced CART cells. Phenotype and functionality were tested using flow cytometry and chromium release assays.

Results: IL-7/IL-15 preferentially induced differentiation into T_N, stem cell memory (T_SCM: naive CD27+ CD95+), CD4+ and CXCR3+ CART cells, while IL-2 increased effector memory (T_EM), CD56+ and CD4+ T regulatory (T_Reg) CART cells. The net amplification of different CART subpopulations derived from HDs and untreated CLL patients was compared. Particularly the expansion of CD4+ CART_N cells differed significantly between the two groups. For HDs, this subtype expanded >60-fold, whereas CD4+ CART_N cells of untreated CLL patients expanded less than 10-fold. Expression of exhaustion marker programmed cell death 1 on CART_N cells on day 10 of culture was significantly higher in patient samples compared to HD samples. As the percentage of malignant B cells was expectedly higher within patient samples, an excessive amount of B cells during culture could account for the reduced expansion potential of CART_N cells in untreated CLL patients. Final T_N/T_E ratio stayed <0.3 despite stimulation condition for patients, whereas this ratio was >2 in samples from HDs stimulated with IL-7/IL-15, thus demonstrating efficient CART_N expansion.

Conclusion: Untreated CLL patients might constitute a challenge for long-lasting CART effects in vivo since only a low number of T_N among the CART product could be generated. Depletion of malignant B cells before starting CART production might be considered to increase the T_N/T_E ratio within the CART product.

Keywords: CD19; T cell expansion; T cell subpopulations; chimeric antigen receptor; chronic lymphocytic leukemia; cytokines; immunotherapy; naive T cells.