Two Ways of Targeting a CD19 Positive Relapse of Acute Lymphoblastic Leukaemia after Anti-CD19 CAR-T Cells

Abstract

Background: Therapeutic options for CD19⁺ relapses after anti-CD19 CAR-T cells are still debated; second infusion of anti-CD19 CAR-T cells, therapeutic antibodies, or targeted therapies can be discussed. Here, we explore the immunophenotyping and lysis sensitivity of CD19⁺ ALL relapse after anti-CD19 CAR-T cells and propose different therapeutic options for such a high-risk disease.

Methods: Cells from successive B-ALL relapses from one patient were collected. A broad immunophenotype analysis was performed. ⁵¹Cr cytotoxic assays, and long-term killing assays were conducted using T-cell effectors that are capable of cytotoxicity through three recognition pathways: antibody-dependent cell-mediated cytotoxicity (ADCC), anti-CD19 CAR-T, and TCR.

Results: Previously targeted antigen expression, even if maintained, decreased in relapses, and new targetable antigens appeared. Cytotoxic assays showed that ALL relapses remained sensitive to lysis mediated either by ADCC, CAR-T, or TCR, even if the lysis kinetics were different depending on the effector used. We also identified an immunosuppressive monocytic population in the last relapse sample that may have led to low persistence of CAR-T.

Conclusion: CD19⁺ relapses of ALL remain sensitive to cell lysis mediated by T-cell effectors. In case of ALL relapses after immunotherapy, a large immunophenotype will make new therapies possible for controlling such high risk ALL.

Keywords: CAR-T cells; acute lymphoblastic leukaemia; immunotherapy; relapse.

Figure 1

Clinical case medical history: disease progression, treatments received, and samples collected (Dg; R2; autologous CAR-T cells, blood samples and R3); ALL: acute lymphoblastic leukaemia; R: rituximab; HSCT: haematopoietic stem cell transplantation; PB: peripheral blood; chemo: chemotherapy.

Figure 2

4h-⁵¹Cr cytotoxic assays over 4 h. Percentage of ALL lysis at four E:T ratio after co-culture with the autologous anti-CD19 CAR-T cell preparation (a); or anti-HLA DPB1*04:01 CD4+ T-clone (b); or anti-CMVpp65 CD8 T-cells (c); or through the ADCC pathway using anti-CD220, anti-CD184, anti-CD24, and anti-CD19 antibodies in combination with mCD16-T lymphocytes (d). Expressions of targeted antigens are, respectively, represented as median of immunofluorescence (MFI) in a log of 10. (BLCL: B cell lineage used as controls). Three experiments in triplicate were performed. Results are given as median/error.

Figure 3

Long-term killing assays. Residual viable ALL cells at 3 time points of co-culture H0 (starting point), after 4 h (H4) of co-culture and after 24 h (H24) of co-culture: (a) with no effector, results showed total viable cells; (b) with a purified autologous anti-CD19 CAR-T cell preparation (percent of residual CD22+ viables cells); (c) with anti-HLA DPB1*04:01 CD4+ T-clone (percent of residual CD22+ viables cells); (d) with anti-CMVpp65 CD8 T-cells (percent of residual CD22+ viables cells). Two experiments in duplicate were performed at effector/ratio = 3:1. Results are given as mean + SD. CMVpp65 peptides: PepTivator CMVpp65 human, 130-093-435, Miltenyi Biotec; T eff: T-cell effectors, here polyclonal CD8+ T cells against CMV peptides.