In Vitro Efficacy of Anti-HER2 CAR T-Cells in HER2 + BCP-ALL
Authors: Le Calvez Baptiste , Grain Audrey , Ollier Jocelyn , Guiet Elodie , Chevallier Patrice , Eveillard Marion , Clemenceau Beatrice 
Published: December 2023

Introduction:
Around 30% of adults and 15% of children with B cell precursor acute lymphoblastic leukemia (BCP-ALL) relapse after a first line of treatment. The prognosis of these patients remains poor. Recent advances have involved targeted therapies with monoclonal antibodies and chimeric antigen receptor (CAR) T-lymphocytes (anti-CD19 CAR T-cells), which have produced very encouraging results in the high risk BCP-ALL setting(Maude et al. 2018). Nevertheless, half of these patients relapse after such therapies. In a third of cases, the disease no longer expresses the antigen previously targeted. New targets are therefore needed. HER2 is expressed in above one third of adult BCP-ALL (Chevallier et al. 2004) and 10% of childhood BCP-ALL (Camuset et al. 2021). HER2 expression is associated with chemoresistance and poor prognosis (Chevallier et al. 2004) (Camuset et al. 2021). The anti-HER2 antibody trastuzumab was prospectively tested in adult patients with HER2+ BCP-ALL, but yielded disappointing results (Chevallier et al. 2012). Anti-HER2 CAR T-cells could be an interesting alternative. Indeed, they have shown encouraging preclinical efficacy in solid tumors expressing HER2/neu (Budi et al. 2022).
The present work was designed to evaluate, in vitro, the sensitivity of HER2+ BCP-ALL to anti-HER2 CAR engineered Epstein Barr virus (EBV) specific T-cells.
Methods:
DMSO-frozen cell-samples from 6 HER2+ BCP-ALL patients were available for this study. Patient characteristics are shown in Table 1. Blast cells from patient samples were preferred to cell lines in order to be more representative of BCP-ALL biology. level of HER2 expression on BCP-ALL samples was determined by flow cytometry and compared to the HER2 expressing breast-cancer cell line BT474.
Anti-EBV cytotoxic T lymphocytes (CTLs), armed with a trastuzumab-based scFv-CH2-CH3-IgG2A-FcεRIγ chimeric receptor as previously described (Clémenceau et al. 2015) were used for long-term cytoxicity assays. For 4 samples, non-transduced anti-EBV CTLs were used as controls. Cytotoxic activity was assessed at a 3-to-1 effector-to-target ratio, in a flow cytometry assay. Cells were harvested at H0, H4 and H24 and stained with a viability stain (VS780). Further staining with anti-CD22 or anti-CD24 (depending on the BCP-ALL immunophenotype) and anti-CD3 antibodies allowed to characterize blast cells and CAR T-cells respectively.
The study was approved by the local review board and two alive patients provided informed consent.
Results:
The level of HER2 expression by the 6 HER2+ BCP-ALL samples was variable with a median relative fluorescence intensity ratio (RFI) of 9 (range 1.8 to 16). These levels of expression were lower than those of the BT474 cell line (RFI= 79).
No BCP-ALL lysis was observed when cells were incubated with trastuzumab alone (not shown). When co-cultured with non-transduced anti-EBV CTLs (CAR negative) (n=4), a decrease of residual viable BCP-ALL cells was observed at variable levels (Figure 1A). The lysis observed at 24h was possibly due to allogeneic recognition since anti-EBV CTLs were not autologous to the leukemias tested.
Conclusion:
This study first reveals that, although CTLs with TCRs restricted to EBV antigens and therefore intrinsically low allo-specificity were used, CAR-independent lysis was observed in 24 hours co-culture with BCP-ALL cells. These results underline the need for cautious interpretation of cytotoxicity test results in an allogeneic setting.
Secondly, these preliminary data suggest that an anti-HER2 CAR T-cell approach could be promising for BCP-ALL even with weak antigen expression.
Anti-EBV CTLs have already been used as a platform for CAR-engineered T-cells (Rossig et al. 2017). Should the cytotoxic activity of allogeneic EBV-CTL be proven to be specifically directed against BCP-ALL, the synergy between TCR and anti-HER2-CAR might be beneficial, and adoptive transfer of anti-HER2 CAR EBV CTLs could be a promising therapeutic option for patients with HER2+ BCP-ALL.
* This research is funded by the non-profit organization Etoile de Martin/SFCE.