CD19 antigen: From tumor target to safety switch able to improve the safety profile of CAR T cell therapy Free

Background

Chimeric antigen receptor (CAR) T cell therapy is an innovative immunotherapeutic approach that has shown a potent and durable anti-tumor effect in hematologic malignancies, such as B cell acute lymphoblastic leukemia (B-ALL), B cell non-Hodgkin lymphoma, and multiple myeloma.More recently, its potential has also been demonstrated in the treatment of solid tumors. A notable example is the academic Phase I–II clinical trial conducted at the Bambino Gesù Children's Hospital in Rome, Italy (NCT03373097), for children with relapsed/refractory neuroblastoma (NB). This study highlighted the strong therapeutic potential of third-generation CAR.GD2 T cell therapy in patients with high-risk NB (PMID: 37018492).

To date, the most relevant adverse effects following infusion of CAR T cells in patients with either hematological or solid tumors, are Cytokine Release Syndrome (CRS) and Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS). CRS is a systemic inflammatory response, while ICANS is a form of neurotoxicity, both stemming from the immune system's reaction to CAR T cells.

Thus, the inclusion in the construct of a suicide gene could represent a relevant mechanism to improve the management of the toxicity.

Methods

Leveraging on our previous experience with donor lymphocyte infusions (DLI) following allogeneic hematopoietic stem cell transplantation (allo-HSCT) to prevent graft-versus-host disease (GvHD) (PMID: 40304059), we have developed a novel and effective strategy for a rapid elimination of CAR T cells based on the expression of the truncated human CD19. This approach was validated in different models, including CAR.GD2 for NB, and CAR.CD123 for Acute Myeloid Leukemia (AML). Specifically, we engineered a bi-cistronic retroviral construct encoding the extracellular domain of human CD19 in frame with a third-generation CAR.GD2 (ΔhCD19.CAR.GD2) or the second-generation CAR.CD123 (ΔhCD19.CAR.CD123). This design enables the expression of the CD19 and thus, the selective depletion of CD19+ CAR T cells upon administration of a bispecific T-cell engager (BiTE) that simultaneously targets hCD19 and CD3. The single-chain variable fragments (scFvs) incorporated in this BiTE are structurally identical to those of Blinatumomab, the first-in-class BiTE antibody approved for refractory B-ALL. While Blinatumomab normally crosslinks CD3 on T cells with CD19 on B cells, in our system, the forced expression of hCD19 on CAR T cells allows the BiTE to mediate their elimination.

Results

In this preclinical study, first, we confirmed that the addition of this safety switch did not impair CAR T cell function by performing in vitro and in vivo assays which demonstrated that ΔhCD19.CAR.GD2 T cells maintain robust anti-tumor activity. Then, we established the efficacy of BiTE-mediated elimination by culturing ΔhCD19.CAR.GD2 T-cells in the presence of anti-hCD19-hCD3 or, as a negative control, of anti-βGal-hCD19 (targeting β-galactosidase and CD19). Exposure to anti-hCD19-hCD3 led to a significant increase in the number of dead CAR T cells (51.8% ± 18.8%) and an increase in apoptosis (52%±6.3%) within 72 hours, whereas no effect was observed with the control BiTE (p=0.02; p=0.001, respectively). Importantly, in vivo studies showed that BiTE-induced depletion of CAR T cells did not permanently compromise their anti-tumor activity. Indeed, after the administration of anti-hCD19-hCD3 BiTE, we observed a significant transient reduction of the CAR T cells in peripheral blood of the treated mice (1.68%±1.96%), compared to control mice receiving anti-βGal-hCD19 BiTE (69.2%±12.6%, p<0.001), followed by CAR T cell re-expansion. Similar results were also observed applying the strategy to CAR.CD123.

Specifically, CAR.CD123 T cells showed a marked and rapid decrease in cell viability observed as early as 72 hours following exposure to hCD19-hCD3 bispecific BiTE (10.6%± 12.9% viable cells) compared to anti-βGal-hCD19 control (62.55%± 28.3% viable cells), highlighting the potential of this approach to mitigate CAR T cell toxicities in patients with aggressive haematologic malignancies.

Conclusions

Altogether, these findings indicate that the truncated human CD19 is a promising approach to integrate a robust and clinically meaningful safety switch into CAR T cell products, enabling control of treatment-related toxicities.