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1511 Accomplices in Cure: Blinatumomab + Dasatinib Reduces TCR Signaling and Effector Function Stronger Than Ponatinib and Ameliorates T-Cell Exhaustion

Program: Oral and Poster Abstracts
Session: 614. Acute Lymphoblastic Leukemias: Therapies, Excluding Transplantation and Cellular Immunotherapies: Poster I
Hematology Disease Topics & Pathways:
Research, Lymphoid Leukemias, ALL, Translational Research, Combination therapy, Diseases, Therapies, Lymphoid Malignancies
Saturday, December 9, 2023, 5:30 PM-7:30 PM

Nora Philipp1,2*, Anetta Marcinek, M.Sc.1,2*, Gerulf Hänel, Ph.D.1,2*, Daniel Nixdorf, MSc1,2*, Veit L. Buecklein, MD1,3* and Marion Subklewe, MD1,2,4

1Laboratory for Translational Cancer Immunology, Gene Center, LMU Munich, Munich, Germany
2Department of Medicine III, LMU University Hospital, LMU Munich, Munich, Germany
3Department of Medicine III, University Hospital, LMU Munich, Munich, Germany
4German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany

The tyrosine kinase inhibitors (TKIs) dasatinib (dasa) and ponatinib (pona) are being tested in clinical trials in philadelphia chromosome-positive (Ph+) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients in combination with the CD19xCD3 bispecific blinatumomab (blina). Pona but not dasa is able to bind the BCR-ABLT315I mutation, which frequently occurs in BCP-ALL patients. Therefore, clinical studies are moving away from dasa and favor pona. Nevertheless, both TKIs in combination with blina have led to striking remission rates in Ph+ BCP-ALL (complete response dasa: 98%, Foá 2020; pona: 92%, Jabbour 2023). Interestingly, increased lymphocyte frequencies were reported in patients under blina + dasa therapy (Puzzolo 2021), indicating that the effect of dasa is not limited to toxicity towards ALL cells conferred by BCR-ABL inhibition. Indeed, dasa modulates T-cell function by interfering with TCR signaling (Mestermann 2019). Also, we have shown that resting of T cells by high-dose dasa treatment ameliorates bispecific-induced T-cell exhaustion (Philipp 2022). In contrast, less is known about the effect of pona on T-cell function. Based on these data, we hypothesize that TKIs fine-tune TCR signaling, which ameliorates T-cell exhaustion during continuous blina infusion. Here, we systematically compared the influence of dasa and pona on blina-mediated TCR signaling and T-cell function.

Inhibition of T-cell activation and function by dasa or pona at 0.3 - 100 nM was tested in vitro over 3 days: (1) CD69 expression, (2) blina-mediated killing as specific lysis of CD19+ tumor cells (3) T-cell expansion analyzed by dye dilution or CD2+ fold change (FC), (4) cytokine secretion measured by cytokine catch or cytometric bead array.

Inhibition of TCR signaling was evaluated using a Jurkat-NFAT T-cell line stimulated for 6h with blina and CD19+ tumor cells. Phosphoflow of pAKT and pZAP70 was performed on CD3/CD28 activated primary human T cells. Toxicity of the TKIs against BCR-ABL+/- cell lines was evaluated after 3 days.

T-cell exhaustion upon continuous blina + dasa stimulation was assessed in an in vitro long-term culture system for continuous bispecific exposure (Philipp 2022). Functional assays as described in (1)-(4) were performed every 7 days with isolated T cells from long-term cultures.

Both TKIs inhibited T-cell function in a dose-dependent manner. Surprisingly, dasa was more potent than pona at low-intermediate doses in reducing T-cell function, shown by CD69 expression (mean % at TKI = 3 nM: basal = 70, dasa = 25, pona = 64, p<0.0001), cytotoxic function (mean % specific lysis at TKI = 12.5 nM: basal = 55, dasa = 0, pona = 54, p<0.0001), T-cell proliferation (mean % proliferated at TKI = 1.5 nM: basal = 62, dasa = 25, pona = 51, p<0.0001), as well as IFN-𝛾 secretion (normalized mean fluorescence intensity (MFI) at TKI = 12.5 nM: basal = 655, dasa = 0, pona = 924, p<0.0001). We also observed that NFAT engagement was significantly lower in dasa-treated Jurkat-NFAT cells (relative luminescence units at TKI 12.5 nM: basal = 1620, dasa = 392, pona = 996, p<0.001). Similarly, phosphoflow analyses revealed lower levels of pAKT and pZAP70 upon CD3/CD28 + dasa versus pona treatment (MFI of pAKT at TKI = 25 nM: basal = 2347, dasa = 1642, pona = 2045). Importantly, both TKIs induced cell death of BCR-ABL+ but not BCR-ABL- cell lines at concentrations >3 nM.

Interestingly, 14 days continuously stimulated T cells with blina + dasa (12.5 nM) did not lead to co-expression of inhibitory checkpoints (mean % of PD-1+Tim-3+LAG-3+ T cells on day 14: blina = 31, +dasa = 0.6, p<0.01). Furthermore, when isolated from long-term cultures and tested in functional assays, blina + dasa treated T cells maintained high cytokine secretion (mean IL-2 in pg/ml on day 14: blina = 730, blina + dasa = 5638, p<0.01), granzyme B expression (mean MFI ratio of CD8+ on day 14: blina = 96, blina + dasa = 335, p<0.01) and cytotoxic activity (mean % specific lysis on day 14: blina = 61, blina + dasa = 80).

Together, these findings indicate that while both TKIs confer comparable toxicity to BCR-ABL+ leukemia cells, dasa dampens TCR signaling and T-cell effector function more potently than pona. Strikingly, dasa can ameliorate T-cell exhaustion upon continuous blina exposure. Our data suggest that low-dose TKIs can fine-tune T-cell activity and might play a considerable role for the clinical success of combinatorial bispecific and TKI therapy in Ph+ BCP-ALL.

Disclosures: Buecklein: Gilead/Kite: Other: Travel Funding, Research Funding; BMS: Research Funding; Miltenyi Biotech: Research Funding; Roche: Honoraria, Research Funding; Pierre Fabre: Other: Travel Funding; Priothera: Consultancy; Pfizer: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy. Subklewe: Seagen: Research Funding; Ichnos Sciences: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Other: Travel Support, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Other: Travel Support, Speakers Bureau; AstraZeneca: Speakers Bureau; Takeda: Consultancy, Honoraria, Research Funding; BMS/Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding; Miltenyi Biotec: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead/Kite: Consultancy, Honoraria, Other: Travel Support, Research Funding, Speakers Bureau; AvenCell: Consultancy, Honoraria; Incyte Biosciences: Consultancy, Honoraria; Molecular Partners: Consultancy, Honoraria, Research Funding; GSK: Speakers Bureau; LAWG: Speakers Bureau; Springer Healthcare: Speakers Bureau; AbbVie: Consultancy, Honoraria; Autolus: Consultancy, Honoraria; advesya (CanCell Therapeutics): Consultancy, Honoraria; Genmab US: Consultancy, Honoraria; Interius BioTherapeutics: Consultancy, Honoraria; Nektar Therapeutics: Consultancy, Honoraria; Orbital Therapeutics: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Scare: Consultancy, Honoraria.

*signifies non-member of ASH