Vecabrutinib Is Efficacious In Vivo in a Preclinical CLL Adoptive Transfer Model
Authors: Jebaraj Billy Michael Chelliah , Scheffold Annika , Tausch Eugen , Fox Judith A. , Taverna Pietro , Stilgenbauer Stephan 
Published: October 2019

Abstract
B cell receptor signaling (BCR) in chronic lymphocytic leukemia (CLL) drives tumor cell proliferation and survival. Inhibition of Bruton's tyrosine kinase (BTK), a key enzyme in the BCR pathway, has proved to be efficacious even in poor-risk and chemo-refractory patients. However resistance to the BTK inhibitor ibrutinib has been shown to emerge in a subset of CLL patients. Of importance, the C481S BTK mutation conferred resistance by preventing the covalent binding of ibrutinib to its target cysteine 481 in BTK. Vecabrutinib (formerly known as SNS-062, a succinate salt) is a novel, highly potent, next generation noncovalent BTK inhibitor which demonstrated biochemical and cellular activity against C481S BTK mutant in vitro. However, the efficacy of vecabrutinib and its impact on the T-cell microenvironment has not been studied in in vivo preclinical CLL models.
In the present study, the efficacy of vecabrutinib was investigated using the Eµ-TCL1 adoptive transfer model. Mice were randomized to treatment with either 40mg/kg vecabrutinib succinate, twice daily by oral gavage (n=6) or vehicle control (n=6). The mice were sacrificed after 2 weeks of treatment and changes in tumor burden as well as alterations in T-cell microenvironment were analysed in detail. Treatment with vecabrutinib decreased tumor burden as observed by a significant decrease in WBC count (36.5 vs. 17.1 giga/L; P=0.002), spleen weight (median 0.56g vs. 0.31g; P=0.005) and liver weight (median 1.5g vs. 1.2g; P=0.005) compared to vehicle treatment. Correspondingly, the CD5+ CD19+ tumor cells were significantly decreased in blood (P=0.002) and spleen (P=0.002) while no significant difference was observed in bone marrow (P=0.818) upon treatment with vecabrutinib.
Since BTK inhibition is known to reshape the tumor microenvironment, we studied the impact of vecabrutinib specifically on T-cell subsets. Firstly, no difference in the proportions of CD4 or CD8 expressing T-cells was observed in mice treated with vehicle or vecabrutinib. However, of interest, the percentage of CD4+ CD25+ FoxP3+ regulatory T cells (Tregs) were significantly decreased upon treatment with vecabrutinib in peripheral blood (P=0.026) and spleen (P=0.009). The decrease in Tregs was due to reduced proliferation of these cells upon exposure to the drug as measured by Ki-67 staining. Also, the Tregs expressing the maturation and activation markers such as CD103 and GITR were significantly decreased in blood and spleen upon drug treatment.
Further, we analysed the changes in CD8 T-cell subsets following treatment with vecabrutinib. Treatment with the drug resulted in expansion of the CD127+ CD44- naïve CD8 T-cells in blood, bone marrow and spleen (all P values 0.002) while the CD127+ CD44+ memory CD8 T-cells were significantly decreased in bone marrow and spleen (all P values 0.009). Also, the CD127low CD44int-hi effector CD8 T-cells were decreased in blood (P=0.004), bone marrow (P=0.004) and spleen (P=0.002) upon vecabrutinib treatment. Therefore, vecabrutinib treatment did not alter the percentage of CD4+ and CD8+ T cells in mice however, significant changes in the subset composition of the CD4 and CD8 T cells were observed.
Lastly, to analyse the impact of vecabrutinib on survival, a cohort of mice (n=12) were transplanted with 5 million splenic tumor cells isolated from Eµ-TCL1 transgenic mice. After allowing for engraftment, the mice were randomized to treatment with the drug (n=6) or vehicle (n=6). Of note, the mice treated with the drug showed a significant increase in survival (median 35 days from transplant; P<0.001) compared to treatment with vehicle (median 28 days).
In summary, vecabrutinib was efficacious in vivo in a preclinical CLL adoptive transfer model, decreasing tumor burden in different organs and significantly improving survival. Treatment with the drug altered the T-cell architecture in vivo. Of interest, the immunosuppressive Tregs, which protect the tumor from immune surveillance were decreased in various tissue compartments; however, a decrease in the effector CD8 T cells might impact anti-tumor immunity if there is a consistent effect upon drug treatment. Vecabrutinib antitumor activity and effects on T-cell populations in vivo in this preclinical CLL model are intriguing, merits further investigation and supports the ongoing phase 1b/2 study in patients with previously treated B-lymphoid malignancies (NCT03037645).

Disclosures
Tausch: AbbVie: Consultancy, Other: Travel grants; Celgene: Consultancy, Other: Travel grants; Gilead: Consultancy, Other: Travel grants. Fox:Sunesis Pharmaceuticals: Employment; Amphivena Therapeutics: Employment. Taverna:Sunesis Pharmaceuticals: Employment. Stilgenbauer:Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.