Background The development of Bruton’s tyrosine kinase inhibitors (BTKi) for the treatment of chronic lymphocytic leukaemia (CLL) has provided a highly effective and relatively non-toxic alternative to conventional chemotherapy. Some studies have shown that BTKi can also lead to improvements in T cell immunity in patients despite in vitro analyses suggesting an immunosuppressive effect of BTKi on T cell function. Methods In this study, we examined both the in vitro effect and long-term in vivo effect of two clinically available BTKi, ibrutinib and zanubrutinib. Additional in vitro assessments were undertaken for a third BTKi, acalabrutinib. Immune subset phenotyping, cytokine secretion, T cell degranulation and proliferation assays were performed on peripheral blood mononuclear cells isolated from untreated CLL patients, and CLL patients on long-term (> 12 months) BTKi treatment. Results Similar to prior studies we observed that long-term BTKi treatment normalises lymphocyte subset frequency and reduces PD-1 expression on T cells. We also observed that T cells from patients taken prior to BTKi therapy showed an abnormal hyper-proliferation pattern typical of senescent T cells, which was normalised by long-term BTKi treatment. Furthermore, BTKi therapy resulted in reduced expression of the T cell exhaustion markers PD-1, TIM3 and LAG3 in late generations of T cells undergoing proliferation. Conclusions Collectively, these findings indicate that there are critical differences between the in vitro effects of BTKi on T cell function and the effects derived from long-term BTKi exposure in vivo. Overall long-term exposure to BTKi, and particularly ibrutinib, resulted in improved T cell fitness in part due to suppressing the abnormal hyper-proliferation of CLL T cells and the associated development of T cell senescence.

Successes with anti-CD20 antibodies in chronic lymphocytic leukemia (CLL) and enhanced activity of Fc-engineered versus unmodified antibody therapy suggest a potentially impactful role for natural killer (NK) cells and other innate immune cells in controlling this disease. Stimulated natural killer cells have shown promise as a cellular therapy but their application has been constrained by limited expansion capacity and low cytotoxic activity against CLL cells. Here, we demonstrate that both healthy donor-derived and CLL patient-derived NK cells expand rapidly when stimulated with feeder cells expressing membrane-bound IL-21 and have potent cytotoxic activity against allogeneic or autologous CLL cells. Combination with anti-CD20 antibodies significantly enhances NK recognition and killing of CLL targets. As any CLL immune therapy would likely be given in combination, we assess commonly-used treatments and demonstrate that ibrutinib has mixed suppressive and protective effects on expanded NK cells whereas expanded NKs are highly resistant to venetoclax. We demonstrate efficacy in vivo in two xenograft mouse models of human CLL that support building upon a regimen of venetoclax and obinutuzumab with mbIL-21-expanded NK cells. Collectively, these data support development of mbIL-21-expanded NKs combined with the CD20 antibody obinutuzumab and venetoclax in the treatment of CLL.

Combination venetoclax plus ibrutinib for the treatment of mantle cell lymphoma (MCL) has demonstrated efficacy in the relapsed or refractory setting; however, the long-term impact on patient immunology is unknown. In this study, changes in immune subsets of MCL patients treated with combination venetoclax and ibrutinib were assessed over a 4-year period. Multiparameter flow cytometry of peripheral blood mononuclear cells showed that ≥12 months of treatment resulted in alterations in the proportions of multiple immune subsets, most notably CD4+ and CD8+ effector and central memory T cells and natural killer cells, and normalization of T-cell cytokine production in response to T-cell receptor stimulation. Gene expression analysis identified upregulation of multiple myeloid genes (including S100 and cathepsin family members) and inflammatory pathways over 12 months. Four patients with deep responses stopped study drugs, resulting in restoration of normal immune subsets for all study parameters except myeloid gene/pathway expression, suggesting long-term combination venetoclax and ibrutinib irreversibly affects this population. Our findings demonstrate that long-term combination therapy is associated with immune recovery in MCL, which may allow responses to subsequent immunotherapies and suggests that this targeted therapy results in beneficial impacts on immunological recovery. This trial was registered at www.clinicaltrials.gov as #NCT02471391.

Optimization of degrader properties is often a challenge due to their beyond-rule-of-5 nature. Given the paucity of known E3 ligases and the often-limited choice of ligands with varied chemical structures for a given protein target, degrader linkers represent the best position within the chimeric molecules to modify their overall physicochemical properties. In this work, a series of AT7519-based CDK9 degraders was assembled using click chemistry, facilitating the tuning of aqueous solubility and lipophilicity while retaining their linker type and molecular weight. Using chromatographic logD and kinetic solubility experiments, we show that degraders with similar chemical constitution but varied position of the embedded triazole demonstrate different lipophilicity and aqueous solubility properties. Overall, this work highlights the impact of triazole placement on linker composition through application of click chemistry for degrader synthesis and its ability to be used to promote the achievement of favorable physicochemical properties.

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Combination Ibrutinib (IB) plus Venetoclax (Ven) for the treatment of Mantle Cell Lymphoma (MCL) has demonstrated efficacy in the relapsed/refractory setting. The AIM trial treated 24 patients with IB monotherapy for 4 weeks (560mg/day) with Ven added (stepwise to 400mg/day) thereafter resulting in an overall response rate of 71% at 16 weeks (Tam et al, NEJM 2018). 5 patients discontinued treatment within 6 months due to resistant disease and 3 patients relapsed. 16 patients remained on treatment for more than 1 year. In this study we examined the longitudinal changes in peripheral blood (PB) immunology in patients treated in the AIM trial in order to identify immunological and inflammatory biomarkers associated with response and to examine if stable control of MCL without further chemotherapy exposure was associated with immunological recovery. Multiparameter flow cytometry of cryopreserved PBMCs collected at enrolment (baseline), and weeks 4, 16, 56 and 110 of treatment was used to assess the proportions of immune subsets (CD4 and CD8 memory subsets, γδ T cells and CD16+ NK cells) in AIM patients and compared to 13 age matched healthy donor (HD) samples. At baseline samples from MCL patients exhibited alterations in multiple immune subsets - most notably skewing in CD8+ memory subsets (Figure 1). Despite reduction of circulating B cell numbers consistent with clinical responses, there were no significant changes to PB immune subset distributions between baseline and 16 weeks. However at the later time points of 56 and 110 weeks there was evidence in responding patients of PB immunology normalisation to HD levels in T cell subsets including a shift in CD8 T cells from Naïve and Central Memory (CM) to Effector Memory (EM) and Terminally differentiated EM (TEMRA) phenotype and a shift in CD4 T cells from Naïve and CM to EM phenotype (Figure 2). Increased proportions of γδ T cells (Baseline 2.8 ± 3.0% week 56 4.4 ± 3.4% P<0.05) and CD16+ NK cells (Baseline 40.1 ± 28.0% week 56 79.1 ± 21.0% P<0.01) were also observed. T cell function assessed by intracellular IFNγ, TNFα and IL-2 in CD4 and CD8 T cells in response to T cell stimulation beads showed no change in IFNγ or TNFα production by CD4 or CD8 cells following therapy. In patients with elevated IL2+ CD4 and CD8 populations at early time points, these normalised by week 56 to HD levels. To further examine changes in immune function pathways, targeted multiplex gene expression profiling using 770 gene NanoString nCounter® PanCancer Immune Profiling Panel was performed on PBMCs at baseline and weeks 4, 16 and 56 and analyzed using nSolver advanced analysis module. Consistent with the flow cytometric findings there were minimal changes in gene expression at week 16 compared to baseline. The greatest changes were observed at week 56 with alterations in multiple pathways including increased signalling through complement, adhesion, TNF superfamily and transporter pathways in responding patients. The top upregulated genes at week 56 were CTSS (Cathepsin S), FCER1G (Fc fragment of IgE), S100A8 (S100 calcium binding protein A8) and CD14 while the top downregulated gene was CD22 (reflecting the change in B cell burden). Increased expression of C1QA and C1QB at week 16 was significantly associated with disease non-response or relapse on IB + Ven and warrants further investigation in an independent cohort to assess its applicability as a predicative biomarker for patient response. Our findings show that long term treatment with combination IB and Ven in MCL is associated with immune recovery and changes in the expression of inflammatory biomarkers. This study demonstrates that while short term assessments can be used to examine clinical disease responses, long term assessments are required to determine the immunological consequences of small molecule inhibitors. Restoration of immune system function in these patients may allow responses to subsequent immunotherapies and suggests that, in contrast to conventional chemotherapy-based regimens, this combination targeted therapy may result in beneficial impacts on immunological recovery. Disclosures Handunnetti: Gilead: Honoraria. Anderson:Walter and Eliza Hall Institute: Employment, Patents & Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.. Roberts:AbbVie: Other: Unremunerated speaker for AbbVie, Research Funding; BeiGene: Research Funding; Janssen: Research Funding; Walter and Eliza Hall Institute: Patents & Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.; Australasian Leukaemia and Lymphoma Group: Membership on an entity's Board of Directors or advisory committees. Seymour:AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding; Roche: Consultancy, Research Funding, Speakers Bureau; Acerta: Consultancy; Celgene: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy. Tam:Janssen: Honoraria, Research Funding; Pharmacyclics LLC, an AbbVie company: Honoraria; Roche: Honoraria; AbbVie: Honoraria, Research Funding; BeiGene: Honoraria; Novartis: Honoraria. Ritchie:Sanofi: Honoraria; Novartis: Honoraria; Imago: Research Funding; Beigene: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy; BMS: Research Funding; Takeda: Research Funding. Koldej:NanoString Technologies: Other: Travel grant.

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Background:The Eμ-TCL1 syngeneic model is the most widely used mouse model of chronic lymphocytic leukemia and has been extensively used to understand the pathogenesis of select genes, the effect of the immune microenvironment and for pre-clinical drug development studies. Recently there has been an increasing awareness of the impact of age and sex differences on not only the development of cancers but also the e cacy and toxicity of speci c cancer therapies. However, despite the predominance of older males in CLL patient demographics, the Eμ-TCL1 adoptive transfer studies have used almost exclusively young female recipient mice.Methods:In this study we performed primary and secondary adoptive transfer experiments in order to understand the impact of recipient age and sex on the development of disease as assessed by ow cytometry and survival in the Eμ-TCL1 adaptive transfer model.Results:We found that young female recipients had pro-longed survival in a primary adoptive transfer, however sex differences were not observed in a subsequent secondary adoptive transfer experiment. Furthermore, while recipient age did not have a signi cant effect on the rate of disease establishment or survival in female mice, aged male mice had a signi cantly decreased rate of Eμ-TCL1 tumor engraftment.Conclusions:These ndings suggest that age and sex differences must be considered in the experimental design of studies using the Eμ-TCL1 adaptive transfer model of CLL.