Christophe Queva

AZD7762, a novel checkpoint kinase inhibitor, drives checkpoint abrogation and potentiates DNA-targeted therapies

Insights from cell cycle research have led to the hypothesis that tumors may be selectively sensitized to DNA-damaging agents resulting in improved antitumor activity and a wider therapeutic margin. The theory relies on the observation that the majority of tumors are deficient in the G1-DNA damage checkpoint pathway resulting in reliance on S and G2 checkpoints for DNA repair and cell survival. The S and G2 checkpoints are regulated by checkpoint kinase 1, a serine/threonine kinase that is activated in response to DNA damage; thus, inhibition of checkpoint kinase 1 signaling impairs DNA repair and increases tumor cell death. Normal tissues, however, have a functioning G1 checkpoint signaling pathway allowing for DNA repair and cell survival. Here, we describe the preclinical profile of AZD7762, a potent ATP-competitive checkpoint kinase inhibitor in clinical trials. AZD7762 has been profiled extensively in vitro and in vivo in combination with DNA-damaging agents and has been shown to potentiate response in several different settings where inhibition of checkpoint kinase results in the abrogation of DNA damage-induced cell cycle arrest. Dose-dependent potentiation of antitumor activity, when AZD7762 is administered in combination with DNA-damaging agents, has been observed in multiple xenograft models with several DNA-damaging agents, further supporting the potential of checkpoint kinase inhibitors to enhance the efficacy of both conventional chemotherapy and radiotherapy and increase patient response rates in a variety of settings. [Mol Cancer Ther 2008;7(9):2955–66]

Effects of GABA agonists on body temperature regulation in GABAB(1)−/− mice

Activation of GABAB receptors evokes hypothermia in wildtype (GABAB(1)+/+) but not in GABAB receptor knockout (GABAB(1)−/−) mice. The aim of the present study was to determine the hypothermic and behavioural effects of the putative GABAB receptor agonist γ‐hydroxybutyrate (GHB), and of the GABAA receptor agonist muscimol. In addition, basal body temperature was determined in GABAB(1)+/+, GABAB(1)+/− and GABAB(1)−/− mice. GABAB(1)−/− mice were generated by homologous recombination in embryonic stem cells. Correct gene targeting was assessed by Southern blotting, PCR and Western blotting. GABAB receptor‐binding sites were quantified with radioligand binding. Measurement of body temperature was done using subcutaneous temperature‐sensitive chips, and behavioural changes after drug administration were scored according to a semiquantitative scale. GABAB(1)−/− mice had a short lifespan, probably caused by generalised seizure activity. No histopathological or blood chemistry changes were seen, but the expression of GABAB(2) receptor protein was below the detection limit in brains from GABAB(1)−/− mice, in the absence of changes in mRNA levels. GABAB receptor‐binding sites were absent in brain membranes from GABAB(1)−/− mice. GABAB(1)−/− mice were hypothermic by approximately 1°C compared to GABAB(1)+/+ and GABAB(1)+/− mice. Injection of baclofen (9.6 mg kg−1) produced a large reduction in body temperature and behavioural effects in GABAB(1)+/+ and in GABAB(1)+/− mice, but GABAB(1)−/− mice were unaffected. The same pattern was seen after administration of GHB (400 mg kg−1). The GABAA receptor agonist muscimol (2 mg kg−1), on the other hand, produced a more pronounced hypothermia in GABAB(1)−/−mice. In GABAB(1)+/+ and GABAB(1)+/− mice, muscimol induced sedation and reduced locomotor activity. However, when given to GABAB(1)−/− mice, muscimol triggered periods of intense jumping and wild running. It is concluded that hypothermia should be added to the characteristics of the GABAB(1)−/−phenotype. Using this model, GHB was shown to be a selective GABAB receptor agonist. In addition, GABAB(1)−/− mice are hypersensitive to GABAA receptor stimulation, indicating that GABAB tone normally balances GABAA‐mediated effects.

Development of a new fully human anti-CD20 monoclonal antibody for the treatment of B-cell malignancies

SummaryDespite the widespread use of rituximab, a chimeric monoclonal antibody with demonstrated efficacy in the treatment of non-Hodgkin’s lymphomas, there is a recognized need to develop new agents with improved efficacy. Towards this end, using XenoMouse® technology, a fully human IgG1 anti-CD20 monoclonal antibody was generated. This antibody, denoted mAb 1.5.3, evoked enhanced pro-apoptotic activity in vitro, as compared to rituximab, in the Ramos lymphoma cell line. Also, mAb 1.5.3 mediated both complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) similar to rituximab in human B-lymphoma lines. Interestingly, mAb 1.5.3 demonstrated superior ADCC compared to rituiximab when FcγRIIIa F/F allotype donors were profiled and superior cytolytic activity across multiple human B-lymphoma and chronic B-cell leukemia lines in an in vitro whole blood assay. Furthermore, mAb 1.5.3 exhibited enhanced anti-tumor activity in Ramos, Daudi, and Namalwa tumour xenograft models. Lastly, mAb 1.5.3 produced a superior B-cell depletion profile in lymph node organs and bone marrow as compared to rituximab in a primate pharmacodynamic (PD) model. These findings underscore the potential of mAb 1.5.3 to exhibit improved clinical activity in the treatment of B-cell malignancies compared to rituximab.

PI3Kδ inhibitor idelalisib in combination with BTK inhibitor ONO/GS-4059 in diffuse large B cell lymphoma with acquired resistance to PI3Kδ and BTK inhibitors

Activated B-cell-like diffuse large B-cell lymphoma relies on B-cell receptor signaling to drive proliferation and survival. Downstream of the B-cell receptor, the key signaling kinases Bruton’s tyrosine kinase and phosphoinositide 3-kinase δ offer opportunities for therapeutic intervention by agents such as ibrutinib, ONO/GS-4059, and idelalisib. Combination therapy with such targeted agents could provide enhanced efficacy due to complimentary mechanisms of action. In this study, we describe both the additive interaction of and resistance mechanisms to idelalisib and ONO/GS-4059 in a model of activated B-cell-like diffuse large B-cell lymphoma. Significant tumor regression was observed with a combination of PI3Kδ and Bruton’s tyrosine kinase inhibitors in the mouse TMD8 xenograft. Acquired resistance to idelalisib in the TMD8 cell line occurred by loss of phosphatase and tensin homolog and phosphoinositide 3-kinase pathway upregulation, but not by mutation of PIK3CD. Sensitivity to idelalisib could be restored by combining idelalisib and ONO/GS-4059. Further evaluation of targeted inhibitors revealed that the combination of idelalisib and the phosphoinositide-dependent kinase-1 inhibitor GSK2334470 or the AKT inhibitor MK-2206 could partially overcome resistance. Characterization of acquired Bruton’s tyrosine kinase inhibitor resistance revealed a novel tumor necrosis factor alpha induced protein 3 mutation (TNFAIP3 Q143*), which led to a loss of A20 protein, and increased p-IκBα. The combination of idelalisib and ONO/GS-4059 partially restored sensitivity in this resistant line. Additionally, a mutation in Bruton’s tyrosine kinase at C481F was identified as a mechanism of resistance. The combination activity observed with idelalisib and ONO/GS-4059, taken together with the ability to overcome resistance, could lead to a new therapeutic option in activated B-cell-like diffuse large B-cell lymphoma. A clinical trial is currently underway to evaluate the combination of idelalisib and ONO/GS-4059 (NCT02457598).

Abstract 2673: Idelalisib has activity at clinically achievable drug concentrations in a subset of ABC and GCB diffuse large B-cell lymphoma and transformed follicular lymphoma cell lines

Diffuse large B-cell lymphoma (DLBCL) is the largest lymphoma subtype representing approximately one-third of all cases of non-Hodgkin9s lymphoma. Gene expression profiling showed that DLBCL can be stratified into activated B-cell (ABC) or germinal center B-cell (GCB) subtypes (Alizadeh et al., Nature 2000). The overall five year survival rate is only approximately 50% (Shaffer et al., Annu. Rev. Immunol. 2012), thus a medical need exits for the treatment of this disease, particularly for patients who relapse after the first line chemoimmunotherapy regimen. Idelalisib is an investigational, highly selective oral inhibitor of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) delta isoform. PI3K delta signaling is required for B lymphocyte activation, proliferation and survival and is dysregulated in several B-cell malignancies. We investigated the activity of idelalisib and ibrutinib, an inhibitor of Bruton9s tyrosine kinase, on the inhibition of proliferation against a panel of 22 DLBCL (9 ABC, 13 GCB) and 3 transformed follicular lymphoma cell lines. Idelalisib has potent activity (EC50 Citation Format: Jia Y. Liu, Tom Kenney, Leslie Butterworth, Adam Kashishian, Sarah Meadows, Peng Yue, Li Li, Kathleen Keegan, Christophe Queva, Stacey Tannheimer. Idelalisib has activity at clinically achievable drug concentrations in a subset of ABC and GCB diffuse large B-cell lymphoma and transformed follicular lymphoma cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2673. doi:10.1158/1538-7445.AM2015-2673

Abstract 1683: A novel adenosine A2A receptor antagonist optimized for high potency in adenosine-rich tumor microenvironment boosts antitumor immunity

High levels of extracellular adenosine in the tumor microenvironment are known to play a significant role in tumor immune evasion and promote tumor growth and metastasis. We defined the receptor(s) required for mediating the effect of adenosine on immune cells within the tumor microenvionment and report the characterization of a novel Immuno-Oncology-dedicated adenosine receptor 2A antagonist that functions in the high adenosine concentration found in tumors. We first explored the expression of the four adenosine receptors in primary human immune cells. A2A receptor was the main adenosine receptor expressed by CD4 and CD8 T lymphocytes and monocytes, and the only one in mature monocyte-derived dendritic cells and NK cells. A2B receptor was poorly detected in T cells and monocytes, while A1 and A3 receptors were never detected. Given these expression patterns, we further studied A2A functions in primary human T lymphocytes and monocytes. Selective A2A agonists such as CGS-21680 strongly suppressed cytokine production by activated primary human T lymphocytes, thus highlighting that A2A is the main effector receptor of the sensing of adenosine in tumors. We further confirmed the elevated extracellular adenosine level in the tumor microenvironment in several mouse and human tumors. High adenosine levels correlated with strong tumoral expression of CD73, the enzyme that converts AMP to adenosine. Interestingly, we showed that A2A receptor antagonists designed for Parkinson’s disease dramatically lost potency in a high adenosine environment ; our data indicated that a 30-fold dose increase may be required for full target inhibition within tumors. Therefore we developed a novel and potent A2A blocker with sub-nanomolar Ki and IC50 in a cAMP assay and a more than 100-fold selectivity over other adenosine receptors. Our lead compound kept a high potency in an adenosine-rich environment and restored cytokine production even in the presence of high concentrations of A2A agonists. iTeos inhibitor also efficiently reversed AMP-mediated T cell suppression. Furthermore, our compound rescued A2A receptor agonist-induced decrease of TNFα production by primary human monocytes, and was able to potently increase CD8 T cell cytotoxicity in a cytotoxicity assay with CD8 T cells as effectors and cancer cells as targets. These results suggest that iTeos new generation of A2A receptor antagonist, designed to keep a high potency in the adenosine-rich tumor microenvironment, may offer a new therapeutic opportunity in Immuno-Oncology. Citation Format: Erica Houthuys, Margreet Brouwer, Florence Nyawouame, Romain Pirson, Reece Marillier, Theo Deregnaucourt, Joao Marchante, Jakub Swiercz, Charlotte Moulin, Vanesa Bol, Gregory Driessens, Michel Detheux, Christophe Queva, Stefano Crosignani, Bruno Gomes. A novel adenosine A2A receptor antagonist optimized for high potency in adenosine-rich tumor microenvironment boosts antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1683. doi:10.1158/1538-7445.AM2017-1683