3 Reasons to Consider this Method in Kinase Inhibitor Drug Development
Basophils are the circulating immune cells that participate in allergic reactions and share many of the signaling pathways that are also present in other types of immune cells that may develop into blood cancers or participate in the development of autoimmune disorders. Therefore, measuring the activation of basophils has become a useful tool in testing for specific allergies and as an in vitro surrogate for drug discovery for agents active in inhibiting allergic reactions and agents active in moderating other disease types that work through these common signaling pathways.
Basophils act by degranulating upon stimulation and activation by allergens, releasing pro-inflammatory mediators. This functional effect can be easily measured in flow cytometry by quantifying the expression of cell surface activation markers such as CD63. The unique properties of basophils as a biomarker of an allergic reaction has promoted the development of Basophil Activation tests (BAT). BATs have been extensively studied as in vitro functional assays and have demonstrated superior accuracy as compared to other blood tests in testing for various allergies (11).
Several drug development projects are focusing on inhibition of the signaling pathways found in immune cells targeting key kinases to slow or prevent progression of diseases caused or modulated through the dysregulated activation of several immune cell types (4).
An example of such immune cell signaling is the B-cell Receptor (BCR) pathway which has emerged as a crucial player in the survival, proliferation and trafficking of malignant B–cells. Key kinases (like BTK, PI3K, and SYK) responsible for constant activation of this pathway have been demonstrated to be concrete targets to treat B-Cell Lymphomas (10). Ibrutinib and Idelalisib, BTK and PI3Kd inhibitors respectively, have been recently approved for the treatment of relapsed or refractory chronic lymphocytic leukemia (CLL), follicular B cell non-Hodgkin’s lymphoma and small lymphocytic lymphoma. Several second generation inhibitors for these kinases as well as SYK inhibitors are at different stages of development (15).
Any functional assay to detect the bioactivity of these kinases in a whole cell system is extremely helpful to screen and evaluate the potency of inhibitor compounds. A basophil activation test perfectly matches the requirements of scientists; first, key kinase targets like SYK, BTK, PI3K δ and γ are also main components in basophil activation signaling pathways and second, the surface expression of CD63 is an extremely easy and clear parameter to quantify. BAT is a fully standardized assay with years of use in allergy labs. Therefore, basophil activation offers a powerful tool, measuring the inhibition of basophil activation by drug treatment as Cromheecke et. al describes “The multitasking capability of basophils enables them to serve a unique role in bridging innate and adaptive immune responses (2).” Measuring basophil activation status is becoming increasingly recognized as a useful model for drug development focused on affecting the activity of various kinases.
In summary, in addition to being a very sensitive tool for allergy testing, BAT can offer solutions in many steps of the Drug Development process, including functioning as a surrogate biomarker for the activity of inhibitors of PI3K, BTK, and SYK. Therefore, this tool can provide a successful mix of easy data analysis, accessible cells, inter-laboratory standardization and the adaptability high throughput methods.
Reasons to Utilize Basophil Activation Testing
Flow cytometry has played an important role in drug development for many years however there have been limitations when performing phenotypic screening (i.e. screening to identify small substances that alter the phenotype of a cell, tissue, or other organism of study). At the time that plate-based sampling was introduced for use on flow cytometers, it added tremendous value to flow cytometry as a drug screening platform and helped to overcome those limitations. In fact, the introduction of faster sampling technologies, peripheral automation, and liquid handling further enhanced the capabilities of flow cytometry as a drug screening tool and converted it to high throughput flow cytometry (HTFC) (4,5).
Wise S. et al. has developed a HTFC screening assay for their compound based on basophil activation by modifying the BUHLMANN Flow CAST® Kinase Inhibition Assay to be scalable for high throughput screening. This scientific poster describes how researchers at Gilead Sciences produced “a robust and automated 384-well whole blood basophil activation assay. The miniaturization was achieved through multiple rounds of red blood cell lysis and washing in deep well 384-well plates using Agilent 70 µL wide-bore 384-well tips. Both lymphocyte integrity and complete red blood cell lysis are maintained as compared to the 96-well format. Inhibitors of basophil activation perform similarly in the 384-well and 96-well formats. This miniaturization and automation allow for increased throughput of compound testing in lead optimization efforts and reduce the FTE resources required (15).”
Several scientists have started to use basophil activation as a surrogate biomarker of the potency and efficacy of their compound in development in order to quantify the inhibition of the basophil activation. Once a potential kinase inhibitor is identified, it must be fully characterized in order to explore its concrete effect on whole cellular functions. In basophils, there are several pathways in which activation results in degranulation and scientists can tightly regulate the activation of these pathways by the use of different activators. For instance, cross-linking the high affinity receptor for the IgE, FceRI results in the activation of a pathway controlled by the PI3Kd and BTK,fMLP on the contrary will activate a PI3Kg basophil degranulation.
Lannutti et al. is an example of research focused on small molecule inhibitors to identify a novel targeted approach for the treatment of patients with B-cell malignancies. One of the first descriptions of the in vitro characterization of the CAL-101, idelalisib (the first in class PI3Kd inhibitors FDA approved for the treatment of CLL) is provided in this study, “Phosphatidylinositol-3-kinase p110δ serves as a central integration point for signaling from cell surface receptors known to promote malignant B-cell proliferation and survival. This provides a rationale for the development of small molecule inhibitors that selectively target p110 as a treatment approach for patients with B-cell malignancies (6).”
This study further explains that the signaling in basophils can be monitored by flow cytometry. It has been demonstrated in recent research that FceRI signals through p110δ while formyl-methionyl-leucyl-phenylalanine (fMLP) signals through G-protein–coupled receptor-p110ɣ. In addition, it has been clear that both stimulus result in the expression of CD63. Since it is known that p110δ promotes malignant B-cell proliferation and survival because it acts a central point for signaling from cell surface receptors, Lannutti et. al focused this study on the blocking effects and capabilities of CAL-101 (a highly selective and potent p110δ small molecule inhibitor). “For the analysis of p110 δ and p110 ɣ signaling, basophil activation was measured in isolated peripheral blood mononuclear cell or whole blood using the Flow CAST® kit according to the manufacturer’s standardized methods (BÜHLMANN Laboratories AG).” Researchers were able to demonstrate that CAL-101 blocked constitutive phosphatidylinositol-3-kinase signaling, resulting in decrease in downstream effectors, increase in poly (ADP-ribose) polymerase and caspase cleavage, and ultimately apoptosis.
More specifically, Lannutti et al. was able to determine the half-maximal concentration at which CAL-101 blocked both FcεRI p110δ mediated CD63 expression and fMLP activation of p110ɣ. Thus, the results of this study clearly provide a rationale for continued evaluation of CAL-101 as a targeted B-cell malignancy treatment (6).
CAL-101, idelalisib is the first in class PI3Kd inhibitors. Second generation inhibitors might demonstrate superiority in activity and selectivity in PI3K inhibition. Basophil activation is a valuable in vitro tool to demonstrate a better potency of new generation inhibitors than idelalisib. O’Farrell M, et al shows data on PWT143, highly selective PI3K delta inhibitor that has been selected as a development candidate. “PWT143 exhibits low nanomolar potency in cellular phosphorylation assays against PI3K delta. PWT143 exhibits cellular selectivity of 2200-, 30- and 700-fold against the alpha, beta and gamma isoforms, with no activity against approximately 500 other kinases tested, including mTOR. Low nM potency has also been demonstrated in a whole blood functional assay of basophil activity which is encouraging for translation to the clinic PWT143 inhibits PI3K delta mediated cellular activity in activated immune cells (Raji cell line, primary basophils) with low nM IC50, even in the presence of whole blood (8)”.
“The vast majority of experimental cancer drugs fail during the later stages of clinical development – after considerable time and expense has already been invested. The right biomarkers could help weed out unsuitable candidates early on, and focus efforts on therapies with real potential.
Critical decision points need to be brought forward, ideally into the initial stages of clinical development (Phase I), before costs, timelines and patient numbers escalate. The shrewd application of biomarkers in early-phase clinical development can help to make these critical go/no-go decisions in time“
Basophil activation as surrogate biomarker of the potency and efficacy of kinase inhibitors is an effective pharmacodynamics (PD) biomarker of drugs in early clinical phase studies. Basophils are easily recovered from the blood of the patients and/or donors before and at time points after the drug intake. Monitoring the ex vivo inhibition of basophil activation during time is a reliable effect on target that help in establishing the right therapy dosage when compared with pharmacokinetic (PK) data (11).
Horak et al demonstrated “in a phase 1 proof-of-concept study idelalisib is well tolerated when administered at a dose of 100 mg twice daily over 7 days in subjects with allergic rhinitis”. They began their study focusing on the preclinical indication that “anti-FcεRI–induced basophil degranulation, as assessed based on surface expression of CD63, is potently inhibited by idelalisib”.” The authors carried out several ex vivo basophil activation assays after patient stimulation with grass allergens in both normal donors and patients treated with several dosage of idelalisib. The data showed that “idelalisib inhibited ex vivo basophil activation” and the PD data was valuable in evaluating the efficacy of the various dosage of the drug tested (3)”.
The use of basophils as PD biomarker is becoming a standard in early phase clinical study, an example can be found in Moreno et al. the “Effect of treatment with a second generation PI3Kd inhibitor, ME-401, on a PD marker for PI3Kδ inhibition: basophil activation assessed via measurement of CD63 expression by flow cytometry, following ex vivo stimulation with an anti-FCeR1 monoclonal antibody (7).”
One common parameter tested in BTK inhibitors is the BTK occupancy that is correlated with the pharmacokinetics of the drug. However, as for PI3K, the evaluation of PD data is extremely helpful to speed up decision process in early clinical phase study. Basophil activation as PD biomarker for the first in class BTK inhibitor, PCI-32765, Ibrutinib, has been extensively evaluated. Advani et al. showed effective “Pharmacodynamic activity of ibrutinib-treated patients in the various cohorts as determined by percentage of CD63+ basophils on a–immunoglobulin E stimulation of whole blood collected at 4 (day 1, day 8) and 24 hours (day 1, day 8, day 15, day 29) after drug administration as compared with predose baseline (set at 100%)”(1).
Other research that has utilized BTK occupancy is Pollyea et al. In this study, researchers correlated BTK occupancy with inhibition of basophil activation “≥99% occupancy of the active site of Btk (left) and inhibition of basophil degranulation (right) by PCI-32765. It was designed so that each point represented a single time point and patient” (9).
Second generation BTK Inhibitors take benefit from the use of Basophils as PD biomarkers. As indicated in Smith et al. “BTK-dependent CD63 expression on basophils occurs upon stimulation of basophils with anti-IgE and thus measurement of CD63 expression by flow cytometry is a PD marker for inhibition of BTK activity in basophils by PRN1008”. In conclusion, Smith et al. demonstrated that BTK occupancy with a daily dose of ≥ 300mg reached therapeutic levels, was safe and well tolerated after single and multiple day dosing and is evident in the amount of inhibition of basophil activation (11).
- Moreno et al. Clinical Pharmacokinetics and Pharmacodynamics of ME-401, an Oral, Potent and Selective Inhibitor of Phosphatidylinositol 3-Kinase P110δ, Following Single Ascending Dose Administration to Healthy Volunteers. AACR 2016. New Orleans, LA.
- O’Farrell M, et al. Preclinical characterization of PWT143, a novel selective and potent phosphatidylinositol 3‑kinase delta (PI3K delta) inhibitor with ex-vivo activity in hematologic malignancies. Blood 2012 120:2907.
- Pollyea D et al. A Phase I Dose Escalation Study of the Btk Inhibitor PCI-32765 in Relapsed and Refractory B Cell Non-Hodgkin Lymphoma. Blood 2009 114:3713
- Rask Andersen et al. Advances in kinase targeting: current clinical use and clinical trials. Trends in Pharmacological Sciences. 2014 Nov;35(11):604-20. doi: 10.1016/j.tips.2014.09.007. PMID: 25312588
- Richie, J. et al. No Biomarker, No Trial? The Translational Scientist. Retrieved in November 2016. 
- Santos, A. et al. Basophil activation test discriminates between allergy and tolerance in peanut-sensitized children. J Allergy Clin Immunol. 2014 Sep;134(3):645-52. doi: 10.1016/j.jaci.2014.04.039. PMID:PMC4164910
- Smith PF et al. SAT0232 A Phase 1 Clinical Trial of PRN1008, an Oral, Reversible, Covalent BTK Inhibitor Demonstrates Clinical Safety and Therapeutic Levels of BTK Occupancy Without Sustained Systemic Exposure. Ann Rheum Dis 2015;74:742 doi:10.1136/annrheumdis-2015-eular.4289.
- Wu et al. Novel agents for chronic lymphocytic leukemia. J Hematol Oncol. 2013 May 16;6:36. doi: 10.1186/1756-8722-6-36. PMCID: PMC3659027
- Wise S. et al. Development of a Robust Automated 384-well Whole Blood Flow Cytometry Assay. SLAS 2014. 3rd Annual Conference & Exhibition. January 18-22, 2014. San Diego, CA