**3.3 Identification of compounds with distinct activation profiles**

Using the multi-plate analysis algorithm and Profile Map Boolean logic algorithm in the iQue Forecyt® software we identified 2 compounds, tunicamycin and an erbastatin analog, that have no, or very moderate, effect on CD69+ while having medium to strong inhibition on CD25 and IFNγ and TNFα (**Figure 4**). Tunicamycin

#### **Figure 4.**

*Kinase inhibitors that barely decreased early activation marker CD69 but did inhibit the expression of the late activation marker CD25 and did decrease the secreted IFNγ and TNFα. (A) 2D plots (CD69 or CD25 vs. SSC) from the screening wells of these 2 inhibitors and the negative control well showed less effect on CD69 expression (see the red numbers) but with drastic decreasing effect on CD25 expression on both CD4+ T helper cells and CD8+ T cytotoxic cells. (B) Table showing the compounds Tunicamycin (EGFR/ERB inhibitor) and an erbstatin analog (EGFR inhibitor) had the significant decreasing effect on the secretion of IFNγ and TNFα. (C) Example of tunicamycin's effect, from a separate dosage test experiment, on the decreasing of other markers except CD69 expression. Each point in the graph represent the mean standard deviation of the duplicate wells. The dash line in each graph shows the baseline level without the compound.*

#### *Developing a Novel Multiplexed Immune Assay Platform to Screen Kinase Modulators… DOI: http://dx.doi.org/10.5772/intechopen.97304*

decreases CD69+ cells in CD4+ and CD8+ cells 2% and 8%, respectively, but significantly decreases CD25+ cells in CD4+ and CD8+ cells 56% and 58%, respectively. The erbastatin analog decreases CD69+ cells in CD4+ and CD8+ cells 6% and 21%, respectively, but significantly decreases CD25+ cells in CD4+ cells and CD8+ cells 47% and 55%. To further confirm this effect, tunicamycin was run in a dosage test and was confirmed that it did not affect CD69 (**Figure 4C**) but inhibited other endpoints in a dose-dependent manner including CD25 expression, and the secretion of IFNγ and TNFα.

Using a similar strategy to modify different thresholds in the multi-plate visualization, we also identified the following 3 distinct kinase inhibitors that have no or very moderate inhibition effect on CD25 expression but more inhibitory effect on all other markers including CD69 and IFNγ and TNFα (data not shown: U0126 (MEK1/2 inhibitor), CAY10621 (SPHK1 inhibitor), and bisindolylmaleimide V (S6K inhibitor)). Collectively, these results suggest two potential unique kinase pathways with different spatiotemporal regulation of the early activation markers CD69, and the late activation marker CD25.

#### *3.3.1 Identification of a cluster of MEK1/2 inhibitors with similar activation profiles*

Of the 152 kinase inhibitors in the library, there were only 4 MEK1/2 inhibitors. The screening results showed that all 4 compounds broadly inhibit the major relevant markers (CD69 and CD25 expression, and IFNγ and TNFα secretion) after 24 hour T cell activation. The exception was U0126, which had less inhibitory effect on CD25 expression (**Supplemental Figure 2**). **Supplemental Figure 2A** represents the inhibitory effect of MEK1/2 inhibitors on the expression of CD69 and CD25 on both T helper and T cytotoxic cells. **Supplemental Figure 2B** illustrates the decreasing effects on the secretion of the cytokines IFNγ and TNFα. In the subsequent dosage test to confirm the screening results (**Supplement Figure 2C**), 24 IC50s were generated from 4 compounds and 6 endpoints. Two MEK1/2 inhibitors AS703026 and PD0325901 showed strong potency in the inhibition of T cell activation across all 6 major endpoints. All 12 IC50s were less than 0.02 μM. Two MEK1/2 inhibitors, U0126 and PD184161, showed moderate inhibition. A total of 11 IC50s from the latter 2 compounds were greater than 0.3 μM. The exception was PD184161/TNFα with and IC50 of 0.072 μM. Although PD184161 had IC50s greater than 10 μM for CD25 expression on T helper and T cytotoxic cells, it does inhibit CD25 expression at the highest tested dose of 10 μM (the dosage curve not shown), similar to the performance in the screening as shown in **Supplement Figure 2A** (see the dark blue columns). High IC50s (greater than the tested highest concentration 10 μM) were due to the lack of the bottom plateau even at the highest dose in the curve fitting.

### *3.3.2 Identification of 3 distinct kinase inhibitors with similar profiles as a well-known Jak1/2 inhibitor*

One commercially valuable compound in the library, ruxolitinib, a myelofibrosis Janus kinase inhibitor with selectivity for subtypes Jak1 and Jak2, is a drug to treat disease. Ruxolitinib was shown as a positive hit in the screening. We used the iQue Forecyt® Profile Map to explore compounds similar to ruxolitinib. In the Profile Map, highlighting the ruxolitinib well triggers the algorithm to place a red tick mark in the slider bars to show the value for each endpoint of the ruxolitinib treatment (**Figure 5A** and **B**). By dragging and minimizing the blue slider bars for each threshold around the red mark (ruxolitinib's position), we identified 3 distinct kinase inhibitors that have profiles similar to ruxolitinib: PKC inhibitor PKC412,

#### **Figure 5.**

*Identification of a cluster of 3 kinase inhibitors with similar phenotypic signatures as a well-known myelofibrosis drug ruxolitinib (Jak1/2 inhibitor). (A) the 11 criteria that were applied to narrow down the hits that had similar phenotypic signature as ruxolitinib. The B10 well (ruxolitinib) in plate 1 was highlighted in the profile map in iQue Forecyt® software and a red dot corresponding to the B10 wells was highlighted automatically in each threshold Bar by the profile map algorithm. By manually dragging the threshold bar from both the left end and the right end to the red dot (ruxolitinib), compounds with similar profile as ruxolitinib can be identified. Please note, these thresholds can be modified, to get more or fewer hits, according to the user's preference. (B) 3 compounds were identified from 2 screening plates with similar phenotypic signature as ruxolitinib by using the thresholds as seen in the criteria panel (A). (C) Bar graph showing the similar decreasing effect of 3 kinase inhibitors on the cell surface activation markers (CD69 and CD25) in the screening. (D) the bar graph showed the similar strong decreasing effect of 3 kinase inhibitors on the secreted cytokines (IFNγ and TNFα) in the screening.*

IKK2 inhibitor CAY10657, and MEK1/2 inhibitor PD184161. It is possible to select a larger or smaller set of compounds with profiles similar to ruxolitinib by choosing a different threshold range for each endpoint. The further quantitative screening results (**Figure 5C** and **D**) suggest these 3 inhibitors, as similar as ruxolitinib, broadly inhibit the T cell activation markers including the expression of CD69 and CD25, and the secretion of cytokine IFNγ and TNFα.

#### **4. Discussion**

We developed a novel, immune assay platform that multiplexes cell and bead measurements in the same assay well. The assay analyzes T cell activation from different angles including cell health, time-dependent expression of early activation marker CD69, late activation marker CD25, even later activation marker HLA-DR, and the effector cytokines IFNγ and TNFα. It is also technically possible to multiplex the measurement of other relevant cytokines in T cell activation such as IL-6, IL-10, and IL-17A, depending on biological relevance. Measuring T cell proliferation in the same assay cells involves staining with a cell tracing fluorescent dye prior to assaying. Based on the decrease of fluorescent intensity with each cell division, T cell proliferation can be simultaneously measured.

T cell activation plays a critical role in T cell-mediated tumor cell killing in cancer immunotherapy. This assay could be adapted to measure T cell activation and tumor cell killing simultaneously by barcoding target cells with a cell tracing dye prior to co-culture. A similar assay with CAR-T and tumor cell co-culture was recently reported [22].

#### *Developing a Novel Multiplexed Immune Assay Platform to Screen Kinase Modulators… DOI: http://dx.doi.org/10.5772/intechopen.97304*

Monitoring the condition of T cell activation during the T cell biomanufacturing process is critical to the success of adoptive T cell therapy. As demonstrated in the results (**Figure 1**), this assay may be used for the daily monitoring of T cell activation, and to acquire time-sensitive activation information by checking simultaneously the early or late activation CD markers as well as the major effector cytokine secretion such as IFNγ and TNFα. As a functional assay, it may also be used for profiling neoantigens, vaccines and other drug candidates such as immune checkpoint inhibitors, bi-specific antibodies, as well as inhibitors against inflammation or autoimmune diseases (**Figure 6**).

Using high throughput flow cytometry, this immune assay may be well-suited to the drug screening environment as we demonstrated in the Z' factor characterization. For a proof of concept, we ran a small screening campaign of a chemogenomic kinase library with 152 kinase inhibitors (each with a known target kinase protein). An iQue Forecyt® Profile Map revealed 25 of the total 152 compounds were identified as hits that broadly inhibited the T cell activation parameters, including the early activation marker CD69, the late activation marker CD25, and secreted cytokines IFNγ and TNFα. Furthermore, the first 7 compounds (**Figure 3C**) showed significant inhibition of all parameters (more than 90%, compared with negative control), which suggest an upstream signal pathway simultaneously regulating the expression of all major activation markers such as CD69 and CD25 and the secretion of cytokines IFNγ and TNFα.

However, it might still be an advantage to include both CD69 and CD25 in the same assay for complete insight into the cell activation. As our results suggest, CD69 and CD25 may be decoupled in the downstream signal pathway. This is supported by the finding that 2 compounds from the library screening only inhibit the expression of the late activation marker CD25, but not the early activation marker CD69 (**Figure 4**). This finding is also supported by a similar kinase inhibitor study describing new modulators of T cell receptor signaling and T cell activation [23]. In addition, we found 3 distinct kinase inhibitors that inhibited the early expression marker CD69, but not the late activation marker CD25, which further supports a theory that expression of CD69 and CD25 are regulated differently in downstream

#### **Figure 6.**

*The potential screening and profiling positions of iQue® human T cell activation kit (TCA assay) in the immune drug discovery workflow. TCA assay may be used as secondary/functional assay to profile the drugs including immune-checkpoint inhibitors, bi-specific antibodies, adoptive TIL cells, CAR-T cells, and inflammation/autoimmune inhibitors. The TCA assay, together with the screening of a chemogenomic library, may be used as primary screening assay for drug repositioning, phenotypic drug discovery and traditional target-based drug discovery.*

pathways. In addition, the secretion of the functional cytokines IFNγ and TNFα may not share the same downstream pathway because we identified 2 different compounds (AG17, an EGFR inhibitor and Indirubin-30 -monoxime, a GSK3β inhibitor) that differentially decreased the secretion of one cytokine more drastically than the other (data not shown). Because 24-hour T cell activation was used as a biology model, it was not critical to analyze HLA-DR as an even later activation marker. This marker may be still useful in monitoring T cell activation for a longer term such as 5 to 10 days. The full analysis of different time-sensitive cell surface markers and the secreted cytokines may provide better insight of the precise T cell activation condition.

Of interest were several classes of compounds identified from the library that suggested drug target potential. A cluster of all four MEK1/2 inhibitors from the library showed a similar inhibition profile (**Supplement Figure 2**). In addition, 4 p38MAPK inhibitors, 3 Src kinase inhibitors, and 3 CaMKII inhibitors also showed similar inhibition profiles within each kinase inhibitor family (data not shown). These data suggest that the T cell activation assay, combined with a chemogenomic library screening, has the potential to identify possible drug targets for immune therapy.

In the kinase library screen, a blockbuster kinase inhibitor drug, ruxolitinib (Jak1/2 inhibitor, a myelofibrosis treatment), showed up as a positive hit. By adjusting the hit identification criteria in the iQue Forecyt® Profile Map algorithm, we identified three distinct inhibitors against three different kinase classes. These compounds showed a very similar T cell activation inhibition profile to ruxolitinib. The selection of criteria in the iQue Forecyt® Profile Map algorithm is subjective. It is possible that choosing a different threshold range for each criterion may result in a larger or smaller set of compounds that have inhibition profiles similar to ruxolitinib. In addition, it may be necessary to run in vitro and in vivo validation tests to further confirm the screening results. This small, proof-of-concept screening campaign showed that the multiplexed immune assay, integrated with a sophisticated data analysis algorithms, may help identify compounds or lesser-known existing drugs similar to a well-known drug. This capability may also provide potential new opportunities for kinase drug repositioning, as protein kinases are major oncology drug targets [24].

Aside from drug repositioning, there exists a need to uncover T cell activation biology as it relates to emerging immune-oncology therapies. While immune checkpoint inhibitors such as PD-1 have shown to be effective in mounting an anti-tumor response for both hematologic and solid tumors, there are further mechanistic details related to T cell activation to be learned. For example, a more complete understanding of the interactions between T cell receptors and ligands, upstream of T cell activation, and the modulation of immune checkpoint inhibition, would further progress the immuno-oncology field [25]. Unfortunately, checkpoint inhibitor therapy can result in tumor resistance, due to changes within the tumor cells and/or host immune response [26]. An option to mitigate checkpoint inhibition resistance is to enhance checkpoint inhibition with robust T cell activation via kinase inhibition [27]. Further advances in uncovering T cell activation dynamics as they relate to checkpoint inhibition therapy may include a more complete understanding of how an individual's microbiome might influence cancer treatment. Specifically, the interplay between microbiome diversity, microbiome metabolic signatures, the alteration of T cell activation and the efficacy of checkpoint inhibition therapy has been documented [25].

*Developing a Novel Multiplexed Immune Assay Platform to Screen Kinase Modulators… DOI: http://dx.doi.org/10.5772/intechopen.97304*

Both opportunities and challenges exist in phenotypic drug discovery. The small, proof-of-concept screening model described here may be extended to screen other chemogenomic libraries, such as ion channel/GPCR inhibitor, for possible target identification (**Figure 6**), or it may be extended to screen FDA-approved drug libraries for potential drug repositioning purposes. The drug candidates from the screening, particularly against immunological or immuno-oncology targets, can be further profiled by using this immune assay platform or a modified format. With all the characteristics of a functional assay, this assay platform may also be adapted for these applications: neoantigen or vaccine profiling; functional profiling of checkpoint inhibitor and bispecific antibodies in T cell activation and immune cell-mediated tumor cell killing; and daily monitoring T cell activation in the bio-manufacturing of the CAR-T and TIL cells used in adoptive T cell therapy.

#### **5. Conclusions**

The ability to readily characterize CD4+ and CD8+ T cell activation state and cytokine secretion is critical for implementing and expanding treatments for cancer, as well as autoimmune and inflammatory conditions. Protein kinase inhibition is an established strategy in oncology treatment, but additional insights are required to expand the portfolio of potential interventions. Key to these efforts is the ability to rapidly and simultaneously monitor cytokines and the temporal expression of T cell activation markers.

This work describes a multiplexed assay screen for protein kinase inhibition to identify compounds that alter T cell activation dynamics. A library of 152 chemogenomic kinase inhibitors was incubated with activated human T cells to determine the expression changes to both early (CD69) and late (CD25) T cell activation markers in conjunction with the cytokine secretion profiles for IFNγ and TNFα from a single assay well. High-throughput flow cytometry screen harnessing integrated, advanced data analytics determined several inhibitors of MEK 1/2 and Jak 1/2 pathways. The existing oncology drug ruxolitinib was identified in the screen and those screen parameters were used to identify 3 additional kinase inhibitors. Importantly, the 3 kinase inhibitor screen hits alter 3 distinct kinase pathways, indicating that this approach is unlikely to show bias for a particular class of kinase activation pathway(s).

#### **6. Future perspectives**

Aside from the far-reaching role of T cell activation in cancer treatment, the COVID-19 pandemic has fueled the need for further research of T cell activation dynamics. There is an unmet need to better understand and identify T cell activation related to COVID-19 infection in order to improve COVID-19 treatment [28]. The extent of T cell activation in COVID-19 is associated with either recovery from infection or poor disease prognosis, such as in the cases of severe COVID-19. Interestingly, there is evidence that the enhanced expression of the late stage T cell activation marker HLA-DR is associated with severe COVID-19 [29]. Thus, the ability to perform drug treatment screens, similar to the approach described in this work, and identifying the modulation of T cell activation while simultaneously quantifying cytokine secretion, represents a potentially useful tool for COVID-19 therapeutics as well as for other emerging infectious diseases.
