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PF-06651600, a dual JAK3/TEC family kinase inhibitor Hua Xu, Michael I. Jesson, Uthpala I. Seneviratne, Tsung H. Lin, M. Nusrat Sharif, Liang Xue, Chuong Nguyen, Robert A. Everley, John I Trujillo, Douglas S. Johnson, Gary R. Point, Atli Thorarensen, Iain Kilty, and Jean-Baptiste Telliez ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00188 • Publication Date (Web): 13 May 2019 Downloaded from http://pubs.acs.org on May 14, 2019

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ACS Chemical Biology

PF-06651600, a dual JAK3/TEC family kinase inhibitor. Hua Xu1,7, Michael I. Jesson2,7, Uthpala I. Seneviratne1,7, Tsung H. Lin3,7, M. Nusrat Sharif3, Liang Xue4, Chuong Nguyen5, Robert A. Everley5, John I. Trujillo5, Douglas S. Johnson1, Gary R. Point6, Atli Thorarensen1, Iain Kilty3, Jean-Baptiste Telliez3,*. 1Medicine

Design, Pfizer Worldwide R&D, 610 Main Street, Cambridge, MA 02139 (USA).

2Drug

Safety R&D, Pfizer Worldwide R&D, 300 Technology Square, Cambridge, MA 02139 (USA). 3Inflammation

and Immunology, Pfizer Worldwide R&D, 610 Main Street, Cambridge, MA

02139 (USA). 4Integrative 5Medicine 6Drug

Biology, Pfizer Worldwide R&D, 610 Main Street, Cambridge, MA 02139 (USA).

Design, Pfizer Worldwide R&D, Eastern Point Road, Groton, CT 06340 (USA).

Safety R&D, Pfizer Worldwide R&D, Eastern Point Road, Groton, CT 06340 (USA).

7These

authors contributed equally to this work.

*Correspondence to: [email protected]. ABSTRACT PF-06651600 was developed as an irreversible inhibitor of JAK3 with selectivity over the other three JAK isoforms. A high level of selectivity towards JAK3 is achieved by the covalent interaction of PF-06651600 with a unique cysteine residue (Cys-909) in the catalytic domain of JAK3, which is replaced by a serine residue in the other JAK isoforms. Importantly, 10 other kinases in the kinome have a cysteine at the equivalent position of Cys-909 in JAK3. Five of those kinases belong to the TEC kinase family including BTK, BMX, ITK, RLK and TEC and are also inhibited by PF-06651600. Preclinical data demonstrates that inhibition of the cytolytic function of CD8+ T cells and NK cells by PF-06651600 is driven by the inhibition of TEC kinases. Based on the underlying pathophysiology of inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, alopecia areata and vitiligo, the dual activity of PF06651600 towards JAK3 and the TEC kinase family may provide a beneficial inhibitory profile for therapeutic intervention. INTRODUCTION Inflammatory and autoimmune diseases can manifest from dysregulated signaling pathways that control immune system homeostatic and environmental responses. Cytokines and their

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downstream pathways are important players in the induction and regulation of immune responses and immune homeostasis1. Type I & II cytokines are dependent on the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway to relay signals from the extracellular environment to the nucleus of immune cells2. Functioning in pairs, the four JAK isoforms; JAK1, JAK2, JAK3 and Tyrosine kinase (TYK) 2, drive signaling through type I/II cytokine receptors3. JAK3 uniquely associates with the common gamma (c) receptor chain shared by interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15 and IL-21 receptors, and it always signals in association with JAK1 bound to beta receptor chains of the aforementioned cytokine receptors. More than twenty years ago, JAK3 was the first JAK family member to generate interest as a potential target due to its causal role in some forms of human severe combined immunodeficiency4. Except for the TYK2 selective allosteric inhibitor BMS-9861655, most JAK inhibitors that have reached clinical development for treatment of inflammatory disease indications have been JAK1 inhibitors with varying selectivity for other JAK isoforms6, 7. Importantly, the clinical consequences of JAK1 inhibition include pharmacodynamic effects such as cholesterol and liver enzyme elevation, and rapid CRP reduction, which are apparent class effects similar to IL-6 inhibitors8. Because JAK3-selective inhibition would only inhibit γcommon chain receptors signaling, it would spare IL-6 signaling, as well as other JAK1dependent immunoregulatory cytokines such as IL-10, IL-27 and IL-35, and could potentially offer better efficacy/safety ratios in the clinic that would be advantageous for the treatment of various inflammatory diseases. PF-06651600 was the first JAK3 inhibitor with high selectivity over the other JAK family members to reach clinical development9. The tyrosine kinase expressed in hepatocellular carcinoma (TEC) family of protein kinases consists of five members (Bruton’s tyrosine kinase (BTK), bone marrow tyrosine kinase on chromosome X (BMX), interleukin 2-inducible T cell kinase (ITK), resting lymphocyte kinase (RLK) and TEC) primarily expressed in hematopoietic cells10, 11. T cells express three TEC kinases, ITK, TEC and RLK that are activated downstream of the T cell receptor (TCR). BTK plays crucial roles in B cell development and function and is activated downstream of the B cell receptor (BCR). BTK and ITK are established therapeutic targets being explored for the treatment of inflammatory diseases because they are involved in transducing signals from antigen receptors on B cells and T cells, respectively12, 13. More broadly, TEC kinases are activated by a variety of signals and are involved in signal transduction pathways regulating various immunological processes in health and disease. PF-06651600 is a recently discovered JAK3 inhibitor with selectivity for JAK3 over the other three JAK isoforms. JAK3 selectivity results from irreversible covalent binding of PF-06651600 to Cys-909 in JAK3 which is replaced with a serine residue at the equivalent position in the other three JAK isoforms. PF-06651600 also irreversibly inhibits the TEC kinase family (BTK, BMX, ITK, RLK, TEC) owing to a Cys residue at the equivalent position of Cys-909 in JAK3 (Telliez et al., 2016 and Table 1). Beyond the inhibition of JAK3 and the TEC kinase family, PF06651600 displays high selectivity over the broader kinome9. Here we describe the cellular


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activity of PF-06651600, assessing effects driven by the inhibition of JAK3 and TEC kinase family members in primary human immune cells, as well as target occupancy of these kinases in primary human immune cells and in spleens from NSG mice engrafted with human peripheral blood mononuclear cells (PBMCs).



RESULTS AND DISCUSSION PF-06651600 inhibits JAK3 and the TEC kinase family. PF-06651600 was originally described as a JAK3-selective inhibitor with cross over activity into the TEC kinase family9. In biochemical assays performed in the presence of 1 mM ATP, PF-06651600 was shown to inhibit JAK3 with a 50% inhibitory concentration (IC50) of 33.1 nM as well as the TEC kinases with IC50s ranging from 155 to 666 nM (Table 2). PF-06651600 was shown to have greater than 10 µM IC50 against JAK1, JAK2 and TYK29. Since PF-06651600 demonstrated the ability to inhibit TEC kinases in biochemical assays, further functional assessment of the inhibition of JAK3 and the TEC kinase family was performed in relevant primary immune cells derived from human whole blood. Due to the nature of the respective signaling pathways and availability of proximal markers specifically related to the activity of the relevant kinases, assessing functional cellular inhibition of JAK3 is easier than for the TEC kinases. In the case of JAK3, activity can be specifically assessed in primary immune cells responsive to c-cytokine receptors by measuring the phosphorylation of STAT proteins downstream of receptor activation. For example, in human whole blood, PF-06651600 inhibited the phosphorylation of STAT5 elicited by IL-2, IL4, IL-7, and IL-15 with IC50 values of 244, 340, 407, and 266 nM, respectively, and it inhibited the phosphorylation of STAT3 elicited by IL-21 with an IC50 of 355 nM9. Similar functional assessments for the TEC kinase family are more challenging because of the paucity of robust proximal readouts that can be confidently attributed to a specific TEC family member14. Nevertheless, using downstream functional readouts and a suite of inhibitors with restricted TEC kinase family member specificity, we were able to characterize the cellular effects of PF06651600 that resulted from the inhibition of TEC kinase members as further described below. Additionally, occupancy data of JAK3 and the TEC kinase family with PF-06651600 was obtained in parallel to functional measurements. To help assess the contribution of the inhibition of TEC kinase family members to the activity of PF-06651600, the data obtained in cellular assays was compared to similar data obtained using fenebrutinib/GDC-0853, a reversible and selective BTK inhibitor15, and JTE-051 a reversible and selective ITK inhibitor (ClinicalTrials.gov Ref NCT02919475). Tofacitinib was also used in these experiments as a selective JAK inhibitor which does not inhibit the TEC kinase family. Inhibition of the eleven kinases in the kinome containing a cysteine residue at the position equivalent to Cys-909 in JAK3 were measured in the presence of 1 mM ATP (data obtained at Carna biosciences) (Table 2). Biochemical inhibition experiments demonstrated that fenebrutinib is a BTK inhibitor with an IC50 of 74 nM and with no significant inhibition of the other ten kinases. JTE-051 inhibits ITK with an IC50 of 4.4 nM with at least 10-fold selectivity over any of the other ten kinases (Table 2).


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PF-06651600 inhibits CD69 expression in T and B cells. BCR cross linking initiates an intracellular signaling cascade that promotes BTK-dependent cluster of differentiation (CD) 69 upregulation13, 16. Freshly isolated human leukocytes pretreated with PF-06651600 or other reference compounds were stimulated with anti-human IgD to cross-link the BCR, and CD69 expression on CD19+ B cells was monitored by FACS. The BTK inhibitor, fenebrutinib potently inhibited anti-IgD-induced CD69 upregulation with an IC50 of 15 nM, while the ITK inhibitor JTE-051 showed weak inhibition with an IC50 of 3.3 µM. These data suggest that anti-human IgD-induced CD69 upregulation on CD19+ B cells is mediated by BTK. PF-06651600 inhibited anti-IgD-induced CD69 upregulation with an IC50 value of 344 nM and the JAK inhibitor, tofacitinib, had no effect in this assay as demonstrated by an IC50 >20 µM (Table 3), suggesting that PF-06651600 activity in these experiments could be mediated through BTK inhibition. ITK is a key component of the TCR signaling pathway12 and TCR activation leads to upregulation of CD69 on the cell surface of T cells16. In these experiments, TCR activation was achieved by treating human CD4+ T cells with a soluble anti-CD3/CD28/CD2 tetrameric antibody complex. Pre-treatment with the ITK inhibitor, JTE-051, resulted in suppression of anti-CD3/CD28/CD2 antibody complex-induced CD69 upregulation with an IC50 of 190 nM, while treatment with the BTK inhibitor, fenebrutinib demonstrated weak inhibition with an IC50 of 8.2 µM. PF-06651600 inhibited this TCR-mediated CD69 upregulation with an IC50 of 380 nM (Table 3). Interestingly, the selective JAK inhibitor, tofacitinib, exhibited inhibition of antiCD3/CD28/CD2 antibody complex-induced CD69 upregulation with an IC50 of 326 nM, suggesting that the JAK-STAT signaling pathways may be at least partially involved in this TCR driven event. Of note, the IC50 for tofacitinib inhibition of CD69 upregulation was 19-fold higher than that for γc cytokine-induced phosphorylation of STAT5 (see Table 3), suggesting that the observed activity may be related to an indirect effect of JAK inhibition. PF-06651600 inhibits cytolytic function of NK cells and CD8+ T cells. Lymphocyte cytolytic function can be assessed by measuring the surface modulation of lysosomal granule protein CD107a (LAMP-1; lysosomal-associated membrane protein 1) and accumulation of intracellular interferon (IFN)-γ following activation of NK cells or CD8+ T cells by tumor cells or antiCD3/CD28 crosslinking, respectively17-19. The pharmacologic activity of JAK and TEC kinase inhibitors was evaluated in PBMCs by measuring the inhibition of cytolytic activity within CD8+ T cell or natural killer (NK) cell populations. In CD8+ T cells, PF-06651600 and the ITK inhibitor, JTE-051, potently inhibited degranulation and IFN-γ production (Figures 1A and 1B) at concentrations consistent with their inhibition of other T cell activation assays. In contrast, there was minimal inhibition of CD8+ T cell cytolytic activity by either tofacitinib or the BTK selective inhibitor, fenebrutinib, at concentrations below 3-10 µM. These data suggest that the activity observed with PF-06651600 does not result from inhibition of JAK3 or BTK but is consistent with inhibition of other TEC family kinases including ITK. In NK cells, PF-06651600 also inhibited degranulation and IFN-γ production at concentrations similar to its inhibition of CD8+ T cells, but JTE-051 inhibitory activity was reduced (Figures 1C and 1D) consistent with differential regulation of NK cell activation by ITK20. Similar to CD8+ T cells, there was



minimal inhibition of NK cell cytolytic activity by either tofacitinib or fenebrutinib at concentrations below 10 µM, demonstrating that the activity of PF-06651600 in NK cells is unrelated to its inhibition of either JAK3 or BTK. IC50 values for all inhibitors are depicted in Table 3. PF-06651600 occupancy of JAK3 and TEC kinases. In order to measure occupancy of JAK3 and TEC kinase family members with PF-06651600, we designed a clickable probe, PF06789402 based on the PF-06651600 scaffold (Figure 2A), which allowed for click chemistry using biotin-TAMRA azide and subsequent detection of labeled or enriched proteins by in-gel fluorescence, immunoblot and mass spectrometry (Figure 2B). PF-06789402 is a close analog of PF-06651600 and binds the same pocket of JAK3 or the TEC kinase family as PF-06651600. In biochemical assays, PF-06789402 displayed similar activities to those of PF-06651600 against JAK3 and TEC kinases (Table S1). Binding of PF-06651600 to these kinases precluded probe binding and subsequent protein enrichment. Therefore, target occupancy with PF-06651600 could be measured by subtracting the amount of probe bound to the enriched kinases from inhibitor-treated samples from the amount obtained in DMSO-treated control samples, as described previously. 21, 22 We first demonstrated a concentration-dependent labeling of ITK by PF-06789402 in Jurkat T cells (Figure S1A). A prominent fluorescent protein band at 70 kDa was observed after click chemistry with biotin-TAMRA-azide. The molecular weight and labeling pattern of the band is similar to the ITK band in the immunoblot analysis of the probe-enriched samples, suggesting that this protein is likely ITK. In addition, the 70 kDa protein was inhibited by PF-06651600 in a concentration-dependent manner, which is almost identical to the competition profile of ITK (Figure S1B), further supporting that the major protein band at 70 kDa is ITK. In subsequent experiments, 1 µM PF-06789402 was used since significantly higher background labeling was seen using 3 µM of the probe (Figure S1A). We then proceeded to use the probe to assess occupancy of ITK and JAK3 by PF-06651600 in anti-CD3/CD28/CD2 stimulated CD4+ T cells. As shown in Figure 2C, PF-06651600 treatment caused a concentration -dependent decrease of the enriched JAK3 and ITK proteins. Western blot results suggest that PF-06651600 had an OC50 (concentration achieving 50% occupancy) of 259 nM against JAK3 and 101 nM against ITK (Figure 2D). In addition, the OC50 for ITK was in agreement with the inhibition of the functional readout measuring CD69 surface expression in these cells (IC50 = 380 nM), further supporting that PF-06651600 binds and inhibits ITK in this system. The same method was applied to measure BTK occupancy in anti-IgD stimulated human leukocytes where PF-06651600 demonstrated an OC50 of 387 nM (Figure 2E and 2F), consistent with the inhibition of CD69 expression measured in these cells (IC50 = 344 nM). We attempted to measure occupancy for other TEC kinases using the western-blot format, however, that was hindered by a lack of reliable antibodies. Encouraged by the observation that PF-06789402 was able to enrich JAK3 and TEC kinases in human PBMCs (Table S2), we went further and employed tandem mass tag 10-plex (TMT-


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10plex) to determine concentration -dependent occupancy of JAK3 and TEC kinase family members by PF-06651600. It is worth noting that by using biotin azide with an acid-cleavable linker, we were able to identify probe-modified peptides in PBMCs, and confirm that the probe labeled active site Cys residues of JAK3 and three of five TEC kinases (Figure S2). Probe modified peptides for RLK and TEC were not detected, possibly due to low protein abundance or poor peptide ionization. We next performed occupancy measurements in PBMC lysates prepared from 5 different healthy human donors, and determined OC50 for PF-06651600 was 73 nM against JAK3, and 58-176 nM against the five TEC kinases (Figure 3), suggesting that PF06651600 has similar inhibitory activities against JAK3 and TEC kinase family members in cellular settings. Thus the occupancy of TEC kinases may explain the activity we observed in the cellular assays described earlier. We also applied the same methodology to measure occupancy of JAK3 and the five TEC kinases by PF-06651600 in NOD-scid IL2r nul mice engrafted for 3-weeks with human PBMCs23, 24. Following a single oral administration of PF-06651600 (30 mg/kg) or vehicle, mice were euthanized at six different time points post-dose (0.25, 3, 5, 12, 24, 48 hr). Spleens were excised and homogenized, then labeled and enriched using the PF-06789402 clickable probe, and analyzed by mass spectrometry (Figure 4A) as described earlier. By comparing the protein abundance of the enriched samples from vehicle and PF-06651600-dosed animals, we monitored the kinase occupancy in a time-dependent manner. As shown in Figure 4B, a single oral dose of 30 mg/kg PF-06651600 leads to nearly 100% occupancy of JAK3 and most of the TEC kinases, except RLK, in the spleen samples from early time points (0.25 to 5 hrs). At later time points occupancy gradually decreased due to compound clearance and protein resynthesis. The relatively large variation in the occupancy measurements likely resulted from differing levels of human PBMC engraftment between individual mice. Interestingly, despite that variation, the time-dependent occupancy patterns appeared consistent with anticipated protein half-life. Specifically, BTK is reported to have a much longer half-life25, 26 than JAK39 or ITK27 (about 12-24 hrs for BTK, 2-3 hr for JAK3 and ITK). Indeed we observed a slower recovery of unbound BTK compared to JAK3 and ITK. This experiment demonstrated that PF-06651600 achieved maximum occupancy of JAK3 and TEC kinases in vivo, further suggesting that the compound has dual JAK3 and TEC family inhibitory activity. CONCLUSION PF-06651600 inhibits JAK3 with high selectivity over other JAK isoforms due to irreversible covalent binding to a Cys residue at position 909 in the catalytic domain that is replaced with a serine residue in other JAKs. PF-06651600 also inhibits TEC kinase family members that possess a Cys residue at the equivalent position to Cys-909 in JAK39. Although the cellular effects of JAK3 inhibition on cytokine signaling have been described previously9, the work described in this study highlight the effects of PF-066516600 on immune cell functions resulting from inhibition of TEC kinase family members rather than those of JAK3. The TEC kinase



family of tyrosine kinases is primarily expressed in immune cells10, 11, and both BTK and ITK have been shown to be essential for antigen receptor signaling in B and T cells respectively12, 13. The functional effect of PF-06651600 on BTK was assessed in B cells by measuring CD69 surface expression following BCR activation with antibodies. PF-06651600 inhibited CD69 surface expression in this assay. In contrast, the JAK inhibitor tofacitinib and the ITK selective inhibitor, JTE-051, either didn’t inhibit or weakly inhibited CD69 surface expression while the BTK selective inhibitor, fenebrutinib potently inhibited CD69 surface expression. These data strongly support that CD69 expression in this assay is BTK-dependent and both JAK and ITKindependent. These data also suggest that the effect of PF-06651600 in this assay is related to BTK inhibition. A similar approach was utilized to assess the effect of PF-06651600 on ITK by using an ITK-dependent T cell assay measuring CD69 surface expression following activation of the TCR on CD4+ T cells. Both PF-06651600 and the ITK selective inhibitor, JTE-051 inhibited CD69 surface expression on CD4+ T cells while the BTK selective inhibitor, fenebrutinib, demonstrated weak inhibitory activity. Interestingly, the selective JAK inhibitor tofacitinib also inhibited CD69 surface expression in this assay, but displayed a shallow curve (data not shown) suggesting that there could be an indirect mechanism involved for this inhibitor. Collectively, these data demonstrate that CD69 surface expression on CD4+ T, following TCR activation, is an ITK-dependent mechanism as demonstrated by the effect of the ITK-selective inhibitor, JTE051. The effect of PF-06651600, in the same assay, is also most likely driven by the inhibition of ITK, although contributions from the inhibition of TEC or RLK cannot be excluded. Further evidence of the effect of PF-066516600 on the TEC kinase family was obtained in functional assays measuring cytolytic activity of CD8+ T cells and NK cells17-19. In CD8+ T cells, PF-06651600 inhibited degranulation and IFN-γ production, both measures of functional activation. In contrast, tofacitinib and fenebrutinib showed minimal inhibition demonstrating the JAK- and BTK-independent nature of the assays. Conversely, the ITK inhibitor, JTE-051, inhibited both degranulation and IFNγ production, demonstrating the ITK-dependence of the assays. Altogether, these data suggest that PF-06651600 inhibits cytolytic functions of CD8+ T cells through inhibition of ITK. In NK cell assays, similar results were observed with PF06651600, as well as with tofacitinib and fenebrutinib. The main difference observed was with JTE-051, which demonstrated weaker inhibition of degranulation and IFNγ production as compared to its activity in CD8+ T cells (Table 3). The fact that PF-06651600 had similar activity in CD8+ T cells and NK cells, while JTE-051 demonstrated a shift in activity between the two cell types, suggests that inhibition of TEC or RLK by PF-06651600 could be responsible for the stronger inhibition in NK cells compared to the ITK-selective inhibitor JTE-051, which does not inhibit TEC or RLK. To assess the occupancy of JAK3 and TEC kinases in a cellular setting, a clickable probe, PF06789402, was designed based on the PF-06651600 scaffold. Since PF-06789402 binds and inhibits JAK3 and the TEC kinases (Table S1), it could be used to enrich probe labeled proteins from cellular lysates. Covalent binding of PF-06651600 prior to the addition of PF-06789402


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precluded probe binding to and subsequent enrichment of target proteins, therefore allowing quantification of the amount of JAK3 and TEC kinases bound by PF-06651600. Measurements in human T cells or PBMC lysates as well as from the spleens of NSG mice engrafted with human PBMCs showed similar levels of occupancy of JAK3 and TEC kinases by PF-06651600 (Figures 2 & 3), which were consistent with the inhibitory activities obtained for these kinases in the various primary human cell assays described in this study (Table 3). Further work will be required to further characterize the relationship between the occupancy of JAK3 and the TEC kinase family members in vivo, and the functional and biological outcomes resulting from the course of their inhibition over time with PF-06651600. In this study we have shown that PF-06651600, a JAK3 and TEC kinase family irreversible inhibitor, blocks immune cellular functions that are dependent on either BTK, ITK or other TEC family members. These results further advance the understanding of the mechanism of action of PF-06651600 in regard to the inhibition of JAK3 and the TEC kinase family at the cellular level. Early clinical pharmacodynamic data with PF-06651600 have shown the absence of several of the effects characteristic of JAK1 inhibitors, consistent with PF-06651600’s selectivity profile over other JAK family members (Pfizer Inc., unpublished data). Further preclinical and clinical studies will be required to further assess the biological impact of the inhibition of JAK3 and TEC kinase family in patients with inflammatory and autoimmune diseases. EXPERIMENTAL PROCEDURES Methods and procedures are provided in the Supporting Information.

ASSOCIATED CONTENT Supporting Information This material is available free of charge via the internet at http://pubs.acs.org Methods; Figures S1 & S2, Tables S1 to S3

ACKNOWLEDGMENTS We want to thank H. Liu for her technical expertise and support with the NSG mice work. All authors of this manuscript are or were employees of Pfizer.

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[20] Khurana, D., Arneson, L. N., Schoon, R. A., Dick, C. J., and Leibson, P. J. (2007) Differential regulation of human NK cell-mediated cytotoxicity by the tyrosine kinase Itk, J Immunol 178, 3575-3582. [21] Xu, H., Gopalsamy, A., Hett, E. C., Salter, S., Aulabaugh, A., Kyne, R. E., Pierce, B., and Jones, L. H. (2016) Cellular thermal shift and clickable chemical probe assays for the determination of drugtarget engagement in live cells, Org Biomol Chem 14, 6179-6183. [22] Xu H, R. L., Chou S, Pierce B, Narayanan A, and Jones, L. H. (2017) Quantitative measurement of intracellular HDAC1/2 drug occupancy using a trans-cyclooctene largazole thiol probe, Med. Chem. Comm. 8, 767-770. [23] Ehx, G., Somja, J., Warnatz, H. J., Ritacco, C., Hannon, M., Delens, L., Fransolet, G., Delvenne, P., Muller, J., Beguin, Y., Lehrach, H., Belle, L., Humblet-Baron, S., and Baron, F. (2018) Xenogeneic Graft-Versus-Host Disease in Humanized NSG and NSG-HLA-A2/HHD Mice, Front Immunol 9, 1943. [24] Ali, N., Flutter, B., Sanchez Rodriguez, R., Sharif-Paghaleh, E., Barber, L. D., Lombardi, G., and Nestle, F. O. (2012) Xenogeneic graft-versus-host-disease in NOD-scid IL-2Rgammanull mice display a Teffector memory phenotype, PLoS One 7, e44219. [25] Evans, E. K., Tester, R., Aslanian, S., Karp, R., Sheets, M., Labenski, M. T., Witowski, S. R., Lounsbury, H., Chaturvedi, P., Mazdiyasni, H., Zhu, Z., Nacht, M., Freed, M. I., Petter, R. C., Dubrovskiy, A., Singh, J., and Westlin, W. F. (2013) Inhibition of Btk with CC-292 provides early pharmacodynamic assessment of activity in mice and humans, J Pharmacol Exp Ther 346, 219228. [26] Saffran, D. C., Parolini, O., Fitch-Hilgenberg, M. E., Rawlings, D. J., Afar, D. E., Witte, O. N., and Conley, M. E. (1994) Brief report: a point mutation in the SH2 domain of Bruton's tyrosine kinase in atypical X-linked agammaglobulinemia, N Engl J Med 330, 1488-1491. [27] Zapf, C. W., Gerstenberger, B. S., Xing, L., Limburg, D. C., Anderson, D. R., Caspers, N., Han, S., Aulabaugh, A., Kurumbail, R., Shakya, S., Li, X., Spaulding, V., Czerwinski, R. M., Seth, N., and Medley, Q. G. (2012) Covalent inhibitors of interleukin-2 inducible T cell kinase (itk) with nanomolar potency in a whole-blood assay, J Med Chem 55, 10047-10063.

FIGURES AND LEGENDS Table 1. Kinases amino acid sequence alignment. Position (in JAK3) JAK3 JAK1 JAK2 TYK2 BLK BMX BTK EGFR HER2 HER4 ITK

904 905 906 907 908 909 912 940 945 947 948 953 954 956 Y L P S G C D L C H R R N L F L P S G S E L Y H R R N L Y L P Y G S D L Y H R R N L Y V P L G S D L Y H R R N L Y M A R G C D I S H R A N L Y I S N G C N L F H R R N L Y M A N G C N L F H R R N L L M P F G C D L L H R R N L L M P Y G C D L L H R R N L L M P H G C E L L H R R N L F M E H G C D L V H R R N L



TEC RLK MAP2K7

F F L

M M M

E E -

R N G

G G T

C C C

N N K

L L L

Page 12 of 17

F Y V

H H H

R R R

R R S

N N N

L L L

Amino acid sequence alignment of the 4 JAK isoforms and 10 other kinases containing a Cys residue at the equivalent position of Cys-909 in JAK3.

Table 2. Kinase inhibition of Cys containing kinases with tofacitinib, JTE-051, fenebrutinib and PF-06651600.

Kinase

Tofacitinib CP-690550

Fenebrutinib RG7845

JTE-051

PF-06651600

BLK

NA

5,120

> 10,000

> 10,000

BMX

> 10,000

174

8,181

666

BTK

> 10,000

469

74

404

EGFR

NA

> 10,000

> 10,000

> 10,000

HER2

NA

> 10,000

> 10,000

> 10,000

HER4

NA

> 10,000

> 10,000

> 10,000

> 10,000

4.4*

> 10,000

395*^

JAK3

55*

> 10,000

> 10,000

33.1*

TEC

> 10,000

486

1,676

403

RLK

> 10,000

323

> 10,000

155

NA

> 10,000

> 10,000

> 10,000

ITK

MAP2K7

The IC50 values for the 4 compounds were determined in the presence of 1 mM ATP at Carna Biosciences. The IC50 values were calculated from concentration vs. % inhibition curve fitting to a four parameter logistic curve with duplicate data points. NA: not available. * Pfizer internal data. ^This IC50 value is different from the previously published data of 8510 nM9 obtained for ITK at Carna Biosciences. We found that IC50 values obtained for ITK at Carna Biosciences were significantly higher than those obtained at Pfizer for multiple compounds including PF06651600 and JTE-051 (Table S3). Generally, values obtained at Pfizer for ITK are in better agreement with cellular data.


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Table 3. Cellular effects of tofacitinib, JTE-051, fenebrutinib and PF-06651600 on immune cell functions.

Readout PF-06651600 Tofacitinib (JAK1/3) Fenebrutinib (BTK) JTE-051 (ITK)

IL-15 induced p-STAT5 in PBMC (JAK1/3)

B lymphocyte anti IgD induced CD69

p-STAT5

CD69

T lymphocy te anti CD2/3/28 induced CD69 CD69

52 (n=13) 17 (n=306) 4900 (n=2)

344 (n=3) >20000 (n=2) 15 (n=2)

3600 (n=2)

3300 (n=2)

CD8+ T lymphocyte CD3/28 induction

NK cell K562 stimulation

CD107a

IFNγ

CD107a

IFNγ

380 (n=18) 326 (n=2) 8200 (n=3)

213 (n=9) 22800 (n=9) 9100 (n=6)

185 (n=9) 21200 (n=9) 12100 (n=6)

509 (n=9) >30000 (n=7) >30000 (n=2)

188 (n=9) 15500 (n=7) 12100 (n=2)

190 (n=7)

120 (n=6)

160 (n=6)

2200 (n=2)

790 (n=2)

IC50 values (nM) represent the geometric mean. (n) Denotes the number of independent experiments. A

B

125

125

PF-06651600

75

Tofacitinib

50

JTE-051

25

Fenebrutinib

100

% of Control

% of Control

100

Tofacitinib

50

JTE-051

25

Fenebrutinib

0

0 -25 0.01

0.1

1

10

PF-06651600

75

-25 0.001

100

0.001

0.01

Concentration (M)

0.1

C

Tofacitinib

50

JTE-051

25

Fenebrutinib

0 -25 0.1

1

10

100

% of Control

PF-06651600

75

0.01

10

100

D

100

0.001

1


125

% of Control

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


125 100 75 50 25 0 -25 -50 -75

PF-06651600 Tofacitinib JTE-051 Fenebrutinib

0.001

0.01


0.1

1

10

100


Figure 1. Inhibition of cytolytic function in activated CD8+ T cells or NK cells.



Inhibition of cytolytic activity in CD8+ T cells as measured by CD107a expression (A) or IFN γ expression (B). Inhibition of cytolytic activity in NK cells as measured by CD107a expression (C) or IFN γ expression (D). Data from representative experiment in n=2 donors.

Figure 2. JAK3, BTK and ITK target occupancy measurement in human CD4+ T cells or human leukocytes. (A) Chemical structures of PF-06651600 and PF-06789402. (B) Workflow of probe-based occupancy assay. (C-D) ITK and JAK3 occupancy in human CD4+ T cells. Representative immunoblot images of concentration-dependent inhibition of probe labeling by


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PF-06651600 in CD4+ T cells (C), and the corresponding occupancy curves (D). (E-F) BTK occupancy by PF-06651600 in human leukocytes. Representative images of concentrationdependent blockage of BTK labeling by PF-06651600 in anti-IgD stimulated human leukocytes (E), and the BTK occupancy curve (F).

Figure 3. Occupancy of JAK3 and TEC kinases by PF-06651600 in human PBMC lysate. (A) Representative dose response occupancy curves for JAK3 and the 5 TEC family members. (B) OC50 values (concentration needed to reach 50% occupancy). N = number of biological replicates; SD = standard deviation; SEM = standard error of the mean.



Figure 4. In vivo occupancy measurements of JAK3 and TEC kinases with PF-06651600 in spleen of NSG mice engrafted with human PBMCs. (A) NSG mice were engrafted with human PBMCs for three weeks. After a single dose of vehicle or PF-06651600 (30 mg/kg, PO), animals were sacrificed and spleens collected at the indicated time points (n=3 per time point per treatment group). Spleen homogenates were then labeled by PF-06789402, and the enriched proteins were further analyzed by mass spectrometry. (B) Percentage of unbound active JAK3 and TEC kinases in human PBMC engrafted NSG mouse model at the indicated time points.


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254x190mm (96 x 96 DPI)


TEC Family Kinase Inhibitor | ACS

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