Discovery of an ITK and TRK kinase inhibitor for the potential topical treatment of atopic dermatitis

Domain: chemistry
Published: 2026-03-09
Editorial outcome: major_revision

Abstract
Interleukin-2-inducible T cell kinase is expressed by T cells and amplifies T cell receptor-dependent signals. Interleukin-2-inducible T cell kinase deletion or inhibition reduces production of interleukin-4 and interleukin-13, key drivers of atopic dermatitis. Nerve growth factor signals via the receptor tropomyosin-related kinase A and may promote pruritus in atopic dermatitis lesions. Here we describe PF-07245303, a compound which potently inhibits interleukin-2-inducible T cell kinase and tropomyosin-related kinase family kinases capable of inhibiting T cell receptor-mediated cytokine production from CD4 and CD8 T cells and suppressing nerve growth factor-induced human basophil activation. In human skin explants, PF-07245303 demonstrates inhibition of tropomyosin-related kinase A phosphorylation, suppresses cytokine expression from T cell receptor-activated resident T cells and reverses the expression of atopic dermatitis associated genes. Topical application of PF-07245303 reduces proinflammatory and epidermal changes in a dermatitis model using female mice. By inhibiting both pathogenic inflammatory mechanisms, PF-07245303 may have therapeutic value for patients with atopic dermatitis.

Introduction
Atopic dermatitis (AD) is the most common chronic inflammatory skin disease with a 25% prevalence in children and a 7% prevalence in adults1. Atopic dermatitis patients suffer from dry and pruritic skin lesions which can affect their quality of life2,3. Genetic and environmental factors can contribute to skin barrier disruption and to immune hyper-activation, which are key drivers of atopic dermatitis pathogenesis3. T cell driven inflammation contributes to atopic dermatitis pathogenesis, and topical agents which can suppress the aberrant T cell activity may provide benefit to patients with this disease1,4.

Interleukin-2-inducible T cell kinase (ITK) is a member of the Tec family of tyrosine kinases5,6. ITK expression is largely limited to immune cells such as T, NK, NKT, and mast cells5,7. In T cells, ITK amplifies T cell receptor (TCR)-dependent signals leading to cytokine production and cell proliferation8–10. Upon TCR activation, lymphocyte-specific protein tyrosine kinase (LCK) phosphorylates and activates ITK which in turn phosphorylates phospholipase C-γ1 (PLC-γ1)10,11. Activated PLC-γ1 generates second messengers diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP ) via the hydrolysis of phosphatidylinositol 4,5-biphosphate (PIP ), and these second messengers activate protein kinase C theta (PKCθ) and calcium mobilization leading to activation of transcription factors NF-κB and NFAT and cytokine expression11,12. ITK is important for in vivo T cell responses, as ITK-deficient mice and/or mice treated with an ITK kinase inhibitor demonstrate reduced disease in models of type I diabetes, multiple sclerosis, lymphoproliferative disease, colitis, allergy/asthma, and airway hyperresponsiveness9,13–18. Transgenic mice expressing kinase-dead ITK are protected from OVA-induced pulmonary inflammation, supporting the role for ITK kinase activity in mediating Th2 inflammation19. Moreover, ITK-deficient mice or mice treated with a reported ITK kinase inhibitor demonstrate reduced skin inflammation in contact hypersensitivity-induced and imiquimod-induced models of dermatitis20–22.

The pathogenic role for T cells and the Th2 cell-derived cytokines IL-4 and IL-13 in atopic dermatitis has been demonstrated through the clinical efficacy of dupilumab, an antibody to the IL-4 receptor that blocks the activity of both IL-4 and IL-131. ITK deletion or inhibition of ITK kinase activity in T cells results in suppression of TCR-induced IL-4 and IL-13 production1,15,23. Elevated levels of ITK were described in peripheral T cells from patients with moderate to severe atopic dermatitis, and ITK expression is elevated in skin lesions from atopic dermatitis patients20,24. Additionally, Th1 cells, Th22 cells, and Th17 cells and the cytokines which they produce, IFNγ, IL-22, and IL-17, respectively, also contribute to atopic dermatitis pathogenesis1,3,4. The inactivation of ITK kinase activity has also been demonstrated to reduce Th1 and Th17 cell cytokine expression25. A therapeutic approach to inhibit the activation of these different T cell subtypes and to suppress the cytokines which they produce might provide efficacy beyond antagonism of only IL-4 and IL-13 signaling in atopic dermatitis patients.

Pruritus is a major factor contributing to reduced quality of life for atopic dermatitis patients2,26. Nerve growth factor (NGF) is a neurotrophic factor which regulates development, growth, and maintenance of neurons, and NGF can sensitize nociceptors and promote pruritus in the skin27–29. NGF signals via the receptor tropomyosin-related kinase A (TRKA), and both NGF and TRKA are present in skin27,28. Expression of NGF and TRKA is enhanced in atopic dermatitis skin lesions, and levels of NGF in skin lesions from atopic dermatitis patients correlate with itch severity27,28,30–32. TRKA and receptor kinases tropomyosin-related kinase B (TRKB) and tropomyosin-related kinase C (TRKC) are expressed by cells in the skin such as keratinocytes, neurons, mast cells, and basophils28,33. NGF induces keratinocyte proliferation, promotes basophil activation, stimulates mast cell degranulation, and contributes to neurogenic itch and inflammation28,31,33,34. A therapy which can suppress pruritus might provide symptomatic relief for patients with AD, and it may break the itch-scratch cycle which contributes to barrier disruption, further exacerbating the course and chronicity of the disease.

Here we describe PF-07245303, a dual inhibitor of ITK and TRK (TRKA, TRKB, and TRKC), kinases with potent activity in biochemical and cell-based assays. PF-07245303 was designed with properties suitable for topical formulation and delivery. This compound demonstrated potent inhibition of TRKA signaling, T cell activation, and cytokine expression in human skin explant models. A compound capable of inhibiting ITK kinase activity and TRK family kinase activity may have therapeutic value in atopic dermatitis and other numerous inflammatory, including allergic skin diseases, such as contact hypersensitivity, by suppressing dermal inflammation.

Results
Identification of PF-07245303, binding profile, and kinase selectivity
Efforts to discover ITK inhibitors have been described35, including Pfizer’s efforts to identify a potential allosteric inhibitor and an ATP competitive covalent inhibitor36,37. The discovery of PF-07245303 included use of structure-based drug design with parallel optimization for desired properties of a soft topical drug, including permeability and solubility to enable access skin tissue compartments and high metabolic clearance to reduce systemic exposure (Figure 1a). Literature examples35,37 were screened in an ITK biochemical assay using 1 mM ATP, and the kinase hinge binding interaction was optimized to lead to the identification of a 2-(1H-pyrazol-5-yl)-1H-benzo[d]imidazole hinge binding moiety. This moiety makes three-point hydrogen bond contacts with the carbonyl of Glu436 and the amide NH and carbonyl of Met438 (Figure 1b). The type 1 ATP competitive inhibitor makes extensive hydrophobic interactions with the ATP site of the kinase: for example, the cyclopropyl group makes contacts with side chains of Val377, Ala389, Lys391 and Phe435. Furthermore, the cyclopropyl moiety reduces planarity in the molecule (Figure 1b) and may contribute to selectivity against other kinases. Structure activity relationship studies identified the 5-methylhexahydrocyclopropane moiety, important for key lipophilic interactions with the p-loop region and improved potency and cellular permeability. Further modulation of physicochemical and ADME properties was achieved by incorporation of a 2-morpholinopyranmide, a weakly basic group with high sp3 character to provide solubility in topical formulations. In addition to potent inhibition of ITK, crossover inhibition of TRKA, B and C kinases was discovered38. The identification of this selective polypharmacology, although not an initial objective, offered the potential value of additional efficacy and warranted further investigation.

The binding kinetics of PF-07245303 to its targets were determined by flowing compound dilutions over immobilized recombinant protein and measuring binding and dissociation. Kinetic rate constants (k and k ), binding affinity (K ), and T were calculated. The affinity for PF-07245303 binding to ITK was 0.71 nM. Binding affinities to TRKA, TRKB, and TRKC were 2.94, 8.73, and 10.0 nM respectively (Figure 1c).

The physicochemical properties of PF-07245303 were tuned for the topical drug space39 (Figure 1d). PF-07245303 demonstrates high permeability as judged by MDCK-LE cell line (13 x 10-6 cm/s) and moderate-to-high clearance in human hepatocyte incubation (30 µL/min/mil)40. The human clearance of PF-07245303 was predicted to be 11 mL/min/kg from scaled human hepatocytes using the parallel tube model. This predicted moderate-to-high human clearance is consistent with a soft drug approach in which the concentration of the drug is high at the site of action, i.e., skin, while the drug is rapidly cleared from systemic circulation41. Additionally, crystalline PF-07245303 displays water solubility of 40.5 µM at pH 7.4.

To confirm that PF-07245303 is a potent and selective inhibitor of ITK and TRK family kinases, it was tested at 1 µM in a screening panel of 218 kinases (Figure 1e, Supplemental Table 1) at 1 mM ATP. PF-07245303 demonstrated the highest percent inhibition for kinases ITK (100%), TRKA (101%), TRKB (99%), and TRKC (97%) (Figure 1e). Follow up IC determinations were performed for kinases with inhibition greater than 50% of maximal response at 1 µM. All kinases identified except for Fms related receptor tyrosine kinase 3 (FLT3) (84%) showed greater than 38-fold selectivity compared to ITK. A follow-up cell-based NanoBRET system using HEK293 cells was used to determine cellular selectivity versus FLT3. Cellular activity in this assay showed ITK potency with an IC of 9.26 nM compared to IC of 1,130 nM (Supplemental Table 2) for FLT3 thereby supporting >100-fold selectivity of PF-07245303 for ITK vs FLT3 in a more physiologically relevant context. In addition, selectivity against related Tec family kinases BTK, TEC and LCK, which is a critical kinase for T cell receptor signaling, was evaluated. As shown in Supplemental Table 2, PF-07245303 demonstrated greater than 100-fold selectivity against these other kinases relative to ITK in the NanoBRET kinase cell-based assay.

PF-07245303 is a potent inhibitor of ITK kinase activity and inhibits ITK-dependent signaling and T cell activation
PF-07245303 demonstrated potent inhibition of ITK kinase activity in a biochemical assay using full-length human ITK protein. PF-07245303 inhibited ITK enzymatic activity in the presence of 1 mM ATP with an IC of 5.62 nM (Figure 2a). PLCγ1 is a substrate for ITK kinase, and PLCγ1 is phosphorylated in response to TCR-dependent signaling6. We assessed the cellular potency of PF-07245303 by measuring inhibition of ITK-mediated phosphorylation of PLCγ1 on residue Y783 in stimulated Jurkat T cells. Immunoblot analysis of lysates from Jurkat T cells stimulated with anti-CD3 and anti-CD28 antibodies in the presence of PF-07245303 demonstrated concentration-dependent inhibition of PLCγ1 phosphorylation with an IC of 154 nM (Figure 2b).

We next evaluated the ability of PF-07245303 to inhibit TCR-induced activation in isolated primary human T cells and T cells in human blood. As shown in Figure 2c, PF-07245303 demonstrated potent inhibition of anti-CD3 and anti-CD28 antibody-mediated stimulation of the T cell derived cytokine IL-2 from isolated human CD4+ T cells with an IC value of 38.5 nM. PF-07245303 was profiled for translation of potency in the highly proteinaceous human blood environment by measuring inhibition of IL-2 production from human blood T cells stimulated in vitro with anti-CD3, anti-CD28 and anti-CD2 antibodies. PF-07245303 inhibited the release of IL-2 in blood with an IC value of 1350 nM (Figure 2d). PF-07245303 was determined to be moderately bound to human plasma protein with mean fraction unbound values of 0.144 as determined by equilibrium dialysis at 2 M with blood to plasma ratio of 1.6042,43. Correcting this whole blood IC value for unbound intrinsic compound results in a potency of 122 nM. To rule out the possibility that the TRK kinase inhibition is responsible for the cytokine inhibition, we tested a compound, PF-0627334044, which selectively inhibits TRK kinases, in the human T cell and whole blood assays. As shown in Supplemental Figure 1, PF-06273340 did not inhibit ITK kinase activity and did not suppress TCR-induced T cell IL-2 production. Thus, PF-07245303 is a potent inhibitor of ITK kinase activity and suppresses T cell activation as demonstrated by inhibition of TCR-induced, ITK-dependent PLCγ1 phosphorylation and suppression of IL-2 production from activated human T cells in vitro.

PF-07245303 inhibits cytokine production from CD4+ T cells, Th1, Th2, and Th17 differentiated cells, and CD8+ T cells
Activated T cells can secrete multiple cytokines which contribute to the pathogenesis of inflammatory diseases including atopic dermatitis1. Therefore, we evaluated the ability of PF-07245303 to suppress the anti-CD3 and anti-CD28 antibody-induced cytokine production from primary human CD4+ T cells. In this assay system, PF-07245303 inhibited the release of pro-inflammatory cytokines IL-4, IL-13, IFNγ, IL-17A, and the anti-inflammatory cytokine IL-10 (Figure 3a – e), with IC values of 73.5 nM, 348 nM, 73.2 nM, 282 nM, and 114.5 nM respectively. TRK family selective compound PF-06273340 did not inhibit T cell production of IL-4, IL-13, IL-17A, nor IFNγ indicating that inhibition of TCR activation-induced cytokines by PF-07245303 is dependent on ITK kinase inhibition (Supplemental Figure 2a-d).

Th1, Th2, and Th17 are subsets of effector CD4+ T cells which produce an array of cytokines which contribute to inflammatory and autoimmune disease5,12. Therefore, we sought to evaluate the impact of ITK kinase inhibition with PF-07245303 on the differentiation of naïve CD4+ T cells to Th1, Th2 and Th17 cells subsets and subsequent cytokine production. In a dose-dependent manner, PF-07245303 inhibited the release of IFNγ, IL-13 and IL-17A from stimulated human naïve CD4+ T cells skewed to Th1, Th2 and Th17 (Figure 3f – h), respectively, with IC values of 35.4 nM for IFNγ release, 60.9 nM for IL-13 release, and 12.5 nM for IL-17A release. In summary, PF-07245303 inhibited the production of pro-inflammatory cytokines IFNγ, IL-13, and IL-17A which can drive inflammatory diseases including vitiligo45, atopic dermatitis1, and psoriasis4, respectively. Moreover, CD8+ T cells and IFNγ can contribute to skin conditions such as vitiligo45 and alopecia areata46. Therefore, we demonstrated that PF-07245303 was also capable of potently inhibiting IFNγ release from TCR-activated human CD8+ T cells with an IC of 18.9 nM (Supplemental Figure 2e).

PF-07245303 is a potent inhibitor of tropomyosin-related kinase receptor TRKA, TRKB, and TRKC kinase activity
Pruritus associated with atopic dermatitis can reduce patient quality of life and contribute to barrier disruption which amplifies disease3,26. As NGF can sensitize nociceptors and promote pruritus in the skin27–29, inhibition of the receptor kinase TRKA which transmits NGF signals may reduce itch and benefit patients with AD. In addition to its ability to inhibit ITK kinase activity, PF-07245303 is a potent inhibitor of tropomyosin-related kinase receptors TRKA, TRKB, and TRKC activity in biochemical assays. Using the cytoplasmic domains of human TRKA, TRKB, and TRKC, PF-07245303 inhibited the kinase activity of these enzymes in the presence of 1 mM ATP with mean IC values of 7.2 nM, 12.0 nM, and 20.7 nM, respectively (Figure 4a – c).

The cell-based potency of PF-07245303 was determined at DiscoverX using their PathHunter® system which uses an enzyme functional complementation technology. This assay was used to assess the inhibitory effects of PF-07245303 and the highly potent and selective pan-TRK inhibitor PF-06273340 on TRKA-, TRKB- or TRKC-dependent phosphorylation. Cells utilized in the assay were U2OS cells which expressed human TRKA, TRKB or TRKC and the low affinity pan-neurotrophin receptor p75 (Figure 4d – f). PF-07245303 demonstrated inhibition of TRKA-, TRKB- or TRKC-dependent phosphorylation in the presence of p75 with IC values of 72.6 nM, 48.7 nM, and 21.7 nM respectively. Similar potencies were observed in the same assay system when the cells expressed TRKA, TRKB, or TRKC in the absence of p75 (Supplemental Figure 3a – c). Furthermore, we sought to evaluate the activity of this compound in primary human cells with endogenous expression of TRKA. To that end we measured the ability of PF-07245303 to suppress NGF-induced activation of human basophils which express TRKA mRNA and protein and are associated with inflammatory skin diseases47,48. PF-07245303 and the selective pan-TRK inhibitor PF-06273340 demonstrated inhibition of NGF-dependent increases in the percent of human blood cells positive for CD203c, a marker of basophil activation and degranulation47,49,50 with IC values of 442 nM and 109 nM, respectively (Figure 4g). Correcting for protein binding yields intrinsic compound potencies of 39.8 nM and 16.3 nM in this assay. We also evaluated the potential role for ITK in IgE receptor-dependent signaling in human basophils. PF-07245303, which does not inhibit BTK in the conditions tested, is effective at reducing IgE receptor-mediated activation of primary human basophils, as measured by IL-4 release (Supplemental Figure 4). Collectively, these data demonstrate that PF-07245303 is also a potent inhibitor of TRKA, TRKB, and TRKC receptor kinases.

Measurement of NGF-induced TRKA phosphorylation in ex vivo human skin tissue and effect with PF-07245303 treatment
As inhibition of ITK and TRK kinase activity might provide utility in the treatment of inflammatory skin diseases, we sought to demonstrate pharmacological activity of PF-07245303 in an ex vivo human skin model. NGF selectively binds to TRKA inducing receptor dimerization and TRKA autophosphorylation51. Quantification of the reduction in NGF-induced TRKA phosphorylation provides a proximal measure of TRKA kinase inhibition and target engagement. To this end, we established a previously described experimental system to measure TRKA phosphorylation and target engagement in human skin explants when the skin tissue is treated with NGF52. We also employed our observations that Th2 cytokines can enhance TRKA expression by pre-treating the skin sections with IL-4 and IL-13 for 24 hours before stimulating the ex vivo skin with NGF. This experimental system allowed for the measurement of compound-dependent inhibition of TRKA phosphorylation in human skin tissue treated with PF-07245303.

Following pretreatment with IL-4 and IL-13, human ex vivo skin sections were treated with NGF for 5 minutes and TRKA detected in skin protein lysates by an ELISA method to capture human TRKA followed by detection of phosphorylated TRKA using a phospho-TRKA antibody. As shown in Figure 4h, we observed an enhancement in levels of phospho-TRKA in skin sections pretreated with Th2 cytokines and stimulated with NGF relative to untreated or only Th2 cytokine treated skin. In skin sections treated with PF-07245303 (10 µM to 0.1 µM) at the time of NGF treatment, we observed concentration-dependent inhibition with almost complete inhibition of phospho-TRKA levels with 10, 3, and 1 µM compound (Figure 4h, Supplemental Table 3). Similarly, 10 µM of the potent and selective pan-TRK inhibitor PF-06273340 decreased TRKA phosphorylation. These results demonstrate that PF-07245303 inhibits activation of TRKA in human skin tissue indicating that adequate concentrations of PF-07245303 were achieved to engage the TRKA target and to demonstrate pharmacology.

PF-07245303 inhibits activation of resident dermal T cells in ex vivo human skin sections stimulated with anti-CD3 and anti-CD28 antibodies
We showed that PF-07245303 could inhibit activation of TRKA kinase in NGF-treated ex vivo human skin. Infiltration of T cells and production of T cell-derived cytokines can contribute to atopic dermatitis, psoriasis, and other dermatological diseases 4. Therefore, we sought to evaluate the capability of the ITK and pan-TRK kinase inhibitor PF-07245303 to suppress activation of skin-resident T cells and reduce the production of pro-inflammatory cytokines. Treatment of human ex vivo skin sections with anti-CD3 and anti-CD28 antibodies activates skin-resident T cells and induces expression of pro-inflammatory cytokines53. As shown in Figure 5a – c, we demonstrated that anti-CD3 and anti-CD28 antibodies induce expression of IFNG, IL13, and IL2 in stimulated ex vivo human skin tissue and enhance production of IL-2 protein detected in the skin culture media relative to unstimulated skin (Figure 5d). With simultaneous addition of 10 or 3 µM PF-07245303 with T cell activating antibodies to the skin culture media, we observe concentration-dependent and almost complete inhibition of anti-CD3 and anti-CD28-induced IFNG, IL13, and IL2 gene expression (Figure 5a – c and Supplemental Table 4). Likewise, PF-07245303 treatment of ex vivo human skin almost completely suppressed the quantity of IL-2 protein detected in the skin culture media at compound concentrations of 10, 3, and 1 µM (Figure 5d, Supplemental Table 5). Similar to data from Smith et al.53, these results indicate that skin-resident T cells can be activated in human skin explants. Furthermore, with addition of PF-07245303 to the culture media exposed to the basolateral side of the skin, adequate concentrations of inhibitor were achieved in skin tissue to attenuate TCR-dependent activation of cytokine expression. To further evaluate the effects of ITK and TRK inhibition in a more physiological model, PF-07245303 was formulated in a fit-for-purpose54 vehicle (0.2% or 1.0%) and applied to the apical side of ex vivo human skin. Topically administered PF-07245303 significantly reduced Th2 cytokine and TCR-stimulated IFNG, IL13, IL2 and IL17A gene expression by 72.8 – 88.0% relative to vehicle control (Figure 6a-d, Supplemental Table 6). These data suggest that in human skin tissue the ITK and pan-TRK kinase inhibitor PF-07245303 can reduce T cell activation-dependent expression of pro-inflammatory cytokines which are known to contribute to the inflammation and pathogenesis of atopic dermatitis and other dermatological diseases.

In the human skin explant model PF-07245303 reverses the expression of many genes associated with atopic dermatitis
We created a disease gene expression signature from human skin explant cultures. Cultures were stimulated with anti-CD3/anti-CD28 and a Th2 cytokine cocktail to mimic the Th2 response seen in atopic dermatitis lesional samples. Anti-CD3/anti-CD28 stimulations with Th2 cytokines regulated the expression of 6128 genes (FDR <0.05) (Figure 7a). Using publicly available RNA sequencing data from atopic dermatitis patient lesional skin55, we show that most genes regulated by anti-CD3/anti-CD28 are also regulated in the same direction in disease tissue (Figure 7b). We also determined the expression profile of skin explant cultures treated with PF-07245303 and show that the majority of genes induced by anti-CD3/anti-CD28 stimulation are reversed by treatment with PF-07245303 (Figure 7c). PF-07245303 treatment reversed anti-CD3/anti-CD28 induced expression of many immune related genes, including CXCL11, IFNG, IL2, IL4, IL13, IL17A, FLG, FLG2, IL31RA, NGF, OSMR and IL4R. (Figure 7d).

PF-07245303 treatment reduces ear thickness, ear tissue cytokines, and disease severity in a mouse model of oxazolone-induced dermatitis
Having demonstrated that both basolateral and topical application of PF-07245303 in human skin explants can inhibit anti-CD3 and anti-CD28-induced activation of resident dermal T cells, we employed the oxazolone-induced contact hypersensitivity mouse model of dermatitis to evaluate the ability for PF-07245303 to reduce skin inflammatory responses in vivo. It is worth noting that the oxazazolone-induced model of dermatitis does not fully mimic the complex features of clinical AD56 . With oxazolone challenge, increased ear thickness was observed in the vehicle group while 43% - 61% inhibition of ear thickness was observed with topically applied 2% PF-07245303 at days 4, 7, 9, and 11 after initiation of oxazolone challenge (Fig. 8a, supplemental table 7). In ear skin sections, typical mixed inflammatory cell infiltration into the dermis and epidermal changes (hyperplasia, ulceration/erosion/thinning, superficial hyperkeratosis/crusting) were observed with oxazolone challenge. Visual and histological composite disease scores were moderately lower in oxazolone-challenged mice treated with PF-07245303 (Fig. 8 b-f, supplemental table 8). Protein levels for IL-4, IL-10, IL-17A, GM-CSF, and TNFα, were moderately decreased while levels of IL-2 and IFNγ were not different with PF-07245303 treatment (Fig. 8 g-j, supplemental Fig. 5, and supplemental table 9). Histological evaluation of PF-07245303 effects on dermal inflammatory cell infiltrates and changes in epithelial endpoints such as hyperplasia, ulceration, thinning, and surface hyperkeratosis shows moderate decrease in disease scores (supplemental Fig. 5). Collectively, these results suggest that application of PF-07245303 can reduce proinflammatory and epidermal barrier changes associated with atopic dermatitis.

Methods
Ethics Statements
Primary human blood samples were obtained with informed written consent from healthy human adult donors in accordance with the Pfizer Inc. Global Colleague Wellness Research Support Program (protocol CW RDP-02, IRB number: 08-33390) approved by the Advarra Institutional Review Board. Human skin tissue was obtained via elective abdominoplasty with written donor consent under Pearl IRB approval in accordance with FDA 21 CFR 56.104 and DHHS 45 CFR 46.101 regulations (Pearl Pathways. Exemption Determination Submission. IRB Study Number: 15-MEDP-101. All procedures performed on animals were in accordance with regulations and established guidelines and were reviewed and approved by a Pfizer’s Institutional Animal Care and Use Committee or through an ethical review process. Pfizer animal care facilities that supported this work are fully accredited by AAALAC International and abide by ARRIVE guidelines.

Crystallography
Crystallization of ITK has been reported previously37 . Crystals of ITK-KD (ITK kinase domain) were grown by the hanging-drop vapour diffusion method at room temperature (22°C). Purified ITK at 3 mg/ml was incubated with 0.5-1 mM inhibitor for approximately 30 minutes prior to crystallization trials. Crystallization drops were set up with 1 µl of protein plus 2 µl of reservoir containing approximately 12% PEG 3350, 0.1 M magnesium acetate and 0.1 M HEPES, pH 7.2. Crystals grew overnight and were flash frozen in liquid nitrogen after transfer to a cryoprotectant solution containing mother liquor plus 20%(v/v) glycerol. Crystal structure data collection and refinement statistics can be found in supplemental section.

Kinase Selectivity Assays
PF-07245303 was evaluated at Carna Biosciences, Inc. (Kobe, Japan) using off-chip mobility shift assays run in duplicate using 218 kinases. Details of kinase assay method available in supplemental methods section.

Measurement of Binding Kinetics using SPR
A CM5 sensor chip was loaded into the T200 Biosensor (Cytiva) and primed with HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% tween-20) buffer. Anti-His antibody (Genscript, Piscataway, NJ) was immobilized onto flowcells 1 and 2 using standard amine coupling conditions resulting in 8-10K RU of immobilized antibody. Recombinant His-tagged human ITK (Lake Pharma, Belmont, CA) was diluted into HBS-EP and injected over a single anti-His flowcell for 30 minutes at 5 µl/min flowrate. The captured ITK was then cross-linked with a 30s injection of 1:1 pre-mixed NHS/EDC, followed by 30s of ethanolamine (amine coupling kit, Cytiva) resulting in a level of captured and cross-linked ITK protein ranging from 7K – 10K RUS. Biotinylated human TRKA, TRKB, and TRKC proteins (Carna Biosciences) were immobilized onto individual flowcells of a Streptavidin (SA) sensor chip using standard immobilization conditions resulting in 4K-10K RU of immobilized TRKA, TRKB, and TRKC proteins. The following was performed on separate sensor chips containing either ITK or TRK proteins. Using the single-cycle kinetics method, compound was tested for binding in a 3-fold serial dilution of five concentrations ranging from 0.49 to 40nM passed over the ligand and reference channel. The association time was 60 seconds, and the dissociation time was 180 seconds for ITK and 60 – 480 seconds for TRKA, TRKB and TRKC at a flowrate of 50 µl/minute for all proteins tested. Buffer injections and DMSO calibration curve were included for referencing the data. Data analysis was performed using Biacore T200 evaluation software version 3.2.2. The double-referenced and solvent corrected data were fit to a 1:1 binding model to calculate rate parameters and affinity constant. T was calculated using the following equation: T = (nl2 / kd)/60 where nl (natural log) of 2 (0.693) is divided by the kd, to calculate T in seconds. This value was then divided by 60 to present the value in minutes.

ITK Isolated Enzyme Potency
The effect of compound on ITK kinase activity was determined by measuring the inhibition of ITK kinase-dependent phosphorylation of a biotin-tagged BTK peptide (CPC # 923799, sequence Biotin-KKVVALYDYMPMN-NH2) as substrate in the presence of ATP detected with time-resolved fluorescence resonance energy transfer (TR-FRET). An 11-point dilution series of compound was prepared to yield final assay concentrations of compound of 30 µM down to 0.0003 µM in 1% DMSO. In 384-well plates, 7.5 µl of assay mixture containing recombinant full-length human ITK protein was added to wells containing 0.1 µL serially diluted compound and the assay plates were incubated for 30 minutes at room temperature. Next, 2.5 µL substrate solution containing ATP (Teknova, A1204) and biotin-BTK peptide (China Peptide Company, CPC # 923799) was added to the wells containing ITK enzyme and compound. The final kinase assay reaction mixture contained 0.25 nM ITK, 1 µM biotin-BTK peptide, 1 mM ATP, 20 mM HEPES pH 7.5, 0.01% gelatin, 10 mM MgCL2, 5 mM GSH, and 0.01% Brij-35. Assay plates were incubated for 60 minutes at room temperature before adding to each well 10 µL of Stop/Detect Buffer (1 nM anti-phospho-tyrosine antibody labeled with Europium (LANCE PT66, Perkin Elmer, AD0069), 16.5 µg/ml Surelight streptavidin APC (Perkin Elmer, CR-130), HEPES pH 7.5, 0.01% gelatin, 10 mM EDTA, 250 mM NaCl) The plate was covered and allowed to incubate overnight at room temperature. Fluorescence was read with an EnVision plate reader with an excitation wavelength of 665 nm and an emission wavelength of 615 nm. The concentrations and resulting effect values for the tested compound were plotted and the concentration of compound required for 50% effect (IC ) was determined with the four-parameter logistic dose response equation. The IC reported is the geometric mean of the IC determined for each individual experiment.

Inhibition of ITK-dependent Proximal Signaling in Jurkat T Cells
Jurkat T cells (Clone E6-1 from ATCC Cat# TIB152) were resuspended into cell media containing RPMI-1640, 10% Heat-inactivated FBS, 100 unit/mL Penicillin-Streptomycin, 2 mM L-glutamine, 10 mM HEPES, 1 mM Sodium pyruvate, MEM non-essential amino acids solution and 5 x 105 cells per well were plated into 12-well plates. Half-log serial dilutions of compound were prepared and added to the cells to yield final assay concentrations of 10 µM down to 0.0003 µM with 0.03% DMSO. Cells were treated with compound for 30 minutes at 37°C followed by activation with ImmunoCult human anti-CD3/CD28 T cell activator (StemCell Tech. 10971) at a 1:100 dilution for 5 minutes at 37°C. Cell pellets were lysed on ice for 30 minutes in Complete Tris lysis buffer containing a 1:100 dilution of protease/phosphatase inhibitor cocktail solution, and 1 mM PMSF. Whole cell lysates were disrupted in SDS-PAGE sample buffer, boiled at 90°C for 5 minutes and loaded on 4-12% polyacrylamide mini-gels. Soluble lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes using the iBlot 2 semi-dry gel transfer system. Membranes were blocked neat with a TBS-based blocking buffer for 1 hour at room temperature and probed overnight at 4°C with specific antibodies to PLCγ1 (1:1,000 dilution, MilliporeSigma 05-366), phospho-PLCγ1 Y783 (1:1,000 dilution, Cell Signaling Tech. 2821L) and β-actin (1:10,000 dilution, Cell Signaling Tech. 4967L). The membranes were washed with TBS with Tween-20 and incubated for 1 hour with IRDye secondary antibodies in TBS with Tween-20. Proteins were detected via infrared fluorescence using the Li-COR Odyssey CLx imaging system. Quantification of total and phosphorylated PLCγ1 protein was performed via densitometry analysis using Image Studio analysis software. Fold change was determined based on the phospho-PLCγ1 to total PLCγ1 signal ratio. Results from four independent analyses were then plotted against each compound concentration and used a three-parameter least squares fit regression model in GraphPad Prism Version 10.4.1 to determine IC .

Compound Potency in Primary Human T Cells
Human CD4+ T cells were isolated, activated with anti-CD3/CD28 for 3 days, and expanded for an additional 4-6 days as follows. Isolated human CD4 T cells were purchased from StemCell Technologies (Cat. 70026) On day 0, CD4+ T cells were thawed, resuspended in assay medium (50% DMEM/50% RPMI with glutamine, 10% heat-inactivated human serum, 1x non-essential amino acids, 2 mM glutamine, and 1x penicillin/streptomycin), treated with anti-CD3/CD28 Dynabeads™ (ThermoFisher, 11161D), and incubated at 37°C/5% CO . On day 3, the beads were removed, and the cells diluted to 5x105 cells/cm2 and placed in G-Rex10™ flasks for incubation at 37°C/5% CO . The expanded CD4+ T-cells were centrifuged and resuspended to 0.5 million cells per ml and 60 µl of CD4+ T cells added per well (30,000 cells/well) to a 384 well plate containing 0.1 µL of an 11-point half log dilution series of compound in 100% DMSO. The final assay compound concentration range was 10 µM to 0.01 nM and 0.13% DMSO. The plates were incubated 15 min at 37°C/5% CO . Twenty µl of diluted ImmunoCultTM human anti-CD3/CD28 T Cell Activator (StemCell Technologies, 10991, 1:12.5 in T cell assay media) was added to all wells of the plate (1:50 final assay concentration). The plates were incubated an additional 20 to 24 hrs at 37°C/5% CO . The plates were centrifuged and 16 µL of supernatant was removed and combined with 4 µl of IL-2 HTRF antibodies (Cisbio kit, 62HIL02PEH). Plates were incubated for 3 hours at room temperature and read with EnVision™ plate reader at 665 nm and 615 nm wavelengths. Using the fluorescence intensity 665/615 ratio data the percent inhibition of each compound dose was evaluated. An unconstrained sigmoid curve was fitted using a 4-parameter logistic equation and an IC reported as the midpoint of the curve. The IC reported is the geometric mean of the IC values determined for each individual experiment.

Compound Potency in Human Whole Blood
Human peripheral blood was collected from donors in the Pfizer Research Support program via venous collection. Sixty µl per well of heparinized human whole blood was added to 384-well, polypropylene plates containing 0.08 µl of an 11-point half log dilution series of compound in 100% DMSO. The final assay compound concentration range was 30 µM to 0.3 nM in 0.1% DMSO. The plates were incubated 45 min at 37°C/5% CO . Twenty µl of diluted ImmunoCult PlusTM (StemCell Technologies, 1:25 in PBS/Glucose Buffer) was added to all wells (1:100 final assay concentration). The plates were incubated 20 to 24 hrs at 37°C/5% CO . The plates were centrifuged and 16 µL of plasma was removed and combined with 4 µl of IL-2 HTRF antibodies (Cisbio kit, 62HIL02PEH). Plates were incubated for 3 hours at room temperature and read with EnVision™ plate reader at 665 nm and 615 nm wavelengths. Using fluorescence intensity 665/615 ratio data the percent inhibition of each compound dose was calculated. An unconstrained sigmoid curve was fitted using a 4-parameter logistic equation and the IC was reported as the midpoint of the curve. The IC reported is the geometric mean of the IC values determined for each individual experiment. Partitioning of PF-07245303 between red blood cells and plasma at a concentration of 1 M (n=4) and fraction unbound at a concentration of 2 mM in human plasma was measured in vitro by equilibrium dialysis (n=4) at York Bioanalytical Solutions, Sandwich UK.

T cell, Th1, Th2, and Th17 cytokine release assays
PBMCs were isolated from either a leukopak (StemCell Technologies or Research Blood Components) or from whole blood obtained with informed written consent from healthy human adult donors using Lymphoprep (StemCell Technologies). Naïve CD4+ T cells were isolated using the EasySep Human Naïve CD4+ T Cell Isolation Kit, according to manufacturer's instructions (StemCell Technologies). CD4+ T cells (StemCell Technologies) were thawed and allowed to rest in X-Vivo 15 + 1% penicillin/streptomycin at approximately 1x106 cells/ml for 2 hours in an incubator at 37°C/5% CO . After two hours the cells were seeded into a 96-well plate at 0.1x106 cells/well in 160µl/well of X-Vivo 15 + 1% Penicillin/streptomycin (0.625x106 cells/ml). Compounds were prepared at 10X and 20µl was added to appropriate wells (0.3% DMSO final) and incubated for 15 minutes at 37°C/5% CO . CD3/CD28 T cell activator Dynabeads were washed in media and then added at 1 bead/cell (20µl of beads/well at 5x106 beads/ml) to appropriate wells. Cells were incubated overnight (20 to 24 hours) at 37°C/ 5% CO . The next day supernatants were harvested and frozen at -80ºC for later analysis. Cytokine content was measured using Meso Scale Diagnostics (MSD, Rockville, MD, USA) Proinflammatory Panel 1 (C-0049-2) and Cytokine Panel 1 (C-0050-2) assay kits per the manufacturer’s protocol.

For Th1/Th2 assays, human naïve CD4+ T cells were either thawed from frozen stock or freshly isolated. The cells were rested in complete media (RPMI +10% FBS + 1% penicillin/streptomycin + 10 ng/mL IL-2 (Peprotech 200-02)) for at least 2 hours at 37°C /5% CO . The cells were then plated at 15,000 cells/well in complete media in a 384-well tissue culture plate. Dynabeads Human T-Activator CD3/CD28 (Invitrogen 11132D) was added to each well at a final concentration of 1 bead/cell. For the Th1 assay a final concentration of 5 ng/mL rhIL-12 (R&D Systems 219-IL/CF) and 5 μg/mL anti-human IL-4 (eBioscience 16-7048-85) were added and for Th2 assay a final concentration of 50 ng/mL rhIL-4 (R&D Systems 204-IL/CF), 10 μg/mL anti-human IFNγ (eBioscience 16-731885) and 5 μg/mL anti-human IL-10 (eBioscience 16-7108-85) were added. Finally, compound/DMSO was added for a final concentration of 0.3% DMSO in a total volume of 100 μL/well and placed in a tissue culture incubator at 37°C / 5% CO . On day 7, supernatants were collected and IFNγ (Th1) and IL-13 (Th2) were measured by MSD following the manufacturer’s protocol (IFNγ and IL-13 Meso Scale Diagnostics K211AEB-2 and B21UB-3).

For the Th17 assay, human naïve CD4+ T cells were rested in complete media (X-VIVO 15 + 1% P/S) for at least 2 hours at 37°C / 5% CO . Meanwhile, a 384-well tissue culture plate was coated with 10 µg/ml anti-human CD3 (eBioscience 16-0037-85) in PBS-CMF for 2 hours at 37°C. The anti-human CD3 was aspirated off the plate and cells were added at a concentration of 20,000 cells/well in complete media. A cytokine skewing cocktail was added with final concentrations of 1000 ng/mL anti-human CD28 (eBioscience 16-0289-85), 25 ng/mL IL-6 (Peprotech AF-200-06), 5 ng/mL TGFβ1 (Peprotech 100-21), 12.5 ng/mL IL-1β (Peprotech AF-200-01B), 25 ng/mL IL-21, and 25 ng/mL IL-23 (R&D Systems 1290-IL). Finally, compound/DMSO was added for a final concentration of 0.3% DMSO in a total of 100 μL/well and placed in a tissue culture incubator at 37°C / 5% CO . On day 6, supernatants were collected and IL-17 measured by MSD following the manufacturer’s protocol (IL-17A: Meso Scale Diagnostics K211ATB-2)

The MSD raw data was converted to pg/mL using the standard curve for each plate, and the pg/ml values were converted to percent inhibition. The compound concentrations and percent inhibition values were entered into GraphPad Prism Version 10.4.1 to determine the IC using a four-parameter fit logistic equation. The IC reported is the arithmetic mean of the IC determined for each individual experiment.

Isolated TRKA, TRKB, and TRKC Enzyme Potency
Potency for PF-07245303 inhibition of TRKA, TRKB, and TRKC kinase activity was performed at Carna Biosciences, Inc. Details of this kinase assay method are available in supplemental methods section.

Whole Cell Potency in Cells Expressing Recombinant TRKA, TRKB, TRKC
The cell-based potency of PF-07245303 was determined in vitro at DiscoveRx using their PathHunter® technology which uses an enzyme functional complementation technology. This assay was used to assess the inhibitory effect of PF-07245303 on β-NGF-induced TRKA-, BDNF-induced TRKB-, and NT-3-induced TRKC dependent phosphorylation in cells. Details of this cellular assay method are available in the supplemental methods section.

Whole Cell Potency in Primary Cells Expressing Native TRKA
Fresh human whole blood was collected into sodium heparin anti-coagulant tubes, plated in a 96- deep-well plate and mixed with the compound dilutions prepared as 10-point, 3-fold dilution series to yield final compound concentrations of 30 µM to 0.002 µM with 0.2% DMSO. The mixture was placed in an incubator at 37°C with 5% CO for 30 minutes followed by stimulation with recombinant human NGF (R&D Systems, 256-GF) at a final concentration 0.1 μg/mL (or PBS as negative control) and the resulting mixtures were further incubated for 20 minutes. Next, anti-CD123-APC (Miltenyi Biotec, 130-115-265) and anti-CD203c-PE (Miltenyi Biotec, 130-092-343) antibodies were added and continued to incubate for another 15 minutes. Optilyse B solution (Beckman Coulter, IMI400) was added to each well to lyse the red blood cells and the plate was incubated at room temperature for 10 minutes. Next, distilled water was added to each well and room temperature incubation continued for an additional 10 minutes. The assay plate was centrifuged, and cell pellets were washed and resuspended in PBS. The labeled samples were acquired using LSRFortessa Flow Cytometer (Becton-Dickinson) and analyzed using FlowJo Version 10 from BD. Forward and side scatter was used to gate on all live non-neutrophil cells, then CD123+/CD203c+ cells were gated as activated basophils (Supplemental Figure 6). Compounds were tested in a dose response manner in five different donors, and the data from each experiment were fit to a four-parameter logistic equation to determine IC values.

Phospho-TRKA Detection in Human Skin Ex Vivo
Freshly excised healthy human abdominal skin from cosmetic reduction surgery was acquired from BioIVT (Westbury, NY). The skin was dermatomed to a thickness of 750 µm before being shipped overnight in skin culture media containing DMEM/F12 with GlutaMax, 180 µM adenine, 0.94 mM calcium chloride, 10 nM 3,3,5 triiodo-L-thyronine, 1X ITS-X, 100x antibiotic/antimycotic, 2% heat-inactivated FBS, 10 µg/ml gentamicin. Seven mm punch biopsies were obtained from the skin and transferred to inserts of a 24 well transwell plate with skin culture media in the bottom. The skin pieces were cultured at air-liquid surface, with the stratum corneum facing up and exposed to air and the dermis of the section was facing down and exposed to media. IL-4 (20 ng/ml) and IL-13 (20 ng/ml) were added to the skin culture media and plates were transferred to a humidified incubator at 37°C/5% CO . After 24 hours, skin pieces were transferred into Eppendorf tubes with or without NGF (10 μg/ml) and compound in DPBS-CMF. Tubes were incubated at 37°C/5% CO for 5 min. Skin tissue was then snap-frozen immediately and stored at -80°C for protein analysis.

Tissue was powderized and homogenized in 1X cell lysis buffer with 1X Halt™ Protease and Phosphatase Inhibitor Cocktail (EDTA-free). Lysates were centrifuged for 15 min at 4°C at maximum speed. Supernatant was collected and quantified using Pierce™ Rapid Gold BCA Protein Assay Kit. Samples were diluted to equal concentrations for pTRKA measurement. An MSD-based assay was performed by using hTRKA mAb (R&D System MAB1751R100) as the capture antibody and phospho-TRKA rabbit mAb anti-TRKA (Tyr674/675)/TRKB (Tyr706/707) (C50F3) Rabbit mAb (Cell Signaling 4621S, lot 10) as the detection antibody. Plate was read with MESO SECTOR S 600 instrument.

Ex Vivo Human Skin Model of Atopic Dermatitis
The human abdominal skin was cut using 7 mm biopsy punches and cultured at air-liquid surface. ImmunoCult™ human anti-CD3/CD28 T Cell Activator and DMSO or compounds were simultaneously delivered basolaterally by addition to the skin culture media. After 24 hours incubation, skin tissue was harvested for gene expression analysis and culture media was collected for protein analysis. Skin tissue was minced, lysed in RLT buffer (Qiagen RNeasy Mini kit) containing 1% betamercaptoethanol (Gibco, 21985-023) and homogenized with Bertin Precylls Cryo Evolution. Lysates were centrifuged for 15 min at 4°C at maximum speed. Supernatants were collected for RNA extraction. RNeasy Mini kit (Qiagen 74106) was used to extract RNA following the manufacturer’s protocol. RNA was quantified using BioTek™ Synergy™ H4 Hybrid Microplate Reader. The qPCR assay was carried out in 384-well plates with TaqMan® RNA-to-Ct™ 1-Step Kit. The qPCR reaction was performed in a Quant7 Studio PCR System with the following incubation sequence: 48°C for 15 min, 95°C for 10 min and 40 cycles each of 95°C for 15 seconds and 60°C for 1 min. For results analysis, each sample was normalized to its own TBP internal standard.

The skin culture media (50 µl/well) was analyzed for IL-2 and other proteins using MSD’s V-PLEX Human Proinflammatory Panel 1 kit (Meso Scale Diagnostics, K15049D) according to manufacturer’s protocols and plates were read on MESO SECTOR S 600. Standards from the kit were used to calculate the concentration of protein levels in the media (Supplemental Table 4).

For topical delivery experiments, human skin explants were freshly obtained from surgical specimens, mounted in Franz cell devices and stimulated with antibodies to CD3 and CD28, which activate the T cell receptor, and a cocktail of Th2 cytokines important for atopic dermatitis pathology. PF-07245303 was formulated in a fit-for-purpose (FFP) vehicle and applied to the apical surface of the skin54. Percent activity on IL2, IL13, IFNG and IL17A gene expression with vehicle or PF-07245303 treatment relative to Th2 stimulation with no treatment were calculated using three control genes (GAPDH, RPLP0, and ACTB) for the data analysis (Supplemental Table 6). The following qPCR Taqman probes from Applied Biosystems (Life Technologies) were used: Hs00174114_m1(IL2), Hs00174379_m1 (IL13), Hs00989291_m1 (IFNG), Hs00174383_m1 (IL17A), Hs00427620_m2 (TBP), Hs02758991_g1 (GAPDH), Hs01060665_g1 (ACTB), and Hs00420895_gH (RPLP0).

RNA Sequencing
The RNA was extracted from skin cultures using isopropanol extraction at MedPharm, Ltd. and sequenced77. RNA was sequenced at Fulgent using an Illumina NovaSeq 6000, the TruSeq Stranded mRNA sample preparation kit with 2 × 150 base pair (bp) paired end reads to a mean read depth of 28 million read pairs per sample. Output BCL files were demultiplexed to fastq using Illumina's bcl2fastq2-2.20 tool. The sequencing reads were mapped to the GRCh38 human reference genome using Star v.2.7.3a78 and subsequently gene counts were generated using Salmon v0.14.279. Transcripts were annotated using Ensemble v98.

Mice
Female BALB/c mice 8-10 weeks of age were obtained from Taconic Biosciences and Charles River Laboratories. All mice were housed in a specific-pathogen-free environment at Pfizer animal facility. Food (2916 Teklad Irradiated Global 16% Protein Rodent diet) and water were provided ad libitum. The vivarium holding rooms were maintained with 12-hour light/dark cycle, 72 °F and 30-70% relative humidity.

Oxazolone-induced Contact Hypersensitivity
Mice were sensitized epicutaneously by application of 100 ml of 4% 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone; SIGMA-Aldrich, St. Louis, MO) diluted in ethanol to the shaved ventral abdomen. Five days later, the mice were challenged by application of 20 µl of 1% oxazolone in ethanol to both sides of the right ear, and 20 µl of vehicle (ethanol) alone to both sides of the left ear at the indicated time points. Ear thickness was measured with an engineering micrometer (Model 7326; Mitutoyo America Corp, IL) at day 0 prior to first challenge (baseline), on each day of the challenge prior to application of oxazolone solution and at the final day of the study. For each timepoint ear swelling was calculated as ear thickness minus baseline value for each ear. One hour after the first challenge and daily after mice received topical 2% PF-07245303 or vehicle applied in 20 µl volume to the front and back of both ears. Twenty-four hours after the last dose, ear skin inflammation was scored using adapted EASI scoring system assessing erythema (redness), induration (thickness) and desquamation (scaling), on a scale of 0-4 each, with maximum score of 16 assigned to each animal. Ears from half of each treatment group were preserved in 10% neutral buffered formalin (Thermo Fisher) for histological assessment, or flash frozen in liquid nitrogen for tissue cytokine analysis. Methods for microscopic review and scoring can be found in the supplemental methods section. Investigators were blinded to group allocation during data collection and analysis.

Measurement of Cytokines
To measure cytokine levels in ear skin tissue frozen samples were homogenized in T-PER buffer supplemented with protease cocktail (both from Thermo Fisher). Frozen tissue was cut in small fragments with scissors in T-PER/proteinase buffer, then one 5mm stainless steel bead (QIAGEN) was added to each sample tube. Sample tubes were homogenized with TissueLyser II (QIAGEN) and total protein concentration was determined by a BCA assay kit (Pierce) following the manufacturer’s protocol. Levels of cytokines in the ear tissue homogenates were determined by MSD kit (U-PLEX T-Cell Combo [mouse], Meso Scale Diagnostics) according to the manufacturer’s instructions. All values were normalized to the total protein concentration per sample.

Statistical Analysis
For analysis of the p-TrkA ex vivo skin assay, all data points were subtracted by the average unstimulated values and then divided by the average stimulated (IL-4, IL-13 and NGF) values to normalize. A one way ANOVA model was utilized with normalized expression as the response variable and treatment as the fixed effect. The model was fit by the lm function in R version 3.5. The least square mean estimates, the estimates of the treatment differences compared to vehicle, and fold change was determined. Adjusted multiple comparison p-values with the FDR method. Notable inhibition was defined as p < 0.05 and F < -0.5.

In the statistical analysis of the ex vivo human skin model of AD, a linear mixed model was fit with ∆∆Ct as the response variable, donor as random effect, and treatment as the fixed effect with or without heterogenous variance for the treatments or a one-way ANOVA. The model was fit by the lme function with the restricted maximum likelihood or lm function in R version 3.5. The least square mean estimates and the estimates of the treatment differences compared to vehicles were determined. Adjusted multiple comparison p-values with the false discovery rate (FDR) method. The adjustment to the p-values was based on 3 or 4 comparisons, gene expression and protein, respectively. The % inhibition for gene expression was calculated using the formula: (1−F)∗100%. F is the fold change which equals 2−estimated ΔΔCt Treatment Difference.

RNAseq gene expression was normalized using edgeR package80 and gene expression differences were evaluated using limma R package81. The P-values were corrected for multiple hypothesis testing using the Benjamini-Hochberg false discovery rate82,81. All analysis were implemented in R version 4.2.1 and all visualizations were created using the EnhancedVolcanoplot R package version 1.14.0 or ggplot2 R package version 4.0.083.

In the statistical analysis of mouse ear thickness, a linear mixed model was fit with ear thickness as the response variable, donor as random effect, and treatment as the fixed effect with heterogenous variance for the treatments. The model was fit by the lme function with the restricted maximum likelihood in R version 3.5. The least square mean estimates and the estimates of the treatment differences compared to vehicles were determined. Adjusted multiple comparison p-values with the false discovery rate (FDR) method. The adjustment to the p-values was based on 2 comparisons.

Data Availability
Source Data has been provided and all primary data generated for this study will be readily shared with researchers for reproducibility purposes upon request. The diffraction data and coordinates of ITK bound to PF-07245303 compound have been deposited in the Protein Data Bank (PDB) under the accession code 9NWX. The RNA sequencing data generated has been deposited in the GEO repository under accession code GSE297645.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript submitted by Duffen and coworkers report the discovery and pharmacology characterisation of PF-07245303, a dual ITK and pan-TRK inhibitor for the treatment of atopic dermatitis. By the inhibition of ITK and TRKs kinases, PF-07245303 acts by both 1) supressing the T cell-dependent inflammation and cytokine production driving atopic dermatitis, and 2) by reducing the pruritis associated with this condition, respectively.\nThe authors have extensively characterised the pharmacology of PF-07245303 for the treatment of atopic dermatitis using biochemical, in vitro and ex vivo models. They have demonstrated the ITK and pan-TRK selectivity of PF-07245303 in vitro and in cell-based assays, shown suitable biochemical downstream response from PF-07245303-derived ITK, TRKA, TRKB and TRKC inhibition, suppression of T cell activation and inhibition of pro-inflammatory cytokine production both in cells and using ex vivo human skin sections. In addition, the authors have shown that treatment with PF-07245303 also reverses the expression of many genes associated with atopic dermatitis.\nThis is an excellent work that will have a significant impact in the field of kinase drug discovery and atopic dermatitis. The results are robust and are presented in a clear manner. We recommend publishing it in Nature Communications provided the authors address the following:\nMajor points\n• The title of the manuscript starts as “Discovery of…”. PF-07245303 is indeed first reported in this work, but the discovery campaign is described very vaguely (Page 5, lines 96 – 108). The authors should expand this section significantly to give information about the medicinal chemistry campaign that led to the discovery of this compound. What kind of screening was used? Which were the initial identified hits? How these initial hits lead to the identification of the 2-(1H-pyrazol-5-yl)-1H-benzo[d]imidazole hinge binding moiety? What does “parallel optimization for soft topical drug properties” mean and how was this carried out? What properties were trying to modulate when selecting the 2-morpholinopyranmide group? This should be accompanied with additional chemical structures and figures if needed. Also, the mode of binding of the drug candidate should be described. Is it a type I inhibitor or displays an alternative binding mode? The drug discovery side of the story should be described in full in this article to increase its impact.\n• The way PF-07245303 was reported to target TRKA, TRKB and TRKC kinases is confusing. In the text, PF-07245303 is described to be designed to inhibit ITK using structure-based drug design methods, but then it is tested against “its targets” ITK, TRKA, TRKB and TRKC kinases. After this, the authors perform a kinome assay, where indeed ITK, TRKA, TRKB and TRKC kinases were the main targets. Was the ITK/pan-TRK inhibitory activity of PF-07245303 intentional? If so, how was this done? If, on the contrary, the polypharmacological properties were a serendipitous discovery, this should be mentioned in the text.\n• The authors claim to have been discovered a “soft” drug, a term that is even included in the tittle of the manuscript. However, they have not explained the rationale behind this. The authors should explain the reasons why a “soft” ITK/Pan-TRK kinase inhibitor is preferable for atopic dermatitis treatment and specify why PF-07245303 is in this category, so the reader can contextualise the advantages of this drug. In the title, the word “soft” before “small molecule” seems out of place. Suggestion: “Discovery of a Soft Topical ITK and Pan-TRK Kinase Inhibitor (PF-07245303) for the Potential Treatment of Atopic Dermatitis”\nAdditional comments to address by the authors:\n1) Page 2, line 38. Replace “inhibitor” with “inhibitors”.\n2) Page 5, lines 107. Replace “physical chemical properties” by “physicochemical properties”.\n3) Page 6, line 117 and page 36, FIGURE 1, panel D. What does “mil” mean in “uL/min/mil”?\n4) Page 6, lines 117 – 119. Remove one of the duplicated phrases.\n5) Page 6, line 126 – 127. Indicate the % of kinase inhibition for FLT3 and ITK.\n6) Page 6, line 132. Replace “TEC family kinases” by “Tec family kinases”.\n7) Page 7, line 142. Change colour of reference 8 to black.\n8) Page 8, line 174. Change “75.8” for “75.8 nM”.\n9) Page 36, FIGURE 1, panel E table and caption. The column tittle “Phys. prop” and the caption “Key physiochemical properties for PF-07245303” are not accurate to describe the parameters shown. MDCK-LE, HLM, hHEP, human PPB Fu and human BPR are assays that define the in vitro DMPK profile of the candidate.\n10) Add the methods for MDCK-LE, HLM, hHEP, human PPB Fu and human BPR determination in the supplemental methods and materials section.\n11) Page 36, FIGURE 1, caption, panel E. Indicate the number of kinases tested. 218?\n12) Additional supp. material is required:\n- Include data table with % kinase inhibition for the kinome selectivity for PF-07245303 shown in FIGURE 1, panel E.\n- Include a measurement of purity (HPLC analysis), 1H-NMR, 13C-NMR spectra of PF-07245303.", "reviewer_2": "In this work, Duffen et al report on the development of what they call a \"soft small molecule\", which is a dual specific kinase inhibitor targeting both the Tec kinase Itk, and members of the Trk family of receptor tyrosine kinases, and tuned for soft topical delivery. They evaluate this inhibitor on its ability to inhibit T cell activation and cytokine production of T cells in vitro in skin explants, as well as on basophil activation. They propose that this compound could be used as a topical treatment for Atopic Dermatitis. The work is interesting, but there are a number of inhibitors against both kinases that have been previously reported, although admittedly none with dual Tec/Trk activity with ability to inhibit both T cells and mast cells. While representing some advance to the field, the work would be significantly strengthened if they had evidence that the compound had an effect on skin inflammation in patients of Atopic Dermatitis. In addition, there are a number of concerns as detailed below:\n1. The authors show that the dual inhibitor acts on both T cells and basophils. However, it would be useful to also compare to treatment with additive combinations of an Itk selective inhibitor and a Trk selective inhibitor (recognizing the caveats of attempting to compare different compounds). This would allow the authors to comment more on the relative importance of the contribution of both kinases to the dual inhibition, especially since Itk has been suggested to play a role in basophil/mast cell activation.\n2. In figure 1B, the structure of the PF-07245303 inhibitor binding to Itk. It would be helpful to determine if there are significant changes in the area of binding to ATP (or in other areas of the kinase), that are induced upon PF-07245303 binding.\n3. Based on figure 1E, the inhibitor seems to also have some effects on other kinases (in yellow shading). Are these the same kinases represented in the NanoBRET kinase assay presented in the supplemental figure? If not, what are those kinases?\n4. Does the inhibitor affect Fc epsilon receptor induced activation of basophils as well?\n5. The data on cytokine expression in figure 5a showing RNA abundance in FKPM vales (of between 7 and 10, or 10-25), those values seem quite low. Can the authors explain the context for this with regards to the level of expression of these cytokines?\n6. The work would be significantly enhanced if the inhibitor could be evaluated using explants from patients with AD, in addition to the data from normal skin explant experiments presented.\n7. The sequence and length of biotinylated peptide used for ITK assays should be included in the methods sections.\n8. In figure 2B, the information included in the figure on the antibody used for the western blotting should be removed and placed in the methods sections.", "reviewer_3": "In their MS, Duffen et al describe the synthesis of an ITK and TRK kinase inhibitor and suggest its potential use in topical treatment of atopic dermatitis based on in vitro assays using human cell cultures and skin models.\nThe authors convincingly demonstrated that their novel compound PF-07245303 is a potent ITK and TRKA/B/C inhibitor both in cell-free and cellular systems in vitro. It is demonstrated that the compound binds to ITK and inhibits ITK-related signaling and inflammatory cytokine production in activated T cells. Moreover, the authors also showed that PF-07245303 binds to TRKA/B/C and inhibits NGF-induced TRKA phosphorylation suggesting that the compound also effectively inhibits TRKs. As ITK inhibition was reported to be beneficial in several T cell-mediated, inflammatory, and allergic conditions, the authors aim at exploiting their compound in attenuating atopic dermatitis-related alterations using human in vitro models.\nAlthough the results clearly indicate a significant improvement of AD-like alterations in the in vitro models, it is a main question why this compound would be superior compared to several available ITK (or TRK) inhibitors. The authors also suggest that the ITK inhibition is responsible for the anti-inflammatory effect and the additional TRK inhibition might evoke an antipruritic effect in AD. However, none of these implications are tested experimentally. It would be important to compare the effect of PF-07245303, as a “dual” kinase inhibitor to selective ITK and selective TRK inhibition in the skin models. RNASeq experiments following new treatments in the skin explants (as shown in Figure 7) would be especially informative.\nThe potential antipruritic activity of the compound should be further dissected, and the expression of additional AD-relevant pruritic mediators (TSLP, endothelin-1) should be analyzed.\nMoreover, testing PF-07245303 in an atopic dermatitis mouse model would be a valuable addition and would make possible to directly assess the claimed antipruritic effect of the compound.\nThe authors argue that the physicochemical properties of PF-07245303 make it optimal for topical application, which might be an advantage over other compounds. However, this potential advantage is not discussed in depth, it is not compared to data available in the literature. Moreover, the compound was applied in the culturing medium of the skin explants, however, in this model, the topical application is also available as the explant is cultured at an air-liquid surface. Are there any data available about the effect of PF-07245303 in “topical application”, i.e. on the surface of the epidermal layer? Alternatively, topical application would be available in animal experiments.\nMinor\nFigure 7D: How to understand the “expression” on the y-axis? What is the unit of “expression”? Are these expression data from the RNA sequencing studies or were determined by qPCR?\nPDB and GEO accession codes should be provided in the final version.\nPrimer/probe sequences or assay IDs used during QPCR experiments should be provided. What gene(s) was(were) used as endogenous control(s)?", "reviewer_4": "Authors synthesized a novel small and soluble compound, PF-07245303, and demonstrated its inhibitory activity on interleukin-2-inducible T cell kinase (ITK) which is essential for Th1, Th2 and Th17 cytokines, and tropomyosin-related kinase (TRK), which is the receptor for nerve growth factor (NGF) and mediates itch. The way of synthesis and biochemical characteristics of this compound was well described and its inhibitory effects on the productions of various inflammatory cytokines increased in atopic dermatitis. were extensively demonstrated using both in vitro and ex vivo models, including isolated T cells, basophiles, whole blood, three-dimensional cultured skin equivalent cell sheets and skin explants. Considering its topical applicability and a new action target which has not been shared by existing medication clinical medicine, the compound appears to have high potential as a new promising medication for various inflammatory skin diseases, such as atopic dermatitis.\nMajor concern:\n1. It is not common that a compound highly specific in inhibition for a certain enzyme shows inhibitory activity to other enzymes. Do ITK and TRK family share molecular similarity in their structures? Authors should explain or discuss about the mechanism or reason of dual effect of PF-07245303.\n2. The potent dual activities of PF-07245303 on ITK and all TRKA, TRKB and TRKC make me concerned about the specificity of its action. It may not only be beneficial in inhibiting non-favorable activities of T cells, but may also suppress favorable anti-inflammatory functions of T cells, such as production of IL-10 and TGFβ, or even may act cytotoxically. Authors should also study the effect of PF-07245303 on such activities and cytotoxicity.\n3. Page 13, line 285 and page 43, line 1004. Please clarify the direction of “basolateral addition”. Was the compound added on the keratinocyte layer as in the case of clinical application? If not, this way of application should also be tested.\n3. Resolution of figures, especially of figure 1C is poor. I cannot read numbers of horizontal axes.\n4. Figure 5 and 6. Table 1, 2 and 3 are supplementary to explain data shown in corresponding figures, i.e. figure 5, figure 6ABC and figure 6D, and preferable to be shown as supplemental materials. Statistical significance of difference (notable inhibition) may be shown in the figures using symbols.\n5. Page 41, line 41. Statistically significant changes in phospho-TRKA levels were NOT observed at ALL 5 compound concentrations tested, but only at 1μM or higher.\n6. Page 45, line 1014-1018. Descriptions of Scatterplot of RNA in (A) and (B) are the same. Description in page 13, line 297 to page 14, line 298 appears to tell that authors prepared figure 7B using data of reference 50. However, figure shown in reference 50 is different, at least in its form, from figure 7B. Authors should describe the method taken for making figure 7B.\nMinor problems:\n1. Page2, line 46, and page 6, line 132. Description of Lck and LCK should be the same, either making capital only top alphabet or all alphabets.\n2. Page 6, line 126. FLT3 should be spelled out on the first appearance.\n3. Page 9, line 205. Is “IC50S” a typo for “IC50 value”?"}, "author_response": "Authors’ response: We appreciate the reviewer’s suggestions. Additional details on the identification of PF-07245303 were added to the manuscript including statements around using literature examples and screening under 1 mM ATP kinase assays. (Page 5, lines 100-104) The description of “soft” drug was also expanded on. (Page 5, lines 96-100, Page 6, lines 125-127) Structural details including being a Type I kinase inhibitor were added to address lack of any observed structures changes expect by that type of inhibition. (Page 5, lines 102-107)\nAuthors’ response: Additional statements were added to clarify that the initial interest was in ITK pharmacology but the identification of TRK pharmacology during the discovery was determined to be a potential value. (Page 5 and 6, lines 112-115) Additional reference added to support the rational of polypharmacology. (Reference # 38)\nAuthors’ response: We added a statement around the desired to have exposure at the site of action thus minimizing any on- or off-target pharmacology (Page 6, lines 125-127).\nAuthors’ response: This statement has been removed for brevity.\nAuthors’ response: We have edited this statement in the revised manuscript as suggested. (Page 5, line 110)\nAuthors’ response: Page 6 line 123 has been updated to “uL/min/million cells”.\nAuthors’ response: We removed the duplication (Page 6, lines 124-125)\nAuthors’ response: We have added the % inhibition values for FLT3 (Page 6, line 134) and ITK (Page 6, line 131) in the text.\nAuthors’ response: We have made this correction. (Page 7, line 140)\nAuthors’ response: We have made the correction.\nAuthors’ response: On page 9 line 183 we have listed the values with units. The values are different from the original manuscript because we performed a new set of experiments in order to simultaneously measure IL-10 in addition to the pro-inflammatory read-outs as suggested by reviewer #4. Inhibition on all cytokines in Figure 3 a – e was evaluated in the same experiments.\nAuthors’ response: We have made this correction to DMPK profile in Figure 1 panel e and in the figure legend page 42, line 1000.\nAuthors’ response: We have added references describing the methods used for these DMPK measurements in the supplemental methods section page 71 lines 1359-1365.\nAuthors’ response: We have indicated the number of kinases (218) in the results (Page 6, line 130) and in the figure legend for Figure 1 panel e (Page 42, line 1001)\nAuthors’ response: We have added Supplemental table 1 to the manuscript which provides the % inhibitions for the panel of kinases tested. (Page 54, line 1174)\nAuthors’ response: We have included this data found on pages 80 and 81.\nAuthors’ response: We agree with the value in benchmarking the activity of our dual ITK-TRK inhibitor versus ITK-selective and TRK-selective inhibitors. Unfortunately, we were not aware of a compound with potent inhibition of ITK and sufficient selectivity of other Tec family kinases which also has sufficient selectivity against TRK kinases when conducting these studies. A compound selective for TRK kinases TRKA, TRKB, and TRKC was available, and we used this compound (PF-06273340) to determine cellular responses which are or are not TRK kinase-dependent.\nTo confirm the ITK kinase-dependence for T cell anti-inflammatory effects, in the original manuscript we did employ the TRK-selective compound PF-06273340 to orthoganaly demonstrate that TCR-dependent T cell stimulation of IL-2 production is not TRK kinase dependent (Supplemental Figure 1). To further address this question, we have included new data demonstrating that the inhibition of additional inflammatory read-outs (IL-13, IL-17A, and IFNγ) are not sensitive to TRK inhibition from anti-CD3 and anti-CD28-stimulated T cells in vitro (supplemental figure 2). These are the same cytokine read-outs which we measured in the anti-CD3, anti-CD28, and Th2 cytokine treated skin explants. This result suggests that the inhibition of IL-2, IL-13, IL-17A, and IFNγ expression from anti-CD3 and anti-CD28 stimulated T cells in the skin is not via TRK kinase inhibition. Unfortunately, we will not be able to test the TRK-selective compound in our human skin explant model as this project has moved to our clinical portfolio, and we no longer have the internal expertise and resources to perform this challenging skin explant model.\nAuthors’ response: We added detail to the manuscript clarifying that the PF-07245303 is a Type 1 kinase inhibitor and as such no observed significant changes to the binding site as typical (Page 5, lines 104-107)\nAuthors’ response: Statement added to the figure legend of Figure 1 page 42 “See Supplemental Data Table 1” for clarity.\nAuthors’ response: We agree that this is an important question for the field. To address this, we performed experiments to evaluate if PF-07245303 inhibits Fc epsilon receptor-mediated activation of basophils. Using human basophils isolated from blood, we observed that PF-07245303, which does not have significant BTK kinase inhibition, is effective at reducing IL-4 release induced via IgE receptor-mediated basophil activation. We have included this data in the manuscript in Supplemental Figure 4 and described in the results section page 11 lines 229-232. We also tested the TRK kinase selective inhibitor PF-06273340 in this assay and did not observe inhibition of IgE receptor-induced IL-4 release from basophils. This result indicates that IgE receptor-mediated basophil IL-4 production is ITK kinase-dependent, possibly with contribution from BTK kinase, but TRK kinase independent.\nAuthors’ response: Figures 5a and 5b has been removed from the manuscript to accommodate the inclusion of new more illustrative data from topical treatment effects in human skin explants and results from mouse dermatitis model to address major reviewer comments, The original Figure 5c is now figure 4h.\nAuthors’ response: We agree that evaluating the effects of PF-07245303 on inflammatory readouts using lesions from AD patients would be a valuable addition to this work. Unfortunately, it is not possible for us to obtain skin lesions in adequate quantities from patients with AD to perform these studies. As another approach, our team tested the efficacy of topically applied PF-07245303 in the oxazolone-induced mouse model of dermatitis. This disease model efficacy data is included in the revised manuscript (Figure 8 and Supplemental figure 5) along with evaluation of histological impacts of PF-07245303 treatment and effects of this treatment on inflammatory cytokines. (Results section pages 14 and 15, lines 308-327)\nAuthors’ response: We have added this information in the revised manuscript as suggested. (Page 21, line 469)\nAuthors’ response: We have removed the antibody information from the figure and included it in the methods section in the revised manuscript as suggested. (Pages 22 and 23, lines 503-507)\nAuthors’ response: We agree with the value in benchmarking the activity with our dual ITK-TRK inhibitor versus ITK-selective and TRK-selective inhibitors in the skin explant model. Unfortunately, when conducting these studies we were not aware of a compound with potent inhibition of ITK and sufficient selectivity of other Tec family kinases which also has sufficient selectivity against TRK kinases.\nAlternatively, to confirm the ITK kinase-dependence for T cell anti-inflammatory effects, in the original manuscript we did employ a TRK-selective compound to demonstrate that TCR-dependent T cell stimulation of IL-2 production is not TRK kinase dependent (Supplemental Figure 1). To further address this question, we have included new data demonstrating that the inhibition of additional inflammatory read-outs (IL-13, IL-17A, and IFNγ) measured in the skin explant are not sensitive to TRK inhibition as shown in Supplemental Figure 2 and described on page 9 lines 183-186.\nAdditionally, it has been demonstrated that basophils are necessary for itch flares in a mouse model of dermatitis. Using a TRK-selective inhibitor we demonstrated that the NGF-induced basophil activation was sensitive to TRK inhibition alone and our dual ITK and TRK kinase inhibitor can also suppress NGF-induced basophil activation (Figure 4 g). However, we are unable to test the TRK-selective compound in our human skin explant model as this project moved to our clinical portfolio, and we no longer have the internal expertise to perform this challenging skin explant model.\nIn the discussion section we reference additional published data from animal models and from two clinical trials testing TRKA or pan-TRK inhibition and observing reduced itching to further support the role for TRK in promoting pruritus. (Pages 17 and 18, lines 385 -391)\nAuthors’ response: We did further evaluate our RNAseq data and demonstrate in the human skin explant model that PF-07245303 inhibited the enhancement of IL-31RA and OSMR which are necessary for IL-31 signaling which is associated with pruritus. Moreover, in the human skin explant model PF-07245303 inhibited the enhancement of NGF which is another driver of pruritus. These data have been added to Figure 7 panel d.\nAuthors’ response: As suggested, our team tested the efficacy of topically applied PF-07245303 in the oxazolone-induced mouse model of dermatitis. We included this disease model efficacy data in the revised manuscript (Figure 8 and Supplemental figure 5). PF-07245303 treatment inhibited oxazolone-induced ear swelling along with reductions in many histological changes associated with AD as well as suppressed multiple inflammatory cytokines. Unfortunately, we do not have the technology and expertise to evaluate itching in the model. Scratching in response to itch can damage skin which can contribute to skin pathology. Our evaluation of affected skin in the dermatitis model by both visual scores and by histological assessment showed significantly reduced evidence of skin pathology with PF-07245303 treatment relative to vehicle control.\nAuthors’ response: As suggested, we have included new data to the manuscript testing the effects of formulated and topically applied PF-07245303 on inflammatory markers in the human skin explant model. Topically applied PF-07245303 significantly reduced Th2 cytokine and TCR-stimulated IFNγ, IL-13, IL-2 and IL-17A gene expression by 72.8 – 88.0% relative to vehicle control. This data is found in the newly added Figure 6 and Supplemental Table 6.\nAs another approach, our team tested the efficacy of topically applied PF-07245303 in the oxazolone-induced mouse model of dermatitis. This disease model efficacy data is included in the revised manuscript (Figure 8 and Supplemental Figure 5) along with evaluation of histological impacts of PF-07245303 treatment and effects of this treatment on inflammatory cytokines.\nAuthors’ response: This is an important point. We have added the statement “Expression of RNA in untreated, anti-CD3/CD28/Th2 stimulated and anti-CD3/CD28/Th2/PF-07245303 treated skin explants for CXCL11, IFNγ, IL13, IL4, IL17A, IL2, FLG, FLG2, IL31RA, NGF, OSMR and IL4R from RNA sequencing studies. Values on the y-axis represent EdgeR normalized expression.” To the legend for Figure 7 to clarify. (Page 51, lines 1118-1121)\nAuthors’ response: These accession codes will be included in the manuscript with acceptance and publication.\nAuthors’ response: The following Applied Biosystems (Life Technologies) assay IDs have been added to the materials & methods section: Hs00174114_m1(IL2), Hs00174379_m1 (IL13), Hs00989291_m1 (IFNγ), Hs00174383_m1 (IL17α), Hs00427620_m2 (TBP), Hs02758991_g1 (GAPDH), Hs01060665_g1 (ACTB), and Hs00420895_gH (RPLP0). Three control genes (GAPDH, RPLP0, and ACTβ) were used for the qPCR data analysis of ex vivo skin assays with topical compound delivery performed at MedPharm Ltd. One control gene (TBP) was used for the qPCR data analysis ex vivo skin assays with basolateral compound delivery performed at Pfizer, Inc. This information was added to the methods section on page 31 lines 690-694.\nAuthors’ response: Thank you for the comments. Additional detail on the inhibitor is a Type 1 kinase inhibitor added. (Page 5, lines 104-115) Unfortunately, we were not successful in acquiring a protein X-ray structure of TRK with this compound.\nAuthors’ response: Consistent with the reviewers’ comment, PF-07245303 inhibits both pro-inflammatory and anti-inflammatory T cell activation. In Figure 3E we have included data demonstrating concentration-dependent inhibition of IL-10 from anti-CD3 and anti-CD28 stimulated T cells treated with PF-07245303.\nTo address the comment on cytotoxicity, we have tested PF-07245303 in an in vitro cytotoxicity assay using THLE cells (Shah F, et al. 2014). The potency of PF-07245303 is very low in the THLE assay (IC50 = 12µM) compared to inhibition of IL-2 release from T cells (IC50 = 38.5 nM, (page 7, line 159 and in figure 2c).\nAuthors’ response: In the Ex Vivo Human Skin Model of Atopic Dermatitis methods section we added more information about the basolateral addition which is delivery to the skin culture media to which the dermis is exposed. The term “basolateral addition” was removed from the sentence in the original manuscript and was replace with “addition of PF-07245303 to the culture media exposed to the basolateral side of the skin” for clarity. (Page 13, lines 281-282) Similar changes were made to the titles for Supplemental Table 2 and Supplemental Table 3 as you highlighted.\nAs suggested, we have included new data to the manuscript testing the effects of formulated and topically applied PF-07245303 on inflammatory markers in the human skin explant model. Topically applied PF-07245303 significantly reduced Th2 cytokine and TCR-stimulated IFNγ, IL-13, IL-2 and IL-17A gene expression by 72.8 – 88.0% relative to vehicle control. This data is found in the newly added Figure 6 and Supplemental Table 6) and results described on page 13, lines 284-288.\nAuthors’ response: We have updated the graphs in Figure 1 panel c with legible y-axis labels.\nAuthors’ response: We have moved the three tables which were associated with figure 5 and figure 6 to the Supplemental materials section (Supplemental Tables 3, 4, and 5), and we have added notation of statistical significance directly to the figures.\nAuthors’ response: We have removed the incorrect statement and appropriately denoted statistically significant changes in phospho-TRKA only with 10, 3, and 1 uM of PF-07245303 in the revised figure 4 panel h.\nAuthors’ response: In Figure 7 similarities in gene signatures of differentially expressed genes in a human skin explant model with similar signatures to that found in skin lesions from AD patients. Treatment of skin explants with PF-07245303 can reverse the disease signature.\n(a,b) Scatterplot of RNA expression differences in anti-CD3/CD28 and Th2 cytokine-stimulated and unstimulated skin explants. Each dot represents a distinct gene.\n(a) Red dots represent genes that are induced by stimulation with q-value < 0.05 and blue dots represent genes that are reduced by stimulation with q-value < 0.05.\n(b) Dots are colored based on regulation in atopic dermatitis skin tissue versus healthy control from GEO dataset GSE121212.\n(c) Scatterplot of RNA expression differences in skin explants treated with PF-07245303 and then stimulated with anti-CD3/CD28 and Th2 cytokines. Dots are colored based on the regulation in (a).\n(d) Expression of RNA in untreated, anti-CD3/CD28/Th2 stimulated and anti-CD3/CD28/Th2/PF-07245303 treated skin explants for CXCL11, IFNγ, IL13, IL4, IL17A, IL2, FLG, FLG2, IL31RA, NGF, OSMR and IL4R from RNA sequencing studies. Values on the y-axis represent EdgeR normalized expression.\nReference 55 in manuscript: Brunner, P.M., et al. Early-onset pediatric atopic dermatitis is characterized by T(H)2/T(H)17/T(H)22-centered inflammation and lipid alterations. J Allergy Clin Immunol 141, 2094-2106 (2018).\nAuthors’ response: We have made this correction. (Page 2, line 46 and page 7, line 140)\nAuthors’ response: We have made this addition. (Page 6, line 134)\nAuthors’ response: We have made this correction. (Page 10, line 218)"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed our suggestions and concerns", "reviewer_2": "Duffen et al’s revised report on the development of a \"soft small molecule\" targeting both Itk and members of the Trk family of TKs has been improved with the inclusion of additional report in actions of the inhibitor in animal models of AD, albeit with modest effect. There are few minor issues that the authors should address as detailed below:\n1. The references to “Jurkat cells” should be to “Jurkat T cells” to make clear that this is a T cell line.\n2. On line 217 of the manuscript, the authors should check to ensure that they are referring the to the correct compound. They should also refer to the Trk selective compound as such to remind the reader and reduce confusion so the reader does not have to recall each time which compound is the dual inhibitor and which is the Trk inhibitor.\n3. The supplementary figures 1 a-c should be labeled.", "reviewer_3": "In the revised version, Duffen et al. provided several new data which significantly advanced their manuscript. Most importantly, they (i) demonstrated the beneficial effect of PF-07245303 on itch-related gene expression upregulated by TH2 cytokines, (ii) demonstrated the efficacy of the compound in “topical” application using the human skin explant model and (iii) showed the positive, anti-inflammatory effect of the compound in the oxazolone-induced contact dermatitis mice model. Although it is not an atopic dermatitis model, which limitation should be shortly discussed, the anti-inflammatory effect of PF-07245303 is convincing in this model.\nMinor comments:\nFigure 1 E – The legend in the figure is incomplete. I assume that red, yellow, and green circles represent 100, 50, and 0 percent inhibition, but it is not indicated. This information should be added to the figure.\nFigure 3 – In the legend, the sentence “PF-07245303 inhibited the release of IFNγ, IL-13, and IL-17 from anti-CD3, anti-CD28 and cytokine cocktails to generate human naïve CD4+ T cells skewed to f Th1, g Th2 and h Th17 T cells.” is hardly understandable and should be reformulated. Why were some donors included in the original version omitted from the revised version?\nFigure 4H shows “representative results for phospho-TRKA activation in human skin explants from one donor.” Would it be possible to include the results of the other 2 donors, or show them in a supplementary figure?\nFigure 7 – RNA Seq data should be uploaded to a public database, and the accession number should be given. It would be nice to see the names of the up- and downregulated genes shown in the volcano plots that could be presented in a list or an interactive figure in the supplementary materials.\nMethods:\n“Crystallization of ITK has been reported previously.” This sentence should be supported with a citation.", "reviewer_4": "Authors report a new compound, PF-07245303, developed by their group with dual inhibitory activity on ITK and TRK, and its potential as a novel topical medication for the treatment of atopic dermatitis and possibly other inflammatory skin disease. The study was comprehensive and well conducted, and provids a promising basis for further clinical studies.\nMajor problems:\n1. As authors stated, the effect of PF-07245303 is not Th2-specific. It is acceptable for authors to argue the potential of this compound as a topical medication to suppress type 2 inflammation of skin diseases, represented by atopic dermatitis. However, current descriptions in the introduction and the discussion of manuscript are substantially biassed to atopic dermatitis, and may be even misleading. Unmet needs in the treatment of atopic dermatitis and functions of PF-07245303 or its applicability should be discussed separately.\n2. Page 45, FIGURE 4. The panel layout is difficult to understand the relations of each panel. How about changing labels as b->c, d->e, d->b, e->d, or move g and h below to align as the third? In addition, if the horizontal length of panels d,e,f are enlarged to make the scale same as those of panels a,b,c, comparisons will be easier.\n3. Page 47, FIGURE 5b. Why statistical tests were conducted for RQ of 1 micro M of PF-07245303, but not for that of DMSO?\n4. The dynamic range of PF-07245303 on biological functions should be discussed more carefully. Since stimuli on ITK and those on TRK are different, it is not easy to compare the efficacies of PF-07245303 on those signalings. Nevertheless, IC50 of PF-07245303 on IL-4 production by basophils induced by anti-IgE receptor antibody and that on CD203c expression induced by NGF are largely different. Moreover, FIGURE 5 showed that the effect of PF-07245303 on cytokine expressions in dermal T cells in human skin sections stimulated by anti-CE3 and anti-CD28 antibodies are different among types of cytokines. For instance, the effect on IL-13 mRNA expression is low and the effect on IL-2 protein release is high.\nMinor problems:\n1. Page 2, line 46. LCK should be spelled out.\n2. Page 13, line 334. A phrase of “inhibition of Th2 cytokines IL-4 and IL-13” should be more accurate, for instance to “inhibition of the production of Th2 cytokines IL-4 and IL-13”.\n3. Page 18, line 403. Reference numbers, “1,56” should be up scribed.\n4. Page 20, line 447-448. Was recombinant human ITK labeled with histidine-tag? If not, please explain how the recombinant human ITK binds to flow cell.\n5. Page 21, line 458 and FIGURE 1c. The dissociation time, 480 seconds seems to be set for TRKA and TRKC, but not for ITK and TRKB.\n6. Page 47, FIGURE 5. The unit of PF-07245303 in panel c should be “micro”, not “u”.\n7. Page 47, line 1081. MSD should be spelled out."}, "author_response": "Authors’ response: Thank you for your time in reviewing this revision.\nAuthors’ response: We appreciate the reviewer’s suggestion, and we have changed “Jurkat cells” to “Jurkat T cells” in the manuscript for clarity.\nAuthors’ response: Thank you. We have made this important correction.\nAuthors’ response: This figure has been labeled as suggested.\nAuthors’ response: We have added the following sentence to the results section for the efficacy in the oxazolone-induced dermatitis model on page 14, lines 315-317 with a relevant reference. “It is worth noting that the oxazazolone-induced model of dermatitis does not fully mimic the complex features of clinical AD”. Reference: https://doi.org/10.3390/jcm10040613\nAuthors’ response: We appreciate the reviewer’s suggestion. The appropriate legend has been added to the figure for clarity.\nAuthors’ response: This sentence in the Figure 3 legend has been re-worded for clarity. Further, to address Reviewer #4’s interest in evaluating the effects of PF-07245303 on IL-10 production from T cells, we performed a new set of experiments in order to simultaneously measure IL-10 in addition to the other cytokine read-outs. Inhibition on all cytokines in Figure 3 a – e was evaluated in the same experiments using the same methods.\nAuthors’ response: The results of the other 2 donors were very similar to the representative donor in Figure 4H. We have included those graphs below. The statistical analysis of data from all 3 donors is found in Supplementary Table 3.\nAuthors’ response: A supplementary table document has been added to the manuscript providing information on the up- and downregulated genes with the gene descriptors and data results that were used to create the volcano plot. RNA Seq data is uploaded to a public database, and the accession number is added and will be released upon publication.\nAuthors’ response: We have added a supportive citation. https://doi.org/10.1042/BJ20131139\nAuthors’ response: Thank you for your time in reviewing this revision.\nAuthors’ response: Our drug discovery effort to develop this topical agent was focused on a treatment for atopic dermatitis considering the well-established role for ITK in modulating Th2 cytokine responses and allergic diseases. As such, we intentionally worked to evaluate the potential for the dual ITK and TRK family kinase inhibitor PF-07245303 to suppress expression of Th2-associated cytokines and we evaluated the compound effects in a human skin explant model where we used the addition of Th2 cytokines to better mimic the environment of atopic dermatitis lesions. For this reason, we have described in the introduction and in the discussion the mechanisms driving AD and how the dual ITK and TRK family kinase inhibitor PF-07245303 might reduce the inflammatory and prurigenic responses responsible for this disease. However, we also appreciate that T cell responses contribute to other dermatological diseases and therefore we have evaluated the effects of PF-07245303 on the production of IFNγ and IL-17 which are cytokines relevant for some forms of AD but also for additional dermatological diseases such as vitiligo and alopecia areata. For these reasons, in the introduction, we stated that “Additionally, Th1 cells, Th22 cells, and Th17 cells and the cytokines which they produce, IFNγ, IL-22, and IL-17, respectively, also contribute to atopic dermatitis pathogenesis1,3,4” (page 3, lines 66-68) and that “A compound capable of inhibiting ITK kinase activity and TRK family kinase activity may have therapeutic value in numerous inflammatory and allergic skin diseases, including atopic dermatitis and contact hypersensitivity, by suppressing dermal inflammation” (page 4, lines 89-92).\nSimilarly, we have included statements in the discussion such as on page 17 lines 373-377: “These results and our demonstration that inhibition of ITK kinase in isolated primary human T cells with PF-07245303 resulted in potent inhibition of Th2 cytokines as well as Th1 and Th17 cytokines IFNγ and IL-17A, and reduced disease severity in a mouse dermatitis model suggest that ITK and TRK kinase inhibition may prove efficacious in many inflammatory dermatological diseases including atopic dermatitis, psoriasis, and vitiligo.”\nThus, we intended to highlight the pharmacology of the dual ITK and TRK family kinase inhibitor PF-07245303 as relevant to suppressing mechanisms driving AD pathogenesis and this was balanced by describing how this topical agent might also be useful for multiple inflammatory skin diseases.\nAuthors’ response: Thank you for this recommendation. We have harmonized the scales of the x-axis in Figure 4 panels a-f. As recommended, we have also moved panels g and h as a third row.\nAuthors’ response: Thank you for identifying this mistake. We have corrected the figure to properly indicate statistical significance relative to DMSO treated control.\nAuthors’ response: We agree that the dynamic range for PF-07245303-mediated inhibition in different cellular systems with different stimuli and different read-outs is important. For this reason, we have factored the protein binding effects on PF-07245303 to determine intrinsic potency in assay systems using whole blood. Therefore, in the isolated basophil assay measuring anti-IgE receptor antibody-mediated IL-4 release we observe an IC50 of 84 nM. In the whole blood basophil assay measuring effects on NGF-induced CD203c expression we measured an IC50 of 442 nM which we state in the manuscript translates to an intrinsic compound potency of 39.8 nM when correcting for protein binding effects (page 8, lines 164-166 and page 10, lines 229-230). These potencies in these two different assay systems with different stimuli are only 2.1-fold different.\nAs you highlighted, we do see differences in sensitivity of different cytokine read-outs to PF-07245303-mediated inhibition of dermal T cells in a human skin explant system with more potent inhibition of IL-2 relative to IL-13 (Figure 5). We observe and report a similar rank-order sensitivity for PF-07245303-mediated IL-2 inhibition versus IL-13 inhibition in our isolated human T cell assays. PF-07245303 demonstrated an IC50 = 38.5 nM for IL-2 production from isolated T cells (Figure 2c) versus an IC50 = 348 nM for IL-13 production (Figure 3b). A similar differential sensitivity for different cytokines to ITK kinase inhibition has been reported by others in a mouse T cell system. Reference: 10.1126/scisignal.aab0949\nAuthors’ response: We have made this correction.\nAuthors’ response: We have made this correction.\nAuthors’ response: We have made these corrections.\nAuthors’ response: The SPR methods explain that an anti-histidine antibody was immobilized on the flowcell to capture the ITK protein.\nAuthors’ response: In the SPR methods (page 21 line 462) we have stated that 480 seconds was the dissociation time for all proteins tested.\nAuthors’ response: We have made this correction.\nAuthors’ response: We have made this correction."}, {"round": 3, "reviewer_comments": {"reviewer_3": "The Authors replied my comments. I do not have any concerns.", "reviewer_4": "I accept most of the replies and rebuttals from the authors. Following minor concerns should be addressed.\n1. Page 4, lines 89-92. It was understood that the focus on a treatment for atopic dermatitis considering the well-established role for ITK in modulating Th2 cytokine responses and allergic diseases. This intension of the authors could be clearer by changing the wording of the last sentence in the introduction, for instance, to “A compound capable of inhibiting ITK kinase activity and TRK family kinase activity may have therapeutic value in atopic dermatitis and other numerous inflammatory, including allergic skin diseases, such as contact hypersensitivity, by suppressing dermal inflammation”.\n2. Page 5, line 451-452. I asked about the labeling of recombinant ITK to be captured by anti-His antibody. I looked into Lake Pharma, but could not find the product information of the recombinant ITK. I believe that ITK was tagged with histidine. If not, it is not likely to be captured by anti-His antibody. Moreover, the company information, including city and country of the provider, should be described.\n3. Page 21, line 462-463 and FIGURE 1c. I now probably understand the meaning of the association time and the dissociation time. The former corresponds to the upper parts of the lines, and the latter to the lower parts of the panels. If so, 480 seconds of the dissociation time seems a mistake of 48 seconds. Please make it sure.\n4. Page 49, line 1062. The information of merchandise for Meso Scale Discovery assay kit (MSD) should be described."}, "author_response": "Authors’ response: Thank you for your time in reviewing this revision.\nAuthors’ response: Thank you for your time in reviewing this revision.\nAuthors’ response: Thank you for the suggested statement and we have updated the sentence above with the reviewer’s suggestion.\nAuthors’ response: Thank you for catching this missing detail. The recombinant ITK protein was produced for Pfizer by Lake Pharma (Belmont, CA USA) as a contract product. The material was His tagged and as such was captured and cross-linked by an anti-His antibody previously immobilized on the sensor chip. We have added this information to the manuscript.\nAuthors’ response: Thank you for your time in reviewing this revision. The reviewer is correct the dissociation time is from the end of the association of the highest concentration to the baseline. We have updated the methods and the text to reflect that the dissociation time for ITK is 180 seconds. We have also included a range for the TRKA,B,C dissociation time to reflect the methods settings across experiments (60 – 480 seconds).\nAuthors’ response: Thank you for catching this missing detail. The MSD kit that was used is the V-PLEX Human Proinflammatory Panel 1, Cat. No. K15049D. We have added this information to the manuscript."}, {"round": 4, "reviewer_comments": {"reviewer_4": "Authors’ replies made in the letter to the reviewers are fine. However, the reply to the first point on writing in the introduction in page 4, line 89-92 was not reflected in the manuscript. In addition, the clarification of MSD as Meso Scale Diagnostics in the reply to the fourth point should be completed by adding the city and country of the manufacturer in page 26 where it first appeared."}, "author_response": "Thank you for your suggestion. The statement has been changed to “A compound capable of inhibiting ITK kinase activity and TRK family kinase activity may have therapeutic value in atopic dermatitis and other numerous inflammatory, including allergic skin diseases, such as contact hypersensitivity, by suppressing dermal inflammation,” as the reviewer suggested.\nThank you for the suggested addition. We have added the city and country to the manufacturer Meso Scale Diagnostics (MSD, Rockville, MD, USA)."}]

Decision letter