Strategies and Case Studies of JAK-STAT Targeted Therapies

Strategies for Novel Autoimmune Drug Development: Preclinical Efficacy and Case Studies of JAK-STAT Targeted Therapies

JAK Family Molecules: Central Hubs in Immune Signaling

 As a family of non-receptor tyrosine kinases, Janus kinases (JAKs) serve as central hubs in immune system signaling ^[1]. This family comprises JAK1, JAK2, JAK3, and TYK2. Their expression patterns and functions in immune cells are both synergistic and highly specific, and their aberrant activation or loss of function is closely associated with the pathogenesis and progression of various autoimmune diseases. The functional roles of individual family members are outlined below (Figure 1):

• JAK1: Primarily mediates the signaling of IL-6, IFN-γ, and the common gamma-chain (γc) family of cytokines, playing an indispensable role in inflammatory responses and immunomodulation ^[2]
• JAK2：Broadly involved in the proliferation and activation of immune cells, mediating the signaling of multiple cytokines, including GM-CSF and IL-3 ^[3]
• JAK3：By associating with the receptors of the common γc cytokine family, it plays a critical role in T cell development, the maintenance of homeostasis, and the function of regulatory T cells (Tregs) ^[2]
• TYK2：Primarily mediates the signal transduction of IFN-α, IL-12, and IL-23 ^[2]

Figure 1. Roles of JAK family members in diverse cytokine receptor signaling

By pairing with various cytokine receptor subunits, these four JAK kinases form an intricate signaling network within the immune system that collectively maintains immune homeostasis. Dysregulation at any point in this network can disrupt this balance. Gain-of-function mutations, loss-of-function mutations, and aberrant signaling involving JAK family members have been demonstrated to be closely associated with the pathogenesis and progression of multiple autoimmune diseases, including rheumatoid arthritis, ankylosing spondylitis, atopic dermatitis, graft-versus-host disease, psoriasis, and inflammatory bowel disease ^{[2, 3]}.

Applications and Novel Drug Development Strategies of JAK Inhibitors in Autoimmune Diseases

Given the pivotal role of the JAK-STAT signaling pathway in the pathogenesis of autoimmune diseases, inhibitors targeting JAK molecules have emerged as a vital class of therapeutics in this field. To date, multiple JAK inhibitors have been approved globally by regulatory authorities, such as the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA), for the treatment of various autoimmune diseases ^[2] (Table 1).

Table 1. Selected JAK inhibitors approved for the treatment of autoimmune diseases ^[2]

Meanwhile, in the current investigational drug pipeline, several JAK inhibitors targeting autoimmune diseases are advancing rapidly and have entered Phase III clinical trials. For example:

• Lynk Pharmaceuticals (LNK01001): As a highly selective JAK1 inhibitor, its development for atopic dermatitis, rheumatoid arthritis, and ankylosing spondylitis has entered Phase III clinical trials in China.
• InnoCare Pharma (ICP-332): An internally developed TYK2 inhibitor, its Phase III registrational clinical trial for the treatment of moderate-to-severe atopic dermatitis has completed patient enrollment.
• Highlightll Pharma (TLL-018): As a dual TYK2/JAK1 inhibitor, it is currently undergoing two Phase III registrational clinical trials in China for chronic spontaneous urticaria and rheumatoid arthritis.

The advancement of these investigational drugs is expected to provide more precise and highly effective treatment options for patients with autoimmune diseases. Currently, the core R&D strategy for novel JAK inhibitors lies in precision and differentiation, which primarily encompasses the following four dimensions:

1. Highly selective inhibitors becoming the mainstream: This approach aims to intuitively enhance efficacy while significantly reducing off-target side effects by precisely targeting specific subtypes (such as JAK1, JAK3, or TYK2) or by targeting driver mutations (such as JAK2 V617F).
2. Exploring novel molecular modalities: To overcome drug resistance and achieve more profound efficacy, the R&D focus has shifted from simple activity inhibition toward new molecular modalities, such as targeted protein degradation (e.g., PROTACs).
3. Target expansion and dual-target synergy: Based on an in-depth understanding of the signaling pathways, efforts are being made to expand therapeutic areas and explore traditional “undruggable” targets like the STAT proteins. Concurrently, designing dual-target inhibitors to leverage synergistic effects has emerged as an important R&D direction.
4. Empowerment through data science-driven algorithms: The deep integration of data science-based algorithms and molecular modeling is accelerating the discovery and optimization of novel compound scaffolds, thereby significantly enhancing R&D efficiency and success rates.

In response to the increasingly broad, in-depth, precise, and differentiated drug R&D demands related to the JAK-STAT signaling pathway, the Immunology Center of WuXi Biology offers comprehensive, one-stop preclinical in vitro and in vivo testing services. Our team provides comprehensive support for the R&D of novel therapeutics targeting various autoimmune and inflammatory diseases associated with the JAK-STAT signaling pathway (Figure 2).

Figure 2. The Immunology Center of WuXi Biology empowers the R&D of novel drugs for autoimmune and inflammatory diseases.

Case Study 1: In Vitro Efficacy Validation of JAK Inhibitors

 WuXi Biology has established a comprehensive and robust molecular and cellular-level validation platform, capable of providing thorough and precise in vitro efficacy evaluations for a diverse array of drug candidates targeting the JAK-STAT signaling pathway.

• Molecular-level validation: Starting with protein interactions and functional assays, the platform can rapidly assess the binding affinity between drugs and JAK molecules (Figure 3A), and their inhibitory effects on kinase activity (Figure 3B). This enables comprehensive and highly efficient early-stage screening and evaluation of potential drug candidates.

Figure 3. In vitro molecular validation for drugs targeting the JAK-STAT pathway.

• Cellular-level validation: Equipped with a comprehensive testing platform and extensive project experience, WuXi Biology can utilize a variety of cell lines to validate the in vitro efficacy of various JAK-STAT-targeted drug candidates across multiple dimensions. These multi-dimensional assessments include the phosphorylation levels of STAT molecules (Figure 4A), downstream mRNA expression within the JAK-STAT signaling pathway (Figure 4B), protein expression levels (Figure 4C), and cellular functions (Figure 4D).

Figure 4. In vitro cellular-level validation for drug candidates targeting the JAK-STAT pathway.

Case Study 2: In Vivo Efficacy Validation for Atopic Dermatitis (AD)

 WuXi Biology has established a comprehensive portfolio of in vivo models for autoimmune diseases, capable of providing in vivo efficacy evaluations across multiple indications for a wide variety of drug candidates targeting the JAK-STAT signaling pathway.

Our team successfully established an animal model of AD by intermittently applying the hapten oxazolone (OXA) topically to the upper back and ears of mice. Compared to the normal control group, mice in the model group exhibited significantly increased clinical ear scores, ear weights, and spleen weights (Figure 5A). Furthermore, the ears of the modeled mice exhibited redness, swelling, increased thickness, and scaling (Figure 5B), indicating the successful establishment of the model. Following the administration of either dexamethasone or the pan-JAK inhibitor tofacitinib, the clinical ear scores, ear weights, and spleen weights of the mice were significantly reduced compared to those in the untreated model group (Figure 5A), and the ear-related symptoms were markedly improved (Figure 5B).

Figure 5. Efficacy of tofacitinib in the OXA-induced AD in vivo model.

Histopathological analysis revealed that, compared to normal control mice, the ear tissues of the model group were significantly thickened, accompanied by pronounced immune cell infiltration and a massive accumulation of mast cells. Conversely, treatment with dexamethasone or tofacitinib reduced ear thickness, attenuated immune cell infiltration, and  decreased the number of mast cells (Figure 6A). Furthermore, flow cytometric analysis of lymphocytes in the ear-draining lymph nodes revealed that animals treated with dexamethasone or tofacitinib exhibited a significant reduction in the total number of leukocytes on Day 26. Additionally, the cell counts of various T cell subpopulations—including total T cells, CD4+ T cells, regulatory T cells (Tregs), and CD8+ T cells—were significantly decreased (Figure 6B), suggesting that tofacitinib may exert its therapeutic effects by inhibiting lymphocyte proliferation. The distinct therapeutic window observed between the model group and the treatment groups indicates that this model is highly suitable for the in vivo efficacy validation of various JAK inhibitors.

Figure 6. Histopathological changes in ear tissue and altered immune cell profiles in the draining lymph nodes within the OXA-induced AD in vivo model.

Case Study 3: In Vivo Efficacy Validation for Psoriatic Arthritis (PsA)

By administering an intraperitoneal injection of mannan, WuXi Biology successfully established an animal model of PsA. Compared to the normal control animals, the clinical scores for both the ears and paws in the model group were significantly elevated (Figure 7A). Histopathological analysis revealed marked skin thickening and immune cell infiltration in the model group, as well as significant immune cell infiltration within the joint cavities (Figure 7B). Furthermore, analysis of ear and joint tissue homogenates demonstrated a significant increase in the expression levels of key pro-inflammatory cytokines, TNFα and IL-17A, indicating the successful establishment of the model (Figures 7C, 7D).

Compared with the untreated model group, treatment with the TYK2-specific inhibitor deucravacitinib effectively improved clinical scores for the ears and paws, as well as skin and joint histopathological scores, and reduced TNFα and IL-17A levels in ear and joint homogenates. This suggests that this model is highly suitable for the in vivo efficacy validation of TYK2-specific inhibitors, as well as various other JAK inhibitors.

Figure 7. Efficacy of deucravacitinib in the mannan-induced PsA in vivo model.

Case Study 4: In Vivo Efficacy Validation for Sjögren’s Syndrome (SS)

By inducing an immune response in rats with injections of salivary gland proteins, the team successfully established an animal model of SS. Compared with normal control animals, the body weight (Figure 8A) and saliva secretion volume (Figure 8B) of the model group decreased significantly, while their salivary gland histopathological scores increased significantly (Figures 8C, D), indicating the successful establishment of the model. Compared with the untreated model group, the administration of upadacitinib and filgotinib resulted in the recovery of salivary secretion to varying degrees (Figure 8B), and  salivary gland histopathological scores were significantly reduced (Figures 8C, D). This suggests that this model is sensitive to JAK inhibitors and is well-suited for the efficacy validation of various JAK inhibitors

Figure 8. Efficacy evaluation of upadacitinib and filgotinib in the salivary gland protein-induced SS in vivo model.

Summary

To support the development of novel drugs targeting the JAK-STAT signaling pathway, the Immunology Center of WuXi Biology has established a comprehensive suite of mature in vitro assay models and in vivo autoimmune disease models. These platforms can be utilized to explore the interactions, safety profiles, and mechanisms of action of JAK-STAT candidate drugs, as well as the pharmacodynamics, pharmacokinetics, and biomarkers of various JAK-STAT pathway-targeted therapeutics across a wide array of autoimmune diseases.

Table 2. Selected in vitro assays and in vivo animal models related to the JAK-STAT signaling pathway established by the Immunology Center of WuXi Biology.

References:

1. Ghoreschi K, Laurence A, O’Shea JJ. Janus kinases in immune cell signaling. Immunol Rev. 2009 Mar;228(1):273-87.
2. Wu, Z., Lian, G., He, L., Hu, P., Guo, Y. and Su, Z. (2026), Targeting JAK–STAT signaling for autoimmune diseases: current understanding, clinical advances, and emerging directions. Immunol Cell Biol, 104: 172-191.
3. Xue, C., Yao, Q., Gu, X. et al.Evolving cognition of the JAK-STAT signaling pathway: autoimmune disorders and cancer. Sig Transduct Target Ther 8, 204 (2023).