TSLP inhibitors in the treatment of asthma

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TSLP inhibitors in the treatment of asthma

TSLP inhibitors in the treatment of asthma.

Research status and prospects of TSLP inhibitors in the treatment of asthma.

The expression of TSLP is increased in the airways of asthmatic patients, and is related to the expression of type 2 chemokines and the severity of the disease.

At present, due to the heterogeneity of airway inflammation, asthma is difficult to treat. There is an urgent need for new treatment methods to treat patients with severe and uncontrolled asthma.

Thymus stromal lymphopoietin (TSLP) is a cytokine mainly expressed by the airway epithelium. It is released when the environment is damaged and triggers a series of downstream inflammatory processes.

Compared with healthy people, the expression of TSLP in the airway of asthma patients is increased, which is related to disease severity and lung function, that is, TSLP gene polymorphism is related to asthma.

It is reported in the literature that TSLP is a key mediator of asthma pathophysiology. It drives airway eosinophilic (allergic and non-allergic) inflammation, non-eosinophilic inflammation and structural changes by acting on a variety of adaptive and innate immune cells and structural cells .

Clinical trials of TSLP blockade through a systemic route have produced positive results in a wide range of asthma patients: it reduces deterioration and multiple inflammatory biomarkers, while improving lung function.

It will be helpful to find excellent TSLP target drugs by understanding the mechanism of TSLP and the development of drugs under research. The following is the detailed content.

1. The role of TSLP in asthma

Thymic stromal lymphopoietin (TSLP) is a short-chain four alpha helix bundle type I interleukin-2 (IL-2) family cytokine, homologous to IL-7.

It was first discovered in 1994 and was first identified Cytokines produced by thymic stromal cells.

TSLP is divided into short subtype and long subtype, which are composed of 60 amino acids and 159 amino acids, respectively.

They are controlled by different gene promoters and controlled by external stimuli.

The short subtype is expressed in a variety of tissues and is related to the steady-state function of TSLP, but its role has not yet been clearly studied.

The expression of the long subtype is inducible, exerts an inflammatory effect, and seems to be related to pathologies such as asthma, atopic dermatitis or psoriasis.

TSLP inhibitors in the treatment of asthma

Toll-like receptor ligands, pro-inflammatory cytokines (IFN-γ, tumor necrosis factor [TNF] and IL-1β) and specific cytokine environment (TNF-α+IL-4+IL-13) or alone TNF-α can up-regulate the long form but not the short form.

It has been documented that the long form contributes to the airway epithelial barrier dysfunction induced by dust mites, and the synthetic short form can prevent these effects.

Similar to IL-25 and IL-33, TSLP is mainly composed of epithelial cells, airway smooth muscle cells, keratinocytes, stromal cells, fibroblasts, mast cells, macrophages/monocytes, granulocytes and dendritic cells (Dc) Secretion.

There is ample evidence that it can differentiate naive CD4+ T lymphocytes in type 2 cells, produce IL-4, IL-5, and IL-13, and reduce interferon gamma (IFN-γ) associated with type 1 cells Expression affects a variety of cells.

The expression of TSLP is increased in the airways of asthmatic patients, and is related to the expression of type 2 chemokines and the severity of the disease.

The cultured primary bronchial epithelial cells spontaneously release TSLP. It suggests that TSLP is involved in the pathogenesis of asthma.

However, there are literatures showing that TSLP signal abnormalities are closely related to other inflammatory allergic diseases, including atopic dermatitis, eosinophilic esophagitis, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis.

In addition, its expression is also triggered by infection by viruses, bacteria and parasitic pathogens. Interestingly, the literature has shown that the respiratory epithelial cells of asthma patients have a strong response to viral double-stranded RNA, and their TSLP levels are higher than those of healthy controls.

TSLP initiates intracellular type 2 signaling by binding to its high-affinity heterodimeric receptor complex, which consists of its specific receptor TSLPR (encoded by CRLF2 located on chromosome 5q22.1) , It has 24% homology with the co-receptor γ chain in IL-2, IL-4, IL-9 and IL-15, and does not include the δ-common chain IL-2 family, and co-expresses TSLPR and IL-7Rα subunit of IL-7Rα (CD127) in cells.

TSLP initially binds to TSLPR and subsequently recruits IL-7Rα chains. TSLP itself has no obvious affinity for IL-7Rα.

TSLP forms a triple repeat of TSLP/TSLPR/IL-7Rα, which activates Janus kinase 1 (JAK1) and JAK2, leading to signal transducers and major transcriptional activators 5A (STAT5A) and STAT5B, as well as STAT1, STAT3 and other STAT proteins.

The degree of phosphorylation is low, mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NF-κB) depend on the cell type.

The production and release of TSLP in airway epithelial cells are usually activated by various dangerous signals, such as mechanical damage, infection-related factors, and allergens, which all support its function as a warning signal for impaired epithelial integrity.

TSLP directly acts on T cells, mast cells and natural killer cells, and indirectly activates CD11c+DC located in the lung or skin epithelium, and then metastasizes to the lymph nodes, activating CD4+ T cells to differentiate into type 2 cells, thereby promoting type 2 immune response.

Type 2 cell differentiation is mediated by TSLP-induced DC expression of TNF superfamily protein OX40 ligand (OX40L; CD252). OX40L expressed by DC interacts with OX40 on primitive T cells to generate type 2 lineage commitment by initiating signaling events.

These events lead to the production of a large number of pro-inflammatory cytokines, such as IL-4, IL-5, and IL-13, which can enhance the production of immunoglobulin (Ig) E, mast cells and mucus, and increase airway height Reactive, but does not enhance IL-10 and IFN-γ of a variety of natural immune cells.

In particular, the interaction between TSLP and CD34+ cells promotes the pro-inflammatory phenotype characterized by type 2 cytokines (IL-5 and IL-13), as well as the proliferation of eosinophils/basophils.

Phenotype. In addition, it increases the migration of eosinophils and may bind to IL-33 via the IL-13 dependent axis, thereby enhancing the production of IL-5 and IL-13.

There is evidence that TSLP released by bronchial epithelial cells drives in situ eosinophilia through the action of allergen proteases and biological components.

Therefore, it can be considered a vital factor in coordinating, persisting and expanding the inflammatory response of asthma.

In animal models, overexpression of TSLP in the lungs develops into asthma-like diseases. In addition, topical application of anti-TSLPR antibodies (Ab) and anti-TSLP antibodies can prevent type 2 mediated airway inflammation.

The importance of TSLP in asthma has been repeatedly proven. There is evidence that inhaled allergen stimulation in patients with mild allergic asthma can induce the expression of TSLPR in CD127 eosinophil progenitor cells in peripheral blood and basophils in blood and airways.

In addition, airway TSLP expression directly increases the degree of allergen challenge associated with an increase in late airway obstruction.

In asthmatic patients, the number of epithelial cells and submucosal cells that express TSLP mRNA is negatively correlated with forced expiratory volume in 1 second (FEV1).

There is also a correlation between airway hyperresponsiveness, serum IgE levels and the number of mast cells expressing TSLP.

Even more convincing is that despite high-dose inhalation or oral corticosteroid treatment, there are still some patients with severe asthma that have increased TSLP expression.

In fact, the type 2 innate lymphocytes (ILC2s) of asthmatic patients with elevated TSLP levels are resistant to steroids.

Therefore, targeting TSLP and TSLP-mediated signal transduction is considered an attractive strategy for the treatment of asthma. Anti-TSLP monoclonal antibodies (mAbs) and TSLP cytokine traps may be effective strategies for TSLP targeted therapy.

2. TSLP target drugs in the clinical stage

At present, many asthma drugs that act on different targets have been marketed, such as Omalizumab targeting IgE, Dupilumab targeting IL-13, ReslizumabMepolizumabBenralizumab targeting IL-5, etc.

However, drugs targeting TSLP are still There is no news of listing.

The TSLP target drugs that have entered the clinical stage are only a few biological drugs, such as the fully human IgG2λ monoclonal antibody Tezepelumab developed by AstraZeneca and Amgen to enter the phase III clinical stage, and the inhalation developed jointly by Novartis and MorphoSys Dosage type—Immunoglobulin G1/λ subtype antibody fragment CSJ-117 has entered phase II clinical phase, and other recombinant humanized IgG1 monoclonal antibodies such as Merck MK-8226, Roche RG7258, Astellas ASP7266 are in phase I Stop development after clinical trials.

Tezepelumab (AMG 157, MEDI9929) is a true first-in-class targeted TSLP monoclonal antibody. As a fully human IgG2λ monoclonal antibody, it can bind to human TSLP and prevent the interaction with its receptor, thereby inhibiting A variety of downstream inflammation pathways. Its currently ongoing clinical trials are shown in the table below.

The phase I clinical study aims to evaluate the initial safety. The pharmacokinetics shows that Tezepelumab is well tolerated in healthy and atopic dermatitis adult subjects, and shows predictable linear pharmacokinetic characteristics and performance. Accepted safety and tolerance.

In a proof-of-concept study, 700 mg of Tezepelumab intravenously every 4 weeks for 12 weeks reduced airway hyperresponsiveness and systemic indicators (compared to placebo, circulating eosinophil levels were reduced by 59% and 21, respectively) %) and airway indicators (the main scoring items are the amount of exhaled nitric oxide and sputum).

After allergen challenge in patients with mild allergic asthma, the eosinophil level dropped from 4.1% at baseline to 0.4% after 6 weeks of inflammation.

Early and late asthmatic responses were significantly reduced. Compared with the placebo group, the asthma response of the placebo group was reduced by 27%, and the asthma response of the placebo group was reduced by 34%.

In a phase IIb, randomized, double-blind, placebo-controlled trial, asthma patients receiving medium and high doses of inhaled corticosteroids/long-acting β2-agonists were given three doses of Tezepelumab subcutaneously for 52 weeks (each 70 mg every 4 weeks, 210 mg every 4 weeks or 280 mg every 2 weeks).

At week 52, regardless of the baseline blood eosinophil count, FeNO or serum IgE level, although continuous decline in blood eosinophil count and FeNO levels were recorded, they led to a 61% and 71% decrease in the annual deterioration rate, respectively And 66%.

Regardless of the dose, the FEV1 of bronchodilators used 52 weeks ago was always higher than that of the placebo group by >100 mL.

Symptom control in the middle and high dose groups was improved, but only the high dose group had improved health-related quality of life. Compared with the placebo group, there was no difference in the overall incidence of adverse events.

The discontinuous incidence of adverse events was 1.1% among patients treated with Tezepelumab and 0.7% in the placebo group.

The post-mortem analysis of this phase II clinical study showed that Tezepelumab not only reduced blood eosinophil count and FeNO levels, but also reduced IL-5, IL-13, periostin, thymus and activated regulatory chemokine (TARC) and The level of IgE.

Research also shows that in patients with severe, uncontrolled asthma, Tezepelumab reduces the frequency of hospitalizations and emergency room visits, and reduces the number of days in the hospital or intensive care unit.

In any case, the exposure-response analysis shows that 210 mg every 4 weeks is the optimal dose for the phase III trial in patients with severe asthma. Tezepelumab’s effect on aggravation and FeNO reduction is not affected by blood eosinophils or other type 2 biomarkers Impact.

It is precisely because of the results of this phase II clinical trial that the FDA approved Tezepelumab as a “breakthrough” biologic drug for the treatment of severe asthma.

Some studies are underway, mainly to verify the long-term effectiveness and safety of Tezepelumab in the control of severe asthma in Caucasian or Asian adults and adolescents, and to evaluate the function and performance of auxiliary prefilled syringes or autoinjectors for subcutaneous injection of Tezepelumab. The patient can self-administer the medicine at home.

In November 2020, the phase III clinical trial NAVIGATOR reached the primary study endpoint, showing that targeted TSLP has therapeutic benefits for severe asthma: in a broad population of severe asthma patients, tezepelumab can make asthma exacerbations statistically significant and clinically significant , Including patients with low eosinophil counts.

The data showed that the study reached the primary endpoint: in the entire patient population, compared with placebo + standard of care (SoC), tezepelumab + SoC treatment made the 52-week asthma exacerbation rate (AAER) statistically significant and clinically significant The reduction.

In this trial, the SoC was a medium-dose or high-dose inhaled corticosteroid (ICS) plus an additional control drug, with or without oral corticosteroids (OCS).

In addition, in the subgroup of patients with a baseline eosinophil count of <300 cells/microliter, the trial also reached the primary endpoint: compared with placebo+SoC, tezepelumab+SoC treatment made AAER statistically significant and The reduction of clinical significance.

Similar reductions in AAER were observed in the subgroup of patients with a baseline eosinophil count of <150 cells/μl.

In terms of safety, tezepelumab is well tolerated in patients with severe asthma.

Preliminary analysis showed that there was no clinically significant difference in safety results between the tezepelumab treatment group and the placebo group.

In December 2020, AstraZeneca/Amgen announced the results of the high-level Phase III SOURCE study.

The study evaluated the efficacy and safety of adding tezepelumab (210mg, once every 4 weeks) vs placebo for 48 weeks in 150 patients with severe asthma who needed standard therapy (LABA) combined with oral glucocorticoid (OCS) maintenance treatment Sexual difference.

The primary end point of the study was the percentage decrease in OCS dosage from baseline when asthma was under continuous control at the 48th week. The secondary end points included annualized asthma exacerbation rate, lung function, asthma control, quality of life, and work efficiency.

The results showed that the SOURCE study failed to reach the primary endpoint, and the tezepelumab treatment group failed to reduce the daily OCS dosage compared with placebo.

Tezepelumab’s other efficacy indicators are consistent with the results of previous studies, including the registration phase III NAVIGATOR study.

The safety results of Tezepelumab are also consistent with previous studies.

3. Future prospects of TSLP target drugs

Increasingly conclusive evidence shows that TSLP is closely related to the pathophysiology of asthma. Although Tezepelumab’s high-level Phase III clinical trial failed to reduce the amount of corticosteroids in asthma patients in December, its good efficacy has shown that people are looking for ways to block TSLP. The new method generated great interest.

The status of potential drugs reported in the literature that have inhibitory activity on TSLP targets is shown in the following table.

Literature has shown that, compared with mouse and humanized antibodies, the fully human single-chain variable region fragments (scfv) of anti-TSLP have higher specificity, show higher affinity, but have lower repellency. scFv29 is screened from a fully human antibody library and is specific for TSLP and does not cross-react with IL-33, IL-4 and IL-13.

Preliminary experiments show that scFv29 has a strong potential to bind TSLP in competition with the TSLP receptor. Can reduce the maturity rate of DC.

Therefore, scFv29 is considered to be a neutralizing antibody that blocks TSLP signaling.

Bifunctional drugs, specifically designed to have two mechanisms of action in the same molecule, provide an exciting new method for the treatment of asthma.

There are also attempts to manufacture bifunctional drugs through the dual targeted combination of cytokines and monoclonal antibodies.

The monovalent bispecific antibody Zweimab and the bivalent bispecific antibody Doppelmab, which are used to target TSLP and IL-13 due to the huge overlap of signal pathways, have been designed, developed and characterized.

The decision to combine anti-IL-13 mAbs with anti-TSLP mAbs in a bispecific format stems from the evidence that these two cytokines are co-expressed in the airway epithelium and lamina propria of severe asthma patients.

Zweimab is a heterodimer with a knobin hole mutation in the heavy chain. Doppelmab has a single-chain antibody portion that is connected to the CH3 domain through a short polypeptide linker. The two have stronger affinity to human targets.

The fusion protein consists of the extracellular domain of TSLPR and IL-7Ra that extends into the extracellular space, also known as the TSLP trap, by combining the extracellular domain of TSLPR and IL-7Rα with a flexible (Gly–Gly–Ser) in two directions.

The linker is formed by fusion and is called TSLP-trap1 and TSLP-trap2. In fact, the “cytokine trap” is formed by the fusion of the constant region of IgG and the extracellular region of two different cytokine receptor components, which bind to cytokines.

The binding strength of the two TSLP traps to TSLP is 250 times that of the unconnected receptor outer domain, and it is 20-30 times stronger than Tezepelumab and its Fab fragment in inhibiting TSLP-induced STAT5. In addition, both TSLP traps can significantly inhibit TSLP-driven DC activation and are as effective as Tezepelumab in this regard.

At the same time, researchers are also trying to find fragments that can destroy the TSLP/TSLPR complex.

Four fragments of different chemical classes have been identified, reducing the formation of TSLP/TSLPR complex to less than 75% with millimolar concentration.

All fragments are positively charged, in line with the cationic nature of the natural ligand TSLP. Fragments combined with TSLP sites undergo a conformational change.

Recently, baicalein was identified as the first small molecule inhibitor of human TSLP signaling pathway.

In vitro studies have shown that the compound blocks the interaction between human TSLP and human TSLPR in a dose-dependent manner. In addition, in the ovalbumin-induced animal model, baicalein monotherapy effectively reduced eosinophil-rich lung inflammation.

The structure-activity relationship study determined that compound 11a is a biphenylflavanone analogue and a new human TSLP inhibitor for the discovery and development of new anti-allergic drugs.

In another study, a new compound PA inhibited the expression and production of TSLP mRNA by blocking caspase-1 in mast cells.

It is well known that caspase-1 is activated by pro-inflammatory stimuli because they increase intracellular calcium levels, which increases intracellular calcium leading to caspase-1 activation.

Linalyl acetate, one of the main components of lavender, inhibits TSLP production and mRNA expression by blocking the caspase-1/NF-κB pathway. In addition, it also reduces intracellular calcium levels.

Catechin is the main active ingredient in green tea. The dosage is 75, 150, 300 mg/kg, which can alleviate allergic symptoms in mice with allergic rhinitis, reduce the levels of IL-5 and IL-13, and affect NF-κB/ The TSLP pathway inhibits the expression of TSLP in epithelial cells and restores the balance of T helper 2/T helper 1 cells.

In contrast, 16D10, a chalcone derivative, in addition to directly inhibiting NF-κB and activating the Kelch-like ECH-related protein 1 (Keap1)-nuclear factor-erythrocytic line-2 related factor 2 (Nrf2) system, it also selects through an unknown mechanism Inhibit the expression and production of TSLP in mouse and human keratinocyte cell lines. Importantly, 16D10 inhibits the expression of TSLP and ovalbumin-specific IgG1 and IgE in vivo.

TSLP inhibitors in the treatment of asthma

(source:chinanet, reference only)

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