An Allosteric Anti-tryptase Antibody for the Treatment of Mast Cell-Mediated Severe Asthma.

Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active ß-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human ß-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a ß-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma.

Inhibiting Isoprenylation Suppresses FcεRI-Mediated Mast Cell Function and Allergic Inflammation.

IgE-mediated mast cell activation is a driving force in allergic disease in need of novel interventions. Statins, long used to lower serum cholesterol, have been shown in multiple large-cohort studies to reduce asthma severity. We previously found that statins inhibit IgE-induced mast cell function, but these effects varied widely among mouse strains and human donors, likely due to the upregulation of the statin target, 3-hydroxy-3-methylgutaryl-CoA reductase. Statin inhibition of mast cell function appeared to be mediated not by cholesterol reduction but by suppressing protein isoprenylation events that use cholesterol pathway intermediates. Therefore, we sought to circumvent statin resistance by targeting isoprenylation. Using genetic depletion of the isoprenylation enzymes farnesyltransferase and geranylgeranyl transferase 1 or their substrate K-Ras, we show a significant reduction in FcεRI-mediated degranulation and cytokine production. Furthermore, similar effects were observed with pharmacological inhibition with the dual farnesyltransferase and geranylgeranyl transferase 1 inhibitor FGTI-2734. Our data indicate that both transferases must be inhibited to reduce mast cell function and that K-Ras is a critical isoprenylation target. Importantly, FGTI-2734 was effective in vivo, suppressing mast cell-dependent anaphylaxis, allergic pulmonary inflammation, and airway hyperresponsiveness. Collectively, these findings suggest that K-Ras is among the isoprenylation substrates critical for FcεRI-induced mast cell function and reveal isoprenylation as a new means of targeting allergic disease.

A randomized double-blinded trial to assess recurrence of systemic allergic reactions following COVID-19 mRNA vaccination.

BACKGROUND: Systemic allergic reactions (sARs) following coronavirus disease 2019 (COVID-19) mRNA vaccines were initially reported at a higher rate than after traditional vaccines. OBJECTIVE: We aimed to evaluate the safety of revaccination in these individuals and to interrogate mechanisms underlying these reactions. METHODS: In this randomized, double-blinded, phase 2 trial, participants aged 16 to 69 years who previously reported a convincing sAR to their first dose of COVID-19 mRNA vaccine were randomly assigned to receive a second dose of BNT162b2 (Comirnaty) vaccine and placebo on consecutive days in a blinded, 1:1 crossover fashion at the National Institutes of Health. An open-label BNT162b2 booster was offered 5 months later if the second dose did not result in severe sAR. None of the participants received the mRNA-1273 (Spikevax) vaccine during the study. The primary end point was recurrence of sAR following second dose and booster vaccination; exploratory end points included biomarker measurements. RESULTS: Of 111 screened participants, 18 were randomly assigned to receive study interventions. Eight received BNT162b2 second dose followed by placebo; 8 received placebo followed by BNT162b2 second dose; 2 withdrew before receiving any study intervention. All 16 participants received the booster dose. Following second dose and booster vaccination, sARs recurred in 2 participants (12.5%; 95% CI, 1.6 to 38.3). No sAR occurred after placebo. An anaphylaxis mimic, immunization stress-related response (ISRR), occurred more commonly than sARs following both vaccine and placebo and was associated with higher predose anxiety scores, paresthesias, and distinct vital sign and biomarker changes. CONCLUSIONS: Our findings support revaccination of individuals who report sARs to COVID-19 mRNA vaccines. Distinct clinical and laboratory features may distinguish sARs from ISRRs.

Defining baseline variability of serum tryptase levels improves accuracy in identifying anaphylaxis.

BACKGROUND: Acute increases of ≥20% + 2 ng/mL (20 + 2 rule) over basal serum tryptase (BST) is the recommended threshold supporting a clinical diagnosis of anaphylaxis. Prospective studies have demonstrated high sensitivity for this algorithm after parenteral exposure, but specificity has not been evaluated. OBJECTIVE: We sought to define a serum tryptase change that distinguishes baseline variability from anaphylaxis on the basis of intraindividual variation in BST. METHODS: Ninety-three total subjects with atopy (n = 62) or hereditary α-tryptasemia (HαT) (n = 31) and ≥2 BST measurements were identified. Sequential BST variability measurements were modeled and threshold ratios that optimized sensitivity and/or specificity determined. Models were tested in 22 individuals with physician-diagnosed anaphylaxis and validated in independent cohorts of individuals with HαT (n = 33), indolent systemic mastocytosis (ISM) (n = 52), and ISM + HαT (n = 12). Mature tryptase levels were measured in HαT (n = 19) and ISM (n = 20). An online application was developed for clinical use. RESULTS: As a result of BST variability, 9.7% (9/93) of primary cohort patients, and 18% (6/33) of HαT, 30% (16/53) of ISM, and 25% (3/12) of ISM + HαT patients from validation cohorts met the 20 + 2 rule despite absent immediate hypersensitivity symptoms; mature tryptase was noncontributory among individuals with HαT or ISM at baseline. A ratio of acute tryptase/BST exceeding 1.685 provided the optimized diagnostic rule for jointly maximizing sensitivity and specificity. Statistically significant improvement in specificity relative to the 20 + 2 rule was observed among individuals with elevated BST caused by HαT and ISM. CONCLUSIONS: Using an acute tryptase/BST ratio of 1.685 improves specificity of measured changes among individuals with HαT and ISM while maintaining high sensitivity for confirmation of anaphylaxis.

Heritable risk for severe anaphylaxis associated with increased α-tryptase-encoding germline copy number at TPSAB1.

BACKGROUND: An elevated basal serum tryptase level is associated with severe systemic anaphylaxis, most notably caused by Hymenoptera envenomation. Although clonal mast cell disease is the culprit in some individuals, it does not fully explain this clinical association. OBJECTIVE: Our aim was to determine the prevalence and associated impact of tryptase genotypes on anaphylaxis in humans. METHODS: Cohorts with systemic mastocytosis (SM) and venom as well as idiopathic anaphylaxis from referral centers in Italy, Slovenia, and the United States, underwent tryptase genotyping by droplet digital PCR. Associated anaphylaxis severity (Mueller scale) was subsequently examined. Healthy volunteers and controls with nonatopic disease were recruited and tryptase was genotyped by droplet digital PCR and in silico analysis of genome sequence, respectively. The effects of pooled and recombinant human tryptases, protease activated receptor 2 agonist and antagonist peptides, and a tryptase-neutralizing mAb on human umbilical vein endothelial cell permeability were assayed using a Transwell system. RESULTS: Hereditary α-tryptasemia (HαT)-a genetic trait caused by increased α-tryptase-encoding Tryptase-α/ß1 (TPSAB1) copy number resulting in elevated BST level-was common in healthy individuals (5.6% [n = 7 of 125]) and controls with nonatopic disease (5.3% [n = 21 of 398]). HαT was associated with grade IV venom anaphylaxis (relative risk = 2.0; P < .05) and more prevalent in both idiopathic anaphylaxis (n = 8 of 47; [17%; P = .006]) and SM (n = 10 of 82 [12.2%; P = .03]) relative to the controls. Among patients with SM, concomitant HαT was associated with increased risk for systemic anaphylaxis (relative risk = 9.5; P = .007). In vitro, protease-activated receptor-2-dependent vascular permeability was induced by pooled mature tryptases but not α- or ß-tryptase homotetramers. CONCLUSIONS: Risk for severe anaphylaxis in humans is associated with inherited differences in α-tryptase-encoding copies at TPSAB1.

Proceedings from the Inaugural American Initiative in Mast Cell Diseases (AIM) Investigator Conference.

The American Initiative in Mast Cell Diseases (AIM) held its inaugural investigator conference at Stanford University School of Medicine in May 2019. The overarching goal of this meeting was to establish a Pan-American organization of physicians and scientists with multidisciplinary expertise in mast cell disease. To serve this unmet need, AIM envisions a network where basic, translational, and clinical researchers could establish collaborations with both academia and biopharma to support the development of new diagnostic methods, enhanced understanding of the biology of mast cells in human health and disease, and the testing of novel therapies. In these AIM proceedings, we highlight selected topics relevant to mast cell biology and provide updates regarding the recently described hereditary alpha-tryptasemia. In addition, we discuss the evaluation and treatment of mast cell activation (syndromes), allergy and anaphylaxis in mast cell disorders, and the clinical and biologic heterogeneity of the more indolent forms of mastocytosis. Because mast cell disorders are relatively rare, AIM hopes to achieve a coordination of scientific efforts not only in the Americas but also in Europe by collaborating with the well-established European Competence Network on Mastocytosis.

Clinical relevance of inherited genetic differences in human tryptases: Hereditary alpha-tryptasemia and beyond.

OBJECTIVE: To describe our current understanding of hereditary α-tryptasemia (HαT), how HαT fits into the evolutionary context of tryptases and contemporary framework of mast cell-associated disorders, and to discuss the future clinical and therapeutic landscape for symptomatic individuals with HαT. DATA SOURCES: Primary peer-reviewed literature. STUDY SELECTIONS: Basic, clinical, and translational studies describing tryptase gene composition, generation, secretion, and elevation and the associated clinical impacts of HαT and treatment of such individuals were reviewed. RESULTS: HαT is a common autosomal dominant genetic trait caused by increased TPSAB1 copy number encoding α-tryptase. Approximately 1 in 20 White individuals have HαT, making it by far the most common cause for elevated basal serum tryptase levels. Although many individuals with HαT may not manifest associated symptoms, the prevalence of HαT is increased in patients with clonal and nonclonal mast cell-associated disorders wherein it is linked to more prevalent and/or severe anaphylaxis and increased mast cell mediator-associated symptoms. Increased generation of mature α/ß-tryptase heterotetramers, and their unique physiochemical properties, may be responsible for some of these clinical findings. CONCLUSION: HαT is a common modifier of mast cell-associated disorders and reactions. Nevertheless, whether HαT may be an independent cause of clinical phenotypes with which it has been associated remains unproven. Correct identification of HαT is critical to accurate interpretation of serum tryptase levels in the clinical evaluation of patients. Beyond HαT, we foresee tryptase genotyping as an important parameter in the standard workup of patients with mast cell-associated disorders and development of therapeutic modalities targeting these patients and associated clinical phenotypes.

Clinical Impact of Inherited and Acquired Genetic Variants in Mastocytosis.

Mastocytosis is a rare and complex disease characterized by expansion of clonal mast cells (MC) in skin and/or various internal organ systems. Involvement of internal organs leads to the diagnosis of systemic mastocytosis (SM). The WHO classification divides SM into indolent SM, smoldering SM and advanced SM variants, including SM with an associated hematologic neoplasm, aggressive SM, and MC leukemia. Historically, genetic analysis of individuals with pure cutaneous mastocytosis (CM) and SM have focused primarily on cohort studies of inherited single nucleotide variants and acquired pathogenic variants. The most prevalent pathogenic variant (mutation) in patients with SM is KIT p.D816V, which is detectable in most adult patients. Other somatic mutations have also been identified-especially in advanced SM-in TET2, SRSF2, ASXL1, RUNX1, CBL and JAK2, and shown to impact clinical and cellular phenotypes. Although only small patient cohorts have been analyzed, disease associations have also been identified in several germline variants within genes encoding certain cytokines or their receptors (IL13, IL6, IL6R, IL31, IL4R) and toll-like receptors. More recently, an increased prevalence of hereditary alpha-tryptasemia (HαT) caused by increased TPSAB1 copy number encoding alpha-tryptase has been described in patients with SM. Whereas HαT is found in 3-6% of general Western populations, it is identified in up to 17% of patients with SM. In the current manuscript we review the prevalence, functional role and clinical impact of various germline and somatic genetic variants in patients with mastocytosis.

Prevention of Hereditary Angioedema Attacks with a Subcutaneous C1 Inhibitor.

BACKGROUND: Hereditary angioedema is a disabling, potentially fatal condition caused by deficiency (type I) or dysfunction (type II) of the C1 inhibitor protein. In a phase 2 trial, the use of CSL830, a nanofiltered C1 inhibitor preparation that is suitable for subcutaneous injection, resulted in functional levels of C1 inhibitor activity that would be expected to provide effective prophylaxis of attacks. METHODS: We conducted an international, prospective, multicenter, randomized, double-blind, placebo-controlled, dose-ranging, phase 3 trial to evaluate the efficacy and safety of self-administered subcutaneous CSL830 in patients with type I or type II hereditary angioedema who had had four or more attacks in a consecutive 2-month period within 3 months before screening. We randomly assigned the patients to one of four treatment sequences in a crossover design, each involving two 16-week treatment periods: either 40 IU or 60 IU of CSL830 per kilogram of body weight twice weekly followed by placebo, or vice versa. The primary efficacy end point was the number of attacks of angioedema. Secondary efficacy end points were the proportion of patients who had a response (≥50% reduction in the number of attacks with CSL830 as compared with placebo) and the number of times that rescue medication was used. RESULTS: Of the 90 patients who underwent randomization, 79 completed the trial. Both doses of CSL830, as compared with placebo, reduced the rate of attacks of hereditary angioedema (mean difference with 40 IU, -2.42 attacks per month; 95% confidence interval [CI], -3.38 to -1.46; and mean difference with 60 IU, -3.51 attacks per month; 95% CI, -4.21 to -2.81; P<0.001 for both comparisons). Response rates were 76% (95% CI, 62 to 87) in the 40-IU group and 90% (95% CI, 77 to 96) in the 60-IU group. The need for rescue medication was reduced from 5.55 uses per month in the placebo group to 1.13 uses per month in the 40-IU group and from 3.89 uses in the placebo group to 0.32 uses per month in the 60-IU group. Adverse events (most commonly mild and transient local site reactions) occurred in similar proportions of patients who received CSL830 and those who received placebo. CONCLUSIONS: In patients with hereditary angioedema, the prophylactic use of a subcutaneous C1 inhibitor twice weekly significantly reduced the frequency of acute attacks. (Funded by CSL Behring; COMPACT EudraCT number, 2013-000916-10 , and ClinicalTrials.gov number, NCT01912456 .).