Interleukin-2 Receptor Alpha Chain Research Articles

Elevated circulating soluble interleukin-2 receptor (sCD25) level is associated with prefrontal excitatory-inhibitory imbalance in individuals with chronic pain: A proton MRS study

Aberrant neuronal excitability in the anterior cingulate cortex (ACC) is implicated in cognitive and affective pain processing. Such excitability may be amplified by activated circulating immune cells, including T lymphocytes, that interact with the central nervous system. Here, we conducted a study of individuals with chronic pain using magnetic resonance spectroscopy (MRS) to investigate the clinical evidence for the interaction between peripheral immune activation and prefrontal excitatory-inhibitory imbalance. In thirty individuals with chronic musculoskeletal pain, we assessed markers of peripheral immune activation, including soluble interleukin-2 receptor alpha chain (sCD25) levels, as well as brain metabolites, including Glx (glutamate + glutamine) to GABA+ (γ-aminobutyric acid + macromolecules/homocarnosine) ratio in the ACC. We found that the circulating level of sCD25 was associated with prefrontal Glx/GABA+. Greater prefrontal Glx/GABA+ was associated with higher pain catastrophizing, evaluative pain ratings, and anxiodepressive symptoms. Further, the interaction effect of sCD25 and prefrontal Glx/GABA+ on pain catastrophizing was significant, indicating the joint association of these two markers with pain catastrophizing. Our results provide the first evidence suggesting that peripheral T cellular activation, as reflected by elevated circulating sCD25 levels, may be linked to prefrontal excitatory-inhibitory imbalance in individuals with chronic pain. The interaction between these two systems may play a role as a potential mechanism underlying pain catastrophizing. Further prospective and treatment studies are needed to elucidate the specific role of the immune and brain interaction in pain catastrophizing.

FEATURES OF THE T-IMMUNE SYSTEM IN PATIENTS WITH GLOMERULONEPHRITISES WITH NEPHROTIC SYNDROME

The study of issues related to glomerulonephritises with nephrotic syndrome is one of the urgent problems of medicine due to their prevalence worldwide, mainly in the young age group. Medical workers distinguish primary (idiopathic) nephrotic syndrome, which occurs in 80–90% of cases, and secondary nephrotic syndrome, mainly associated with systemic autoimmune diseases, diabetes mellitus and neoplasms. Glomerulonephritises, manifested by nephrotic syndrome (membranous nephropathy, focal segmental glomerulosclerosis, nephropathy with minimal changes), are known to be autoimmune diseases. To date, the immunological mechanisms of the pathogenesis of glomerulonephritises with nephrotic syndrome associated with the T-system of adaptive immunity remain unexplored. The aim of the study was to study the role of the T–immune system in the pathogenesis of primary nephrotic syndrome based on the study of immunoregulatory, activated T-cell subsets in patients with this pathology. Material and methods. 136 patients with chronic glomerulonephritis with nephrotic syndrome were selected for the study. The assessment of the T-immune system included determination of the lymphocyte phenotype of immunoregulatory T-cell subsets (T-helper/inducers, cytotoxic T-lymphocytes), various subpopulations of activated T-cells (activated T-lymphocytes; activated T-lymphocytes expressing CD25–alpha chain of IL-2 receptor; activated cytotoxic T-lymphocytes expressing HLA-DR and CD38) and regulatory T-cells (Treg cells). Study results. In the patients of the examined cohort, an increase in the number of T-lymphocytes and T-helper cells, as well as activated T-lymphocytes expressing HLA-DR antigens, was found. At this, the content of cytotoxic T-cells and the number of activated T-cells expressing the IL-2 – CD25 receptor did not differ from similar indicators in healthy individuals. The levels of Treg cells and activated cytotoxic T-lymphocytes with the CD3+CD8brightCD38+ phenotype were reduced. The immunoregulatory index (T-helpers/cytotoxic T-lymphocytes) was increased, due to an increase in the number of T-helper cells against the background of an unchanged number of cytotoxic T-lymphocytes. Conclusions. The results of the study indicate that the main features of the T-system of the immune response in primary nephrotic syndrome are imbalance in the ratio of the content of immunoregulatory cells due to predominance of T-helper cells and a decrease in the number of Treg cells.

Abstract 4079: EGL-001 is a novel immunocytokine designed to specifically target and disarm T regulatory cells in the tumor microenvironment

Abstract Introduction Immune checkpoint blockade (ICB) therapies targeting PD-1/PD-L1 or CTLA-4 have greatly improved clinical outcome in many cancers. However, multiple mechanisms of resistance to ICB severely limit the number of patients experiencing long-term survival benefits. Overcoming resistance to ICB represents a significant challenge for drug development. Regulatory T cells (Tregs) in the tumor microenvironment (TME) prevent effector T cells (Teffs) from mounting a productive immune response and have been implicated in ICB resistance. Novel therapeutic strategies aimed at disarming Tregs in the TME could restore anti-tumor responses and sensitize patients to ICB therapy. Treg survival is dependent on interleukin-2 (IL-2), which signals in Tregs via the high affinity IL-2 receptor (IL-2R). Tumor-associated Tregs differentiate from tumor-associated Teffs and from peripheral Tregs by higher expression of CD25, the IL-2R alpha chain. Tregs in the TME also express higher surface levels of CTLA-4 as compared to peripheral Tregs. Thus, CD25 and CTLA-4 overexpression could allow preferential targeting of tumor-associated Tregs. Methods and Results We used single-cell RNA sequencing data from cancer patients and flow cytometry analysis of human tumor samples to verify that CD25 and CTLA-4 are selectively overexpressed in tumor-associated Tregs as compared with peripheral Tregs or other T cells. Based on these findings, we developed EGL-001, a novel immunocytokine (ICK) comprising a humanized IgG1 directed against CTLA-4 fused to a Treg-specific IL-2R antagonist IL-2 mutein, to selectively target tumor infiltrating Tregs. EGL-001 IgG1 moiety binds membrane CTLA-4, as demonstrated by flow cytometry, and competes with CTLA-4-CD80/86 interaction, as evaluated by ELISA. EGL-001 IL-2 mutein moiety binds to CD25, as demonstrated by bio-layer interferometry, and specifically blocks high-affinity IL-2R signaling in Tregs. Signaling blockade is further potentiated by a CTLA-4 dependent CD25 downmodulation process evidenced by confocal microscopy and flow cytometry. We demonstrate that EGL-001 unique tumor-associated Treg-targeted dual mechanism of action results in increased Treg apoptosis and decreased Treg frequencies in multiple in vitro, ex vivo and in vivo systems. Importantly, EGL-001 only minimally affects IL-2 signaling and survival of Teffs, which are then expected to be unleashed from Treg cell-mediated suppression. Accordingly, EGL-001 induces anti-tumor responses in mice in combination with anti-PD1 therapy. Conclusions EGL-001 is an ICK with a novel and unique mechanism of action combining ICB and Treg selective IL-2 starvation, designed to specifically disarm and deplete tumor-associated Tregs. Based on our preclinical data, EGL-001 is being developed as a cancer immunotherapeutic to improve patients’ response to ICB and counteract ICB resistance. Citation Format: Fiorella Kotsias, Caroline Janot-Sardet, Pamela Pamela Caudana, Erica Russo, Marine Dubois, Andres Zucchetti, Mercedes Tkach, Sebastien Lemoine, Ilgar Z. Mamedov, Alvaro Lladser, Jordan Denizeau, Annalisa Meola, Christine Sedlik, Edith Borcoman, Felix Rey, Pablo Guardado-Calvo, Bernard Vanhove, Monica Gostissa, Eliane PIaggio. EGL-001 is a novel immunocytokine designed to specifically target and disarm T regulatory cells in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4079.

Estimation of plasma soluble interleukin-2 receptor alpha chain level in adults with acute myeloid leukemia

Abstract: BACKGROUND: Acute myeloid leukemia (AML) is a clonal malignant condition of immature hematopoietic cells, characterized by clonal proliferation of abnormal cells (blasts) in the marrow leading to impairment of the normal blood cell production giving rise to failure of the bone marrow. Soluble interleukin-2 (IL-2) receptor alpha chain is a protein that is involved in the assembly of the high-affinity IL-2 receptor, and it has a critical role in controlling immune system homeostasis. The overexpression of sIL-2RA was investigated in many hematopoietic malignancies, and it was correlated with poor outcome. OJECTIVES: The aim of this study was to assess the sIL-2RA level as a prognostic factor and assess its impact on survival and if it can be used as a targeted treatment for a better outcome. MATERIALS AND METHODS: Sixty newly diagnosed adults with AML before starting therapy were included in the study, and they were followed up for 6 months to document survival status. Thirty healthy adults were taken as a control group. Using an enzyme-linked immunosorbent assay, the plasma sIL-2RA level was measured. Statistical analysis was done using Microsoft Excel 2019 and version 26 SPSS statistical software. P <0.05 was considered statistically significant. RESULTS: A considerable difference in the plasma sIL-2RA level between AML patients and controls also was more elevated in patients who died after 6-month follow-up. According to the blast percentage, total white blood cell count, and M0-M2 subgroups, the sIL-2RA level correlated positively. Irrelevant association was found regarding the patients’ age, the count of platelet, and the hemoglobin. CONCLUSIONS: Plasma sIL-2RA level is higher in AML patients than the control group at the time of diagnosis. Patients with a high level of plasma sIL-2RA have an inferior (overall survival) and poor outcome. SIL-2RA level is higher in M0-M2 subgroups than other subtypes. There is a significant association between sIL-2RA level and the absolute count of leukemic blasts.

Identification of immune cell markers associated with ulcerative colitis histological disease activity in colonic biopsies

AimsAccurate determination of histological activity in ulcerative colitis (UC) is essential given its diagnostic and prognostic importance. Data on the relationship between histology and immune cell markers are limited. We...

Altered IL-7 signaling in CD4+ T cells from patients with visceral leishmaniasis.

CD4+ T cells play a central role in control of L. donovani infection, through IFN-γ production required for activation of macrophages and killing of intracellular parasites. Impaired control of parasites can in part be explained by hampered CD4+ T cells effector functions in visceral leishmaniasis (VL) patients. In a recent studies that defined transcriptional signatures for CD4+ T cells from active VL patients, we found that expression of the IL-7 receptor alpha chain (IL-7Rα; CD127) was downregulated, compared to CD4+ T cells from endemic controls (ECs). Since IL-7 signaling is critical for the survival and homeostatic maintenance of CD4+ T cells, we investigated this signaling pathway in VL patients, relative to ECs. CD4+ T cells were enriched from peripheral blood collected from VL patients and EC subjects and expression of IL7 and IL7RA mRNA was measured by real time qPCR. IL-7 signaling potential and surface expression of CD127 and CD132 on CD4+ T cell was analyzed by multicolor flow cytometry. Plasma levels of soluble IL-7 and sIL-7Rα were measured by ELISA. Transcriptional profiling data sets generated previously from our group showed lower IL7RA mRNA expression in VL CD4+ T cells as compared to EC. A significant reduction was, however not seen when assessing IL7RA mRNA by RT-qPCR. Yet, the levels of soluble IL-7Rα (sIL-7Rα) were reduced in plasma of VL patients compared to ECs. Furthermore, the levels of soluble IL-7 were higher in plasma from VL patients compared to ECs. Interestingly, expression of the IL-7Rα protein was higher on VL patient CD4+ T cells as compared to EC, with activated CD38+ CD4+ T cells showing higher surface expression of IL-7Rα compared to CD38- CD4+ T cells in VL patients. CD4+ T cells from VL patients had higher signaling potential baseline and after stimulation with recombinant human IL-7 (rhIL-7) compared to EC, as measured by phosphorylation of STAT5 (pSTAT5). Interestingly, it was the CD38 negative cells that had the highest level of pSTAT5 in VL patient CD4+ T cells after IL-7 stimulation. Thus, despite unaltered or potentially lowered IL7RA mRNA expression by CD4+ T cells from VL patients, the surface expression of the IL-7Rα was higher compared to EC and increased pSTAT5 was seen following exposure to rhIL-7. Accordingly, IL-7 signaling appears to be functional and even enhanced in VL CD4+ T cells and cannot explain the impaired effector function of VL CD4+ T cells. The enhanced plasma IL-7 may serve as part of homeostatic feedback mechanism regulating IL7RA expression in CD4+ T cells.

Assessing the association between the circulating levels of inflammatory cytokines and the risk of tuberculosis: A bidirectional two-sample mendelian randomization study

BackgroundNumerous observational studies have previously reported an association between inflammatory cytokines and tuberculosis (TB). However, the causal relationship between these factors remains unclear. Consequently, we conducted two-sample Mendelian randomization (MR) analyses to ascertain the causal link between levels of inflammatory cytokines and the risk of TB. MethodsSingle nucleotide polymorphisms (SNPs) robustly associated with the cytokines, located in or close to their coding gene. SNP was obtained from genome-wide association studies (GWAS) of 8293 individuals of Finnish. TB data was obtained from the UK Biobank, which included 46,293 individuals of European ancestry (comprising 2277 TB cases and 46,056 controls). Two-sample, bi-directional MR analyses using inverse-variance weighted (IVW) method as the primary analysis. Followed by comprehensive sensitivity analyses to validate the robustness of results. ResultThe study showed that the causal relationship between circulating levels of interleukin (IL)-7 and risk of TB (odds ratio [OR] = 1.001, 95% confidence intervals [CIs]: 1.000, 1.003. p = 0.047). No causal associations were observed between other influencing factors and the occurrence of TB. Furthermore, the analysis revealed that TB infection exhibited negative causal associations with macrophage inflammatory protein 1 alpha ([MIP-1α], OR = 0.007, 95% CI: 0.000, 0.192. p = 0.004), IL-2 (OR = 0.014, 95% CI: 0.010, 0.427. p = 0.014), interleukin-2 receptor alpha chain([IL-2rα], OR = 0.019, 95% CI: 0.001, 0.525. p = 0.019) and basic fibroblast growth factor ([bFGF], OR = 0.066, 95% CI: 0.006, 0.700. p = 0.024). ConclusionThe study has illuminated the causal link between inflammatory cytokines and TB, thereby enhancing our comprehension of the potential mechanisms underlying TB pathogenesis. This discovery offers promising avenues for the identification of novel therapeutic targets in TB treatment. These insights may ultimately pave the way for more effective treatment approaches, thereby improving patient outcomes.

Genetic deficiencies of both IL-4 receptor alpha chain and IL-10 trigger early onset of severe colitis in mice

Inflammatory bowel diseases are associated with dysregulated inflammatory immune responses in the gastrointestinal tract. We found that deficiencies of both IL-4 receptor alpha chain (IL-4Rα) and IL-10 in BALB/c mice (IL-4Rα × IL-10 KO mice) highly induced spontaneous rectal prolapse and diarrhea. These mice also exhibited severe colitis in their cecum and colon and marked elevation of serum proinflammatory cytokines including TNFα and IFNγ. These pathologies were transmittable with their cecal contents containing Helicobacter spp. Their mesenteric LN cells produced TNFα and IFNγ in response to soluble H. hepaticus antigens and high titers of H. hepaticus-specific serum IgG were also detected. These results suggested the important function of IL-4Rα signaling in controlling the intestinal inflammation and the susceptibility to intestinal microbes including H. hepaticus. Therefore, these IL-4Rα × IL-10 KO mice potentially provide the significant murine model for clarifying the causes and control of spontaneous colitis and intestinal inflammation.

Epitope-engineered human hematopoietic stem cells are shielded from CD123-targeted immunotherapy.

Targeted eradication of transformed or otherwise dysregulated cells using monoclonal antibodies (mAb), antibody-drug conjugates (ADC), T cell engagers (TCE), or chimeric antigen receptor (CAR) cells is very effective for hematologic diseases. Unlike the breakthrough progress achieved for B cell malignancies, there is a pressing need to find suitable antigens for myeloid malignancies. CD123, the interleukin-3 (IL-3) receptor alpha-chain, is highly expressed in various hematological malignancies, including acute myeloid leukemia (AML). However, shared CD123 expression on healthy hematopoietic stem and progenitor cells (HSPCs) bears the risk for myelotoxicity. We demonstrate that epitope-engineered HSPCs were shielded from CD123-targeted immunotherapy but remained functional, while CD123-deficient HSPCs displayed a competitive disadvantage. Transplantation of genome-edited HSPCs could enable tumor-selective targeted immunotherapy while rebuilding a fully functional hematopoietic system. We envision that this approach is broadly applicable to other targets and cells, could render hitherto undruggable targets accessible to immunotherapy, and will allow continued posttransplant therapy, for instance, to treat minimal residual disease (MRD).

AI-driven Discovery of Celecoxib and Dexamethasone for Exploring their Mode of Action as Human Interleukin (IL-6) Inhibitors to Treat COVID-19-induced Cytokine Storm in Humans.

In the case of COVID-19 patients, it has been observed that the immune system of the infected person exhibits an extreme inflammatory response known as cytokine release syndrome (CRS) where the inflammatory cytokines are swiftly produced in quite large amounts in response to infective stimuli. Numerous case studies of COVID-19 patients with severe symptoms have documented the presence of higher plasma concentrations of human interleukin-6 (IL-6), which suggests that IL-6 is a crucial factor in the pathophysiology of the disease. In order to prevent CRS in COVID-19 patients, the drugs that can exhibit binding interactions with IL-6 and block the signaling pathways to decrease the IL-6 activity may be repurposed. This research work focused on molecular docking-based screening of the drugs celecoxib (CXB) and dexamethasone (DME) to explore their potential to interact with the binding sites of IL-6 protein and reduce the hyper-activation of IL-6 in the infected personnel. Both of the drugs were observed to bind with the IL-6 (IL-6 receptor alpha chain) and IL-6Rα receptor with the respective affinities of -7.3 kcal/mol and -6.3 kcal/mol, respectively, for CXB and DME. Moreover, various types of binding interactions of the drugs with the target proteins were also observed in the docking studies. The dynamic behaviors of IL-6/IL-6Rα in complex with the drugs were also explored through molecular dynamics simulation analysis. The results indicated significant stabilities of the acquired drug-protein complexes up to 100 ns. The findings of this study have suggested the potential of the drugs studied to be utilized as antagonists for countering CRS in COVID-19 ailment. This study presents the studied drugs as promising candidates both for the clinical and pre-clinical treatment of COVID-19.

Thymic Stromal Lymphopoietin (TSLP), Its Isoforms and the Interplay with the Epithelium in Allergy and Asthma.

Thymic stromal lymphopoietin (TSLP) is a pleiotropic cytokine that has emerged as a critical player in the development and progression of allergy and asthma. It is primarily produced by epithelial cells and functions as a potent immune system activator. TSLP acts through interaction with its receptor complex, composed of the TSLP receptor (TSLPR) and interleukin-7 receptor alpha chain (IL-7Rα), activating downstream complex signalling pathways. The TSLP major isoform, known as long-form TSLP (lfTSLP), is upregulated in the airway epithelium of patients with allergic diseases. More research is warranted to explore the precise mechanisms by which short-form TSLP (sfTSLP) regulates immune responses. Understanding the dynamic interplay between TSLP and the dysfunctional epithelium provides insights into the mechanisms underlying allergy and asthma pathogenesis. Targeting TSLP represents an important therapeutic strategy, as it may upstream disrupt the inflammatory cascade and alleviate symptoms associated with allergic inflammation.

Dupilumab in complex therapy for chronic rhinosinusitis with nasal polyps and asthma

Chronic Rhinosinusitis with Nasal Polyps (CRwNP) caused by T2 inflammation has a complex pathogenesis, for which treatment options include medical therapy (topical and systemic glucocorticosteroids, leuktriene inhibitors, etc.) and surgical therapy, often recurs and significantly reduces the quality of life for patients. Chronic rhinosinusitis with nasal polyps is common in patients with asthma and, particularly, severe asthma and an allergic reaction to nonsteroidal anti-inflammatory drugs. These conditions are in most cases associated with a common pathogenesis, including a type II immune response, hyperproduction of interleukins (IL) 4, 5 and 13, activation of eosinophils and tissue remodeling. Biological therapy which block the effect of IL-4, 5, 13 can significantly improve the condition of such patients. Dupilumab is a humanized IgG4 monoclonal antibody that targets the IL-4 receptor alpha chain (IL-4Rα), thereby blocking the action of IL-4 and IL-13. Currently, dupilumab is approved for asthma, CRwNP and atopic dermatitis. Dupilumab can reduce the number of exacerbations, treatment with systemic corticosteroids, improve the condition of the upper and lower respiratory tract and achieve an increase in FEV1 by 0.2–0.3 L, which leads to a significant control of these diseases. One of the topical issues is the choice of tactics choice of treatment tactics in patients with CRwNP, asthma and nasal septum deviation. This article presents a clinical case of a patient with severe asthma, CRwNP and deviated nasal septum receiving dupilumab for 4 months.

Control of the temporal development of Alzheimer’s disease pathology by the MR1/MAIT cell axis

BackgroundNeuroinflammation is an important feature of Alzheimer’s disease (AD). Understanding which aspects of the immune system are important in AD may lead to new therapeutic approaches. We study the major histocompatibility complex class I-related immune molecule, MR1, which is recognized by an innate-like T cell population called mucosal-associated invariant T (MAIT) cells.MethodsHaving found that MR1 gene expression is elevated in the brain tissue of AD patients by mining the Agora database, we sought to examine the role of the MR1/MAIT cell axis in AD pathology. Brain tissue from AD patients and the 5XFAD mouse model of AD were used to analyze MR1 expression through qPCR, immunofluorescence, and flow cytometry. Furthermore, mice deficient in MR1 and MAIT cells were crossed with the 5XFAD mice to produce a model to study how the loss of this innate immune axis alters AD progression. Moreover, 5XFAD mice were also used to study brain-resident MAIT cells over time.ResultsIn tissue samples from AD patients and 5XFAD mice, MR1 expression was substantially elevated in the microglia surrounding plaques vs. those that are further away (human AD: P < 0.05; 5XFAD: P < 0.001). In 5XFAD mice lacking the MR1/MAIT cell axis, the development of amyloid-beta plaque pathology occurred at a significantly slower rate than in those mice with MR1 and MAIT cells. Furthermore, in brain tissue from 5XFAD mice, there was a temporal increase in MAIT cell numbers (P < 0.01) and their activation state, the latter determined by detecting an upregulation of both CD69 (P < 0.05) and the interleukin-2 receptor alpha chain (P < 0.05) via flow cytometry.ConclusionsTogether, these data reveal a previously unknown role for the MR1/MAIT cell innate immune axis in AD pathology and its potential utility as a novel therapeutic target.

IL-4 RECEPTOR ALPHA CHAIN Q576R RR GENOTYPE IS ASSOCIATED WITH INCREASED MARKERS OF TYPE 2 INFLAMMATION IN ASPIRIN EXACERBATED RESPIRATORY DISEASE

In the US, chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by elevated levels of IL-4 and IL-13. Several single nucleotide polymorphisms within the gene encoding for the shared IL-4 receptor alpha chain have been identified, including Q576R (c.1727A>G). The frequency of the RR genotype was significantly higher among asthmatics and the variant was associated with more severe lower airway disease. The impact of the RR genotype in CRSwNP is unclear.

IL-15 Prevents the Development of T-ALL from Aberrant Thymocytes with Impaired DNA Repair Functions and Increased NOTCH1 Activation.

We previously reported that NOD.Scid mice lacking interleukin-15 (IL-15), or IL-15 receptor alpha-chain, develop T-acute lymphoblastic leukemia (T-ALL). To understand the mechanisms by which IL-15 signaling controls T-ALL development, we studied the thymocyte developmental events in IL-15-deficient Scid mice from NOD and C57BL/6 genetic backgrounds. Both kinds of mice develop T-ALL characterized by circulating TCR-negative cells expressing CD4, CD8 or both. Analyses of thymocytes in NOD.Scid.Il15-/- mice prior to T-ALL development revealed discernible changes within the CD4-CD8- double-negative (DN) thymocyte developmental stages and increased frequencies of CD4+CD8+ double-positive cells with a high proportion of TCR-negative CD4+ and CD8+ cells. The DN cells also showed elevated expressions of CXCR4 and CD117, molecules implicated in the expansion of DN thymocytes. T-ALL cell lines and primary leukemic cells from IL-15-deficient NOD.Scid and C57BL/6.Scid mice displayed increased NOTCH1 activation that was inhibited by NOTCH1 inhibitors and blockers of the PI3K/AKT pathway. Primary leukemic cells from NOD.Scid.Il15-/- mice survived and expanded when cultured with MS5 thymic stromal cells expressing Delta-like ligand 4 and supplemented with IL-7 and FLT3 ligand. These findings suggest that IL-15 signaling in the thymus controls T-ALL development from aberrant thymocytes with an impaired DNA repair capacity and increased NOTCH1 activation.

The IL-4Rα Q576R polymorphism is associated with increased severity of atopic dermatitis and exaggerates allergic skin inflammation in mice

Atopic dermatitis (AD) is characterized by TH2-dominated skin inflammation and systemic response to cutaneously encountered antigens. The TH2 cytokines IL-4 and IL-13 play a critical role in the pathogenesis of AD. The Q576->R576 polymorphism in the IL-4 receptor alpha (IL-4Rα) chain common to IL-4 and IL-13 receptors alters IL-4 signaling and is associated with asthma severity. We sought to investigate whether the IL-4Rα R576 polymorphism is associated with AD severity and exaggerates allergic skin inflammation in mice. Nighttime itching interfering with sleep, Rajka-Langeland, and Eczema Area and Severity Index scores were used to assess AD severity. Allergic skin inflammation following epicutaneous sensitization of mice 1 or 2 IL-4Rα R576 alleles (QR and RR) and IL-4Rα Q576 (QQ) controls was assessed by flow cytometric analysis of cells and quantitative RT-PCR analysis of cytokines in skin. The frequency of nighttime itching in 190 asthmatic inner-city children with AD, as well as Rajka-Langeland and Eczema Area and Severity Index scores in 1116 White patients with AD enrolled in the Atopic Dermatitis Research Network, was higher in subjects with the IL-4Rα R576 polymorphism compared with those without, with statistical significance for the Rajka-Langeland score. Following epicutaneous sensitization of mice with ovalbumin or house dust mite, skin infiltration by CD4+ cells and eosinophils, cutaneous expression of Il4 and Il13, transepidermal water loss, antigen-specific IgE antibody levels, and IL-13 secretion by antigen-stimulated splenocytes were significantly higher in RR and QR mice compared with QQ controls. Bone marrow radiation chimeras demonstrated that both hematopoietic cells and stromal cells contribute to the mutants' exaggerated allergic skin inflammation. The IL-4Rα R576 polymorphism predisposes to more severe AD and increases allergic skin inflammation in mice.

CD123 Directed IgM Antibody-based T-cell Engager, IGM-2537, Demonstrates Potent in vitro and in vivo Activity with Minimal Cytokine Release

Introduction: Despite recent progress, the outlook for patients with acute myeloid leukemia (AML) remains poor. Novel therapies are needed for effective treatment of AML, especially in the relapsed/refractory setting. Evidence suggests that incomplete depletion of leukemia stem cells (LSCs) causes relapse. CD123, the IL-3 receptor alpha chain, is highly expressed on both leukemic blasts and LSCs and thus is a suitable therapeutic target in AML. CD123-based immunotherapies including bispecific antibodies or CAR T cells have shown promise, but cytokine release syndrome (CRS) is a major safety concern with dose limiting toxicity (DLT). We are developing IGM-2537, a novel IgM antibody-based CD123xCD3 bispecific T cell engager (TCE), designed with potent anti-tumor activity and lower cytokine release. IGM-2537 has 10 binding sites for CD123, and single binding site for CD3 via single chain Fv domain (scFv) fused to a joining (J) chain, thus providing high affinity, high avidity binding to CD123-positive malignant cells while co-engagement of CD3 T cells leading to T cell induced cytotoxicity with reduced cytokine release. Here we report functional characterization of IGM-2537 in a broad range of in vitro, ex vivo and in vivo anti-tumor efficacy studies. The improved tolerability, good pharmacodynamic (PD) response and lower cytokine release profile of a benchmark IgM TCE comparator in cynomolgus monkeys will be discussed. Materials and Methods: IGM-2537 was constructed using the pentameric human IgM framework. Recombinant IgM heavy chains and light chains with high affinity variable domains derived from an anti-CD123 IgG were co-expressed with a J-chain fused to anti-CD3 scFv. Surface plasma resonance (SPR), cell membrane protein array, and alanine scanning were utilized to characterize human CD123 binding affinity, specificity, and epitope mapping, respectively. Bispecific enzyme-linked immunosorbent assay (ELISA), T-cell NFAT reporter assay and T-cell dependent cellular cytotoxicity (TDCC) assays were employed to evaluate the potency of IGM-2537 to co-engage both CD123 and CD3 antigens, to induce T cell activation and target cytotoxicity, respectively. Ex vivo AML colony formation assay was applied to assess its activity to eliminate AML blasts. Human MV4-11 xenograft tumor model in humanized NSGdKO mice were utilized to determine anti-tumor efficacy of IGM-2537. Lastly, to confirm that CD123 directed IgM-based TCE molecule can potentially provide better safety profile, a benchmark IgM TCE comparator was assessed in cynomolgus monkeys for tolerability and PK/PD responses. Results and Conclusions: IGM-2537 bound to human CD123 with high affinity, avidity, and specificity. Furthermore, IGM-2537 bound a unique epitope of human CD123 in domain 2 which does not overlap with IL-3. IGM-2537 co-engaged with both CD123 and CD3 antigens, leading to T cell-redirected killing of AML cell lines with concomitant T cell activation. Moreover, IGM-2537 eliminated AML blast cells at physiologically relevant effector/target (E/T) ratios in an ex vivo assay. Most importantly, IGM-2537 demonstrated comparable maximal killing activity to a benchmark IgG TCE molecule but shows significant reduction of cytokine release exemplified by IFN-γ, TNF-α and IL-6, which provide initial evidence of better preclinical safety profile for a CD123 directed IgM-based T-cell engager. As expected, the efficient in vitro TDCC activity of IGM-2537 translated into in vivo anti-tumor efficacy with tumor growth inhibition observed at dose ranges from 1 to 10 mg/kg. To further support that the IgM TCE format can provide a better safety window, a benchmark IgM TCE comparator was evaluated for tolerability (including cytokine production), PK and PD responses in cynomolgus monkeys. All animals tolerated the benchmark comparator well at dose levels up to 10 mg/kg (the maximal dose level evaluated); however, the tolerability observed with IgM TCE was 100x greater compared to IgG TCE. Complete depletion of CD123-positive basophils and substantial reduction of plasmacytoid dendritic cells (pDC) were seen in blood and bone marrow with minimal to no cytokine induction. In summary, our IGM-2537 demonstrates potent in vitro and in vivo activity with minimal cytokine induction. IgM as the framework of a TCE molecule can potentially provide better safety profile and an improved therapeutic window.

Engineered Single Amino Acid Substitutions Protect Hematopoietic Stem and Progenitor Cells from CD123 Targeted Immunotherapy

Targeted eradication of transformed or otherwise dysregulated cells using monoclonal antibodies (mAb), T cell engagers (TCE) or chimeric antigen receptor (CAR) cells is very effective for hematologic diseases. CD123, the interleukin-3 (IL-3) receptor alpha-chain, is highly expressed in various hematological malignancies, including acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN). However, shared expression of CD123 on hematopoietic stem and progenitor cells (HSPCs) bears the risk for extensive myelotoxicity upon targeted depletion. To enable therapeutic targeting of non-essential proteins post HSC transplantation (HSCT), it was proposed to engineer HSPCs that lack the target protein. Here, we show that expression and function of a genetically modified CD123 receptor can be preserved in genome engineered therapeutic cells despite full shielding from targeted therapy. We used the known co-crystal structure of CD123 bound to the mAb CSL362 (talacotuzumab) for computer-aided protein engineering. In silico, 28 candidate single amino acid (aa) substitutions at 3 key residues were designed to interfere with CSL362 binding while potentially preserving its function. Flow cytometry confirmed no binding of a CSL362 biosimilar to 13 variants and weak binding to 12 variants while 3 variants showed similar binding as wildtype (wt) CD123. Protein expression of all variants was comparable to wt CD123. Using real-time, label-free protein interaction measurements, we could not detect interactions between the non-binding variant receptors and the CSL362 biosimilar, even at high protein concentrations. Despite this, the non-binding variants preserved the normal binding of IL-3. Protein characterization of selected non- and weak binding variants demonstrated normal biophysical properties of all but one variant. All 13 non-binding variants abolished antibody dependent cellular cytotoxicity (ADCC) of the CSL362 IgG1 biosimilar in a surrogate reporter assay. In addition, a CSL362-derived TCE resulted in T cell activation and target cell killing of wt and strong binding CD123 variants while non-binding variants neither activated the T cells nor resulted in cell killing. Finally, the majority of non-binding variants (12/13) were completely protected from CSL362-CAR T cell cytotoxicity. To assess the function of the variant receptors we engineered the top 2 variants into the IL-3 dependent cell line TF-1 using CRISPR/Cas9-mediated homology directed repair (HDR). Engineered variant TF-1 cells demonstrated IL-3 dose-dependent growth comparable to their unedited counterparts. The addition of the CSL362 biosimilar inhibited IL-3 dependent cell growth in unedited TF-1 cells but not cells expressing variant receptors. Similarly, CRISPR/Cas9-engineered human CD34+ HSPC knock-in (KI) cells expressing the selected variants (Fig. 1a) were shielded from TCE killing using HLA-matched T cells in vitro (Fig. 1b). In addition, a set of in vitro experiments performed with CD34+ HSPC KI cells suggested normal differentiation potential and intact IL-3 signaling as measured by pSTAT5. In summary, our data demonstrate that single aa substitutions in CD123 introduced to HSPCs by CRISPR/Cas9-mediated HDR enable selective shielding from various immunotherapies, while preserving biophysical and functional properties. Variant IL-3 receptor engineered HSPC's and paired immunotherapies could enable novel and targeted therapeutic interventions post HSCT to either treat minimal residual disease or be used as a salvage therapy. Such approaches could expand the therapeutic window and broaden indications and applications for targeted immunotherapies for various malignant and benign conditions. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Phase 1, First-in-Human, Dose-Escalation Study of MGD024, a CD123 x CD3 Bispecific Dart® Molecule, in Patients with Relapsed or Refractory CD123-Positive (+) Hematologic Malignancies

Background The interleukin-3 receptor alpha chain CD123 is overexpressed in a number of hematologic malignancies and is particularly upregulated in leukemia stem cells, which are believed to serve as a reservoir for relapse (El Achi Cancers 2020). Several investigational agents targeting CD123 with diverse mechanisms of action including bispecific T-cell engagers, antibody-drug conjugates, and Chimeric Antigen Receptor (CAR) T-cell therapies, are being investigated in these malignancies, with many showing preliminary clinical anticancer activity (El Achi Cancers 2020). Of note, the CD123-directed therapy tagraxofusp has now become the standard of care (SoC) for blastic plasmacytoid dendritic cell neoplasm (BPDCN) (Pemmaraju NEJM 2019). MGD024 is an investigational, second-generation CD123 × CD3 DART molecule that can simultaneously bind CD123 on malignant cells and CD3 on T cells, targeting CD123+ leukemic cells for recognition and elimination by CD3+ T lymphocytes as effector cells. MGD024 was engineered to minimize cytokine release syndrome (CRS), while maintaining antitumor activity. This new construct permits intermittent intravenous (IV) dosing due to a longer half-life (Alderson ASH 2021) compared with the first-generation DART molecule flotetuzumab, a continuous-infusion molecule that showed preliminary single-agent activity in refractory acute myeloid leukemia (AML) (Uy Blood 2021). Moreover, preliminary in vitro and in vivo data supported MGD024 clinical investigation as a single agent or in combination with SoC in patients with CD123+ hematologic malignancies (Alderson ASH 2021). Study Design and Method This is a Phase 1, first-in-human, dose-escalation study (CP-MGD024-01; NCT05362773) of MGD024 in patients with the following relapsed or refractory hematologic malignancies: AML, myelodysplastic syndrome (MDS), classical Hodgkin's lymphoma (cHL), chronic myeloid leukemia (CML), B-cell acute lymphoid leukemia (B-ALL), hairy cell leukemia (HCL), advanced systemic mastocytosis (ASM), and BPDCN. Eligible patients must have CD123+ malignant cells by flow cytometry or immunohistochemistry, Eastern Cooperative Oncology Group (ECOG) performance status ≤2, and no history or current evidence of any condition, therapy, or laboratory abnormality that may confound trial results. Primary objectives are to assess the safety, tolerability, dose-limiting toxicities (DLTs), maximum tolerated dose (MTD) or maximum administered dose (if no MTD is defined) and identify a recommended Phase 2 dose and schedule of MGD024. Secondary objectives include pharmacokinetics, immunogenicity, preliminary assessment of MGD024 clinical activity, and preliminary evaluation of safety/efficacy of tocilizumab in the management of MGD024-induced CRS. Patients receive MGD024 via intermittent intravenous infusion for approximately one year or until discontinuation for unacceptable toxicity or disease progression, whichever occurs first. The dose escalation begins with an accelerated titration phase (1+3 design) and converts to a conventional 3+3 design after the first patient experiences a DLT or a Grade ≥2 nonhematologic adverse event (AE) considered at least possibly related to MGD024 during Cycle 1, whichever occurs first. Safety assessments are based on AEs occurring from the first dose until 30 days after the last dose or until starting another anticancer therapy. Response assessments are performed after Cycle 1, then after every even numbered cycle starting with Cycle 2 until progression or discontinuation, whichever occurs first, and at the end of treatment visit. Response evaluation is determined using modified European LeukemiaNet (ELN) 2017 criteria for ALM and ALL; International Working Group (IWG) 2006 criteria for MDS; Lugano 2014 criteria for cHL; ELN 2020 criteria for CML; 2017 consensus criteria for HCL; IWG-Myeloproliferative Neoplasms Research and Treatment (MRT) & European Competence Network on Mastocytosis (ECNM) 2013 criteria for ASM; and modified criteria from Pemmaraju NEJM 2019 for BPDCN. Patients benefitting from MGD024 may continue to receive MGD024 after completion of the study treatment period, at physician discretion.

AML-397 Integrated Safety Analysis of Tagraxofusp, a CD123-Directed Targeted Therapy, in Patients With Hematologic Malignancies

The interleukin-3 receptor alpha chain (CD123), a cell-surface target, is aberrantly expressed on various myeloid neoplasms, including blastic plasmacytoid dendritic cell neoplasm (BPDCN), acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), and myelofibrosis (MF). Tagraxofusp (TAG, SL-401), a first-in-class CD123-targeted therapy, is the only treatment approved by the FDA and EMA for patients with BPDCN and is being investigated in AML, CMML, and MF. To report the aggregated safety data for TAG monotherapy from trials in BPDCN, AML, CMML, and MF. An integrated safety analysis was performed for patients who received TAG monotherapy in three phase 1/2, multicenter clinical studies: Study 0114 (NCT02113982; BPDCN/AML), Study 0214 (NCT02270463; AML), and Study 0314 (NCT02268253; CMML/MF). Hospitals and medical research institutions. In total, 201 patients were included: BPDCN, n=86; AML, n=36; CMML, n=33; and MF, n=36. Patients received the recommended phase 2 TAG dose of 12 mcg/kg intravenously on days 1-3 (MF and CMML) or days 1-5 (BPDCN and AML). Treatment-related adverse events (TRAEs), adverse events (AEs) of interest, and discontinuations. As of July 2021, 11 (6%) patients discontinued TAG due to any-grade TRAEs. The most common any-grade TRAEs included hypoalbuminemia (41%), increased alanine (ALT; 40%) and aspartate (AST; 39%) aminotransferases, and thrombocytopenia (26%). The most common grade ≥3 TRAEs were thrombocytopenia (20%), increased AST (20%), and increased ALT (17%). Prolonged bone marrow suppression was not observed. Overall, 23% of patients experienced ≥1 serious TRAE. The onset of most TRAEs occurred in cycle 1 and was resolved by cycle 2. Capillary leak syndrome (CLS) occurred in 35 patients (17%), with onset usually within cycle 1. Most CLS events were non-severe (grade ≤2); 2 of 201 (1%) patients had a grade 5 event. This integrated analysis is the largest collation of safety data following treatment with TAG monotherapy. Most TRAEs were transient, and their frequency/severity decreased with increasing cycles. No myelosuppression or cumulative toxicity was reported following treatment over multiple cycles. These data confirm that the established and manageable safety profile of TAG monotherapy has been maintained in the 3 years following US approval.