Ig Isotypes Research Articles

The Janus (dual) model of immunoglobulin isotype evolution: Conservation and plasticity are the defining paradigms.

The study of antibodies in jawed vertebrates (gnathostomes) provides every immunologist with a bird's eye view of how human immunoglobulins (Igs) came into existence and subsequently evolved into their present forms. It is a fascinating Darwinian history of conservation on the one hand and flexibility on the other, exemplified by the Ig heavy chain (H) isotypes IgM and IgD/W, respectively. The cartilaginous fish (e.g., sharks) Igs provide a glimpse of "how everything got off the ground," while the amphibians (e.g., the model Xenopus) reveal how the adaptive immune system made an about face with the emergence of Ig isotype switching and IgG-like structure/function. The evolution of mucosal Igs is a captivating account of malleability, convergence, and conservation, and a call to arms for future study! In between there are spellbinding chronicles of antibody evolution in each class of vertebrates and rather incredible stories of how antibodies can adapt to occupy niches, for example, single-domain variable regions, cold-adapted Igs, convergent mechanisms to dampen antibody function, provision of mucosal defense, and many more. The purpose here is not to provide an encyclopedic examination of antibody evolution, but rather to hit the high points and entice readers to appreciate how things "came to be."

Serum immunoglobulin levels in group E of chronic obstructive pulmonary disease: insights for clinical management and immunoglobulin therapy strategies

ObjectiveThe study aimed to characterize serum immunoglobulin (Ig) concentrations and their relationship with clinical and paraclinical features in patients with COPD group E in the stable stage. Additionally, the study focused on evaluating the relationship between serum Ig levels and the risk of exacerbations over the next 12 months, thereby clarifying the role of serum Ig deficiency in affecting the future risk for these patients.MethodsA prospective observational study assessed IgG, IgA, IgM, and IgE levels in 67 COPD patients and 30 healthy controls at Military Hospital 103 from October 2017 to August 2020. Primary outcomes included Ig isotype levels in COPD patients, with secondary outcomes exploring differences compared to controls and associations with clinical variables.ResultsCOPD patients showed significantly lower IgG concentrations and higher IgA levels than controls. IgM and IgE levels did not differ significantly. Subgroup analysis revealed notable decreases in IgG1 and IgG3 concentrations, with 10.4% of patients exhibiting reduced IgG levels and 0.3% diagnosed with common variable immunodeficiency. No significant associations were found between Ig levels and exacerbation risk or clinical variables.ConclusionsSerum IgG and IgM concentrations were significantly reduced in COPD patients compared to normal individuals, with IgG1 and IgG3 concentrations notably low. Serum IgA levels were significantly higher in COPD patients compared with normal controls. However, no significant association was found between Ig concentrations, particularly serum IgG deficiency and its subclasses, with the frequency and risk of exacerbations during 12 months of longitudinal follow-up. Caution is warranted in the use of immunoglobulin therapy in the treatment of COPD patients.Trial registrationAn independent ethics committee approved the study (Ethics Committee of Military Hospital 103 (No. 57/2014/VMMU-IRB), which was performed in accordance with the Declaration of Helsinki, Guidelines for Good Clinical Practice.

Immunoglobulin class-switch recombination: Mechanism, regulation, and related diseases.

Maturation of the secondary antibody repertoire requires class-switch recombination (CSR), which switches IgM to other immunoglobulins (Igs), and somatic hypermutation, which promotes the production of high-affinity antibodies. Following immune response or infection within the body, activation of T cell-dependent and T cell-independent antigens triggers the activation of activation-induced cytidine deaminase, initiating the CSR process. CSR has the capacity to modify the functional properties of antibodies, thereby contributing to the adaptive immune response in the organism. Ig CSR defects, characterized by an abnormal relative frequency of Ig isotypes, represent a rare form of primary immunodeficiency. Elucidating the molecular basis of Ig diversification is essential for a better understanding of diseases related to Ig CSR defects and could provide clues for clinical diagnosis and therapeutic approaches. Here, we review the most recent insights on the diversification of five Ig isotypes and choose several classic diseases, including hyper-IgM syndrome, Waldenström macroglobulinemia, hyper-IgD syndrome, selective IgA deficiency, hyper-IgE syndrome, multiple myeloma, and Burkitt lymphoma, to illustrate the mechanism of Ig CSR deficiency. The investigation into the underlying mechanism of Ig CSR holds significant potential for the advancement of increasingly precise diagnostic and therapeutic approaches.

Aspergillus fumigatus binding IgA and IgG1 are increased in bronchoalveolar lavage fluid of horses with neutrophilic asthma.

Equine asthma (EA) is a common lower airway disease in horses, but whether its pathogenesis is allergic is ambiguous. Extrinsic stimuli like hay dust induce acute exacerbation of clinical signs and sustained local neutrophilic inflammation in susceptible horses. Aspergillus fumigatus is an EA stimulus, but it is unclear if it merely acts as an IgE-provoking allergen. We aimed to comprehensively analyze immunoglobulin (Ig) isotypes in EA, elucidating their binding to different A. fumigatus antigens, and their quantities systemically in serum and locally in bronchoalveolar lavage fluid (BALF). Serum and BALF from healthy horses (HE, n = 18) and horses with mild-moderate asthma (MEA, n = 20) or severe asthma (SEA, n = 24) were compared. Ig isotype (IgG1, IgG3/5, IgG4/7, IgG6, IgA, and IgE) binding to nine antigens (A.fumigatus lysate, and recombinant Asp f 1, Asp f 7, Asp f 8, dipeptidyl-peptidase 5, class II aldolase/adducin domain protein, glucoamylase, beta-hexosaminidase, and peptide hydrolase) was compared by enzyme-linked immunosorbent assays. Total Ig isotype contents were determined by bead-based assays. MEA and SEA differed from HE but hardly from each other. Compared to HE, asthmatic horses showed increased anti-A. fumigatus binding of IgG (BALF and serum) and IgA (BALF). Serum and BALF IgE binding and total IgE contents were similar between HE and EA. Single antigens, as well as A. fumigatus lysate, yielded similar Ig binding patterns. Serum and BALF IgG1 binding to all antigens was increased in SEA and to several antigens in MEA. Serum IgG4/7 binding to two antigens was increased in SEA. BALF IgA binding to all antigens was increased in SEA and MEA. Total BALF IgG1 and IgG4/7 contents were increased in SEA, and serum IgG4/7 content was increased in MEA compared to HE. Yet, total isotype contents differentiated EA and HE less clearly than antigen-binding Ig. A. fumigatus immunogenicity was confirmed without identification of single dominant antigens here. A. fumigatus provoked elevated BALF IgG1 and IgA binding, and these isotypes appear relevant for neutrophilic EA, which does not support allergy. BALF Ig isotype differentiation beyond IgE is crucial for a comprehensive analysis of immune responses to fungi in EA pathogenesis.

Effects of house-cultivated edible bird's nest on immunoglobulin and cytokine release in vitro.

Edible bird's nest (EBN) is known as the "Caviar of the East" because of its high nutritional and medicinal values. Nevertheless, its effect on human immunity is yet to be explored. This study examined the effects of EBN's aqueous extract (EBNE) on human immunity through the modular immune in vitro construct (MIMIC) model consisting of peripheral tissue equivalent (PTE) and lymphoid tissue equivalent (LTE) modules. One hundred twenty mL of full blood was obtained from four healthy human volunteers. The human immune system was simulated using an in vitro model, called MIMIC. Under EBNE treatment, monocyte transendothelial migration through reversed endothelial layers was observed. Using PTE and LTE modules, monocytes were differentiated into dendritic cells with lipopolysaccharide, then co-cultured with T- and B-cells for cytokine and immunoglobulin (Ig) production. The human cytokine array G2000 and quantitative human Ig isotyping array were used to identify the cytokine profile and Ig isotypes, respectively. IgE, IgA, and IgG3 levels were significantly raised by EBNE. These cytokines, including brain-derived neurotrophic factor, ciliary neurotrophic factor, glial cell line-derivative neurotrophic factor, insulin-like growth factor 1, and insulin-like growth factor binding protein 4, were generated. For the first time, this work uses a MIMIC model to illustrate the impact of EBNE on human immune response. This new understanding of EBN's immunoregulatory effect allows for further exploration of how EBN interacts with the human immune system.

Nasal and Pharyngeal Mucosal Immunity to Poliovirus in Children Following Routine Immunization With Inactivated Polio Vaccine in the United States.

Although polioviruses (PVs) replicate in lymphoid tissue of both the pharynx and ileum, research on polio vaccine-induced mucosal immunity has predominantly focused on intestinal neutralizing and binding antibody levels measured in stool. To investigate the extent to which routine immunization with intramuscularly injected inactivated polio vaccine (IPV) may induce nasal and pharyngeal mucosal immunity, we measured PV type-specific neutralization and immunoglobulin (Ig) G, IgA, and IgM levels in nasal secretions, adenoid cell supernatants, and sera collected from 12 children, aged 2-5 years, undergoing planned adenoidectomies. All participants were routinely immunized with IPV and had no known contact with live PVs. PV-specific mucosal neutralization was detected in nasal and adenoid samples, mostly from children who had previously received 4 IPV doses. Across the 3 PV serotypes, both nasal (Spearman ρ ≥ 0.87, P ≤ .0003 for all) and adenoid (Spearman ρ ≥ 0.57, P ≤ .05 for all) neutralization titers correlated with serum neutralization titers. In this small study sample, there was insufficient evidence to determine which Ig isotype(s) was correlated with neutralization. Our findings provide policy-relevant evidence that routine immunization with IPV may induce nasal and pharyngeal mucosal immunity. The observed correlations of nasal and pharyngeal mucosal neutralization with serum neutralization contrast with previous observations of distinct intestinal and serum responses to PV vaccines. Further research is warranted to determine which antibody isotype(s) correlate with polio vaccine-induced nasal and pharyngeal mucosal neutralizing activity and to understand the differences from intestinal mucosal immunity.

Regulation of BCR-mediated Ca2+ mobilization by MIZ1-TMBIM4 safeguards IgG1+ GC B cell-positive selection.

The transition from immunoglobulin M (IgM) to affinity-matured IgG antibodies is vital for effective humoral immunity. This is facilitated by germinal centers (GCs) through affinity maturation and preferential maintenance of IgG+ B cells over IgM+ B cells. However, it is not known whether the positive selection of the different Ig isotypes within GCs is dependent on specific transcriptional mechanisms. Here, we explored IgG1+ GC B cell transcription factor dependency using a CRISPR-Cas9 screen and conditional mouse genetics. We found that MIZ1 was specifically required for IgG1+ GC B cell survival during positive selection, whereas IgM+ GC B cells were largely independent. Mechanistically, MIZ1 induced TMBIM4, an ancestral anti-apoptotic protein that regulated inositol trisphosphate receptor (IP3R)-mediated calcium (Ca2+) mobilization downstream of B cell receptor (BCR) signaling in IgG1+ B cells. The MIZ1-TMBIM4 axis prevented mitochondrial dysfunction-induced IgG1+ GC cell death caused by excessive Ca2+ accumulation. This study uncovers a unique Ig isotype-specific dependency on a hitherto unidentified mechanism in GC-positive selection.

IgG Isotypes Targeting a Recombinant Chimeric Protein of Trypanosoma cruzi in Different Clinical Presentations of Chronic Chagas Disease.

Chagas disease (CD) is caused by the protozoan Trypanosoma cruzi, which leads to a spectrum of clinical presentations that range from asymptomatic to severe cardiac involvement. The host immune response plays a pivotal role in disease progression. Ig isotypes may contribute to disease pathogenesis. Investigating these components can provide insights into the immunopathogenic mechanisms underlying CD. This cross-sectional study aims to establish a correlation between the Ig profile of individuals infected with T. cruzi with the clinical forms of chronic CD. Serum samples were collected from partner institutions in different states of Brazil. Individuals diagnosed with chronic CD were categorized based on the clinical form of the disease. The indirect ELISA method using the recombinant chimeric Molecular Biology Institute of Paraná membrane protein 8.4 as the antigen was used to determine the Ig profile, including total IgG, IgG1, IgG2, IgG3, and IgG4. Ninety-seven serum samples from patients classified as negative (NEG, n = 38), indeterminate (IND, n = 24), mild cardiac (MC, n = 20), and severe cardiac (SC, n = 15) forms were analyzed. IgG1 exhibited greater levels compared with the other isotypes, showing a significant difference between the MC and IND groups. IgG3 levels were greater in individuals from the MC group compared with the SC group. IgG1 and IgG3 isotypes can serve as biomarkers to evaluate the progression of CD because they exhibit variations across clinical groups. Additional longitudinal studies are necessary to explore the relationship between antibody kinetics and the development of tissue damage.

The aryl hydrocarbon receptor differentially modulates the expression profile of antibody isotypes in a human B-cell line.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant and high affinity ligand for the aryl hydrocarbon receptor (AhR). In animal models, AhR activation by TCDD generally inhibits antibody secretion. However, it is less clear if this translates to human antibody production. Using a human Burkitt lymphoma B-cell line (CL-01) that can be stimulated to secrete Ig and undergo class switch recombination to other Ig isotypes, the current study evaluated the effects of AhR activation or antagonism on the human Ig isotypic expression profile with CD40L+IL-4 stimulation. Our results suggest that AhR agonists (TCDD and indirubin) have little to no effect on IgM or IgA secretion, which were also not induced with stimulation. However, AhR activation significantly inhibited stimulation-induced IgG secretion, an effect reversed by the AhR antagonist CH223191. Evaluation of Ig heavy chain (IgH) constant region gene expression (ie Cμ, Cγ1-4, Cα1-2, and Cε that encode for IgM, IgG1-4, IgA1-2, and IgE, respectively) demonstrated differential effects. While Cμ and Cα2 transcripts were unaffected by stimulation or AhR agonists, AhR activation significantly inhibited stimulation-induced Cγ2-4 and Cε mRNA transcripts, which was reversed by AhR antagonism. Notably, AhR antagonism in the absence of exogenous AhR ligands significantly increased IgG and IgA secretion as well as the expression of Cγ2-4 and Cε. These results suggest that modulation of AhR activity differentially alters the IgH isotypic expression profile and antibody secretion that may be partly dependent on cellular stimulation. Since a variety of chemicals from anthropogenic, industrial, pharmaceutical, dietary, and bacterial sources bind the AhR, the ability of environmental exposures to alter AhR activity (i.e. activate or inhibit) may have a direct influence on immune function and antibody-relevant disease conditions.

Lifetime Exposure to Cigarette Smoke, B-Cell Tumor Immune Infiltration, and Immunoglobulin Abundance in Ovarian Tumors.

Cigarette smoke exposure has been linked to systemic immune dysfunction, including for B-cell and immunoglobulin (Ig) production, and poor outcomes in patients with ovarian cancer. No study has evaluated the impact of smoke exposure across the life-course on B-cell infiltration and Ig abundance in ovarian tumors. We measured markers of B and plasma cells and Ig isotypes using multiplex immunofluorescence on 395 ovarian cancer tumors in the Nurses' Health Study (NHS)/NHSII. We conducted beta-binomial analyses evaluating odds ratios (OR) and 95% confidence intervals (CI) for positivity of immune markers by cigarette exposure among cases and Cox proportional hazards models to evaluate hazard ratios (HR) and 95% CI for developing tumors with low (<median) or high (≥median) immune cell/Ig percentage. There were no associations between smoke exposure and B-cell or IgM infiltration in ovarian tumors. Among cases, we observed higher odds of IgA+ among ever smokers (OR, 1.54; 95% CI, 1.14-2.07) and ever smokers with no parental smoke exposure (OR, 2.03; 95% CI, 1.18-3.49) versus never smokers. Women with parental cigarette smoke exposure versus not had higher risk of developing ovarian cancer with low IgG+ (HR, 1.51; 95% CI, 1.10-2.09), whereas ever versus never smokers had a lower risk (HR, 0.74; 95% CI, 0.56-0.99). Ever smoking was associated with increased odds of IgA in ovarian tumors. IgA has been associated with improved ovarian cancer outcomes, suggesting that although smoking is associated with poor outcomes in patients with ovarian cancer, it may lead to improved tumor immunogenicity.

A structural peculiarity of Antarctic fish IgM drives the generation of an engineered mAb by CRISPR/Cas9.

IgM is the major circulating Ig isotype in teleost fish, showing in Antarctic fish unique features such as an extraordinary long hinge region, which plays a crucial role in antibody structure and function. In this work, we describe the replacement of the hinge region of a murine monoclonal antibody (mAb) with the peculiar hinge from Antarctic fish IgM. We use the CRISPR/Cas9 system as a powerful tool for generating the engineered mAb. Then, we assessed its functionality by using an innovative plasmonic substrate based on bimetallic nanoislands (AgAuNIs). The affinity constant of the modified mAb was 2.5-fold higher than that obtained from wild-type mAb against the specific antigen. Here, we show the suitability of the CRISPR/Cas9 method for modifying a precise region in immunoglobulin gene loci. The overall results could open a frontier in further structural modifications of mAbs for biomedical and diagnostic purposes.

The relationship between the indicators of natural antibodies, total immunoglobulins and immunocompetent blood cells of sheep in the process of post-vaccination immune response

Innate immunity plays an important role in protecting the body from pathogenic microorganisms. The significance of its structures in vaccination is currently widely discussed. The purpose of the research was to study the dynamics of immunological parameters during the immune response to various types of vaccines.The paper analyzes the levels of natural antibodies in the blood serum of sheep and considers possible correlations with indicators of innate immunity during the post-vaccination immune response. After blood sampling (background), the animals were immunized with rabies (n=5) and anthrax (n=5) vaccines. On the first day of the immune response in the blood of the sheep of the experimental groups, there was a slight increase in tot- Ig, the number of neutrophils increased, and the number of lymphocytes decreased. It was found that on the seven day the concentration of tot- Ig, which also includes specific antibodies to the antigens of vaccines against rabies and anthrax, increased by 2 times compared to the background values. However, the NAb concentration slightly increased only on the first day of the immune response to the anthrax vaccine. In animals, regardless of the vaccine administered, a negative correlation between the number of lymphocytes and neutrophils was constantly maintained (r= -0.88±0.02). As well as in the control (r= - 0.48), the negative correlation between neutrophils /NAb (r= - 0.52; r= -0.76) remained, which indicates the activation of phagocytes and the synthesis of natural antibodies. On the other hand, a correlation relationship between lymphocytes/NAb was established during the immune response to the vaccine against rabies (r=0.5) and anthrax (r=0.7), and the absence of this relationship in the control (r=0.16), which indicates the reaction of natural antibodies to a foreign antigen. The correlations of lymphocytes /NAb (r=0.7) and lymphocytes/tot- Ig (r=0.8) were higher in the dynamics of the immune response to the anthrax vaccine than to the rabies vaccine (r=0.5). It appears that serum anti- KLH NAbs, which include various Ig isotypes, may re ect innate humoral immune competence.

Immunoproteomics reveal increased serum IgG3/5 binding to Dermatophagoides and yeast protein antigens in severe equine asthma in a preliminary study.

Severe equine asthma (SEA) is a common, chronic respiratory disease of horses characterized by hyperreactivity to hay dust which has many similarities to severe neutrophilic asthma in humans. SEA-provoking antigens have not been comprehensively characterized, but molds and mites have been suggested as relevant sources. Here, we identified relevant antigen candidates using immunoproteomics with IgG isotype-binding analyses. Proteins from Dermatophagoides pteronyssinus (Der p) were separated by two-dimensional gel electrophoresis followed by immunoblotting (2D immunoblots) resulting in a characteristic pattern of 440 spots. After serum incubation, antibody (Ig)-binding of all Ig (Pan-Ig) and IgG isotypes (type-2-associated IgG3/5, type-1-associated IgG4/7) was quantified per each spot and compared between asthmatic and healthy horses' sera (n=5 per group). Ig binding differences were detected in 30 spots. Pan-Ig binding was higher with asthmatics compared to healthy horses' sera on four spots, and IgG3/5 binding was higher on 18 spots. Small IgG4/7 binding differences were detected on 10 spots with higher binding with asthmatics' sera on four but higher binding with healthy horses' sera on six spots. Proteins from the spots with group differences including mite and yeast proteins were identified by liquid chromatography mass spectrometry. The latter likely originated from the feeding substrate of the Der p culture. Prioritized antigen candidates amongst the proteins identified were Der p 1, Der p 11, group 15 allergens, myosin heavy chain, and uncharacterized Der p proteins. Additionally, yeast enolases, alcohol dehydrogenase (ADH), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase, and heat shock proteins were prioritized. Eleven antigen candidates were tested for confirmation by ELISAs using the respective proteins separately. Differences in asthmatics vs. healthy horses' serum Ig binding to Der p 1, Der p 18, and three yeast enzymes (enolase, ADH, and PGK) confirmed these as promising antigens of immune responses in SEA. Antigens with relevance in SEA were newly identified by immunoproteomics, and yeast antigens were considered for SEA for the first time. Serum IgG3/5 binding to relevant antigens was increased in SEA and is a novel feature that points to increased type-2 responses in SEA but requires confirmation of the corresponding cellular responses.

Landscape of immune cells in systematic lupus erythematosus patients with Epstein-Barr virus infection: assessed by single-cell sequencing.

To analyse the immune cell and B-cell receptor (BCR) profiles of patients with SLE, with or without EBV infection, and identify the differences between them. We included two patients with SLE and positive detection of EBV infections (SLE-EBV+), four with SLE with negative detection of EBV infections (SLE-EBV-) and two healthy controls. Single-cell RNA sequencing was used to investigate the heterogeneity of cell populations by combining the transcriptomic profiles and BCR repertoires. A total of 83 478 cells were obtained and divided into 31 subtypes. The proportion of CD8+ proliferation T cells was higher in the SLE-EBV+ group than in the SLE-EBV- group. The IFN-α/β pathways were upregulated in most T cells, monocytes and B cells in the SLE-EBV+ group, compared with the SLE-EBV- group. Moreover, T-cell trajectory indicated CD4+ Tregs may play crucial roles in SLE combined with EBV infection. In the BCR heavy chain, the IGHV3 and IGHV4 gene families were frequently present in all groups. Additionally, IgM was the largest component of five Ig isotypes, but its proportion was significantly decreased in the SLE-EBV+ group. This study provides a comprehensive characterization of the immune cell profiles and BCR repertoires of patients with SLE, both with and without concurrent EBV infections, contributing to a better understanding of the mechanism underlying the immune response to EBV infection in patients with SLE.

The distribution and function of teleost IgT

Given the uniquely close relationship between fish and aquatic environments, fish mucosal tissues are constantly exposed to a wide array of pathogenic microorganisms in the surrounding water. To maintain mucosal homeostasis, fish have evolved a distinct mucosal immune system known as mucosal-associated lymphoid tissues (MALTs). These MALTs consist of key effector cells and molecules from the adaptive immune system, such as B cells and immunoglobulins (Igs), which play crucial roles in maintaining mucosal homeostasis and defending against external pathogen infections. Until recently, three primary Ig isotypes, IgM, IgD, and IgT, have been identified in varying proportions within the mucosal secretions of teleost fish. Similar to the role of mucosal IgA in mammals and birds, teleost IgT plays a predominant role in mucosal immunity. Following the identification of the IgT gene in 2005, significant advances have been made in researching the origin, evolution, structure, and function of teleost IgT. Multiple IgT variants have been identified in various species of teleost fish, underscoring the remarkable complexity of IgT in fish. Therefore, this study provides a comprehensive review of the recent advances in various aspects of teleost IgT, including its genomic and structural features, the diverse distribution patterns within various fish mucosal tissues (the skin, gills, gut, nasal, buccal, pharyngeal, and swim bladder mucosa), its interaction with mucosal symbiotic microorganisms, and its immune responses towards diverse pathogens, including bacteria, viruses, and parasites. We also highlight the existing research gaps in the study of teleost IgT, suggesting the need for further investigation into the functional aspects of IgT and IgT+ B cells. This research is aimed at providing valuable insights into the immune functions of IgT and the mechanisms underlying the immune responses of fish against infections.

Real-World Utilization of Daratumumab in Front-Line Treatment of Newly Diagnosed Multiple Myeloma: A Retrospective Observational Study

Introduction: Daratumumab (DARA), a CD38 monoclonal antibody, has demonstrated remarkable efficacy among newly diagnosed (ND) and relapsed/refractory multiple myeloma (MM) patients. The introduction of a quadruplet therapy with an addition of DARA per the GRIFFIN trial has improved the treatment paradigm. However, the real-world data on treatment patterns and utilization of DARA in front-line treatment are limited. This study aimed to evaluate the real-world utilization of DARA in front-line treatment and examine factors that may impact the utilization of DARA-based regimen in NDMM. Methods: We conducted a retrospective observational study of all patients, age ≥ 18, with clinical MM who were newly diagnosed and treated within the Yale New Haven Health (YNHH) system between August 1, 2020 and August 31, 2022. We manually reviewed clinical presentation, age at diagnosis, gender, race, ethnicity, Revised International Staging System (R-ISS) stage, Eastern Cooperative Oncology Group (ECOG) performance status (PS), insurance type, practice setting, immunoglobulin (Ig) isotype, hemoglobin (Hb), creatinine (Cr), and calcium (Ca) at diagnosis, presence of bone lesion, bone marrow plasma cell (BMPC) %, cytogenetic abnormalities, transplant eligibility, and treatment response based on the International Myeloma Working Group (IMWG) criteria at 3 months after front-line therapy. Descriptive statistics were performed with Pearson's χ²-test using GraphPad PRISM version 9.0.0. Results: We identified 241 patients with NDMM during the study period within the YNHH system. Overall median age at diagnosis was 69 years (interquartile range: 61-78). 60.2% (n=145) were male, 68% (n=164) identified themselves as white and 90% (n=217) as non-Hispanic. 51.9% (n=125) had R-ISS stage II and 65.6% (n=158) had ECOG PS of 0-1. 63.1% (n=152) were treated at a community practice setting and 53.9% (n=130) had commercial insurance. 55.6% (n=134) had IgG isotype and 41.1% (n=99) had unfavorable risk cytogenetics which was defined as a 17p deletion, t(4;14), t(14;16), t(14;20), gain or amplification of 1q, 1p deletion, and chromosome 1 translocations. 28.2% (n=68) received front-line quadruplet therapy: DARA+lenalidomide (LEN)+bortezomib (BORTE)+dexamethasone (DEX) (n=54), DARA+cyclophosphamide+BORTE+DEX (n=10), or DARA+LEN+carfilzomib (CARFIL)+DEX (n=4). 58.5% (n=141) received triplet therapy, 8.3% (n=20) received doublet therapy, 0.83% (n=2) received monotherapy, and 4.1% (n=10) opted for hospice care. 41.5% (n=100) received DARA in front-line treatment: quadruplet as above (n=68), triplet of DARA+LEN+DEX, DARA+BORTE+DEX, or DARA+CARFIL+DEX (n=31), and doublet of DARA+DEX (n=1). Factors including gender (male vs. female, p=0.018), practice setting (academic vs community, p=0.014), R-ISS stage (I vs II vs III vs unknown, p=0.003), Ig isotype (IgG vs IgA vs light chain vs other, p=0.006), cytogenetic abnormalities (standard risk vs unfavorable risk, p=0.02), and transplant eligibility (transplant eligible vs transplant ineligible, p=0.001) were associated with the adoption of DARA in front-line treatment. While 51.7% (n=46) of the patients treated at an academic practice setting received DARA in front-line treatment, only 35.5% (n=54) of the patients treated at a community practice setting received such treatment. 69% (n=69) of those who received DARA-based regimen in front-line treatment achieved at least a very good partial response (VGPR) at 3 months compared to 54.6% (n=77) of those who received non-DARA-based regimen. There were no significant differences in age, race, ethnicity, ECOG PS, insurance type, laboratory data (Hb, Cr, and Ca) at diagnosis, presence of bone lesion, and BMPC % between the group treated with DARA-based regimen and the group treated with non-DARA-based regimen. Conclusion: This analysis of real-world data from patients with NDMM highlighted underutilization of DARA in front-line treatment, especially at a community practice setting. Gender, practice setting, R-ISS stage, Ig isotype, cytogenetic abnormalities, and transplant eligibility were significantly associated with the adoption of DARA in front-line treatment. Additionally, utilization of DARA in front-line treatment was associated with a better treatment response at 3 months, suggesting that interventions to promote the utilization of DARA will help improve outcomes of NDMM patients.

A PRoliferation-Inducing Ligand (APRIL) in the Pathogenesis of Immunoglobulin A Nephropathy: A Review of the Evidence.

A PRoliferation-Inducing Ligand (APRIL), the thirteenth member of the tumor necrosis factor superfamily, plays a key role in the regulation of activated B cells, the survival of long-lived plasma cells, and immunoglobulin (Ig) isotype class switching. Several lines of evidence have implicated APRIL in the pathogenesis of IgA nephropathy (IgAN). Globally, IgAN is the most common primary glomerulonephritis, and it can progress to end-stage kidney disease; yet, disease-modifying treatments for this condition have historically been lacking. The preliminary data in ongoing clinical trials indicate that APRIL inhibition can reduce proteinuria and slow the rate of kidney disease progression by acting at an upstream level in IgAN pathogenesis. In this review, we examine what is known about the physiologic roles of APRIL and evaluate the experimental and epidemiological evidence describing how these normal biologic processes are thought to be subverted in IgAN. The weight of the preclinical, clinical, and genetic data supporting a key role for APRIL in IgAN has galvanized pharmacologic research, and several anti-APRIL drug candidates have now entered clinical development for IgAN. Herein, we present an overview of the clinical results to date. Finally, we explore where more research and evidence are needed to transform potential therapies into clinical benefits for patients with IgAN.

Vaccine-induced SARS-CoV-2 antibody response: the comparability of S1-specific binding assays depends on epitope and isotype discrimination.

Quantification of the SARS-CoV-2-specific immune response by serological immunoassays is critical for the management of the COVID-19 pandemic. In particular, neutralizing antibody titers to the viral spike (S) protein have been proposed as a correlate of protection (CoP). The WHO established the First International Standard (WHO IS) for anti-SARS-CoV-2 immunoglobulin (Ig) (NIBSC 20/136) to harmonize binding assays with the same antigen specificity by assigning the same unitage in binding antibody units (BAU)/ml. In this study, we analyzed the S1-specific antibody response in a cohort of healthcare workers in Germany (n = 76) during a three-dose vaccination course over 8.5 months. Subjects received either heterologous or homologous prime-boost vaccination with ChAdOx1 nCoV-19 (AstraZeneca) and BNT162b2 (Pfizer-BioNTech) or three doses of BNT162b2. Antibodies were quantified using three anti-S1 binding assays (ELISA, ECLIA, and PETIA) harmonized to the WHO IS. Serum levels of neutralizing antibodies were determined using a surrogate virus neutralization test (sVNT). Binding assays were compared using Spearman's rank correlation and Passing-Bablok regression. All assays showed good correlation and similar antibody kinetics correlating with neutralizing potential. However, the assays show large proportional differences in BAU/ml. ECLIA and PETIA, which detect total antibodies against the receptor- binding domain (RBD) within the S1 subunit, interact similarly with the convalescent plasma-derived WHO IS but differently with vaccine serum, indicating a high sensitivity to the IgG/IgM/IgA ratio. All three binding assays allow monitoring of the antibody response in COVID-19-vaccinated individuals. However, the assay-specific differences hinder the definition of a common protective threshold in BAU/ml. Our results highlight the need for the thoughtful use of conversion factors and consideration of method-specific differences. To improve the management of future pandemics and harmonize total antibody assays, we should strive for reference material with a well-characterized Ig isotype composition.

Pharmacogenomic Analyses Implicate B Cell Developmental Status and MKL1 as Determinants of Sensitivity toward Anti-CD20 Monoclonal Antibody Therapy.

Monoclonal antibody (mAb) therapy directed against CD20 is an important tool in the treatment of B cell disorders. However, variable patient response and acquired resistance remain important clinical challenges. To identify genetic factors that may influence sensitivity to treatment, the cytotoxic activity of three CD20 mAbs: rituximab; ofatumumab; and obinutuzumab, were screened in high-throughput assays using 680 ethnically diverse lymphoblastoid cell lines (LCLs) followed by a pharmacogenomic assessment. GWAS analysis identified several novel gene candidates. The most significant SNP, rs58600101, in the gene MKL1 displayed ethnic stratification, with the variant being significantly more prevalent in the African cohort and resulting in reduced transcript levels as measured by qPCR. Functional validation of MKL1 by shRNA-mediated knockdown of MKL1 resulted in a more resistant phenotype. Gene expression analysis identified the developmentally associated TGFB1I1 as the most significant gene associated with sensitivity. qPCR among a panel of sensitive and resistant LCLs revealed immunoglobulin class-switching as well as differences in the expression of B cell activation markers. Flow cytometry showed heterogeneity within some cell lines relative to surface Ig isotype with a shift to more IgG+ cells among the resistant lines. Pretreatment with prednisolone could partly reverse the resistant phenotype. Results suggest that the efficacy of anti-CD20 mAb therapy may be influenced by B cell developmental status as well as polymorphism in the MKL1 gene. A clinical benefit may be achieved by pretreatment with corticosteroids such as prednisolone followed by mAb therapy.

Identifying next generation Ig selective cleaving enzymes for treatment of autoimmune diseases using IMPACT platform

Abstract Dysregulated humoral immune mechanisms result in immunoglobulin production that is pathogenic and contributes to a wide range of autoimmune diseases and transplant rejection. Proteases derived from human pathogens can specifically cleave these immunoglobulins (Ig) and can potentially eliminate circulating, cell-surface and immune-complex Ig classes and modulate Ig-mediated autoimmunity and inflammation, providing a novel opportunity to treat antibody-mediated diseases. We describe here the discovery, generation, and functional characterization of Ig specific proteases. These proteases were identified using Seismic Therapeutic’s IMPACT platform and tested for their ability to cleave intact Ig subclasses and Ig isotypes in a high-throughput CE-SDS cleavage assay. Additionally, we found that these enzymes cleave soluble and cell bound Ig in human blood, dramatically reducing effector function. We believe that these subclass and isotype specific cleaving proteases will provide an opportunity to develop novel targeted therapies for various autoimmune diseases where autoantibodies are the key drivers of the disease. In addition, we believe that they will be potentially better tolerated compared to current therapeutic interventions such as plasma exchange and other immunosuppressive therapies.