Heavy Chain Constant Region Research Articles

Site-Specific N-Glycoprofiling of Immunoglobulin G Subtypes from Donkey Milk.

Donkey milk IgG was probed for the site-specific N-glycosylation pattern through RP-UHPLC-MS/MS. The affinity-purified milk IgG was subjected to SDS-PAGE and proteomic analysis, which revealed the presence of subtypes. Multiple N-glycopeptides arising from the predicted donkey IgG1, IgG2, IgG3, IgG5, IgG6, and IgG7 subtypes' heavy-chain constant region were shown to contain glycans at the highly conserved glycosylation site NST in the CH2 domain. Differences in the peptide backbone with the NST site among subtypes generated after trypsin digestion resulted in the evaluation of the subtype-specific glycan pattern. Glycan sequence analysis indicated predominantly biantennary complex types with core fucosylation at the site NST. Interestingly, an additional site NQT in the CH1 domain of the heavy-chain constant region of IgG5 was found to possess mainly sialylated biantennary complex glycans with NeuAc and NeuGc. Structural diversity of glycans was mainly observed in the predicted donkey IgG1, IgG5, and IgG7, whereas IgG2, IgG3, and IgG6 resulted in the glycopeptides that are of low abundance in the analyzed samples. These findings would pave the way for a better understanding of donkey milk functional properties.

Single domain antibody: Development and application in biotechnology and biopharma.

Heavy-chain antibodies (HCAbs) are a unique type of antibodies devoid of light chains, and comprised of two heavy chains-only that recognize their cognate antigen by virtue of a single variable domain also referred to as VHH, single domain antibody (sdAb), or nanobody (Nb). These functional HCAbs, serendipitous discovered about three decades ago, are exclusively found in camelids, comprising dromedaries, camels, llamas, and vicugnas. Nanobodies have become an essential tool in biomedical research and medicine, both in diagnostics and therapeutics due to their beneficial properties: small size, high stability, strong antigen-binding affinity, low immunogenicity, low production cost, and straightforward engineering into more potent affinity reagents. The occurrence of HCAbs in camelids remains intriguing. It is believed to be an evolutionary adaptation, equipping camelids with a robust adaptive immune defense suitable to respond to the pressure from a pathogenic invasion necessitating a more profound antigen recognition and neutralization. This evolutionary innovation led to a simplified HCAb structure, possibly supported by genetic mutations and drift, allowing adaptive mutation and diversification in the heavy chain variable gene and constant gene regions. Beyond understanding their origins, the application of nanobodies has significantly advanced over the past 30 years. Alongside expanding laboratory research, there has been a rapid increase in patent application for nanobodies. The introduction of commercial nanobody drugs such as Cablivi, Nanozora, Envafolimab, and Carvykti has boosted confidence among in their potential. This review explores the evolutionary history of HCAbs, their ontogeny, and applications in biotechnology and pharmaceuticals, focusing on approved and ongoing medical research pipelines.

Proteomic Profiling of HDL in Newly Diagnosed Breast Cancer Based on Tumor Molecular Classification and Clinical Stage of Disease.

The association between high-density lipoprotein (HDL) cholesterol and breast cancer (BC) remains controversial due to the high complexity of the HDL particle and its functionality. The HDL proteome was determined in newly diagnosed BC classified according to the molecular type [luminal A or B (LA or LB), HER2, and triple-negative (TN)] and clinical stage of the disease. Women (n = 141) aged between 18 and 80 years with BC, treatment-naïve, and healthy women [n = 103; control group (CT)], matched by age and body mass index, were included. Data-independent acquisition (DIA) proteomics was performed in isolated HDL (D = 1.063-1.21 g/mL). Results: Paraoxonase1, carnosine dipeptidase1, immunoglobulin mMu heavy chain constant region (IGHM), apoA-4, and transthyretin were reduced, and serum amyloid A2 and tetranectin were higher in BC compared to CT. In TNBC, apoA-1, apoA-2, apoC-2, and apoC-4 were reduced compared to LA, LB, and HER2, and apoA-4 compared to LA and HER2. ComplementC3, lambda immunoglobulin2/3, serpin3, IGHM, complement9, alpha2 lysine rich-glycoprotein1, and complement4B were higher in TNBC in comparison to all other types; complement factor B and vitamin D-binding protein were in contrast to LA and HER2, and plasminogen compared to LA and LB. In grouped stages III + IV, tetranectin and alpha2-macroglobulin were reduced, and haptoglobin-related protein; lecithin cholesterol acyltransferase, serum amyloid A1, and IGHM were increased compared to stages I + II. Conclusions: A differential proteomic profile of HDL in BC based on tumor molecular classification and the clinical stage of the disease may contribute to a better understanding of the association of HDL with BC pathophysiology, treatment, and outcomes.

Genetic targeting of the mouse immunoglobulin locus to permit the expression of heavy-chain only antibodies against viral antigens

Abstract Conventional antibodies are composed of two identical sets of heavy and light chains, forming a dimer. The CH1 domain of the antibody heavy chain is essential for the pairing of heavy and light chains. However, certain animals, such as camels and sharks, evolved to produce heavy-chain-only antibodies (HCAbs) by losing this crucial CH1 domain in some of their antibody heavy chain constant regions. These HCAbs boast unique physicochemical properties that confer significant advantages: their smaller size allows for improved access to target antigens, increased hydrophilicity, enhanced tissue penetration, and greater structural stability compared to standard antibodies. Our research has successfully established a mouse model that intrinsically produces HCAbs from the mouse's own immunoglobulin heavy chain locus. Our findings demonstrate that heavy-chain-only B cell receptors, once introduced, facilitate normal B cell development, promote germinal center formation, and enhance plasma cell differentiation relative to B cells producing conventional antibodies. These genetically modified mice were vaccinated with viral antigens, resulting in the production of HCAbs that specifically target the SARS-CoV-2 spike protein and the HIV-1 Env trimer. Through single-cell sequencing, we have identified unique nucleotide sequences of clonotypes; the HCAbs are being synthesized to assess their viral neutralization potential and antigen-binding affinity to highlight the efficacy of our model.

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.

A Novel Heterozygous Variant in AICDA Impairs Ig Class Switching and Somatic Hypermutation in Human B Cells and is Associated with Autosomal Dominant HIGM2 Syndrome

B cells and their secreted antibodies are fundamental for host-defense against pathogens. The generation of high-affinity class switched antibodies results from both somatic hypermutation (SHM) of the immunoglobulin (Ig) variable region genes of the B-cell receptor and class switch recombination (CSR) which alters the Ig heavy chain constant region. Both of these processes are initiated by the enzyme activation-induced cytidine deaminase (AID), encoded by AICDA. Deleterious variants in AICDA are causal of hyper-IgM syndrome type 2 (HIGM2), a B-cell intrinsic primary immunodeficiency characterised by recurrent infections and low serum IgG and IgA levels. Biallelic variants affecting exons 2, 3 or 4 of AICDA have been identified that impair both CSR and SHM in patients with autosomal recessive HIGM2. Interestingly, B cells from patients with autosomal dominant HIGM2, caused by heterozygous variants (V186X, R190X) located in AICDA exon 5 encoding the nuclear export signal (NES) domain, show abolished CSR but variable SHM. We herein report the immunological and functional phenotype of two related patients presenting with common variable immunodeficiency who were found to have a novel heterozygous variant in AICDA (L189X). This variant led to a truncated AID protein lacking the last 10 amino acids of the NES at the C-terminal domain. Interestingly, patients’ B cells carrying the L189X variant exhibited not only greatly impaired CSR but also SHM in vivo, as well as CSR and production of IgG and IgA in vitro. Our findings demonstrate that the NES domain of AID can be essential for SHM, as well as for CSR, thereby refining the correlation between AICDA genotype and SHM phenotype as well as broadening our understanding of the pathophysiology of HIGM disorders.

Sequence and structure statistics of the nanobody database and molecular dynamics simulation analysis of the nanobody VHH

Antibodies are immunoglobulins produced in vivo by immune cells stimulated by exogenous molecules, and nano-antibodies are a class of molecules that are similar to conventional antibodies but smaller in size. Originally discovered as an antibody in camelidsand cartilaginous fishes, they are called heavy chain antibodies due to the lack of light and heavy chain constant regions in the CH1 region except for the retained heavy chain, and their binding region to the antigen consists of the heavy chain variable region only, making them the smallest antibodies available with complete antibody functional properties. In this experiment, we analyzed the commonalities and differences of the nanobodies by extracting all their amino acid sequences, performed protein modeling of the relevant nanobodies of coronaviruses among them, and then analyzed the data of RMSD and RMSF by using molecular dynamics simulation, and finally predicted and analyzed the protein conformation and the structures of VHH at all levels.

Development and evaluation of a bivalent vaccine based on recombinant newcastle disease virus expressing infectious bursal disease virus VP2L-CH3-CH4 in SPF chickens

Newcastle disease (ND) and infectious bursal disease (IBD) are two viral infectious diseases that are extremely damaging to the poultry industry and are widespread throughout the world. It is necessary to develop a safe and effective vaccine against IBD and ND because vaccination is an effective preventive measure. It has been discovered that recombinant proteins expressed by an expression system in which a fragment of mammalian Immunoglobulin G (IgG) Fragment crystallizable (Fc) is linked to a segment of a gene have antibody-like properties that increase the exogenous protein's serum half-life. Heavy chain constant region 3 and heavy chain constant region 4 (CH3-CH4) of Avian Immunoglobulin Y (IgY) is structurally very similar to mammalian Ig G Fc. In this study, a bivalent vaccine rClone30-VP2L-CH3-CH4-GMCSF was developed by using NDV rClone30-chGM-CSF vector to produce VP2L-CH3-CH4 fusion protein. The vaccine has been given to 14-day-old specific pathogen free (SPF) free chickens to test whether it has the potential to prevent IBD and ND. Anti-IBDV and anti-NDV antibody levels in serum were evaluated using ELISA and HI, respectively, and the contents of CD4+ T, CD8+ T, and B cells in leukocytes were determined via flow cytometry. The contents and mRNA transcription levels of four inflammatory factors, IL-1β, IL-4, IFN-γ and chGM-CSF, were detected by ELISA and real-time PCR respectively. The results showed that after vaccination with the rClone30-VP2L-CH3-CH4-GMCSF vaccine, the levels of anti NDV and anti IBDV antibodies in chickens were significantly higher than those of the rClone30 vaccine and commercial vaccines. Meanwhile, the contents and transcription levels of inflammatory factors in chickens inoculated with rClone30-VP2L-CH3-CH4-GMCSF were significantly increased, and the proliferation response of B cells, CD4+ and CD8+ T cells was also stronger. However, the rClone30-VP2L-CH3-CH4-GMCSF vaccine had no significant advantage over the rClone30-VP2L-GMCSF vaccine in any of the above-mentioned features. In summary, rClone30-VP2L-CH3-CH4-GMCSF can stimulate the body to produce a stronger immune response, showing its potential to be considered as vaccine against IBD and ND, but the addition of CH3-CH4 did not improve the vaccine's immune effect as expected. The research lays the foundation for developing vaccines for other infectious viral diseases and avoids a unrealistic vaccine optimization method.

One Case of Heavy Chain Deposition Disease Treated with Dara+KD Regimen and Literature Review

Background: Heavy chain deposition disease (HCDD) is a systemic disease caused by the deposition of monoclonal immunoglobulin heavy chains in tissues.It is a rare monoclonal immunoglobulin deposition disease. The mechanism of HCDD is still unclear, which may be related to the deletion of heavy chain constant region 1 and the activation of the Complement system by monoclonal heavy chains. Renal biopsy remains the gold standard for diagnosing HCDD at present. There are significant individual differences in the formation of abnormal heavy chains in HCDD patients, and FLC, M protein, and complement detection have certain reference significance for the evaluation of diagnosis and treatment processes. The chemotherapy scheme represented by Bortezomib has a certain effect. HDM/ASCT and kidney transplantation can be used as second-line treatment for some patients. At present, there is no report on the use of CD38 monoclonal antibody combined with KD regimen. Objective: To explore the efficacy and toxicity of CD38 monoclonal antibody combined with KD regimen in the treatment of heavy chain deposition disease. Methods: The diagnosis, treatment and efficacy of HCDD who hospitalized in the Hematology Department of the First Affiliated Hospital of Sun Yat-sen University was analyzed, and the value of CD38 monoclonal antibody combined with KD regimen in the treatment of HCDD was discussed. The patient was male, 44 years old, due to “depressed edema of both lower limbs and eyelid edema for more than two months”. Results: Heavy chain deposition disease is a rare malignant Plasma cell disease. CD38 monoclonal antibody combined with KD regimen can rapidly alleviate the disease,.This patient got CR and obvious improvement of clinical symptoms after 1 cycle chemotherapy, and has no obvious side effects.We be expected to do 4 cycle chemothetaphy and would do ASCT.until now,this patient has got 3 cycles. Conclusion: Heavy chain deposition disease is a rare malignant Plasma cell disease. CD38 monoclonal antibody combined with KD regimen can rapidly alleviate the disease, with fast onset time, high CR rate, obvious improvement of clinical symptoms, and no obvious side effects.

B-179 Development of a Reflex Mass Spectrometric Workflow for Confirmation of IgE and IgD Specificity Paraproteins Utilising the EXENT® Platform

Abstract Background The EXENT® (GAM) assay is a MALDI-TOF (Matrix Assisted Laser Desorption Ionisation Time of Flight) mass spectrometry-based solution which utilises IgG, IgA, IgM, total kappa and total lambda specific paramagnetic beads, which could be used in the investigation of monoclonal proteins in multiple myeloma and other plasma cell disorders (in development by The Binding Site (TBS), part of Thermo Fisher Scientific). Rare cases of IgD and IgE myelomas will be partially typed with the current iteration of the EXENT (GAM) assay. We present here a workflow to confirm the heavy chain specificity of such paraproteins as IgD or IgE using reflex LC-MS analysis of EXENT eluates. Methods Samples known to contain IgD (2) or IgE (3) paraproteins were provided from either routine workload at the immunology laboratory at Birmingham Heartlands Hospital, UK or an internal library of historical myeloma samples. Capillary zone electrophoresis (CZE) and immunofixation (IFE) were performed on Capillarys2™ and Hydrasys2 Scan™ platforms, respectively (Sebia, France). Serum free light chain (sFLC) measurement was performed using Freelite® reagents (The Binding Site, UK) on a Cobas c501 turbidimeter (Roche, Switzerland). Samples were prepared via EXENT (GAM) assay, by separately incubating diluted serum with antisera specific for IgG, IgA, IgM, total kappa and total lambda conjugated to paramagnetic beads. After washes, purified immunoglobulins were treated to reduce and elute the light chain component. Samples were mixed with matrix, spotted onto a MALDI target plate and acquired by MALDI-TOF MS. Once light-chain specificity of the suspected IgD or IgE paraprotein was determined, repeat immuno-precipitation with the same light-chain bead was carried out. Eluted samples were neutralised, transferred to fresh tubes, reduced, alkylated and digested with trypsin for 3 h. After acidification digested samples were analysed by C18 reverse phase UPLC on a BioAccord LC-MS system (Waters, UK). Data was then screened against a database of known immunoglobulin heavy chain peptides to assess the presence of heavy chain peptides specific for each immunoglobulin isotype. Results In each sample tested, the EXENT solution identified at least one monoclonal light chain that was only seen in the total kappa or total lambda immunoprecipitations with no corresponding monoclonal peak(s) at the same mass(es) in the IgG, IgA or IgM specificities. This finding would be consistent with the presence of clonal plasma cells producing either monoclonal free light chains only, or IgD and IgE paraproteins. Subsequent LC-MS and LC-MSe (tandem MS) analysis of reduced, alkylated and tryptically digested eluates corresponding to these suspected IgD and IgE samples produced high quality peptide sequencing data. The data identified multiple IgD or IgE specific peptides in each sample and showed between 40% and 66% sequence coverage for IgD or IgE heavy-chain constant regions. Thus, confirming the presence of IgD or IgE in these samples which had been partially typed by the EXENT solution. Conclusion This data demonstrates that the EXENT (GAM) assay could potentially aid in detection of IgD and IgE paraproteins when combined with LC-MS proteomic techniques, as it allows efficient and facile confirmation of these rare heavy chain isotypes.

Abstract LB210: RenNano® mice: A heavy-chain-only antibody platform for the generation of nanobody therapeutics

Abstract Monoclonal antibodies have been used to successfully treat various diseases, including tumors, autoimmune diseases, and infectious diseases. Traditional antibodies are comprised of a tetramer of two heavy chains and two light chains, totaling 150 kDa in molecular weight. However, the large size of antibodies can limit the therapeutic application; in particular, the penetration of tumors and the blood brain barrier (BBB) is not always feasible. In contrast to traditional antibodies, heavy-chain-only antibodies (HCAbs) are significantly smaller (~75 kDa), as they contain only two heavy chains. Since the heavy chain variable domain of HCAbs (i.e., VHH or single domain antibody, sdAb, or nanobody) is solely responsible for antigen recognition, nanobodies can function independently as a therapeutic molecule, which may be advantageous for penetrating tumors or the BBB. Previously, we generated a fully human antibody mouse platform, RenMabTM, in which the murine heavy chain and kappa light chain variable domains were replaced by the full human heavy chain and kappa light chain V(D)J loci in situ. Here, we have further modified the RenMabTM model to generate a fully human heavy-chain-only antibody mouse model, termed RenNano®. The modified heavy chain constant regions of RenNano® mice allow them to spontaneously produce HCAbs. Flow cytometry and biolayer interferometry confirmed that RenNano®-derived HCAbs can bind antigens without light chains. Despite this reliance on the heavy chain only variable regions for antigen specificity, RenNano® mice can generate antigen-specific antibodies with high affinity (10−8 -10−9 KD) upon immunization with various antigens. In addition, many RenNano®-derived HCAbs exhibited a longer CDR3 length, which could promote the recognition of difficult-to-reach epitopes. Furthermore, RenNano®-derived HCAbs have favorable diversity, and excellent developability properties such as a higher degree of hydrophilicity. Anti-4-1BB HCAbs can also activate 4-1BB-NF-κB signaling in a dose-dependent manner, as demonstrated in reporter assays. In summary, the full human heavy-chain-only antibody mice, RenNano®, can produce human HCAb with high affinity and good efficacy. Thus, RenNano® is a powerful platform to discover HCAb/nanobodies for various therapeutic applications. Citation Format: Yiqing Hu, Qi Zhang, Lijun Zhang, Yabo Zhang, Huizhen Zhao, Jiawei Yao, Liu Yang, Baihong Liu, Shensen Wang, Zhengfang Su, Li Hui, Qingcong Lin, Qiangqiang Ma, Yuelei Shen. RenNano® mice: A heavy-chain-only antibody platform for the generation of nanobody therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB210.

The Effects of IGHG1 on the Proliferation and Apoptosis of Human Acute Myeloid Leukemia THP-1 Cells

To investigate the effects of the immunoglobulin G1 heavy chain constant region (IGHG1) on the proliferation and apoptosis of acute myeloid leukemia (AML) THP-1 cells and its possible mechanism of action. Human AML THP-1 cells were cultured in vitro and divided into control (normally cultured THP-1 cells), pcDNA3.1 ［THP-1 cells transfected with IGHG1 overexpression (pcDNA3.1-IGHG1) negative control plasmid］, pcDNA3.1-IGHG1 (THP-1 cells transfected with plasmid containing pcDNA3.1-IGHG1), LY364947 ［transforming growth factor-β (TGF-β)/signal transduction protein (Smad) inhibitor LY364947 20 μmol/L treated THP-1 cells］, si-NC ［THP-1 cells transfected with IGHG1-small interfering RNA (siRNA) negative control］, si-IGHG1 (THP-1 cells transfected with IGHG1-siRNA), and si-IGHG1+LY364947 (IGHG1-siRNA and LY364947 co-treated THP-1 cells) a total of 7 groups. Fluorescence quantitative PCR was used to detect the expression of IGHG1 and immunoglobulin G (IgG) mRNA of THP-1 cells in each group; CCK-8 was used to detect THP-1 cells proliferation activity; flow cytometry was used to detect THP-1 cells apoptosis and cell cycle in each group; Western blot was used to detect the THP-1 cells proliferation, apoptosis and the expression of TGF-β/Smad signaling pathway related proteins. Compared with the control group, after overexpression of IGHG1, the expression of IGHG1 and IgG mRNA, cell proliferation viability, S phase cell ratio, expressions of Cyclin D1, B cell lymphoma-2 (Bcl-2), IgG, TGF-β1, phosphorylated Smad3 (p-Smad3)/Smad3 protein in THP-1 cells were significantly increased (P<0.05), the apoptosis rate, G0/G1 phase cell ratio, expression of p21, Bcl-2 related X protein (Bax), Caspase-3 protein were significantly reduced (P<0.05); after inhibiting TGF-β/Smad signaling pathway or silencing IGHG1, the expression of IGHG1 and IgG mRNA, cell proliferation viability, S phase cell ratio, expression of Cyclin D1, Bcl-2, IgG, TGF-β1, p-Smad3/Smad3 protein in THP-1 cells were significantly reduced (P<0.05), the apoptosis rate, G0/G1 phase cell ratio, expressions of p21, Bax, and Caspase-3 protein were significantly increased (P<0.05); and compared with silencing IGHG1, after co-treatment of IGHG1 gene silencing and TGF-β/Smad pathway inhibition, the expression of IGHG1 and IgG mRNA, cell proliferation viability, S phase cell ratio, expressions of Cyclin D1, Bcl-2, IgG, TGF-β1, p-Smad3/Smad3 protein in THP-1 cells were significantly reduced (P<0.05), the apoptosis rate, G0/G1 phase cell ratio, expression of p21, Bax, and Caspase-3 protein were significantly increased (P<0.05). Silencing IGHG1 gene can down-regulate the expression of IgG, inhibit the proliferation of human AML THP-1 cells, block cell cycle progression, and induce cell apoptosis; its mechanism may be related to the inhibition of the TGF-β/Smad pathway activation.

Preparation of IgE Antibody and Distribution of IgE+ Secretory Cells in the Palatine Tonsil of Bactrian Camel.

Allergic diseases induced by dust have seriously threatened human health, while Bactrian camels can live in a sandy environment for a long time. To prepare rabbit anti-Bactrian camel IgE antibody and explore the distribution characteristics of IgE+ secretory cells in the palatine tonsils, which lays a theoretical foundation for the distribution of local antibodies in the palatal tonsils of Bactrian camel and the study of immune function. In this study, the amino acid sequences of Bactrian camel IgE, IgA, IgM and IgG heavy chain constant regions were compared, and a specific IgE gene fragment were selected (447 bp). The recombinant plasmid pET-28a-IgE was induced in Escherichia coli BL21(DE3) by IPTG and its expression conditions were optimized. The antibody was prepared by immunizing rabbits with purified IgE recombinant protein, its titer and specificity were detected by indirect ELISA and Western blotting. Immunohistochemical and statistical methods investigated the distribution of IgE+ secretory cells in the palatine tonsils. The IgE recombinant protein was expressed in the form of inclusion bodies with a size of 16 kDa. The optimal IPTG induction concentration was 0.7 mmol/L and the induction time was 8 h. The titer of the antibody was 1:16000 by ELISA, and the antibody could specifically bind to the recombinant protein by Western blotting. IgE+ secretory cells were mainly distributed in the subepithelial compartments of reticulated crypt epithelium of the palatine tonsil of the Bactrian camel, followed by the subepithelial compartments of stratified squamous epithelium and occasionally in the extrafollicular region. The rabbit anti-Bactrian camel IgE polyclonal antibody was successfully prepared. It is confirmed that IgE exists in the palatine tonsils of Bactrian camels under normal living conditions. In addition, IgE+ secretory cells are mainly distributed in the subepithelial compartments of reticulated crypt epithelium of the palatine tonsil, which is consistent with the distribution characteristics of IgG+ and sIgA+ secretory cells in the palatal tonsils of the Bactrian camel.

Prevention of Shiga toxin 1-caused colon injury by plant-derived recombinant IgA

Immunoglobulin A (IgA) is a candidate antibody for oral passive immunization against mucosal pathogens like Shiga toxin-producing Escherichia coli (STEC). We previously established a mouse IgG monoclonal antibody (mAb) neutralizing Shiga toxin 1 (Stx1), a bacterial toxin secreted by STEC. We designed cDNA encoding an anti-Stx1 antibody, in which variable regions were from the IgG mAb and all domains of the heavy chain constant region from a mouse IgA mAb. Considering oral administration, we expressed the cDNA in a plant expression system aiming at the production of enough IgA at low cost. The recombinant-IgA expressed in Arabidopsis thaliana formed the dimeric IgA, bound to the B subunit of Stx1, and neutralized Stx1 toxicity to Vero cells. Colon injury was examined by exposing BALB/c mice to Stx1 via the intrarectal route. Epithelial cell death, loss of crypt and goblet cells from the distal colon were observed by electron microscopy. A loss of secretory granules containing MUC2 mucin and activation of caspase-3 were observed by immunohistochemical methods. Pretreatment of Stx1 with the plant-based recombinant IgA completely suppressed caspase-3 activation and loss of secretory granules. The results indicate that a plant-based recombinant IgA prevented colon damage caused by Stx1 in vivo.

Heavy Chain Constant Region Usage in Antibodies to Peptidylarginine Deiminase 4 as a Marker of Disease Subsets in Rheumatoid Arthritis.

The study of autoantibody isotypes in autoimmune diseases is useful for identifying clinically relevant endotypes. This study was undertaken to study the prevalence and clinical significance of different isotypes and IgG subclasses of anti-peptidylarginine deiminase 4 (anti-PAD4) autoantibodies in individuals with rheumatoid arthritis (RA). In 196 RA subjects and 64 healthy controls, anti-PAD4 antibody types were determined using enzyme-linked immunosorbent assay. We investigated associations between anti-PAD4 antibodies and clinical outcomes, and relevant features were confirmed in an independent RA cohort. Anti-PAD4 IgG1, anti-PAD4 IgG2, anti-PAD4 IgG3, anti-PAD4 IgG4, anti-PAD4 IgA, and anti-PAD4 IgE antibodies were more frequent in RA patients than healthy controls (P < 0.001). Anti-PAD4 IgG1, anti-PAD4 IgG3, and anti-PAD4 IgE were associated with distinct clinical features. Anti-PAD4 IgG1 was predictive of progressive radiographic joint damage (odds ratio [OR] 4.88, P=0.005), especially in RA patients without baseline joint damage (40% versus 0%, P=0.003) or in those negative for anti-cyclic citrullinated peptide and/or rheumatoid factor (OR 32; P=0.009). IgG1 was also associated with higher levels of C-reactive protein (P=0.006) and interleukin-6 (P=0.021). RA patients with anti-PAD4 IgG3 had higher baseline joint damage scores (median Sharp/van der Heijde score 13 versus 7, P=0.046), while those with anti-PAD4 IgE had higher Disease Activity Score in 28 joints (median 4.0 versus 3.5, P=0.025), more frequent rheumatoid nodules (31% versus 16%, P=0.025), and more frequent interstitial lung disease (ground-glass opacification) (24% versus 9%, P=0.014). Anti-PAD4 IgG1 antibody associations with joint damage were corroborated in an independent RA cohort. Anti-PAD4 IgG1, anti-PAD4 IgG3, and anti-PAD4 IgE antibodies identify discrete disease subsets in RA, suggesting that heavy chain usage drives distinct effector mechanisms of anti-PAD4 antibodies in RA.

Immunoglobulin heavy constant gamma gene evolution is modulated by both the divergent and birth-and-death evolutionary models.

Immunoglobulin G (IgG) is one of the five antibody classes produced in mammals as part of the humoral responses accountable for protecting the organisms from infection. Its antibody heavy chain constant region is encoded by the Ig heavy-chain gamma gene (IGHG). In humans, there are four IGHG genes which encode the four subclasses, each with a specialized effector function. Although four subclasses of IgG proteins have also been reported in macaques, this does not appear to be the rule for all primates. In Platyrrhini, IgG has been stated to be encoded by a single-copy gene. To date, it remains unknown how the IGHG has expanded or contracted in the primate order; consequently, we have analyzed data from 38 primate genome sequences to identify IGHG genes and describe the evolution of IGHG genes in primate order. IGHG belongs to a multigene family that evolves by the birth-death evolutionary model in primates. Whereas Strepsirrhini and Platyrrhini have a single-copy gene, in Catarrhini, it has expanded to several paralogs in their genomes; some deleted and others pseudogenized. Furthermore, episodic positive selection may have promoted a species-specific IgG effector function. We propose that IgG evolved to reach an optimal number of copies per genome to adapt their humoral immune responses to different environmental conditions. This study has implications for biomedical trials using non-human primates.

Abstract 313: Novel bioengineered monoclonal antibodies targeting oxidized macrophage migration inhibitory factor as anti-cancer therapeutics and companion diagnostics

Abstract Background: Macrophage Migration Inhibitory Factor (MIF) has been described as a pleiotropic cytokine known to exacerbate tumor growth (Chesney and Mitchell, 2015). Due to its ubiquitous nature, MIF can be considered as an inappropriate target for therapeutic intervention. However, we discovered oxidized MIF (oxMIF) as the disease-related isoform of MIF, which is specifically present in tumor tissue (Schinagl A et al., 2016). A 1st generation anti-oxMIF monoclonal antibody (mAb) demonstrated an acceptable safety profile and efficacy signal in a phase 1 clinical trial (Mahalingam D et al., 2020). Two bioengineered 2nd generation anti-oxMIF mAbs with highly improved biophysicochemical and biological properties were generated and compared to the 1st generation anti-oxMIF mAb. Both bioengineered mAbs share identical variable domains, but distinct heavy-chain constant regions, to increase effector functions and efficacy of the therapeutic mAb ON203 and to reduce interactions with FcγRs for the radio diagnostic mAb ON102. Methods: Hydrophobicity and stability were determined by HIC and SEC. ADCC and ADCP activity were investigated by reporter and PBMC-mediated cell killing assays. Tumor penetration was assessed using IRDye 800CW or Zr89-labeled mAb in tumor-bearing Balb/c or Balb/c nude mice. PK and bioavailability were assessed in Balb/c nude mice. Efficacy was determined in PC3 xenograft models in NMRI nude mice. Results: Bioengineering significantly reduced hydrophobicity of ON203 and ON102, leading to improved stability and strongly reduced aggregation but retaining the low nM affinity for oxMIF. This further resulted in a two-fold improved bioavailability and tumor accumulation after 24-48h and a three-fold enhanced retention on day 7 in mice harboring solid tumors of the colon, when compared to the 1st generation anti-oxMIF mAb. In contrast to previously described anti-oxMIF reference, ON203 and ON102 did not mediate any unspecific release of MCP-1, IL-6, or TNF-α from PBMCs. Reporter and PBMC-mediated cell killing assays proved ADCC activity of ON203 with EC50 values of 0.1-0.8 nM and ADCP activity with EC50 values of 2.3 nM, whereas the 1st generation anti-oxMIF mAb was at least 10-fold less potent. Our findings for ON203 translated into abolishment of tumor growth in NMRI nude mice harboring human PC3 prostate cancer cells, showing superior efficacy compared to the 1st generation anti-oxMIF mAb. Conclusion: ON203 has a high potential to significantly improve efficacy compared to the 1st generation anti-oxMIF mAb, with Zr89-ON102 as companion diagnostic for patient stratification. We aim to develop these anti-oxMIF mAbs together for clinical use to create new treatment options for patients with solid tumors, with a clear rationale to combine ON203 with other immunotherapies or checkpoint inhibitors (Noe and Mitchell, 2020). Citation Format: Alexander Schinagl, Michael Thiele, Irina Mirkina, Gregor Rossmueller, Alejandro A. Puchol Tarazona, Randolf J. Kerschbaumer. Novel bioengineered monoclonal antibodies targeting oxidized macrophage migration inhibitory factor as anti-cancer therapeutics and companion diagnostics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 313.

Intravenous immunoglobulin induces IgG internalization by tolerogenic myeloid dendritic cells that secrete IL-10 and expand Fc-specific regulatory T cells

Intravenous immunoglobulin (IVIG) is used as an immunomodulatory agent in many inflammatory conditions including Multisystem Inflammatory Syndrome-Children (MIS-C) and Kawasaki disease (KD). However, the exact mechanisms underlying its anti-inflammatory action are incompletely characterized. Here, we show that in KD, a pediatric acute vasculitis that affects the coronary arteries, IVIG induces a repertoire of natural Treg that recognize immunodominant peptides in the Fc heavy chain constant region. To address which antigen-presenting cell (APC) populations present Fc peptides to Treg, we studied the uptake of IgG by innate cells in subacute KD patients 2 weeks after IVIG and in children 1.6–14 years after KD. Healthy adults served as controls. IgG at high concentrations was internalized predominantly by two myeloid dendritic cell (DC) lineages, CD14+ cDC2 and ILT-4+ CD4+ tmDC mostly through Fcγ receptor (R) II and to a lesser extent FcγRIII. Following IgG internalization, these two DC lineages secreted IL-10 and presented processed Fc peptides to Treg. The validation of IVIG function in expanding Fc-specific Treg presented by CD14+ cDC2 and ILT-4+ CD4+ tmDC was addressed in a small cohort of patients with MIS-C. Taken together, these results suggest a novel immune regulatory function of IgG in activating tolerogenic innate cells and expanding Treg, which reveals an important anti-inflammatory mechanism of action of IVIG.

Characterization and determination of bovine immunoglobulin G subtypes in milk and dairy products by UPLC-MS

In this study, ultra-high performance liquid chromatography mass spectrometry (UPLC-MS) method was established for the characterization and quantitative determination of immunoglobulin G (IgG) subtypes (IgG1, IgG2, IgG3) in bovine dairy products. High-resolution mass spectrometry (HRMS) was applied to qualitatively confirm the theoretical peptides with specificity, enzymatic hydrolysis curve and stability among in heavy chain constant (CH1, CH2 and CH3) regions. The characteristic peptides VHNEGLPAPIVR, EPSVFIFPPKPK, GLPAPIVR, VVSALR were screened to quantitative analysis bovine IgG1, IgG2, IgG3 and the total amount of bovine IgG1 and IgG3, respectively. Isotope-labeled peptides were obtained by isotope dimethylation reaction, which aimed to correct the matrix effects. The results showed that the recovery was between 98.7% and 103.5%, and the precision of inter-day and intra-day was less than 6.8%. Moreover, this method had good linearity (R2 ≥ 0.999). Therefore, this research provided an effective method for quantitatively detecting bovine IgG subtypes in milk and dairy products.

AID function in somatic hypermutation and class switch recombination.

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation of immunoglobulin (Ig) gene variable regions and class switch recombination (CSR) of Ig heavy chain constant regions. Two decades of intensive research has greatly expanded our knowledge of how AID functions in peripheral B cells to optimize antibody responses against infections, while maintaining tight regulation of AID to restrain its activity to protect B cell genomic integrity. The many exciting recent advances in the field include: 1) the first description of AID's molecular structure, 2) remarkable advances in high throughput approaches that precisely track AID targeting genome-wide, and 3) the discovery that the cohesion-mediate loop extrusion mechanism [initially discovered in V(D)J recombination studies] also governs AID-medicated CSR. These advances have significantly advanced our understanding of AID's biochemical properties in vitro and AID's function and regulation in vivo. This mini review will discuss these recent discoveries and outline the challenges and questions that remain to be addressed.