Constant Domains Research Articles

Molecular design of the γδT cell receptor ectodomain encodes biologically fit ligand recognition in the absence of mechanosensing

High-acuity αβT cell receptor (TCR) recognition of peptides bound to major histocompatibility complex molecules (pMHCs) requires mechanosensing, a process whereby piconewton (pN) bioforces exert physical load on αβTCR-pMHC bonds to dynamically alter their lifetimes and foster digital sensitivity cellular signaling. While mechanotransduction is operative for both αβTCRs and pre-TCRs within the αβT lineage, its role in γδT cells is unknown. Here, we show that the human DP10.7 γδTCR specific for the sulfoglycolipid sulfatide bound to CD1d only sustains a significant load and undergoes force-induced structural transitions when the binding interface-distal γδ constant domain (C) module is replaced with that of αβ. The chimeric γδ-αβTCR also signals more robustly than does the wild-type (WT) γδTCR, as revealed by RNA-sequencing (RNA-seq) analysis of TCR-transduced Rag2-/- thymocytes, consistent with structural, single-molecule, and molecular dynamics studies reflective of γδTCRs as mediating recognition via a more canonical immunoglobulin-like receptor interaction. Absence of robust, force-related catch bonds, as well as γδTCR structural transitions, implies that γδT cells do not use mechanosensing for ligand recognition. This distinction is consonant with the fact that their innate-type ligands, including markers of cellular stress, are expressed at a high copy number relative to the sparse pMHC ligands of αβT cells arrayed on activating target cells. We posit that mechanosensing emerged over ∼200 million years of vertebrate evolution to fulfill indispensable adaptive immune recognition requirements for pMHC in the αβT cell lineage that are unnecessary for the γδT cell lineage mechanism of non-pMHC ligand detection.

Characterization and modulation of anti-αβTCR antibodies and their respective binding sites at the βTCR chain to enrich engineered T cells

T cell engineering strategies offer cures to patients and have entered clinical practice with chimeric antibody-based receptors; αβT cell receptor (αβTCR)-based strategies are, however, lagging behind. To allow a more rapid and successful translation to successful concepts also using αβTCRs for engineering, incorporating a method for the purification of genetically modified T cells, as well as engineered T cell deletion after transfer into patients, could be beneficial. This would allow increased efficacy, reduced potential side effects, and improved safety of newly to-be-tested lead structures. By characterizing the antigen-binding interface of a good manufacturing process (GMP)-grade anti-αβTCR antibody, usually used for depletion of αβT cells from stem cell transplantation products, we developed a strategy that allows for the purification of untouched αβTCR-engineered immune cells by changing 2 amino acids only in the TCRβ chain constant domain of introduced TCR chains. Alternatively, we engineered an antibody that targets an extended mutated interface of 9 amino acids in the TCRβ chain constant domain and provides the opportunity to further develop depletion strategies of engineered immune cells.

A comprehensive analysis of novel disulfide bond introduction site into the constant domain of human Fab

Generally, intermolecular disulfide bond contribute to the conformational protein stability. To identify sites where intermolecular disulfide bond can be introduced into the Fab’s constant domain of the therapeutic IgG, Fab mutants were predicted using the MOE software, a molecular simulator, and expressed in Pichia pastoris. SDS-PAGE analysis of the prepared Fab mutants from P. pastoris indicated that among the nine analyzed Fab mutants, the F130C(H):Q124C(L), F174C(H):S176C(L), V177C(H):Q160C(L), F174C(H):S162C(L), F130C(H):S121C(L), and A145C(H):F116C(L) mutants mostly formed intermolecular disulfide bond. All these mutants showed increased thermal stability compared to that of Fab without intermolecular disulfide bond. In the other mutants, the intermolecular disulfide bond could not be completely formed, and the L132C(H):F118C(L) mutant showed only a slight decrease in binding activity and β-helix content, owing to the exertion of adverse intermolecular disulfide bond effects. Thus, our comprehensive analysis reveals that the introduction of intermolecular disulfide bond in the Fab’s constant domain is possible at various locations. These findings provide important insights for accomplishing human Fab stabilization.

C1q binding to surface-bound IgG is stabilized by C1r2s2 proteases

Complement is an important effector mechanism for antibody-mediated clearance of infections and tumor cells. Upon binding to target cells, the antibody's constant (Fc) domain recruits complement component C1 to initiate a proteolytic cascade that generates lytic pores and stimulates phagocytosis. The C1 complex (C1qr2s2) consists of the large recognition protein C1q and a heterotetramer of proteases C1r and C1s (C1r2s2). While interactions between C1 and IgG-Fc are believed to be mediated by the globular heads of C1q, we here find that C1r2s2 proteases affect the capacity of C1q to form an avid complex with surface-bound IgG molecules (on various 2,4-dinitrophenol [DNP]-coated surfaces and pathogenic Staphylococcus aureus). The extent to which C1r2s2 contributes to C1q-IgG stability strongly differs between human IgG subclasses. Using antibody engineering of monoclonal IgG, we reveal that hexamer-enhancing mutations improve C1q-IgG stability, both in the absence and presence of C1r2s2 In addition, hexamer-enhanced IgGs targeting S. aureus mediate improved complement-dependent phagocytosis by human neutrophils. Altogether, these molecular insights into complement binding to surface-bound IgGs could be important for optimal design of antibody therapies.

Formaldehyde sensing with a parts-per-billion limit of detection by dielectric properties and crystal symmetry optimization in BiFeO3-based p-type solid solution

Regarding sensing properties, a well-accepted theory is that the dielectric constant plays a crucial role in the depletion layer width and sensing response. Nevertheless, this theory has not yet been experimentally well demonstrated and applied in the design of high-performance sensors due to the incompatibility in conductivity, because traditional dielectrics are insulating while sensors are semiconducting. Herein, a very promising formaldehyde sensor based on new types of dielectrics (multiferroics) was designed via the strategy of improving the dielectric properties. The as-prepared Bi0.95Sm0.05FeO3 sensors show a decent sensing response in detecting ppb level formaldehyde, which is the best-known indoor carcinogenic air pollutant. It also shows low humidity dependence without sacrificing the gas response, making it an excellent sensor in indoor air quality monitoring. The sensing mechanism is attributed to the noncentrosymmetric R3c phase, which exhibits a higher dielectric constant and conductive domain walls, favoring a thicker hole accumulation layer and high carrier mobility, respectively. In stark contrast, the centrosymmetric Pbnm samples exhibit poor sensing responses, which hardly respond to formaldehyde because of the lower dielectric constant and cancellation of the domain structure, even though they possess a smaller particle size.

Differential response of oxidative and glycolytic skeletal muscle fibers to mesterolone

Oxidative and glycolytic muscle fibers differ in their ultrastructure, metabolism, and responses to physiological stimuli and pathological insults. We examined whether these fibers respond differentially to exogenous anabolic androgenic steroids (AASs) by comparing morphological and histological changes between the oxidative anterior latissimus dorsi (ALD) and glycolytic pectoralis major (PM) fibers in adult avian muscles. Adult female White Leghorn chickens (Gallus gallus) were randomly divided into five groups: a vehicle control and four mesterolone treatment groups (4, 8, 12, and 16 mg/kg). Mesterolone was administered orally every three days for four weeks. Immunocytochemical techniques and morphometric analyses were employed to measure the changes in muscle weight, fiber size, satellite cell (SC) composition, and number of myonuclei. Mesterolone increased both body and muscle weights and induced hypertrophy in glycolytic PM fibers but not in oxidative ALD fibers. Mesterolone induced SC proliferation in both muscles; however, the myonuclear accretion was noticeable only in the PM muscle. In both muscles, the collective changes maintained a constant myonuclear domain size and the changes were dose independent. In conclusion, mesterolone induced distinct dose-independent effects in avian oxidative and glycolytic skeletal muscle fibers; these findings might be clinically valuable in the treatment of age-related sarcopenia.

Removing a light-chain missing byproduct by MMC ImpRes mixed-mode chromatography under weak partitioning mode in purifying a WuXiBody-based bispecific antibody

WuXiBody is a bispecific antibody (bsAb) platform developed by WuXi Biologics. Its key feature is the replacement of one parental antibody's CH1/CL region with the T cell receptor (TCR) constant domain, a design that promotes cognate heavy chain (HC)-light chain (LC) pairing. BsAbs based on WuXiBody can adopt either asymmetric or symmetric format. For purifying a WuXiBody-based symmetric bsAb, we identified a LC-missing species as a major byproduct. While for bsAbs based on other platforms removal of such byproduct can pose considerable challenge to the downstream team, in this case WuXiBody's unique design makes separation relatively straightforward. We previously showed that Capto MMC ImpRes mixed-mode chromatography under bind-elute mode can effectively remove this LC-missing species. However, the dynamic binding capacity (DBC) of Capto MMC ImpRes is relatively low under the selected condition, making the process less desirable for large-scale manufacturing. In this study, we demonstrated that when Capto MMC ImpRes chromatography is conducted under weak partitioning mode, high throughput, good yield, and effective byproduct removal are simultaneously achieved.

Identity in Mares-Goldblatt Models for Quantified Relevant Logic

Mares and Goldblatt (The Journal of Symbolic Logic, 71(01), 163–187, 2006) provided an alternative frame semantics for two quantified extensions of the relevant logic R. In this paper, I show how to extend the Mares-Goldblatt frames to accommodate identity. Simpler frames are provided for two zero-order logics en route to the full logic in order to clarify what is needed for identity and substitution, as opposed to quantification. I close with a comparison of this work with the Fine-Mares models for relevant logics with identity and a discussion of constant and variable domains.

One-variable fragments of intermediate logics over linear frames

A correspondence is established between one-variable fragments of (first-order) intermediate logics defined over a fixed countable linear frame and Gödel modal logics defined over many-valued equivalence relations with values in a closed subset of the real unit interval. It is also shown that each of these logics can be interpreted in the one-variable fragment of the corresponding constant domain intermediate logic, which is equivalent to a Gödel modal logic defined over (crisp) equivalence relations. Although the latter modal logics in general lack the finite model property with respect to their frame semantics, an alternative semantics is defined that has this property and used to establish co-NP-completeness results for the one-variable fragments of the corresponding intermediate logics both with and without constant domains.

Unraveling the Allosteric Communication Mechanisms in T-Cell Receptor-Peptide-Loaded Major Histocompatibility Complex Dynamics Using Molecular Dynamics Simulations: An Approach Based on Dynamic Cross Correlation Maps and Residue Interaction Energy Calculations.

Antigen presentation by major histocompatibility complex (MHC) proteins to T-cell receptors (TCRs) plays a crucial role in triggering the adaptive immune response. Most of our knowledge on TCR-peptide-loaded major histocompatibility complex (pMHC) interaction stemmed from experiments yielding static structures, yet the dynamic aspects of this molecular interaction are equally important to understand the underlying molecular mechanisms and to develop treatment strategies against diseases such as cancer and autoimmune diseases. To this end, computational biophysics studies including all-atom molecular dynamics simulations have provided useful insights; however, we still lack a basic understanding of an overall allosteric mechanism that results in conformational changes in the TCR and subsequent T-cell activation. Previous hydrogen-deuterium exchange and nuclear magnetic resonance studies provided clues regarding these molecular mechanisms, including global rigidification and allosteric effects on the constant domain of TCRs away from the pMHC interaction site. Here, we show that molecular dynamics simulations can be used to identify how this overall rigidification may be related to the allosteric communication within TCRs upon pMHC interaction via essential dynamics and nonbonded residue-residue interaction energy analyses. The residues taking part in the rigidification effect are highlighted with an intricate analysis on residue interaction changes, which lead to a detailed outline of the complex formation event. Our results indicate that residues of the Cβ domain of TCRs show significant differences in their nonbonded interactions upon complex formation. Moreover, the dynamic cross correlations between these residues are also increased, in line with their nonbonded interaction energy changes. Altogether, our approach may be valuable for elucidating intramolecular allosteric changes in the TCR structure upon pMHC interaction in molecular dynamics simulations.

Higher-order immunoglobulin repertoire restrictions in CLL: the illustrative case of stereotyped subsets 2 and 169

Chronic lymphocytic leukemia (CLL) major stereotyped subset 2 (IGHV3-21/IGLV3-21, ∼2.5% of all cases of CLL) is an aggressive disease variant, irrespective of the somatic hypermutation (SHM) status of the clonotypic IGHV gene. Minor stereotyped subset 169 (IGHV3-48/IGLV3-21, ∼0.2% of all cases of CLL) is related to subset 2, as it displays a highly similar variable antigen-binding site. We further explored this relationship through next-generation sequencing and crystallographic analysis of the clonotypic B-cell receptor immunoglobulin. Branching evolution of the predominant clonotype through intraclonal diversification in the context of ongoing SHM was evident in both heavy and light chain genes of both subsets. Molecular similarities between the 2 subsets were highlighted by the finding of shared SHMs within both the heavy and light chain genes in all analyzed cases at either the clonal or subclonal level. Particularly noteworthy in this respect was a ubiquitous SHM at the linker region between the variable and the constant domain of the IGLV3-21 light chains, previously reported as critical for immunoglobulin homotypic interactions underlying cell-autonomous signaling capacity. Notably, crystallographic analysis revealed that the IGLV3-21–bearing CLL subset 169 immunoglobulin retains the same geometry and contact residues for the homotypic intermolecular interaction observed in subset 2, including the SHM at the linker region, and, from a molecular standpoint, belong to a common structural mode of autologous recognition. Collectively, our findings document that stereotyped subsets 2 and 169 are very closely related, displaying shared immunoglobulin features that can be explained only in the context of shared functional selection.

A Fc engineering approach to define functional humoral correlates of immunity against Ebola virus

Protective Ebola virus (EBOV) antibodies have neutralizing activity and induction of antibody constant domain (Fc)-mediated innate immune effector functions. Efforts to enhance Fc effector functionality often focus on maximizing antibody-dependent cellular cytotoxicity, yet distinct combinations of functions could be critical for antibody-mediated protection. As neutralizing antibodies have been cloned from EBOV disease survivors, we sought to identify survivor Fc effector profiles to help guide Fc optimization strategies. Survivors developed a range of functional antibody responses, and we therefore applied a rapid, high-throughput Fc engineering platform to define the most protective profiles. We generated a library of Fc variants with identical antigen-binding fragments (Fabs) from an EBOV neutralizing antibody. Fc variants with antibody-mediated complement deposition and moderate natural killer (NK) cell activity demonstrated complete protective activity in a stringent invivo mouse model. Our findings highlight the importance of specific effector functions in antibody-mediated protection, and the experimental platform presents a generalizable resource for identifying correlates of immunity to guide therapeutic antibody design.

Communication Strategy of Aceh Government to Handling Covid-19 Pandemic in Aceh Province

Corona Virus Disease 2019 or Covid-19 that firstly identified in Wuhan-China rapidly spreaded to 215 countries over the world and has not ended yet. In Indonesia, the first Covid-19 outbreak case was on March 2020 and also has spread rapidly to all provinces. Aceh is noted to be more successful in dealing with the pandemic compared to other provinces. Aceh achieved appreciation from central government and asked to share successful experiences with other provinces. This study is aimed to identify and analyze the Communication Strategy applied by Aceh Government and the challenges faced while handling the Covid-19 Pandemic in Aceh. This descriptive-analytic research uses interview, observation and documentation study techniques as data collection techniques. Furthermore, the data is processed using Constant Comparative Techniques and Domain Analysis in order to obtain relatively valid conclusions. The results of the study concluded that there were 4 (four) Communication Strategies implemented by the Aceh Government, namely: Motivational Communication Strategy, Quick Response Strategy, Leadership Commitment Communication Strategy and Mass Communication Strategy. Meanwhile, there are various challenges faced by the Aceh Government in implementing a communication strategy while handling the Covid-19 pandemic, that is; Regional Accessibility, Socio-Economic, Socio-Cultural and Religious as well as Local Political Dynamics.

Structural Analysis of Anti-Hapten Antibodies to Identify Long-Range Structural Movements Induced by Hapten Binding.

Antibodies are well known for their high specificity that has enabled them to be of significant use in both therapeutic and diagnostic applications. Antibodies can recognize different antigens, including proteins, carbohydrates, peptides, nucleic acids, lipids, and small molecular weight haptens that are abundantly available as hormones, pharmaceuticals, and pesticides. Here we focus on a structural analysis of hapten-antibody couples and identify potential structural movements originating from the hapten binding by comparison with unbound antibody, utilizing 40 crystal structures from the Protein Data Bank. Our analysis reveals three binding surface trends; S1 where a pocket forms to accommodate the hapten, S2 where a pocket is removed when the hapten binds, and S3 where no pockets changes are found. S1 and S2 are expected for induced-fit binding, whereas S3 indicates that a pre-existing population of optimal binding antibody conformation exists. The structural analysis reveals four classifications of structural reorganization, some of which correlate to S2 but not to the other binding surface changes. These observations demonstrate the complexity of the antibody-antigen interaction, where structural changes can be restricted to the binding sites, or extend through the constant domains to propagate structural changes. This highlights the importance of structural analysis to ensure successful and compatible transformation of small antibody fragments at the early discovery stage into full antibodies during the subsequent development stages, where long-range structural changes are required for an Fc effector response.

Bispecific antibodies with Fab-arms featuring exchanged antigen-binding constant domains

Monoclonal antibodies can acquire the property of engagement of a second antigen via fusion methods or modification of their CDR loops, but also by modification of their constant domains, such as in the mAb2 format where a set of mutated amino acid residues in the CH3 domains enables a high-affinity specific interaction with the second antigen. We tested the possibility of introducing multiple binding sites for the second antigen by replacing the Fab CH1/CL domain pair with a pair of antigen-binding CH3 domains in a model scaffold with trastuzumab variable domains and VEGF-binding CH3 domains. Such bispecific molecules were produced in a “Fab-like” format and in a full-length antibody format. Novel constructs were of expected molecular composition using mass spectrometry. They were expressed at a high level in standard laboratory conditions, purified as monomers with Protein A and gel filtration and were of high thermostability. Their high-affinity binding to both target antigens was retained. Finally, the Her2/VEGF binding domain-exchanged bispecific antibody was able to mediate a potentiated surface Her2-internalization effect on the Her2-overexpressing cell line SK-BR-3 due to improved level of cross-linking with the endogenously secreted cytokine. To conclude, bispecific antibodies with Fabs featuring exchanged antigen-binding CH3 domains offer an alternative solution in positioning and valency of antigen binding sites.

Antibody-mediated delivery of T-cell epitopes to antigen-presenting cells induce strong CD4 and CD8 T-cell responses

Targeted delivery of antigen to antigen-presenting cells (APCs) enhances antigen presentation and thus, is a potent strategy for making more efficacious vaccines. This can be achieved by use of antibodies with specificity for endocytic surface molecules expressed on the APC. We aimed to compare two different antibody-antigen fusion modes in their ability to induce T-cell responses; first, exchange of immunoglobulin (Ig) constant domain loops with a T-cell epitope (Troybody), and second, fusion of T-cell epitope or whole antigen to the antibody C-terminus. Although both strategies are well-established, they have not previously been compared using the same system. We found that both antibody-antigen fusion modes led to presentation of the T-cell epitope. The strength of the T-cell responses varied, however, with the most efficient Troybody inducing CD4 T-cell proliferation and cytokine secretion at 10–100-fold lower concentration than the antibodies carrying antigen fused to the C-terminus, both in vitro and after intravenous injection in mice. Furthermore, we exchanged this loop with an MHCI-restricted T-cell epitope, and the resulting antibody enabled efficient cross-presentation to CD8 T cells in vivo. Targeting of antigen to APCs by use of such antibody-antigen fusions is thus an attractive vaccination strategy for increased activation of both CD4 and CD8 peptide-specific T cells.

On Origin and Evolution of the Antibody Molecule.

Simple SummaryLike many other molecules playing vital functions in animals, the antibody molecule possesses a complex structure with distinctive features. The structure of the basic unit, i.e., the immunoglobulin domain of very ancient origin is substantially simple. However, high complexity resides in the types and numbers of the domains composing the whole molecule. The emergence of the antibody molecule during evolution overturned the effectiveness of the organisms’ defense system. The particular organization of the coding genes, the mechanisms generating antibody diversity, and the plasticity of the overall protein structure, attest to an extraordinary successful evolutionary history. Here, we attempt to trace, across the evolutionary scale, the very early origins of the most significant features characterizing the structure of the antibody molecule and of the molecular mechanisms underlying its major role in recognizing an almost unlimited number of pathogens.The vertebrate immune system provides a powerful defense because of the ability to potentially recognize an unlimited number of pathogens. The antibody molecule, also termed immunoglobulin (Ig) is one of the major mediators of the immune response. It is built up from two types of Ig domains: the variable domain, which provides the capability to recognize and bind a potentially infinite range of foreign substances, and the constant domains, which exert the effector functions. In the last 20 years, advances in our understanding of the molecular mechanisms and structural features of antibody in mammals and in a variety of other organisms have uncovered the underlying principles and complexity of this fundamental molecule. One notable evolutionary topic is the origin and evolution of antibody. Many aspects have been clearly stated, but some others remain limited or obscure. By considering a wide range of prokaryotic and eukaryotic organisms through a literature survey about the topic, we have provided an integrated view of the emergence of antibodies in evolution and underlined the very ancient origins.

An expanded genetic code facilitates antibody chemical conjugation involving the lambda light chain

Most of the currently approved therapeutic antibodies are of the immunoglobulin gamma (IgG) κ isotype, leaving a vast opportunity for the use of IgGλ in medical treatments. The incorporation of designer amino acids into antibodies enables efficient and precise manufacturing of antibody chemical conjugates. Useful conjugation sites have been explored in the constant domain of the human κ-light chain (LCκ), which is no more than 38% identical to its LCλ counterpart in amino acid sequence. In the present study, we used an expanded genetic code for site-specifically incorporating Nε-(o-azidobenzyloxycarbonyl)-l-lysine (o-Az-Z-Lys) into the antigen-binding fragment (Fab) of an IgGλ, cixutumumab. Ten sites in the LCλ constant domain were found to support efficient chemical conjugation exploiting the bio-orthogonal azido chemistry. Most of the identified positions are located in regions that differ between the two light chain isotypes, thus being specific to the λ isotype. Finally, o-Az-Z-Lys was incorporated into the Fab fragments of cixutumumab and trastuzumab to chemically combine them; the resulting bispecific Fab-dimers showed a strong antagonistic activity against a cancer cell line. The present results expand the utility of the chemical conjugation method to the whole spectrum of humanized antibodies, including the λ isotype.

Abstract PO020: Anti-membrane-IgM monoclonal antibody, mAb4, inhibits the BCRC, modulating downstream signaling pathways

Abstract The concept that the B-cell Receptor Complex (BCRC) initiates a driver pathway in leukemia-lymphoma has been validated by clinical data. Previously, we reported the generation of a first-in-class antibody, mAb4, to a neo-epitope specific to the BCRC’s membrane IgM (mIgM) subunit, which inhibits cell growth and induces apoptosis (Welt, et al. 2016; US Patent Nos. 9,926,381 and 10,227,419). mAb4 epitope mapping demonstrates that it does not bind to the complementarity-determining regions (CDRs), nor to the micro-clustering site of mIgM that is believed to play a role in BCRC signal amplification. To characterize the mechanism of action of this antibody, we are investigating the inhibition of tyrosine kinase activity of several BCRC downstream signaling pathways. We hypothesize that mAb4 inhibits signal transduction of multiple BCRC-dependent downstream pathways. We are currently evaluating kinase activity across three sites on BTK (Ser180, Tyr223, and Tyr551). Phosphorylation of other common clinical targets, including Syk, Lyn, BCL2, and PI3K, will also be studied. To do this, we are using antibody reagents against phosphorylation sites to assess activation or inhibition of these kinases by mAb4 compared to control mAb-treated IgM-expressing B-cells, and, where available, cells treated with drugs against these targets. In preliminary ELISA analyses we find that mAb4 and a BTK inhibitor drug, inhibit BTK phosphorylation at Tyr223 and Tyr551, but not Ser180, across all anti-phospho-tyrosine/serine antibody reagents tested (Ser180: clones 3i5 and 3D3; Tyr223: clones A16128B and 720101; and Tyr 551: clones A16064A, 797837, and M4G3LN). In vitro, mAb4-treated leukemia and lymphoma cells undergo growth inhibition and apoptosis while the BTK inhibitor drug mediates only cell growth inhibition, as reported by others. Here we show that mAb4 inhibits phosphorylation of BTK tyrosine upon binding to a determinant that spans the extracellular proximal domain and the constant domain 4 (μC4) regions of mIgM. While BTK inhibitor drugs induce a cell growth inhibitory effect, we find that mAb4 mediates BCRC internalization, and in low-density cultures, cell growth inhibition, anti-clonogenic activity, and apoptosis, and may therefore represent a next generation of BTK inhibitor. Though mAb4 does not interact with the mIgM’s CDRs nor the micro-clustering site on μC4, it modulates transmembrane signal transduction. We predict that continued analyses of additional downstream kinases (Syk, Lyn, BCL2, and PI3K) will similarly show an effect following BCRC inhibition by mAb4. These finding indicate that mAb4 may target mIgM at a critical structural site, which interrupts specific ligand binding signal transduction. Furthermore, mAb4 is highly specific to IgM-expressing B-cells, thus this novel approach to modulating B-cell signaling to induce a cytotoxic response may represent an exciting new direction in clinical development. Citation Format: Rachel S. Welt, Jonathan A. Welt, Virginia Raymond, David Kostyal, Sydney Welt. Anti-membrane-IgM monoclonal antibody, mAb4, inhibits the BCRC, modulating downstream signaling pathways [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO020.

IgG1 Subclass Restriction and Biochemical Peculiarities of Monoclonal Immunoglobulins in Scleromyxedema.

Scleromyxedema (SME) is a rare mucinosis associated with monoclonal gammopathy. Several biochemical peculiarities of monoclonal immunoglobulins (Ig) in SME patients were reported in case reports or short series, such as IgGλ over-representation, cationic migration, and partial deletion. Monoclonal immunoglobulins (Ig) from the serum of 12 consecutive patients diagnosed with scleromyxedema (SME) were analyzed using electrophoretic and immunoblotting techniques. All monoclonal Ig from 12 SME were of IgG1 subclass, with an overrepresentation of the lambda-type light chain and a cationic mobility on standard zone electrophoresis, as compared with 21 cases of monoclonal gammopathy of undetermined significance (MGUS) of IgG1 subclass. Reactivity with specific monoclonal antibodies demonstrated no evident deletion of the heavy chain constant domains, which was also confirmed by analysis of Ig heavy chain molecular weight on a purified monoclonal component from one case. Significant isotype restriction of both heavy and light chains, and peculiar biochemical properties suggest that monoclonal Ig might be involved in pathophysiological events of SME.