Fc Portion Of IgM Research Articles

Physiological and Pathophysiological Roles of IgM Fc Receptor (FcµR) Isoforms

IgM is the first antibody to emerge during phylogeny, ontogeny, and immune responses and serves as a first line of defense. Effector proteins interacting with the Fc portion of IgM, such as complement and its receptors, have been extensively studied for their functions. IgM Fc receptor (FcµR), identified in 2009, is the newest member of the FcR family and is intriguingly expressed by lymphocytes only, suggesting the existence of distinct functions as compared to the FcRs for switched Ig isotypes, which are expressed by various immune and non-hematopoietic cells as central mediators of antibody-triggered responses by coupling the adaptive and innate immune responses. Results from FcµR-deficient mice suggest a regulatory function of FcµR in B cell tolerance, as evidenced by their propensity to produce autoantibodies of both IgM and IgG isotypes. In this article, we discuss conflicting views about the cellular distribution and potential functions of FcµR. The signaling function of the Ig-tail tyrosine-like motif in the FcµR cytoplasmic domain is now formally shown by substitutional experiments with the IgG2 B cell receptor. The potential adaptor protein associating with FcµR and the potential cleavage of its C-terminal cytoplasmic tail after IgM binding are still enigmatic. Critical amino acid residues in the Ig-like domain of FcµR for interacting with the IgM Cµ4 domain and the mode of interaction are now defined by crystallographic and cryo-electron microscopic analyses. Some discrepancies on these interactions are discussed. Finally, elevated levels of a soluble FcµR isoform in serum samples are described as the consequence of persistent B cell receptor stimulation, as seen in chronic lymphocytic leukemia and probably in antibody-mediated autoimmune disorders.

The long elusive IgM Fc receptor, FcμR.

IgM exists as both a monomer on the surface of B cells and a pentamer secreted by plasma cells. Both pre-immune "natural" and antigen-induced "immune" IgM antibodies are important for protective immunity and for immune regulation of autoimmune processes by recognizing pathogens and self-antigens. Effector proteins interacting with the Fc portion of IgM, such as complement and complement receptors, have thus far been proposed but fail to fully account for the IgM-mediated protection and regulation. A major reason for this deficit in our understanding of IgM function seems to be lack of data on a long elusive Fc receptor for IgM (FcμR). We have recently identified a bona fide FcμR in both humans and mice. In this article we briefly review what we have learned so far about FcμR.

The old but new IgM Fc receptor (FcμR).

IgM is the first Ig isotype to appear during phylogeny, ontogeny and the immune response. The importance of both pre-immune "natural" and antigen-induced "immune" IgM antibodies in immune responses to pathogens and self-antigens has been established by studies of mutant mice deficient in IgM secretion. Effector proteins interacting with the Fc portion of IgM, such as complement and complement receptors, have thus far been proposed, but fail to fully account for the IgM-mediated immune protection and regulation of immune responses. Particularly, the role of the Fc receptor for IgM (FcμR) in such effector functions has not been explored until recently. We have identified an authentic FcμR in humans using a functional cloning strategy and subsequently in mice by RT-PCR and describe here its salient features and the immunological consequences of FcμR deficiency in mice. Since the FcμR we cloned was identical to Toso or Fas inhibitory molecule 3 (FAIM3), there have been spirited debates regarding the real function of FcμR/Toso/FAIM3 and we will also comment on this topic.

Fcμ-Receptor (FcμR; TOSO/FAIM3) Mediated Internalization of Antibody-Drug Conjugates: A Novel Approach to Selectively Target Chronic Lymphocytic Leukemia Cells

Abstract 734The therapeutic principle of antibody-drug conjugates (ADCs) in cancer relies on an antibody to deliver a cytotoxic agent into malignant cells that express a tumor-associated antigen. Recent progress in the field has shown that the use of such conjugates can increase drug activity and reduce cytotoxicity through selective targeting. Recently, TOSO/FAIM3 was identified as the long sought FcR for IgM (FcμR). FcμR is a transmembrane protein expressed on CD19+ B cells, on some CD4+/CD8+ T cells, and weakly on CD56+/CD3− NK cells. We and others have shown that FcμR is consistently overexpressed on chronic lymphocytic leukemia (CLL) cells compared to normal B-cells. In addition, we detected FcμR expression in the mantle cell lymphoma cell line Mino. Using immunofluorescence staining, we found that FcμR can rapidly internalize IgM and transport it through the endocytic pathway to the lysosome. Interestingly, aggregation of FcμR with IgM lead to rapid internalization (>80% internalized within 5 minutes, n=5) whereas mAb bound FcμR was not internalized (n=3). Because of its restricted expression on CLL cells, its cell surface localization and its rapid internalization, FcμR represents an excellent target to deliver a cytotoxic drug into CLL cells using an ADC. To make use of the rapid internalization of FcμR when bound by the Fc-portion of IgM, we engineered a protein scaffold derived from the CH3-CH4 IgM constant region, and inserted a C-terminal selenocysteine, that allows site specific and covalent conjugation of drugs to the protein scaffold. Using gel filtration, we found that purified CH3-CH4 exists mostly as a multimer including pentameric and hexameric forms, similar to native IgM. First, we verified that the CH3-CH4 protein scaffold also binds FcμR, is internalized and addressed to the lysosomes. Next, we conjugated the CH3-CH4 protein scaffold to cemadotin, a synthetic anti-mitotic agent that inhibits tubulin polymerization. This CH3-CH4-cemadotin conjugate was designed to specifically release the drug inside the cell by inserting a peptide linker that can by cleaved by cathepsin B in the lysosomal compartment. In vitro cell killing showed that this CH3-CH4-cemadotin conjugate potently and selectively killed FcμR expressing cells (IC(50) <0.16 μM) while it was about 200-fold less efficient for cells with undetectable FcμR (IC(50) >31 μM). Control experiments showed equal, i.e. non-selective cytotoxicity of free cemadotin against FcμR expressing and non-expressing cells and absence of cell killing with the unconjugated CH3-CH4 protein scaffold. Taken together, these data identify FcμR as a promising therapeutic target in CLL and possibly select other malignanices. IgM-derived scaffolds constitute ideal carriers for drugs or toxins as they are rapidly internalized. In addition, trafficking of IgM scaffolds to the lysosome provides an additional layer of selectivity as a precursor inactive drug can be activated through lysosomal peptidases. Ongoing efforts aim to test modifications of the scaffold and evaluate additional cytotoxic drugs. Furthermore, we are scaling up production of the lead scaffold drug conjugate in order to test its efficacy in xenograft mouse models.This work was supported by the Intramural Research Program of the National, Heart, Lung and Blood Institute. Disclosures:No relevant conflicts of interest to declare.

Fragmentation of Immunoglobulin M

Fragmentation of IgM antibodies may be necessary because of the large molecular weight of the native molecule (900 kDa). IgMs fragments resemble IgG in size and structure, but they may have a decreased binding affinity. The Fc portion of IgM can have powerful biological effector functions such as complement activation. Because some T cells have receptors for IgM, it may be desirable to produce fragments of IgM for both cytotoxicity studies and for in vivo use. A protocol is presented for digestion of IgM with pepsin to produce F(ab')(2)micro. The fragment can be reduced to produce the monovalent F(ab')u, if desired. IgM can also be reduced and alkylated in a single step, as described, using cysteine to produce IgMs, the bivalent monomer or subunit of IgM.

Characterization of an IgM Fc-binding receptor on human T cells.

Abstract The existence of an IgM receptor on human T cells has been suggested by T cell rosetting with IgM-coated erythrocytes. In this study we used immunofluorocytometry to demonstrate that 1) after short-term culture, a majority of the T cells can bind IgM at easily detectable levels, 2) human and mouse IgM preparations bind to human T cells but other Ig isotypes do not, 3) the IgM binding is saturable and inhibitable only by Ig of IgM isotype and 4) the Fc portion of IgM is involved in the binding to a protease-sensitive cell surface protein. Biochemical analysis of the T cell receptor for IgM reveals a cell surface protein of 60 kDa in comparison with the 58 kDa Fc mu receptors (Fc mu R) on B-lineage cells. Although Fc mu R expression is up-regulated after B cell activation, the reverse is true after T cell activation. In addition, the T cell Fc mu R is relatively resistant to phospholipase C treatment. These results indicate that T- and B-lineage cells express Fc mu R with different biologic characteristics.

Leucocyte chemotactic factor generated by tryptic digestion of human IgM

Extensively purified human IgM protein was digested with trypsin. One of the resultant split-fragments, was found to have a mol. wt of about 12,000 and strong chemotactic activity on rabbit peritoneal neutrophils. This chemotactic substance proved to be derived from the Fc portion of IgM, and not from (Fab) 2μ or Fabμ. (Fab) 2μ, Fabμ, (Fc) 5μ and unchanged IgM, which are produced from tryptic digestion of IgM, were shown to be devoid of chemotactic activity.