Human Fc Receptor Research Articles

A PrimatizedMAb to Human CD4 Causes Receptor Modulation, without Marked Reduction in CD4+T Cells in Chimpanzees:In Vitroandin VivoCharacterization of a MAb (IDEC-CE9.1) to Human CD4

A Primatized anti-CD4 monoclonal antibody (MAb), CE9.1, with V-domain from cynomolgus macaque (showing 92% homology with human consensus sequence V-domains), and a human IgG1 constant region, was characterizedin vitroandin vivoin chimpanzees. This MAb binds human CD4 withKdof 1.0 nMand was also able to bind to human IgG Fc receptors (FcγR). However, despite being of the IgG1 subclass, CE9.1 did not bind to complement component C1q, nor did it mediate complement-dependent cytotoxicity. Examination of T cells from a number of species showed restricted reactivity for CE9.1, recognizing only human and chimpanzee CD4. In both human and chimpanzee MLRs, it had an IC50of about 10.0 ng/mL. Therefore, a chimpanzeein vivomodel was used to characterize CE9.1. CE9.1 caused transient decrease in the number of lymphocytes bearing the CD4 receptor starting at doses of 0.3 mg/kg in anin vivodose ranging study in one chimpanzee. This effect was reversed within approximately 7 days. In a multiple high-dose study in which 10.0 mg/kg of CE9.1 was administered at intervals of 1–3 months, there was a dramatic loss of CD4 marker with a reciprocal increase in the number of CD3+CD8−CD4−cells. The CD4 receptor was totally undetectable on these lymphocytes for 1–2 weeks, with a gradual, but complete, reversal within 4 weeks. We interpret these observations as receptor modulation because, although there was apparent loss of CD4+lymphocytes, an equivalent number of CD3+CD8−T lymphocytes were present in circulation in all four chimpanzees treated with 10.0 mg/kg CE9.1. Even at this high dose, only limited reduction of CD4+T lymphocytes was observed in these animals. These observations are in sharp contrast to what has been reported in rodents or in human clinical studies using other IgG1 mAbs to human CD4. CD8 counts, although variable, remained unaffected by CE9.1 treatment. No adverse events were observed following administration of CE9.1 to chimpanzees, and there was no detectable host immune responses to the Primatized MAb.

Multiple transcripts of human IgA Fc receptor CD89 in neutrophils, eosinophils and the monocyte-like cell line THP-1.

Multiple transcripts of human IgA Fc receptor CD89 in neutrophils, eosinophils and the monocyte-like cell line THP-1.

Human cytomegalovirus induces an Fc gamma receptor (Fc gammaR) in endothelial cells and fibroblasts that is distinct from the human cellular Fc gammaRs.

The expression of a trypsin-sensitive receptor for the Fc portion of IgG (Fc gammaR) was demonstrated by flow cytometry on the surface of human umbilical vein endothelial cells and fibroblasts infected with human cytomegalovirus (CMV). Double-labeling experiments showed strong expression of the CMV Fc gammaR in a perinuclear region of infected cells but not in bystander uninfected cells. The CMV Fc gammaR did not react with a panel of murine monoclonal antibodies directed against the known human IgG Fc receptors, Fc gammaRI, Fc gammaRII, and Fc gammaRIII. The cytoplasmic form but not the cell surface form of CMV Fc gammaR bound murine IgG3 moderately and murine IgG1 more weakly, while both forms bound rabbit IgG almost as strongly as human IgG. The function of CMV Fc gammaR is unclear, but it may allow CMV to evade host antibody responses. However, the binding of immune complexes to infected endothelium might also contribute to immunopathology.

The use of chimeric human Fc(epsilon) receptor I to redirect cytotoxic T lymphocytes to tumors.

Chimeric receptors that redirect effector cell function to tumor cells or virus-infected cells have received much attention. Given the high affinity of Fc(epsilon)RI for immunoglobulin E (IgE) and low serum IgE levels, redirection of effector cells using Fc(epsilon) receptor may provide a novel, versatile, and effective anti-tumor strategy. We have used a mouse perforin 5'-promoter to express a single-chain human Fc(epsilon) receptor in the mouse cytotoxic T lymphocyte cell line, CTLL-R8. Upon ligation of the chimeric Fc(epsilon) receptors by IgE, a signal for effector function is transmitted via the intracellular domain of CD3zeta. Selection in G418-containing medium produced CTLLR8 transfectant clones that: (1) expressed chimeric Fc(epsilon) receptor as determined by flow cytometry; (2) bound human IgE antibodies with high affinity as determined by Scatchard analysis; (3) specifically rosetted IgE-coated SRBC; (4) lysed target cells in IgE-mediated ADCC and reverse ADCC assays; and (5) retarded tumor growth in a Winn assay. Therefore these chimeric Fc(epsilon) receptors can effectively redirect cytotoxicity to tumor cells. Future efforts will assess the versatility and efficacy of these IgE-binding chimeric receptors to redirect killer cell function in animal tumor models.

Mouse mast cell gp49B1 contains two immunoreceptor tyrosine-based inhibition motifs and suppresses mast cell activation when coligated with the high-affinity Fc receptor for IgE.

Mouse mast cells express gp49B1, a cell-surface member of the Ig superfamily encoded by the gp49B gene. We now report that by ALIGN comparison of the amino acid sequence of gp49B1 with numerous receptors of the Ig superfamily, a newly recognized family has been established that includes gp49B1, the human myeloid cell Fc receptor for IgA, the bovine myeloid cell Fc receptor for IgG2, and the human killer cell inhibitory receptors expressed on natural killer cells and T lymphocyte subsets. Furthermore, the cytoplasmic domain of gp49B1 contains two immunoreceptor tyrosine-based inhibition motifs that are also present in killer cell inhibitory receptors; these motifs downregulate natural killer cell and T-cell activation signals that lead to cytotoxic activity. As assessed by flow cytometry with transfectants that express either gp49B1 or gp49A, which are 89% identical in the amino acid sequences of their extracellular domains, mAb B23.1 was shown to recognize only gp49B1. Coligation of mAb B23.1 bound to gp49B1 and IgE fixed to the high-affinity Fc receptor for IgE on the surface of mouse bone marrow-derived mast cells inhibited exocytosis in a dose-related manner, as defined by the release of the secretory granule constituent beta-hexosaminidase, as well as the generation of the membrane-derived lipid mediator, leukotriene C4. Thus, gp49B1 is an immunoreceptor tyrosine-based inhibition motif-containing integral cell-surface protein that downregulates the high-affinity Fc receptor for IgE-mediated release of proinflammatory mediators from mast cells. Our findings establish a novel counterregulatory transmembrane pathway by which mast cell activation can be inhibited.

Alternative splicing of the human IgA Fc receptor CD89 in neutrophils and eosinophils.

Receptors for the Fc portion of IgA (Fc alpha R) trigger important immunological elimination processes against IgA-coated targets. Investigation of human Fc alpha R (CD89) transcripts in neutrophils, eosinophils and a monocyte-like cell line, THP-1, with the use of reverse transcriptase PCR, Northern blotting and RNase protection analysis, has provided evidence in these cell types for at least two distinct transcripts generated by alternative splicing. The cDNAs derived from the two major transcripts of both neutrophils and eosinophils have been cloned and sequenced. For both cell types, the larger clone represents the previously described full-length receptor, whereas the second, shorter, splice variant lacks the entire second, membrane-proximal, Ig-like domain. Stable CHO-K1 transfectants have been obtained for both full-length and truncated variant neutrophil receptors. Whereas the full-length receptor is recognized by a panel of five anti-Fc alpha R monoclonal antibodies (mAbs), the shorter variant is bound weakly by only two of the antibodies, suggesting that the epitopes recognized by the majority of the mAbs lie at least in part in the second Ig-like domain of Fc alpha R. Both full-length and splice variant forms of the receptor bind secretory IgA, but the weak binding to serum IgA seen with the full-length receptor is not evident with the shorter variant. Alternative splicing might therefore serve as a means of diversifying Fc alpha R structure and function.

Characterization of a series of isotype switch variants of a new CD20 monoclonal antibody.

A series of heavy chain switch variants has been isolated from a new B cell-specific monoclonal antibody belonging in the CD20 cluster. The antibodies NKI-B20/1, NKI-B20/2b, and NKI-B20/2a (of isotype IgG1, IgG2b, and IgG2a, respectively) have been used to study the influence of isotype and of the target antigen on the capacity to mediate cytotoxicity with a number of effector mechanisms. Unlike many mouse MAbs, NKI-B20/2b and NKI-B20/2a were cytolytic with human complement on human target cells that did not express the complement regulatory factor HRF20. All 3 isotypes of NKI-B20 mediated antibody-dependent cell-mediated cytotoxicity (ADCC) with rIL-2-activated NK cells from mouse spleen. Here the antigen density seemed the most important factor in determining the level of cell kill. With mouse peritoneal macrophages as effector cells again all 3 isotypes of NKI-B20 mediated cytotoxicity. For the IgG1 and IgG2b variants of NKI-B20 this is at variance to what has been reported for MAbs of other specificities. Despite the high activity with murine effector cells none of the NKI-B20 MAbs mediated ADCC with human peripheral blood NK cells, with or without stimulation with rIL-2, due to the lack of interaction of the murine MAbs with the human Fc receptor. The CD20 antigen appears to be a good target antigen for various forms of cytotoxicity, to which its relatively high antigenic density, its resistance to antibody-induced modulation, and its unusual structure all contribute.

The role of the CD40 antigen on malignant B cells.

An increasing amount of literature has been published concerning the interaction of the CD40 antigen and its ligand with regard to normal B cell ontogeny. In this review, an overview of the CD40 antigen and the CD40 ligand is given, focussing on their possible role in B cell malignancies. Data on the expression of the CD40 antigen on various B cell malignancies (acute and chronic leukemias, non-Hodgkin's lymphoma and multiple myeloma) are presented. The recently developed novel culture "CD40 system" is described. This system is a powerful tool used to culture normal B cells, but also most malignant B cells. We demonstrate in addition a more prominent role of the human Fc receptor presenting murine fibroblasts in the "CD40 system", especially in relation to cultured plasma cells. Finally, some important applications of the "CD40 system" are also summarized.

An IgG3-IL2 fusion protein activates complement, binds Fc gamma RI, generates LAK activity and shows enhanced binding to the high affinity IL-2R.

The therapeutic value of Interleukin 2 (IL-2) is limited by its short half life and systemic toxicity. One approach to overcoming these problems is to fuse this protein to an antibody, a protein with a long half life and the ability to target a unique antigen within the body. To examine the biochemical properties of such a molecule a fusion protein was constructed linking the N-terminus of human IL-2 to the C-terminus of IgG3. A similar fusion between IgG1 and IL-2 has previously been shown to bind antigen, generate antibody-dependent cellular cytotoxicity (ADCC) and stimulate T cell proliferation and cytotoxicity. We now extend these studies and show that the fusion protein, termed IgG3-IL2, is appropriately N-glycosylated within the IgG3 CH2 domain, binds the human high affinity Fc receptor (Fc gamma RI) with an affinity slightly lower than that of IgG3, and is able to activate complement via the classical pathway to lyse antigen coated sheep red blood cells (SRBC). When used to stimulate the proliferation of the IL-2 dependent cell line CTLL-2, IgG3-IL2 has a specific activity slightly lower than that of human recombinant IL-2 (hrIL-2). In marked contrast, when comparable unit concentrations, as defined by the standard CTLL-2 proliferation assay, are used to stimulate human peripheral blood lymphocytes (PBL), IgG3-IL2 generates significantly greater lymphokine activated killer (LAK) cell cytotoxicity than does hrIL-2. Competition studies show that IgG3-IL2 binds the intermediate affinity form of the IL-2 receptor (IL-2R), consisting of the beta and gamma subunits, with an affinity slightly less than that of hrIL-2. In contrast, IgG3-IL2 shows a greater affinity than hrIL-2 for the high affinity IL-2R, consisting of alpha, beta and gamma subunits. Our studies show that the IgG3-IL2 fusion protein possesses a combination of the biological properties of IgG3 and IL-2 including antigen binding, complement activation, Fc gamma RI binding, IL-2R binding and stimulation of both proliferation and LAK activity. This combination of activities may allow IgG3-IL2 to target humoral and cell-mediated immune activation to the site of an antigen of interest or target an antigen to IL-2R bearing cells or organs.

Temperature-dependent binding of human IgG 1 to a human high affinity Fc receptor

We have measured the kinetics of binding and unbinding of human IgG 1 to a human high affinity Fc receptor (FcγI) at several temperatures. The association rate constant ( k f ) and the dissociation rate ( k r ) of this complex was determined with 125I-IgG 1 monomer and FcγI on U937 cells. At 37°C, k f = 2.7 × 10 5 M −1 s −1 and k r = 4.5 × 10 −4 s −1. Both rates decreased with decreasing temperature. However, the equilibrium association constant, K a , increased with decreasing temperature. From the temperature dependence of K a we determined that the binding of IgG 1 to FcγI is driven largely by enthalpic forces and that a small but positive entropic contribution to free energy leads to a tighter complex at lower temperature.

Enhanced metabolism of LDL aggregates mediated by specific human monocyte IgG Fc receptors.

Macrophage-derived foam cells are important constituents of atheromatous lesions. In addition to the scavenger receptor pathway, uptake of immune complexed lipoproteins through IgG Fc receptors (Fc gamma receptors) represents an additional pathway of macrophage foam cell development that may be important during atherogenesis. The importance of this mechanism is suggested by studies showing that the titer of autoantibodies to modified lipoproteins correlated with the extent of occlusive disease in patients, and that those antibodies exist in human lesions. Human mononuclear phagocytes possess three structurally and functionally distinct classes of Fc gamma receptors, each of which could be associated with a unique pathway of lipoprotein metabolism. In order to determine whether uptake of an acute lipid load through each type of Fc gamma receptor was associated with foam cell development, we used bispecific antibodies consisting of anti-LDL monoclonal antibodies conjugated to anti-Fc gamma receptor monoclonal antibodies to study the effects of targeting LDL aggregates to each specific type of Fc gamma receptor on freshly isolated adherent human monocytes. Relative to appropriate controls, LDL degradation, cellular sterol mass, and foam cell development of monocytes were enhanced by targeting LDL aggregates to Fc gamma RI or Fc gamma RII, and this was accompanied by an apparent impairment of LDL degradation. Uptake was specific to the Fc gamma receptors and was not influenced by the presence of scavenger receptor ligands. Thus, with the bispecific approach, the functions of each class of Fc gamma receptor can be studied on an individual basis with respect to several aspects of cellular cholesterol metabolism. This will be critical for determining which of these receptors are potentially most important in the clearance of lipoprotein immune complexes during atherogenesis.

Interaction of human monocyte Fc gamma receptors with rat IgG2b. A new indicator for the Fc gamma RIIa (R-H131) polymorphism.

Rat mAbs receive considerable interest for immunologic intervention in man. The rat IgG2b isotype has previously been found to be optimally active both in vivo and in vitro. We found that both a rat IgG2b CD3 mAb and a monovalent hybrid rat IgG2b-mouse IgG1 bispecific Ab triggered T cell activation in PBMC. Inhibition analyses with mAb blocking different human IgG Fc receptors (Fc gamma R) showed a dimorphic pattern. In donors expressing an Fc gamma RIIa-R/R131 allotype (previously defined on the basis of interaction with mouse (m) IgG1 as "high responder") anti-Fc gamma RI mAb 197 inhibited rat IgG2b induced T cell mitogenesis almost completely. In Fc gamma RIIa-H/H131 ("low responder" allotype) donors, however, both anti-Fc gamma RI mAb 197 and anti-Fc gamma RII mAb IV.3 were essential for optimal inhibition of mitogenesis. T cell proliferation experiments performed with the use of Fc gamma R-transfected fibroblasts as accessory cells showed the high affinity Fc gamma RIa (CD64) to interact with both rat IgG2b and rat IgG2b-mlgG1 hybrid CD3 mAb. The use of the two types of Fc gamma RIIa (CD32)-transfectants instead showed rat IgG2b CD3 mAb to interact solely with the IIa-H/H131 allotype. Interestingly, rat IgG2b-mlgG1 hybrid mAb did not interact effectively with this low affinity Fc gamma R. This suggests a requirement for only one rat IgG2b H chain for Fc gamma RIa-mediated binding, whereas two identical H chains seem to be necessary for proper interaction with Fc gamma RIIa. Ab-sensitized RBC-rosette experiments performed with the use of a rat IgG2b anti-NIP mAb confirmed the interaction pattern observed with rat CD3 mAb, supporting the phenomena to be isotype-, and not mAb-, dependent. These analyses point to a unique reactivity pattern for rat IgG2b Abs, interacting both with the high affinity Fc gamma RIa in all donors and Fc gamma RIIa of individuals expressing the IIa-H131 allotype.

The Human Low Affinity Immunoglobulin G Fc Receptor III-A and III-B Genes: MOLECULAR CHARACTERIZATION OF THE PROMOTER REGIONS

The human Fc receptor with low affinity for IgG (Fc gamma RIII, CD16) is encoded by two nearly identical genes, Fc gamma RIII-A and Fc gamma RIII-B, resulting in tissue-specific expression of alternative membrane-anchored isoforms. The transmembrane CD16 receptor forms a heteromeric structure with the Fc epsilon RI (gamma) and/or CD3 (zeta) subunits on the surface of activated monocytes/macrophages, NK cells, and a subset of T cells. The expression of the glycosylphosphatidylinositol-anchored CD16 isoform encoded by the Fc gamma RIII-B gene is restricted to polymorphonuclear leukocytes and can be induced by Me2SO differentiation of HL60 cells. We have isolated and sequenced genomic clones of the human Fc gamma RIII-A and Fc gamma RIII-B genes, located their transcription initiation sites, identified a different organization of their 5' regions, and demonstrated four distinct classes of Fc gamma RIII-A transcripts (a1-a4) compared with a single class of Fc gamma RIII-Bb1 transcripts. Both CD16 promoters (positions -198 to -10) lack the classical "TATA" positioning consensus sequence but confer transcriptional activity when coupled to the human lysozyme enhancer. Both promoters also display different tissue-specific transcriptional activities reflecting the expected gene expression of Fc gamma RIII-A and Fc gamma RIII-B in NK cells versus polymorphonuclear leukocytes. Within the -198/-10 fragments, the sequences of the two CD16 genes have been identified to differ in 10 positions. It is suggested that these nucleotide differences might contribute to cell type-specific transcription of Fc gamma RIII genes.

Identification of novel CD23 transcripts on human T and B lymphocytes and eosinophil cell line

The main aim of the present studies was to investigate the structure of the human low-affinity IgE Fc receptor (CD23) present on T and B lymphoid cells and eosinophil cell line. A novel finding in these studies has been the detection and sequence analysis of CD23 transcripts in human T lymphocytes. These studies have established that some of the human T-cell populations analyzed express CD23 mRNA and that its structure is quite similar to that previously described for human B lymphocytes. A second major finding in these studies is that some human T- and B-cell lines and eosinophil cell line contain multiple forms of CD23 transcripts. These appear to be generated via alternative splicing, resulting in transcripts that may encode a truncated, possibly secretory form of CD23. These findings in human T and B lymphocytes and eosinophils provide new information about the structure of lymphocyte CD23 and suggest that alternative processing of transcripts generates CD23 mRNA that encodes CD23 isoforms. These studies are the first experimental evidence showing that CD23 isoforms may occur in the human and are the first direct evidence for production of CD23 by human T lymphocytes. In addition, these studies provide the first experimental evidence that T and B lymphocytes express CD23 transcripts lacking exon 3-encoded sequences, raising the possibility that a secretory form of CD23 may be synthesized by human T and B lymphocytes, and eosinophils.

Receptor-mediated endocytosis is sensitive to antibodies against the uncoating ATPase (hsc70).

We have investigated the functional role of the coated vesicle-uncoating ATPase (UA), a cognate heat shock protein (hsc70), in receptor-mediated endocytosis. A monoclonal antibody against bovine brain UA/hsc70 was generated that recognizes a 26 kDa proteolytic fragment harbouring the putative clathrin-binding site. In vitro, this antibody blocked the UA/hsc70-mediated release of clathrin from isolated coated vesicles (CVs). Upon microinjection into tissue culture cells, it specifically inhibited the heat shock-induced nuclear migration of UA/hsc70. This antibody also interfered with endocytosis of ligand-receptor complexes in injected cells. Two different systems were studied: the uptake of aggregated human IgG by BHK cells transfected with a human Fc receptor (FcRII), and the internalization of LDL by human fibroblasts. Injection of the monoclonal antibody in concentrations yielding approximately equal molar ratios of antibody to enzyme resulted in a reduction of endocytosis to 20-30% of control values, as seen by conventional light and confocal laser scanning microscopy, and by electron microscopy. In the transfected BHK cells, the endocytosed ligand remained associated with the labeling for clathrin and was not delivered to the endosomal compartment within the period expected from control serum- or non-injected cells. Thin sections revealed an accumulation of coated structures in the antibody-injected cells as compared to controls. Thus, our data show that UA is essential for normal receptor-mediated endocytosis, and is presumably involved in the uncoating of CVs preceding their fusion with endosomes.

Heterogeneity of Human IgG Fc Receptors

The complex family of human IgG Fc receptors show a wide cellular distribution and a strong functional heterogeneity. To date, eight different genes that are transcribed into at least 12 different mRNAs have been recognized. Although corresponding products have been identified for only some of the transcripts, in vivo at least six different FcγR isoforms are shown to be present on the surface of all kinds of leukocytes. Upon interaction with immunoglobulin, FcγR mediate a variety of biological responses such as phagocytosis, endocytosis, release of inflammatory mediators, and antibody-dependent cellular cytotoxicity. One of the main questions in FcγR research is determining the specific relationship between a particular FcγR isoform and a particular effector mechanism. The availability of the cDNA and genomic clones corresponding to the different isoforms, combined with different gene transfer systems in vitro and in vivo and the recently developed procedure of gene knockout by homologous recombination, allows the generation of stably transfected cell lines and transgenic animals that express one particular FcγR isoform. These model systems enable us to study the function of a particular FcγR isoform in the context of a particular cell type in vitro and in the context of the intact immune system in vivo.

Methods for Studying Fc Receptor Expression

In the past two decades, rapid progress has been made in the field of Fc receptors. One of the reasons for this development has been the advances and improvements in the methodologies used to detect and analyze Fc receptors. This review describes the methods used to detect the murine and human Fc receptors of the various immunoglobulin isotypes at the RNA, protein, and functional level. We have given special attention to the binding assays used to detect Fc receptors through immunoglobulins or anti-Fc receptor monoclonal antibodies as probes. The various Fc receptor types exhibit a marked heterogeneity and we present a list of reagents that can differentiate between the different Fc receptor forms. These methods can detect Fc receptors on the cell membrane, as well as the cytoplasmic and secreted forms. Recent advances in the use of PCR and RNA/DNA analyses to study Fc receptor detection, expression, and function are also reviewed. The advantages and drawbacks of the various experimental approaches are presented. In addition, an appendix provides detailed protocols for the detection of Fc receptors by confocal immunofluorescence microscopy, rosetting, cytofluorometry, immunoprecipitation, and PCR

CAMPATH-1 Monoclonal Antibodies in Bone Marrow Transplantation

CAMPATH-1 (CDw52) antibodies recognize a very small lipid-anchored glycoprotein that is expressed on the surface of human lymphocytes. They are remarkably lytic with human complement. In addition, CAMPATH-1G (rat IgG2b) and CAMPATH-1H (human IgG1) bind to human Fc receptors and are very effective for cell lysis in vivo. CAMPATH-1M (rat IgM) and CAMPATH-1G have been used to control GVHD and graft rejection in bone marrow transplantation by depletion of the T cells of the donor and recipient. Depletion of donor T cells alone gave excellent control of GVHD but up to 20% of the patients transplanted from HLA-matched siblings, and 51% of those transplanted from nonsibling donors, experienced graft failure caused by immunological rejection. Graft rejection could be partly overcome by additional immunosuppression either with CsA or total lymphoid irradiation (TLI). More effective was the use of CAMPATH-1G in vivo to deplete residual host lymphocytes. Preliminary results from current protocols of antibody depletion give two year actuarial leukemia-free survival as good as or better than similar studies with conventional GVHD prophylaxis, as well as a decreased morbidity from chronic GVHD, although engraftment was delayed by about 5 days. We propose that prophylactic T cell depletion with CAMPATH-1 antibodies is a simple and valid alternative to drug-based immunosuppression that may be particularly applicable to patients with acute leukemia or nonmalignant diseases transplanted from HLA-matched siblings as well as any patients transplanted from unrelated donors. Future developments of antibody-based immunosuppression may allow the extension of marrow transplantation for tolerance induction to organ transplants or in autoimmune diseases.

Clinical implications of the polymorphic interaction of murine IgG2b and IgG1 with human Fc receptors

When monoclonal antibodies (mAbs) became available as an option for immunotherapy, it soon became evident that, apart from specificity and affinity, the isotype of the mAb was important for its effectiveness. Different isotypes of murine (m) immunoglobulin exhibited significant differences in their capability to interact with human effector systems. Differences in their binding to human Fc receptors for IgG (Fcy receptors) were especially prominent. Human monocytes could perform antibody-dependent cellular cytotoxicity (ADCC) against tumour cells in the presence of murine IgG2a mAB, but only slightly or not at all with the mIgG1 or mIgG2b subclasses 1. Subsequently, it was shown that monomeric mIgG2a (and mIgG3) could bind to human monocyte Fc receptors whereas no binding occured with monomeric mIgG1 or mIgG2b 2.

Proteolysis increases the Fc-mediated binding of murine IgG2b to human EBV-transformed B cells, but decreases the expression of Fc gamma RII and Fc epsilon RII.

We have previously described a polymorphic human Fc receptor for murine IgG2b (mIgG2b). This receptor was defined by the binding of (complexed) mIgG2b to monocytes and Epstein-Barr virus (EBV)-transformed B cells. Three per cent of normal individuals were high responders with respect to mIgG2b (mIgG2b-HR), whereas the other individuals were low responders for mIgG2b (mIgG2b-LR). In the present study we investigated the effect of proteolytic enzymes on the Fc-mediated binding of mIgG2b to EBV-B cells. Pronase, human leucocyte elastase and cathepsin G caused an increased binding (in an EA-rosetting assay) of mIgG2b to EBV-B cells from mIgG2b-HR, but not from mIgG2b-LR. Simultaneous immunofluorescence studies demonstrated that these proteolytic enzymes strongly reduced the expression of Fc gamma RII and Fc epsilon RII on these cells, whereas HLA class I or HLA class II molecules were not affected. These findings strongly suggest that binding of mIgG2b is not mediated by Fc gamma RII or Fc epsilon RII. We also studied the effect of proteolysis on mIgG2b-HR EBV-B cells from an HLA class II-negative individual. In this case EA-mIgG2b rosetting was decreased after proteolysis, suggesting that HLA class II molecules may have a role in protecting the binding site for mIgG2b against proteolytic destruction.