Activating and inhibitory Fcγ receptors in immunotherapy: being the actor or being the target

Membrane Fcγ receptors (FcγRs) can act either as potent activators of effector cell functions or as inhibitors of receptor-mediated cell activation following engagement by IgG antibodies bound to their target molecules. The remarkable ability of activating FcγRs to trigger antibody-dependent cellular cytotoxicity, cytokine release and phagocytosis/endocytosis followed by antigen presentation has stimulated the development of a number of therapeutic monoclonal antibodies whose Fc regions have been engineered to optimize their effector functions, mostly their killing activities. Conversely, the demonstration that inhibitory FcγRs can block or downmodulate effector functions has led to the concept that targeting these receptors is of interest in a number of pathologies. The use of bispecific antibodies leading to the crosslinking of FcγRIIB with activating receptors could induce immunomodulation in autoimmune or allergic diseases. Alternatively, the use of cytotoxic/antagonist anti-FcγRIIB antibodies could kill FcγRIIB-positive tumor cells or prevent the downmodulation of activating receptors. Thus, antibodies engineered to preferentially target activating or inhibitory FcγRs are currently being designed for therapeutic use.     

Measurement of the binding activity of defined IgG aggregates to macrophage Fc receptors

Small soluble IgG aggregates of defined size were prepared from pooled human IgG by gel filtration chromatography, and examined by analytical ultracentrifugation. Three such fractions, dimer-rich, trimer-rich and 25S aggregate were used to inhibit IgG monomer binding in a study of the influence of aggregation in the binding of human IgG1 to mouse macrophage Fc receptors. Of the polymers tested, IgG in the trimeric form was found to bind with the greatest avidity, being 158 times more active than monomeric IgG, whereas IgG as a larger 25S aggregate had an increased binding activity of 80 times; the avidity of IgG as dimer was increased by a factor of 2 over monomeric IgG. The possible mechanisms involved in achieving enhanced binding are discussed.     

Fc Receptors as Targets for Immunotherapy

Human membrane and soluble Fee receptors (FcεRI, FcεRII/CD23) and Fcγ receptors (FcγRI/CD64, FCγRII/CD32, FcγRIII/CD 16) have been implicated in a number of diseases. Their functional roles such as capture and clearance of immune complexes, antibody-dependent cell cytotoxicity, or cytokine or inflammatory mediator release, make them potential targets for immuno-intervention. In the present review, we will describe how membrane and soluble human FcεR and FcγR have been already used as targets/tools for immuno-interventions by using monoclonal and bispecific engineered antibodies. Some therapeutic uses of these molecules both in cancer, infectious, and auto-immune diseases are presented.     

Low‐affinity FcγR interactions can decide the fate of novel human IgG‐sensitised red blood cells and platelets

G1Δnab is a mutant human IgG1 constant region with a lower ability to interact with FcγR than the natural IgG constant regions. Radiolabelled RBCs and platelets sensitised with specific G1Δnab Abs were cleared more slowly from human circulation than IgG1‐sensitised counterparts. However, non‐destructive splenic retention of G1Δnab‐coated RBCs required investigation and plasma radioactivities now suggest this also occurred for platelets sensitised with an IgG1/G1Δnab mixture. In vitro assays with human cells showed that G1Δnab‐sensitised RBCs did not cause FcγRI‐mediated monocyte activation, FcγRIIIa‐mediated antibody‐dependent cell‐mediated cytotoxicity (ADCC) or macrophage phagocytosis although they did adhere to macrophages. Thus, FcγRII was implicated in the adhesion despite the Δnab mutation reducing the already low‐affinity binding to this receptor class. Additional contacts via P‐selectin enhance the interaction of sensitised platelets with monocytes and this system provided evidence of FcγRII‐dependent activation by G1Δnab. These results emphasise the physiological relevance of low‐affinity interactions: It appears that FcγRII interactions of G1Δnab allowed splenic retention of G1Δnab‐coated RBCs with inhibitory FcγRIIb binding preventing RBC destruction and that FcγRIIb engagement by G1Δnab on IgG1/G1Δnab‐sensitised platelets overcame activation by IgG1. Considering therapeutic blocking Abs, G1Δnab offers lower FcγR binding and a greater bias towards inhibition than IgG2 and IgG4 constant regions.     

Human NK cells express Fc receptors for IgA which mediate signal transduction and target cell killing

Receptors for the Fc region of IgG (FcgammaRIIIa, FcgammaRIIc) and IgM (FcmicroR) were previously described on NK cells. In this work the expression of Fc receptors for IgA (FcalphaR) on human NK cells and the signaling events were investigated. The FcalphaR was demonstrated by flow cytometry using secretory IgA (sIgA) and anti-human IgA antibody. The percentage of NK cells (CD3(-)CD56(+)CD16(+)) expressing FcalphaR ranged between 55.7% and 95.7%, with a mean +/- SD of 75.2+/-11.8. The association constant and the number of (125)I-labeled sIgA ((125)I-sIgA) molecules bound per cell, calculated by Scatchard analysis, were 2 x 10(7) M(-1) and 1.7 x 10(4), respectively. The binding specificity was proved by inhibition experiments. Cold sIgA but not IgA Fab fragments were able to inhibit (125)I-sIgA binding in a concentration-dependent manner. Binding of sIgA to NK cells was neither inhibited by anti-mannose receptor antibody, nor by L-fucose, D-galactose, D-glucose, D-mannose or N-acetyl-D-glucosamine. Pretreatment of NK cells with polymeric IgA inhibited their capacity to kill (51)Cr-labeled K562 target cells by 34.8%, whereas with monomeric IgA only by 13.1%. Ligand-induced clustering of the FcalphaR resulted in activation of tyrosine kinases Lck, Syk and phosphatidylinositol 3-kinase. The present studies support the concept that human NK cells bind preferentially sIgA and polymeric IgA with moderate affinity via FcalphaR, which is different from the FcalphaRI/CD89 and other carbohydrate-recognizing receptors like mannose receptor/CD206. This novel structure mediates signal transduction and cell killing.     

IgA Fc受体研究进展

IgA Fc受体(FcαRI,CD89)属于免疫受体家族成员,主要表达于单核-巨噬细胞、中性粒细胞、嗜酸性粒细胞和树突状细胞等免疫效应细胞表面。FcαRI能特异的与血清型和分泌型IgA结合,并通过γ链介导一系列免疫反应,包括抗体依赖细胞介导的细胞毒性作用(ADCC)、补体依赖的细胞毒性(CDC)及细胞吞噬作用等。FcαRI是一种双功能受体,在不同的生理条件下可以介导免疫系统的活化和抑制反应。FcαRI在机体免疫防御和在维持系统免疫平衡方面扮演着重要的角色,有望成为治疗人类疾病的新靶点。本文对FcαRI的结构、功能及其应用等研究现状进行综述。     

Size-dependent effect of IgA on the IgA Fc receptor (CD89)

The IgA Fc receptor (FcR; CD89) is expressed on several types of cells of the myeloid cell lineage. We investigated whether different sizes of heat-aggregated IgA (aIgA) bind to CD89 and subsequently induce cellular activation. As a model we used the murine B cell line IIA1.6 transfected with CD89 or IIA1.6 cells transfected with CD89 as well as with the FcR γ chain to study the binding of IgA to CD89. When these cells expressing CD89 were incubated with monomeric IgA, no significant binding of IgA to the cells was detectable by fluorescence-activated cell sorter analysis; however, incubation of the cells with aggregated IgA resulted in 93 ± 2% positive cells. Incubation of the cells with different sizes of IgA-containing aggregates revealed optimal binding with aggregates containing five to six molecules of IgA per aggregate. No difference was observed between the binding to CD89 of both IgA1- or IgA2-containing aggregates. Furthermore, the binding of aIgA was found to be CD89-specific, since the binding of IgA was completely inhibited by the CD89-specific monoclonal antibody My43 and no detectable binding occurred to the IIA1.6 parent cell line. Activation studies using interleukin-2 (IL-2) production as a marker, showed that the FcR γ chain is necessary to induce cellular activation. Only cells transfected with both CD89 and the FcR γ chain (CD89⁺/γ⁺) enhance the IL-2 production 10–12-fold upon stimulation with aggregates of IgA. Furthermore, triggering of CD89 only results in increase of intracellular calcium concentration ([Ca²⁺]_i) in cells co-expressing FcR γ chain. Mutation of the tyrosine residues in the FcR γ chain immunoreceptor tyrosine-based activation motif of the FcR γ chain abolishes this increase in [Ca²⁺]_i, indicating association and involvement of the FcR γ chain in CD89-mediated signaling.     

人IgG Fc基因的扩增及其在真核细胞中的分泌性表达

目的:通过扩增人IgG Fc基因并与人CD5基因信号肽序列融合,构建真核分泌型表达载体,实现在真核细胞中高效表达,为利用Fc基因制备融合抗原,研究抗原基因和抗原蛋白的免疫治疗奠定了基础.方法:从外科手术切除的扁桃体中分离人淋巴细胞,用Trizol试剂提取淋巴细胞的总RNA,通过RT-PCR方法从淋巴细胞中扩增人IgG Fc 片段的基因序列,并将其克隆到pMD-T18 质粒载体上.然后以上述含有Fc片段序列的质粒载体和含有人CD5基因的质粒为模板,通过重叠延伸PCR方法将Fc片段与CD5信号肽基因序列进行融合,并将其插入到pcDNA3.1A 真核表达载体上,构建成与CD5信号肽融合的Fc片段基因的分泌型表达载体.经磷酸钙介导转染293T细胞使其表达.结果:测序表明扩增的Fc基因序列与GeneBank发表的序列完全一致,Western blot检测结果表明,本实验构建的Fc基因分泌型表达载体能够在真核细胞进行高效分泌性表达,表达水平在转染后48小时可达50 μg/1×106细胞.结论:采用RT-PCR扩增了人IgG Fc cDNA 基因,经过与人CD5信号肽序列融合构建了分泌型表达载体,实现了在293T细胞中的高效表达,这为今后构建特定抗原基因与IgG Fc基因融合表达载体,研究DNA疫苗及Fc的生物佐剂提供了实验依据.     

IgA receptors in health and disease

The varied interaction of the Fc region of IgA with receptors confers this antibody class with many of its unique properties. The epithelial polymeric Ig receptor on mucosal epithelial cells transports polymeric immunoglobulin A (pIgA) produced by mucosal B cells to the mucosal surface where, in complex with the secretory component (SC), this secretory immunoglobulin A (SIgA) excludes the multitude of dietary, environmental, and microbial antigens that continuously bombard the mucosae. In health, this IgA-mediated exclusion not only forms the initial defence against infection, it also spares the systemic immune system from potentially deleterious responses to innocuous antigens which can otherwise culminate in inflammatory bowel disease or asthma. Beyond antigen exclusion, in closer encounters with antigens, IgA receptors play roles in protective immunity and disease. FcaRI is the principal myeloid IgA receptor and is responsible for differing IgA-mediated effector responses such as respiratory burst, degranulation, and phagocytosis variously by granulyoctes, monocytes, and macrophages. Furthermore an unknown IgA receptor specific for the secretory component (SC) elicits powerful effector responses from eosinophils. On dendritic cells, FcaRI participates in antigen presentation while on microfold cells, key cells in mucosal antigen presentation, another unknown IgA receptor functions in the transport of antigens across the mucosal epithelial barrier. The activity of another uncharacterized IgA1/IgD receptor on T cells may affect autoimmune disorders. The interplay of different IgA receptors affects immune complex deposition in the common renal disease immunoglobulin A nephropathy (IgAN). Finally, the therapeutic application of various IgA receptors has been sought in the areas of infectious disease, vaccines, and cancer.     

Fc receptors are major mediators of antibody based inflammation in autoimmunity

There is now renewed interest in the role of antibodies in autoimmunity. Recent compelling evidence indicates that autoantibodies and the effector mechanisms they induce, for example, Fc receptor activation of leukocytes and/or the complement cascade, are central players in the development of autoimmunity, by perpetuating inflammation and perhaps even regulating the process itself. Of increasing interest are Fc receptors, which have been more closely investigated in the past decade using recombinant proteins, gene deficient mice and mouse models of human disease. These analyses point towards major roles of Fc receptors in antibody hypersensitivity reactions and by extension autoimmune disease, and they reveal opportunities in the development of novel therapeutic approaches in the treatment of autoimmune diseases.     

Cross-talk between pathogen recognizing Toll-like receptors and immunoglobulin Fc receptors in immunity

The individual role of pathogen-binding Toll-like receptors (TLRs) and antibody-binding Fc receptors (FcRs) during pathogenic infections has been studied extensively. However, combined activation of these different receptor classes has received little attention, even though they are triggered simultaneously when immune cells bind antibody-opsonized pathogens. In the last few years, it has become evident that joined activation of TLRs and FcRs substantially tailors inflammatory immune responses, which is an efficient and controlled mechanism of the host to act upon invading pathogens. In this review, we discuss the mechanisms of cross-talk between different TLRs and FcRs and the resulting inflammatory immune responses. Furthermore, we propose how chronic activation via this cross-talk might be detrimental in inflammatory (auto) immune diseases. We conclude with the potential exploitation of the interplay between TLRs and FcRs for monoclonal antibody therapy to target tumors. Future interests in this field of research include establishing a more detailed and mechanistic understanding of the mode of action of TLR and FcR cross-talk and exploration of its physiological importance in health and disease. This may furthermore open up novel therapeutic options for intervention in inflammatory diseases or cancer.     

Identification of an Oolemmal IgG Fc Receptor: Its Role in Promoting Binding of Antibody-Labelled Human Sperm to Zona-Free Hamster Eggs

ABSTRACT: Antisperm antibodies (ASAs) present in sera of infertile men and women have been shown either to promote or inhibit penetration of zona-free hamster eggs by antibody-labelled human spermatozoa. Increased numbers of oolemmal-bound sperm have been noted in association with increased sperm penetration frequencies, following antibody labelling, when compared with antibody-free sperm. The promotion of adherence of ASA-labelled sperm to the oolemma could be mediated through the binding of antibodies to common epitopes present on the sperm and egg surfaces or through Fc-mediated binding to an oolemmal Fc receptor. In support of the latter hypothesis, we report that zona-free hamster eggs bind aggregated human IgG and IgG Fc fragments. The presence of an oolemmal IgG Fc receptor has been confirmed using a rat monoclonal antibody (2.4G2) directed against a murine IgG Fc receptor (Fc γ R_II) as judged both by indirect immunofluorescence and by immunobead binding. In addition, the pre-incubation of zona-free hamster eggs with IgG Fc diminished both adhesion to and penetration of the oolemma by human spermatozoa.     

Activation-induced proteolysis of the cytoplasmic domain of ζ in T cell receptors and Fc receptors

The CD3-T cell receptor (TCR) complex on T cells and the Fcγ receptor type III (FcγRIII)-ζ-γ complex on natural killer cells are functionally analogous activation receptors that associate with a family of disulfide-linked dimers composed of the related subunits ζ and γ. Immunochemical analysis of receptor complexes separated on two-dimensional diagonal gels allowed the identification of a previously uncharacterized ζ-p14 heterodimer. ζ-p14 is a component of both CD3-TCR and FcγRIII-ζ-γ. Peptide mapping analysis shows that p14 is structurally related to ζ, suggesting that it is either: (i) derived from ζ proteolytically or (ii) the product of an alternatively spliced mRNA. The observation that COS cells transformed with a cDNA encoding ζ express ζ-p14 supports the former possibility. The expression of CD3-TCR complexes including ζ-p14 increases following activation with phorbol 12-myristate 13-acetate or concanavalin A, suggesting that proteolysis of ζ may contribute to receptor modulation or desensitization.     

The role of immunoglobulin therapy in allergic diseases

Intravenous immunoglobulin (IVIG) has been used for many years to treat patients with primary immunodeficiencies. More recently, IVIG has been shown to have anti-inflammatory activity when used at substantially higher concentrations. A number of studies have examined the efficacy of IVIG in allergic diseases. For patients with severe refractory asthma, sinusitis, atopic dermatitis, and urticaria, IVIG offers an alternative therapy with relatively few side-effects. Although the mechanism by which IVIG may attenuate the allergic response is still undetermined, clinical studies have shown that immunoglobulin therapy can decrease serum IgE levels and increase glucocorticoid binding affinity, while in vitro studies have shown that IVIG can decrease T-cell secretion of TH2 cytokines. Further studies are needed to confirm the initial encouraging results seen in allergic patients with severe, resistant disease.     

抗人IgGFc和Fab单克隆抗体的鉴定及应用

本文将采用木瓜蛋白酶水解和ＳＰＡ－Ｓｅｐｈａｒｏｓｅ４Ｂ柱亲和层析等手段获得的人ＩｇＧＦｃ和Ｆａｂ，以抗人ＩｇＧＦｃ和抗人ＩｇＧＦａｂ单抗为参比品（Ｓｉｇｍａ）．鉴定了细胞库中抗人ＩｇＧ系列的部分细胞林，得到分泌特异性抗人ＩｇＧＦｅ和抗人ＩｇＧＦａｂ单抗的细胞各一株。在此基础上。应用抗人ＩｇＧＦｃ及抗人ＩｇＧＦａｂ单抗分别制备了Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析柱，纯化了酶解的人ＩｇＧＦｃ和Ｆａｂ。经ＥＬＩＳＡ法鉴定，相互间无交叉反应。同时用此方法还制备了人抗ＨＢｅＦａｂ，并将此Ｆａｂ标记过氧化物酶。配制ＨＢｅＥＬＩＳＡ诊断盒，证明其生物学活性未受影响，而且消除了类风湿因子引起的ＨＢｅＡｇ假阳性现象。     

On the interaction between agalactosyl IgG and Fcγ receptors

One of the serum abnormalities observed in autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) is the occurrence of IgG that lacks the terminal galactose on asparagine-linked biantennary complex type oligosaccharides [Gal(0)-IgG] located in the CH2 domain. Additionally, IgG without glycosylation is known to be defective in several effector functions due to a reduced ability to bind to its specific receptors (FcγR). It has thus been speculated that, by analogy with unglycosylated IgG, Gal(0)-IgG may also be functionally impaired or exert altered effector mechanisms. If this were true, Gal(0)-IgG could contribute to the phenotype of above-mentioned autoimmune diseases, like impaired immune complex clearance and defective down-regulation of activated B cells. Here, we show by three different methods that the interaction of Gal(0)-IgG and normally glycosylated IgG with the low-affinity FcγRII (CD32) is indistinguishable with respect both to binding and receptor-mediated signalling. These data argue against a prominent role for FcγR-dependent Gal(0)-IgG interactions in the etiology or pathogenesis of autoimmune diseases.     

Cytomegalovirus IgG and IgA serum antibodies in a study of HIV infection and HIV related diseases in homosexual men.

The significance of serum IgG and IgA antibodies to cytomegalovirus (CMV) at various stages of human immune deficiency virus (HIV) infection was studied in 175 homosexual men. Sera were obtained from 123 HIV seropositives [41 asymptomatic, 29 with lymphadenopathy associated syndrome (LAS), 22 with AIDS related complex (ARC), and 31 AIDS patients], 17 HIV seroconverters, and 35 HIV asymptomatic seronegatives. The sera were tested blindly for CMV IgA and IgG antibodies using the immunoperoxidase assay (IPA) and CMV infected human embryo cells. Cross-sectional analysis of CMV IgG antibodies at a titer of greater than or equal to 20 showed 87% and 100% prevalence in the HIV seronegative groups and in the HIV seropositive groups, respectively (P less than 0.05). CMV IgG antibodies at a titer of greater than or equal to 80 were present in significantly higher proportions among the HIV seropositive subjects of the various groups as compared with the HIV seronegative homosexual men. However, in the HIV seronegatives who later seroconverted to HIV, a significantly higher prevalence of CMV antibodies (35%) was detected before HIV seroconversion, as compared with the persistently HIV seronegative subjects (14.3%) (P less than 0.05). The HIV seronegatives pre-HIV seroconversion also exhibited a significantly higher geometric mean titer (GMT) of CMV IgG antibodies (62.17 +/- 0.64) as compared with the persistently HIV seronegatives (34.0 +/- 0.6) (P = 0.03). Significantly higher GMTs of CMV IgG antibodies were detected in all the HIV seropositive groups as compared with the persistently HIV seronegative group. CMV IgG antibodies were not detected in the HIV seronegative subjects.(ABSTRACT TRUNCATED AT 250 WORDS)