Aglycosylation of human IgG1 and IgG3 monoclonal antibodies can eliminate recognition by human cells expressing Fc gamma RI and/or Fc gamma RII receptors.

Aglycosylated human IgG1 and IgG3 monoclonal anti-D (Rh) and human IgG1 and IgG3 chimaeric anti-5-iodo-4-hydroxy-3-nitrophenacetyl (anti-NIP) monoclonal antibodies produced in the presence of tunicamycin have been compared with the native glycosylated proteins with respect to recognition by human Fc gamma RI and/or Fc gamma RII receptors on U937, Daudi or K562 cells. Human red cells sensitized with glycosylated IgG3 form rosettes via Fc gamma RI with 60% of U937 cells. Inhibition of rosette formation required greater than 35-fold concentrated more aglycosylated than glycosylated human monoclonal anti-D (Rh) antibody. Unlabelled polyclonal human IgG and glycosylated monoclonal IgG1 and anti-D (Rh) antibody inhibited the binding of 125I-labelled monomeric human IgG binding by U937 Fc gamma RI at concentrations greater than 50-fold lower than the aglycosylated monoclonal IgG1 anti-D (Rh) (K50 approximately 3 x 10(-9) M and approximately 6 x 10(-7) M respectively). Similar results were obtained using glycosylated and aglycosylated monoclonal human IgG1 or IgG3 chimaeric anti-NIP antibody-sensitized red cells rosetting with Fc gamma RI-/Fc gamma RII+ Daudi and K562 cells. Rosette formation could be inhibited by the glycosylated form (at greater than 10(-6) M) but not by the aglycosylated form. Haemagglutination analysis using a panel of murine monoclonal antibodies specific for epitopes located on C gamma 2, C gamma 3 or C gamma 2/C gamma 3 interface regions did not demonstrate differences in Fc conformation between the glycosylated or aglycosylated human monoclonal antibodies. These data suggest that the Fc gamma RI and Fc gamma RII sites on human IgG are highly conformation-dependent and that the carbohydrate moiety serves to stabilize the Fc structure rather than interacting directly with Fc receptors.     

Suppressor cell activity in mice bearing a progressively growing Simian virus 40-induced sarcoma

Summary General and cell-mediated immunity (CMI) were investigated in BALB/c mice bearing progressively growing Simian virus 40-induced (mKSA) sarcoma by means of the Winn tumor cell neutralization (WN), ¹²⁵I isotopic footpad (IFP), lymphoproliferative (LP) and plaque-forming cell (PFC) assays. Correlation between depressed antitumor immunity and the IFP responses was observed in tumor-bearing (TB) mice. Depressed LP responses to both T- and B-cell mitogens were observed in both early and late stages of tumor growth. Results obtained with the PFC assay similarly demonstrated depressed humoral immunity to sheep red blood cells (SRBC). Suppressor cell activity was demonstrated in cocultivation experiments in which spleen cells of TB mice were mixed with normal spleen cells. Treatment of TB spleen cells by passage through Sephadex G-10 columns or incubation on plastic surfaces to deplete the adherent cells restored LP responses. Cocultivation of Sephadex G-10- or plastic-adherent cells from TB mice with normal spleen cells significantly reduced mitogen-induced LP responses of normal cells. Examination of cell surface markers indicated an increase in the proportion of spleen cells bearing Fc receptors, which correlated with progressive mKSA tumor growth. There was also a correlation between Fc receptor-bearing spleen cells and macrophages, as shown by nonspecific esterase staining. These results indicate that depressed LP and PFC responses and the appearance of suppressor cells in mKSA tumor-bearing mice parallel an impaired ability to recognize (IFP responses) and neutralize (WN responses) tumor cells.     

Significance of Rh-sensitization during Pregnancy: Its Relation to a Preventive Programme

A study of the incidence of Rh-sensitization showed a 6.5% incidence of anti-D appearing for the first time during the last trimester of pregnancy. In 24 of the 29 subjects these antibodies were weak, and were found only when a sensitive technique using enzyme-treated Rh-deletion test cells was employed. The remaining five, however, had high-titre antibodies, which were associated with a positive Coombs test on cord blood.Rh-immune globulin administered at delivery resulted in disappearance of the anti-D in all but one of the subjects with weak antibody to whom it was given, suggesting that this treatment can reverse early sensitization. There was no effect when Rh-immune globulin was given to one subject with a high anti-D titre.Since sensitization has been found to occur frequently during the last trimester of pregnancy, an antenatal schedule of prophylaxis is advocated.     

T cell activation via CD2 [T, gp50] molecules: accessory cells are required to trigger T cell activation via CD2-D66 plus CD2-9.6/T11(1) epitopes

Binding monoclonal antibodies (MAb) both to D66 and 9.6/T11(1) epitopes on the CD2 [T,gp50]-defined molecule produces a high level of T cell mitosis. This was observed with a battery of MAb of different isotypes. In contrast, none of the anti-D66 or anti-9.6/T11(1)Ab could trigger T cell proliferation in combination with anti-T11(3). Moreover, all anti-D66-9.6/T11(1) pairs of MAb tested required monocytes to activate T cells which were recruited through their Fc receptors. Variations among normal individuals were observed in the level of response to anti-D66-9.6/T11(1) pairs of Ab, 75% of a population of French Caucasians giving a high response. The level of response of a given individual was determined by his accessory cells. However, the level of response of an individual appeared to be minimally influenced by the isotype of a peculiar anti-D66 or anti-9.6/T11(1) Ab. The addition of exogeneous IL 2 could overcome the removal of accessory cells or the modulation of CD3 molecules. In contrast, IL 2 receptor appearance was not overcome by removal of monocytes. Thus, T cell activation via CD2 seems to be produced by "touching" several definite regions of this molecule which trigger a cascade of events similar to those produced by mitogenic lectins. One can assume that the appropriate conformational changes of the CD2 molecule induced by anti-D66-9.6/T11(1) pairs of Ab are solely produced when they are presented by accessory cells. This leaves open the question of whether accessory cells would also play a more active role.     

鱼类Fc受体研究

Fc受体是免疫细胞表面一种重要受体分子,通过与免疫球蛋白Fc段结合触发多种生物学功能,是联系体液免疫和细胞免疫的桥梁。部分硬骨鱼中已经发现了Fc受体,在斑马鱼、斑点又尾鲴和鲤鱼中都克隆到了Fc受体的γ亚基,在鲨鱼和大西洋鲑中证明有能够与免疫球蛋白结合的Fc受体存在,并在斑点叉尾鲴、河豚和虹鳟中存在着类似α亚基的Fc受体。对鱼类Fc受体的发现和研究必将为了解鱼类的免疫机制及免疫进化提供重要的资料。     

Contribution of humoral immune responses to the antitumor effects mediated by anthracyclines

Immunogenic cell death induced by cytotoxic compounds contributes to the success of selected chemotherapies by eliciting a protective anticancer immune response, which is mediated by CD4⁺ and CD8⁺ T cells producing interferon-γ. In many instances, cancer progression is associated with high titers of tumor-specific antibodies, which become detectable in the serum, but whose functional relevance is elusive. Here, we explored the role of humoral immune responses in the anticancer efficacy of anthracyclines. Chemotherapy reduced the number of tumor-infiltrating B cells, and failed to promote humoral responses against immunodominant tumor antigens. Although anthracycline-based anticancer chemotherapies failed in T cell-deficient mice, they successfully reduced the growth of cancers developing in mice lacking B lymphocytes (due to the injection of a B-cell-depleting anti-CD20 antibody), immunoglobulins (Igs) or Ig receptors (Fc receptor) due to genetic manipulations. These results suggest that the humoral arm of antitumor immunity is dispensable for the immune-dependent therapeutic effect of anthracyclines against mouse sarcoma. In addition, we show here that the titers of IgA and IgG antibodies directed against an autoantigen appearing at the cell surface of tumor cells post chemotherapy (calreticulin, CRT) did not significantly increase in patients treated with anthracyclines, and that anti-CRT antibodies had no prognostic or predictive significance. Collectively, our data indicate that humoral anticancer immune responses differ from cellular responses in, thus far, that they do not contribute to the success of anthracycline-mediated anticancer therapies in human breast cancers and mouse sarcomas.     

Natural cytotoxicity

Summary The microcytotoxicity assay was used to examine lymphocyte subpopulations derived from healthy donors or persons suffering from non-malignant diseases for natural cytotoxicity.Part of the natural cytotoxicity may be caused by cells devoid of conventional surface markers. However, most of the natural cytotoxicity was found to be associated with cells bearing Fc receptors, irrespective of T and non-T identity.The Fc receptors appeared to be directly involved in the mechanism of natural cytotoxicity, since blocking of these receptors with Fc fragments or immune complexes significantly reduced this reactivity.Sponsored by the Danish Cancer Society     

Synthesis of natural allohemagglutinins of the ABO system in healthy children aged 3 months to 3 years

The work was carried out to establish the titre and score of haemagglutination of natural anti-A and anti-B antibodies in healthy children during the first three years of life. The material studied included 900 healthy children aged between 3 months and 3 years and 100 adults serving as controls. The method of test tube haemagglutination was used for determining the titre and score of alloagglutinins in relation to standard erythrocytes always obtained from the same donors. In addition, in 72 children and 10 adults the levels of IgG, IgA and IgM were determined quantitatively. A statistical analysis of the results showed that the levels of anti-A and anti-B alloagglutinins were relatively high at the age of 3 months / about 25% of the adult levels / and increased very rapidly in the first years of life reaching about 90% of the adult level at the end of the 3rd year of life. Besides that, it was demonstrated that it is useful in clinical practice to use the titre and score of natural alloagglutinins as indicators of humoral immunity, especially in children in the first years of life. Acceleration was demonstrated in the intensity of haemagglutination of natural antibodies in the last 40 years since their titre in the contemporary infantile population / up to the age of 1 year / is about 50% higher than that found in 1929. These findings suggest that increased immune reactivity of children observed presently may be due to prophylactic vaccinations.     

The anti-lymphoma activities of anti-CD137 monoclonal antibodies are enhanced in FcγRIII−/− mice

Agonistic monoclonal antibodies (mAbs) directed against the co-signaling molecule CD137 (4-1BB) elicit potent anti-tumor immunity in mice. This anti-tumor immunity has traditionally been thought to result from the ability of the Fab portion of anti-CD137 to function as an analog for CD137L. Although binding of CD137 by anti-CD137 mAbs has the potential to cross-link the Fc fragments, enabling Fc engagement of low to moderate affinity Fc gamma receptors (FcγR), the relative import of such Fc–FcγR interactions in mediating anti-CD137 associated anti-tumor immunity is unknown. We studied the ability of a rat anti-mouse CD137 mAb (2A) to mediate the anti-tumor response against the EL4E7 lymphoma in WT and FcγR^?/? strains. 2A-treated FcRγ^?/? mice had improved anti-tumor immunity against EL4E7, which could be completely recapitulated in FcγRIII^?/? animals. These improved anti-tumor responses were associated with increased splenic CD8β T cell and dendritic cell (DC) populations. Furthermore, there was an increase in the number of DCs expressing high levels of the CD40, CD80, and CD86 molecules that are associated with more effective antigen presentation. Our results demonstrate an unexpected inhibitory role for FcγRIII in the anti-tumor function of anti-CD137 and underscore the need to consider antibody isotype when engineering therapeutic mAbs.     

Engineering an aglycosylated Fc variant for enhanced FcγRI engagement and pH-dependent human FcRn binding

The clinical use of therapeutic antibodies has increased sharply because of their many advantages over conventional small molecule drugs, particularly with respect to their affinity, specificity, and serum stability. Tumor or infected cells are removed by the binding of antibody Fc regions to Fc gamma receptors (FcγRs), which stimulate the activation of immune effector cells. Aglycosylated full-length IgG antibodies expressed in bacteria have different Fc conformations compared to their glycosylated counterparts produced in mammalian cells. As a result, they are unable to bind FcγRs, resulting in little to no activation of immune effector cells. In this study, we created a combinatorial library randomized at the upper CH2 loops of an aglycosylated Fc variant (Fc5: E382V/M428) and used a high-throughput flow cytometry library screening method, combined with bacterial display of homodimeric Fc domains for enhanced FcγR binding affinity. The trastuzumab Fc variant containing the identified mutations (Q295R, L328W, A330V, P331A, I332Y, E382V, M428I) not only exhibited over 120 fold higher affinity of specific binding to FcγRI than wild type aglycosylated Fc, but also retained pH-dependent FcRn binding. These results show that an aglycosylated antibody expressed in bacteria can be evolved for novel FcγR affinity and specificity.     

Animal models for IgE-meditated cancer immunotherapy

Although most monoclonal antibodies developed for cancer therapy are of the IgG class, antibodies of the IgE class have certain properties that make them attractive as cancer therapeutics. These properties include the superior affinity for the Fc epsilon receptors (FcεRs), the low serum level of IgE that minimizes competition of endogenous IgE for FcεR occupancy, and the ability to induce a broad and vigorous immune response through the interaction with multiple cells including mast cells, basophils, monocytes, macrophages, dendritic cells, and eosinophils. Tumor-targeted IgE antibodies are expected to harness the allergic response against tumors and activate a secondary, T-cell-mediated immune response. Importantly, the IgE antibody can be used for passive immunotherapy and as an adjuvant of cancer vaccines. However, there are important limitations in the use of animal models including the fact that human IgE does not interact with rodent FcεRs and that there is a different cellular distribution of FcεRs in humans and rodents. Despite these limitations, different murine models have been used with success to evaluate the in vivo anti-cancer activity of several IgE antibodies. These models include wild-type immunocompetent animals bearing syngeneic tumors, xenograft models using immunocompromised mice bearing human tumors and reconstituted with human effector cells, and human FcεRIα transgenic mice bearing syngeneic tumors. In addition, non-human primates such as cynomolgus monkeys can be potentially used for toxicological and pharmacokinetic studies. This article describes the advantages and disadvantages of these models and their use in evaluating the in vivo properties of IgE antibodies for cancer therapy.     

High affinity allele for the gene of FCGR3A is risk factor for HIV infection and progression

Background

We investigated the genetics of Fc receptors, which function as activating receptors on immune cells and help to control HIV through antibody-mediated cellular cytotoxicity. Thus, Fc receptors may be important for virus immunity but might also promote immune hyperactivation that would enhance infection.

Methodology/Principal Findings

We measured abundance of low and high activity alleles in two Fc receptor genes, FCGR2A and FCGR3A, for persons with HIV disease, natural virus suppressors (HIV+, without disease) and healthy controls to show whether genotypes were associated with infection and disease. Individuals homozygous for the high activity allele of FCGR3A (158VV) were predominantly found among HIV progressors and this group was also skewed toward higher allele frequencies for the V158 variant. Both of the HIV positive groups (progressors and natural virus suppressors) had significantly higher frequencies of the V158 allele compared with uninfected controls. There were no apparent associations among FCGR2A alleles and HIV status.

Conclusions/Significance

Our results indicate that high activity alleles of FCGR3A may be risk factors for HIV infection or progression and we need to understand how allelic variants affect the balance between virus control and immune activation.     

Neutralized adenovirus-immune complexes can mediate effective gene transfer via an Fc receptor-dependent infection pathway

Neutralization of adenovirus (Ad) by anti-Ad neutralizing antibodies in serum involves formation of Ad-immune complexes that prevent the virus from interacting with target cells. We hypothesized that Ad-immune complexes likely contain viable Ad vectors which, although no longer capable of gaining access to receptors on target cells, may be able to express transgenes in cells bearing Fc receptors for immunoglobulins, i.e., that antibody-based "neutralization" of Ad vectors may be circumvented by the Fc receptor pathway. To test this hypothesis, we expressed the Fcgamma receptor IIA (FcgammaR) in A549 lung epithelial cells or human dermal fibroblasts and evaluated gene transfer in the presence of human neutralizing anti-Ad serum. FcgammaR-expressing cells bound and internalized copious amounts of Ad, with a distinct population of internalized Ad trafficking to the nucleus. The dose-response curves for inhibition of gene transfer revealed that FcgammaR-expressing cells required a more-than-10-fold higher concentration of anti-Ad serum to achieve 50% inhibition of Ad-encoded beta-galactosidase expression compared with non-FcgammaR-expressing cells. The discrepancy between neutralization of Ad during infection of FcgammaR-expressing cells and neutralization of Ad during infection of non-FcgammaR-expressing cells occurred with either heat-inactivated or non-heat-inactivated sera, was blocked by addition of purified Fc domain protein, and did not require the cytoplasmic domain of FcgammaR, suggesting that immune complex internalization proceeded via endocytosis rather than phagocytosis. FcgammaR-mediated infection by Ad-immune complexes did not require expression of the coxsackie virus-Ad receptor (CAR) since similar data were obtained when CAR-deficient human dermal fibroblasts were engineered to express FcgammaR. However, interaction of the Ad penton base with cell surface integrins contributed to the difference in neutralization between FcgammaR-expressing and non-FcgammaR-expressing cells. The data indicate that complexes formed from Ad and anti-Ad neutralizing antibodies, while compromised with respect to infection of non-FcgammaR-expressing target cells, maintain the potential to transfer genes to FcgammaR-expressing cells, with consequent expression of the transgene. The formation of Ad-immune complexes that can target viable virus to antigen-presenting cells may account for the success of Ad-based vaccines administered in the presence of low levels of neutralizing anti-Ad antibody.     

Human alveolar macrophage FcR-mediated cytotoxicity. Heteroantibody- versus conventional antibody-mediated target cell lysis

Human alveolar macrophage have three distinct receptors for IgG: FcRI, FcRII, and FcRIII. In order to compare the ability of these receptors to mediate target cell lysis, three different assay systems were examined. First, we studied lysis of chicken E (CE) opsonized with heteroantibodies, which are synthetic antibodies composed of Fab fragments with anti-FcR activity covalently linked to Fab fragments with anti-CE activity. We found alveolar macrophage readily lysed heteroantibody-opsonized CE via each of the three FcR classes (FcRI, 20 +/- 5%; FcRII, 27 +/- 7%; and FcRIII, 13 +/- 13%, p less than 0.05). Non-FcR-dependent lysis of anti-beta 2-microglobulin x anti-CE heteroantibody-opsonized CE was not detected. Second, lysis of hybridoma cell lines bearing anti-FcR antibodies on their cell surface was examined to assess killing of "tumor-like" target cells. Whereas peripheral blood monocytes and lymphocytes were able to lyse hybridoma cell lines bearing surface anti-FcR mAb, alveolar macrophages were not. Third, activity of alveolar macrophage FcR was examined in a conventional antibody-dependent cellular cytotoxicity assay by using O+ (R1,R2) human RBC opsonized with human anti-D and anti-CD serum as target cells. We found lysis of anti-D and anti-CD opsonized human RBC was mediated exclusively via FcRI. No activity of FcRII or FcRIII was detected in these latter assays even if performed under conditions that impair FcRI activity. Thus, all three FcR present on alveolar macrophage mediate lysis of heteroantibody-opsonized CE; in contrast, with the use of a conventional antibody-dependent cellular cytotoxicity assay, only FcRI activity was detected. We were unable to demonstrate lysis of anti-FcR-bearing hybridoma cell lines by alveolar macrophages.     

TNF receptor II fusion protein with tandemly repeated Fc domains

The extracellular domain of tumour necrosis factor (TNF) receptor II fused with the human IgG1 Fc region (TNFRII-Fc), as well as antibodies against TNF, has been used to treat rheumatoid arthritis. However, TNFRII-Fc is less effective than these antibodies in terms of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against cells bearing TNF on the cell surface. We hypothesized that these activities could be increased by fusing TNFRII with tandemly repeated Fc (TNFRII-Fc-Fc). The affinities of TNFRII-Fc-Fc for soluble TNF-α and transmembrane TNF-α and the TNF-α cytotoxicity-inhibitory activity were as potent as those of TNFRII-Fc. TNFRII-Fc-Fc showed much higher binding avidity for Fcγ receptors than TNFRII-Fc and was more potent in terms of both ADCC and CDC against cells expressing transmembrane TNF-α. TNFRII-Fc-Fc of 80 kDa, as well as TNFRII-Fc-Fc of 200 kDa, was detected. TNFRII-Fc-Fc (80 kDa) was as potent as TNFRII-Fc in terms of both ADCC and CDC. These results suggest that Fc multimerization of receptor-Fc fusion proteins can augment effector functions such as ADCC and CDC, and thereby have the potential to provide a superior therapeutic effect. This may be the case not only for TNFRII-Fc but also for other receptor-Fc fusion proteins.     

Effect of "rasayanas", a herbal drug preparation on immune responses and its significance in cancer treatment.

Rasayanas are considered to be immunostimulating preparations used extensively in indigenous medical practice. However there are only very few reports to substantiate this claim, and this paper gives preliminary evidence for the potentiation of immunity by Rasayanas given to mice orally. Administration of Rasayanas were found to enhance the proliferation of spleen cells significantly especially in the presence of mitogen. Similar result was also seen with bone marrow cells; however mitogenic stimulation could not be observed. Esterase activity was found to be enhanced in bone marrow cells indicating increased maturation of cells of lymphoid linkage. Rasayanas also enhanced humoral immune response as seen from the increased number of antibody forming cells and circulating antibody titre. These results indicate the usefulness of Rasayana as immunostimulating agent.     

Staphylococcal lipase affects phagocytosis of Staphylococcus aureus by human granulocytes and monocytes.

The rate of immunological and non-immunological phagocytosis of staphylococci by lipase pre-treated human granulocytes and monocytes was compared. It was found that the effect of this enzyme on two types of cells is opposite. Lipase decreases phagocytosis by granulocytes and increases by monocytes. The revealed differences between phagocytosing cells studied prompted us to investigate the influence of lipase on Fc receptors on these cells (rosette EA test). The different susceptibility of Fc receptors on non-activated phagocytes to lipase was found. This could be at least partially responsible for the difference observed between phagocytic activity of granulocytes (decreased) and monocytes (increased) pretreated with staphylococcal lipase. Inactivated enzyme showed a similar effect as active enzyme in the case of granulocytes. However, inactivated enzyme had no effect on rosette formation by lipase pretreated monocytes, indicating an enzymatic effect.     

Engineering host cell lines to reduce terminal sialylation of secreted antibodies

Covalently-linked glycans on proteins have many functional roles, some of which are still not completely understood. Antibodies have a very specific glycan modification in the Fc region that is required for mediating immune effector functions. These Fc glycans are typically highly heterogeneous in structure, and this heterogeneity is influenced by many factors, such as type of cellular host and rate of Ab secretion. Glycan heterogeneity can affect the Fc-dependent activities of antibodies. It has been shown recently that increased Fc sialylation can result in decreased binding to immobilized antigens and some Fcγ receptors, as well as decreased antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In contrast, increased Fc sialylation enhances the anti-inflammatory activity of antibodies. To produce antibodies with increased effector functions, we developed host cell lines that would limit the degree of sialylation of recombinantly-expressed antibodies. Towards this end, the catalytic domain of the Arthrobacter ureafaciens sialidase (sialidase A) was engineered for secreted expression in mammalian cell lines. Expression of this sialidase A gene in mammalian cells resulted in secreted expression of soluble enzyme that was capable of removing sialic acid from antibodies secreted into the medium. Purified antibodies secreted from these cells were found to possess very low levels of sialylation compared with the same antibodies purified from unmodified host cells. The low sialylated antibodies exhibited similar binding affinity to soluble antigens, improved ADCC activity, and they possessed pharmacokinetic properties comparable to their more sialylated counterparts. Further, it was observed that the amount of sialidase A expressed was sufficient to thoroughly remove sialic acid from Abs made in high-producing cell lines. Thus, engineering host cells to express sialidase A enzyme can be used to produce recombinant antibodies with very low levels of sialylation.Key words: antibodies, IgGs, glycans, oligosaccharides, sialic acid, sialidase, ADCC, CDC, effector functions, cells, Fc receptors, proteases     

Role of IgE receptors in effector function of human eosinophils

After analysis of the technical parameters of the rosette assay with human IgE-coated erythrocytes, Fc epsilon receptors for IgE (Fc epsilon R) on human peripheral blood eosinophils were compared to Fc epsilon R on lymphocytes and monocytes. Antibodies directed against Fc epsilon R on lymphocytes and monocytes inhibited the IgE rosettes formed by eosinophils from hypereosinophilic patients, which suggests that Fc epsilon R on eosinophils were antigenically related to Fc epsilon R on lymphocytes and monocytes. Fc epsilon R on human eosinophils were shown to participate in the killing effect of Schistosoma mansoni schistosomula in vitro in the presence of purified eosinophils from highly hypereosinophilic patients (blood counts greater than 3000/mm3) and anti-schistosomula IgE antibodies present in S. mansoni-infected patient sera. Similar levels of inhibition of cytotoxicity were obtained after preincubation of eosinophils with aggregated human IgE or with anti-Fc epsilon R antibodies, whereas preincubation with aggregated IgG or with anti-C3b receptor antibodies did not decrease the killing effect for schistosomula targets. This IgE-dependent cytotoxic capacity seemed restricted to eosinophils with an abnormally low density ("hypodense" cells) present only in highly hypereosinophilic patients. These observations might be related to nonparasitic situations in which increased levels of IgE and tissue or blood eosinophils are observed.     

Dimers and multimers of monoclonal IgG1 exhibit higher in vitro binding affinities to Fcγ receptors

The in vitro binding of monomeric, dimeric and multimeric forms of monoclonal IgG1 molecules, designated mAb1 and mAb2, to the extracellular domains of Fcγ receptors RI, RIIA and RIIIB were investigated using a surface plasmon resonance (SPR) based biosensor technique. Stable noncovalent and covalent dimers of mAb1 and mAb2, respectively, were isolated from CHO cell expressed materials. The dissociation constants of monomeric mAb1 and mAb2 were determined to be 1 nM for the FcγRI-binding and 6–12 μM for the FcγRIIA- and FcγRIIIB-binding. Dimeric mAb1 and mAb2 exhibited increased affinities, by 2-3 fold for FcγRI and 200-800 fold for FcγRIIA and FcγRIIIB. Further increases in binding were observed when the antibodies formed large immune complexes with multivalent antigens, but not in a linear relation with size. The binding properties of monomeric mAb2 were identical with and without a bound monovalent antigen, indicating that antigen-binding alone does not induce measurable change in binding of antibodies to Fcγ receptors. Dimerization is sufficient to show enhancement in the receptor binding. Given the wide distribution of the low-affinity Fcγ receptors on immune effector cells, the increased affinities to aggregated IgG may lead to some biological consequences, depending on the subsequent signal transduction events. The SPR-based in vitro binding assay is useful in evaluating Fcγ receptor binding of various species in antibody-based biotherapeutics.