IgG2a Monoclonal Antibody Research Articles

Protective, anti-tumor monoclonal antibody recognizes a conformational epitope similar to melibiose at the surface of invasive murine melanoma cells.

Polyclonal and monoclonal antibodies (MAbs) have been raised against B16F10 cells collected from growing tumors in vivo or grown in culture media supplemented with normal mouse serum to avoid xenogeneic reactivity. Antibody binding to glutaraldehyde-fixed melanoma cells and Melan A melanocytes was assayed using chemiluminescent-enzyme-linked immunosorbent assay (CL-ELISA) for increased sensitivity. Most of the reactivity of antitumor polyclonal IgG (92%) was inhibitable by a carbohydrate pool consisting of melibiose, mannose, lactose, and sialic acid. Two monoclonal IgG(2a) antibodies, A4 and B11, had their reactivity to melanoma cells completely and specifically inhibited by melibiose. MAb A4 did not bind to alpha-galactosyl residues abundantly expressed in a protozoan mucin used as substrate, and its binding to the tumor cells was not affected by alpha-galactosidase treatment or addition of alpha-methyl-galactopyranoside or raffinose. Recognition of a mimotope similar to melibiose is suggested. MAb is cytotoxic in vitro in a complement-mediated reaction and effectively neutralizes melanoma cells protecting syngeneic mice against tumor development in vivo. This MAb is thus an important tool for further studies on antitumor adjuvant therapy combined with other agents associated with immuno- and chemotherapy of invasive melanoma.

Monoclonal antibody to stage-specific fetal brain 68-kDa glycoprotein (FGP68) revealed increased FGP68 expression in human primary brain tumors.

We have produced a novel rat IgG(2a) monoclonal antibody against a stage-specific fetal brain glycoprotein of 68 kDa (FGP68), and succeeded in applying it to staining paraffin sections. To gain some insight into the pathobiological significance of this FGP68, this monoclonal antibody was used in immunohistochemical studies to compare the expression of FGP68 and Ki-67 antigen (MIB-1) in 235 primary brain tumors. Approximately half of the glioblastomas multiforme (GBMs) (44/75) and anaplastic astrocytomas (9/17) as well as some astrocytomas (5/30), medulloblastomas (2/14) and primitive neuroectodermal tumors (2/10) had tumor cells that expressed FGP68; however, pilocytic astrocytomas (0/7), oligodendrogliomas (0/15), ependymomas (0/6), schwannomas (0/21), meningiomas (0/22) and pituitary adenomas (0/18) did not express FGP68. The values of the MIB-1 labeling index were statistically higher in GBMs (0.005< P<0.01, Wilcoxon rank-sum test) and anaplastic astrocytomas (0.025< P<0.05) that expressed FGP68 than in those that did not. Normal brain tissue from 20 individuals aged 3-75 years was negative for FGP68 and MIB-1. We conclude that primary brain tumors express FGP68, one of the oncofetal proteins derived from fetal brain, and that FGP68 expression in certain brain tumor cells may depend, in part, on proliferation potential. Based on the possibility that the stage-specific FGP68 plays an important role in brain embryogenesis, some of FGP68-expressing tumor cells might phylogenetically revert to more primitive cells.

Inhibition of tumour growth and metastasis of human fibrosarcoma cells HT-1080 by monoclonal antibody BCD-F9

BCD-F9 is a murine IgG 2a monoclonal antibody (mAb) that recognises a conformational epitope found on the surface of many human tumour cells. The aim of this study was to investigate the ability of BCD-F9 to recognise a variety of neoplastic cell lines and to test BCD-F9 in vivo for anticancer activity in subcutaneous (s.c.) and metastatic tumour models. Intravenous (i.v.) administration of BCD-F9 in CD-1 nude mice xenografted s.c. with human HT-1080 cells led to a significant inhibition of tumour growth. We demonstrated that BCD-F9 administrated i.v. significantly prolonged the life-span of CD-1 nude mice inoculated i.v. with the same tumour cell line that induces aggressive lung metastases in the untreated mice. We also investigated the antitumour activity of BCD-F9 in vitro in antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ADCMC) assays. The effector cell subpopulations were obtained by macrophage stimulation (thioglycollate) or natural killer (NK) cell enrichment (negative selection). BCD-F9 was found to be effective in mediating tumour cell killing in vitro by ADCC and ADCMC mechanisms. These results suggest that the mAb BCD-F9 can have a potential use in immunotherapy for treatment of tumours of different origin.

Both Th1 and Th2 Cytokines Affect the Ability of Monoclonal Antibodies To Protect Mice againstCryptococcus neoformans

Variable-region-identical mouse immunoglobulin G1 (IgG1), IgG2b, and IgG2a monoclonal antibodies to the capsular polysaccharide of Cryptococcus neoformans prolong the lives of mice infected with this fungus, while IgG3 is either not protective or enhances infection. CD4+ T cells are required for IgG1-mediated protection, and CD8+ T cells are required for IgG3-mediated enhancement. Gamma interferon is required for both effects. These findings revealed that T cells and cytokines play a role in the modulation of cryptococcal infection by antibodies and suggested that it was important to more fully define the cytokine requirements of each of the antibody isotypes. We therefore investigated the efficacy of passively administered variable-region-identical IgG1, IgG2a, IgG2b, and IgG3 monoclonal antibodies against intravenous infection with C. neoformans in mice genetically deficient in interleukin-12 (IL-12), IL-6, IL-4, or IL-10, as well as in the parental C57BL/6J strain. The relative inherent susceptibilities of these mouse strains to C. neoformans were as follows: IL-12(-/-) > IL-6(-/-) > C57BL/6J approximately IL-4(-/-) >> IL-10(-/-). This is consistent with the notion that a Th1 response is necessary for natural immunity against cryptococcal infection. However, none of the IgG isotypes prolonged survival in IL-12(-/-), IL-6(-/-), or IL-4(-/-) mice, and all isotypes significantly enhanced infection in IL-10(-/-) mice. These results indicate that passive antibody-mediated protection against C. neoformans requires both Th1- and Th2-associated cytokines and reveal the complexity of the mechanisms through which antibodies modulate infection with this organism.

Relationship between complement activation and renal deposition of immune complexes made with IgG2a monoclonal antibodies.

Previous studies identified a single murine monoclonal IgG2a anti-dinitrophenyl antibody that, when combined with the antigen, formed immune complexes (IC) that were preferentially deposited in glomeruli. The present study examined the clearance and organ localization in Balb/c mice of expanded panels of radiolabeled IC containing murine monoclonal antibodies. The results identified a second IgG2a antibody that formed IC with a predilection for renal deposition. IC made with the two IgG2a antibodies that were preferentially deposited in the kidney were the least efficient binders of human C1q or homologous murine C3b and C4b within the IgG2a panel. These observations suggest a new model of IC-mediated renal disease initiation in which relatively weak complement activation leads to inefficient IC clearance by complement receptor-bearing circulating cells and consequent IC deposition in tissues susceptible to IC-mediated injury.

Monoclonal anti-double stranded DNA antibody is a leucocyte-binding protein to up-regulate interleukin-8 gene expression and elicit apoptosis of normal human polymorphonuclear neutrophils.

To determine whether anti-double stranded DNA (anti-dsDNA) autoantibody could bind and affect the functions of normal human polymorphonuclear neutrophils (PMN). Normal human PMN were incubated with different concentrations of a monoclonal mouse anti-dsDNA antibody (12B3) or mouse isotype-matched IgG2a. The binding of anti-dsDNA and PMN was measured by flow cytometry and interleukin-8 (IL-8) gene expression in PMN was detected by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). PMN apoptosis was justified by morphological changes. The cognate antigen(s) of anti-dsDNA on the PMN surface was identified by membrane biotinylation, immunoprecipitation and Western blot. The binding of PMN with anti-dsDNA was much higher than with non-specific mouse IgG2a (70.8 vs 2.0%). Anti-dsDNA at concentrations higher than 12.5 ng/ml significantly enhanced the production and mRNA expression of IL-8 by PMN. However, anti-dsDNA facilitated PMN apoptosis after 3 h incubation. Western blot analysis of biotinylated PMN cell lysates demonstrated that a 50-52 kDa membrane molecule is the cognate antigen of anti-dsDNA. Anti-dsDNA autoantibody up-regulates IL-8 gene expression and elicits activation-induced cell death (AICD) of human PMN via binding to a 50-52 kDa membrane-expressed molecule.

DNA and Heparin Alter the Internalization Process of Anti-DNA Monoclonal Antibodies According to Patterns Typical of Both the Charged Molecule and the Antibody

The internalization into CHO-K1 fibroblasts of three polyreactive monoclonal IgG2a anti-DNA autoantibodies (mAbs), F14.6, J20.8 and F4.1, isolated from the same unimmunized (NZB×NZW) F1 mouse, and synthetic peptides derived from F4.1 was studied using a technique which quantifies nuclear accumulation. The localization of the mAbs was intranuclear. We compared the influence of two negatively-charged molecules, DNA or heparin. At low concentrations, DNA had dual effects—inhibitory or stimulatory—depending on the mAb. Heparin was inhibitory or had no effect. The possibility that proteoglycans are ‘receptors’ recognized by anti-DNA mAbs which bind through heparin-sensitive reactions, was explored. Only F4.1 internalization was partly inhibited in glycosaminoglycan-deficient cells. We propose that the complex alterations of internalization patterns of these polyreactive mAbs by the two negatively charged molecules can be explained by (a) the potential of polyreactive mAbs to bind to various charge (or conformation-) dependent ‘receptors’, (b) the potential of a subclass of mAbs complexed with DNA to utilize additional ‘receptor(s)’. Glycosaminoglycans were required for internalization of F4.1-derived peptides, which remained extranuclear, suggesting that nuclear internalization of mAb F4.1 is a multistep process that requires certain sequences present on the intact mAb.

No evidence for a role of FcgammaRIIB in suppression of in vivo antibody responses to erythrocytes by passively administered IgG.

SETTING THE STAGEIn a paper in this issue of Scandinavian Journal ofImmunology, Sinclair addresses the role of Fc-signalling inthe modulation of immune responses by passive antibody [1].This is a broad field, encompassing aspects of immuneresponses from autoimmune disease models to cell signallingin vitro. A substantial part of the paper deals in a critical waywith recent results from our laboratory concerning the abilityof immunoglobulin (Ig)G to feedback-regulate in vivo anti-body responses in mice [2,3] and we will limit our reply tothis area. In a field as complicated as antibody feedbackregulation, it is important to define exactly what is discussed.Antibodies of many classes have the capacity to modulateantibody responses, either positively or negatively [4]. IgGhas a dual role, suppressing the response to particulateantigens such as erythrocytes [5–9], and enhancing theresponse to soluble antigens such as keyhole limpethemocyanine (KLH), bovine serum albumin (BSA), andovalbumin (OVA) [3,9–11]. The same monoclonal IgG anti-trinitriphenyl (anti-TNP) can suppress or enhance, dependingon whether TNP is conjugated to an erythrocyte or a solubleantigen [9,11]. Considering the strikingly different structure ofthese two types of immune complexes this is not surprising.While an IgG molecule is much smaller than an erythrocyte(10 nm versus 7 mm in diameter), IgG is on average twice aslarge as BSA. When discussing IgG-mediated immunoregula-tion, it is therefore vital to keep apart studies using particulateversus soluble antigens. Major differences between in vitroand in vivo systems for studying antibody responses exist(e.g., lack of lympoid organ structure in vitro) and unlessspecifically stated, only in vivo systems will be consideredhere.NO EVIDENCE FOR A ROLE OF FCgRIIB INSUPPRESSION OF IN VIVO ANTIBODYRESPONSES TO SRBC BY PASSIVELYADMINISTERED IgGWhy IgG has the capacity to completely inhibit a response toSRBC is a question that has been debated extensively over theyears. Hypotheses put forward are: (i) epitope masking by IgG,preventing recognition of the antigen by B cells, (ii) effectiveelimination of IgG-coated antigen via Fc-dependent phago-cytosis, and (iii) central inhibition of B-cell activation viacocrosslinking of the B-cell receptor and the inhibitoryFcgRIIB. Sinclair argues that the mechanism behind the IgG-mediated suppression of erythrocyte-specific responses wasresolved 30 years ago and that his so called TripartiteInactivation Model ((iii) see above) explains the phenomenon[12]. Some textbooks and reviews also state for a fact that themechanism behind the IgG-mediated suppression has beenproven to be FcgRIIB-mediated negative signalling to B cells.We do not share this opinion. Although this receptorundoubtedly plays an inhibitory role in many experimentalsystems, there are many observations calling for scepticismabout its role in inhibition of erythrocyte responses by passivelyadministered IgG. Firstly, data regarding the Fc-dependence ofsuppression are conflicting. The efficiency of F(ab

Update of clinical trials with edrecolomab: A monoclonal antibody therapy for colorectal cancer

Edrecolomab is a murine IgG2a monoclonal antibody that recognizes the tumor-associated antigen Ep-CAM. Its antitumor effects are mediated through antibody-dependent cellular cytotoxicity, complement-mediated cytolysis, and the induction of an anti-idiotypic network. An initial study of 189 patients with resected stage III colorectal cancer showed that edrecolomab reduced the relative risk of mortality by 32% compared with observation alone (P < .01). Edrecolomab has now been investigated in two large phase III studies of patients with stage III colon cancer, either as a single agent or in combination with 5-FU-based chemotherapy. Preliminary safety data have shown that edrecolomab is well tolerated when used as monotherapy and adds little to chemotherapy-related side effects when used in combination. Edrecolomab is also being studied as monotherapy following resection of stage II colon cancer, and in combination with chemotherapy in patients with resected stage II or III rectal cancer. In conclusion, edrecolomab is a novel biological therapy for the adjuvant treatment of colorectal cancer. Completed and ongoing trials may support its use as monotherapy in stage II colon cancer or in combination with chemotherapy in stage III colon cancer and stage II/III rectal cancer.

Transfer of idiotypic protein primed allogeneic marrow grafts elicits potent graft-versus-myeloma effects in mice.

The active immunization of bone marrow (BM) donors with myeloma immunoglobulin (Ig) results in an idiotypic T cell response that can be transferred to the recipient. Using a murine model we evaluated the effectiveness, side-effects and underlying mechanisms of this approach. Balb/c (H-2d) mice were given a dose of HOPC-1F myeloma cells secreting the monoclonal IgG2a followed by lethal total body irradiation (7.5 Gy) 2 days later and a subsequent transplantation of 2 x 10(7) allogeneic MHC-matched DBA/2-derived marrow cells. Donors were pre-immunized with three i.p. injections of HOPC(IgG2a) or control Ig given with incomplete Freund's adjuvants (IFA) spaced 1 week apart. In some experiments, donor-spleen cells were additionally transferred 2 h post transplant. Injection of HOPC-myeloma led to death of all animals after a median survival time (MST) of 42 days. A lethal dose of TBI followed by transfer of unmanipulated marrow grafts plus splenocytes resulted in moderate antimyeloma effects with 8% of mice achieving long-term survival. Nearly the same results were obtained after transplantation of BM immunized with the control Ig. In contrast, transplantation of marrow grafts from HOPC(IgG2a) immunized donors exerted a significant GVM effect with 63% long-term survival for more than 180 days. The additional transfer of 2 x 10(7) immune splenocytes derived from the same donor resulted in even stronger anti-myeloma effects (FFR 87%). No increase in the incidence of severe acute GVHD was observed. In vitro data suggest that allogeneic CD8+ idiotype-specific T cells may be the major effector cells. Our results demonstrate that active immunization of the donor with the myeloma-specific Ig can induce powerful graft-versus-myeloma effects after allogeneic BMT.

Characterization of monoclonal antibody MEST-2 specific to glucosylceramide of fungi and plants.

An IgG2a monoclonal antibody anti-glucosylceramide was established and termed MEST-2. High performance thin layer chromatography immunostaining, and solid-phase radioimmunoassay showed that MEST-2 reacts with glucosylceramide from yeast and mycelium forms of Paracoccidioides brasiliensis, Histoplasma capsulatum, and Sporothrix schenckii; from hyphae of Aspergillus fumigatus; and from yeast forms of Candida albicans, Cryptococcus neoformans, Cryptococcus laurentii, and Cryptococcus albidus. Studies on the fine specificity of MEST-2 showed that it recognizes the beta-D-glucose residue, and that the 2-hydroxy group present in the fatty acid is an important auxiliary feature for the antibody binding. It was also demonstrated that phosphatidylcholine and ergosterol modulate MEST-2 reactivity to glucosylceramide, by solid-phase radioimmunoassay. Indirect immunofluorescence showed that MEST-2 reacts with the surface of yeast forms of P. brasiliensis, H. capsulatum and S. schenckii. Weak staining of mycelial forms of P. brasiliensis and hyphae of A. fumigatus was also observed. The availability of a monoclonal antibody specific to fungal glucosylceramide, and its potential use in analyzing biological roles attributed to glucosylceramide in fungi are discussed.

Divergent expression of midkine in the human fetal liver and kidney: immunohistochemical analysis of developmental changes in hilar primitive bile ducts and hepatocytes

Midkine (MK) is a novel heparin-binding growth factor whose gene has been identified in embryonal carcinoma cells in early stages of retinoic acid-induced differentiation. In this study, we investigated the developmental expression of MK protein in the human fetal liver and kidney. Twenty-one specimens each of the liver and kidney from fetuses (gestational weeks from 9 to 40) and neonates less than 4 weeks old were examined. Immunohistochemical and Western blot analyses were performed using a rat IgG2a monoclonal antibody against the carboxyl terminal region of human MK. Immunohistochemical analysis revealed MK expression in the human fetal liver and kidney. The MK expression in the fetal liver showed a strong reaction from 9 to 16 gestational weeks. MK was expressed in the ductal plate, migrating biliary cells and newly formed bile ducts, and in hepatocytes of the hilar region in all specimens in the first and second trimesters. By contrast, the MK expression decreased gradually and was weak or not detected in the third trimester and neonatal period. However, MK expression in the kidney was found at 16 gestational weeks, as well as during both gestation and the neonatal period. Divergent MK-expression was detected in the human fetal liver and kidney, and its expression may be related to fetal development, maturation, and functions of the liver and kidney.

131I-Lym-1 in mice implanted with human Burkitt's lymphoma (Raji) tumors: loss of tumor specificity due to radiolysis.

Preliminary evaluations of 125I-labeled Lym-1, an anti-lymphoma mouse IgG2a monoclonal antibody, demonstrated favorable tumor uptake in mice bearing human Burkitt's lymphoma (Raji) tumors. In this study, the pharmacokinetics of 125I- and 131I-Lym-1, and the dosimetry, efficacy, and toxicity of 131I-Lym-1 in Raji-tumored mice were evaluated. Lym-1 was radioiodinated by the chloramine-T method and analyzed for monomeric fraction and immunoreactivity (antigen cell binding, relative to unmodified Lym-1). Nude mice bearing Raji tumors (20-500 mm3) received 1.5 MBq (40 microCi) 125I-Lym-1, or 1.5, 7.4, 14.8, or 18.5 MBq (40, 200, 400, or 500 microCi) 131I-Lym-1. Pharmacokinetic data (total body and blood clearance and biodistribution) were used to estimate radiation dosimetry. Mini-thermoluminescent dosimetry (TLD) was also used to measure radiation dosimetry directly for 7 days after injection of 131I-Lym-1. Tumor size, survival, body weight, and blood counts were monitored for 60 days to evaluate therapeutic efficacy and toxicity of 131I-Lym-1. At the time of injection, the mean quality assurance (QA) values for 125I-Lym-1 were 100% monomer and 100% relative immunoreactivity; the corresponding values for 131I-Lym-1 were 73% and 66%, indicating that radiolysis had occurred during the interval between radiolabeling and injection. 125I-Lym-1 exhibited high and sustained concentration in tumors relative to normal organs, whereas 131I-Lym-1 did not. Assuming identical pharmacokinetic behavior to 125I-Lym-1, 131I-Lym-1 would deliver radiation doses of 3.45, 0.83, 1.03, 0.34, and 0.56 Gy per MBq injected (12.8, 3.1, 3.8, 1.3, and 2.1 rad/microCi), to tumor, liver, lungs, total body, and marrow, respectively. When the actual pharmacokinetic data for 131I-Lym-1 (1.5 MBq) were used to estimate dosimetry, corresponding values of 0.51, 0.72, 0.49, 0.31, and 0.41 Gy/MBq (1.9, 2.7, 1.8, 1.1, and 1.5 rad/microCi) were obtained. Similar values were obtained for mice receiving 7.4 or 14.8 MBq of 131I-Lym-1. Similarly, TLD data indicated little preferential radiation dosimetry to tumor. Response rates (cure + CR + PR) for mice receiving 0, 7.4, 14.8, and 18.5 MBq of 131I-Lym-1 were 8%, 7%, 21%, and 45%, respectively. The LD50/30 dose of 131I-Lym-1 was 12.7 MBq (343 microCi). 125I-Lym-1 exhibited high and sustained concentration in Raji tumors in mice, indicating excellent therapeutic potential for 131I-Lym-1. However, in vitro QA results for 131I-Lym-1 indicated that radiolysis had occurred, and 131I-Lym-1 demonstrated little accumulation in tumor, or preferential radiation dosimetry to tumor in the same model.

Immunohistochemical and In Situ Hybridization Analyses of Midkine Expression in Thyroid Papillary Carcinoma

Midkine (MK) is a novel heparin-binding growth factor whose gene has been identified in embryonal carcinoma cells in early stages of retinoic acid-induced differentiation. We immunohistochemically examined 90 thyroid papillary carcinomas (85 invasive type and five encapsulated type), using a rat IgG2a monoclonal antibody against the carboxyl terminal region of human MK in archival paraffin sections. The thyroid tumors exhibited an intense reaction in the cytoplasm. Most of the papillary carcinomas (77/90), had tumor cells that expressed MK. These were classified into the following two types: invasive type (76/85) and encapsulated type (1/5). Notably, the intensity of MK was stronger at the invading border area of the tumors than in the center. In tissues adjacent to the cancer tissues, normal follicular epithelial cells expressed MK very faintly or not at all. The in situ hybridization analysis revealed that the signals of MK transcripts were found in the cytoplasm of the cancer cells. In the noncancerous follicular epithelial cells adjacent to neoplasm the signals of MK transcripts were detected very weakly or not at all. The distribution and localization of the MK-transcript signals determined by in situ hybridization analysis were similar to those obtained by immunohistochemical analysis. We conclude that thyroid papillary carcinoma strongly expresses MK protein and messenger RNA, and that this overexpression may relate to the development and invasion of these carcinomas.

Increased midkine expression in intrahepatic cholangiocarcinoma: immunohistochemical and in situ hybridization analyses.

Midkine (MK) is a novel heparin-binding growth factor whose gene was identified in embryonal carcinoma cells in the early stages of retinoic acid-induced differentiation. This study investigates the overexpression of MK in intrahepatic cholangiocarcinoma (CC). Forty-five primary CC specimens from patients (aged 19-81 years, 24 males and 21 females) were examined. Histologically, 17 cases of CC were classified as the well-differentiated type, 19 as moderately-differentiated and 9 as poorly-differentiated. Immunohistochemical analysis was performed using a rat IgG2a monoclonal antibody against the carboxyl terminal region of human MK. We successfully applied this monoclonal antibody against MK to analyze archival paraffin sections. The cancer tissues showed a positive reaction to this antibody, and there was an intense reaction in their cytoplasm. Approximately 40% of individuals with CC (17/45) had tumor cells that expressed MK, and these were classified into the following types: moderately-differentiated type (9/19), well-differentiated type (8/17) and poorly-differentiated type (0/ 9). In situ hybridization analysis revealed that signals of MK transcripts were found in the cytoplasm of the cancer cells; the distribution and localization of the MK-transcript signals determined by in situ hybridization analysis were similar to those obtained by immunohistochemical analysis. These findings revealed that CC express increased MK at the messenger RNA and protein levels.

Low frequency vibrations and structural characterization of a murine IgG2a monoclonal antibody studied by Raman and IR spectroscopies

The Raman and ir spectra of a murine IgG2a monoclonal antibody molecule are reported. In accordance with previous studies on immunoglobulins, the secondary structure is predominantly of the β-sheet type. The low frequency region of the Raman spectrum was also analyzed in detail. A structured band with two maxima near 43 and 94 cm−1 was observed. This band has been attributed to vibrations of elastic body. The results are interpreted using a simple elastic model. © 2000 John Wiley & Sons, Inc. Biopoly 53: 342–349, 2000

Low frequency vibrations and structural characterization of a murine IgG2a monoclonal antibody studied by raman and IR spectroscopies.

The Raman and ir spectra of a murine IgG2a monoclonal antibody molecule are reported. In accordance with previous studies on immunoglobulins, the secondary structure is predominantly of the beta-sheet type. The low frequency region of the Raman spectrum was also analyzed in detail. A structured band with two maxima near 43 and 94 cm(-1) was observed. This band has been attributed to vibrations of elastic body. The results are interpreted using a simple elastic model.

Glomerular deposition of immune complexes made with IgG2a monoclonal antibodies.

The factors that determine whether immune complexes (IC) are cleared safely from the circulation or are deposited in vulnerable tissues such as glomeruli are not well defined. To better understand how IC are handled, the present study examined the fate in vivo of three model IC preparations with different immunochemical characteristics. Radiolabeled IC were constructed with murine IgG1, IgG2a, or IgG3 anti-DNP mAbs bound to DNP-BSA, designated IgG1 IC, IgG2a IC, and IgG3 IC, respectively. The IC were infused i.v. into BALB/c mice, and clearance and tissue localization of the three IC probes were compared. The results indicate that the major portion of each IC preparation was cleared from the circulation by the liver. However, compared with the other two probes, IgG2a IC were preferentially deposited in the kidney. Histologic examination revealed the presence of IgG2a IC in glomeruli. The enhanced renal uptake of IgG2a IC could not be attributed solely to such characteristics as IC size, Ag/Ab ratio, Ab charge, or affinity. However, the preferential renal deposition of IgG2a IC was abrogated by complement depletion. Thus, enhanced renal uptake in normal mice was complement dependent. These data suggest that interactions between IC and the complement system can influence the propensity of IC to deposit in tissues susceptible to IC-mediated injury.

High pathogenic potential of low-affinity autoantibodies in experimental autoimmune hemolytic anemia.

To assess the potency of low-affinity anti-red blood cell (RBC) autoantibodies in the induction of anemia, we generated an immunoglobulin (Ig)G2a class-switch variant of a 4C8 IgM anti-mouse RBC autoantibody, and compared its pathogenic potential with that of its IgM isotype and a high-affinity 34-3C IgG2a autoantibody. The RBC-binding activity of the 4C8 IgG2a variant was barely detectable, at least 1,000 times lower than that of its IgM isotype, having a high-binding avidity, and that of the 34-3C IgG2a monoclonal antibody (mAb). This low-affinity feature of the 4C8 mAb was consistent with the lack of detection of opsonized RBCs in the circulating blood from the 4C8 IgG2a-injected mice. However, the 4C8 IgG2a variant was highly pathogenic, as potent as its IgM isotype and the 34-3C IgG2a mAb, due to its capacity to interact with Fc receptors involved in erythrophagocytosis. In addition, our results indicated that the pentameric form of the low-affinity IgM isotype, by promoting the binding and agglutination of RBCs, is critical for its pathogenic activity. Demonstration of the remarkably high pathogenic potency of low-affinity autoantibodies, if combined with appropriate heavy chain effector functions, highlights the critical role of the Ig heavy chain constant regions, but the relatively minor role of autoantigen-binding affinities, in autoimmune hemolytic anemia.

IgG-Mediated Enhancement of Antibody Responses Is Low in Fc Receptor γ Chain-Deficient Mice and Increased in FcγRII-Deficient Mice

AbstractImmunization with IgG/Ag or IgE/Ag complexes leads to a higher production of specific Abs than immunization with Ag alone. The enhancing effect of IgE is exclusively dependent upon the low-affinity receptor for IgE, FcεRII, whereas the mechanism behind IgG-mediated enhancement is unknown. We have investigated whether receptors for the Fc part of IgG are required for responses to IgG/Ag. Mice lacking the γ subunit of Fc receptors (FcRs) (FcRγ−/−), FcγRII (FcγRII−/−), or FcγRIII (FcγRIII−/−) were immunized with BSA-2,4,6-trinitrophenyl (TNP) alone or BSA-TNP complexed to monoclonal TNP-specific IgG1, IgG2a, or IgG2b. As expected, all subclasses enhanced the Ab-response to BSA in wild-type mice. Enhancement was in the same order of magnitude in FcγRIII−/− mice (≤177-fold of controls administered Ag alone), whereas it was abrogated in FcRγ−/− mice and augmented in FcγRII−/− mice (≤5147-fold of controls). The response to IgE/Ag complexes in FcRγ−/− and FcγRII−/− mice was similar to that seen for wild-type mice, demonstrating that non-FcγR-dependent responses were normal. Our observations suggest that IgG/Ag complexes enhance Ab responses via FcγRs. Moreover, they reveal a strong negative regulation of Ab responses to IgG/Ag exerted by FcγRII.