mAb 3G8 Research Articles

In vitro elimination of epidermal growth factor receptor‐overexpressing cancer cells by CD32A‐chimeric receptor T cells in combination with cetuximab or panitumumab

Cetuximab and panitumumab bind the human epidermal growth factor receptor (EGFR). Although the chimeric cetuximab (IgG1) triggers antibody-dependent-cellular-cytotoxicity (ADCC) of EGFR positive target cells, panitumumab (a human IgG2) does not. The inability of panitumumab to trigger ADCC reflects the poor binding affinity of human IgG2 Fc for the FcγRIII (CD16) on natural killer (NK) cells. However, both human IgG1 and IgG2 bind the FcγRII (CD32A) to a similar extent. Our study compares the ability of T cells, engineered with a novel low-affinity CD32A131R -chimeric receptor (CR), and those engineered with the low-affinity CD16158F -CR T cells, in eliminating EGFR positive epithelial cancer cells (ECCs) in combination with cetuximab or panitumumab. After T-cell transduction, the percentage of CD32A131R -CR T cells was 74 ± 10%, whereas the percentage of CD16158F -CR T cells was 46 ± 15%. Only CD32A131R -CR T cells bound panitumumab. CD32A131R -CR T cells combined with the mAb 8.26 (anti-CD32) and CD16158F -CR T cells combined with the mAb 3g8 (anti-CD16) eliminated colorectal carcinoma (CRC), HCT116FcγR+ cells, in a reverse ADCC assay in vitro. Crosslinking of CD32A131R -CR on T cells by cetuximab or panitumumab and CD16158F -CR T cells by cetuximab induced elimination of triple negative breast cancer (TNBC) MDA-MB-468 cells, and the secretion of interferon gamma and tumor necrosis factor alpha. Neither cetuximab nor panitumumab induced Fcγ-CR T antitumor activity against Kirsten rat sarcoma (KRAS)-mutated HCT116, nonsmall-cell-lung-cancer, A549 and TNBC, MDA-MB-231 cells. The ADCC of Fcγ-CR T cells was associated with the overexpression of EGFR on ECCs. In conclusion, CD32A131R -CR T cells are efficiently redirected by cetuximab or panitumumab against breast cancer cells overexpressing EGFR.

Fine epitope mapping of monoclonal antibodies 9B9 and 3G8 to the N domain of angiotensin-converting enzyme (CD143) defines a region involved in regulating angiotensin-converting enzyme dimerization and shedding

A panel of monoclonal antibodies (mAbs) raised against both the N and C domains of angiotensin-I-converting enzyme (ACE, peptidyl dipeptidase, EC 3.4.15.2) have been extensively mapped and have facilitated the study of various aspects of ACE structure and biology. In this study, we characterize two mAbs, 9B9 and 3G8, that recognize the N domain of ACE and that influence shedding and dimerization. Fine epitope mapping was performed, which mapped the epitopes for these mAbs to the N terminal region of the N domain where they overlap to a large extent, despite having different effects on ACE processing. The mAb 3G8 epitope appears to be shielded by the C domain and to be carbohydrate dependent as binding increased significantly as a result of underglycosylation, whereas these factors did not influence mAb 9B9 recognition. Three mutations within the overlapping region of these two epitopes, Q18H, L19E, and Q22A, which decreased mAb 3G8 binding to the soluble N domain, were introduced into full-length somatic ACE (sACE) to determine their influence on ACE expression and processing. Increased ACE expression, cell surface expression, and basal shedding were observed with all three mutations. Furthermore, cross-linking and western blotting of Chinese hamster ovary (CHO) cell lysates detected two distinct ACE dimers, a native and cross-linked dimer. Increasing amounts of the cross-linked dimer were observed for the mutant sACEQ22A, further implicating the overlapping region of the mAb 9B9 and 3G8 epitopes in ACE processing.

Ofatumumab, a Human Mab Targeting a Membrane-Proximal Small-Loop Epitope On CD20, Induces Potent NK Cell-Mediated ADCC.

Abstract 1725Poster Board I-751 IntroductionCD20 represents a well established target for immunotherapy of B-cell malignancies such as follicular lymphoma (FL). The standard therapy for FL is the monoclonal antibody (mAb) rituximab given as a single agent or combined with chemotherapy. Antibody-dependent cytotoxicity (ADCC) has been suggested to be an important in vivo mechanism of action of CD20 mAb as differences in response rates to rituximab exist between individuals expressing different allotypes of the polymorphic Fc receptor FcgRIIIa. Individuals homozygous for the FcgRIIIa 158V allotype respond significantly better than individuals expressing the FcgRIIIa 158F allotype. Ofatumumab, a unique human mAb targeting a novel membrane-proximal small-loop epitope on CD20, has been shown to be associated with highly efficient killing of primary tumor cells through complement-dependent cytotoxicity. Here we studied the ability of ofatumumab to induce ADCC by NK cells from FcgRIIIa 158V and 158F homozygous healthy donors and compared it to that of rituximab. MethodsBlood was drawn (with informed consent) from ten healthy volunteers homozygous for FcgRIIIa 158V, and ten homozygous for FcgRIIIa 158F, selected from a panel of 479 donors and matched for age, sex and race. NK cells were purified, after which we assessed Fc-mediated antibody binding to NK cells, and ADCC, in a blinded study. Glycan profiling was performed for ofatumumab and rituximab by HPAEC-PAD analysis. MAb binding was measured by flow cytometry in a competition binding assay in which binding of FITC-labelled CD16 mAb 3G8 was blocked by the binding of ofatumumab or rituximab. ADCC was assessed by measuring Europium release from the B cell line ARH77 target cells upon incubation with a concentration curve of ofatumumab or rituximab in the presence of NK effector cells (effector:target ratio 5:1).Statistical analysis was performed using a non-linear mixed effect model fitting sigmoidal concentration-responses curves to data from each donor for each antibody. The model provided estimates of mean EC50 for each antibody and donor group, and allowed differences between these means to be tested for statistical significance. ResultsBiochemical analysis of clinical grade ofatumumab and rituximab batches indicated their quality to be comparable with similar amounts of aggregates present. The level of core-fucosylation was determined to be similar and ranged from 4 – 6 % for both mAbs. Monomeric ofatumumab bound more strongly to NK cells expressing FcgRIIIa 158 V/V (EC50: 900 mg/ml [95% CI 680-1200 mg/ml]) than FcgRIIIa 158 F/F (EC50: 3970 mg/ml [95% CI 2940-5370 mg/ml]). The approximately 4.4 fold difference in affinity was statistically significant (p<0.0001). A similar 4.2 fold difference (p<0.0001) was found for rituximab (FcgRIIIa 158 V/V EC50: 1370 mg/ml [95% CI 1020-1850 mg/ml] and FcgRIIIa 158 F/F EC50: 5720 mg/ml [95% CI 4170-7850 mg/ml]). Rituximab bound 1.4 fold less tightly to both FcgRIIIa allotypes than ofatumumab (p<0.0001).Ofatumumab induced a potent NK-mediated ADCC with both FcgRIIIa 158V/V expressing (EC50: 6.4 ng/ml [95% CI 4.8-8.5 ng/ml]) as well as FcgRIIIa 158 F/F expressing NK cells (EC50: 17.6 ng/ml [95% CI 13.4-23.1 ng/ml]). The observed ∼2.7 fold difference in potency was significant (p< 0.0001). Notably, higher concentrations of rituximab compared to ofatumumab were required to induce ADCC by NK cells obtained from both types of donors. Thus, rituximab induced ADCC by FcgRIIIa 158V/V and 158F/F expressing NK cells at EC50s of 12 ng/ml [95% CI 11-13 ng/ml] and 31 ng/ml [95% CI 28-34 ng/ml], respectively. This 1.8 fold difference in ADCC potency between ofatumumab and rituximab was statistically significant (p<0.0001). ConclusionsWe assessed FcgRIIIa affinity and potency to induce ADCC by purified NK cells for ofatumumab and rituximab in a blinded study. Expected differences in affinity for the 158V and 158F allotypes of FcgRIIIa were observed for both ofatumumab and rituximab. These differences correlated with a stronger ADCC by FcgRIIIa 158V/V compared to 158F/F expressing NK cells. Significantly, ofatumumab was able to induce ADCC more potently than rituximab for both Fc receptor allotypes. Ofatumumab binds CD20 stably and at a distinct membrane-proximal epitope compared to rituximab. Our data suggest that these binding characteristics may positively impact ofatumumab's ability to direct killing of tumor cells via ADCC. DisclosuresCraigen:GSK: Employment, Equity Ownership. Mackus:Genmab: Employment, Equity Ownership. Engleberts:Genmab: Employment, Equity Ownership. Miller:GSK: Employment, Equity Ownership. Speller:GSK: Employment, Equity Ownership. Chamberlain:GSK: Employment, Equity Ownership. Davis:GSK: Employment, Equity Ownership. McHugh:GSK: Employment, Equity Ownership. Bullmore:GSK: Employment, Equity Ownership. Cox:GSK: Employment, Equity Ownership. Wetten:GSK: Employment, Equity Ownership. Perdock:Genmab: Employment, Equity Ownership. Bakker:Genmab: Employment. van de Winkel:Genmab: Employment, Equity Ownership. Parren:Genmab: Employment.

FcγRIIA and FcγRIIIB Mediate Nuclear Factor Activation through Separate Signaling Pathways in Human Neutrophils

Receptors for IgG Abs (Fcgamma receptors) are capable of triggering diverse cell responses in leukocytes. In neutrophils, two Fcgamma receptors, namely FcgammaRIIA and FcgammaRIIIB, are constitutively expressed. The signaling pathways that regulate FcgammaRIIA-mediated phagocytosis have been relatively well described. However, the different signaling pathways that lead to NF activation after engagement of each Fcgamma receptor have only been partially described. To address this problem, neutrophils were stimulated by cross-linking selectively each type of Fcgamma receptor with specific mAbs, and NF activation was then analyzed. FcgammaRIIIB, but not FcgammaRIIA, promoted a robust increase in phosphorylated ERK in the nucleus, and also efficient phosphorylation of the NF Elk-1. Complete mAb 3G8 (anti-FcgammaRIIIB) induced a higher response than did F(ab')(2) fragments of mAb 3G8, suggesting a possible synergistic effect of both FcgammaR receptors. However, mAb IV.3 (anti-FcgammaRIIA) alone did not cause an increase of phosphorylated ERK in the nucleus. FcgammaRIIIB-induced nuclear phosphorylation of ERK, and of Elk-1, was not affected by Syk, PI3K, or MEK inhibitors. In contrast, FcgammaRIIA- or FcgammaRIIIB-mediated phosphorylation of cytoplasmic ERK depended on Syk, PI3K, and MEK. Also, ERK, but not MEK, was constitutively present in the nucleus, and FcgammaRIIIB cross-linking did not increase the levels of nuclear ERK or MEK. These data clearly show that different neutrophil Fcgamma receptors possess different signaling capabilities. FcgammaRIIIB, but not FcgammaRIIA, activates a unique signaling pathway leading to the nuclear-restricted phosphorylation of ERK and Elk-1, independently of Syk, PI3K, or MEK.

FcγRIIIa Expression Is Not Increased on Natural Killer Cells Expressing the FcγRIIIa-158V Allotype

The presence of a valine (V) versus a phenylanaline (F) at position 158 of Fc gamma RIIIa/CD16a improves the affinity for IgG and is associated with higher therapeutic response to rituximab. Increased CD16 expression on natural killer (NK) cells from donors with the VV or VF versus FF genotype has recently been reported. We indeed observed higher binding of the anti-CD16 monoclonal antibody (mAb) 3G8 on NK cells from V carriers (VV = VF > FF). However, the binding of two other anti-CD16 mAbs, LNK16 and DJ130c, decreased with the number of V allele (VV < VF < FF). CD16 transcript levels were independent on the genotype. Rituximab binding to NK cells from V carriers was higher than its binding to FF NK cells at low concentrations (10 and 100 microg/mL). However, the difference was nearly completely abolished at saturating concentrations (>or=1,000 microg/mL). Finally, nearly 100% of CD16-expressing NK cells displayed a complete down-modulation of the receptor after optimal engagement by plate-bound 3G8, whatever the genotype. By contrast, the percentages of NK cells down-modulating CD16 after competitive engagement of the receptor by plate-bound rituximab increased with the number of V allele (FF, 18.2 +/- 8.6%; VF, 32.0 +/- 4.9%; and VV, 42.4 +/- 9.9%). These results are in discrepancy with the expected increased competition that would result from an increased expression of CD16 on VV and VF NK cells. We conclude that increased binding and functional and clinical responses associated with the high-affinity Fc gamma RIIIa-158V are unrelated to an increased expression of this allotype.

IgG Fc Receptor III Homologues in Nonhuman Primate Species: Genetic Characterization and Ligand Interactions

Ig Fc receptors bind to immune complexes through interactions with the Fc regions of specific Ab subclasses to initiate or inhibit the defense mechanisms of the leukocytes on which they are expressed. The mechanism of action of IgG-based therapeutic molecules, which are routinely evaluated in nonhuman primate models, involves binding to the low-affinity FcRIII (CD16). The premise that IgG/CD16 interactions in nonhuman primates mimic those present in humans has not been evaluated. Therefore, we have identified and characterized CD16 and associated TCR zeta-chain homologues in rhesus macaques, cynomolgus macaques, baboons, and sooty mangabeys. Similar to humans, CD16 expression was detected on a lymphocyte subpopulation, on monocytes, and on neutrophils of sooty mangabeys. However, CD16 was detected only on a lymphocyte subpopulation and on monocytes in macaques and baboons. A nonhuman primate rCD16 generated in HeLa cells interacted with human IgG1 and IgG2. By contrast, human CD16 binds to IgG1 and IgG3. As shown for humans, the mAb 3G8 was able to block IgG binding to nonhuman primate CD16 and inhibition of nonhuman primate CD16 N-glycosylation enhanced IgG binding. Clearly, differences in interaction with IgG subclasses and in cell-type expression should be considered when using these models for in vivo evaluation of therapeutic Abs.

Localization of an N-Domain Region of Angiotensin-Converting Enzyme Involved in the Regulation of Ectodomain Shedding Using Monoclonal Antibodies

ACE chimeric proteins and N domain monoclonal antibodies (mAbs) were used to determine the influence of the N domain, and particular regions thereof, on the rate of ACE ectodomain shedding. Somatic ACE (having both N and C domains) was shed at a rate of 20%/24 h. Deletion of the C domain of somatic ACE generated an N domain construct (ACEDeltaC) which demonstrated the lowest rate of shedding (12%). However, deletion of the N domain of somatic ACE (ACEDeltaN) dramatically increased shedding (212%). Testicular ACE (tACE) having 36 amino acid residues (heavily O-glycosylated) at the N-terminus of the C domain shows a 4-fold decrease in the rate of shedding (49%) compared to that of ACEDeltaN. When the N-terminal region of the C domain was replaced with the corresponding homologous 141 amino acids of the N domain (N-delACE) the rate of shedding of the ACEDeltaN was only slightly decreased (174%), but shedding was still 3.5-fold more efficient than wild-type testicular ACE. Monoclonal antibodies specific for distinct, but overlapping, N-domain epitopes altered the rate of ACE shedding. The mAb 3G8 decreased the rate of shedding by 30%, whereas mAbs 9B9 and 3A5 stimulated ACE shedding 2- to 4-fold. Epitope mapping of these mAbs in conjunction with a homology model of ACE N domain structure, localized a region in the N-domain that may play a role in determining the relatively low rate of shedding of somatic ACE from the cell surface.

Epitope-dependent blocking of the angiotensin-converting enzyme dimerization by monoclonal antibodies to the N-terminal domain of ACE: possible link of ACE dimerization and shedding from the cell surface.

In a biomembrane modeling system, reverse micelles, somatic ACE forms dimers via carbohydrate-mediated interaction, providing evidence for the existence of a carbohydrate-recognizing domain on the ACE molecule. We localized this putative region on the N-domain of ACE using monoclonal antibodies (mAbs) to seven different epitopes of ACE. Two mAbs, 9B9 and 3G8, directed to distinct, but overlapping, epitopes of the N-domain of ACE shielded the CRD. Only "simple" ACE-antibody complexes were found in the system. Five mAbs allowed the formation of "double" antibody-ACE-ACE-antibody complexes via carbohydrate-mediated interactions. The results were confirmed using the ACE N- and C-domains. Testicular ACE was unable to form carbohydrate-mediated ACE dimers in the reverse micelles, while the N-domain of ACE, obtained by limited proteolysis of the parent full-length ACE, retained the ability to form dimers. Furthermore, mAb 3G8, which blocked ACE dimerization in micelles, significantly inhibited ACE shedding from the surface of ACE-expressing cells. Galactose prevented ACE dimerization in reverse micelles and also affected antibody-induced ACE shedding in an epitope-dependent manner. Restricted glycosylation of somatic ACE, obtained by the treatment of CHO-ACE cells with the glucosidase inhibitor N-butyldeoxynojirimycin, significantly increased the rate of basal ACE shedding and altered antibody-induced ACE shedding. A chemical cross-linking approach was used to show that ACE is present (at least in part) as noncovalently linked dimers on the surface of CHO-ACE cells. These results suggest a possible link between putative ACE dimerization on the cell surface and the proteolytic cleavage (shedding) of ACE.

Epitope-specific antibody-induced cleavage of angiotensin-converting enzyme from the cell surface

Angiotensin I-converting enzyme (ACE; CD143, EC 3.4.15.1) is a type-1 integral membrane protein that can also be released into extracellular fluids (such as plasma, and seminal and cerebrospinal fluids) as a soluble enzyme following cleavage mediated by an unidentified protease(s), referred to as ACE secretase, in a process known as ‘shedding'. The effects of monoclonal antibodies (mAbs) to eight different epitopes on the N-terminal domain of ACE on shedding was investigated using Chinese hamster ovary cells (CHO cells) expressing an ACE transgene and using human umbilical vein endothelial cells. Antibody-induced shedding of ACE was strongly epitope-specific: most of the antibodies increased the shedding by 20–40%, mAbs 9B9 and 3A5 increased the shedding by 270 and 410% respectively, whereas binding of mAb 3G8 decreased ACE shedding by 36%. The ACE released following mAb treatment lacked a hydrophobic transmembrane domain anchor. The antibody-induced shedding was completely inhibited at 4°C and by zinc chelation using 1,10-phenanthroline, suggesting involvement of a metalloprotease in this process. A hydroxamate-based metalloprotease inhibitor (batimastat, BB-94) was 15 times more efficacious in inhibiting mAb-induced ACE shedding than basal (constitutive) ACE release. Treatment of CHO-ACE cells with BB-94 more effectively prevented elevation in antibody-dependent (but not basal) ACE release induced by 3,4-dichloroisocoumarin and iodoacetamide. These data suggest that different secretases might be responsible for ACE release under basal compared with antibody-induced shedding. Further experiments with more than 40 protease inhibitors suggest that calpains, furin and the proteasome may participate in this process.

Epitope-specific antibody-induced cleavage of angiotensin-converting enzyme from the cell surface

Angiotensin I-converting enzyme (ACE; CD143, EC 3.4.15.1) is a type-1 integral membrane protein that can also be released into extracellular fluids (such as plasma, and seminal and cerebrospinal fluids) as a soluble enzyme following cleavage mediated by an unidentified protease(s), referred to as ACE secretase, in a process known as "shedding". The effects of monoclonal antibodies (mAbs) to eight different epitopes on the N-terminal domain of ACE on shedding was investigated using Chinese hamster ovary cells (CHO cells) expressing an ACE transgene and using human umbilical vein endothelial cells. Antibody-induced shedding of ACE was strongly epitope-specific: most of the antibodies increased the shedding by 20-40%, mAbs 9B9 and 3A5 increased the shedding by 270 and 410% respectively, whereas binding of mAb 3G8 decreased ACE shedding by 36%. The ACE released following mAb treatment lacked a hydrophobic transmembrane domain anchor. The antibody-induced shedding was completely inhibited at 4 degrees C and by zinc chelation using 1,10-phenanthroline, suggesting involvement of a metalloprotease in this process. A hydroxamate-based metalloprotease inhibitor (batimastat, BB-94) was 15 times more efficacious in inhibiting mAb-induced ACE shedding than basal (constitutive) ACE release. Treatment of CHO-ACE cells with BB-94 more effectively prevented elevation in antibody-dependent (but not basal) ACE release induced by 3,4-dichloroisocoumarin and iodoacetamide. These data suggest that different secretases might be responsible for ACE release under basal compared with antibody-induced shedding. Further experiments with more than 40 protease inhibitors suggest that calpains, furin and the proteasome may participate in this process.

Chimaeric Lym-1 monoclonal antibody-mediated cytolysis by neutrophils from G-CSF-treated patients: stimulation by GM-CSF and role of Fcγ-receptors

Chimaeric Lym-1 (chLym-1) is a monoclonal antibody generated by fusing the variable region genes of murine Lym-1 to human γ1 and κ constant regions. Owing to its selectivity and avidity for human malignant B cells, it is an attractive candidate for developing immune-interventions in B-lymphomas. In the attempt to identify rational bases for optimizing potential chLym-1 related therapeutic approaches, we studied the ability of this ch-mAb to trigger neutrophil-mediated Raji cell cytolysis in cooperation with two neutrophil-related cytokines, G-CSF and GM-CSF. ChLym-1 triggered low levels of cytolysis by normal neutrophils but induced consistent cytolysis in neutrophils from individuals treated with G-CSF. When exposed to GM-CSF, neutrophils from subjects treated with G-CSF became potent effectors, also leading to 75% lysis. By using mAbs specific for distinct FcγRs, normal neutrophils were inhibited by mAb IV.3, suggesting the intervention of FcγRII, constitutively expressed on the cells. On the other hand, neutrophils from patients treated with G-CSF were inhibited by mAb IV.3 plus mAb 197, a finding consistent with a cooperative intervention of FCγRII and G-CSF-induced FcγRI. The anti-FcγRIII mAb 3G8 promoted significant enhancement of the neutrophil cytolytic efficiency. Therefore, neutrophil FcγRIII behaves as a down-regulator of the cytolytic potential. The present findings suggest new attempts to develop mAb-based and G-CSF/GM-CSF combined immune-interventions in B lymphomas. © 2001 Cancer Research Campaign http://www.bjcancer.com

Chimaeric Lym-1 monoclonal antibody-mediated cytolysis by neutrophils from G-CSF-treated patients: stimulation by GM-CSF and role of Fc gamma -receptors.

Chimaeric Lym-1 (chLym-1) is a monoclonal antibody generated by fusing the variable region genes of murine Lym-1 to human gamma1 and kappa constant regions. Owing to its selectivity and avidity for human malignant B cells, it is an attractive candidate for developing immune-interventions in B-lymphomas. In the attempt to identify rational bases for optimizing potential chLym-1 related therapeutic approaches, we studied the ability of this ch-mAb to trigger neutrophil-mediated Raji cell cytolysis in cooperation with two neutrophil-related cytokines, G-CSF and GM-CSF. ChLym-1 triggered low levels of cytolysis by normal neutrophils but induced consistent cytolysis in neutrophils from individuals treated with G-CSF. When exposed to GM-CSF, neutrophils from subjects treated with G-CSF became potent effectors, also leading to 75% lysis. By using mAbs specific for distinct FcgammaRs, normal neutrophils were inhibited by mAb IV.3, suggesting the intervention of FcgammaRII, constitutively expressed on the cells. On the other hand, neutrophils from patients treated with G-CSF were inhibited by mAb IV.3 plus mAb 197, a finding consistent with a cooperative intervention of FCgammaRII and G-CSF-induced FcgammaRI. The anti-FcgammaRIII mAb 3G8 promoted significant enhancement of the neutrophil cytolytic efficiency. Therefore, neutrophil FcgammaRIII behaves as a down-regulator of the cytolytic potential. The present findings suggest new attempts to develop mAb-based and G-CSF/GM-CSF combined immune-interventions in B lymphomas.

Immune complex stimulation of neutrophil apoptosis: investigating the involvement of oxidative and nonoxidative pathways

Neutrophils are involved in the pathogenesis of various inflammatory diseases. One of the mechanisms by which neutrophilic inflammation is generated is immune complex (IC) deposition in tissue. As the clearance of apoptotic neutrophils from inflamed sites is considered a crucial determinant for the resolution of inflammation, we investigated the effects of IC-induced neutrophil activation on apoptosis and the mechanisms regulating neutrophil survival. Our results show that IC stimulated apoptosis efficiently. The percentage of apoptotic neutrophils was reduced by the anti-FcγRII mAb IV.3, but not by anti-FcγRIII mAb 3G8. The spontaneous apoptosis was completely inhibited by the antioxidant compound catalase, which in turn prevented only partially the apoptosis in presence of IC. The oxidative metabolism triggered by IC was inhibited only blocking both FcγRII and FcγRIII. Neutrophils from patients with chronic granulomatous disease, congenitally incapable of producing oxidants, showed low level of spontaneous apoptosis, but underwent a nearly 3-fold increment in the apoptosis rate when incubated with IC. In conclusion, neutrophil apoptosis appears to be a process governed by multiple pathways, some of which are strictly ROS-dependent, others acting in a nonoxidative manner. In particular, the herein shown FcγRII-dependent, ROS-independent, signal-inducing neutrophil apoptosis may uncover new pharmacological targets for the promotion of cell removal from sites of inflammation, thereby favoring the resolution of the inflammatory process.

Zebrafish no isthmus reveals a role for pax2.1 in tubule differentiation and patterning events in the pronephric primordia.

Pax genes are important developmental regulators and function at multiple stages of vertebrate kidney organogenesis. In this report, we have used the zebrafish pax2.1 mutant no isthmus to investigate the role for pax2.1 in development of the pronephros. We demonstrate a requirement for pax2.1 in multiple aspects of pronephric development including tubule and duct epithelial differentiation and cloaca morphogenesis. Morphological analysis demonstrates that noi(- )larvae specifically lack pronephric tubules while glomerular cell differentiation is unaffected. In addition, pax2.1 expression in the lateral cells of the pronephric primordium is required to restrict the domains of Wilms' tumor suppressor (wt1) and vascular endothelial growth factor (VEGF) gene expression to medial podocyte progenitors. Ectopic podocyte-specific marker expression in pronephric duct cells correlates with loss of expression of the pronephric tubule and duct-specific markers mAb 3G8 and a Na(+)/K(+) ATPase (&agr;)1 subunit. The results suggest that the failure in pronephric tubule differentiation in noi arises from a patterning defect during differentiation of the pronephric primordium and that mutually inhibitory regulatory interactions play an important role in defining the boundary between glomerular and tubule progenitors in the forming nephron.

Effect of Allotype on Activation of Neutrophils by FcγRIIIB Cross-Linking

Human neutrophils constitutively synthesize two receptors for the constant region of IgG, FcγRII, and FcγRIIIB. Fluo-3-loaded neutrophils were treated with biotinylated Fab fragments of anti-FcγR antibodies and cross-linked with streptavidin, and intracellular calcium ([Ca2+]i) was monitored by flow cytometry. Polymerization of filamentous actin was quantitated by NBD–phallacidin using flow cytometry. Cross-linking of FcγRII by monoclonal antibody (mAb) IV.3 induces an increase in [Ca2+]i, superoxide generation, and the polymerization of actin. [Ca2+]i responses from cross-linking of FcγRIIIB by mAb 3G8 varied from minimal to no release. To determine whether discrepancies in 3G8-induced [Ca2+]i release were due to allotype variation, we selected five donors who were homozygous for the NA1 allotype of FcγRIIIB and five who were either heterozygous or homozygous for the NA2 allotype and compared their [Ca2+]i response and actin polymerization induced by FcγRIIIB cross-linking. Cross-linking of FcγRIIIB by 3G8 produced minimal [Ca2+]i release and polymerization of actin irrespective of donor allotype.

FMLP- and TNF-stimulated monoclonal Lym-1 antibody-dependent lysis of B lymphoblastoid tumour targets by neutrophils.

Human neutrophils, incubated with Cr51-labelled B lymphoblastoid Raji cells in the presence of the anti-target monoclonal antibody (mAb) Lym-1 plus formyl-methionyl-leucyl-phenylalanine (FMLP) or tumour necrosis factor alpha (TNF-α), were found to induce significant Cr51 release, i.e. significant cytolysis. The lytic process was inhibited by mAb IV.3, specific for the Fcγ receptor (FcγR) type II. The mAb 3G8, which reacts with FcγR type III, was ineffective. Moreover, the lysis was inhibited by the anti-CD18 mAb MEM-48. These data suggest that FMLP/Lym-1 as well as TNF-α/Lym-1 cytolytic systems strictly require FcγRII and CD18 integrins. As the lysis induced by TNF-α/Lym-1 was prevented by pertussis toxin (PT), PT-sensitive G-proteins are likely to intervene in post-FcγRII signal transduction. Both the FMLP- and the TNF-α-dependent systems were also found to be equally susceptible to inhibition by various inhibitors of kinases (genistein, staurosporin, 1-(5-isoquinolinnylsulphonyl)-2-methylpiperazine and wortmannin). On the contrary, an inhibitor of protein kinase C (bis-indolyl-maleimide, BIM) was effective only in the FMLP/Lym-1 cytolytic system. Therefore, it appears that signals delivered by FMLP or TNF-α, BIM-sensitive and insensitive respectively, converge and synergize with those from G-protein-coupled FcγRII and, probably, CD18-integrins to promote the expression of the neutrophil cytolytic potential. © 1999 Cancer Research Campaign

Cell type-specific glycoforms of Fc gamma RIIIa (CD16): differential ligand binding.

Fc gamma RIIIa, considered an intermediate affinity receptor, can variably bind monomeric IgG and appears to have a higher affinity for IgG than the lower affinity Fc gamma Rs, Fc gamma RII and Fc gamma RIIIb. We explored this property for both NK cell and monocyte Fc gamma RIIIa and found higher affinity ligand binding by Fc gamma RIIIa expressed on NK cells compared with Fc gamma RIIIa on monocytes. In normal whole blood or plasma (containing 8-11 mg/ml IgG), NK cell Fc gamma RIIIa was fully blocked, but in monocytes Fc gamma RIIIa showed approximately 60% blockade of the binding of mAb 3G8, which binds in or near the ligand binding site. The ligand binding site of NK cell Fc gamma RIIIa was blocked with as little as 2 mg/ml of human IgG, while monocyte Fc gamma RIIIa was only partially (30%) blocked by 2 mg/ml of human IgG. In contrast, plasma containing approximately 26 mg/ml of IgG (obtained from patients receiving therapeutic gamma-globulin) showed complete saturation of monocyte Fc gamma RIIIa with blockade of mAb 3G8 binding. These binding differences are not due to allelic polymorphisms or primary sequence differences between donors. Although NK cell and monocyte Fc gamma RIIIa have identical protein cores, they each undergo differential cell type-specific glycosylation. NK cell Fc gamma RIIIa is glycosylated with high mannose- and complex-type oligosaccharides, while monocyte Fc gamma RIIIa has no high mannose-type oligosaccharides. These results indicate that natural glycoforms of Fc gamma RIIIa (cell type-specific glycosylation variants) bind ligand differently, conferring a lower affinity on monocyte/macrophage Fc gamma RIIIa, which makes the receptor ideal for initial immune complex capture and sensitive to moderate changes in serum IgG levels.

Distribution of epitopes of Trypanosoma cruzi amastigotes during the intracellular life cycle within mammalian cells.

In this study we have examined the distribution of epitopes defined by monoclonal antibodies raised against Trypanosoma cruzi amastigotes during the intracellular life cycle of the parasite. We have raised monoclonal antibodies towards amastigote forms and performed preliminary immunochemical characterization of their reactivities. MAB 1D9, 3G8, 2B7, 3B9, and 4B9 react with carbohydrate epitopes of the parasite major surface glycoprotein--Ssp-4 defined by MAB 2C2 [5]; MAB 4B5 reacts with a noncarbohydrate epitope in all developmental stages of the parasite, and MAB 3B2 also detects a noncarbohydrate epitope preferentially in T. cruzi flagellated forms. Vero cells infected with tissue culture-derived trypomastigotes of clone D11 (G strain) were fixed at different times during the intracellular proliferation of parasites, and processed for immuno-electron microscopy and confocal immunofluorescence with the different monoclonal antibodies. We observed that while the surface distribution of MAB 2C2 and 4B9 epitopes was uniform throughout the cycle, MAB 1D9, 3G8, and 2B7 reacted with cytoplasmic membrane-bound compartments of the amastigotes. MAB 3B9 displayed a unique surface dentate pattern in some amastigotes. MAB 4B5 recognized a curved-shaped structure at the flagellar pocket region in some intracellular amastigotes and localized to the membrane in dividing forms. In intracellular trypomastigotes, MAB 4B5 also displayed a punctate pattern near the flagellar pocket.

The anti-FcγRIII MAb 3G8 induces neutrophil activation via a cooperative action of FcγRIIIb and FcγRIIa

Human neutrophils express two types of Fcγ receptors, the transmembrane FcγRIIa and the glycan-phosphatidylinositol-anchored FcγRIIIb, that show synergism in provoking a cellular response. To analyse further the requirements for this synergism to occur we used the monoclonal antibody 3G8, directed against FcγRIII This antibody is able to induce neutrophil activation, as measured by an increase in the intracellular free Ca 2+ concentration and homotypic neutrophil aggregation, but only when the Fe part of the antibody is able to interact with FcγRIIa. We observed that binding of the Fab parts of 3G8 mAb to two FcγRIIIb molecules and binding of the Fc part to one FcγRIIa molecule is required, because a bispecific antibody, 2B1, in which only one 3G8 Fab is present, did not induce neutrophil activation. Moreover, engagement of one FcγRIIa molecule and two FcγRIIIb molecules on the same cell is instrumental to achieve activation of the mAb 3G8. The activation of neutrophils by the 3G8 antibody represents a further example of synergistic activation of neutrophils via Fcγ receptors.

DIFFERING USE OF NEUTROPHIL (PMN) RECEPTORS BY IMMUNOGLOBULIN G (IgG) ELICITED IN RESPONSE TO GROUP B STREPTOCOCCAL (GBS) TYPE III POLYSACCHARIDE(III-PS) OR GBS III-PS TETANUS TOXOID (III-TT) CONJUGATE VACCINES.

Previously, we have shown an interactive effect for PMN Fc and complement receptors (CR) in phagocytosis of type III GBS. We proposed that antibodies elicited to GBS III-PS or III-TT vaccines might differ in PMN receptor use. Sera from 10 adults with low preimmunization levels of III GBS-specific antibody (<1.0 μg/ml, IgG ELISA) were collected 1 yr after receipt of III-PS or III-TT vaccines when antibody levels were 7-213 μg/ml. The lowest concentration of purified IgG that would support 90% killing of III GBS in an opsonophagocytic (OP) assay, 0.7 μg/ml of antibody to III GBS, was used in further testing of IgG from each of the individual vaccinees. Blockade of CR3 by the monoclonal antibody (mAb) Leu 15 significantly inhibited OP mediated by IgG from III-PS recipients but not for III-TT recipients (83.3 ± 21.8% vs 35 ± 10.1%, P = 0.009, unpaired t test). Neither showed significant inhibition by mAb CD35, directed against CR1. In contrast, uptake of radiolabeled type III GBS was significantly impaired for IgG from III-TT but not for that from III-PS recipients when FcRII was blocked using Fab fragments of mAb IV.3 (74.0 ± 16.5% vs 16.2 ± 19.0% inhibition, P = 0.002) or when FcRIII was blocked by Fab′2 fragments of mAb 3G8 (45.5± 14.3% vs 7.9 ± 9.4% inhibition, P = 0.003). There was no difference in the IgG subclass responses to type III GBS-PS whether it was conjugated to TT or not; IgG2 predominated for both groups. These results indicate reduced use of CR3 and increased use of FcR for IgG elicited in response to III-TT when compared to IgG from III-PS recipients. Differing use of PMN FcR or CR by IgG elicited in response to III-PS vs III-TT hasimplications important for host defense.