Characterization of the human immunoglobulin G Fc-binding activity in Prevotella intermedia

Many pathogenic bacteria possess cell surface receptors which can bind immunoglobulins via the Fc portion. The aim of this study was to characterize the human immunoglobulin G (IgG) Fc-binding activity of Prevotella intermedia, a suspected etiologic agent of adult chronic periodontitis. The Fc-binding activity of P. intermedia on whole cells and on extracellular vesicles was demonstrated. Incubation of P. intermedia cells in the presence of Zwittergent 3-14 allowed complete solubilization of the Fc receptor from the cell surface. This cell envelope extract was thus used to characterize the Fc-binding activity. A microtiter plate assay using alkaline phosphatase-labeled Fc fragments showed that preincubation of the cell envelope extract with human IgG, human IgG Fc fragments, or human serum completely inhibited the Fc-binding activity. Partial inhibition was obtained with human IgG F(ab')2 fragments, whereas no inhibition occurred following preincubation with human IgA, carbohydrates, and selected proteins. Preincubation of the cell envelope extract with IgG from a variety of animals demonstrated that rabbit, mouse, rat, goat, and sheep IgG did not inhibit Fc-binding activity, whereas cow, pig, and dog IgG partially inhibited Fc-binding activity. A strong inhibition comparable to that obtained with human IgG was noted with monkey IgG. The Fc receptor of P. intermedia is thus different from the six types previously reported in other nonoral bacteria. Polyacrylamide gel electrophoresis and Western blotting (immunoblotting) analysis of the cell envelope extract revealed a major band with a molecular mass of approximately 65 kDa which reacted with peroxidase-labeled human IgG Fe fragments. Transmission electron microscopy showed a uniform distribution of the Fc receptor on the bacterial surface, as revealed by gold labeling. The Fc-binding activity demonstrated in this study may act as an additional virulence factor for P. intermedia by reducing IgG reactions with the bacterial cell.     

Bispecific-armed, interferon gamma-primed macrophage-mediated phagocytosis of malignant non-Hodgkin's lymphoma

To show that macrophages can be effectively targeted against malignant B cells, bispecific antibodies (BsAb) were constructed from two antibodies having specificity for the high-affinity Fc receptor for IgG (Fc gamma RI/CD64) and the B-cell differentiation antigens CD19 and CD37. Using a flow cytometry-based assay and confocal imaging, we show that these constructs mediated significant phagocytosis of B lymphocytes by macrophages that could be enhanced with interferon gamma (IFN gamma) and IFN gamma in combination with macrophage colony-stimulating factor. BsAb-dependent phagocytosis was triggered through Fc gamma RI and could be blocked only by using F(ab')2 fragments from the parent molecule or by cross-linking Fc gamma RI. BsAb-dependent phagocytosis was not blocked by antibodies to the other Fc receptors, Fc gamma RII and Fc gamma RIII. Because these antibody constructs bind to an epitope outside the Fc gamma RI ligand binding site, we show that autologous serum, polyclonal IgG, and monomeric IgG1 did not block BsAb-dependent phagocytosis, whereas autologous serum and the IgG fractions blocked parent molecule monoclonal antibody-dependent phagocytosis due to the avid binding of monomeric IgG to Fc gamma RI. Finally, BsAb-mediated phagocytosis was effective against the malignant B cells of patients with mantle cell lymphoma, prolymphocytic leukemia, and chronic lymphocytic leukemia. Based on these studies, we propose that BsAbs may provide an effective means of immunomodulation for patients with B-cell malignancies.     

Immune modulatory effects of immunoglobulins on cell-mediated immune responses in vitro

Intravenous Immunoglobulin (IVIG) at a concentration of 5 mg/ml, significantly inhibited mitogenic responses to phytohaemagglutinin (PHA), concanavalin A (conA) and pokeweed mitogen (PWM) by peripheral blood cells from healthy donors. No difference in inhibition by IVIG was seen when stimulating different T-lymphocyte cell subsets. Inhibition by IVIG was dose-dependent. An increased response was observed when IVIG was added more than 12 h after PHA compared to adding 1 h before [P = 0.05]. Intravenous immunoglobulin added to mixed lymphocyte cultures (MLC), reduced the median response by more than 60% (range 14-89%; P = 0.03) and almost completely abrogated the lymphocyte response to Staphylococcus aureus protein A (SPA), whose median inhibition was 94% (range 90-99%; P = 0.02). When comparing 12 different commercial IVIG preparations at a concentration of 2.5 mg/ml, the median inhibition of the PHA stimulation ranged from 4% to 35% and the MLC response from 0% to 66%. In the presence of IVIG the lymphocyte response to different herpes virus antigens was reduced by > 50%. No difference in inhibitory effect was seen when comparing IVIG and cytomegalovirus (CMV) hyper Ig, but CMV negative Ig resulted in lower inhibition [P = 0.05]. Three out of five IgG preparations (2.5 mg/ml) made from single donors inhibited PHA stimulation significantly more than commercial IVIG [P < 0.05]. Mean inhibition was 61% compared to 35%. Inhibition by pooled IgG from five donors was 56%. F(ab')2 fragments of IVIG inhibited the MLC response by more than 50% (range 34-75%), SPA stimulation by 97% (83-104%) and PHA stimulation by more than 30% (26-37%). One of two Fc preparations tested had an inhibitory effect, but the inhibition was less than that obtained with the F(ab')2 fragments [P = 0.04]. These results further strengthen the notion that IVIG exerts its immune modulatory effect by binding to leukocyte surface receptors. A clear inhibition was obtained with concentrations corresponding to the serum levels obtained when IVIG is given 250-500 mg/kg bodyweight. F(ab')2 fragments have the same inhibitory effect as intact IgG molecules but the role of Fc fragments still remains unclear. Differences in the immunosuppressive effect of various IVIG preparations may be associated with the method of preparation.     

Mechanism of platelet aggregation induced by a monoclonal antibody requiring Fc portion

A monoclonal antibody designated Apt4, which is IgG1, was produced by fusion of mouse myeloma cells to spleen cells from a BALB/c mouse immunized with normal human platelets. Apt4 whole IgG caused the aggregation of both platelet rich plasma (PRP) and washed platelets from normal subjects and a patient with Bernard Soulier syndrome but not those from two patients with the Type 1 Glanzmann's thrombasthenia. No aggregation was observed when Apt4 F(ab')2 fragments were used. Immunofluorescence study showed that both whole IgG and F(ab')2 fragments of Apt4 bound to fresh or formalin fixed platelets from normal subjects and a patient with Bernard Soulier syndrome but not to those from two patients with Glanzmann's thrombasthenia. Aggregation induced by Apt4 IgG was inhibited by EDTA (10 mM), PGE1 (1 mM), 2-deoxy-D-glucose/antimycin (1.4 uM), and apyrase (20 units/ml). Preincubation of normal PRP with monoclonal anti-GPIIb/IIIa or anti-GPIb antibodies completely or partially inhibited the Apt4-induced aggregation, whereas anti-GPIIIa antibodies have no effects on this activation. Monoclonal ant-Fc gamma RII antibody (IV.3) inhibited Apt4 induced aggregation. Immunoprecipitation of 125I-labeled platelet membrane lysate by Apt4 IgG showed two protein bands with a molecular weight of 145,000 and 95,000 daltons respectively under non-reducing condition, which are corresponding to GPIIb and GPIIIa. In conclusion, Apt4 antibody binds to GPIIb/IIIa complex and induces aggregation, requiring energy metabolism, calcium, ADP release and Fc portion of IgG to interact with Fc receptor, but independent of thromboxane A2 formation.     

Anti-idiotypic activity against anti-myeloperoxidase antibodies in pooled human immunoglobulin

We investigated the ability of six different pooled human immunoglobulin (PHIG) preparations to inhibit the binding of anti-myeloperoxidase (MPO) antibodies to MPO. All six PHIG preparations inhibited the binding of anti-MPO antibodies from six sera to MPO in a concentration-dependent manner in the concentration range 0.016-10 mg/ml. There was considerable variation in the ability of each PHIG preparation to inhibit the binding of anti-MPO antibody in a given serum. Further differences were seen in the ability of a given PHIG to inhibit anti-MPO binding in different sera. F(ab')2 fragments from two PHIG preparations also inhibited in a concentration-dependent manner anti-MPO binding to MPO in all six sera in the concentration range 0.002-2.65 mg/ml, with a maximum inhibition of 42%. Little inhibition was seen with F(ab')2 of normal human IgG from individual donors (1.8-12.2% at the maximum concentration of 2 mg/ml). F(ab')2 fragments from three anti-MPO containing sera and two affinity-purified anti-MPO antibodies were eluted by affinity chromatography from Sepharose-bound PHIG F(ab')2 and showed anti-MPO antibody activity. We have shown that PHIG and F(ab')2 fragments of PHIG inhibit anti-MPO binding to MPO, and further that F(ab')2 fragments of PHIG bind to F(ab')2 fragments of anti-MPO antibodies. These observations indicate binding between the variable regions of PHIG and the antigen binding site of anti-MPO antibodies, and are consistent with an anti-idiotypic reaction. The variability seen in the inhibitory effect of the different PHIG preparations in anti-MPO-positive sera implies differences in their anti-idiotype content, while the variability of the inhibitory effect of a particular PHIG preparation between different sera suggests heterogeneity in the idiotypic repertoire of anti-MPO antibodies. Such variations in the inhibitory effect of different PHIG preparations on antibody binding may be an important determinant of their therapeutic effect.     

Lasers in pediatric surgery

OBJECTIVE: To characterize immunologic specificity and possible antiidiotype activity of IgG anti-F(ab')2 in normal subjects as well as in patients with active and inactive systemic lupus erythematosus (SLE). METHODS: IgG anti-F(ab')2 and anti-double-stranded DNA (anti-dsDNA) were affinity isolated from immunoadsorption columns of F(ab')2 and dsDNA linked to Sepharose 4B. Affinity-purified IgG anti-F(ab')2 (APAF) and affinity-isolated IgG anti-dsDNA (APAD) were tested by enzyme-linked immunosorbent assay (ELISA) for other cross-reacting specificities including anti-Sm, anti-Sm/RNP, and anti- Crithidia binding. Anti-DNA specificity of APAF and APAD was assayed by S1 nuclease treatment of heat-denatured DNA. Rabbit antiidiotypic antisera were prepared by immunization with APAF and APAD from normal subjects and SLE patients and absorption with insolubilized human Cohn fraction II (Fr II). VL and VH regions of 5 monoclonal IgM antibodies with anti-F(ab')2/anti-DNA specificity generated by Epstein-Barr virus B cell stimulation were sequenced by polymerase chain reaction and characterized for VH and VL subgroup. APAF and APAD were also examined by high-resolution electron microscopy for possible ring forms indicative of antiidiotypic V-region interactions. RESULTS: APAF from normal subjects, representing 0.08-0.18% of serum IgG, showed striking relative concentrations of both anti-F(ab')2 and anti-DNA, as well as anti-Sm and anti-Sm/RNP ELISA reactivity. Both APAF and APAD reacting with F(ab')2 or dsDNA on the ELISA plate could be cross-inhibited by F(ab')2 or DNA in solution. Anti-DNA reactivity in normal APAF and APAD was much more sensitive to S1 nuclease treatment than similar fractions from SLE patients. Neither APAF nor APAD from controls produced positive antinuclear immunofluorescence or positive Crithidia staining, whereas these were strongly positive using SLE APAF and APAD. Absorbed rabbit antisera against normal or SLE APAF and APAD showed strong ELISA reactivity against both APAF and APAD, but no residual reactivity with normal Fr II. VL and VH sequencing of monoclonal human IgM antibodies showing both anti-F(ab')2 and anti-DNA reactivity showed relative VH3, V kappa 1 or VH1, V kappa 3 restriction. No evidence of ring forms or V-region "kissing" dimers was obtained when normal or SLE APAD or APAF was examined by high-resolution electron microscopy. CONCLUSION: IgG anti-F(ab')2 in both normal subjects and SLE patients represents a polyreactive Ig subfraction with concomitant anti-DNA, anti-Sm, and anti-Sm/RNP specificities. Anti-DNA reactivity in SLE is qualitatively different from that in normal APAD and APAF since normal APAD and APAF anti-DNA is much more sensitive to S1 nuclease digestion of denatured dsDNA. APAF and APAD share distinct V-region antigens which may be related to prominent VH3 or VH1 antigenic components. No evidence for in vivo complexing of anti-DNA and anti-F(ab')2 as ring forms or antiidiotype-IgG complexes was observed during ultrastructural studies. In both normal individuals and SLE patients, APAF may represent a small polyreactive IgG subfraction which also contains antinuclear and anti-DNA specificities.     

Blockade of epidermal growth factor receptor function by bivalent and monovalent fragments of 225 anti-epidermal growth factor receptor monoclonal antibodies

We have previously described anti-epidermal growth factor (EGF) receptor monoclonal antibodies (MAbs) which can block binding of transforming growth factor alpha (TGF-alpha) and EGF to receptors and inhibit activation of receptor tyrosine kinase. Studies with these MAbs involving cell cultures and nude mouse xenografts demonstrated their capacity to inhibit the growth of a variety of tumor cell lines, which express EGF receptors and TGF-alpha and appear to depend upon receptor activation for cell proliferation. To explore the mechanism(s) by which anti-EGF receptor 225 MAb inhibits cell proliferation, we have compared the activity of native 225 MAb with the response to bivalent 225 F(ab')2 and monovalent 225 Fab' fragments. Both native 225 MAb and its fragments could inhibit the binding of 125I-EGF to EGF receptors. Scatchard analysis revealed that the Kd of 225 F(ab')2 is comparable to that of 225 MAb (1 nM), whereas the Kd of 225 Fab' is 5 nM. Both bivalent 225 MAb and 225 F(ab')2 and monovalent 225 Fab' were able to completely inhibit TGF-alpha-induced EGF receptor tyrosine kinase activation, as assayed by autophosphorylation of tyrosine residues of EGF receptors on MCF10A nonmalignant human mammary cells, MDA468 human breast adenocarcinoma cells, and A431 human vulvar squamous carcinoma cells. The bivalent forms of MAb could inhibit proliferation stimulated by endogenous (autocrine) TGF-alpha in cultures of these three cell lines. They also blocked growth stimulation by added exogenous TGF-alpha in cultures of MCF10A cells and the growth-inhibitory effect of exogenous TGF-alpha upon MDA468 and A431 cell cultures. Monovalent 225 Fab' had weaker inhibitory effects upon the proliferation of these cell lines. To determine whether the in vivo antiproliferative activity of anti-EGF receptor MAb can occur without the participation of the Fc portion of MAb, the capacities of 225 F(ab')2 and native 225 MAb to inhibit growth of s.c. A431 cell xenografts were compared. Equimolar amounts of either 225 MAb or 225 F(ab')2 were administered at intervals equivalent to the half-lives of the molecules, to attempt to maintain comparable plasma levels. Both 225 MAb and 225 F(ab')2 inhibited A431 cell xenograft growth in a dose-dependent manner, with a more sustained response in the case of the intact antibody.(ABSTRACT TRUNCATED AT 400 WORDS)     

Receptor activity on some mesenchymal cells in CNS of normal rabbits. Indications of the monocytic origin of intracerebral perivascular cells, epiplexus cells and mononuclear phagocytes in the subarachnoid space

The surface receptor activity for various cell types within rabbit CNS was investigated. Sheep red blood cells (E) used as markers were washed, sensitized with the IgG-fraction of E-antibodies (EA) or additionally coated with complement (EAC) and incubated with CNS cells. The inhibitory effect produced by the addition of soluble IgG was investigated. Incubation (1 h) with red cells was undertaken in three ways: 1. Rabbit leptomeninx was stripped and incubated. 2. Rabbits were killed, the brain was perfused with warm buffer and red cell complexes were then injected intracerebrally, intrathecally and intraventricularly into the perfused brains. 3. E, EA and EAC were injected intracerebrally, intrathecally and intra-ventricularly into living rabbits. Using these methods, receptor sites for IgG and complement of mononuclear cells from the subarachnoid space, epiplexus cells and perivascular cells from intracerebral vessels could be demonstrated. In other areas of the body, these receptors have been demonstrated using similar methods only on cells from the monocyte-macrophage series. The common derivation of these three cell types from the monocytes as well as their comparable function within the immune response is discussed.     

Accurate measurement of intrinsic positive end-expiratory pressure: how to detect and correct for expiratory muscle activity

The basic amino acid L-lysine was administered to mice in an attempt to circumvent unwanted renal accumulation of 67Cu-labelled F(ab')2 fragments derived from the anti-NCAM IgG1, SEN7 and anti-CEA IgG1 monoclonal antibody (MAb)35. In control experiments, significant renal uptake of both 67Cu-labelled F(ab')2 fragments was observed, radiolabel being primarily localised to proximal tubules in the renal cortex. Following optimised L-lysine dosing protocols, renal uptake of 67Cu-MAb35 F(ab')2 was inhibited by up to 42%. Surprisingly, little inhibition (< 10%) of 67Cu-SEN7 F(ab')2 uptake was observed. Experiments to investigate this differential inhibition indicated that inhibition of MAb35 F(ab')2 uptake was relatively short-lived (approx. 6 hr), whilst no apparent differences were found in blood clearance rates between either 67Cu-F(ab')2 fragment. L-lysine administration caused a significant diuresis with high levels of intact 67Cu-labelled SEN7 and MAb35 F(ab')2 appearing in the urine, possibly due to blockade of renal uptake and lysine-induced increases in glomerular membrane permeability. Iso-electric focusing studies failed to identify any charge differences between the 67Cu-labelled F(ab')2 fragments, although a cathodal migration of all 67Cu-labelled samples, presumably due to the net positive charge conferred by addition of 67Cu2+ ions, was observed. Our results demonstrate that in addition to net charge, other unidentified characteristics may influence renal accumulation of radiometal-labelled F(ab')2 fragments and their inhibition by L-lysine.     

A small domain (6.5 kDa) of bacterial protein G inhibits C3 covalent binding to the Fc region of IgG immune complexes

Attachment of the complement component C3 to antigen-antibody (Ag-Ab) complexes (immune complexes, IC) is the key molecular event responsible for the elimination of many Ag in the form of Ag-Ab-C3b. The CH1 domain and the Fc region of the Ab, which have previously been involved in the binding of C3b, are also the targets of several bacterial IgG-binding proteins, particularly proteins G and A. Here we describe the ability of a small recombinant protein G domain (B2; 6.5 kDa) to inhibit the covalent binding of C3b to the Fc portion of IgG without affecting the binding to the Fab part. Protein G (B2 domain) produced a remarkable inhibition of covalent binding of C3b to IC formed with rabbit IgG, but none with the F(ab')2 fragment, indicating that B2 interferes with the C3b binding to the Fc region. A weak inhibition was observed with IC formed with mouse IgG2b which preferentially binds B2 domain on the CH1 domain of the Fab. To confirm these data, recombinant single-chain Ab devoid of CH1 domains (scAb), and including the rabbit or human Fc portion (hinge-CH2-CH3), were produced and used to form IC. Protein G-B2 domain inhibited C3b binding to IC formed with scAb of either human or rabbit constant regions, supporting the view of a specific blockade of C3b binding to the Fc region. A similar inhibition of C3b binding was observed using protein A instead of protein G B2 domain and the same set of IC. On the CH1 domain, C3b and B2 bind on opposite faces, and therefore do not interfere with each other in their binding. However, B2 domain bound to the inter-CH2-CH3 region impedes the C3b binding to the Fc. This inhibition clarifies the specificity of C3b for the different regions of IgG and explains how bacterial IgG-binding proteins provide the bacteria with a mechanism of evasion from the opsonizing action of complement and contribute to the virulence. This could be a general mechanism of escape because protein G binds the majority of mammalian Ig.     

Increased tumor localization by monoclonal antibody A7 after F(ab')2 fragmentation in athymic nude mice bearing human pancreatic carcinomas

Much recent research has been directed toward the use of monoclonal antibodies (MoAbs) for the immunodetection of solid tumors. In pancreatic cancer, conventional immunoscintigraphy using intact MoAbs remains disappointing. In this study, 125I-labeled F(ab')2 fragments produced by pepsin digestion of MoAb A7 were injected intravenously into nude mice bearing human pancreatic cancer, HPC-YS, xenografts that have previously been shown to react specifically with MoAb A7. The tumor tissue/blood ratio of 125I-labeled F(ab')2 fragments of MoAb A7 increased with time and was much higher than those for normal tissues. Moreover, the tumor tissue/blood ratio of 125I-labeled F(ab')2 fragments was greater than that of intact MoAb A7, although the F(ab')2 accumulation was less than that of intact MoAb A7 in the tumor. These results suggest that F(ab')2 fragments of MoAb A7 may be suitable carriers of radionuclides for immunodetection of human pancreatic cancer.     

FcR-mediated inhibition of cell activation and other forms of coinhibition

The tripartite inactivation model proposed that coaggregation of the B cell antigen receptor (BCR) with the Fc receptor (FcR) by antigen and specific IgG antibody complexes explained the Fc-dependent inhibition of immune responses by antibody. This model has since been substantiated by many observations and its impact on studies of immune regulation has been threefold: (1) IgG antibody, via Fc gamma RIIB, mediates inhibition of cell activation in many cell types, demonstrating the general importance of this mechanism in immune regulation; (2) Fc gamma RIIB was the first receptor described that regulates immune responses by coinhibition, that is, regulation as a result of interaction between activating receptors (BCR, TCR, Fc epsilon RI, Fc gamma RIII, Fc gamma RIIA) and inhibitory receptors (Fc gamma RIIB, CTLA4, CD5, CD22, p58/70/140 KIR, gp49B1/gp91, Ly49A/C/E/F/G, NKG2-A/B, APCR, Fas (CD95), TGF beta-R, TNF-R, IFN gamma-R, and others). The list of coinhibitors is expanding, just as the list of costimulators has grown. Tolerance through multiple coinhibitors implies that Signal 1 alone is not tolerogenic; and (3) Studies of Fc gamma RIIB coinhibitory mechanisms have pointed the way to potential general inhibitory signaling pathways used by many receptors, involving the competing effects of various kinases and phosphatases, and other competitive events. Investigations of Fc gamma RIIB physiologic function and of other coinhibitory receptors, together with recent biochemical analyses, give an initial understanding of the biology of these inhibitory receptory receptors. Paradoxes within and between theoretical constructs, functional observations, and mechanistic studies point to critical questions for future study.     

The antigen-binding domain of a human IgG-anti-F(ab')2 autoantibody

Recent studies revealed an immunoregulatory role of natural IgG-anti-F(ab')2 antibodies in both healthy individuals and patients with certain diseases. The implication of anti-F(ab')2 antibodies in the pathogenesis of diseases prompted us to study the gene segment structure of their antigen-binding domains and their binding characteristics. cDNA was prepared from the lymphocytes of a patient with a high IgG-anti-F(ab')2 serum titer. Variable heavy and light gene segments were amplified by PCR and inserted into a phagemid surface expression vector. Single-chain antibodies displayed on the phage surface were screened for binding to F(ab')2 fragments. The subsequent analysis of 95 single clones demonstrated that they all bound specifically to F(ab')2. Sequence analyses of 12 clones showed that 11 were identical and 1 contained a silent point mutation in the heavy chain and three amino acid exchanges in the light chain. The heavy chains belonged to the V(H)3 and the light chains to the V(kappa)2 gene family. The 11 identical light-chain genes were completely homologous to a germ-line sequence (DPK-15). Binding assays showed that the single-chain antibodies bind to F(ab')2, but not to Fab, Fc, or intact IgG. This binding pattern was confirmed by surface plasmon resonance studies, which revealed a relatively high affinity (Ka = 2.8 x 10(7) M(-1)). The strong binding capacity was further demonstrated by competitive inhibition of the serum anti-IgG antibody's interaction with antigen. The present study defines for the first time to our knowledge the gene segment structure of the antigen-binding domain of two human IgG-anti-F(ab')2 autoantibody clones and describes the binding kinetics of the purified monomeric fragments.     

Immunohistochemical metallothionein expression in thymoma: correlation with histological types and cellular origin

In radioimmunotherapy, the long circulation times of antibody radioconjugates correlate with high relative radiation doses to nontumor tissues. Tumor/normal tissue ratios can be significantly improved by using targeting molecules with shorter circulation times. IFabs are multimers of VH-CH1-linker-VK-CK monomers. The lack of the Fc region in IFabs should lead to circulation times that are shorter than those of IgG molecules. The monomers assemble into disulfide-bond-stabilized multimers, 90% of which are 100 kDa dimers (IFab2). IFab2s should not be rapidly eliminated through kidney filtration because their molecular weight is above the threshold for renal passage. We report the first experimental in vivo tests for 125I-IFab radioconjugates derived from a humanized version of the anti-breast mucin monoclonal antibody BrE-3. Biodistributions are reported for athymic nude mice carrying human mammary tumor MX-1 xenografts. The T1/2 beta's for the different tissues ranged from 13.3 h for blood to 19.9 h for tumor. Therefore, IFab radioconjugates cleared the body with a rate comparable to that of F(ab')2 fragments. Except for stomach, tumor/nontumor dose ratios were significantly better for IFabs than for the parent antibody (BrE-3)4 days after injection.     

Bispecific molecules directed to the Fc receptor for IgA (Fc alpha RI, CD89) and tumor antigens efficiently promote cell-mediated cytotoxicity of tumor targets in whole blood

The FcR for IgA (Fc alpha RI, CD89) is primarily expressed on cytotoxic immune effector cells. By chemically cross-linking F(ab') fragments of the FcR for IgA (Fc alpha RI)-specific mAb (A77) with tumor Ag-specific mAb (anti-HER2/neu and anti-epidermal growth factor receptor), we have developed bispecific molecules (BSM) that simultaneously bind to respective tumor Ags and Fc alpha RI-expressing effector cells in whole blood. These BSM mediated up to 55% of specific lysis of appropriate tumor Ag-expressing target cells (from a variety of tumors) with purified polymorphonuclear leukocytes, monocytes, or whole blood effector cells without preactivation with exogenous cytokines. To our knowledge, this is the first demonstration of Ab-dependent cell-mediated cytotoxic activity via Fc alpha RI in whole blood. Also, monocyte-derived macrophages mediated phagocytosis of HER2/neu-expressing tumor cells (>95% tumor cell loss). These BSM-mediated cytotoxic activities were completely inhibited by F(ab')2 of A77, demonstrating the specific role of Fc alpha RI as a trigger molecule. Furthermore, the binding of these BSM to monocytes or polymorphonuclear leukocytes in whole blood did not induce modulation of Fc alpha RI in the absence of the target Ag. Therefore, immune effector cells may be "armed" with Fc alpha RI-directed BSM in whole blood. These Fc alpha RI-directed BSM may offer new treatment options for various malignancies and other disease conditions.     

Staphylococcal neutral phosphatase. A highly cationic molecule with binding properties for immunoglobulin

Staphylococcal neutral phosphatase (NPtase) was purified from two Staphylococcus aureus strains by sequential high salt extraction, ultracentrifugation and ion exchange chromatography. The enzyme showed maximum phosphatase activity at neutral pH, appeared as two bands in SDS-PAGE (31 and 32 kDa), and the isoelectric point was > 10. No close similarity between NPtase and other known bacterial proteins in respect of their N-terminal amino acid sequences was found. Purified NPtase bound rat and human polyclonal IgG [intact and F(ab')2 fragments], IgM, IgA, intact myeloma immunoglobulins, myeloma light chains, gamma heavy chain and, with a much lower affinity, Fc fragments. Furthermore, NPtase can bind serum albumin. Heparin, a highly negatively charged molecule, significantly inhibited NPtase binding to immunoglobulins and HSA, but did not inhibit the binding of specific antibodies to NPtase; this indicates that charge interactions are important. The newly characterized staphylococcal phosphatase with binding properties for immunoglobulin is an interesting bacterial protein that could be involved in post-infectious sequelae.     

Functional expression of the MHC class I-related receptor, FcRn, in endothelial cells of mice

Our recent data indicate that the MHC class I-related receptor, FcRn, plays a role in regulating serum IgG levels, in addition to its known role in transferring IgG from mother to young. In the current study, the distribution of FcRn in adult mice has been investigated using several approaches. First, tissue distribution of anti-FcRn F(ab')2, murine IgG1 and recombinant, IgG1-derived Fc-hinge fragments has been analyzed, and these FcRn binding proteins localize predominantly in skin and muscle with lesser amounts in liver and adipose tissue. Second, histochemical analyses of muscle and liver with anti-FcRn F(ab')2 indicate that FcRn is expressed in the endothelium of small arterioles and capillaries, but not in larger vessels such as the central vein and portal vasculature. Third, immunoprecipitation and immunofluorescence studies of cultured murine endothelial cells show that functional FcRn is expressed in these cells, and is located within vesicular structures in the cytosol and not on the membrane. Taken together the data demonstrate that FcRn is expressed in functionally active form in endothelial cells, indicating that these cells are a possible site at which serum IgG homeostasis is maintained.     

Transfer of human serum IgG to nonobese diabetic Igmu null mice reveals a role for autoantibodies in the loss of secretory function of exocrine tissues in Sj?gren's syndrome

The NOD (nonobese diabetic) mouse has been studied as an animal model for autoimmune insulin-dependent diabetes and Sj?gren's syndrome. NOD.Igmu null mice, which lack functional B lymphocytes, develop progressive histopathologic lesions of the submandibular and lachrymal glands similar to NOD mice, but in the absence of autoimmune insulitis and diabetes. Despite the focal appearance of T cells in salivary and lachrymal tissues, NOD.Igmu null mice fail to lose secretory function as determined by stimulation of the muscarinic/cholinergic receptor by the agonist pilocarpine, suggesting a role for B cell autoantibodies in mediating exocrine dryness. Infusion of purified serum IgG or F(ab')2 fragments from parental NOD mice or human primary Sj?gren's syndrome patients, but not serum IgG from healthy controls, alters stimulated saliva production, an observation consistent with antibody binding to neural receptors. Furthermore, human patient IgG fractions competitively inhibited the binding of the muscarinic receptor agonist, [3H]quinuclidinyl benzilate, to salivary gland membranes. This autoantibody activity is lost after preadsorption with intact salivary cells. These findings indicate that autoantibodies play an important part in the functional impairment of secretory processes seen in connection with the autoimmune exocrinopathy of Sj?gren's syndrome.     

Two dual-specific (anti-IgG and anti-dsDNA) monoclonal autoantibodies derived from the NZB/NZW F1 recognize an epitope in the hinge region

The anti-IgG properties of two dual-specific (anti-dsDNA and anti-IgG) monoclonal NZB/NZW F1-derived autoantibodies, BV 17-45 and BV 16-13, were studied to resolve the location and possible commonality of the IgG epitope. To determine if BV 17-45 and BV 16-13 recognized the same IgG epitope, the relative temperature sensitivity of the conformational IgG epitopes were evaluated using the conformational sensitive immunoassay. Comparison of the temperature sensitivity of the conformational immunoglobulin epitopes over a temperature range of 25-100 degrees C suggested that the epitope recognized by BV 17-45 was the same as the IgG epitope recognized by BV 16-13. Further studies with papain- and pepsin-generated F(ab')2, Fab, and Fc fragments of BV 17-45 and BV 16-13 revealed that the dual-specific autoantibodies BV 17-45 and BV 16-13 both bound an epitope in the hinge region of the IgG molecule. The potential correlation between these studies and the pathogenic nature of dual-specific autoantibodies is discussed.