The visualization of receptors for the Fc portion of the IgG molecule on human neutrophil leukocytes

Human neutrophils treated with either Fc or with intact IgG and subsequently with fluorescein-labelled anti-IgG showed binding of the Fc or the IgG to the cell membrane. Neutrophils did not appear to bind F(ab′)₂ fragments. Under suitable conditions, polar capping of fluorescence was seen. The data suggest receptors for Fc on the neutrophil membrane and mobility of these receptors.     

Evading pre-existing anti-hinge antibody binding by hinge engineering

Antigen-binding fragments (Fab) and F(ab′)₂ antibodies serve as alternative formats to full-length anti-bodies in therapeutic and immune assays. They provide the advantage of small size, short serum half-life, and lack of effector function. Several proteases associated with invasive diseases are known to cleave antibodies in the hinge-region, and this results in anti-hinge antibodies (AHA) toward the neoepitopes. The AHA can act as surrogate Fc and reintroduce the properties of the Fc that are otherwise lacking in antibody fragments. While this response is desired during the natural process of fighting disease, it is commonly unwanted for therapeutic antibody fragments. In our study, we identify a truncation in the lower hinge region of the antibody that maintains efficient proteolytic cleavage by IdeS protease. The resulting neoepitope at the F(ab′)₂ C-terminus does not have detectable binding of pre-existing AHA, providing a practical route to produce F(ab′)₂ in vitro by proteolytic digestion when the binding of pre-existing AHA is undesired. We extend our studies to the upper hinge region of the antibody and provide a detailed analysis of the contribution of C-terminal residues of the upper hinge of human IgG1, IgG2 and IgG4 to pre-existing AHA reactivity in human serum. While no pre-existing antibodies are observed toward the Fab of IgG2 and IgG4 isotype, a significant response is observed toward most residues of the upper hinge of human IgG1. We identify a T₂₂₅L variant and the natural C-terminal D₂₂₁ as solutions with minimal serum reactivity. Our work now enables the production of Fab and F(ab′)₂ for therapeutic and diagnostic immune assays that have minimal reactivity toward pre-existing AHA.     

Neutralizing Activities of Early and Late IgG Fragments from Rabbits Immunized with Herpes Simplex Virus

Rabbits immunized with herpes virus were bled periodically and bivalent and univalent fragments of IgG from each serum sample were prepared by enzymatic digestion. The 2-week F(ab′)₂ showed a low neutralizing activity only after addition of anti-IgG. F(ab′)₂ of the 4-week serum retained almost all of the neutralizing activity of IgG, while its univalent fragments demonstrated none even when tested with anti-IgG. In contrast to these early IgG fragments, univalent fragments of the 9-week and 20-week IgG neutralized the virus to considerable extents in the absence of anti-IgG; after addition of anti-IgG the activity equaled that of intact IgG in the cases of Fab′ and Fab-II, though the activity of Fab-I was relatively low. The three univalent fragments were all sensitive to heating at 70 C and to ultraviolet irradiation, whereas intact IgG resisted these treatments. F(ab′)₂ was resistant to the heating and less sensitive to ultraviolet irradiation than univalent fragments. Neutralization kinetic curve experiments to test blocking effects of IgG fragments against the neutralization by intact IgG suggested that the early Fab′ did combine with the virus and that the late Fab′ exerted a higher blocking effect than the early Fab′.     

Free flow electrophoresis of chloroplasts

Highly purified intact chloroplasts were isolated from spinach (Spinacia oleracea L.) leaves by free flow electrophoresis. Morphological and biochemical studies showed that the fraction enriched in intact chloroplasts has a higher protein to chlorophyll ratio and a higher linolenic acid content than the broken organelles of the other fraction. The intact chloroplasts prepared by electrophoresis retained their capacity for CO₂ fixation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that this fraction was rich in stroma and lamellae proteins. Free flow electrophoresis, which separates organelles and molecules according to their surface charges, is a good technique for producing purified chloroplasts with complete physiological activities.     

Targeting of the antiviral protein from Phytolaccaamericana with an antibody

The antiviral protein (PAP) of $\text{Phytolacca}\text{americana}$ was conjugated with the Fab' fragment of IgG from a rabbit antiserum against murine leukemia L1210 cells via a disulfide bond employing $\text{N}$-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) as the coupling agent. The conjugate showed a potent $\text{in}\text{vitro}$ cytotoxicity against L1210 cells which was competitively blocked by F(ab′)₂ directed against L1210 cells. PAP itself did not exhibit the cytotoxicity at the concentration corresponding to the PAP content in the conjugate concurrently tested.     

The equine protease inhibitory system (Pi): Abnormal expressions of PiF,PiL, and PiS

Three cases of abnormal expression of the equine protease inhibitory alleles, Pi F, L, and S₁, were observed following the examination of 30,000 plasma samples by one-dimensional acid (pH 4.6) polyacrylamide gel electrophoresis. Characterization of the abnormal proteins in terms of isoelectric point, molecular mass, inhibitory spectra, and sialic acid content was performed using one- and two-dimensional electrophoretic techniques. The Pi F and S₁ abnormalities were postulated to be the result of amino acid substitutions causing alterations in the processing of the carbohydrate side chains. No explanation could be offered for the Pi L abnormality other than a charge shift mutation. Abnormal types, F*, L*, and S*₁ behaved as alleles but the distribution of L* in offspring from one stallion (present in only 6 of 83 offspring) differed significantly from expectation.This work was supported by a grant from the Australian Stud Book, Alison Road, Randwick, N.S.W. 2031.     

Transchelation of 99mTc from low affinity sites to high affinity sites of antibody

An exclusive labeling of high affinity sites of IgG and its F(ab′)₂ fragments with ^99mTc was accomplished. Antibody was first labeled in 0.1 M acetate buffer at pH 4.5, using stannous chloride as a reducing agent. Thus, high capacity, low affinity sites and low capacity, high affinity sites were both labeled. These ^99mTc complexes were stable at pH 4.5 and 7.0; however, they became destabilized at pH 8.2 and 9.0. Transchelation of ^99mTc to DTPA took place at the higher pH values and leveled off at 54% ^99mTc-F(ab′)₂ and 73% ^99mTc-IgG. These results indicate that the majority of ^99mTc bound to the low affinity sites was transchelated to the high affinity sites rather than to DTPA since low affinity sites account for 84% of total F(ab′)₂ sites and 76% of IgG sites. Biodistribution data in mice at 2.5 h postinjection were consistent with this hypothesis in that tissue concentrations of ¹¹¹In-DTPA-F(ab′)₂ were similar to the reequilibrated ^99mTc-F(ab′)₂ but were much higher than that of the unequilibrated ^99mTc-F(ab′)₂.     

Rabbit antiserum to human B-cell alloantigens. II. Mechanism of inhibition of stimulation in the mixed lymphocyte response.

The process by which a rabbit antiserum to human B-cell alloantigens blocks stimulation in the mixed lymphocyte response (MLR) was investigated. A functional mammalian Fc region was necessary for the antiserum to be inhibitory, since F(ab′)₂ fragments failed to inhibit and a chicken antiserum with similar specificity to the rabbit anti-B-cell serum did not effectively block the response. Immune elimination of the stimulating cell population possibly via antibody dependent cell-mediated cytotoxicity (ADCC) or phagocytosis by macrophages was suggested by the observation that the addition of aggregated IgG to the MLR reduced the level of inhibition. It was also found that the number of immunoglobulin positive cells decreased in cultures treated with intact rabbit anti-B-cell serum, but not the corresponding F(ab′)₂ fragments, whether the cells were from a single individual or an allogeneic cell mixture. ADCC appears to be involved in the blocking process, as demonstrated by the marked reduction in MLR suppression when the MLR was initiated in the absence of ADCC effector cells. Removal or inhibition of monocytes in the MLR partially restored the response in experiments where the stimulator cells were pretreated with the antiserum, but not when the antiserum was present throughout the MLR.     

Biological Activities of Murine Low-Affinity Fc Receptors for IgG

Murine low-affinity Fc receptors for IgG (FcγRIIbl, FcγRIIb2, and FcγRIII) bind the same IgG subclasses and are not distinguished by available anti-FcγRII/III mAbs (2.4G2). They trigger various biological activities, among which are the internalization of soluble and particulate immune complexes, cell activation, and its regulation. To determine the biological properties of the three murine receptors, each was expressed by stable transfection of corresponding cDNAs in two model cells: the murine lymphoma B cell IIA1.6 and the rat basophilic leukemia cell RBL-2H3. Biological activities of recombinant receptors were triggered with soluble immune complexes or 2.4G2 IgG in IIA1.6 cells, which express no FcγR, and with 2.4G2 Fab or F(ab′)₂, cross-linked with mouse anti-rat F(ab′)₂ in RBL, which express rat FcγR. Conditions for studying cell activation and endocytosis in both cell models are described, as are conditions for studying phagocytosis in RBL cells and antigen presentation or regulation of cell activation in IIA1.6 cells. Internalization of immune complexes was triggered by FcγRIIb2 and FcγRIII, but not by FcγRIIb1. Intracytoplasmic sequences required for phagocytosis and endocytosis could be distinguished in FcγRIIb2, but not in FcγRIII. Cell activation was restricted to FcγRIII. FcγRIII-mediated endocytosis, phagocytosis, and cell activation involved the consensus tyrosine-containing activation motif found in the intracytoplasmic domain of the γ subunit. Regulation of cell activation was induced by both FcγRII isoforms and depended on the same sequence as endocytosis. As a consequence, a single motif can determine more than one biological response of the cell, and a given response may be triggered by several motifs, borne by different FcγR.     

Disposition of glycoproteins in plasma membrane of cultured rat embryonic fibroblasts

Plasma membrane fractions were isolated from untreated and trypsin- or neuraminidase-treated rat embryo fibroblasts and their sialic acids contents per mg membrane protein were determined. The difference represented enzyme releasable sialic acid exposed on the medium side of the cell mambrane. It was 14 to 23% of the total membrane bound sialic acid. Isolated plasma membrane fraction from entreated and enzyme treated cells were then subjected to trypsin or neuraminidase treatment to obtain enzyme-releasable sialic acid from both faces and from the cytoplasmic face of the membrane respectively. Between 30 and 50% of the total membrane bound sialic was released from both the faces and 14 to 30% from the cytoplasmic face. An average of 59% was insusceptible to these enzymes. As an alternative to a cytoplasmic location of sialic acid containing membrane constituents, inaccessibility of enzymes to some of these constituents present on the surface of intact cells is considered.Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of plasma membrane fractions isolated from untreated and trypsin treated cells and of trysinized plasma membrane fraction was carried out to know the number and gel migration of proteins and glycoproteins which are exposed on each of the two faces of the plasma membrane and are sensitive or insensitive to trypsin. The resilts obtained were confirmed by SDS-polyacrylamide gel electrophoresis of untreated and trypsin-treated cells and of isolated plasma membrane fraction after subjecting them to enzymatic radioiodination.     

Effects of antibodies to MHC gene products on different Fc-receptor-mediated cell functions

The effects of rabbit anti-HLA-DR and anti-β₂-microglobulin (anti-β₂m) antibodies on three different Fc-receptor-mediated cell functions of peripheral blood mononuclear leukocytes were studied. Both IgG and F(ab′)₂ fragments of anti-HLA-DR antibodies inhibited the cytotoxicity against a monolayer of antibody-sensitized sheep erythrocytes (plaque formation) whereas no effect was observed on the cytotoxicity against antibody-sensitized chicken erythrocytes in suspension (⁵¹Cr release), or on EA-rosette formation. On the other hand, all the three functions were inhibited by the IgG fraction of anti-β₂m antibodies but not by the corresponding F(ab′)₂ fragments. The results demonstrate that the plaque-forming cells (PFC) carry HLA-DR-like antigens. Furthermore, a closer association exists between the HLA-DR-like antigens and the Fc receptors than between the β₂m molecules and the Fc receptors on the PFC. The results further support our earlier investigations suggesting that the PFC are of monocytic origin.     

Isolation of a murine leukemia FC receptor by selective release induced by surface redistribution.

A protein which binds to the Fc region of IgG has been isolated from the murine leukemia L1210. The isolation technique involves surface cross-linking of the cells's Fc receptors with the use of aggregated human IgG and anti-human IgG. This results in the redistribution (patch formation and capping) of the cells's Fc receptors. Lactoperoxidase-catalyzed radioiodination of the cells before complex binding indicates that Fc receptor redistribution results in the selective release of surface proteins. SDS-PAGE analyses of the supernatants from cells thus treated reveals a major peak corresponding to a molecular weight of 45,000 daltons. This protein has been purified from the cell supernatants by immunoprecipitation and chromatography of the percipitates on Sephadex G-200 under dissociating conditions. After separation from the immune complex this protein can be bound to heat-aggregated IgG, but not aggregated F(ab')2 fragments. The 45,000 dalton protein appears to be the Fc receptor which has been released from the cell surface in association with the complex.     

Molecular interactions in human T-cell-mediated cytotoxicity to Epstein-Barr virus. I. Blocking of effector cell function by monoclonal antibody OKT3

The ability of different anti-human T-cell lymphocyte monoclonal antibodies to inhibit the effector function of the cytotoxic T-cell response against autologous Epstein-Barr virus (EBV)-infected B-cell targets has been tested. It was found that monoclonal antibody, OKT3, which reacts with most human T cells, blocks the effector cell function in the absence of complement, an effect that was dose dependent. When monoclonal antibody OKT3 was tested at a concentration of 1 μg/ml, inhibition of cytotoxicity ranged between 50 and 80%. The F(ab′)₂ fragment of OKT3 inhibited as well as the intact IgG molecule, indicating that the Fc portion of the antibody is not necessary for the cytotoxicity blocking. The Fab fragment of OKT3 had lower blocking activity per microgram of protein tested. Antibodies SC1, OKT11 (anti-pan T cell), OKT8 (anti-cytotoxic/suppressor subset), and L368 (anti-HLA) did not have any discernible blocking effects. However, antibodies SC1, OKT8, and L368 could abrogate the cytotoxic activity in the presence of complement. Blocking by OKT3 was not due to its being present on the cell surface in higher concentrations than the other monoclonal antibodies since cytofluorographic analysis demonstrated that the amount of OKT8 or L368 antibodies bound on the cells was greater than OKT3. In addition, blocking was not due to antigenic modulation since incubation with antibody OKT3-F(ab′)₂ was not associated with a significant decrease in the amount of its reactive antigen. Under the conditions tested OKT3 did not affect cell viability or cause agglutination.     

Relative susceptibilities to neuraminidase of the glycoproteins of the human erythrorcyte

Release of sialic acid from the glycoproteins of the normal human erythrocyte surface by neuraminidase was investigated. The glycoproteins of the membrane were separated by electrophoresis in sodium dodecylsulfate polyacrylamide gels. Sialic acid was determined in the sliced gel by a modification of the 2-thiobarbituric acid method, revealing three sialic acid-containing glycoproteins. Treatment of intact erythrocytes with neuraminidase to remove varying amounts of sialic acid indicates that all the glycoproteins are essentially equally accessible to the neuraminidase when 20%–60% of the sialic acid is removed. Similar but not quite identical results were obtained with isolated erythrocyte membranes.Treatment of intact cells with the lectins concanavalin A or phytohemagglutinin-P resulted in shielding of about 25% and 50%, respectively, of the sialic acid from neuraminidase. Concanavalin A blocked sialic acid release over long time periods and with high concentrations of neuraminidase. In contrast, the sialic acid shielding by phytohemagglutinin-P can be overcome by high concentrations of neuraminidase. Both lectins were found to shield the various glycoproteins selectively, with different patterns of shielding. Wheat germ agglutinin exhibited no detectable effect on the susceptibility of the erythrocyte sialic acid to neuraminidase.     

An immunoblotting technique for the detection of bound and secreted bacterial Fc receptors

A rapid semiquantitative procedure that enables bacteria to be screened for surface or secreted receptors for the Fc region of human IgG is described. Surface Fc receptors were detected by direct transfer of bacterial colonies to nitrocellulose by electroblotting and then probing with ¹²⁵I-labeled human IgG in the presence of a two fold molar excess of unlabeled F(ab′)₂fragments. The blots were exposed to X-ray film and the intensity of the resulting autoradiograph was a measure of surface Fc receptors expression. This procedure reliably distinguished Staphylococcus aureus strains which expressed different levels of surface Fc receptors. When applied to the study of group A streptococci, a number of Fc receptor-positive strains were identified. Unlike the homogeneous Fc receptor expression on individual colonies of the staphylococcal strains, a wide variation in the level of Fc receptor expression was observed within a given streptococcal strain. Group A streptococcal substrains which expressed high and low levels of surface Fc receptors could be isolated from replica plates.Secreted Fc receptors were measured by a simple modification of the blotting procedure in which the nitrocellulose was placed on the opposite side of the agar from the bacterial colonies. Secreted Fc receptors was electroblotted through the agar onto nitrocellulose and probed as described above. This approach readily detected nanogram quantities of secreted type I Fc receptor (protein A) from the Staphylococcus aureus Cowan strain. None of the group A streptococcal strains tested were found to secrete detectable quantities of Fc receptors.     

Isolation of an Fcgamma-binding protein from the cell membrane of a macrophage-like cell line (P388D1) after detergent solubilization

Lactoperoxidase-catalyzed iodination, NP-40 lysis, and subsequent affinity chromatography on IgG-Sepharose were used in an attempt to define some of the molecular properties of the Fc receptor of P388D1, a macrophage-like mouse tumor line. Radioiodinated material retained on columns of Sepharose coupled either to monomeric mouse IgG2a or monomeric human IgG1 appeared on SDS polyacrylamide gel electrophoresis to contain principally three labeled components, a major band of about 57,000 m.w. and two minor bands of 28,000 and 24,000 m.w. The mobilities of these components changed little on reduction, which suggested that they represented single polypeptide chains, An identical pattern was obtained with Sepharose-linked Fc fragments of human IgG1, but neither Fab fragments of IgG1 nor IgM appeared to bind these components. Since the specificity of binding to the immobilized proteins is the same as that observed in vivo, it is postulated that these proteins represent either all or some portion of the P388D1 Fc receptor.     

Roles of plasma proteins and surface negative charge of erythrocytes in erythrocyte aggregation

The effect of plasma proteins (and IgG fragments) and sialic acid content of erythrocytes on the aggregation of human erythrocytes was quantitatively examined by using a rheoscope combined with a television image analyser and a computer. (1) The velocity of erythrocyte aggregation by plasma proteins was increased with increasing in their molecular weight, i.e., IgG less than IgA less than fibrinogen less than IgM. F(ab')2. Fab and Fc could not induce the aggregation. (2) The aggregation induced by fibrinogen was accelerated by IgG and its peptic fragment, F(ab')2, but was unaffected by the plasmic fragments, Fab and Fc. The accelerating effect by IgG and F(ab')2 was inhibited by Fab and Fc. (3) The aggregation of erythrocytes was accelerated by decreasing the sialic acid content (due to the reduction of the electrostatic repulsive force among erythrocytes), and the effect of desialylation on the IgG-induced aggregation was greater than that of desialylation on the fibrinogen-induced aggregation. (4) The roles of plasma proteins and of sialic acid content of erythrocytes on the aggregation of erythrocytes were discussed.     

Structural studies of Fc receptors. III. Isolation and molecular weight analysis of the component chain of Fc gamma receptor of macrophage

Surface receptors of guinea pig peritoneal macrophages specific for the Fc region of IgG (Fc gamma receptor) were isolated and identified as a surface-radioiodinated component with a molecular weight of 44,000 that bound in an Fc-specific manner to IgG2 of guinea pig immunoglobulin immobilized in any of the following three different ways: IgG2 antibody in insoluble immune complex, IgG2 antibody bound to antigen-coupled Sepharose, and IgG2 covalently coupled to Sepharose. In order to obtain the Fc gamma receptor retaining the binding activity, the Fc-binding component was isolated by IgG2 affinity chromatography in which mild acidic buffer (pH 5.0-4.0) was chosen to elute the component bound to the affinity column. Forty-five to sixty-two percent of the eluted radioactivity was shown to rebind to the IgG2-affinity column. The bound fraction showed a single radioactive peak of 44,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Fc-binding component isolated by the affinity chromatography behaved similarly in gel filtration in the presence of a detergent, as did the detergent-solubilized Fc gamma receptor before isolation by affinity chromatography. These results suggested that the Fc gamma receptor was isolated in a native form. Furthermore, it was confirmed that the isolated Fc gamma receptor is distinct from actin or the actin-like protein (DNase I-binding protein) which had been reported to bind to IgG-affinity column.     

The preparation of rat liver lysosome membranes. Several membrane proteins contain complex oligosaccharides

Lysosome membranes were isolated, and membrane proteins and glycoproteins were characterized by electrophoresis and lectin probes of nitrocellulose blots. Rat liver lysosomes were isolated on a discontinuous metrizamide gradient and characterized by subcellular marker enzymes. Lysosomes were lysed by hypotonic freeze-thaw shock and membranes were isolated. The release of beta-N-acetylhexosaminidase was used to monitor the disruption of the lysosomes. Proteins of lysosome membranes were analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis. There were at least 30 proteins present and several were glycoproteins. Nitrocellulose blots of lysosome membrane proteins were probed with a panel of lectins, including concanavalin A, Ulex europaeus agglutinin I, Lotus tetragonolobus agglutinin, soybean agglutinin, peanut agglutinin, and Ricinus communis agglutinin I. Peanut agglutinin and Ricinus communis agglutinin I binding were also examined after neuramidase treatment of lysosome membranes. Ten proteins bound concanavalin A, and neuraminidase pretreatment revealed six proteins that bound Ricinus communis agglutinin I and three proteins that bound peanut agglutinin. The other lectins tested did not bind to any lysosome membrane proteins. These results indicate that lysosome membranes contain several glycoproteins, some of which contain sialic acid terminating complex oligosaccharides.     

A rapid method for the isolation and characterization of a homogeneous population of sterptococcal Fc receptors

Immunoblotting techniques were developed and used to determine the most suitable conditions for extracting bacterial receptors for the Fc region of human IgG. Crude extracts of a group C streptococcus were separated on 10% polyacrylamide SDS slab gels, electroblotted onto a nitrocellulose membrane, probed with radioiodinated human IgG containing unlabeled F(ab)₂ fragments and visualized by autoradiography. This procedure enabled us to compare size, heterogeneity and quantity of functionally active Fc receptors in crude extracts. Although more total Fc receptor could be extracted by phage lysis or mutanolysin treatment, only treatment of the group C streptococcus with trypsin, under suboptimal pH conditions for enzyme activity, resulted in a homogeneous product. The yield of affinity purified Fc receptor was 64 μg/g (wet weight) of bacteria. The affinity purified protein had a molecular weight of 40 000 and retained its ability to bond to the Fc region of IgG. The methods described are also applicable to the isolation of Fc receptors from other bacterial sources.