Passive enhancement of mouse skin allografts by alloantibodies is Fc dependent.

The capacity of F(ab')2 fragments of alloantibodies to enhance mouse allografts was studied in B6AF1 recipients of B10.D2 skin grafts. F(ab')2 obtained by digestion of B6AF1 anti-B10.D2 antibodies was purified by means of affinity chromatography, with anti-subclass antisera and protein A. The degree of contaminating IgG was less than 0.02%. Administration of F(ab')2 with an antigen-binding capacity similar to the IgG from which it originated, inhibited acute antibody-mediated graft rejection but was unable to induce enhancement. Even a dose that was 130 times the molar amount of the minimal enhancing dose of undigested IgG2 was ineffective. We conclude, therefore, that passive enhancement of mouse skin allografts by alloantibodies requires the Fc part.     

Evolution of alloantibodies and suppressor cells in allografted mice treated for passive enhancement

The kinetics and quality of the alloimmune reaction were studied in CBA (H-2k) mice treated for passive enhancement of tumor allografts (Sa 1 indigenous of A/J (H-2a or H-2k/d) mice). Serum samples of treated animals were tested for their biological properties relevant to different antibody isotypes in vitro (hemagglutination, complement-dependent cytotoxicity, and anaphylaxis, i.e., mast cell degranulation involving all main Ig isotypes; IgM, IgG2, and IgG1, IgE, respectively) as well as in vivo (allograft enhancement). Spleen cells from these treated animals were examined for their capacity to interfere with the rejection of tumor allografts by adoptive transfers into syngeneic recipients. In vitro, 51Cr release cytolysis assays were performed in order to test their cytolytic and regulatory activities in comparison to rejecting control animals. It has been shown that: grafted mice, pretreated for passive enhancement, kept their grafts longer and synthetized anaphylactic antibodies (mainly IgG1) earlier and at higher titers than normal serum controls, which rejected the same Sa 1 allografts. Mice with enhanced tumors synthetized cytotoxic antibodies (mainly IgG2) later than rejecting controls. Serum samples from treated and control animals, harvested 10 days (early sera) and 30 days (late sera) after grafting, were injected with a "normal dose" (0.2 ml) and a "high" dose (0.4 ml) to new CBA recipients grafted with Sa 1. Early immune sera were only enhancing at high doses when derived from animals previously treated for enhancement (at the low dose both immune sera were enhancing). Late sera, presenting both complement-fixing, cytotoxic (predominantly IgG2), and IgG1 anaphylactic alloantibodies in the two groups, induced enhancement in all cases, but more strongly when derived from the group treated for Sa 1 enhancement. Adoptive transfer of spleen cells from animals treated for passive enhancement were able either to inhibit the accelerated rejection (Day 10) or to promote enhancement of Sa 1 allogeneic cells (Day 30) while similar cells taken (Day 10 and Day 30) from control graft-rejecting mice transferred accelerated rejection. Among the transferred T-cell sub-populations, the suppressive effect was mediated by Lyt 2 T cells. In vitro, these spleen cells showed a weaker cytolytic activity than those of allograft-rejecting mice. Moreover, they were able to regulate the cytolytic activity of cytotoxic effector cells from specifically immunized CBA mice.     

Enhancement of antigen-induced interleukin 4 and IgE production by specific IgG1 in murine lymphocytes.

Conalbumin (CA)-specific type 2 helper T cell (Th2) clone, D10G4.1 (D10) produces IL4 when stimulated with varying doses of TNP-CA in the presence of mitomycin C-treated C3H spleen cells or purified B cells as antigen-presenting cells (APC). The production of IL4 was assessed by bioassay and by expression of IL4 mRNA. IL4 production reached maximum at 100 micrograms/ml of TNP-CA, whereas 1 microgram/ml of the antigen induced less than 10% of the maximum level of IL4. This lower level of IL4 production was augmented to the maximum level when monoclonal anti-TNP IgG1 was added to the culture at 0.5-1 microgram/ml. Anti-TNP IgE, but not anti-TNP IgM, was also effective, though IgE was 1/10 as effective as IgG1. IgG1 with an irrelevant specificity and F(ab')2 of anti-TNP IgG1 did not show augmenting effects. Moreover, the enhancement by anti-TNP IgG1 was completely abolished by monoclonal antibody against murine Fc gamma RII, 2.4G2. These results suggest that a low dose of the antigen complexed with IgG1 is focused on APC by means of Fc gamma RII, processed, and presented efficiently to the Th2 clone. On the other hand, the co-culture of D10 with normal C3H B cells in the presence of 1-100 micrograms/ml TNP-CA resulted in polyclonal IgE production. Anti-TNP IgG1 markedly augmented the lower level of IgE production induced by a suboptimal dose of the antigen (1 microgram/ml). This augmentation was shown to be dependent on endogenous IL4 because the enhancement was abolished by monoclonal anti-IL4 (11B11).     

Triggering of the superoxide generation of macrophages by crosslinking of Fc gamma receptor

Crosslinking of monomeric IgG2 molecules bound to the Fc gamma receptors on the cell surface of guinea pig macrophages generated the triggering signal for the superoxide-generating system. A binding experiment indicated that macrophages have saturable binding sites for monomeric IgG2. Scatchard analysis of the binding data showed that macrophages have an average of 4 X 10(5) binding sites per cell and the association constant for the binding was 4.2 X 10(6) M-1. Binding of monomeric IgG2 to macrophages could be detected by subsequent reaction with the 125I-labeled F(ab')2 fragment of rabbit antibody specific for guinea pig Fab. Although binding of IgG2 monomer to Fc receptor did not stimulate superoxide release, further addition of the F(ab')2 fragment of anti-guinea pig Fab antibody did induce generation and release of superoxide, and the amount released was dependent on the dose of cell-bound IgG2. When macrophages were bound with a constant dose of IgG2 monomer in the first step, the superoxide release triggered by the addition of the F(ab')2 of anti-guinea pig Fab was dependent on the dose of the F(ab')2 fragment added. These results show that crosslinking of Fc receptors triggers the superoxide generation.     

Inhibition of anti-IgM and growth factor-induced proliferation of guinea pig B cells by one of two distinct Fc gamma receptors on the cells

Guinea pig B cells were found to proliferate when co-stimulated with F(ab')2 of rabbit anti-guinea pig IgM and human 12-kDa B cell growth factor (BCGF), though the proliferation did not occur with the replacement of the F(ab')2 by its parent IgG antibody. In addition, the intact antibody inhibited the proliferation induced by F(ab')2 of anti-IgM and BCGF. Because both two distinct types of FcR for IgG on the B cells, one specific for IgG2 (Fc gamma 2R) and the other for both IgG2 and IgG1 (Fc gamma 1/gamma 2R), can bind rabbit IgG, we determined whether they participate in the inhibition of the B cell proliferation by intact anti-guinea pig IgM antibody. Blocking Fc gamma 1/gamma 2R by F(ab')2 of anti-Fc gamma 1/gamma 2R mAb significantly reversed the inhibitory effect of intact anti-IgM antibody. F(ab')2 of anti-Fc gamma 2R mAb, however, was not effective. Furthermore, guinea pig IgG1 and IgG2 anti-rabbit IgG antibodies suppressed similarly the B cell proliferation induced by F(ab')2 of rabbit anti-IgM and BCGF. These results show that between these two types of Fc gamma R on B cells, Fc gamma 1/gamma 2R alone is involved in the regulation of anti-IgM and BCGF-induced B cell proliferation, and inhibits the response when cross-linked to the surface IgM.     

Cross-linking of B lymphocyte Fc gamma receptors and membrane immunoglobulin inhibits anti-immunoglobulin-induced blastogenesis

The Fc portion of rabbit anti-mouse immunoglobulin (Ig) antibodies interferes with anti-Ig-induced B lymphocyte activation as measured by DNA synthesis on day 3 of culture or maturation to Ig-secreting cells in the presence of soluble helper factors on day 4 or 5. To investigate this Fc-dependent effect at an earlier stage in B cell activation, rabbit IgG anti-mouse mu-chain- or delta-chain-specific antibodies were compared with their F(ab')2 fragments for the ability to induce mouse B cells to undergo blast transformation, as defined by an increase in cell volume during the first 24 hr of culture. Both F(ab')2 anti-Ig reagents induce blast transformation, although F(ab')2 anti-mu antibodies induce a greater size change than F(ab')2 anti-delta antibodies. Whole anti-mu or anti-delta antibodies do not induce blast transformation; however, in the presence of a monoclonal anti-mouse Fc gamma receptor antibody that blocks IgG binding to Fc gamma receptors (Fc gamma R), whole anti-mu or anti-delta antibodies induce blast transformation as well as their F(ab')2 fragments. Because the anti-Fc gamma R antibody alone has no effect on blast transformation, it appears that the simultaneous binding of membrane IgM (or IgD) and Fc gamma R by whole anti-Ig antibodies prevents this early event in membrane Ig-induced B cell activation.     

Absence of auto-antiidiotypic activity between the IgM and IgG fractions of human mixed cryoglobulins

Experimental animal models and observations in humans suggest that levels of Id and auto-anti-Id fluctuate reciprocally after Ag stimulation. In human monoclonal B cell disorders, however, the co-existence of paraprotein Id and its auto-anti-Id has been described in essential mixed cryoglobulinemia and in association with acquired C1 inhibitor deficiency. Because the majority of cryoglobulin IgM possess rheumatoid factor activity and thus bind the Fc region of IgG, we examined potential idiotypic interactions between cryoglobulin IgM and F(ab')2 fragments of autologous cryoglobulin IgG fractions. A rabbit antibody to the pepsin agglutinator site of human F(ab')2 was used as detection reagent. By recognizing epitopes exposed on F(ab')2 after the removal of Fc determinants by pepsin digestion, this reagent eliminates the detection of contaminating intact IgG. In a sensitive assay, we were unable to detect idiotypic interactions between the separated IgM and pepsin-digested IgG fractions of 10 mixed cryoglobulins. On the basis of these results, we suggest that in mixed cryoglobulinemia, the coexistence of paraprotein Id and its auto-anti-Id is unlikely.     

Further studies on the biologic properties of guinea pig IgG1 antibodies. II. In vivo activation of C3 by anti-glomerular basement membrane antibodies.

Normal rats were injected with guinea pig anti-rat glomerular basement membrane antibodies of the IgG1 or IgG2 class or with their F (ab') 2 fragments, in order to study which antibody site triggers the alternate complement pathway in vivo. Both IgG classes were able to induce a heavy proteinuria and led to C3 deposition in the glomeruli in a pattern similar to their own distribution along the glomerular basement membrane, as shown by the immunofluorescence technique. The Fab(ab')2 fragment of IgG2 did not produce C3 binding or proteinuria. The F(ab')2 fragment of IgG1 was difficult to obtain devoid of Fc determinants. A F(ab')2 fragment of IgG1 still bearing Fc determinants led to C3 binding and proteinuria, whereas the true F(ab')2 fragment of IgG1 had none of these effects in two out of three animals.     

The use of haptenated immunoglobulins to induce B cell tolerance in vitro. The roles of hapten density and the Fc portion of the immunoglobulin carrier

The relationship between the Fc region of trinitrophenylated (TNP)-immunoglobulins (Ig), and their ability to induce tolerance was examined. It was found that adult B cells responding to a T-independent (TI) antigen were tolerized by TNP11 human gamma globulin (HGG), but not by TNP10F(ab')2 fragments of HGG. Increasing the hapten density on the F(ab')2 fragments overcame their inability to induce tolerance. Thus, a TNP17-F(ab')2 was an effective tolerogen. Murine myeloma proteins of different IgG subclasses were similarly tested. A TNP12-IgG2a and a TNP11-IgG1 induced tolerance, whereas two TNP11-12-IgG3 did not. However, a more heavily haptenated TNP18-IgG3 was tolerogenic. These results suggest that lightly haptenated immunoglobulins depend upon Fc receptor binding to induce tolerance in adult B cells. Non-Fc receptor-binding carriers are not tolerogenic unless they are more heavily haptenated. Finally, T cell and macrophage depletion experiments suggest that the tolerogens act directly on the B cells.     

Structural effect of a recombinant monoclonal antibody on hinge region peptide bond hydrolysis

IgG hinge region peptide bonds are susceptible to degradation by hydrolysis. To study the effect of Fab and Fc on hinge region peptide bond hydrolysis, a recombinant humanized monoclonal IgG1 antibody, its F(ab')2 fragment, and a model peptide with amino acid sequence corresponding to the hinge region were incubated at 40 degrees C in formulation buffer including complete protease inhibitor and EDTA for 0, 2, 4, 6 and 8 weeks. Two major cleavage sites were identified in the hinge region of the intact recombinant humanized monoclonal antibody and its F(ab')2 fragment, but only one major cleavage site of the model peptide was identified. Hinge region peptide bond hydrolysis of the intact antibody and its F(ab')2 fragment degraded at comparable rates, while the model peptide degraded much faster. It was concluded that Fab region of the IgG, but not Fc portion had significant effect on preventing peptide bond cleavage by direct hydrolysis. Hydrolysis of hinge region peptide bonds was accelerated under both acidic and basic conditions.     

Mechanism of action of anti-IL-2R monoclonal antibodies. ART-18 prolongs cardiac allograft survival in rats by elimination of IL-2R+ mononuclear cells

ART-18, a mouse IgG1 mAb recognizing the IL-2 binding domain of the rat p55 subunit IL-2R molecule, prevents graft rejection in various experimental models, although its mechanism of action in vivo, like that of anti-IL-2R mAb generally, remains elusive. These studies were designed to define whether IL-2R+ T effector cells were actually eliminated or their function merely inhibited by comparing directly the in vitro and in vivo efficacy of intact ART-18 and its F(ab)/F(ab')2 fragments. Addition of each mAb preparation profoundly suppressed MLR set up between naive LEW responders and x-radiated BN stimulators, suggesting that mAb fragments retained Ag binding functions in vitro. However, both ART-18 F(ab) and F(ab')2 were ineffectual in vivo as judged by their inability to affect acute (8 days) rejection of (LEW X BN)F1 cardiac allografts in LEW recipients (graft survival ca. 11 and 9 days, respectively, compared to ca. 21 days after therapy with intact ART-18, p less than 0.001). The sera levels of ART-18 and ART-18 F(ab')2 were 4 to 5 micrograms/ml, but only less than 0.5 micrograms/ml of F(ab) could be detected. The therapeutic failure of ART-18 fragments was unrelated to potential host sensitization, as rat antimouse F(ab) or F(ab')2 serum IgG titers remained in the same range as those against intact ART-18. The role of the Fc portion of Ig in the mode of action of ART-18 was then tested further by flow microfluorimetry analysis of host mononuclear spleen cells and immunoperoxidase stains of the graft infiltrate. IL-2R+ cells were abundant in rats treated with ART-18 fragments, comparable to acutely rejecting controls. In contrast, IL-2R expression was abolished in animals undergoing ART-18 therapy. The elimination of IL-2R+ cells is required to prolong cardiac allograft survival in rats after IL-2R targeted treatment with ART-18 mAb.     

Induction of apoptosis by monoclonal antibody anti-APO-1 class switch variants is dependent on cross-linking of APO-1 cell surface antigens.

Apoptosis, programmed cell death, was previously shown to be induced by the mAb anti-APO-1 (IgG3, kappa) by binding to the APO-1 cell surface Ag, a new member of the nerve growth factor/TNF receptor superfamily. To investigate the role of the Ig H chain Fc regions we compared induction of apoptosis by the original mAb IgG3 anti-APO-1 with anti-APO-1 F(ab')2 fragments and different anti-APO-1 isotypes (IgG1, IgG2b, IgG2a, and IgA) isolated by sequential sublining. We found that IgG3 was the most active isotype; IgG1, IgG2a, and IgA showed intermediate activity, and IgG2b and F(ab')2 were inactive. Cytotoxic activity of the inactive or less active antibody preparations was fully reconstituted by protein A, anti-mouse Ig, or anti-mouse Ig F(ab')2, respectively. Thus, APO-1-mediated induction of apoptosis was dependent on efficient cross-linking of APO-1 cell surface Ag, indirectly augmented by anti-APO-1 Fc-Fc self-aggregation. Because of their different in vitro activity we selected IgG3-, IgG2b-, and IgA anti-APO-1 to test their antitumor activity against solid human B lymphoblastoid tumors in SCID mice. The isotypes showed a different serum half-life (IgG3: 9.2-10.4 days, IgG2b: 1.9-2.6 days, and IgA: 14.1-29.2 h) and a different initial tumor localization 4 h after i.p. injection (IgG3 around the blood vessels, IgG2b homogeneously, and IgA heterogeneously distributed in the tumor). All antibody preparations induced tumor regression by induction of apoptosis, even IgG2b anti-APO-1 inactive in vitro without cross-linking. The activity of IgA anti-APO-1, which did not mediate complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity indicates that apoptosis may be used as the main if not the only mechanism of induction of tumor regression in vivo. As with in vitro, IgG3 anti-APO-1 was the most effective isotype also in vivo. This result suggests that cross-linking of APO-1 on the tumor cell surface may also be required for tumor regression by apoptosis in vivo. Taken together, our data show that selective targeting of apoptosis to tumors may be an efficient antitumor mechanism.     

Studies of allogeneic tumor transplants: induced rejection of advanced tumors by immune alteration of recipients

In the present experiments, a methylcholanthrene-induced sarcoma (S-702) of B10.D2 origin was found to grow rapidly in B6AF1 mice leading to the death of all recipients in 5 to 9 wk. Nevertheless, immunity to MHC antigens presented by the tumor was readily demonstrable in tumor-bearing mice by their responses to donor strain skin grafts until late in the course of tumor growth, when a nonspecific form of immune suppression developed. In addition, B6AF1 mice preimmunized by exposure to B10.D2 donor strain antigens did not permit tumor growth. Treatment of tumor-bearing B6AF1 mice with CY at 18 days, when the tumors measured over 12-mm in diameter, followed by the i.p. injection of B10.D2 lymphoid cells (at a dosage of from 1.2 to 2.5 X 10(8) cells) resulted in the complete regression of 100% of these large tumors. CY treatment combined with localized immune stimuli in the form of donor strain skin grafts or secondary tumor implants was incapable of producing a sufficiently heightened immune response to cause tumor rejection. A dose of CY temporarily retarded tumor growth in most mice, and in a minority of animals so treated (less than 25%) tumors regressed completely. In syngeneic (B10.D2) animals, CY also temporarily slowed tumor growth, but total regression was never observed. An effective B10.D2 cell inoculum could consist not only of living lymphoid cells but of irradiated (1000 rad) cells as well. Tumor cell suspensions (after irradiation, 10,000 rad) were also effective. These observations suggest local immune factors at the host-tumor interface may have been of importance in the survival of these allogeneic tumor transplants and that CY influenced this state, perhaps through an influence on suppressor cells, allowing subsequent administration of donor strain cellular antigens to induce an effective tumor rejection response.     

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.     

Interactions between lymphocyte membrane molecules. II. Characterization of an interaction between B lymphocyte surface IgD and Fc IgG receptors that differs from the surface IgM-Fc IgG receptor interaction

The B lymphocyte surface membrane receptors IgD (sigD) and Fc IgG receptors (Fc gamma R) were evaluated for interactions by means of immunofluorescence. Ligand-(F(ab')2 anti-delta) induced capping of sIgD resulted in co-capping of Fc gamma R if the latter were occupied during the capping process by soluble antigen-antibody complexes (which themselves provided insufficient cross-linking to result in capping), but not if the Fc gamma R were occupied by monomeric IgG or unoccupied. Capping of Fc gamma R by highly cross-linked complexes did not cause co-capping of sIgD occupied by monomeric F(ab') anti-delta. The interaction between sIgD and Fc gamma R was specific in that cross-reactions between ligands were excluded and ligand-induced capping of sIgD did not cause co-capping of ligand-occupied sIgM or I-A antigens. The sIgD-Fc gamma R interaction occurred on only approximately 60% of B lymphocytes, and this B cell subpopulation did not correlate with other B cell subpopulations (CBA/N strain B cells and B cells bearing either large or small amounts of sIgD). The sIgD-Fc gamma R interaction differed from the sIgM-Fc gamma R interaction in that co-redistribution of the Fc gamma R was occupied by monomeric IgG and involved nearly all B lymphocytes. The qualitative and quantitative differences between the sIgD-Fc gamma R and sIgM-Fc gamma R interactions suggest a mechanism whereby the two antigen receptors could provide different signals to the B lymphocyte.     

The use of haptenated-immunoglobulin molecules to induce tolerance in B cells from neonatal mice

The role of the Fc region of trinitrophenylated (TNP)-immunoglobulins (Ig) in their ability to induce tolerance in immature B cells was examined. With the use of B cells from neonatal mice, tolerogens that could or could not bind to Fc receptors were assessed for their ability to induce tolerance. This was accomplished by tolerizing spleen cells in bulk culture and assessing the degree of tolerance by challenging the cells with the thymus-independent antigen TNP-Brucella abortus (TNP-BA) in limiting dilution cultures. It was found that by using tolerogens containing 10 to 11 haptens per Ig molecule, immature B cells were very susceptible to tolerance induction. Mature B cells were not as susceptible. This increased susceptibility was independent of the Fc portion of the tolerogen, because TNP11-HGG and a TNP10-F(ab')2 induced equivalent degrees of unresponsiveness. When the TNP density was lowered to approximately five haptens per Ig molecule, those Ig molecules that contained Fc portions were superior tolerogens with the use of B cells from 6-day-old mice. Thus, a TNP4-HGG, TNP7-mouse IgG1, and TNP6-mouse IgG2a were more effective tolerogens than either TNP5-F(ab')2 or TNP6-mouse IgG3. These results confirm previous findings that immature B cells are inherently more susceptible to tolerance induction than mature B cells. They also suggest that very lightly haptenated Ig molecules may depend on Fc receptor-binding for effective tolerance induction. Finally, by means of a cytofluorograph, the surface IgD (sIgD) and sIgM phenotypes of splenic B cells from neonates of increasing age were determined. When comparing the phenotype of maturing cells with their tolerance susceptibilities, a correlation between the appearance of sIgD and the acquisition of resistance to tolerance was observed.     

Immunization strategies to block the herpes simplex virus type 1 immunoglobulin G Fc receptor

Herpes simplex virus type 1 (HSV-1) glycoprotein gE functions as an immunoglobulin G (IgG) Fc receptor (FcgammaR) that promotes immune evasion. When an IgG antibody binds by the F(ab')(2) domain to an HSV antigen, the Fc domain of some of the same antibody molecules binds to the FcgammaR, which blocks Fc-mediated functions. gE is a type 1 membrane glycoprotein with a large ectodomain that is expressed on the virion envelope and infected-cell surface. Our goal was to determine if immunizing with gE protein fragments could produce antibodies that bind by the F(ab')(2) domain to gE and block the FcgammaR, as measured by competitively inhibiting nonimmune human IgG binding to the FcgammaR. Three gE peptides were constructed in baculovirus spanning almost the entire ectodomain and used to immunize mice and rabbits. Two fragments were highly effective at producing antibodies that bind by the F(ab')(2) domain and block the FcgammaR. The most potent of these two antibodies was far more effective at blocking the FcgammaR than antibodies that are only capable of binding by the Fc domains to the FcgammaR, including anti-gC, anti-gD, and nonimmune IgG. These results suggest that immunizing with gE fragments has potential for preventing immune evasion by blocking activities mediated by the HSV-1 FcgammaR.     

Thioether-bonded constructs of Fab'gamma and Fc gamma modules utilizing differential reduction of interchain disulfide bonds

We describe a two-stage preparation of chemically engineered Ab constructs, employing as modules Fab'gamma from mAb or rAb, and Fc from human normal IgG1. A multivalent, optionally multispecific F(ab')(n) core is formed in stage one, and one or more Fc modules added in stage two. Examples include bispecific Fab(2)Fc(2) (for simplicity, primes and Greek letters are omitted from names of final constructs) and trivalent Fab(3)Fc(2), which are designed to kill neoplastic cells. An essential element in the construction is the availability of the Fab' in two reduced forms, Fab'(-sulfhydryl (SH))(5) and Fab'-SH. The first is obtained by full reduction of the interchain disulfide bonds (SS) in the F(ab')(2) fragment of IgG. Fab'-SH is obtained by disulfide-interchange reactions on Fab'(-SH)(5), whereby the gamma-light SS is reconstituted, an unusual intrachain SS forms in the gamma-chain hinge, and one hinge SH remains. F(ab')(2) and F(ab')(3) cores are built using partially reduced modules, being given intermodular thioether links that resist reduction. These cores are then fully reduced, making available SH groups for addition of the Fcgamma modules. In the final constructs, all intermodular links embody tandem thioether bonds arising at hinge-region cysteines. Cytotoxic activities of representative constructs, and some enhancements deriving from multiple modules, are assessed. In guinea pigs, catabolism of Fab(2)Fc(2) yielded a t(1/2) similar to that of human IgG1, although the serum Fab(2)Fc(2) revealed some proteolytic breakdown not shown by the IgG1. Immunotherapy of a guinea-pig leukemia confirmed the ability of these constructs to kill target cells in vivo.     

Dichotomous effect of monocyte Fc receptor interaction on anti-CD3-induced immunoglobulin synthesis

Monoclonal antibodies against the TCR/CD3 complex are capable of activating T cells which in turn may induce immunoglobulin synthesis in B cells under appropriate conditions. Here we present evidence that distinct immune responses, induced by four commonly used TCR/CD3 mAb (Leu4, OKT3, BMA030, BMA031) were related to the mAb interaction with monocyte Fc receptors for IgG. Depending on their isotype and on the technique by which they were crosslinked, TCR/CD3 mAb induced variable IgM and IgG synthesis in PBMC: If the mAb were crosslinked by monocyte IgG-Fc receptors they induced a high Ig production, while crosslinking the same mAb by plastic-bound goat anti-mouse antibodies (panning) failed to do so. Nevertheless, both crosslinking techniques triggered a strong proliferation and IL-2, IL-4, and IFN gamma lymphokine gene expression. The lack of Ig production under panning conditions was due to an additional IgG-Fc receptor interaction with monocytes: (a) If namely mAb F(ab')2 fragments, or mAb isotypes unable to bind to monocyte Fc receptors (IgG2b, IgG1 in nonresponders) were crosslinked by panning, both a good proliferation as well as Ig production ensued; (b) if TCR/CD3 mAb isotypes which could additionally bind to monocyte Fc receptor (IgG2a) were crosslinked, no Ig production occurred; (c) if mAb F(ab')2 fragments were crosslinked with a second anti-T cell antibody of IgG2a isotype, which could bind to monocyte Fc receptors, Ig synthesis was reduced. Interestingly enough, this diminishing effect, due to monocyte Fc receptor interaction, was only observed if CD4-positive cells were proliferating, but not if CD8-positive cells were activated.     

Characterization of human T lymphocytes that express the C3b receptor

The presence of the C3b receptor (C3bR) on human peripheral blood T lymphocytes was recognized by the capacity of rabbit F(ab')2 anti-C3bR and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat F(ab')2 anti-rabbit F(ab')2 to stain 14.5 +/- 3.7% (mean +/- SEM; n = 5) of lymphocytes forming rosettes with sheep erythrocytes (E). The F(ab')2 anti-C3bR also blocked the capacity of peripheral blood lymphocytes stained with OKT11 to form rosettes with bovine E bearing C3b and immunoprecipitated a single membrane protein having a m.w. of approximately 250,000 from detergent lysates of 125I-labeled, purified T cells. Measurement by fluorescent flow cytometry of the quantitative expression of the C3bR indicated that T cells had slightly more antigenic sites/cell than did E and approximately 10-fold fewer sites than were present on B cells. The surface constituents of the peripheral blood T cells expressing the C3bR were assessed in an assay that employed simultaneously three markers: rosette formation with sheep E, TRITC staining with anti-C3bR and fluorescein isothiocyanate (FITC)-staining with a panel of monoclonal antibodies or with aggregated IgG. Among lymphocytes forming rosettes with sheep E and expressing the C3bR, 99.6 +/- 0.4%, 65.0 +/- 5.8%, 17.2 +/- 6.2%, and 15.3 +/- 5.0% of the cells expressed antigens detected by OKT3, OKT4, OKT8, and OKM1 monoclonal antibodies, respectively. Ninety-seven per cent of the C3bR-bearing T cells were also capable of specifically binding aggregated IgG, indicating the presence of Fc receptors for IgG (Fc gamma R) on these cells. The T cells expressing the C3bR had large nuclei, thin rims of basophilic cytoplasm and no azurophilic granules. Thus, the C3bR is present on some T cells, all of which have a typical lymphocyte morphology, the T3 antigen and the Fc gamma R.