Purification of ricin A1, A2, and B chains and characterization of their toxicity

This paper describes a protocol for the preparation of highly purified A (A1 and A2) and B chains of the plant toxin, ricin, and biochemical and biological characterization of these proteins. Intact ricin was bound to acid-treated Sepharose 4B and was split on the column into A and B chains with 2-mercaptoethanol. The A chains were eluted with borate buffer containing 2-mercaptoethanol. A1 and A2 were then partially separated by cation exchange chromatography and the contaminating B chain was removed by affinity chromatography on Sepharose-asialofetuin and Sepharose-monoclonal anti-B chain. The B chain was eluted from the Sepharose 4B column by treatment with galactose and was further purified by cation and anion exchange chromatography; contaminating A chains were removed by affinity chromatography on Sepharose-monoclonal anti-A chain. The purified A and B chains were active as determined by their ability to inhibit protein synthesis in a cell-free assay and their binding to asialofetuin, respectively. Furthermore, by polyacrylamide gel electrophoresis, toxicity in mice, and toxicity on several different cell types, both A and B chains were shown to be minimally cross-contaminated. Finally, it was shown that ammonium chloride significantly enhanced the nonspecific toxicity of B chains for cells in vitro. In contrast, ammonium chloride did not enhance either the nonspecific toxicity of A chains in vitro or the specific toxicity of A chain-containing immunotoxins prepared with the highly purified A1, A2 chains.     

Whole ricin and recombinant ricin A chain idiotype-specific immunotoxins for therapy of the guinea pig L2C B cell leukemia

The therapeutic efficacy of whole ricin, or recombinant ricin A chain, coupled to a monoclonal antibody that reacts with the idiotype of the surface IgM expressed on guinea pig L2C lymphoblasts, was assessed. In vitro studies were done to characterize the immunotoxins (IT) and to demonstrate their specificity before use in vivo. The concentration of whole ricin IT (M6-Ricin) that inhibited protein synthesis by 50% (IC50) in L2C cells was 1.4 X 10(-9) M, in a 5-hr assay, in the presence of lactose to block non-antibody-directed toxicity. M6-Ricin did not inhibit protein synthesis in two control guinea pig cell lines that did not express the idiotype, nor did a whole ricin IT prepared with an isotype-matched monoclonal antibody of irrelevant specificity inhibit protein synthesis in L2C cells. Two recombinant ricin A chain IT, which differed from one another by a factor of 2 to 3 in the number of A chains conjugated per antibody molecule, were less effective in vitro than M6-Ricin (IC50 of greater than 5 X 10(-8) M). For in vivo experiments, the IT were given by the i.p. route 24 hr after the i.p. inoculation of 1 X 10(5) L2C cells. The highest doses of M6-Ricin and M6-Ricin A chain IT tested, 30 micrograms/kg and 3000 micrograms/kg, respectively, were within fourfold to fivefold of their maximum tolerated doses; no deaths or ill effects due to ricin toxicity were noted. These doses increased the median survival time of L2C-bearing guinea pigs to 31 to 34 days, compared with 15 days for untreated animals. This magnitude of increase in survival indicates that 99.999% (5 logs) of injected tumor cells were eliminated, thus accounting for the 12% long-term survival rate obtained. Median survival times for guinea pigs treated with 30 micrograms/kg of the A chain IT were 18 and 21 days for the two conjugates tested, and the median survival for guinea pigs treated with 3000 micrograms/kg of unconjugated antibody was 18 days. Our data demonstrate that recombinant A chain IT are active in vivo and that the B chain of ricin can potentiate IT activity in vivo. Although the potency differs by 100-fold, the therapeutic index of the intact ricin IT is similar to that of the ricin A chain IT.     

Identification and characterization of a monoclonal antibody recognizing a galactose-binding domain of the toxin ricin

A monoclonal antibody raised against purified ricin B chain, 75/3B12, blocked ricin toxicity 30- to 100-fold in vitro. The 75/3B12 IgG and F(ab')2 blocked ricin binding to cell surface galactose-containing receptors. The 75/3B12 Fab bound ricin D with a Ka of 10(7) M-1, and this binding was blocked by asialofetuin, lactose, and N-acetylgalactosamine--molecules which interact with the ricin galactose-binding site--but not by fetuin, sucrose, or glucose. The 75/3B12 Fab contained no detectable carbohydrate and, according to several lines of evidence, did not bind ricin via Ig carbohydrate determinants. The monoclonal antibody appears to recognize a galactose-binding site on ricin D via the variable region of the antibody. The 75/3B12 Fab bound ricin E only 1/50 as well as ricin D and bound the Ricinus agglutinin only 1/80 as well as ricin D. The antibody specificity indicates that structural differences exist in the galactose-binding sites of the Ricinus communis lectins. Abrin and other lectins which bind galactose or N-acetylgalactosamine were not significantly bound by the monoclonal antibody. In vitro, the antibody blocked the nontarget toxicity of immunotoxins similarly to lactose. However, in vivo, unlike lactose, the 75/3B12 antibody protected mice from ricin toxicity.     

Synergy between immunotoxins prepared with native ricin A chains and chemically-modified ricin B chains

Ricin B chains treated with chloramine-T in the presence or absence of NaI show a 100-fold to 200-fold reduction in their ability to bind to the galactose-containing protein asialofetuin. Such treated B chains do not form covalently associated homodimers with treated B chains or heterodimers with native ricin A chains. Furthermore, they cannot enhance the toxicity of a ricin A chain-containing rabbit anti-human immunoglobulin (RAHIg-A) for Daudi cells. However, when such B chains are coupled to goat anti-rabbit Ig (GARIg), they potentiate the killing of RAHIg-A-treated Daudi cells only slightly less effectively than GARIg coupled to native B chains. Furthermore, if GARIg-B chain conjugates are treated with chloramine-T after coupling, they fail to bind to asialofetuin but enhance the killing of Daudi cells treated with RAHIg-A. These results demonstrate that the ability of ricin B chains to bind to galactose and to enhance the toxicity of ricin A chains (in the form of an antibody-A chain) can be operationally separated. Thus, the two functions of the B chain may reside on separate domains of the molecule.     

Inhibition of the helper function of murine T cells with Fab'-anti-L3T4 ricin A chain immunotoxin

The ability of Fab'-anti-L3T4 A chain-containing immunotoxins to inhibit the helper function of keyhole limpet hemocyanin-specific T helper lymphocytes was evaluated. Keyhole limpet hemocyanin-specific T helper cells were prepared from the lymph nodes of primed mice and were enriched for T cells. Enriched populations of trinitrophenyl-specific B cells were prepared from spleens of normal mice. In the presence of antigen, the keyhole limpet hemocyanin-specific T helper cells were able to induce proliferation and differentiation of the trinitrophenyl-specific B cells. However, when the T helper cells were first treated with an immunotoxin composed of Fab' fragments of anti-L3T4 antibody coupled to ricin A chain (Fab'-anti-L3T4-A), they failed to induce proliferation and differentiation of the antigen-specific B cells. The concentrations of Fab'-anti-L3T4-A required to inhibit T cell help for the proliferation and differentiation of trinitrophenyl-specific B cells by 50% were 1 X 10(-9)M and 4 X 10(-10) M, respectively. Fab'-anti-L3T4 antibody alone did not inhibit T cell-induced B cell proliferation and differentiation by 50% at greater than 100-fold higher concentrations.     

Expression of an anti-CD3 single-chain immunotoxin with a truncated diphtheria toxin in a mutant CHO cell line

ADP-ribosylating immunotoxins are generally expressed in Escherichia coli and then refolded in vitro. Because the efficiency of the in vitro refolding process decreases with the number of protein domains and internal disulfide bonds, these immunotoxins have been generally limited to single-chain monovalent structures. We now show that using the hamster cell line CHO K1 RE1.22c (J. M. Moehring and T. J. Moehring, 1979, Somat. Cell Genet. 5, 453-468) that has been mutated to ADP-ribosylation insensitivity, a level of 4 microg/ml of a truncated anti-T cell immunotoxin, DT390-scFvUCHT1, can be secreted into the medium. This immunotoxin is glycosylated at the two potential N-linked glycosylation sites in the toxin moiety: positions 16-18 in the A chain and residues 235-237 in the B chain. The glycosylated immunotoxin is relatively nontoxic (IC(50) 4.8 x 10(-10) M). Removal of the N-linked oligosaccharides by N-glycosidase F treatment or mutations at the two N-linked glycosylation sites results in a highly active immunotoxin with an IC(50) of 4 x 10(-12) M toward CD3(+) Jurkat cells. This is a 12-fold increase in toxicity over the same immunotoxin harvested from E. coli periplasm without refolding. A single Asn(235) Ala mutation that removed the B chain glycosylation was nearly as toxic as the double mutant. This suggests that B chain glycosylation is the major cause for the loss of toxicity.     

Comparison of the cytotoxic potency of T101 Fab, F(ab')2 and whole IgG immunotoxins

The in vitro killing of the human CEM cell line was studied by using ricin A-chain immunotoxins constructed with either the whole IgG or the Fab and F(ab')2 fragments of the same T101 (anti-CD5) antibody. In the presence of ammonium chloride as an activator, the "whole" immunotoxin as well as the "fragment" immunotoxins did not show any significant difference in the cell killing efficacy. In contrast, without the activator, the efficacy of the T101 immunotoxin was greatly improved when fragments were used. Indeed, at a saturating dose, a cytoreduction of three orders of magnitude was obtained with the fragment immunotoxins vs less than one order of magnitude for the whole immunotoxin, as assessed in a clonogenic assay. This enhancing effect was related to better cell killing kinetics, because with a similar amount of A-chain molecules bound per cell, T101 fragment immunotoxins achieved a twofold faster protein synthesis inactivation rate than the corresponding whole IgG immunotoxin. No significant difference in activity was shown between monovalent (Fab) and divalent (F(ab')2) forms of fragment immunotoxins. The observation that T101 fragment immunotoxins were more potent than intact immunotoxins was extended to another fragment immunotoxin constructed with an antibody (F111.98) directed against a different epitope of the CD5 Ag. In another model (anti-CD22 1G11 antibody on Raji cells), the fragment immunotoxin did not show any superiority over the IgG immunotoxin which was by itself very potent, strongly suggesting an Ag-dependent phenomenon.     

Blocked and non-blocked ricin immunotoxins against the CD4 antigen exhibit higher cytotoxic potency than a ricin A chain immunotoxin potentiated with ricin B chain or with a ricin B chain immunotoxin

Summary An immunotoxin consisting of ricin A chain linked to the monoclonal antibody M-T151, recognising the CD4 antigen, was weakly toxic to the human T-lymphoblastoid cell line CEM in tissue culture. The incorporation of [³H]leucine by CEM cells was inhibited by 50% at an M-T151-ricin-A-chain concentration (IC₅₀) of 4.6 nM compared with an IC₅₀ of 1.0 pM for ricin. In contrast, immunotoxins made by linking intact ricin to M-T151 in such a way that the galactose-binding sites of the B chain subunit were either blocked sterically by the antibody component or were left unblocked, were both powerfully cytotoxic with IC₅₀ values of 20–30 pM. The addition of ricin B chain to CEM cells treated with M-T151—ricin-A-chain enhanced cytotoxicity by only eight-fold indicating that isolated B chain potentiated the action of the A chain less effectively than it did as an integral component of an intact ricin immunotoxin. Ricin B chain linked to goat anti-(mouse immunoglobulin) also potentiated weakly.Lactose completely inhibited the ability of isolated ricin B chain to potentiate the cytotoxicity of M-T151—ricin-A-chain and partially (3- to 4-fold) inhibited the cytotoxicity of the blocked and non-blocked ricin immunotoxins. Thus, in this system, the galactose-binding sites of the B chain contributed to cell killing regardless of whether isolated B chain was associated with the A chain immunotoxin or was present in blocked or non-blocked form as part of an intact ricin immunotoxin. The findings suggest that the blocked ricin immunotoxin may become unblocked after binding to the target antigen to re-expose the cryptic galactose-binding sites. However, the unblocking cannot be complete because the maximal inhibition of [³H]leucine incorporation by the blocked immunotoxin was only 80% compared with greater than 99% inhibition by the non-blocked immunotoxin.     

Expression of ricin A chain and ricin A chain-KDEL in Escherichia coli

Ricin and its A chains can be used to conjugate with monoclonal antibodies to prepare immunotoxins. Ricin A chain (RTA) and its modification RTA-KDEL (ER-retrieval signal) were expressed with the pKK223.3 system in Escherichia coli under control of a tac promoter. The recombinant proteins can be purified by one-step affinity chromatography on a column of Blue-Sepharose 6B. The toxicities of RTA and its mutant RTA-KDEL were evaluated by the MTT assay in HeLa, MCF, and ECV-304 cells following fluid-phase endocytosis. RTA-KDEL was somewhat more cytotoxic than RTA itself in the different cell lines. The results suggest that rRTA-KDEL may be useful for the synthesis of more potent immunotoxins.     

Identification of carbohydrate moieties involved in EDTA-stable or EDTA-sensitive cell contact of Dictyostelium discoideum

Antisera against purified contact site A glycoprotein, with an apparent molecular weight of 80 X 10(3) (80 kDa), from Dictyostelium discoideum were raised by using Freund's adjuvant (antiserum-A) and by using Alu-Gel-S (antiserum-B) as immunoadjuvants. They were converted into Fab fragments for the cell agglutination assay. Fab fragments of antiserum-B inhibited only EDTA-stable cell contact, whereas Fab fragments of antiserum-A (Fab-A) inhibited EDTA-sensitive cell contact as well as EDTA-stable cell contact. We prepared several cell types in order to identify target antigens for the adhesion-blocking Fab-A in EDTA-sensitive cell contact or EDTA-stable cell contact. One of these cell types produced contact site A without N-glycosidically-linked carbohydrate chains. It is known that contact site A contains two kinds of N-glycosidically-linked carbohydrate chains (carbohydrates I and II, Yoshida, M., Stadler, J., Bertholdt, G., and Gerisch, G. (1984) EMBO J. 3, 2653-2670). When growth-phase cells were treated with tunicamycin (TM) at a final concentration of 2 micrograms/ml in nutrient medium (TM-pretreated cells), the cells produced contact site A without N-glycosidically-linked carbohydrate chains (53 kDa) at the normal developmental stage. These cells lacked EDTA-sensitive cell contact as well as EDTA-stable cell contact. The neutralization of the adhesion-blocking Fab-A was done by using particulate fractions from each cell type. The blocking activity in EDTA-stable cell contact was neutralized by the cell type with carbohydrate II. Taking these results into consideration, EDTA-stable cell contact may be formed by the interaction between protein moieties of contact site A and carbohydrate II. Concerning EDTA-sensitive cell contact, the blocking activity was neutralized by each cell type irrespective of TM treatment. This suggests that O-glycosidically-linked carbohydrate chains play a role in EDTA-sensitive cell contact. Moreover, the biological activity in EDTA-sensitive cell contact of TM-pretreated cells suggests that N-glycosidically-linked carbohydrate chains may also be involved in this contact.     

Arming alpha 2-macroglobulin with ricin A chain forms a cytotoxic conjugate that inhibits protein synthesis and kills human fibroblasts

A conjugate containing alpha 2-macroglobulin and highly purified ricin A chain was made using N-succinimidyl-3-(2-pyridyldithio)propionate. Radioimmunoassay indicated that it contained 1.2 mol A chain per mol alpha 2-macroglobulin. The conjugate inhibited polyuridylic-acid directed translation by rat liver ribosomes and protein synthesis in human fibroblasts. There was a 90 min lag period before the beginning of inhibition in fibroblasts, but complete inhibition could be achieved. By measuring protein synthesis as a function of protein concentration, it was demonstrated that 8.25 X 10(-9) M conjugate was required to inhibit 50% of protein synthesis in 6 h. To achieve the same level of inhibition, 165-times more (1.3 X 10(-6) M) unconjugated A chain was required, and 180-times less ricin (4.6 X 10(-11) M). Ricin was more than 28 000 times more inhibitory than A chain alone. The presence of alpha 2-macroglobulin did not increase the cytotoxicity of unconjugated A chain, and it even protected the cells to a slight extent. The inhibitory action of the conjugate was blocked by antibodies specific for alpha 2-macroglobulin or ricin, and it was not prevented by galactose or antibodies specific for ricin B chain. Electron microscopy of the conjugate indirectly labelled with ferritin demonstrated that it was internalized by receptor mediated endocytosis through coated pits. These data indicate that the A chain portion of the conjugate survives the conditions in the lysosomes to the extent that it retains its ability to inactivate cytoplasmic ribosomes.     

Ricin B chain-containing immunotoxins prepared with heat-denatured B chain lacking galactose-binding ability potentiate the cytotoxicity of a cell-reactive ricin A chain immunotoxin

Ricin B chain incubated at 37 degrees C in the absence of lactose loses its ability to bind the galactose-containing protein, asialofetuin. Circular dichroism analysis of the B chain during thermal denaturation indicates that the loss of galactose-binding ability by the B chain correlates with limited unfolding of the molecule. As a result of this conformational change, disulfide bonds that are shielded from the solvent by the compact folded structure of the B chain become exposed and the chitobiosyl cores of both N-linked oligomannose chains become susceptible to cleavage by endoglycosidases. The heat-denatured B chain does not enhance the toxicity of a ricin A chain-containing rabbit anti-human immunoglobulin (RAHIg-A) to Daudi cells. However, when heat-denatured B chain is coupled to goat anti-rabbit immunoglobulin (GARIg), the resulting immunotoxin, GARIg-hdB, potentiates the killing of RAHIg-A-treated Daudi cells to an extent similar to that of an immunotoxin prepared with GARIg and native B chain. These results indicate that the native, galactose-binding structure of the B chain is not necessary to enhance the cytotoxicity of the cell-reactive A chain immunotoxin (IT-A) and suggests that regions of the B chain exposed by unfolding the molecule may mediate potentiation of cytotoxicity.     

Antigen-driven oligoclonal expansion of tumor-infiltrating B cells in infiltrating ductal carcinoma of the breast

The objective of this study was to determine whether tumor-infiltrating B cells (TIL-B) of infiltrating ductal carcinoma (IDC) of the breast represent a tumor-specific humoral immune response. Immunohistochemical analysis of three Her-2/neu-negative IDC tumors from geriatric patients showed that TIL-B cluster in structures similar to germinal centers containing CD20(+) B lymphocyte and CD3(+) T lymphocyte zones with interdigitating CD21(+) follicular dendritic cells, suggesting an in situ immune response. A total of 29, 31, and 58 IgG1 H chain clones was sequenced from the three IDC tumors, respectively. Intratumoral oligoclonal expansion of TIL-B was demonstrated by a preponderance (45-68%) of clonal B cells. In contrast, only 7% of tumor-draining lymph node and 0% of healthy donor PBL IgG H chains were clonal, consistent with the larger repertoires of node and peripheral populations. Patterns and levels of TIL-B IgG H chain somatic hypermutation suggested affinity maturation in intratumoral germinal centers. To examine the specificity of TIL-B Ig, a phage-displayed Fab library was generated from the TIL-B of one IDC tumor. Panning with an allogeneic breast cancer cell line enriched Fab binding to breast cancer cells, but not nonmalignant cell lines tested. However, panning with autologous tumor tissue lysate increased binding of Fab to both tumor tissue lysate and healthy breast tissue lysate. These data suggest an in situ Ag-driven oligoclonal B cell response to a variety of tumor- and breast-associated Ags.     

The role of ricin B chain in the intracellular trafficking of anti-CD5 immunotoxins

The mAb anti-CD5 was linked to purified ricin A chain (RTA) or intact ricin (Rc) containing B chain to determine the role of ricin B chain in the intracellular trafficking of anti-CD5 immunotoxins (IT). IT were radiolabeled with iodine-125 and then studied for their subcellular compartmentalization in an acute lymphoblastic leukemia T cell line, CEM. Ricin A chain IT was not as toxic to CEM cells as Rc-IT in protein synthesis inhibition assays. This difference was not attributed to differential binding or modulation of the CD5 determinant from the cell surface as measured by FACS analysis. However, we found a relationship between the toxicity of anti-CD5-Rc and anti-CD5-RTA and their ability to traffic to CEM lysosomes. Kinetic analysis of the transfer of radioimmunotoxin to the lysosomes showed that anti-CD5-Rc was trafficked significantly more slowly than anti-CD5-RTA, perhaps due to an extended period of time in the Golgi compartment. The possibility of a Golgi interaction was tested by adding monensin, a carboxylic ionophore that interrupts trafficking through the Golgi, to cells treated with anti-CD5-RTA. The addition of monensin caused anti-CD5-RTA to traffic in a manner identical to anti-CD5-Rc. We conclude that 1) B chain slows trafficking of anti-CD5-Rc to the lysosomes; 2) the rate-limiting step in the toxicity difference between anti-CD5-Rc and anti-CD5-RTA is the rate of transfer to the lysosomes; and 3) trafficking through the Golgi may be important for anti-CD5-IT toxicity.     

抗人IgG Fc片段及Fab段单抗的鉴定及应用

本实验采用木瓜酶水解,SPA柱亲合层析等手段得到人IgGFc段及Fab段,以Sigma抗人IgGfFc段和抗人IgG Fab段单抗为标准品,鉴定了细胞库中抗人IgG系列的部分细胞株,得到特异性分泌抗人IgG Fc段和抗人IgG Fab段单抗的细胞各一株。 在上述实验基础上,用抗人IgG Fc及抗人IgG Fab单抗分别制备了Sepharose4B亲合层析柱,提纯了酶解人IgG Fc、Fab片段,经ELISA法鉴定,相互之间无交叉反应。同时用此方法制备了人抗HBe Fab片段,并将该片段进行了过氧化物酶标记,用来配制HBe ELISA诊断盒,证明其生物活性未受影响,而且消除了类风湿因子引起的HBe Ag假阳性现象。因抗HBe单抗来源困难,如采用HBe多抗制备ELISA试剂,本法将是提高质量的一个好方法。     

Mannose receptor-mediated uptake of ricin toxin and ricin A chain by macrophages. Multiple intracellular pathways for a chain translocation

The role of the high mannose carbohydrate chains in the mechanism of action of ricin toxin was investigated. Ricin is taken up by two routes in macrophages, by binding to cell surface mannose receptors, or by binding of the ricin galactose receptor to cell surface glycoproteins. Removal of carbohydrate from ricin by periodate oxidation led to a large loss in toxicity via both routes of uptake by an effect on the B chain not due to a loss of galactose binding affinity. These data suggest that the carbohydrate chains of ricin B chain may be required for full toxicity. The pathway of uptake of ricin by the macrophage mannose receptor was found to differ in several respects from uptake via the galactose-specific pathway. Analysis of intoxication of macrophages by ricin in the presence of ammonium chloride suggested that mannose receptor bound ligand passes through acidic vesicles prior to translocation, unlike galactose bound ligand. Intoxication by ricin via galactose-specific uptake was potentiated by swainsonine but not by castanospermine, suggesting that ricin may be attacked by an endogenous mannosidase within the cell, and that ricin passes through either a lysosomal or a Golgi compartment prior to translocation.     

Human monocytes and U937 cells bear two distinct Fc receptors for IgG

Several convergent lines of evidence have led us to propose that human monocytes and the related cell line U937 possess a second class of IgG Fc receptor (FcR) in addition to the 72-Kd high affinity FcR previously described. IgG affinity purification from detergent lysates of surface radiolabeled U937 cells has yielded both a 40-Kd IgG-binding membrane protein (p40) and the 72-Kd FcR protein. By the same procedure, only the p40 was isolated from the erythroblast cell line K562 and from the B cell lines, Daudi and Raji. Serologic cross-reactivity between the 40-Kd FcR on U937 and Daudi cells was demonstrated using a goat anti-FcR antiserum. A murine (m) monoclonal antibody, raised against the FcR of K562 cells, precipitated the 40-Kd FcR from lysates of U937 and K562 cells but not from Daudi or Raji cells. This antibody, referred to as anti-p40 (IV.3), selectively inhibited the binding of murine IgG1-coated erythrocytes to U937 cells, whereas monomeric human IgG selectively inhibited binding of human anti-Rh(D)-coated erythrocytes to U937 cells. Both Daudi and U937 cells mediated mIgG1 anti-T3 (Leu-4)-induced stimulation of T lymphocytes. In contrast, mIgG2a anti-T3 (OKT3)-induced stimulation was supported effectively by U937 cells but only modestly by Daudi cells. Intact IgG or Fab fragments of anti-p40 (IV.3) blocked mIgG1 anti-T3 (Leu-4) stimulation but not mIgG2a anti-T3 (OKT3) stimulation of T cells; monomeric human IgG blocked only OKT3-induced stimulation. The simplest interpretation of these results is that human monocytes and U937 cells bear two classes of IgG FcR, one of 72 Kd and the other, as described above, of 40 Kd. We propose that the 72-Kd FcR mediates rosette formation with red cells coated by human anti-Rh IgG as well as T cell stimulation by mIgG2a anti-T3 (OKT3) and that the 40-Kd FcR mediates rosette formation with erythrocytes bearing mIgG1 as well as T cell stimulation by mIgG1 anti-T3 (Leu-4). Furthermore, we suggest that these two FcR are the human homologues of the murine macrophage FcRI (binding mIgG2a) and FcRII (binding mIgG2b/1).     

Selective in vitro inhibition of an antibody response to purified acetylcholine receptor by using anti-idiotypic antibodies coupled to the A chain of ricin

In vitro antibody responses by rat lymph node cells against purified acetylcholine receptor (AChR) were shown to be inhibitable by protein conjugates prepared with anti-idiotypic antibody and the toxic A chain of ricin. The idiotype specificity of the cytotoxicity was demonstrated by the inability of the same immunotoxin to inhibit an unrelated antibody response (anti-KLH) and by abrogation of specific toxicity in the presence of unconjugated anti-idiotype or antigen (AChR). Furthermore, immunotoxin prepared with an irrelevant antibody specificity failed to significantly inhibit either the anti-AChR or control antibody responses. Therefore, we suggest that idiotype-specific immunotoxins may be a useful addition or alternative to presently employed immunotherapies for autoimmune disease.     

Evaluation of ricin A-chain immunotoxins directed against human T cells

We have synthesized four immunotoxins (ITs) by covalently coupling the A chain of ricin to murine monoclonal antibodies that recognize surface antigens on human T cells. Treatment of human peripheral blood lymphocytes with either 10.2-A, directed against the CD5 (Tp67) antigen, or 64.1-A, directed against the CD3 (Tp19) antigen, abolished protein synthesis in cells subsequently cultured with phytohemagglutinin (PHA). In contrast, two other ITs (9.6-A and 35.1-A), both directed against the CD2 (Tp50) antigen, had minimal effects on protein synthesis in PHA-stimulated cells. The binding of each IT to T cells was shown by immunofluorescence with fluorescein-conjugated goat anti-mouse immunoglobulin (FITC-GAMIg) and fluorescein-conjugated rabbit anti-ricin A-chain (FITC-RAR) antibodies. Activity of the ricin A chain in each IT was demonstrated by its ability to inhibit protein synthesis in a cell-free reticulocyte lysate assay. Ultrastructural immunoperoxidase analysis of IT internalization showed that ineffective and effective ITs were endocytosed at the same rate (50% of cells had labeled endosomes after 15 min). However, ineffective IT 35.1-A was more rapidly delivered to lysosomes (15-30 min) than effective ITs (10.2-A and 64.1-A) (greater than or equal to 30 min). The data support the hypothesis that there are several distinct pathways for internalization of ITs and that the ability of ricin A chain to reach and inactivate ribosomes may depend upon the specific membrane receptor involved in binding a given IT, its route of internalization, and the rate of entry of the IT into lysosomes.     

Interaction of antibody-bearing small unilamellar liposomes with target free antigen in vitro and in vivo. Some influencing factors

Affinity chromatography-purified and non-purified rabbit immunoglobulin G (IgG) raised against human immunoglobulin M (IgM) or kappa chain was incorporated into carboxyfluorescein-containing small unilamellar liposomes composed of egg phosphatidylcholine, cholesterol and phosphatidic acid (molar proportions 7:7:1). IgG incorporation was carried out by co-sonicating the immunoglobulin with the lipids (30% incorporated) (method A) or by interacting it with preformed liposomes bearing goat anti-(rabbit IgG) IgG (63 and 70% incorporated) (method B). (1) Judging from liposomal carboxyfluorescein-latency values, incorporation of IgG by either method did not affect liposomal stability. Furthermore, treatment of liposomes with papain released 75.1% (method A) and 93.3% and 95.1% (method B) of the IgG, suggesting that most of its antigen-recognizing Fab regions were available on the liposomal surface. This was strongly supported by the immunoelectrophoretic detection of Fab in papain-released products. (2) Liposomes bearing purified anti-IgM IgG bound 30%, (method A) and 45% (method B) of IgM in buffer. These values wee about 6-fold greater (both methods) than those obtained with corresponding liposomes bearing non-purified IgG. Binding of liposomes bearing anti-(kappa chain) IgG to kappa chain in buffer was 37% of that added. In the presence of mouse blood or serum, binding of IgM to liposomes bearing purified anti-IgM IgG was decreased slightly (24 and 30% for methods A and B). However, because of the nearly complete abolition of IgM binding to liposomes bearing non-purified IgG, these values were now 20–25-fold greater than those obtained with liposomes bearing non-purified IgG. (3) In mice pre-injected with IgM, at least 36.1% and 37.7% of the antigen was bound to subsequently injected liposomes bearing anti-IgM IgG incorporated by methods A and B respectively. No binding occurred with liposomes bearing the non-purified IgG. (4) Cholesterol-rich small unilamellar liposomes bearing affinity chromatography-purified antibodies may prove useful for the specific binding of free antigens in vivo.