High specific cytotoxicity of antibody-toxin hybrid molecules (immunotoxins) for target cells

In an attempt to develop highly efficient antibody—drug conjugates for passive immunotherapy of cancer the A-chain of the potent toxin, ricin, was coupled to antibodies in order to render them specifically cytotoxic for target cells without the participation of complement. The antibody—toxin conjugates (immunotoxins) showed no loss of antibody or A-chain activity. In vitro, highly purified A-chain was about 5000 times less toxic on HeLa cells than whole ricin. Unconjugated A-chain made no difference between TNP-HeLa and HeLa cells but when coupled to anti-DPN antibodies it became about 500 times more cytotoxic to TNP-HeLa cells than to HeLa cells. In vivo A-chain (LD₅₀: 20 mg/kg) was about 3000 times less toxic than whole ricin and treatment with immunotoxin significantly inhibited tumor take and tumor growth of TNP-HeLa cells in nude mice.     

Monoclonal antibodies to purified ricin A-chain: production and properties

Ricin A-chain was purified from native ricin using lactosyl-Sepharose. It was non-toxic to whole cells at a concentration of 1 microM yet nearly as effective as an equimolar concentration of ricin in blocking in vitro protein synthesis. Hybridomas secreting monoclonal antibodies to ricin A-chain were produced using the murine myeloma cell line NS-1. These anti-ricin A-chain antibodies cross-reacted with whole ricin but exhibited little cross-reactivity with purified ricin B-chain. Antibodies 2F2 and 2F5 both immunoprecipitated ricin A-chain. Both antibodies also precipitated ricin B-chain, as did the irrelevant control antibodies MOPC-21 and MPC-11. Pre-incubation of B-chain with 0.1 M galactose eliminated greater than 90% of precipitation by 2F2, MOPC-21 and MPC-11 but effected minimally precipitation by 2F5. Enzyme-linked immunosorbent assays using antibodies 2F2 and 2F5 to detect ricin A-chain in murine or human serum were linear between 40 and 800 ng ricin A-chain per ml. Anti-ricin A-chain antibodies 2F2 and 2F5 produced some inhibition of in vitro A-chain catalytic activity. Specific monoclonal antibodies to A-chain hemitoxin will be useful for characterization of functional hemitoxin domains, in in vitro assays for the stability of A-chain immunotoxins, and in characterizing the cellular internalization and processing of conjugates containing ricin or ricin A-chain.     

Actin-specific ADP-ribosyltransferase produced by a Clostridium difficile strain.

By screening possible ADP-ribosyltransferase activities in culture supernatants from various Clostridium species, we have found one Clostridium difficile strain (CD196) (isolated in our laboratory) that is able to produce, in addition to toxins A and B, a new ADP-ribosyltransferase that was shown to covalently modify cell actin as Clostridium botulinum C2 or Clostridium perfringens E iota toxins do. The molecular weight of the CD196 ADP-ribosyltransferase (CDT) was determined to be 43 kilodaltons, and its isoelectric point was 7.8. No cytotoxic activity on Vero cells or lethal activity upon injection in mice was associated with this enzyme. CDT was neither related to C. difficile A or B toxins nor to C. botulinum C2 toxin component I. However, Vero cells cultivated in the presence of C. difficile B toxin had a lower amount of actin able to be ADP-ribosylated by CDT or C2 toxin in vitro. Antibodies raised against CDT reacted by immunoblot analysis with a 43-kilodalton protein of C. perfringens type E culture supernatant producing the iota toxin.     

Immunotoxin with mistletoe lectin I A-chain and ricin A-chain directed against CD5 antigen of human T-lymphocytes; comparison of efficiency and specificity.

Monoclonal anti-CD5 antibody was coupled to the enzymatically active subunit of plant toxin [either mistletoe lectin I (ML) or ricin]. The obtained conjugates proved to be selectively toxic to CD5-bearing target cells. The immunotoxin prepared from ML A-chain (MLA) was as toxic as native ML and approximately 80-fold more active than the corresponding conjugate with ricin A-chain (RTA). The comparative studies of the structural properties of isolated MLA and RTA were carried out using intrinsic fluorescence spectroscopy. The results showed similar properties for both proteins. No antigenic cross-reactivity against both toxins was detected when using polyclonal antibodies. The results suggest that MLA-antibody conjugates may be potential candidates for therapeutical use.     

Immunoglobulin A antibodies against ricin A and B subunits protect epithelial cells from ricin intoxication

Epithelial cells of the respiratory and gastrointestinal tracts are extremely vulnerable to the cytotoxic effects of ricin, a Shiga-like toxin with ribosome-inactivating properties. While mucosal immunity to ricin correlates with secretory immunoglobulin A (IgA) antibody levels in vivo, the potential of IgA to protect epithelial cells from ricin in vitro has not been examined due to the unavailability of well-defined antitoxin IgA antibodies. Here we report the characterization of four monoclonal IgA antibodies (IgA MAbs) produced from the Peyer's patches and mesenteric lymph nodes of BALB/c mice immunized intragastrically with ricin toxoid. Two IgA MAbs (33G2 and 35H6) were active against ricin's lectin subunit (RTB), and two (23D7 and 25A4) reacted with the toxin's enzymatic subunit (RTA). All four IgA MAbs neutralized ricin in a Vero cell cytotoxicity assay, blocked toxin-induced interleukin-8 release by the human monocyte/macrophage cell line 28SC, and protected polarized epithelial cell monolayers from ricin-mediated protein synthesis inhibition. 33G2 and 35H6 reduced ricin binding to the luminal surfaces of human intestinal epithelial cells to undetectable levels in tissue section overlay assays, whereas 23D7 had no effect on toxin attachment. 23D7 and 25A4 did, however, reduce ricin transcytosis across MDCK II cell monolayers, possibly by interfering with intracellular toxin transport. We conclude that IgA antibodies against RTA and RTB can protect mucosal epithelial cells from ricin intoxication.     

In vivo follow-up of the cytotoxic effect of protein A--ricin toxin conjugate, on antibody-coated target cells

Antibodies of the IgG class, specifically interacted with H-2 antigens of murine leukemia EL4 cells, were used to bind the ricin toxin covalently linked to protein A of Staphylococcus aureus. The toxin thus complexed, introduced in the cytoplasm by endocytosis, was able to kill the leukemic cells inoculated in animals. The interaction of immunotoxin with the leukemic cells was performed in vitro using one, two or three treatments and the cytotoxic effect on the target cells was followed up in vivo. The time interval between immunotoxin treatments was indicated by the membrane turn-over study of EL4 cells coated with specific antibodies in their monomeric form, complexed by protein A or interacted with protein A--ricin toxin conjugate. A proportion of 99.8% cells killed was obtained after three treatments.     

Epitope specificity of three anti-pertussis toxin monoclonal antibodies with dissimilar effects in assays of toxin neutralizing activity.

The epitope specificity of two monoclonal antibodies against the S1 subunit (A4, A12) and one MAb against the S3 subunit (B9) of pertussis toxin, all protective in the mouse aerosol model of B. pertussis infection, but with different effects in assays of toxin-neutralizing activity, was examined in competitive binding enzyme immunoassays using biotinylated anti-pertussis toxin monoclonal antibodies or biotinylated goat anti-pertussis toxin polyclonal antibody after preincubation with unlabelled antibody. Biotinylated A4 was blocked by A4, A12, and B9; A12 was blocked by A4, A12, and B9. In contrast, biotinylated B9 was blocked by B9 and A4, but not by A12. All three monoclonal antibodies successfully blocked the anti-pertussis toxin polyclonal antibody; a mixture of the three anti-pertussis toxin monoclonal antibodies was more effective than any monoclonal antibody alone P less than or equal to 0.01). These data suggest that these three anti-pertussis toxin monoclonal antibodies recognize separate, but closely linked epitopes on pertussis toxin, and that epitopes on the S1 subunit and B-oligomer may induce protective immunity.     

Cytotoxic effect of ricin A-chain conjugates containing monoclonal antibodies against human erythroid cells

Selective elimination of human erythroblastoid cells by the conjugate of the A-chain of a plant toxin ricin (R_A) and monoclonal antibody (MAb) HAE9 (IgM) directed against human erythroblast antigen (Ag-Eb) has been demonstrated. In comparative experiments, MAb HAE3 (IgM) against human glycophorin-A was used. On average, the conjugates obtained contained two A-chain molecules and one antibody molecule. Efficiency of cytotoxic action of native ricin and conjugates was compared both with the amount of binding sites on the surface of K562 cells and the internalization rate of these proteins. The association constants of the proteins proved to be almost the same (k_a = 10⁸ M⁻¹). The ID₅₀ values were 1.1 × 10⁻¹¹, 3.2 × 10⁻¹⁰ and 3.1 × 10⁻⁹ M for ricin, HAE9/R_A and HAE3/R_A, respectively. Ammonium chloride at a concentration of 10 mM increases the cytotoxic effect of the HAE9/R_A conjugate approximately 10 times and does not change the activity of the HAE3/R_A conjugate.     

Specific killing of mouse leukemic cells with ricin A-chain immunotoxin

Monoclonal IgM antibody against L1210V leukemia was coupled with ricin A-chain using N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) as a cross--linking agent. The coniugate had potent concentration--dependent cytotoxicity against L1210V, L1210 and RL male 1 cells being completely non toxic to EL-4, P388, RPC-5 and mouse bone marrow cells. The minimum time required for killing L120V leukemia cells was 30h of in vitro exposure, at a concentration 10(-6) M (as assessed by trypan blue test). However, 1h contact of L1210V cells with immunotoxin was sufficient to completely inhibit proliferation of leukemic cells subsequently inoculated into compatible mice. The toxicity could be potentiated by addition of NH4Cl, that shortened minimum exposure time to 18h and 45 min respectively.     

Monoclonal antibodies specific for Clostridium difficile toxin B and their use in immunoassays.

Five mouse monoclonal antibodies (MAbs) against Clostridium difficile toxin B have been raised and characterized. Three of them were immunoglobulin M (IgM) antibodies (6B10, 6G3, and 10B9), and the other two were of the IgG1 isotype (9E5 and 17G2), recognizing specifically two distinct epitopes on the toxin B molecule. No MAb was able to neutralize cytotoxic activity significantly. The two IgG1 MAbs were purified and applied to various immunodiagnostic assays. MAbs coupled to latex beads were used for specific removal of toxin B from cytotoxic samples and for agglutination assay. An indirect sandwich enzyme-linked immunosorbent assay with MAb 9E5 or 17G2 as the capture antibody was established for identification of toxin B with a lower detection limit of 5 ng/ml.     

Monoclonal antibody against pertussis toxin: effect on toxin activity and pertussis infections.

Antibody-producing hybridomas of myeloma SP2/O and spleen cells of BALB/c mouse immunized with pertussis toxoid and pertussis toxin were selected by the binding ability of the monoclonal antibody to the subunit protein of the toxin. Two monoclonal antibodies, 1B7 and 3F10, specific for a subunit which has no binding activity to haptoglobin and sheep erythrocytes, named S1, and one antibody, 1H2, for a subunit related to the binding activity of the pertussis toxin molecule to haptoglobin or sheep erythrocytes, named S4, were examined for mouse protective activity against pertussis infection. Antibody 1B7 not only neutralized leukocytosis-promoting and islet-activating activities of the toxin but also protected mice against intracerebral and aerosol challenge with Bordetella pertussis. The antibody, furthermore, showed therapeutic effects on mice showing severe clinical signs with pertussis infection. The other two antibodies, 3F10 and 1H2, showed neither neutralizing nor protecting activity, nor significant synergistic effects on antibody 1B7.     

抗蓖麻毒素A链人源化单域抗体的设计、原核表达及生物活性检测

目的 运用计算机辅助蛋白质分子设计的方法设计针对蓖麻毒素A链(RTA)的拮抗肽,实现在大肠杆菌B121中的可溶性表达,并对其生物学活性进行评价.方法 根据RTA的晶体结构、RTA-rRNA相互作用复合物模型,在CVFF(consistent-valence force field)、Amber力场下,对RTA的空间构象进行理论模拟,初步确定其生物活性功能域;然后针对该功能域设计小分子拮抗肽,并借助人抗体重链町变区骨架,在CDR3区对拮抗肽进行展示,用重叠延伸PCR全基因合成人源化的单域抗体并克隆至载体pET-32a(+);双酶切和DNA测序技术对构建的载体进行鉴定;IPrG诱导人源化的单域抗体表达,用镍离子亲和层析纯化,竞争ELISA和MTT法分别进行结合和中和活性检测.结果 从头搭建并设计合成了人源化的单域抗体,实现了其原核表达,并进行了牛物活性检测;建立了基于人源化的单域抗体的RTA和蓖麻毒素检测方法.结论 研究结果为新型蓖麻毒素小分子拮抗剂的研制奠定了理论和实验基础.     

In vivo follow up of the cytotoxic effect of ricin toxin vectorized by multivalent hybrid antibody on target cells

A multivalent hybrid antibody complex composed of two IgG molecules specific for ricin toxin and two specific for the H-2 antigens of murine leukemia EL4 cells, cross-linked by SpA, was used as vector of the toxin to the target cells. The high affinity of the hybrid antibody for the specific antigens achieved an efficient attachment to the EL4 cell membrane and binding of ricin toxin; this high-molecular-weight complex, introduced by endocytosis into the leukemic cell cytoplasm, was able to specifically deliver the toxin to the target cells. The effect of multivalent hybrid antibody-vectorized toxin was followed up in vivo. This method enabled determination of the proportion of killed cells (over 90% after a single treatment of leukemic cells or about 99% after double treatment). The presence of a low proportion of tumoral cells maintaining their proliferative capacity is discussed.     

肝癌单抗—蓖麻毒素A链结合物的制备及其细胞毒性质

本文应用异型双功能基化合物SPDP与单克隆抗体反应,产生单克隆抗体-PDP,然后再与暴露巯基的RTA结合,制备肝癌单抗-RTA结合物。ELISA及毒素重组试验鉴定表明:结合物在10~(-9)mol/L浓度时与靶细胞仍有较好的结合能力,结合物中RTA保持良好活力。体外细胞毒结果表明:结合物对靶细胞具有良好的导向杀伤作用,在10~(-9)mol/L浓度时对人肝癌细胞株BEL7404的杀伤率为41.2％,对正常细胞SL_7的杀伤力很弱。     

Characterization of monoclonal antibodies to heat-labile enterotoxin encoded by a plasmid from a clinical isolate of Escherichia coli.

Eight selected hybridoma cell lines that produced monoclonal antibodies against heat-labile enterotoxin from an Escherichia coli strain of human origin (LTh) were characterized. Antibodies produced by these cell lines were tested for binding specificity in a series of solid-phase radioimmunoassays and Western blots by using as test antigens LTh, the A, A1, A2, and B polypeptides of LTh, the heat-labile enterotoxin from an E. coli strain of porcine origin, and cholera toxin. The monoclonal antibodies were also tested for isotype and ability to neutralize LTh. Two of the anti-LTh monoclonal antibodies cross-reacted with cholera toxin, and six were specific for determinants of LTh that were not present on cholera toxin. One was specific for a unique epitope of LTh that was not shared by the heat-labile enterotoxin from an E. coli strain of porcine origin or cholera toxin. Four antibodies specific for epitopes on the B subunit of LTh (LTh-B) reacted with pentameric LTh-B but did not react in Western blots with monomeric LTh-B. The remaining four antibodies were specific for epitopes on LTh-A; two of these antibodies bound to A1, one reacted with A2, and one recognized only intact LTh-A. Only one monoclonal antibody had detectable neutralizing activity, and it was specific for LTh-A.     

Enzyme-linked immunosorbent assay (ELISA) for antibodies to Clostridium difficile toxins in patients with pseudomembranous colitis and antibiotic-associated diarrhoea

Enzyme-linked immunosorbent assay (ELISA) was established with purified toxins from Clostridium difficile as antigene to measure antibody response in patiensts with pseudomembranous colitis (PMC) and prolonged antibiotic-associated diarrhoea (AAD). Positive ELISA titres were defined in a control population. Antibodies of IgG class against toxin B were demonstrated in 6/88 (7%) control sera and in 31/61 (51%) sera from 11/19 (58%) patients. Antibodies of IgA class were found in one patient while antibodies of IgM class were not demonstrated. ELISA antibodies against toxin A were not demonstrated. For comparison a neutralization test was performed and neutralizing antibodies to toxin B but not to toxin A were demonstrated in 10/61 (16%) sera from 4/19 (21%) patients and in none of the controls. ELISA was found to be a more sensitive assay than neutralization. ELISA antibodies were detected from the third week of the disease while neutralizing antibodies appeared after 5 weeks. Lack of an antibody response in ELISA seemed to correlate to a more severe colitis.     

Mice are actively immunized after passive monoclonal antibody prophylaxis and ricin toxin challenge.

Mice passively immunized by a protective, anti-ricin A-chain monoclonal antibody, then challenged intravenously with ricin, were protected from a subsequent ricin challenge, and were actively immunized. Two significant advantages accrued from this experiment: the monoclonal antibody neutralized the toxicity of the ricin immunogen, and active immunization was achieved with very low antigen load (approximately 0.5 microgram/mouse). We ruled out the possibility that residual monoclonal antibody provided the protection by using three independent criteria. There was significant (four orders of magnitude) enhancement of the immune response in the presence of the monoclonal antibody; control immunizations of mice with ricin A-chain, ricin B-chain or either chain with the monoclonal antibody did not induce active immunity; and the active immunization could not be replicated when protective goat polyclonal antibody was substituted for the monoclonal antibody. Because high titres were achieved rapidly without any adjuvant, we are currently investigating haptenized ricin to determine if anti-hapten monoclonal antibodies can be produced by this refined procedure.     

Effect of monoclonal antibody to pertussis toxin on toxin activity.

Two distinct monoclonal antibodies, one to pertussis toxin subunit S2, called 9G8, and another to subunits S2 and S3, called 11E6, were generated from the hybridomas of myeloma SP2/0 and spleen cells of BALB/c mice immunized mainly with the subunit S234 complex. Binding ability of 9G8 and 11E6 to the subunits was confirmed by the enzyme-linked immunosorbent assay and immunoblotting analysis. Generation of 11E6 bound to both S2 and S3 might mean that there is common antigenicity between S2 and S3. Neutralizing activities of 9G8 and 11E6 on various biological activities of pertussis toxin, including ADP-ribosyltransferase and leukocytosis-promoting, islet-activating, permeability-increasing. Chinese hamster ovary (CHO) cell-clustering, and hemagglutinating activities, were compared with those of anti-S1 monoclonal antibodies 1B7 and 3F10, which were isolated and characterized in a previous study (H. Sato, A. Ito, J. Chiba, and Y. Sato, Infect. Immun. 46:422-428, 1984). 1B7 and 3F10 neutralized ADP-ribosyltransferase activity of pertussis toxin or S1, but 9G8 and 11E6 did not. 1B7 showed very potent neutralization against leukocytosis-promoting, islet-activating, permeability-increasing, and CHO cell-clustering activities of pertussis toxin, but 3F10 did not, although anti-ADP-ribosyltransferase activities of both antibodies were identical. 11E6 neutralized leukocytosis-promoting, islet-activating, CHO cell-clustering, and hemagglutinating activities but not permeability-increasing activity. 9G8 showed slight neutralization of leukocytosis-promoting and CHO cell-clustering activities. Specific activities of 1B7 and 11E6 in each neutralization test were higher than or almost comparable to those of polyclonal antibodies to pertussis toxin. The neutralizing mechanism of 1B7 and 11E6 in leukocytosis-promoting activity was compared. 11E6 seemed to interfere with the binding of pertussis toxin to receptors on mouse spleen cells.     

Antibody convergence along a common idiotypic axis in immunodeficiency virus and hepatitis C virus infections.

The anti-idiotypic antibody 1F7 selectively binds antibodies against human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) gag, pol, and env proteins. We tested anti-hepatitis C virus (HCV) antibodies to investigate selection of the 1F7 idiotype on antibodies against other chronic pathogens. Twelve of 15 HCV-seropositive individuals co-infected with HIV had detectable antibodies against recombinant HCV core, 4 against HCV NS4 protein, and 3 against HCV NS3 protein. All four HCV-seropositive, non-HIV-infected individuals had antibodies against HCV core and NS4, while 3 had antibodies against NS3. The 1F7 idiotype was frequently present on antibodies against each of the HCV antigens in the HIV co-infected and non-HIV-infected groups. Antibodies against HCV, including antibodies recognizing the putative principal neutralizing determinant of HCV E2 protein, displayed skewed kappa/lambda light chain usage consistent with clonal dominance. These observations extend the association between expression of the 1F7 idiotype and abnormal B cell clonal dominance in HIV and SIV infection to HCV infection and suggest that early establishment of an oligoclonal antibody response against HCV may freeze the B cell repertoire, impair adaptation to emergent HCV variants, and favor escape from neutralizing antibodies. We also demonstrated that expression of the 1F7 idiotype extends beyond antibodies against multiple antigens of AIDS-causing retroviruses to include antibodies against multiple antigens of an unrelated chronic hepatitis virus. Thus, distinct pathogens establishing chronic infection in the face of strong humoral immune responses select antibodies along a common idiotypic axis of the immune network.     

Neutralizing Activity Against Clostridium difficile Toxin in the Supernatants of Cultured Colostral Cells

Human colostral specimens were obtained from 60 Japanese postpartum women within the first 3 days after delivery. Neutralizing activity against Clostridium difficile toxin was evaluated with Y1 adrenal cells in miniculture. When Y1 adrenal cells were exposed briefly to the toxin, they showed a rounding response in culture, resembling that effected by Escherichia coli enterotoxin; however, preincubation of the toxin with aqueous phase of colostrum significantly reduced its cytopathic effect on Y1 adrenal cells. Of 60 colostral specimens, 17 samples had neutralizing activity against the toxin. Cell-free supernatants of colostral cells cultured for 7 days without mitogens contained significant amounts of both immunoglobulin A (IgA) and IgM, but very small amounts of IgG. Neutralizing activity of cell-free supernatants of cultured colostral cells was evaluated as described above. Neutralizing activity against the toxin was identified in five samples of culture supernatants out of 60 colostral cell specimens. In all five cases, the aqueous phase of colostrum also had a neutralizing effect against C. difficile toxin. Neutralizing activity against the toxin found in five supernatants of cultured colostral cells was completely abolished only by anti-human IgA antibody as assessed by immune precipitation.