Large scale preparation of immunotoxins constructed with the Fab' fragment of IgG1 murine monoclonal antibodies and chemically deglycosylated ricin A chain

In this report, we describe a method for the preparation of large amounts (grams) of immunotoxins (ITs) consisting of Fab' fragments of murine IgG1 monoclonal antibodies conjugated to chemically deglycosylated ricin A chain (dgA). The preparation of Fab' and dgA chain and the purification of the Fab'-dgA IT were accomplished by gel filtrations and affinity chromatography utilizing six Pharmacia Bioprocess columns (Sephadex G-25M, Sephacryl S-200HR and Blue Sepharose CL-4B) integrated into a semi-automatic chromatography system controlled by a Pharmacia C3-process controller. The final Fab'-dgA ITs were highly purified, potent, sterile and low in endotoxin concentration.     

Large scale preparation of an immunoconjugate constructed with human recombinant CD4 and deglycosylated ricin A chain

A method for the preparation and purification of large amounts (grams) of a conjugate containing recombinant CD4 antigen (rCD4) and chemically deglycosylated ricin A chain (dgA) is described. The cross-linking of rCD4 and dgA molecules was accomplished with N-succinimidyl-oxycarbonyl-alpha-methyl-(2-pyridyldithio)toluene (SMPT). The rCD4-dgA conjugate was purified by an automatic liquid chromatography system consisting of Blue-Sepharose CL-4B and Sephacryl S-200HR Pharmacia Bioprocess columns. The purified, endotoxin-free rCD4-dgA conjugate had a stable (hindered) disfulfide bond between rCD4 and dgA and was able to efficiently kill a human T cell line infected with HIV-1.     

The toxicity of chemically deglycosylated ricin A-chain in mice.

Tumor-reactive antibodies coupled to ricin or its A-chain (immunotoxins) have been used in rodents and humans to treat a variety of neoplastic diseases. Side-effects of such treatment include hepatotoxicity, vascular leak syndrome, myalgia and low grade fever. At high doses, severe toxicities include liver damage, pulmonary edema, aphasia, rhabdomyolysis and kidney failure. There have been a limited number of toxicologic studies on uncoupled ricin or its A-chain and none on deglycosylated A-chain. Since the latter has been utilized in "second generation" immunotoxins, the current studies were carried out to evaluate the toxicities induced by deglycosylated ricin A-chain (dgA) in mice. The administration of dgA to normal BALB/c mice causes early (24 h) weight loss and late (10 day) accumulation of ascites. These effects could be partially altered by changing the route of injection of dgA from i.v. to i.p. Thus, i.p. administration caused weight loss but not ascites, whereas i.v. administration caused both. Weight loss was associated with reduced fluid intake by the treated mice, and was not associated with increased levels of serum TNF-alpha. SCID mice injected with the same dose of dgA as normal BALB/c mice developed ascites, but it was of lesser severity, suggesting that a functional immune system, differences in microbial flora, or strain differences may be involved in the development of ascites.     

The effects of aromatic and aliphatic maleimide crosslinkers on anti-CD5 ricin immunotoxins

The aromatic maleimide crosslinkers m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfosuccinimidyl 4-(p-maleimidophenyl) butyrate (S-SMPB) and m-maleimidobenzoylsulfosuccinimide ester (S-MBS) and the aliphatic crosslinker N-gamma-maleimidobutyryloxysuccinimide ester (GMBS) were used to make anti-CD5 intact ricin immunotoxins (IT). IT made with the various crosslinkers were compared under standard conjugation conditions for differences in yield, toxicity of the toxin moiety, binding of the antibody moiety, IT activity, and IT specificity. Our findings showed that IT yield was dramatically improved using crosslinkers with an aromatic, rather than an aliphatic configuration. Gel analysis showed that all IT were of similar, but not identical composition. Conjugation resulted in several IT species including antibody linked to one or two molecules of ricin. For MBS IT and S-SMPB IT, differences in amounts of IT in final fractions and IT in fractions after removal of IT species containing galactose binding sites showed that differences in yield may be attributable to the formation of IT species with obstructed galactose binding sites. All IT bound selectively by FACS analysis and blocking studies. The aliphatic GMBS crosslinker yielded the most toxic IT in cell-free translation assays as well as in shorter-term protein synthesis inhibition and mitogen assays. However, evaluation in the longer-term, more sensitive clonogenic assay showed that at 1000 ng/ml there were no differences in potency between any of the IT. We conclude that the yield of intact ricin IT can be improved using aromatic maleimide crosslinkers without sacrificing IT potency.     

A comparison of anti-lymphocyte immunotoxins containing different ribosome-inactivating proteins and antibodies.

Immunotoxins were prepared with several single-chain ribosome-inactivating proteins (RIPs type 1) and with the A-chain of ricin linked to the F(ab')2 fragment of sheep anti-mouse IgG. The cytotoxic activity of these conjugates was tested on human lymphocytes pretreated with an anti-CD3 murine MoAb. The immunotoxins inhibited DNA synthesis in phytohaemagglutinin (PHA)-stimulated lymphocytes with IC50S (concentrations causing 50% inhibition) ranging from 8.9 x 10(-13) to 5.7 x 10(-11) M (immunotoxins containing dianthin 32, saporin, pokeweed antiviral protein from seeds (PAP-S), bryodin, momordin, momorcochin, and trichokirin), 1 x 10(-8) M (immunotoxin containing gelonin) and 5 x 10(-9) M (immunotoxin containing ricin A-chain). The immunotoxin containing saporin linked to the anti-mouse IgG F(ab')2 fragment was also highly toxic to human lymphocytes pretreated with anti-CD2, -CD3, -CD5 and -CD45 MoAbs, with IC50S less than or equal to 10(-11) M. Immunotoxins were prepared also with saporin linked to MoAbs against various CD antigens. The immunotoxin prepared with the anti-CD3 antibody had the highest specific cytotoxicity to human lymphocytes.     

Kinetics of inter-heavy chain disulfide bond formation of liganded and unliganded human immunoglobulin G by radioimmunoassay.

Monoclonal antibodies (mAbs) to the hinge region of human IgG1 immunoglobulins were prepared by immunization with a proteolytic fragment of hinge segment coupled to keyhole limpet hemocyanin. A mAb, 4G3, was obtained capable of binding to intact IgG but not to partially reduced IgG. Using this mAb, inter-heavy (H) chain disulfide bond formation from partially reduced anti-tetanus antibodies (Abs) in the presence or absence of antigens (Ags) was studied by solid phase radioimmunoassay. When the Abs were partially reduced in solution and then coated to plastic plates, only 10% regeneration of inter-H chain disulfide bonds occurred after reoxidation, although 100% formation occurred in solution [Kishida et al., J. Biochem. (Tokyo) 79, 91-105 (1976)]. This difference in the extent of disulfide bond formation can be explained by the fact that there are two convertible isomers in solution, Conformer I and II, one of which (Conformer I) can form disulfide bonds but is present as a minor component. Since the motion of IgG molecules on plates is restricted by hydrophobic interactions, the two conformers are not convertible as in solution. Therefore only Conformer I which existed before coating formed inter-H chain disulfide bonds. Similar kinetic measurements were performed using plates coated with tetanus toxoid. Abs, partially reduced in solution and then allowed to react with Ags on the plate were able to completely regenerate their inter-H chain disulfide bonds, although the rate of reaction was slow. These results can also be explained by the fact that the two isomers are convertible since Ag-Ab complexes are dissociable. Ag-binding therefore did not significantly perturb conformation of the hinge segments. In addition, no difference between liganded and unliganded Abs was observed in the binding of anti-hinge mAbs. These results imply little or no contribution of the hinge region to transmission of the signal produced by Ag-binding to Fc.     

Potentiation of a weakly active ricin A chain immunotoxin recognizing the neural cell adhesion molecule.

A ricin A chain immunotoxin, SEN36-ricin A chain, directed against the neural cell adhesion molecule (N-CAM) had no selective cytotoxic activity against three different small cell lung cancer (SCLC) cell lines in tissue culture despite expression of the target antigen on more than 98% of cells in each line detected by indirect immunofluorescence. Treatment of the SW2 SCLC cell line with suramin and interferons alpha and gamma increased the level of N-CAM expression only slightly and had no significant effect on the cytotoxic activity of the SEN36 immunotoxin. In the presence of the carboxylic ionophore monensin at a concentration of 0.1 microM, the toxicity of SEN36-ricin A chain to the SW2 cell line was enhanced by 12,000-fold. In contrast, lysosomotropic amines showed little or no potentiation of activity, suggesting that lysosomal degradation was not the major factor limiting the action of the anti-N-CAM immunotoxin. The findings of this study indicate that ricin A chain immunotoxins directed against N-CAM on SCLC are unlikely to have sufficient activity to be useful therapeutic agents in the absence of potentiating agents such as monensin, which can interfere with the normal intracellular pathways of antigen routing.     

蓖麻毒蛋白A链与Ia抗原单抗偶联物对肿瘤细胞的作用

用ε—氨己酰半乳糖胺—琼脂糖和去唾液酰胎儿球蛋白—Sepharose 4B柱层析纯化蓖麻毒蛋白A链,以DE—52离子交换层析法纯化得Ia抗原MAb—HB_(55)5。将二者通过二硫键交联成免疫毒素RTA—HB_(55)。在体外条件下,RTA—HB_(55)对Raji细胞杀伤的IC_(50)为3×10~(-10)M,游离RTA的IC_(50)为10~(-8)M,RTA—HB_(55)对K_(562)细胞的IC_(50)0则为2×10~(-8)M。表明RTA—HB_(55)能选择性地杀伤靶细胞而不杀伤非靶细胞,可望在B淋巴细胞白血病和淋巴瘤的治疗中获得应用。     

Cytotoxic properties of a conjugate of ricin a chain and antiallotypic monoclonal antibodies

Laboratory of Immunology, All-Union Research Institute of Genetics and Selection of Industrial Microorganisms, Ministry of the Medical and Biological Industry of the USSR. Laboratory of Immunology, All-Union Research Institute of Experimental Cardiology, All-Union Cardiologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. N. Smirnov.) Translated from Byulleten' Éksperimental'noi Biologii Meditsiny, Vol. 105, No. 5, pp. 568–570, May, 1988.     

计算机辅助设计使重组ricin—A链在E.coli中的高效表达

基于对螺旋区随机堆积的ＲＮＡ二级结构的预测和密码子偏性计算，建立了ｐＢＶ２２０载体中外源基因高效表达的判别模型。该模型认为，外源基因的表达水平，与其５′端３０３９区域，以及３′端３０３９区域的二级结构自由能具有显著的统计学意义；其次与３′端９ｂｐ的局部密码子偏性相关。据此模型，我们设计了用于克隆表达ｒｉｃｉｎＡ链的引物。将ＰＣＲ扩增的基因克隆入ｐＢＶ２２０载体中，在ＤＨ５α宿主中获得了高效表达（表达水平在２０％以上），而且在ｐＥｘＳｅｃＩ载体系统中亦获得了高效表达。     

The nature and importance of the inter-epsilon chain disulfide bonds in human IgE.

IgE antibodies are best known for their pathological role in allergy. The class-specific effector sites are located in the epsilon chains; these form covalent dimers via two cystine residues (Cys241 and Cys328) linking opposite C epsilon 2 domains. The nature and biological significance of the inter-epsilon chain disulfide-bond arrangement is unresolved. For structural and functional analysis site-specific mutations were introduced into the C epsilon 2 domain of recombinant human IgE. The introduction of an additional cyanogen bromide cleavage site (His246----Met) facilitated the identification of parallel disulfide bond pairing. This linkage was also confirmed for myeloma IgE PS by sequence determination of disulfide-linked C epsilon 2 dimers. Substitution of Cys241 and Cys328 by Ser does not destroy receptor binding, but reductive alkylation, or the replacement of Cys328 by Met, leads to loss of activity. This shows that covalent dimerization is not essential for IgE/receptor interaction and points to the importance of the structural integrity of the site surrounding Cys328, visualized in a new model of human Fc epsilon.     

Characterization of the increased cytotoxicity of gelonin anti-T cell immunoconjugates compared with ricin A chain immunoconjugates.

Ribosomal inactivating proteins such as gelonin (Gel) and ricin A chain (RTA) conjugated to MoAbs bind to specific target cells, and upon internalization inhibit protein synthesis, ultimately resulting in cell death. We report here that Gel anti-T cell MoAb conjugates are more cytotoxic than RTA conjugates when tested against human peripheral blood mononuclear cells (PBMC). This increased cytotoxicity is observed whether Gel is conjugated to the anti-T cell MoAb or to an anti-mouse immunoglobulin Fab' fragment which then binds to the murine anti-human T cell MoAb. Gel conjugates are not only effective at lower concentrations, but also produce a greater extent of inhibition of cellular proliferation. Moreover, a 10 min exposure to a Gel conjugate is as effective as a 90 h exposure to an RTA conjugate. When part of anti-T cell F(ab')2 or Fab' conjugates, Gel affects the early steps in cellular intoxication more than RTA; Gel conjugates bind more avidly and accelerate the modulation of antigen. In contrast, when part of whole IgG conjugates, Gel does not affect the binding to or modulation of surface antigen compared with RTA, while it does increase conjugate cytotoxicity. These observations suggest that Gel may be delivered more efficiently into the cytosol than RTA. A divergent intracellular pathway for Gel is also supported by the inability of chemical potentiators, which strongly enhance RTA potency, to affect Gel potency. These properties of Gel might also be advantageous for immunoconjugates made with other MoAbs or receptor-binding molecules.     

Synthesis and biological characterization of a covalent conjugate between interferon and ricin toxin B chain

Human interferon (IFN)-α has been covalently conjugated to ricin toxin B chain (RTB) using the heterobifunctional crosslinking reagent, N-succinimidyl-3(2-pyridyldi-thio)propionate (SPDP). Conjugate molecules (RTB:IFN) were purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and assayed for antiviral activity on human GM-258 fibroblasts in the presence or absence of competing concentrations of galactose, the specific sugar employed by RTB for cell surface binding. While the antiviral activity of native IFN-α was unchanged in the presence of galactose, the antiviral activity of RTB:IFN was markedly diminished in the presence of galactose at concentrations ranging from 1 to 100 mM. These studies demonstrate that IFN-α can exert an antiviral effect on human fibroblasts after binding to the receptor of another ligand molecule (i.e., RTB).     

The preparation of human red cell ghosts containing calcium buffers.

1. Ca buffers may be introduced into human red cells by reversible haemolysis. The resealed ghosts retain Ca and chelating anions in the same ratio as in the haemolysing solution, enabling the intracellular Ca2+ concentration to be calculated simply. 2. The passive permeability of the ghosts to Na and Cl is unaffected by intracellular Ca2+ concentrations in the 10(-8)-10(-4) M range, whereas the K permeability is greatly increased at concentrations above 10(-7) M. 3. These preparations enable Ca-dependent K movements to be studied under stable conditions. When the ghosts contain about 5 X 10(-6) M-Ca2+, over 96% of K transport occurs via the Ca-sensitive route.     

The rapid and large scale separation of peritoneal macrophages

A simple method for rapid and large scale (over 10⁹ cells) separation of peritoneal macrophages by incubation in a glass-bead column and elution with buffered ethylenediamine tetraacetic acid (EDTA) solution is described. Macrophages obtained were about 90 per cent pure with the recovery rates of 38–53 per cent depending upon the sample size, and 83 per cent viability. Lymphocyte contamination was reduced to 5 per cent or less.     

Effective avidin coating of erythrocytes by disulfide bond formation. Production of a stable rosetting agent for multiple applications

A method is described for covalently coupling avidin to the membrane of glutaraldehyde-treated erythrocytes. The method is based on the use of the heterobifunctional coupling reagent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and appears to be more effective than other protein coupling techniques. The resulting avidin-coated red cells are very stable and can be stored for more than 1 year without aggregation or loss of biotin-binding activity. They can be used in various rosetting assays and were tested for T cell depletion or negative cell selection with five biotinylated monoclonal antibodies.     

Notes on the large scale preparation and on the properties of anti-lymphocyte serum for use in mice

Mouse spleen and thymus cells have been used in the preparation of horse anti-mouse anti-lymphocyte serum (HAMLS). The cells were used either separately or in a mixture and three types of immunization schedules were used, viz. two-pulse, extended and chronic.

Antisera of marked immunosuppressive activity, as measured by the ability to prolong the life of skin homografts in mice, were obtained using all three schedules, the median survival time being, at best, 27·5 days for the chronic schedule, 26·7 days for the two-pulse schedule and 25·8 days for the extended schedule. The two-pulse and the extended schedules produced non-toxic antisera in a relatively short period of time but were uneconomic in terms of antigen and horses. The chronic schedule was preferred but after 10 weeks the development of unwanted antibodies precluded the further useful immunization of the horses.

     

Effects of disulfide bond reduction on the excitatory and inhibitory postsynaptic responses of Aplysia ganglion cells.

Three kinds of the cholinoceptive neurons, nicotinic depolarizing (D)-, nicotinic hyperpolarizing (H)-, and muscarinic H-tyes, as well as two other kinds of neurons, GABA H- and dopamine H-types, were identified in Aplysia abdominal ganglion, and the effects of disulfide bond reduction and reoxidation on their postsynape acetylcholine-induced responses of both nicotinic types (D- and H-) were depressed by reducing the disulfide bonds with dithiothreitol (DTT) and restored by reoxidizing with 5, 5' -dithiobis-(2-nitrobenzoic acid): (DTNB), whereas the responses of the muscarinic H-, GABA H-, and dopamine H-cells were not affected at all by either DTT or DTNB. In contrast to the results obtained from the electroplax, the cholinergic receptors in our preparation showed neither the activation by hexamethonium nor the augmentation of decamethonium-induced responses after reduction of disulfide bonds. In addition, our preparation did not demonstrate the long-lasting responses to bromoaTT-induced depression of the nicotinic responses was studied on the dose-response curves; the mode of receptor inhibition was rather complexed, being neither type of competitive nor non-competitive. We concluded that the disulfide bond is a crucial element in both types of nicotinic receptors (D and H), and that this bond is related to the activation process of the receptors regardless of their ionic specificities.     

Synthesis of block copolymers containing a carbon chain and a polypeptide block

The bifunctional initiators, bis(2-glycylaminophenyl) disulfide ( 1 ) and 2, 2′-azo-2,2′-dimethyl-dipropionohydrazide ( 2 ), were used to obtain polymeric initiators of two types: carbon chain initiators with amino end groups initiating the polymerization of amino acid N-carboxyanhydrides (NCAs) and symmetric polypeptide initiators containing ? S? S? or ? N?N? groups, which decompose on exposure to UV light or elevated temperature forming free radicals. These polymeric initiators were used to synthesize block copolymers (BC) containing carbon chain and polypeptide blocks of ABA and AB structures. It is shown that AB block copolymers are capable of aggregation to a much higher extent than those of ABA type. These BC can form liquid-crystalline polymer structures and undergo controlled biodegradation by enzymes.