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At least 19 recordsLinked to original sources
PubMed1991

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.

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.

Animals↗
PubMed1986

Study of the plasma clearance of antibody--ricin-A-chain immunotoxins. Evidence for specific recognition sites on the A chain that mediate rapid clearance of the immunotoxin.

In recent years, antibody--ricin-A-chain immunotoxins have been investigated as anti-neoplastic agents. To achieve in vivo therapy it is necessary that the immunotoxin remains in circulation at a sufficiently high level for a sufficiently long time to allow binding to tumor cells to occur. Therefore, examination of the pharmacology of immunotoxins may elucidate the reasons for the poor in vivo tumoricidal effect of immunotoxin described before. In this study the plasma clearance of antibody--ricin-A-chain immunotoxins, after intravenous injection in animals of different species, has been examined. Sensitive and reproducible techniques were developed to monitor the level of immunotoxin and its constituents in the blood. It is shown that immunotoxins are rapidly eliminated from the bloodstream. Neither the properties of the antibody moiety nor the nature of the linkage binding ricin A-chain to antibody account for the disappearance of immunotoxin from the plasma. On the other hand, we found that the rapid clearance of immunotoxin is due to the mannose residues on the ricin A-chain moiety which are specifically recognized by liver cells. When immunotoxin is administrated together with yeast mannan, which enhances the level of active immunotoxin in circulation by inhibition of liver uptake, the anti-cancer efficacy of immunotoxin in vivo is drastically improved.

Animals↗
PubMed1997

Comparison of the potency and therapeutic efficacy of the anti-CD7 immunotoxin HB2-saporin constructed with one or two saporin moieties per immunotoxin molecule.

Immunotoxins that carry two toxin molecules to the target cell should in theory have a greater anti-tumour effect than those that carry just one. We have investigated the therapeutic efficacy of two anti-CD7-saporin immunotoxins constructed with one saporin (HB2-Sap 1-mer) or two saporin molecules (HB2-Sap 2-mer) per immunotoxin molecule. In vitro, the 2-mer immunotoxin was 5.6 times more effective than the 1-mer immunotoxin at inhibiting protein synthesis in the CD7+ human T-cell acute lymphoblastic leukaemia (T-ALL) cell line HSB-2 and was also more effective at inhibiting HSB-2 cell proliferation. Flow cytometry revealed that the 2-mer immunotoxin had a reduced binding capacity to HSB-2 cells compared with the 1-mer immunotoxin or native HB2 antibody. In therapy studies in SCID mice with disseminated HSB-2 human leukaemia, the 2-mer immunotoxin performed marginally better than the 1-mer immunotoxin, but log-rank analysis did not reveal any significant differences between the two therapy groups. We therefore conclude that, although the 2-mer immunotoxin performed better than the 1-mer immunotoxin against target HSB-2 cells in vitro, this improved performance was not reflected as an improved in vivo therapeutic outcome in the SCID mouse model.

Animals↗
PubMed1996

Antibody engineering of recombinant Fv immunotoxins for improved targeting of cancer: disulfide-stabilized Fv immunotoxins.

Recombinant immunotoxins are chimeric proteins in which a truncated toxin is fused to a recombinant antigen-binding domain such as a recombinant Fv or Fab. Recombinant immunotoxins target cell surface receptors and other antigens on tumor cells. The antigen-binding and -targeting domains in recombinant immunotoxins are usually single-chain Fvs (scFv), which are the antibody variable regions connected by a flexible peptide linker and fused directly to a bacterial toxin. However, Fabs have also been used. Recombinant immunotoxins have very good activity in vitro on cultured human tumor cell lines and have produced complete regressions and cures of established tumor xenografts in nude mouse models. Problems with the stability and binding of some scFv immunotoxins as well as scFvs not linked to toxin led to the development of a new type of recombinant Fv immunotoxin in which the targeting variable domains of the Fv are stabilized by an interchain disulfide bond located in structurally conserved framework positions of the VH and VL domains. These are termed disulfide-stabilized Fvs (dsFv) or dsFv immunotoxins. dsFvs and dsFv immunotoxins have several advantages over scFv immunotoxins. This review summarizes the design, construction, activities in vitro and in vivo, and biochemical characteristics of dsFv immunotoxins and compares them with scFv immunotoxins.

Animals↗
PubMed1983

Synergy of ricin A chain-containing immunotoxins and ricin B chain-containing immunotoxins in in vitro killing of neoplastic human B cells.

A strategy is described for improving the efficacy of ricin A chain-containing immunotoxins. Highly purified preparations of ricin A chain or ricin B chain were separately coupled to anti-human immunoglobulin antibodies and the conjugates (immunotoxins) were affinity purified to eliminate free chains. Mixtures of anti-Ig-A and anti-Ig-B immunotoxins markedly synergized in vitro in their ability to kill the Ig-bearing human lymphoma cell line Daudi. In contrast, A-chain- or B-chain-containing immunotoxins of irrelevant specificity did not synergize with anti-Ig-A or anti-Ig-B immunotoxins. This finding indicates that free A or B chains do not play a major role in the synergy and that the synergy is specific. Thus, synergy depends on the specificity of the two antibodies; the lectin binding ability of a B-chain-containing immunotoxin of irrelevant specificity does not suffice. This approach of delivering ricin A chain and B chain separately to a target cell may have significant advantages in killing cells that are not effectively killed by A-chain-containing immunotoxins alone.

Burkitt Lymphoma↗
PubMed1996

Immunotoxins containing A-chain of mistletoe lectin I are more active than immunotoxins with ricin A-chain.

Conjugates of anti-CD25 monoclonal antibodies against cell surface IL-2 receptor with MLIA and RTA were prepared and investigated. Both of the immunotoxins had high specific cytotoxic activity on target cells. The IC50 value of the anti-CD25/MLIA immunotoxin was 15-fold greater than that of the anti-CD25/RTA. Previous studies of the anti-CD5 immunotoxins with MLIA and RTA showed that the anti-CD5/MLIA IT was 80-fold more active than anti-CD5/RTA IT [Tonevitsky et al. (1991) Int. J. Immunopharmacol. 13, 1037-1041]. The surface hydrophobicity of the MLI A-chain was 4-fold higher than that of the ricin A-chain as estimated by binding with ANS. In model experiments with small unilamellar DMPC liposomes, MLIA but not RTA increased the turbidity of liposome suspensions at pH 4.5. Our results indicate that the greater cytotoxic activity of the MLI A-chain immunotoxin probably provided a higher surface hydrophobicity of the protein and the ability to interact with phospholipid membranes.

Anilino Naphthalenesulfonates↗
PubMed1989

Experimental model for testing the efficiency of immunotoxins administered in vivo: evaluation of two ricin A-chain--multivalent antibody immunotoxins.

Two immunotoxins, the ricin A chain-multivalent hybrid antibody (750 kDa) and the complex A chain-staphylococcal protein A-rabbit IgG antibody (370 kDa), were prepared. A simple method was elaborated to test the immunotoxins' efficiency in selectively killing target cells in tumor-bearing mice. The target cell (murine EL4 leukemia) was coated with a xenogenic molecule by a method conserving its ability to proliferate and kill the inoculated animals. When the challenged animals were treated with these immunotoxins, which were specific for the antigenic molecule coating the tumor cells, survival time was lengthened compared with that of untreated animals, corresponding to a proportion of over 90% cells killed. This demonstrates the efficiency of the immunotoxins and the validity of the method elaborated.

Animals↗
PubMed1993

The current status of immunotoxins: an overview of experimental and clinical studies as presented at the Third International Symposium on Immunotoxins.

The Third International Symposium on Immunotoxins was held on June 19-21, 1992 in Orlando, Florida. This symposium was sponsored by NATO, NIH, Pierce Chemical Company, Walt Disney Cancer Institute at Florida Hospital, Duke Comprehensive Cancer Center, Xoma, Immunogen, Seragen, Bristol-Myers Squibb, Chiron, Ortho Biotech, Upjohn, Merck Sharp & Dohme Research Laboratories, Abbot Laboratories, Lilly Research Laboratories, and Evans & Sutherland. The Pierce Immunotoxin Award which recognizes outstanding contributions to immunotoxin research and development, was presented to Drs David FitzGerald, Fatih Uckun, David Eisenberg, and Ira Wool, for their contributions to the immunotoxin field.

Clinical Trials as Topic↗
PubMed1988

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.

Adjuvants, Immunologic↗
PubMed1995

Therapy of human B-cell lymphoma bearing SCID mice is more effective with anti-CD19- and anti-CD38-saporin immunotoxins used in combination than with either immunotoxin used alone.

The CD19+ CD38+ human Burkitt's lymphoma cell line Ramos grows aggressively when injected intravenously (i.v.) into severe combined immunodeficient (SCID) mice, killing 100% of animals within a 33-42 day period with widely disseminated disease. Treatment commencing 7 days after i.v. injection of Ramos cells, with 3 doses of an anti-CD19 immunotoxin (IT; BU12-SAPORIN) or an anti-CD38IT (OKT10-SAPORIN) led to a significant prolongation of survival compared with sham-treated controls; the anti-CD38 IT gave the greatest prolongation of survival, but all treated animals eventually succumbed to disease. When both ITs were used in combination at equivalent dose levels, the therapeutic outcome was significantly improved over that obtained for single IT therapy, with 20% of animals surviving disease-free to 300 days. When anti-CD38 IT was given in combination with anti-CD19 antibody there was no therapeutic improvement over anti-CD38 IT used alone. However, when anti-CD19 IT was given in combination with CD38 antibody, a significant prolongation of survival ensued over that obtained with anti-CD19 IT alone, though this was not as significantly pronounced as that obtained when both ITs were used in combination and was only as good as the survival obtained with OKT10 antibody used alone. CD19 and CD38 are expressed on the surface of the vast majority of B-cell lymphoma and common acute lymphoblastic leukaemia cells, and our findings provide a sound rationale for a combination immunotoxin trial in these diseases directed against both these target molecules.

ADP-ribosyl Cyclase↗
PubMed1992

The antitumor activity of an anti-CD22 immunotoxin in SCID mice with disseminated Daudi lymphoma is enhanced by either an anti-CD19 antibody or an anti-CD19 immunotoxin.

The antitumor activities of immunotoxins (ITs) constructed with deglycosylated ricin A chain (dgA) and either anti-CD19 (HD37) or anti-CD22 (RFB4) monoclonal antibodies were compared in SCID mice with disseminated human Daudi lymphoma (SCID/Daudi). As reported previously, after intravenous injection with Daudi cells, SCID mice develop disseminated lymphoma, which infiltrates the vertebral column and causes paralysis of the hind legs before death. The mean paralysis time (MPT) has been taken as an end point in this tumor model. We have previously reported that early treatment of SCID/Daudi mice with RFB4 coupled to dgA prolongs the MPT in a manner consistent with the killing of 4 logs of tumor cells. In the present study, we show that HD37-dgA kills 2 logs of tumor cells. The lower potency of the HD37-dgA is consistent with its lower IC50 on Daudi cells in vitro. We further show that the antitumor activity of a mixture of HD37-dgA and RFB4-dgA is significantly enhanced in SCID/Daudi mice and is consistent with the killing in excess of 5 logs of tumor cells. However, identical enhancement was observed when a mixture of the RFB4-dgA and the HD37 antibody was administered. In contrast, enhancement was not observed when mice were injected with a mixture of the RFB4 antibody and the HD37-dgA. The results indicate that a "cocktail" of HD37 antibody and RFB4-dgA immunotoxin can have significant antitumor activity in this mouse model of lymphoma and suggest that combinations of particular antibodies and ITs may have cooperative antitumor activity.

Animals↗
PubMed2001

Therapy of human T-cell acute lymphoblastic leukaemia with a combination of anti-CD7 and anti-CD38-SAPORIN immunotoxins is significantly better than therapy with each individual immunotoxin.

Severe combined immunodeficient (SCID) mice injected i.v. with the human T-ALL cell line CCRF CEM (SCID-CEM mice) develop within 50 days life-threatening multi-organ growth of leukaemia cells. The development of leukaemia in SCID-CEM mice treated with three 10 microg i.v. doses of the anti-CD7 immunotoxin (IT) HB2-SAPORIN or the anti-CD38 IT OKT10-SAPORIN was significantly delayed compared with PBS sham-treated animals but 90% of animals treated with either IT eventually developed disseminated leukaemia cell growth. In contrast treatment of SCID-CEM mice with a combination of both ITs led not only to a significantly greater delay in time to leukaemia development but also in the numbers of animals remaining leukaemia free (60%). The native HB2 and OKT10 antibodies (both murine IgG1antibodies) exerted significant, though relatively weak therapeutic effects, probably mediated through an antibody-dependent cellular cytotoxicity (ADCC) mechanism. Moreover, there was no in vivo additivity of therapeutic effect when both antibodies were used in combination. Apparent, however, was that the combination of HB2-SAPORIN IT with OKT10 antibody led to an intermediate therapeutic effect that was significantly greater than that obtained when either was used alone but significantly less than that obtained when the two IT combination was utilized. This was similarly the case for the combination of OKT10-SAPORIN IT with HB2 antibody though the effect was less pronounced in this instance. This result suggests that the therapeutic effect of IT + antibody treatment results from an additivity between antibody-mediated delivery of saporin combined with a SCID mouse NK cell-mediated ADCC attack on the target cell directed through target cell bound antibody Fc engagement with FcgammaRIII on the NK cell surface. The combination of both ITs however gave the best therapeutic outcome in SCID-CEM mice probably as the result of (i) delivery of greater amounts of saporin to target CEM cells positive for both CD7 and CD38, (ii) delivery of an effective dose of saporin to CEM cells downregulated or negative for one of the target antigens and (iii) through ADCC mechanisms that interact additively with IT action. We have previously proposed that combination IT therapy would be one means of overcoming the problem of heterogeneity of antigen expression within a global tumour cell population and these additional findings support this and provide a further strengthening of the rationale for employing cocktails of ITs for the treatment of human malignancies.

ADP-ribosyl Cyclase↗
PubMed1990

Marked difference in the in vivo antitumor efficacy between two immunotoxins targeted to different epitopes of common acute lymphoblastic leukemia antigen (CD10). Mechanisms involved in the differential activities of immunotoxins.

In the present study, two isotype-matching mAb, SN5d and SN5, which are directed toward two distinctively different epitopes of common acute lymphoblastic leukemia Ag (CD10) but show a very similar binding affinity to leukemia cells, were compared for their in vivo antitumor activity after conjugated to ricin A chain (RA). Our recently established nude mouse model carrying an ascitic tumor of NALM-6 human pre-B leukemia cells was used as the tumor model. A marked difference was observed in the in vivo antitumor efficacy between SN5d-RA and SN5-RA; SN5d-RA was much more effective than SN5-RA. Several experiments were carried out to gain information concerning the mechanisms involved in the different antitumor efficacy of the two immunotoxins. Although naked (unconjugated) mAb SN5d was much less effective than SN5d-RA conjugates in the in vivo tumor suppression, mAb SN5d was more effective than mAb SN5 in the in vivo tumor suppression. Additionally, marked differences were found between SN5d and SN5 in the induction of antigenic modulation and in the regulation of Ag biosynthesis and expression. Binding of SN5 to NALM-6 leukemia cells caused strong antigenic modulation (down-regulation of Ag expression) and strongly down-regulated Ag biosynthesis and cell surface expression of new Ag. In contrast, binding of SN5d to NALM-6 leukemia cells caused little modulation of overall cell surface expression of common acute lymphoblastic leukemia Ag; the decrease of old Ag by endocytosis after binding to mAb SN5d was compensated by newly exocytosed cell-surface expressed Ag. The present results appear to reveal a novel mechanism which regulates cytotoxic activities of antibodies and immunoconjugates.

Animals↗
PubMed1984

Cytotoxicity of a cell-reactive immunotoxin containing ricin A chain is potentiated by an anti-immunotoxin containing ricin B chain.

In vitro killing of the human Daudi cell line by either univalent [F(ab')] or divalent (IgG) forms of rabbit anti-human Ig (RAHIg) coupled to ricin A chain can be specifically potentiated by a "piggyback" treatment with ricin B chain coupled to goat anti-rabbit Ig (GARIg). When cells are treated with univalent immunotoxin (IT) [F(ab') RAHIg-A] and then cultured, IT can be detected on the cell surface for at least 5 h, since GARIg-B can still enhance killing at this time. These results provide a strategy for in vivo use of A chain- and B chain-containing IT.

Adjuvants, Immunologic↗
PubMed1988

Comparison of the pharmacokinetics and hepatotoxic effects of saporin and ricin A-chain immunotoxins on murine liver parenchymal cells.

Immunotoxins containing the ribosome-inactivating protein, saporin, are very effective antitumor agents but are highly toxic to mice. They induce severe necrotic lesions in the liver parenchyma of the recipients. Such extensive damage to the liver parenchyma is not observed with ricin A-chain immunotoxins even at 5-fold higher dosage. The hepatotoxicity of the saporin immunotoxins was found in the present study to arise from a combination of two effects. First, saporin and saporin immunotoxins were 30- and 6-fold more toxic to primary cultures of mouse liver parenchymal cells than were ricin A-chain and ricin A-chain immunotoxins, respectively. This was despite the fact that the cells bound 4- to 5-fold less saporin or saporin immunotoxins than ricin A-chain or ricin A-chain immunotoxins. The binding of ricin A-chain and its immunotoxin to the cells was mediated through the carbohydrate residues present on the A-chain whereas saporin is not glycosylated and thus must bind to other sites on the cell surface which result in transport of saporin relatively efficiently to the cytosol. The second reason for the hepatotoxic action of the saporin immunotoxin was that it had a longer blood half-life (t 1/2 alpha = 1.1 h; t 1/2 beta = 17.1 h) than the ricin A-chain immunotoxin (t 1/2 = 0.52 h; t 1/2 beta = 9.7 h). Analyses using a two-compartment pharmacokinetic model showed that the two immunotoxins broke down in vivo to give free antibody at a similar rate (t 1/2 = 10-12 h) but that the ricin A-chain immunotoxin was eliminated 11 times more rapidly than the saporin immunotoxin by routes other than breakdown. It was calculated that, in mice given a median lethal dose of saporin immunotoxin, the blood levels of immunotoxin remained above the concentration that killed 50% of parenchymal cells in vitro for more than 48 h. In mice given a median lethal dose of ricin A-chain immunotoxin, the blood levels fell below the concentration that was toxic to parenchymal cells in vitro within 4 h. The longer blood half-life of the saporin immunotoxin may also explain our previous finding that it had antitumor activity superior to that of a ricin A-chain immunotoxin in mice.

Animals↗
PubMed1994

Antitumor activity and pharmacokinetics in mice of a recombinant immunotoxin containing a disulfide-stabilized Fv fragment.

Disulfide-stabilized Fvs (dsFv) are recombinant Fv fragments of antibodies in which the inherently unstable VH-VL heterodimer is stabilized by a disulfide bond engineered between structurally conserved framework positions of VH and VL. We have recently described a recombinant immunotoxin, B3(dsFv)-PE38KDEL, that is composed of such a dsFv connected to a truncated form of Pseudomonas exotoxin (PE38KDEL). This disulfide-stabilized immunotoxin is indistinguishable in activity and specificity from its single-chain immunotoxin counterpart (Brinkmann et al., Proc. Natl. Acad. Sci. USA, 90: 7538-7542, 1993). We have now constructed and evaluated the stability, pharmacokinetics, and antitumor effect of a very similar disulfide-stabilized immunotoxin B3(dsFv)-PE38. This immunotoxin is specifically cytotoxic to human cancer cell lines such as A431 that express the B3 antigen on their surface. In addition, the dsFv-immunotoxin is more stable at 37 degrees C in human serum than the corresponding single-chain immunotoxin B3(Fv)-PE38. The survival of the disulfide-stabilized immunotoxin in the circulation of mice was determined by a bioassay on cultured A431 cells after administering the immunotoxin i.v. The half-life in blood was 23 min. To determine the therapeutic effects of the disulfide-stabilized immunotoxin, it was given i.v. to immunodeficient mice bearing s.c. human epidermoid carcinomas. The dsFv-immunotoxin caused complete regression of tumors with no toxic effect on mice. The antitumor effect was similar to that reported for the single-chain Fv-immunotoxin. Our data show that dsFv-immunotoxins retain full in vitro as well as in vivo activity when compared to scFv-immunotoxins. Because dsFv-immunotoxins have full activity, are more stable, and can be produced with significantly improved yields compared to scFv-immunotoxins, the dsFv-immunotoxins may be more useful for therapeutic applications than scFv-immunotoxins.

Animals↗