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.     

Comparison of multiple anti-CEA immunotoxins active against human adenocarcinoma cells

Summary Anti-carcinoembryonic antigen (CEA) immunotoxins constructed with multiple anti-CEA antibodies (goat and baboon polyclonal, and three murine monoclonal antibodies) by covalently linking them to the A chain of ricin via a disulfide bond all function as potent and specific toxins for CEA-bearing cells, suggesting that the CEA molecule is capable of directing productive internalization of ricin A chain. The high potency of anti-CEA immunotoxins apparently makes addition of ricin B chain unnecessary for high toxic efficiency, as in some other systems, because presence of the B chain reduces target cell specificity. Several characteristics of the immunotoxins which might account for their cytotoxic potency were studied. Equilibrium association constants of the goat, baboon, and murine monoclonal C-19 antibodies with fluid-phase CEA were determined by using Langmuir plots and were found to be 8.79, 6.61, and 8.13×10⁹ M ^–1, respectively, indicating the high and similar affinities of the three antibodies toward CEA. Radioimmunoassay binding studies of the three immunotoxins with ¹²⁵I-CEA showed that the antibody portions of the molecules retained the ability to form complexes with CEA after conjugation to ricin A chain. The maximum number of anti-CEA antibody molecules bound per cell, as demonstrated by ¹¹¹In-labeled C-19 binding assays with CEA-bearing cell lines, varied from 2.65×10⁵ per cell for HT29 to 2.01×10⁶ for LoVo, with an intermediate value of 1.17×10⁶ per cell for WiDr. Cytotoxicity of the immunotoxins was assessed by inhibition of protein synthesis and expressed as a median inhibitory dose (ID₅₀). Comparison of the ID₅₀'s of each immunotoxin on the three cell lines has shown that the immunotoxin made of the monoclonal C-19 antibody is in general 6 to 7 times more cytotoxic than the goat and baboon antibody immunotoxins. The affinity of CEA-antibody binding is probably an important, but not a sole factor in determining the immunotoxin potency. The fact that the antibodies with very similar affinity toward fluid phase CEA make immunotoxins of different potency might indicate that interactions with membrane-bound CEA are more complex and/or the efficiency of internalization of various immunotoxins is different. An important factor in immunotoxin action appears to be the CEA content in target adenocarcinoma cells.Supported in part by the NIH BRSG grant SO7RRO5712, the American Cancer Society, Mass. Div. Research Grant 1543-C-1, and by the Aid for Cancer Research (Boston) award to L. V. L., and by RO1 CA 29160 and RO1 CA 39748 grants to T. W. G.     

Blocked ricin-conjugated T cell immunotoxins: Effect of anti-CD6-blocked ricin on normal T cell function

Summary The biological properties of an immunotoxin composed of an anti-CD6 monoclonal antibody conjugated to whole ricin, which had been modified so that the galactose-binding sites of the B chain were blocked (blocked ricin), were examined. Treatment of peripheral blood lymphocytes with anti-CD6-blocked ricin for a 24-h period prevented T cell proliferation induced by phytohemagglutinin in a dose-dependent manner with concentrations causing 50% inhibition (IC₅₀) ranging from 5 pM to 30 pM. In contrast, treatment with either blocked ricin alone or with a control immunotoxin prepared with a B-cell-lineage-restricted monoclonal antibody gave IC₅₀ values of approximately 2 nM. Although shortening the duration of the anti-CD6-blocked ricin treatment to as little as 3 h had little significant effect on the observed inhibition, T cell viability experiments demonstrated that the magnitude of immunotoxin-induced killing after a given time period is significantly higher when the target cells become activated. Thus, from the initial concentration of cells treated with anti-CD6-blocked ricin placed in culture, 40%–45% viable cells remained after 2 days yet only 3%–9% remained if phorbol ester and Ca²⁺ ionophore were added; activation of T cells after mock treatment using blocked ricin plus nonconjugated anti-CD6 demonstrated that this effect was not the result of activation alone. The toxicity of anti-CD6-blocked ricin was also measured by inhibition of PHA-induced clonogenic growth of normal T cells. Continuous treatment of the cells using anti-CD6-blocked ricin at 0.1 nM resulted in a surviving fraction of about 3.5 × 10^–3; when immunotoxin treatment was for 24 h or less, the surviving fraction was only about 10^–1. As an indication of the unique specificity of anti-CD6-blocked ricin, immunotoxin pretreatment of potential responder cells prevented the generation of allogeneic cytolytic T lymphocytes in mixed lymphocyte cultures yet had little effect on the generation of interleukin-2-induced lymphokine-activated killer cell activity. We conclude that anti-CD6-blocked ricin demonstrates a cellular specificity and potency that make it a highly promising anti-T cell reagent.     

Immunotoxin sensitivity of Chinese hamster ovary cells expressing human transferrin receptors with differing internalization rates

 Previous studies have shown that immunotoxin action is dependent upon selective binding to the target cell, internalization and then passage into the cytosol. It is important to define precisely how these critical steps are controlled so that the underlying relationship of each to high cytotoxic effectiveness is understood. In order to evaluate the contribution of internalization rate and receptor number on immunotoxin potency, the effects of an anti-(transferrin receptor, TfR)/ricin A chain immunotoxin, 7D3-A, were assessed on a parent Chinese hamster ovary cell line developed in our laboratory with no TfR (TfR^neg) and two lines transfected with either wild-type TfR (Tfr^wt) or an internalization-deficient (TfR^{δ7 – 58del}) mutated human TfR. Potent, receptor-mediated cytotoxicity resulted from the action of 7D3-A on TfR^wt cells (ID₅₀<1 nM) while both TfR^neg cells and TfR^{δ7 – 58del} were only minimally affected (ID₅₀>100 nM). Butyrate up-regulation substantially increased receptor expression on the TfR^wt and TfR^{δ7 – 58del} cells, but no corresponding rise in sensitivity to 7D3-A was observed. In contrast, immunotoxin potency was increased by co-treatment of TfR^wt cells with the carboxylic ionophore monensin and the effect was even more pronounced for TfR^{δ7 – 58del} cells. We conclude that internalization rate or intracellular destination is a much more important determinant of immunotoxin efficacy than receptor number. Received: 15 March 1996 / Accepted: 28 May 1996     

Human antibody responses to components of the monoclonal antimelanoma antibody ricin A chain immunotoxin XomaZyme-MEL

Human antibody responses to immunotoxin components were evaluated in 21 melanoma patients who were treated with XomaZyme-MEL, a murine monoclonal antimelanoma antibody-ricin A chain conjugate. Twenty of the 21 melanoma patients produced antibodies against ricin A chain, while 15 of 21 produced antibodies reactive with the murine monoclonal antibody component. Both IgM and IgG antibody responses were produced. Immunoglobulin responses were usually detected 1 to 2 weeks following initiation of therapy, with peak levels generally attained 2 to 4 weeks posttherapy. Titers of the anti-ricin A chain antibodies were generally higher than those of the antimurine monoclonal antibodies for the dose range tested. There was no clear correlation between the dose of immunotoxin administered and the antibody titer. By use of a competitive flow cytometry assay, antiidiotype responses were demonstrated in eight of 10 melanoma patients who had antimurine antibodies. Both the kinetics of appearance and the relative titers of the antiidiotype responses generally corresponded to the antimurine responses. The development of antimmunotoxin antibodies can reduce the therapeutic potential of immunotoxins through several mechanisms. The development of antibodies in a significant number of patients suggests that optimally effective, repeated courses of therapy will require some procedure for suppressing or abrogating the response against the immunotoxin.     

Cytotoxic properties of a ricin A chain immunotoxin recognising the cluster-5A antigen associated with human small-cell lung cancer

Summary The cytotoxic properties of a ricin A chain immunotoxin made with the mouse monoclonal antibody SWA20, recognising a family of sialoglycoprotein antigens selectively expressed by human small-cell lung cancer (SCLC), were examined using a panel of tumour cell lines in tissue culture. SWA20—ricin-A-chain was selectively toxic to the SW2, NCI-H69 and GLC-8 SCLC cell lines, inhibiting the incorporation of [³H]leucine by 50% at a concentration of 0.2–2 nM, but had no selective activity against the NCI-H23 and NCI-H125 lung adenocarcinoma or the control CEM T-lymphoblastoid cell lines. The SWA20 immunotoxin intoxicated the SW2 cell line rapidly, inhibiting [³H]leucine incorporation by 50% within 2 h compared with 0.5 h for ricin. Analysis of the effects of SWA20—ricin-A-chain on the growth of SW2 cells using a limiting-dilution clonogenic assay revealed that the immunotoxin could eliminate 95% of clonogenic malignant cells. Although SWA20—ricin-A-chain was found to be rapidly active against the majority of tumour cells, its action was limited by the presence of insensitive cells expressing low levels of the target antigen.     

In vitro and in vivo properties of an anti-CD5—momordin immunotoxin on normal and neoplastic T lymphocytes

An anti-CD5 monoclonal antibody (mAb) was linked to the plant toxin momordin, a type-1 ribosome-inactivating protein purified fromMomordica charantia. The in vitro cytotoxicity of the immunotoxin was evaluated as the inhibition of protein and/or DNA synthesis on isolated peripheral blood mononuclear cells (PBMC) and on human T cell leukemia Jurkat. The potency of the immunotoxin on PBMC was very high (IC₅₀ = 1–10 pM) and was not affected by blood components. The conjugate was also very efficient in the inhibition of the proliferative response in a mixed lymphocyte reaction (IC₅₀ = 10 pM). Moreover, the in vitro performances of the immunotoxin compared favourably with those reported for other anti-CD5-based immunoconjugates containing ricin A chain. The in vivo activity of the immunotoxin was assessed in the model ofnu/nu mice bearing Jurkat leukemia. A significant inhibition of the tumour development (80%,P <0.01) in the animals treated with immunotoxin was observed. Taken together, the in vitro and in vivo results suggest that the anti-CD5-momordin conjugate may be useful for graft-versus-host disease therapy and potentially in the treatment of CD5-positive leukemias and lymphomas.     

An ineffective monoclonal antibody-ricin A chain conjugate is converted to a tumouricidal agent in vivo by subsequent systemic administration of ricin B chain

Summary An immunotoxin comprising a tumour-specific monoclonal antibody (11/160) coupled to ricin A chain, although inactive in in vitro cytotoxicity assays against HSN_tc sarcoma target cells, was found to be capable of significant tumouricidal activity in syngeneic rats if potentiated by ricin B chain. The 11/160-ricin A, when bound to tumour cells prior to their inoculation, led to a slight inhibition of tumour growth s. c. compared with untreated sarcoma cells or those coated with antibody alone. However, all tumours in these groups developed progressively (69/69), whereas in those rats receiving 15 g or 150 g ricin B chain i. v. 5 min after tumour cell inoculation, the take rate was reduced to 75% and 30% respectively, and significantly longer latent periods were evident for those tumours which did develop. Ricin B chain similarly inhibited, in a dose-dependent manner, the lung colonisation potential of 11/160-ricin A coated HSN_tc cells. No effects were obtained if the B chain treatment followed inoculation of untreated or antibody-coated cells, suggesting that systemically administered B chain is capable of gaining access to and activating antibody-ricin A chain conjugates bound to the surface of syngeneic sarcoma cells in lung or subcutaneous sites. Tumour inhibition was obtained in some instances with intervals of up to 24 h between inoculation of conjugate-coated tumour cells and B chain. Experiments are in progress to determine if such potentiation may be feasible in a therapeutic rather than a prophylactic setting using this syngeneic solid tumour system.     

Immunotoxins constructed with chimeric,short-lived anti-CD22 monoclonal antibodies induce less vascular leak without loss of cytotoxicity

An immunotoxin (IT) constructed with RFB4, a murine anti-CD22 monoclonal antibody, and the “deglycosylated” A chain of ricin has shown activity at safe doses in patients with non-Hodgkin lymphoma and in children with acute lymphoblastic leukemia. The dose limiting toxicity is vascular leak syndrome (VLS), which appears to be due to a unique amino acid motif in the ricin toxin A (RTA) chain that damages vascular endothelial cells. We mutated recombinant (r) RTA to disable this site, but await testing of the IT prepared with this mutant RTA in humans. Another possible approach to reducing IT-induced VLS is to shorten the half-life of the IT in vivo. We previously constructed a mouse-human chimeric RFB4 by grafting the variable genes of RFB4 onto the human IgG1k constant regions. Here, we report the expansion of our panel of mutant chimeric RFB4s (mcRFB4s) that lack the ability to bind to the neonatal Fc receptor (FcRn). In comparison with cRFB4, which had a T_1/2 of 263 h, the mcRFB4s had T_1/2s ranging from 39–106 h. ITs were constructed with these mcRFB4s and rRTA. The mcRFB4-RTA ITs retained their cytotoxicity in vitro and had shorter half lives than the parental cRFB4-RTA IT. In addition, the mcRFB4 IT with the shortest T_1/2 induced less pulmonary vascular leak in mice, which we have postulated is a surrogate marker for VLS in humans.Key words: chimeric, anti-CD22, monoclonal antibody, Fc mutations, ricin A chain, immunotoxins     

蓖麻毒素与其单克隆抗体相互作用动力学研究

表面等离子体激元共振(SPR)是一种可微量、实时、动态地监测生物分子相互作用的生物传感技术。蓖麻毒素为核糖体失活蛋白,具有很强的细胞毒性作用。通过SPR技术研究了两种抗蓖麻毒素的单克隆抗体C5、D12与蓖麻毒素相互作用的动力学,计算出两者的亲和常数分别为2.49×108mol-1·L和7.9×108mol-1·L,并对两种抗体的抗原表位进行了分析。     

Could dual G-protein coupling explain [d-Met]FMRFamide's mixed action in vivo?

In vitro studies have demonstrated that FMRFamide-related peptide receptors can be coupled to different G-proteins, mediating opposite stimulatory and inhibitory effects. The present study tested whether this duality might extend to effects in vivo. Antinociception in mice of ICV [d-Met²]FMRFamide, which produced agonist [ED₅₀ = 36.3 μg (61.6 nmol)] and antagonist [ID₅₀ = 0.72 μg (1.22 nmol)] actions, was attenuated by 24-h pretreatment with ICV pertussis toxin (ID₅₀ = 0.55 μg) or cholera toxin (ID₅₀ = 0.09 μg), suggesting that [d-Met²]FMRFamide in vivo effects might also be explained by dual coupling.     

In vivo cytotoxic efficacy of immunotoxins prepared from anti-CD5 antibody linked to ricin A-chain

Summary The antitumoral efficacy of various anti-CD5 immunotoxins, prepared with whole monoclonal antibody (mAb), F(ab)₂ or Fab fragment linked to native ricin A-chain (RTA) or partially deglycosylated ricin A-chain (dRTA), was examined in vivo in ascitic nude mice bearing a large burden of Ichikawa human tumour cells. We first demonstrated that after systemic administration of IgG-RTA or F(ab)₂-dRTA, the cytotoxic activity of immunotoxin molecules specifically bound to tumour cells was preserved. Secondly we showed, by using different immunotoxins with various targeting capacities, that their cytotoxic effect in vivo was related to the number of immunotoxin molecules bound per cell. However, even when antigen saturation was achieved after i.p. injection, the cytotoxic effect did not exceed 53% of the tumour burden. By contrast, when the immunotoxin was administered i.p. or i.v. with the enhancer monensin conjugated to human serum albumin and injected i.p., 90% of the tumour cells were killed. This potentiating effect was demonstrated even when the tumour localisation was as low as 5% of the saturation level. Such an effect could be completely prevented by addition of unconjugated monoclonal antibody, demonstrating the specificity of the immunotoxin-induced cytotoxicity in the presence of the enhancer. However this enhancement was demonstrated whatever the route of immunotoxin administration, i.p. or i.v., but was only observed when the enhancer was injected i.p. and not i.v.. These results emphasize the importance of optimizing the therapeutic course to improve the antitumoral efficacy of immunotoxins.     

An immunotoxin containing a rat IgM monoclonal antibody (Campath 1) and saporin 6: effect on T lymphocytes and hemopoietic cells

Summary The elimination of the cells responsible for graft-versus-host disease in allogeneic bone marrow transplantation has been attempted with a variety of methods, including the use of the ribosome-inactivating toxin ricin bound to monoclonal antibodies acting as carriers. However the high nonspecific toxicity of these immunotoxins containing the whole toxin greatly limited clinical application. Toxicity can be reduced using the A-chain of ricin or other ribosome-inactivating proteins (RIPs) which are devoid of a B-chain with lectin properties. We used saporin 6 purified from Saponaria officinalis seeds, which was conjugated with the rat IgM monoclonal antibody Campath 1 specific for mature T and B lymphocytes as well as for monocytes. The immunotoxin retained both RIP and antibody activity, inhibiting protein synthesis both in a cellfree system and in cells bearing the Campath 1 antigen; it also abolished methyl ³H-thymidine uptake in phytohemagglutinin-stimulated T lymphocytes. Myeloid progenitors were largely spared as shown by myeloid stem cell (CFUGM) growth which was scarcely affected. Toxicity of the immunotoxin to cell lines not expressing the antigen recognized by Campath 1 monoclonal antibody was not greater than the toxicity due to free saporin 6, while the immunotoxin was more toxic to mice than free saporin.Work supported by grants of Regione Emilia Romagna, delibera n. 1970, 13/5/86, by the Italian National Research Council, Roma, Finalized Project Oncologia, contracts n. 86.00589.44 and n. 86.00603.44 and by the Pallotti's Legacy for Cancer ResearchAngelo Dinota is supported by a grant from Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan, Italy.     

The improvement of an anti-CD22 immunotoxin: Conversion to single-chain and disulfide stabilized form and affinity maturation by alanine scan

HA22-LR is a recombinant immunotoxin for the treatment of B-cell malignancies that contains the Fv portion of an anti-CD22 antibody fused to a functional portion of Pseudomonas exotoxin A. In the present study, we attempted to improve this molecule. First, we produced a single-chain version of HA22-LR (scdsFv-HA22-LR) in which a peptide linker was introduced between the disulfide-linked light and heavy chains to enable production via single fermentation. No difference in cytotoxic activity was observed between scdsFv-HA22-LR and prototype HA22-LR. Next, we attempted to increase the affinity of scdsFv-HA22-LR by using alanine scanning mutagenesis of complementarity determining regions (CDRs) to assess the specific contribution of each CDR residue to the antigen binding. We found that mutation of asparagine 34 in V_LCDR1, which is located at the V_L/V_H interface, to alanine (N34A) caused a substantial increase in affinity and activity. Estimated K_D values measured by fluorescence-activated cell sorting were lowered by 10-fold: 0.056 nM in the N34A mutant compared to 0.58 nM in wild type (WT). Cell viability assays of CD22-positive B-cell lymphoma and leukemia cell lines showed that the N34A mutant had increased cytotoxicity ranging from ∼2 (HAL-1, IC₅₀(WT): 2.37 ± 0.62 ng/ml, IC₅₀(N34A): 1.32 ± 0.41 ng/ml) to 10 (SUDHL-6, IC₅₀(WT): 0.47 ± 0.090 ng/ml, IC₅₀(N34A): 0.048 ± 0.018 ng/ml)-fold compared to WT immunotoxin. The present study suggests that the N34A mutant of scdsFv-HA22-LR could have important consequences in a clinical setting.Key words: immunotoxin, HA22, affinity-maturation, alanine scan, V_H/V_L interface     

The effects of cyclophosphamide on the toxicity and immunogenicity of ricin A chain immunotoxin in rats

We conducted a study to determine if treatment with cyclophosphamide (CY) could suppress the formation of anti-murine and anti-ricin A chain antibodies in rats treated with a murine monoclonal antibody-ricin A chain immunotoxin (IT). Female Sprague-Dawley rats received intravenous doses of IT at a dose of 5 mg/kg body weight alone or in combination with CY at a dose level of either 10 or 20 mg/kg body weight. The IT was given as one or two courses consisting of five consecutive daily intravenous injections (days 0 to 4, or days 0 to 4 and days 21 to 25 of the study). Cyclophosphamide was given on days 2, 4, 6, 13, and 17 of the study to the group receiving a single course of IT; additional doses of CY were administered on days 23, 25, and 27 to the group receiving two courses of IT. On days 4, 14, 21, 28, and 35, animals from each group were evaluated for antibodies to murine IgG and ricin A chain, and for clinical laboratory parameters and histopathology. Animals receiving IT alone developed significant titers of both anti-murine and anti-ricin A chain antibodies. Compared with the response in the animals receiving single-course IT, the response to both of the components of the IT was significantly increased on days 28 and 35 in the animals receiving a second course of IT. The groups receiving a combination of either one or two courses of CY and IT demonstrated a significantly decreased antibody response to both the murine IgG and the ricin A chain compared with the group receiving IT alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurements of auto‐antibodies to α‐synuclein in the serum and cerebral spinal fluids of patients with Parkinson's disease

Biomarkers for α‐synuclein are needed for diagnosis and prognosis in Parkinson's disease (PD ). Endogenous auto‐antibodies to α‐synuclein could serve as biomarkers for underlying synucleinopathy, but previous assessments of auto‐antibodies have shown variability and inconsistent clinical correlations. We hypothesized that auto‐antibodies to α‐synuclein could be diagnostic for PD and explain its clinical heterogeneity. To test this hypothesis, we developed an enzyme‐linked immunosorbent assay for measuring α‐synuclein auto‐antibodies in human samples. We evaluated 69 serum samples (16 healthy controls (HC ) and 53 PD patients) and 145 CSF samples (52 HC and 93 PD patients) from our Institution. Both serum and CSF were available for 24 participants. Males had higher auto‐antibody levels than females in both fluids. CSF auto‐antibody levels were significantly higher in PD patients as compared with HC , whereas serum levels were not significantly different. CSF auto‐antibody levels did not associate with amyloid‐β_1–42, total tau, or phosphorylated tau. CSF auto‐antibody levels correlated with performance on the Montreal Cognitive Assessment, even when controlled for CSF amyloidβ_1–42. CSF hemoglobin levels, as a proxy for contamination of CSF by blood during lumbar puncture, did not influence these observations. Using recombinant α‐synuclein with N‐ and C‐terminal truncations, we found that CSF auto‐antibodies target amino acids 100 through 120 of α‐synuclein. We conclude that endogenous CSF auto‐antibodies are significantly higher in PD patients as compared with HC , suggesting that they could indicate the presence of underlying synucleinopathy. These auto‐antibodies associate with poor cognition, independently of CSF amyloidβ_1–42, and target a select C‐terminal region of α‐synuclein.

Read the Editorial Highlight for this article on page 433 .

     

Intratumoral anti-HuD immunotoxin therapy for small cell lung cancer and neuroblastoma

Background

Most patients with small cell lung cancer (SCLC) or neuroblastoma (NB) already show clinically detectable metastases at diagnosis and have an extremely poor prognosis even when treated with combined modalities. The HuD-antigen is a neuronal RNA-binding protein that is expressed in 100% of SCLC tumor cells and over 50% of neuroblastoma cells. The correlation between high titers of circulating anti-HuD antibodies in patients and spontaneous tumor remission suggests that the HuD-antigen might be a potential molecular target for immunotherapy.

Methods

We have constructed a new antibody-toxin compound (called BW-2) by assembling a mouse anti-human-HuD monoclonal antibody onto streptavidin/saporin complexes.

Results

We found that the immunotoxin BW-2 specifically killed HuD-positive human SCLC and NB cancer cells at very low concentrations in vitro. Moreover, intratumoral immunotoxin therapy in a nude mouse model of human SCLC (n?=?6) significantly reduced local tumor progression without causing toxicity. When the same intratumoral immunotoxin protocol was applied to an immunocompetent A/J mouse model of NB, significant inhibition of local tumor growth was also observed. In neuroblastoma allografted A/J mice (n?=?5) treated twice with intratumoral immunotoxin, significant tumor regression occurred in over 80% of the animals and their duration of tumor response was significantly prolonged.

Conclusions

Our study suggests that anti-HuD based immunotoxin therapy may prove to be an effective alternative treatment for patients with SCLC and NB.

     

Effects of BCL-2 overexpression on the sensitivity of MCF-7 breast cancer cells to ricin, diphtheria and Pseudomonas toxin and immunotoxins

Immunotoxins are presently being evaluated as novel agents for cancer therapy. The direct mechanism by which immunotoxins kill cancer cells is inhibition of protein synthesis, but cytotoxicity due to induction of apoptosis has also been observed with these agents. Some cancers that express high levels of BCL-2 are relatively resistant to apoptosis inducing agents. It is therefore important to determine to what degree the toxicity of ricin, diphtheria toxin, Pseudomonas exotoxin and Pseudomonas exotoxin derived immunotoxins towards cancer cells can be attributed to inhibition of protein synthesis, and to what degree to subsequent induction of apoptosis. We compared the sensitivity of MCF-7 breast cancer cells that were stably transfected with a BCL-2 expression plasmid and thus protected against apoptosis and of MCF-7 cells transfected with a control plasmid towards ricin, diphtheria and Pseudomonas toxin, a Pseudomonas toxin-derived immunotoxin (LMB-7) and tumour necrosis factor (TNF). We found that BCL-2 mediated inhibition of apoptosis renders the cells almost completely resistant (1000-fold) to tumour necrosis factor, but the same cells were only 3–10 fold more resistant to cytotoxicity induced by immunotoxin LMB-7 as well as Pseudo-monas exotoxin, diphtheria toxin and ricin. We next studied several leukaemia cell lines with variable levels of BCL-2 expression and found them quite sensitive to a Pseudomonas exotoxin containing immunotoxin independent of the level of BCL-2. Our data indicate that although BCL-2 overexpression can have a modest effect on sensitivity to an immunotoxin, cell lines derived from patients are still very sensitive to immunotoxins.     

Different cytotoxic activity and intracellular fate of an anti-CD5-momordin immunotoxin in normal compared to tumour cells

We investigated the different sensitivity of peripheral blood mononuclear cells (PBMC) and human T cell leukaemias (Jurkat and CEM) to an anti-CD5-momordin immunotoxin. In a short-term assay, the immunotoxin displayed different cytotoxic activity on normal and tumour cells: for leukaemic cell lines an incubation time of 72 h was necessary for the immunotoxin to reach the IC₅₀ of 41–53 pM, compared to the 1 h sufficient for 6 pM immunotoxin to inhibit 50% of PBMC protein synthesis. In a long-term clonogenic assay (15 days), the immunotoxin demonstrated a compareble efficacy of clonogenic cell killing for both cell types. We investigated the immunotoxin internalization pathway by a flow-cytometric method and our data seem to indicate that the molecules meet a different intracellular fate in the two cell populations. It may be assumed that the low cytotoxic activity of immunotoxins on tumour cells, detected in the shortterm assay, is due to infefficient delivery to their cytoplasmatic target, while a longer exposure of the cells to the immunotoxin promotes adequate intracellular distribution.     

In vitro vomitoxin exposure alters IgA and IgM secretion by CH12LX B cells

The CH12LX cell line was used as a clonal model to assess the direct effects of vomitoxin on IgM and IgA secretion in B cells. When vomitoxin was included in LPS-driven CH12LX B cell cultures, it had multiple effects on Ig secretion. Whereas vomitoxin doses of 115 and 120 ng/ml caused 50% inhibition(ID₅₀) of IgA and IgM production, respectively, toxin concentrations in the 5 to 50 ng/ml range slightly stimulated IgA production. However, low vomitoxin doses did not induce switching of membrane IgM+ CH12LX B cells to membrane IgA+. Total cell number was unaffected at vomitoxin concentrations up to 100 ng/ml but dropped markedly at 200 ng/ml (ID₅₀=170 ng/ml). Using the MTT reduction assay as another measure of viability and cell function, vomitoxin was also inhibitory (ID₅₀=130 ng/ml). Both thymidine incorporation and leucine incorporation were also inhibited by the toxin with estimated ID₅₀s being 120 and 110 ng/ml, respectively. The results indicate that although at high doses, vomitoxin inhibits proliferation, Ig secretion and DNA/protein synthesis in the clonal B cell model, the toxin marginally stimulated IgA secretion at lower doses.