Antidisialoganglioside ricin A-chain immunotoxins show potent antitumor effects in vitro and in a disseminated human neuroblastoma severe combined immunodeficiency mouse model.

Several monoclonal antibodies (mAbs) were screened on different neuroblastoma cell lines to evaluate ricin A-chain immunotoxins for possible use against human neuroblastoma. Four mAbs were identified that exhibited high antitumor activity against neuroblastoma cell lines as measured in an indirect cytotoxicity assay. These mAbs, including 14G2a (antidisialoganglioside), ch14.18 (a humanized switch variant), BW704 (antidisialoganglioside), and chCE7 (anti-glycoprotein of M(r) 190,000), were subsequently linked via the bivalent linker N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-piridyldithio++ +)toluene to deglycosylated ricin A chain. The most potent immunotoxin, 14G2a.dgA, inhibited the protein synthesis of neuroblastoma cell lines IMR5 and NMB by 50% at concentrations of 6 x 10(-12) M. To test the antitumor efficacy of these immunotoxins in vivo, we developed a disseminated human neuroblastoma model in severe combined immunodeficiency mice. Treatment of tumor-bearing mice with 14G2a.dgA 12 days after tumor challenge resulted in a significant prolongation of survival as compared with phosphate-buffered saline-treated controls (16.8 versus 6.5 weeks). We conclude that ricin A-chain immunotoxins might be of potential use in the treatment of human neuroblastoma.

Generation and characterization of recombinant vascular targeting agents from hybridoma cell lines.

Vascular targeting agents (VTAs) can be produced by linking antibodies or antibody fragments directed against endothelial cell markers to effector moieties. So far, it has been necessary to produce the components of VTAs (antibody, antibody fragment, linker, and effector) separately and, subsequently, to conjugate them by biochemical reactions. We devised a cloning and expression system to allow rapid generation of recombinant VTAs from hybridoma cell lines. The VTAs consist of a single chain Fv antibody fragment as a targeting moiety and either truncated Pseudomonas exotoxin (resulting in immunotoxins) or truncated human tissue factor (resulting in coaguligands) as effectors. The system was applied to generate recombinant immunotoxins and coaguligands directed against endoglin, vascular endothelial growth factor (VEGF):VEGF receptor (VEGFR) complex and vascular cell adhesion molecule 1 (VCAM-1). The fusion proteins exhibited similar functional activity to analogous biochemical constructs. This is the first report to describe the generation and characterization of recombinant coaguligands.

Experimental treatment of human Hodgkin's disease with ricin A-chain immunotoxins.

In the present paper we describe the evaluation of ricin A-chain immunotoxins for clinical application in Hodgkin's disease. The immunotoxins were constructed by chemically linking deglycosylated ricin-A to monoclonal antibodies (MoAb) recognising lymphocyte activation markers CD25, CD30, or IRac, which are expressed by Hodgkin's and Reed-Sternberg (H-RS) cells. The cytotoxic effects of the immunotoxins were investigated in vitro against L540Cy Hodgkin cells and in vivo against Hodgkin's tumors in nude mice and disseminated Hodgkin's tumors in SCID mice. MoAbs were evaluated for crossreactivity with normal human tissues and staining of sections from Hodgkin's disease tissue. Of 32 MoAbs, eight showed little crossreactivity with vital human organs and produced highly active immunotoxins. The most effective immunotoxin, RFT5 gamma l.dgA (CD25), inhibits the growth of H-RS cells at concentrations of 7 x 10(-12) M. RFT5 gamma l.dgA destroys about 60% of solid Hodgkin's tumors of 0.5 cm diameter in nude mice and induces complete remissions in 95% of SCID mice with disseminated Hodgkin's tumors when administered one day after tumor challenge. This immunotoxin binds to all H-RS cells in more than 90% of patients with Hodgkin's disease. Patients with refractory Hodgkin's disease are currently being treated in a phase-I/II clinical trial.

An anti-GD2 single chain Fv selected by phage display and fused to Pseudomonas exotoxin A develops specific cytotoxic activity against neuroblastoma derived cell lines.

Since the disialoganglioside GD2 is abundantly present on the surface of neuroblastoma cells, we constructed a new recombinant immunotoxin for possible clinical use in patients with neuroblastoma. A functional 14.18 scFv-phage was obtained by selection of an anti-GD2 hybridoma derived phage antibody mini-library on the neuroblastoma-derived, GD2-expressing cell line IMR5. By insertion into the bacterial expression vector pBM1.1 the selected scFv was fused to a deletion mutant of Pseudomonas exotoxin A (ETA'). Periplasmically expressed 14.18(scFv)-ETA' bound to the GD2 expressing cell line IMR5, but not to the GD2 negative Hodgkin-derived cell line L540Cy as documented by ELISA and flow cytometry. The recombinant immunotoxin (rIT) inhibited cell viability of IMR5 cells by 50% at concentrations (IC(50)) of 0.326 microg/ml. This recombinant immunotoxin will be further investigated in vivo for its value as a new immunotherapeutic agent for the treatment of patients with neuroblastoma.

[Molecular mechanisms of vasculogenesis and angiogenesis. What regulates vascular growth?]. / Molekulare Mechanismen der Vaskulogenese und Angiogenese. Möglichkeiten antiangiogener Therapie.

The basic mechanisms governing how endothelial cells, periendothelial cells, matrix molecules and blood constituents interact with each other are discussed. The many insights gained from this basic knowledge are being extended to further understand physiological and pathological features of vascular sprouting and maintenance. Understanding these basic principles that drive angiogenesis and vasculogenesis will lead to a more specific therapy of many disorders in ophthalmology and other fields, such as arteriosclerosis, tumor growth, myocardial ischemia and tissue repair.

[Anti-angiogenesis: a new approach to tumor therapy?]. / Antiangiogenese: ein neuer Ansatz in der Tumortherapie?

BACKGROUND: The overall mortality due to metastatic cancer has not or only minimally been reduced in spite of intensive research and many innovations in the field of conventional antineoplastic therapy in the past decade. In the last years it has become a fact that tumor growth is angiogenesis-dependent. Therefore, inhibitors of angiogenesis are a new class of antineoplastic substances with a novel mechanism of action that might be a powerful complement to conventional cytostatic therapy. SUBSTANCES AND CLINICAL TRIALS: Inhibitors of tumor-angiogenesis which have entered clinical trials, with their results published until December 1998 are discussed here. Most results originate from phase-I or phase-II clinical trials. They are discussed and compared in respect to toxicity and response. Also some substances with high therapeutic potential which are still in preclinical testing are discussed. RESULTS: Many of the investigated angiogenesis inhibitors demonstrated anti-tumor effects in phase-I or phase-II clinical trials. The commonest manifestation was stable disease, followed by partial remissions. In a few cases, complete remissions were observed. The toxicities of these substances differ both in type and degree of side effects. CONCLUSION: Some antiangiogenic drugs appear to be promising candidates for a clinical use in the therapy of solid tumors. Further conclusions can only be drawn after evaluation of the results of ongoing phase-III clinical trials.

[New perspectives in oncology: is selective destruction of tumor cells with immunotoxins in Hodgkin's disease an additional therapeutic alternative?]. / Neue Perspektiven in der Onkologie: Bietet die gezielte Zerstörung von Tumorzellen durch Immuntoxine beim Morbus Hodgkin eine zusätzliche therapeutische Alternative?

In the present paper, the authors describe the production and testing of immunotoxins for clinical application in Hodgkin's disease. The immunotoxins were constructed by chemical coupling of deglycolysated ricin-A to monoclonal antibodies against antigens on Hodgkin's and Reed-Sternberg cells (CD25, CD30, IRac). The cytotoxic effect of the immunotoxins was investigated in vitro against Hodgkin's and Reed-Sternberg cells (H-RS) and in vivo against solid Hodgkin's tumors in nude mice and disseminated Hodgkin's tumors in SCID mice. Cross-reactivity with normal tissue and the staining behaviour observed in sections of Hodgkin's tissue of various subtypes proved important parameters for the assessment of clinical applicability. Of more than 30 evaluated MoAb's, eight immunotoxins were produced, of which six showed both, cytotoxic effects of considerable potency against Hodgkin's tumor cells and low cross-reactivity with vital human organs. The most effective immunotoxin, RFT5 gamma 1.dgA, (CD25) inhibits the growth of H-RS cells at concentrations of 7 pMol and destroys about 60% of solid Hodgkin's tumors of 0.5 cm in diameter in nude mice. This immunotoxin binds to virtually all tumor cells in more than 90% of patients with Hodgkin's disease. Sufficient quantities of RFT5 gamma 1.dgA were produced for the treatment of patients with refractory Hodgkin's disease. These patients are currently being treated in a phase I clinical trial.

Affinity chromatography system for parallel purification of recombinant protein samples.

In protein engineering, the tasks of generating and testing a large number of variants of a molecule and of optimizing expression conditions for one distinct molecule create the need for purification methods that can handle a large number of samples simultaneously. We describe the development and some application results of a simple affinity chromatography system that can be used for the parallel purification of 24 protein samples, yielding sufficient quantities for biochemical and functional analysis. Advantages of this system over existing systems are as follows. Compared with commercially available complete chromatography systems, the costs of this system are minimal. In comparison with vacuum systems with various outlets, and with batch purification systems where centrifugation is necessary, this system allows gentler processing of the samples. This could be important for proteins that are easily damaged.

Expression of soluble VEGF receptor 2 and characterization of its binding by surface plasmon resonance.

Vascular endothelial growth factor (VEGF) is an endothelial cell specific mitogen that induces angiogenesis in several pathological conditions. To block angiogenesis, soluble VEGF receptor can be used. In this study, we describe a method for high yield expression of soluble VEGF receptor 2 (sFlk-1) in a baculovirus expression system (30 mg purified sFlk-1 per L of insect cell supernatant). We also determined the binding constants for both human and mouse VEGF to the recombinant receptor by surface plasmon resonance. In this cell-free assay, under the given experimental conditions, the on-rate ka was 0.5-2.2 x 10(6) M-1s-1 and the off-rate kd was 2-4 x 10(-4) s-1 (KD = 2-6 x 10(-10) M). To our knowledge this is the first study to report on- and off-rates for the VEGF:sFlk-1 interaction. Heparin was not required for the binding of VEGF to sFlk-1 in this assay. The obtained values will serve as baseline parameters for the design of improved versions of recombinant soluble VEGF receptor.

Immunotoxins: is there a clinical value?

Drug targeting is an attractive new approach to killing malignant cells, thereby leaving normal tissue unharmed. A decisive breakthrough was the advent of hybridoma technology, making monoclonal antibodies (MoAb) available in limitless supply. To construct reagents with selectivity for certain tumor cells, MoAbs or Fab' fragments were chemically linked to ribosome-damaging toxins derived from plants or bacteria like ricin, abrin, saporin, Pseudomonas exotoxin (PE), and diphtheria toxin (DT) to form immunotoxins, which combined the selectivity of the carrier moiety with the potency of the toxin moiety. The first generation of these immunotoxins showed impressive results in vitro but in most cases disappointing antitumour effects in animals or humans. By contrast, the second generation of immunotoxins, consisting of either A chain immunotoxins with a greatly improved stability in vivo or so-called 'blocked' ricin immunotoxins, have been demonstrated to be extremely effective in several animal models. Preliminary results of the current clinical trials suggest a possible clinical use of immunotoxins in leukemia and lymphoma patients. Genetically engineered fusion toxins have become available, which consist of a growth factor or a cytokine fused to a toxin moiety. In this paper, we will review the features of the three groups of immunotoxins which are most frequently used, i.e., ricin A chain and similar immunotoxins, blocked ricin immunotoxins, and recombinant toxins constructed with Pseudomonas exotoxin or diphtheria toxin.

Cocktails of ricin A-chain immunotoxins against different antigens on Hodgkin and Sternberg-Reed cells have superior anti-tumor effects against H-RS cells in vitro and solid Hodgkin tumors in mice.

Three ricin A-chain immunotoxins (ITs) recognizing different antigens on Hodgkin-Reed/Sternberg (H-RS) cells were evaluated for their anti-tumor effects when used in combination as "cocktails". These ITs, BB10.dgA (CD25), HRS3.dgA (CD30), and IRac.dgA (70 kDa), strongly inhibited the growth of L540Cy H-RS cells in vitro. The protein synthesis of this cell line was reduced more efficiently by the combination of 2 of these ITs than by BB10.dgA, HRS3.dgA or IRac.dgA alone. A cocktail of all 3 ITs was most effective in vitro. This was at least in part due to the non-homogeneous distribution of CD25, CD30 or IRac on the L540Cy H-RS target cells and to the fact that subpopulations deficient in one antigen nevertheless expressed appreciable levels of the other target antigens. IT cocktails were also superior as anti-tumor agents in nude mice with solid L540Cy tumors. Ninety percent of mice treated with cocktails containing 2 or 3 ITs had continuous complete remissions (CCR), as compared with only 40% of mice treated with the same dose of a single IT. Analysis of 7 L540Cy sub-lines re-established ex vivo from mice that relapsed after having achieved complete remission (CR) after therapy with a single IT showed that the surviving tumor cells were antigen-deficient variants which were resistant to the original IT, but which could be killed by ITs directed against other target antigens. Thus, IT cocktails give superior results against human H-RS cells, both in vitro and in vivo.

Successful treatment of disseminated human Hodgkin's disease in SCID mice with deglycosylated ricin A-chain immunotoxins.

To evaluate the effects of deglycosylated ricin A-chain (dgA) immunotoxins against disseminated Hodgkin's lymphoma, we used RFT5.dgA (CD25) and IRac.dgA (70 kD) to treat L540Cy Hodgkin cells in severely immunodeficient SCID mice. In this model, more than 90% of the animals developed multiple lymphomas in various organs such as the lymph nodes, liver, bone marrow, and extranodal sites that killed untreated animals after a mean survival time (MST) of 36.3 days. A single intraperitoneal injection of 8 micrograms of either immunotoxin rendered 95% (RFT5.dgA) and 93% (IRac.dgA), respectively, of mice tumor-free when applied 1 day after tumor challenge. The MST of the RFT5.dgA-treated group was extended by more than 80 days (P < .00001). SCID mice treated 12 days after tumor challenge had lower remission rates (46%), suggesting that the antitumor effect of the immunotoxins depends on the number of tumor cells present. We conclude that ricin A-chain immunotoxins have potent antitumor effects against disseminated Hodgkin's tumors in SCID mice and that this model is ideally suited for the evaluation of different immunotoxin treatment modalities.

Disseminated growth of Hodgkin's-derived cell lines L540 and L540cy in immune-deficient SCID mice.

Local tumor growth has been reported after subcutaneous and intraperitoneal injection of Hodgkin's disease (HD) derived cell lines into different immunodeficient mouse strains. An animal model with disseminated growth of tumor cells would be useful for studying the in vivo biology of HD cells as well as for preclinical testing of new therapeutic regimens. For this purpose the HD-derived cell lines L540, L540cy, L428, and KM-H2 were injected intravenously into SCID mice. In contrast to L428 and KM-H2, widespread neoplasia occurred after a period of four to six weeks following injection of L540 and the subline L540cy. Lymph nodes were found to be the preferred site of tumor growth. CD30 surface antigen expression on Hodgkin cells and the karyotype of the tumor cells were preserved in the animal host. Thus, to a large extent, the SCID mouse model mimics the dissemination pattern of Hodgkin's disease in man. To evaluate the role of adhesion molecule expression in the dissemination of HD-derived cell lines, CD44 and members of the immunoglobulin, integrin, selectin, and Fc receptor families were quantified by flow cytometry. CD30 expression was also measured. Although CD44 expression has been correlated with dissemination in non-Hodgkin's lymphoma (NHL), this was not the case in the Hodgkin's SCID mouse model. CD44 was not expressed on the disseminating cell lines L540 and L540cy but was expressed in the nondisseminating lines L428 and KM-H2.