F. Stirpe

Response of refractory Hodgkin's disease to monoclonal anti-CD30 immunotoxin

In Hodgkins disease, Hodgkin and Reed-Sternberg cells consistently express the antigen CD30. We investigated the possible therapeutic role of an immunotoxin prepared by covalent linking of an anti-CD30 monoclonal antibody (Ber-H2) to saporin (SO6), a type-1 ribosome-inactivating protein. The immunotoxin (0.8 mg/kg in one or two doses) was given to four patients with advanced refractory Hodgkins disease. In three, there was rapid and substantial reduction in tumour mass (50% to greater than 75%). Clinical responses were transient (6-10 weeks). In-vivo binding of the immunotoxin to tumour cells was shown by immunohistology in two patients. Antibodies to both parts of the immunotoxin developed in all patients.

The conjugate Rituximab/saporin-S6 completely inhibits clonogenic growth of CD20-expressing cells and produces a synergistic toxic effect with Fludarabine

Immunotoxins are chimeric proteins consisting of a toxin coupled to an antibody. To date, several clinical trials have been conducted, and some are still ongoing, to evaluate their anti-tumor efficacy. In this view, we chemically constructed an anti-CD20 immunotoxin with the mAb Rituximab and the type 1 ribosome-inactivating protein (RIP) saporin-S6, designed for B cells non-Hodgkins lymphoma (NHL) therapy. This immunotoxin showed a specific cytotoxicity for the CD20+ cell lines Raji and D430B, evidenced by inhibition of protein synthesis, evaluation of apoptosis and clonogenic assay. Upon conjugation, saporin-S6 increased its toxicity on target cells by at least 2 logs, with IC50 values of 0.1–0.3 nM. The percentage of AnnexinV+ cells was over 95% in both cell lines treated with 10 nM immunotoxin. A complete elimination of Raji clones was reached with the 10 nM immunotoxin, whereas a mixture of free RIP and mAb gave about 90% of clonogenic growth. Rituximab/saporin-S6, at 10 nM concentration, also induced apoptosis in 80% of lymphoma cells from NHL patients. Moreover, sensitivity of Raji to Rituximab/saporin-S6 was augmented when cells were coincubated with Fludarabine. The synergistic toxic effect of the two drugs led to a total elimination of the neoplastic population.

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 × 10−13 to 5.7 × 10−11m (immunotoxins containing dianthin 32, saporin, pokeweed antiviral protein from seeds (PAP‐S), bryodin, momordin, momorcochin, and trichokirin), 1 × 10−8m (immunotoxin containing gelonin) and 5 × 10−9m (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 × 10−11m. 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.

An immunotoxin containing momordin suitable for bone marrow purging in multiple myeloma patients.

Attempts have been made by a number of methods to eliminate minimal residual disease from bone marrow to be reinfused in autologous transplantation. In this paper we describe a conjugate containing a monoclonal antibody, named 8A, recognising a plasma cell-associated antigen, and momordin, a ribosome-inactivating protein similar to the ricin A-chain. This immunotoxin is active on target cell lines and on neoplastic plasma cells, while myeloid progenitors are fairly resistant. The conjugate is shown to be acceptable for ex vivo purging in autologous bone marrow transplantation in multiple myeloma patients.

Efficacy and toxicity of plasma-cell-reactive monoclonal antibodies B-B2 and B-B4 and their immunotoxins

Abstract Immunotherapy based on the delivery of toxic agents to the tumor site using monoclonal antibodies (mAb) may be a promising modality in the treatment of hematological malignancies. In the selection of mAb, both for ex vivo but even more for in vivo therapy, not only their reactivity to the neoplastic cells should be considered, but also reactivity to other body constituents. Here we describe the screening of two human plasma-cell-reactive mAb B-B2 and B-B4, which may be used for immunotherapy of multiple myeloma. Cross-reactivity of B-B2 and B-B4 was determined by immunohistochemistry on a series of tissues. This revealed for both B-B2 and B-B4 a strong staining of epithelial cells in various organs, e.g. lung, liver, skin, kidney and gut, while only a weak and diffuse staining was seen with endothelial cells. In bone marrow reactivity was only found with plasma cells and not with hemopoietic precursors (CD34+ cells). Immunotoxins from B-B2 and B-B4 were constructed by coupling them to the plant-derived ribosome-inactivating protein saporin. Both B-B2 and B-B4 immunotoxins appeared to be efficient in specific inhibition of protein synthesis in plasma cell lines (IC50 respectively 1 nM and 0.1 nm). The immunotoxins were also tested on epithelial cell line A431, on liver cell line HepG2 and on human umbilical vein endothelial cells. The epithelial cell line A431 was reactive with both B-B2 and B-B4, but was only inhibited by B-B4 immunotoxin. Cell line HepG2 was reactive with both mAb, but was not inhibited by either immunotoxin. The endothelial cells showed no reactivity with B-B2 and B-B4 and were not inhibited by either immunotoxin. Bone marrow treated with B-B2 and B-B4 immunotoxin did not show a decrease in colonies of hemopoietic precursor cells. Incubation of multiple-myeloma-derived bone marrow with these immunotoxin resulted in a clear decrease of the number of plasma cells.

Polynucleotide: Adenosine Glycosidase Activity of Immunotoxins Containing Ribosome-Inactivating Proteins

Abstract Polynucleotide:adenosine glycosidases (rRNA N-glycosidases, EC 3.2.2.22, more commonly known as ribosome-inactivating proteins, RIP) are a numerous family of plant and bacterial enzymes, shown to release also adenine from DNA in vitro. They are well suited for the preparation of specifically toxic conjugates with several carriers, including monoclonal antibodies (immunotoxins). Here we show that (i) immunotoxins containing various PNAG (dianthin, gelonin, momordin I, PAP-S, PDS-2, ricin A-chain, saporin-L1, saporin-S6) all act on DNA; (ii) activity on DNA in vitro is less compromised by disulphide linkage to antibody than is inhibition of cell-free protein translation; and (iii) specific cytotoxicity of immunotoxin does not correlate with substrate specificity.

Immunoconjugates made of an anti-EGF receptor monoclonal antibody and type 1 ribosome-inactivating proteins from Saponaria ocymoides or Vaccaria pyramidata.

The present paper describes two immunoconjugates consisting of an anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MAb), named Mint5, covalently linked to the type 1 ribosome-inactivating proteins (RIPs) ocymoidine (Ocy) and pyramidatine (Pyra) from Saponaria ocymoides and Vaccaria pyramidata respectively. Both antibody and toxins are shown to retain their respective biological properties upon chemical conjugation. The immunoconjugates exert specific inhibition of EGFR expressing target cell proliferation and protein synthesis in in vitro assays and also inhibit the growth of grafted human tumour cells in nude mice.

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.

Immunotoxins containing saporin 6 and monoclonal antibodies recognizing plasma cell-associated antigens: effects on target cells and on normal myeloid precursors (CFU-GM)

Monoclonal antibodies 8A and 62B1, recognizing plasma cell‐associated antigens, were covalently linked to saporin 6, a ribosome‐inactivating protein similar to the A‐chain of ricin. Both immunotoxins were tested on target human cell lines U266 and Raji, on non‐target K562 cell line and on myeloid CFU‐GM progenitors. The cloning efficiency and viability of target cells were strongly reduced by 8A‐saporin 6 and 62B1‐saporin 6 immunotoxins, with an ID50 up to 200000‐fold lower than free saporin 6, whilst the K562 non‐target cell line was unaffected. Normal human myeloid precursors (CFU‐GM) were inhibited by immunotoxins only to a limited extent. An application of this model for autologous bone marrow transplantation in multiple myeloma patients is proposed. Since no eradication of cloning target cells was achieved by a single immunotoxin, mixtures made with different antibodies could help to reach this goal.

Occupational sensitization to ribosome-inactivating proteins in researchers

Background Ribosome‐inactivating proteins (RIPs) are expressed in many plants. Because of their anti‐infectious and anti‐proliferative effects, intensive research is going on for applying these toxins in therapy against viral infections or malignancies. Recently, we demonstrated that type I allergy against RIPs from elderberry can occur.