An anti-CD5 immunotoxin for chronic lymphocytic leukemia: enhancement of cytotoxicity with human serum albumin-monensin.

Five patients with B-cell chronic lymphocytic leukemia (B-CLL) were treated with 6 courses of the anti-CD5 immunotoxin T101-ricin A chain (T101-RTA). Each course consisted of 8 bi-weekly infusions of T101-RTA (7 or 14 mg/m2). The immunotoxin was well tolerated in all cases with no major toxicities. Though saturation of circulating leukemic cell-associated target antigen was demonstrated by FACS analysis in all patients, no intact immunotoxin was detected in bone-marrow or lymph-node aspirates. Pharmacokinetic studies revealed rapid clearance of T101-RTA, with a half-life of 43 min. None of the patients developed detectable titers of antibody against either T101 murine antibody or ricin A chain. Clinical response was limited to a rapid and transient fall in WBC count lasting less than 24 hr, most likely secondary to the antibody portion of the conjugate. In vitro, fresh B-CLL cells were resistant to T101-RTA at concentrations up to 10(-8)M, while fresh malignant T-cells with a 10-fold increase in expression of CD5 antigen were sensitive. In the presence of the enhancing agent human serum albumin-monensin, fresh B-CLL cells were sensitive to T101-RTA, with an ID50 more than 2 logs below the maximal concentration of immunotoxin achieved in vivo. We conclude that T101-RTA is a potentially useful agent in the treatment of T-cell leukemias. In the presence of HSA-monensin, this spectrum of activity may be extended to B-CLL.

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.

Thy 1.2+ leukemia cells eradicated from in vitro leukemia-bone marrow cell mixtures by antibody-toxin conjugates.

A conjugate constituted by a monoclonal anti-Thy 1.2 antibody chemically coupled to the toxic subunit of ricin was synthesized. The conjugate was specifically cytotoxic for Thy 1.2+ (EL 4) but not for Thy 1.2- (BW5147) thymoma cells. In vitro treatment of bone marrow-leukemia cell mixtures (10:1 and 1:1 bone marrow to EL 4 cell ratios) with anti-Thy 1.2-ricin A chain totally eradicated EL 4 tumor cells, while the number of myeloid colony-forming units and pluripotent colony-forming units developed in vitro and in vivo was unaffected, which suggested that the treatment had no toxic side effects at least on the precursor cells examined.

Present potential of immunotoxins.

Immuno-a-toxins (I-a-T) are hybrid molecules designed for a more selective therapy of cancer, which combine an antibody preferentially directed against tumour cells and the A-chain of the toxin ricin. Although a majority of them are highly and selectively cytotoxic to their target cells in vitro, only some of them gave rise to a therapeutic effect in animal models. In vitro kinetics studies suggested the importance of a rapid mode of action for in vivo efficacy, which is not the property of all I-a-T. Ways of accelerating and potentiating such conjugates are proposed, such as the use of lysosomotropic amines like ammonium chloride. Whereas the in vivo use of these activators is still under research, their in vitro use gives rise to a 99.99% cytoreduction of leukemic cells and thus is directly applicable to clinical situations such as bone marrow transplantations.

[Immunotoxins]. / Les immunotoxines.

Immunotoxins are conjugates between antibodies especially directed against cancer cells and a subunit of a powerful toxin. We used the A-chain of ricin. These conjugates are specifically cytotoxic when used at very low concentrations in vitro and can destroy more than 99.99% of clonogenic cells. The efficacy of immunotoxins was also demonstrated in vivo but is inferior to its in vitro potency. For this reason the first use of immunotoxins in man can be the cleaning up of bone marrow from leukemic cells in the near future.

Immunotoxins: hybrid molecules of monoclonal antibodies and a toxin subunit specifically kill tumour cells.

Several attempts to attack tumours in experimental systems have been made using conjugates of chemotherapeutic agents or potent toxins with antibodies (immunotoxins). In vitro studies have been highly successful, showing target specificity of a high order in some cases. However, so far, such conjugates have been inadequate in vivo, probably for two main reasons. First, conventional heteroclonal antibodies are perhaps inappropriate, because purification by biochemical methods leaves a large amount of non-antibody gamma-globulins. The use of monoclonal antibodies may overcome this problem. Second, when whole toxins have been conjugated to antibodies there has been a strong residual nonspecific cytotoxicity due to the binding capacity of a subunit, the B-piece of the toxin. (Diphtheria toxin or ricin consist of two polypeptide subunits. The A-piece is responsible for inhibition of protein synthesis on ribosomes, and the B-piece binds to galactose residues on the cell membrane and facilitates the transmembrane passage of the A-piece.) In the present work the problem of nonspecific binding by the B-piece has been circumvented by using the A-piece only as the toxin component of immunotoxins; these immunotoxins are active both in vitro and in vivo.

Effect of early bursectomy on germinal centre and immunoglobulin production in chickens.

Chickens were bursectomized in ovo on days 18, 19 or 20 of incubation or within 6 h of hatch and immunized at day 28 after hatch by an intravenous injection of sheep red blood cells (SRBC). The immune response in the blood was measured by haemagglutination tests on the serum and by immunocyto-adherence tests for enumeration of rosette-forming cells with SRBC. Total serum 19S and 7S immunoglobulins were determined by radial immunodiffusion and the number of germinal centres per mm2 of fixed and stained spleen tissue sections was determined. In ovo bursectomy produced undetectably low serum levels of haemagglutinins, a reduction in SRBC rosette-forming cells with normal or increased 19S and reduced 7S immunoglobulins together with a complete disappearance (absence) of germinal centres in the spleen. The role of 19S antibody in the generation of germinal centres is discussed. The finding that birds which are equipped with serum 19S but no 7S Ig lack splenic germinal centres casts doubt on the hypothesis that the switch 19S leads to 7S Ig is necessarily an intrabursal event.