Improved pharmacokinetics and tumor localization of immunotoxins constructed with the Mr 30,000 form of ricin A chain.

The two naturally occurring forms of ricin A chain, Mr 33,000 and Mr 30,000 (RTA33 and RTA30) have been purified, and their chemical compositions, toxicities, and tissue distributions have been determined. As reported previously, the in vitro and in vivo toxicities of RTA30 and RTA33 are similar. However, RTA30, which contains less carbohydrate with a lower mannose content than RTA33, accumulated less in the liver than did RTA33. Monoconjugate immunotoxins (i.e., containing one RTA per monoclonal antibody molecule) were constructed between RTA30 or RTA33 and the antitumor monoclonal antibody 791T/36, which recognizes a Mr 72,000 antigen on osteosarcoma and colon carcinoma cells. The two immunotoxins had similar cytoxicities in vitro but differed substantially in their pharmacokinetics and tissue distributions in vivo in nude mice bearing C170 human colorectal carcinoma xenografts. The immunotoxin derived from RTA30 (IT30) accumulated less in the liver than the immunotoxin derived from RTA33 (IT33) and cleared more slowly from the blood; the alpha and beta half-lives for IT30 and IT33 were 0.50 and 20.5 versus 0.17 and 14.6 h, respectively. As a probable consequence, IT30 accumulated to approximately 3-fold higher levels in the C170 xenografts than IT33. The reduced clearance of IT30 by the reticuloendothelial system thus resulted in prolonged survival in the blood and enhanced tumor localization relative to IT33.

The use of surface tension measurements in the design of antibody-based product formulations.

Many proteins in aqueous solution are susceptible to interfacial denaturation and precipitation during mechanical agitation. With the large number of protein parenteral products currently in research or clinical testing, it is important not only to understand this denaturation process but also to develop effective methods for stabilizing the products. Surfactants, such as Polysorbate 80 or sodium dodecyl sulfate (SDS), are frequently used to stabilize parenteral products. While it is a commonly accepted technique, little has been published about the precipitation and stabilization processes in general. We describe the stabilization of antibody products in solution by preferential adsorption of a water-soluble, non-ionic surfactant at the air-liquid interface. Data are presented from antibody and immunotoxin solution shake studies and surface tension measurements to support the utility of surface tension measurements in formulation design.

Specific inhibition of in vitro lymphocyte transformation by an anti-pan T cell (gp67) ricin A chain immunotoxin.

The toxin A chain of ricin has been conjugated by a disulfide bond to a murine monoclonal antibody that recognizes the gp67kD antigen present on 95% of peripheral T lymphocytes. The immunotoxin retains both functions of its component parts: it binds to human peripheral blood lymphocytes, and it inhibits protein synthesis in a cellfree reticulocyte system. The immunotoxin has been evaluated for its ability to inhibit in vitro T lymphocyte transformation. In the presence of 20 mM NH4Cl, the immunotoxin decreases lymphocyte proliferation in response to phytohemagglutinin to less than 8% of untreated controls. The proliferative response in mixed lymphocyte culture and the development of allocytotoxic T cells is also dramatically inhibited by this immunotoxin. Monoclonal antibody alone does not inhibit these responses. Specificity of the immunotoxin has been established: the effect of the immunotoxin can be blocked by unconjugated monoclonal antibody, but not by a control monoclonal antibody that recognizes another T lymphocyte differentiation antigen or by a control monoclonal antibody that does not recognize human peripheral blood leukocytes. Treatment of human bone marrow cells with the immunotoxin preserves hematopoietic progenitor cells, as measured by granulocyte-macrophage, erythroid, and multipotential hematopoietic progenitor cell assays. These results indicate that an anti-pan T lymphocyte-ricin A chain immunotoxin is an effective agent against immunocompetent T lymphocytes in vitro, and may be an effective agent for use in clinical bone marrow transplantation.

Purification of monoclonal antibodies from mouse ascites eliminates contaminating infectious mouse type C viruses and nucleic acids.

We examined the fate of infectious mouse type C viruses (MuLV) and nucleic acids during the purification of monoclonal antibodies (MoAbs) from mouse ascites. Hybridoma cell lines and their cell fluids can contain infectious ecotropic and xenotropic MuLV whose presence in ascitic fluid may be masked by the presence of anti-viral factors, particularly the anti-xenotropic virus neutralizing factor. They can also contain DNA and RNA. The specific techniques we used for the purification of MoAbs from the ascitic fluid completely eliminated all detectable infectious MuLV, anti-viral factors, and nucleic acids, even when these agents were added in quantities far in excess of those normally expected. Viral proteins, potentially contaminating the MoAb preparations, were also substantially reduced by our procedures. Because infectious MuLV or nucleic acids may have adverse effects on human subjects, attention should be given to each MoAb purification process to insure their effective removal.

Inhibition of human lymphoma DNA-dependent RNA polymerase activity by 6-mercaptopurine ribonucleoside triphosphate.

6-Mercaptopurine was found to inhibit the growth of cultured human lymphoma P3HR-1 cells and the incorporation of [3H]-uridine into trichloroacetic acid-precipitable materials of the cells. One of the derivatives of 6-mercaptopurine, 6-mercaptopurine ribonucleoside triphosphate (6-thio-ITP), was found to inhibit in vitro RNA synthesis (both engaged and free enzyme activities) of the isolated nuclei from P3HR-1 cells. The alpha-amanitin-resistant RNA polymerase (polymerase I) and alpha-amanitin-sensitive RNA polymerase (polymerase II) of the cells were isolated and partially purified by either diethylaminoethyl cellulose or diethylaminoethyl Sephadex column chromatography, followed by DNA-cellulose affinity chromatography. It was found that these partially purified enzymes were also sensitive to 6-thio-ITP inhibition. Kinetic studies showed that the inhibition of RNA polymerase activities by 6-thio-ITP could be reversed by increasing concentrations of guanosine 5'-triphosphate in the reaction mixture, indicating that 6-thio-ITP may act as a competitive inhibitor of the enzymes by competing with guanosine 5'-triphosphate for its enzyme-binding site. These data suggest that inhibition of RNA transcription by 6-thio-ITP may be considered as one of the mechanisms of the cytotoxic action of 6-mercaptopurine in human tumor cells.

Effect of N-trifluoroacetyladriamycin-14-valerate on [3H]thymidine uptake and DNA synthesis of human lymphoma cells.

The effects of N-trifluoroacetyladriamycin-14-valerate on the uptake of [3H]thymidine and its incorporation into DNA of human P3HR-1 lymphoma cells were studied. In the absence of the drug, at 0 degrees C, [3H]thymidine was transported into the cells but not incorporated into DNA, as determined by both the trichloroacetic acid-soluble and -precipitable counts obtained with the cells. At 37 degrees C, [3H]thymidine was readily transported into the cells and incorporated into DNA. In the presence of the drug, both [3H]thymidine uptake (as shown by acid-soluble counts) and the amount of its incorporation into acid-precipitable materials were markedly reduced. However, the uptake of [3H]thymidine at 0 degrees C was found to be equally sensitive to drug inhibition as at 37 degrees C. The incorporation at 37 degrees C of [3H]thymidine into acid-precipitable materials of the cells, which had been prelabeled at 0 degrees C with [3H]thymidine, was found to be insensitive to inhibition by the drug. The in vitro activities of DNA polymerases alpha and beta purified from human P3HR-1 cells were also found not to be susceptible to inhibition. Nuclei purified from cells pretreated with the drug continued to synthesize DNA. The cytofluorograms of the cells treated with the drug indicated that the treated cells accumulated at the G2/M phase, whereas the S phase of the cells was not arrested. These results suggest that N-trifluoroacetyladriamycin-14-valerate inhibits [3H]thymidine uptake but not cellular DNA synthesis in human P3HR-1 lymphoma cells.