Monoclonal antibody L6-daunomycin conjugates constructed to release free drug at the lower pH of tumor tissue

Abstract

Measurements in cancer patients showed that the pH of tumors averages 0.8 unit lower than that of the surrounding normal tissues, confirming published work. Based on this, the anti-carcinoma monoclonal antibody (mAb) L6 was used to prepare immunoconjugates with daunomycin (DM), the drug being released at the acidic pH of the tumor. A direct linking of the aconitic derivative of DM (AcoDM) to mAb L6 led to conjugates that either had a low drug/antibody ratio (less than 5:1) or precipitated in vitro. In order to increase the drug load and avoid precipitation, several biopolymers were tested as spacers between the drug and the L6. To attach the polymer derivative to the mAb, the former was maleimidized and the mAb was thiolated. The AcoM/mAb ratio obtained was 20, and the mAb retained its highly specific binding to tumor cells. At pH 6 the AcoDM-L6 conjugate was toxic to cultured C-3347 carcinoma cells with an inhibitory concentration (IC50) of 5 micrograms/ml. The conjugate was less effective than the free DM with an IC50 of 0.2 micrograms/ml. The L6 alone was not toxic. At a tumor pH of 6.5, 15% of the AcoDM was released. The amount of released drug reached a maximum 24-48 h after exposure to the acidic medium. In vivo localization studies demonstrated a similar tumor uptake of the conjugate and mAb L6 with 18% of the injected dose/g tumor and a maximum uptake in tumor 48 h after injection. Our data indicate that it is possible to construct conjugates based on a pH-sensitive linker that can be targeted successfully to a tumor with release of a portion of the drug at the tumor site, but testing is needed to establish whether such release has anti-tumor activity in vivo and offers an advantage over treatment with unconjugated drug.

pH in human tumour xenografts: effect of intravenous administration of glucose

“…In view of the potential clinical use of pH-sensitive anticancer agents (Connors et al, 1964;Yatvin et al, 1980;Wike-Hooley et al, 1984;Ward & Jain, 1988;Lavie et al, 1991;Hiroaka & Hahn, 1989;Tannock & Rotin, 1989;Tietze et al, 1989;Jahde et al, 1989) but from mean values of 6.73 and 6.77, respectively, to 6.12 and 6.13 in pancreatic carcinoma STO and breast cancer SE. This hetereogeneity of the pH response, which in individual tumours could not be predicted from the H+ ion activity at normoglycemia, is due to cell type-specific differences in the rate of lactic acid production as well as tissue-specific differences in the transport of glucose and acidic metabolites.…”

“…The predictive value of the results presented here for the pH response to glucose of human tumours in situ depends, therefore, on the validity of xenografts as models of cellular metabolism as well as histomorphological parameters governing substrate and metabolite transport in tissues. Both in vitro and in vivo, human tumour cells, with rare exceptions, respond to elevated extracellular concentrations of glucose by increased lactic acid production (Naeslund & Swenson, 1953;Eagle et al, 1958;Aisenberg, 1961;Ashby, 1966;Thistlethwaite, 1987;Lavie et al, 1991). It is more difficult to predict whether acidic metabolites, once their production is stimulated, will indeed accumulate in human tumours in situ to a similar extent as observed in human tumour xenografts.…”

“…Moreover, H+ ions are potent modulators of various types of chemical reactions. Several classes of anti-cancer agents with different mechanisms of action, as diverse as alkylating drugs, pH-sensitive immunoconjugates or hyperthermia, have been identified whose cytotoxicity is sensitive to changes in microenvironmental pH (Yatvin et al, 1980;Lavie et al, 1991;Hiroaka & Hahn, 1989;Jahde et al, 1989). Acid-labile prodrugs of cytotoxic agents have been synthesised which are stable, and hence nontoxic at physiological pH Gluisenkamp et al, 1992).…”

“…Since H+ ions are potent modulators of various types of chemical reactions, tumour-selective acidification can be made use of, in a second step, to increase the target cell-directed cytotoxicity of anticancer agents, e.g. alkylating drugs, acid-labile prodrugs, pHsensitive immunoconjugates, hyperthermia, or acid-labile liposomes Tietze et al, 1989;Lavie et al 1991;Hiroaka & Hahn, 1989;Yatvin et al, 1980).…”

“…With the use of invasive as well as noninvasive techniques for measuring pH, the H+ ion activity in a wide variety of animal tumours was shown to be increased following stimulation of aerobic glycolysis (Wike-Hooley et al, 1984; Ward & Jain, 1988;Jahde et al, 1982b;Jahde et al, 1989). We are, however, only aware of few investigations analysing the H + ion activity in human tumours in situ following glucose-mediated stimulation of lactic acid production (Naeslund & Swenson, 1953;Ashby, 1966;Thistlethwaite, 1987;Lavie et al, 1991). In these studies, only 25 individual tumours, including ten malignant melanomas, five female genital cancers and ten tumours of varying histology were analysed.…”