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The internalizing anti-Le(y) monoclonal antibody (MAb) BR64 was conjugated to the anticancer drug doxorubicin (DOX) using an acid-labile hydrazone bond to the DOX and either a disulfide or thioether bond to the MAb. The resulting disulfide (BR64-SS-DOX) and thioether (BR64-S-DOX) conjugates were evaluated for stability, potency, and antigen-specific activity in both in vitro and in vivo model systems. The BR64-SS-DOX conjugates demonstrated antigen-specific activity both in vitro and when evaluated against antigen-expressing, DOX-sensitive human carcinoma xenografts. However, the stability and potency of disulfide conjugates were poor, and in vivo activity superior to unconjugated DOX was seen only at doses approaching the maximum tolerated dose. Furthermore, BR64-SS-DOX conjugates were not active against antigen-expressing, DOX-insensitive colon tumor xenografts. In contrast, the BR64-S-DOX conjugates demonstrated good stability both in vitro and in vivo. The increased stability of the BR64-S-DOX conjugates resulted in the delivery of more biologically active DOX to tumors with a concomitant increase in potency and efficacy over that which could be achieved with either unconjugated DOX or BR64-SS-DOX conjugates. Delivery of DOX by BR64-SS-DOX conjugates resulted in complete regressions and cures of both DOX-sensitive lung xenografts and DOX-intensitive colon tumor xenografts. These results demonstrate the importance of linker stability when delivering drugs such as DOX to carcinomas via internalizing antibodies and are likely to have direct relevance to the clinical utility of MAb-directed delivery.

CTLs specific for tumor antigens play a major role in the immunity against cancer. We have shown that class I-restricted CTLs can be induced by injecting soluble antigens mixed in an antigen formulation (AF) that consists of squalane, Tween 80, and Pluronic L121 (S. Raychaudhuri et al., Proc. Natl. Acad. Sci. USA, 89: 8308-8312, 1992). In this study, using ovalbumin and the ovalbumin-expressing transfectoma (EG7) as a tumor model system, we examined the in vivo antitumor effect of antigen-AF mixture. Vaccination of mice with ovalbumin in AF 2 or 3 days after EG7 tumor challenge showed significant inhibition of tumor growth compared to mice vaccinated with ovalbumin in alum or in saline. Depletion of CD8+ cells at the time of immunization completely abrogated the AF-induced tumor protection, indicating that CD8+ T cells are the major effectors in tumor protection in vivo. Depletion of CD4+ cells led to a marginal loss of tumor protection, which may be the result of inhibition of ovalbumin-specific CTL response due to the lack of T-helper activity. Our results demonstrate that AF can be used in subunit vaccines to stimulate CTLs and tumor regression in vivo.

The anticarcinoma antibody BR64 was conjugated to a doxorubicin derivative, doxorubicin 13-[3-(2-pyridyldithio)propionyl]hydrazone, and the resulting conjugates (BR64-DOX) were evaluated for activity and immunological specificity in vitro and in human tumor xenograft models. The BR64-DOX immunoconjugates retained immunoreactivity and cytotoxicity and demonstrated antigen-specific cytotoxicity in vitro. The potency of BR64-DOX immunoconjugates in vitro was related to the drug:monoclonal antibody mole ratio of the conjugates. The antitumor activity of BR64-DOX conjugates was consistently superior to the maximal activity obtained with the parent drug, doxorubicin (DOX), in established human lung and human breast carcinoma xenograft models. The superior antitumor activity of BR64-DOX conjugates was reflected both in tumor growth inhibition and in regressions and cures of established tumors following the administration of tolerated doses of BR64-DOX. The antitumor activity of BR64-DOX conjugates was not the result of synergism between monoclonal antibody BR64 and DOX, because mixtures consisting of monoclonal antibody and optimized DOX were not more active than an equivalent dose of DOX administered alone. The antitumor activity of BR64-DOX conjugates was antigen specific; equivalent doses of nonbinding isotype-matched conjugates were not active against established tumor xenografts.

Adriamycin (ADM) was chemically coupled to two monoclonal antibodies (MAb) expressed on human B-cell lymphomas. Immunoconjugates were prepared by linking to the MAb an ADM derivative, Adriamycin 13-[3-(2-pyridyldithio)propionyl]hydrazone (ADM-HZN), which releases ADM under mild acidic conditions (see preceding article). The (ADM-HZN) conjugates were tested for antitumor activity on two human B-lymphoma xenografts, Daudi and Ramos, which were growing as solid tumors in athymic mice. The conjugates, injected i.p., significantly inhibited tumor growth when antibody protein doses were greater than or equal to 500 mg/kg (approximately 10 mg/mouse). At these input antibody doses, (ADM-HZN) conjugates were more potent and had greater antitumor activity than free ADM given at an optimized dose and schedule. MAb-conjugated ADM was also tolerated to much higher levels than unconjugated drug. Antitumor activity was not obtained using mixtures of MAb plus free drug or with MAb-drug conjugates that did not bind to the tumor target cell. Thus, the antitumor activity of the immunoconjugate was directed by binding of the MAb portion of the conjugate to target tumor cells.

A novel method for linking Adriamycin (ADM) to monoclonal antibodies is described in which the 13-keto position of the anthracycline is used as the attachment site to the linker arm. A new ADM acylhydrazone derivative, Adriamycin 13-[3-(2-pyridyldithio)propionyl]hydrazone hydrochloride, which contains a pyridyl-protected disulfide, was synthesized and used for conjugation to monoclonal antibodies (MAbs) that were thiolated with N-succinimidyl 3-(pyridyldithiol)propionate or 2-iminothiolane. This resulted in formation of a linker between MAb and drug that contained a disulfide bond. Conjugation conditions were optimized to yield conjugates with high ADM:MAb molar ratios. The final immunoconjugate yields were found to decrease as the ADM:MAb molar ratio of the conjugates increased. Stability studies indicated that ADM was released from the immunoconjugates at mildly acidic pHs ranging from 4.5-6.5. Treatment of immunoconjugates with mild reducing agent dithiothreitol resulted in release of an acylhydrazone derivative of ADM. Flow-cytometric studies showed that the binding activity of various MAbs following conjugation to ADM was preserved at ADM:MAb molar ratios up to 10. Antibody-directed cytotoxicity was demonstrated under several assay conditions using combinations of antigen-positive and antigen-negative cells and binding and nonbinding immunoconjugates. In several experiments, ADM immunoconjugates were more potent than equivalent amounts of unconjugated ADM.