Pamela A. Trail

Monoclonal antibody drug immunoconjugates for targeted treatment of cancer

Abstract. Monoclonal antibodies (mAb) directed to tumor-associated antigens (TAA) or antigens differentially expressed on the tumor vasculature have been covalently linked to drugs that have different mechanisms of action and various levels of potency. The use of these mAb immunoconjugates to selectively deliver drugs to tumors has the potential to both improve antitumor efficacy and reduce the systemic toxicity of therapy. Several immunoconjugates, particularly those that incorporate internalizing antibodies and tumor-selective linkers, have demonstrated impressive activity in preclinical models. Immunoconjugates that deliver doxorubicin, maytansine and calicheamicin are currently being evaluated in clinical trials. The feasibility of using immunoconjugates as cancer therapeutics has been clearly demonstrated. Gemtuzumab ozogamicin, a calicheamicin conjugate that targets CD33, has recently been approved by the Food and Drug Administration (FDA) for treatment of acute myelogenous leukemia (AML). This review concentrates on the properties of the tumor and the characteristics of the mAb, linker, and drugs that influence the efficacy, potency, and selectivity of immunconjugates selected for cancer treatment.

Characterization of kringle domains of angiostatin as antagonists of endothelial cell migration, an important process in angiogenesis.

Angiogenesis is a complex process that involves endothelial cell proliferation, migration, basement membrane degradation, and neovessel organization. Angiostatin, consisting of four homologous triple‐disulfide bridged kringle domains, has previously been shown to exhibit profound inhibition of endothelial cell proliferation in vitro and angiogenesis in vivo. It was also demonstrated that angiostatin could suppress the growth of a variety of tumors via the blocking of angiogenesis. The primary aim of our study was to characterize the kringle domains of angiostatin for their inhibitory activities of endothelial cell migration in order to elucidate their contributions to the anti‐angiogenic function of angiostatin. In this report, we demonstrate for the first time that the kringles of angiostatin play different roles in inhibiting endothelial cell migration, a crucial process in angiogenesis. Kringle 4, which has only marginal anti‐proliferative activity, is among the most potent fragments in inhibiting endothelial cell migration (IC50 of approximately 500 nM). In contrast, kringle 1–3, which is equivalent to angiostatin in inhibiting endothelial cell proliferation, manifests only a modest anti‐migratory effect. The combination of kringle 1–3 and kringle 4 results in an anti‐migratory activity comparable to that of angiostatin. When kringle 1 is removed from kringle 1–3, the resulting kringle 2–3 becomes more potent than kringle 1–3. This implies that kringle 1, although virtually ineffective in inhibiting endothelial cell migration, may influence the conformation of kringle 1–3 to alter its anti‐migratory activity. We also show that disruption of the kringle structure by reducing/alkylating agents markedly attenuates the anti‐migratory activity of angiostatin, demonstrating the significance of kringle conformation in maintaining the anti‐angiogenic activity of angiostatin. Our data suggest that different kringle domains may contribute to the overall anti‐angiogenic function of angiostatin by their distinct anti‐migratory activities.—Ji, W. R., Castellino, F. J., Chang, Y., DeFord, M. E., Gray, H., Villarreal, X., Kondri, M. E., Marti, D. N., Llinás, M., Schaller, J., Kramer, R. A., and Trail, P. A. Characterization of kringle domains of angiostatin as antagonists of endothelial cell migration, an important process in angiogenesis. FASEB J. 12, 1731–1738 (1998)

Monoclonal antibody drug conjugates in the treatment of cancer.

Monoclonal antibodies directed to tumor-associated antigens have been chemically conjugated to drugs with different mechanisms of action and different levels of potency. Monoclonal-antibody-directed drug delivery has the potential to both improve efficacy and reduce systemic toxicity. Several immunoconjugates have demonstrated impressive antigen-specific antitumor activity in preclinical models. Phase I trials of a calicheamicin immunoconjugate for treatment of acute myeloid leukemia and a doxorubicin immunoconjugate for treatment of carcinoma have recently been completed.

Dual IGF-I/II-neutralizing antibody MEDI-573 potently inhibits IGF signaling and tumor growth.

Insulin-like growth factors (IGF), IGF-I and IGF-II, are small polypeptides involved in regulating cell proliferation, survival, differentiation, and transformation. IGF activities are mediated through binding and activation of IGF-1R or insulin receptor isoform A (IR-A). The role of the IGF-1R pathway in promoting tumor growth and survival is well documented. Overexpression of IGF-II and IR-A is reported in multiple types of cancer and is proposed as a potential mechanism for cancer cells to develop resistance to IGF-1R-targeting therapy. MEDI-573 is a fully human antibody that neutralizes both IGF-I and IGF-II and inhibits IGF signaling through both the IGF-1R and IR-A pathways. Here, we show that MEDI-573 blocks the binding of IGF-I and IGF-II to IGF-1R or IR-A, leading to the inhibition of IGF-induced signaling pathways and cell proliferation. MEDI-573 significantly inhibited the in vivo growth of IGF-I- or IGF-II-driven tumors. Pharmacodynamic analysis demonstrated inhibition of IGF-1R phosphorylation in tumors in mice dosed with MEDI-573, indicating that the antitumor activity is mediated via inhibition of IGF-1R signaling pathways. Finally, MEDI-573 significantly decreased (18)F-fluorodeoxyglucose ((18)F-FDG) uptake in IGF-driven tumor models, highlighting the potential utility of (18)F-FDG-PET as a noninvasive pharmacodynamic readout for evaluating the use of MEDI-573 in the clinic. Taken together, these results demonstrate that the inhibition of IGF-I and IGF-II ligands by MEDI-573 results in potent antitumor activity and offers an effective approach to selectively target both the IGF-1R and IR-A signaling pathways.

Constitutive Expression of Functional 4-1BB (CD137) Ligand on Carcinoma Cells

Members of the TNF superfamily, including Fas, Fas ligand, and CD40, have been shown to be expressed on tumor cells. In the studies described in this work, we report that another family member, the ligand for 4-1BB (CD137), is expressed on various human carcinoma cell lines, on cells of solid tumors derived from these cell lines, and cells obtained from human tumors. Expression of 4-1BB ligand (4-1BBL) mRNA was detected by both RT-PCR and Northern blot analysis, and expression of 4-1BBL protein was detected by Western blot analysis of whole cell lysates and by FACS analysis of tumor cells and cell lines. Incubation of tumor cells with a 4-1BB-Ig fusion protein led to the production of IL-8 by the cells, demonstrating that the 4-1BBL is functionally active and signals back into the tumor cells. Furthermore, 4-1BBL expressed on the carcinoma cells functioned as a costimulatory molecule for the production of cytokines (most notably IFN-γ) in cocultures of T cells and tumor cells. These findings suggest that 4-1BBL expressed on carcinoma cells may significantly influence the outcome of a T cell-tumor cell interaction.

Doxorubicin immunoconjugates containing bivalent, lysosomally-cleavable dipeptide linkages

Bivalent doxorubicin (DOX)-dipeptides (16a-c) were prepared and conjugated to the monoclonal antibody BR96. The dipeptides are cleaved by lysosomal proteases following internalization of the resulting immunoconjugates. Conjugate 18b demonstrated antigen-specific in vitro tumor cell killing activity (IC(50)=0.2 microM) that was equipotent to DOX with a near doubling of drug molecules/MAb. Size exclusion chromatography showed 18b to be a noncovalent dimer that was formed immediately upon conjugation.

Enhanced delivery improves the efficacy of a tumor-specific doxorubicin immunoconjugate in a human brain tumor xenograft model

OBJECTIVE To evaluate dose intensification with osmotic blood-brain barrier disruption (BBBD) and the potential use of drug targeting with monoclonal antibody (MAb) BR96 conjugated to doxorubicin (BR96-DOX, now called SGN15) for treatment of intracerebral and subcutaneous human LX-1 small cell lung carcinoma xenografts in rats. METHODS LX-1 tumors with high, low, or heterogeneous levels of the Lewis(y) antigen for BR96 were evaluated. Rats were treated with intracarotid or intravenous BR96-DOX, with or without osmotic BBBD. RESULTS Both BR96-DOX and MAb BR96 treatment resulted in significant regression of subcutaneous tumors, in contrast to control groups including doxorubicin alone, saline, or nonbinding doxorubicin immunoconjugate. BR96-DOX delivered with BBBD to brain tumors with low antigen expression resulted in significantly (P < 0.001) increased rat survival time compared with animals that received intravenous or intra-arterial BR96-DOX. CONCLUSION The combination of an effective drug such as doxorubicin with a MAb to facilitate tumor-selective localization and osmotic BBBD to increase tumor delivery may have practical application in the clinic, because an increased delivery of drug to tumor can be obtained without increasing the dose of systemic drug.

Synthesis of an Immunoconjugate of Camptothecin

The first immunoconjugate of camptothecin has been synthesized wherein the drug is attached to the tumor-recognizing antibody BR96 via a Cathepsin B cleavable linker. Endocytosis of the immunoconjugate upon binding to the tumor cell followed by enzymatic cleavage of the linker inside the endosome ensures tumor-specific release of the drug. In this way, it is hoped that the dose-limiting side effects associated with camptothecin can be eliminated while the antitumor activity is preserved.

Selective tumor sensitization to taxanes with the MAB-drug conjugate CBR96-doxorubicin

The chimeric monoclonal antibody cBR96 conjugated to doxorubicin (cBR96‐Dox) is selectively internalized by a wide variety of human carcinomas expressing an extended form of Lewis Y antigen (Ley). Endocytosis is followed by cleavage and release of free doxorubicin from the endocytic vesicles and subsequent cytotoxicity. Combination studies with standard anti‐cancer agents, undertaken to further increase the potency of this targeted therapy, identified significant synergistic anti‐tumor activity of cBR96‐Dox and either of the taxanes paclitaxel or docetaxel. Treatment with cBR96‐Dox 24 hr prior to paclitaxel resulted in a steady increase in the percentage of G2 tumor cells and corresponding increase in sensitivity to taxanes. Cell cycle analysis indicated the cBR96‐delivered doxorubicin was most effective against S‐phase cells, yet cells exposed to even subtoxic levels progressed to and arrested in G2, at a point of high sensitivity to the anti‐tubulin agent paclitaxel. The synergy obtained by staged combination of cBR96‐Dox and paclitaxel in vitro was reflected in significant anti‐tumor efficacy in vivo against xenograft models of human lung and breast tumors that could not be achieved by either agent alone. The staged combination elicited significant or complete regressions of established human Ley‐positive tumor xenografts using significantly reduced drug levels. Taken together, these data demonstrate a mechanistic approach to the selective elimination of Ley‐positive tumors by using targeted doxorubicin followed by taxane treatment.

Synthesis and antitumor activity of the immunoconjugate BR96-Dox

Abstract BR96-Dox is an immunoconjugate (IC) in which doxorubicin (8 equivalents) is linked via an acid-labile hydrazone to the chimeric MAb BR96. It binds to a modified Le y Ag on tumor cells, which then internalize it via endocytosis into lysosomes. There, the acidic milieu hydrolyzes the hydrazone link, releasing free Dox. In vivo, it is more active and less toxic than untargeted Dox, producing complete remissions and many cures of subcutaneous human breast, lung and colon tumors, as well as disseminated lung tumors. In vivo, only BR96 + and not BR96 − tumors respond, and ICs with nonbinding Abs are inactive.