Although TNF and FasL have potent cytotoxic activity against many types of tumor cells, the application of these death ligands to cancer therapy has been restricted by their severe toxicity to normal tissues. The discovery of Apo2L as a death ligand, with its wide tissue-mRNA distribution and its unique receptor system, suggested that this ligand might be more suitable than TNF or FasL for systemic cancer therapy.
Our initial goal toward preclinical assessment of Apo2L was to generate a recombinant molecule that forms stable, biologically active trimers, without fusion to foreign sequences. Previous work suggested that soluble versions of Apo2L require fusion to epitope tags and/or aggregation with antibodies directed to the tags for potent biological activity (7
). A recent report describes a biologically active fusion protein that contains Apo2L extracellular sequence (amino acids 95–281) linked to a trimerizing leucine zipper (LZ-TRAIL); this protein was expressed in Chinese hamster ovary cells (28
). Our data show that active, homotrimeric soluble Apo2L (amino acids 114–281) can be made in bacteria without fusion to heterologous sequences; this recombinant protein is likely to be less immunogenic in humans than Apo2L fusion proteins and thus, perhaps, more suitable for repeated dosing. Consistent with this notion, 7 daily injections of soluble human Apo2L in nonhuman primates did not induce a detectable antibody response by the end of study on day 9.
Given the severe systemic toxicity of TNF and FasL, our next goal was to test for any toxicity that might be associated with Apo2L injection. Much of TNF’s toxicity is caused by activation of the proinflammatory transcription factor NF-κB (5
). The NF-κB–inducing activity of Apo2L in cultured vascular endothelial cells was markedly weaker than that of TNF, suggesting that Apo2L may not have significant proinflammatory activity. Furthermore, Apo2L was not cytotoxic toward cultured normal cells from endothelial, epithelial, fibroblastic, smooth muscle, or astrocytic origin, supporting the notion that this ligand is not generally cytotoxic to nontransformed cells. Walczak et al. reported resistance of several normal cell types to LZ-TRAIL, with the exception of cultured astrocytes (28
We investigated also whether intravenous injection of Apo2L causes toxicity in nonhuman primates. Repeated systemic exposure of cynomolgus monkeys to significant doses of Apo2L did not cause detectable changes in an extensive set of clinical and histopathological tests, suggesting that the administration of this protein is remarkably safe. These results are in agreement with recent findings in mice (28
). Given that rodents are much less sensitive to the toxic effects of TNF than are humans (31
), the apparent safety of Apo2L in primates is a key advance toward the testing of this molecule in humans.
Our third goal was to assess the antitumor activity of Apo2L in models of cancer. A broad spectrum of cancer cell lines, including many that have p53 mutations, exhibited sensitivity in vitro to Apo2L. This finding is consistent with previous reports that Apo2L is cytotoxic toward cell lines from glioma (34
), melanoma (35
), and Kaposi’s sarcoma (36
), and against cells of certain hematological malignancies (37
). Our studies in colon tumor xenograft models indicate that Apo2L can inhibit establishment de novo of tumors. In mice bearing sizable subcutaneous tumors, Apo2L initiated tumor cell apoptosis, consistent with its apoptosis-inducing activity in vitro. Exposure to Apo2L caused tumor regression or suppressed tumor growth, which resulted in long-term inhibition of tumor progression. These results agree with the findings of Walczak et al., who studied mainly a xenograft model based on the MDA-MB-231 breast carcinoma cell line (28
We tested Apo2L’s ability to cooperate with established chemotherapeutic drugs to exert antitumor activity. Apo2L acted synergistically with 5-FU, and particularly with CPT-11, to cause either tumor regression or complete tumor remission. Perhaps activation of distinct apoptosis-signaling mechanisms by the death ligand and the DNA-damaging agents (3
) contributes to their cooperation. In addition, 5-FU can upregulate mRNA expression of DR5 (12
), which may augment apoptosis induction by Apo2L.
In conclusion, we have generated a potently active recombinant soluble human Apo2L molecule that is devoid of exogenous sequences. Repeated administration of this molecule to nonhuman primates appeared remarkably safe and nonimmunogenic. In vitro, soluble Apo2L exerted cytostatic or cytotoxic effects on a wide variety of tumor cell lines, but not on several types of normal cells. In vivo, soluble Apo2L showed substantial antitumor activity in xenograft models based on several colon carcinoma cell lines, and exhibited synergistic activity with 5-FU or CPT-11, causing marked regression or complete remission of tumors. Thus, Apo2L may prove to be a useful new tool to fight cancer cells, leaving normal cells unharmed.