Conventional immunoconjugate is composed of cell-targeting mAb, ACA as payload and linker for the conjugation. The linker technology is an important part of the immunoconjugate strategy, and various linkers have been exploited to date. Among them, acid labile hydrazine linkage, thiol reduction of disulfide linkers, and lysosomal peptidase proteolysis of peptide linkers were favorably applied to ensure stability in blood.[6-8] For these types of linkers, cell-mediated endocytosis of antibody (antibody-internalization) and intracellular biochemical (enzymatic) processing of the immunoconjugate were indispensable to make the active ACA work. Our carbamate-bond based linker, which is used in a clinically approved anticancer prodrug CPT-11 to release an active component SN-38 within the tumor cell but not in blood circulation[29, 31, 32] can be classified into the conventional type mentioned above. Anti-CD 20 Immunoconjugate-PEG-SN-38 via carbamate-bond showed strong anti-tumor activity against malignant lymphoma, in which the distribution within the tumor tissue and antibody-internalization into tumor cells occur effectively. Although there were negative reports concerning the internalization of anti-CD20 mAbs, several authors, recently demonstrated internalization of anti-CD20 mAbs including rituximab in malignant lymphoma and leukemia cells.[33-36] These conflicting results might reflect the differences of cell types, mAbs or methodologies used.[35, 36] In contrast to malignant lymphoma, most human solid tumors possess abundant stroma that hinders the tissue-distribution of antibodies.[17-22] Cell–cell interaction between malignant epithelial cells also inhibits the penetration of mAb besides tumor-stroma.[18, 37] Moreover, heterogeneity of the cells in the tumor prevents development of immunoconjugate therapy based on cancer cell-specific antigen.[9-12] This led us to design an anti-stromal targeting immunoconjugate strategy using the tumor stroma both as a scaffold for binding and assembling immunoconjugates and as a relay base for a second attack by payload-ACA persistently released from the scaffold.[21, 22] In this drug design we selected a specially selected linker using ester-bond, which can release SN-38 in physiological condition (non-enzymatic hydrolysis) outside the cells. Both ester-bond and carbamate-bond were concerned to be cleaved by plasma carboxyl-esterase in the circulation after the injection. Cleavages of our conjugates were very low in mouse plasma, which has much higher levels of carboxyl-esterase activity than in human. Recently, we conducted clinical trials of NK012, a SN-38 incorporating polymeric micelle. In this formulation, SN-38 was conjugated to poly-Glu-chain via ester-bond. From these trials, we learned that human blood also contains high amounts of carboxylesterase. Nevertheless, NK012 proved good stability in human blood circulation.[38, 39]
Anti-collagen 4 immunoconjugate exiting from vessel can bind to the outer vessel wall and cells surrounding the stroma. Anti-collagen 4 immunoconjugate via carbamate-bond was not useful because it can scarcely release SN-38 outside the cells. On the other hand, anti-collagen 4 immunoconjugate via ester-bond can release SN-38 on the stroma. Low molecular weight agent SN-38 can penetrate through stroma into the cells. SN-38 released from the scaffold of adjacent collagen-4-positive vascular wall also attack tumor endothelium. Anti-collagen 4 mAb-SN-38 via ester-bond exerted more potent antitumor activity compared to anti-EpCAM mAb-SN-38 via carbamate-bond or ester-bond. It is too complicated to explain these data. However, we speculate that in addition to the insufficient attainability of anti-EpCAM mAb to tumor cells by stromal barrier and its low internalization into the cells, the retention of anti-EpCAM mAb within the tumor cell lesion is lower than that of anti-collagen 4 mAb within the tumor stroma. Consequently, the amount of SN-38 released inside of the cells from anti-EpCAM mAb-SN-38 via carbamate-bond or outside of the cells from anti-EpCAM mAb-SN-38 via ester-bond, may be less than that of SN-38 released outside of the cells from anti-collagen 4 mAb-SN-38 via ester-bond.
Although there had been a concern about the influence of anti-collagen 4 immunoconjugate on normal tissues having high level of collagen 4, we observed the safety of the immunoconjugate in several mouse models. We think that cancer stromal targeting (CAST) therapy is dependent on the fundamental concept that antibodies or immunoconjugates are generally too large to pass through the normal vessel walls, whereas they can extravasate from leaky tumor vessels to achieve tumor selective targeting by using EPR effect and bind to collagen 4, a plentiful component of the tumor stroma.[1-4, 40] We also speculate that such a passive targeting effect is one of the reasons why recent anti-EGFR antibody therapies show no serious adverse effects in spite of high level EGFR expression in normal tissues including intestinal mucosa, dermis and others.[9, 10, 41]
In general, human cancer is classified into three types according to the tissue component. One is hypervascular stroma-poor tumor such as malignant lymphoma, the second is hypovascular stroma-rich tumor such as pancreatic cancer and stomach cancer, and the third is intermediated tumor between the two types such as breast cancer and colorectal cancer. We thus propose the new therapeutic strategy of immunoconjugates to the feature of individual tumor as tissue stromal component: (i) cell-targeting mAb conjugated with ACAs via carbamate-bond for hypervascular and stroma-poor tumor; (ii) stroma-targeting mAb conjugated with ACAs via ester-bond for hypovascular and stroma-rich tumor; (iii) both cell-targeting immunoconjugate via carbamate-bond and stroma-tgargeting via ester-bond for intermediated type of tumor (Fig. 5).