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- Material and methods
Monoclonal antibody (mAb) 38C2 belongs to a group of catalytic antibodies that were generated by reactive immunization and contains a reactive lysine. 38C2 catalyzes aldol and retro-aldol reactions, using an enamine mechanism, and mechanistically mimics natural aldolase enzymes. In addition, mAb 38C2 can be redirected to target integrins αvβ3 and αvβ5 through the formation of a covalent bond between a β-diketone derivative of an arginine–glycine–aspartic acid (RGD) peptidomimetic and the reactive lysine residue in the antibody combining site to provide the chemically programmed mAb cp38C2. In this study, we investigated the potential of enhancing the activity of receptor-binding small molecule drug (SCS-873) through antibody conjugation. Using a M21 human melanoma xenograft model in nude mice, cp38C2 inhibited the growth of the tumor by ˜81%. The chemically programmed antibody was shown to be highly active at a low concentration while SCS-873 alone was ineffective even at dosages ˜1,000-fold higher than those used for the chemically programmed antibody. In vitro programming of the catalytic antibody was shown to be as effective as in vivo programming. In an experimental metastasis assay, treatment with mAb cp38C2 significantly prolonged overall survival of tumor-bearing severe combined immuno-deficient (SCID) mice when compared to treatment with unprogrammed mAb 38C2, SCS-873 alone or the integrin-specific monoclonal antibody LM609. In vitro, cp38C2 inhibited human and mouse endothelial and human melanoma cell adhesion, migration and invasion. Additionally, cp38C2 inhibited human and mouse endothelial cell proliferation and was active in complement-dependent cytotoxicity assays. These studies establish the potential of chemically programmed monoclonal antibodies as a novel and effective class of immunotherapeutics that combine the merits of traditional small molecule drug design with immunotherapy. © 2006 Wiley-Liss, Inc.
The discovery that tumor growth and metastasis are codependent on the formation of neovascularization1 revealed many potential protein targets for cancer treatment.2, 3, 4 The angiogenic process depends on proliferation of vascular endothelial cells, migration of tumor cells to the vascular endothelium followed by cell adhesion, and finally invasion of the endothelium.5 A family of highly conserved adhesion molecules, known as integrins, is central to the regulation of these processes. Integrins are heterodimeric transmembrane receptor complexes composed of noncovalently associated α and β chains, and recognize the arginine–glycine–aspartic acid (RGD) sequence present in their extracellular matrix (ECM) ligands.6 At present, 18 α and 8 β subunits are known; these form 24 different αβ heterodimers with different specificity for various ECM cell-adhesive proteins.7 Ligands for various integrins include fibronectin, collagen, laminin, von Willebrand factor, osteopontin, thrombospondin and vitronectin, all components of the ECM. Certain integrins can also bind to soluble ligands, such as fibrinogen or to other adhesion molecules on adjacent cells.7
Integrin αvβ3 is one of the most well-characterized integrin heterodimers and is one of the several heterodimers that have been implicated in tumor-induced angiogenesis.8, 9 While sparingly expressed in mature blood vessels, αvβ3 is significantly upregulated during angiogenesis in vivo.10 The expression of αvβ3 correlates with the aggressiveness of the disease in breast and cervical cancer as well as in malignant melanoma,11, 12, 13 and recent studies have suggested that αvβ3 may be useful as a diagnostic or prognostic indicator for some tumors.14 Integrin αvβ3 is expressed on some invasive tumors, including metastatic melanoma15, 16 and late-stage glioblastoma,17 thus contributing to their malignant phenotype. Integrin αvβ3 is particularly attractive as a therapeutic target because of its relatively limited cellular distribution. It is not generally expressed on epithelial cells, and minimally expressed on other cell types.18, 19, 20 Furthermore, αvβ3 antagonists, including both cyclic RGD peptides and monoclonal antibodies (mAbs), significantly inhibit cytokine-induced angiogenesis and the growth of solid tumor on the chick chorioallantoic membrane.21
Another αv integrin heterodimer, αvβ5, is more widely expressed on malignant tumor cells and is quite likely involved in VEGF-mediated angiogenesis.8, 22 It has been shown that αvβ3 and αvβ5 promote angiogenesis via distinct pathways: αvβ3 through bFGF and TNF-α, and αvβ5 through VEGF and TGF-α.8 Another study has shown that inhibition of Src kinase can block VEGF-induced, but not bFGF-induced, angiogenesis.23 These results strongly imply that bFGF and VEGF activate different angiogenic pathways that require αvβ3 and αvβ5, respectively. Therefore, αvβ3 and αvβ5 are attractive targets for antitumor therapies, and may provide therapeutic benefit in the treatment of solid tumors.
A number of integrin αvβ3 and αvβ5 antagonists are being developed for use as angiogenesis inhibitors.24 These include Vitaxin, a humanized form of mouse anti-human αvβ3 mAb LM609,25, 26, 27 a fully human anti-human αv mAb CNTO95,28 cyclic RGD peptides,29 and synthetic small molecule RGD mimetics.30, 31 We have developed a chemically programmed antibody approach that is unique, wherein small synthetic molecules and catalytic mAb form a reversible covalent bond that results in the reprogramming of the specificity of the antibody both in vitro and in vivo.32 The chemically programmed antibody (cp38C2) is prepared by covalently linking a small synthetic molecule, RGD mimetic SCS-873, which binds specifically to integrins αvβ3 and αvβ5, to aldolase mAb 38C2 (Fig. 1). The complex of the SCS-873 mimetic and mAb 38C2 was spontaneously formed in vivo, the half-life of the SCS-873 mimetic in the cp38C2 complex was increased in circulation by more than 2 orders of magnitude relative to the SCS-873 alone, and cp38C2 effectively reduced tumor growth in mouse models.
Figure 1. Chemical programming. (a) Using chemical adaptor SCS-873, catalytic mAb 38C2 can be chemically programmed through formation of a covalent bond between a β-diketone derivative of an RGD peptidomimetic and a reactive lysine residue in the antibody combining site. The resulting chemically programmed mAb 38C2 (cp38C2) manifests double targeting of cancer cells and tumor-induced endothelial cells via dual integrin αvβ3 and αvβ5 recognition on cell surfaces. (b) Structure of SCS-873. A 1,3-diketone derivative of an RGD peptidomimetic developed for high specificity and affinity to integrins αvβ3 and αvβ5. A long spacer between RGD peptidomimetic core and 1,3-diketone group was designed to allow recognition of both moieties at the same time.
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In the current study, we evaluated the effect of the preformed cp38C2 complex on the growth and metastases of M21 human melanoma in mouse models. Further, advancing our earlier study, the current study employs the preformed complex of small molecule and antibody which is, as we discuss, a more relevant format for the anticipated clinical studies. In addition, this mode of administration allows for a direct comparison of the therapeutic effect of small molecule antibody, small molecule complex, and conventional antibody, all of which is addressed in extensive detail in the current study.
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- Material and methods
In the current study, we have characterized the efficacy of chemically programmed mAb cp38C2 against human melanoma and human or mouse endothelial cells in vivo and in vitro. In vivo, growth of human melanoma tumors in nude mice was significantly reduced by treatment with cp38C2, using 2 distinct therapeutic regimens. The first therapeutic regimen studied involved the separate administration of SCS-873 and mAb 38C2 to examine the prospect of in vivo self-assembly of the cp38C2 complex, since it may be possible in future studies to deliver chemical programming agents into a pre-existing reservoir of 38C2-like antibody, prepared through immunization or gene delivery. The second therapeutic regimen studied involved in vitro preparation or programming of the cp38C2 complex that was then administered as a conventional mAb therapeutic. Delivery of the preformed complex is a substantially more controlled treatment than in vivo self-assembly, and would likely be the administration route of first choice if this drug were to enter clinical studies.
Self-assembly of cp38C2 in vivo provided an 81% mean reduction in tumor growth, while the preformed complex provided a 68% mean reduction in tumor growth relative to the PBS-treated control groups. Although the mean difference in tumor growth inhibition seen in the 2 therapeutic regimens was not statistically significant, the overall effect was achieved by injecting 300 μg of total cp38C2 (3.6 μg of SCS-873) preformed in vitro, when compared with the injection of 2.5 μg total 38C2 and of 3.5 mg total SCS-873 to form the complex in vivo. In addition, treatment with cp38C2 significantly improved the survival of mice-bearing melanoma metastases in lungs. After 200 days, all the survivors had lungs free of tumor metastases, thus highlighting the potential importance of targeting more than one protein for antitumor efficacy. In all in vivo studies, the chemically programmed antibody demonstrated a therapeutic effect that was far superior to that observed following treatment with the SCS-873 programming agent itself, even when SCS-873 was administered at an overwhelming ˜1,000-fold molar excess when compared with cp38C2, 3.5 mg of SCS-873 vs. 3.6 μg of SCS-873 found in the cp38C2 complex.
In vitro results demonstrated that cp38C2 has potent antiangiogenic properties in a variety of assays. Through dual αvβ3 and αvβ5 integrin targeting, cp38C2 was effective in the inhibition of adhesion of M21 melanoma cells and endothelial cells to the integrin ligand, vitronectin. cp38C2 also inhibited proliferation of human and mouse endothelial cells, but not M21 tumor cells. Previous studies demonstrated that anti-integrin mAb or RGD peptides reduced cell growth by inhibiting integrin ligation, detaching cells from the ECM and inducing an anchorage-dependent apoptosis, termed as anoikis.21, 42, 43 Our proliferation and adhesion data show that anchorage loss did not trigger any pro-apoptotic signals in human M21 melanoma cells. In addition, cp38C2 also inhibited migration and blocked invasion of M21 melanoma tumor and human and mouse endothelial cells. These studies are in agreement with previous observations of the abilities of antibody- and RGD-based peptide antagonists of integrin αvβ3/αvβ5 to inhibit angiogenesis, thus leading to regression of tumors in animal models.9, 28, 31, 44, 45
During angiogenesis, αvβ3 is upregulated on the surface of activated endothelial cells, enhancing migration, proliferation and invasion.9, 46 There is also evidence that αvβ5, one of the most ubiquitous members of the integrin family,22 may play a distinct yet important role in angiogenesis. Using two different models of angiogenesis, it was shown that mAb LM609 that binds to αvβ3 selectively inhibited bFGF-stimulated angiogenesis. In contrast, mAb P1F6, an antibody that blocks αvβ5, selectively inhibited angiogenesis stimulated by VEGF.8 This suggests that the 2 integrins participate in angiogenesis through distinct pathways. Trikha et al.28 found that antibody blockage of either αvβ3 by LM609 or αvβ5 by P1F6 reduced endothelial cell adhesion and migration in vitro, but that the 2 agents had to be combined to reach the level of inhibition achieved by the mAb CNTO95 that binds to all αv integrins. The ability to block tumor growth through multiple pathways may prove critical to effectively inhibiting angiogenesis and tumor growth in vivo. In the same study, CNTO95 displayed potent antiangiogenic effects in both rodent model and novel nonhuman primate model in cynomolgus monkeys.28 In the study by Rader et al.,32 we showed that cp38C2 could inhibit tumor growth through an antiangiogenic effect. In that study, αvβ3/αvβ5-negative human colon carcinoma cells SW1222 were injected into nude mice. Administration of cp38C2 significantly inhibited the growth of αvβ3/αvβ5-negative tumors by blocking the growth of mouse blood vessels. The hypothesis that cp38C2 can inhibit tumor growth indirectly, presumably via inhibition of angiogenesis, is also supported by the observation that cp38C2 has no effect on M21 cell proliferation, but inhibits endothelial cell proliferation in vitro.
Other synthetic dual αvβ3/αvβ5 integrin inhibitors have been created and have demonstrated antiangiogenic and antitumor efficacy in vivo. For example, the peptidomimetic compound S247 demonstrated antiangiogenic and antimetastatic effects against colon tumor xenografts in mice dosed with 70 mg/kg daily.45 Similar results were reported for the RGD mimetic SCH 221153, which inhibited human melanoma tumor growth and angiogenesis in mice, when dosed twice daily at 20 and 50 mg/kg.31 A cyclic peptide EMD 121974 is currently in clinical trials in patients with advanced solid tumors.47 These observations point to the potential importance of dual integrin antagonism in inhibiting tumor growth and angiogenesis. Moreover, it should be noted that the chemically programmed antibody approach demonstrated here allows for a once a week dosing schedule, making compliance in chronic therapy more likely.
The ability to inhibit integrin function on tumor cells directly, in addition to blocking integrins on angiogenic endothelium, may prove critical in halting the growth of some tumors. Many αv integrins have been suggested to play critical roles in the biology of tumors. For example, changes in expression of αvβ3 and αvβ5 have been associated with the progression, growth and dissemination of melanomas.12, 48 In the present study, cp38C2 was equally effective against tumor and mouse endothelial cells in all in vitro assays, except proliferation. In our mouse lung metastasis model, however, where tumor growth is most likely angiogenesis-independent, treatment with cp38C2 significantly prolonged the overall survival of mice, suggesting the direct inhibition of αvβ3/αvβ5-positive melanoma tumor growth or lung engraftment. In the same model, we observed an effect of SCS-873 alone on survival of the mice, although the effect was much less pronounced than that of cp38C2. Taken together with the fact that SCS-873 did not affect primary tumor growth in vivo, we theorize that the major antitumor effect was its ability to inhibit tumor growth directly.
In the mouse model assay of solid tumor growth, cp38C2 was able to bind and block integrins on both tumor cells and angiogenic endothelium, and caused a marked reduction in tumor size by the end of the study. In this model, dosing with SCS-873 alone, at ˜1,000-times the molar dosage of the compound present in cp38C2, was ineffective. The lower amount of SCS-873 antagonist required in the chemically programmed approach may also be significant with some compounds, where excessive dosing leads to the generation of toxic metabolites. Both in vivo and in vitro data collectively suggest that through combined blockade of integrins αvβ3 and αvβ5 on tumor and endothelial cells, cp38C2 may have multiple mechanisms of action that contribute to its observed antitumor efficacy in animal models. In in vitro experiments, cp38C2 exhibited very high CDC activity against M21 tumor cells, and also showed a fairly high level of ADCC activity when tested in combination with nude NK cells. Our demonstration that chemically programmed antibodies can functionally direct CDC as well as ADCC indicates that the full range of antibody Fc functions should be accessible, and that these 2 mechanisms may contribute to the in vivo antitumor activity of cp38C2 in mouse models.
Chemically programmed antibodies may have a number of advantages over conventional antibody-based approaches. One of the most important features of this methodology is that the antibody can be chemically programmed to target proteins other than αvβ3 and αvβ5. For example, using appropriate compounds linked to β-diketone, cp38C2 can be programmed to bind other receptors. Multiple programming should also be available for 38C2 by using a mixture of different compounds to provide tumor and tumor-activated endothelial cell targeting through different receptors. Therefore, this approach may offer significant features related to multiple specificities not found in conventional antibodies.
Another important feature of 38C2 is that it has been already humanized using a rational design strategy in preparation for human studies.49 Two antibody-based integrin antagonists are currently being developed for cancer treatment. The first is Vitaxin, the humanized form of the murine anti-human αvβ3 antibody LM609.26, 27 A dose-escalating phase I study in cancer patients demonstrated that it was safe.25 The other antibody is CNTO95, a fully human mAb that recognizes the family of αv integrins, developed at Medarex (Princeton, NJ) by immunizing mice that were transgenic for part of the human immunoglobulin repertoire.28 A phase I clinical trial of CNTO95 is ongoing in patients with solid tumors. Cilengitide, a peptide antagonist of αvβ3 and αvβ5, has also proven safe in phase I trials.47 These preclinical and clinical observations demonstrate the importance of targeting multiple integrin pathways to inhibit angiogenesis.
Finally, human/mouse crossreactivity of SCS-873 and cp38C2 is also very important. The vast majority of anti-human integrin specific antibodies were raised in mice and do not react with mouse integrins. This makes their preclinical evaluation in mouse models difficult or impossible. The in vivo antitumor effect of LM609 was evaluated by using a SCID mouse model of human angiogenesis.21 In this system, αvβ3-negative human melanoma cells were injected into full thickness human skin, grafted onto SCID mice. Because LM609 does not crossreact with mouse integrins, its antiangiogenic activity was attributed to blockade of human αvβ3 receptors in the vasculature of the human skin.21 One limitation of this study is that xenografted tumors would grow even in the absence of human vasculature, because the mouse vasculature can sustain tumor growth. Like LM609, chimeric anti-human αvβ3 and αIIbβ3 mAb 7E3 (Abciximab) and fully human anti-human αv integrins mAb CNTO95 do not crossreact with mouse integrins.
In conclusion, chemically programmed mAb cp38C2 designed to target αvβ3 and αvβ5 integrins is a unique and promising immunotherapeutic. cp38C2 inhibited tumor growth and angiogenesis in vitro and in vivo. A combination of cp38C2 and additional programming for other known tumor receptors may be promising as a strategy to inhibit the growth of primary tumors and metastases in patients.