Southwestern In Vivo Cellular and Molecular Imaging Program

Project 1
Novel Strategies for Therapeutic Vascular Targeting Agents

Investigators and Areas of Expertise:

Philip Thorpe, Ph.D. (Drug delivery expert, who has developed, patented, and implemented tumor specific infarcts, as selective approach to therapy), Sophia Ran, Ph.D. (Tumor biologist with expertise in endothelial cell biology and vascular targeting). Drs. Thorpe and Ran are co-authors of patents describing phosphatidylserine as a specific marker of tumor vasculature and use of anti-phospholipid antibodies for tumor therapy and imaging. Dr. Thorpe currently has two RO1's based on development of tumor infarct techniques. Within the Program, Ralph Mason, Ph.D. (Tumor physiologist and MR expert) will coordinate small animal studies with respect to this Development Project, and Pamela Nurenberg, M. D. (Radiologist physician with expertise in MR and clinical trials) will coordinate progression to clinical trials. Anca Constantinescu, Ph.D. (Radio-chemist) will implement the labeling and ensure quality control for animal and patient drug development.

Hypothesis and Specific Aims:

Background and Significance:

A number of non-invasive techniques are available to assess tumor physiology, particular blood flow and oxygenation (Core 1). We will assess the ability to detect thrombosis in tumors resulting from the application of targeted coagulating agents (Coaguligands). Combined with pharmacokinetic studies to assess and screen new anti-vascular and coagulating agents, this will help to accelerate development and evaluation of novel formulations in mice with ultimate transfer of the imaging modalities to appropriate clinical trials.

Targeting the vasculature of solid tumors represents an innovative strategy for treatment of cancer. In contrast to traditional approaches that aim at destruction of tumor cells, the goal of vascular targeting is a selective killing of tumor blood vessels through formation of occlusive thrombi. This concept offers several advantages. Firstly, the tumor endothelial cells are directly accessible to intravenously administrated therapeutic agents, permitting rapid localization of a high percentage of the injected dose. Secondly, since each capillary provides oxygen and nutrients for thousands of tumor cells, even limited damage to the tumor vasculature could produce an avalanche of tumor cells death. Thirdly, the outgrowth of mutant endothelial cells lacking the target antigen is unlikely because they represent a normal, genetically stable cell population. Finally, since all tumor vessels share common morphological and biochemical properties, this strategy should be applicable to different tumor types.

Coagulation of tumor vasculature has been achieved by targeting human tissue factor (a major initiator of coagulation cascade to an experimentally induced marker (MHC class II) on tumor vasculature in mice. Once it forms a functional complex with factor VII/VIIa on the surface of negatively charged phospholipid membranes, it activates factors IX and X leading to the formation of thrombin, and ultimately, a blood clot. The recombinant, truncated form of tissue factor (tTF) lacks the cytosolic and transmembrane domains and has a significantly diminished capacity to initiate thrombosis. However, when a directing antibody delivers tTF to the endothelial cell surface, it regains thrombogenic activity. Although tTF is a human protein, making immune reactions in human patients less likely; it is fully functional as a coagulant in rodents, enabling a realistic evaluation of the construct in animal tumor models. To target TF to tumor vasculature, a bispecific antibody was constructed: one arm recognized the MHC class II antigen and the other was directed against a non-inhibitory epitope on tTF. The antibody was mixed with tTF prior to injection, thus creating a complex termed a "coaguligand". When mice bearing large tumors (0.8 cm in diameter) were injected with anti-class II-coaguligand, thrombosis of tumor vasculature followed by dramatic tumor regression was observed

VCAM-1 and E-selectin represent additional tumor endothelial markers, which are strongly up regulated on vessels in various human solid tumors.

Potential obstacles to successful targeting with anti-VCAM-1 or anti-E-selectin coaguligands are that these are classic markers of acute and chronic inflammation and their expression is strongly up-regulated in non-malignant disorders associated with inflammatory conditions, angiogenesis and vascular injury (e.g., wound healing, rheumatoid arthritis, ischemia-reperfusion injury and atherosclerosis. Such conditions might be present in elderly cancer patients, who are likely to be candidates for vascular targeting therapies, raising questions of safety. It should be noted, however, that inflamed, non-malignant endothelium might not externalize PS, thus preventing activity of a coaguligand bound to target-positive vessels. In fact, our preliminary experiments in vitro showed that TNF-stimulated HUVEC bind anti-human E-selectin coaguligand, but are unable to support it activity as long as PS is segregated to the cytosolic side of the membrane (Ran, unpublished results). Moreover, anti-VCAM-1 coaguligand injected into Apo E mice, which are prone to develop artherosclerotic lesions similar to human disease, did not cause mortality or any apparent toxicity. These studies addressing concerns of using coaguligands under non-malignant inflammatory condition are still in progress, but the initial results support the idea that these coaguligands can be tumor-selective and safe agents.

In addition to exploring VCAM-1 and E-selectin as tumor vascular targets, we recently discovered that PS itself is a specific marker of tumor endothelium, found in a variety of animal solid tumors. We have generated monoclonal anti-PS antibodies that home specifically to tumor, but not normal vessels. Anti-PS antibodies also localize to apoptotic and necrotic tumor cells and tumor stroma. Since tumor environment induces massive exposure of PS on different cellular components, anti-PS antibodies are expected to be highly sensitive reagents for tumor imaging and therapy.

Dr. Thorpe is a leading expert on tumor targeting and induction of specific thromboses. Hitherto, the work has relied on histological and gross anatomical evaluation to establish the efficacy of novel coaguligands. The collaboration will provide non-invasive imaging techniques, which should accelerate the discovery process for targets, efficacious conjugates and successful development of drugs. The extensive availability of small animal imaging devices, as well as clinical units will facilitate the development of this therapeutic approach from test tube to bedside at a single institution with close interaction of basic and imaging scientists together with physicians.

Assessment of tumor infaction (Figure).

References

1. Burrows, FJ, Thorpe PE. Vascular-targeting- a new approach to the therapy of solid tumors. Pharmacol. Ther. 1994, 64, 155-74.

2. Huang, X, Molema G, King S, Watkins L, Edington TS, Thorpe PE. Tumor infarction in mice by anti-body directed targeting of tissue factor to tumor vasculature. Science 1997, 275, 547-550.

3. Mason, RP, Constantinescu A, Ran S, Thorpe PE. Oxygenation in a human tumor xenograft: manipulation through respiratory challenge and anti-body directed infarction. Proc 27th ISOTT. Dartmouth, NH, 1999, 43.

4. Mason, RP, Constantinescu A, Ran S, Thorpe PE. Oxygenation in a human tumor xenograft: manipulation through respiratory challenge and anti-body directed infarction. In Dunn JF, Swartz HM, eds. Oxygen Transport to Tissue XXII. Proceedings of the 27th annual meeting of the International Society on Oxygen Transport to Tissue, 2001, in the press.

5. Ran, S, Gao B, Duffy S, Watkins L, Rote N, Thorpe PE. Infarction of solid Hodgkin's tumors in mice by antibody-directed targeting of tissue factor to tumor vasculature. Cancer Res. 1998, 58, 4646-53.

For Further Information Contact: RALPH  P. MASON, Ph.D.
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