hI-con1™ AND CANCER

Recent experiments conducted at Yale University demonstrated that the novel recombinant protein, hI-con1™, triggers immune cell-mediated killing of TF-expressing USPC and clear cell ovarian cancer cells. Because hI-con1 has the ability to trigger destruction of cells that highly express tissue factor, including certain aggressive and chemotherapy-refractory cancers AND the pathologic blood vessels (PBVs) essential for their survival, hI-con1 holds promise as a novel therapeutic agent for treating cancer patients and could be particularly effective in treatment of cancers that fail to respond adequately to chemotherapy.

Background

Because solid cancers often contain rapidly growing cells, they require a source of nutrition and a means of getting rid of waste products. Both of these functions are provided by PBV that form as the tumor grows. Yale University investigators Drs. Zhiwei Hu and Alan Garen, as well as others, have demonstrated that the cells lining these PBV express tissue factor (TF) (Hu, Z. et al, 1999; Contrino et. al. 2009) making them a target for hI-con1 . In addition, many aggressive tumor cells, including metastatic cancer cells, express surface TF (Contrino et. al. 2009; Cocco et. al. 2010).

Because PBV are leaky, molecules such as hI-con1 can pass through tumor blood vessels more easily than blood vessels in normal tissues. Thus, if the cells of the cancer being treated with hI-con1 express TF, then this agent could target both pathological blood vessel cells and cancer cells, leading to a two-pronged attack:

• hI-con1 could attack PBV, and in doing so, cause cell death in cancers by depriving the cells of the nutrients they need to grow and by eliminating their ability to dispose of their waste;

• hI-con1 could leak through PBV and carry out a direct attack on the cancer;

On the other hand, because the cells lining blood vessels in normal tissues of adults are contiguous and do not display TF, hI-con1 should neither destroy normal vasculature nor pass through it to reach the tissue surrounding normal blood vessels. So even though cells of healthy tissues might possess TF, they should be protected from destruction (See Figure 1).

Figure 1: THEORETICAL SELECTIVE ACTION OF hI-con1

Theoretical Selection Action of hI-con1

Early Work with I-con1

A number of studies have been carried out which demonstrate the remarkable promise of I-con1 as an anticancer agent. In the earliest of these studies, Drs. Hu and Garen tested the anti-cancer effects of I-con1 by gene therapy, which involved delivering the gene for I-con1 protein directly into human tumors growing in mice by injection with adenovirus containing the gene for I-con1. Once inside the tumor cells, the virus expresses the I-con1 protein, allowing it to elicit its anticancer effects. As the example in Illustration 2 demonstrates, after treatment with virus containing I-con1 , human melanomas lacked PBVs and were much smaller and paler than tumors injected with virus not containing the gene for I-con1 (See Figure 2).

FIGURE 2: EFFECTS OF ADENOVIRUS ENCODING I-con1
ON HUMAN MELANOMAS IN MICE

Effects of Adenovirus Encoding hI-con1 on Human Melanomas in Mice

In additional viral therapy studies (see References), Drs. Hu and Garen demonstrated that if the test animal possessed two tumors and the virus encoding I-con1 was injected into one of them, both tumors were successfully treated with no evidence of adverse side effects. The interpretation of these experiments is that cells in the injected tumor produce I-con1 protein and that the protein is released into the bloodstream. Normal blood vessels are unaffected, but once the I-con1 protein reaches PBV serving the second tumor, it can attach to and destroy the cells lining those blood vessels, thus causing destruction of the second tumor. In this way, I-con1 could be effective in combating metastases.

The dramatic antitumor response of I-con1 has been demonstrated by Drs. Hu and Garen against two different kinds of human cancers growing in mice, melanoma and prostate cancer, suggesting the general effectiveness of I-con1 in destroying solid tumors since they all require PBV.

In Vivo Studies Using I-con1 Protein

Once Iconic licensed hI-con1 from Yale, it set about to make sufficient quantities of the purified hI-con1 protein from mammalian cells. In a study sponsored by Iconic, this purified hI-con1 protein was tested directly as an anti-cancer therapeutic agent by Dr. Hu. Intravenous injections of this protein were found to cause a dramatic inhibition of growth of human prostate cancers in mice (See Figure 3).

FIGURE 3: EFFECTS OF I-con1 PROTEIN ON HUMAN PROSTATE CANCER IN MICE

Immunosuppressed (SCID) mice were injected with LnCap human prostate cancer cells. When the tumors were palpable the mice received two intravenous injections, on days 0 and 8, of saline (control) or the indicated amount of I-con1 protein. Tumor volumes were measured on the indicated days.

In Vitro Studies Using hI-con1 Protein

In a recent series of studies, Dr. Alessandro Santin, in the Department of Obstetrics, Gynecology & Reproductive Sciences at the Yale University School of Medicine evaluated the effect in vitro of hI-con1 protein prepared by Iconic on uterine serous papillary carcinoma (USPC) cells. These endometrial carcinomas are highly aggressive and show an inherent resistance to chemotherapy. Dr. Santin and his colleagues found that cells derived from a number of human USPC tumors expressed TF, suggesting that they would be appropriate targets for hI-con1 . They treated these cells with hI-con1 in the presence of immune cells (peripheral blood lymphocytes) and the immunostimulant Interleukin 2 in order to evaluate whether hI-con1 could trigger immune cell-mediated killing of the USPC cells. The results in Illustration 4 confirm that hI-con1 promotes immune killing of cells from six different USPC tumors, all of which were resistant to rituxan (i.e., control antibody). Notably, tumor cells expressing higher levels of TF on their surface were particularly susceptible to the destructive effect of hI-con1 (See Figure 4).

FIGURE 4: EFFECT OF hI-con1 PROTEIN ON USPC CELLS IN VITRO

Representative effect of low doses of interleukin-2 (IL-2) in combination with hI-con1 (30µg/ml) on ADCC against USPC-ARK-3 primary cell line (Effectors to target ratio 25:1). PBL from healthy donors were incubated for 4 hours in the presence of 100 IU/ml of IL-2. hI-con1 -mediated ADCC was significantly increased in the presence of low doses of IL-2. No significant increase in cytotoxicity was detected after 4-h IL-2 treatment in the absence of hI-con1 or in the presence of the rituximab isotype control mAb.