Core-concept preclinical studies

Both the core-concept and the COMBIG-concept have undergone several important preclinical studies.

In order to be able to patent its core-concept, Immunicum had to first verify its vaccine in preclinical studies. All studies (both in vitro and in animal models) were conducted with vaccine cells that were loaded with whole tumor cells.

The initial studies were performed in vitro with allogeneic DCs and set the foundation for Immunicum’s first patent application in 2002. Data was published in Scandinavian Journal of Immunology (62, 234–242) and shows that allogeneic DCs are capable of inducing an immune response in test tube. The patent covers the use of both allogeneic DCs and allogeneic monocytes as vaccine cells. Being that Immunicum had now verified the potential of using allogeneic DCs as vaccine cells in test tube, the next step was to verify the potential of using allogeneic monocytes as vaccine cells.

Immunicum’s Norwegian research partners and part owners, Professor Gustav Gaudernack and Professor Gunnar Kvalheim at Radiumhospitalet in Oslo performed in vitro-studies that verified the potential of using allogeneic monocytes for activation of the immune system against cancer. The results have been published in a PhD thesis entitled “Immuno-Gene Therapy in Cancer Patients: Pre-clinical and Clinical Development of Dendritic Cell-based Vaccines” by Li-Jun Mu.

Following the successful in vitro-studies, Immunicum moved on to perform studies in animal models. Rats were injected subcutaneously (under the skin) with a breast cancer cell line and were vaccinated in both prophylactic and therapeutic settings. Activated allogeneic monocytes or monocyte-derived allogeneic DCs were loaded with apoptotic tumor cells and subsequently used as vaccine cells. Prophylactic vaccinations reduced tumor take from 80% in non-vaccinated rats to 20% in vaccinated rats. This immunity was long-lasting since re-challenge of tumor-rejecting rats with tumor cells 6 weeks later failed to induce any tumor growth. In the therapeutic setting all rats developed tumors, but tumor growth was significantly reduced in rats given tumor-loaded and activted allogeneic monocytes (p<0.05) (see picture 2 on page 12) or monocyte-derived DCs (p<0.001) (see picture 1 on page 12). Data was published in the scientific journal Cancer Immunology and Immunotherapy (2008, 57 suppl1, p10.).

A proof-of-concept (PoC) study (VP10-14), a pre-toxicology study (VP10-17), and a larger toxicology study (VP10-43) have been performed with COMBIG-DCs. COMBIG-DCs of human origin cannot be used in animal models since species’ specific DCs are required. In order to mimic the human situation as closely as possible, vaccine cells derived from circulating monocytes were used. The rat was preferred over the mouse as test specie, since monocytes from peripheral mouse blood are hard to collect.

The manufacturing process intended for human studies was followed as closely as possible. Allogeneic rat monocytes were cultivated and activated using the same agents as for human cells. The donor animals were female Sprague-Dawley rat in study VP10-14, and female Male Brown Norway rats in study VP10-17. Female Male Brown Norway rats were also used for the toxicology study.

Proof-of-concept study

The main objective of Immunicum’s PoC-study (VP10-14) was to investigate if intratumoral injection of allogeneic DCs produced inhibitory effects on subcutaneous tumor growth on distant tumors in the rats. Each rat was injected with tumor cells subcutaneously on two different locations in order to allow one tumor (the “first tumor”) to later be injected with vaccine cells and let the second tumor (inoculated at a later time and at a distant site from the first tumor) function as a test on whether the induced anti-cancer immune response was systemic. Due to the vaccine's capability of generating a strong inflammatory response, as demonstrated by the intratumoral infiltration of different leukocyte subsets, the injected/vaccinated tumors initially increased in size. Due to the rapid growth of the first tumor, growth studies on the second, non-treated, tumor had to be conducted during a limited time period. However, the results showed an increased recruitment of NK-cells, macrophages/DCs and cytotoxic T-cells (CTLs) to the injected/vaccinated tumors with a following significant reduction in volume and weight of the second, distant, tumor. This indicates that COMBIG-vaccine cells are capable of mounting a systemic immune response against cancer sufficient to combat tumor cell growth.

Recently published data from others (Qu et al, J Immunol 2010 Sept 1;185(5):2895-902);Mito et al, Cancer Research, 2010,Sept 15;70(18):7093-101) are fully in line with our COMBIG data, thus indicating that intratumoral delivery of properly activated DCs may induce a strong anti-cancer immune response. Notably, intra-tumoral injection of DCs in a dog tumor model by Mito et al was found to elicit significant clinical responses in the majority of seven vaccinated dogs, including four complete responses and two partial responses against malignant tumors. Based on the traditional assumption that the injected DCs have to directly activate tumor-specific CTLs, the injected DCs in the dog cancer study were autologous.

However, contrary to previous assumptions, Bhardwaj and collegues, recently demonstrated that injected autologous DCs have an insignificant role in direct priming of antigen-specific CTLs in vivo, but instead function as an adjuvant for transferring antigens to endogenous DCs which are responsible for subsequent activation of T-cells (Yewdall et al, PLoS One,2010 Jun 16;5(6):e11144). These data from Bhardwaj and colleagues are thus fully in concert with Immunicum’s COMBIG-concept which is based on allogeneic DCs.

Toxicology studies

Before initiating its upcoming clinical trials, Immunicum performed two toxicity studies (VP10-17 and VP10-43) to investigate whether injection of activated allogeneic DCs into the kidney produced toxic reactions after repeated administrations into the kidneys of rats. In conclusion, it was possible to inject activated allogeneic DCs two times into the kidney, without any major sequels. The rats tolerated the surgery well and on most of the parameters investigated no difference between vehicle treated and vaccine cell treated animals were found. As expected, the kidneys injected directly with the vaccine cells did exhibit a slightly more severe inflammatory response than the vehicle injected kidneys. The lack of inflammatory responses in the right kidneys was of greater importance and suggests that no autoimmune reaction against kidney tissue was induced by the two injections during the time period of the study.