About the vaccine

Experimental results from the studies with inbred mice and their autologous tumors indicate that the inoculation of semi-allogeneic cell hybrids (derived from the fusion between autologous tumor cells and an allogeneic cell line) protects the animal host from a subsequent lethal challenge with unmodified autologous tumor cells. Later studies confirmed the validity of these observations by showing that the adoptive transfer of immunity induced by semi-allogeneic tumor cells required T lymphocytes and that the enhanced immunity was not due simply to an allogeneic effect. In fact, co-administration (injection) into experimental mice of allogeneic cells together with irradiated autologous tumor cells (i.e., without fusion) did not protect them from a subsequent challenge with autologous tumor cells, supporting the conclusion that, in order to achieve anti-tumor protection, the tumor-associated antigen and the alloantigen needed to be on the same cell, ass shown below in a model of patient-tailored, semi-allogeneic vaccine.

The vaccine mimics antigen-presenting cells (APC). Tumor-specific antigenic peptides expressed by the vaccine are processed for presentation by major histocompatibility complex (MHC) class I and II surface molecules. At the same time, vaccine-associated allo-antigens (transplantation antigens) constitute the only human cell line specifically engineered for the purpose of generating patient-tailored, semi-allogeneic vaccines (US Patent # 6,063,375 granted on May 16, 2000).

Fo1-12 cells have been certified for human use according to Food an Drug Administration (FDA) requirements and a master cell bank has been established for manufacturing cell vaccines for human studies. Patient-tailored cells hybrids can be irradiated and injected as customized immuno-therapeutics in patients affected by virtually any type of cancer. These vaccines are easy to produce and have already been tested in two pilot clinical studies.

In pre-clinical studies, we observed that lymphocytes from HLA-A2+ melanoma and HIV+ patients, stimulated *in vitro with HLA-A2-restricted antigenic peptides in the presence of irradiated semi-allogeneic cell hybrids killed target cells presenting the same peptide more efficiently than lymphocytes stimulated with antigenic peptide alone. Subsequently, we were allowed by the FDA, under BB-IND 6266, to conduct two Phase I studies in patients with disseminated melanoma and metastic adenocarcinoma. We determined that treatment of cancer patients with irradiated semi-allogeneic vaccines derived from the fusion of their tumor cells with Fo1-12 cells is associated with minimal or no toxicity, and can induce a specific anti-tumor immune response, measured by a positive delayed-type hypersensitivity (DTH) to irradiated autologous tumor cells injected intra-dermally. In one melanoma patient with stage III disease and treated with semi-allogeneic vaccine, we observed the reduction in size of two lymph nodes with melanoma cells and the patient is still free of disease almost ten years after vaccination. 00000 Taken together, these reports demonstrated that treatment with semi-allogeneic cancer vaccines can lead to the acquisition of a specific immune response against a variety of tumor types, including melanoma and colon cancer.

The present goal of SemiAlloGen is to validate the semi-allogeneic vaccine platform in animal models of “liquid tumors” such as lymphoma and leukemia, and of prostate cancer, because these malignancies often have an indolent course in humans and would be well suited for an immunotherapeutic approach that in our experience is safe, eminently feasible, effective, and can be used in combination with standard anti-cancer therapies.

In vivo animal studies with EL-4  T lymphoma cells: For animal studies involving EL-4 cells, ten week-old C57BL/6 male mice were injected intraperitoneally (i.p.) with 1x10⁶ RAG x EL-4 semi-allogeneic somatic cell hybrids in 0.5 mL phosphate buffered saline (PBS). As a control, age-matched mice were either mock-vaccinated i.p. with 0.5 mL PBS or vaccinated i.p. with 1x10⁶ RAG cells in 0.5 mL PBS. Three to four weeks after vaccination or mock-vaccination each mouse was challenged by i.p. injection with either 2x10⁴ or 3X10⁴ EL-4 lymphoma cells in 0.5 mL PBS. Mice were then monitored very closely for growth of i.p. tumors and sacrificed when the abdomen became clearly extended, generally within three weeks. Necropsy was performed on each animal and EL-4 abdominal tumors were carefully dissected and weighed out.

All mock-vaccinated mice challenged with EL-4 cells developed large tumors. The mean tumor size of mice injected i.p. with 2x10⁴ cells was 3.9 grams and the mean tumor size of mice injected i.p. with 2x10⁴ cells was 4.3 grams. In contrast, three of five vaccinated mice that were challenged i.p. with 2x10⁴ EL-4 cells showed no evidence of abdominal tumor growth at time of sacrifice and the mean tumor size in this group of mice was 0.2 grams. Similarly, one of five vaccinated mice that were challenged i.p. with 2x10⁴ EL-4 cells showed no evidence of abdominal tumor growth at time of sacrifice and the mean tumor size in this group of mice was 1.4 grams. Differences in tumor size between vaccinated and mock-vaccinated mice were statistically significant for both challenges (p-values were <0.0001 and 0.0017 for the 2x10⁴ and the 3x10⁴ EL-4 lymphoma cells challenge, respectively).

A second set of animal experiments was performed to investigate whether the anti-tumor protection was the result of an allogeneic effect. Three groups of mice were utilized: 1) mock-vaccinated with 0.5 mL PBS alone (Non-Imm); 2) vaccinated with 1x10⁶ RAG cells in 0.5 mL PBS (Allo); 3) vaccinated with 1x10⁶ RAG x EL-4 semi-allogeneic somatic cell hybrids in 0.5 mL PBS (Semi-Allo). After three weeks each mouse was challenged by i.p. injection with 2x10⁴ EL-4 lymphoma cells in 0.5 mL PBS and mock-vaccinated mice showed an extended abdomen because of i.p. tumor growth within three weeks from the day of challenge.

At the time of sacrifice, all mock-vaccinated mice challenged with EL-4 cells had developed large tumors and the mean tumor size was 5.1 grams; also, four of five mice vaccinated with allogeneic RAG cells and challenged with EL-4 cells had i.p. tumors (mean tumor size: 2.0 grams). In contrast, four of ten mice vaccinated with RAG x EL-4 semi-allogeneic somatic cell hybrids and subsequently challenged i.p. with 2x10⁴ EL-4 cells showed no evidence of abdominal tumor growth at time of sacrifice (mean tumor size in this group of mice was 0.7 grams). The results of these animal studies clearly indicate that although the i.p. injection of allogeneic cells (RAG) conferred some degree of anti-tumor protection compared to mock-vaccinated mice, semi-allogeneic vaccines were significantly better at inducing specific anti-tumor protection.

These experimental observations are entirely consistent with the results of previous studies showing that, although vaccination with allogeneic cells provides some degree of anti-tumor protection, semi-allogeneic vaccines are significantly more effective at conferring highly specific anti-tumor immunity.

Studies with focused microarrays: Total RNA was isolated from spleens of untreated mice, mock-vaccinated and vaccinated mice. The spleens of non-immune mice appeared to be smaller than those of immune mice. Spleen RNA from both experiments was subjected to focused microarray analyses of murine Th1-Th2-Th3 immune function (Superarray Bioscience Corp., cat.# OMM-034). All data from these experiments were normalized using minimum value background subtraction and expression of house-keeping genes, before being compared. Scatter plot comparisons included only genes that showed either up-regulation or down-regulation by 1.5 fold or more. The results obtained with both sets of samples were remarkably consistent. Compared to mock-vaccinated mice, the spleen of mice vaccinated with semi-allogeneic somatic cell hybrids expressed significantly higher levels of CD80, CD 86, C2ta (class II transactivator), Jund1 (Jun proto-oncogene related gene d1), Stat1 and Stat4 (signal transducer and activator of transcription 1 and 4). CD40, FASLG (Fas ligand), and HAVCR2 (hepatitis A virus cellular receptor 2). In contrast, the following genes appeared to be consistently under-expressed in the spleen of immune mice compared to non-immune mice: GATA3 (GATA binding protein 3), Gfi1 (growth factor independent 1), LAT (linker for activation of T cells), and SOCS3 (suppressor of cytokine signaling 3). Table I below summarizes these observations.

Genes that were specifically over-expressed in mice immunized with semi--allogeneic vaccines included: CD80 and CD86, both expressed by activated APC; Stat4, a member of the Stat family of transcription factors that is essential for mediating responses to IL-12 in lymphocytes and for regulating the differentiation of T helper cells; and CD40, a co-stimulator molecule found on the surface of B lymphocytes, dendritic cells, and follicular dendritic cells.

Mice immunized with either semi-allogeneic or allogeneic vaccines overexpressed: C2ta, enhancer of T-cell activation and proliferation and suppressor of Th2-tye cytokine expression; Stat1, activated by various ligands including interferon α (IFN - α) and IFN - γ; FASLG, critical in triggering apoptosis of some types of cells following interaction with FAS; and HAVCR2, over-expressed by activated CD4 Th1 cells and CD11b+ macrophages.

Non-immune mice over-expressed: LAT, involved in T-cell receptor (TCR)-initiated, T cell-specific signaling events and possibly associated with over expression of Th2-type cytokines; SOCS3, a suppressor of cytokine signaling; and Gfi1, a transcriptional repressor essential during myeloid differentiation (Gfi1-/- mice exhibit diminished monocyte-derived dendritic cells and disturbed cytokine production by macrophages in response to LPS).

Taken together the results of these animal studies demonstrate that semi-allogeneic vaccines specifically induce a Th-1-type immune response, exactly what is required to elicit an anti-tumor immunity that can lead to a beneficial clinical response.