Genovax has two projects in the development phase: here is a short outline.

GX 101

Treatment of systemic lupus erythematosus with a tolerogenic gene product constituted by a Consensus peptide-Fc immunoglobulin chimera.

Systemic lupus erythematosus (SLE) is characterized by the reactivity of lymphocytes against endogenous antigens with the subsequent production of autoantibodies.

Some of these autoantibodies are pathogenic inducing tissue damage by different mechanisms. It has been shown that autoreactive B lymphocytes (the cells producing autoantibodies) can process and present in a MHC class II context peptides derived from their own immunoglobulin idiotypes and that some of these idiotypes are able to activate autoreactive, disease-inducing T cells.

Thus, both nuclear antigens and specific autoantibody-derived V regions can function as autoantigens inducing and maintaining the autoimmune response in SLE.

An artificial peptide has been synthesized which encompasses sequences of variable region-derived peptides from different anti-dsDNA autoantibodies. This synthetic "consensus" peptide (pCons) has been shown to effectively prevent disease onset when injected in young mice and to arrest the disease in about 50% of mice with already developed disease.

This effect has been shown to be mediated by induction of regulatory T lymphocyte activation, including both CD4+CD25+ and CD8+CD28- T cells.

In order to increase peptide stability and its availability for antigen presenting cell capture, processing and presentation to regulatory T lymphocytes a molecular chimera has been produced assembling the pCons sequence with that of a human IgG Fc region (Ig-pCons).

The gene coding for such engineered construct has been inserted in a plasmid under the control of a CMV promoter (pCMV vector from Stratagene).

Control plasmids, coding for the Fc IgG region alone or for a negative gene construct (Ig-pNeg) constituted by an unrelated, non-tolerogenic peptide (pNeg) fused to the Fc IgG region, have been also produced.

The efficient expression of all peptides when transfected in COS-7 cells at both transcriptional and transduction levels was demostrated. The antigenic specificity of the Ig-pCons gene product compared with that of control gene products was also demonstrated by the specific reactivity of a pCons specific T cell line toward the Ig-pCons molecule.

In order to test the tolerogenic therapeutic effect in vivo of Ig-pCons gene product in a model of gene vaccination, the plasmid coding for Ig-pCons has been transfected into spleen B lymphocytes of lupus prone mice by spontaneous transgenesis.

Such a procedure allows B lymphocyte plasmid transfection without any cell manipulation taking advantage of the capacity of B cells to spontaneously endocytose and express external DNA. Hence, this procedure is optimal for gene vaccination protocols in humans as demonstrated by the absence of side effects observed in a clinical trial performed in prostate cancer patients after FDA approval (ClinicalTrials.gov identifier  NCT00061035) (Zanetti M. Protocol #0207-545: a phase I/II, escalating dose, open-label evaluation of safety, feasibility, and tolerability of transgenic lymphocyte immunization (TLI) vaccine subjects with histologically proven prostate adenocarcinoma. Hum Gene Ther. 2003 Feb 10;14(3):301-2).

Transfected cells were then i.v. injected in syngenic mice at a pre-nephritic age (20-22 weeks old). Control groups of mice were constituted by untreated mice or mice receiving syngenic B cells transfected with empty plasmid, plasmid coding for Fc Ig or for Ig-pNeg.

Treatment with cells transfected with Ig-pCons was associated with protection from renal disease and significantly increased survival. Ig-pCons treated mice also shown reduced circulating immunoglobulin levels and increased concentration of TGF-Β secreting regulatory CD8+CD28- T lymphocytes. Interestingly, these cells, purified from Ig-pCons treated mice and injected into pre-nephritic mice were able to efficiently protect them from nephritis onset.

The final goal of the project is to achieve a new biological treatment for human lupus. Ig-pCons gene construct appears to be a good candidate for this purpose since it is able to prevent disease onset in lupus-prone mice and pCons induces activation of human regulatory T lymphocytes. Aims of this project are:

1. To repeat the experiments of protection from SLE development in lupus-prone mice by gene vaccination using a modified form of the gene construct including the signal peptide in the molecule with the aim of increasing its secretion by transfected cells and its availability to other antigen presenting cells.
2. To produce and purify Ig-pCons gene product in order to obtain amounts of protein sufficient to perform the experiments of protection and survival evaluation directly immunizing lupus-prone mice with the protein.
3. To perform all the tests necessary to achieve the approval by AIFA and EMEA for clinical application in human SLE patients for both the plasmid and protein forms of Ig-pCons.

GX 301

Phase I/II clinical trial in patients affected with stage IV renal or prostate cancer with active immunization to telomerase-derived peptides.

Renal cell carcinoma

Renal cell carcinoma (RCC) represents the 3% of cancers in adults and its incidence increases at a rate of 1.5-5.9% per year. The mortality rate for this disease is increasing proportionally to its incidence. About 25-30% of patients has advanced, metastatic disease at diagnosis, and several patients without clinical evidence of metastasis at the time of diagnosis have already disseminated micrometastases.

This fact is responsible for the poor efficacy of actual therapeutic approaches and for the low survival rates (0 -13% of metastatic patients survive after 5 years from diagnosis).

Prostate cancer is a major medical problem. In Europe there are 2.6 millions of new cases every year. It constitutes the 11% of cancer cases in males, and it is responsible for the 9% of deaths due to cancer in Europe. Moreover, its incidence in male population is the double of that of lung cancer; prostate cancer represents 30% of new cancer cases in males with a elevated mortality rate.

At diagnosis 45% of patients have already an advanced disease with a survival rate < 50% at five years independently from the therapeutic option.

Telomerase is the reverse transcriptase of eukaryotic cells deputed to synthesis of the telomeric regions of chromosomes. Telomerase is present in embryonic but not in adult somatic cells with few exceptions constituted by germinal cells and actively proliferating cells. However, the rate of telomerase expression in these cells is so low that telomerase can not be recognize by telomerase-specific cytotoxic lymphocytes (CTL).

It is essential for cell immortalization: this explains why it is expressed by about 90% of tumours (independently on the histological type). For this reason, telomerase is now considered a potential "universal" TAA. This view is further supported by the finding that cytotoxic T lymphocytes (CTL) able to kill cancer cells through the specific recognition of peptides derived from the catalytic subunit of the human telomerase reverse transcriptase (hTERT) have been identified in the peripheral blood of both healthy subjects and cancer patients.

In particular, about 90% of cancer patients has telomerase-specific CTL. On these bases, clinical trials have been performed (and are ongoing) to analyze the safety and the immunological effects of immunization of cancer patients with single telomerase peptides.

These trials have demonstrated that telomerase immunization is a safe procedure: the vaccination has no detectable effect on SCID-repopulating and colony-forming activities in the bone marrow of cancer patients and may induce telomerase-specific immune responses in several cancer patients.

Adjuvants are substances able to provide a pro-inflammatory support to immunization protocols. Imiquimod is an imidazoquinoline that has intrinsic biological activities requested to an ideal adjuvant. It binds to TLR 7 and 8 on human dendritic cells (DC) inducing maturation of these cells toward antigen presentation by increasing expression of HLA and co-stimulatory molecules, by increasing expression of chemokine receptors such as CCR7 and inducing secretion of Th1 cytokines (IFNΓ, IL12, TNFΑ).

Hence, imiquimod induces activation of effector pathways of immune responses, an event necessary to effectively support immunization by tumor-derived peptides.

On the basis of the above considerations, we plan to activate a clinical trial entitled "Phase I/II clinical trial aimed to treat patients affected with stage IV renal or prostate cancer by active immunization to telomerase-derived peptides". The trial has already received the approval from the Ethics Committee of the San Martino University Hospital in Genova, and from the Competent Authority, which in Italy for Phase I trials is the National Institute of Health.