Preliminary safety assessment of CIGB-210, an investigational peptide for HIV infection.

Current human immunodeficiency virus treatments need to be periodically administered lifelong. In this study we assess the effect of repeated doses of an anti-HIV peptide drug candidate in C57BL6 strain. Two schemes of up to 15 administrations and one of 30, daily dosing for 5 days per week, all by the subcutaneous route were evaluated. Different dose concentrations of the peptide were assayed. CIGB-210 treated animals showed no symptoms or abnormal behavior as compared with placebo. All the animals gained weight during the study. Macroscopic evaluation showed no alterations in any of the organs studied. Microscopic analysis of the tissues did not show morphological changes in thymus, stomach, small and large intestines, kidney, brain, or cerebellum. The proliferative response of splenocytes and their capacity to secrete gamma interferon were not compromised by the repeated administration of CIGB-210. There were not statistically significant differences for any of the parameters evaluated during the study among treated and non-treated groups. We can conclude that CIGB-210 is well tolerated in C57BL6 mice in the dose concentration range explored and merits subsequent toxicological studies.

Safety and Therapeutic Profile of a GnRH-Based Vaccine Candidate Directed to Prostate Cancer. A 10-Year Follow-Up of Patients Vaccinated With Heberprovac.

Heberprovac is a GnRH based vaccine candidate containing 2.4 mg of the GnRHm1-TT peptide as the main active principle; 245 µg of the very small size proteoliposomes adjuvant (VSSP); and 350 µL of Montanide ISA 51 VG oil adjuvant. The aim of this study was to assess the safety and tolerance of the Heberprovac in advanced prostate cancer patients as well as its capacity to induce anti-GnRH antibodies, the subsequent effects on serum levels of testosterone and PSA and the patient overall survival. The study included eight patients with histologically-proven advanced prostate cancer with indication for hormonal therapy, who received seven intramuscular immunizations with Heberprovac within 18 weeks. Anti-GnRH antibody titers, testosterone and PSA levels, as well as clinical parameters were recorded and evaluated. The vaccine was well tolerated. Significant reductions in serum levels of testosterone and PSA were seen after four immunizations. Castrate levels of testosterone were observed in all patients at the end of the immunization schedule, which remained at the lowest level for at least 20 months. In a 10-year follow-up three out of six patients who completed the entire trial survived. In contrast only one out eight patients survived in the same period in a matched randomly selected group receiving standard anti-hormonal treatment. Heberprovac vaccination showed a good security profile, as well as immunological, biochemical and, most importantly, clinical benefit. The vaccinated group displayed survival advantage compared with the reference group that received standard treatment. These results warrant further clinical trials with Heberprovac involving a larger cohort.

Vaccine CIGB 247 is potentially safe for use as a novel therapeutic vaccine against cancer in Chlorocebus aethiops monkeys.

CIGB 247 is a novel cancer therapeutic vaccine based on human vascular endothelial growth factor (VEGF) variant molecule as antigen, in combination with a bacterial adjuvant. This vaccine candidate has previously demonstrated efficacy and safety in mice, rats, rabbits and non-human primates. In the present study we evaluated the effects on the clinical, hematological and biochemical parameters of CIGB 247 vaccine in Chlorocebus aethiops monkeys. Three groups of monkeys were immunized with three doses of vaccine formulation to measure physiological values of clinical, hematological and serum biochemical parameters. Monkeys' body weight and temperature were kept stable and close to standard values throughout the study. Variations in the levels of red blood cells and hemoglobin were observed among the different groups for all injected doses, but these hematological parameters recovered normal values at the end of the study. On the other hand, biochemical parameters such as the total bilirubin and total protein counts showed variations along the study, while they were not associated with the test substance. In summary, no negative effects on clinical, hematological and biochemical parameters were detected. Together, our results put forward the potential and support the safety of the CIGB 247 vaccine candidate for use in clinical applications. The data presented here can be used to estimate a human dosing regimen from preclinical data.

Antigen dose escalation study of a VEGF-based therapeutic cancer vaccine in non human primates.

CIGB-247 is a cancer therapeutic, based on recombinant modified human vascular endothelial growth factor (VEGF) as antigen, in combination with the oil free adjuvant VSSP (very small sized proteoliposomes of Neisseria meningitidis outer membrane). Our previous experimental studies in mice with CIGB-247 have shown that the vaccine has both anti-tumoral and anti-metastatic activity, and produces both antibodies that block VEGF-VEGF receptor interaction, and a specific T-cell cytotoxic response against tumor cells. CIGB-247, with an antigen dose of 100 µg, has been characterized by an excellent safety profile in mice, rats, rabbits, and non human primates. In this article we extend the immunogenicity and safety studies of CIGB-247 in non human primates, scaling the antigen dose from 100 µg to 200 and 400 µg/vaccination. Our results indicate that such dose escalation did not affect animal behavior, clinical status, and blood parameters and biochemistry. Also, vaccination did not interfere with skin deep skin wound healing. Anti-VEGF IgG antibodies and specific T-cell mediated responses were documented at all three studied doses. Antigen dose apparently did not determine differences in maximum antibody titer during the 8 weekly immunization induction phase, or the subsequent increase in antibodies seen for monthly boosters delivered afterwards. Higher antigen doses had a positive influence in antibody titer maintenance, after cessation of immunizations. Boosters were important to achieve maximum antibody VEGF blocking activity, and specific T-cell responses in all individuals. Purified IgG from CIGB-247 immunized monkey sera was able to impair proliferation and formation of capillary-like structures in Matrigel, for HMEC cells in culture. Altogether, these results support the further clinical development of the CIGB-247 therapeutic cancer vaccine, and inform on the potential mechanisms involved in its effect.

Ratio of HCV structural antigens in protein-based vaccine formulations is critical for functional immune response induction.

HCV (hepatitis C virus) infection is among the leading causes of chronic liver disease, but currently there is no vaccine available. Data have accumulated about the importance of targeting different HCV antigens in vaccine candidate preparations. Here, a surface response study to select the optimal ratio of recombinant HCV structural antigens in a vaccine preparation, capable of generating in vivo functional cellular immune response in mice, was performed. The immunogenicity of the selected HCV structural protein mixture (Co-E1-E2) in mice and African green monkeys, after five doses of immunization, was also demonstrated. Specific T-cell proliferative response against HCV structural antigens was induced in vaccinated mice. Moreover, on challenge with recombinant HCV VV (vaccinia virus), all mice controlled the viraemia and 80% were protected. On the other hand, monkeys immunized with Co-E1-E2 developed antibodies, specifically directed to region 412-438 of E2 protein, that include an epitope implicated in HCV neutralization, in addition to a specific proliferative response against HCV Core and E2 proteins. These results indicated that the optimal amount and ratio of HCV recombinant proteins should be taken into account to elicit a successful immune response against HCV and therefore have important implications for vaccine design.

Immunogenicity and some safety features of a VEGF-based cancer therapeutic vaccine in rats, rabbits and non-human primates.

We have developed a cancer vaccine candidate (hereafter denominated CIGB-247), based on recombinant modified human vascular endothelial growth factor (VEGF) as antigen, and the adjuvant VSSP (very small sized proteoliposomes of Neisseria meningitidis outer membrane). In mice, previous work of our group had shown that vaccination with CIGB-247 extended tumor-take time, slowed tumor growth, and increased animal survival. Immunization elicited anti-human and murine VEGF-neutralizing antibodies, and spleen cells of vaccinated mice are cytotoxic in vitro to tumor cells that produce VEGF. We have now tested the immunogenicity of CIGB-247 in Wistar rats, New Zealand White rabbits and the non-human primate Chlorocebus aethiops sabaeus. Using weekly, biweekly and biweekly plus montanide immunization schemes, all three species develop antigen-specific IgG antibodies that can block the interaction of VEGF and VEGF receptor 2 in an ELISA assay. Antibody titers decline after vaccination stops, but can be boosted with new immunizations. In monkeys, DTH and direct cell cytotoxicity experiments suggest that specific T-cell responses are elicited by vaccination. Immunization with CIGB-247 had no effect on normal behavior, hematology, blood biochemistry and histology of critical organs, in the tested animals. Skin deep wound healing was not affected in vaccinated rats and monkeys.

Toxicology and biodistribution study of CIGB-230, a DNA vaccine against hepatitis C virus.

CIGB-230, a mixture of a DNA plasmid expressing hepatitis C virus (HCV) structural antigens and a HCV recombinant capsid protein, has demonstrated to elicit strong immune responses in animals. The present study evaluated the plasmid biodistribution after the administration of CIGB-230 in mice, as well as toxicity of this vaccine candidate in rats. In the biodistribution study, mice received single or repeated intramuscular injections of CIGB-230, 50 microg of plasmid DNA mixed with 5 microg of Co.120 protein. Plasmid presence was assessed in ovaries, kidney, liver, pancreas, mesenteric ganglion, blood, and muscle of the injection site by a qualitative polymerase chain reaction. The toxicology evaluation included treatment groups receiving doses 5, 15, or 50 times higher, according to the body weight, than the expected therapeutic clinical dose. During the first hour after repeated inoculation, a promiscuous distribution was observed. However, 3 months later, plasmid could not be detected in any tissue. There was an absence of detectable adverse effects on key toxicology parameters and no damage evidenced in inspected organs and tissues. These results indicate that CIGB-230 is nontoxic at local and systemic levels and no concerns about persistence are observed, which support clinical testing of this vaccine candidate against HCV.

CIGB-300, a novel proapoptotic peptide that impairs the CK2 phosphorylation and exhibits anticancer properties both in vitro and in vivo.

Protein Kinase CK2 is a serine-threonine kinase frequently deregulated in many human tumors. Here, we hypothesized that a peptide binder to the CK2 phosphoacceptor site could exhibit anticancer properties in vitro, in tumor animal models, and in cancer patients. By screening a random cyclic peptide phage display library, we identified the CIGB-300 (formerly P15-Tat), a cyclic peptide which abrogates the CK2 phosphorylation by blocking recombinant substrates in vitro. Interestingly, synthetic CIGB-300 led to a dose-dependent antiproliferative effect in a variety of tumor cell lines and induced apoptosis as evidenced by rapid caspase activation. Importantly, CIGB-300 elicited significant antitumor effect both by local and systemic administration in murine syngenic tumors and human tumors xenografted in nude mice. Finally, we performed a First-in-Man trial with CIGB 300 in patients with cervical malignancies. The peptide was found to be safe and well tolerated in the dose range studied. Likewise, signs of clinical benefit were clearly identified after the CIGB-300 treatment as evidenced by significant decrease of the tumor lesion area and histological examination. Our results provide an early proof-of-principle of clinical benefit by using an anti-CK2 approach in cancer. Furthermore, this is the first clinical trial where an investigational drug has been used to target the CK2 phosphorylation domain.

Immunization with a recombinant fowlpox virus expressing a hepatitis C virus core-E1 polyprotein variant, protects mice and African green monkeys (Chlorocebus aethiops sabaeus) against challenge with a surrogate vaccinia virus.

HCV (hepatitis C virus) is a worldwide health problem nowadays. No preventive vaccine is available against this pathogen, and therapeutic treatments currently in use have important drawbacks, including limited efficacy. In the present work a recombinant fowlpox virus, FPCoE1, expressing a truncated HCV core-E1 polyprotein, was generated. FPCoE1 virus generally failed to elicit a humoral immune response against HCV antigens in BALB/c mice. By contrast, mice inoculated with FPCoE1 elicited a positive interferon-gamma secretion response against HCV core in ex-vivo ELISPOT (enzyme-linked immunospot) assays. Remarkably, mice inoculated with FPCoE1 significantly controlled viraemia in a surrogate challenge model with vvRE, a recombinant vaccinia virus expressing HCV structural antigens. In fact, 40% of the mice had no detectable levels of vvRE in their ovaries. Administration of FPCoE1 in vervet monkeys [Chlorocebus (formerly Cercophitecus) aethiops sabaeus] induced lymphoproliferative response against HCV core and E1 proteins in 50% of immunized animals. Monkeys immunized with FPCoE1 had no detectable levels of vvRE in their blood, whereas monkeys inoculated with FP9, the negative control virus, had detectable levels of vvRE in blood up to 7 days after challenge. In conclusion, recombinant fowlpox virus FPCoE1 is able to induce an anti-HCV immune response in mice and monkeys. This ability could be rationally employed to develop effective strategies against HCV infection by using FPCoE1 in combination with other vaccine candidates or antiviral treatments.