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.

Specific humoral and cellular immune responses in cancer patients undergoing chronic immunization with a VEGF-based therapeutic vaccine.

CIGB-247 is a cancer therapeutic vaccine, based on recombinant modified human vascular endothelial growth factor (VEGF) as antigen, in combination with the adjuvant VSSP, a bacterially-derived adjuvant. The vaccine have demonstrated efficacy in several murine malignancy models. These studies supported the rationale for a phase I clinical trial where safety, tolerance, and immunogenicity of CIGB-247 was studied in patients with advanced solid tumors at three antigen dose level. Surviving individuals of this clinical trial were eligible to receive off-trial voluntary re-immunizations. The present work is focus in the immunological follow up of these patients after approximately three years of immunizations, without additional oncological treatments. Long term vaccination was feasible and safe. Our results indicated that after sustained vaccination most of the patients conserved their seroconversion status. The specific anti-VEGF IgG titer diminished, but in all the cases keeps values up from the pre-vaccination levels. Continued vaccination was also important to produce a gradual shift in the anti-VEGF IgG response from IgG1 to Ig4. Outstanding, our results indicated that long-term off-trial vaccination could be associated with the maintaining of one reserve of antibodies able to interfere with the VEGF/Receptor interaction and the production of IFNγ secretion in CD8+ cells. The results derived from the study of this series of patients suggest that long term therapeutic vaccination is a feasible strategy, and highlight the importance of continuing the clinical development program of this novel cancer therapeutic vaccine candidate. We also highlight the future clinical applications of CIGB-247 in cancer and explain knowledge gaps that future studies may address. Registration number and name of trial registry: RPCEC00000102. Cuban Public Clinical Trial Registry (WHO accepted Primary Registry). Available from: http://registroclinico.sld.cu/.

Vaccination with a mutated variant of human Vascular Endothelial Growth Factor (VEGF) blocks VEGF-induced retinal neovascularization in a rabbit experimental model.

Vascular Endothelial Growth Factor (VEGF) is a key driver of the neovascularization and vascular permeability that leads to the loss of visual acuity of eye diseases like wet age-related macular degeneration, diabetic macular edema, and retinopathy of premature. Among the several anti-VEGF therapies under investigation for the treatment of neovascular eye diseases, our group has developed the vaccine candidate CIGB-247-V that uses a mutated form of human VEGF as antigen. In this work we evaluated if the vaccine could prevent or attenuate VEGF-induced retinal neovascularization in the course of a rabbit eye neovascularization model, based on direct intravitreal injection of human VEGF. Our experimental findings have shown that anti-VEGF IgG antibodies induced by the vaccine were available in the retina blood circulation, and could neutralize in situ the neovascularization effect of VEGF. CIGB-247-V vaccination proved to effectively reduce retinal neovascularization caused by intravitreal VEGF injection. Altogether, these results open the way for human studies of the vaccine in neovascular eye syndromes, and inform on the potential mechanisms involved in its effect.

CIGB-247: a VEGF-based therapeutic vaccine that reduces experimental and spontaneous lung metastasis of C57Bl/6 and BALB/c mouse tumors.

CIGB-247 is a novel cancer therapeutic vaccine that uses a mutated form of human VEGF as antigen. Being metastatic disease the most dramatic factor of tumor biology affecting patient survival and cure, preclinical evaluation of the impact of CIGB-247 vaccination on experimental metastasis mouse models is highly relevant, and constitutes the focus of this work. CIGB-247 was administered in a weekly schedule known to effectively reduce primary tumor growth. The vaccine was tested in experimental and spontaneous metastasis models of colon (CT26), lung (3LL-D122) and breast (F3II) carcinomas growing in C57Bl/6 or BALB/c mice. Primary tumor growth parameters, metastatic counts, and/or animal survival were recorded. Histology and specific humoral and cellular responses to the vaccine were evaluated. As compared to control groups, CIGB-247 vaccination significantly reduced the number and size of metastatic tumor foci in lungs after intravenous inoculation of CT26 and 3LL-D122 tumor cells. Spontaneous lung dissemination from 3LL-D122 and F3II breast tumor cells implanted in the footpad, or subcutaneously, was also reduced by immunization with CIGB-247. The vaccine elicited in both mouse strains antibodies specific for human and murine VEGF that effectively blocked the interaction of VEGF with VEGF receptor 2. Differing from other experimental reports that describe the use of VEGF for active tumor immunotherapy, CIGB-247 elicited a specific cellular response, measured both by a DTH increment and the induction of spleen cells cytotoxic to syngeneic tumor cells producing murine VEGF. In summary our results reinforce the potential of CIGB-247 vaccination to reduce both tumor growth and the number and size of tumor metastasis in lungs, the latter both after direct inoculations of cells in the blood stream, or as part of primary tumor progression in immunocompetent mice.

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.

Isolation of a novel neutralizing antibody fragment against human vascular endothelial growth factor from a phage-displayed human antibody repertoire using an epitope disturbing strategy.

Following the clinical success of Bevacizumab, a humanized monoclonal antibody that blocks the interaction between vascular endothelial growth factor (VEGF) and its receptors, the search for new neutralizing antibodies targeting this molecule has continued until now. We used a human VEGF variant containing three mutations in the region recognized by Bevacizumab to direct antibody selection towards recognition of other epitopes. A total of seven phage-displayed antibody fragments with diverse binding properties in terms of inter-species cross-reactivity and sensitivity to chemical modifications of the antigen were obtained from a human phage display library. All of them were able to recognize not only the selector mutated antigen, but also native VEGF. One of these phage-displayed antibody fragments, denominated 2H1, was shown to compete with the VEGF receptor 2 for VEGF binding. Purified soluble 2H1 inhibited in a dose dependent manner the ligand-receptor interaction and abolished VEGF-dependent proliferation of human umbilical vein endothelial cells. Our epitope disturbing strategy based on a triple mutant target antigen was successful to focus selection on epitopes different from a known one. Similar approaches could be used to direct phage isolation towards the desired specificity in other antigenic systems.

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.

Anti-tumoral effect of active immunotherapy in C57BL/6 mice using a recombinant human VEGF protein as antigen and three chemically unrelated adjuvants.

Following the clinical success of Bevacizumab, a humanized monoclonal antibody that affects the interaction between vascular endothelial growth factor (VEGF) and its receptors, blocking tumor-induced angiogenesis has become one of the most important targets for the development of new cancer therapeutic drugs and procedures. Among the latter, therapeutic vaccination using VEGF as antigen presents itself as very attractive, with the potential of generating not only a growth factor blocking antibody response but also a cellular response against tumor cells and stromal elements, which appear to be a major source of tumor VEGF. In this paper, we report the development of a protein vaccine candidate, based on a human modified VEGF antigen that is expressed at high levels in E. coli. With respect to controls, immunization experiments in C57BL/6 mice using weekly doses of this antigen and three adjuvants of different chemical natures show that time for tumor development after subcutaneous injection of Melanoma B16-F10 cells increases, tumors that develop grow slower, and overall animal survival is higher. Immunization also prevents tumor development in some mice, making them resistant to second tumor challenges. Vaccination of mice with the human modified VEGF recombinant antigen produces antibodies against the human antigen and the homologous mouse VEGF molecule. We also show that sera from immunized mice block human VEGF-induced HUVEC proliferation. Finally, a possible contribution of T cell cytotoxicity to the overall anti-tumor effect is suggested from the results of vaccination experiments where CD8+ lymphocytes were impaired using neutralizing rat antibodies.

Biologically active vascular endothelial growth factor as a bacterial recombinant glutathione S-transferase fusion protein.

Human VEGF121 (vascular endothelial growth factor isoform 121) was produced as a recombinant fusion protein with GST (glutathione S-transferase) in Escherichia coli. After affinity purification with glutathione, the GST-VEGF121 fusion protein preparation was used to obtain antibodies in mice against commercial hrVEGF (human recombinant VEGF) through immunization. It was also employed successfully to select specific antihuman VEGF antibody fragments of human origin employing phage-display technology. The fusion protein preparation was separated in monomeric, dimeric and oligomeric forms using size-exclusion chromatography. The dimers were recognized by a soluble VEGF receptor 2-Fc chimaera, and stimulated the growth of human umbilical-vein endothelial cells in vitro in a similar fashion to a commercial hrVEGF. The presence of GST in the fusion protein apparently did not affect the correct assembly of dimers and display of residues critical for receptor recognition. The two-step purification method reported in the present paper involves no laborious renaturalization methods, yields 10 mg/l of the mixture of different aggregation states after affinity chromatography, and 5 mg/l of the biologically active dimer after gel filtration, thus providing a source of material for the development of new anti-angiogenic therapeutic molecules.

In vitro assembly into virus-like particles is an intrinsic quality of Pichia pastoris derived HCV core protein.

Different variants of hepatitis C virus core protein (HCcAg) have proved to self-assemble in vitro into virus-like particles (VLPs). However, difficulties in obtaining purified mature HCcAg have limited these studies. In this study, a high degree of monomeric HCcAg purification was accomplished using chromatographic procedures under denaturing conditions. Size exclusion chromatography and sucrose density gradient centrifugation of renatured HCcAg (in the absence of structured RNA) under reducing conditions suggested that it assembled into empty capsids. The electron microscopy analysis of renatured HCcAg showed the presence of spherical VLPs with irregular shapes and an average diameter of 35nm. Data indicated that HCcAg monomers assembled in vitro into VLPs in the absence of structured RNA, suggesting that recombinant HCcAg used in this work contains all the information necessary for the assembly process. However, they also suggest that some cellular factors might be required for the proper in vitro assembly of capsids.