Belinda Sánchez Ramírez

Active antimetastatic immunotherapy in Lewis lung carcinoma with self EGFR extracellular domain protein in VSSP adjuvant

The epidermal growth factor receptor (EGFR) plays a central role in regulating neoplastic processes. The EGFR overexpression in many human epithelial tumors has been correlated with disease progression and bad prognosis. Passive EGFR‐directed immunotherapy, but not active specific approaches, has already been introduced in medical oncology practice. Then we wonder if mice immunization with the extracellular domain of murine EGFR (mEGFR‐ECD) in adjuvants can circumvent tolerance to self EGFR, by inducing an immune response with consequent antitumor effect. The present study demonstrated that despite mEGFR expression in thymus, strong DTH response was induced by inoculation of mice with the mEGFR‐ECD. This self‐immunization, using both CFA and very small sized proteoliposomes from Neisseria meningitidis (VSSP), promoted highly specific IgG titers, predominantly IgG2a and IgG2b. Sera from mice immunized with mEGFR‐ECD/VSSP not only recognized EGFR+ tumor cell lines by FACS, but also inhibited their in vitro growth, even in the absence of complement. Noteworthy, vaccination of mice with mEGFR‐ECD/VSSP stimulated a potent antimetastatic effect in the EGFR+ Lewis lung carcinoma model, while reproduction‐associated side effects were absent. Curiously, mice immunized with the human EGFR‐ECD (Her1‐ECD) in VSSP though induced highly specific IgG antibodies with strong in vitro cytotoxic effect over EGFR+ human cell lines, showed low cross‐reactivity with the mEGFR‐ECD. These results further encouraged the development of the Her1‐ECD/VSSP vaccine project for patients with EGFR+ tumors.

Anti-EGFR activation, anti-proliferative and pro-apoptotic effects of polyclonal antibodies induced by EGFR-based cancer vaccine.

Up to now clinical experiences focusing EGF receptor, an attractive target for cancer therapy, have been limited to passive therapies, suggesting that therapeutic cancer vaccines inducing anti-epidermal growth factor receptor (EGFR) antibodies could also work. Here, the humoral immune response induced in mice with a vaccine formulation containing the human EGFR-extracellular domain and very small-sized proteoliposomes (VSSP), a novel nanoparticulated adjuvant was assessed. In vaccinated mice sera average of the specific polyclonal antibodies (PAb) titers was 10(-5). Anti-EGFR PAb were able to bind EGFR+ tumor cell lines, expressing different levels of the molecule. Noteworthy, the presence of Cetuximab only partially inhibited the vaccine-induced antibodies binding to H125 cells. Anti-EGFR PAb abrogated ligands-dependent EGFR phosphorylation, provoking tumor cells apoptosis. The described EGFR-based vaccine might be a superior therapeutic approach for patients with EGFR+ tumors.

Lentiviral vector followed by protein immunisation breaks tolerance against the self‐antigen Her1 and results in lung cancer immunotherapy

Lung cancer remains a leading cause of cancer mortality, and so the aim of the present study was to develop a therapeutic vaccine protocol.

Non-clinical immuno-toxicological evaluation of HER1 cancer vaccine in non-human primates: a 12-month study.

Human epidermal growth factor receptor (HER1) constitutes a tumor associated antigen. Its overexpression in many epithelial tumors has been associated with bad prognosis and poor survival. Cancer vaccine based on the extracellular domain (ECD) of HER1 and adjuvated in very small sized proteoliposomes (VSSP) and Montanide ISA 51-VG is a new and complementary approach for the treatment of epithelial tumors. The present study deals with the immunogenicity of this vaccine in Macaca fascicularis monkeys and evaluation of its toxicity during 12 months. Twelve monkeys were randomized into two groups of 3 animals per sex: control and vaccinated. Treated monkeys received 9 doses of vaccination and were daily inspected for clinical signs. Body weight, rectal temperature, cardiac and respiratory rates were measured during the study. Humoral immune response, clinical pathology parameters and delayed type hypensensitivity were analyzed. Skin biopsy was performed at the end of the study in all animals. Animals survival in the study was 100% (n=12). Local reactions were observed at the administration site of four treated animals (n=6), with two showing slight inflammatory cutaneous damage. Clinical pathology parameters were not affected. HER1 vaccine induced high IgG antibodies titers in the treated animals even when DTH was not observed. The induced antibodies recognized HER1+ tumor cell lines, decreased HER1 phosphorylation and showed anti-proliferative and pro-apoptotic effects in H125 cells. In general the present study showed that HER1 vaccine induced specific immune response in M. fascicularis monkeys and was well tolerated, suggesting it could be safely used in clinical studies in epithelial cancer patients.

Pharmacokinetics and Biodistribution Study of 7A7 Anti-Mouse Epidermal Growth Factor Receptor Monoclonal Antibody and Its F(ab')2 Fragment in an Immunocompetent Mouse Model

Immunocompetent mice, Fc receptor γ-chain deficient mice (Fcer1g−/−), and molecular tools as F(ab′)2 bivalent fragments appear as the most suitable biological models to study the mechanisms of the action of anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs). In vivo experiments contrasting antitumor effects of whole Abs and their bivalent fragments commonly involve a previous comparative pharmacokinetics study. In this paper, pharmacokinetics and biodistribution of an anti-mouse EGFR Ab were assessed using immunocompetent mice. 125I-labeled 7A7 mAb holds an elimination half-life (t1/2β) of 23.1 h in C57BL/6 mice. Accumulation of mAb was found in liver, spleen, kidneys, and mostly in lungs. We used an ELISA method to determine the t1/2β of a 7A7 mAb using the same experimental setting. Results from this new analysis revealed a t1/2β of 23.9 h, supporting this method as a safer and easier system to evaluate pharmacokinetics parameters of mAbs targeting mouse EGFR. Using this system we also studied pharmacokinetics of 7A7 F(ab′)2 fragment. A tenfold difference between the mAb and fragment t1/2β was found. These data support the use of the 7A7 F(ab′)2 fragment in in vivo studies to explore the contribution of the EGFR signaling blockade and the Fc region to the antitumor effect of 7A7 mAb in this autologous scenario.

HER1-ECD vaccination dispenses with emulsification to elicit HER1-specific anti-proliferative effects.

EGFR (HER1) highlights as one of the most relevant tumour associated antigen in epithelial malignant cells. Monoclonal antibodies and tyrosine kinase inhibitors against EGFR remain as the most advanced approaches in clinical trials. More recently, an active immunotherapy using the HER1 extracellular domain (ECD) adjuvated in very small sized proteoliposomes (VSSP) and emulsified in Montanide ISA-51 demonstrated its strength to inhibit tumor cell line proliferation by arresting cells in G0/G1 stage and induction of apoptosis. In this study, we present a simpler HER1-ECD-based formulation, which is lacking the oily component Montanide ISA-51. Generated antibodies following non-emulsive formulation immunization recognized membrane EGFR; avoid EGF and TGFα coupling to EGFR leading to a marked abrogation of EGFR phosphorylation levels. Non-emulsive formulation also arrests cell cycle in G0/G1 stage, demonstrating it preserves previous formulation quality in a newer and simpler formulation.

Assessment of the impact of manufacturing changes on the physicochemical properties and biological activity of Her1-ECD vaccine during product development.

Vaccine preparations based on the extracellular domain of Her1 (Her1-ECD) have demonstrated, in vitro and in vivo, a potent antimetastatic effect on EGFR(+) Lewis lung carcinoma model, while associated side effects were absent. The Her1-ECD is a glycoprotein with a molecular weight of 105 kDa and has 11 potential sites for N-glycosylation. Currently Her1-ECD based vaccine has been evaluated in patients with hormone refractory prostate cancer. Her1-ECD molecule used for in clinical trials was obtained from culture supernatant of HEK 293 transfectomes used the protein free culture media and is purified by immunoaffinity chromatography. In order to increase the cell growth and productivity, new defined culture media have been developed (alternative culture media) in Her1-ECD vaccine production process. In this work, a comparability study was performed to evaluate the impact of process changes in the characteristics physic-chemical and biologicals of the Her1-ECD protein and the degree of similitude between both variants. Techniques such as: SDS-PAGE, SEC-HPLC, isoelectric point, peptide mapping, mass spectrometric, SCX-HPLC, oligosaccharide map, ELISA and flow cytometric were used with this aim. Results indicated that this process change decreases the degree of sialylation of the protein but does not affect its biological activity (measured as titers of Abs and recognition for A431 cell line).

HER1-based vaccine: Simultaneous activation of humoral and cellular immune response

The human epidermal growth factor receptor 1 (HER1) is a tumor-associated antigen that has been validated as a clinical target for several passive, non-immune therapies currently approved for the treatment of epithelial tumors. HER1 is an oncogene that not only promotes tumor progression and survival, but also immune escape. Its overexpression in some epithelial malignancies has been correlated with a poor prognosis. We developed an approach to target HER1 by specific active immunotherapy, recognizing the extracellular domain of the receptor, using a combination of VSSP and Montanide ISA 51 as adjuvants. We summarize the results obtained with this vaccine in both the preclinical and clinical settings, emphasizing the importance of the induction of both humoral and cellular responses for the success of cancer vaccines as safe therapeutic alternatives for the treatment of cancer.

Anti-proliferative and pro-apoptotic effects induced by simultaneous inactivation of HER1 and HER2 through endogenous polyclonal antibodies

The human epidermal growth factor receptor (HER1) and its partner HER2 are extensively described oncogenes and validated targets for cancer therapy. However, the effectiveness of monospecific therapies targeting these receptors is hampered by resistance emergence, which is frequently associated with the upregulation of other members of HER family. Combined therapies using monoclonal antibodies or tyrosine kinase inhibitors have been suggested as a promising strategy to circumvent this resistance mechanism. We propose an alternative approach based on simultaneous inactivation of HER1 and HER2 by multi-epitope blockade with specific polyclonal antibodies induced by vaccination. Elicited antibodies impaired both receptors activation and induced their degradation, which caused the inhibition of down-signaling cascades. This effect was translated into cell cycle arrest and apoptosis induction of human tumor cells. Elicited antibodies were able to reduce the viability of a panel of human tumor lines with differential expression levels of HER1 and HER2. The most significant effects were obtained in the tumor lines with lower expression levels of both receptors. These new insights would contribute to the rational design of HER receptors targeting multivalent vaccines, as an encouraging approach for the treatment of cancer patients.