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 EGFR1 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 EGFR1 Lewis lung carcinoma model, while reproductionassociated 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 EGFR1 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 EGFR1 tumors. ' 2006 Wiley-Liss, Inc.

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Summary Serological (Western blot) detection of bovine immunodeficiency‐like virus (BIV) in Holstein dairy herds is reported in Costa Rica for the first time, as well as the isolation of the virus, from a seropositive bovine, by cocultivation of peripheral blood mononuclear cells (PBMC) with embryonic rabbit epithelial (EREp) cells. The isolated strain, BIV CR1, reacted similarly in Western blot as the reference strain BIV R29 and is clearly distinguishable from bovine leukaemia virus (BLV). The data suggest an association between BIV infections and BLV infections, as it has been reported elsewhere. From these results it can be concluded that BIV is present in Costa Rica and it is suggested that these viral infections will probably follow the epidemiological parameters of BLV infections in Costa Rica, reaching high infection rates in dairy herds.

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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.

Summary Experiments were designed to evaluate the effect of BLV on mitogen‐stimulated peripheral blood mononuclear cells (PBMC) from naturally infected cattle. BIV was also taken into consideration due to a recent report showing that in Costa Rica, most of the BLV‐infected animals are also seropositive for BIV. The methodology was based on a non‐radioactive technique to determine lymphoproliferation. A colourimetric assay using XTT (formazan salt) to measure cell multiplication was adapted for bovine PBMC. ELISA and Western blotting were used to determine the serologic status of the cattle. PCR was only available for BIV detection. Our results show clearly that, dually‐infected cattle (BIV‐BLV) have reduced lymphoproliferative responses to the mitogen Con A. Haematological abnormalities associated with viral infections were also observed, specially leukocytosis and lymphocytosis. Cows with lymphosarcomas are severely affected. The specific antibody response to different viral proteins could not be associated with the suppressive status of the animals. Due to the high rate of dual infections observed in Costa Rica, these results are not sufficient to clarify which virus is responsible for the suppressive activity, if one or both viruses are necessary, or if they act synergistically.

Transforming growth factor alpha (TGFa) is an important epidermal growth factor receptor (EGFR) ligand. Over-expression of both molecules in epithelial tumors has been correlated with poor prognosis and disease progression. Due to the importance of TGFa in tumorigenesis, this molecule has great potential for cancer immunotherapy. We previously designed a TGFa-based vaccine consisting of a fusion protein between human TGFa (hTGFa) and P64k protein from Neisseria meningitidis expressed in Escherichia coli. However, this protein was obtained highly aggregated, which hampered its introduction into clinical use. In this study, we demonstrate that this aggregation state is not a consequence of IMAC purification, but is formed after bacterial disruption. To obtain this protein as a monomer, we designed a procedure that included an unfolding/refolding step at the end of purification. We verified that hTGFa in the refolded fusion protein (hTGFa-P64k-r) is immunogenic in mice. The latter was capable of inducing a humoral immune response against hTGFa identical to that generated with the aggregated fusion protein, demonstrating that the aggregation level has no influence on hTGFa immunogenicity. We also showed that TGFa-directed antibodies induced apoptosis in A431 cells. The present results also validated the potential use of this vaccine in cancer patients with tumors overexpressing TGFa. Drug Dev Res 69 : 481-494, 2008.

Correlation between “clonal” neoantigen burden (neoantigens generated early during cancer development and therefore represented at high frequency in all tumor lesions) and responsiveness to checkpoint blockade therapy has underscored the importance of neoantigens in promoting tumor immunogenicity, and has spurred a broad effort to develop methods to identify neoantigens for use in vaccination protocols. Yet, the majority of patients do not express, or express low numbers of, clonal neoantigens, and consequently will be less likely to benefit from checkpoint blockade and other forms of immune potentiating therapies. Here we describe several strategies to generate de novo neoantigens in the patient' disseminated tumor lesions and show that in murine tumor models they potentiate checkpoint blockade therapy. We have previously shown that oligonucleotide aptamer-targeted siRNA inhibition of the Nonsense-mediated mRNA (NMD) process in tumor cells in situ results in the induction of neoatigens and inhibition of tumor growth (Pastor et al., Nature, 2010, 465:227). We now demonstrate that tumor-targeted NMD inhibition potentiates PD-1 blockade therapy. A general concern and potential limitation of the NMD approach is that a significant proportion of NMD inhibition-induced neoantigens are generated as a result of random mutations or aberrant splicing and hence will not be represented at high frequency in all the tumor lesions of the patient. To that end, we are exploring alternative strategies to generate clonal neoantigens by targeting key components of antigen presentation pathways, specifically the TAP transporter, ERAAP peptidase, and Invariant chain, the latter to generate class II-restricted neoantigens. Studies have shown that downregulation of these products, using genetic means or antisense RNA, not only inhibits the canonical antigen presentation pathway but also upregulates alternative pathways that present new, otherwise silent or subdominant, epitopes. Since such epitopes are not generated as a result of random mutations they are more likely to represent “clonal” neoepitopes generated in every cell in which the target was downregulated. Here we are developing clinically feasible broadly applicable approaches to inhibit the aforementioned mediators of antigen presentation using corresponding siRNAs targeted to tumor cells in situ by conjugation to a nucleolin-binding aptamer. Nucleolin, a nucleolar product, is translocated to the surface the majority of tumor cells of both human and murine origin and thereby serves as a broad, if not universal, target to deliver therapeutic cargo to the disseminated tumor cells of the patient. We show that nucleolin aptamer-targeted downregulation of ERAAP or Invariant chain in tumor-bearing mice inhibits tumor growth and potentiates PD-1 blockade therapy. Ongoing studies explore the combinatorial use of neoantigen induction methods and combination of neoantigen induction with immune potentiating strategies including but not limited to checkpoint blockade. Citation Format: Greta Garrido Hidalgo, Agata Levay, Alexey Berezhnoy, Brett Schrand, Eli Gilboa. Inducing neoantigens in disseminated tumor lesions to enhance their susceptibility to PD-1 blockade therapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A023.