No abstract

Antitumor strategies based on positive modulation of the immune system currently represent therapeutic options with prominent acceptance for cancer patients' treatment due to its selectivity and higher tolerance compared to chemotherapy. Racotumomab is an anti-idiotype (anti-Id) monoclonal antibody (mAb) directed to NeuGc-containing gangliosides such as NeuGcGM3, a widely reported tumor-specific neoantigen in many human cancers. Racotumomab has been approved in Latin American countries as an active immunotherapy for advanced non-small cell lung cancer (NSCLC) treatment. In this work, we evaluated the induction of Ab-dependent cell-mediated cytotoxicity (ADCC) in NSCLC patients included in a phase III clinical trial, in response to vaccination with racotumomab. The development of anti-NeuGcGM3 antibodies (Abs) in serum samples of immunized patients was first evaluated using the NeuGcGM3-expressing X63 cells, showing that racotumomab vaccination developed antigen-specific Abs that are able to recognize NeuGcGM3 expressed in tumor cell membranes. ADCC response against NeuGcGM3-expressing X63 (target) was observed in racotumomab-treated- but not in control group patients. When target cells were depleted of gangliosides by treatment with a glucosylceramide synthase inhibitor, we observed a significant reduction of the ADCC activity developed by sera from racotumomab-vaccinated patients, suggesting a target-specific response. Our data demonstrate that anti-NeuGcGM3 Abs induced by racotumomab vaccination are able to mediate an antigen-specific ADCC response against tumor cells in NSCLC patients.

Neuroblastoma (NB) is the most common pediatric malignancy diagnosed before the first birthday in which MYCN oncogene amplification is associated with poor prognosis. Although aberrant glycosylation is an important actor in cell biology, little is known about its role in pediatric cancers such as NB. In this work we characterized the glycophenotype and the enzyme expression involved in glycans biosynthesis in five established human NB cell lines and in patient-derived primary tumors with different MYCN status. Our results show a high expression of Lewis glycan family both in MYCN-amplified cell lines and patient samples. Additionally, we report that MYCN-amplified cells overexpressed Core 2-initiating glycosyltransferase C2GNT1 in association with specific ST3Gals and FUTs, and showed increased binding to E- and P- selectins. Silencing of C2GNT1 expression in NB cells diminished expression of Lewis glycans, decreased the E- and P-selectin binding, and reduced cell adhesion, migration and proliferation in vitro. Treatment of MYCN-non-amplified cells with Trichostatin A (TSA), an histone deacetylase inhibitor, increased the expression of Lewis glycans and the enzymes involved in their biosynthesis. Our results demonstrate that MYCN-amplified NB cells overexpress Lewis family glycans, which belong to the Core 2 O-glycans group. Their expression plays a key role in the malignant behaviour of the NB cells and it is modulated by epigenetic mechanisms.

Modulation of the immune system has been demonstrated as a powerful approach to treating cancer. Immunotherapies are generally classified as active or passive according to their ability to trigger the immune system. During the last decades, information regarding the relevance of aberrant glycosylation as a major player in tumour biology encouraged expectations for the development of new therapeutic strategies directed at glycans. Several tumour-associated carbohydrate antigens (TACAs) have been identified and validated as suitable immunotherapeutic targets, leading to promising therapeutic developments. It is known that TACAs are poorly immunogenic since they are unable to trigger a proper immune response. Given that they are not presented by major histocompatibility complex (MHC) molecules and that they induce immune tolerance, the development of active immunotherapeutic strategies against TACAs is a real challenge. However, antitumor strategies based on mimetics of TACAs have been developed and show promising results. Active immunotherapies based on TACAs mimicry can currently be grouped into strategies based on the use of mimetic peptides and anti-idiotype (Id) antibodies. In this review, we discussed the scientific basis on which these strategies are based and the available therapeutic options that have shown the best results in preclinical studies and in clinical practice.

N-glycolylneuraminic acid (NeuGc) is a sialic acid molecule present in mammalian cells as terminal constituents of membrane glycoconjugates such as gangliosides and glycoproteins. Although the role of NeuGc-containing glycoconjugates in human cancer is still under investigation, gangliosides such as NeuGcGM3 has been consistently reported as a tumor antigen in various epithelial and neuroectodermal malignancies, including non-small cell lung cancer (NSCLC). Interestingly, NeuGc-glycoconjugates are abundant in aggressive neoplasms, but they are usually absent in normal human tissues. In contrasts to most mammals, human beings lack the key enzyme that catalyzes N-glycolylation of sialic acid. Cancer cells can favor NeuGc intake from diet sources, thus allowing the expression of NeuGc-containing glycoconjugates. Racotumomab is an anti-NeuGc anti-idiotype monoclonal antibody that has been approved in Latin American countries as maintenance immunotherapy for advanced NSCLC. Combinatorial approaches involving long-term immunization against tumor-specific antigens together with the anti-PD1 immune-checkpoint inhibitor pembrolizumab is an attractive strategy for immunotherapy. In this work, we have examined the antitumor activity of racotumomab in combination with anti-PD1 therapy, using the 3LL Lewis lung carcinoma as a preclinical model of NSCLC in C57BL/6 mice. Immunization with either weekly or biweekly s.c. doses of racotumomab at 50-200 μg/dose formulated in aluminum hydroxide (racotumomab-alum) demonstrated a significant antitumor effect against the progression of lung tumor nodules (p<0.05, ANOVA followed by Tukey). Similarly, checkpoint blockade with an anti-mouse PD1 monoclonal antibody injected i.p. at 200 μg/dose exerted a comparable antitumor effect in this 3LL lung model (p<0.01, unpaired t-test). Interestingly, sequential administration of anti-PD1 therapy followed by repeated immunizations with racotumomab-alum was highly effective against lung nodules and well tolerated, showing a reduction in nodules formation of 62 and 45% compared to anti-PD1 or racotumomab-vaccinated groups, respectively (p<0.01, ANOVA followed by Tukey). Our preclinical data provide support for the combination of anti-PD1 checkpoint blockade with the anti-idiotype monoclonal antibody racotumomab in advanced NSCLC, since combination treatment has a significant additive antitumor effect compared to each individual treatment. Citation Format: Valeria I. Segatori, Cynthia A. Gulino, Carla S. Capobianco, Selene Rojo, Gretel M. Ferreira, Héctor A. Cuello, Mariano R. Gabri, Daniel A. Alonso. Racotumomab and PD-1 blockade combination exhibits an additive antitumor effect in a non-small cell lung cancer model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6648.