American Association for Cancer Research
00085472can201072-sup-240540_2_supp_6405211_qd1hgf.docx (1.27 MB)

Supplementary Data from A Genetic Vaccine Encoding Shared Cancer Neoantigens to Treat Tumors with Microsatellite Instability

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journal contribution
posted on 2023-03-31, 03:48 authored by Guido Leoni, Anna Morena D'Alise, Gabriella Cotugno, Francesca Langone, Irene Garzia, Maria De Lucia, Imma Fichera, Rosa Vitale, Veronica Bignone, Fabio Giovanni Tucci, Federica Mori, Adriano Leuzzi, Elena Di Matteo, Fulvia Troise, Adele Abbate, Rossella Merone, Valentino Ruzza, Maria Grazia Diodoro, Mahesh Yadav, Monica Gordon-Alonso, Cristophe Vanhaver, Maddalena Panigada, Elisa Soprana, Antonio Siccardi, Antonella Folgori, Stefano Colloca, Pierre van der Bruggen, Alfredo Nicosia, Armin Lahm, Maria Teresa Catanese, Elisa Scarselli

Fig. S1: Number of immunogenic epitopes in healthy donors vaccinated with viral antigens. Fig. S2: Size distribution of FSPs in Nous-209. Fig. S3: Detection of Nous-209 FSMs in early and late stage MSI tumors. Fig.S4: Mutation allele frequency of Nous-209 FSMs detected in tumor and healthy mucosa of 20 CRC MSI patients. Fig. S5: Schematic of Nous-209 vaccine. Fig. S6: Overview of the procedure to isolate T cells recognizing peptide Ly4_24.



Tumors with microsatellite instability (MSI) are caused by a defective DNA mismatch repair system that leads to the accumulation of mutations within microsatellite regions. Indels in microsatellites of coding genes can result in the synthesis of frameshift peptides (FSP). FSPs are tumor-specific neoantigens shared across patients with MSI. In this study, we developed a neoantigen-based vaccine for the treatment of MSI tumors. Genetic sequences from 320 MSI tumor biopsies and matched healthy tissues in The Cancer Genome Atlas database were analyzed to select shared FSPs. Two hundred nine FSPs were selected and cloned into nonhuman Great Ape Adenoviral and Modified Vaccinia Ankara vectors to generate a viral-vectored vaccine, referred to as Nous-209. Sequencing tumor biopsies of 20 independent patients with MSI colorectal cancer revealed that a median number of 31 FSPs out of the 209 encoded by the vaccine was detected both in DNA and mRNA extracted from each tumor biopsy. A relevant number of peptides encoded by the vaccine were predicted to bind patient HLA haplotypes. Vaccine immunogenicity was demonstrated in mice with potent and broad induction of FSP-specific CD8 and CD4 T-cell responses. Moreover, a vaccine-encoded FSP was processed in vitro by human antigen-presenting cells and was subsequently able to activate human CD8 T cells. Nous-209 is an “off-the-shelf” cancer vaccine encoding many neoantigens shared across sporadic and hereditary MSI tumors. These results indicate that Nous-209 can induce the optimal breadth of immune responses that might achieve clinical benefit to treat and prevent MSI tumors. These findings demonstrate the feasibility of an “off-the-shelf” vaccine for treatment and prevention of tumors harboring frameshift mutations and neoantigenic peptides as a result of microsatellite instability.

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