<div>Abstract<p>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 <i>in vitro</i> 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.</p>Significance:<p>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.</p></div>
// Emily Capone 1, 2 , Enza Piccolo 1 , Imma Fichera 1, 6 , Paolo Ciufici 3 , Daniela Barcaroli 3 , Arturo Sala 3, 4 , Vincenzo De Laurenzi 2 , Valentina Iacobelli 1, 5 , Stefano Iacobelli 1, 2, * and Gianluca Sala 1, 2, * 1 MediaPharma s.r.l., 66100, Chieti, Italy 2 Dipartimento di Scienze Mediche, Orali e Biotecnologiche; University "G. d'Annunzio" Chieti-Pescara, Centro Studi sull'Invecchiamento, CESI-MeT, 66100 - Chieti, Italy 3 Dipartimento di Scienze Psicologiche, della Salute e del Territorio, University "G. d'Annunzio" Chieti-Pescara, Centro Studi sull'Invecchiamento, CESI-MeT, 66100 - Chieti, Italy 4 College of Health and Life Sciences, Brunel University London, UK 5 Department of Gynecology and Obstetrics, University of Rome "La Sapienza", 00100 - Rome, Italy 6 Current address: Nouscom SRL 100 I-00128 Rome, Italy * These authors share senior authorship Correspondence to: Stefano Iacobelli, email: s.iacobelli@mediapharma.it Keywords: endosialin, ADC, sarcoma, duocarmycin, target therapy Received: May 11, 2017 Accepted: June 19, 2017 Published: July 22, 2017 ABSTRACT The endosialin/CD248/TEM1 receptor is expressed on the cell surface of tumor-associated stroma cells as well as in sarcoma and neuroblastoma cells. This receptor is emerging as an attractive molecule in diagnostics and therapeutics because of its expression across the stroma of many human tumors, the low to absent expression in normal tissues and accessibility from the vascular circulation. In this study, we present evidence of the preclinical efficacy of a novel Antibody-Drug Conjugate (ENDOS/ADC). It consists of a humanized endosialin monoclonal antibody, named hMP-E-8.3, conjugated to a potent duocarmycin derivative. In endosialin expressing cancer cell lines, this ENDOS/ADC showed a powerful, specific and target-dependent killing activity. High expression levels of endosialin in cells correlated with efficient internalization and cytotoxic effects in vitro . Efficacy studies demonstrated that ENDOS/ADC treatment led to a long-lasting tumor growth inhibition of a cell line-based model of human osteosarcoma. Taken together, our results demonstrate that endosialin is an attractive target in sarcoma and suggest that ENDOS/ADC has the potential to be developed into a bio-therapeutic agent for these malignancies.
Cadherin-16 was originally identified as a tissue-specific cadherin present exclusively in kidney. Only recently, Cadherin-16 has been detected also on the plasma membrane of mouse thyrocytes. This last finding prompted us to note that the expression profile of Cadherin-16 resembles that of the transcription factor Pax8, a member of the Pax (paired-box) gene family, predominantly expressed in the developing and adult kidney and thyroid. Pax8 has been extensively characterized in the thyroid and shown to be a master gene for thyroid development and differentiation. In this study, we determined the role of the transcription factor Pax8 in the regulation of Cadherin-16 expression. We demonstrate that the Cadherin-16 minimal promoter is transcriptionally active in thyroid cells as well as in kidney cells, that Pax8 is able to activate transcription from a Cadherin-16 promoter reporter construct, and more importantly, that indeed Pax8 is able to bind in vivo the Cadherin-16 promoter region. In addition, by means of Pax8 RNA interference in thyroid cells and by analyzing Pax8 null mice, we demonstrate that Pax8 regulates also in vivo the expression of Cadherin-16. Finally, we reveal that the expression of Cadherin-16 is TSH dependent in FRTL-5 thyroid cells and significantly reduced in mouse thyroid carcinomas. Therefore, we conclude that Cadherin-16 is a novel downstream target of the transcription factor Pax8, likely since the early steps of thyroid development, and that its expression is associated with the fully differentiated state of the thyroid cell.
<p>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.</p>
<div>Abstract<p>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 <i>in vitro</i> 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.</p>Significance:<p>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.</p></div>
<p>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.</p>
