1 ). Targeting neoantigens derived from driver mutations ensures that the vaccine should preferentially target clinically relevant outgrowing tumor cell clones, which are at risk of developing into manifest cancer. At the same time, this strategy limits the likelihood ofimmune evasion through antigen loss. Although frameshift mutations in cMS can result in two possible novel reading frames, mutation data of MSI cancers demonstrate that the reading frame resulting from a single nucleotide deletion ( /C01) is clearly the predominant type of mutation (www.seltarbase.org), enhancing the potential coverage of the FSP vaccine. Importantly, mutations giving rise to the FSP antigens in the vaccine are also present in extracolonic tumors such as endo- metrial cancer (35); therefore, these tumor types may also be prevent- able by the FSP vaccine. Immune evasion mediated through loss of the tumor cells ’capacity to present antigens to the immune system has been reported previously in MSI cancers (36, 37). It is conceivable that a potent FSP vaccine may enhance the immunoselective pressure and thereby increase the probability of immune evasion through Beta2-microglobulin (B2M ) mutations (38). Interestingly, patients with B2M -mutant cancers apparently have an excellent prognosis (39 –42) and do not develop tumor relapses or distant metastases. Hence, FSP vaccination may not only reduce the cancer incidence in Lynch syndrome individuals directly by elimination of emerging MSI cell clones, but also help to make MSI cancer a surgically curable disease. The study results demonstrate that FSP vaccination has low toxicity, as systemic adverse effects were rare and most likely unrelated to the vaccine agents. However, local reactions at the injection site were commonly observed (grade 2 in three patients). This most likely results from one or more of the following factors: (i) Injection site reactions were most likely triggered or potentiated by Montanide ISA 51 VG, as redness, swelling, and granuloma formation have previously been described as typical side effects (43). In contrast to the present trial, in which 19 of 22 participants were tumor-free at the time point of inclusion, most cancer vaccine trials using Montanide were performed in patients with manifest advanced-stage cancer and therefore poten- tially a compromised immune status. (ii) It seems plausible that Figure 4. Immune responses against FSP antigens. FSP-speci ﬁc immune responses (CD4, CD8, and antibody responses) of all 22 study patients (pt) at baseline (week 1, w1), during vaccination (week 3 to 25, w3 to w25) and 2 months after last vaccination (follow-up, FU). White boxes indicate negative, black cells indicate positive immune responses. Gray boxes, not assessable due to study discontinuation.Kloor et al. Clin Cancer Res; 26(17) September 1, 2020 CLINICAL CANCER RESEARCH 4508Downloaded from http://aacrjournals.org/clincancerres/article-pdf/26/17/4503/2062752/4503.pdf by guest on 29 November 2024 preexisting FSP-speci ﬁc immune responses, which have previously been detected in Lynch syndrome mutation carriers and patients with MSI cancer (16), have enhanced the local immune responses upon vaccination. This hypothesis is supported by the fact that the patient who developed most pronounced injection site reactions had strong preexisting DTH responses (patient 5) against all vaccine peptides. (3) In comparison to most tumor antigens previously used in Montanide- containing formulations, FSP antigens are long neoantigens capable of inducing more pronounced immune responses (1). Thus, the observed side effects underline the immunogenicity of FSP neoantigens and their great potential as vaccine agents. The relevance of FSP neoanti- gens for antitumor immune responses is underlined by the recent observation that the extent of responses to PD-1 immune checkpoint blockade is related to the insertion/deletion (indel) mutation load in MSI tumors (44). For future FSP-based vaccine applications, DTH status should be considered as a potential predictor of local adverse effects, and alternative adjuvant combinations should be considered. In summary, we demonstrate in a ﬁrst-in-man study that vaccina- tion with MSI-induced FSP neoantigens is well tolerated and induces T-cell-based and humoral immune responses. This study marks an important step towards the establishment of a ﬁrst neoantigen-based nonviral vaccine for the prevention of human cancers. Lynch syn- drome is a unique scenario to achieve this goal, because the success of a vaccine can be monitored in a de ﬁned high-risk population. Disclosure of Potential Con ﬂicts of Interest M. Kloor reports grants from Oryx GmbH und Co KG during the conduct of the study. M. Reuschenbach reports grants from Oryx GmbH und Co KG (sponsor of the study) during the conduct of the study, patent for content related to this study pending (as inventor), and reports employment with MSD Sharp & Dohme GmbH. M. Reuschenbach's contribution to the submitted work fully originated from thetime she was a full-time employee of Heidelberg University Hospital. M. Tariverdian reports grants from Oryx GmbH und Co KG during the conduct of the study. M. von Knebel Doeberitz reports grants from Oryx GmbH und CoKG during the conduct of the study. No potential con ﬂicts of interest were disclosed by the other authors. Authors ’Contributions M. Kloor: Conceptualization, data curation, supervision, investigation, visualization, writing-original draft, writing-review and editing. M. Reuschenbach: Conceptualization, data curation, investigation, methodology, writing-review and editing. C. Pauligk: Data curation, formal analysis, validation, visualization, writing- review and editing. J. Karbach: Data curation, formal analysis, validation, investigation, writing-review and editing. M.-R. Ra ﬁyan: Data curation, investigation, writing-review and editing. S.-E. Al-Batran: Supervision, investigation, project administration, writing-review and editing. M. Tariverdian: Validation, investigation, writing-review and editing. E. Jaeger: Conceptualization, resources, supervision, funding acquisition, validation, investigation, project administration, writing-review and editing. M. von Knebel Doeberitz: Conceptualization, resources, supervision, funding acquisition, validation, investigation, project administration, writing-review and editing. Acknowledgments Oryx GmbH und Co KG of Baldham, Germany, was the sponsor of the study according to the German Pharmaceutical Law (Arzneimittelgesetz). Oryx GmbH und Co KG also provided research funds to the Heidelberg University Hospital (to M. von Knebel Doeberitz) and the Northwest Hospital Frankfurt (to E. Jaeger and S.A. Al- Batran). The authors thank Claudia Wahle, Jonathan D €orre, Beate Kuchenbuch, Nina Nelius, and Petra H €oﬂer for excellent technical assistance. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received December 10, 2019; revised April 2, 2020; accepted June 10, 2020; published ﬁrst June 15, 2020. References 1.