16, 19). Here we present the ﬁrst-in-human step toward the goal of a cancer- preventive vaccine in Lynch syndrome. We report the results of a vaccine trial with three shared FSP antigens (Micoryx). The results of the trial demonstrate that FSP antigen vaccination is safe and induces cellular and humoral immune responses in all patients vaccinated per protocol. Patients and Methods Investigational agents The following peptides were used for vaccination: AIM2(-1) HSTIKVIKAKKKHREVKRTNSSQLV, HT001(-1) EIFLPKGRSN- SKKKGRRNRIPAVLRTEGEPLHTPSVGMRETTGLGC, TAF1B(-1) NTQIKALNRGLKKKTILKKAGIGMCVKVSSIFFINKQKP. FSPs were synthesized, formulated, packed, and distributed in accordance with current GMP protocols by Bachem. Montanide ISA-51 VG was supplied by SEPPIC GmbH. Before vaccination, the lyophilized pep- tides (150 mg) were removed from the freezer and dissolved separately in 300- mL water [HT001(-1), TAF1B(-1)] or 300- mL PBS [AIM2( /C01)]. Two hundred microliters of each of the peptide solutions (100 mg) was mixed with 200- mL Montanide ISA-51 VG until both liquids were intermixed and a thick, creamy, opaque, and consistent emulsion was generated. Four hundred microliters of the emulsions (100- mg peptide each) was administered separately subcutaneously. Vaccination dosing was selected according to previous peptide vaccination trials (20). For DTH, 90 mL of the dissolved peptides (30- mg peptide) was mixedwith an additional 210- mL PBS and injected intradermally at separate sites distant from the vaccination sites to generate a visible and palpable skin depot. Study endpoints and inclusion criteria This study was an open-label single-arm phase I/IIa study of immunization with MSI-induced FSPs combined with MONTANIDE ISA-51 VG in patients with advanced MSI colorectal cancer. Primary endpoints were safety (phase I) and immunogenicity (phase IIa). Secondary endpoints were tumor response (both phases) and immu- nogenicity (phase I) and safety (phase IIa), following a study scheme described previously (21). The study started as phase I with six patients. Interim safety results of phase I were submitted to the regulatory authorities, the Paul-Ehrlich Institute and the responsible IRB (Ethics Committee Landes €arztekammer Hessen), after approval the study continued as phase IIa ( n¼16). For both study phases, patients were eligible for inclusion if they fulﬁlled the following criteria: (i) history of histologically con ﬁrmed Lynch syndrome-related or sporadic MSI colorectal cancer of advanced-stage (UICC stage III/UICC stage IV), comprising patients with lymph node metasta ses (UICC III) and patients with metastasis to distant organs or peritoneal carcinomatosis (UICC IV); (ii) prior adjuvant standard the rapy or prior palliative standard therapy in the ﬁrst-, second-, and third-line or patient has refused adjuvant or palliative standard th erapy; (iii) expected survival of at least 6 months, (iv) full recovery from surgery, chemotherapy or r a d i a t i o nt h e r a p y ;( v )E C O Gp e r f o r m a n c es t a t u s0 ,1 ,o r2 ;( v i ) adequate laboratory values (neutrophil count ≥1.5/C2109/L, lymphocyte count ≥0.5/C2109/L, platelet count ≥100/C2109/L, serum bilirubin <2 mg/dL). In certain cases ( n¼3), patients affected by Lynch syndrome and being at a high risk of disease relapse or secondary tumors were included if they developed recurrent cancers of stages lower than UICC III. MSI status was determined by PCR ampli ﬁcation of the Bethesda marker panel recommended by the NCI (BAT25, BAT26, D2S123, D5S346, D17S250; ref. 22) complemented by the mononucleotide marker CAT25 (22). Fluorescently labeled oligonucleotide primers were used for ampli ﬁcation, and ampli ﬁed fragments were visualized on an ABI3100 sequencer as described previously (23). When two or more of the markers displayed novel alleles, a tumor was classi ﬁed as MSI. Patients were excluded if they had chemotherapy, radiation therapy, or immunotherapy within 4 weeks before study entry, untreated brain metastases, immunode ﬁciency syndromes or autoimmune disease, or did not give written informed consent. The study was conducted in accordance with the Declaration of Helsinki. The study was performed after approval by regulatory authorities and an institutional review board (Paul-Ehrlich institute and ethics committee of the Landes €arz- tekammer Hessen) and registered (ClinicalTrials.gov Identi ﬁer: NCT01461148, EudraCT No. 2011-000765-12). All study patients gave written informed consent. The study was sponsored by Oryx GmbH und Co KG. All authors had access to the study data and reviewed and approved the ﬁnal manuscript. Statistical considerations Assuming a likelihood of 10% for a vaccine-induced immune response (de ﬁned as positive response against one vaccine peptide for at least one assay modality) and a binomial distribution for the number of responses, a total of 22 patients (six patients from phase I, 16 patients from phase IIa) was required to obtain 80% power. Therefore, a sample size of 16 patients was set for phase IIa. For a likelihood ofTranslational Relevance Effective cancer-preventive vaccines would mark a revolution in medicine. Demonstration of their feasibility is a major milestone on the way towards tailored prevention approaches. This is only possible in de ﬁned high-risk populations. We demonstrate in the setting of Lynch syndrome and history of mismatch repair- deﬁcient colorectal cancer that vaccination with shared and pre- dictable neoantigens is clinically safe and does not induce systemic side effects. Moreover, our data provide evidence that a combina- torial vaccine of three recurrent mutation-induced neoantigens leads to the induction of measurable immune responses in all patients vaccinated per protocol. Our study underlines the feasi- bility of vaccination with mutational neoantigens for cancer pre- vention in Lynch syndrome. These results strongly call for clinical trials evaluating possible cancer-preventive effects of this approach.Kloor et al. Clin Cancer Res; 26(17) September 1, 2020 CLINICAL CANCER RESEARCH 4504Downloaded from http://aacrjournals.org/clincancerres/article-pdf/26/17/4503/2062752/4503.pdf by guest on 29 November 2024 13.5% for a patient to display a response, the power in this scenario is 90%. Vaccine scheme Patients were vaccinated once a week for 4 weeks, followed by a 4-week rest period (one cycle). This schedule was repeated up to a total of three cycles (12 vaccinations; Fig. 2 ). Vaccinations were performed alternately on the right and left upper arm. Study treatment was discontinued in case of progression or intolerable toxicity. Toxicity assessment Patients were considered evaluable for toxicity if they received at least one vaccination. Patients were seen at baseline/screening and at every vaccination visit for safety assessment until disease progression or discontinuation for other reasons. Patients were assessed for adverseevents by nondirective questioning. Adverse events were also recorded when reported by the patient during or between visits or through physical examination, laboratory test, or other assessments. Toxicity assessment was performed according to the protocols of previous peptide vaccination studies (21). Laboratory tests included autoanti- body status (ANA, ANCA, AMA, microsomal antibodies, TSH recep- tor antibodies), regular complete blood cell counts, and chemistry. All adverse events were documented according to the Common Toxicity Criteria for Adverse Events (CTCAE, version 4.0, https://ctep.cancer. gov/protocolDevelopment/electronic_applications/ctc.htm). Poten- tial relationships of adverse events to the investigational agents were determined by the investigator. Immunogenicity assessment Patients were considered evaluable for immunologic ef ﬁcacy if they completed at least one study cycle with subsequent immune assess- ment (week 9). Antigen-speci ﬁce fﬁcacy of the vaccine was de ﬁned as the induction of immune responses against each FSP. Immunologic responses were evaluated in weeks 9, 17, and 25 (T cells by DTH and ELISpot) and in weeks 3, 9, 11, 17, 19, and 25 (antibodies by ELISA) and were compared with the antigen-speci ﬁc immune response mea- sured prior to vaccination (week 1). Immune responses were reas- sessed 2 months after end of the study. For the assessment of DTH, 30mg of each FSP was administered intradermally without adjuvant, and reactions were documented after 48 hours. DTH was considered positive if an area of redness and induration of >4 mm in diameter ( þ) or an enlarged area of redness and palpable induration of >8m mi n Figure 1. A,Concept of FSP vaccines against MSI can- cer. Tumor initiation and progression of MSI cancers are triggered by DNA MMR de ﬁcien- cy. MMR de ﬁciency leaves errors in DNA, which result from polymerase slippage events during DNA replication, unrepaired. Therefore, MSI cells accumulate a high num- ber of somatic mutations. Most mutations are incompatible with cell survival; however, if the right combination of mutations occurs in an MMR-de ﬁcient cell, the respective clone can progress to manifest cancer. Selection of cell clones capable of progressing into cancer results in the occurrence of identical muta- tions across individual MSI cancers. Most mutations in MMR-de ﬁcient cells result from polymerase slippage and represent insertion/ deletion (indel) mutations at repetitive sequence stretches. Indels affecting coding microsatellites can give rise to predictable neoantigens, termed FSP neoantigens. Vaccination with such FSP neoantigens holds the potential of sensitizing the immune system speci ﬁcally against neoan- tigens associated with cancer initiation and progression. B,The FSP vaccine encom- passes peptides de rived from the ( /C01) frameshift sequence of the cMS-bearing genes TAF1B, HT001, AIM2 .T y p eo f the coding microsatellite and mutation frequency in MSI colorectal cancer and MSI endometrial cancer are provided in the table. Amino acid sequences of the peptides are provided in the bottom panel. x, Mutation frequencies derived from Ballhausen, et al (24). Figure 2. Vaccination scheme. A total of 12 applications of the three study FSP antigens were administered together with Montanide ISA 51 VG in three cycles over a 6-month period.Frameshift Peptide Vaccine for MSI Cancer AACRJournals.org Clin Cancer Res; 26(17) September 1, 2020 4505Downloaded from http://aacrjournals.org/clincancerres/article-pdf/26/17/4503/2062752/4503.pdf by guest on 29 November 2024 diameter and/or central necrosis ( þþ) were observed, following criteria described previously (21). To determine the presence of FSP-speci ﬁc T cells in the peripheral blood, CD4-positive and CD8-positive T cells were separated from peripheral blood lymphocytes by antibody-coated magnetic beads (Minimacs; Miltenyi Biotec). IFN gELISpot was performed as reported previously (20). ELISpot responses were considered positive if the number of spots in peptide-exposed wells was at least two times higher than the number of spots in unstimulated wells, with a minimum of 10 peptide-speci ﬁc spots per 25,000 T cells. FSP-speci ﬁc antibodies were determined by peptide ELISA from serum samples as described previously (25). Brie ﬂy, peptides were coated to 96-well polystyrol microtiter plates “Maxisorp ”(Nunc) at a concentration of 40 mg/mL in PBS. Peptide binding and saturating peptide concentrations were determined by alkaline phosphatase peptide competition. Sera were diluted 1:100 in PBS and 0.5% casein. Antibodies were detected using an HRP-labeled rabbit anti –human-IgG antibody (Jackson Immunor- esearch; 1:10,000 in PBS, 0.5% casein) and TMB as a substrate (Sigma). Optical density (450 nm) was measured after adding 1 N H 2SO4. Positive immune responses were de ﬁned as positive DTH response and/or humoral immune response and/or CD8 and/or CD4 cellular immune response against at least one of the FSPs exceeding the assay- speciﬁc cut-off values. Tumor status assessment and clinical response Assessment of tumor status and clinical response was performed according to previously described study protocols (21). Patients were considered evaluable for tumor response if they had measurable tumors with radiological tumor imaging (CT, MRI) at baseline. For those patients, imaging was performed every 8 weeks until end or discontinuation of study. Post-study follow-up was performed after 6 months. Tumor status was classi ﬁed according to RECIST whenever applicable. According to the protocol, signs of progressive disease, and death events were assessed and recorded during the trial and at study follow-up. Progression-free survival (PFS) was de ﬁned as the time period between the day of ﬁrst study treatment and the detection of progressive disease, or death from any cause, whichever occurred ﬁrst. Overall survival (OS) was de ﬁned as the time period between the day of ﬁrst study treatment and death from any cause. Patients without the respective event being observed at the time of maximum follow-up were censored at this time point. Results Patients Patients with MSI colorectal cancer ( n¼22) were included in the study (phase I: n¼6, phase II: n¼16; consort diagram is provided inFig. 3 ). Patients' baseline characteristics are provided in Table 1 . The majority of patients had UICC stage III cancer ( n¼15), three patients had UICC stage IV cancer, two patients had UICC stage II cancer, one patient had UICC stage I cancer at ﬁrst cancer diagnosis. In 20 patients (90.9%), the primary tumor was localized in the colon, and two patients had a history of rectal cancer. Nineteen patients were tumor-free at study inclusion, thus three patients were evaluable for assessing tumor response. All patients had received at least one of the three common modalities of solid tumor treatment, that is, surgery, irradiation, and systemic therapy prior to study inclusion. The majority of patients was male ( n¼15), median age was 54.8 years with only ﬁve (23%) patients being older than 60 years at study inclusion. Most patients had a normal or only slightly impaired ECOG performance status (ECOG 0, n¼15; ECOG 1, n¼6). Only one patient was included into the studywith ECOG 2. In 18 patients, systemic chemotherapy had been applied prior to study inclusion (for details see Supplementary Data S2). For 16 patients, information about MMR gene germline sequencing was available; 13 patients harbored a pathogenic germline variant in one of the MMR genes, in the remaining three patients, no germline variant had been identi ﬁed in the four MMR genes. All 22 patients received at least one application of the FSP vaccine, 19 completed the ﬁrst treatment cycle (four applications) and were available for ﬁrst analysis of immunogenicity. Sixteen patients com- pleted the entire vaccination scheme of three cycles (12 applications). Premature discontinuation occurred in six patients and was due to progressive disease ( n¼1), toxicity ( n¼3), or patient ’s wish ( n¼3) with two simultaneous reasons for discontinuation in one patient. Toxicity During phase I of the trial, seven SAEs occurred in two patients; however, all SAEs were restricted to patients with UICC stage IV cancer, and none of the SAEs was considered as possibly related to the study treatment. In none of the vaccinated patients, any evidence of autoimmunity was detectable. Therefore, the safety pro ﬁle of the vaccine was considered favorable, and continuation of the study with phase IIa was approved. In the combined analysis of phase I and phase IIa, all 22 patients were evaluable for toxicity. No SAR or SUSAR were documented, and no new or unexpected risks appeared in the study. No deaths with possible relation to study therapy occurred. Reported death events during the study (two patients) were related to progression of the underlying tumor disease and not connected to the study medication. All reported SAEs were unrelated to the study treatment. Adverse drug reactions, that is, events reported to have an at least possible Figure 3. Trial pro ﬁle./C3, One patient had two reasons for study discontinuation: Phase I: progressive disease, n¼1; toxicity, n¼1; patient's wish, n¼1; toxicity and patient's wish, phase II: toxicity, n¼1; patient's wish, n¼1.Kloor et al. Clin Cancer Res; 26(17) September 1, 2020 CLINICAL CANCER RESEARCH 4506Downloaded from http://aacrjournals.org/clincancerres/article-pdf/26/17/4503/2062752/4503.pdf by guest on 29 November 2024 relationship to the administration of the study drug, had a maximum of grade 2 ( Table 2 ). Apart from injection site reactions in 17 (78%) of patients (grade 1 or 2), adverse drug reactions were rare. Three patients discontinued trial participation because of toxicity, one of these was dose limiting (allergic reaction, NCI grade 2). Two patients showed prolonged redness, induration, and swelling at the injection sites, which persisted more than 1 year after the last vaccine administration.Immunogenicity In total, 19 patients were evaluable for immunologic ef ﬁcacy of the FSP vaccine, because they completed at least one treatment cycle followed by at least one subsequent assessment of immune responses (week 9 or later). FSP-speci ﬁc immune responses were monitored by delayed-type hypersensitivity reaction (DTH), IFN gELISpot (CD4 and CD8 T cells separately), and ELISA (humoral immune responses). Although baseline immune responses (week 1) against the vaccine peptides were detected in 11 of 22 patients (50%), all evaluable patients showed an immune response against at least one of the vaccine peptides during and after vaccination ( Fig. 4 ). All 16 patients who were vaccinated per protocol and completed the entire vaccination scheme developed an immune response against at least one of the vaccine peptides. In most patients, positive immune reactions emerged at week 9 and remained detectable until the end of treatment. The most frequent type of detectable immune response was of antibody nature, followed by CD4 T cells, and CD8 cell responses ( Fig. 4 ). A subset of patients ( n¼5) showed a delayed time hypersensitivity (DTH) response upon vaccination. In detail, two (10.5%) of the evaluable patients showed induction of a DTH reaction upon vacci- nation for the peptides AIM2 (patients 8 and 10) and HT001 (patients 10 and 12), 3 (15.7%) for the peptide TAF1B (patients 3, 10, and 11). Strong baseline DTH reactions were seen in one patient (patient 5) for all three peptides. Full response data for the intention-to-treat population are pro- vided as Supplementary Data S1. Tumor response Tumor response according to RECIST criteria could be assessed in three patients. Two (66.6%) of these patients showed stable disease as best overall response. One heavily pretreated patient with bulky metastases (patient 12) showed a stable disease and stable CEA levels over 7 months under the study treatment. In one patient, study participation was discontinued because of toxicity, and no further restaging was performed. No secondary tumor events were recorded during the study period. Discussion Of 19 analyzed patients (per protocol set), all patients (100%; 95% conﬁdence interval, 82.35% –100%) showed FSP-speci ﬁc immune responses after vaccination, demonstrating that vaccination with FSP antigens induces signi ﬁcant humoral and cellular immune responses. Notably, also the majority of patients with baseline immu- nity against at least one of the FSP antigens (11 patients) developed additional immune responses in another category after vaccination (10 patients, 91%). Compared with other vaccine trials (26) with cancer antigens, the frequency of induced immune responses was high. Although quan- tiﬁcation of ELISpot data after in vitro stimulation with peptides should be performed with caution, substantially elevated spot numbers were observed in patients after vaccination compared with baseline (Supplementary Data S3). This might re ﬂect the fact that mutational FSP neoantigens rarely induce immune tolerance (27), which is a common problem in cancer vaccines based on self-antigens (28). In addition, the FSP neoantigens evaluated in the Micoryx study encom- pass long neopeptide stretches ranging between 13 [AIM2( /C01)] and 32 [HT001( /C01)] amino acids, potentially encompassing several immune- relevant epitopes. Although no HLA typing was performed in this study due to limited sample availability, we speculate that the lack of CD8-positive T-cell responses in nine of 16 patients vaccinated perTable 1. Patient baseline characteristics. Characteristics Total n¼22 Age, median (range), years 54.8 (31.0 –80.7) <45,n( % ) 4 (18) 45 to ≤50 3 (14) 50 to ≤55 3 (14) 55 to ≤60 7 (32) >60 5 (23) Gender, n(%) Female 7 (32) Male 15 (68) Tumor location, n(%) Colon 20 (91) Rectum 2 (9) Initial UICC stage, n(%) I 1 (5) II 2 (9) III 15 (68) IV 3 (14) Unknown 1 (5) Tumor status at study inclusion, n(%) Tumor-free 19 (86) Not tumor-free 3 (14) Time since initial diagnosis, n(%) <5 years 8 (36) 5t o≤10 years 4 (18) 10 to ≤15 years 4 (18) 15 to ≤20 years 2 (9) >20 years 4 (18) MMR gene germline mutation, n(%) MLH1 6 (27) MSH2 6 (27) MSH6 1 (5) PMS2 0 None in the genes above 3 (14) No germline data available 6 (27) Performance status (ECOG), n(%) 0 15 (68) 1 6 (27) 2 1 (5) Table 2. Adverse drug reactions, maximum CTC severity grade by patient and by category (CTCAE 4.0). Event Total n¼22 Grade 1 2 3 4 Allergic reaction, n(%) 1 (5) — 1 —— Dizziness 1 (5) 1 (5) —— — Dyspnea 1 (5) — 1 —— Injection site reaction 17 (78) 14 (64) 3 (14) —— Peripheral sensory neuropathy 1 (5) 1 (5) —— — Rash 3 (14) 3 (14) —— — Skin hyperpigmentation 1 (5) 1 (5) —— —Frameshift Peptide Vaccine for MSI Cancer AACRJournals.org Clin Cancer Res; 26(17) September 1, 2020 4507Downloaded from http://aacrjournals.org/clincancerres/article-pdf/26/17/4503/2062752/4503.pdf by guest on 29 November 2024 protocol may re ﬂect the fact that no HLA class I-binding peptide derived from the FSPs in the vaccine was processed and presented in these patients. Additional studies are required to speci ﬁcally determine relevant epitopes contained in FSP neoantigens and to compare the efﬁcacy of different vaccination approaches, accounting for the prev- alence of different HLA types in target populations (29, 30). Clinical ef ﬁcacy of the vaccine could not be examined in frame of this study, because 19 patients were tumor-free at study inclusion. Future clinical studies are currently planned to examine the ef ﬁcacy of the FSP vaccine in a larger set of patients in a therapeutic and preventive setting. Potential future applications of the FSP vaccine in a therapeutic setting comprise combinations with novel immune-modulatory agents such as anti –PD-1 or PD-L1 antibodies that have shown high efﬁcacy in a subset of patients with MSI cancer (31, 32). In contrast to immune checkpoint modulators, FSP vaccination also holds high potential for tumor prevention in Lynch syndrome (33), due to the much less pronounced side effect pro ﬁle. The high-risk population of Lynch syndrome mutation carriers represents a unique scenario to evaluate the concept of tumor antigen-speci ﬁc vaccines for cancer prevention. Such a tumor-preventive vaccine may be applied alone or in combination with chemopreventive agents such as aspirin (34). Important from the perspective of prevention, the vaccine specif- ically targets FSP neoantigens derived from mutations with a potential role as functionally relevant drivers. A driver function of mutations inactivating the genes AIM2, HT001 (ASTE1), TAF1B would be compatible with the fact that they occur signi ﬁcantly more frequently in MSI colorectal cancer than expected by chance (www.seltarbase. org; Fig.