Vol.:(0123456789)1 3Cancer Immunology, Immunotherapy (2023) 72:2045–2056 https://doi.org/10.1007/s00262-023-03386-7 RESEARCH Preliminary clinical study of personalized neoantigen vaccine therapy for microsatellite stability (MSS)‑advanced colorectal cancer Yao‑Jun Yu1 · Na Shan2 · Li‑Yi Li1 · Yue‑Sheng Zhu3 · Li‑Miao Lin3 · Chen‑Chen Mao1 · Ting‑Ting Hu4 · Xiang‑Yang Xue5 · Xiao‑Ping Su3,5 · Xian Shen1,6,7 · Zhen‑Zhai Cai3 Received: 28 November 2022 / Accepted: 25 January 2023 / Published online: 16 February 2023 © The Author(s) 2023 Abstract Immunotherapy based on immune checkpoint inhibitors (ICIs) has provided revolutionary results in treating various cancers. However, its efficacy in colorectal cancer (CRC), especially in microsatellite stability-CRC, is limited. This study aimed to observe the efficacy of personalized neoantigen vaccine in treating MSS–CRC patients with recurrence or metastasis after surgery and chemotherapy. Candidate neoantigens were analyzed from whole-exome and RNA sequencing of tumor tissues. The safety and immune response were assessed through adverse events and ELISpot. The clinical response was evaluated by progression-free survival (PFS), imaging examination, clinical tumor marker detection, circulating tumor DNA (ctDNA) sequencing. Changes in health-related quality of life were measured by the FACT-C scale. A total of six MSS–CRC patients with recurrence or metastasis after surgery and chemotherapy were administered with personalized neoantigen vaccines. Neoantigen-specific immune response was observed in 66.67% of the vaccinated patients. Four patients remained progression- free up to the completion of clinical trial. They also had a significantly longer progression-free survival time than the other two patients without neoantigen-specific immune response (19 vs. 11 months). Changes in health-related quality of life improved for almost all patients after the vaccine treatment. Our results shown that personalized neoantigen vaccine therapy is likely to be a safe, feasible and effective strategy for MSS–CRC patients with postoperative recurrence or metastasis. Keywords Colorectal cancer · Immunotherapy · MSS · Neoantigen · Personalized vaccine Introduction Colorectal cancer (CRC) is one of the most common malignant tumors in the world, accounting for approxi - mately 1.2 million diagnoses and over 600,000 direct or Implication: Personalized neoantigen vaccine therapy is expected to bring a major breakthrough in treating patients with MSS–CRC. Yao-Jun Yu and Na Shan are the same first authors. * Xiao-Ping Su bloooge@163.com * Xian Shen 13968888872@163.com * Zhen-Zhai Cai caizhenzhai@wmu.edu.cn 1 Department of Gastrointestinal Surgery, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, People’s Republic of China 2 Department of Gastroenterology, Shaoxing People’s Hospital, Shaoxing 312000, People’s Republic of China 3 Department of Gastroenterology, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, People’s Republic of China4 Department of Gastroenterology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, People’s Republic of China 5 School of Basic Medicine, Wenzhou Medical University, Wenzhou 325000, People’s Republic of China 6 Department of Gastrointestinal Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, People’s Republic of China 7 Department of General Surgery, The Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province, People’s Republic of China 2046 Cancer Immunology, Immunotherapy (2023) 72:2045–2056 1 3 indirect deaths each year [1 ]. The prognosis of advanced CRC is poor, and the current regimens mainly consist of surgical resection, chemoradiotherapy, and targeted ther - apy [2 ].The majority of the patients with metastatic colo- rectal cancer (mCRC) lose the opportunity of resection surgery at the initial diagnosis, while several problems have been encountered in the selection of the treatment population and the course of disease in chemoradiotherapy and targeted therapy [3 , 4].There is no doubt that immu- notherapy is now a standard treatment along with sur - gery, chemotherapy, radiotherapy and targeting therapy; and the field of cancer immunotherapy is continuing to develop [5 ]. In 2017, immune checkpoint inhibitors (ICIs) were approved for clinical use in patients with microsat- ellite instability-high (MSI-H) CRC. However, only 5% of advanced CRC patients with MSI-H can benefit from ICIs therapy. Most advanced CRC patients with micros- atellite stability (MSS) fail to benefit from this treatment [6–8]. Therefore, there is an urgent need to develop a novel immunotherapy strategy for patients with MSS-CRC and to facilitate personalized treatment of MSS–CRC. Tumor vaccines play an important role in amplifying the anti-tumor immune response in cancer patients. Neoanti - gens are tumor-specific antigens that arise due to somatic mutations in the tumor genome. Neoantigens possess sig- nificant immunogenic potential as they are not expressed in normal tissues. Because of their strong affinity for major histocompatibility complex (MHC), neoantigen-based ther - apy can be recognized by the host's immune system as non- self, which would prevent them from inducing central and peripheral immune tolerance mechanisms [9 , 10]. Personal- ized vaccines have achieved therapeutic effects in a variety of tumors. In July 2017, two clinical studies demonstrated the successful treatment of advanced melanoma based on personalized neoantigens [11, 12]. Maria et al. identified neoantigen-reactive tumor-infiltrating lymphocytes (TILs) in patients with common gastrointestinal tumors; they also identified the immunogenicity of gene products encoded by somatic nonsynonymous mutations based on in vitro T-cell recognition assays [ 13]. Eric Tran et al. identified a poly - clonal CD8 + T-cell response against mutant KRAS G12D in tumor-infiltrating lymphocytes obtained from a patient with metastatic colorectal cancer [14].These findings demonstrate that neoantigen vaccines may hold great potential for the treatment of a variety of tumors, including colorectal cancer. Our previous study confirmed the effectiveness of the neo- antigen vaccine in a mouse CRC model [15].However, the role of neoantigens in MSS–CRC patients remains unclear. In this study, we conducted a clinical research of neoan- tigen vaccines for MSS–CRC patients with recurrence or metastasis after surgery and chemotherapy and explored the identification and effective screening of neoantigens. Our results preliminarily demonstrate the feasibility, safety and immunological activity of the neoantigen vaccine, which is expected to bring a major breakthrough in treat- ing patients with MSS–CRC, prolonging the progression- free survival time, and improving the treatment and quality of life of these patients. Patients and methods Study oversight This study is a single-arm, single-center clinical study conducted at the Second Affiliated Hospital of Wen- zhou Medical University in China and was registered at the Chinese Clinical Trial Registry (http:// www. chictr. org. cn/; ChiCTR1900022372; The registration date was April 8, 2019). The corresponding design, protocol, and modification of this study, involving human samples, were approved by the Medical Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University (MEC numbers: LCKY2018-67). The protocols were strictly performed according to the Helsinki Declaration, and the study adhered to the privacy rights of humans. All enrolled patients signed a written informed consent form. The clini - cal trial was conducted following the agreement and its revised scheme, and all authors can view the experimental data and ensure the accuracy and integrity of the data and analysis. Patients The patients were included based on the following inclu- sion criteria: (1) diagnosed with CRC, with TNM stages of IIIb–IV; (2) 18 to 75 years old male or female, with at least one radiographically measurable lesion; (3) patients had tumor recurrence or metastasis after surgery and chem- otherapy, and their life expectancy should be at least 3 months; and (4) serum bilirubin not higher than 1.5 times the upper limit of normal (ULN), ALT or AST not higher than 2.5 times ULN, and creatinine clearance higher than 50 mL/min. For chemotherapy and targeted drug therapy, patients were required to have a washout period of at least 2 weeks. Patients were excluded from this study based on the following criteria: (1) patients with HIV infection, HCV infection, serious coronary artery disease, or other dis - eases deemed unsuitable for inclusion in this study by the researchers; (2) patients with a history of bone marrow or organ transplantation; (3) patients with coagulation dys- function; (4) drug abuse, alcohol abuse, clinical or psycho- logical or social factors affecting informed consent or study 2047 Cancer Immunology, Immunotherapy (2023) 72:2045–2056 1 3 implementation; (5) patients with secondary brain metas- tasis; and (6) any uncertain factors affecting the safety or compliance of patients. Neoantigen identification Sample collection and DNA/RNA preparation All tumor tissues were obtained from paraffin sections retained after surgery (patients 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11). Blood samples were collected and mixed with an EDTA-based anticoagulant. The extraction of DNA and RNA was performed from the paraffin sections of the post- operative tumor tissues with the All Prep DNA/RNA Mini Kit (Qiagen, Valencia, CA, USA/Illumina, San Diego, CA, USA); DNA from EDTA-anti coagulated peripheral blood samples was extracted with the QIAamp DNA Blood Mini Kit (Qiagen). Whole‑exome DNA and RNA sequencing DNA sequencing was performed by generating two groups of double-ended sequencing results, corresponding to the collected tumor and blood cells (normal samples), in FASTQ format in Illumina HiSeq X10 platform (paired-end run, 150 bp). Fastp (Fastp v0.16.0) was used to control the qual- ity of the original sequencing data and filter the low-quality data. Burrows-Wheeler Aligner (BWA-MEM, v.0.7.17- r1188) was used to compare the filtered clean reads with GRCh38/hg38 human reference genome to generate the sequence alignment map (SAM) file. Two groups of BAM files were obtained by utilizing SAMtools (v1.7–2) to con- vert SAM files into binary alignment map (BAM) files and establish indexes. The Genome Analysis Toolkit (GATK, v.4.1.3.0) was utilized for the removal of the variations from the DNA sequencing results. For RNA sequencing, terminal sequencing results, cor - responding to the collected tumor tissues, in FASTQ format were generated in Illumina HiSeq X10 platform (paired-end run, 150 bp). Fastp (Fastp v0.16.0) was utilized to control the quality of RNA sequencing data and filter low-quality data. The STAR software (2.6.0a) was utilized to compare the filtered clean reads with GRCh38/hg38 human reference genome to generate a SAM file. Based on SAMtools (V1.7), the SAM file was converted into a BAM file, and indexes were established. The Genome Analysis Toolkit (GATK, v.4.1.3.0) was utilized for detecting the variations in the RNA sequencing results. Human leukocyte antigen (HLA) typing Based on the DNA sequencing data, patients’ HLA class I genotypes (HLA-A, HLA-B, and HLA-C) were assessed using the OptiType software. Based on the RNA clean reads, HLA class II genes (DQA1, DQB1, DRB1, DRA, DPA1, and DPB1) encoding MHC class II receptors were analyzed based on Seq2HLA (V 2.3). Neoantigen filtering Integration analysis of DNA mutations and HLA was con - ducted based on MuPeXI (version 1.2.0) [16], NetMHCpan 4.0, and the variant effect predictor (VEP) (version ensembl- vep-release-92) databases [17]. The HLA-binding affinity scores (IC50) were obtained for all 8–11-mer variants for MHC class I molecules and 12–15-mer variants for MHC class II molecules. Expression level of mutant genes was determined from RNA-seq data in transcripts per million. Each peptide was given a priority score on the basis of HLA- binding affinity, expression level, similarity to self-peptides, and mutant allele frequency. Peptides with a priority score larger than 0 were selected as neoantigen candidates. Neo- antigen candidates with high priority score will be finally selected to synthetic vaccine. MSI testing MSI sensor scans a given reference genome to find the loca- tions of homologous polymers and microsatellites. In this study, homopolymers of at least 5-bp length and microsatel- lites of maximum repeat unit length of five were recorded from the reference genome, and the location and flanking sequence of each site were saved in a site file for subsequent analysis. A standard χ2 test was performed at each site hav - ing at least 20 reads in both the tumor and normal samples to assess the goodness-of-fit between their respective k -mer distributions (Sokal and Rohlf, 2012). For each sample, the total number of sites with sufficient data (at least 20 cross reads both in normal and tumor) and the number of somatic sites were noted; the percentage of somatic sites gave the score of MSI. Personalized neoantigen long‑peptide vaccine synthesis and vaccination The personalized neoantigen peptides were synthesized by the standard solid-phase synthetic peptide chemistry on CS 536 peptide synthesizer. The polypeptide is 27 amino acids long, with the mutation site as the center, and 13 amino acids are added before and after the polypeptide [18, 19]. The purification and purity analysis of the polypeptide were performed by reversed phase-high performance liq - uid chromatography (RP-HPLC) (> 98% purity, endotoxin concentration was less than 0.01 EU/g and trifluoroacetic acid (TFA) residue was less than 1%).The quality inspec- tion of the vaccine was carried out through the detection 2048 Cancer Immunology, Immunotherapy (2023) 72:2045–2056 1 3 of bacterial endotoxin, fungal D-glucan, bacterial and fun- gal smear, and acute toxicity test in mice. Then, the neo- antigen vaccine pools were mixed with 0.5 mg poly I:C (polyinosinic:polycytidylic acid) (Guangdong South China Pharmaceutical Co., Ltd.) and subcutaneously injected into both axilla and groin. Patients received the neoantigen vac- cine on days 1, 4, 8, 15, and 22 (priming phase) and at weeks 12 and 20 (boosting phase). Safety assay and changes in health‑related quality of life Safety was assessed by an evaluation of the clinical adverse events, which were graded according to the Common Ter - minology Criteria for Adverse Events (CTCAE) (version 4.0) published by the National Cancer Institute (NCI). In addition, the changes in blood routine, urine routine, liver and kidney functions, and electrolyte and coagulation func - tions before and after vaccination were also employed as an objective for safety evaluation. Changes in health-related quality of life were measured by the Functional Assessment of Cancer Therapy-Colorectal cancer (FACT-C) scale (version 4.0) which has been well- tested in cancer survivors and found to be reliable, valid, and responsive [20]. Clinical tumor response monitoring To complete clinical tumor marker detection, sufficient blood samples were collected at multiple time points before and after neoantigen vaccine therapy. Patients were evalu- ated for measurable tumor metastases with an enhanced CT scan every 2 months. Interferon‑gamma (IFN‑γ) enzyme‑linked immunospot (ELISpot) assay IFN-γ ELISPOT kit (Shenzhen Dakwei Biotechnology Co., Ltd, Anshan, China) was used to perform the ELISpot assay for detection of IFN-γ in neoantigen-specific T cells. Periph- eral blood mononuclear cells (PBMC) separation was per - formed with 10 mL of peripheral blood, while dendritic cells (DC) and T cells were separated by a 6-well plate culture. Thereafter, DC and T cells were mixed in a ratio of 10:1 and aliquoted into 96-well plates for co-stimulation culture; 200 µL Rosewell Park Memorial Institute medium (RPMI 1640) containing 100 U/mL IL-2, 10% fetal bovine serum, and 4 µg polypeptide was added to each well. The co-stimu- lated cells were then transferred to the Elispot plate. While 1/1000 CD3 and phosphate-buffered saline (PBS) solution were applied to the positive control and negative control wells, 4 µg polypeptide was added to the polypeptide well. The cells were grown in a CO2 incubator at 37 °C for 24 h. The cells were then rinsed, and 100 µL of fresh medium containing 1 µg/mL 7-B6-1-biotin was added to each well before incubating for 2 h at room temperature. Cells in each well were rinsed five times with PBS buffer before being incubated with 100 µL of 3, 3’,5,5’-tetramethylbenzidine (TMB) color-developing solution. Once spots in the well were visible, the color-developing reaction was stopped by rinsing with water. Detection of the neoantigen mutation in cfDNA Cell-free DNA (cfDNA) was extracted from serial plasma samples of the enrolled patients using QIAamp Circulating Nucleic Acid Kit (Qiagen). Detection of neoantigen muta- tion in cfDNA was performed as in the previous study with some changes [ 21]. We designed one reverse primer with variations in its 3′ nucleotide and one forward primer (Sup- plementary Table 1) for each neoantigen mutation. Poly - merase chain reaction (PCR) amplifications were performed using the LC480 SYBR Green mix (Roche Diagnostics, Meylan, France). The cycling conditions were as follows: denaturation for 10 min at 95 °C; amplification for 45 cycles, with denaturation for 10 s at 95 °C, annealing for 15 s at 65 °C, and extension for 20 s at 72 °C. Statistical analysis Data from all enrolled patients received at least 5 planned prime phase vaccinations were included in the safety and clinical outcome evaluation. Descriptive statistics were used to determine safety of neoantigen vaccine and changes in health-related quality of life. Bar chart of immune response and ctDNA dynamic curves were obtained using GraphPad Prism 8. Results Study design and patient characteristics This single-arm and single-center clinical trial evaluated the safety and feasibility of personalized neoantigen vaccines for the treatment of MSS–CRC patients with postoperative recurrence or metastasis. In this study, the safety and feasi- bility of personalized neoantigen vaccines were the primary endpoints, and the corresponding immune response and pro- gression-free survival (PFS) were the secondary endpoints. This study analyzed the personalized neoantigens based on the whole-exome sequencing and transcriptome sequencing data of tumor tissue sections and peripheral blood samples retained after the operation (Fig. 1a). The neoantigen vac- cine was mixed with poly I:C and subcutaneously injected 2049 Cancer Immunology, Immunotherapy (2023) 72:2045–2056 1 3 into the patients' axilla and groin regions (Fig. 1b). Accord- ing to the injection treatment plan, the priming-phase injec- tions were administered on days 1, 4, 8, 15, and 22, whereas the boosting-phase injections were administered on weeks 12 and 20 (Fig. 1c). After preliminary screening and clinician evaluation, 11 patients with postoperative recurrence or metastasis of CRC were initially included in the neoantigen screening. The postoperative tumor pathological tissues provided by four patients (patients W04, W05, W08, and W11) were screened out as they exhibited serious RNA degradation due to their long retention time, and the measured RNA data could not be used for the analysis of neoantigens. Also, patient W07 was screened out because the measured neoantigens were less consistent with the RNA transcription data. Finally, a total of 6 CRC patients with postoperative recurrence or metastasis were included to evaluate the safety and efficacy of neoantigen vaccine. After MSI detection, it was found Fig. 1 Vaccine preparation and injection schedule. a Tumor neo- antigen analysis. The flow chart illustrates the whole process of tumor neoantigen analysis. b Personalized vaccination. Personal- ized peptide vaccines and polyinosinic-polycytidylic acid injection were co-injected subcutaneously. Each injection was subcutaneously administered in the bilateral axilla and bilateral groin. c Vaccine administration. The patients received the neoantigen peptide vaccine at days 1, 4, 8, 15, and 22 (the initial phase), and weeks 12 and 20 (the enhancement phase), respectively. The black arrows indicate the time points at which the injection was scheduled Table 1 Baseline clinical characteristics of the patients M male; F female; L-OHP oxaliplatin; CAPE capecitabine; 5-FU 5-Fluorouracil; CPT-11 Irinotecan; BEV bevacizumab; C225 cetuximab; BAI, BAE bronchial artery infusion and embolization; MWA microwave ablation, MSI microsatellite instability; MSS microsatellite stabilityPatient ID Age Sex Cancer diagnosis Prior therapy Tumor stage Intervals between surgery and vac- cinationDistant metastases Tumor differentia- tionMSI W01 45 M Rectal Surgery, L-OHP, 5-FU, CAPE, CPT-11, BEVT4N2bM1c 9 months Peritoneal metas- tasisLow-medium dif- ferentiationMSS W02 58 M Colon Surgery, L-OHP, CAPET2N2aM1a 28 months Liver metastasis High-medium dif- ferentiationMSS W03 37 M Colon Surgery, MWA, BAI, BAET2N2aM1b 117 months Liver and lung metastasisMedium differen- tiationMSS W06 45 F Colon Surgery, MWA, CPT-11, 5-FU, BEV, Fruquin- tinibT3N1bM1a 23 months Liver metastasis Medium differen- tiationMSS W09 43 F Sigmoid colon L-OHP, CAPE, Surgery, MWA, CPT-11, 5-FU, BEVT3N1aM1a 19 months Liver and lung metastasisMedium differen- tiationMSS W10 72 M Rectal Surgery, L-OHP, CAPE, CPT-11, 5-FU, C225T4N2aM0 20 months Pelvic lymph nodes metastasesMedium differen- tiationMSS 2050 Cancer Immunology, Immunotherapy (2023) 72:2045–2056 1 3 that all 6 patients were MSS–CRC. The demographics and baseline clinical characteristics of the six patients are listed in Table 1. A total of 3192 nonsynonymous somatic single-nucle- otide variants (SNVs) were identified based on whole- exome sequencing of the tumor and matched peritumor tissues, with an average of 456 mutations (range 196–802) in each patient. Only 29.2% (range 7.08–48.43%) of these somatic mutations were confirmed for expression by transcriptome sequencing analysis and identified as neo- antigens by epitope prediction (Fig. 2a). Of all the can- didate neoantigens, only the neoantigen with high prior - ity score can be finally selected to synthetic vaccine. For each patient, 7–19 personalized neoantigen long-peptides derived from somatic point mutations were synthesized for vaccine manufacture. The neoantigens finally selected to synthetic vaccine for 6 patients are detailed in Supplemen- tary Table 2. It was observed that 66.67% (4/6) of patients had neoantigens derived from TTN, OBSCN, MUC16, and MUC3A mutations. But even if different patients have neoantigens derived from the same mutations, their specific mutation sites are different. We also performed statistical analysis for mutations with a frequency greater than 33.33% (2/6) (Fig. 2b). The results showed that these neoantigens emerged with high frequency often had low priority score, and only a few were selected for vaccine preparation. Fig. 2 The overview of neo- antigen profiles. a Number of candidate neoantigens (priority score > 0) and non-candidate neoantigens (priority score < 0) detected in each of the six CRC patient's tissue samples. b Heat- map summarizing the somatic/ neoantigen hotspot gene profile in the enrolled CRC patients 2051 Cancer Immunology, Immunotherapy (2023) 72:2045–2056 1 3 The safety of neoantigen vaccine and changes in health‑related quality of life Since there was a drug washout period of at least 2 weeks between vaccine treatment and conventional treatment, we could distinguish the clinical adverse reactions after vac- cine treatment from those of conventional treatment. All six enrolled patients received five planned priming phase vaccinations. As for booster vaccinations, patients W01, W02, W03, and W10 received four, seven, three, and two booster vaccinations, respectively. No obvious treatment- related adverse events were observed, and routine blood/ biochemical tests did not indicate any obvious abnormali- ties during vaccinations. After vaccination, all patients had mild injection-site reaction (Grade 1). Patient W02 experienced minor itching on his back following the third vaccination treatment, and patient W09 experienced minor headache following the first vaccination (Table 2). These symptoms went away quickly without treatment and did not reoccur during the follow-up injection therapy. Changes in health-related quality of life were measured on the FACT-C as listed in Table 3. Results indicated that Table 2 Treatment-related adverse events in the patients *Including all patients who received at least one dose of trial treat- mentAll treated patients (n = 6) Grade 1–2 Grade 3–4 Constitutional Injection-site reaction 6 (100%) 0 Flu-like symptoms 0 0 Fever 0 0 Fatigue 0 0 Chills 0 0 Dizziness 0 0 Gastrointestinal Nausea 0 0 Constipation 0 0 Vomiting 0 0 Diarrhea 0 0 Dry mouth 0 0 Respiratory Cough 0 0 Dyspnea 0 0 Laboratory Anemia 0 0 Neutropenia 0 0 Other Rash 1(16.7%) 0 Headache 1(16.7%) 0 Table 3 Quality of life at baseline and at 20 weeks (n = 6) All treated patients (n = 6) Baseline 20 weeks W01 W02 W03 W06 W09 W10 W01 W02 W03 W06 W09 W10 FACT-C (0–132) 94 92 93 96 110 102 94 97 92 101 109 101 Physical well-being (0–28)25 25 23 19 28 26 22 23 23 19 27 25 Social/family well-being (0–24)14 15 16 19 17 15 15 19 16 21 17 17 Emotional well-being (0–24)20 16 16 22 23 21 17 16 16 19 21 20 Functional well-being (0–28)18 16 17 18 23 21 20 16 18 19 24 21 CRC subscale (0–28) 17 20 21 18 19 19 20 23 19 23 20 18 2052 Cancer Immunology, Immunotherapy (2023) 72:2045–2056 1 3 all patients had varying degrees of improvement in their quality of life. Clinical and immune response monitoring during vaccination and follow‑up The detailed timeline presentation of clinical treatments, vaccinations, and clinical outcomes is shown in Fig. 3a. The median follow-up time of six patients was 17 months (range 11–24 months). During the clinical trial, two of the six patients were confirmed with clinical relapse by path- ological biopsy or MRI/CT scan, with a median PFS of 11 months; the other four patients remained relapse-free. (Mean follow-up time was 19 months.) After 14 months of follow-up, patient W01 developed a new tumor in the intes- tinal wall, which was pathologically confirmed as colonic adenocarcinoma. After 8 months of follow-up, the patient W10's spinal metastasis was confirmed by MRI/CT scan. Patient W03 is resting at home and being closely monitored among the four patients who have not progressed in their dis- ease; patients W02, W06 and W09 are currently undergoing chemotherapy combined with targeted therapy. During vaccination and follow-up, in vitro IFN-γ ELISpot assay was performed to monitor immune response by using Fig. 3 Clinical outcome and immune response monitoring in the enrolled CRC patients during vaccination and follow-up. a The detailed timeline presentation of clinical treatments, vaccinations, and clinical outcomes for six enrolled CRC patients from the begin-ning of vaccine treatment until the deadline of the clinical trial. B The ex vivo IFN-γ enzyme-linked immunoblot (ELISPOT) responses for PBMCs stimulated by personalized neoantigen pools before and after the neoantigen vaccination 2053 Cancer Immunology, Immunotherapy (2023) 72:2045–2056 1 3 autologous PBMCs stimulated by neoantigen pools or each neoantigen individually. IFN-γ ELISpot assay indicated that our standard vaccination protocol is efficient in the induc- tion of antigen-specific immune response in CRC patients. As shown in Fig. 3b, the neoantigen-specific activation of PBMCs induced by neoantigen vaccines was observed in four out of six patients after vaccination. The ELISpot results showed that the neoantigen-reactive T cells (NRTs) in patients W02, W03, W06 and W09 are significantly more than that in patients W01 and W10 (Fig. 3b). Importantly, the PFS of these patients (Patients W02, W03, W06 and W09) was significantly longer than that of the other two (Patients W01 and W10) (19 vs 11 months). Discussion This clinical trial was a single-arm, single-center study, which included six MSS–CRC patients with postopera- tive recurrence or metastasis. After the personalized neo- antigen vaccination, four of six patients exhibited a posi- tive immune response to the neoantigen vaccine and had significantly longer PFS time than the other two patients with negative neoantigen response (19 vs 11 months). No significant adverse events were observed in the six enrolled patients during the neoantigen vaccine administration. All patients reported that the quality of life had improved dur - ing the vaccine treatment. According to the analysis of the above results, even MSS–CRC patients with postoperative recurrence or metastasis are expected to benefit from this immunotherapy. The most surprising thing was the perfor - mance of patient W02. The patient received chemotherapy again when liver metastasis occurred after colorectal cancer surgery during which time there was no significant decrease in carcinoembryonic antigen (CEA). The patient started the single-drug treatment of vaccine after stopping the chemo- therapy treatment. After four injections, the patient's CEA dropped from an initial 126.7 to 82.73 ng/mL (Fig. 4a). Abdominal-enhanced CT images at three different time points before and after treatment demonstrated that multiple space-occupying liver lesions decreased or even disappeared in varying degrees during the treatment (Fig. 4b). The IFN-γ spots per 105 PBMCs of the peptide or peptide pool with the best response are shown in Fig. 4c. As shown in Fig. 4d, all monitored personalized nonsynonymous somatic muta - tions decreased to varying degrees after receiving the vac- cine treatment. In China, the incidence rate of CRC is fifth among all malignancies [22]; about 20% of CRC is first diagnosed at stage IV, and its 5-year survival rate is only about 13% [23–25]. At present, immunotherapy based on ICIs has been proven to be successful in treating lung cancer, metastatic melanoma, esophageal cancer, and other tumors [26– 28]. Keynote-177 randomized phase III clinical trial demon- strated that PFS of patients with MSI-H metastatic CRC using pembrolizumab was better than chemotherapy. The imaging-based evaluation indicated that the total and com- plete remission rates of the pembrolizumab group were higher [29], thereby reinforcing the clinical value of immu- nization therapy based on ICIs in treating MSI-H metastatic CRC. In 2015 and 2016, the American Society of Clinical Oncology (ASCO) published clinical trials of programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) inhibitors, pembrolizumab, and nivolumab in the treatment of advanced CRC. It was observed that the objec- tive remission rate of MSI-H CRC patients was excellent; the remission time was long, and the adverse drug reactions were controllable. However, the vast majority of advanced patients with MSS do not benefit from ICIs-based immuno- therapy [30]. Therefore, breaking the immune resistance of patients with MSS–CRC or proposing new immunotherapy methods has become a hot spot in the treatment of CRC. Our research included six MSS–CRC patients with post- operative recurrence or metastasis for the clinical trial of individualized tumor vaccine treatment. The preliminary results demonstrated that the personalized neoantigen vac- cine is a safe, feasible, and effective strategy for MSS–CRC treatment and provided additional data on individualized medicine for MSS–CRC. Also, it can improve the quality of life of patients with MSS–CRC to a certain extent. Previous studies had proved the safety and effectiveness of personalized tumor vaccines based on neoantigens in the treatment of melanoma and advanced lung cancer [18, 31]. Also, the literature indicated that vaccines based on indi- vidual mutations and predicted neo-epitopes could induce T-cell infiltration [32], which also proved the possibility of killing tumor cells by utilizing the autoimmune system. The existence of tumor-reactive CD8 (+) cells in gastrointestinal tumors had been confirmed at the molecular level, which also provided a basis for the development of immunotherapy for tumor patients [33]. Eric Tran et al. observed that TILs from nine out of ten patients with metastatic gastrointesti- nal cancers contained CD4( +) and/or CD8( +) T cells that recognized one to three neo-epitopes derived from somatic mutations expressed by the patient's own tumor [34]. Thus, a high frequency of patients with common gastrointestinal cancers harbor immunogenic mutations that can potentially be exploited for the development of highly personalized immunotherapies. Some clinical trials had proved the safety and effectiveness of vaccines based on tumor neoantigen [35]; however, there are few studies on tumor vaccine ther - apy in MSS–CRC. Importantly, our previous study found adoptive transfer of neoantigen-reactive T cells induced by vaccination with two mutant peptides could effectively inhibit tumor growth in tumor-bearing mouse models [15]. As tumors with a large number of mutations are linked to 2054 Cancer Immunology, Immunotherapy (2023) 72:2045–2056 1 3 Fig. 4 Case report of advanced colorectal cancer patient W02. a Clinical vaccine treatment timeline and corresponding tumor marker of patient W02. b Enhanced CT findings of liver metastases during the neoanti- gen vaccination and follow-up. c Monitoring of ex vivo IFN-γ ELISPOT response for PBMCs stimulated by personalized neoantigen in patient W02. d The performances of ctDNA for CRC monitoring in patient W02 2055 Cancer Immunology, Immunotherapy (2023) 72:2045–2056 1 3 response to immunotherapy due to the larger number of potential neoantigens, we had a closer look at the number of neoantigens identified in six patients with MSS–CRC. Sur - prisingly, even patients with MSS–CRC have a considerable amount of neoantigens. Similar to the mutational rate, some studies had shown that the median number of neoantigens in CRC patients with MSI-H tumors was around 20 times higher than in MSS tumors [36].The amount of neoantigens may become a key factor in the benefit of tumor immuno- therapy. However, the number of neoantigens selected for the personalized vaccine of patient W02 was the smallest among all patients, but the number of immune response cells in his IFN-γ ELISpot assay was the largest. We believe that the amount of neoantigens is not necessarily the only decisive factor to determine the benefit of immunotherapy. Screen- ing neoantigens with strong immunogenicity for patients with MSS–CRC may greatly confer the greatest benefits in tumor immunotherapy. In our clinical trial, the four patients with great neoantigen-specific immune response had a sig- nificantly longer PFS than the other two patients without neoantigen-specific immune response (19 vs. 11 months). The strong neoantigen-specific immune response may be associated with longer PFS. In summary, our results preliminarily prove that person- alized neoantigen vaccine therapy is a safe, feasible and immunological effective strategy for MSS–CRC patients with postoperative recurrence or metastasis and may pro- vide clinical benefit. Patients who exhibit stronger positive immune responses to the neoantigen vaccines tend to have better clinical outcomes. However, this study still has the following shortcomings: Firstly, we failed to detect the inten - sity of the immune response quantitatively and accurately determine the duration of the immune response; secondly, due to limited sample size enrolled in this study, the cor - responding findings will still need to be validated in large- scale clinical trials. Supplementary Information The online version contains supplemen- tary material available at https:// doi. org/ 10. 1007/ s00262- 023- 03386-7. Acknowledgements We appreciate the help of neoantigen screening and data analysis by the P&PMed Biotechnology Co. Ltd.