BMJ Open 2022;12:e055742. doi:10.1136/bmjopen-2021-055742Open access in- depth study of the molecular mechanisms of tumour occurrence and development, biotherapy has become the fourth mode of comprehensive tumour treatment and has received more and more attention.2–4 Tumour biotherapy is a new therapy that applies modern biotechnology and its products for tumour prevention and treatment. It achieves antitumour effects by mobilising the host’s natural defence mechanism or giving natural (or genetic engineering) highly targeted substances. It mainly includes somatic cell therapy, cytokine therapy, tumour peptides therapy, molecular targeted therapy, radioimmune targeted therapy, tumour gene therapy and biological chemotherapy.3–9 Among them, tumour neoantigen study is one of the hotspots in domestic and foreign research in recent years.10 Individ- ualised neoantigen tumour immunotherapy has brought inspiring therapeutic effects. Several studies have shown that tumour neoantigen therapy can produce miraculous effects, inhibiting tumour progression and even achieving complete tumour regression.11–13 Tumour neoantigens are a series of immunogenic substance derived from tumour- specific mutations, instead of from the normal human genome,14–17 which presented on the surface of tumour cells by major histocompatibility complex (MHC). The new antigen can be transferred into the patient in the form of synthetic long peptide, RNA, activated dendritic cell and DNA.18–21 Neoantigens are not present in normal cells, so the immune system can recognise them as ‘non- self’ antigens.22 The principle is to eliminate or control tumours by activating the patient’s own immune system.20 In the body, neoantigen epitopes can bind to MHC-Ⅰ to activate CD8 +T lymphocytes, which can differentiate CD8 +T lymphocytes into cyto- toxic T lymphocytes (CTL) and exert cytotoxic effects. Also, neoantigen epitopes can combine with MHC-Ⅱ to activate CD4 +T lymphocytes to differentiate into helper T cells, such as Th1 and Th2 cells, which interact with macrophages and B lymphocytes, respectively.23 24 Tumour neoantigens have been studied in a wide range of tumours, including lung, breast, glioma, liver, kidney, ovarian, pseudomyxoma peritonei and pancreatic cancer, etc.5 12 18 25–28 Most of research on neoantigens has focused just on a unique tumour and a single mutated gene.5 11 29 The safety and efficacy of neoantigen peptide mixtures derived from multiple gene mutation sites in the treat- ment of advanced solid malignancies with genomic insta- bility are still unclear. The trial aims to evaluate the safety and efficacy of indi- vidualised tumour neoantigen peptide mixtures in the treatment of genomic unstable advanced solid malignant tumours. METHODS Study design overview This is an open- label, non- randomised, prospective, single- arm trial, which will be conducted at phase I Trial Ward of Tertiary Class A Cancer Hospital.Objectives The trial aims to evaluate the safety and efficacy of indi- vidualised tumour neoantigen peptides mixtures in the treatment of genomic unstable advanced solid malignant tumours. Endpoints The primary endpoint is to observe and record all adverse events (AEs) and the incidence of serious AEs (SAE) (grade 3 or above AE) associated with the study drug within 24 weeks after the administration of the individualised neoantigen tumour polypeptide (Refer to National Cancer Institute Common Terminology Criteria for Adverse Events (NCI) version 4.03). After that, the subjects will continue to be followed at the preset follow- up points for safety. Secondary endpoints are to evaluate the progression- free survival (PFS), objective response rate (ORR), objective duration of remission (DOR), 1- year survival rate and overall survival (based on Enhanced CT/MRI). The efficiency of neoantigen peptides in inducing specific antitumour immunity is assessed from peripheral blood samples (based on IFN-γ, CD4 +T cells, CD8 +T cells determined by flow cytometry and enzyme- linked immuneospot (ELISPOT) technique. In addition, the efficacy evaluation also includes the detection of circulating tumour DNA (ctDNA) and the quality of life of cancer patients. Additional scientific programme Genome sequencing Whole exome and transcriptome sequencing will be tested on the patient’s biopsy or surgically removed tumour tissue, and whole exome sequencing will also be performed on the patient’s blood sample. This gene sequencing can obtain comprehensive genomic infor - mation such as the mutation status of all genes, human leucocyte antigen (HLA) genotypes and expression levels of mutated genes in the patients, which can be used to confirm patient’s entry criteria and synthesise neoantigen tumour peptides. Design and synthesise of neoantigen tumour peptides The main body of the neoantigen design process in this study refers to the proposal published in Nature in 2018 by Neon Therapeutics and Harvard Medical School.28 On this basis, the unique analysis technology of Beijing Xink- angyuan Biotechnology will be applied to further ensure the quality of tumour neoantigen peptides. Beijing Neoan- tigen Biotechnology owns a series of software copyrights such as NeoRQC, NeoAlign, NeoHLAType, NeoCVC, NeoVQC, NeoANN, NeoGEP, NeoDsign and other soft- wares which are under the NeoOne platform specially designed for neoantigen design and synthesis. They will be used for gene mutation analysis, gene expression anal- ysis, HLA typing, neoantigen design and multidirectional quality control to ensure that the neoantigen design in this study meets the world’s leading clinical requirements. The whole design process is shown in figure 1.