Work

Vaccination targeting the novel endogenous tumor-associated antigen Gpr149 induces protective immunity against checkpoint inhibitor-refractory SB28 glioblastoma

Year: 2026

Type: preprint

Abstract: Background Glioblastoma is a lethal brain tumor notable for its limited spontaneous induction of CD8 + T cell responses. Vaccination may overcome this limitation by generating tumor-specific CD8 + T cells capable of mediating anti-tumor immunity in the brain. However, preclinical vaccine development is constrained by a lack of immunologically stringent glioblastoma models with defined endogenous antigens. We therefore sought to identify endogenous, vaccine-targetable antigens expressed by the immune checkpoint-refractory SB28 syngeneic mouse glioblastoma model and to evaluate their targetability using a heterologous prime-boost regimen with the chimpanzee adenovirus ChAdOx1 and the modified vaccinia Ankara (MVA) viral vector-based vaccines. Methods RNA sequencing and whole-exome sequencing were used to identify candidate tumor-associated antigens (TAAs) and neoantigens, and tumor-specific expression was validated by RT-qPCR. ChAdOx1 and MVA vectors encoding candidate antigens were generated and evaluated for immunogenicity and anti-tumor efficacy. Antigen-specific CD8 + T cell responses and minimal epitopes were characterized by ex vivo restimulation, MHC-I binding assays, tetramer staining, and tumor cytotoxicity assays. Results We identified five candidate TAAs ( Gpr149, Cdh6, Fbn2, Igf2bp1, and Shox2) but no suitable neoantigen targets in SB28. Vaccination induced CD8 + T cell responses against the candidate TAAs, and immunogenic minimal epitopes were identified for each. Among the candidates, Gpr149 elicited the strongest vaccine-induced CD8 + T cell responses. In mice challenged intracranially with SB28, Gpr149-targeted ChAdOx1/MVA vaccination combined with immune checkpoint blockade significantly prolonged median survival and resulted in long-term survival in 50% of mice, whereas vaccination targeting the other TAAs did not improve survival. CD8 + T cells recognizing the immunodominant Gpr149 128-135 epitope (VCYNFYTM) specifically recognized and lysed wild-type SB28 cells, but not Gpr149-knockout SB28 cells, confirming endogenous processing and presentation of the targeted epitope. Conclusions Gpr149 is an endogenous SB28 tumor antigen that can be effectively targeted by ChAdOx1/MVA vaccination to generate protective anti-tumor immunity against intracranial glioblastoma. Protection was associated with a dominant CD8 + T cell response to the Gpr149 128-135 epitope VCYNFYTM. These findings establish endogenously expressed Gpr149 in SB28 as a physiologically relevant model for evaluating antigen-targeted immunotherapies and provide a systematic approach for identifying endogenous vaccine targets in other preclinical models. Key Messages What is already known on this topic – The immune checkpoint-refractory SB28 glioblastoma model provides a stringent setting for preclinical immunotherapy studies, but endogenous tumor antigens suitable for antigen-targeted vaccination have not been defined, requiring studies of antigen-specific immunity to rely largely on transgenic foreign antigens. What this study adds – We identify Gpr149 as an endogenous SB28 tumor antigen and show that ChAdOx1/MVA vaccination generates functional Gpr149-specific CD8+ T cells that confer protective immunity against intracranial tumor challenge. How this study might affect research, practice or policy – Gpr149 provides an endogenous-antigen system for more physiologically relevant testing of antigen-targeted immunotherapies in immunologically cold tumors, while the antigen-discovery approach and ChAdOx1/MVA platform provide a framework for evaluating clinically relevant glioblastoma antigens in future therapeutic studies.

Source: bioRxiv (Cold Spring Harbor Laboratory)

DOI: 10.64898/2026.10.29.760792