Accumulating evidence suggests that the efficacy of cancer immunotherapies, such as immune checkpoint blockade, is to a large extent driven by T-cells that recognize cancer neoantigens – T-cell antigens that arise as a result of patient-specific nonsynonymous tumor mutations. Based on these data, a major effort has been initiated to develop approaches that can be used to specifically boost the activity of neoantigen-reactive CD8+ and CD4+ T-cells in individual patients. However, tumor mutations and their associated neoantigens are, with few exceptions, unique to individual cancer patients, underscoring the need for technologies that enable the comprehensive discovery of both CD4+ and CD8+ T-cell-recognized neoantigens in a truly personalized fashion.
We present HANSolo (HLA-Agnostic Neoantigen Screening), a high-throughput genetic platform for the personalized identification of CD4+ and CD8+ T-cell-recognized (neo)antigens. In this method, patient-matched, Bcl-6/xL-immortalized B cell lines are engineered to express large libraries of minigenes that encode candidate T-cell antigens. As the resulting B cells are fully MHC class I and class II proficient, this enables the unbiased screening of T-cell specificities across the complete MHC class I and class II genotypes of individual patients. To this purpose, antigen library-expressing B cells are co-incubated with patient T-cells, and antigen hits are identified by next-generation sequencing to measure the depletion of those B cells that express T-cell-recognized epitopes.
We benchmark the feasibility and sensitivity of our genetic screening method, and illustrate the potential of our approach by profiling the neoantigen-specificities of patient-derived T-cell populations in a setting in which neoantigen-specific T-cells represent only a minor fraction of the T-cell population.
Collectively, these data demonstrate the feasibility of personalized and HLA-agnostic discovery of CD4+ and CD8+ T-cell neoantigens from large genetic libraries. Thus, this technology should facilitate the development of personalized neoantigen-based cancer immunotherapies, such as neoantigen vaccines or neoantigen TCR gene therapies.
Legal entity responsible for the study
Dutch Cancer Society Young Investigator Grant (grant No. 2020-1 / 12977); ZonMw Translational Research Program 2 (grant No. 446002001); Queen Wilhelmina Cancer Research Award ERC AdG SENSIT (grant agreement No. 742259); NWO Gravitation program (NWO 2012-2022); Oncode Institute.
All authors have declared no conflicts of interest.