Personalized cancer immunotherapies such as therapeutic vaccines and adoptive transfer of T cell receptor-transgenic T cells rely on the presentation of tumor-specific peptides by human leukocyte antigen class I molecules to cytotoxic T cells. Such neoepitopes can for example arise from somatic mutations and their identification is crucial for the rational design of new therapeutic interventions. Liquid chromatography mass spectrometry (LC-MS)-based immunopeptidomics is the only method to directly prove actual peptide presentation and we have developed a parameter optimization workflow to tune targeted assays for maximum detection sensitivity on a per peptide basis, termed optiPRM. Optimization of collision energy using optiPRM allows for the improved detection of low abundant peptides that are very hard to detect using standard parameters. Applying this to immunopeptidomics, we detected a neoepitope in a patient-derived xenograft from as little as 2.5 × 10⁶ cells input. Application of the workflow on small patient tumor samples allowed for the detection of five mutation-derived neoepitopes in three patients. One neoepitope was confirmed to be recognized by patient T cells. In conclusion, optiPRM, a targeted MS workflow reaching ultra-high sensitivity by per peptide parameter optimization, makes the identification of actionable neoepitopes possible from sample sizes usually available in the clinic.

Peptide-loaded MHC class I (pMHC-I) multimers have revolutionized our capabilities to monitor disease-associated T cell responses with high sensitivity and specificity. To improve the discovery of T cell receptors (TCR) targeting neoantigens of individual tumor patients with recombinant MHC molecules, we developed a peptide-loadable MHC class I platform termed MediMer. MediMers are based on soluble disulfide-stabilized β₂-microglobulin/heavy chain ectodomain single-chain dimers (dsSCD) that can be easily produced in large quantities in eukaryotic cells and tailored to individual patients’ HLA allotypes with only little hands-on time. Upon transient expression in CHO-S cells together with ER-targeted BirA biotin ligase, biotinylated dsSCD are purified from the cell supernatant and are ready to use. We show that CHO-produced dsSCD are free of endogenous peptide ligands. Empty dsSCD from more than 30 different HLA-A,B,C allotypes, that were produced and validated so far, can be loaded with synthetic peptides matching the known binding criteria of the respective allotypes, and stored at low temperature without loss of binding activity. We demonstrate the usability of peptide-loaded dsSCD multimers for the detection of human antigen-specific T cells with comparable sensitivities as multimers generated with peptide-tethered β₂m-HLA heavy chain single-chain trimers (SCT) and wild-type peptide-MHC-I complexes prior formed in small-scale refolding reactions. Using allotype-specific, fluorophore-labeled competitor peptides, we present a novel dsSCD-based peptide binding assay capable of interrogating large libraries of in silico predicted neoepitope peptides by flow cytometry in a high-throughput and rapid format. We discovered rare T cell populations with specificity for tumor neoepitopes and epitopes from shared tumor-associated antigens in peripheral blood of a melanoma patient including a so far unreported HLA-C*08:02-restricted NY-ESO-1-specific CD8⁺ T cell population. Two representative TCR of this T cell population, which could be of potential value for a broader spectrum of patients, were identified by dsSCD-guided single-cell sequencing and were validated by cognate pMHC-I multimer staining and functional responses to autologous peptide-pulsed antigen presenting cells. By deploying the technically accessible dsSCD MHC-I MediMer platform, we hope to significantly improve success rates for the discovery of personalized neoepitope-specific TCR in the future by being able to also cover rare HLA allotypes.

Background: Methods to identify and characterize antigen-reactive T cell receptors (TCRs) represent important tools to understand and exploit T cell responses in patients with cancer and beyond. Current methods are hampered by the rarity of individual T cell clones and limited applicability to monitor both major histocompatibility complex (MHC) class I-restricted and MHC class II-restricted responses, hence insufficiently reflecting the entire antigen-reactive repertoire of a patient. To obtain broad and deep insight into polyclonal, antigen-specific TCR repertoires, we developed the ‘epitope-specific expansion culture with subsequent identification of TCRs’ (ESPEC-SUIT) assay to identify and track antigen-specific TCRs. Methods: In vitro stimulation of peripheral blood mononuclear cells with (vaccine-targeted) neoantigens was verified in cytokine secretion assays and read-out by TCRβ repertoire sequencing (TCRseq). Candidate antigen-reactive clonotypes were defined by specific expansion in cultures stimulated with relevant antigen, followed by TCR cloning and validation in co-cultures of TCR transgenic effector cells and peptide-presenting targets. Using TCRseq information, candidate and validated clonotypes were traced and characterized in bulk and single-cell repertoire sequencing data of longitudinally collected blood samples and tumor tissue. Results: In a cohort of 32 patients with cancer, we demonstrate that ESPEC-SUIT supports strong, robust and reproducible expansion of CD4 ⁺ and CD8 ⁺ T cells in response to various antigens. TCRseq revealed highly polyclonal neoepitope-specific T cell responses, which can be further characterized with respect to cross-reactivity, affinity or human leukocyte antigen (HLA) restriction. In a subcohort of 10 patients, we selected 341 ESPEC-SUIT-derived TCRs for cloning and in vitro functional validation from >2000 candidates and confirmed antigen-reactivity for >75%. We exemplify the usefulness of this TCR discovery method for downstream analysis in neoepitope vaccinated patients with glioma, where we found longitudinal changes in candidate TCR frequencies in blood mirroring antigen-specific ex vivo Enzyme-Linked ImmunoSpot (ELISpot) responses. Furthermore, up to 67% of candidates could be detected in on-treatment brain tumor tissue and exhibited gene expression signatures overlapping with clonotypes of confirmed specificity to the vaccine antigen. Conclusion: ESPEC-SUIT provides unprecedented insight into highly polyclonal, antigen-specific T cell responses and enables discovery of large numbers of TCRs for a given antigen. It represents an efficient, cost-effective and scalable framework for the interrogation of tumor-reactive T cell responses in patients with cancer.

HLA-E presenting the HLA-G leader peptide VMAPRTLFL (HLA-E[pHLA-G]) on tumor cells plays a crucial role in suppressing natural killer (NK) and cytotoxic CD8+ T cells through NKG2A interaction. While blocking HLA-E:NKG2A is a promising immune checkpoint (IC) approach in cancer therapy, toxicity remains a major clinical concern. We developed a novel IC inhibitor that selectively prevents HLA-E:NKG2A interaction, a monoclonal antibody that selectively targets the HLA-E[pHLA-G] complex, distinguishing cancerous from noncancerous cells. In clinical bone marrow samples from patients with multiple myeloma (MM), 4D7 specifically recognized tumor-associated HLA-E–peptide complexes. Using NK cells from healthy donors, 4D7 effectively blocked the HLA-E:NKG2A interaction, and enhanced NKG2A-positive NK cell activity in autologous MM cell cocultures. Importantly, 4D7 did not inhibit NKG2C-positive NK cells, preserving their activity, even though NKG2C also interacts with HLA-E. In MM-bearing mice treated with human NK cells, 4D7 significantly reduced tumor growth. This targeted approach activates NK cells only against tumor cells presenting HLA-E–peptide complexes, potentially minimizing toxicity compared with current NKG2A inhibitors. The development of 4D7 highlights a promising advancement in immunotherapy for hematologic malignancies, offering improved outcomes for patients with MM, and a foundation for broader application across cancer types.

Objectives: Reduced expression of adhesion molecules in tumor vasculature can limit infiltration of effector T cells. To improve T cell adhesion to tumor endothelial cell (EC) antigens and enhance transendothelial migration, we developed bispecific, T-cell engaging antibodies (bsAb) that activate T cells after cross-linking with EC cell surface antigens. Methods: Recombinant T-cell stimulatory anti-VEGFR2–anti-CD3 and costimulatory anti-TIE2–anti-CD28 or anti-PD-L1–anti-CD28 bsAb were engineered and expressed. Primary lines of human umbilical vein endothelial cells (HUVEC) that constitutively express VEGFR2 and TIE2 growth factor receptors and PD-L1, but very low levels of adhesion molecules, served as models for anergic tumor EC. Results: In cocultures with HUVEC, anti-VEGFR2–anti-CD3 bsAb increased T cell binding and elicited rapid T cell activation. The release of proinflammatory cytokines TNF-α, IFN-γ, and IL-6 was greatly augmented by the addition of anti-TIE2–anti-CD28 or anti-PD-L1–anti-CD28 costimulatory bsAb. Concomitantly, T cell-released cytokines upregulated E-selectin, ICAM1, and VCAM1 adhesion molecules on HUVEC. HUVEC cultured in breast cancer cell-conditioned medium to mimic the influence of tumor-secreted factors were similarly activated by T cell-engaging bsAb. Migration of T cells in transwell assays was significantly increased by anti-VEGFR2–anti-CD3 bsAb. The combination with costimulatory anti-TIE2–anti-CD28 bsAb augmented activation and proliferation of migrated T cells and their cytotoxic capacity against spheroids of the MCF-7 breast cancer cell line seeded in the lower transwell chamber. Conclusions: T cells activated by anti-VEGFR2–anti-CD3 and costimulatory EC-targeting bsAb can reverse the energy of quiescent EC in vitro, resulting in improved T cell migration through an EC layer.