Generation of Tumor-Specific Cytotoxic T Cells From Blood via In Vitro Expansion Using Autologous Dendritic Cells Pulsed With Neoantigen-Coupled Microbeads

Abstract

For the past decade, adoptive cell therapy including tumor-infiltrating lymphocytes, genetically modified cytotoxic lymphocytes expressing a chimeric antigen receptor, or a novel T-cell receptor has revolutionized the treatment of many cancers. Progress within exome sequencing and neoantigen prediction technologies provides opportunities for further development of personalized immunotherapies. In this study, we present a novel strategy to deliver in silico predicted neoantigens to autologous dendritic cells (DCs) using paramagnetic beads (EpiTCer beads). DCs pulsed with EpiTCer beads are superior in enriching for healthy donor and patient blood-derived tumor-specific CD8+ T cells compared to DC loaded with whole-tumor lysate or 9mer neoantigen peptides. A dose-dependent effect was observed, with higher EpiTCer bead per DC being favorable. We concluded that CD8+ T cells enriched by DC loaded with EpiTCer beads are tumor specific with limited tumor cross-reactivity and low recognition of autologous non-activated monocytes or CD8+ T cells. Furthermore, tumor specificity and recognition were improved and preserved after additional expansion using our Good Manufacturing Process (GMP)-compatible rapid expansion protocol. Phenotypic analysis of patient-derived EpiTCer DC expanded CD8+ T cells revealed efficient maturation, with high frequencies of central memory and effector memory T cells, similar to those observed in autologous expanded tumor-infiltrating lymphocytes. These results indicate that DC pulsed with EpiTCer beads enrich for a T-cell population with high capacity of tumor recognition and elimination, which are features needed for a T-cell product to be used for personalized adoptive cell therapy.

Non-classical HLA-E restricted CMV 15-mer peptides are recognized by adaptive NK cells and induce memory responses

“…Following overnight maturation, DC were washed, counted, and co-cultured with NK cells in 96-well plates at a concentration of 0.05-0.15 x 10 6 cells and 1:10 ratio per well in RPMI-1640 with 10 ng/ml IL-15 for 14 days. In further experiments, NK cells were cultured with mDC+pp65-peptides for 14 days in addition (5 mg/ ml) of either a control isotype-matched antibody IgG (clone Poly4053), anti-NKG2C (clone 134522), anti-NKG2A (clone 131411), or anti-HLA-E (clone 3D12), anti-MHC I (clone W6/ 32), and MHC II (Tü39) blocking antibodies (27)(28)(29)(30) (BioLegend, R&D systems) at the primary phase (day 0 and 7) or at the secondary restimulation phase (at day 14, during 6h stimulation).…”

Combination of multiple omics techniques for a personalized therapy or treatment selection

“…Next to the improved identification of possible neoAg, there are also studies to improve their clinical implementation with better strategies for loading DC with polypeptides ( 33 ) or optimal spacers for multi-epitope constructs to allow processing into the single peptides ( 34 ). Moreover, a genetic and proteomic signature for neoAg-specific CD4 + and CD8 + T cells has been identified, which could allow the isolation of neoAg-specific T cells from patients’ TIL without the need of previous in vitro expansion ( 35 ).…”

Mesothelin- and nucleolin-specific T cells from combined short peptides effectively kill triple-negative breast cancer cells

“…Adoptive cell immunotherapy, characterized by the ex vivo expansion of antigen-speci c T cells, presents a promising therapeutic avenue due to its speci city, capacity for self-replication, and minimal toxicity [9]. To generate antigenspeci c T cells, various methodologies have been investigated, including the transfection of dendritic cells with DNA constructs encoding tumor antigen epitopes [10], the pulsing of dendritic cells with antigen peptides [11], as well as the stimulation of peripheral blood mononuclear cells (PBMCs) with synthetic tumor-associated antigen (TAA) or tumor-speci c antigen (TSA) peptides [12,13].…”

“…Adoptive cell immunotherapy, characterized by the ex vivo expansion of antigen-speci c T cells, presents a promising therapeutic avenue due to its speci city, capacity for self-replication, and minimal toxicity [9]. To generate antigenspeci c T cells, various methodologies have been investigated, including the transfection of dendritic cells with DNA constructs encoding tumor antigen epitopes [10], the pulsing of dendritic cells with antigen peptides [11], as well as the stimulation of peripheral blood mononuclear cells (PBMCs) with synthetic tumor-associated antigen (TAA) or tumor-speci c antigen (TSA) peptides [12,13].Recent reports have disclosed the successful generation of multi-peptide-speci c T cells through the stimulation of PBMCs with multi-peptides in the context of hematopoietic malignancies [13][14][15]. These multi-peptide-speci c T cells, stimulated by multipeptides, resulted in an overexpression of TAAs in multiple myeloma, and they were capable of producing interferon-gamma (IFNγ), granzyme B, and perforin, serving as surrogates of cytolytic activity [13].…”