Defining the Diversity and Specificity of Neoantigen-Specific T Cells in Chronic Lymphocytic Leukemia

Recent studies have highlighted the promise of targeting tumor neoantigens but little is known about the clonal composition of neoantigen-specific T cells. Knowledge of the number and diversity of neoantigen-specific T cell receptors (TCRs), their cognate antigens and how these change over time are informative parameters to consider in the rational design of future immunotherapy. TCR repertoire studies of bulk T cells can provide measures of diversity, but generally mask the antigen-specificity information encoded by TCRαβ pairs, and are insufficient for directly linking particular TCRs to individual antigens.

To address these challenges, we developed an approach to discover TCRαβ sequence from single cells, use this information to reconstitute any TCR, and to test the reconstructed TCRs for reactivity against candidate antigens. As a proof-of concept, we sought to identify TCRs against a pool of well-characterized viral peptides (CMV, EBV, Influenza) of defined HLA restriction (called 'CEF'). We performed one round of in vitro stimulation of PBMC from 2 healthy volunteers with CEF peptides, and then isolated CEF-specific T cells based on IFN-γ production. These cells underwent single cell targeted sequencing of TCRα/β chains, using 45 Vα and 50 Vβ primers specific for the variable regions and a single constant region primer. Compared to unstimulated PBMCs, the CEF-stimulated PBMCs were clonally expanded. For donors 1 and 2, 57 and 74 total clonotypes were identified, of which 16 and 13 constituted between 1.7-34% and 1.5-18% of total T cells, respectively.

To interrogate the antigen-specificity of the identified TCR sequences, we cloned TCRs of interest. As all TCRs share the same modular structure, we designed a plasmid library to include all Vα or β and constant regions. Based on the discovered CDR3-α and -β sequences, oligonucleotides for these regions critical for antigen recognition were custom-synthesized, and cloned by Golden Gate assembly of selected library components. In this fashion, we rapidly cloned the 9 most dominant TCR clones per donor and lentivirally transduced them into a TCR-deficient Jurkat cell line. Upon screening these lines against individual CEF peptides, we confirmed that 4 TCRs per donor had specificities to CEF peptides, previously characterized to stimulate donor-specific reactivity by IFN-γ ELISPOT assay. Screening of the remaining reconstructed TCRs is ongoing.

Having demonstrated the ability to link TCRs with cognate antigen, we next applied this approach to study neoantigen-specific T cells from patients with chronic lymphocytic leukemia (CLL). We have previously reported the results of somatic mutation detection from whole-exome sequencing data from 157 CLL patients, from which we predicted 1851 neoantigens with high binding affinity (IC₅₀ < 150 nM). Of these, 326 neoantigens had predicted high binding affinity to HLA-A2. From this collection, we focused on the identification of TCRs specific to neoantigens predicted to be generated by mutation in 2 genes. The first was FNDC3B, a point mutation of which we previously showed to generate a neoepitope that stimulated expansion of CD8⁺ T cells in a transplanted patient in association with durable remission, and which displayed cytotoxic potential against autologous tumor. The second was MGA, a putative CLL driver, wherein an indel was predicted to generate a novel open reading frame. To identify the number of T cell clones that could be stimulated by these candidate neoantigens, we stimulated HLA-A2+ PBMC with predicted neopeptides arising from these genes, and flow cytometrically isolated peptide-reactive CD8⁺ T cells for TCR sequencing. 148 of 250 (57%) mut-FNDC3B-specific T cells had the same CDR3 sequences, with 5 dominant clones identified. Three of 5 T cell clones (constituting 57%, 3% and 2% of total T cells) recognized the neopeptide. Similarly, out of 43 MGA-specific CD8⁺ T cells, 4 dominant clones were identified, 1 of which we confirmed as specific to MGA.

Altogether, these studies demonstrate our capability to effectively identify paired TCRαβ sequences, express them on demand and probe antigen specificity. Ongoing studies are focused on further defining the antigen specificity and avidity of engineered T cells, including reactivity against autologous leukemia cells. These studies are expected to provide an assessment of the diversity and function of neoantigen-reactive TCRs.