Alexander B. Afeyan

Human leukocyte antigen (HLA)-bound tumor peptides can be routinely isolated from cancer samples and identified using mass spectrometry (MS). However, MS approaches can be stochastic or rely on spectral libraries, which are not customarily available for individual-specific peptides, thus limiting the ability to discover novel peptides. Here, we introduce Pepyrus, which generates user-defined, individual-specific or disease-specific peptide libraries in Escherichia coli to improve the sensitivity and confidence of MS peptide identification, including lowly abundant neoantigens. Using Pepyrus-generated peptide libraries paired with an HLA-specific data-independent acquisition strategy, we recover >75% of the expected sequences per single injection for libraries of >10,000 peptides and identify 0.1 fmol of spiked-in peptides in a complex background. We apply Pepyrus to create personalized libraries, facilitating identification of clinically relevant HLA peptides, including several novel peptides from cell lines derived from persons with melanoma and renal cell carcinoma. Pepyrus enables identification of rare HLA-bound peptides and provides the ability to generate large training datasets to improve spectra, retention time and ion mobility prediction tools.

The ability to screen the reactivity of T cell receptors (TCRs) is essential to understanding how antigen-specific T cells drive productive or dysfunctional immune responses during infections, cancer and autoimmune diseases. Methods to profile large numbers of TCRs are critical for characterizing immune responses sustained by diverse T cell clones. Here we provide a medium-throughput approach to reconstruct dozens to hundreds of TCRs in parallel, which can be simultaneously screened against primary human tissues and broad curated panels of antigenic targets. Using Gibson assembly and miniaturized lentiviral transduction, individual TCRs are rapidly cloned and expressed in T cells; before screening, TCR cell lines undergo combinatorial labeling with dilutions of three fluorescent dyes, which allows retrieval of the identity of individual T cell effectors when they are organized and tested in pools using flow cytometry. Upon incubation with target cells, we measure the upregulation of CD137 on T cells as a readout of TCR activation. This approach is scalable and simultaneously captures the reactivity of pooled TCR cell lines, whose activation can be deconvoluted in real time, thus providing a path for screening the reactivity of dozens of TCRs against broad panels of synthetic antigens or against cellular targets, such as human tumor cells. We applied this pipeline to systematically deconvolute the antitumoral and antiviral reactivity and antigenic specificity of TCRs from human tumor-infiltrating lymphocytes. This protocol takes ~2 months, from experimental design to data analysis, and requires standard expertise in cloning, cell culture and flow cytometry.

About 50% of patients with locally advanced head and neck squamous cell carcinoma (HNSCC) experience recurrences after definitive therapy. The presurgical administration of anti–programmed cell death protein 1 (PD-1) immunotherapy results in substantial pathologic tumor responses (pTR) within the tumor microenvironment (TME). However, the mechanisms underlying the dynamics of antitumor T cells upon neoadjuvant PD-1 blockade remain unresolved, and approaches to increase pathologic responses are lacking. In a phase 2 trial (NCT02296684), we observed that 45% of patients treated with two doses of neoadjuvant pembrolizumab experienced marked pTRs (≥50%). Single-cell analysis of 17,158 CD8 ⁺ T cells from 14 tumor biopsies, including 6 matched pre-post neoadjuvant treatment, revealed that responding tumors had clonally expanded putative tumor-specific exhausted CD8 ⁺ tumor-infiltrating lymphocytes (TILs) with a tissue-resident memory program, characterized by high cytotoxic potential (CTX ⁺ ) and ZNF683 expression, within the baseline TME. Pathologic responses after 5 weeks of PD-1 blockade were consistent with activation of preexisting CTX ⁺ ZNF683 ⁺ CD8 ⁺ TILs, paralleling loss of viable tumor and associated tumor antigens. Response was associated with high numbers of CD103 ⁺ PD-1 ⁺ CD8 ⁺ T cells infiltrating pretreatment lesions, whereas revival of nonexhausted persisting clones and clonal replacement were modest. By contrast, nonresponder baseline TME exhibited a relative absence of ZNF683 ⁺ CTX ⁺ TILs and subsequent accumulation of highly exhausted clones. In HNSCC, revival of preexisting ZNF683 ⁺ CTX ⁺ TILs is a major mechanism of response in the immediate postneoadjuvant setting.