Physical and in silico immunopeptidomic profiling of a cancer antigen prostatic acid phosphatase reveals targets enabling TCR isolation

Abstract

Tissue-specific antigens can serve as targets for adoptive T cell transfer-based cancer immunotherapy. Recognition of tumor by T cells is mediated by interaction between peptide–major histocompatibility complexes (pMHCs) and T cell receptors (TCRs). Revealing the identity of peptides bound to MHC is critical in discovering cognate TCRs and predicting potential toxicity. We performed multimodal immunopeptidomic analyses for human prostatic acid phosphatase (PAP), a well-recognized tissue antigen. Three physical methods, including mild acid elution, coimmunoprecipitation, and secreted MHC precipitation, were used to capture a thorough signature of PAP on HLA-A*02:01. Eleven PAP peptides that are potentially A*02:01-restricted were identified, including five predicted strong binders by NetMHCpan 4.0. Peripheral blood mononuclear cells (PBMCs) from more than 20 healthy donors were screened with the PAP peptides. Seven cognate TCRs were isolated which can recognize three distinct epitopes when expressed in PBMCs. One TCR shows reactivity toward cell lines expressing both full-length PAP and HLA-A*02:01. Our results show that a combined multimodal immunopeptidomic approach is productive in revealing target peptides and defining the cloned TCR sequences reactive with prostatic acid phosphatase epitopes.

Defining T cell receptor repertoires using nanovial-based affinity and functional screening

“…We then sought to test if the multiplexed secretion assay can also be applied to low-potency TCRs targeting tissue antigens since high-affinity T cell clones might get removed during thymic negative selection ( 18 ). Our previous work led to the discovery of a set of TCRs targeting prostatic acid phosphatase (PAP), a prostate tissue antigen ( 19 ). Those candidates showed encouraging peptide recognition but had limited cytotoxicity on processed epitopes ( 19 ).…”

“…Our previous work led to the discovery of a set of TCRs targeting prostatic acid phosphatase (PAP), a prostate tissue antigen ( 19 ). Those candidates showed encouraging peptide recognition but had limited cytotoxicity on processed epitopes ( 19 ). In the context of HLA-A*02:01, TCR128 and 218 transduced PBMCs were loaded onto nanovials conjugated with PAP21 pMHC.…”

“…All sets of functionalized nanovials were pooled together as one nanovial suspension (a total of 0.75 million nanovials). PBMCs were activated for 7 days with peptides associated with each antigen (CMV1: pp65/NLVPMVATV, CMV2: IE1/VLEETSVML, EBV: BMLF1/GLCTLVAML) as reported previously ( 19 ). 5 million activated PBMCs were loaded onto the pooled nanovial suspension.…”

“…Human donor PBMCs. To prime naïve T cells with peptides of interest, PBMCs from commercial vendors (AllCells) were cultured and processed as previously described with chemically synthesized peptides (>80% purity, Elim Biopharm) (19).…”

“…Multiplexed secretion-based profiling of cancer-specific cognate T cells. PBMCs were transduced with prostate acid phosphatase specific TCRs (TCR128, 156, 218) as previously described (19).…”