Synthetic Peptides with Inadvertent Chemical Modifications Can Activate Potentially Autoreactive T Cells.

The human CD8+ T cell clone 6C5 has previously been shown to recognize the tert-butyl-modified Bax161-170 peptide LLSY(3-tBu)FGTPT presented by HLA-A*02:01. This nonnatural epitope was likely created as a by-product of fluorenylmethoxycarbonyl protecting group peptide synthesis and bound poorly to HLA-A*02:01. In this study, we used a systematic approach to identify and characterize natural ligands for the 6C5 TCR. Functional analyses revealed that 6C5 T cells only recognized the LLSYFGTPT peptide when tBu was added to the tyrosine residue and did not recognize the LLSYFGTPT peptide modified with larger (di-tBu) or smaller chemical groups (Me). Combinatorial peptide library screening further showed that 6C5 T cells recognized a series of self-derived peptides with dissimilar amino acid sequences to LLSY(3-tBu)FGTPT. Structural studies of LLSY(3-tBu)FGTPT and two other activating nonamers (IIGWMWIPV and LLGWVFAQV) in complex with HLA-A*02:01 demonstrated similar overall peptide conformations and highlighted the importance of the position (P) 4 residue for T cell recognition, particularly the capacity of the bulky amino acid tryptophan to substitute for the tBu-modified tyrosine residue in conjunction with other changes at P5 and P6. Collectively, these results indicated that chemical modifications directly altered the immunogenicity of a synthetic peptide via molecular mimicry, leading to the inadvertent activation of a T cell clone with unexpected and potentially autoreactive specificities.

CD8+ T-cell recognition of a synthetic epitope formed by t-butyl modification.

We set out to clone Bax-specific CD8+ T-cells from peripheral blood samples of primary chronic lymphocytic leukemia patients. A number of clones were generated using a Bax peptide pool and their T-cell epitope was mapped to two peptides sharing a common 9-aa sequence (LLSYFGTPT), restricted by HLA-A*0201. However, when these T-cell clones were tested against highly purified syntheses (>95%) of the same peptide sequence, there was no functional response. Subsequent mass spectrometric analysis and HPLC fractionation suggested that the active component in the original crude peptide preparations (77% pure) was a peptide with a tert-butyl (tBu) modification of the tyrosine residue. This was confirmed by modification of the inactive wild type (wt) sequence to generate functionally active peptides. Computer modeling of peptide:HLA-A*0201 structures predicted that the tBu modification would not affect interactions between peptide residues and the HLA binding site. However these models did predict that the tBu modification of tyrosine would result in an extension of the side chain out of the peptide-binding groove up towards the TCR. This modified product formed <1% of the original P603 crude peptide preparation and <0.05% of the original 23 peptide mixture used for T-cell stimulation. The data presented here, illustrates the potential for chemical modifications to change the immunogenicity of synthetic peptides, and highlights the exquisite capacity of TCR to discriminate between structurally similar peptide sequences. Furthermore this study highlights potential pitfalls associated with the use of synthetic peptides for the monitoring and modulating of human immune responses. This article is protected by copyright. All rights reserved.

Checkpoint Blockade Reverses Anergy in IL-13Rα2 Humanized scFv-Based CAR T Cells to Treat Murine and Canine Gliomas.

We generated two humanized interleukin-13 receptor α2 (IL-13Rα2) chimeric antigen receptors (CARs), Hu07BBz and Hu08BBz, that recognized human IL-13Rα2, but not IL-13Rα1. Hu08BBz also recognized canine IL-13Rα2. Both of these CAR T cell constructs demonstrated superior tumor inhibitory effects in a subcutaneous xenograft model of human glioma compared with a humanized EGFRvIII CAR T construct used in a recent phase 1 clinical trial (ClinicalTrials.gov: NCT02209376). The Hu08BBz demonstrated a 75% reduction in orthotopic tumor growth using low-dose CAR T cell infusion. Using combination therapy with immune checkpoint blockade, humanized IL-13Rα2 CAR T cells performed significantly better when combined with CTLA-4 blockade, and humanized EGFRvIII CAR T cells' efficacy was improved by PD-1 and TIM-3 blockade in the same mouse model, which was correlated with the levels of checkpoint molecule expression in co-cultures with the same tumor in vitro. Humanized IL-13Rα2 CAR T cells also demonstrated benefit from a self-secreted anti-CTLA-4 minibody in the same mouse model. In addition to a canine glioma cell line (J3T), canine osteosarcoma lung cancer and leukemia cell lines also express IL-13Rα2 and were recognized by Hu08BBz. Canine IL-13Rα2 CAR T cell was also generated and tested in vitro by co-culture with canine tumor cells and in vivo in an orthotopic model of canine glioma. Based on these results, we are designing a pre-clinical trial to evaluate the safety of canine IL-13Rα2 CAR T cells in dog with spontaneous IL-13Rα2-positive glioma, which will help to inform a human clinical trial design for glioblastoma using humanized scFv-based IL-13Rα2 targeting CAR T cells.