Crystallization and preliminary X-ray structural studies of a Melan-A pMHC-TCR complex

Melanocytes are specialized pigmented cells that are found in all healthy skin tissue. In certain individuals, diseased melanocytes can form malignant tumours, melanomas, which cause the majority of skin-cancer-related deaths. The melanoma-associated antigenic peptides are presented on cell surfaces via the class I major histocompatibility complex (MHC). Among the melanoma-associated antigens, the melanoma self-antigen A/melanoma antigen recognized by T cells (Melan-A/MART-1) has attracted attention because of its wide expression in primary and metastatic melanomas. Here, a preliminary X-ray crystal structural study of a soluble cognate T-cell receptor (TCR) in complex with a pMHC presenting the Melan-A peptide (ELAGIGILTV) is reported. The TCR and pMHC were refolded, purified and mixed together to form complexes, which were crystallized using the sitting-drop vapour-diffusion method. Single TCR–pMHC complex crystals were cryocooled and used for data collection. Diffraction data showed that these crystals belonged to space group P4(1)/P4(3), with unit-cell parameters a = b = 120.4, c = 81.6 Å. A complete data set was collected to 3.1 Å and the structure is currently being analysed

Increased peptide contacts govern high affinity binding of a modified TCR whilst maintaining a native pMHC docking mode

NaturalT cell receptors (TCRs) generally bind to their cognate pMHC molecules with weak affinity and fast kinetics, limiting their use as therapeutic agents. Using phage display, we have engineered a high affinity version of the A6 wild-type TCR (A6wt), specific for the human leukocyte antigen (HLA-A�0201) complexed with human T cell lymphotropic virus type 111–19 peptide (A2-Tax). Mutations in just 4 residues in the CDR3b loop region of the A6wt TCR were selected that improved binding to A2-Tax by nearly 1000-fold. Biophysical measurements of this mutant TCR (A6c134) demonstrated that the enhanced binding was derived through favorable enthalpy and a slower off-rate. The structure of the free A6c134 TCR and the A6c134/A2-Tax complex revealed a native binding mode, similar to the A6wt/A2-Tax complex. However, concordant with the more favorable binding enthalpy, the A6c134TCR made increased contacts with theTax peptide compared with the A6wt/A2- Tax complex, demonstrating a peptide-focused mechanism for the enhanced affinity that directly involved the mutated residues in the A6c134TCR CDR3b loop.This peptide-focused enhancedTCR binding may represent an important approach for developing antigen specific high affinity TCR reagents for use in T cell based therapies

Structural and kinetic basis for heightened immunogenicity of T cell vaccines

Analogue peptides with enhanced binding affinity to major histocompatibility class (MHC) I molecules are currently being used in cancer patients to elicit stronger T cell responses. However, it remains unclear as to how alterations of anchor residues may affect T cell receptor (TCR) recognition. We correlate functional, thermodynamic, and structural parameters of TCR–peptide–MHC binding and demonstrate the effect of anchor residue modifications of the human histocompatibility leukocyte antigens (HLA)–A2 tumor epitope NY–ESO-1157–165–SLLMWITQC on TCR recognition. The crystal structure of the wild-type peptide complexed with a specific TCR shows that TCR binding centers on two prominent, sequential, peptide sidechains, methionine–tryptophan. Cysteine-to-valine substitution at peptide position 9, while optimizing peptide binding to the MHC, repositions the peptide main chain and generates subtly enhanced interactions between the analogue peptide and the TCR. Binding analyses confirm tighter binding of the analogue peptide to HLA–A2 and improved soluble TCR binding. Recognition of analogue peptide stimulates faster polarization of lytic granules to the immunological synapse, reduces dependence on CD8 binding, and induces greater numbers of cross-reactive cytotoxic T lymphocyte to SLLMWITQC. These results provide important insights into heightened immunogenicity of analogue peptides and highlight the importance of incorporating structural data into the process of rational optimization of superagonist peptides for clinical trials

Direct molecular mimicry enables off-target cardiovascular toxicity by an enhanced affinity TCR designed for cancer immunotherapy

Natural T-cell responses generally lack the potency to eradicate cancer. Enhanced affinity T-cell receptors (TCRs) provide an ideal approach to target cancer cells, with emerging clinical data showing significant promise. Nevertheless, the risk of off target reactivity remains a key concern, as exemplified in a recent clinical report describing fatal cardiac toxicity, following administration of MAGE-A3 specific TCR-engineered T-cells, mediated through cross-reactivity with an unrelated epitope from the Titin protein presented on cardiac tissue. Here, we investigated the structural mechanism enabling TCR cross-recognition of MAGE-A3 and Titin, and applied the resulting data to rationally design mutants with improved antigen discrimination, providing a proof-of-concept strategy for altering the fine specificity of a TCR towards an intended target antigen. This study represents the first example of direct molecular mimicry leading to clinically relevant fatal toxicity, mediated by a modified enhanced affinity TCR designed for cancer immunotherapy. Furthermore, these data demonstrate that self-antigens that are expressed at high levels on healthy tissue should be treated with extreme caution when designing immuno-therapeutics

Specificity of bispecific T cell receptors and antibodies targeting peptide-HLA

Tumor-associated peptide–human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface–expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents

TCR‐induced alteration of primary MHC peptide anchor residue

The HLA‐A*02:01‐restricted decapeptide EAAGIGILTV, derived from melanoma antigen recognized by T‐cells‐1 (MART‐1) protein, represents one of the best‐studied tumor associated T‐cell epitopes, but clinical results targeting this peptide have been disappointing. This limitation may reflect the dominance of the nonapeptide, AAGIGILTV, at the melanoma cell surface. The decapeptide and nonapeptide are presented in distinct conformations by HLA‐A*02:01 and TCRs from clinically relevant T‐cell clones recognize the nonapeptide poorly. Here, we studied the MEL5 TCR that potently recognizes the nonapeptide. The structure of the MEL5‐HLA‐A*02:01‐AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide–MHC anchoring. This “flexing” at the TCR–peptide–MHC interface to accommodate the peptide antigen explains previously observed incongruences in this well‐studied system and has important implications for future therapeutic approaches. Finally, this study expands upon the mechanisms by which molecular plasticity can influence antigen recognition by T cells

Modification of iron binding ligands in isopenicillin n synthase

Isopenicillin N synthase (IPNS) is a non-haem iron dependent dioxygenase which catalyses the oxidative conversion of anddelta;-(L-andalpha;-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N (IPN). Sequence comparisons between IPNS isozymes reveal the complete conservation of two histidine (His214, His270), one aspartate (Asp216) [also known as the '2-His-l-carboxylate' motif] and one glutamine (Gln330) residue. The crystal structure of IPNS (Aspergillus nidulans) active site (in the absence of ACV) revealed an octahedrally coordinated manganese atom surrounded by these four protein ligands and two water molecules. The role of the four conserved metal binding ligands was investigated using site directed mutagenesis. The results demonstrated that ligation of the iron with Gln330 was not essential for the catalytic activity of IPNS. In contrast, ligation of the iron with the three remaining metal ligands was indispensable for catalytic activity. Additionally, it was demonstrated that the conserved Asp216 residue may be substituted by a glutamate residue (D216E) with significant retention of catalytic activity. Crystallographic and spectroscopic evidence suggested that the D216E mutant bound both iron and ACV in a similar way to wild-type IPNS. The inactivation of wild-type IPNS was examined under in vitro assay conditions. This study showed that inactivation of IPNS results (minimally) from a slow non-oxidative pathway (in buffer alone) and a fast oxidative pathway via Udenfriend's chemistry (ferrous iron, ascorbate, and oxygen). The oxidative inactivation pathway was substantially reduced by the inclusion of catalase in the assay mixture, thus indicating that oxidative IPNS inactivation results (in part) from the generation of hydrogen peroxide in solution. Inactivation was also accompanied by a slow fragmentation of intact IPNS into (at least) five oligopeptides (observed by sodium dodecyl sulphate polyacrylamide gel electrophoresis). N-Terminal sequencing analyses confirmed that the fragmentation resulted from at least two cleavage sites within the active site (between Asp216-Val217 and Val272-Lys273).</p

Modification of iron binding ligands in isopenicillin n synthase

Isopenicillin N synthase (IPNS) is a non-haem iron dependent dioxygenase which catalyses the oxidative conversion of anddelta;-(L-andalpha;-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N (IPN). Sequence comparisons between IPNS isozymes reveal the complete conservation of two histidine (His214, His270), one aspartate (Asp216) [also known as the '2-His-l-carboxylate' motif] and one glutamine (Gln330) residue. The crystal structure of IPNS (Aspergillus nidulans) active site (in the absence of ACV) revealed an octahedrally coordinated manganese atom surrounded by these four protein ligands and two water molecules. The role of the four conserved metal binding ligands was investigated using site directed mutagenesis. The results demonstrated that ligation of the iron with Gln330 was not essential for the catalytic activity of IPNS. In contrast, ligation of the iron with the three remaining metal ligands was indispensable for catalytic activity. Additionally, it was demonstrated that the conserved Asp216 residue may be substituted by a glutamate residue (D216E) with significant retention of catalytic activity. Crystallographic and spectroscopic evidence suggested that the D216E mutant bound both iron and ACV in a similar way to wild-type IPNS. The inactivation of wild-type IPNS was examined under in vitro assay conditions. This study showed that inactivation of IPNS results (minimally) from a slow non-oxidative pathway (in buffer alone) and a fast oxidative pathway via Udenfriend's chemistry (ferrous iron, ascorbate, and oxygen). The oxidative inactivation pathway was substantially reduced by the inclusion of catalase in the assay mixture, thus indicating that oxidative IPNS inactivation results (in part) from the generation of hydrogen peroxide in solution. Inactivation was also accompanied by a slow fragmentation of intact IPNS into (at least) five oligopeptides (observed by sodium dodecyl sulphate polyacrylamide gel electrophoresis). N-Terminal sequencing analyses confirmed that the fragmentation resulted from at least two cleavage sites within the active site (between Asp216-Val217 and Val272-Lys273).</p