Targeting of intracellular oncoproteins with peptide-centric CARs.

The majority of oncogenic drivers are intracellular proteins, constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient for generating responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins essential for tumorigenesis. We focused on targeting the unmutated peptide QYNPIRTTF discovered on HLA-A*24:02, which is derived from the neuroblastoma-dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (PC-CARs) through a counter panning strategy using predicted potentially cross-reactive peptides. We further proposed that PC-CARs can recognize peptides on additional HLA allotypes when presenting a similar overall molecular surface. Informed by our computational modelling results, we show that PHOX2B PC-CARs also recognize QYNPIRTTF presented by HLA-A*23:01, the most common non-A2 allele in people with African ancestry. Finally, we demonstrate potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that PC-CARs have the potential to expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and allow targeting through additional HLA allotypes in a clinical setting.

Cross-HLA targeting of intracellular oncoproteins with peptide-centric CARs.

The majority of oncogenic drivers are intracellular proteins, thus constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient to generate responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins that are essential for tumourigenesis and focus on targeting the unmutated peptide QYNPIRTTF, discovered on HLA-A*24:02, which is derived from the neuroblastoma dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (CARs) using a counter-panning strategy with predicted potentially cross-reactive peptides. We further hypothesized that peptide-centric CARs could recognize peptides on additional HLA allotypes when presented in a similar manner. Informed by computational modelling, we showed that PHOX2B peptide-centric CARs also recognize QYNPIRTTF presented by HLA-A*23:01 and the highly divergent HLA-B*14:02. Finally, we demonstrated potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that peptide-centric CARs have the potential to vastly expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and widen the population of patients who would benefit from such therapy by breaking conventional HLA restriction.

Universal open MHC-I molecules for rapid peptide loading and enhanced complex stability across HLA allotypes.

The polymorphic nature and intrinsic instability of class I major histocompatibility complex (MHC-I) and MHC-like molecules loaded with suboptimal peptides, metabolites, or glycolipids presents a fundamental challenge for identifying disease-relevant antigens and antigen-specific T cell receptors (TCRs), hindering the development of autologous therapeutics. Here, we leverage the positive allosteric coupling between the peptide and light chain (ß2 microglobulin, ß2m) subunits for binding to the MHC-I heavy chain (HC) through an engineered disulfide bond bridging conserved epitopes across the HC/ß2m interface, to generate conformationally stable, peptide-receptive molecules named "open MHC-I." Biophysical characterization shows that open MHC-I molecules are properly folded protein complexes of enhanced thermal stability compared to the wild type when loaded with low- to moderate-affinity peptides. Using solution NMR, we characterize the effects of the disulfide bond on the conformation and dynamics of the MHC-I structure, ranging from local changes in ß2m-interacting sites of the peptide-binding groove to long-range effects on the α2-1 helix and α3 domain. The interchain disulfide bond stabilizes MHC-I molecules in an open conformation to promote peptide exchange across multiple human leukocyte antigen (HLA) allotypes, covering representatives from five HLA-A supertypes, six HLA-B supertypes, and oligomorphic HLA-Ib molecules. Our structure-guided design, combined with conditional ß-peptide ligands, provides a universal platform to generate ready-to-load MHC-I systems of enhanced stability, enabling a range of approaches to screen antigenic epitope libraries and probe polyclonal TCR repertoires covering highly polymorphic HLA-I allotypes, as well as oligomorphic nonclassical molecules.

Universal open MHC-I molecules for rapid peptide loading and enhanced complex stability across HLA allotypes.

The polymorphic nature and intrinsic instability of class I major histocompatibility complex (MHC-I) and MHC-like molecules loaded with suboptimal peptides, metabolites, or glycolipids presents a fundamental challenge for identifying disease-relevant antigens and antigen-specific T cell receptors (TCRs), hindering the development of autologous therapeutics. Here, we leverage the positive allosteric coupling between the peptide and light chain (ß 2 microglobulin, ß 2 m) subunits for binding to the MHC-I heavy chain (HC) through an engineered disulfide bond bridging conserved epitopes across the HC/ß 2 m interface, to generate conformationally stable, open MHC-I molecules. Biophysical characterization shows that open MHC-I molecules are properly folded protein complexes of enhanced thermal stability compared to the wild type, when loaded with low- to intermediate-affinity peptides. Using solution NMR, we characterize the effects of the disulfide bond on the conformation and dynamics of the MHC-I structure, ranging from local changes in ß 2 m interacting sites of the peptide binding groove to long-range effects on the α 2-1 helix and α 3 domain. The interchain disulfide bond stabilizes empty MHC-I molecules in a peptide-receptive, open conformation to promote peptide exchange across multiple human leucocyte antigen (HLA) allotypes, covering representatives from five HLA-A, six HLA-B supertypes, and oligomorphic HLA-Ib molecules. Our structural design, combined with conditional ß-peptide ligands, provides a universal platform for generating ready-to-load MHC-I systems of enhanced stability, enabling a range of approaches to screen antigenic epitope libraries and probe polyclonal TCR repertoires in the context of highly polymorphic HLA-I allotypes, as well as oligomorphic nonclassical molecules. Significance Statement: We outline a structure-guided approach for generating conformationally stable, open MHC-I molecules with enhanced ligand exchange kinetics spanning five HLA-A, all HLA-B supertypes, and oligomorphic HLA-Ib allotypes. We present direct evidence of positive allosteric cooperativity between peptide binding and ß 2 m association with the heavy chain by solution NMR and HDX-MS spectroscopy. We demonstrate that covalently linked ß 2 m serves as a conformational chaperone to stabilize empty MHC-I molecules in a peptide-receptive state, by inducing an open conformation and preventing intrinsically unstable heterodimers from irreversible aggregation. Our study provides structural and biophysical insights into the conformational properties of MHC-I ternary complexes, which can be further applied to improve the design of ultra-stable, universal ligand exchange systems in a pan-HLA allelic setting.

Structural principles of peptide-centric chimeric antigen receptor recognition guide therapeutic expansion.

Peptide-centric chimeric antigen receptors (PC-CARs) recognize oncoprotein epitopes displayed by cell-surface human leukocyte antigens (HLAs) and offer a promising strategy for targeted cancer therapy. We have previously developed a PC-CAR targeting a neuroblastoma-associated PHOX2B peptide, leading to robust tumor cell lysis restricted by two common HLA allotypes. Here, we determine the 2.1-angstrom crystal structure of the PC-CAR-PHOX2B-HLA-A*24:02-ß2m complex, which reveals the basis for antigen-specific recognition through interactions with CAR complementarity-determining regions (CDRs). This PC-CAR adopts a diagonal docking mode, where interactions with both conserved and polymorphic HLA framework residues permit recognition of multiple HLA allotypes from the A9 serological cross-reactive group, covering a combined global population frequency of up to 46.7%. Biochemical binding assays, molecular dynamics simulations, and structural and functional analyses demonstrate that high-affinity PC-CAR recognition of cross-reactive pHLAs necessitates the presentation of a specific peptide backbone, where subtle structural adaptations of the peptide are critical for high-affinity complex formation, and CAR T cell killing. Our results provide a molecular blueprint for engineering CARs with optimal recognition of tumor-associated antigens in the context of different HLAs, while minimizing cross-reactivity with self-epitopes.

Structural principles of peptide-centric Chimeric Antigen Receptor recognition guide therapeutic expansion.

Peptide-Centric Chimeric Antigen Receptors (PC-CARs), which recognize oncoprotein epitopes displayed by human leukocyte antigens (HLAs) on the cell surface, offer a promising strategy for targeted cancer therapy 1 . We have previously developed a PC-CAR targeting a neuroblastoma- associated PHOX2B peptide, leading to robust tumor cell lysis restricted by two common HLA allotypes 2 . Here, we determine the 2.1 Å structure of the PC-CAR:PHOX2B/HLA-A*24:02/ß2m complex, which reveals the basis for antigen-specific recognition through interactions with CAR complementarity-determining regions (CDRs). The PC-CAR adopts a diagonal docking mode, where interactions with both conserved and polymorphic HLA framework residues permit recognition of multiple HLA allotypes from the A9 serological cross-reactivity group, covering a combined American population frequency of up to 25.2%. Comprehensive characterization using biochemical binding assays, molecular dynamics simulations, and structural and functional analyses demonstrate that high-affinity PC-CAR recognition of cross-reactive pHLAs necessitates the presentation of a specific peptide backbone, where subtle structural adaptations of the peptide are critical for high-affinity complex formation and CAR-T cell killing. Our results provide a molecular blueprint for engineering CARs with optimal recognition of tumor-associated antigens in the context of different HLAs, while minimizing cross-reactivity with self-epitopes.

Structural mechanism of tapasin-mediated MHC-I peptide loading in antigen presentation.

Loading of MHC-I molecules with peptide by the catalytic chaperone tapasin in the peptide loading complex plays a critical role in antigen presentation and immune recognition. Mechanistic insight has been hampered by the lack of detailed structural information concerning tapasin-MHC-I. We present here crystal structures of human tapasin complexed with the MHC-I molecule HLA-B*44:05, and with each of two anti-tapasin antibodies. The tapasin-stabilized peptide-receptive state of HLA-B*44:05 is characterized by distortion of the peptide binding groove and destabilization of the ß2-microglobulin interaction, leading to release of peptide. Movements of the membrane proximal Ig-like domains of tapasin, HLA-B*44:05, and ß2-microglobulin accompany the transition to a peptide-receptive state. Together this ensemble of crystal structures provides insights into a distinct mechanism of tapasin-mediated peptide exchange.

Dynamics of MHC-I molecules in the antigen processing and presentation pathway.

The endogenous antigen processing and presentation (APP) is a fundamental pathway found in jawed vertebrates, which allows for a set of epitope peptides sampled from the intracellular proteome to be assembled and displayed on class I proteins of the major histocompatibility complex (MHC-I). Peptide/MHC-I antigens enable different aspects of adaptive immunity to emerge, by providing a basis for recognition of self vs. non-self by T cells and Natural Killer (NK) cells. Pioneering studies of pMHC-I molecules and their higher-order protein complexes with molecular chaperones and membrane receptors have gleaned important insights into the peptide loading and antigen recognition mechanisms. While X-ray and cryoEM structures have provided us with static snapshots of different MHC-I assembly stages, complementary biophysical techniques have revealed that MHC-I molecules are highly mobile on a range of biologically relevant timescales, which bears importance for their assembly, peptide repertoire selection, membrane display and turnover. This review summarizes insights gained from experimental and simulation studies aimed at investigating MHC-I dynamics, and their functional implications.

Computational structural enzymology methodologies for the study and engineering of fatty acid synthases, polyketide synthases and nonribosomal peptide synthetases.

Various computational methodologies can be applied to enzymological studies on enzymes in the fatty acid, polyketide, and non-ribosomal peptide biosynthetic pathways. These multi-domain complexes are called fatty acid synthases, polyketide synthases, and non-ribosomal peptide synthetases. These mega-synthases biosynthesize chemically diverse and complex bioactive molecules, with the intermediates being chauffeured between catalytic partners via a carrier protein. Recent efforts have been made to engineer these systems to expand their product diversity. A major stumbling block is our poor understanding of the transient protein-protein and protein-substrate interactions between the carrier protein and its many catalytic partner domains and product intermediates. The innate reactivity of pathway intermediates in two major classes of polyketide synthases has frustrated our mechanistic understanding of these interactions during the biosynthesis of these natural products, ultimately impeding the engineering of these systems for the generation of engineered natural products. Computational techniques described in this chapter can aid data interpretation or used to generate testable models of these experimentally intractable transient interactions, thereby providing insight into key interactions that are difficult to capture otherwise, with the potential to expand the diversity in these systems.