Structural basis for self-discrimination by neoantigen-specific TCRs.

T cell receptors (TCR) are pivotal in mediating tumour cell cytolysis via recognition of mutation-derived tumour neoantigens (neoAgs) presented by major histocompatibility class-I (MHC-I). Understanding the factors governing the emergence of neoAg from somatic mutations is a major focus of current research. However, the structural and cellular determinants controlling TCR recognition of neoAgs remain poorly understood. This study describes the multi-level analysis of a model neoAg from the B16F10 murine melanoma, H2-Db/Hsf2 p.K72N68-76, as well as its cognate TCR 47BE7. Through cellular, molecular and structural studies we demonstrate that the p.K72N mutation enhances H2-Db binding, thereby improving cell surface presentation and stabilizing the TCR 47BE7 epitope. Furthermore, TCR 47BE7 exhibited high functional avidity and selectivity, attributable to a broad, stringent, binding interface enabling recognition of native B16F10 despite low antigen density. Our findings provide insight into the generation of anchor-residue modified neoAg, and emphasize the value of molecular and structural investigations of neoAg in diverse MHC-I contexts for advancing the understanding of neoAg immunogenicity.

Molecular mechanism of phosphopeptide neoantigen immunogenicity.

Altered protein phosphorylation in cancer cells often leads to surface presentation of phosphopeptide neoantigens. However, their role in cancer immunogenicity remains unclear. Here we describe a mechanism by which an HLA-B*0702-specific acute myeloid leukemia phosphoneoantigen, pMLL747-755 (EPR(pS)PSHSM), is recognized by a cognate T cell receptor named TCR27, a candidate for cancer immunotherapy. We show that the replacement of phosphoserine P4 with serine or phosphomimetics does not affect pMHC conformation or peptide-MHC affinity but abrogates TCR27-dependent T cell activation and weakens binding between TCR27 and pMHC. Here we describe the crystal structures for TCR27 and cognate pMHC, map of the interface produced by nuclear magnetic resonance, and a ternary complex generated using information-driven protein docking. Our data show that non-covalent interactions between the epitope phosphate group and TCR27 are crucial for TCR specificity. This study supports development of new treatment options for cancer patients through target expansion and TCR optimization.

Structural Basis for Self-Discrimination by Neoantigen-Specific TCRs.

Physical interactions between T cell receptors (TCRs) and mutation-derived tumour neoantigens (neoAg) presented by major histocompatibility class-I (MHC-I) enable sensitive and specific cytolysis of tumour cells. Adoptive transfer of neoAg-reactive T cells in patients is correlated with response to immunotherapy; however, the structural and cellular mechanisms of neoAg recognition remain poorly understood. We have identified multiple cognate neoAg:TCRs from B16F10, a common murine implantable tumour model of melanoma. We identified a high affinity TCR targeting H2-Db-restricted Hsf2K72N that conferred specific recognition of B16F10 in vitro and in vivo. Structural characterization of the peptide-MHC (pMHC) binary and pMHC:TCR ternary complexes yielded high-resolution crystal structures, revealing the formation of a solvent-exposed hydrophobic arch in H2-Db that enables multiple intermolecular contacts between pMHC and the TCR. These features of structural stability strikingly mimic that of a previously published influenza peptide-H2-Db complex and its corresponding TCR, suggesting that there are shared structural motifs between neoantigens and viral peptides that explain their shared immunogenicity.

Discovery of GBT440, an Orally Bioavailable R-State Stabilizer of Sickle Cell Hemoglobin.

We report the discovery of a new potent allosteric effector of sickle cell hemoglobin, GBT440 (36), that increases the affinity of hemoglobin for oxygen and consequently inhibits its polymerization when subjected to hypoxic conditions. Unlike earlier allosteric activators that bind covalently to hemoglobin in a 2:1 stoichiometry, 36 binds with a 1:1 stoichiometry. Compound 36 is orally bioavailable and partitions highly and favorably into the red blood cell with a RBC/plasma ratio of ∼150. This partitioning onto the target protein is anticipated to allow therapeutic concentrations to be achieved in the red blood cell at low plasma concentrations. GBT440 (36) is in Phase 3 clinical trials for the treatment of sickle cell disease (NCT03036813).

GBT440 increases haemoglobin oxygen affinity, reduces sickling and prolongs RBC half-life in a murine model of sickle cell disease.

A major driver of the pathophysiology of sickle cell disease (SCD) is polymerization of deoxygenated haemoglobin S (HbS), which leads to sickling and destruction of red blood cells (RBCs) and end-organ damage. Pharmacologically increasing the proportion of oxygenated HbS in RBCs may inhibit polymerization, prevent sickling and provide long term disease modification. We report that GBT440, a small molecule which binds to the N-terminal α chain of Hb, increases HbS affinity for oxygen, delays in vitro HbS polymerization and prevents sickling of RBCs. Moreover, in a murine model of SCD, GBT440 extends the half-life of RBCs, reduces reticulocyte counts and prevents ex vivo RBC sickling. Importantly, oral dosing of GBT440 in animals demonstrates suitability for once daily dosing in humans and a highly selective partitioning into RBCs, which is a key therapeutic safety attribute. Thus, GBT440 has the potential for clinical use as a disease-modifying agent in sickle cell patients.

Transition state model and mechanism of nucleophilic aromatic substitution reactions catalyzed by human glutathione S-transferase M1a-1a.

An active site His107 residue distinguishes human glutathione S-transferase hGSTM1-1 from other mammalian Mu-class GSTs. The crystal structure of hGSTM1a-1a with bound glutathione (GSH) was solved to 1.9 A resolution, and site-directed mutagenesis supports the conclusion that a proton transfer occurs in which bound water at the catalytic site acts as a primary proton acceptor from the GSH thiol group to transfer the proton to His107. The structure of the second substrate-binding site (H-site) was determined from hGSTM1a-1a complexed with 1-glutathionyl-2,4-dinitrobenzene (GS-DNB) formed by a reaction in the crystal between GSH and 1-chloro-2,4-dinitrobenzene (CDNB). In that structure, the GSH-binding site (G-site) is occupied by the GSH moiety of the product in the same configuration as that of the enzyme-GSH complex, and the dinitrobenzene ring is anchored between the side chains of Tyr6, Leu12, His107, Met108, and Tyr115. This orientation suggested a distinct transition state that was substantiated from the structure of hGSTM1a-1a complexed with transition state analogue 1-S-(glutathionyl)-2,4,6-trinitrocyclohexadienate (Meisenheimer complex). Kinetic data for GSTM1a-1a indicate that kcat(CDNB) for the reaction is more than 3 times greater than kcat(FDNB), even though the nonenzymatic second-order rate constant is more than 50-fold greater for 1-fluoro-2,4-dinitrobenzene (FDNB), and the product is the same for both substrates. In addition, Km(FDNB) is about 20 times less than Km(CDNB). The results are consistent with a mechanism in which the formation of the transition state is rate-limiting in the nucleophilic aromatic substitution reactions. Data obtained with active-site mutants support transition states in which Tyr115, Tyr6, and His107 side chains are involved in the stabilization of the Meisenheimer complex via interactions with the ortho nitro group of CDNB or FDNB and provide insight into the means by which GSTs adapt to accommodate different substrates.

COHbC and COHbS crystallize in the R2 quaternary state at neutral pH in the presence of PEG 4000.

Human hemoglobin binds oxygen cooperatively and functions as a tetramer composed of two identical alphabeta heterodimers. While human hemoglobin is the best characterized allosteric protein, the quaternary R (oxygenated or liganded) to T (deoxygenated) structural transition remains controversial. The R2 state has been postulated to represent either an intermediate or final quaternary state elicited by ligand binding. However, the biological relevance of the R2 state has been questioned as it has not been observed crystallographically under physiological conditions. The high-resolution R2 quaternary structures of human COHbC (betaE6K) and COHbS (betaE6V) are reported at neutral pH and low ionic strength using PEG 4000 as a precipitant. Crystals of COHbC, COHbS and their mixtures are isomorphous, indicating that they share the same tertiary and quaternary structures. In contrast, oxyHbA or COHbA did not yield crystals at neutral pH under similar conditions. Solubility studies and modeling suggest that at neutral pH and low ionic strength the beta6 mutant hemoglobins crystallize (betaK6 > betaV6) as a result of more favorable lattice contacts.

Structure of mutant human carbonmonoxyhemoglobin C (betaE6K) at 2.0 A resolution.

Previous studies have demonstrated that in vitro crystallization of R-state liganded hemoglobin C (HbC), a naturally occurring mutant human hemoglobin (betaE6K), in high-phosphate buffer solutions provides a potential model system for the intracellular crystallization of HbC associated with chronic hemolytic anemia in CC disease. The first high-resolution crystal structure of liganded HbC is reported here. HbC was crystallized from high phosphate and the structure of the carbonmonoxy-liganded R-state form was refined at 2.0 A resolution. Crystals exhibit diffraction consistent with the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = 54.16, c = 195.30 A. The structure was solved by difference Fourier techniques and refinement by simulated annealing and restrained least-squares yielded a final R of 0.183 and an R(free) of 0.238 for all 19,382 unique reflections. The side chain of betaK6 exhibits very weak electron density consistent with significant mobility within the crystalline lattice. The highly dynamic nature of the side chain could potentially support a number of specific polar interactions that might reduce the barrier to crystallization and thus result in enhanced crystallization kinetics for HbC relative to HbA. Specifically, the NZ atom of the BK6 side chain could participate in an amino-aromatic hydrogen bond with the pi-electron cloud of betaH116 in a symmetry-related tetramer. BetaK6 NZ might also interact with the main-chain carbonyl O atom of betaH117 and the carboxylate group of betaE22 from a symmetry-related tetramer.