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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., Introduction The ability to selectively target tumor-specific antigens holds great promise for the development of specific cancer treatments, but their identification remains a key challenge. Peptide fragments presented on the [...]

T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)-mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagentstermed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy., Harnessing the power of adaptive immunity to combat cancer has been a long-term goal of translational immunotherapy. However, although many avenues in this field have been explored over the past [...]

Affinity chromotography is used to purify human erythrocyte band 3 and band 4.2 peripheral proteins. Liposomes of the lipid group are formed from band 3 protein and exhibit a ratio resembling that of erythrocyte membranes. The working and proper organization of the protein are examined by employing stilbenedisulfonate prevention of band 3 controlled efflux of radiolabeled sulfate from preloaded liposomes. A distinct variation in efflux is observed when sulfate efflux controlled by purified band 3 and incompletely purified band 3 constituting sample quantities of bands 4.1 and 4. 2 are compared.

The mammalian α/β T cell receptor (TCR) repertoire plays a pivotal role in adaptive immunity by recognizing short, processed, peptide antigens bound in the context of a highly diverse family of cell-surface major histocompatibility complexes (pMHCs). Despite the extensive TCR-MHC interaction surface, peptide-independent cross-reactivity of native TCRs is generally avoided through cell-mediated selection of molecules with low inherent affinity for MHC. Here we show that, contrary to expectations, the germ line-encoded complementarity determining regions (CDRs) of human TCRs, namely the CDR2s, which appear to contact only the MHC surface and not the bound peptide, can be engineered to yield soluble low nanomolar affinity ligands that retain a surprisingly high degree of specificity for the cognate pMHC target. Structural investigation of one such CDR2 mutant implicates shape complementarity of the mutant CDR2 contact interfaces as being a key determinant of the increased affinity. Our results suggest that manipulation of germ line CDR2 loops may provide a useful route to the production of high-affinity TCRs with therapeutic and diagnostic potential. [ABSTRACT FROM AUTHOR]

HLA-A*2402 is the most commonly expressed HLA allele in oriental populations. It is also widely expressed in the Caucasian population, making it one of, if not the most abundant HLA I types. In order to study its structure in terms of overall fold and peptide presentation, a soluble form of this HLA I (α1, α2, α3 and βm domains) has been expressed, refolded and crystallized in complex with a cancer-related telomerase peptide (VYGFVRACL), and its structure has been solved to 2.8 Å resolution. The overall structure of HLA-A*2402 is virtually identical to other reported peptide-HLA I structures. However, there are distinct features observable from this structure at the HLA I peptide binding pockets. The size and depth of pocket B makes it highly suitable for binding to large aromatic side chains, which explains the high prevalence of tyrosine at peptide position 2. Also, for HLA binding at peptide position 5, there is an additional anchor point, which allows the proximal amino acids to protrude out, providing a prominent feature for TCR interaction. Finally, pocket F allows the anchor residue at position 9 to be bound unusually deeply within the HLA structure. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 P41/ P43, 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. [ABSTRACT FROM AUTHOR]

Nitrous oxide reductase is a periplasmic respiratory protein with a novel copper catalytic centre; it catalyses the terminal step, reduction of nitrous oxide to nitrogen, of the bacterial denitrification process. Nitrous oxide reductase from Paracoccus pantotrophus has been crystallized by the hanging-drop method. A prerequisite for crystallization was the oxidation of the enzyme with potassium ferricyanide in order to obtain homogenous oxidation states of the copper centres. The crystals belong to space group P212121, with unit-cell parameters a = 116.4, b 118.3, c 267.0 Å. Two homodimers, of approximate molecular weight 67 kDa per subunit, probably constitute the asymmetric unit and give a Matthews coefficient, Vm, of 3.4 Å3 Da-1 and a solvent content of 59% by volume. The crystals diffract X-rays to 3.0 Å resolution on an in-house source and are suitable for structure determination. [ABSTRACT FROM AUTHOR]

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., (© 2019 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Isopenicillin N synthase (IPNS) catalyses the four-electron oxidation of a tripeptide, l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV), to give isopenicillin N (IPN), the first-formed β-lactam in penicillin and cephalosporin biosynthesis. IPNS catalysis is dependent upon an iron(II) cofactor and oxygen as a co-substrate. In the absence of substrate, the carbonyl oxygen of the side-chain amide of the penultimate residue, Gln330, co-ordinates to the active-site metal iron. Substrate binding ablates the interaction between Gln330 and the metal, triggering rearrangement of seven C-terminal residues, which move to take up a conformation that extends the final α-helix and encloses ACV in the active site. Mutagenesis studies are reported, which probe the role of the C-terminal and other aspects of the substrate binding pocket in IPNS. The hydrophobic nature of amino acid side-chains around the ACV binding pocket is important in catalysis. Deletion of seven C-terminal residues exposes the active site and leads to formation of a new type of thiol oxidation product. The isolated product is shown by LC-MS and NMR analyses to be the ene-thiol tautomer of a dithioester, made up from two molecules of ACV linked between the thiol sulfur of one tripeptide and the oxidised cysteinyl β-carbon of the other. A mechanism for its formation is proposed, supported by an X-ray crystal structure, which shows the substrate ACV bound at the active site, its cysteinyl β-carbon exposed to attack by a second molecule of substrate, adjacent. Formation of this product constitutes a new mode of reaction for IPNS and non-heme iron oxidases in general., (© 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Natural T 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 CDR3β 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 A6c134 TCR made increased contacts with the Tax 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 A6c134 TCR CDR3β loop. This peptide-focused enhanced TCR binding may represent an important approach for developing antigen specific high affinity TCR reagents for use in T cell based therapies.

Human CD8 is a T cell coreceptor, which binds to pHLA I and plays a pivotal role in the activation of cytotoxic T lymphocytes. Soluble recombinant CD8 alphaalpha has been shown to antagonize T cell activation, both in vitro and in vivo. However, because of a very low affinity for pHLA I, high concentrations of soluble CD8 alphaalpha are required for efficient inhibition. Based upon our knowledge of the wild-type CD8/pHLA I structure, we have designed and produced a mutated form of soluble CD8 alphaalpha that binds to pHLA I with approximately fourfold higher affinity. We have characterized the binding of the high affinity CD8 mutant using surface plasmon resonance and determined its structure at 2.1 A resolution using X-ray crystallography. The analysis of this structure suggests that the higher affinity is achieved by providing a larger side chain that allows for an optimal contact to be made between the HLA alpha3 loop and the mutated CDR-like loops of CD8., ((c) 2007 Wiley-Liss, Inc.)

The mammalian alpha/beta T cell receptor (TCR) repertoire plays a pivotal role in adaptive immunity by recognizing short, processed, peptide antigens bound in the context of a highly diverse family of cell-surface major histocompatibility complexes (pMHCs). Despite the extensive TCR-MHC interaction surface, peptide-independent cross-reactivity of native TCRs is generally avoided through cell-mediated selection of molecules with low inherent affinity for MHC. Here we show that, contrary to expectations, the germ line-encoded complementarity determining regions (CDRs) of human TCRs, namely the CDR2s, which appear to contact only the MHC surface and not the bound peptide, can be engineered to yield soluble low nanomolar affinity ligands that retain a surprisingly high degree of specificity for the cognate pMHC target. Structural investigation of one such CDR2 mutant implicates shape complementarity of the mutant CDR2 contact interfaces as being a key determinant of the increased affinity. Our results suggest that manipulation of germ line CDR2 loops may provide a useful route to the production of high-affinity TCRs with therapeutic and diagnostic potential.