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In human celiac disease (CeD) HLA-DQ2.5 presents gluten peptides to antigen-specific CD4 + T cells, thereby instigating immune activation and enteropathy. Targeting HLA-DQ2.5 with neutralizing antibody for treating CeD may be plausible, yet using pan-HLA-DQ antibody risks affecting systemic immunity, while targeting selected gluten peptide:HLA-DQ2.5 complex (pHLA-DQ2.5) may be insufficient. Here we generate a TCR-like, neutralizing antibody (DONQ52) that broadly recognizes more than twenty-five distinct gluten pHLA-DQ2.5 through rabbit immunization with multi-epitope gluten pHLA-DQ2.5 and multidimensional optimization. Structural analyses show that the proline-rich and glutamine-rich motif of gluten epitopes critical for pathogenesis is flexibly recognized by multiple tyrosine residues present in the antibody paratope, implicating the mechanisms for the broad reactivity. In HLA-DQ2.5 transgenic mice, DONQ52 demonstrates favorable pharmacokinetics with high subcutaneous bioavailability, and blocks immunity to gluten while not affecting systemic immunity. Our results thus provide a rationale for clinical testing of DONQ52 in CeD., (© 2023. The Author(s).)

Establishing a technological platform for creating clinical compounds inhibiting intracellular protein-protein interactions (PPIs) can open the door to many valuable drugs. Although small molecules and antibodies are mainstream modalities, they are not suitable for a target protein that lacks a deep cavity for a small molecule to bind or a protein found in intracellular space out of an antibody's reach. One possible approach to access these targets is to utilize so-called middle-size cyclic peptides (defined here as those with a molecular weight of 1000-2000 g/mol). In this study, we validated a new methodology to create oral drugs beyond the rule of 5 for intracellular tough targets by elucidating structural features and physicochemical properties for drug-like cyclic peptides and developing library technologies to afford highly N -alkylated cyclic peptide hits. We discovered a KRAS inhibitory clinical compound (LUNA18) as the first example of our platform technology.

Efficient production of bispecific antibodies (BsAbs) in single mammalian cells is essential for basic research and industrial manufacturing. However, preventing unwanted pairing of heavy chains (HCs) and light chains (LCs) is a challenging task. To address this, we created an engineering technology for preferential cognate HC/LC and HC/HC paring called FAST-Ig (Four-chain Assembly by electrostatic Steering Technology - Immunoglobulin), and applied it to NXT007, a BsAb for the treatment of hemophilia A. We introduced charged amino-acid substitutions at the HC/LC interface to facilitate the proper assembly for manufacturing a standard IgG-type BsAb. We generated CH1/CL interface-engineered antibody variants that achieved > 95% correct HC/LC pairing efficiency with favorable pharmacological properties and developability. Among these, we selected a design (C3) that allowed us to separate the mis-paired species with an unintended pharmacological profile using ion-exchange chromatography. Crystal structure analysis demonstrated that the C3 design did not affect the overall structure of both Fabs. To determine the final design for HCs-heterodimerization, we compared the stability of charge-based and knobs into hole-based Fc formats in acidic conditions and selected the more stable charge-based format. FAST-Ig was also applicable to stable CHO cell lines for industrial production and demonstrated robust chain pairing with different subclasses of parent BsAbs. Thus, it can be applied to a wide variety of BsAbs both preclinically and clinically.

Osimertinib is the only EGFR-tyrosine kinase inhibitor (TKI) capable of overcoming EGFR-T790M-mutated NSCLC, but osimertinib-resistant EGFR triple mutations (Del19/T790M/C797S or L858R/T790M/C797S) have been reported. Although allosteric EGFR TKIs (e.g., EAI-045) that potentially overcome L858R/T790M/C797S have been identified, there are no effective inhibitors against Del19/T790M/C797S. In this study, we identified CH7233163 as having the potential to overcome EGFR-Del19/T790M/C797S. CH7233163 showed potent antitumor activities against tumor with EGFR-Del19/T790M/C797S in vitro and in vivo In addition to EGFR-Del19/T790M/C797S, the characterization assays showed that CH7233163 more selectively inhibits various types of EGFR mutants (e.g., L858R/T790M/C797S, L858R/T790M, Del19/T790M, Del19, and L858R) over wild type. Furthermore, crystal structure analysis suggested that CH7233163 is a noncovalent ATP-competitive inhibitor for EGFR-Del19/T790M/C797S that utilizes multiple interactions with the EGFR's αC-helix-in conformation to achieve potent inhibitory activity and mutant selectivity. Therefore, we conclude that CH7233163 is a potentially effective therapy for osimertinib-resistant patients, especially in cases of EGFR-Del19/T790M/C797S., (©2020 American Association for Cancer Research.)

Noninvasive evaluation of tertiary structures is fundamental to the research, development, and use of the biologics. However, few methodologies are currently available for evaluating large molecular weight (MW) biologics, such as therapeutic monoclonal antibodies (mAbs; 150 kDa). Here, we have newly developed a 15 N direct detection nuclear magnetic resonance (NMR) technique, the 15 N direct detection CRINEPT, which allows the observation of the main chain amide resonances of a nondeuterated protein with MW 150 kDa. The technique not only substantially expands the range of proteins applicable to solution NMR studies but also allows the noninvasive structural analyses of intact mAbs in a wide range of temperature and solvent conditions. As a proof of principle, we successfully acquired the 15 N-detected CRINEPT spectra of an intact mAb in its formulated solution at 4 °C. The technique was able to discriminate heterogeneous galactosylation states, demonstrating the benefit of high resolution of the 15 N direct detection.

Purpose: To investigate mechanisms governing the stabilization and destabilization of immunoglobulin (IgG1) by arginine (Arg)., Methods: The effects of Arg on the aggregation/degradation, thermodynamic stability, hydrophobicity, and aromatic residues of IgG1 were respectively investigated by size-exclusion chromatography, differential scanning calorimetry, probe fluorescence, and intrinsic fluorescence., Results: Arg monohydrochloride (Arg-HCl) suppressed IgG1 aggregation at near-neutral pH, but facilitated aggregation and degradation at acidic pH or at high storage temperature. Equimolar mixtures of Arg and aspartic acid (Asp) or glutamic acid (Glu) suppressed aggregation without facilitating degradation even at high temperature. Arg-HCl decreased the thermodynamic stability of IgG1 by enthalpic loss, which was counteracted by using Asp or Glu as a counterion for Arg. The suppression of aggregation by Arg-HCl was well correlated with the decrease in hydrophobicity of IgG1. The intrinsic fluorescence of IgG1 was unaffected by Arg-HCl., Conclusions: Suppression of IgG1 aggregation can be attributed to the interaction between Arg and hydrophobic residues; on the other hand, facilitation of aggregation and degradation is presumably due to the interaction between Arg and some acidic residues, which could be competitively inhibited by simultaneously adding either Asp or Glu.

The product of gene At3g16450.1 from Arabidopsis thaliana is a 32 kDa, 299-residue protein classified as resembling a myrosinase-binding protein (MyroBP). MyroBPs are found in plants as part of a complex with the glucosinolate-degrading enzyme myrosinase, and are suspected to play a role in myrosinase-dependent defense against pathogens. Many MyroBPs and MyroBP-related proteins are composed of repeated homologous sequences with unknown structure. We report here the three-dimensional structure of the At3g16450.1 protein from Arabidopsis, which consists of two tandem repeats. Because the size of the protein is larger than that amenable to high-throughput analysis by uniform (13)C/(15)N labeling methods, we used stereo-array isotope labeling (SAIL) technology to prepare an optimally (2)H/(13)C/(15)N-labeled sample. NMR data sets collected using the SAIL protein enabled us to assign (1)H, (13)C and (15)N chemical shifts to 95.5% of all atoms, even at a low concentration (0.2 mm) of protein product. We collected additional NOESY data and determined the three-dimensional structure using the cyana software package. The structure, the first for a MyroBP family member, revealed that the At3g16450.1 protein consists of two independent but similar lectin-fold domains, each composed of three beta-sheets.

Nuclear-magnetic-resonance spectroscopy can determine the three-dimensional structure of proteins in solution. However, its potential has been limited by the difficulty of interpreting NMR spectra in the presence of broadened and overlapping resonance lines and low signal-to-noise ratios. Here we present stereo-array isotope labelling (SAIL), a technique that can overcome many of these problems by applying a complete stereospecific and regiospecific pattern of stable isotopes that is optimal with regard to the quality and information content of the resulting NMR spectra. SAIL uses exclusively chemically and enzymatically synthesized amino acids for cell-free protein expression. We demonstrate for the 17-kDa protein calmodulin and the 41-kDa maltodextrin-binding protein that SAIL offers sharpened lines, spectral simplification without loss of information, and the ability to rapidly collect the structural restraints required to solve a high-quality solution structure for proteins twice as large as commonly solved by NMR. It thus makes a large class of proteins newly accessible to detailed solution structure determination.

A monoclonal antibody (DEM-1) specific for the Dewar photoproduct is used for detection and quantification of photolesions in DNA. To help understand the molecular recognition of damaged DNA by the antibody protein, we have cloned and sequenced the variable region genes of DEM-1. We have also prepared Fab fragments of DEM-1 (DEM1Fab), and synthesized two kinds of 3'-biotinylated oligonucleotides of different lengths containing a central Dewar photoproduct of TpT to analyze the effects of the antigen size on the binding rates by means of surface plasmon resonance (SPR). Results obtained from SPR analyses suggest that DEM1Fab may recognize tetranucleotide unit as the epitope.

Mdm2 is a cellular antagonist of p53 that keeps a balanced cellular level of p53. The two proteins are linked by a negative regulatory feedback loop and physically bind to each other via a putative helix formed by residues 18-26 of p53 transactivation domain (TAD) and its binding pocket located within the N-terminal 100-residue domain of mdm2 (Kussie, P. H., Gorina, S., Marechal, V., Elenbaas, B., Moreau, J., Levine, A. J., and Pavletich, N. P. (1996) Science 274, 948-953). In a previous report we demonstrated that p53 TAD in the mdm2-freee state is mostly unstructured but contains two nascent turns in addition to a "preformed" helix that is the same as the putative helix mediating p53-mdm2 binding. Here, using heteronuclear multidimensional NMR methods, we show that the two nascent turn motifs in p53 TAD, turn I (residues 40-45) and turn II (residues 49-54), are also capable of binding to mdm2. In particular, the turn II motif has a higher mdm2 binding affinity ( approximately 20 mum) than the turn I and targets the same site in mdm2 as the helix. Upon mdm2 binding this motif becomes a well defined full helix turn whose hydrophobic face formed by the side chains of Ile-50, Trp-53, and Phe-54 inserts deeply into the helix binding pocket. Our results suggest that p53-mdm2 binding is subtler than previously thought and involves global contacts such as multiple "non-contiguous" minimally structured motifs instead of being localized to one small helix mini-domain in p53 TAD.

The unambiguous assignment of the aromatic ring resonances in proteins has been severely hampered by the inherently poor sensitivities of the currently available methodologies developed for uniformly 13C/15N-labeled proteins. Especially, the small chemical shift differences between aromatic ring carbons and protons for phenylalanine residues in proteins have prevented the selective observation and unambiguous assignment of each signal. We have solved all of the difficulties due to the tightly coupled spin systems by preparing regio-/stereoselectively 13C/2H/15N-labeled phenylalanine (Phe) and tyrosine (Tyr) to avoid the presence of directly connected 13C-1H pairs in the aromatic rings. The superiority of the new labeling schemes for the assignment of aromatic ring signals is clearly demonstrated for a 17 kDa calcium binding protein, calmodulin.

Cyclobutane pyrimidine dimer (CPD) photolyases, which contain FAD as a cofactor, use light to repair CPDs. We performed structural analyses of the catalytic site of the Thermus thermophilus CPD photolyase-DNA complex, using FAD-induced paramagnetic relaxation enhancement (PRE). The distances between the tryptophan residues and the FAD calculated from the PRE agree well with those observed in the x-ray structure (with an error of <3 A). Subsequently, a single-stranded DNA containing 13C-labeled CPD was prepared, and the FAD-induced PRE of the NMR resonances from the CPD lesion in complex with the CPD photolyase was investigated. The distance between the FAD and the CPD calculated from the PRE is 16 +/- 3 A. The FAD-induced PRE was also observed in the CPD photolyase-double-stranded DNA complex. Based on these results, a model of the CPD photolyase-DNA complex was constructed, and the roles of Arg-201, Lys-240, Trp-247, and Trp-353 in the CPD-repair reaction are discussed.

We provide detailed descriptions of our refined protocols for the cell-free production of labeled protein samples for NMR spectroscopy. These methods are efficient and overcome two critical problems associated with the use of conventional Escherichia coli extract systems. Endogenous amino acids normally present in E. coli S30 extracts dilute the added labeled amino acids and degrade the quality of NMR spectra of the target protein. This problem was solved by altering the protocol used in preparing the S30 extract so as to minimize the content of endogenous amino acids. The second problem encountered in conventional E. coli cell-free protein production is non-uniformity in the N-terminus of the target protein, which can complicate the NMR spectra. This problem was solved by adding a DNA sequence to the construct that codes for a cleavable N-terminal peptide tag. Addition of the tag serves to increase the yield of the protein as well as to ensure a homogeneous protein product following tag cleavage. We illustrate the method by describing its stepwise application to the production of calmodulin samples with different stable isotope labeling patterns for NMR analysis.

The cyclobutane pyrimidine dimer (CPD) is one of the major forms of DNA damage caused by irradiation with ultraviolet (UV) light. CPD photolyases recognize and repair UV-damaged DNA. The DNA recognition mechanism of the CPD photolyase has remained obscure because of a lack of structural information about DNA-CPD photolyase complexes. In order to elucidate the CPD photolyase DNA binding mode, we performed NMR analyses of the DNA-CPD photolyase complex. Based upon results from (31)P NMR measurements, in combination with site-directed mutagenesis, we have demonstrated the orientation of CPD-containing single-stranded DNA (ssDNA) on the CPD photolyase. In addition, chemical shift perturbation analyses, using stable isotope-labeled DNA, revealed that the CPD is buried in a cavity within CPD photolyase. Finally, NMR analyses of a double-stranded DNA (dsDNA)-CPD photolyase complex indicated that the CPD is flipped out of the dsDNA by the enzyme, to gain access to the active site.

The antigen binding site of monoclonal antibody 64M5, which possesses a high degree of affinity for DNA containing pyrimidine (6-4) pyrimidone photoproducts, were investigated by use of stable-isotope-assisted NMR spectroscopy. A variety of 64M5 Fab fragments specifically labeled with 13C and 15N at backbone amide groups were prepared. Extensive assignments of amide resonances originating from the variable region of 64M5 were made by using 2D-HN(CO) measurements along with recombination of the heavy and light chains of 64M5. On the basis of chemical shift changes of the amide resonances caused upon addition of d(T[6-4]T) and d(GTAT[6-4]TATG), the binding sites of 64M5 Fab for the (6-4) photodimer and for the oligodeoxynucleotides flanking it were identified. It was revealed that the L1 and L3 segments, which are responsible for the binding to (6-4) photodimer, exhibit conformational multiplicities in the absence of antigens, and take different conformations between the d(T[6-4]T) and d(GTAT[6-4]TATG)-bound forms. On the basis of spectral comparison with another Fab fragment with a similarity in the amino acid sequence of the VL domain of 64M5, we suggest that the conformational multiplicities observed in the present study is caused by a substitution of an amino acid residue at the position of a key residue in L3 canonical structure, which leads to a preferable effect on the antigen binding, and by a specific combination of L1 and L3 canonical structures., (Copyright 1998 Academic Press.)

We have used site-directed mutagenesis to uncover the origin of the binding affinity differences exhibited by a series of monoclonal antibodies that recognize pyrimidine (6-4) pyrimidone photoproducts in the context of single- or double-stranded DNA. In this study, we have focused on two antibodies-64M3 and 64M5-that share the same binding specificity but differ in their affinities. We used single-chain Fv (scFv) derivatives for these studies since they can be easily expressed in Escherichia coli. To facilitate this, we also developed a simple, on-column refolding procedure for scFvs that is rapid and does not require high dilution. We took several precautions to ensure that the scFvs faithfully reflected the behavior of the parent monoclonal antibodies. Results obtained from chimeric scFvs constructed from 64M3 and 64M5 suggested that the higher affinity of the 64M5 antibody was mainly due to its VL region. Loop-grafting studies in which VH CDR loops of 64M3 were individually transplanted into 64M5 were consistent with this hypothesis. Since the VL sequences of 64M3 and 64M5 differ at only three positions (L30, L50, and L90), alanine-scanning mutagenesis was used to assess the importance of these three residues in DNA binding by 64M5. These studies highlighted the importance of all three VL CDR loops; furthermore, they suggested that photoproduct binding involved conformational changes within the VL region.

A 31P nuclear magnetic resonance (NMR) study of the interactions between oligonucleotides containing the (6-4) photoproduct and the Fab fragments of monoclonal antibodies (64M3 and 64M5) recognizing the (6-4) photoproduct is reported. The 31P chemical shift data indicate that backbone conformation of (64) adduct is affected by the presence of flanking oligodeoxynucleotides, and (6-4) adducts with different backbone conformations are accommodated in the antigen binding sites of these antibodies. It was also revealed that epitopes for these antibodies consist of not only the (6-4) adduct but the flanking di- or tri-deoxynucleotides on both the 5' and 3' sides as well.

The amino acid sequence of a mouse monoclonal antibody Mab13-1, a catalytic antibody against TCPP (meso-tetrakis(4-carboxyphenyl)porphyrin), was confirmed by mass spectrometric (MS) peptide mapping. The amino-terminal sequence of the heavy chain was established by MS/MS analysis of the isolated N-terminal peptide. The presence of a unique disulfide bond between Cys93H and Cys102H was identified by MS peptide mapping and sequence analysis of an S-S containing peptide. Positions of other disulfide bonds were identified to be conserved. The non-conserved disulfide bridge was found to be resistant as other intra-chain disulfide bonds against reduction under non-denaturing condition, and to be buried inside the molecule. This extra disulfide bond is expected to support antigen-binding by restricting the flexibility of CDR-H3 loop, and it might be favorable for the recognition of a plane antigen, a porphyrin derivative.