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Abstract Allergens from domestic cats (Felis catus) cause allergy-related health problems worldwide. Fel d 1 is a major allergen that causes severe allergic reactions in humans, including rhinitis, conjunctivitis, and life-threatening asthma. Therefore, patients with cat allergies anticipate hypoallergenic cats. We successfully generated Fel d 1 chain 2 (CH2) genome-edited cats using the CRISPR-Cas9 system in this study. T7 endonuclease 1 assay and Sanger sequencing were used to confirm the mutation in CH2 genome-edited cats. Fel d 1 level in CH2 genome-edited cats were assessed by enzyme-linked immunosorbent assay (ELISA). Remarkably, ELISA showed that the level of Fel d 1 in the CH2 homozygous genome-edited cat (Name: Alsik) was extremely low compared with that in wild type domestic cats and could be hypoallergenic cats. Additionally, we successfully cloned the CH2 homozygous genome-edited cat using cytoplasm injection clone technology. The cloned CH2 homozygous genome-edited cat was verified using microsatellite analysis. Creating hypoallergenic cats using the CRISPR-Cas9 system is a significant step forward because these cats can safely approach allergic patients.

In this work, we report rovibrational energy levels for four isotopologues of methylene (CH2, CHD, CD2, and 13CH2) in their ground triplet electronic state (X~3B1) from variational calculation up to ~10,000 cm−1 and using a new accurate ab initio potential energy surface (PES). Triplet methylene exhibits a large‐amplitude bending vibration and can reach a quasilinear configuration due to its low barrier (~2000 cm−1). To construct the ab initio PES, the Dunning's augmented correlation‐consistent core‐valence orbital basis sets were employed up to the sextuple‐ζ quality [aug‐cc‐pCVXZ, X = T, Q, 5, and 6] combined with the single‐ and double‐excitation unrestricted coupled cluster approach with a perturbative treatment of triple excitations [RHF‐UCCSD(T)]. We have shown that the accuracy of the ab initio energies is further improved by including the corrections due to the scalar relativistic effects, DBOC and high‐order electronic correlations. For the first time, all the available experimental rovibrational transitions were reproduced with errors less than 0.12 cm−1, without any empirical corrections. Unlike more "traditional" nonlinear triatomic molecules, we have shown that even the energies of the ground vibrational state (000) with rather small rotational quantum numbers are strongly affected by the very pronounced rovibrational resonance interactions. Accordingly, the polyad structure of the vibrational levels of CH2 and CD2 was analyzed and discussed. The comparison between the energy levels obtained from the effective Watson A‐reduced Hamiltonian, from the generating‐function approach and from a variational calculation was given. [ABSTRACT FROM AUTHOR]

Protein aggregation is a major challenge in the development of therapeutic monoclonal antibodies (mAbs). Several stressors can cause protein aggregation, including temperature shifts, mechanical forces, freezing-thawing cycles, oxidants, reductants, and extreme pH. When antibodies are exposed to low pH conditions, aggregation increases dramatically. However, low pH treatment is widely used in protein A affinity chromatography and low pH viral inactivation procedures. In the development of an IgG4 subclass antibody, mAb1-IgG4 showed a strong tendency to aggregate when temporarily exposed to low pH conditions. Our findings showed that the aggregation of mAb1-IgG4 under low pH conditions is determined by the stability of the Fc. The CH2 domain is the least stable domain in mAb1-IgG4. The L309E, Q311D, and Q311E mutations in the CH2 domain significantly reduced the aggregation propensity, which could be attributed to a reduction in the hydrophobicity of the CH2 domain. Protein stabilizers, such as sucrose and mannose, could also attenuate low pH-induced mAb1-IgG4 aggregation by shielding hydrophobic areas and increasing protein stability. Our findings provide valuable strategies for managing the aggregation of protein therapeutics with a human IgG4 backbone.

Among the immunoglobulin domains, the CH2 domain has the lowest thermal stability, which also depends on amino acid sequence and buffer conditions. To further identify factors that influence CH2 folding and stability, we characterized the domain in the reduced form using differential scanning fluorimetry and nuclear magnetic resonance. We show that the CH2 domain can fold, similarly to the disulfide‐bridged form, without forming a disulfide‐bridge, even though the protein contains two Cys residues. Although the reduced form exhibits thermal stability more than 15°C lower than the disulfide‐bridged form, it does not undergo immediate full oxidization. To explain this phenomenon, we compared CH2 oxidization at different conditions and demonstrate a need for significant fluctuation of the folded conformation to enhance CH2 disulfide‐bridge formation. We conclude that, since CH2 can be purified as a folded, semi‐stable, reduced protein that can coexist with the oxidized form, verification of the level of oxidization at each step is critical in CH2 engineering studies. [ABSTRACT FROM AUTHOR]

In this work, we report rovibrational energy levels for four isotopologues of methylene (CH 2 , CHD, CD 2 , and 13 CH 2 ) in their ground triplet electronic state ( X ~ 3 B 1 ) from variational calculation up to ~10,000 cm -1 and using a new accurate ab initio potential energy surface (PES). Triplet methylene exhibits a large-amplitude bending vibration and can reach a quasilinear configuration due to its low barrier (~2000 cm -1 ). To construct the ab initio PES, the Dunning's augmented correlation-consistent core-valence orbital basis sets were employed up to the sextuple-ζ quality [aug-cc-pCVXZ, X = T, Q, 5, and 6] combined with the single- and double-excitation unrestricted coupled cluster approach with a perturbative treatment of triple excitations [RHF-UCCSD(T)]. We have shown that the accuracy of the ab initio energies is further improved by including the corrections due to the scalar relativistic effects, DBOC and high-order electronic correlations. For the first time, all the available experimental rovibrational transitions were reproduced with errors less than 0.12 cm -1 , without any empirical corrections. Unlike more "traditional" nonlinear triatomic molecules, we have shown that even the energies of the ground vibrational state (000) with rather small rotational quantum numbers are strongly affected by the very pronounced rovibrational resonance interactions. Accordingly, the polyad structure of the vibrational levels of CH 2 and CD 2 was analyzed and discussed. The comparison between the energy levels obtained from the effective Watson A-reduced Hamiltonian, from the generating-function approach and from a variational calculation was given., (© 2023 Wiley Periodicals LLC.)

Smaller recombinant antibody fragments are now emerging as alternatives of conventional antibodies. Especially, immunoglobulin (Ig) constant CH2 domain and engineered CH2 with improved stability are promising as scaffolds for selection of specific binders to various antigens. We constructed a yeast display library based on an engineered human IgG1 CH2 scaffold with diversified loop regions. A group of CH2 binders were isolated from this yeast display library by panning against nucleolin, which is a tumor-associated antigen involved in cell proliferation, tumor cell growth and angiogenesis. Out of 20 mutants, we selected 3 clones exhibiting relatively high affinities to nucleolin on yeasts. However, recombinant CH2 mutants aggregated when they were expressed. To find the mechanism of the aggregation, we employed computational prediction approaches through structural homology models of CH2 binders. The analysis of potential aggregation prone regions (APRs) and solvent accessible surface areas (ASAs) indicated two hydrophobic residues, Val 264 and Leu 309 , in the β-sheet, in which replacement of both charged residues led to significant decrease of the protein aggregation. The newly identified CH2 binders could be improved to use as candidate therapeutics or research reagents in the future. [ABSTRACT FROM AUTHOR]

Protein aggregation is a major challenge in the development of therapeutic monoclonal antibodies (mAbs). Several stressors can cause protein aggregation, including temperature shifts, mechanical forces, freezing-thawing cycles, oxidants, reductants, and extreme pH. When antibodies are exposed to low pH conditions, aggregation increases dramatically. However, low pH treatment is widely used in protein A affinity chromatography and low pH viral inactivation procedures. In the development of an IgG4 subclass antibody, mAb1-IgG4 showed a strong tendency to aggregate when temporarily exposed to low pH conditions. Our findings showed that the aggregation of mAb1-IgG4 under low pH conditions is determined by the stability of the Fc. The CH2 domain is the least stable domain in mAb1-IgG4. The L309E, Q311D, and Q311E mutations in the CH2 domain significantly reduced the aggregation propensity, which could be attributed to a reduction in the hydrophobicity of the CH2 domain. Protein stabilizers, such as sucrose and mannose, could also attenuate low pH-induced mAb1-IgG4 aggregation by shielding hydrophobic areas and increasing protein stability. Our findings provide valuable strategies for managing the aggregation of protein therapeutics with a human IgG4 backbone., Competing Interests: QW, CC, KC, LS, QL, and JL were employed by the company Zhuhai United Laboratories Co., Ltd. CC, SW, and HH were employed by the company The United Biotechnology (Zhuhai Hengqin) Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wu, Cao, Wei, He, Chen, Su, Liu, Li, Lai and Li.)

Using the completed active space second-order perturbation (CASPT2) method, valence and Rydberg excited states of CH2 molecule are probed with the large atomic natural orbital (ANO-L) basis set. Five states are optimized and the geometric parameters are in good agreement with the available data in literatures, furthermore, the state of 21 B1 is obtained for the first time. Valence and Rydberg excited states of CH2 are also calculated for the vertical transitions with the ANO-L+ basis set that is constructed by adding a set of 1s1p1d Rydberg orbitals into the ANO-L basis set. Two Rydberg states of the p̃3 A2 and r̃3 B1 at 9.88 and 10.50 eV are obtained for the first time, and the 3a1 → 3d yz nature of the state p̃3 A2 and the 3a1 → d x2− y2 nature of the state r̃3 B1 are confirmed. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: Isolated immunoglobulin CH2 domains were proposed as scaffolds for selection of binders with potential effector functions. We tested the feasibility of this approach by constructing a large (size 5×1010) library where all amino acids in two loops (BC and FG) were mutated to four residues (Y, A, D, or S). Three binders were selected from this library by panning against a gp120–CD4 complex. The strongest binder, m1a1, recognized specifically a highly conserved CD4i epitope and inhibited to various extents seven out of nine HIV-1 isolates from different clades. The loop BC and the conformational state of the scaffold are critical for its binding. These results provide a proof of concept for the potential of CH2 as a scaffold for construction of libraries containing potentially useful binders. The newly identified HIV-1 inhibitors could be further improved to candidate therapeutics and/or used as research reagents for exploration of conserved gp120 structures. [Copyright &y& Elsevier]

Infrared is the most widely used analytical method for interpreting the structure of petroleum fractions. In this study, IR spectra of standard normal nonadecane were used to get ε for methyl and methylene groups. A linear relationship was obtained by plotting the peak intensities at 2,954, 2,926, 2,854, 1,466, and 1,376 cm-1 against different amounts of N-nonadecane. The R2 obtained for most of the plots was in the range of 0.988-0.997. The Beer-Lambert's law was used for the first time to quantitatively estimate the weight percent of methylene and methyl groups present in the different fractions of asphalt. [ABSTRACT FROM AUTHOR]

Abstract: The inner capsid protein VP6 of group A rotavirus possesses group and subgroup epitope specificities. Avian rotaviruses have a unique VP6 that is antigenically different from its mammalian counterpart. The lack of information on the VP6 protein of chicken rotavirus strain, CH2, at the genetic and antigenic level was a major motivation for this work. Sequencing of the complete cDNA of the VP6 gene of CH2, revealed a nucleotide (amino acid) identity that varied from 78.3 to 98.5% (86.4–98.2%) when compared with other avian rotaviruses. Regardless of its host origin dissimilarity, CH2 VP6 showed a close sequence homology (97.4–98.2%) with turkey and pigeon rotaviruses. Homology-based modeling of the CH2 VP6 from the corresponding crystal structure of the bovine rotavirus, RF strain, demonstrated that the hypervariable region (residue 228–240) does have a critical role in strain specific antigenic characteristics of avian and mammalian rotaviruses. A predicted conformational epitope encompasses experimentally characterized group and subgroup epitopes suggesting that it is a major antibody binding site on the VP6 protein. The VP6 structure modeling and conformational epitope prediction together with enzyme immuno assay of SG MAbs placed CH2 in SGI/II. The study may be helpful in designing peptides for group A rotavirus diagnostic assays and to achieve heterotypic protection against rotavirus serotypes. [Copyright &y& Elsevier]

Experimental absorption spectra of benzene, isooctane, and their mixtures are obtained in the wavelength range λ = 1620–1820 nm in which the first overtones of vibrational frequencies of CH, CH2, and CH3 hydrocarbon groups are located. Positions of fundamental absorption bands of benzene are refined. Absorption spectra of benzene-isooctane mixtures are shown to intersect in a narrow area near λ ≈ 1695 nm. The main maximum of benzene absorption at λ = 1671.5 ± 0.5 nm, where the influence of isooctane absorption is practically absent, is proposed for determining the content of benzene in benzene-isooctane mixtures. A linear calibration curve for λ = 1671.5 nm that encompasses the full range of benzene concentrations (0–100%) is presented. [ABSTRACT FROM AUTHOR]

The immunoglobulin CH2 domain is a simple model system suitable for the study of the folding of all-β-proteins. Its structure consists of two β-sheets forming a greek-key β-barrel, which is stabilized by an internal disulfide bridge located in the hydrophobic core. Crystal structures of various antibodies suggest that the CH2 domains of the two heavy chains interact with their sugar moieties and form a homodimer. Here, we show that the isolated, unglycosylated CH2 domain is a monomeric protein. Equilibrium unfolding was a two-state process, and the conformational stability is remarkably low compared to other antibody domains. Folding kinetics of CH2 were found to consist of several phases. The reactions could be mapped to three parallel pathways, two of which are generated by prolyl isomerizations in the unfolded state. The slowest folding reaction, which was observed only after long-term denaturation, could be catalyzed by a prolyl isomerase. The majority of the unfolded molecules, however, folded more rapidly, on a time-scale of minutes. Presumably, these molecules also have to undergo prolyl isomerization before reaching the native state. In addition, we detected a small number of fast-folding molecules in which all proline residues appear to be in the correct conformation. On both prolyl isomerization limited pathways, the formation of partly structured intermediates could be observed. [Copyright &y& Elsevier]

The first four states of the CH2 molecule (X3B1, ã1 A1, b1A1, and c1A1) are examined using state-of-the-art ab initio methods and basis sets. The construction of potential energy curves with respect to the C + H2 and CH + H channels provides significant clues to understanding the geometric and electronic structure of the above states. All of our numerical findings are in excellent agreement with the existing experimental data. [ABSTRACT FROM AUTHOR]

The reaction singlet potential energy curves of reactions Ni(d9 s1 3D)+CH2(3B1)→H2+NiC and Ni(d9s1 3D)+CH2(3B1)→trans-HNiCH, and the electronic and geometric structures and vibrational frequencies of all intermediates and transition states in the reactions above have been studied at B3LYP/6-311+G(2d,2p) and B3LYP/6-311++G(3df,2p) levels. The former reaction is predicted to be endothermic by about 32.9 kJ mol−1, and the latter exothermic by about 65.9 kJ mol−1. At low energy levels, the formation of trans-HNiCH would be the dominant process, while at high energy levels, the formation of H2+NiC would be the dominant process. [Copyright &y& Elsevier]

The importance of Sn to improve the performance of noble metal-based catalysts has been extensively discussed in the literature. However, a detailed discussion about the effect of Sn on the electronic structure of the metals has not been reported so far. In this work, density functional theory (DFT) calculations have been performed on the M2 and MSn (M=Ru, Rh, Pd, Sn) dimers and their interaction with CH2 and CF2 carbenes. The electronic structure, geometry, and harmonic frequencies are reported and compared with the available experimental data and to the previous published high level ab initio and DFT calculations. The calculated values of M2 are in good agreement with the experimental and theoretical results. The exception is the binding energy of the Sn dimer, which can be different from the experimental estimates as much as 16 kcal · mol−1. It has been stressed that this difficulty is related to the atomic energy reference used to estimate the binding energy. Transition metal elements are still a difficult task for DFT methods. Calculations on MSn—CH2 and MSn—CF2 species with different multiplicities and conformations have been performed. Bonding analyses on the M—Sn metal dimers showed that carbenes prefer to adsorb in bridge sites favoring a donating/backdonating mechanism of interaction that is enhanced with the presence of the Sn atom. The MSn—CH2 binding energy is predicted to be 40% smaller than the MSn—CF2 binding energy, following the same trends observed for the first-row transition-metal M—CH2 and M—CF2 complexes. © 2003 Wiley Periodicals, Inc. Int J Quantum Chem 95: 164–176, 2003 [ABSTRACT FROM AUTHOR]

Among the immunoglobulin domains, the CH2 domain has the lowest thermal stability, which also depends on amino acid sequence and buffer conditions. To further identify factors that influence CH2 folding and stability, we characterized the domain in the reduced form using differential scanning fluorimetry and nuclear magnetic resonance. We show that the CH2 domain can fold, similarly to the disulfide-bridged form, without forming a disulfide-bridge, even though the protein contains two Cys residues. Although the reduced form exhibits thermal stability more than 15°C lower than the disulfide-bridged form, it does not undergo immediate full oxidization. To explain this phenomenon, we compared CH2 oxidization at different conditions and demonstrate a need for significant fluctuation of the folded conformation to enhance CH2 disulfide-bridge formation. We conclude that, since CH2 can be purified as a folded, semi-stable, reduced protein that can coexist with the oxidized form, verification of the level of oxidization at each step is critical in CH2 engineering studies., (© 2021 The Protein Society.)

Engineered antibody domains (eAds) are promising candidate therapeutics but their half-life is relatively short partly due to weak or absent binding to the neonatal Fc receptor (FcRn). We developed a novel approach to increase the eAd binding to FcRn based on a combination of structure-based design, computational modeling and phage display methodologies. By using this approach, we identified 2 IgG1 CH2-derived eAds fused to a short FcRn-binding motif derived from IgG1 CH3 that exhibited greatly enhanced FcRn binding with strict pH dependency. Importantly, the increased affinity resulted in significantly enhanced FcRn-mediated epithelial transcytosis and prolonged elimination half-life (mean 44.1 hours) in cynomolgus macaques. These results demonstrate for the first time that the half-life of isolated eAds can be prolonged (optimized) by increasing their binding to FcRn while maintaining their small size, which has important implications for development of therapeutics, including eAd-drug conjugates with enhanced penetration in solid tissues.

High viscosity of monoclonal antibody formulations at concentrations ≥100 mg/mL can impede their development as products suitable for subcutaneous delivery. The effects of hydrophobic and electrostatic intermolecular interactions on the solution behavior of MAB 1, which becomes unacceptably viscous at high concentrations, was studied by testing 5 single point mutants. The mutations were designed to reduce viscosity by disrupting either an aggregation prone region (APR), which also participates in 2 hydrophobic surface patches, or a negatively charged surface patch in the variable region. The disruption of an APR that lies at the interface of light and heavy chain variable domains, VH and VL, via L45K mutation destabilized MAB 1 and abolished antigen binding. However, mutation at the preceding residue (V44K), which also lies in the same APR, increased apparent solubility and reduced viscosity of MAB 1 without sacrificing antigen binding or thermal stability. Neutralizing the negatively charged surface patch (E59Y) also increased apparent solubility and reduced viscosity of MAB 1, but charge reversal at the same position (E59K/R) caused destabilization, decreased solubility and led to difficulties in sample manipulation that precluded their viscosity measurements at high concentrations. Both V44K and E59Y mutations showed similar increase in apparent solubility. However, the viscosity profile of E59Y was considerably better than that of the V44K, providing evidence that inter-molecular interactions in MAB 1 are electrostatically driven. In conclusion, neutralizing negatively charged surface patches may be more beneficial toward reducing viscosity of highly concentrated antibody solutions than charge reversal or aggregation prone motif disruption.

The transcriptional co-regulator CBP (CREB-binding protein) has a highly conserved cysteine/histidine-rich region (CH2) whose structure and function remain uncharacterized. Using nuclear magnetic resonance (NMR spectroscopy), sequence alignment, mass spectrometry, and mutagenesis, we show that the CH2 domain is not a canonical plant homeodomain (PHD) finger, as previously proposed, but binds an additional zinc atom through the region N-terminal to the putative PHD motif. The CH2 domain and the preceding bromodomain interact and mutually stabilize each other, implying a cooperative function. We tested the hypothesis that the bromodomain and the CH2 domain can interact with histones, but found that the CH2 does not participate in histone-recognition., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Currently, almost all FDA approved therapeutic antibodies (except ReoPro, Lucentis and Cimzia which are Fabs), and the vast majority of those in clinical trials are full-size antibodies mostly in IgG1 format of about 150 kDa size. A fundamental problem for such large molecules is their poor penetration into tissues (e.g., solid tumors) and poor or absent binding to regions on the surface of some molecules (e.g., on the HIV envelope glycoprotein) which are fully accessible only by molecules of smaller size. Therefore, much work especially during the last decade has been aimed at developing novel scaffolds of much smaller size and high stability. Here I briefly describe a proposition to use the immunoglobulin (Ig) constant CH2 domain (CH3 for IgE and IgM) as a scaffold. CH2 is critical for the Ig effector functions. Isolated CH2 is stable monomer in contrast to all other constant domains and most of the variable domains. CH2 and engineered CH2 domains with improved stability can be used as scaffolds for construction of libraries containing diverse binders to various antigens. Such binders based on a CH2 scaffold could also confer some effector functions. Because the CH2 domains are the smallest independently folded antibody domains that can be engineered to contain simultaneously antigen-binding sites and binding sites mediating effector and stability functions, and to distinguish them from domain antibodies which are used to denote engineered VH or VL domains or nanobodies which are used to denote camelid VHH, I termed them nanoantibodies (nAbs).