Fast-tracking antibody maturation using a B cell-based display system.

Affinity maturation, an essential component of antibody engineering, is crucial for developing therapeutic antibodies. Cell display system coupled with somatic hypermutation (SHM) initiated by activation-induced cytidine deaminase (AID) is a commonly used technique for affinity maturation. AID introduces targeted DNA lesions into hotspots of immunoglobulin (Ig) gene loci followed by erroneous DNA repair, leading to biased mutations in the complementary determining regions. However, systems that use an in vivo mimicking mechanism often require several rounds of selection to enrich clones possessing accumulated mutations. We previously described the human ADLib® system, which features autonomous, AID-mediated diversification in Ig gene loci of a chicken B cell line DT40 and streamlines human antibody generation and optimization in one integrated platform. In this study, we further engineered DT40 capable of receiving exogenous antibody genes and examined whether the antibody could be affinity matured. The Ig genes of three representative anti-hVEGF-A antibodies originating from the human ADLib® were introduced; the resulting human IgG1 antibodies had up to 76.4-fold improvement in binding affinities (sub-picomolar KD) within just one round of optimization, owing to efficient accumulation of functional mutations. Moreover, we successfully improved the affinity of a mouse hybridoma-derived anti-hCDCP1 antibody using the engineered DT40, and the observed mutations remained effective in the post-humanized antibody as exhibited by an 8.2-fold increase of in vitro cytotoxicity without compromised physical stability. These results demonstrated the versatility of the novel B cell-based affinity maturation system as an easy-to-use antibody optimization tool regardless of the species of origin.Abbreviations: ADLib®: Autonomously diversifying library, ADLib® KI-AMP: ADLib® knock-in affinity maturation platform, AID: activation-induced cytidine deaminase, CDRs: complementary-determining regions, DIVAC: diversification activator, ECD: extracellular domain, FACS: fluorescence-activated cell sorting, FCM: flow cytometry, HC: heavy chainIg: immunoglobulin, LC: light chain, NGS: next-generation sequencing, PBD: pyrrolobenzodiazepine, SHM: somatic hypermutation, SPR: surface plasmon resonance.

Streamlined human antibody generation and optimization by exploiting designed immunoglobulin loci in a B cell line.

Monoclonal antibodies (mAbs) are widely utilized as therapeutic drugs for various diseases, such as cancer, autoimmune diseases, and infectious diseases. Using the avian-derived B cell line DT40, we previously developed an antibody display technology, namely, the ADLib system, which rapidly generates antigen-specific mAbs. Here, we report the development of a human version of the ADLib system and showcase the streamlined generation and optimization of functional human mAbs. Tailored libraries were first constructed by replacing endogenous immunoglobulin genes with designed human counterparts. From these libraries, clones producing full-length human IgGs against distinct antigens can be isolated, as exemplified by the selection of antagonistic mAbs. Taking advantage of avian biology, effective affinity maturation was achieved in a straightforward manner by seamless diversification of the parental clones into secondary libraries followed by single-cell sorting, quickly affording mAbs with improved affinities and functionalities. Collectively, we demonstrate that the human ADLib system could serve as an integrative platform with unique diversity for rapid de novo generation and optimization of therapeutic or diagnostic antibody leads. Furthermore, our results suggest that libraries can be constructed by introducing exogenous genes into DT40 cells, indicating that the ADLib system has the potential to be applied for the rapid and effective directed evolution and optimization of proteins in various fields beyond biomedicine.

Probing the unfolding of myoglobin and domain C of PARP-1 with covalent labeling and top-down ultraviolet photodissociation mass spectrometry.

Ultraviolet photodissocation (UVPD) mass spectrometry was used for high mass accuracy top-down characterization of two proteins labeled by the chemical probe, S-ethylacetimidate (SETA), in order to evaluate conformational changes as a function of denaturation. The SETA labeling/UVPD-MS methodology was used to monitor the mild denaturation of horse heart myoglobin by acetonitrile, and the results showed good agreement with known acetonitrile and acid unfolding pathways of myoglobin. UVPD outperformed electron transfer dissociation (ETD) in terms of sequence coverage, allowing the SETA reactivity of greater number of lysine amines to be monitored and thus providing a more detailed map of myoglobin. This strategy was applied to the third zinc-finger binding domain, domain C, of PARP-1 (PARP-C), to evaluate the discrepancies between the NMR and crystal structures which reported monomer and dimer forms of the protein, respectively. The trends reflected from the reactivity of each lysine as a function of acetonitrile denaturation in the present study support that PARP-C exists as a monomer in solution with a close-packed C-terminal α helix. Additionally, those lysines for which the SETA reactivity increased under denaturing conditions were found to engage in tertiary polar contacts such as salt bridging and hydrogen bonding, providing evidence that the SETA/UVPD-MS approach offers a versatile means to probe the interactions responsible for conformational changes in proteins.

Construction of the octose 8-phosphate intermediate in lincomycin A biosynthesis: characterization of the reactions catalyzed by LmbR and LmbN.

Lincomycin A is a potent antimicrobial agent noted for its unusual C1 methylmercapto-substituted 8-carbon sugar. Despite its long clinical history for the treatment of Gram-positive infections, the biosynthesis of the C(8)-sugar, methylthiolincosamide (MTL), is poorly understood. Here, we report our studies of the two initial enzymatic steps in the MTL biosynthetic pathway leading to the identification of D-erythro-D-gluco-octose 8-phosphate as a key intermediate. Our experiments demonstrate that this intermediate is formed via a transaldol reaction catalyzed by LmbR using D-fructose 6-phosphate or D-sedoheptulose 7-phosphate as the C(3) donor and D-ribose 5-phosphate as the C(5) acceptor. Subsequent 1,2-isomerization catalyzed by LmbN converts the resulting 2-keto C(8)-sugar (octulose 8-phosphate) to octose 8-phosphate. These results provide, for the first time, in vitro evidence for the biosynthetic origin of the C(8) backbone of MTL.

[Renal protective effects of sulodexide in diabetic rats and its anti-oxidative mechanism].

OBJECTIVE: To investigate the renal protective effects of sulodexide and its anti-oxidative stress mechanism in diabetic rats. METHOD: Thirty male SD rats were randomized into 3 equal groups, namely the control group, diabetic group, and sulodexide treatment group. Twelve weeks after establishment of rat diabetic models and administration of sulodexide, the rats were sacrificed for measurement of the urine volume, body mass, kidney mass/body weight ratio, plasma glucose, and glycosylated hemoglobin (HbA1c). Malondialdehyde (MDA) levels and superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX) activities in the renal tissue or serum were tested. Electron microscopy was performed to observe the pathological changes in the kidneys. RESULTS: The urine volume, renal mass/body mass ratio, serum glucose, HbA1C, and serum and renal MDA levels all significantly increased in the diabetic rats in comparison with the normal controls (P<0.05). But the body weight and activities of SOD, CAT, and GSH-PX in the renal tissue in the normal control group were significantly higher than those in the diabetic and sulodexide group. After 12 weeks of sulodexide treatment, SOD, CAT, and GSH-PX activities in the renal tissue of rats were significantly increased in comparison with those in the diabetic rats (P<0.05). Electron microscopy showed obvious irregular thickening of the glomerular capillary basement membrane in the diabetic group with vacuolization in the mitochondria in the epithelial cells, and such pathological changes were significantly alleviated in the sulodexide treatment group. CONCLUSIONS: Sulodexide can effectively lower the urinary albumin excretion rate, improve the ultrastructural renal pathologies and prevent glomerular basement membrane thickening in diabetic rats, probably in association with the reduction of the MDA levels and enhancement of SOD, CAT, and GSH-PX activities.