Affinity Improvement of a Therapeutic Antibody by Structure-Based Computational Design: Generation of Electrostatic Interactions in the Transition State Stabilizes the Antibody-Antigen Complex

The optimization of antibodies is a desirable goal towards the development of better therapeutic strategies. The antibody 11K2 was previously developed as a therapeutic tool for inflammatory diseases, and displays very high affinity (4.6 pM) for its antigen the chemokine MCP-1 (monocyte chemo-attractant protein-1). We have employed a virtual library of mutations of 11K2 to identify antibody variants of potentially higher affinity, and to establish benchmarks in the engineering of a mature therapeutic antibody. The most promising candidates identified in the virtual screening were examined by surface plasmon resonance to validate the computational predictions, and to characterize their binding affinity and key thermodynamic properties in detail. Only mutations in the light-chain of the antibody are effective at enhancing its affinity for the antigen in vitro, suggesting that the interaction surface of the heavy-chain (dominated by the hot-spot residue Phe101) is not amenable to optimization. The single-mutation with the highest affinity is L-N31R (4.6-fold higher affinity than wild-type antibody). Importantly, all the single-mutations showing increase affinity incorporate a charged residue (Arg, Asp, or Glu). The characterization of the relevant thermodynamic parameters clarifies the energetic mechanism. Essentially, the formation of new electrostatic interactions early in the binding reaction coordinate (transition state or earlier) benefits the durability of the antibody-antigen complex. The combination of in silico calculations and thermodynamic analysis is an effective strategy to improve the affinity of a matured therapeutic antibody.     

Overcoming Antigenic Diversity by Enhancing the Immunogenicity of Conserved Epitopes on the Malaria Vaccine Candidate Apical Membrane Antigen-1

Malaria vaccine candidate Apical Membrane Antigen-1 (AMA1) induces protection, but only against parasite strains that are closely related to the vaccine. Overcoming the AMA1 diversity problem will require an understanding of the structural basis of cross-strain invasion inhibition. A vaccine containing four diverse allelic proteins 3D7, FVO, HB3 and W2mef (AMA1 Quadvax or QV) elicited polyclonal rabbit antibodies that similarly inhibited the invasion of four vaccine and 22 non-vaccine strains of P. falciparum. Comparing polyclonal anti-QV with antibodies against a strain-specific, monovalent, 3D7 AMA1 vaccine revealed that QV induced higher levels of broadly inhibitory antibodies which were associated with increased conserved face and domain-3 responses and reduced domain-2 response. Inhibitory monoclonal antibodies (mAb) raised against the QV reacted with a novel cross-reactive epitope at the rim of the hydrophobic trough on domain-1; this epitope mapped to the conserved face of AMA1 and it encompassed the 1e-loop. MAbs binding to the 1e-loop region (1B10, 4E8 and 4E11) were ∼10-fold more potent than previously characterized AMA1-inhibitory mAbs and a mode of action of these 1e-loop mAbs was the inhibition of AMA1 binding to its ligand RON2. Unlike the epitope of a previously characterized 3D7-specific mAb, 1F9, the 1e-loop inhibitory epitope was partially conserved across strains. Another novel mAb, 1E10, which bound to domain-3, was broadly inhibitory and it blocked the proteolytic processing of AMA1. By itself mAb 1E10 was weakly inhibitory but it synergized with a previously characterized, strain-transcending mAb, 4G2, which binds close to the hydrophobic trough on the conserved face and inhibits RON2 binding to AMA1. Novel inhibition susceptible regions and epitopes, identified here, can form the basis for improving the antigenic breadth and inhibitory response of AMA1 vaccines. Vaccination with a few diverse antigenic proteins could provide universal coverage by redirecting the immune response towards conserved epitopes.     

Mouse Oocyte Methylomes at Base Resolution Reveal Genome-Wide Accumulation of Non-CpG Methylation and Role of DNA Methyltransferases

DNA methylation is an epigenetic modification that plays a crucial role in normal mammalian development, retrotransposon silencing, and cellular reprogramming. Although methylation mainly occurs on the cytosine in a CG site, non-CG methylation is prevalent in pluripotent stem cells, brain, and oocytes. We previously identified non-CG methylation in several CG-rich regions in mouse germinal vesicle oocytes (GVOs), but the overall distribution of non-CG methylation and the enzymes responsible for this modification are unknown. Using amplification-free whole-genome bisulfite sequencing, which can be used with minute amounts of DNA, we constructed the base-resolution methylome maps of GVOs, non-growing oocytes (NGOs), and mutant GVOs lacking the DNA methyltransferase Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3L. We found that nearly two-thirds of all methylcytosines occur in a non-CG context in GVOs. The distribution of non-CG methylation closely resembled that of CG methylation throughout the genome and showed clear enrichment in gene bodies. Compared to NGOs, GVOs were over four times more methylated at non-CG sites, indicating that non-CG methylation accumulates during oocyte growth. Lack of Dnmt3a or Dnmt3L resulted in a global reduction in both CG and non-CG methylation, showing that non-CG methylation depends on the Dnmt3a-Dnmt3L complex. Dnmt3b was dispensable. Of note, lack of Dnmt1 resulted in a slight decrease in CG methylation, suggesting that this maintenance enzyme plays a role in non-dividing oocytes. Dnmt1 may act on CG sites that remain hemimethylated in the de novo methylation process. Our results provide a basis for understanding the mechanisms and significance of non-CG methylation in mammalian oocytes.     

Efficacy and safety of paromomycin for treating amebiasis in Japan

The clinical management of amebiasis is a growing concern, particularly among human immunodeficiency virus (HIV)-infected individuals who are predisposed to severe illness. Treatment with a luminal amebicide is strongly recommended following acute-stage treatment with a nitroimidazole. In 2004, the Japanese Research Group on Chemotherapy of Tropical Diseases introduced paromomycin, which was not nationally licensed, and offered it to a number of patients. From 2004 to 2011, 143 case records of amebiasis (123 with amebic colitis, 16 with amebic liver abscess, and 4 with both) in which patients were treated with paromomycin, mainly 1500 mg/day for 9 or 10 days following metronidazole treatment, were submitted. Among 123 evaluable cases, 23 (18.7%) experienced possible adverse effects, the most common being diarrhea (17/123, 13.8%) and other gastrointestinal problems that were resolved after the completion or discontinuation of treatment. In addition, single cases of bloody stools associated with Clostridium difficile colitis, skin rash, and the elevation of liver enzymes were also reported, although the causal relationship was not clear. HIV infection did not appear to increase the incidence of adverse drug effects. Each of the 11 asymptomatic or mildly symptomatic amebic colitis cases became negative for stool cysts after paromomycin treatment. Paromomycin was shown to be safe and well tolerated, as well as effective in a special subset of amebic colitis cases.     

Thiol Oxidation Induced by Oxidative Action of Adriamycin

To clarify the mechanism of the cardiotoxic action of adriamycin (ADM), the participation of free radicals from ADM in cardiotoxicity was investigated through the protective action of glutathione (GSH) or by using electron spin resonance (ESR). Oxidation of ADM by horseradish peroxidase and H₂O₂ (HRP-H₂O₂) was blocked by GSH concentration dependently. Inactivation of creatine kinase (CK) induced during interaction of ADM with HRP-H₂O₂ was also protected by GSH. Other anthracycline antitumor drugs that have a p-hydroquinone structure in the B ring also inactivated CK, and GSH inhibited the inactivation of CK. These results suggest that ADM was activated through oxidation of the p-hydroquinone in the B ring by HRP-H₂O₂. Although ESR signals of the oxidative ADM B ring semiquinone were not detected, glutathionyl radicals were formed during the interaction of ADM with HRP-H₂O₂ in the presence of GSH. ADM may be oxidized to the ADM B ring semiquinone and then reacts with the SH group. However, ESR signals of ADM C ring semiquinone, which was reductively formed by xanthine oxidase (XO) and hypoxanthine (HX) under anaerobic conditions, were not diminished by GSH, but they completely disappeared with ferric ion. These results indicate that oxidative ADM B ring semiquinones oxidized the SH group in CK, but reductive ADM C ring semiquinone radicals may participate in the oxidation of lipids or DNA and not of the SH group.     

Failure of mycoplasma lipoprotein MALP-2 to induce NK cell activation through dendritic cell TLR2

Macrophage-activating lipopeptide 2 (MALP-2), a mycoplasmal diacylated lipopeptide with palmitic acid moiety (Pam2), activates Toll-like receptor (TLR) 2 to induce inflammatory cytokines. TLR2 is known to mature myeloid dendritic cells (mDC) to drive mDC contact-mediated natural killer (NK) cell activation. Here we tested if MALP-2 activates NK cells through stimulation of TLR2 on mDC. Although synthetic MALP-2 with 6 or 14 amino acids (a.a.) stretch (designated as s and f) matured mDC to induce IL-6, IL-12p40 and TNF-α to a similar extent, they far less activated NK cells than Pam2CSK4, a positive control of 6 a.a.-containing diacyl lipopeptide. MALP-2s and f were TLR2/6 agonists and activate the MyD88 pathway similar to Pam2CSK4, but MALP-2s having the CGNNDE sequence acted on mDC TLR2 to barely induce external NK activation. Even the s form, with slightly high induction of IL-6 compared to the f form, barely induced in vivo growth retardation of NK-sensitive implant tumor. Pam2CSK4 and MALP-2 have the common lipid moiety but different peptides, which are crucial for NK cell activation. The results infer that MALP-2 is applicable to a cytokine inducer but not to an adjuvant for antitumor NK immunotherapy.     

The role of QTLs in the breeding of high-yielding rice

Food shortages have once again become a serious problem, not only because of world population growth but also as a result of escalating demand for crops as a substrate for biofuels. The production of improved plant varieties, especially major crops such as rice, is urgently required to increase yield. The completion of the sequencing of the rice genome has made it possible to clone and analyze quantitative trait loci (QTLs). Furthermore, the development of high-throughput sequencing and genotyping technologies has improved spectacularly the accuracy of QTL analysis. In this review article, we focus on the potential roles of major QTLs in the selection for agronomic traits in rice and discuss the application of high-throughput technologies for high-resolution genetic analysis.     

Induction of apoptosis and lipogenesis in human preadipocyte cell line by n −3 PUFAs

We examined the effect of n ?3 PUFAs (polyunsaturated fatty acids) on the growth and maturation of human preadipocyte cell line AML‐I. On day 3 of the culture, n ?3 fatty acids such as DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid), but not n ?6 fatty acid LA (linoleic acid), induced growth arrest accompanied by the appearance of characteristics of apoptosis in AML‐I cells at concentrations between 250 and 500 μM by Annexin V‐FITC staining. In Western blotting analysis, the loss of NF‐κB, Bcl‐2 and p‐Akt and the accumulation of Bad and Akt were observed in the cytoplasmic protein from the EPA‐treated cells. Exposure of AML‐I to EPA or DHA increased the cytoplasmic lipid accumulation compared with the vehicle‐treated cells in a time‐dependent manner during 4 and 6 days culture period by Oil Red O staining. The expression of FAS (fatty acid synthase) and PPAR‐γ (peroxisome proliferator‐activated receptor‐γ) were increased in EPA‐treated cells. These results suggest that EPA and DHA promote differentiation, inhibit proliferation and induce apoptosis in preadipocyte cell line AML‐I.