De novo design of modular and tunable protein biosensors.

Naturally occurring protein switches have been repurposed for the development of biosensors and reporters for cellular and clinical applications1. However, the number of such switches is limited, and reengineering them is challenging. Here we show that a general class of protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which the binding of a peptide key triggers biological outputs of interest2. The designed sensors are modular molecular devices with a closed dark state and an open luminescent state; analyte binding drives the switch from the closed to the open state. Because the sensor is based on the thermodynamic coupling of analyte binding to sensor activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We create biosensors that can sensitively detect the anti-apoptosis protein BCL-2, the IgG1 Fc domain, the HER2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac troponin I and an anti-hepatitis B virus antibody with the high sensitivity required to detect these molecules clinically. Given the need for diagnostic tools to track the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)3, we used the approach to design sensors for the SARS-CoV-2 spike protein and antibodies against the membrane and nucleocapsid proteins. The former, which incorporates a de novo designed spike receptor binding domain (RBD) binder4, has a limit of detection of 15 pM and a luminescence signal 50-fold higher than the background level. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes, and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.

RNA recognition by a human antibody against brain cytoplasmic 200 RNA.

Diverse functional RNAs participate in a wide range of cellular processes. The RNA structure is critical for function, either on its own or as a complex form with proteins and other ligands. Therefore, analysis of the RNA conformation in cells is essential for understanding their functional mechanisms. However, no appropriate methods have been established as yet. Here, we developed an efficient strategy for panning and affinity maturation of anti-RNA human monoclonal antibodies from a naïve antigen binding fragment (Fab) combinatorial phage library. Brain cytoplasmic 200 (BC200) RNA, which is also highly expressed in some tumors, was used as an RNA antigen. We identified MabBC200-A3 as the optimal binding antibody. Mutagenesis and SELEX experiments showed that the antibody recognized a domain of BC200 in a structure- and sequence-dependent manner. Various breast cancer cell lines were further examined for BC200 RNA expression using conventional hybridization and immunoanalysis with MabBC200-A3 to see whether the antibody specifically recognizes BC200 RNA among the total purified RNAs. The amounts of antibody-recognizable BC200 RNA were consistent with hybridization signals among the cell lines. Furthermore, the antibody was able to discriminate BC200 RNA from other RNAs, supporting the utility of this antibody as a specific RNA structure-recognizing probe. Intriguingly, however, when permeabilized cells were subjected to immunoanalysis instead of purified total RNA, the amount of antibody-recognizable RNA was not correlated with the cellular level of BC200 RNA, indicating that BC200 RNA exists as two distinct forms (antibody-recognizable and nonrecognizable) in breast cancer cells and that their distribution depends on the cell type. Our results clearly demonstrate that anti-RNA antibodies provide an effective novel tool for detecting and analyzing RNA conformation.

Block of T cell development in P53-deficient mice accelerates development of lymphomas with characteristic RAG-dependent cytogenetic alterations.

Mice deficient in the DNA damage sensor P53 display normal T cell development but eventually succumb to thymic lymphomas. Here, we show that inactivation of the TCR beta gene enhancer (E beta) results in a block of T cell development at stages where recombination-activating genes (RAG) are expressed. Introduction of the E beta mutation into p53-/- mice dramatically accelerates the onset of lethal thymic lymphomas that harbor RAG-dependent aberrant rearrangements, chromosome 14 and 12 translocations, and amplification of the chromosomal region 9A1-A5.3. Phenotypic and genetic analyses suggest that lymphomas emerge through a normal thymocyte development pathway. These findings provide genetic evidence that block of lymphocyte development at stages with RAG endonuclease activity can provoke lymphomagenesis on a background with deficient DNA damage responses.

Risk factors associated with death due to severe fever with thrombocytopenia syndrome in hospitalized Korean patients (2018-2022).

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) has no vaccine or treatment and an extremely high fatality rate. We aimed to analyze and evaluate the risk factors for death associated with SFTS. METHODS: Among reports from 2018 to 2022, we compared and analyzed 1,034 inpatients aged 18 years or older with laboratory-confirmed SFTS who underwent complete epidemiological investigations. RESULTS: Most of the inpatients with SFTS were aged 50 years or older (average age, 67.6 years). The median time from symptom onset to death was 9 days, and the average case fatality rate was 18.5%. Risk factors for death included age of 70 years or older (odds ratio [OR], 4.82); agriculture-related occupation (OR, 2.01); underlying disease (OR, 7.20); delayed diagnosis (OR, 1.28 per day); decreased level of consciousness (OR, 5.53); fever/chills (OR, 20.52); prolonged activated partial thromboplastin time (OR, 4.19); and elevated levels of aspartate aminotransferase (OR, 2.91), blood urea nitrogen (OR, 2.62), and creatine (OR, 3.21). CONCLUSION: The risk factors for death in patients with SFTS were old age; agriculture-related occupation; underlying disease; delayed clinical suspicion; fever/chills; decreased level of consciousness; and elevated activated partial thromboplastin time, aspartate aminotransferase, blood urea nitrogen, and creatine levels.

Longitudinal kinetics of neutralizing antibodies against circulating SARS-CoV-2 variants and estimated level of group immunity of booster-vaccinated individuals during omicron-dominated COVID-19 outbreaks in the Republic of Korea, 2022.

The coronavirus disease 2019 pandemic persisted for 3 years and is now transitioning to endemicity. We illustrated the change in group immunity induced by vaccination (monovalent vaccines) and breakthrough infections (BIs) in a healthcare worker (HCW) cohort. Five sampling points were analyzed: before the third dose and 1, 3, 5, and 8 months after the vaccination. The last two points corresponded roughly to 1 and 4 months after omicron BA.1/BA.2 BI. A semi-quantitative anti-spike binding antibody (Sab) assay and plaque reduction neutralization test (PRNT) against circulating variants were conducted. A linear regression model was utilized to deduce correlation equations. Baseline characteristics and antibody titers after the third dose were not different between 106 HCWs with or without BI (54/52). One month after the third dose, BA.1 PRNT increased with wild-type (WT), but 3 months after the third dose, it decreased more rapidly than WT PRNT. After BI, BA.1 PRNT increased robustly and waned slower than WT. A linear equation of waning kinetics was deduced between log10Sab and months, and the slope became gradual after BI. The estimated BA.5 PRNT titers at the beginning of the BA.5 outbreak were significantly higher than the BA.1 PRNT titers of the initial BA.1/BA.2 wave, which might be associated with the smaller size of the BA.5 wave. BA.1/BA.2 BI after the third dose elicited robust and broad neutralizing activity, preferentially maintaining cross-neutralizing longevity against BA.1 and BA.5. The estimated kinetics provide an overview of group immunity through the third vaccination and BA.1/BA.2 BI, correlating with the actual outbreaks. IMPORTANCE This study analyzed changes in group immunity induced by coronavirus disease 2019 (COVID-19) vaccination and BA.1/BA.2 breakthrough infections (BIs) in a healthcare worker cohort. We investigated the longitudinal kinetics of neutralizing antibodies against circulating variants and confirmed that BA.1/BA.2 BIs enhance the magnitude and durability of cross-neutralization against BA.1 and BA.5. Correlation equations between semi-quantitative anti-spike antibody and plaque reduction neutralization test titers were deduced from the measured values using a linear regression model. Based on the equations, group immunity was estimated to last up to 11 months following the third dose of the COVID-19 vaccine. The estimated group immunity suggests that the augmented immunity and flattened waning slope through BI could correlate with the overall outbreak size. Our findings could provide a better understanding to establish public health strategies against future endemicity.

Brief report: L1 cell adhesion molecule, a novel surface molecule of human embryonic stem cells, is essential for self-renewal and pluripotency.

Despite the recent identification of surface markers of undifferentiated human embryonic stem cells (hESCs), the crucial cell-surface molecules that regulate the self-renewal capacity of hESCs remain largely undefined. Here, we generated monoclonal antibodies (MAbs) that specifically bind to undifferentiated hESCs but not to mouse embryonic stem cells. Among these antibodies, we selected a novel MAb, 4-63, and identified its target antigen as the L1 cell adhesion molecule (L1CAM) isoform 2. Notably, L1CAM expressed in hESCs lacked the neuron-specific YEGHH and RSLE peptides encoded by exons 2 and 27, respectively. L1CAM colocalized with hESC-specific cell-surface markers, and its expression was markedly downregulated on differentiation. Stable L1CAM depletion markedly decreased hESC proliferation, whereas L1CAM overexpression increased proliferation. In addition, the expression of octamer-binding transcription factor 4, Nanog, sex-determining region Y-box 2, and stage-specific embryonic antigen (SSEA)-3 was markedly downregulated, whereas lineage-specific markers and SSEA-1 were upregulated in L1CAM-depleted hESCs. Interestingly, the actions of L1CAM in regulating the proliferation and differentiation of hESCs were exerted predominantly through the fibroblast growth factor receptor 1 signaling pathway. Taken together, our results suggest that L1CAM is a novel cell-surface molecule that plays an important role in the maintenance of self-renewal and pluripotency in hESCs.

Neutralizing Activity Against SARS-CoV-2 Delta and Omicron Variants Following a Third BNT162b2 Booster Dose According to Three Homologous or Heterologous COVID-19 Vaccination Schedules.

With the emergence and rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta and Omicron variants, escaping vaccine-induced immunity is a concern. Three vaccination schedules, homologous or heterologous, have been initially applied due to an insufficient supply of vaccines in Korea. We investigated neutralizing activities against Omicron and Delta variants in each schedule. Three schedules using three doses of the BNT162b2 (BNT) or the ChAdOx1 (ChAd) vaccines include ChAd-ChAd-BNT, ChAd-BNT-BNT, and BNT-BNT-BNT. Neutralizing activities were evaluated using plaque-reduction neutralization test (PRNT) against wild type (WT) SARS-CoV-2, Delta variant, and Omicron variant. A total of 170 sera from 75 participants were tested, and the baseline characteristics of participants were not significantly different between groups. After the 2nd vaccine dose, geometric mean titers of PRNT ND50 against WT, Delta, and Omicron were highest after ChAd-BNT vaccination (2,463, 1,097, and 107) followed by BNT-BNT (2,364, 674, and 38) and ChAd-ChAd (449, 163, and 25). After the 3rd dose of BNT, the increase of PRNT ND50 against WT, Delta, and Omicron was most robust in ChAd-ChAd-BNT (4,632, 988, and 260), while the BNT-BNT-BNT group showed the most augmented neutralizing activity against Delta and Omicron variants (2,315 and 628). ChAd-BNT-BNT showed a slight increase of PRNT ND50 against WT, Delta, and Omicron (2,757, 1,279, and 230) compared to the 2nd dose. The results suggest that a 3rd BNT booster dose induced strengthened neutralizing activity against Delta and Omicron variants. The waning of cross-reactive neutralizing antibodies after the 3rd dose and the need for additional boosting should be further investigated.

Acquisition of chemoresistance in intrahepatic cholangiocarcinoma cells by activation of AKT and extracellular signal-regulated kinase (ERK)1/2.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignant tumor and is refractory to conventional chemotherapy. The aim of this study is therefore to elucidate the mechanism of chemoresistance in ICC which is not fully understood. We generated cisplatin resistant ICC cells via long term exposure to cisplatin and found that these cells are also resistant to 5-fluorouracil (5-FU) and gemcitabine. The chemoresistant cells showed enhanced Bcl-2 expression and reduced Bax expression compared to parental ICC cells. In addition, the resistant cells showed enhanced activation of AKT and extracellular signal-regulated kinase (ERK) 1/2. Inhibition of AKT activation by phosphoinocitide 3-kinase (PI3K) inhibitor LY294002 resulted in reduced Bcl-2 expression and enhanced Bax expression and thus induced apoptosis in the resistant cells, whereas inhibition of ERK1/2 activation by mitogen-activated protein kinase (MEK) inhibitor U0126 did not induce apoptosis without affecting the expression of Bcl-2 and Bax but decreased cell growth. Moreover, the inhibition of AKT or ERK1/2 sensitized the resistant cells to cisplatin and therefore resulted in greatly enhanced cisplatin-induced apoptosis and growth inhibition in the cells. The results indicate that AKT and ERK1/2 signaling mediate chemoresistance in the cells and could be important therapeutic targets for overcoming chemoresistance in ICC.

Epitope-Paratope Interaction of a Neutralizing Human Anti-Hepatitis B Virus PreS1 Antibody That Recognizes the Receptor-Binding Motif.

Hepatitis B virus (HBV) is a global health burden that causes acute and chronic hepatitis. To develop an HBV-neutralizing antibody that effectively prevents HBV infection, we previously generated a human anti-preS1 monoclonal antibody (1A8) that binds to genotypes A-D and validated its HBV-neutralizing activity in vitro. In the present study, we aimed to determine the fine epitope and paratope of 1A8 to understand the mechanism of HBV neutralization. We performed alanine-scanning mutagenesis on the preS1 (aa 19-34, genotype C) and the heavy (HCDR) and light (LCDR) chain complementarity-determining regions. The 1A8 recognized the three residues (Leu22, Gly23, and Phe25) within the highly conserved receptor-binding motif (NPLGFFP) of the preS1, while four CDR residues of 1A8 were critical in antigen binding. Structural analysis of the epitope-paratope interaction by molecular modeling revealed that Leu100 in the HCDR3, Ala50 in the HCDR2, and Tyr96 in the LCDR3 closely interacted with Leu22, Gly23, and Phe25 of the preS1. Additionally, we found that 1A8 also binds to the receptor-binding motif (NPLGFLP) of infrequently occurring HBV. The results suggest that 1A8 may broadly and effectively block HBV entry and thus have potential as a promising candidate for the prevention and treatment of HBV infection.

An antibody against L1 cell adhesion molecule inhibits cardiotoxicity by regulating persistent DNA damage.

Targeting the molecular pathways underlying the cardiotoxicity associated with thoracic irradiation and doxorubicin (Dox) could reduce the morbidity and mortality associated with these anticancer treatments. Here, we find that vascular endothelial cells (ECs) with persistent DNA damage induced by irradiation and Dox treatment exhibit a fibrotic phenotype (endothelial-mesenchymal transition, EndMT) correlating with the colocalization of L1CAM and persistent DNA damage foci. We demonstrate that treatment with the anti-L1CAM antibody Ab417 decreases L1CAM overexpression and nuclear translocation and persistent DNA damage foci. We show that in whole-heart-irradiated mice, EC-specific p53 deletion increases vascular fibrosis and the colocalization of L1CAM and DNA damage foci, while Ab417 attenuates these effects. We also demonstrate that Ab417 prevents cardiac dysfunction-related decrease in fractional shortening and prolongs survival after whole-heart irradiation or Dox treatment. We show that cardiomyopathy patient-derived cardiovascular ECs with persistent DNA damage show upregulated L1CAM and EndMT, indicating clinical applicability of Ab417. We conclude that controlling vascular DNA damage by inhibiting nuclear L1CAM translocation might effectively prevent anticancer therapy-associated cardiotoxicity.

Construction of a humanized antibody to hepatitis B surface antigen by specificity-determining residues (SDR)-grafting and de-immunization.

We previously constructed a humanized antibody, HuS10, by grafting the complementarity-determining regions (CDRs) of a parental murine monoclonal antibody into the homologous human antibody sequences. This process is termed CDR grafting. Some residues that were thought to affect the CDR loops and stabilize the structure of the variable regions were retained in the framework region. HuS10 exhibited in vivo virus-neutralizing activity, but its murine content had the potential to elicit immune responses in patients. In this study, to minimize the immunogenic potential of HuS10, we replaced 17 mouse residues in HuS10 with the comparable human residues using specificity-determining residue (SDR)-grafting and de-immunization methods. The resultant humanized antibody, HzS-III, had the same affinity and epitope specificity as HuS10 and had reduced immunogenic potential, as assessed by T-cell epitope analysis. Thus, SDR grafting in combination with de-immunization may be a useful strategy for minimizing the immunogenicity of humanized antibodies. In addition, HzS-III may be a good candidate for immunoprophylaxis of HBV infection.

Generation and Characterization of Monoclonal Antibodies to the Ogawa Lipopolysaccharide of Vibrio cholerae O1 from Phage-Displayed Human Synthetic Fab Library.

Vibrio cholerae, cause of the life-threatening diarrheal disease cholera, can be divided into different serogroups based on the structure of its lipopolysaccharide (LPS), which consists of lipid-A, corepolysaccharide and O-antigen polysaccharide (O-PS). The O1 serogroup, the predominant cause of cholera, includes two major serotypes, Inaba and Ogawa. These serotypes are differentiated by the presence of a single 2-O-methyl group in the upstream terminal perosamine of the Ogawa O-PS, which is absent in the Inaba O-PS. To ensure the consistent quality and efficacy of the current cholera vaccines, accurate measurement and characterization of each of these two serotypes is highly important. In this study, we efficiently screened a phage-displayed human synthetic Fab library by bio-panning against Ogawa LPS and finally selected three unique mAbs (D9, E11, and F7) that specifically react with Ogawa LPS. The mAbs bound to Vibrio cholerae vaccine in a dose-dependent fashion. Sequence and structure analyses of antibody paratopes suggest that IgG D9 might have the same fine specificity as that of the murine mAbs, which were shown to bind to the upstream terminal perosamine of Ogawa O-PS, whereas IgGs F7 and E11 showed some different characteristics in the paratopes. To our knowledge, this study is the first to demonstrate the generation of Ogawa-specific mAbs using phage display technology. The mAbs will be useful for identification and quantification of Ogawa LPS in multivalent V. cholerae vaccines.

De novo design of modular and tunable allosteric biosensors.

Naturally occurring allosteric protein switches have been repurposed for developing novel biosensors and reporters for cellular and clinical applications 1 , but the number of such switches is limited, and engineering them is often challenging as each is different. Here, we show that a very general class of allosteric protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which binding of a peptide key triggers biological outputs of interest 2 . Using broadly applicable design principles, we allosterically couple binding of protein analytes of interest to the reconstitution of luciferase activity and a bioluminescent readout through the association of designed lock and key proteins. Because the sensor is based purely on thermodynamic coupling of analyte binding to switch activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We demonstrate the modularity of this platform by creating biosensors that, with little optimization, sensitively detect the anti-apoptosis protein Bcl-2, the hIgG1 Fc domain, the Her2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac Troponin I and an anti-Hepatitis B virus (HBV) antibody that achieve the sub-nanomolar sensitivity necessary to detect clinically relevant concentrations of these molecules. Given the current need for diagnostic tools for tracking COVID-19 3 , we use the approach to design sensors of antibodies against SARS-CoV-2 protein epitopes and of the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. The latter, which incorporates a de novo designed RBD binder, has a limit of detection of 15pM with an up to seventeen fold increase in luminescence upon addition of RBD. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.

Development of a Theranostic Convergence Bioradiopharmaceutical for Immuno-PET Based Radioimmunotherapy of L1CAM in Cholangiocarcinoma Model.

PURPOSE: Cholangiocarcinoma is a malignancy of bile duct with a poor prognosis. Conventional chemotherapy and radiotherapy are generally ineffective, and surgical resection is the only curative treatment for cholangiocarcinoma. L1-cell adhesion molecule (L1CAM) has been known as a novel prognostic marker and therapeutic target for cholangiocarcinoma. This study aimed to evaluate the feasibility of immuno-PET imaging-based radioimmunotherapy using radiolabeled anti-L1CAM antibody in cholangiocarcinoma xenograft model. EXPERIMENTAL DESIGN: We prepared a theranostic convergence bioradiopharmaceutical using chimeric anti-L1CAM antibody (cA10-A3) conjugated with 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) chelator and labeled with 64Cu or 177Lu and evaluated the immuno-PET or SPECT/CT imaging and biodistribution with 64Cu-/177Lu-cA10-A3 in various cholangiocarcinoma xenograft models. Therapeutic efficacy and response monitoring were performed by 177Lu-cA10-A3 and 18F-FDG-PET, respectively, and immunohistochemistry was done by TUNEL and Ki-67. RESULTS: Radiolabeled cA10-A3 antibodies specifically recognized L1CAM in vitro, clearly visualized cholangiocarcinoma tumors in immuno-PET and SPECT/CT imaging, and differentiated the L1CAM expression level in cholangiocarcinoma xenograft models. 177Lu-cA10-A3 (12.95 MBq/100 µg) showed statistically significant reduction in tumor volumes (P < 0.05) and decreased glucose metabolism (P < 0.01). IHC analysis revealed 177Lu-cA10-A3 treatment increased TUNEL-positive and decreased Ki-67-positive cells, compared with saline, cA10-A3, or 177Lu-isotype. CONCLUSIONS: Anti-L1CAM immuno-PET imaging using 64Cu-cA10-A3 could be translated into the clinic for characterizing the pharmacokinetics and selecting appropriate patients for radioimmunotherapy. Radioimmunotherapy using 177Lu-cA10-A3 may provide survival benefit in L1CAM-expressing cholangiocarcinoma tumor. Theranostic convergence bioradiopharmaceutical strategy would be applied as imaging biomarker-based personalized medicine in L1CAM-expressing patients with cholangiocarcinoma.

Selection of an affinity-matured antibody against a defined epitope by phage display of an immune antibody library.

In a previous study, we generated a murine hepatitis B virus (HBV)-neutralizing monoclonal antibody (mAb), KR127, that binds to an epitope (amino acids 37-45, NSNNPDWDF) of the preS1 antigen. Furthermore, an epitope tag, S1 (NANNPDWDF), was developed for protein tagging. The aim of the present study was to develop a high-affinity antibody to the same preS1 epitope. Mice were immunized with the N-terminal domain of human thrombopoietin fused to the S1 tag (nTPO-S1), and a phage-displayed chimeric Fab library was constructed and screened by panning against nTPO-S1. A high-affinity antibody (3-34) was selected that binds to the preS1 antigen. The IgG molecules of 3-34 showed approximately nine-fold higher affinity (K(D) 1.2 nM) for preS1 compared with KR127 (K(D) 10.4 nM), competed with KR127 for binding to the epitope, and bound to HBV particles. This study provides a simple and efficient way to develop a high-affinity antibody to a defined epitope by phage display of an immune antibody library.

In vivo hepatitis B virus-neutralizing activity of an anti-HBsAg humanized antibody in chimpanzees.

Previously, we constructed a humanized antibody (HuS10) that binds to the common a antigenic determinant on the S protein of HBV. In this study, we evaluated its HBV-neutralizing activity in chimpanzees. A study chimpanzee was intravenously administered with a single dose of HuS10, followed by intravenous challenge with the adr subtype of HBV, while a control chimpanzee was only challenged with the virus. The result showed that the control chimpanzee was infected by the virus, and thus serum HBV surface antigen (HBsAg) became positive from the 14(th) to 20(th) week and actively acquired serum anti-HBc and anti-HBs antibodies appeared from the 19(th) and 23(rd) week, respectively. However, in the case of the study chimpanzee, serum HBsAg became positive from the 34(th) to 37(th) week, while actively acquired serum anti-HBc and anti-HBs antibodies appeared from the 37(th) and 40(th) week, respectively, indicating that HuS10 neutralized the virus in vivo and thus delayed the HBV infection. This novel humanized antibody will be useful in the immunoprophylaxis of HBV infection.

Generation and Characterization of a Neutralizing Human Monoclonal Antibody to Hepatitis B Virus PreS1 from a Phage-Displayed Human Synthetic Fab Library.

The hepatitis B virus (HBV) envelope contains small (S), middle (M), and large (L) proteins. PreS1 of the L protein contains a receptor-binding motif crucial for HBV infection. This motif is highly conserved among 10 HBV genotypes (A-J), making it a potential target for the prevention of HBV infection. In this study, we successfully generated a neutralizing human monoclonal antibody (mAb), 1A8 (IgG1), that recognizes the receptor-binding motif of preS1 using a phage-displayed human synthetic Fab library. Analysis of the antigen-binding activity of 1A8 for different genotypes indicated that it can specifically bind to the preS1 of major HBV genotypes (A-D). Based on Bio-Layer interferometry, the affinity (KD) of 1A8 for the preS1 of genotype C was 3.55 nM. 1A8 immunoprecipitated the hepatitis B virions of genotypes C and D. In an in vitro neutralization assay using HepG2 cells overexpressing the cellular receptor sodium taurocholate cotransporting polypeptide, 1A8 effectively neutralized HBV infection with genotype D. Taken together, the results suggest that 1A8 may neutralize the four HBV genotypes. Considering that genotypes A-D are most prevalent, 1A8 may be a neutralizing human mAb with promising potential in the prevention and treatment of HBV infection.

Generation of a Human Monoclonal Antibody to Cross-Reactive Material 197 (CRM197) and Development of a Sandwich ELISA for CRM197 Conjugate Vaccines.

Cross-reactive material 197 (CRM197) is a non-toxic mutant of diphtheria toxin containing a single amino acid substitution of glycine 52 with glutamic acid. CRM197 has been used as a carrier protein for poorly immunogenic polysaccharide antigens to improve immune responses. In this study, to develop a sandwich ELISA that can detect CRM197 and CRM197 conjugate vaccines, we generated a human anti-CRM197 monoclonal antibody (mAb) 3F9 using a phage-displayed human synthetic Fab library and produced mouse anti-CRM197 polyclonal antibody. The affinity (KD) of 3F9 for CRM197 was 3.55 nM, based on Bio-Layer interferometry, and it bound specifically to the B fragment of CRM197. The sandwich ELISA was carried out using 3F9 as a capture antibody and the mouse polyclonal antibody as a detection antibody. The detection limit of the sandwich ELISA was ＜1 ng/ml CRM197. In addition, the 3F9 antibody bound to the CRM197-polysaccharide conjugates tested in a dose-dependent manner. This ELISA system will be useful for the quantification and characterization of CRM197 and CRM197 conjugate vaccines. To our knowledge, this study is the first to generate a human monoclonal antibody against CRM197 and to develop a sandwich ELISA for CRM197 conjugate vaccines.

Multi-paratopic VEGF decoy receptor have superior anti-tumor effects through anti-EGFRs and targeted anti-angiogenic activities.

Limitation of current anti-Vascular Endothelial Growth Factor (VEGF) cancer therapy is transitory responses, inevitable relapses and its insufficient tumor-targeting. Thus, multifaceted approaches, including the development of bispecific antibodies and combination strategies targeting different pathways have been proposed as an alternative. Here, we developed a novel multi-paratopic VEGF decoy receptor, Cetuximab-VEGF-Grab and Trastuzumab-VEGF-Grab, by genetically fusing VEGF decoy receptor (VEGF-Grab) to a single chain Fv of anti-Epidermal Growth Factor Receptor (EGFR) antibody (Cetuximab and Trastuzumab). These multi-paratopic VEGF decoy receptor, which recognize VEGF and EGFR family (EGFR or HER2), effectively suppressed both VEGF and EGFR pathways in vitro, to levels similar to those of the parental VEGF-Grab and anti-EGFR antibodies. In addition, the concurrent binding of multi-paratopic VEGF decoy receptor to VEGF and EGFR family enabled their specific localization to EGFR + tumor in vitro and in vivo. Furthermore, Cetuximab-VEGF-Grab and Trastuzumab-VEGF-Grab exhibited the enhanced anti-tumor activities compared to VEGF-Grab in EGFR + tumor xenograft mouse model via anti-EGFR and the targeted anti-angiogenic activities. These results indicate that multi-paratopic VEGF decoy receptor can be a promising agent, combining tumor-targeted anti-angiogenic therapy with efficient blockade of proliferative signals mediated by EGFR family.

The T-cell receptor beta variable gene promoter is required for efficient V beta rearrangement but not allelic exclusion.

To investigate the role of promoters in regulating variable gene rearrangement and allelic exclusion, we constructed mutant mice in which a 1.2-kb region of the V beta 13 promoter was either deleted (P13(-/-)) or replaced with the simian virus 40 minimal promoter plus five copies of Gal4 DNA sequences (P13(R/R)). In P13(-/-) mice, cleavage, rearrangement, and transcription of V beta 13, but not the flanking V beta gene segments, were significantly inhibited. In P13(R/R) mice, inhibition of V beta 13 rearrangement was less severe and was not associated with any apparent reduction in V beta 13 cleavage. Expression of a T-cell receptor (TCR) transgene blocked cleavages at the normal V beta 13-recombination signal sequence junction and V beta 13 coding joint formation of both wild-type and mutant V beta 13 alleles. However, a low level of aberrant V beta 13 cleavage was consistently detected, especially in TCR transgenic P13(R/R) mice. These findings suggest that the variable gene promoter is required for promoting local recombination accessibility of the associated V beta gene segment. Although the promoter is dispensable for allelic exclusion, it appears to suppress aberrant V beta cleavages during allelic exclusion.