Stability of murine, chimeric and humanized antibodies against pre-S2 surface antigen of hepatitis B virus

We have constructed a humanized antibody with specificity for the pre-S2 surface antigen of hepatitis B virus (HBV) by grafting the complementarity determining regions (CDRs) of parental murine monoclonal antibody (mAb) into human anti-Sm antibody framework regions. The humanized antibody has a substitution at position 94 in a framework region of the heavy chain variable region, and exhibits the same antigen binding affinity as the parental murine monoclonal and chimeric antibodies. In order to assess the stability of these antibodies, thermal inactivation of the parental, chimeric and humanized antibodies was analyzed. Fifty percent inactivation of the chimeric and humanized antibodies was observed at 63.7 degrees C and 68.7 degrees C, respectively, compared to 55.0 degrees C for murine antibody. The humanized antibody also exhibited increased stability against denaturant. Guanidine-induced unfolding monitored by the changes in fluorescence intensity at 360 nm showed that midpoints of the transition of the chimeric and humanized antibodies were 2.47 M and 2.56 M, respectively, whereas that of the murine antibody was 1.36 M.     

待孕夫妇乙肝表面抗原及乙肝表面抗体阳性情况分析

目的 分析与探讨待孕夫妇乙肝表面抗原及乙肝表面抗体检测结果,并研究其对临床孕前检查的影响及评价。方法 随机选取2015‒2017年度在我院进行孕前检查的夫妇2440对（4 880例）为研究对象,按照年度将待孕夫妇分为两组,每组2 440例,两组均加强孕前检查中的乙肝表面抗原（HBsAg）及乙肝表面抗体（HBsAb）的检测。A组为2015年3月‒2016年2月在我院进行乙肝表面抗原及乙肝表面抗体检查的待孕夫妇;B组为2016年3月‒2017年2月在我院进行乙肝表面抗原及乙肝表面抗体检查的待孕夫妇。比较两组待孕夫妇乙肝表面抗原及乙肝表面抗体检测的阳性结果。结果 B组HBsAg阳性率、HBsAb阳性率明显高于A组（6.43% vs 4.63%;62.99% vs 58.44%）,差异有统计学意义（P＜0.05）。B组、A组男性HBsAg阳性率明显高于同组女性（59.87% vs 40.13%;60.18% vs 39.82%）,HBsAb阳性率低于同组女性（46.52% vs 53.48%;47.41% vs 52.59%）,差异均有统计学意义（P＜0.05）。B组、A组高中及以上学历HBsAg阳性率明显低于同组高中以下学历（38.85% vs 61.95%;38.05% vs 61.15%）,高中及以上学历HBsAb阳性率高于同组高中以下学历（53.15% vs 46.84%;51.75% vs 48.25%）,差异均有统计学意义（P＜0.05）结论 目前夫妇乙肝感染仍处于增高趋势,对于进行孕前检查的待孕夫妇加强乙肝表面抗原及乙肝表面抗体的检测,有助于疾病的早期诊断、干预及治疗,能够减少乙肝传播,可有效降低新生儿乙肝发病率,促进优生优育,提高出生人口整体素质。     

Standardization and validation of an alkaline phosphatase-linked immunoassay to quantify a plant-derived antibody directed against the hepatitis B virus surface antigen

Immunopurification is one of the most effective chromatography steps to purify the hepatitis B surface antigen, which have successfully been used as an active pharmaceutical ingredient of hepatitis B vaccines. Plant-derived antibodies could be an appropriated ligand for such purposes because plants are the most cost-effective production systems and have the additional advantage that plant viruses cannot infect humans. In this work, a polyclonal antibody alkaline phosphatase-linked immunoassay was standardized and validated to quantify a plant-derived antibody directed against the HBsAg. The validation of an immunoassay to quantify plantibodies is a relatively complex task due to the complexity of the plant extract, the low level of expression of this molecule, and the potential interferences of endogenous peroxidases contributed by plants. These results allow estimating the plant-derived antibody concentration up to 3.81 ng/mL with high specificity, precision, and repeatability. The working range of the standard curve was between 3.81 and 60 ng/mL, and the intra- and inter-variation coefficients were between 10% and 20% in a production process's sample dependent way. This enzyme-linked immunosorbent assay is considered valuable to improve the design of the purification process and also to obtain a better estimation of the antibody expression level and process's recovery.     

Age-specific prevalence of hepatitis B e antigen (HBeAg) and antibody to HBeAg (anti-HBe) among asymptomatic hepatitis B surface antigen (HBsAg) carriers in Okinawa and Kyushu, Japan

A total of 1,741 asymptomatic hepatitis B surface antigen (HBsAg) carriers in two areas (Okinawa and Kyushu) in Japan were surveyed for the presence of hepatitis B e antigen (HBeAg) and the corresponding antibody (anti-HBe) to determine the age-specific prevalence of these markers and the mean age of carriers with HBeAg. Prevalence of HBeAg was significantly higher in Kyushu (36.4% of 755 carriers) than in Okinawa (20.0% of 986 carriers) (P less than 0.001). The mean age of carriers with HBeAg was 25.5 years in Kyushu and 16.1 years in Okinawa, suggesting that HBeAg converted to anti-HBe earlier in Okinawa than in Kyushu. In contrast, the prevalence of anti-HBe was significantly higher in Okinawa (74.6% of 986) than in Kyushu (56.3% of 755) (P less than 0.001). The prevalence of HBeAg decreased with age up to 40-49 years of age and then increased in both areas. Prevalence of anti-HBe was inversely related to the prevalence of HBeAg in both areas. These data suggest that HBeAg and anti-HBe are chronological markers of chronic hepatitis B virus infection and that the duration of HBeAg persistence can be different in different area, even in the same country.     

Therapeutic inhibition of hepatitis B virus surface antigen expression by RNA interference

RNA interference (RNAi) mediated inhibition of virus-specific genes has emerged as a potential therapeutic strategy against virus induced diseases. Human hepatitis B virus (HBV) surface antigen (HBsAg) has proven to be a significant risk factor in HBV induced liver diseases, and an increasing number of mutations in HBsAg are known to enhance the difficulty in therapeutic interventions. The key challenge for achieving effective gene silencing in particular for the purpose of the therapeutics is primarily based on the effectiveness and specificity of the RNAi targeting sequence. To explore the therapeutic potential of RNAi on HBV induced diseases in particular resulted from aberrant or persistent expression of HBsAg, we have especially screened and identified the most potent and specific RNAi targeting sequence that directly mediated inhibition of the HBsAg expression. Using an effective DNA vector-based shRNA expression system, we have screened 10 RNAi targeting sequences (HBsAg-1 to 10) that were chosen from HBsAg coding region, in particular the major S region, and have identified four targeting sequences that could mediate sequence specific inhibition of the HBsAg expression. Among these four shRNAs, an extremely potent and highly sequence specific HBsAg-3 shRNA was found to inhibit HBsAg expression in mouse HBV model. The inhibition was not only preventive in cotransfection experiments, but also had therapeutic effect as assessed by post-treatment protocols. Moreover, this HBsAg-3 shRNA also exhibited a great potency of inhibition in transgenic mice that constitutively expressed HBsAg. These results indicate that HBsAg-3 shRNA can be considered as a powerful therapeutic agent on HBsAg induced diseases.     

Grafting of "abbreviated" complementarity-determining regions containing specificity-determining residues essential for ligand contact to engineer a less immunogenic humanized monoclonal antibody

Murine mAb COL-1 reacts with carcinoembryonic Ag (CEA), expressed on a wide range of human carcinomas. In preclinical studies in animals and clinical trials in patients, murine COL-1 showed excellent tumor localization. To circumvent the problem of immunogenicity of the murine Ab in patients, a humanized COL-1 (HuCOL-1) was generated by grafting the complementarity-determining regions (CDRs) of COL-1 onto the frameworks of the variable light and variable heavy regions of human mAbs. To minimize anti-V region responses, a variant of HuCOL-1 was generated by grafting onto the human frameworks only the "abbreviated" CDRs, the stretches of CDR residues that contain the specificity-determining residues that are essential for the surface complementarity of the Ab and its ligand. In competition RIAs, the recombinant variant completely inhibited the binding of radiolabeled murine and humanized COL-1 to CEA. The HuCOL-1 and its variant showed no difference in their binding ability to the CEA expressed on the surface of a CEA-transduced tumor cell line. Compared with HuCOL-1, the HuCOL-1 variant showed lower reactivity to patients' sera carrying anti-V region Abs to COL-1. The final variant of the HuCOL-1, which retains its Ag-binding reactivity and shows significantly lower serum reactivity than that of the parental Ab, can serve as a prototype for the development of a potentially useful clinical reagent.     

Hepatitis B surface antigen immunopurification using a plant-derived specific antibody produced in large scale

This paper provides an evaluation of a plant-derived HBsAg-specific antibody in the immunopurification of the recombinant HBsAg for vaccine purposes. This plant-derived antibody was obtained from different batches of 100-200kg of tobacco leaves and coupled to Sepharose CL-4B with high efficiency. The plant-derived antibody immunoaffinity matrix purification behavior (elution capacity, antigen purity, purification cycles, and ligand leakage) was comparable to that of its mouse-derived monoclonal antibody homolog. This result supports the feasibility of using this plant-derived antibody for the immunopurification of the Hepatitis B surface antigen for human use, opening a new possibility to overcome the constrain of monoclonal antibody production in mice.     

Antibody-targeted chemotherapy of B-cell lymphoma using calicheamicin conjugated to murine or humanized antibody against CD22

Antibody-targeted chemotherapy with immunoconjugates of calicheamicin is a clinically validated strategy in cancer therapy. This study describes the selection of a murine anti-CD22 mAb, m5/44, as a targeting agent, its conjugation to a derivative of calicheamicin (CalichDM) via either acid-labile or acid-stable linkers, the antitumor activity of CalichDM conjugated to m5/44, and its subsequent humanization by CDR grafting. Murine IgG1 mAb m5/44 was selected based on its subnanomolar affinity for CD22 and ability to be internalized into B cells. CalichDM conjugated to m5/44 caused potent growth inhibition of CD22⁺ human B-cell lymphomas (BCLs) in vitro. The conjugate of m5/44 with an acid-labile linker was more potent than an acid-stable conjugate, a nonbinding conjugate with a similar acid-labile linker, or unconjugated CalichDMH in inhibiting BCL growth. CalichDM conjugated to m5/44 caused regression of established BCL xenografts in nude mice. In contrast, both unconjugated m5/44 and a nonbinding conjugate were ineffective against these xenografts. Based on the potent antitumor activity of m5/44-CalichDM conjugates, m5/44 was humanized by CDR grafting to create g5/44, an IgG4 anti-CD22 antibody. Both m5/44 and g5/44 bound CD22 with subnanomolar affinity. Competitive blocking with previously characterized murine anti-CD22 mAbs suggested that g5/44 recognizes epitope A located within the first N-terminal Ig-like domain of human CD22. Antitumor efficacy of CalichDM conjugated to g5/44 against BCL xenografts was more potent than its murine counterpart. Based on these results, a calicheamicin conjugate of g5/44, CMC-544, was selected for further development as a targeted chemotherapeutic agent for the treatment of B-cell malignancies.Abbreviations AcBut 4-(4-Acetylphenoxy) butanoic acid - AcPAc (3-Acetylphenyl) acetic acid - ATC Antibody-targeted chemotherapy - BCL B-cell lymphoma - CalichDM N-Acetyl--calicheamicin dimethyl disulfide derivative(s) - CalichDMA CalichDM acid - CalichDMH CalichDM hydrazide - CDR Complementarity determining region - NHL Non-Hodgkins lymphoma - PBMC Peripheral blood mononuclear cell - TAA Tumor-associated antigen     

A simple purification procedure for hepatitis B surface antigen (HBs) by monoclonal antibody immunosorbent affinity chromatography

Using immobilized monoclonal antibodies (anti-HBs) to hepatitis B surface antigen (HBs) as an immunosorbent affinity column, a simple and effective procedure for HBs purification has been developed. A serum sample containing high titer HBs (i.e. HBe-positive serum) is passed through the column without prior treatment. The HBs is further purified by cesium chloride gradient ultracentrifugation. The recovery of HBs is greater than 70% while the purity is very comparable to those obtained from several isopycnic and rate zonal ultracentrifugation procedures (Dreesman et al., 1972). Over a period of two months, the column was used repeatedly for thirty cycles without any noticeable deterioration.     

Expression and characterization of a humanized cocaine-binding antibody

The murine immunoglobulin G (IgG) cocaine-binding monoclonal antibody (mAb), GNC92H2, is notable for its exquisite specificity for cocaine, as opposed to chemically-related cocaine metabolites, and for its moderately high affinity (K(d) approximately 200 nM) for cocaine. Recently, we described the crystal structure of a mouse/human chimeric Fab construct at 2.3 A resolution. Herein, we report the successful framework humanization of a single-chain Fv (scFv) GNC92H2 construct without loss of affinity for cocaine. In brief, we compared the mAb GNC92H2 sequence to human antibody sequences, and used structure-based design to incorporate mutations (total = 49) that would humanize the framework region without affecting the overall shape of the binding pocket or the key cocaine-contact residues. The codons of the rationally designed sequence were optimized for E. coli expression, and the gene was synthesized by a de novo PCR reaction using 14 overlapping primers. Expression of the scFv construct was significantly improved in E. coli by fusion to thioredoxin. Intriguingly, this construct apparently refolds to form soluble active antibody in the reducing environment of the cytoplasm. Competitive ELISA and equilibrium dialysis demonstrated comparable binding activity between the humanized scFv and the whole IgG. The successful humanization of mAb GNC92H2 should enhance its potential therapeutic value by reducing its overall. immunogenicity.     

Human antibody immunosorption of hepatitis B surface antigen from blood and plasma

Removal of the Hepatitis B surface antigen (HBsAg) from whole blood and blood products, using human antibody (HBAb) immunosorbent, was studied and kinetics of complexing were monitored using radioimmunoassay (RIA). An intermittent complexing process was developed that minimizes damage to the cellular components of blood. HBsAg concentration in blood was reduced 1.5 to 2 logarithmic cycles in 3 hr with this system. Free HBsAg remaining in solution at equilibrium was further reduced by transferring the blood to a vessel containing unused immunosorbent. Through multiple stage treatment of a blood sample, it may be possible to reduce the probability of contamination with HBsAg to below the infectious level. This process may be applied to the selective removal of other proteins from blood and plasma.     

Enhanced chemiluminescence ELISA for the detection of antibody to hepatitis B virus surface antigen

A chemiluminescent enzyme linked immunosorbent assay (ELISA) for the detection of antibody to hepatitis B virus surface antigen (anti-HBs) in human serum has been developed. Polystyrene microtitre plates were coated with recombinant, yeast-derived hepatitis B surface antigen (rec-HBsAg). Patient serum samples and appropriate controls were added to the rec-HBsAg-coated wells and incubated to bind anti-HBs. The wells were then washed and a fluorescein isothiocyanate (FITC) conjugate of a human plasma-derived hepatitis B surface antigen (HBsAg) was added. Following incubation and further washing the bound FITC-labelled HBsAg was detected after addition of a horseradish peroxidase (HRP) conjugate of a monoclonal anti-FITC antibody and assaying for the enzyme. The activity of the HRP was measured using luminol and hydrogen peroxide as substrates and iodophenol as a chemiluminescence enhancer. The luminescence was recorded using a camera luminometer. Preliminary tests have shown the assay to be suitable for the detection of antibody in sera from both vaccinees and also from individuals with a past hepatitis B virus infection. The use of the FITC-anti-FITC system together with the measurement of a chemiluminescence signal makes possible the completion of this assay in a few hours. The assay has been shown to be both specific and sensitive and provides a permanent photographic record.     

Production of antibody to individual polypeptides derived from purified hepatitis B surface antigen.

Purified preparations of hepatitis B surface antigen (HBsAg) were solubilized with sodium dodecyl sulfate and urea under reducing conditions and subsequently fractionated by preparative sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis (PAGE). Pools of the individual fractions eluted from the preparative PAGE were concentrated and purified further by analytical PAGE. Five purified polypeptides were isolated from HBsAg, types adw and ayw, with molecular weights of 19,000, 24,000, 27,000, 35,000, and 40,000. Each preparations was emulsified in Freund complete adjuvant and injected into guinea pigs. Antibody to each HBsAg type was measured by radioimmunoassay. The 19,000 molecular weight polypeptide derived from ayw particles and the 27,000 molecular weight subunit obtained from both types failed to elicit an antibody response. The other three polypeptides derived from the ayw particles elicited group-specific antibody responses. Similar group-specific reactivities were observed in the testing of anti-adw 35,000 and anti-adw 40,000 molecular weight polypeptide sera. However, guinea pigs immunized with the 19,000 and the 24,000 molecular weight polypeptides of the adw type produced antibody that reacted preferentially with adw particles. This indicates that either these subunits carry predominately d determinants or that, because of the low levels of material used for inoculation, no immune response or an undetectable one was elicited to the a or w components.     

The structure of an antitumor C(H)2-domain-deleted humanized antibody

C(H)2-domain-deleted CC49 (HuCC49DeltaCH2), a recombinant humanized antibody that recognizes the TAG-72 antigen expressed on a variety of human carcinomas, is secreted from cultured cells as a mixture of two homodimeric isoforms. Isoform A contains two covalent interchain disulfide bonds at heavy chain positions 239 and 242, while isoform B fails to develop any interchain disulfide bonds but has 239-242 intrachain disulfide bonds instead. Form A is currently in preclinical development as a therapeutic agent for treating colorectal carcinoma, though form B shows equal efficacy. HuCC49DeltaCH2 form B can be crystallized from sodium formate only in the presence of detergents. X-ray diffraction data were collected on a single cryo-cooled crystal grown with Triton X-100 and the structure was solved by molecular replacement. The model has refined to R=0.246 (R(free)=0.297) for 2.8A data. The antibodies pack in the crystal around crystallographic 2-fold axes as tetramers with approximate 222 symmetry. Atomic force microscopy studies show that this tetrameric structure is the crystal building block and also exists free in the mother liquor. The tetramer is composed of two rings, back-to-back, with a thickness of approximately 83A. Each ring is composed of two antibodies with the complementarity-determining regions (CDR) of the two Fabs of one antibody interacting with the CDR regions of the second antibody in a head-to-head fashion. These rings are approximately 167A long and 112A wide. The C(H)3 domain is inverted with respect to the Fabs when compared to the usual orientation found in conventional antibodies. The polypeptides joining the C(H)3 domains to the Fab portions of the antibody are not seen and are almost certainly disordered. The antigen combining site of HuCC49DeltaCH2 is very similar, but not identical, in topology and charge distribution to that of antibody B72.3, which binds a similar epitope on TAG-72. The combining site consists of a deep cleft, heavily lined with aromatic amino acid side-chains but bounded by numerous charged groups.     

Construction and characterization of a high-affinity humanized SM5-1 monoclonal antibody

SM5-1 is a mouse monoclonal antibody which has a high specificity for melanoma, hepatocellular carcinoma, and breast cancer, making it a promising candidate for cancer targeting therapy. We have therefore attempted to construct a humanized antibody of SM5-1 to minimize its immunogenicity for potential clinical use. Using a molecular model of SM5-1 built by computer-assisted homology modeling, framework region (FR) residues of potential importance to the antigen binding were identified. Then, a humanized version of SM5-1 was generated by transferring these mouse key FR residues onto a human framework that was selected based on homology to the mouse framework, together with the mouse complementarity-determining region (CDR) residues. This humanized antibody retained only six murine residues outside of the CDRs but was shown to possess affinity and specificity comparable to that of the parental antibody, suggesting that it might have the potential to be developed for future clinical use.     

A mimotope of Pre-S_2 region of surface antigen of viral hepatitis B screened by phage display

INTRODUCTIONHepatitis B virus (HBV) infection is the mostcommon viral infection in humans especially inChina and East Asia. At present, the most effi-cient method to control the disease is vaccinationof new-borns. Both blood derived vaccine and re-combinant vaccine are hepatitis B surface alltigen(HBsAg)-based and useful in the prevention of thedisease[1-3]. HBsAg could evoke protective humoralimmune response in vivo, but the immune memoryonly last for 5 years or so[3, 4]. How to des…     

A rat oocyte differentiation antigen (OA-1) detected by a monoclonal antibody

Cell surface antigenic changes associated with differentiation of the rat oocyte and early embryo have been demonstrated with a monoclonal antibody (anti-OA-1). Antigen is first detectable coincident with initiation of oocyte growth, is a constant feature of all growing oocytes and displays a redistribution during meiotic maturation. Following fertilization, antigen is detectable on the surface of the embryo through the four-cell stage. This first monospecific marker for the rat oocyte and embryo should prove useful in probing structure/function relationships in oocyte growth, meiotic maturation fertilization, and/or early embryonic development.     

Galactose-regulated expression of hepatitis B surface antigen by a recombinant yeast

Summary A yeast expressing the hepatitis B surface antigen (HBsAg) gene under the control of a regulated promoter was grown in batch and fed-batch modes. In batch fermentations, the addition of galactose caused the rapid production of HBsAg. Cells grown in fed-batch mode did not produce HBsAg unless the conditioned medium was replaced prior to induction.