Single chain antibody fragment display systems: a review / 生物工程学报

Single chain antibody fragment (scFv) is a small molecule composed of a variable region of heavy chain (VH) and a variable region of light chain (VL) of an antibody, and these two chains are connected by a flexible short peptide. scFv is the smallest functional fragment with complete antigen-binding activity, which contains both the antibody-recognizing site and the antigen-binding site. Compared with other antibodies, scFv has the advantages of small molecular weight, strong penetration, low immunogenicity, and easy expression. Currently, the most commonly used display systems for scFv mainly include the phage display system, ribosome display system, mRNA display system, yeast cell surface display system and mammalian cell display system. In recent years, with the development of scFv in the field of medicine, biology, and food safety, they have also attracted much attention in the sectors of biosynthesis and applied research. This review summarizes the advances of scFv display systems in recent years in order to facilitate scFv screening and application.

Phage antibody library technology in tumor therapy: a review / 生物工程学报

Tumor is a serious threat to human health. At present, surgical resection, chemoradiotherapy, targeted therapy and immunotherapy are the main therapeutic strategies. Monoclonal antibody has gradually become an indispensable drug type in the clinical treatment of cancer due to its high efficiency and low toxicity. Phage antibody library technology (PALT) is a novel monoclonal antibody preparation technique. The recombinant immunoglobulin variable region of heavy chain (VH)/variable region of light chain (VL) gene is integrated into the phage vector, and the antibody is expressed on the phage surface in the form of fusion protein to obtain a diverse antibody library. Through the process of adsorption-elution-amplification, the antibody library can be screened to obtain the antibody molecule with specific binding antigen as well as its gene sequence. PALT has the advantages of short antibody production cycle, strong plasticity of antibody structure, large antibody yield, high diversity and direct production of humanized antibodies. It has been used in screening tumor markers and preparation of antibody drugs for breast cancer, gastric cancer, lung cancer and liver cancer. This article reviews the recent progress and the application of PALT in tumor therapy.

BRAF V600E and MAP2K1 Mutations in Hairy Cell Leukemia and Splenic Marginal Zone Lymphoma Cases

No abstract available.

Comprehensiveness and programmatic vulnerability to stds/hiv/aids in primary care / La integralidad y vulnerabilidad programática de las ets/vih/sida en la atención básica / A integralidade e a vulnerabilidade programática às DST/HIV/AIDS na Atenção Básica




This study aimed to identify programmatic vulnerability to STDs/HIV/AIDS in primary health centers (PHCs). This is a descrip - tive and quantitative study carried out in the city of São Paulo. An online survey was applied (FormSUS platform), involving administrators from 442 PHCs in the city, with responses received from 328 of them (74.2%), of which 53.6% were nurses. At - tention was raised in relation to program - matic vulnerability in the PHCs regarding certain items of infrastructure, prevention, treatment, prenatal care and integration among services on STDs/HIV/AIDS care. It was concluded that in order to reach comprehensiveness of actions for HIV/ AIDS in primary health care, it is necessary to consider programmatic vulnerability, in addition to more investment and reor - ganization of services in a dialogue with the stakeholders (users, multidisciplinary teams, and managers, among others).


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Objetivo Fue identificar la vulnerabilidad programática de las Unidades Básicas de Salud con la atención a las ETS/VIH/SIDA. Método Es un estudio descriptivo con un abordaje cuantitativo llevado a cabo en el Municipio de San Pablo. Fue utilizado un formulario online (el FormSUS) con los gerentes de las 442 Unidades Básicas de Salud del Municipio de San Pablo. Participaran en el estudio 74.2% de los gerentes, estos 53.6% eran enfermeros. Resultados Se destaca la vulnerabilidad programática de las Unidades Básicas de Salud en relación a algunos elementos de la infraestructura, acciones de prevención, tratamiento, prenatal y la integración entre los servicios en la atención a las ETS/VIH/SIDA. Conclusión La construcción de tales marcadores constituye un instrumento, presentado en otro artículo, el cual puede ayudar a apoyar la captura de vulnerabilidades de las mujeres en relación a las ETS/VIH en el contexto de los servicios de Atención Primaria de Salud. Los marcadores constituyen importante herramienta para operacionalizar el concepto de vulnerabilidad en la Atención Primaria. Además, promueven procesos de trabajo inter e multidisciplinar e inter e multisectorial. La propuesta de un instrumento basado en dichos marcadores puede apoyar la captura de la vulnerabilidad de las mujeres en relación a las ETS/VIH. .

Objetivo Identificar a vulnerabilidade programática às DST/HIV/aids na Atenção Básica para o enfrentamento do HIV/Aids. Método Estudo descritivo, com abordagem quantitativa, realizado no Município de São Paulo (MSP). Utilizou-se formulário online (FormSUS), com gerentes das 442 Unidades Básicas de Saúde (UBS) do MSP. Participaram do estudo 74,2% gerentes, dos quais 53,6% eram enfermeiros. Resultados Destaca-se a vulnerabilidade programática nas UBS com relação a alguns itens de infraestrutura, ações de prevenção, de tratamento, no pré-natal e de integração entre os serviços na atenção às DST/HIV/aids. Conclusão Para a efetivação da integralidade no enfrentamento do HIV/aids na Atenção Básica é necessário atentar para a vulnerabilidade programática, além de mais investimentos e reorganização dos serviços, num diálogo com os atores sociais envolvidos (usuários, equipe multiprofissional, gerentes, gestores, entre outros).


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Recent advances in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) was largely considered to be a disease of slow progression, standard treatment with Chlorambucil and having almost similar prognosis. With the introduction of molecular methods for understanding the disease pathophysiology in CLL there has been a remarkable change in the approach towards the disease. The variation in B-cell receptor response and immunoglobulin heavy chain variable region (IGHV) mutation, genetic aberration and defect in apoptosis and proliferation has had an impact on therapy initiation and prognosis. Early diagnosis of molecular variant is therefore necessary in CLL.

Immunoglobulin VH Chain Gene Analysis of Peripheral Blood IgM-Producing B Cells in Patients with Kawasaki Disease

PURPOSE: Kawasaki disease is a systemic vasculitis, and its etiology and pathogenesis are still not clear. Our study was undertaken to investigate the characteristics of the activation of B cells in the peripheral blood of Kawasaki disease (KD) patients and evidence of stimulation by superantigens. MATERIALS AND METHODS: Blood samples were obtained from three patients (2 males, 1 female) with KD, who were admitted to our Hospital, Seoul, Korea. The mean age was 1.2 years. Distribution of B cells was studied in the acute and subacute phases of KD patients. From the RNA of B cells, we obtained complementary DNA (cDNA) and performed polymerase chain reaction (PCR). To determine the oligoclonal expansion of immunoglobulin M (IgM) VH family, we cloned and sequenced the PCR products from each group and analyzed DNA. RESULTS: In the peripheral blood of acute phase patients, T cells were significantly decreased (p < 0.05), whereas B cells were significantly increased (p < 0.05). When the first PCR was done on the B cell chains, VH1 to VH6 were all found to be expressed. The number of micro gene clones obtained from 3 patients was 312, and they belonged to VH3, VH4 and VH5 family. M99686 germ line was most frequently used and the next most frequently used, were X92224/J, L21967 and L21964. A similar order was seen in patients. Among the clones, 20 sets of clones showed the same base sequence and this was frequent between VH2 and VH5. There was one set, which showed almost the same base sequence between different patients, and the homology was 99.5%. Twenty sets of clones that had the same base sequence showed high similarity to the germ line (94 - 100%). Among these, the clones that utilized the M99686 germ line were 4 sets which were most frequent. The 3-dimensional structure of one of these clones showed typical beta, sheet structure of immunoglobulin chains. CONCLUSION: The IgM transcripts expressed by the B cells in the peripheral blood of KD patients in the acute phase of the disease clearly showed an oligoclonal expansion, suggesting that KD is caused not by stimulation of a superantigen, but rather by a conventional antigen.

Immunoglobulin VH Chain Gene Analysis of Peripheral Blood IgM-Producing B Cells in Patients with Kawasaki Disease

PURPOSE: Kawasaki disease is a systemic vasculitis, and its etiology and pathogenesis are still not clear. Our study was undertaken to investigate the characteristics of the activation of B cells in the peripheral blood of Kawasaki disease (KD) patients and evidence of stimulation by superantigens. MATERIALS AND METHODS: Blood samples were obtained from three patients (2 males, 1 female) with KD, who were admitted to our Hospital, Seoul, Korea. The mean age was 1.2 years. Distribution of B cells was studied in the acute and subacute phases of KD patients. From the RNA of B cells, we obtained complementary DNA (cDNA) and performed polymerase chain reaction (PCR). To determine the oligoclonal expansion of immunoglobulin M (IgM) VH family, we cloned and sequenced the PCR products from each group and analyzed DNA. RESULTS: In the peripheral blood of acute phase patients, T cells were significantly decreased (p < 0.05), whereas B cells were significantly increased (p < 0.05). When the first PCR was done on the B cell chains, VH1 to VH6 were all found to be expressed. The number of micro gene clones obtained from 3 patients was 312, and they belonged to VH3, VH4 and VH5 family. M99686 germ line was most frequently used and the next most frequently used, were X92224/J, L21967 and L21964. A similar order was seen in patients. Among the clones, 20 sets of clones showed the same base sequence and this was frequent between VH2 and VH5. There was one set, which showed almost the same base sequence between different patients, and the homology was 99.5%. Twenty sets of clones that had the same base sequence showed high similarity to the germ line (94 - 100%). Among these, the clones that utilized the M99686 germ line were 4 sets which were most frequent. The 3-dimensional structure of one of these clones showed typical beta, sheet structure of immunoglobulin chains. CONCLUSION: The IgM transcripts expressed by the B cells in the peripheral blood of KD patients in the acute phase of the disease clearly showed an oligoclonal expansion, suggesting that KD is caused not by stimulation of a superantigen, but rather by a conventional antigen.

Human monoclonal single chain antibodies (HuScFv) that bind to the polymerase proteins of influenza A virus.

Current anti-influenza drugs target the viral neuraminidase or inhibit the function of the ion channel M2 protein. Not only is the supply of these drugs unlikely to meet the demand during a large influenza epidemic/ pandemic, but also has an emergence of drug resistant influenza virus variants been documented. Thus a new effective drug or antiviral alternative is required. The influenza virus RNA polymerase complex consists of nucleoproteins (NP) that bind to three polymerase subunits: two basic polymerases, PB1 and PB2, and an acidic polymerase (PA). These proteins play a pivotal role in the virus life cycle; thus they are potential targets for the development of new anti-influenza agents. In this study, we produced human monoclonal antibodies that bound to the influenza A polymerase proteins by using a human antibody phage display library. Complementary DNA was prepared from the total RNA of a highly pathogenic avian influenza (HPAI) virus: A/duck/Thailand/144/2005(H5N1). The cDNA synthesized from the total virus RNA was used as template for the amplification of the gene segments encoding the N-terminal halves of the PB1, PB2 and PA polymerase proteins which encompassed the biologically active portions of the respective proteins. The cDNA amplicons were individually cloned into appropriate vectors and the recombinant vectors were introduced into Escherichia coli bacteria. Transformed E. coli clones were selected, and induced to express the recombinant proteins. Individually purified proteins were used as antigens in bio-panning to select the phage clones displaying specific human monoclonal single chain variable fragments (HuScFv) from a human antibody phage display library constructed from Thai blood donors in our laboratory. The purified HuScFv that bound specifically to the recombinant polymerase proteins were prepared. The inhibitory effects on the biological functions of the respective polymerase proteins should be tested. We envisage the use of the HuScFv in their cell penetrating version (transbodies) as an alternative influenza therapeutic to current anti-virus drugs.

The C-terminal variable domain of LigB from Leptospira mediates binding to fibronectin

Adhesion through microbial surface components that recognize adhesive matrix molecules is an essential step in infection for most pathogenic bacteria. In this study, we report that LigB interacts with fibronectin (Fn) through its variable region. A possible role for LigB in bacterial attachment to host cells during the course of infection is supported by the following observations: (i) binding of the variable region of LigB to Madin-Darby canine kidney (MDCK) cells in a dose-dependent manner reduces the adhesion of Leptospira, (ii) inhibition of leptospiral attachment to Fn by the variable region of LigB, and (iii) decrease in binding of the variable region of LigB to the MDCK cells in the presence of Fn. Furthermore, we found a significant reduction in binding of the variable region of LigB to Fn using small interfering RNA (siRNA). Finally, the isothermal titration calorimetric results confirmed the interaction between the variable region of LigB and Fn. This is the first report to demonstrate that LigB binds to MDCK cells. In addition, the reduction of Fn expression in the MDCK cells, by siRNA, reduced the binding of LigB. Taken together, the data from the present study showed that LigB is a Fn-binding protein of pathogenic Leptospira spp. and may play a pivotal role in Leptospira-host interaction during the initial stage of infection.

Characterization of a novel anti-human TNF-alpha murine monoclonal antibody with high binding affinity and neutralizing activity

In order to develop an anti-human TNF-alpha mAb, mice were immunized with recombinant human TNF-alpha. A murine mAb, TSK114, which showed the highest binding activity for human TNF-alpha was selected and characterized. TSK114 specifically bound to human TNF-alpha without cross-reactivity with the homologous murine TNF-alpha and human TNF-beta TSK114 was found to be of IgG1 isotype with kappa light chain. The nucleotide sequences of the variable regions of TSK114 heavy and light chains were determined and analyzed for the usage of gene families for the variable (V), diversity (D), and joining (J) segments. Kinetic analysis of TSK114 binding to human TNF-alpha by surface plasmon resonance technique revealed a binding affinity (KD) of ~5.3 pM, which is about 1,000- and 100-fold higher than those of clinically relevant infliximab (Remicade) and adalimumab (Humira) mAbs, respectively. TSK114 neutralized human TNF-alpha-mediated cytotoxicity in proportion to the concentration, exhibiting about 4-fold greater efficiency than those of infliximab and adalimumab in WEHI 164 cells used as an in vitro model system. These results suggest that TSK114 has the potential to be developed into a therapeutic TNF-alpha-neutralizing antibody with picomolar affinity.

Intracellular Antibody Fragment Against Hepatitis B Virus X Protein Does Not Inhibit Viral Replication

Replication of the hepatitis B virus is suppressed by deficiency of the X protein. Although several molecules that block cellular targets of X protein reduce the production of hepatitis B virus progeny, the effect of a specific inhibitor of X protein on viral replication has not been investigated. To block X protein specifically, we adopted an intracellular expression approach using H7 single chain variable fragment (H7scFv), an antibody fragment against X protein. We previously demonstrated that cytoplasmic expression of H7scFv inhibits X protein-induced tumorigenicity and transactivation. In this study, intracellular H7scFv expression inhibits reporter gene transactivation but not viral replication determined by endogenous hepatitis B virus polymerase activity assay and real-time PCR. Our findings imply that intracellular expression of antibody fragment against X protein may not be an alternative therapeutic modality for inhibition of hepatitis B virus replication.

Production of human single-chain variable fragment (scFv) antibody specific for digoxin by ribosome display.

Ribosome display was applied in vitro to select single-chain variable fragment (scFv) antibody specific for digoxin from a human non-immune naive scFv library. A cell-free system was used to produce stable antibody-ribosome-mRNA (ARM) complexes to provide the linkage of genotype and phenotype, allowing simultaneous selection of a desired antibody together with its encoding mRNA. The mRNA was then recovered and amplified as DNA by reverse transcriptase-polymerase chain reaction (RT-PCR). Repeating the display cycle enriched the selected molecules, enabling rare species to be isolated. In this study, digoxin-binding segments were selected over four cycles of ARM display and the selected DNA was cloned and expressed as a single-chain variable fragment antibody (the best scFv, A3) in Escherichia coli. The affinity (equilibrium dissociation constant Kd) of digoxin was 8.3 x 10(-8) M for A3, which validated construction of the naïve library and the power of ribosome display lending to the evolution of functional characteristics, such as potency of leading candidate antibodies to provide therapeutic antibodies. A3 was purified using affinity chromatography and determined by Western blot. The results indicate that ribosome display technnique can be efficiently used to isolate specific antibody fragments from a naive library.

Mutation analysis of IgVH gene in B-cell chronic lymphocytic leukemia / 华中科技大学学报（医学）（英德文版）

The variable heavy chain region (VH) genes of 3 untreated patients with B-cell chronic lymphocytic leukemia (B-CLL) were cloned and analyzed. The VH family used was VH3-11, VH3-72 and VH3-33. More than 2% difference from the corresponding germline gene was detected in all the 3 obtained potential functional genes (average 16.7). Mutation pattern analysis indicated evidence of antigen selective pressure observed in 1 of 3 cases. Our findings suggested that the tumor cells originate from post-GC cells.

Genetic relationships among serogroup B: serotype 4 Neisseria meningitidis strains

We compared the results obtained by serotyping of PorB epitopes using an expanded panel of monoclonal antibodies (mAb) including mAb 7 and mAb 10, with results obtained by RFLP of rRNA genes (ribotyping). The purpose of this study was to assess the correlation between phenotypic- and genotypic- methods for typing N. meningitidis. The ribotypes obtained using ClaI or EcoRV endonucleases grouped the strains in seven and two different patterns, respectively. This additional characterization of PorB epitopes improved the correlation between these two methods of typing N. meningitidis

Sequence analysis of the V1-V2 hypervariable region of the HIV-1 envelope gene from pediatric patients

The presence of HIV-1 DNA sequence variants of pediatric AIDS patients was investigated in a two-stage polymerase chain reaction (PCR) using nested primers for the first and second (V1-V2) hypervariable regions of the proviral envelope (gp 120) gene (env1). Gel electrophoresis analysis yielded amplified DNA bands which exhibited length variations which were characteristic for each child. The PCR products were cloned and the resulting clones demonstrated inserts of different lengths in a patient and between patients. Analysis of five clones from two different patients at the level of DNA sequencing indicates an extreme heterogeneity in the V1-V2 region. DNA maximum similarity between all of the isolated clones ranged between 69 to 96 percent. Comparison between DNA sequences of the isolated clones and HIV-1 strains of other parts of the world was also performed. The highest percentage of similarity that was found with known HIV variants included the following strains: HIVADA, HIVJFL, HIVSW42, HIVSWB83, HIVTRA2, HIVWMJ22. The sequences also showed a high degree of similarity to the clade B virus HIVSF162. The analyzed HIV-1 sequences demonstrated the expected high degree of variation in the V1-V2 region of the envelope gene and in some cases that the variation between isolates from the same patient may exceed the variation between the individual clones and the reference HIV-1 strains