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Affinity Maturation

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Min Chen – One of the best experts on this subject based on the ideXlab platform.

  • Deficient for endoplasmic reticulum calcium sensors Stim1 and Stim2 affects aberrant antibody Affinity Maturation in B cells
    Oncotarget, 2016
    Co-Authors: Jianfeng Zhang, Chao Luan, Yu Hu, Min Chen

    Abstract:

    // Xuhua Mao 1 , Jianfeng Zhang 2 , Yue Han 3 , Chao Luan 3 , Yu Hu 3 , Zhimin Hao 3 , Min Chen 3 1 Department of Clinical Laboratory, Yixing People’s Hospital, China 2 Department of Preventive Health Care, the Second Affiliated Hospital of Southeast University, China 3 Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Institute of Dermatology, Chinese Academy of Medical Sciences, China Correspondence to: Min Chen, email: // Keywords : B cells, calcium influx, Stim1, Stim2, antibody Affinity, Immunology and Microbiology Section, Immune response, Immunity Received : June 01, 2016 Accepted : August 13, 2016 Published : August 27, 2016 Abstract Antigen specific B cells undergo a process termed Affinity Maturation in the germinal centers of secondary lymphoid organs where B cells with high Affinity receptors are selected to mature into antibody-producing cells or to the memory B cell pool. It is known that B cell antigen receptor (BCR) signaling plays pivotal role in this selection process. Calcium influx is an essential component of BCR signaling. The current report is to determine the effect of calcium influx on antibody Affinity Maturation. In our studies, mice deficient for both endoplasmic reticulum calciumsensor Stim1 and Stim2 was immunized with T-cell dependent and independent antigens. Antibody Affinity was measured by ELISA. We demonstrated that Stim1 &Stim2 deficient B cells exhibit accelerated pace of Affinity Maturation compared to wild type controls while the overall antibody production was not dramatically impaired to T-independent antigen immunization. In conclusion, calcium influx plays an important role in antibody Affinity Maturation in humoral immune responses. The knowledge can be used in manipulate humoral immune response for the design of effective vaccines.

  • Deficient for endoplasmic reticulum calcium sensors Stim1 and Stim2 affects aberrant antibody Affinity Maturation in B cells
    Oncotarget, 2016
    Co-Authors: Jianfeng Zhang, Chao Luan, Yu Hu, Min Chen

    Abstract:

    // Xuhua Mao 1 , Jianfeng Zhang 2 , Yue Han 3 , Chao Luan 3 , Yu Hu 3 , Zhimin Hao 3 , Min Chen 3 1 Department of Clinical Laboratory, Yixing People’s Hospital, China 2 Department of Preventive Health Care, the Second Affiliated Hospital of Southeast University, China 3 Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Institute of Dermatology, Chinese Academy of Medical Sciences, China Correspondence to: Min Chen, email: // Keywords : B cells, calcium influx, Stim1, Stim2, antibody Affinity, Immunology and Microbiology Section, Immune response, Immunity Received : June 01, 2016 Accepted : August 13, 2016 Published : August 27, 2016 Abstract Antigen specific B cells undergo a process termed Affinity Maturation in the germinal centers of secondary lymphoid organs where B cells with high Affinity receptors are selected to mature into antibody-producing cells or to the memory B cell pool. It is known that B cell antigen receptor (BCR) signaling plays pivotal role in this selection process. Calcium influx is an essential component of BCR signaling. The current report is to determine the effect of calcium influx on antibody Affinity Maturation. In our studies, mice deficient for both endoplasmic reticulum calciumsensor Stim1 and Stim2 was immunized with T-cell dependent and independent antigens. Antibody Affinity was measured by ELISA. We demonstrated that Stim1 &Stim2 deficient B cells exhibit accelerated pace of Affinity Maturation compared to wild type controls while the overall antibody production was not dramatically impaired to T-independent antigen immunization. In conclusion, calcium influx plays an important role in antibody Affinity Maturation in humoral immune responses. The knowledge can be used in manipulate humoral immune response for the design of effective vaccines.

Haiying Hang – One of the best experts on this subject based on the ideXlab platform.

  • In vitro Affinity Maturation of antibody against membrane-bound GPCR molecules
    Applied Microbiology and Biotechnology, 2019
    Co-Authors: Jie Wang, Lili An, Yun Zhao, Cheng Zhang, Shengnan Li, Chen Ye, Shuqian Jing, Haiying Hang

    Abstract:

    G protein-coupled receptors (GPCRs), also known as seven-transmembrane domain receptors, are among the most important targets against which many small molecule drugs have been developed. However, only two antibody drugs targeting GPCRs have been approved for clinical use although many antibody drugs against non-GPCR protein targets have been successfully developed for various disease indications. One of the challenges for developing anti-GPCR drugs is the high difficulty to perform Affinity Maturation due to their insolubility in aqueous solutions. To address this issue, CHO cell display libraries of single-chain variable fragments (scFvs) and full-length antibodies were maturated directly against vesicle probes prepared from CHO cells displaying the endothelin A receptor (ETaR) GPCR. The probe in the vesicle form ensures the physiological conformation and functional activity of the protein and avoids issues with membrane protein insolubility. The size of the vesicle had a clear effect on protein-ligand interaction; we used small-sized vesicles with low expression levels of GPCRs for the Affinity Maturation. Four rounds of Affinity Maturation combining vesicles as probes with the CHO cell display platform improved Affinity by 13.58-fold for scFvs and 5.05-fold for full-length antibodies. We expect that this method will not only be used for the Affinity Maturation of antibodies against GPCRs but will also be used to mature antibodies for other types of proteins where the conformation/activity of which depends on the proper membrane environment.

  • coupling recombinase mediated cassette exchange with somatic hypermutation for antibody Affinity Maturation in cho cells
    Biotechnology and Bioengineering, 2016
    Co-Authors: Chuan Chen, Yun Zhao, Nan Li, Haiying Hang

    Abstract:

    Heterologous expression of activation-induced cytidine deaminase (AID) can induce somatic hypermutation (SHM) for genes of interest in various cells, and several research groups (including ours) have successfully improved antibody Affinity in mammalian or chicken cells using AID-induced SHM. These Affinity Maturation systems are time-consuming and inefficient. In this study, we developed an antibody Affinity Maturation platform in Chinese hamster ovary (CHO) cells by coupling recombinase-mediated cassette exchange (RMCE) with SHM. Stable CHO cell clones containing a single copy puromycin resistance gene (PuroR) expression cassette flanked by recombination target sequences (FRT and loxP) being able to highly express a gene of interest placed in the cassette were developed. The PuroR gene was replaced with an antibody gene by RMCE, and the antibody was displayed on the cell surface. Cells displaying antibodies on their membrane were transfected with the AID gene, and mutations of the antibody gene were accumulated by AID-mediated hypermutation during cell proliferation followed by flow cytometric cell sorting for cells bearing antibody mutants with improved Affinity. Affinity improvements were detected after only one round of cell sorting and proliferation, mutant clones with 15-fold Affinity improvement were isolated within five rounds of Maturation (within 2 months). CHO cells are fast growing, stress-resistant and produce antibody with glycosylations suitable for therapy. Our antibody-evolution platform based on CHO cells makes antibody-Affinity Maturation more efficient and is especially convenient for therapeutic antibody Affinity improvement. Biotechnol. Bioeng. 2015;9999: 1–14. © 2015 Wiley Periodicals, Inc.

  • Coupling recombinase-mediated cassette exchange with somatic hypermutation for antibody Affinity Maturation in CHO cells.
    Biotechnology and bioengineering, 2015
    Co-Authors: Chuan Chen, Yun Zhao, Haiying Hang

    Abstract:

    Heterologous expression of activation-induced cytidine deaminase (AID) can induce somatic hypermutation (SHM) for genes of interest in various cells, and several research groups (including ours) have successfully improved antibody Affinity in mammalian or chicken cells using AID-induced SHM. These Affinity Maturation systems are time-consuming and inefficient. In this study, we developed an antibody Affinity Maturation platform in Chinese hamster ovary (CHO) cells by coupling recombinase-mediated cassette exchange (RMCE) with SHM. Stable CHO cell clones containing a single copy puromycin resistance gene (PuroR) expression cassette flanked by recombination target sequences (FRT and loxP) being able to highly express a gene of interest placed in the cassette were developed. The PuroR gene was replaced with an antibody gene by RMCE, and the antibody was displayed on the cell surface. Cells displaying antibodies on their membrane were transfected with the AID gene, and mutations of the antibody gene were accumulated by AID-mediated hypermutation during cell proliferation followed by flow cytometric cell sorting for cells bearing antibody mutants with improved Affinity. Affinity improvements were detected after only one round of cell sorting and proliferation, mutant clones with 15-fold Affinity improvement were isolated within five rounds of Maturation (within 2 months). CHO cells are fast growing, stress-resistant and produce antibody with glycosylations suitable for therapy. Our antibody-evolution platform based on CHO cells makes antibody-Affinity Maturation more efficient and is especially convenient for therapeutic antibody Affinity improvement.

Tanja Lövgren – One of the best experts on this subject based on the ideXlab platform.

  • synthetic single framework antibody library integrated with rapid Affinity Maturation by vl shuffling
    Protein Engineering Design & Selection, 2011
    Co-Authors: Eeva-christine Brockmann, Sultana Akter, T. Savukoski, Tuomas Huovinen, A. Lehmusvuori, Janne Leivo, O. Saavalainen, Alex Azhayev, Tanja Lövgren

    Abstract:

    Affinity Maturation is often applied to improve the properties of antibodies isolated from universal antibody libraries in vitro. A synthetic human scFv antibody library was constructed in single immunoglobulin framework to enable rapid Affinity Maturation by updated Kunkel’s mutagenesis. The initial diversity was generated predominantly in the V(H) domain combined with only 36 V(L) domain variants yielding 3 × 10(10) unique members in the phage-displayed library. After three rounds of panning the enriched V(H) genes from the primary library selections against lysozyme were incorporated into a ready-made circular single-stranded Affinity Maturation library containing 7 × 10(8) V(L) gene variants. Several unique antibodies with 0.8-10 nM (K(d), dissociation constant) affinities against lysozyme were found after panning from the Affinity Maturation library, contrasted by only one anti-lysozyme scFv clone with K(d) <20 nM among the clones panned from the primary universal library. The presented single-framework strategy provides a way to convey significant amount of functional V(H) domain diversity to Affinity Maturation without bimolecular ligation leading to a diverse set of antibodies with binding affinities in the low nanomolar range.

  • Synthetic single-framework antibody library integrated with rapid Affinity Maturation by VL shuffling
    Protein Engineering Design and Selection, 2011
    Co-Authors: Eeva-christine Brockmann, Sultana Akter, Shahriar Akter, T. Savukoski, Tuomas Huovinen, A. Lehmusvuori, Janne Leivo, O. Saavalainen, Alex Azhayev, Tanja Lövgren, Jukka Hellman

    Abstract:

    Affinity Maturation is often applied to improve the properties of antibodies isolated from universal antibody libraries in vitro. A synthetic human scFv antibody library was constructed in single immunoglobulin framework to enable rapid Affinity Maturation by updated Kunkel’s mutagenesis. The initial diversity was generated predominantly in the V(H) domain combined with only 36 V(L) domain variants yielding 3 × 10(10) unique members in the phage-displayed library. After three rounds of panning the enriched V(H) genes from the primary library selections against lysozyme were incorporated into a ready-made circular single-stranded Affinity Maturation library containing 7 × 10(8) V(L) gene variants. Several unique antibodies with 0.8-10 nM (K(d), dissociation constant) affinities against lysozyme were found after panning from the Affinity Maturation library, contrasted by only one anti-lysozyme scFv clone with K(d)