The Experts below are selected from a list of 1506 Experts worldwide ranked by ideXlab platform
Lee R. Swem - One of the best experts on this subject based on the ideXlab platform.
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In vivo antigen-driven plasmablast enrichment in combination with antigen-specific cell sorting to facilitate the isolation of rare monoclonal antibodies from human B cells
Nature Protocols, 2014Co-Authors: Zhonghua Lin, Ning Chai, Dhaya Seshasayee, Wyne P. Lee, Mercedesz Balazs, Nancy Y Chiang, Gerald Nakamura, Lee R. SwemAbstract:The authors of this Protocol describe a system wherein human antigen-specific antibody secreting plasmablasts are enriched in vivo in a Mouse host. The enriched plasmablasts can then be sorted by flow cytometry for subsequent IgG expression. The ability to rapidly generate large panels of antigen-specific human antibodies in a rodent would enable the efficient discovery of novel therapeutically useful antibodies. We have developed a system wherein human antigen-specific antibody–secreting plasmablasts can be enriched in vivo , in a severe combined immunodeficient (SCID)/Beige Mouse host. The antigen-specific plasmablasts can then be sorted by flow cytometry, enabling single-cell cloning and expression of fully human immunoglobulin-G. By using this technique, we have generated four broadly reactive anti–influenza A antibodies. Therefore, the method described here is useful for the identification of rare functional antibodies. This protocol takes ∼1 month to complete, from the time of human vaccination to the cloning of heavy- and light-chain genes. For additional small-scale transient expression, purification and binding analysis, the protocol would take an additional month.
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In vivo antigen-driven plasmablast enrichment in combination with antigen-specific cell sorting to facilitate the isolation of rare monoclonal antibodies from human B cells
Nature protocols, 2014Co-Authors: Zhonghua Lin, Nan Chiang, Ning Chai, Dhaya Seshasayee, Wyne P. Lee, Mercedesz Balazs, Gerald R. Nakamura, Lee R. SwemAbstract:The ability to rapidly generate large panels of antigen-specific human antibodies in a rodent would enable the efficient discovery of novel therapeutically useful antibodies. We have developed a system wherein human antigen-specific antibody-secreting plasmablasts can be enriched in vivo, in a severe combined immunodeficient (SCID)/Beige Mouse host. The antigen-specific plasmablasts can then be sorted by flow cytometry, enabling single-cell cloning and expression of fully human immunoglobulin-G. By using this technique, we have generated four broadly reactive anti-influenza A antibodies. Therefore, the method described here is useful for the identification of rare functional antibodies. This protocol takes ∼1 month to complete, from the time of human vaccination to the cloning of heavy- and light-chain genes. For additional small-scale transient expression, purification and binding analysis, the protocol would take an additional month.
Zhonghua Lin - One of the best experts on this subject based on the ideXlab platform.
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In vivo antigen-driven plasmablast enrichment in combination with antigen-specific cell sorting to facilitate the isolation of rare monoclonal antibodies from human B cells
Nature Protocols, 2014Co-Authors: Zhonghua Lin, Ning Chai, Dhaya Seshasayee, Wyne P. Lee, Mercedesz Balazs, Nancy Y Chiang, Gerald Nakamura, Lee R. SwemAbstract:The authors of this Protocol describe a system wherein human antigen-specific antibody secreting plasmablasts are enriched in vivo in a Mouse host. The enriched plasmablasts can then be sorted by flow cytometry for subsequent IgG expression. The ability to rapidly generate large panels of antigen-specific human antibodies in a rodent would enable the efficient discovery of novel therapeutically useful antibodies. We have developed a system wherein human antigen-specific antibody–secreting plasmablasts can be enriched in vivo , in a severe combined immunodeficient (SCID)/Beige Mouse host. The antigen-specific plasmablasts can then be sorted by flow cytometry, enabling single-cell cloning and expression of fully human immunoglobulin-G. By using this technique, we have generated four broadly reactive anti–influenza A antibodies. Therefore, the method described here is useful for the identification of rare functional antibodies. This protocol takes ∼1 month to complete, from the time of human vaccination to the cloning of heavy- and light-chain genes. For additional small-scale transient expression, purification and binding analysis, the protocol would take an additional month.
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In vivo antigen-driven plasmablast enrichment in combination with antigen-specific cell sorting to facilitate the isolation of rare monoclonal antibodies from human B cells
Nature protocols, 2014Co-Authors: Zhonghua Lin, Nan Chiang, Ning Chai, Dhaya Seshasayee, Wyne P. Lee, Mercedesz Balazs, Gerald R. Nakamura, Lee R. SwemAbstract:The ability to rapidly generate large panels of antigen-specific human antibodies in a rodent would enable the efficient discovery of novel therapeutically useful antibodies. We have developed a system wherein human antigen-specific antibody-secreting plasmablasts can be enriched in vivo, in a severe combined immunodeficient (SCID)/Beige Mouse host. The antigen-specific plasmablasts can then be sorted by flow cytometry, enabling single-cell cloning and expression of fully human immunoglobulin-G. By using this technique, we have generated four broadly reactive anti-influenza A antibodies. Therefore, the method described here is useful for the identification of rare functional antibodies. This protocol takes ∼1 month to complete, from the time of human vaccination to the cloning of heavy- and light-chain genes. For additional small-scale transient expression, purification and binding analysis, the protocol would take an additional month.
Renier J Brentjens - One of the best experts on this subject based on the ideXlab platform.
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393 engineering armored t cell receptor mimic tcrm chimeric antigen receptor car t cells specific for the intracellular protein wilms tumor 1 wt1 for treatment of hematologic and solid malignancies
Molecular Therapy, 2016Co-Authors: Sarwish Rafiq, Terence J Purdon, Tao Dao, Chen Liu, David A Scheinberg, Renier J BrentjensAbstract:Adoptive therapy with chimeric antigen receptor (CAR) T cells specific for CD19 is clinically successful in a limited set of leukemias and most CARs studied are targeted against external antigens. Wilms Tumor Antigen 1 (WT1) protein (WT1) is an intracellular antigen overexpressed in many cancers, including leukemias and solid malignancies and is thus an appealing, broadly applicable target. We have engineered the first armored T cell receptor-mimic (TCRm) CAR against WT1. Derived from the ESK1 antibody, the second generation CAR, WT1-28z, is reactive with the RMFPNAPYL peptide of WT1 that is processed and presented on the surface of cells in the context of HLA-A*02:01. WT1-28z was further modified to secrete human IL-12 cytokine, thus creating the armored CAR WT1-28z/IL-12. T cells expressing WT1-28z or WT1-28z/IL-12 are cytotoxic against a range of both hematological and solid tumors. Importantly, both WT1-directed T cells are specific for the WT1-HLA-A*02:01 complex and are not reactive against cells that do not express both HLA-A*02:01 and WT1. In established SCID/Beige Mouse models of either acute leukemia or ovarian cancer, one dose of WT1-28z T cells prolongs survival of mice over untreated or irrelevant antigen-specific CAR T cell treated mice. Furthermore, one dose of the armored WT1-28z/IL12 CAR T cells further significantly prolongs survival of mice in both models over WT1-28z CAR T cell treated mice, with a subset of mice whose disease was eradicated. The armored TCRm CAR T cells against WT1 are effective in eradicating disease in both hematologic and solid tumors and may hold great clinical potential to expand on the success of CAR T cell therapy.
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CD19 Targeted Cord Blood Derived T Cells for Cancer Immunotherapy.
Blood, 2010Co-Authors: Hollie J. Pegram, Vincent Larussa, Renier J BrentjensAbstract:Abstract 3767 Transplantation of unrelated umbilical cord blood (CB) derived stem cells is often used to treat adult patients with B-cell acute lymphoblastic leukemia (B-ALL). However, many patients relapse and overall prognosis is poor. We hypothesize that additional therapy involving adoptive transfer of CB derived T cells modified to express a CD19-specific chimeric antigen receptor (CAR) could improve patient outcome following allogeneic CB transplant. To this end, we have previously demonstrated that human T cells which express the anti-CD19 19–28ζ CAR, containing the signaling domains of the co-stimulatory CD28 receptor and CD3ζ chain, effectively eradicate CD19 + tumors both in vitro as well as in vivo in SCID-Beige mice. Herein, we demonstrate the ability to effectively isolate and expand T cells from CB samples using magnetic beads coated with agonistic CD3 and CD28 antibodies (Invitrogen) and comparing subsequent T cell expansions in in vitro cultures with varying additions of exogenous stimulatory cytokines, including IL-2, a combination of IL-2 and IL-7, IL-12 or IL-15. We demonstrate that in vitro culture in the context of exogenous IL-12 (10 ng/ml) resulted in optimal expansion of CB T cells (150-fold). In addition, expansion of T cells in the context of exogenous IL-12 resulted in a favorable phenotype for adoptive cell transfer, with T cells expressing high levels of Granzyme B and Perforin while retaining a “memory” phenotype as assessed by persistent expression of CD62L. This combination of cytotoxic and memory phenotype is optimal for adoptive cell therapy. T cells expanded in this manner were subsequently efficiently retrovirally transduced to express the 19–28ζ CAR. The resulting CD19-specific CB derived T cells were able to specifically lyse CD19 + tumor targets as assessed by standard 51 Cr release cytotoxicity assays. We are currently investigating the in vivo anti-tumor function of these modified cells in our previously established preclinical SCID-Beige Mouse tumor model and ultimately plan to conduct a Phase 1 clinical trial with these modified T cells in patients with B-ALL undergoing CB transplant. Disclosures: No relevant conflicts of interest to declare.
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Virus Specific T-Lymphocytes Genetically Modified to Target the CD19 Antigen Eradicates Systemic Lymphoma In Mice
Blood, 2010Co-Authors: Kevin J. Curran, Renier J Brentjens, Michel Sadelain, Clare Taylor, Ekaterina Doubrovina, Xiuyan Wang, Richard J. O'reilly, Nancy A. Kernan, Isabelle RiviereAbstract:Abstract 2092 Human T-cells can be genetically modified to target tumor antigens through tumor antigen-specific artificial T-cell receptors termed chimeric antigen receptors (CARs). To provide a therapeutic option for patients with relapsed leukemia following allogeneic stem cell transplant (allo-SCT) we have developed a novel immunotherapy utilizing donor derived virus specific cytotoxic T-lymphocytes genetically modified to target the CD19 antigen expressed on most B-cell acute lymphoblastic leukemias (B-ALL). We have previously demonstrated that donor T-cells modified to express a CAR specific to the B-cell antigen CD19, termed 19–28z, traffic to systemic sites of tumor and successfully eradicate human CD19 + tumors in a SCID-Beige Mouse model. This therapy is currently under clinical investigation using autologous T-cells for adults with chronic lymphocytic leukemia (CLL) and B-ALL (BB-IND 13266). However, in the setting of allo-SCT, a lymphocyte infusion of genetically modified donor T-cells has the potential to cause graft versus host disease (GVHD). In our center9s experience with infusions of donor derived EBV-CTLs and EBV-CTLs derived from third party donors for treatment of EBV associated lymphoma we have noted no alloreactivity or development of GVHD in the recipient. Furthermore, we and others have shown EBV-CTLs have long term persistence following adoptive transfer which may enhance the anti-tumor efficacy of genetically modified T-lymphocytes. To this end we postulate the therapeutic use of infusions of donor derived EBV-CTLs genetically modified to target the CD19 antigen in patients with relapsed leukemia post allo-SCT. To investigate the ability of EBV-CTLs to be genetically modified to express our anti-CD19 CAR (19-28z) via gammaretroviral vector gene transfer we tested 3 established EBV-CTL donor cell lines. We compared EBV-CTL activation using autologous EBV B-cell lymphoblastoid cell lines (EBV-BLCL), beads coated with agonistic CD3 + CD28 monoclonal antibodies (Invitrogen Carlsbad, CA), or a combination of BLCL + beads. Transduction efficiency ranged from 25–75% and was consistently higher in the EBV-CTL groups activated using EBV-BLCL alone. In standard 51 Cr release cytotoxicity assay 19–28z + EBV-CTLs exhibited specific cytotoxicity against the CD19 + human tumor cell lines BA-25 (B-ALL), Raji (Burkitt 9s lymphoma) and the Mouse thymoma cell line EL4 modified to express the human CD19 antigen (EL4-hCD19 + ). In contrast the untransduced EBV-CTLs failed to lyse these targets. However, both 19–28z + EBV-CTLs and the untransduced EBV-CTLs retained the ability to specifically lyse autologous BLCL but not autologous PHA-blasts showing retained EBV specificity. Finally we tested the ability of 19–28z + EBV-CTLs to eradicate established systemic Raji tumor in our SCID-Beige model of disease. Mice were injected with 5×10 5 Raji-eGFP-fire fly luciferase (Raji-eGFP-FFLuc) tumor cells via tail vein injection six days prior to T-lymphocyte injection. Established tumor was confirmed using bioluminescence imaging (BLI) prior to T-lymphocyte infusion. Mice were treated via tail vein injection with 7.5 × 10 6 19–28z + EBV-CTL (n = 5) or control EBV-CTLs (n= 4). Control mice all died of systemic disease ( 100 days). These results validate the therapeutic potential of tumor targeted genetically modified EBV-specific T-lymphocytes which may provide a therapeutic option for patients with relapsed CD19+ B-ALL following allo-SCT. Disclosures: No relevant conflicts of interest to declare.
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Abstract B255: Discovery and validation of a novel class of small molecule inhibitors of the CDC7 kinase: Modulation of tumor cell growth in vitro and in vivo
Therapeutic Agents - Small Molecule Kinase Inhibitors, 2009Co-Authors: Mark G. Frattini, Renier J Brentjens, David Shum, Kristen M. O'dwyer, Peter Maslak, Mark L. Heaney, Joseph G. Jurcic, Hakim Djaballah, Thomas J. KellyAbstract:High throughput screening of compounds comprising the Memorial Sloan Kettering chemical library resulted in several confirmed hits against the recombinant Cdc7:Dbf4 heterodimeric kinase, a key regulator in the initiation of DNA replication and the G1 to S phase transition. Chemoinformatic analysis of the hits revealed an enrichment in one chemical cluster made up of several naturally occurring compounds, of which the most potent compound, CKI‐7, was selected for further investigation. First, CKI‐7 was found to be a non competitive inhibitor for ATP and prompted us to prolife it against a panel of 200 known kinases in order to assess its selectivity profile. The results were as predicted and very few kinases were specifically affected. Second, CKI‐7 cytotoxic activity was assessed against a panel of well established cancer cell lines representing both hematopoietic and solid tumor malignancies as well as against a panel of primary hematopoietic cells derived from leukemia patients (both chemotherapy naive and relapsed/refractory samples) and was found to be a very effective agent with potencies in the low nanomolar range. Subsequent studies using an isogenic pair of cell lines with one over expressing the Bcl_xL anti‐apoptotic protein further confirmed the induction of the intrinsic apoptotic pathway via caspase‐3 activation in the absence and attenuation of the activity in the presence of Bcl_xL. This was further demonstrated through standard cell cycle synchronization studies revealing that exposure to the Cdc7 inhibitor results in an S phase arrest, cell cycle dependent caspase‐3 activation, and apoptotic cell death. This cell death is the direct result of Cdc7 kinase inhibition by CKI‐7 as demonstrated using a Cdc7 substrate biomarker assay. Third, the physicochemical properties of this class of naturally occurring compounds also prompted us to investigate their effect on several multidrug resistence (MDR) over‐expressing cell lines. We found that CKI‐7 was not a substrate for the efflux pumps demonstrating that this novel compound can overcome a major mechanism of chemotherapy resistence in human tumor cells. Based of the above observations, in vivo dose‐dependent anti‐tumor activity of CKI‐7 was subsequently demonstrated in a SCID‐Beige Mouse systemic tumor model utilizing a recently isolated Philadelphia chromosome positive acute lymphoblastic leukemia cell line (PhALL3.1) in addition to melanoma and non small cell lung cancer xenograft models. Taken together, our data confirm that Cdc7 is a new promising target for cancer therapy, and that the newly discovered inhibitor CKI‐7, a naturally occurring selective small molecule inhibitor of this enzyme, is an equally promising novel cancer therapeutic agent. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B255.
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Discovery and Validation of a Novel Class of Small Molecule Inhibitors of the CDC7 Kinase: Modulation of Tumor Cell Growth in Vitro and In Vivo.
Blood, 2009Co-Authors: Mark G. Frattini, Renier J Brentjens, David Shum, Kristen M. O'dwyer, Peter Maslak, Mark L. Heaney, Joseph G. Jurcic, Hakim Djaballah, Thomas J. KellyAbstract:Abstract 3771 Poster Board III-707 High throughput screening of compounds comprising the Memorial Sloan Kettering chemical library resulted in several confirmed hits against the recombinant Cdc7:Dbf4 heterodimeric kinase, a key regulator in the initiation of DNA replication and the G1 to S phase transition. Chemoinformatic analysis of the hits revealed an enrichment in one chemical cluster made up of several naturally occurring compounds, of which the most potent compound, CKI-7, was selected for further investigation. First, CKI-7 was found to be a non competitive inhibitor for ATP and prompted us to prolife it against a panel of 200 known kinases in order to assess its selectivity profile. The results were as predicted and very few kinases were specifically affected. Second, CKI-7 cytotoxic activity was assessed against a panel of well established cancer cell lines representing both hematopoietic and solid tumor malignancies as well as against a panel of primary hematopoietic cells derived from leukemia patients (both chemotherapy naive and relapsed/refractory samples) and was found to be a very effective agent with potencies in the low nanomolar range. Subsequent studies using an isogenic pair of cell lines with one over expressing the Bcl\_xL anti-apoptotic protein further confirmed the induction of the intrinsic apoptotic pathway via caspase-3 activation in the absence and attenuation of the activity in the presence of Bcl\_xL. This was further demonstrated through standard cell cycle synchronization studies revealing that exposure to the Cdc7 inhibitor results in an S phase arrest, cell cycle dependent caspase-3 activation, and apoptotic cell death. This cell death is the direct result of Cdc7 kinase inhibition by CKI-7 as demonstrated using a Cdc7 substrate biomarker assay. Third, the physicochemical properties of this class of naturally occurring compounds also prompted us to investigate their effect on several multidrug resistence (MDR) over-expressing cell lines. We found that CKI-7 was not a substrate for the efflux pumps demonstrating that this novel compound can overcome a major mechanism of chemotherapy resistence in human tumor cells. Based of the above observations, in vivo dose-dependent anti-tumor activity of CKI-7 was subsequently demonstrated in a SCID-Beige Mouse systemic tumor model utilizing a recently isolated Philadelphia chromosome positive acute lymphoblastic leukemia cell line (PhALL3.1). Taken together, our data confirm that Cdc7 is a new promising target for cancer therapy, and that the newly discovered inhibitor CKI-7, a naturally occurring selective small molecule inhibitor of this enzyme, is an equally promising novel cancer therapeutic agent. Disclosures: No relevant conflicts of interest to declare.
Mercedesz Balazs - One of the best experts on this subject based on the ideXlab platform.
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In vivo antigen-driven plasmablast enrichment in combination with antigen-specific cell sorting to facilitate the isolation of rare monoclonal antibodies from human B cells
Nature Protocols, 2014Co-Authors: Zhonghua Lin, Ning Chai, Dhaya Seshasayee, Wyne P. Lee, Mercedesz Balazs, Nancy Y Chiang, Gerald Nakamura, Lee R. SwemAbstract:The authors of this Protocol describe a system wherein human antigen-specific antibody secreting plasmablasts are enriched in vivo in a Mouse host. The enriched plasmablasts can then be sorted by flow cytometry for subsequent IgG expression. The ability to rapidly generate large panels of antigen-specific human antibodies in a rodent would enable the efficient discovery of novel therapeutically useful antibodies. We have developed a system wherein human antigen-specific antibody–secreting plasmablasts can be enriched in vivo , in a severe combined immunodeficient (SCID)/Beige Mouse host. The antigen-specific plasmablasts can then be sorted by flow cytometry, enabling single-cell cloning and expression of fully human immunoglobulin-G. By using this technique, we have generated four broadly reactive anti–influenza A antibodies. Therefore, the method described here is useful for the identification of rare functional antibodies. This protocol takes ∼1 month to complete, from the time of human vaccination to the cloning of heavy- and light-chain genes. For additional small-scale transient expression, purification and binding analysis, the protocol would take an additional month.
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In vivo antigen-driven plasmablast enrichment in combination with antigen-specific cell sorting to facilitate the isolation of rare monoclonal antibodies from human B cells
Nature protocols, 2014Co-Authors: Zhonghua Lin, Nan Chiang, Ning Chai, Dhaya Seshasayee, Wyne P. Lee, Mercedesz Balazs, Gerald R. Nakamura, Lee R. SwemAbstract:The ability to rapidly generate large panels of antigen-specific human antibodies in a rodent would enable the efficient discovery of novel therapeutically useful antibodies. We have developed a system wherein human antigen-specific antibody-secreting plasmablasts can be enriched in vivo, in a severe combined immunodeficient (SCID)/Beige Mouse host. The antigen-specific plasmablasts can then be sorted by flow cytometry, enabling single-cell cloning and expression of fully human immunoglobulin-G. By using this technique, we have generated four broadly reactive anti-influenza A antibodies. Therefore, the method described here is useful for the identification of rare functional antibodies. This protocol takes ∼1 month to complete, from the time of human vaccination to the cloning of heavy- and light-chain genes. For additional small-scale transient expression, purification and binding analysis, the protocol would take an additional month.
Wyne P. Lee - One of the best experts on this subject based on the ideXlab platform.
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In vivo antigen-driven plasmablast enrichment in combination with antigen-specific cell sorting to facilitate the isolation of rare monoclonal antibodies from human B cells
Nature Protocols, 2014Co-Authors: Zhonghua Lin, Ning Chai, Dhaya Seshasayee, Wyne P. Lee, Mercedesz Balazs, Nancy Y Chiang, Gerald Nakamura, Lee R. SwemAbstract:The authors of this Protocol describe a system wherein human antigen-specific antibody secreting plasmablasts are enriched in vivo in a Mouse host. The enriched plasmablasts can then be sorted by flow cytometry for subsequent IgG expression. The ability to rapidly generate large panels of antigen-specific human antibodies in a rodent would enable the efficient discovery of novel therapeutically useful antibodies. We have developed a system wherein human antigen-specific antibody–secreting plasmablasts can be enriched in vivo , in a severe combined immunodeficient (SCID)/Beige Mouse host. The antigen-specific plasmablasts can then be sorted by flow cytometry, enabling single-cell cloning and expression of fully human immunoglobulin-G. By using this technique, we have generated four broadly reactive anti–influenza A antibodies. Therefore, the method described here is useful for the identification of rare functional antibodies. This protocol takes ∼1 month to complete, from the time of human vaccination to the cloning of heavy- and light-chain genes. For additional small-scale transient expression, purification and binding analysis, the protocol would take an additional month.
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In vivo antigen-driven plasmablast enrichment in combination with antigen-specific cell sorting to facilitate the isolation of rare monoclonal antibodies from human B cells
Nature protocols, 2014Co-Authors: Zhonghua Lin, Nan Chiang, Ning Chai, Dhaya Seshasayee, Wyne P. Lee, Mercedesz Balazs, Gerald R. Nakamura, Lee R. SwemAbstract:The ability to rapidly generate large panels of antigen-specific human antibodies in a rodent would enable the efficient discovery of novel therapeutically useful antibodies. We have developed a system wherein human antigen-specific antibody-secreting plasmablasts can be enriched in vivo, in a severe combined immunodeficient (SCID)/Beige Mouse host. The antigen-specific plasmablasts can then be sorted by flow cytometry, enabling single-cell cloning and expression of fully human immunoglobulin-G. By using this technique, we have generated four broadly reactive anti-influenza A antibodies. Therefore, the method described here is useful for the identification of rare functional antibodies. This protocol takes ∼1 month to complete, from the time of human vaccination to the cloning of heavy- and light-chain genes. For additional small-scale transient expression, purification and binding analysis, the protocol would take an additional month.