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1.
Front Immunol ; 15: 1340619, 2024.
Article in English | MEDLINE | ID: mdl-38711498

ABSTRACT

To design new CARs targeting hepatitis B virus (HBV), we isolated human monoclonal antibodies recognizing the HBV envelope proteins from single B cells of a patient with a resolved infection. HBV-specific memory B cells were isolated by incubating peripheral blood mononuclear cells with biotinylated hepatitis B surface antigen (HBsAg), followed by single-cell flow cytometry-based sorting of live, CD19+ IgG+ HBsAg+ cells. Amplification and sequencing of immunoglobulin genes from single memory B cells identified variable heavy and light chain sequences. Corresponding immunoglobulin chains were cloned into IgG1 expression vectors and expressed in mammalian cells. Two antibodies named 4D06 and 4D08 were found to be highly specific for HBsAg, recognized a conformational and a linear epitope, respectively, and showed broad reactivity and neutralization capacity against all major HBV genotypes. 4D06 and 4D08 variable chain fragments were cloned into a 2nd generation CAR format with CD28 and CD3zeta intracellular signaling domains. The new CAR constructs displayed a high functional avidity when expressed on primary human T cells. CAR-grafted T cells proved to be polyfunctional regarding cytokine secretion and killed HBV-positive target cells. Interestingly, background activation of the 4D08-CAR recognizing a linear instead of a conformational epitope was consistently low. In a preclinical model of chronic HBV infection, murine T cells grafted with the 4D06 and the 4D08 CAR showed on target activity indicated by a transient increase in serum transaminases, and a lower number of HBV-positive hepatocytes in the mice treated. This study demonstrates an efficient and fast approach to identifying pathogen-specific monoclonal human antibodies from small donor cell numbers for the subsequent generation of new CARs.


Subject(s)
Hepatitis B Surface Antigens , Hepatitis B virus , Humans , Hepatitis B virus/immunology , Hepatitis B virus/genetics , Animals , Mice , Hepatitis B Surface Antigens/immunology , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/metabolism , Antibodies, Monoclonal/immunology , Immunotherapy, Adoptive , Hepatitis B/immunology , Hepatitis B/virology , Broadly Neutralizing Antibodies/immunology , B-Lymphocytes/immunology , T-Lymphocytes/immunology
2.
Article in Chinese | WPRIM (Western Pacific) | ID: wpr-806846

ABSTRACT

Objective@#To amplify and identify monoclonal antibody genes from HIV-1-infected patients.@*Methods@#Single cell sorting was used to isolate antigen-specific single B cells. Sequence Identity Matrix and the international ImMunoGeneTics information system were used to analyze antibody variable region genes. Binding abilities were detected by enzyme linked immunosorbent assay. Neutralizing activities were tested by TZM-bl/pseudovirus assay.@*Results@#The heavy and light chain genes of four, seven, and eleven antibodies were amplified and sequenced from three HIV-1-infected patients, respectively. They were derived from various germline genes with flexible CDR3 lengths and somatic mutations. A1 and B3 antibodies bound to HIV-1 clade B, CRF01_AE, and CRF07_BC antigens. The half maximal inhibitory concentration values of A1 and B3 against MW965 virus were 0.04 μg/ml and 37.34 μg/ml.@*Conclusion@#In this study, we acquired a lot of monoclonal antibody genes and two HIV-1 monoclonal binding and neutralizing antibodies, which would provide basic data for further research on monoclonal antibody identification.

3.
Braz. J. Pharm. Sci. (Online) ; 54(spe): e01007, 2018. graf, ilus
Article in English | LILACS | ID: biblio-974431

ABSTRACT

The use of serum containing polyclonal antibodies from animals immunized with toxins marked the beginning of the application of antibody-based therapy in late nineteenth century. Advances in basic research led to the development of the hybridoma technology in 1975. Eleven years later, the first therapeutic monoclonal antibody (mAb) was approved, and since then, driven by technological advances, the development of mAbs has played a prominent role in the pharmaceutical industry. In this review, we present the developments to circumvent problems of safety and efficacy arising from the murine origin of the first mAbs and generate structures more similar to human antibodies. As of October 2017, there are 61 mAbs and 11 Fc-fusion proteins in clinical use. An overview of all mAbs currently approved is provided, showing the development of sophisticated mAbs formats that were engineered based on the challenges posed by therapeutic indications, including antibody-drug conjugates (ADC) and glycoengineered mAbs. In the field of immunotherapy, the use of immunomodulators, bispecific mAbs and CAR-T cells are highlighted. As an example of promising therapy to treat infectious diseases, we discuss the generation of neutralizing monoclonal-oligoclonal antibodies obtained from human B cells. Scientific and technological advances represent mAbs successful translation to the clinic


Subject(s)
Animals , Mice , Technological Development/classification , Antibodies , Antibodies, Monoclonal/analysis , Mice, Transgenic/classification , Immunotherapy/adverse effects
4.
Braz J Pharm Sci, v. 54, n. spe , 2018
Article in English | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-2610

ABSTRACT

The use of serum containing polyclonal antibodies from animals immunized with toxins marked the beginning of the application of antibody-based therapy in late nineteenth century. Advances in basic research led to the development of the hybridoma technology in 1975. Eleven years later, the first therapeutic monoclonal antibody (mAb) was approved, and since then, driven by technological advances, the development of mAbs has played a prominent role in the pharmaceutical industry. In this review, we present the developments to circumvent problems of safety and efficacy arising from the murine origin of the first mAbs and generate structures more similar to human antibodies. As of October 2017, there are 61 mAbs and 11 Fc-fusion proteins in clinical use. An overview of all mAbs currently approved is provided, showing the development of sophisticated mAbs formats that were engineered based on the challenges posed by therapeutic indications, including antibody-drug conjugates (ADC) and glycoengineered mAbs. In the field of immunotherapy, the use of immunomodulators, bispecific mAbs and CAR-T cells are highlighted. As an example of promising therapy to treat infectious diseases, we discuss the generation of neutralizing monoclonal-oligoclonal antibodies obtained from human B cells. Scientific and technological advances represent mAbs successful translation to the clinic.

5.
Clin Vaccine Immunol ; 24(5)2017 May.
Article in English | MEDLINE | ID: mdl-28356257

ABSTRACT

Analysis of monoclonal antibodies (MAbs) derived from single B cell cloning has been highly beneficial for antimicrobial immunotherapy, vaccine design, and advancing our understanding of pathogen-triggered effects on the human immunoglobulin repertoire. Sequencing of variable domains of single B cells, and characterization of binding and functional activities of MAbs derived from those sequences, provides in-depth insight not only into sites of susceptibility for antibody-mediated neutralization or opsonization of the pathogen but also into the dynamics of protective antibody evolution during infection. This information can be utilized to rapidly develop novel immunotherapies of completely human origin and provides a roadmap for structure-based vaccine design that aims to elicit similar protective antibody responses. Here, we summarize recent aspects of the single B cell cloning approach.


Subject(s)
Anti-Infective Agents/immunology , Antibodies, Monoclonal/immunology , B-Lymphocytes/immunology , Antibodies, Neutralizing/immunology , Clone Cells , Humans
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