Generation Of Human Monoclonal Antibodies Research Articles

38227 Specific and highly potent human monoclonal antibodies against SARS-CoV-2

ABSTRACT IMPACT: We devised a new method to produce highly potent SARS-CoV2-specific that can be used to treat severely ill patients with Covid-19. OBJECTIVES/GOALS: Neutralizing antibodies against SARS-CoV-2 are thought to offer the most immediate and effective treatment for those severely afflicted by Covid-19. We devised an approach for rapid and efficient generation of human monoclonal antibodies with neutralizing activity against SARS-CoV-2. METHODS/STUDY POPULATION: SARS-CoV-2 S1 spike protein-specific memory B cells were isolated from 12 subjects recovering from infection with that virus. Paired end single index sequencing was performed using up to 10,000 antigen-specific B cells per subject. Antigen-specific B cell clones were identified by unique diversity and joining gene V(D)J rearrangements and the CDR3 regions. VH and VL regions were cloned and the products expressed in 293T/17 cells to generate spike-specific human monoclonal antibodies. RESULTS/ANTICIPATED RESULTS: Forty-three human monoclonal antibodies were produced. Every monoclonal antibody so generated neutralized viruses pseudotyped with Spike protein of the Wuhan-1 strain. Eighteen monoclonal antibodies neutralized pseudotyped viruses with half-maximal inhibitory concentration (IC50s) between 1 pg/mL and 1 ng/mL (6.7 x 10E-15 M to 6.7 x 10E-12 M), exceeding by 10-100-fold the potency of previously reported anti-SARS-CoV-2-neutralizing monoclonal antibodies. Eight monoclonal antibodies neutralized viruses pseudotyped with mutant spike proteins previously identified in clinical isolates, including receptor binding domain mutants and the C-terminal D614G mutant with IC50<6.7 x10E-12M. DISCUSSION/SIGNIFICANCE OF FINDINGS: We show that SARS-CoV-2 evokes high affinity B cell responses. Some B cells produce antibodies that are broadly neutralizing; others produce strain-specific antibodies. However, antigenic variants that would potentially escape control by immunity or vaccination were nonetheless identified.

DSab-origin: a novel IGHD sensitive VDJ mapping method and its application on antibody response after influenza vaccination

BackgroundFunctional antibody genes are often assembled by VDJ recombination and then diversified by somatic hypermutation. Identifying the combination of sourcing germline genes is critical to understand the process of antibody maturation, which may facilitate the diagnostics and rapid generation of human monoclonal antibodies in therapeutics. Despite of successful efforts in V and J fragment assignment, method in D segment tracing remains weak for immunoglobulin heavy diversity (IGHD).ResultsIn this paper, we presented a D-sensitive mapping method called DSab-origin with accuracies around 90% in human monoclonal antibody data and average 95.8% in mouse data. Besides, DSab-origin achieved the best performance in holistic prediction of VDJ segments assignment comparing with other methods commonly used in simulation data. After that, an application example was explored on the antibody response based on a time-series antibody sequencing data after influenza vaccination. The result indicated that, despite the personal response among different donors, IGHV3–7 and IGHD4–17 were likely to be dominated gene segments in these three donors.ConclusionsThis work filled in a computational gap in D segment assignment for VDJ germline gene identification in antibody research. And it offered an application example of DSab-origin for studying the antibody maturation process after influenza vaccination.

Construction of Naive and Immune Human Fab Phage-Display Library.

This protocol describes the processes involved in the generation of human antibody libraries in Fab format. The antibody repertoire is derived from peripheral blood mononucleocytes focusing on different immunoglobulin isotypes. A two-step cloning process was used to generate a diverse human Fab library for subsequent selection by phage display. The method can be applied for the generation of both naive and immune antibody libraries. The naive repertoire allows for the library to be applied for the generation of human monoclonal antibodies against a broad range of target antigens making it a useful resource for antibody generation. However, the immune repertoire will be focused against target antigens from a particular disease. The protocol will focus on the generation of the library including the panning process.

The Cloning and Expression of Human Monoclonal Antibodies: Implications for Allergen Immunotherapy.

Allergic responses are dependent on the highly specific effector functions of IgE antibodies. Conversely, antibodies that block the activity of IgE can mediate tolerance to allergen. Technologies that harness the unparalleled specificity of antibody responses have revolutionized the way that we diagnose and treat human disease. This area of research continues to advance at a rapid pace and has had a significant impact on our understanding of allergic disease. This review will present an overview of humoral responses and provide an up-to-date summary of technologies used in the generation of human monoclonal antibodies. The impact that monoclonal antibodies have on allergic disease will be discussed, with a particular focus on allergen immunotherapy, which remains the only form of treatment that can modulate the underlying immune mechanisms and induce long-term clinical tolerance.

Analytic Vaccinology: Antibody-Driven Design of a Human Cytomegalovirus Subunit Vaccine.

Identification of the most relevant protective antigens has represented a considerable obstacle for the development of subunit vaccines against viral infections, including human cytomegalovirus (HCMV) infection. This chapter describes the method of analytic vaccinology, centered on the clonal analysis of human B cell response to HCMV, which represents an essential tool for assessing the impact of individual viral antigens in the antiviral antibody response. By providing key information on the immunogenicity and protective properties of the antibodies elicited by viral proteins, the analytic vaccinology method guides the selection of the most appropriate vaccine candidates. Here we discuss methodologies for the generation of human monoclonal antibodies from B cells of immune donors, antibody screening in in vitro assays of antigen binding and virus neutralization, and strategies of animal immunization useful for the preclinical evaluation of selected viral antigens. The approach of analytic vaccinology could be universally applied to the characterization of B-cell immune response against any virus of interest and ultimately used for vaccine development.

J-GRID and SATREPS programs in Thailand

Thailand-Japan Research Collaboration Center on Emerging and Re-emerging Infections (RCC-ERI) was established in 2005 as a research collaboration center cooperated by Research Institute for Microbial Diseases (RIMD), Osaka University and Department of Medical Sciences, Ministry of Public Health of Thailand. In addition, Mahidol-Osaka Center for Infectious Diseases (MOCID) was established in 2010, also as another research collaboration center cooperated by RIMD, Osaka University and Faculty of Tropical Medicine, Mahidol University. Epidemiological and basic studies on human pathogenic viruses have been conducted in RCC-ERI and MOCID. In this report, brief overviews of the research activities of both centers, as well as the phenotypic studies on the envelope glycoproteins of HIV-1 Thai strains that have been performed at RCC-ERI, are presented. SATREPS is a Japanese government program by the Japan Science and Technology Agency (JST) and the Japan International Cooperation Agency (JICA) that promotes international joint research targeting global issues. Our research group at RIMD have collaborated with the Ministry of Public Health - National Institute of Health (NIH) and Mahidol University in Thailand from 2008 as a four-years project. Our aim on this collaboration is to generate human monoclonal antibodies neutralizing infectious viral agents, hopefully to apply them to clinical field as "therapeutic antibodies". Here we introduce our trials in this project, especially on the generation of human monoclonal antibodies against dengue virus as well as against influenza virus that have been performed as collaboration between Thai and Japanese research groups.

Accessing of recombinant human monoclonal antibodies from patient libraries by eukaryotic ribosome display

What are effective antibodies and when do they arise to prevent or delay disease onset during a natural infection or in the course of vaccination? To address these questions at a molecular level requires longitudinal studies, capturing and analyzing the antibody repertoire at regular intervals following exposure or sero-conversion. Such studies require a method that allows the rapid generation and evaluation of monoclonal antibodies from relatively small volumes of blood. Here we describe an approach for rapidly generating human monoclonal antibodies in vitro by directly screening single-chain antibody repertories derived from donor peripheral blood mononuclear cells using ribosome display. Two single-chain antibody libraries were constructed using RNA extracted from peripheral blood mononuclear cells of two HIV-1 long-term non-progressor donors (K530 and M325). Both libraries were subjected to a single round of in vitro ribosome display for enrichment of human monoclonal antibodies against recombinant gp120(K530), derived from virus isolated from donor K530. This study has validated a novel, in vitro method for the rapid generation of human monoclonal antibodies. An antibody library could be constructed from as little as 3 μg of total RNA, the equivalent of 3-5 mL of human blood.

Neutralizing human monoclonal antibodies to severe acute respiratory syndrome coronavirus: target, mechanism of action, and therapeutic potential

SUMMARYThe emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS‐CoV) led to a rapid response not only to contain the outbreak but also to identify possible therapeutic interventions, including the generation of human monoclonal antibodies (hmAbs). hmAbs may be used therapeutically without the drawbacks of chimeric or animal Abs. Several different methods have been used to generate SARS‐CoV specific neutralizing hmAbs including the immunization of transgenic mice, cloning of small chain variable regions from naïve and convalescent patients, and the immortalization of convalescent B cells. Irrespective of the techniques used, the majority of hmAbs specifically reacted with the receptor binding domain (RBD) of the spike (S) protein and likely prevented receptor binding. However, several hmAbs that can bind to epitopes either within the RBD, located N terminal of the RBD or in the S2 domain, and neutralize the virus with or without inhibiting receptor binding have been identified. Therapeutic utility of hmAbs has been further elucidated through the identification of potential combinations of hmAbs that could neutralize viral variants including escape mutants selected using hmAbs. These results suggest that a cocktail of hmAbs that can bind to unique epitopes and have different mechanisms of action might be of clinical utility against SARS‐CoV infection, and indicate that a similar approach may be applied to treat other viral infections. Copyright © 2011 John Wiley & Sons, Ltd.

Hybridoma Technologies for Antibody Production

Hybridoma technology features effective usage of innate functions of both immune cells and cancers, allowing production of hybridoma cells, which continuously generate monoclonal antibodies specific to antigens of interest. For standard generation of hybridoma cells, B lymphocytes must be somatically fused with myeloma cells using various technologies. However, the methods generally do not necessarily result in selective fusion of target B lymphocytes with myeloma cells. To overcome this problem, we have developed a new hybridoma technology that involves preselection of B lymphocytes with target antigens based on immunoglobulin receptors and selective fusion of B cell-myeloma cell complexes with electrical pulses. The advanced methodology, termed B-cell targeting, multitargeting and stereospecific targeting, may be applicable to simultaneous production of monoclonal antibodies, selective production of stereospecific monoclonal antibodies, and also to efficient generation of human monoclonal antibodies for clinical purposes.

Generation of human monoclonal antibodies against Propionibacterium acnes by applying the phage display method to human peripheral blood mononuclear cells immunized in vitro

Propionibacterium acnes is a gram-positive, non-spore-forming, rod-shaped bacterium that is often detected in normal human skin flora. P. acnes has been associated with many diseases. In this study, we attempted to generate anti-P. acnes human monoclonal antibodies. A phage antibody library was first generated from human peripheral blood mononuclear cells immunized in vitro with P. acnes using the phage display method, and P. acnes-specific phage antibodies were obtained using solid phase panning. Antigen-specific variable region genes were then amplified and recombined into vectors expressing human IgG antibodies. The results indicated that the recombinant human IgG antibodies exhibited P. acnes-specific binding. This study demonstrates that the combined use of an in vitro immunization protocol and the phage display method enables the generation of human monoclonal antibodies against pathogenic bacteria and toxic antigens.

Longitudinal Determination of Hepatitis B Surface Antigen-Specific B Lymphocyte Frequency in Healthy High Responder Adults after Booster Vaccination

Objectives: The influence of booster vaccination on hepatitis B surface antigen (HBsAg)-specific B lymphocytes in humans has not been well characterized. Considering the low frequency of circulating B cells specific for HBsAg in vaccine high responder subjects, determination of this frequency at different time intervals after booster dose injection may provide invaluable information for evaluation of immune response to rHBsAg and identification of the most appropriate timing for isolation of specific B cells and generation of human monoclonal antibodies. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from 7 healthy high responder adults at 1, 2, 4, 8 and 16 weeks following administration of booster vaccination with an rHBsAg. The cells were transformed with Epstein-Barr virus and cultured at different cell densities over a feeder of human fetal foreskin fibroblasts. Following transformation, total hIg and HBsAg-specific antibody were screened in culture supernatant using ELISA, and primary frequency of specific B cells was calculated by limiting dilution assay based on Poisson analysis. Actual frequency was determined taking into consideration the percent of B cells in each PBMC population and efficiency of EBV transformation. Results: The mean frequencies of specific B cells after booster vaccination were found to be 1/13,462, 1/3,318, 1/5,224, 1/8,861 and 1/10,714 for the specified time intervals, respectively. Significant differences were observed between the frequencies of samples collected at all time intervals with the exception of week 1 versus weeks 8 and 16, week 2 versus week 4, and week 8 versus week 16. Conclusions: Our results may provide an indirect measure for immunological memory and may help optimize immunization strategies for novel vaccines and generate human monoclonal antibodies.

Recent advances in the generation of human monoclonal antibody

The use of monoclonal antibodies (mAbs) has now gained a niche as an epochal breakthrough in medicine. Engineered antibodies (Abs) currently account for over 30% of biopharmaceuticals in clinical trials. Several methods to generate human mAbs have evolved, such as (1) immortalization of antigen-specific human B cell hybridoma technology, (2) generation of chimeric and humanized antibody (Ab) from mouse Ab by genetic engineering, (3) acquisition of antigen-specific human B cells by the phage display method, and (4) development of transgenic mice for producing human mAbs. Besides these technologies, we have independently developed a method to generate human mAbs by combining the method of in vitro immunization using peripheral blood mononuclear cells and the phage display method. In this paper, we review the developments in these technologies for generating human mAbs.

Phage versus Phagemid Libraries for Generation of Human Monoclonal Antibodies

Non-immune (naı̈ve) phage antibody libraries have become an important source of antibodies for reagent, diagnostic, and therapeutic use. To date, reported naı̈ve libraries have been constructed in phagemid vectors as fusions to pIII, yielding primarily single copy (monovalent) display of antibody fragments. For this work, we subcloned the single chain Fv (scFv) gene repertoire from a naı̈ve phagemid antibody library into a true phage vector to create a multivalently displayed scFv phage library. Compared to monovalently displayed scFv, multivalent phage display resulted in improved efficiency of display as well as antibody selection. A greater number of antibodies were obtained and at earlier rounds of selection. Such increased efficiency allows the screening for binding antibodies after a single round of selection, greatly facilitating automation. Expression levels of antigen-binding scFv were also higher than from the phagemid library. In contrast, the affinities of scFv from the phage library were lower than from the phagemid library. This could be overcome by utilizing the scFv in a multivalent format, by affinity maturation, or by converting the library to monovalent display after the first round of selection.

Generation of heterohybridomas secreting human immunoglobulins; pokeweed mitogen prestimulation is highly effective but phytohemagglutinin drives most B cells into apoptosis

Human monoclonal antibodies have commonly been generated by forming hybridomas of stable lymphoblastoid cell lines and Epstein–Barr virus (EBV)-transformed human B cells that have been exposed to phytohaemagglutin (PHA)-stimulated T cells. However, this technique has predominantly given rise to IgM- but very rarely IgG- or IgA-producing clones. We now report that, regardless of prior EBV infection, pokeweed mitogen (PWM) stimulation of human peripheral blood mononuclear cells (PBMCs) generated much higher numbers of IgM-, IgA- and IgG-producing B cells than did stimulation with PHA. Fusion of PWM-stimulated PBMCs with a mouse myeloma cell line also gave rise to 7- to 12-fold higher numbers of IgG- and IgA-producing heterohybridomas than PBMCs that were prestimulated with PHA. Judged by Annexin V staining, stimulation with PHA induced a very high rate of B cell apoptosis within 24 h, whereas, even after 7 days, PWM stimulation only induced marginal B cell apoptosis. This should explain why PHA is much inferior to PWM in stimulating immunoglobulin (Ig) production in vitro and in generating immunoglobulin-producing human B cell hybridomas. It is concluded that PWM stimulation may greatly facilitate the generation of human monoclonal antibodies of all isotypes.

A human myeloma cell line suitable for the generation of human monoclonal antibodies.

Ever since monoclonal antibodies were produced in 1975 with mouse myeloma cells there has been interest in developing human myeloma cultures for the production of monoclonal antibodies. However, despite multiple attempts, no human myeloma line suitable for hybridoma production has been described. Here we report the derivation of a hypoxanthine-aminopterin-thymidine-sensitive and ouabain-resistant human myeloma cell line (Karpas 707H) that contains unique genetic markers. We show that this line is useful for the generation of stable human hybridomas. It can easily be fused with ouabain-sensitive Epstein-Barr virus-transformed cells as well as with fresh tonsil and blood lymphocytes, giving rise to stable hybrids that continuously secrete very large quantities of human immunoglobulins. The derived hybrids do not lose immunoglobulin secretion over many months of continuous growth. The availability of this cell line should enable the in vitro immortalization of human antibody-producing B cells that are formed in vivo. The monoclonal antibodies produced may have advantages in immunotherapy.

Synovial tissue B-lymphocytes as source for the generation of human monoclonal antibodies

Synovial tissue B-lymphocytes as source for the generation of human monoclonal antibodies

Synovial tissue B-lymphocytes as source for the generation of human monoclonal antibodies

Synovial tissue B-lymphocytes as source for the generation of human monoclonal antibodies

Incorporation of T cell counterparts in the fusion partners for generation of human monoclonal antibodies from Staphylococcus aureus stimulated B lymphocytes

B cell growth and differentiation into immunoglobulin secreting cells is controlled by various cytokines and cell to cell contact with T cells. Fusion partner for human hybridoma therefore should accommodate all or some of these signaling systems to overcome the unique situation of MHC incompatibility, need for specific growth factors simultaneously taking into consideration the downstream processing of the product for the clinical use. We have thus directed our efforts towards the development of a fusion partner which would not need Epstein-Barr virus transformation of B cells prior to fusion. A nontransforming mitogen, formalinized Staphylococcus aureus (FSTA) was used for stimulating human B cells. Successful production of human IgM monoclonal antibody was achieved by incorporating Jurkat-4 cells in existing mouse human heterohybrid through fusion of these cells followed by fusion with human B cells. To accommodate chromosomes of both T and B cells after fusion, human myeloid precursor cells KG1a, and to incorporate T cell, HuT78 cells were fused. CD34+ and CD4+ hybrid of KG1a and HuT 78 cells-434 AM-when used as fusion partner could allow secretion of MAbs, however growth potential was low. SP2/0 cells were then incorporated in 434 AM cells to give myeloma environment to fused human B cells. Rabies virus neutralizing human IgG MAb secreting clone was generated by fusing FSTA stimulated human B cells with this fusion partner.

Generation of human monoclonal antibodies against ganglioside antigens and their applications in the diagnosis and therapy of cancer.

Different approaches to generating human monoclonal antibodies (MAbs) against tumor-associated ganglioside antigens have been carried out in several laboratories. A specific goal addressed by our laboratory is to produce human MAbs to several ganglioside antigens of relevance as therapeutic targets, such as the GM2, GD2, GD3 and GM3 gangliosides in melanoma. In vitro immunization of human B lymphocytes from normal donors was performed using liposomes containing gangliosides as the immunizing antigen combined with either complete tetanus toxoid or a synthetic peptide corresponding to a T helper epitope to stimulate in vitro immunization. Specific human anti-ganglioside antibodies were obtained, indicating that the antibody response found in vitro was antigen-driven. To overcome the widely reported problems concerning stability of immunoglobulin production by the antibody-secreting cell lines, a method of positive selection using GM3-coated magnetic beads has been developed in order to rescue unstable clones. Development of new methods to reproducibly generate ganglioside-specific human MAbs will amplify the possibilities for diagnostic and therapeutic applications.

A new and efficient method to generate human IgG monoclonal antibodies reactive to cancer cells using SCID-hu mice

Human monoclonal antibodies (mAbs) are very useful for treatment of cancer, but they are difficult to obtain since immunization of humans is not a practical proposition at present. As an approach to circumvent this problem, we have simultaneously inoculated cancer tissues and regional lymphnode cells obtained from lung cancer patients into SCID mice to allow in vivo stimulation of human lymphocytes against autologous cancer tissues. Human immunoglobulins, especially IgG, were observed in the SCID-hu sera, and some showed high reactivity to lung cancer cell lines. Testing of human B-lymphoblastoid cell lines obtained from SCID-hu spleen and thymus for antibody activity revealed 16–45% of them to be reactive to lung cancer cells. These percentages are high as compared with previous reports. Furthermore, we could establish 4 human IgG mAbs reactive to lung cancer cell lines. These results indicate successful stimulation of specific human lymphocytes in vivo, which thereby enables efficient generation of human monoclonal antibodies using SCID-hu mice.