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PloS One 2019The diversity of antibody variable regions makes cDNA sequencing challenging, and conventional monoclonal antibody cDNA amplification requires the use of degenerate...
The diversity of antibody variable regions makes cDNA sequencing challenging, and conventional monoclonal antibody cDNA amplification requires the use of degenerate primers. Here, we describe a simplified workflow for amplification of IgG antibody variable regions from hybridoma RNA by a specialized RT-PCR followed by Sanger sequencing. We perform three separate reactions for each hybridoma: one each for kappa, lambda, and heavy chain transcripts. We prime reverse transcription with a primer specific to the respective constant region and use a template-switch oligonucleotide, which creates a custom sequence at the 5' end of the antibody cDNA. This template-switching circumvents the issue of low sequence homology and the need for degenerate primers. Instead, subsequent PCR amplification of the antibody cDNA molecules requires only two primers: one primer specific for the template-switch oligonucleotide sequence and a nested primer to the respective constant region. We successfully sequenced the variable regions of five mouse monoclonal IgG antibodies using this method, which enabled us to design chimeric mouse/human antibody expression plasmids for recombinant antibody production in mammalian cell culture expression systems. All five recombinant antibodies bind their respective antigens with high affinity, confirming that the amino acid sequences determined by our method are correct and demonstrating the high success rate of our method. Furthermore, we also designed RT-PCR primers and amplified the variable regions from RNA of cells transfected with chimeric mouse/human antibody expression plasmids, showing that our approach is also applicable to IgG antibodies of human origin. Our monoclonal antibody sequencing method is highly accurate, user-friendly, and very cost-effective.
Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antigen-Antibody Reactions; DNA Primers; DNA, Complementary; HEK293 Cells; Humans; Hybridomas; Immunoglobulin G; Immunoglobulin Variable Region; Mice; Recombinant Fusion Proteins; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, Protein; Workflow
PubMed: 31233538
DOI: 10.1371/journal.pone.0218717 -
Nature Sep 2023Certain bacterial strains from the microbiome induce a potent, antigen-specific T cell response. However, the specificity of microbiome-induced T cells has not been...
Certain bacterial strains from the microbiome induce a potent, antigen-specific T cell response. However, the specificity of microbiome-induced T cells has not been explored at the strain level across the gut community. Here, we colonize germ-free mice with complex defined communities (roughly 100 bacterial strains) and profile T cell responses to each strain. The pattern of responses suggests that many T cells in the gut repertoire recognize several bacterial strains from the community. We constructed T cell hybridomas from 92 T cell receptor (TCR) clonotypes; by screening every strain in the community against each hybridoma, we find that nearly all the bacteria-specific TCRs show a one-to-many TCR-to-strain relationship, including 13 abundant TCR clonotypes that each recognize 18 Firmicutes. By screening three pooled bacterial genomic libraries, we discover that these 13 clonotypes share a single target: a conserved substrate-binding protein from an ATP-binding cassette transport system. Peripheral regulatory T cells and T helper 17 cells specific for an epitope from this protein are abundant in community-colonized and specific pathogen-free mice. Our work reveals that T cell recognition of commensals is focused on widely conserved, highly expressed cell-surface antigens, opening the door to new therapeutic strategies in which colonist-specific immune responses are rationally altered or redirected.
Topics: Animals; Mice; Antigens, Surface; Bacteria; Firmicutes; Gastrointestinal Microbiome; T-Lymphocytes, Regulatory; Th17 Cells; T-Lymphocytes; Symbiosis; Germ-Free Life; Receptors, Antigen, T-Cell; Hybridomas; Cell Separation
PubMed: 37587342
DOI: 10.1038/s41586-023-06431-8 -
International Immunopharmacology Aug 2020The advancements in technology and manufacturing processes have allowed the development of new derivatives, biosimilar or advanced improved versions for approved... (Review)
Review
The advancements in technology and manufacturing processes have allowed the development of new derivatives, biosimilar or advanced improved versions for approved antibodies each year for treatment regimen. There are more than 700 antibody-based molecules that are in different stages of phase I/II/ III clinical trials targeting new unique targets. To date, approximately more than 80 monoclonal antibodies (mAbs) have been approved. A total of 7 novel antibody therapeutics had been granted the first approval either in the United States or European Union in the year 2019, representing approximately 20% of the total number of approved drugs. Most of these licenced mAbs or their derivatives are either of hybridoma origin or their improvised engineered versions. Even with the recent development of high throughput mAb generation technologies, hybridoma is the most favoured method due to its indigenous nature to preserve natural cognate antibody pairing information and preserves innate functions of immune cells. The recent advent of antibody engineering technology has superseded the species level barriers and has shown success in isolation of hybridoma across phylogenetically distinct species. This has led to the isolation of monoclonal antibodies against human targets that are conserved and non-immunogenic in the rodent. In this review, we have discussed in detail about hybridoma technology, its expansion towards different animal species, the importance of antibodies isolated from different animal sources that are useful in biological applications, advantages, and limitations. This review also summarizes the challenges and recent progress associated with hybridoma development, and how it has been overcome in these years to provide new insights for the isolation of mAbs.
Topics: Animals; Antibodies, Monoclonal; Humans; Hybridomas
PubMed: 32473573
DOI: 10.1016/j.intimp.2020.106639 -
Current Research in Immunology 2021The isolation of single monoclonal antibodies (mAbs) against a given antigen was only possible with the introduction of the hybridoma technology, which is based on the... (Review)
Review
The isolation of single monoclonal antibodies (mAbs) against a given antigen was only possible with the introduction of the hybridoma technology, which is based on the fusion of specific B lymphocytes with myeloma cells. Since then, several mAbs were described for therapeutic, diagnostic, and research purposes. Despite being an old technique with low complexity, hybridoma-based strategies have limitations that include the low efficiency on B lymphocyte-myeloma cell fusion step, and the need to use experimental animals. In face of that, several methods have been developed to improve mAb generation, ranging from changes in hybridoma technique to the advent of completely new technologies, such as the antibody phage display and the single B cell antibody ones. In this review, we discuss the hybridoma technology along with emerging mAb isolation approaches, taking into account their advantages and limitations. Finally, we explore the usefulness of the hybridoma technology nowadays.
PubMed: 35492397
DOI: 10.1016/j.crimmu.2021.03.002 -
Journal of Hematology & Oncology Sep 2020Recent evidence suggests that resistance to CD19 chimeric antigen receptor (CAR)-modified T cell therapy may be due to the presence of CD19 isoforms that lose binding to... (Clinical Trial)
Clinical Trial
BACKGROUND
Recent evidence suggests that resistance to CD19 chimeric antigen receptor (CAR)-modified T cell therapy may be due to the presence of CD19 isoforms that lose binding to the single-chain variable fragment (scFv) in current use. As such, further investigation of CARs recognize different epitopes of CD19 antigen may be necessary.
METHODS
We generated a new CD19 CAR T (HI19α-4-1BB-ζ CAR T, or CNCT19) that includes an scFv that interacts with an epitope of the human CD19 antigen that can be distinguished from that recognized by the current FMC63 clone. A pilot study was undertaken to assess the safety and feasibility of CNCT19-based therapy in both pediatric and adult patients with relapsed/refractory acute lymphoblastic leukemia (R/R B-ALL).
RESULTS
Data from our study suggested that 90% of the 20 patients treated with infusions of CNCT19 cells reached complete remission or complete remission with incomplete count recovery (CR/CRi) within 28 days. The CR/CRi rate was 82% when we took into account the fully enrolled 22 patients in an intention-to-treat analysis. Of note, extramedullary leukemia disease of two relapsed patients disappeared completely after CNCT19 cell infusion. After a median follow-up of 10.09 months (range, 0.49-24.02 months), the median overall survival and relapse-free survival for the 20 patients treated with CNCT19 cells was 12.91 months (95% confidence interval [CI], 7.74-18.08 months) and 6.93 months (95% CI, 3.13-10.73 months), respectively. Differences with respect to immune profiles associated with a long-term response following CAR T cell therapy were also addressed. Our results revealed that a relatively low percentage of CD8 naïve T cells was an independent factor associated with a shorter period of relapse-free survival (p = 0.012, 95% CI, 0.017-0.601).
CONCLUSIONS
The results presented in this study indicate that CNCT19 cells have potent anti-leukemic activities in patients with R/R B-ALL. Furthermore, our findings suggest that the percentage of CD8 naïve T cells may be a useful biomarker to predict the long-term prognosis for patients undergoing CAR T cell therapy.
TRIAL REGISTRATION
ClinicalTrials.gov : NCT02975687; registered 29 November, 2016. https://clinicaltrials.gov/ct2/keydates/NCT02975687.
Topics: Adult; Amino Acid Sequence; Antigen-Antibody Reactions; Antigens, CD19; Antigens, Neoplasm; Cell Line, Tumor; Child; Clone Cells; Epitopes; Female; Follow-Up Studies; Humans; Hybridomas; Immunotherapy, Adoptive; Kaplan-Meier Estimate; Male; Models, Molecular; Pilot Projects; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Progression-Free Survival; Proportional Hazards Models; Prospective Studies; Protein Conformation; Receptors, Chimeric Antigen; Recurrence; Sequence Alignment; Single-Chain Antibodies
PubMed: 32894185
DOI: 10.1186/s13045-020-00953-8 -
BioTechniques Sep 2018The benefits of polyclonal antibodies as tools for assay-specific target discovery and detection are numerous. As the future of basic research, diagnostics and biomarker... (Review)
Review
The benefits of polyclonal antibodies as tools for assay-specific target discovery and detection are numerous. As the future of basic research, diagnostics and biomarker discovery is dependent on high-quality reproducible data, there is a need to understand the importance and benefits of these valuable tools. All antibody forms - polyclonal, hybridoma-based monoclonal and recombinant monoclonal - have pros and cons for development, validation and use. Yet, polyclonal antibodies are embroiled in a firestorm of controversy concerning data reproducibility. We address best practices for developing and using polyclonal antibodies, pitfalls to their use and how to avoid them, and benefits to the life science community. Eliminating their use risks overlooking the unique benefits of polyclonal antibodies as 'fit-for-purpose' life science tools.
Topics: Antibodies; Antibody Affinity; Binding Sites, Antibody; Reproducibility of Results; Research Design
PubMed: 30089399
DOI: 10.2144/btn-2018-0065 -
Current Opinion in Biotechnology Dec 2019Antibodies (Abs) are ubiquitous reagents for biological and biochemical research and are rapidly expanding into new therapeutic areas. They are one of the most important... (Review)
Review
Antibodies (Abs) are ubiquitous reagents for biological and biochemical research and are rapidly expanding into new therapeutic areas. They are one of the most important probes for determining how proteins function under normal and pathophysiological conditions. Abs are required for the quantification of targets, detection of temporal and spatial patterns of protein expression in cells and tissues, and identification of interacting partners and their biological activities. Their remarkable specificity and unique binding properties can facilitate three-dimensional structure determination using X-ray crystallography and electron cryomicroscopy. While hybridoma technology that involves animal immunization is often productive, many antigen targets do not generate useful Abs. This is particularly true if unique states of the target or critical non-immunogenic target sequences need to be recognized by the Abs. By using the methods of recombinant antibody generation, identification, and engineering, these 'hybridoma-refractory' antigens can be readily targeted. Specific, reproducible, and renewable recombinant Abs are proving to be invaluable reagents in applications ranging from biological discovery to structure determination of challenging macromolecules.
Topics: Animals; Antibodies; Immunization; Recombinant Proteins
PubMed: 30849700
DOI: 10.1016/j.copbio.2019.01.012 -
Frontiers in Immunology 2019Each year, millions of humans fall victim to animal envenomings, which may either be deadly or cause permanent disability to the effected individuals. The Nobel... (Review)
Review
Each year, millions of humans fall victim to animal envenomings, which may either be deadly or cause permanent disability to the effected individuals. The Nobel Prize-winning discovery of serum therapy for the treatment of bacterial infections (tetanus and diphtheria) paved the way for the introduction of antivenom therapies for envenomings caused by venomous animals. These antivenoms are based on polyclonal antibodies derived from the plasma of hyperimmunized animals and remain the only specific treatment against animal envenomings. Following the initial development of serum therapy for snakebite envenoming by French scientists in 1894, other countries with high incidences of animal envenomings, including Brazil, Australia, South Africa, Costa Rica, and Mexico, started taking up antivenom production against local venomous animals over the course of the twentieth century. These undertakings revolutionized envenoming therapy and have saved innumerous patients worldwide during the last 100 years. This review describes in detail the above-mentioned historical events surrounding the discovery and the application of serum therapy for envenomings, as well as it provides an overview of important developments and scientific breakthroughs that were of importance for antibody-based therapies in general. This begins with discoveries concerning the characterization of antibodies, including the events leading up to the elucidation of the antibody structure. These discoveries further paved the way for other milestones in antibody-based therapies, such as the introduction of hybridoma technology in 1975. Hybridoma technology enabled the expression and isolation of monoclonal antibodies, which in turn formed the basis for the development of phage display technology and transgenic mice, which can be harnessed to directly obtain fully human monoclonal antibodies. These developments were driven by the ultimate goal of producing potent neutralizing monoclonal antibodies with optimal pharmacokinetic properties and low immunogenicity. This review then provides an outline of the most recent achievements in antivenom research, which include the application of new biotechnologies, the development of the first human monoclonal antibodies that can neutralize animal toxins, and efforts toward creating fully recombinant antivenoms. Lastly, future perspectives in the field of envenoming therapies are discussed, including rational engineering of antibody cross-reactivity and the use of oligoclonal antibody mixtures.
Topics: Allergens; Animals; Antivenins; Desensitization, Immunologic; History, 19th Century; History, 20th Century; History, 21st Century; Humans; Hypersensitivity; Nobel Prize; Snakes; Venoms
PubMed: 31354735
DOI: 10.3389/fimmu.2019.01598 -
Frontiers in Microbiology 2017Since the development of antibody-production techniques, a number of immunoglobulins have been developed on a large scale using conventional methods. Hybridoma... (Review)
Review
Since the development of antibody-production techniques, a number of immunoglobulins have been developed on a large scale using conventional methods. Hybridoma technology opened a new horizon in the production of antibodies against target antigens of infectious pathogens, malignant diseases including autoimmune disorders, and numerous potent toxins. However, these clinical humanized or chimeric murine antibodies have several limitations and complexities. Therefore, to overcome these difficulties, recent advances in genetic engineering techniques and phage display technique have allowed the production of highly specific recombinant antibodies. These engineered antibodies have been constructed in the hunt for novel therapeutic drugs equipped with enhanced immunoprotective abilities, such as engaging immune effector functions, effective development of fusion proteins, efficient tumor and tissue penetration, and high-affinity antibodies directed against conserved targets. Advanced antibody engineering techniques have extensive applications in the fields of immunology, biotechnology, diagnostics, and therapeutic medicines. However, there is limited knowledge regarding dynamic antibody development approaches. Therefore, this review extends beyond our understanding of conventional polyclonal and monoclonal antibodies. Furthermore, recent advances in antibody engineering techniques together with antibody fragments, display technologies, immunomodulation, and broad applications of antibodies are discussed to enhance innovative antibody production in pursuit of a healthier future for humans.
PubMed: 28400756
DOI: 10.3389/fmicb.2017.00495