Antibody Library Research Articles

A dual-targeting approach using a human bispecific antibody against the receptor-binding domain of the Middle East Respiratory Syndrome Coronavirus

The emergence of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) has posed a significant global health concern due to its severe respiratory illness and high fatality rate. Currently, despite the potential for resurgence, there are no specific treatments for MERS-CoV, and only supportive care is available. Our study aimed to address this therapeutic gap by developing a potent neutralizing bispecific antibody (bsAb) against MERS-CoV. Initially, we isolated four human monoclonal antibodies (mAbs) that specifically target the MERS-CoV receptor-binding domain (RBD) using phage display technology and an established human antibody library. Among these four selected mAbs, our intensive in vitro functional analyses showed that the MERS-CoV RBD-specific mAb K111.3 exhibited the most potent neutralizing activity against MERS-CoV pseudoviral infection and the molecular interaction between MERS-CoV RBD and human dipeptidyl peptidase 4. Consequently, we engineered a novel bsAb, K207.C, by utilizing K111.3 as the IgG base and fusing it with the single-chain variable fragment of its non-competing pair, K111.1. This engineered bsAb showed significantly enhanced neutralization potential against MERS-CoV compared to its parental mAb. These findings suggest that K207.C may serve as a potential candidate for effective MERS-CoV neutralization, further highlighting the promise of the bsAb dual-targeting approach in MERS-CoV neutralization.

Development, High-Throughput Profiling, and Biopanning of a Large Phage Display Single-Domain Antibody Library.

Immunoglobulin G-based monoclonal antibodies (mAbs) have been effective in treating various diseases, but their large molecular size can limit their penetration of tissue and efficacy in multifactorial diseases, necessitating the exploration of alternative forms. In this study, we constructed a phage display library comprising single-domain antibodies (sdAbs; or "VHHs"), known for their small size and remarkable stability, using a total of 1.6 × 109 lymphocytes collected from 20 different alpacas, resulting in approximately 7.16 × 1010 colonies. To assess the quality of the constructed library, next-generation sequencing-based high-throughput profiling was performed, analyzing approximately 5.65 × 106 full-length VHH sequences, revealing 92% uniqueness and confirming the library's diverse composition. Systematic characterization of the library revealed multiple sdAbs with high affinity for three therapeutically relevant antigens. In conclusion, our alpaca sdAb phage display library provides a versatile resource for diagnostics and therapeutics. Furthermore, the library's vast natural VHH antibody repertoire offers insights for generating humanized synthetic sdAb libraries, further advancing sdAb-based therapeutics.

Unraveling the Properties of Phage Display Fab Libraries and Their Use in the Selection of Gliadin-Specific Probes by Applying High-Throughput Nanopore Sequencing.

Directed evolution is a pivotal strategy for new antibody discovery, which allowed the generation of high-affinity Fabs against gliadin from two antibody libraries in our previous studies. One of the libraries was exclusively derived from celiac patients' mRNA (immune library) while the other was obtained through a protein engineering approach (semi-immune library). Recent advances in high-throughput DNA sequencing techniques are revolutionizing research across genomics, epigenomics, and transcriptomics. In the present work, an Oxford Nanopore in-lab sequencing device was used to comprehensively characterize the composition of the constructed libraries, both at the beginning and throughout the phage-mediated selection processes against gliadin. A customized analysis pipeline was used to select high-quality reads, annotate chain distribution, perform sequence analysis, and conduct statistical comparisons between the different selection rounds. Some immunological attributes of the most representative phage variants after the selection process were also determined. Sequencing results revealed the successful transfer of the celiac immune response features to the immune library and the antibodies derived from it, suggesting the crucial role of these features in guiding the selection of high-affinity recombinant Fabs against gliadin. In summary, high-throughput DNA sequencing has improved our understanding of the selection processes aimed at generating molecular binders against gliadin.

Combining Cellular Immunization and Phage Display Screening Results in Novel, FcγRI-Specific Antibodies.

Antibodies that specifically bind to individual human fragment crystallizable γ receptors (FcγRs) are of interest as research tools in studying immune cell functions, as well as components in bispecific antibodies for immune cell engagement in cancer therapy. Monoclonal antibodies for human low-affinity FcγRs have been successfully generated by hybridoma technology and are widely used in pre-clinical research. However, the generation of monoclonal antibodies by hybridoma technology that specifically bind to the high-affinity receptor FcγRI is challenging. Monomeric mouse IgG2a, IgG2b, and IgG3 bind human FcγRI with high affinity via the Fc part, leading to an Fc-mediated rather than a fragment for antigen binding (Fab)-mediated selection of monoclonal antibodies. Blocking the Fc-binding site of FcγRI with an excess of human IgG or Fc during screening decreases the risk of Fc-mediated interactions but can also block the potential epitopes of new antibody candidates. Therefore, we replaced hybridoma technology with phage display of a single-chain fragment variable (scFv) antibody library that was generated from mice immunized with FcγRI-positive cells and screened it with a cellular panning approach assisted by next-generation sequencing (NGS). Seven new FcγRI-specific antibody sequences were selected with this methodology, which were produced as Fc-silent antibodies showing FcγRI-restricted specificity.

Problems of creating antibody phage libraries and their solutions.

Phage display has become an efficient, reliable and popular molecular technique for generating libraries encompassing millions or even billions of clones of divergent peptides or proteins. The method is based on the correspondence between phage genotype and phenotype, which ensures the presentation of recombinant proteins of known amino acid composition on the surface of phage particles. The use of affinity selection allows one to choose variants with affinity for different targets from phage libraries. The implementation of the antibody phage display technique has revolutionized the field of clinical immunology, both for developing tools to diagnose infectious diseases and for producing therapeutic agents. It has also become the basis for efficient and relatively inexpensive methods for studying protein-protein interactions, receptor binding sites, as well as epitope and mimotope identification. The antibody phage display technique involves a number of steps, and the final result depends on their successful implementation. The diversity, whether natural or obtained by combinatorial chemistry, is the basis of any library. The choice of molecular techniques is critical to ensure that this diversity is maintained during the phage library preparation step and during the transformation of E. coli cells. After a helper phage is added to the suspension of transformed E. coli cells, a bacteriophage library is formed, which is a working tool for performing the affinity selection procedure and searching for individual molecules. Despite the apparent simplicity of generating phage antibody libraries, a number of subtleties need to be taken into account. First, there are the features of phage vector preparation. Currently, a large number of phagemid vectors have been developed, and their selection is also of great importance. The key step is preparing competent E. coli cells and the technology of their transformation. The choice of a helper phage and the method used to generate it is also important. This article discusses the key challenges faced by researchers in constructing phage antibody libraries.

Advancement in the development of single chain antibodies using phage display technology.

Phage display technology has become an important research tool in biological research, fundamentally changing the traditional monoclonal antibody preparation process, and has been widely used in the establishment of antigen-antibody libraries, drug design, vaccine research, pathogen detection, gene therapy, antigenic epitope research, and cellular signal transduction research.The phage display is a powerful platform for technology development. Using phage display technology, single chain fragment variable (scFv) can be screened, replacing the disadvantage of the large size of traditional antibodies. Phage display single chain antibody libraries have significant biological implications. Here we describe the types of antibodies, including chimeric antibodies, bispecific antibodies, and scFvs. In addition, we describe the phage display system, phage display single chain antibody libraries, screening of specific antibodies by phage libraries and the application of phage libraries.

Structural Survey of Antigen Recognition by Synthetic Human Antibodies.

Synthetic antibody libraries have been used extensively to isolate and optimize antibodies. To generate these libraries, the immunological diversity and the antibody framework(s) that supports it outside of the binding regions are carefully designed/chosen to ensure favorable functional and biophysical properties. In particular, minimalist, single-framework synthetic libraries pioneered by our group have yielded a vast trove of antibodies to a broad array of antigens. Here, we review their systematic and iterative development to provide insights into the design principles that make them a powerful tool for drug discovery. In addition, the ongoing accumulation of crystal structures of antigen-binding fragment (Fab)-antigen complexes generated with synthetic antibodies enables a deepening understanding of the structural determinants of antigen recognition and usage of immunoglobulin sequence diversity, which can assist in developing new strategies for antibody and library optimization. Toward this, we also survey here the structural landscape of a comprehensive and unbiased set of 50 distinct complexes derived from these libraries and compare it to a similar set of natural antibodies with the goal of better understanding how each achieves molecular recognition and whether opportunities exist for iterative improvement of synthetic libraries. From this survey, we conclude that despite the minimalist strategies used for design of these synthetic antibody libraries, the overall structural interaction landscapes are highly similar to natural repertoires. We also found, however, some key differences that can help guide the iterative design of new synthetic libraries via the introduction of positionally tailored diversity.

Beyond Natural Immune Repertoires: Synthetic Antibodies.

Synthetic antibody libraries, in which the antigen-binding sites are precisely designed, offer unparalleled precision in antibody engineering, exceeding the potential of natural immune repertoires and constituting a novel generation of research tools and therapeutics. Recent advances in artificial intelligence-driven technologies and their integration into synthetic antibody discovery campaigns hold the promise to further streamline and effectively develop antibodies. Here, we provide an overview of synthetic antibodies. Our associated protocol describes how to develop highly diverse and functional synthetic antibody phage display libraries.

Disulfide-constrained peptide scaffolds enable a robust peptide-therapeutic discovery platform.

Peptides present an alternative modality to immunoglobulin domains or small molecules for developing therapeutics to either agonize or antagonize cellular pathways associated with diseases. However, peptides often suffer from poor chemical and physical stability, limiting their therapeutic potential. Disulfide-constrained peptides (DCP) are naturally occurring and possess numerous desirable properties, such as high stability, that qualify them as drug-like scaffolds for peptide therapeutics. DCPs contain loop regions protruding from the core of the molecule that are amenable to peptide engineering via direct evolution by use of phage display technology. In this study, we have established a robust platform for the discovery of peptide therapeutics using various DCPs as scaffolds. We created diverse libraries comprising seven different DCP scaffolds, resulting in an overall diversity of 2 x 1011. The effectiveness of this platform for functional hit discovery has been extensively evaluated, demonstrating a hit rate comparable to that of synthetic antibody libraries. By utilizing chemically synthesized and in vitro folded peptides derived from selections of phage displayed DCP libraries, we have successfully generated functional inhibitors targeting the HtrA1 protease. Through affinity maturation strategies, we have transformed initially weak binders against Notch2 with micromolar Kd values to high-affinity ligands in the nanomolar range. This process highlights a viable hit-to-lead progression. Overall, our platform holds significant potential to greatly enhance the discovery of peptide therapeutics.

Abstract 7546: Targeting NSD2 to reverse lineage plasticity and drug resistance in neuroendocrine prostate cancer and subtypes of mCRPC

Abstract Treatment with highly potent anti-androgens, such as enzalutamide and abiraterone promotes lineage plasticity in metastatic castration-resistant prostate cancer (mCRPC), which results in intra-tumor heterogeneity and emergence of mCRPC subtypes. The histological transformation from adenocarcinoma to aggressive neuroendocrine prostate cancer (CRPC-NE) has been associated with a loss of dependency on lineage-survival signals, leading to targeted drug resistance. Epigenomic reprogramming might be a fundamental driver of lineage plasticity. To determine CRPC-NE vulnerabilities, we have performed image-based screening using a small library of antibodies targeting different histone marks on CRPC-NE organoids derived from genetically engineered mice. We find that the histone mark H3K36me2 and the histone methyltransferase NSD2 play important roles in maintain the CRPC-NE epigenetic state. Knockout of NSD2 or ablation of H3K36me2 using an oncohistone mutant H3.3K36M reverted CRPC-NE to an adenocarcinoma phenotype. Simultaneous profiling of the transcriptome and epigenome from single cells verified this lineage conversion and the generation of cell states with canonical AR signaling. Moreover, H3K36me2 or NSD2 depleted mouse and human CRPC-NE organoids responded to enzalutamide treatment in vitro and in vivo, suggesting a reversal of castration-resistance. Most importantly, a small molecule inhibitor of NSD2 in combination with enzalutamide leads to growth suppression or apoptosis of mouse and human CRPC-NE organoids, as well as organoids of other CRPC subtypes. In conclusion, inhibition of NSD2 reverts the epigenomic state of CRPC-NE, reversing lineage plasticity and restoring anti-androgen sensitivity. Thus, we suggest that the combination of NSD2 inhibition with AR inhibition may represent a novel therapeutic approach for patients with CRPC-NE or even other CRPC subtypes. Citation Format: Jia J. Li, Alessandro Vasciaveo, Dimitrios Karagiannis, Xiao Chen, Chen Yu, Andrea Califano, Chao Lu, Michael M. Shen. Targeting NSD2 to reverse lineage plasticity and drug resistance in neuroendocrine prostate cancer and subtypes of mCRPC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7546.

Abstract 3107: SMASH: Single molecule antibody screening with high-throughput imaging system

Abstract Larger, more diverse antibody libraries are being screened in recent years. Despite enabling the high-throughput screening of extensive libraries, the process for identification and characterization of antibodies remains laborious and time-intensive. Here we report a platform that not only compiles hit finding and validation processes, but also provides high-resolution binding affinity as well as rank order among the library being screened. We streamlined the library construction and affinity screening via microscale transient expression in HEK293T cells with designed antibody sequence and direct quantification of antibody-antigen binding affinity which reduced the timeline from multiple weeks to a single week. We constructed the single-chain fragment antigen binding (scFab) libraries by replacing the duplex DNA fragment in CDR of interest while preserving the human framework of Fab. We have demonstrated that the SPID technique allows to detect antibody-antigen interactions from just dozens of pg of scFabs in total. This allowed us to bypass the conventional DNA cloning or even PCR amplification to achieve the minimum amount of DNA plasmids for expressing scFabs to be analyzed such as BLI and SPR. Therefore, we assembled the DNA plasmids through in vitro ligation with high efficiency and directly introduced it to HEK293T in 96-well microplate with microscale (200 ul) for transient transfection, which only takes three days for library generation. Next, we utilized SPID to quantify the number of single molecules between free and antigen-bound fractions of scFab, resulting in occupancy at the antibody-antigen binding equilibrium. This technique facilitated the determination of binding affinity (KD) for 200 antibody-antigen pairs within a 6-hour TAT encompassing both measurement and analysis. In total, we utilized 4,000 times less antibody for characterization while achieving enhanced sensitivity compared to both BLI and SPR methods. As a proof-of-concept, we generated a library consisting of 720 variants of Trastuzumab in scFab format to probe the landscape of CDRs affecting affinities against HER2 showing nM to low uM dynamic range of KD. Out of 720 analyzed variants, 5% were deleterious, 38% provided substantial changes, 40% showed little/no change, 17% enhanced affinity to HER2. To validate the robustness of the platform, we extensively expanded the library screening, assessing over 2,000 antibody variants for their affinity against more than three different therapeutic targets. To conclude, we demonstrated an efficient approach for monitoring changes in affinity resulting from single mutations in scFab. This rapid screening of site-saturated libraries would lead to a combinational variant library that our platform can screen. Ultimately, our platform aims to identify potential candidates for therapeutic antibody development throughput the phases of antibody discovery, maturation, and engineering. Citation Format: Jihye Jo, Changju Chun, Byeong-Kwon Sohn, Booyoung Yu, Jiyu Lee. SMASH: Single molecule antibody screening with high-throughput imaging system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3107.

Abstract 2670: Development of novel anti-SIRPα/β dual antibody with single-agent phagocytosis activity

Abstract Signal-regulatory protein alpha (SIRPα) is an inhibitory receptor expressed on macrophages that interacts with CD47, resulting a "don't eat me" signal on cancer cells. This interaction suppresses macrophage dependent phagocytosis and contributes to the evasion of host immune system. Consequently, the SIRPα-CD47 axis has become a well sought after and established therapeutic angle to treat cancer. While CD47-targeting agents have shown promise in clinical trials, the ubiquitous expression of CD47 raises the concerns about hematologic toxicity, including anaemia and thrombocytopenia, hindering the development of CD47-targeting therapies. This has prompted exploration of another end of the axis i.e., targeting SIRPα. Moreover, targeting SIRPβ protein, expressed on myleoid cells, via agonistic approach would generate ITAM-mediated activating signal through DAP12 protein contributing to cancer cell death. Considering the limited expression of SIRPα and β, a dual antibody capable of blocking the CD47-SIRPα pathway and activating SIRPβ downstream signaling may result in a desirable efficacy and safety profile. In this study, we present novel anti-SIRP α/β dual antibody clones discovered from a proprietary human antibody discovery platform consisting of a large human antibody library, along with in vitro and in silico screening technologies. The platform was explored against native SIRPα/β antigens to identify novel human antibody clones based on affinity-based epitope coverage. Subsequently, antibody clones were produced and evaluated for preclinical functional relevance. Novel full-length SIRPα/β dual antibody clones demonstrated high affinity binding to soluble human SIRPα and SIRPβ proteins as well as these receptors expressed on CHO-K1 cells and macrophages. The antibody clones effectively disrupted CD47-SIRPα interaction in biochemical assays and significantly enhanced activation of macrophages as observed by increased cytokine release. Most importantly, the dual antibody clones demonstrated potent in vitro single-agent phagocytosis activity against tumor cells. In summary, the novel SIRPα/β dual antibodies activating macrophage mediated phagocytosis of tumor cells without the need for tumor opsonizing antibodies or agents capable of inducing “eat-me-signals” highlight their potential as a single-agent therapy. These findings prompt us to continue the development of the anti-SIRP α/β dual antibody for use in cancer therapy. Citation Format: Subhra Prakash Chakrabarty, Sandeep Sadashiv Patil, Sunaina Singh Rajput, Amrita Mukerji, Sandhya Aurumugam, Pooja Balu Gade, Sidhesha S, Anup Kumar Patra, Shivaranjini C, Vaishnavi Shivaji Khamkar, Lavanya P, Lakshmeesha K N, Ujjwal Kapoor, Jagadeesh R, Jwala Nagaraj, Vijaya Shankar Nataraj, Kavitha Nellore, Thomas Antony, Susanta Samajdar, Murali Ramachandra. Development of novel anti-SIRPα/β dual antibody with single-agent phagocytosis activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2670.

Development of Shark Single Domain Antibodies Specific for Human α-Fetoprotein and the Multimerization Strategy in Serum Detection.

Sensitive detection of cancer biomarkers can contribute to the timely diagnosis and treatment of diseases. In this study, the whitespotted bamboo sharks were immunized with human α-fetoprotein (AFP), and a phage-displayed variable new antigen receptor (VNAR) single domain antibody library was constructed. Then four unique VNARs (VNAR1, VNAR11, VNAR21, and VNAR25) against AFP were isolated from the library by biopanning for the first time. All of the sequences belong to type II of VNAR, and the VNAR11 was much different from the rest of the three sequences. Then VNAR1 and VNAR11 were selected to fuse with the C4-binding protein α chain (C4bpα) sequence and efficiently expressed in the Escherichia coli system. Furthermore, a VNAR-C4bpα-mediated sandwich chemiluminescence immunoassay (VSCLIA) was developed for the detection of AFP in human serum samples. After optimization, the VSCLIA showed a limit of detection of 0.74 ng/mL with good selectivity and accuracy. Moreover, the results of clinical serum samples detected by the VSCLIA were confirmed by an automatic immunoanalyzer in the hospital, indicating its practical application in actual samples. In conclusion, the novel antibody element VNAR exhibits great potential for immunodiagnosis, and this study also provides a new direction and experimental basis for AFP detection.

Synthetic development of a broadly neutralizing antibody against snake venom long-chain α-neurotoxins.

Snakebite envenoming is a major global public health concern for which improved therapies are urgently needed. The antigenic diversity present in snake venom toxins from various species presents a considerable challenge to the development of a universal antivenom. Here, we used a synthetic human antibody library to find and develop an antibody that neutralizes long-chain three-finger α-neurotoxins produced by numerous medically relevant snakes. Our antibody bound diverse toxin variants with high affinity, blocked toxin binding to the nicotinic acetylcholine receptor in vitro, and protected mice from lethal venom challenge. Structural analysis of the antibody-toxin complex revealed a binding mode that mimics the receptor-toxin interaction. The overall workflow presented is generalizable for the development of antibodies that target conserved epitopes among antigenically diverse targets, and it offers a promising framework for the creation of a monoclonal antibody-based universal antivenom to treat snakebite envenoming.

Deep mining of antibody phage-display selections using Oxford Nanopore Technologies and Dual Unique Molecular Identifiers

Antibody phage-display technology identifies antibody-antigen interactions through multiple panning rounds, but traditional screening gives no information on enrichment or diversity throughout the process. This results in the loss of valuable binders. Next Generation Sequencing can overcome this problem. We introduce a high accuracy long-read sequencing method based on the recent Oxford Nanopore Technologies (ONT) Q20 + chemistry in combination with dual unique molecular identifiers (UMIs) and an optimized bioinformatic analysis pipeline to monitor the selections. We identified binders from two single-domain antibody libraries selected against a model protein. Traditional colony-picking was compared with our ONT-UMI method. ONT-UMI enabled monitoring of diversity and enrichment before and after each selection round. By combining phage antibody selections with ONT-UMIs, deep mining of output selections is possible. The approach provides an alternative to traditional screening, enabling diversity quantification after each selection round and rare binder recovery, even when the dominating binder was > 99% abundant. Moreover, it can give insights on binding motifs for further affinity maturation and specificity optimizations. Our results demonstrate a platform for future data guided selection strategies.

Characterization of chicken-derived antibody against Alpha-Enolase of Streptococcus pneumoniae

Streptococcus pneumoniae is a clinically relevant pathogen notorious for causing pneumonia, meningitis, and otitis media in immunocompromised patients. Currently, antibiotic therapy is the most efficient treatment for fighting pneumococcal infections. However, an arise in antimicrobial resistance in S. pneumoniae has become a serious health issue globally. To resolve the problem, alternative and cost-effective strategies, such as monoclonal antibody-based targeted therapy, are needed for combating bacterial infection. S. pneumoniae alpha-enolase (spEno1), which is thought to be a great target, is a surface protein that binds and converts human plasminogen to plasmin, leading to accelerated bacterial infections. We first purified recombinant spEno1 protein for chicken immunization to generate specific IgY antibodies. We next constructed two single-chain variable fragments (scFv) antibody libraries by phage display technology, containing 7.2 × 107 and 4.8 × 107 transformants. After bio-panning, ten scFv antibodies were obtained, and their binding activities to spEno1 were evaluated on ELISA, Western blot and IFA. The epitopes of spEno1 were identified by these scFv antibodies, which binding affinities were determined by competitive ELISA. Moreover, inhibition assay displayed that the scFv antibodies effectively inhibit the binding between spEno1 and human plasminogen. Overall, the results suggested that these scFv antibodies have the potential to serve as an immunotherapeutic drug against S. pneumoniae infections.

Generation of a Naïve Human scFv Phage Display Library and Panning Selection.

Phage display antibody libraries have been successfully used as the essential tool to produce monoclonal antibodies against a plethora of targets ranging from diseases to native biologically important proteins as well as small molecules. It is well documented that diverse antibody genes are the major genetic source for the construction of a high-quality antibody library and selection of high-affinity antibodies. Naïve antibody libraries are derived using the IgM repertoire of healthy donors obtained from B-cells isolated from human peripheral blood mononuclear cell (PBMC). Single-chain fragment variable (scFv) is a routinely used format due to its smaller size and preference for phage display. The process involves the use of a two-step cloning method for library construction. The protocol also covers the biopanning process for target positive clone selection.

Cell Cycle Mapping Using Multiplexed Immunofluorescence.

The development of technologies that allow measurement of the cell cycle at the single-cell level has revealed novel insights into the mechanisms that regulate cell cycle commitment and progression through DNA replication and cell division. These studies have also provided evidence of heterogeneity in cell cycle regulation among individual cells, even within a genetically identical population. Cell cycle mapping combines highly multiplexed imaging with manifold learning to visualize the diversity of "paths" that cells can take through the proliferative cell cycle or into various states of cell cycle arrest. In this chapter, we describe a general protocol of the experimental and computational components of cell cycle mapping. We also provide a comprehensive guide for the design and analysis of experiments, discussing key considerations in detail (e.g., antibody library preparation, analysis strategies, etc.) that may vary depending on the research question being addressed.

Recombinant human scFv antibody fragments against phospholipase A2 from Naja naja and Echis carinatus snake venoms: In vivo neutralization and mechanistic insights

Snake envenomation results in a range of clinical sequelae, and widely used animal-based conventional antivenoms exhibit several limitations including the adverse immunological effects in human snake bite victims. Therefore, human monoclonal anti-snake venom antibodies or fragments can be an alternate therapy for overcoming the existing limitations. We developed venom-neutralizing humanized scFv antibodies and analyzed biochemical mechanisms associated with the inhibition of toxicity. Tomlinson I and J human scFv antibody libraries were screened against Naja naja and Echis carinatus venoms, and seven unique scFv antibodies were obtained. Further, specific toxins of snake venom interacting with each of these scFvs were identified, and phospholipase A2 (PLA2) was found to be prominently captured by the phage-anchored scFv antibodies. Our study indicated PLA2 to be one of the abundant toxins in Naja naja and Echis carinatus venom samples. The scFvs binding to PLA2 were used to perform in vivo survival assay using the mouse model and in vitro toxin inhibition assays. scFv N194, which binds to acidic PLA2, protected 50% of mice treated with Naja naja venom. Significant prolongation of survival time and 16% survival were observed in Echis carinatus venom-challenged mice treated with scFv E113 and scFv E10, respectively. However, a combination comprised of an equal amount of two scFvs, E113 and E10, both interacting with basic PLA2, exhibited synergistically enhanced survival of 33% in Echis carinatus venom-challenged mice. No such synergistically enhanced survival was observed in the case of combinatorial treatment with anti-Naja naja scFvs, N194, and N248. These scFvs demonstrated partial inhibition of venom-induced myotoxicity, and E113 also inhibited hemolysis by 50%, which corroborates the enhanced survival during combinatorial treatment in Echis carinatus venom-challenged mice.

Structural trends in antibody-antigen binding interfaces: a computational analysis of 1833 experimentally determined 3D structures

Antibodies are attractive therapeutic candidates due to their ability to bind cognate antigens with high affinity and specificity. Still, the underlying molecular rules governing the antibody-antigen interface remain poorly understood, making in silico antibody design inherently difficult and keeping the discovery and design of novel antibodies a costly and laborious process. This study investigates the characteristics of antibody-antigen binding interfaces through a computational analysis of more than 850,000 atom-atom contacts from the largest reported set of antibody-antigen complexes with 1833 nonredundant, experimentally determined structures. The analysis compares binding characteristics of conventional antibodies and single-domain antibodies (sdAbs) targeting both protein- and peptide antigens. We find clear patterns in the number antibody-antigen contacts and amino acid frequencies in the paratope. The direct comparison of sdAbs and conventional antibodies helps elucidate the mechanisms employed by sdAbs to compensate for their smaller size and the fact that they harbor only half the number of complementarity-determining regions compared to conventional antibodies. Furthermore, we pinpoint antibody interface hotspot residues that are often found at the binding interface and the amino acid frequencies at these positions. These findings have direct potential applications in antibody engineering and the design of improved antibody libraries.