Discovery Of Antibodies Research Articles

Language model-based B cell receptor sequence embeddings can effectively encode receptor specificity.

High throughput sequencing of B cell receptors (BCRs) is increasingly applied to study the immense diversity of antibodies. Learning biologically meaningful embeddings of BCR sequences is beneficial for predictive modeling. Several embedding methods have been developed for BCRs, but no direct performance benchmarking exists. Moreover, the impact of the input sequence length and paired-chain information on the prediction remains to be explored. We evaluated the performance of multiple embedding models to predict BCR sequence properties and receptor specificity. Despite the differences in model architectures, most embeddings effectively capture BCR sequence properties and specificity. BCR-specific embeddings slightly outperform general protein language models in predicting specificity. In addition, incorporating full-length heavy chains and paired light chain sequences improves the prediction performance of all embeddings. This study provides insights into the properties of BCR embeddings to improve downstream prediction applications for antibody analysis and discovery.

Difficult-to-express antigen generation through a co-expression and disassociation methodology.

Extracellular domain (ECD) antigens are crucial components for antibody discovery, in vitro assays, and epitope mapping during therapeutical antibody development. Oftentimes, those antigens are difficult to produce while retaining the biologic function/activity upon extracellular secretion in commonly used expression systems. We have developed an effective method to cope with the challenge of generating quality antigen ECDs. In this method, a monoclonal antibody (Mab) or antibody fragment antigen-binding (Fab) region acts as a "chaperone" to stabilize the antigen ECD through forming an antibody:antigen complex. This methodology includes transient co-expression of the complex in Chinese hamster ovary cells and then dissociation of the purified complex into individual components by low pH treatment in the presence of arginine. The antigen is then separated from the chaperone on a preparative size exclusion chromatography (pSEC) followed by an optional affinity chromatography process to remove residual Mab or Fab. We demonstrate this co-expression/disassociation methodology on two difficult-to-express antigen ECDs from cluster-of-differentiation/cytokine family and were successful in producing stable, biologically active antigens when the common methods using Histidine-tagged and/or Fc-fused protein failed. This can be applied as a general approach for antigen production if a Mab or binding partner is available.

Development of LIBRA-seq for the guinea pig model system as a tool for the evaluation of antibody responses to multivalent HIV-1 vaccines.

Consistent elicitation of serum antibody responses that neutralize diverse clades of HIV-1 remains a primary goal of HIV-1 vaccine research. Prior work has defined key features of soluble HIV-1 Envelope (Env) immunogen cocktails that influence the neutralization breadth and potency of multivalent vaccine-elicited antibody responses including the number of Env strains in the regimen. We designed immunization groups that consisted of different numbers of SOSIP Env strains to be used in a cocktail immunization strategy: the smallest cocktail (group 2) consisted of a set of two Env strains, which were a subset of the three Env strains that made up group 3, which, in turn, were a subset of the six Env strains that made up group 4. Serum neutralizing titers were modestly broader in guinea pigs that were immunized with a cocktail of three Envs compared to cocktails of two and six, suggesting that multivalent Env immunization could provide a benefit but may be detrimental when the cocktail size is too large. We then adapted the LIBRA-seq platform for antibody discovery to be compatible with guinea pigs, and isolated several tier 2 neutralizing monoclonal antibodies. Three antibodies isolated from two separate guinea pigs were similar in their gene usage and CDR3s, establishing evidence for a guinea pig public clonotype elicited through vaccination. Taken together, this work investigated multivalent HIV-1 Env immunization strategies and provides a novel methodology for screening guinea pig B cell receptor antigen specificity at a high-throughput level using LIBRA-seq.IMPORTANCEMultivalent vaccination with soluble Env immunogens is at the forefront of HIV-1 vaccination strategies but little is known about the influence of the number of Env strains included in vaccine cocktails. Our results suggest that adding more strains is sometimes beneficial but may be detrimental when the number of strains is too high. In addition, we adapted the LIBRA-seq platform to be compatible with guinea pig samples and isolated several tier 2 neutralizing monoclonal antibodies, some of which share V and J gene usage and >70% CDR3 identity, thus establishing the existence of public clonotypes in guinea pigs elicited through vaccination.

Matrixed CDR grafting: A neoclassical framework for antibody humanization and developability

Discovery and optimization of a biotherapeutic monoclonal antibody requires a careful balance of target engagement and physicochemical developability properties. To take full advantage of the sequence diversity provided by different antibody discovery platforms, a rapid and reliable process for humanization of antibodies from nonhuman sources is required. Canonically, maximizing homology of the human variable region (V-region) to the original germline was believed to result in preservation of binding, often without much consideration for inherent molecular properties. We expand on this approach by grafting the complementary determining regions (CDRs) of a mouse anti-LAG3 antibody into an extensive matrix of human variable heavy chain (VH) and variable light chain (VL) framework regions with substantially broader sequence homology to assess the impact on complementary determining region-framework compatibility through progressive evaluation of expression, affinity, biophysical developability, and function. Specific VH and VL framework sequences were associated with major expression and purification phenotypes. Greater VL sequence conservation was correlated with retained or improved affinity. Analysis of grafts that bound the target demonstrated that initial developability criteria were significantly impacted by VH, but not VL. In contrast, cell binding and functional characteristics were significantly impacted by VL, but not VH. Principal component analysis of all factors identified multiple grafts that exhibited more favorable antibody properties, notably with nonoptimal sequence conservation. Overall, this study demonstrates that modern throughput systems enable a more thorough, customizable, and systematic analysis of graft-framework combinations, resulting in humanized antibodies with improved global properties that may progress through development more quickly and with a greater probability of success.

Towards the Application of a Label-Free Approach for Anti-CD47/PD-L1 Bispecific Antibody Discovery.

The engineering of bispecific antibodies that exhibit optimal affinity and functional activity presents a significant scientific challenge. To tackle this, investigators employ an assortment of protein assay techniques, such as label-free interaction methodologies, which offer rapidity and convenience for the evaluation of extensive sample sets. These assays yield intricate data pertaining to the affinity towards target antigens and Fc-receptors, instrumental in predicting cellular test outcomes. Nevertheless, the fine-tuning of affinity is of paramount importance to mitigate potential adverse effects while maintaining efficient obstruction of ligand-receptor interactions. In this research, biolayer interferometry (BLI) was utilized to probe the functional characteristics of bispecific antibodies targeting cluster of differentiation 47 (CD47) and programmed death-ligand 1 (PD-L1) antigens, encompassing affinity, concurrent binding to two disparate antigens, and the inhibition of ligand-receptor interactions. The findings derived from BLI were juxtaposed with data from in vitro signal regulatory protein-α (SIRP-α)/CD47 blockade reporter bioassays for two leading bispecific antibody candidates, each demonstrating distinct affinity to CD47.

Discovery and characterization of cross-reactive intrahepatic antibodies in severe alcoholic hepatitis.

The pathogenesis of antibodies in severe alcoholic hepatitis (SAH) remains unknown. We analyzed immunoglobulins (Ig) in explanted livers from SAH patients (n=45) undergoing liver transplantation and tissues from corresponding healthy donors (HD, n=10) and found massive deposition of IgG and IgA isotype antibodies associated with complement fragment C3d and C4d staining in ballooned hepatocytes in SAH livers. Ig extracted from SAH livers, but not patient serum exhibited hepatocyte killing efficacy. Employing human and Escherichia coli K12 proteome arrays, we profiled the antibodies extracted from explanted SAH, livers with other diseases, and HD livers. Compared with their counterparts extracted from livers with other diseases and HD, antibodies of IgG and IgA isotypes were highly accumulated in SAH and recognized a unique set of human proteins and E. coli antigens. Further, both Ig- and E. coli-captured Ig from SAH livers recognized common autoantigens enriched in several cellular components including cytosol and cytoplasm (IgG and IgA), nucleus, mitochondrion, and focal adhesion (IgG). Except IgM from primary biliary cholangitis livers, no common autoantigen was recognized by Ig- and E. coli-captured Ig from livers with other diseases. These findings demonstrate the presence of cross-reacting anti-bacterial IgG and IgA autoantibodies in SAH livers.

Discovery and characterization of cross-reactive intrahepatic antibodies in severe alcoholic hepatitis

The pathogenesis of antibodies in severe alcoholic hepatitis (SAH) remains unknown. We analyzed immunoglobulins (Ig) in explanted livers from SAH patients (n=45) undergoing liver transplantation and tissues from corresponding healthy donors (HD, n=10) and found massive deposition of IgG and IgA isotype antibodies associated with complement fragment C3d and C4d staining in ballooned hepatocytes in SAH livers. Ig extracted from SAH livers, but not patient serum exhibited hepatocyte killing efficacy. Employing human and Escherichia coli K12 proteome arrays, we profiled the antibodies extracted from explanted SAH, livers with other diseases, and HD livers. Compared with their counterparts extracted from livers with other diseases and HD, antibodies of IgG and IgA isotypes were highly accumulated in SAH and recognized a unique set of human proteins and E. coli antigens. Further, both Ig- and E. coli-captured Ig from SAH livers recognized common autoantigens enriched in several cellular components including cytosol and cytoplasm (IgG and IgA), nucleus, mitochondrion, and focal adhesion (IgG). Except IgM from primary biliary cholangitis livers, no common autoantigen was recognized by Ig- and E. coli-captured Ig from livers with other diseases. These findings demonstrate the presence of cross-reacting anti-bacterial IgG and IgA autoantibodies in SAH livers.

Busting the Breast Cancer with AstraZeneca’s Gefitinib

Breast cancer is the most common cancer diagnosed in women, and in 2020, there were 684, 996 deaths due to this disease. Epidermal growth factor receptors (EGFRs) and their respective ligands have been blamed for the pathogenesis and resistance to treatment in specific breast cancer cases. With EGFR having four homologues: EGFR1, EGFR2, EGFR3, and EGFR4, in-depth understanding of EGFR biology led to the discovery of small-molecule inhibitors and antibodies against this receptor. Gefitinib (GEF), a tyrosine kinase inhibitor of EGFR1, possesses a vast potential for treatment against breast cancer and is supported by a multiplicity of experiments. Unfortunately, in clinical trials, GEF did not show the outcomes expected with complete response and disease progress. This is due to incomplete understanding of the molecular mechanisms involved in EGFR signaling and endocrine sensitivity. Hence, additional in-depth experiments are needed regarding various molecular pathways and crosstalk pathways to comprehend GEF’s action mechanism thoroughly in breast cancer patients. In this review, the role of EGFR in the development and pathogenesis of breast cancer and the pharmacokinetics and pharmacotherapy of GEF for the treatment of breast cancer have been elaborated. Nanomedicines synthesized with GEF have shown positive experimental response, paving a promising path for GEF against breast cancer.

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.

Paraneoplastic Neurologic Syndromes.

Progress is ongoing in understanding paraneoplastic neurologic disorders, with new syndromes and antibodies being described and more detailed evidence available to guide workup for diagnosis and treatment to improve outcomes. Many excellent reviews have summarized the molecular features of different antibodies, but this article emphasizes the clinical features of each syndrome that may help guide initial diagnosis and treatment, which often should occur before an antibody or cancer is found to confirm the diagnosis. Recent findings include updated diagnostic criteria with validated sensitivity and specificity, discovery of novel antibodies, and clinical findings that increase the likelihood of an underlying paraneoplastic disorder. Suggestive syndromes that have been recently identified include faciobrachial dystonic seizures and pilomotor auras in anti-leucine-rich glioma inactivated protein 1 encephalitis, extreme delta brush on EEG in N-methyl-d-aspartate (NMDA)-receptor encephalitis, déjà vu aura in anti-glutamic acid decarboxylase 65 (GAD65) encephalitis, and sleep disturbances in several disorders. In addition, there is confirmed utility of brain positron emission tomography (PET) and CSF markers, including carcinoembryonic antigen and oligoclonal bands, as well as improved tests for the presence of leptomeningeal cancer cells in CSF. Associations of cancer immunotherapies with paraneoplastic syndromes and herpes simplex virus encephalitis (and COVID-19) with NMDA-receptor encephalitis have been described. All neurologists should be aware of advances regarding paraneoplastic neurologic syndromes, as patients can present with a wide variety of neurologic symptoms and earlier diagnosis and treatment can improve outcomes.

APNmAb005, an anti‐tau antibody targeting synaptic tau oligomers, in Phase 1 for treatment of Alzheimer’s Disease and primary tauopathies

AbstractBackgroundAccumulation of tau oligomers at the synapses in Alzheimer’s disease (AD) disrupts neuronal activities and leads to neurodegeneration. Tau immunotherapy has been actively pursued as a treatment for tauopathies in the past decade. Despite several failed Phase 2 trials of N‐terminus binding antibodies a variety of tau epitopes are being investigated at different stages of clinical trials. APNmAb005 is a humanized monoclonal antibody designed to block synaptic toxicity elicited by tau oligomers.MethodAPNmAb005 was selected as a clinical candidate from APRINOIA’s antibody discovery platform based on its unique binding properties to pathological tau in vitro immunoassays as well in vivo efficacy studies in rTg4510 mice. Humanized APNmAb005 is in IgG4 backbone and the production was scaled up successfully with good stability and solubility to support preclinical and clinical development. GLP safety study was conducted in cynomolgus monkeys. A Phase 1a randomized, double‐blind, placebo‐controlled study of APNmAb005 in healthy subjects is underway.ResultEpitope‐mapping study reveals that APNmAb005 binds to a conformation‐dependent epitope in the mid‐region, which results in a preferable recognition of pathological tau in early Braak stage (III‐IV) AD brains as well as tau aggregates in astrocytes and oligodendrocytes in brains from progressive supranuclear palsy (PSP) patients but not monomeric tau in normal brains. APNmAb005 blocks in vitro tau seeding at nanomolar concentrations and rescues neuronal loss in rTG4510 mice. Four‐week repeat dosing toxicology study shows no observed adverse effects.ConclusionAPNmAb005 is a conformation selective antibody that binds to tau oligomers and aggregates preferentiually in synapses in pathological brain tissues and inhibits tau spreading. The mid‐region conformation dependent epitope is distinct from all other tau antibodies currently in clinical development. APNmAb005 offers a unique tau immunotherapy for neurodegenerative disorders and is currently in Phase 1 to evaluate its safety and tolerability.

140P Eliciting mAbs targeting MHC-bound peptides with a novel antibody discovery platform

140P Eliciting mAbs targeting MHC-bound peptides with a novel antibody discovery platform

An image-based Abplex method for high-throughput GPCRs antibody discovery.

As the field of antibody therapeutics advances rapidly, membrane proteins, particularly G protein-coupled receptors (GPCRs), have emerged as highly sought-after drug targets. However, the challenges associated with extracting membrane proteins have created a demand for effective antibody screening systems targeting these proteins. In this study, we propose developing an innovative antibody screening strategy (Abplex) based on high-content imaging. This approach leverages intact cells that express target membrane proteins, facilitating the presentation of proteins in their native conformation. Furthermore, it acquires both specific and non-specific binding signals in a single well, thereby bolstering the robustness of the outcomes. The technique involves just one step and can be completed within 50 min, enabling the analysis of a single sample in just one second. The amalgamation of dependable experimental findings, a simplified workflow, reduced hands-on time, and a swift analytical pace positions our method for superior throughput and precision when juxtaposed with traditional techniques such as CbELISA and FACS. Moreover, we introduce the concept of cell barcoding, wherein cells are labeled with different fluorescence spatial patterns. This feature allows for multiplexed detection to meet the needs of various experiments. The characteristics of Abplex promise to expedite GPCR-targeting antibody discovery, advance therapeutics and enable new disease treatments.

Deciphering the antigen specificities of antibodies by clustering their complementarity determining region sequences.

Determining antigen and epitope specificity is an essential step in the discovery of therapeutic antibodies as well as in the analysis adaptive immune responses to disease or vaccination. Despite extensive efforts, deciphering antigen specificity solely from BCR amino acid sequence remains a challenging task, requiring a combination of experimental and computational approaches. Here, we describe and experimentally validate a simple and straightforward approach for grouping antibodies that share antigen and epitope specificities based on their CDR sequence similarity. This approach allows us to identify the specificities of a large number of antibodies whose antigen targets are unknown, using a small fraction of antibodies with well-annotated binding specificities.

Invited Symposium 3: Pediatric Neuropsychology Medical Advances and Life Span Outcomes

Abstract & Learning Objectives:Medical advances continue to improve the outlook for pediatric patients with a variety of acquired and congenital medical conditions. Such critical advances have an impact on lifespan outcomes for affected individuals. Neuropsychology plays a critical role in evaluating outcomes and informing clinical care for pediatric patients, with an increasing role in prevention. Neuropsychologists are essential members of interdisciplinary teams and ongoing medical management. Our symposium will present examples of the latest progress made over the last decade in the areas of sickle cell disease, demyelinating disorders, congenital heart disease, and cancer. Highlights include recent research on neurocognitive surveillance for pediatric patients with sickle cell disease including identification of risk and resilience factors and ways to reduce cognitive decline; discovery of the anti-MOG antibody in patients with demyelinating disorders and associated neuropsychological outcomes; advances in the understanding of congenital heart disease, the latest initiatives in the field, and discussion of neuropsychology’s role in the care of these patients; and advances in targeted therapies for childhood cancer, risks associated with cancer and its treatment into adulthood, and an application of a developmental, lifespan approach in the care of childhood cancer survivors. Following each presentation, there will be an opportunity for discussion and questions. Upon conclusion of this course, learners will be able to: 1.Describe recent advances in medical care for pediatric sickle cell disease, demyelinating disorders, congenital heart disease, and cancer2.Explain long-term neuropsychological outcomes in pediatric medical disorders3.Assess the role of neuropsychology in advancing the field across pediatric medical disorders

IgLM: Infilling language modeling for antibody sequence design

Discovery and optimization of monoclonal antibodies for therapeutic applications relies on large sequence libraries but is hindered by developability issues such as low solubility, high aggregation, and high immunogenicity. Generative language models, trained on millions of protein sequences, are a powerful tool for the on-demand generation of realistic, diverse sequences. We present the Immunoglobulin Language Model (IgLM), a deep generative language model for creating synthetic antibody libraries. Compared with prior methods that leverage unidirectional context for sequence generation, IgLM formulates antibody design based on text-infilling in natural language, allowing it to re-design variable-length spans within antibody sequences using bidirectional context. We trained IgLM on 558 million (M) antibody heavy- and light-chain variable sequences, conditioning on each sequence's chain type and species of origin. We demonstrate that IgLM can generate full-length antibody sequences from a variety of species and its infilling formulation allows it to generate infilled complementarity-determining region (CDR) loop libraries with improved in silico developability profiles. A record of this paper's transparent peer review process is included in the supplemental information.

Insights into next generation sequencing guided antibody selection strategies

Therapeutic antibody discovery often relies on in-vitro display methods to identify lead candidates. Assessing selected output diversity traditionally involves random colony picking and Sanger sequencing, which has limitations. Next-generation sequencing (NGS) offers a cost-effective solution with increased read depth, allowing a comprehensive understanding of diversity. Our study establishes NGS guidelines for antibody drug discovery, demonstrating its advantages in expanding the number of unique HCDR3 clusters, broadening the number of high affinity antibodies, expanding the total number of antibodies recognizing different epitopes, and improving lead prioritization. Surprisingly, our investigation into the correlation between NGS-derived frequencies of CDRs and affinity revealed a lack of association, although this limitation could be moderately mitigated by leveraging NGS clustering, enrichment and/or relative abundance across different regions to enhance lead prioritization. This study highlights NGS benefits, offering insights, recommendations, and the most effective approach to leverage NGS in therapeutic antibody discovery.

Rapid discovery of high-affinity antibodies via massively parallel sequencing, ribosome display and affinity screening

Developing therapeutic antibodies is laborious and costly. Here we report a method for antibody discovery that leverages the Illumina HiSeq platform to, within 3 days, screen in the order of 108 antibody–antigen interactions. The method, which we named ‘deep screening’, involves the clustering and sequencing of antibody libraries, the conversion of the DNA clusters into complementary RNA clusters covalently linked to the instrument’s flow-cell surface on the same location, the in situ translation of the clusters into antibodies tethered via ribosome display, and their screening via fluorescently labelled antigens. By using deep screening, we discovered low-nanomolar nanobodies to a model antigen using 4 × 106 unique variants from yeast-display-enriched libraries, and high-picomolar single-chain antibody fragment leads for human interleukin-7 directly from unselected synthetic repertoires. We also leveraged deep screening of a library of 2.4 × 105 sequences of the third complementarity-determining region of the heavy chain of an anti-human epidermal growth factor receptor 2 (HER2) antibody as input for a large language model that generated new single-chain antibody fragment sequences with higher affinity for HER2 than those in the original library.

Discovery of a human monoclonal antibody that cross-neutralizes venom phospholipase A2s from three different snake genera

Despite the considerable global impact of snakebite envenoming, available treatments remain suboptimal. Here, we report the discovery of a broadly-neutralizing human monoclonal antibody, using a phage display-based cross-panning strategy, capable of reducing the cytotoxic effects of venom phospholipase A2s from three different snake genera from different continents. This highlights the potential of utilizing monoclonal antibodies to develop more effective, safer, and globally accessible polyvalent antivenoms that can be widely used to treat snakebite envenoming.

Widespread impact of immunoglobulin V-gene allelic polymorphisms on antibody reactivity

The ability of the human immune system to generate antibodies to any given antigen can be strongly influenced by immunoglobulin V-gene allelic polymorphisms. However, previous studies have provided only limited examples. Therefore, the prevalence of this phenomenon has been unclear. By analyzing >1,000 publicly available antibody-antigen structures, we show that many V-gene allelic polymorphisms in antibody paratopes are determinants for antibody binding activity. Biolayer interferometry experiments further demonstrate that paratope allelic polymorphisms on both heavy and light chains often abolish antibody binding. We also illustrate the importance of minor V-gene allelic polymorphisms with low frequency in several broadly neutralizing antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus. Overall, this study not only highlights the pervasive impact of V-gene allelic polymorphisms on antibody binding but also provides mechanistic insights into the variability of antibody repertoires across individuals, which in turn have important implications for vaccine development and antibody discovery.