Surface patches induce nonspecific binding and phase separation of antibodies.

Nonspecific interactions are a key challenge in the successful development of therapeutic antibodies. The tendency for nonspecific binding of antibodies is often difficult to reduce by rational design, and instead, it is necessary to rely on comprehensive screening campaigns. To address this issue, we performed a systematic analysis of the impact of surface patch properties on antibody nonspecificity using a designer antibody library as a model system and single-stranded DNA as a nonspecificity ligand. Using an in-solution microfluidic approach, we find that the antibodies tested bind to single-stranded DNA with affinities as high as KD = 1 µM. We show that DNA binding is driven primarily by a hydrophobic patch in the complementarity-determining regions. By quantifying the surface patches across the library, the nonspecific binding affinity is shown to correlate with a trade-off between the hydrophobic and total charged patch areas. Moreover, we show that a change in formulation conditions at low ionic strengths leads to DNA-induced antibody phase separation as a manifestation of nonspecific binding at low micromolar antibody concentrations. We highlight that phase separation is driven by a cooperative electrostatic network assembly mechanism of antibodies with DNA, which correlates with a balance between positive and negative charged patches. Importantly, our study demonstrates that both nonspecific binding and phase separation are controlled by the size of the surface patches. Taken together, these findings highlight the importance of surface patches and their role in conferring antibody nonspecificity and its macroscopic manifestation in phase separation.

Mechanism of an RBM-targeted rabbit monoclonal antibody 9H1 neutralizing SARS-CoV-2.

The COVID-19 pandemic, caused by SARS-CoV-2, has led to over 750 million infections and 6.8 million deaths worldwide since late 2019. Due to the continuous evolution of SARS-CoV-2, many significant variants have emerged, creating ongoing challenges to the prevention and treatment of the pandemic. Therefore, the study of antibody responses against SARS-CoV-2 is essential for the development of vaccines and therapeutics. Here we perform single particle cryo-electron microscopy (cryo-EM) structure determination of a rabbit monoclonal antibody (RmAb) 9H1 in complex with the SARS-CoV-2 wild-type (WT) spike trimer. Our structural analysis shows that 9H1 interacts with the receptor-binding motif (RBM) region of the receptor-binding domain (RBD) on the spike protein and by directly competing with angiotensin-converting enzyme 2 (ACE2), it blocks the binding of the virus to the receptor and achieves neutralization. Our findings suggest that utilizing rabbit-derived mAbs provides valuable insights into the molecular interactions between neutralizing antibodies and spike proteins and may also facilitate the development of therapeutic antibodies and expand the antibody library.

A phase I study assessing the safety, tolerability, immunogenicity, and low-density lipoprotein cholesterol-lowering activity of immunotherapeutics targeting PCSK9.

PURPOSE: AT04A and AT06A are two AFFITOPE® peptide vaccine candidates being developed for the treatment of hypercholesterolemia by inducing proprotein convertase subtilisin/kexin type 9 (PCSK9)-specific antibodies. This study aimed to investigate safety, tolerability, antibody development, and reduction of low-density lipoprotein cholesterol (LDLc) following four subcutaneous immunizations. METHODS: This phase I, single-blind, randomized, placebo-controlled study was conducted in a total of 72 healthy subjects with a mean fasting LDLc level at baseline of 117.1 mg/dL (range 77-196 mg/dL). Each cohort enrolled 24 subjects to receive three priming immunizations at weeks 0, 4, and 8 and to receive a single booster immunization at week 60 of either AT04A, AT06A, or placebo. In addition to safety (primary objective), the antigenic peptide- and PCSK9-specific antibody response and the impact on LDLc were evaluated over a period of 90 weeks. RESULTS: The most common systemic treatment-related adverse events (AEs) reported were fatigue, headache, and myalgia in 75% of subjects in the AT06A group and 58% and 46% of subjects in the placebo and AT04A groups, respectively. Injection site reactions (ISR) representing 63% of all treatment-emergent adverse events (TEAEs), were transient and mostly of mild or moderate intensity and rarely severe (3%). Both active treatments triggered a robust, long-lasting antibody response towards the antigenic peptides used for immunization that optimally cross-reacted with the target epitope on PCSK9. In the AT04A group, a reduction in serum LDLc was observed with a mean peak reduction of 11.2% and 13.3% from baseline compared to placebo at week 20 and 70 respectively, and over the whole study period, the mean LDLc reduction for the AT04A group vs. placebo was -7.2% (95% CI [-10.4 to -3.9], P < 0.0001). In this group, PCSK9 target epitope titers above 50 were associated with clinically relevant LDLc reductions with an individual maximal decrease of 39%. CONCLUSIONS: Although both AT04A and AT06 were safe and immunogenic, only AT04A demonstrated significant LDLc-lowering activity, justifying further development. TRIAL REGISTRATION: EudraCT: 2015-001719-11. ClinicalTrials.gov Identifier: NCT02508896.

Longitudinal Antibody Responses in People Who Inject Drugs Infected With Similar Human Immunodeficiency Virus Strains.

BACKGROUND: Multiple factors influence the human immunodeficiency virus (HIV) antibody response produced during natural infection, leading to responses that can vary in specificity, strength, and breadth. METHODS: People who inject drugs identified as recently infected with HIV (n = 23) were analyzed for clustering of their viral sequences (genetic distance, <2%). Longitudinal antibody responses were identified for neutralizing antibody (Nab) potential, and differences in antibody subclass, specificity, and Fc receptor ligation using pseudovirus entry and multiplexed Fc array assays, respectively. Responses were analyzed for differences between subject groups, defined by similarity in the sequence of the infecting virus. RESULTS: Viral sequences from infected individuals were grouped into 3 distinct clusters with 7 unclustered individuals. Subjects in cluster 1 generally had lower antibody response magnitudes, except for antibodies targeting the V1/V2 region. Subjects in clusters 2 and 3 typically had higher antibody response magnitudes, with the Fv specificity of cluster 2 favoring gp140 recognition. NAb responses differed significantly between clusters for 3 of 18 pseudoviruses examined (P < .05), but there were no differences in overall NAb breadth (P = .62). DISCUSSION: These data demonstrate that individuals infected with similar viral strains can generate partially similar antibody responses, but these do not drastically differ from those in individuals infected with relatively unrelated strains.

DOTAD: A Database of Therapeutic Antibody Developability.

The development of therapeutic antibodies is an important aspect of new drug discovery pipelines. The assessment of an antibody's developability-its suitability for large-scale production and therapeutic use-is a particularly important step in this process. Given that experimental assays to assess antibody developability in large scale are expensive and time-consuming, computational methods have been a more efficient alternative. However, the antibody research community faces significant challenges due to the scarcity of readily accessible data on antibody developability, which is essential for training and validating computational models. To address this gap, DOTAD (Database Of Therapeutic Antibody Developability) has been built as the first database dedicated exclusively to the curation of therapeutic antibody developability information. DOTAD aggregates all available therapeutic antibody sequence data along with various developability metrics from the scientific literature, offering researchers a robust platform for data storage, retrieval, exploration, and downloading. In addition to serving as a comprehensive repository, DOTAD enhances its utility by integrating a web-based interface that features state-of-the-art tools for the assessment of antibody developability. This ensures that users not only have access to critical data but also have the convenience of analyzing and interpreting this information. The DOTAD database represents a valuable resource for the scientific community, facilitating the advancement of therapeutic antibody research. It is freely accessible at http://i.uestc.edu.cn/DOTAD/ , providing an open data platform that supports the continuous growth and evolution of computational methods in the field of antibody development.

A mini review on recent progress of microfluidic systems for antibody development.

Objectives: Antibody is specific reagent that be utilized in various field of biomedical research. Monoclonal antibodies are mostly produced using two common techniques namely hybridoma and antibody engineering, which suffer from some limitations such as boring screening procedures, long production time, low efficacy and a degree of automation. To address these limitations, various microfluidics techniques have been developed for the antibody isolation and screening. Methods: This study specifically investigates nearly recent reports published in peer-reviewed journals indexed in various databases including Web of Science, Scopus, PubMed, Google Scholar, and Science Direct. Results: In this study, we identified a total of seventy papers from a pool of 130 articles. These papers focus on the application of three major groups of microfluidic platforms, namely valves, microwells, and droplets, in the development of antibodies using hybridoma method and phage display technology. We provide a summary of these applications and also discuss the key findings in this field. Additionally, we illustrate our discussion with several examples to enhance understanding. Conclusions: Microfluidics has the potential to serve as a valuable tool in streamlining complex laboratory procedures involved in antibody discovery. However, it is important to note that microfluidics is limited to laboratory settings. Further enhancements are needed to address existing challenges and to make microfluidics a reliable, accurate, and cost-effective tool for antibody discovery.

Shrimp hemocyanin elicits a potent humoral response in mammals and is favorable to hapten conjugation.

Conjugation to a carrier protein is essential to give rise to the antigenicity of hapten. Three carrier proteins e.g. KLH (Keyhole Limpet hemocyanin), BSA (bovine serum albumin), and OVA (Ovalbumin) were used mostly. KLH is advantageous to the others, majorly owing to its strong immunogenicity and limited usage in other biological assays. However, the cost of obtaining Keyhole Limpet is high and the solubility of KLH is not as well as the other carriers, especially after hapten conjugation. Here, we extracted the shrimp hemocyanin (SHC) from Litopenaeus vannamei (L. vannamei), which is a commonly sea product worldwide. The high pure SHC could be acquired by two-step purification, with a production yield of > 1 g proteins (98% pure) per 1 kg shrimp. Compared to KLH, the peptide-SHC conjugates exhibit higher solubility after hapten conjugation. Meanwhile, compared with KLH, SHC induces comparable antibody production efficiency in mammals, with or without conjugation. Furthermore, rabbit polyclonal antibodies or mouse monoclonal antibodies were generated by immunizing SHC-peptide conjugates, and the subsequent antibodies were confirmed to be used in western blot, immunofluorescence and immunohistochemistry. Therefore, we demonstrated that SHC may be used as a substitute for KLH in future antibody and vaccine development.

Early determination of potential critical quality attributes of therapeutic antibodies in developability studies through surface plasmon resonance-based relative binding activity assessment.

Precise measurement of the binding activity changes of therapeutic antibodies is important to determine the potential critical quality attributes (CQAs) in developability assessment at the early stage of antibody development. Here, we report a surface plasmon resonance (SPR)-based relative binding activity method, which incorporates both binding affinity and binding response and allows us to determine relative binding activity of antibodies with high accuracy and precision. We applied the SPR-based relative binding activity method in multiple forced degradation studies of antibody developability assessment. The current developability assessment strategy provided comprehensive, precise characterization of antibody binding activity in the stability studies, enabling us to perform correlation analysis and establish the structure-function relationship between relative binding activity and quality attributes. The impact of a given quality attribute on binding activity could be confidently determined without isolating antibody variants. We identified several potential CQAs, including Asp isomerization, Asn deamidation, and fragmentation. Some potential CQAs affected binding affinity of antibody and resulted in a reduction of binding activity. Certain potential CQAs impaired antibody binding to antigen and led to a loss of binding activity. A few potential CQAs could influence both binding affinity and binding response and cause a substantial decrease in antibody binding activity. Specifically, we identified low abundance Asn33 deamidation in the light chain complementarity-determining region as a potential CQA, in which all the stressed antibody samples showed Asn33 deamidation abundances ranging from 4.2% to 27.5% and a mild binding affinity change from 1.76 nM to 2.16 nM.

Computational and artificial intelligence-based methods for antibody development.

Due to their high target specificity and binding affinity, therapeutic antibodies are currently the largest class of biotherapeutics. The traditional largely empirical antibody development process is, while mature and robust, cumbersome and has significant limitations. Substantial recent advances in computational and artificial intelligence (AI) technologies are now starting to overcome many of these limitations and are increasingly integrated into development pipelines. Here, we provide an overview of AI methods relevant for antibody development, including databases, computational predictors of antibody properties and structure, and computational antibody design methods with an emphasis on machine learning (ML) models, and the design of complementarity-determining region (CDR) loops, antibody structural components critical for binding.

Optimizing a therapeutic humanized follicle-stimulating hormone-blocking antibody formulation by protein thermal shift assay.

Biopharmaceutical products are formulated using several Food and Drug Administration (FDA) approved excipients within the inactive ingredient limit to maintain their storage stability and shelf life. Here, we have screened and optimized different sets of excipient combinations to yield a thermally stable formulation for the humanized follicle-stimulating hormone (FSH)-blocking antibody, MS-Hu6. We used a protein thermal shift assay in which rising temperatures resulted in the maximal unfolding of the protein at the melting temperature (Tm ). To determine the buffer and pH for a stable solution, four different buffers with a pH range from 3 to 8 were screened. This resulted in maximal Tm s at pH 5.62 for Fab in phosphate buffer and at pH 6.85 for Fc in histidine buffer. Upon testing a range of salt concentrations, MS-Hu6 was found to be more stable at lower concentrations, likely due to reduced hydrophobic effects. Molecular dynamics simulations revealed a higher root-mean-square deviation with 1 mM than with 100 mM salt, indicating enhanced stability, as noted experimentally. Among the stabilizers tested, Tween 20 was found to yield the highest Tm and reversed the salt effect. Among several polyols/sugars, trehalose and sucrose were found to produce higher thermal stabilities. Finally, binding of recombinant human FSH to MS-Hu6 in a final formulation (20 mM phosphate buffer, 1 mM NaCl, 0.001% w/v Tween 20, and 260 mM trehalose) resulted in a thermal shift (increase in Tm ) for the Fab, but expectedly not in the Fc domain. Given that we used a low dose of MS-Hu6 (1 µM), the next challenge would be to determine whether 100-fold higher, industry-standard concentrations are equally stable.

Construction of a TRFIC strip for rapid and sensitive detection of Ralstoniasolanacearum.

The development of a sensitive and rapid screening method for Ralstonia solanacearum is critical for the control of tobacco wilt. In the present study, tissue homogenates of three tobacco varieties (Honda, Yunnan 87 and K326) with different resistance to R. solanacearum, were individually used as additives to the bacteria culture medium. The changes in R. solanacearum secretome were investigated and one of the most abundant secretary proteins with increased expression, polygalacturonase (PG), was selected as a marker for R. solanacearum identification. Then PG gene was cloned into E. coli, and the expressed protein was used as the immunogen to develop monoclonal antibodies. Subsequently, the monoclonal antibody against PG was coupled with synthesized polystyrene microspheres, and a rapid test strip system was developed for the detection of R. solanacearum based on time-resolved fluorescent immunochromatographic (TRFIC) method. Under optimal conditions, the detection limit of the strips could reach 72 cells/mL; while it was 422 cells/mL with a linear range from 4 × 102 to 5.12 × 104 cells/mL when testing tobacco samples, which is 1000 times lower than that of colloidal gold-labeled strips. Notably, no cross-reactivity was observed with nine tobacco-related pathogens. Finally, this TRFIC strips was applied to detect R. solanacearum existed in the tobacco and soils of fields with or without bacterial wilt. The results demonstrated that this TRFIC strips could distinguish the difference in bacterial concentration existed in tobacco and soil between the two fields. In summary, this test strip is suitable for sensitive, quick screening of R. solanacearum in tobacco.

Development and Validation of Antibodies Targeting Site-Specific Histone Methylation.

The development of specific anti-modification antibodies as research tools has revolutionized the way histone methylation is studied. Lack of stringent quality controls, however, led to the development of nonspecific antibodies, compromising their use. In this chapter, we provide a series of protocols that collectively will help those studying histone methylation to develop and thoroughly validate high-end sequence-specific and methylation-dependent antibodies.

In silico prediction of post-translational modifications in therapeutic antibodies.

Monoclonal antibodies are susceptible to chemical and enzymatic modifications during manufacturing, storage, and shipping. Deamidation, isomerization, and oxidation can compromise the potency, efficacy, and safety of therapeutic antibodies. Recently, in silico tools have been used to identify liable residues and engineer antibodies with better chemical stability. Computational approaches for predicting deamidation, isomerization, oxidation, glycation, carbonylation, sulfation, and hydroxylation are reviewed here. Although liable motifs have been used to improve the chemical stability of antibodies, the accuracy of in silico predictions can be improved using machine learning and molecular dynamic simulations. In addition, there are opportunities to improve predictions for specific stress conditions, develop in silico prediction of novel modifications in antibodies, and predict the impact of modifications on physical stability and antigen-binding.

Rare SARS-CoV-2 antibody development in cancer patients.

SARS-CoV-2 antibody development and immunity will be crucial for the further course of the pandemic. Until now, it has been assumed that patients who are infected with SARS-CoV-2 will develop antibodies as has been the case with other coronaviruses, like MERS-CoV and SARS-CoV. In the present study, we analyzed the development of antibodies in 77 patients with an oncologic diagnosis 26 days after positive RT-qPCR testing for SARS-CoV2. RT-qPCR and anti-SARS-CoV2-antibody methods from BGI (MGIEasy Magnetic Beads Virus DNA/RNA Extraction Kit) and Roche (Elecsys Anti-SARS-CoV-2 immunoassay) were used, respectively, according to the manufacturers' specifications. Surprisingly, antibody development was detected in only 6 of 77 individuals with a confirmed history of COVID-19. Despite multiple testing, the remaining patients did not show measurable antibody concentrations in subsequent tests. These results undermine the previous hypothesis that SARS-CoV2 infections are regularly associated with antibody development and cast doubt on the provided immunity to COVID-19. Understanding the adaptive and humoral response to SARS-CoV2 will play a key role in vaccine development and gaining further knowledge on the pathogenesis.

Development of antibody-dependent cell cytotoxicity function in HIV-1 antibodies.

A prerequisite for the design of an HIV vaccine that elicits protective antibodies is understanding the developmental pathways that result in desirable antibody features. The development of antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC) is particularly relevant because such antibodies have been associated with HIV protection in humans. We reconstructed the developmental pathways of six human HIV-specific ADCC antibodies using longitudinal antibody sequencing data. Most of the inferred naive antibodies did not mediate detectable ADCC. Gain of antigen binding and ADCC function typically required mutations in complementarity determining regions of one or both chains. Enhancement of ADCC potency often required additional mutations in framework regions. Antigen binding affinity and ADCC activity were correlated, but affinity alone was not sufficient to predict ADCC potency. Thus, elicitation of broadly active ADCC antibodies may require mutations that enable high-affinity antigen recognition along with mutations that optimize factors contributing to functional ADCC activity.

Nearly four decades after the human immunodeficiency virus (HIV for short) was first identified, the search for a vaccine still continues. An effective immunisation would require elements that coax the human immune system into making HIV-specific antibodies ­ the proteins that can recognise, bind to and deactivate the virus. Crucially, antibodies can also help white blood cells to target and destroy cells infected with HIV. This 'antibody-dependent cellular cytotoxicity' could be a key element of a successful vaccine, yet it has received less attention than the ability for antibodies to directly neutralize the virus. In particular, it is still unclear how antibodies develop the ability to flag HIV-infected cells for killing. Indeed, over the course of an HIV infection, an immune cell goes through genetic changes that tweak the 3D structure of the antibodies it manufactures. This process can improve the antibodies' ability to fight off the virus, but it was still unclear how it would shape antibody-dependent cellular cytotoxicity. To investigate this question, Doepker et al. retraced how the genes coding for six antibody families changed over time in an HIV-carrying individual. This revealed that antibodies could not initially trigger antibody-dependent cellular cytotoxicity. The property emerged and improved thanks to two types of alterations in the genetic sequences. One set of changes increased how tightly the antibodies could bind to the virus, targeting sections of the antibodies that can often vary. The second set likely altered the 3D structure in others ways, potentially affecting how antibodies bind the virus or how they interact with components of the immune system that help to kill HIV-infected cells. These alterations took place in segments of the antibodies that undergo less change over time. Ultimately, the findings by Doepker et al. suggest that an efficient HIV vaccine may rely on helping antibodies to evolve so they can bind more tightly to the virus and trigger cellular cytotoxicity more strongly.

Computational modeling - an approach to the development of blood grouping reagents.

Introduction: Blood group antigens are defined by an immune response that generates antibodies against a red blood cell molecule. Antibodies against these antigens can be associated with hemolytic transfusion reactions. However, difficulties can arise when developing antibodies against antigens through the use of peptide sequences alone. Three-dimensional representations (models) of the molecular structure of antigen-bearing proteins can provide valuable insights into tertiary structures and their consequent antigenicity. This can be achieved through predictive computational modeling to produce both structural and molecular dynamics models of blood group proteins.Areas covered: Authors discuss the use of molecular dynamic simulations on existing structures, as well as the use of computational modeling techniques in the development of protein models lacking preexisting data. Finally, the authors discuss specific examples of the use of computationally derived models of the MNS blood group system and its use in attempts to produce antibodies against MNS proteins.Expert opinion: Although in silico techniques have limitations, computer-based predictive models can inform the direction of research into blood group proteins. It is to be expected that as computer-based techniques grow more powerful these contributions will be even more significant.

Clinical evaluation of commercial automated SARS-CoV-2 immunoassays.

OBJECTIVE: Numerous immunoassays for detecting antibodies directed against SARS-CoV-2 have been rapidly developed and released. Validations of these have been performed with a limited number of samples. The lack of standardisation might lead to significantly different results. This study compared ten automated assays from six vendors in terms of sensitivity, specificity and reproducibility. METHODS: This study compared ten fully automated immunoassays from the following vendors: Diasorin, Epitope Diagnostics, Euroimmun, Roche, YHLO, and Snibe. The retrospective part of the study included patients with a laboratory-confirmed COVID-19 infection, and controls comprised patients with a suspected infection, in whom the disease was excluded. Furthermore, biobanked sera were taken as negative controls (n = 97). The retrospective part involved four groups: (1) laboratory-confirmed COVID-19 infection (n = 183); (1B) suspected COVID-19 infection (n = 167) without a qRT-PCR result but positive serological results from at least two different assays, and suspected COVID-19 infection due to a positive serological result from the Roche assay (n = 295); (2) biobanked sera obtained from patients before the emergence of SARS-CoV-2 (n = 97) as negative controls; and (2A) probably COVID-19-negative sera with negative serological results from at least two different assays (n = 152). RESULTS: Overall diagnostic sensitivities were: Euroimmun (IgA) 87%; Epitope Diagnostics (IgG) 83%; YHLO (IgG) 77%; Roche (IgM/IgG) 77%; Euroimmun (IgG) 75%; Diasorin (IgG) 53%; Epitope Diagnostics (IgM) 52%; Snibe (IgG) 47%; YHLO (IgM) 35%; and Snibe (IgM) 26%. Diagnostic specificities were: YHLO (IgG) 100%; Roche, 100%; Snibe (IgM/IgG) 100%; Diasorin (IgG) 97%; Euroimmun (IgG) 94%; YHLO (IgM) 94%; Euroimmun (IgA) 83%. CONCLUSION: Assays from different vendors substantially varied in terms of their performance. These findings might facilitate selection of appropriate serological assays.

On the interactions of the receptor-binding domain of SARS-CoV-1 and SARS-CoV-2 spike proteins with monoclonal antibodies and the receptor ACE2.

A new betacoronavirus named SARS-CoV-2 has emerged as a new threat to global health and economy. A promising target for both diagnosis and therapeutics treatments of the new disease named COVID-19 is the coronavirus (CoV) spike (S) glycoprotein. By constant-pH Monte Carlo simulations and the PROCEEDpKa method, we have mapped the electrostatic epitopes for four monoclonal antibodies and the angiotensin-converting enzyme 2 (ACE2) on both SARS-CoV-1 and the new SARS-CoV-2 S receptor binding domain (RBD) proteins. We also calculated free energy of interactions and shown that the S RBD proteins from both SARS viruses binds to ACE2 with similar affinities. However, the affinity between the S RBD protein from the new SARS-CoV-2 and ACE2 is higher than for any studied antibody previously found complexed with SARS-CoV-1. Based on physical chemical analysis and free energies estimates, we can shed some light on the involved molecular recognition processes, their clinical aspects, the implications for drug developments, and suggest structural modifications on the CR3022 antibody that would improve its binding affinities for SARS-CoV-2 and contribute to address the ongoing international health crisis.

A critical challenge in the development of antibody: Selecting the appropriate fragment of the target protein as an antigen based on various epitopes or similar structure.

The main challenge in the development of antibody is to select the appropriate antigen particularly when a truncated protein is used for immunization or as vaccine antigen. In previous studies, fragment selection was mainly based on epitopes and less often on the structure. Fewer studies have paid attention to the prediction of the truncated protein 3D structure and retained its similarity in the native and truncated proteins. Here we used in silico analysis to select two fragments of Pyruvate Kinase M2 (PKM2), as a tumor marker. One fragment, M-tPKM2, had a shorter sequence with one epitope although the predicted 3D structure was similar to the native PKM2. The other fragment, R-tPKM2, had a longer sequence and thus more epitopes, but had a different structure from the native PKM2. Recombinant truncated proteins were expressed in E. coli and purified via affinity chromatography. Secondary structure elements in purified proteins were determined by Circular Dichroism, then they were utilized to develop antibodies in mice. Both antigens could elicit high immune response against themselves (OD450 = 3.326 ± 0.562 for M-tPKM2; OD450 = 3.562 ± 0.110 for R-tPKM2). However, significantly higher response against PKM2 was observed among the mice immunized with M-tPKM2 (p < 0.0001 by One way ANOVA followed by Tukey's post hoc comparison). Also, the monoclonal antibody produced against the M-tPKM2 could recognize the native PKM2 in the MCF7 cells. Our finding suggested that for the purpose of designing an antigen with the ability to produce a potent antibody against the target protein, it is better to select sequences which have a similar structure in truncated and native proteins, even at the cost of having shorter sequences and fewer epitopes.

High-throughput screening of antibody variants for chemical stability: identification of deamidation-resistant mutants.

Developability assessment of therapeutic antibody candidates assists drug discovery by enabling early identification of undesirable instabilities. Rapid chemical stability screening of antibody variants can accelerate the identification of potential solutions. We describe here the development of a high-throughput assay to characterize asparagine deamidation. We applied the assay to identify a mutation that unexpectedly stabilizes a critical asparagine. Ninety antibody variants were incubated under thermal stress in order to induce deamidation and screened for both affinity and total binding capacity. Surprisingly, a mutation five residues downstream from the unstable asparagine greatly reduced deamidation. Detailed assessment by LC-MS analysis confirmed the predicted improvement. This work describes both a high-throughput method for antibody stability screening during the early stages of antibody discovery and highlights the value of broad searches of antibody sequence space.