Heavy Chain CDR3 Research Articles

Scifer: An R/Bioconductor package for large-scale integration of Sanger sequencing and flow cytometry data of index-sorted single cells

Sanger sequencing remains widely used in various experimental contexts, often in combination with flow cytometry for indexing specific cell populations. However, existing software lacks the capability to automate quality control (QC) of raw Sanger sequencing data and integrate it with flow cytometry information on a large scale. Here, we introduce scifer, an R package now available in the latest release of Bioconductor (3.20) showcasing its effectiveness in seamlessly integrating these types of data as demonstrated by analyses of B cell and T cell receptor sequences. Scifer preprocesses raw data from index sorts and immune receptor Sanger sequencing. It identifies high-quality sequences based on selected parameters, such as length, Phred scores, and heavy-chain complementarity-determining region 3 (HCDR3) quality. As a result, the quality of germline assignments is significantly increased and spurious variable gene mutations are reduced. Scifer is automated and can process thousands of sequences in less than an hour. Its output provides quality control reports, FASTA files, summarized tables, and electropherograms for manual inspection. In summary, scifer is a user-friendly software that speeds up the analysis of immune receptor repertoire sequences, offering wide applicability.

Development, physico-chemical characterization, and in vivo stability of a novel aglycosylated monoclonal antibody targeting FAM19A5

AbstractIntroducing aglycosylation into therapeutic monoclonal antibodies (mAbs) can prevent side effects associated with fragment crystallizable (Fc)-mediated effector functions. This modification induces structural changes in the heavy chain constant domain 2-constant domain 3 within Fc regions, which decreases antibody stability at acidic pH and high temperature. In this study, NS101, a novel aglycosylated mAb targeting family with sequence similarity 19, A5 (FAM19A5) for neurological diseases was evaluated with respect to its developability and in vivo stability as therapeutics. When recombinant CHO cells producing NS101 were cultivated using a fed-batch mode in a 500 L bioreactor, cell growth and mAb production profiles were consistent across three consecutive runs. NS101, thus produced, features an additional intra-disulfide bond in the heavy chain complementarity-determining region 3, contributing strong and sophisticated binding to the cryptic epitope. The melting temperature (Tm) of NS101 was lower than that of commercial glycosylated therapeutic mAbs, but NS101 showed better stability at 4 °C for 36 months. The binding affinity of NS101 to FAM19A5 and neonatal Fc receptor were comparable to those of glycosylated NS101. In addition, in three human cohort groups receiving 6, 12, and 24 mg/kg of NS101, the mean half-life was 22 days, and NS101 exhibited in vivo stability, considering that the half-lives of commercialized therapeutic mAbs and endogenous IgGs are 2–4 weeks and 21 days, respectively. Taken together, the results obtained here demonstrate that NS101, a novel aglycosylated mAb, has potential as a therapeutic agent for neurological diseases.

Germline-targeting HIV vaccination induces neutralizing antibodies to the CD4 binding site.

Eliciting potent and broadly neutralizing antibodies (bnAbs) is a major goal in HIV-1 vaccine development. Here, we describe how germline-targeting immunogen BG505 SOSIP germline trimer 1.1 (GT1.1), generated through structure-based design, engages a diverse range of VRC01-class bnAb precursors. A single immunization with GT1.1 expands CD4 binding site (CD4bs)-specific VRC01-class B cells in knock-in mice and drives VRC01-class maturation. In nonhuman primates (NHPs), GT1.1 primes CD4bs-specific neutralizing serum responses. Selected monoclonal antibodies (mAbs) isolated from GT1.1-immunized NHPs neutralize fully glycosylated BG505 virus. Two mAbs, 12C11 and 21N13, neutralize subsets of diverse heterologous neutralization-resistant viruses. High-resolution structures revealed that 21N13 targets the same conserved residues in the CD4bs as VRC01-class and CH235-class bnAbs despite its low sequence similarity (~40%), whereas mAb 12C11 binds predominantly through its heavy chain complementarity-determining region 3. These preclinical data underpin the ongoing evaluation of GT1.1 in a phase 1 clinical trial in healthy volunteers.

Generation of Chicken Antibody Libraries and Selection of Antigen Binders.

Chicken antibodies have been widely used for research and diagnostic purposes. Chicken antibodies are often cross-reactive to epitopes shared by humans, nonhuman primates, and other mammals, and can be tested in many mouse disease models, which provides an advantage for their preclinical study and evaluation. In addition, the variable region of chicken antibodies has unique structural characteristics, including noncanonical cysteine residues in the heavy chain complementarity-determining region (CDR)3 and a long heavy chain CDR3, which together with a short light chain CDR enable the formation of unconventional antibody paratopes. As chickens have single functional copies of the V H and J H genes, and the somatic gene conversion process usually involves D H genes, all functional VDJ gene fragments can be obtained from the B-cell repertoire using a single PCR primer set, without any primer bias. As for the light chain, chickens only have a V λ light chain, composed of a single V λ and J λ gene pair. Therefore, the chicken light chain repertoire can also be accurately amplified using a single primer set. This unbiased reconstitution of the chicken B-cell repertoire provides a great advantage not only in the construction of phage display libraries but also for the in silico selection of antigen binders from a virtual B-cell receptor repertoire. Here, we introduce the use of chicken antibodies in research, diagnostic, and therapeutic fields. In addition, the chromosomal organization of chicken immunoglobulin genes and its diversification mechanisms for shaping the antibody repertoire are also discussed.

De novo generation of SARS-CoV-2 antibody CDRH3 with a pre-trained generative large language model

Artificial Intelligence (AI) techniques have made great advances in assisting antibody design. However, antibody design still heavily relies on isolating antigen-specific antibodies from serum, which is a resource-intensive and time-consuming process. To address this issue, we propose a Pre-trained Antibody generative large Language Model (PALM-H3) for the de novo generation of artificial antibodies heavy chain complementarity-determining region 3 (CDRH3) with desired antigen-binding specificity, reducing the reliance on natural antibodies. We also build a high-precision model antigen-antibody binder (A2binder) that pairs antigen epitope sequences with antibody sequences to predict binding specificity and affinity. PALM-H3-generated antibodies exhibit binding ability to SARS-CoV-2 antigens, including the emerging XBB variant, as confirmed through in-silico analysis and in-vitro assays. The in-vitro assays validate that PALM-H3-generated antibodies achieve high binding affinity and potent neutralization capability against spike proteins of SARS-CoV-2 wild-type, Alpha, Delta, and the emerging XBB variant. Meanwhile, A2binder demonstrates exceptional predictive performance on binding specificity for various epitopes and variants. Furthermore, by incorporating the attention mechanism inherent in the Roformer architecture into the PALM-H3 model, we improve its interpretability, providing crucial insights into the fundamental principles of antibody design.

Systematic characterization of immunoglobulin loci and deep sequencing of the expressed repertoire in the Atlantic cod (Gadus morhua)

BackgroundThe Atlantic cod is a prolific species in the Atlantic, despite its inconsistent specific antibody response. It presents a peculiar case within vertebrate immunology due to its distinct immune system, characterized by the absence of MHCII antigen presentation pathway, required for T cell-dependent antibody responses. Thorough characterisation of immunoglobulin loci and analysis of the antibody repertoire is necessary to further our understanding of the Atlantic cod’s immune response on a molecular level.ResultsA comprehensive search of the cod genome (gadmor3.0) identified the complete set of IgH genes organized into three sequential translocons on chromosome 2, while IgL genes were located on chromosomes 2 and 5. The Atlantic cod displayed a moderate germline V gene diversity, comprising four V gene families for both IgH and IgL, each with distinct chromosomal locations and organizational structures. 5’RACE sequencing revealed a diverse range of heavy chain CDR3 sequences and relatively limited CDR3 diversity in light chains. The analysis highlighted a differential impact of V-gene germline CDR3 length on receptor CDR3 length between heavy and light chains, underlining different recombination processes.ConclusionsThis study reveals that the Atlantic cod, despite its inconsistent antibody response, maintains a level of immunoglobulin diversity comparable to other fish species. The findings suggest that the extensive recent duplications of kappa light chain genes do not result in increased repertoire diversity. This research provides a comprehensive view of the Atlantic cod's immunoglobulin gene organization and repertoire, necessary for future studies of antibody responses at the molecular level.

Vaccination induces broadly neutralizing antibody precursors to HIV gp41

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.

Human CD4-binding site antibody elicited by polyvalent DNA prime-protein boost vaccine neutralizes cross-clade tier-2-HIV strains

The vaccine elicitation of HIV tier-2-neutralization antibodies has been a challenge. Here, we report the isolation and characterization of a CD4-binding site (CD4bs) specific monoclonal antibody, HmAb64, from a human volunteer immunized with a polyvalent DNA prime-protein boost HIV vaccine. HmAb64 is derived from heavy chain variable germline gene IGHV1-18 and light chain germline gene IGKV1-39. It has a third heavy chain complementarity-determining region (CDR H3) of 15 amino acids. On a cross-clade panel of 208 HIV-1 pseudo-virus strains, HmAb64 neutralized 20 (10%), including tier-2 strains from clades B, BC, C, and G. The cryo-EM structure of the antigen-binding fragment of HmAb64 in complex with a CNE40 SOSIP trimer revealed details of its recognition; HmAb64 uses both heavy and light CDR3s to recognize the CD4-binding loop, a critical component of the CD4bs. This study demonstrates that a gp120-based vaccine can elicit antibodies capable of tier 2-HIV neutralization.

Vaccine priming of rare HIV broadly neutralizing antibody precursors in nonhuman primates.

Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to HIV and other pathogens. However, antibody-antigen recognition is typically dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3-dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. In this work, immunization with N332-GT5, an HIV envelope trimer designed to target precursors of the HCDR3-dominant bnAb BG18, primed bnAb-precursor B cells in eight of eight rhesus macaques to substantial frequencies and with diverse lineages in germinal center and memory B cells. We confirmed bnAb-mimicking, HCDR3-dominant, trimer-binding interactions with cryo-electron microscopy. Our results demonstrate proof of principle for HCDR3-dominant bnAb-precursor priming in outbred animals and suggest that N332-GT5 holds promise for the induction of similar responses in humans.

The D Gene in CDR H3 Determines a Public Class of Human Antibodies to SARS-CoV-2.

Public antibody responses have been found against many infectious agents. Structural convergence of public antibodies is usually determined by immunoglobulin V genes. Recently, a human antibody public class against SARS-CoV-2 was reported, where the D gene (IGHD3-22) encodes a common YYDxxG motif in heavy-chain complementarity-determining region 3 (CDR H3), which determines specificity for the receptor-binding domain (RBD). In this review, we discuss the isolation, structural characterization, and genetic analyses of this class of antibodies, which have been isolated from various cohorts of COVID-19 convalescents and vaccinees. All eleven YYDxxG antibodies with available structures target the SARS-CoV-2 RBD in a similar binding mode, where the CDR H3 dominates the interaction with antigen. The antibodies target a conserved site on the RBD that does not overlap with the receptor-binding site, but their particular angle of approach results in direct steric hindrance to receptor binding, which enables both neutralization potency and breadth. We also review the properties of CDR H3-dominant antibodies that target other human viruses. Overall, unlike most public antibodies, which are identified by their V gene usage, this newly discovered public class of YYDxxG antibodies is dominated by a D-gene-encoded motif and uncovers further opportunities for germline-targeting vaccine design.

Extraction of the CDRH3 sequence of the mouse antibody repertoire selected upon influenza virus infection by subtraction of the background antibody repertoire.

As specific interactions between antigens and cell-surface antibodies trigger the proliferation of B-cell clones, the frequency of each antibody sequence in the samples reflects the size of each clonal population. Nevertheless, it is extremely difficult to extract antigen-specific antibody sequences from the comprehensive bulk antibody sequences obtained from blood samples due to repertoire bias influenced by exposure to dietary antigens and other infectious agents. This issue can be addressed by subtracting the background noise from the post-immunization or post-infection repertoire data. In the present study, we propose a method to quantify repertoire data from comprehensive repertoire data. This method allowed subtraction of the background repertoire, resulting in more accurate extraction of expanded antibody repertoires upon influenza virus infection. This accurate extraction of antigen- or infection-specific repertoire information is a useful tool for vaccine evaluation.

Preparation and characterization of monoclonal antibodies against porcine gasdermin D protein

Pyroptosis is a newly discovered type of pro-inflammatory programmed cell death that plays a vital role in various processes such as inflammations, immune responses, and pathogen infections. As one of the main executioners of pyroptosis, gasdermin D (GSDMD) is a membrane pore-forming protein that typically exists in a self-inhibitory state. Once activated, GSDMD will be cleaved into an N-terminal fragment with pore-forming activity, becoming the key indicator of pyroptosis activation, and a C-terminal fragment. Although commercial antibodies against human and murine GSDMD proteins are currently available, their reactivity with porcine GSDMD (pGSDMD) is poor, which limits research on the biological functions of pGSDMD and pyroptosis in pigs in vivo and in vitro. Here, five monoclonal antibodies (mAbs) were prepared by immunizing BALB/c mice with procaryotically expressed full-length pGSDMD, all of which did not cross react with human and murine GSDMD proteins. Epitope mapping demonstrated that 15H6 recognizes amino acids (aa) at positions 28–34 of pGSDMD (LQTSDRF), 19H3 recognizes 257–260aa (PPQF), 23H10 and 27A10 recognize 78–82aa (GPFYF), and 25E2 recognizes 429–435aa (PPTLLGS). The affinity constant and isotype of 15H6, 19H3, 23H10, 27A10, and 25E2 mAbs were determined to be 1.32 × 10−9, 3.66 × 10−9, 9.04 × 10−9, 1.83 × 10−9, and 8.00 × 10−8 mol/L and IgG1/κ, IgG2a/κ, IgG2a/κ, IgG1/κ, and IgG1/κ, respectively. Heavy- and light-chain variable regions sequencing showed that the heavy-chain complementarity-determining region (CDR) sequences of all five mAbs are completely different, while the light-chain CDR sequences of the four mAbs that recognize the N-terminus of pGSDMD are identical. Our prepared mAbs provide valuable materials for studying pGSDMD function and pyroptosis.Key points• A total of five mouse anti-pGSDMD mAbs were prepared, of which four recognize the N-terminus of pGSDMD and one recognize its C-terminus.• The main performance parameters of the five mAbs, including epitope, antibody titer, affinity constant, isotype, and heavy- and light-chain CDR, were characterized.• All five mAbs specifically recognize pGSDMD protein and do not cross react with human and murine GSDMD proteins.

Anti-idiotype isolation of a broad and potent influenza A virus-neutralizing human antibody.

The VH6-1 class of antibodies includes some of the broadest and most potent antibodies that neutralize influenza A virus. Here, we elicit and isolate anti-idiotype antibodies against germline versions of VH6-1 antibodies, use these to sort human leukocytes, and isolate a new VH6-1-class member, antibody L5A7, which potently neutralized diverse group 1 and group 2 influenza A strains. While its heavy chain derived from the canonical IGHV6-1 heavy chain gene used by the class, L5A7 utilized a light chain gene, IGKV1-9, which had not been previously observed in other VH6-1-class antibodies. The cryo-EM structure of L5A7 in complex with Indonesia 2005 hemagglutinin revealed a nearly identical binding mode to other VH6-1-class members. The structure of L5A7 bound to the isolating anti-idiotype antibody, 28H6E11, revealed a shared surface for binding anti-idiotype and hemagglutinin that included two critical L5A7 regions: an FG motif in the third heavy chain-complementary determining region (CDR H3) and the CDR L1 loop. Surprisingly, the chemistries of L5A7 interactions with hemagglutinin and with anti-idiotype were substantially different. Overall, we demonstrate anti-idiotype-based isolation of a broad and potent influenza A virus-neutralizing antibody, revealing that anti-idiotypic selection of antibodies can involve features other than chemical mimicry of the target antigen.

Cold-blooded vertebrates evolved organized germinal center-like structures.

Germinal centers (GCs) or analogous secondary lymphoid microstructures (SLMs) are thought to have evolved in endothermic species. However, living representatives of their ectothermic ancestors can mount potent secondary antibody responses upon infection or immunization, despite the apparent lack of SLMs in these cold-blooded vertebrates. How and where adaptive immune responses are induced in ectothermic species in the absence of GCs or analogous SLMs remain poorly understood. Here, we infected a teleost fish (trout) with the parasite Ichthyophthirius multifiliis (Ich) and identified the formation of large aggregates of highly proliferating IgM+ B cells and CD4+ T cells, contiguous to splenic melanomacrophage centers (MMCs). Most of these MMC-associated lymphoid aggregates (M-LAs) contained numerous antigen (Ag)-specific B cells. Analysis of the IgM heavy chain CDR3 repertoire of microdissected splenic M-LAs and non-M-LA areas revealed that the most frequent B cell clones induced after Ich infection were highly shared only within the M-LAs of infected animals. These M-LAs represented highly polyclonal SLMs in which Ag-specific B cell clonal expansion occurred. M-LA-associated B cells expressed high levels of activation-induced cytidine deaminase and underwent significant apoptosis, and somatic hypermutation of Igμ genes occurred prevalently in these cells. Our findings demonstrate that ectotherms evolved organized SLMs with GC-like roles. Moreover, our results also point to primordially conserved mechanisms by which M-LAs and mammalian polyclonal GCs develop and function.

Molecular interaction analysis of anti-IL-8 scFv-10F8-6His against IL-8 monomer through molecular docking and molecular dynamic simulations

Elevated interleukin 8 (IL-8) expression has been linked to unfavorable outcomes in a range of inflammatory conditions, such as rheumatoid arthritis, psoriasis, and cancer. The human monoclonal antibody (HuMab) 10F8 and the hybridoma 35B11-B bind to an epitope on human IL-8, respectively. 10F8 inhibited interaction between IL-8 and neutrophils in eczema and pustulosis palmoplantaris patients while 35B11-B decreased size lesion in rat model. The binding interaction of monoclonal antibodies and IL-8, especially how complementarity-determining region (CDR) loops could bind the N-terminal of IL-8, has not been fully deliberated at molecular-level. Here, we used a combination of molecular docking, heated and long coarse-grained molecular dynamics simulations to identify key residues of established interaction. Based on heated MD simulation, docked pose of complexes generated by ClusPro showed good binding stability throughout of 70 ns simulation. Based on long molecular dynamic simulations, key residues for the binding were identified throughout of 1000 ns simulation. TYR-53, ASP-99, and ARG-100 of heavy chain CDR together with TYR-33 of light chain CDR are among the highest contributing energy residues within the binding interaction. Meanwhile, LYS11 and TYR13 of IL-8 are important for the determination of overall binding energy. Furthermore, the result of decomposition residues analysis is in good agreement with the interaction analysis data. Current study provides a list of important interacting residues and further scrutiny on these residues is essential for future development and design of a new and stable recombinant antibody against IL-8. Communicated by Ramaswamy H. Sarma

Potent cross-neutralization of respiratory syncytial virus and human metapneumovirus through a structurally conserved antibody recognition mode

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) infections pose a significant health burden. Using pre-fusion conformation fusion (F)proteins, we isolated a panel of anti-F antibodies from a human donor. One antibody (RSV-199) potently cross-neutralized 8 RSV and hMPV strains by recognizing antigenic site III, which is partially conserved in RSV and hMPV F. Next, we determined the cryoelectron microscopy (cryo-EM) structures of RSV-199 bound to RSV F trimers, hMPV F monomers, and an unexpected dimeric form of hMPV F. These structures revealed how RSV-199 engages both RSV and hMPV F proteins through conserved interactions of the antibody heavy-chain variable region and how variability within heavy-chain complementarity-determining region 3 (HCDR3) can be accommodated at the F protein interface in site-III-directed antibodies. Furthermore, RSV-199 offered enhanced protection against RSV A and B strains and hMPV in cotton rats. These findings highlight the mechanisms of broad neutralization and therapeutic potential of RSV-199.

The molecular basis of the neutralization breadth of the RBD-specific antibody CoV11.

SARS-CoV-2, the virus behind the COVID-19 pandemic, has changed over time to the extent that the current virus is substantially different from what originally led to the pandemic in 2019-2020. Viral variants have modified the severity and transmissibility of the disease and continue do so. How much of this change is due to viral fitness versus a response to immune pressure is hard to define. One class of antibodies that continues to afford some level of protection from emerging variants are those that closely overlap the binding site for angiotensin-converting enzyme 2 (ACE2) on the receptor binding domain (RBD). Some members of this class that were identified early in the course of the pandemic arose from the VH 3-53 germline gene (IGHV3-53*01) and had short heavy chain complementarity-determining region 3s (CDR H3s). Here, we describe the molecular basis of the SARS-CoV-2 RBD recognition by the anti-RBD monoclonal antibody CoV11 isolated early in the COVID-19 pandemic and show how its unique mode of binding the RBD determines its neutralization breadth. CoV11 utilizes a heavy chain VH 3-53 and a light chain VK 3-20 germline sequence to bind to the RBD. Two of CoV11's four heavy chain changes from the VH 3-53 germline sequence, to Ile and to Arg, and some unique features in its CDR H3 increase its affinity to the RBD, while the four light chain changes from the VK 3-20 germline sequence sit outside of the RBD binding site. Antibodies of this type can retain significant affinity and neutralization potency against variants of concern (VOCs) that have diverged significantly from original virus lineage such as the prevalent omicron variant. We also discuss the mechanism by which VH 3-53 encoded antibodies recognize spike antigen and show how minimal changes to their sequence, their choice of light chain, and their mode of binding influence their affinity and impact their neutralization breadth.

Identification of CDRH3 loops in the B cell receptor repertoire that can be engaged by candidate immunogens.

A major goal for the development of vaccines against rapidly mutating viruses, such as influenza or HIV, is to elicit antibodies with broad neutralization capacity. However, B cell precursors capable of maturing into broadly neutralizing antibodies (bnAbs) can be rare in the immune repertoire. Due to the stochastic nature of B cell receptor (BCR) rearrangement, a limited number of third heavy chain complementary determining region (CDRH3) sequences are identical between different individuals. Thus, in order to successfully engage broadly neutralizing antibody precursors that rely on their CDRH3 loop for antigen recognition, immunogens must be able to tolerate sequence diversity in the B cell receptor repertoire across an entire vaccinated population. Here, we present a combined experimental and computational approach to identify BCRs in the human repertoire with CDRH3 loops predicted to be engaged by a target immunogen. For a given antibody/antigen pair, deep mutational scanning was first used to measure the effect of CDRH3 loop substitution on binding. BCR sequences, isolated experimentally or generated in silico, were subsequently evaluated to identify CDRH3 loops expected to be bound by the candidate immunogen. We applied this method to characterize two HIV-1 germline-targeting immunogens and found differences in the frequencies with which they are expected to engage target B cells, thus illustrating how this approach can be used to evaluate candidate immunogens towards B cell precursors engagement and to inform immunogen optimization strategies for more effective vaccine design.

Mechanism of Insoluble Aggregate Formation in a Reconstituted Solution of Spray-Dried Protein Powder

BackgroundSpray-drying is considered a promising alternative drying method to lyophilization (freeze-drying) for therapeutic proteins. Particle counts in reconstituted solutions of dried solid dosage forms of biologic drug products are closely monitored to ensure product quality. We found that high levels of particles formed after reconstitution of protein powders that had been spray-dried under suboptimal conditions.MethodsVisible and subvisible particles were evaluated. Soluble proteins in solution before spray-drying and in the reconstituted solution of spray-dried powder were analyzed for their monomer content levels and melting temperatures. Insoluble particles were collected and analyzed by Fourier transform infrared microscopy (FTIR), and further analyzed with hydrogen-deuterium exchange (HDX).ResultsParticles observed after reconstitution were shown not to be undissolved excipients. FTIR confirmed their identity as proteinaceous in nature. These particles were therefore considered to be insoluble protein aggregates, and HDX was applied to investigate the mechanism underlying aggregate formation. Heavy-chain complementarity-determining region 1 (CDR-1) in the aggregates showed significant protection by HDX, suggesting CDR-1 was critical for aggregate formation. In contrast, various regions became more conformationally dynamic globally, suggesting the aggregates have lost protein structural integrity and partially unfolded after spray-drying.DiscussionThe spray-drying process could have disrupted the higher-order structure of proteins and exposed the hydrophobic residues in CDR-1 of the heavy chain, contributing to the formation of aggregate through hydrophobic interactions upon reconstitution of spray-dried powder. These results can contribute to efforts to design spray-dry resilient protein constructs and improve the robustness of the spray-drying process.

Engineering of conserved residues near antibody heavy chain complementary determining region 3 (HCDR3) improves both affinity and stability

Affinity and stability are crucial parameters in antibody development and engineering approaches. Although improvement in both metrics is desirable, trade-offs are almost unavoidable. Heavy chain complementarity determining region 3 (HCDR3) is the best-known region for antibody affinity but its impact on stability is often neglected. Here, we present a mutagenesis study of conserved residues near HCDR3 to elicit the role of this region in the affinity-stability trade-off. These key residues are positioned around the conserved salt bridge between VH-K94 and VH-D101 which is crucial for HCDR3 integrity. We show that the additional salt bridge at the stem of HCDR3 (VH-K94:VH-D101:VH-D102) has an extensive impact on this loop's conformation, therefore simultaneous improvement in both affinity and stability. We find that the disruption of π-π stacking near HCDR3 (VH-Y100E:VL-Y49) at the VH-VL interface cause an irrecoverable loss in stability even if it improves the affinity. Molecular simulations of putative rescue mutants exhibit complex and often non-additive effects. We confirm that our experimental measurements agree with the molecular dynamic simulations providing detailed insights for the spatial orientation of HCDR3. VH-V102 right next to HCDR3 salt bridge might be an ideal candidate to overcome affinity-stability trade-off.