Antibody Variable Domains Research Articles

Humanization of antibodies using a machine learning approach on large-scale repertoire data.

MotivationMonoclonal antibody (mAb) therapeutics are often produced from non-human sources (typically murine), and can therefore generate immunogenic responses in humans. Humanization procedures aim to produce antibody therapeutics that do not elicit an immune response and are safe for human use, without impacting efficacy. Humanization is normally carried out in a largely trial-and-error experimental process. We have built machine learning classifiers that can discriminate between human and non-human antibody variable domain sequences using the large amount of repertoire data now available.ResultsOur classifiers consistently outperform the current best-in-class model for distinguishing human from murine sequences, and our output scores exhibit a negative relationship with the experimental immunogenicity of existing antibody therapeutics. We used our classifiers to develop a novel, computational humanization tool, Hu-mAb, that suggests mutations to an input sequence to reduce its immunogenicity. For a set of therapeutic antibodies with known precursor sequences, the mutations suggested by Hu-mAb show substantial overlap with those deduced experimentally. Hu-mAb is therefore an effective replacement for trial-and-error humanization experiments, producing similar results in a fraction of the time.Availability and implementationHu-mAb (humanness scoring and humanization) is freely available to use at opig.stats.ox.ac.uk/webapps/humab.Supplementary information Supplementary data are available at Bioinformatics online.

A 15-min non-competitive homogeneous assay for microcystin and nodularin based on time-resolved F\xf6rster resonance energy transfer (TR-FRET)

Simple and rapid methods are required for screening and analysis of water samples to detect cyanobacterial cyclic peptide hepatotoxins: microcystin/nodularin. Previously, we reported a highly sensitive non-competitive heterogeneous assay for microcystin/nodularin utilizing a generic anti-immunocomplex (anti-IC) single-chain fragment of antibody variable domains (scFv) isolated from a synthetic antibody library together with a generic adda ((2S,3S,4E,6E,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid)-specific monoclonal antibody (Mab) recognizing the common adda part of the microcystin/nodularin. Using the same antibody pair, here we report a homogeneous non-competitive assay for microcystin/nodularin based on TR-FRET (time-resolved Förster resonance energy transfer) measurement. The anti-IC scFv labeled with Alexa Fluor 680 and the Mab labeled with europium enabled the FRET process to occur in the presence of microcystin/nodularin. The TR-FRET signal is proportional to the toxin concentration in the sample. The rapid (15 min) homogeneous assay without requiring any washing step detected all the tested nine toxin variants (microcystin-LR, -dmLR, -RR, -dmRR, -YR, -LY, -LF -LW, and nodularin-R). Very good signal to blank ratio (~13) was achieved using microcystin-LR and the sample detection limit (blank+3SD of blank) for microcystin-LR was ~0.3 μg/L (~0.08 μg/L in 80-μL reaction well). The practical application of the TR-FRET assay was demonstrated with water samples spiked with microcystin-LR as well as with environmental water. The average recoveries of microcystin-LR from spiked water ranged from 65 to 123%. Good correlation (r2 = 0.73 to 0.99) with other methods (liquid chromatography-mass spectrometry and previously reported heterogeneous assay) was found when environmental samples were analyzed. The developed wash-free assay has the potential to play as a quick screening tool to detect microcystin/nodularin from water below the World Health Organization’s guideline limit (1 μg/L of microcystin-LR).Graphical abstract

The Nanobody JK36 circumvents the epitope overlapping commonly observed between therapeutic anti-CD38 antibodies and commercially available analytical mouse antibodies

Abstract Therapeutic anti-CD38 antibodies have been demonstrated to elicit immune-mediated elimination of plasma cells and other CD38-expressing cells. Epitope overlapping with commercially available analytical monoclonal mouse anti-CD38 antibodies limits the possibility to study surface CD38 expression in the presence of these therapeutic antibodies. Nanobodies, derived from heavy-chain antibodies that naturally occur in llamas and other Camelids, can circumvent these undesired interferences. Nanobodies are single variable domain antibody fragments (VHH) that often expose a long complementarity-determining region 3 (CDR3). This feature allows them to recognize hidden epitopes, e.g. in molecular cavities, that are inaccessible to the CDRs in conventional analytical and therapeutic antibodies. In a model of anti-CD38 occupation with a therapeutic antibody, we have evaluated JK36, an anti-CD38 nanobody recognizing a cryptic epitope. Normal whole blood samples were pre-incubated with Daratumumab, a therapeutic humanized anti-CD38 mouse antibody. Binding of the Rabbit-JK36 (Rb-JK36) construct was compared to the binding observed with the conventional mouse antibodies, clones LS198-4-3 and T16. LS198-4-3 and T16 antibodies partially or completely failed to label the CD38 epitope, in case of being masked with Daratumumab. In contrast, the Rb-JK36 staining pattern and intensity remained unchanged, confirming that Rb-JK36 binds to an epitope which is not masked by Daratumumab. This study confirms that JK36 is a well-suited probe for research studies on CD38 expression in the presence of therapeutic or commonly used analytical anti-CD38 antibodies, opening new methodical approaches in plasma cell immunotherapy research.

Shark Antibody Variable Domains Rigidify Upon Affinity Maturation-Understanding the Potential of Shark Immunoglobulins as Therapeutics.

Sharks and other cartilaginous fish are the phylogenetically oldest living organisms that have antibodies as part of their adaptive immune system. As part of their humoral adaptive immune response, they produce an immunoglobulin, the so-called immunoglobulin new antigen receptor (IgNAR), a heavy-chain only antibody. The variable domain of an IgNAR, also known as VNAR, binds the antigen as an independent soluble domain. In this study, we structurally and dynamically characterized the affinity maturation mechanism of the germline and somatically matured (PBLA8) VNAR to better understand their function and their applicability as therapeutics. We observed a substantial rigidification upon affinity maturation, which is accompanied by a higher number of contacts, thereby contributing to the decrease in flexibility. Considering the static x-ray structures, the observed rigidification is not obvious, as especially the mutated residues undergo conformational changes during the simulation, resulting in an even stronger network of stabilizing interactions. Additionally, the simulations of the VNAR in complex with the hen egg-white lysozyme show that the VNAR antibodies evidently follow the concept of conformational selection, as the binding-competent state already preexisted even without the presence of the antigen. To have a more detailed description of antibody–antigen recognition, we also present here the binding/unbinding mechanism between the hen egg-white lysozyme and both the germline and matured VNARs. Upon maturation, we observed a substantial increase in the resulting dissociation-free energy barrier. Furthermore, we were able to kinetically and thermodynamically describe the binding process and did not only identify a two-step binding mechanism, but we also found a strong population shift upon affinity maturation toward the native binding pose.

High-efficacy, high-manufacturability human VH domain antibody therapeutics from transgenic sources.

Interest in single-domain antibodies (sdAbs) stems from their unique structural/pronounced, hence therapeutically desirable, features. From the outset-as therapeutic modalities-human antibody heavy chain variable domains (VHs) attracted a particular attention compared with 'naturally-occurring' camelid and shark heavy-chain-only antibody variable domains (VHHs and VNARs, respectively) due to their perceived lack of immunogenicity. However, they have not quite lived up to their initial promise as the VH hits, primarily mined from synthetic VH phage display libraries, have too often been plagued with aggregation tendencies, low solubility and low affinity. Largely unexplored, synthetic camelized human VH display libraries appeared to have remediated the aggregation problem, but the low affinity of the VH hits still persisted, requiring undertaking additional, laborious affinity maturation steps to render VHs therapeutically feasible. A wholesome resolution has recently emerged with the development of non-canonical transgenic rodent antibody discovery platforms that appear to facilely and profusely generate high affinity, high solubility and aggregation-resistant human VHs.

A conformation-specific antibody against oligomeric β-amyloid restores neuronal integrity in a mouse model of Alzheimer's disease

Conformationally distinct aggregates of the amyloid β (Aβ) peptide accumulate in brains of patients with Alzheimer's disease (AD), but the roles of the different aggregates in disease progression are not clear. We previously isolated two single-chain variable domain antibody fragments (scFvs), C6T and A4, that selectively bind different toxic conformational variants of oligomeric Aβ. Here, we utilize these scFvs to localize the presence of these Aβ variants in human AD brain and to demonstrate their potential as therapeutic agents for treating AD. Both A4 and C6T label oligomeric Aβ in extracellular amyloid plaques, whereas C6T also labels intracellular oligomeric Aβ in human AD brain tissue and in an AD mouse model. For therapeutic studies, the A4 and C6T scFvs were expressed in the AD mice by viral infection of liver cells. The scFvs were administered at 2 months of age, and mice sacrificed at 9 months. The scFvs contained a peptide tag to facilitate transport across the blood brain barrier. While treatment with C6T only slightly decreased Aβ deposits and plaque-associated inflammation, it restored neuronal integrity to WT levels, significantly promoted growth of new neurons, and impressively rescued survival rates to WT levels. Treatment with A4 on the other hand significantly decreased Aβ deposits but did not significantly decrease neuroinflammation or promote neuronal integrity, neurogenesis, or survival rate. These results suggest that the specific Aβ conformation targeted in therapeutic applications greatly affects the outcome, and the location of the targeted Aβ variants may also play a critical factor.

Antibody Fragments as Tools for Elucidating Structure-Toxicity Relationships and for Diagnostic/Therapeutic Targeting of Neurotoxic Amyloid Oligomers.

The accumulation of amyloid protein aggregates in tissues is the basis for the onset of diseases known as amyloidoses. Intriguingly, many amyloidoses impact the central nervous system (CNS) and usually are devastating diseases. It is increasingly apparent that neurotoxic soluble oligomers formed by amyloidogenic proteins are the primary molecular drivers of these diseases, making them lucrative diagnostic and therapeutic targets. One promising diagnostic/therapeutic strategy has been the development of antibody fragments against amyloid oligomers. Antibody fragments, such as fragment antigen-binding (Fab), scFv (single chain variable fragments), and VHH (heavy chain variable domain or single-domain antibodies) are an alternative to full-length IgGs as diagnostics and therapeutics for a variety of diseases, mainly because of their increased tissue penetration (lower MW compared to IgG), decreased inflammatory potential (lack of Fc domain), and facile production (low structural complexity). Furthermore, through the use of in vitro-based ligand selection, it has been possible to identify antibody fragments presenting marked conformational selectivity. In this review, we summarize significant reports on antibody fragments selective for oligomers associated with prevalent CNS amyloidoses. We discuss promising results obtained using antibody fragments as both diagnostic and therapeutic agents against these diseases. In addition, the use of antibody fragments, particularly scFv and VHH, in the isolation of unique oligomeric assemblies is discussed as a strategy to unravel conformational moieties responsible for neurotoxicity. We envision that advances in this field may lead to the development of novel oligomer-selective antibody fragments with superior selectivity and, hopefully, good clinical outcomes.

RNase H-dependent PCR enables highly specific amplification of antibody variable domains from single B-cells.

Immunization-based antibody discovery platforms require robust and effective protocols for the amplification, cloning, expression, and screening of antibodies from large numbers of B-cells in order to effectively capture the diversity of an experienced Ig-repertoire. Multiplex PCR using a series of forward and reverse primers designed to recover antibodies from a range of different germline sequences is challenging because primer design requires the recovery of full length antibody sequences, low starting template concentrations, and the need for all the primers to function under the same PCR conditions. Here we demonstrate several advantages to incorporating RNase H2-dependent PCR (rh-PCR) into a high-throughput, antibody-discovery platform. Firstly, rh-PCR eliminated primer dimer synthesis to below detectable levels, thereby eliminating clones with a false positive antibody titer. Secondly, by increasing the specificity of PCR, the rh-PCR primers increased the recovery of cognate antibody variable regions from single B-cells, as well as downstream recombinant antibody titers. Finally, we demonstrate that rh-PCR primers provide a more homogeneous sample pool and greater sequence quality in a Next Generation Sequencing-based approach to obtaining DNA sequence information from large numbers of cloned antibody cognate pairs. Furthermore, the higher specificity of the rh-PCR primers allowed for a better match between native antibody germline sequences and the VL/VH fragments amplified from single B-cells.

The Effects of Framework Mutations at the Variable Domain Interface on Antibody Affinity Maturation in an HIV-1 Broadly Neutralizing Antibody Lineage.

Understanding affinity maturation of antibodies that can target many variants of HIV-1 is important for vaccine development. While the antigen-binding site of antibodies is known to mutate throughout the co-evolution of antibodies and viruses in infected individuals, the roles of the mutations in the antibody framework region are not well understood. Throughout affinity maturation, the CH103 broadly neutralizing antibody lineage, from an individual designated CH505, altered the orientation of one of its antibody variable domains. The change in orientation was a response to insertions in the variable loop 5 (V5) of the HIV envelope. In this study, we generated CH103 lineage antibody variants in which residues in the variable domain interface were mutated, and measured the binding to both autologous and heterologous HIV-1 envelopes. Our data show that very few mutations in an early intermediate antibody of the lineage can improve binding toward both autologous and heterologous HIV-1 envelopes. We also crystallized an antibody mutant to show that framework mutations alone can result in a shift in relative orientations of the variable domains. Taken together, our results demonstrate the functional importance of residues located outside the antigen-binding site in affinity maturation.

Dynamics of heavy chain junctional length biases in antibody repertoires

Antibody variable domain sequence diversity is generated by recombination of germline segments. The third complementarity-determining region of the heavy chain (CDR H3) is the region of highest sequence diversity and is formed by the joining of heavy chain VH, DH and JH germline segments combined with random nucleotide trimming and additions between these segments. We show that CDR H3 and junctional segment length distributions are biased in human antibody repertoires as a function of VH, VL and JH germline segment utilization. Most length biases are apparent in the naive and antigen experienced B cell compartments but not in nonproductive recombination products, indicating B cell selection as a major driver of these biases. Our findings reveal biases in the antibody CDR H3 diversity landscape shaped by VH, VL, and JH germline segment use during naive and antigen-experienced repertoire selection.

Generation and validation of structurally defined antibody-siRNA conjugates.

Gene silencing by RNA interference (RNAi) has emerged as a powerful treatment strategy across a potentially broad range of diseases. Tailoring siRNAs to silence genes vital for cancer cell growth and function could be an effective treatment, but there are several challenges which must be overcome to enable their use as a therapeutic modality, among which efficient and selective delivery to cancer cells remains paramount. Attempts to use antibodies for siRNA delivery have been reported but these strategies use either nonspecific conjugation resulting in mixtures, or site-specific methods that require multiple steps, introduction of mutations, or use of enzymes. Here, we report a method to generate antibody–siRNA (1:2) conjugates (ARCs) that are structurally defined and easy to assemble. This ARC platform is based on engineered dual variable domain (DVD) antibodies containing a natural uniquely reactive lysine residue for site-specific conjugation to β-lactam linker-functionalized siRNA. The conjugation is efficient, does not compromise the affinity of the parental antibody, and utilizes chemically stabilized siRNA. For proof-of-concept, we generated DVD-ARCs targeting various cell surface antigens on multiple myeloma cells for the selective delivery of siRNA targeting β-catenin (CTNNB1). A set of BCMA-targeting DVD-ARCs at concentrations as low as 10 nM revealed significant CTNNB1 mRNA and protein knockdown.

Understanding the role of antibody glycosylation through the lens of severe viral and bacterial diseases.

Abundant evidence points to a critical role for antibodies in protection and pathology across infectious diseases. While the antibody variable domain facilitates antibody binding and the blockade of infection, the constant domain (Fc) mediates cross talk with the innate immune system. The biological activity of the Fc region is controlled genetically via class switch recombination, resulting in the selection of distinct antibody isotypes and subclasses. However, a second modification is made to all antibodies, via post-translational changes in antibody glycosylation. Studies from autoimmunity and oncology have established the role of immunoglobulin G (IgG) Fc glycosylation as a key regulator of humoral immune activity. However, a growing body of literature, exploring IgG Fc glycosylation through the lens of infectious diseases, points to the role of inflammation in shaping Fc-glycan profiles, the remarkable immune plasticity in antibody glycosylation across pathogen-exposed populations, the canonical and noncanonical functions of glycans and the existence of antigen-specific control over antibody Fc glycosylation. Ultimately, this work provides critical new insights into the functional roles for antibody glycosylation as well as lays the foundation for leveraging antibody glycosylation to drive prevention or control across diseases.

VH -VL interdomain dynamics observed by computer simulations and NMR.

The relative orientation of the two variable domains, VH and VL, influences the shape of the antigen binding site, that is, the paratope, and is essential to understand antigen specificity. ABangle characterizes the VH‐VL orientation by using five angles and a distance and compares it to other known structures. Molecular dynamics simulations of antibody variable domains (Fvs) reveal fluctuations in the relative domain orientations. The observed dynamics between these domains are confirmed by NMR experiments on a single‐chain variable fragment antibody (scFv) in complex with IL‐1β and an antigen‐binding fragment (Fab). The variability of these relative domain orientations can be interpreted as a structural feature of antibodies, which increases the antibody repertoire significantly and can enlarge the number of possible binding partners substantially. The movements of the VH and VL domains are well sampled with molecular dynamics simulations and are in agreement with the NMR ensemble. Fast Fourier transformation of the ABangle metrics allows to assign timescales of 0.1‐10 GHz to the fastest collective interdomain movements. The results clearly show the necessity of dynamics to understand and characterize the favorable orientations of the VH and VL domains implying a considerable binding interface flexibility and reveal in all antibody fragments (Fab, scFv, and Fv) very similar VH‐VL interdomain variations comparable to the distributions observed for known X‐ray structures of antibodies.Significance StatementAntibodies have become key players as therapeutic agents. The binding ability of antibodies is determined by the antigen‐binding fragment (Fab), in particular the variable fragment region (Fv). Antigen‐binding is mediated by the complementarity‐determining regions consisting of six loops, each three of the heavy and light chain variable domain VH and VL. The relative orientation of the VH and VL domains influences the shape of the antigen‐binding site and is a major objective in antibody design. In agreement with NMR experiments and molecular dynamics simulations, we show a considerable binding site flexibility in the low nanosecond timescale. Thus we suggest that this flexibility and its implications for binding and specificity should be considered when designing and optimizing therapeutic antibodies.

N-Linked Glycans in the Variable Domain of IgG Anti-Citrullinated Protein Antibodies Predict the Development of Rheumatoid Arthritis.

ObjectiveAnti–citrullinated protein antibodies (ACPAs) are disease‐specific biomarkers in rheumatoid arthritis (RA). More than 90% of IgG ACPAs harbor N‐linked glycans in the antibody variable (V) domain. The corresponding N‐glycosylation sites in ACPA V‐region sequences result from somatic hypermutation, a T cell–dependent process. As ample evidence indicates that T cells drive the maturation of the ACPA response prior to arthritis onset, we undertook this study to investigate whether the presence of glycans in IgG ACPA V domains predicts the transition from predisease autoimmunity to overt RA.MethodsWe analyzed 2 independent sets of serum samples obtained from 126 ACPA‐positive first‐degree relatives (FDRs) of RA patients. Both sets originated from an Indigenous North American population and comprised cross‐sectional and longitudinal samples of individuals who did or did not develop inflammatory arthritis. Serum IgG ACPAs were affinity‐purified and subjected to ultra high‐performance liquid chromatography–based glycan analysis.ResultsIn both data sets, FDR‐derived IgG ACPA displayed markedly lower levels of V domain glycans (<50%) compared to IgG ACPA from RA patients. Notably, FDRs who later developed RA showed extensive V‐domain glycosylation before the onset of arthritis. Moreover, IgG ACPA V‐domain glycosylation was strongly associated with future development of RA (hazard ratio 6.07 [95% confidence interval 1.46–25.2]; P = 0.013).ConclusionExtensive glycosylation of the IgG ACPA V domain is present in a subset of predisposed FDRs of Indigenous North American RA patients. The presence of this feature substantially increases the risk of RA development. Based on these findings, we propose that glycosylation of the IgG ACPA V domain represents a predictive marker for RA development in ACPA‐positive individuals and may serve to better target prevention measures.

Abstract 3189: CAB-CAR-T: A novel conditionally active biologics approach to minimize on-target off-tumor effects in adoptive immunotherapy

Abstract Adoptive cellular therapy (ACT) using chimeric antigen receptor (CAR) modified T cells has demonstrated promising antitumor effects in hematologic malignancies leading to the recent approval of two CAR-T products targeting the B cell receptor associated protein CD19, however the safe development of CAR-T therapies for solid tumor malignancies remains less advanced in part due to the lack of precision in targeting solid tumor antigens. Identification of clinically viable tumor restricted target antigens while avoiding on-target off-tumor toxicities in normal tissue presents a significant challenge in the solid tumor CAR-T therapeutic scenario. The altered glycolytic pathway of the Warburg effect within the tumor microenvironment (TME) drives lactic acid production leading to an opportunity to develop novel CARs with functional TME dependent switching ability. The resulting acidic extracellular pH is a major feature of the TME and this unique property represents one novel mechanism by which target specificity may be achieved. Here we describe a novel Conditionally Active Biologics approach in adoptive immunotherapy termed CAB-CAR-T. By genetically engineering combinations of amino acid mutations into the antibody variable antigen recognition domains (scFv) of CAR constructs, we demonstrate that TME restricted target antigen engagement can be engineered into chimeric antigen receptors. The described CAB-CARs display minimal antigen dependent activity at the physiological pH of normal tissue. Under TME conditions (low pH) the affinity of CAB-CAR scFv domains against their antigen increases, such that tumor target recognition and CAR-T activity become pH dependent. Thus CAB-CAR-T cells display reversible “AND logic gate” properties, requiring both antigen presence and TME conditions for activity. Comparisons of target cell killing under TME permissive and physiologic pH conditions in a cellular impedance based kinetic killing assay demonstrate that CAB-CAR-T cells efficiently lyse antigen positive target cells at pH 6.7 (TME) with minimal activity at pH 7.4 (normal tissue). pH profiles of CAB-CAR-T were examined through buffering the culture medium down from physiologic pH in 0.1 pH increments, and the data demonstrated that the cytolytic activity of CAB-CAR-T incrementally increased as extracellular pH was decreased compared to control T cells. Similar pH dependence was observed for other measures of CAB-CAR-T activation including the early T cell surface activation marker CD69, the degranulation marker CD107a and cytokine release (IL-2 and IFNγ). Conditionally active chimeric antigen receptor T cells (CAB-CAR-T) harness unique properties of the tumor microenvironment to provide an opportunity to develop safer CAR-T therapeutics for tumor antigen targets also expressed on healthy tissues by minimizing on-target off-tumor activity. Citation Format: Jianfang Hu, Benjamin Lopez, Tiffany Lam, Anirban Kundu, Timothy Mayall, Gregory Schreiber, Farzad Haerizadeh, James Onuffer, Gregory Frost. CAB-CAR-T: A novel conditionally active biologics approach to minimize on-target off-tumor effects in adoptive immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3189.

AbRSA: A robust tool for antibody numbering.

The remarkable progress in cancer immunotherapy in recent years has led to the heat of great development for therapeutic antibodies. Antibody numbering, which standardizes a residue index at each position of an antibody variable domain, is an important step in immunoinformatic analysis. It provides an equivalent index for the comparison of sequences or structures, which is particularly valuable for antibody modeling and engineering. However, due to the extremely high diversity of antibody sequences, antibody-numbering tools cannot work in all cases. This article introduces a new antibody-numbering tool named AbRSA, which integrates heuristic knowledge of region-specific features into sequence mapping to enhance the robustness. The benchmarks demonstrate that, AbRSA exhibits robust performance in numbering sequences with diverse lengths and patterns compared with the state-of-the-art tools. AbRSA offers a user-friendly interface for antibody numbering, complementarity-determining region delimitation, and 3D structure rendering. It is freely available at http://cao.labshare.cn/AbRSA.

Bispecific Antibody Fragment Targeting APP and Inducing α-Site Cleavage Restores Neuronal Health in an Alzheimer's Mouse Model.

The amyloid β (Aβ) peptide, correlated with development of Alzheimer's disease (AD), is produced by sequential proteolytic cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. Alternative proteolytic cleavage of APP by α-secretase prevents formation of Aβ peptide and produces a neuroprotective protein, a soluble fragment of APPα (sAPPα). We previously generated a single-chain variable domain antibody fragment (scFv) that binds APP at the β-secretase cleavage site and blocks cleavage of APP (iBsec1), and a second scFv which has been engineered to have α-secretase-like activity that increases α-secretase cleavage of APP (Asec1a) and showed that a bispecific antibody (Diab) combining both iBsec1 and Asec1a constructs protects mammalian cells from oxidative stress. Here, we show that the diabody is an effective therapeutic agent in a mouse model of AD. An apolipoprotein B (ApoB) binding domain peptide was genetically added to the diabody to facilitate transfer across the blood-brain barrier, and a recombinant human adeno-associated virus 2/8 (rAAV2/8) was used as a vector to express the gene constructs in a APP/PS1 mouse model of AD. The diabody increased levels of sAPPα, decreased Aβ deposits and levels of oligomeric Aβ, increased neuronal health as indicated by MAP2 and synaptophysin staining, increased hippocampal neurogenesis, and most importantly dramatically increased survival rates compared with untreated mice or mice treated only with the β-secretase inhibitor. These results indicate that altering APP processing to inhibit β-site activity while simultaneously promoting α-secretase processing provides substantially increased neuronal benefits compared with inhibition of β-secretase processing alone and represents a promising new therapeutic approach for treating AD.

Yeast-secreted, dried and food-admixed monomeric IgA prevents gastrointestinal infection in a piglet model.

A scalably manufacturable oral antibody technology that can interfere with gastrointestinal (GI) targets is needed. Contrary to the complex native secretory IgA, we achieve this using a single-gene encoded monomeric-IgA-like antibody, composed of camelid VHH fused to IgA Fc (mVHH-IgA). This can be produced in soybean seeds or secreted from Pichia pastoris yeast, freeze-or spray-dried, and when delivered in food prevents enterotoxigenic Escherichia coli (F4-ETEC) infection in piglets.

Evaluation of a noncanonical Cys40-Cys55 disulfide linkage for stabilization of single-domain antibodies.

Incorporation of noncanonical disulfide linkages into single-domain antibodies (sdAbs) has been shown to enhance thermostability and other properties. Here, we evaluated the effects of introducing a novel disulfide linkage formed between Cys residues at IMGT positions 40 and 55 on the melting temperatures (T m s), reversibility of thermal unfolding, solubility, and antigen-binding affinities of three types of sdAbs (VH H, VH , and VL domains). The Cys40-Cys55 disulfide linkage was tolerated by 9/9 VH Hs, 12/12 VH s, and 2/11 VL s tested and its formation was confirmed by mass spectrometry. Using circular dichroism, we found that the Cys40-Cys55 disulfide linkage increased sdAb T m by an average of 10.0°C (range: 0-21.8°C). However, enhanced thermostability came at the cost of a partial loss of refolding ability upon thermal denaturation as well as, for some sdAbs, significantly decreased solubility and antigen-binding affinity. Thus, Cys40/Cys55 can be added to the panel of known locations for introducing stabilizing noncanonical disulfide linkages into antibody variable domains, although its effects should be tested empirically for individual sdAbs.

A Precisely Designed Immunotoxin Against VCAM1 Consisting of a Humanized Antibody Variable Domain Fused to Granzyme: An In Silico Approach

Atherosclerosis is a complex disease related to cardiovascular disorders and is one of the most considerable causes of mortality worldwide. It has been shown that the immune system plays a pivotal role in generating atherosclerosis plaques. The atherosclerosis plaques form via activation of endothelial cells by recruiting adhesion molecules such as VCAM1. Therefore, VCAM1 could be considered as a suitable target to design immune-therapeutics. In this regard, we have lunched an in silico approach to design an immunotoxin against VCAM1. The structure of an anti-VCAM1 antibody was predicted using homology modeling software. The structure of the predicted chains were linked by (Gly4Ser)3 linker. Then, using various modeling software the structure of the GrB was linked to the antibody structure via an adaptor sequence. The final structure was energy minimized, evaluated for accuracy and characterized for its biological properties. Our results indicated that the employed structure prediction method has successfully managed to model the immunotoxin structure. Moreover, our results indicated that the designed immunotoxin is capable of VCAM1 interaction with proper orientation involving its C–D loop. In conclusion it should be pointed out that, the employed in silico approach could pave the way for precise and accurate design of immune-therapeutic agents against cancer. Using this approach we have designed an immunotoxin capable of VCAM1 targeting in a proper orientation and following cancer cell destruction by the toxin domain of the molecule.