Extensive sequence and structural evolution of Arginase 2 inhibitory antibodies enabled by an unbiased approach to affinity maturation.

Affinity maturation is a powerful technique in antibody engineering for the in vitro evolution of antigen binding interactions. Key to the success of this process is the expansion of sequence and combinatorial diversity to increase the structural repertoire from which superior binding variants may be selected. However, conventional strategies are often restrictive and only focus on small regions of the antibody at a time. In this study, we used a method that combined antibody chain shuffling and a staggered-extension process to produce unbiased libraries, which recombined beneficial mutations from all six complementarity-determining regions (CDRs) in the affinity maturation of an inhibitory antibody to Arginase 2 (ARG2). We made use of the vast display capacity of ribosome display to accommodate the sequence space required for the diverse library builds. Further diversity was introduced through pool maturation to optimize seven leads of interest simultaneously. This resulted in antibodies with substantial improvements in binding properties and inhibition potency. The extensive sequence changes resulting from this approach were translated into striking structural changes for parent and affinity-matured antibodies bound to ARG2, with a large reorientation of the binding paratope facilitating increases in contact surface and shape complementarity to the antigen. The considerable gains in therapeutic properties seen from extensive sequence and structural evolution of the parent ARG2 inhibitory antibody clearly illustrate the advantages of the unbiased approach developed, which was key to the identification of high-affinity antibodies with the desired inhibitory potency and specificity.

Naturally Acquired Human Antibodies Against Reticulocyte-Binding Domains of Plasmodium vivax Proteins, PvRBP2c and PvRBP1a, Exhibit Binding-Inhibitory Activity.

Background: Crucial gaps in our understanding of Plasmodium vivax reticulocyte invasion and protective immunity have hampered development of vivax vaccines. P. vivax exclusively invades reticulocytes that is mediated by the P. vivax reticulocyte-binding proteins (PvRBPs) specifically PvRBP2c and PvRBP1a. Vivax infections in Duffy-null individuals have suggested the evolution of alternate invasion pathways that may be mediated by the PvRBPs. Thus, PvRBPs appear as potential targets for efficacious P. vivax neutralization. However, there are limited data validating their vaccine efficacy. In the absence of vivax invasion assays, binding-inhibitory activity of antibodies has been reported to be associated with protection and a measure of vaccine potential. Methods: -based analysis was performed of the PvRBP reticulocyte-binding properties and binding-inhibitory activity of specific anti-PvRBP2c/PvRBP1a human antibodies. Results: PvRBP2c and PvRBP1a displayed a distinct reticulocyte-binding specificity, and their specific reticulocyte-binding domains were mapped within their N-terminal regions. Importantly, naturally acquired antibodies against the reticulocyte-binding domains efficaciously blocked reticulocyte binding of native PvRBPs, suggesting that the human immune system produced functional binding-inhibitory antibodies through exposure to vivax malaria. Conclusions: Reticulocyte-binding domains of PvRBP2c/PvRBP1a are targets of naturally acquired binding-inhibitory antibodies, substantiating their promise as candidate antigens against which vaccine-inducible immunity could potentially be boosted through natural infections.

Hemophilia A gene therapy via intraosseous delivery of factor VIII-lentiviral vectors.

Current treatment of hemophilia A (HemA) patients with repeated infusions of factor VIII (FVIII; abbreviated as F8 in constructs) is costly, inconvenient, and incompletely effective. In addition, approximately 25 % of treated patients develop anti-factor VIII immune responses. Gene therapy that can achieve long-term phenotypic correction without the complication of anti-factor VIII antibody formation is highly desired. Lentiviral vector (LV)-mediated gene transfer into hematopoietic stem cells (HSCs) results in stable integration of FVIII gene into the host genome, leading to persistent therapeutic effect. However, ex vivo HSC gene therapy requires pre-conditioning which is highly undesirable for hemophilia patients. The recently developed novel methodology of direct intraosseous (IO) delivery of LVs can efficiently transduce bone marrow cells, generating high levels of transgene expression in HSCs. IO delivery of E-F8-LV utilizing a ubiquitous EF1α promoter generated initially therapeutic levels of FVIII, however, robust anti-FVIII antibody responses ensued neutralized functional FVIII activity in the circulation. In contrast, a single IO delivery of G-FVIII-LV utilizing a megakaryocytic-specific GP1bα promoter achieved platelet-specific FVIII expression, leading to persistent, partial correction of HemA in treated animals. Most interestingly, comparable therapeutic benefit with G-F8-LV was obtained in HemA mice with pre-existing anti-FVIII inhibitors. Platelets is an ideal IO delivery vehicle since FVIII stored in α-granules of platelets is protected from high-titer anti-FVIII antibodies; and that even relatively small numbers of activated platelets that locally excrete FVIII may be sufficient to promote efficient clot formation during bleeding. Additionally, combination of pharmacological agents improved transduction of LVs and persistence of transduced cells and transgene expression. Overall, a single IO infusion of G-F8-LV can generate long-term stable expression of hFVIII in platelets and correct hemophilia phenotype for long term. This approach has high potential to permanently treat FVIII deficiency with and without pre-existing anti-FVIII antibodies.

Delta-like 1-Expressing Cells at the Gland Base Promote Proliferation of Gastric Antral Stem Cells in Mouse.

BACKGROUND & AIMS: Notch pathway signaling maintains gastric epithelial cell homeostasis by regulating stem cell proliferation and differentiation. We previously identified NOTCH1 and NOTCH2 as the key Notch receptors controlling gastric stem cell function. Here, we identify the niche cells and critical Notch ligand responsible for regulating stem cell proliferation in the distal mouse stomach. METHODS: Expression of Notch ligands in the gastric antrum was determined by quantitative reverse-transcriptase polymerase chain reaction and cellular localization was determined by in situ hybridization and immunostaining. The contribution of specific Notch ligands to regulate epithelial cell proliferation in adult mice was determined by inducible gene deletion, or by pharmacologic inhibition using antibodies directed against specific Notch ligands. Mouse gastric organoid cultures were used to confirm that Notch ligand signaling was epithelial specific. RESULTS: Delta-like 1 (DLL1) and Jagged 1 (JAG1) were the most abundantly expressed Notch ligands in the adult mouse stomach, with DLL1 restricted to the antral gland base and JAG1 localized to the upper gland region. Inhibition of DLL1 alone or in combination with other Notch ligands significantly reduced epithelial cell proliferation and the growth of gastric antral organoids, while inhibition of the other Notch ligands, DLL4, JAG1, and JAG2, did not affect proliferation or organoid growth. Similarly, DLL1, and not DLL4, regulated proliferation of LGR5+ antral stem cells, which express the NOTCH1 receptor. CONCLUSIONS: DLL1 is the key Notch ligand regulating epithelial cell proliferation in the gastric antrum. We propose that DLL1-expressing cells at the gland base are Notch niche cells that signal to adjacent LGR5+ antral stem cells to regulate stem cell proliferation and epithelial homeostasis.

Multiplexed Microsphere-Based Flow Cytometric Assay to Assess Strain Transcending Antibodies to Plasmodium vivax Duffy Binding Protein II Reveals an Efficient Tool to Identify Binding-Inhibitory Antibody Responders.

Malaria remains a major public health problem worldwide, and Plasmodium vivax is the most widely distributed malaria parasite. Naturally acquired binding inhibitory antibodies (BIAbs) to region II of the Duffy binding protein (DBPII), a P. vivax ligand that is critical for reticulocyte invasion, are associated with a reduced risk of clinical malaria. Owing to methodological issues in evaluating antibodies that inhibit the DBPII-DARC interaction, a limited number of studies have investigated DBPII BIAbs in P. vivax-exposed populations. Based on the assumption that individuals with a consistent BIAb response are characterized by strain-transcending immune responses, we hypothesized that detecting broadly reactive DBPII antibodies would indicate the presence of BIAb response. By taking advantage of an engineered DBPII immunogen targeting conserved DBPII neutralizing epitopes (DEKnull-2), we standardized a multiplex flow cytometry-based serological assay to detect broadly neutralizing IgG antibodies. For this study, a standard in vitro cytoadherence assay with COS-7 cells expressing DBPII was used to test for DBPII BIAb response in long-term P. vivax-exposed Amazonian individuals. Taken together, the results demonstrate that this DBPII-based multiplex assay facilitates identifying DBPII BIAb carriers. Of relevance, the ability of the multiplex assay to identify BIAb responders was highly accurate when the positivity for all antigens was considered. In conclusion, the standardized DBPII-based flow cytometric assay confirmed that DBPII-BIAb activity was associated with the breadth rather than the magnitude of anti-DBPII antibodies. Altogether, our results suggest that multiplex detection of broadly DBPII-reactive antibodies facilitates preliminary screening of BIAb responders.

Modulation of Gonadotropins Activity by Antibodies.

Gonadotropins are essential for reproduction control in humans as well as in animals. They are widely used all over the world for ovarian stimulation in women, spermatogenesis stimulation in men, and ovulation induction and superovulation in animals. Despite the availability of many different preparations, all are made of the native hormones. Having different ligands with a wide activity range for a given receptor helps better understand its molecular and cellular signaling mechanisms as well as its physiological functions, and thus helps the development of more specific and adapted medicines. One way to control the gonadotropins' activity could be the use of modulating antibodies. Antibodies are powerful tools that were largely used to decipher gonadotropins' actions and they have shown their utility as therapeutics in several other indications such as cancer. In this review, we summarize the inhibitory and potentiating antibodies to gonadotropins, and their potential therapeutic applications.

Measurement of Allergen-Specific Inhibitory Antibody Activity.

Specific allergen immunotherapy (AIT) is an effective treatment for IgE-mediated allergic diseases and involves T- and B-cell-mediated events. IgE receptors on the surface of antigen-presenting cells facilitate the presentation of allergens in the presence of specific IgE antibody resulting in T-cell activation. Interference with these IgE-dependent mechanisms by "blocking" IgG antibodies suppresses pro-inflammatory Th2 cell responses and manifests as a reduction in allergic responses in vivo.In vitro assays used to measure the inhibition of binding of allergen-IgE complexes have previously utilized proliferation of antigen-specific T-cell clones as an assay readout. Here we describe two simplified assays to measure allergen binding without the complexity of generating T-cell clones. The IgE-facilitated allergen binding assay (IgE-FAB) utilizes flow cytometry to measure the binding of allergen-IgE complexes to EBV-transformed B cells. The enzyme-linked immunosorbent-facilitated antigen binding (ELIFAB) assay uses standard ELISA-based techniques to measure allergen-IgE binding to plate-bound CD23, the low-affinity IgE receptor expressed on B cells.

New application of anti-TLR monoclonal antibodies: detection, inhibition and protection.

Monoclonal antibody (mAb) is an essential tool for the analysis in various fields of biology. In the field of innate immunology, mAbs have been established and used for the study of Toll-like receptors (TLRs), a family of pathogen sensors that induces cytokine production and activate immune responses. TLRs play the role as a frontline of protection against pathogens, whereas excessive activation of TLRs has been implicated in a variety of infectious diseases and inflammatory diseases. For example, TLR7 and TLR9 sense not only pathogen-derived nucleic acids, but also self-derived nucleic acids in noninfectious inflammatory diseases such as systemic lupus erythematosus (SLE) or hepatitis. Consequently, it is important to clarify the molecular mechanisms of TLRs for therapeutic intervention in these diseases. For analysis of the molecular mechanisms of TLRs, mAbs to nucleic acid-sensing TLRs were developed recently. These mAbs revealed that TLR7 and TLR9 are localized also in the plasma membrane, while TLR7 and TLR9 were thought to be localized in endosomes and lysosomes. Among these mAbs, antagonistic mAbs to TLR7 or TLR9 are able to inhibit in vitro responses to synthetic ligands. Furthermore, antagonistic mAbs mitigate inflammatory disorders caused by TLR7 or TLR9 in mice. These results suggest that antagonistic mAbs to nucleic acid-sensing TLRs are a promising tool for therapeutic intervention in inflammatory disorders caused by excessive activation of nucleic acid-sensing TLRs. Here, we summarize the molecular mechanisms of TLRs and recent progresses in the trials targeting TLRs with mAbs to control inflammatory diseases.

Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B.

Existing antibodies (Abs) used to treat botulism cannot enter the cytosol of neurons and bind to botulinum neurotoxin (BoNT) at its site of action, and thus cannot reverse paralysis. However, Abs targeting the proteolytic domain of the toxin could inhibit the proteolytic activity of the toxin intracellularly and potentially reverse intoxication, if they could be delivered intracellularly. As such, antibodies that neutralize toxin activity could serve as potent inhibitory cargos for therapeutic antitoxins against botulism. BoNT serotype B (BoNT/B) contains a zinc endopeptidase light chain (LC) domain that cleaves synaoptobrevin-2, a SNARE protein responsible for vesicle fusion and acetylcholine vesicle release. To generate monoclonal Abs (mAbs) that could reverse paralysis, we targeted the protease domain for Ab generation. Single-chain variable fragment (scFv) libraries from immunized mice or humans were displayed on yeast, and 19 unique BoNT/B LC-specific mAbs isolated by fluorescence-activated cell sorting (FACS). The equilibrium dissociation constants (KD) of these mAbs for BoNT/B LC ranged from 0.24 nM to 14.3 nM (mean KD 3.27 nM). Eleven mAbs inhibited BoNT/B LC proteolytic activity. The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site. The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors.

Coagulation factor VII variants resistant to inhibitory antibodies.

Replacement therapy is currently used to prevent and treat bleeding episodes in coagulation factor deficiencies. However, structural differences between the endogenous and therapeutic proteins might increase the risk for immune complications. This study was aimed at identifying factor (F)VII variants resistant to inhibitory antibodies developed after treatment with recombinant activated factor VII (rFVIIa) in a FVII-deficient patient homozygous for the p.A354V-p.P464Hfs mutation, which predicts trace levels of an elongated FVII variant in plasma. We performed fluorescent bead-based binding, ELISA-based competition as well as fluorogenic functional (activated FX and thrombin generation) assays in plasma and with recombinant proteins. We found that antibodies displayed higher affinity for the active than for the zymogen FVII (half-maximal binding at 0.54 ± 0.04 and 0.78 ± 0.07 BU/ml, respectively), and inhibited the coagulation initiation phase with a second-order kinetics. Isotypic analysis showed a polyclonal response with a large predominance of IgG1. We hypothesised that structural differences in the carboxyl-terminus between the inherited FVII and the therapeutic molecules contributed to the immune response. Intriguingly, a naturally-occurring, poorly secreted and 5-residue truncated FVII (FVII-462X) escaped inhibition. Among a series of truncated rFVII molecules, we identified a well-secreted and catalytically competent variant (rFVII-464X) with reduced binding to antibodies (half-maximal binding at 0.198 ± 0.003 BU/ml) as compared to the rFVII-wt (0.032 ± 0.002 BU/ml), which led to a 40-time reduced inhibition in activated FX generation assays. Taken together our results provide a paradigmatic example of mutation-related inhibitory antibodies, strongly support the FVII carboxyl-terminus as their main target and identify inhibitor-resistant FVII variants.

Which Antibody Functions are Important for an HIV Vaccine?

HIV antibody (Ab) functions capable of preventing mucosal cell-free or cell-to-cell HIV transmission are critical for the development of effective prophylactic and therapeutic vaccines. In addition to CD4(+) T cells, other potential HIV-target cell types including antigen-presenting cells (APCs) (dendritic cells, macrophages) residing at mucosal sites are infected. Moreover, the interactions between APCs and HIV lead to HIV cell-to-cell transmission. Recently discovered broadly neutralizing antibodies (NAbs) are able to neutralize a broad spectrum of HIV strains, inhibit cell-to-cell transfer, and efficiently protect from infection in the experimentally challenged macaque model. However, the 31% protection observed in the RV144 vaccine trial in the absence of detectable NAbs in blood samples pointed to the possible role of additional Ab inhibitory functions. Increasing evidence suggests that IgG Fcγ receptor (FcγR)-mediated inhibition of Abs present at the mucosal site may play a role in protection against HIV mucosal transmission. Moreover, mucosal IgA Abs may be determinant in protection against HIV sexual transmission. Therefore, defining Ab inhibitory functions that could lead to protection is critical for further HIV vaccine design. Here, we review different inhibitory properties of HIV-specific Abs and discuss their potential role in protection against HIV sexual transmission.

Antibody-Mediated Fcγ Receptor-Based Mechanisms of HIV Inhibition: Recent Findings and New Vaccination Strategies.

The HIV/AIDS pandemic is one of the most devastating pandemics worldwide. Today, the major route of infection by HIV is sexual transmission. One of the most promising strategies for vaccination against HIV sexual infection is the development of a mucosal vaccine, which should be able to induce strong local and systemic protective immunity. It is believed that both humoral and cellular immune responses are needed for inducing a sterilizing protection against HIV. Recently, passive administration of monoclonal neutralizing antibodies in macaques infected by vaginal challenge demonstrated a crucial role of FcγRs in the protection afforded by these antibodies. This questioned about the role of innate and adaptive immune functions, including ADCC, ADCVI, phagocytosis of opsonized HIV particles and the production of inflammatory cytokines and chemokines, in the mechanism of HIV inhibition in vivo. Other monoclonal antibodies - non-neutralizing inhibitory antibodies - which recognize immunogenic epitopes, have been shown to display potent FcγRs-dependent inhibition of HIV replication in vitro. The potential role of these antibodies in protection against sexual transmission of HIV and their biological relevance for the development of an HIV vaccine therefore need to be determined. This review highlights the potential role of FcγRs-mediated innate and adaptive immune functions in the mechanism of HIV protection.