Fab Fragments Research Articles

Engineering and physicochemical characterization of a novel, stable, symmetric bispecific antibody with dual target-binding using a common light chain.

Bispecific antibodies (BsAbs) have emerged as a major class of antibody therapeutics owing to their substantial potential in disease treatment. While several BsAbs have been successfully approved in recent years, ongoing development efforts continue to focus on optimizing various BsAbs tailored to particular antigens and action mechanisms, aiming to achieve favorable physicochemical properties. BsAbs generally encounter challenges due to their unfavorable physicochemical characteristics and poor manufacturing efficiencies, highlighting the need for optimization to achieve reliable productivity and developability. Herein, we describe the development of a novel symmetric BsAb, REGULGENT™ (N-term/C-term), comprising two Fab domains, using a common light chain. The heavy chain fragment encoded two antigen-binding determinants in one chain. The design and production of REGULGENT™ (N-term/C-term) are simple owing to the use of the same light chain, which does not induce heavy and light chain mispairing, frequently observed with the asymmetric BsAb format. REGULGENT™ (N-term/C-term) exhibited high expression and low aggregation characteristics during cell culture and stress treatment under low pH conditions. Differential scanning calorimetric data indicated that REGULGENT™ molecules had high conformational stability, similar to that of stabilized monoclonal antibodies. Surface plasmon resonance data showed that REGULGENT™ (N-term/C-term) could bind to two antigens simultaneously and exhibited a high affinity for two antigens. In summary, the symmetric BsAb format of REGULGENT™ confers its desirable IgG-like physicochemical properties, thus making it an excellent candidate for commercial development. The findings demonstrate a novel BsAb with substantial development potential for clinical applications.

Suppression of matrigel-induced choroidal neovascularization by AAV delivery of a novel anti-Scg3 antibody.

Efforts to develop gene therapy for long-term treatment of neovascular disease are hampered by ongoing concerns that biologics against vascular endothelial growth factor (VEGF) inhibit both physiological and pathological angiogenesis and are therefore at elevated risk of adverse side effects. A potential solution is to develop disease-targeted gene therapy. Secretogranin III (Scg3), a unique disease-restricted angiogenic factor described by our group, contributes significantly to ocular neovascular disease. We have shown that Scg3 blockade with a monoclonal antibody Fab fragment (Fab) stringently inhibits pathological angiogenesis without affecting healthy vessels. Here we tested the therapeutic efficacy of adeno-associated virus (AAV)-anti-Scg3Fab to block choroidal neovascularization (CNV) induced by subretinal injection of Matrigel in a mouse model. Intravitreal AAV-anti-Scg3Fab significantly reduced CNV and suppressed CNV-associated leukocyte infiltration and macrophage activation. The efficacy and anti-inflammatory effects were equivalent to those achieved by positive control AAV-aflibercept against VEGF. Efficacies of AAV-anti-Scg3Fab and AAV-aflibercept were sustained over 4 months post AAV delivery. The findings support development of AAV-anti-Scg3 as an alternative to AAV-anti-VEGF with equivalent efficacy and potentially safer mechanism of action.

Estimating the Concentration of Heavy Metals in Oleander/Pink and white Oleander Plants

Oleander is a large ornamental evergreen shrub that may grow 20–25 ft in height. During the summer months’ large clusters of white, pink, red, or yellow (yellow oleander) flowers appear at the ends of the branches. All parts of the plant contain cardiac glycosides. Oleander is a common source of serious plant poisoning. In South Asia, particularly Sri Lanka, also present in Libya desert and some house gardens with white and pink types, oleander has become a notorious method of suicide. Toxicity is largely characterized by gastrointestinal symptoms and cardiac abnormalities. Tachy or bradydysthythmias may be present in addition to slowed conduction and heart block. Hyperkalemia is common and correlates with severity of poisoning. A specific antidote—digoxin-specific fragment antigen-binding region (Fab region)- is available. In Mediterranean climates oleanders can be expected to bloom from April through October, with the heaviest bloom usually occurring between May and June. Free-flowering varieties like 'Petite Salmon' or 'Mont Blanc' require no period of rest and can flower continuously throughout the year if the weather remains warm. The exact nature of plant poisoning varies from region to region, but certain plants are almost ubiquitous in distribution and among these, Oleander is a garden plant that features in many homes. Incidents of poisoning from these plants are therefore not uncommon and may be the result of accidental exposure or deliberate, suicidal ingestion of the toxic parts. An attempt has been made to review the management principles with regard to toxicity of these plants and survey the literature in order to highlight current concepts in the treatment of poisoning resulting from both plants. Ingestion of its seeds results in a clinical picture similar to digoxin toxicity. It contains cardiac glycosides that are toxic to cardiac myocytes and autonomic system. Cardiac glycosides of oleander cause poisoning by inhibiting plasmalemmal Na+/K+-ATPase. Results: The poisoning effects of plant or their active alkaloids induced infiltration of cells with hemorrhage and sever negative changes in the lung, induce lesions, and infiltration of inflammatory cells into the portal spaces with scattered necrosis of hepatocytes in the liver, cardiac toxicity of the plant in the heart were included, induced varying degrees of hemorrhage, myocardial degeneration, and necrosis. It also induced arrhythmia, sinus bradycardia, and prolonged P-R interval in electrocardiographic records. Conclusions: The toxic effects of N. oleander are mostly related to its inhibitory effects on the Naþ-Kþ ATPase pump in the cellular membrane. However, the exact molecular mechanism involved in the toxicity of N. oleander is not clear.

In vitro immuno-prevention of nitration/dysfunction of myogenic stem cell activator HGF, towards developing a strategy for age-related muscle atrophy.

In response to peroxynitrite (ONOO-) generation, myogenic stem satellite cell activator HGF (hepatocyte growth factor) undergoes nitration of tyrosine residues (Y198 and Y250) predominantly on fast IIa and IIx myofibers to lose its binding to the signaling receptor c-met, thereby disturbing muscle homeostasis during aging. Here we show that rat anti-HGF monoclonal antibody (mAb) 1H41C10, which was raised in-house against a synthetic peptide FTSNPEVRnitroY198EV, a site well-conserved in mammals, functions to confer resistance to nitration dysfunction on HGF. 1H41C10 was characterized by recognizing both nitrated and non-nitrated HGF with different affinities as revealed by Western blotting, indicating that the paratope of 1H41C10 may bind to the immediate vicinity of Y198. Subsequent experiments showed that 1H41C10-bound HGF resists peroxynitrite-induced nitration of Y198. A companion mAb-1H42F4 presented similar immuno-reactivity, but did not protect Y198 nitration, and thus served as the control. Importantly, 1H41C10-HGF also withstood Y250 nitration to retain c-met binding and satellite cell activation functions in culture. The Fab region of 1H41C10 exerts resistivity to Y250 nitration possibly due to its localization in the immediate vicinity to Y250, as supported by an additional set of experiments showing that the 1H41C10-Fab confers Y250-nitration resistance which the Fc segment does not. Findings highlight the invitro preventive impact of 1H41C10 on HGF nitration-dysfunction that strongly impairs myogenic stem cell dynamics, potentially pioneering cogent strategies for counteracting or treating age-related muscle atrophy with fibrosis (including sarcopenia and frailty) and the therapeutic application of investigational HGF drugs.

Cryo-EM Structure of Phospholipase Cε Defines N-terminal Domains and their Roles in Activity.

Phospholipase Cε (PLCε) increases intracellular Ca 2+ and protein kinase C (PKC) activity in the cardiovascular system in response to stimulation of G protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). The ability of PLCε to respond to these diverse inputs is due, in part, to multiple, conformationally dynamic regulatory domains. However, this heterogeneity has also limited structural studies of the lipase to either individual domains or its catalytic core. Here, we report the 3.9 Å reconstruction of the largest fragment of PLCε to date in complex with an antigen binding fragment (Fab). The structure reveals that PLCε contains a pleckstrin homology (PH) domain and four tandem EF hands, including subfamily-specific insertions and intramolecular interactions with the catalytic core. The structure, together with a model of the holoenzyme, suggest that part of the N-terminus and PH domain form a continuous surface that could engage cytoplasmic leaflets of the plasma and perinuclear membranes, contributing to activity. Functional characterization of this surface confirm it is critical for maximum basal and G protein-stimulated activities. This study provides new insights into the autoinhibited, basal conformation of PLCε and the first mechanistic insights into how it engages cellular membranes for activity.

Investigating the Mechanisms of Antibody Binding to Alpha-Synuclein for the Treatment of Parkinson's Disease.

Parkinson's disease (PD) is an idiopathic neurodegenerative disorder with the second-highest prevalence rate behind Alzheimer's disease. The pathophysiological hallmarks of PD are both degeneration of dopaminergic neurons in the substantia nigra pars compacta and the inclusion of misfolded α-synuclein (α-syn) aggregates known as Lewy bodies. Despite decades of research for potential PD treatments, none have been developed, and developing new therapeutic agents is a time-consuming and expensive process. Computational methods can be used to investigate the properties of drug candidates currently undergoing clinical trials to determine their theoretical efficiency at targeting α-syn. Monoclonal antibodies (mAbs) are biological drugs with high specificity, and Prasinezumab (PRX002) is an mAb currently in Phase II, which targets the C-terminus (AA 118-126) of α-syn. We utilized BioLuminate and PyMol for the structure prediction and preparation of the fragment antigen-binding (Fab) region of PRX002 and 34 different conformations of α-syn. Protein-protein docking simulations were performed using PIPER, and 3 of the docking poses were selected based on the best fit. Molecular dynamics simulations were conducted on the docked protein structures in triplicate for 1000 ns, and hydrogen bonds and electrostatic and hydrophobic interactions were analyzed using MDAnalysis to determine which residues were interacting and how often. Hydrogen bonds were shown to form frequently between the HCDR2 region of PRX002 and α-syn. Free energy was calculated to determine the binding affinity. The predicted binding affinity shows a strong antibody-antigen attraction between PRX002 and α-syn. RMSD was calculated to determine the conformational change of these regions throughout the simulation. The mAb's developability was determined using computational screening methods. Our results demonstrate the efficiency and developability of this therapeutic agent.

Optimal Humanized Scg3-Neutralizing Antibodies for Anti-Angiogenic Therapy of Diabetic Retinopathy.

Secretogranin III (Scg3) is a diabetic retinopathy (DR)-restricted angiogenic factor identified in preclinical studies as a target for DR therapy. Previously, our group generated and characterized ML49.3, an anti-Scg3 monoclonal antibody (mAb) which we then converted into an EBP2 humanized antibody Fab fragment (hFab) with potential for clinical application. We also generated anti-Scg3 mT4 mAb and related EBP3 hFab. In this study, to identify the preferred hFab for DR therapy, we compared all four antibodies for binding, neutralizing and therapeutic activities in vitro and in vivo. Octet binding kinetics analyses revealed that ML49.3 mAb, EBP2 hFab, mT4 mAb and EBP3 hFab have Scg3-binding affinities of 35, 8.7, 0.859 and 0.116 nM, respectively. Both anti-Scg3 EBP2 and EBP3 hFabs significantly inhibited Scg3-induced proliferation and migration of human umbilical vein endothelial cells in vitro, and alleviated DR vascular leakage and choroidal neovascularization with high efficacy. Paired assays in DR mice revealed that intravitreally injected EBP3 hFab is 26.4% and 10.3% more effective than EBP2 hFab and aflibercept, respectively, for ameliorating DR leakage. In conclusion, this study confirms the markedly improved binding affinities of hFabs compared to mAbs and further identifies EBP3 hFab as the preferred antibody to develop for anti-Scg3 therapy.

Administration of anti-HIV-1 broadly neutralizing monoclonal antibodies with increased affinity to Fcγ receptors during acute SHIVAD8-EO infection

Anti-HIV-1 broadly neutralizing antibodies (bNAbs) have the dual potential of mediating virus neutralization and antiviral effector functions through their Fab and Fc domains, respectively. So far, bNAbs with enhanced Fc effector functions in vitro have only been tested in NHPs during chronic simian-HIV (SHIV) infection. Here, we investigate the effects of administering in acute SHIVAD8-EO infection either wild-type (WT) bNAbs or bNAbs carrying the S239D/I332E/A330L (DEL) mutation, which increases binding to FcγRs. Emergence of virus in plasma and lymph nodes (LNs) was delayed by bNAb treatment and occurred earlier in monkeys given DEL bNAbs than in those given WT bNAbs, consistent with faster clearance of DEL bNAbs from plasma. DEL bNAb-treated monkeys had higher levels of circulating virus-specific IFNγ single-producing CD8+ CD69+ T cells than the other groups. In LNs, WT bNAbs were evenly distributed between follicular and extrafollicular areas, but DEL bNAbs predominated in the latter. At week 8 post-challenge, LN monocytes and NK cells from DEL bNAb-treated monkeys upregulated proinflammatory signaling pathways and LN T cells downregulated TNF signaling via NF-κB. Overall, bNAbs with increased affinity to FcγRs shape innate and adaptive cellular immunity, which may be important to consider in future strategies of passive bNAb therapy.

Allosteric inhibition of IgE–FcεRI interactions by simultaneous targeting of IgE F(ab’)2 epitopes

Immunoglobulin E (IgE) plays pivotal roles in allergic diseases through interaction with a high-affinity receptor (FcεRI). We established that Fab fragments of anti-IgE antibodies (HMK-12 Fab) rapidly dissociate preformed IgE-FcεRI complexes in a temperature-dependent manner and inhibit IgE-mediated anaphylactic reactions, even after allergen challenge. X-ray crystallographic studies revealed that HMK-12 Fab interacts with each of two equivalent epitopes on the Cε2 homodimer domain involved in IgE F(ab’)2. Consequently, HMK-12 Fab-mediated targeting of Cε2 reduced the binding affinity of Fc domains and resulted in rapid removal of IgE from the receptor complex. This unexpected finding of allosteric inhibition of IgE-FcεRI interactions by simultaneous targeting of two epitope sites on the Cε2 homodimer domain of IgE F(ab’)2 may have implications for the development of novel therapies for allergic disease.

Combination of Anti-CD40 and Anti-CD40L Antibodies as Co-Stimulation Blockade in Preclinical Cardiac Xenotransplantation.

The blockade of the CD40/CD40L immune checkpoint is considered essential for cardiac xenotransplantation. However, it is still unclear which single antibody directed against CD40 or CD40L (CD154), or which combination of antibodies, is better at preventing organ rejection. For example, the high doses of antibody administered in previous experiments might not be feasible for the treatment of humans, while thrombotic side effects were described for first-generation anti-CD40L antibodies. To address these issues, we conducted six orthotopic pig-to-baboon cardiac xenotransplantation experiments, combining a chimeric anti-CD40 antibody with an investigational long-acting PASylated anti-CD40L Fab fragment. The combination therapy effectively resulted in animal survival with a rate comparable to a previous study that utilized anti-CD40 monotherapy. Importantly, no incidence of thromboembolic events associated with the administration of the anti-CD40L PAS-Fab was observed. Two experiments failed early because of technical reasons, two were terminated deliberately after 90 days with the baboons in excellent condition and two were extended to 120 and 170 days, respectively. Unexpectedly, and despite the absence of any clinical signs, histopathology revealed fungal infections in all four recipients. This study provides, for the first time, insights into a combination therapy with anti-CD40/anti-CD40L antibodies to block this immune checkpoint.

Structural Basis for the Recognition of GPRC5D by Talquetamab, a Bispecific Antibody for Multiple Myeloma

Multiple myeloma (MM) is a complex hematological malignancy characterized by abnormal antibody production from plasma cells. Despite advances in the treatment, many patients experience disease relapse or become refractory to treatment. G-protein-coupled receptor class C group 5 member D (GPRC5D), an orphan GPCR predominantly expressed in MM cells, is emerging as a promising target for MM immunotherapy. Talquetamab, a Food and Drug Administration-approved T-cell-directing bispecific antibody developed for treatment of MM, targets GPRC5D. Here, we elucidate the structure of GPRC5D complexed with the Fab fragment of talquetamab, using cryo-electron microscopy, providing the basis for recognition of GPRC5D by the bispecific antibody. GPRC5D forms a symmetric homodimer with the interface between transmembrane helix (TM) 4 of one protomer and TM4/5 of the other protomer. A single talquetamab Fab interacts with the GPRC5D dimer with its orientation toward the dimer interface. All six complementarity-determining regions of talquetamab engage with extracellular loops and TM3/5/7. In particular, the side-chain of an arginine residue from the antibody penetrates into a shallow pocket on the extracellular surface of GPRC5D. The structure offers insights for optimizing antibody design against GPRC5D for relapsed or refractory MM therapy.

Rational design of environmentally responsive antibodies with pH-sensing synthetic amino acids

Antibodies are widely used as therapeutic agents to tackle various diseases. In the present study, to enhance their clinical values, we rationally designed pH-responsivity by exploiting the idiosyncratic protonation/deprotonation profiles of non-natural amino acids. 3-Nitro-l-tyrosine, 3-cyano-l-tyrosine, and 3, 5-halogenated-l-tyrosine, each with near neutral pKa, were thus incorporated into Fab fragments in place of tyrosines and other residues in the variable regions. Cell-based assays showed that these modifications achieved up to 140-fold tighter binding to antigens and several-fold tighter cytotoxicity to antigen-expressing cell at pH 6.0 than pH 7.4. The pH-dependent binding effect was retained in full-length antibodies. In silico structural analyses revealed electrostatic repulsion at neutral pH between antigens and antibodies or inside the antibody as the underlying mechanisms of the acid preference, and this finding increases the designability of pH-dependent antigen binding. The development of antibodies responsive to the microenvironments of diseased tissues will allow more disease-related antigens to be targeted in treatments, because of the reduced cross-reactivity toward healthy tissues.

Antibody-mediated control mechanisms of viral infections.

Antibodies generated after vaccination or natural pathogen exposure are essential mediators of protection against many infections. Most studies with viruses have focused on antibody neutralization, in which protection is conferred by the fragment antigen binding region (Fab) through targeting of different steps in the viral lifecycle including attachment, internalization, fusion, and egress. Beyond neutralization, the fragment crystallizable (Fc) region of antibodies can integrate innate and adaptive immune responses by engaging complement components and distinct Fc gamma receptors (FcγR) on different host immune cells. In this review, we discuss recent advances in our understanding of antibody neutralization and Fc effector functions, and the assays used to measure them. Additionally, we describe the contexts in which these mechanisms are associated with protection against viruses and highlight how Fc-FcγR interactions can improve the potency of antibody-based therapies.

Potent AMA1-specific human monoclonal antibody against P. vivax Pre-erythrocytic and Blood Stages.

New therapeutics are necessary for preventing Plasmodium vivax malaria due to easy transmissibility and dormancy in the liver that increases the clinical burden due to recurrent relapse. We isolated 12 Pv Apical Membrane Antigen 1 (PvAMA1) specific human monoclonal antibodies from Peripheral Blood Mononuclear Cells of a Pv exposed individual. PvAMA1 is essential for sporozoite and merozoite invasion, making it a unique therapeutic target. HumAb 826827 blocked the invasion of human erythrocytes using Pv clinical isolates and inhibited sporozoite invasion of human hepatocytes in vitro (IC50 of 0.3 to 3.7 ug/mL). It also significantly reduced liver infection of chimeric FRG humHep mice in vivo. The crystal structure of rPvAMA1 bound to 826827 shows that 826827 partially occupies the highly conserved hydrophobic groove in PvAMA1 that binds its known receptor, RON2. We have isolated a potent humAb that is isolate transcendent, blocks both pre erythrocytic and blood stage infection, and could be a new therapy for Pv.

1H, 13C and 15N resonance assignments of a shark variable new antigen receptor against hyaluronan synthase.

Single domain antibody (sdAb) is only composed of a variable domain of the heavy-chain-only antibody, which is devoid of light chain and naturally occurring in camelids and cartilaginous fishes. Variable New Antigen Receptor (VNAR), a type of single domain antibody present in cartilaginous fishes such as sharks, is the smallest functional antigen-binding fragment found in nature. The unique features, including flexible paratope, high solubility and outstanding stability make VNAR a promising prospect in antibody drug development and structural biology research. However, VNAR's research has lagged behind camelid-derived sdAb, especially in the field of structural research. Here we report the 1H,15N,13C resonance assignments of a VNAR derived from the immune library of Chiloscyllium plagiosum, termed B2-3, which recognizes the hyaluronan synthase. Analysis of the backbone chemical shifts demonstrates that the secondary structure of VNAR is predominately composed of β-sheets corresponding to around 40% of the B2-3 backbone. The Cβ chemical shift values of cysteine residues, combined with mass spectrometry data, clearly shows that B2-3 contains two pairs of disulfide bonds, which is import for protein stability. The assignments will be essential for determining the high resolution solution structure of B2-3 by NMR spectroscopy.

Self-reporting photodynamic nanobody conjugate for precise and sustainable large-volume tumor treatment

Nanobodies (Nbs), the smallest antigen-binding fragments with high stability and affinity derived from the variable domain of naturally occurring heavy-chain-only antibodies in camelids, have been shown as an efficient way to improve the specificity to tumors for photodynamic therapy (PDT). Nonetheless, the rapid clearance of Nbs in vivo restricts the accumulation and retention of the photosensitizer at the tumor site causing insufficient therapeutic outcome, especially in large-volume tumors. Herein, we develop photodynamic conjugates, MNB-Pyra Nbs, through site-specific conjugation between 7D12 Nbs and type I photosensitizer MNB-Pyra (morpholine-modified nile blue structure connected to pyrazolinone) in a 1:2 ratio. The photosensitizers with long-term retention can be released at the tumor site by reactive oxygen species cleavage after illumination, accompanied with fluorescence recovery for self-reporting the occurrence of PDT. Ultimately, a single dose of MNB-Pyra Nbs demonstrate highly effective tumor suppression with high biosafety in the large-volume tumor models after three rounds of PDT. This nanobody conjugate provides a paradigm for the design of precise long-time retention photosensitizers and is expected to promote the development of PDT.

The Structural Basis for Recognition of Human Leukocyte Antigen Class I Molecules by the Pan-HLA Antibody W6/32.

The central immunological role of HLA class I (HLA-I) in presenting peptide Ags to cellular components of the immune system has been the focus of intense study for >60 y. A confounding factor in the study of HLA-I has been the extreme polymorphism of these molecules. The mAb W6/32 has been a fundamental reagent bypassing the issue of polymorphism by recognizing an epitope that is conserved across diverse HLA-I allotypes. However, despite the widespread use of W6/32, the epitope of this Ab has not been definitively mapped. In this study, we present the crystal structure of the Fab fragment of W6/32 in complex with peptide-HLA-B*27:05. W6/32 bound to HLA-B*27:05 beneath the Ag-binding groove, recognizing a discontinuous epitope comprised of the α1, α2, and α3 domains of HLA-I and β2-microglobulin. The epitope comprises a region of low polymorphism reflecting the pan-HLA-I nature of the binding. Notably, the W6/32 epitope neither overlaps the HLA-I binding sites of either T cell Ag receptors or killer cell Ig-like receptors. However, it does coincide with the binding sites for leukocyte Ig-like receptors and CD8 coreceptors. Consistent with this, the use of W6/32 to block the interaction of NK cells with HLA-I only weakly impaired inhibition mediated by KIR3DL1, but impacted HLA-LILR recognition.

A Case Report of Crotalidae Immune F(ab')2-associated Coagulopathy Recurrence in a Preschool-age Child.

Pit viper envenomation may cause coagulopathy. The coagulopathy has been treated with crotalidae polyvalent immune fragment antigen-binding (Fab) ovine antivenom for the last few decades in the United States and usually corrects the acute coagulopathy within hours. Days after receiving Fab, coagulopathy may recur in approximately half of the patients. Another divalent antivenom, crotalidae immune F(ab')2 (equine)-F(ab')2-was approved by the US Food and Drug Administration for the treatment of pit viper envenomation. F(ab')2 is composed of two linked antigen-binding fragments of immunoglobulin G. Several studies have demonstrated that F(ab')2 is less likely to be associated with recurrence. There is no reported case of F(ab')2-associated late coagulopathy in very young patients. We report the first case of recurrence associated with F(ab')2 use in a preschool-age child. A preschool-age male developed leg swelling and hypofibrinogenemia after rattlesnake envenomation. F(ab')2 was administered to stabilize the leg edema and to correct the hypofibrinogenemia. The patient improved clinically and was discharged on hospital day five. Seven days after the rattlesnake envenomation, he returned to the emergency department as instructed. Laboratory data revealed recurrent hypofibrinogenemia. There are two antivenoms available in the US to treat crotalid envenomation, Fab and F(ab')2. F(ab')2 is less likely to be associated with recurrent coagulopathy in comparison to Fab. We report the first case of recurrence associated with F(ab')2 in a preschool-age child. It is important that the emergency physician be aware of potential F(ab')2-associated recurrent coagulopathy. Adult and pediatric patients may need to follow up to be evaluated for hypofibrinogenemia and/or thrombocytopenia after receiving F(ab')2.

Molecular insight into the neutralization mechanism of human-origin monoclonal antibody AH100 against Hantaan virus.

Both Old World and New World hantaviruses are transmitted through rodents and can lead to hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome in humans without the availability of specific therapeutics. The square-shaped surface spikes of hantaviruses consist of four Gn-Gc heterodimers that are pivotal for viral entry into host cells and serve as targets for the immune system. Previously, a human-derived neutralizing monoclonal antibody, AH100, demonstrated specific neutralization against the Old World hantavirus, Hantaan virus. However, the precise mode binding of this neutralizing monoclonal antibody remains unclear. In the present study, we determined the structure of the Hantaan virus Gn-AH100 antigen-binding fragment complex and identified its epitope. Crystallography revealed that AH100 targeted the epitopes on domain A and b-ribbon and E3-like domain. Epitope mapping onto a model of the higher order (Gn-Gc)4 spike revealed its localization between neighboring Gn protomers, distinguishing this epitope as a unique site compared to the previously reported monoclonal antibodies. This study provides crucial insights into the structural basis of hantavirus neutralizing antibody epitopes, thereby facilitating the development of therapeutic antibodies.IMPORTANCEHantaan virus (HTNV) poses a significant threat to humans by causing hemorrhagic fever with renal syndrome with high mortality rates. In the absence of FDA-approved drugs or vaccines, it is urgent to develop specific therapeutics. Here, we elucidated the epitope of a human-derived neutralizing antibody, AH100, by determining the HTNV glycoprotein Gn-AH100 antigen-binding fragment (Fab) complex structure. Our findings revealed that the epitopes situated on the domain A and b-ribbon and E3-like domain of the HTNV Gn head. By modeling the complex structure in the viral lattice, we propose that AH100 neutralizes the virus by impeding conformational changes of Gn protomer, which is crucial for viral entry. Additionally, sequence analysis of all reported natural isolates indicated the absence of mutations in epitope residues, suggesting the potential neutralization ability of AH100 in diverse isolates. Therefore, our results provide novel insights into the epitope and the molecular basis of AH100 neutralization.

Exploring How Antibody Format Drives Clearance from the Brain.

Monoclonal antibodies (mAbs) have high binding specificity and affinity, making them attractive for treating brain diseases. However, their effectiveness is limited by poor blood-brain barrier (BBB) penetration and rapid central nervous system (CNS) clearance. Our group identified blood-brain barrier modulator (BBBM) peptides that improved mAb penetration across the BBB into the brain. In this study, we investigated the pharmacokinetics of a mAb delivered to the brain using BBBMs after intravenous (IV) administration and explored the impact of antibody format (size, neonatal Fc receptor (FcRn) binding, hyaluronic acid binding) on brain clearance following direct injection into the central nervous system (CNS) via intracerebroventricular (ICV) injection. IRDye800CW-labeled antibodies were administered into C57BL/6 mice via ICV or IV injection, and organ concentrations were measured after various time points. When a mAb was coadministered with a BBBM peptide, the permeation of mAb across the BBB was increased compared to mAb alone at early time points; however, the mAb was cleared within 2 h from the brain. ICV experiments revealed that an antibody Fab fragment had a higher brain exposure than a mAb, and that a Fab fused to a hyaluronic acid binding domain (Fab-VG1) showed remarkable improvement in brain exposure. These findings suggest that BBBMs and antibody format optimization may be promising strategies for enhancing brain retention of therapeutic antibodies.