Development Of Therapeutic Antibodies Research Articles

Hierarchically Structured Molecularly Imprinted Nanotransducers for Truncated HER2-Targeted Photodynamic Therapy of Therapeutic Antibody-Resistant Breast Cancer.

Antibodies have been a mainstream class of therapeutics for clinical treatment of various diseases, especially cancers. However, mutation in cancer cells leads to resistance to therapeutic antibodies, hyperactivity of proliferation of cancer cells, and difficulty in the development of therapeutic antibodies. Herein, we present a strategy termed molecularly imprinted nanotransducer (MINT) for targeted photodynamic therapy (PDT) of mutated cancers. The MINT is a rationally engineered nanocomposite featuring a core of an upconversion nanoparticle, a shell of a thin layer of molecularly imprinted polymer, and a photosensitizer modified on the surface. As a proof-of-principle, truncated HER2 (P95HER2) overexpressed breast cancer, a challenging cancer lacking effective targeted therapeutics, was used as the cancer model. The designed structure, properties, functions, and anticancer efficacy of MINT were systematically investigated and experimentally confirmed. The MINT could not only specifically target P95HER2+ cancer cells in vitro and in vivo but also efficiently transfer the irradiated light and generate excited-state oxygen, resulting in efficient targeted cancer killing. Therefore, the MINT strategy provides a promising therapeutic for targeted PDT of drug-resistant cancers caused by target mutation.

Delivery of a novel membrane-anchored Fc chimera enhances NK cell-mediated killing of tumor cells and persistently virus-infected cells.

Antibody-dependent cellular cytotoxicity (ADCC) is one of the most powerful mechanisms for Natural Killer (NK) cells to kill cancer cells or virus-infected cells. A novel chimeric protein (NA-Fc) was created, which when expressed in cells, positions an IgG Fc domain on the plasma membrane, mimicking the orientation of IgG bound to the cell surface. This NA-Fc chimera was tested with PM21-NK cells, produced through a previously developed particle-based method which yields superior NK cells for immunotherapeutic applications. Real time viability assays revealed higher PM21-NK killing of both ovarian and lung cancer cells expressing NA-Fc, which correlated with increased release of TNF-α and IFN-γ cytokines from NK cells and was dependent on CD16-Fc interactions. Lentivirus delivery of NA-Fc to target cells increased the rate of PM21-NK cell killing of A549 and H1299 lung, SKOV3 ovarian and A375 melanoma cancer cells. This NA-Fc-directed killing was extended to virus infected cells, where delivery of NA-Fc to lung cells that were persistently infected with Parainfluenza virus resulted in increased killing by PM21-NK cells. In contrast to its effect on PM21-NK cells, the NA-Fc molecule did not enhance complement mediated lysis of lung cancer cells. Our study lays the foundation for application of the novel NA-Fc chimera that could be delivered specifically to tumors during oncolytic virotherapy to mark target cells for ADCC by co-treatment with adoptive NK cells. This strategy would potentially eliminate the need to search for unique cancer specific antigens for development of new antibody therapeutics.

Drugs under Development (Neurology)

Neurological diseases, especially degenerative diseases, have been mainly treated symptomatically with small molecule drugs. In recent years, however, the development of antibody therapeutics, nucleic acid therapeutics, and gene therapies that selectively act on specific proteins, RNA, and DNA has been underway to identify disease-modifying drugs that improve disease outcomes by acting on the underlying pathogenic mechanisms of diseases. It is expected to enable disease-modifying therapy not only for neuroimmunological and functional diseases, but also for neurodegenerative diseases caused by loss of protein function and accumulation of abnormal proteins.

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

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

A procedure for producing an anti-AXL nanobody in E. coli

As one of the receptors of the TAM family, AXL plays a vital role in stem cell maintenance, angiogenesis, immune escape of viruses and drug resistance against tumors. In this study, the truncated extracellular segment containing two immunoglobulin-like domains of human AXL (AXL-IG), which has been confirmed to bind growth arrest specific 6 (GAS6) by structural studies [1], was expressed in a prokaryotic expression system and then purified. Immunizing camelid with the purified AXL-IG as antigen could lead to the production of unique nanobodies composed of only variable domain of heavy chain of heavy-chain antibody (VHH), which are around 15 kD and stable. We screened out a nanobody A-LY01 specific binding to AXL-IG. We further determined the affinity of A-LY01 to AXL-IG and revealed that A-LY01 could specifically recognize full-length AXL on the surface of HEK 293T/17 cells. Our study provides appropriate support for the development of diagnostic reagents and antibody therapeutics targeting AXL.

Generation of a symmetrical trispecific NK cell engager based on a two-in-one antibody.

To construct a trispecific IgG-like antibody at least three different binding moieties need to be combined, which results in a complex architecture and challenging production of these molecules. Here we report for the first time the construction of trispecific natural killer cell engagers based on a previously reported two-in-one antibody combined with a novel anti-CD16a common light chain module identified by yeast surface display (YSD) screening of chicken-derived immune libraries. The resulting antibodies simultaneously target epidermal growth factor receptor (EGFR), programmed death-ligand 1 (PD-L1) and CD16a with two Fab fragments, resulting in specific cellular binding properties on EGFR/PD-L1 double positive tumor cells and a potent ADCC effect. This study paves the way for further development of multispecific therapeutic antibodies derived from avian immunization with desired target combinations, valencies, molecular symmetries and architectures.

Surface patches induce nonspecific binding and phase separation of antibodies

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

Structural constraints link differences in neutralization potency of human anti-Eastern equine encephalitis virus monoclonal antibodies

Selection and development of monoclonal antibody (mAb) therapeutics against pathogenic viruses depends on certain functional characteristics. Neutralization potency, or the half-maximal inhibitory concentration (IC50) values, is an important characteristic of candidate therapeutic antibodies. Structural insights into the bases of neutralization potency differences between antiviral neutralizing mAbs are lacking. In this report, we present cryo-electron microscopy (EM) reconstructions of three anti-Eastern equine encephalitis virus (EEEV) neutralizing human mAbs targeting overlapping epitopes on the E2 protein, with greater than 20-fold differences in their respective IC50 values. From our structural and biophysical analyses, we identify several constraints that contribute to the observed differences in the neutralization potencies. Cryo-EM reconstructions of EEEV in complex with these Fab fragments reveal structural constraints that dictate intravirion or intervirion cross-linking of glycoprotein spikes by their IgG counterparts as a mechanism of neutralization. Additionally, we describe critical features for the recognition of EEEV by these mAbs including the epitope-paratope interaction surface, occupancy, and kinetic differences in on-rate for binding to the E2 protein. Each constraint contributes to the extent of EEEV inhibition for blockade of virus entry, fusion, and/or egress. These findings provide structural and biophysical insights into the differences in mechanism and neutralization potencies of these antibodies, which help inform rational design principles for candidate vaccines and therapeutic antibodies for all icosahedral viruses.

IgG1 Is the Optimal Subtype for Treating Atherosclerosis by Inducing M2 Macrophage Differentiation, and Is Independent of the FcγRIIA Gene Polymorphism

In recent years, it has been established that atherosclerosis is an autoimmune disease. However, little is currently known about the role of FcγRIIA in atherosclerosis. Herein, we sought to investigate the relationship between FcγRIIA genotypes and the effectiveness of different IgG subclasses in treating atherosclerosis. We constructed and produced different subtypes of IgG and Fc-engineered antibodies. In vitro, we observed the effect of different subtypes of IgG and Fc-engineered antibodies on the differentiation of CD14+ monocytes from patients or healthy individuals. In vivo, Apoe-/- mice were fed a high-fat diet (HFD) for 20 weeks and administered injections of different CVI-IgG subclasses or Fc-engineered antibodies. Flow cytometry was used to assess the polarization of monocytes and macrophages. Although CVI-IgG4 reduced the release of MCP-1 compared to the other subtypes, IgG4 did not yield an anti-inflammatory effect by induction of human monocyte and macrophage differentiation in vitro. Furthermore, genetic polymorphisms of FcγRIIA were not associated with different CVI-IgG subclasses during the treatment of atherosclerosis. In vivo, CVI-IgG1 decreased Ly6Chigh monocyte differentiation and promoted M2 macrophage polarization. We also found that the secretion of IL-10 was upregulated in the CVI-IgG1-treated group, whereas V11 and GAALIE exerted no significant effect. These findings highlight that IgG1 is the optimal subtype for treating atherosclerosis, and CVI-IgG1 can induce monocyte/macrophage polarization. Overall, these results have important implications for the development of therapeutic antibodies.

Process development of a SARS-CoV-2 nanoparticle vaccine

One of the outcomes from the global COVID-19 pandemic caused by SARS-CoV-2 has been an acceleration of development timelines to provide treatments in a timely manner. For example, it has recently been demonstrated that the development of monoclonal antibody therapeutics from vector construction to IND submission can be achieved in five to six months rather than the traditional ten-to-twelve-month timeline using CHO cells [1,2]. This timeline is predicated on leveraging existing, robust platforms for upstream and downstream processes, analytical methods, and formulation. These platforms also reduce; the requirement for ancillary studies such as cell line stability, or long-term product stability studies. Timeline duration was further reduced by employing a transient cell line for early material supply and using a stable cell pool to manufacture toxicology study materials. The development of non-antibody biologics utilizing traditional biomanufacturing processes in CHO cells within a similar timeline presents additional challenges, such as the lack of platform processes and additional analytical assay development. In this manuscript, we describe the rapid development of a robust and reproducible process for a two-component self-assembling protein nanoparticle vaccine for SARS-CoV-2. Our work has demonstrated a successful academia-industry partnership model that responded to the COVID-19 global pandemic quickly and efficiently and could improve our preparedness for future pandemic threats.

Harnessing Fc/FcRn Affinity Data from Patents with Different Machine Learning Methods.

Monoclonal antibodies are biopharmaceuticals with a very long half-life due to the binding of their Fc portion to the neonatal receptor (FcRn), a pharmacokinetic property that can be further improved through engineering of the Fc portion, as demonstrated by the approval of several new drugs. Many Fc variants with increased binding to FcRn have been found using different methods, such as structure-guided design, random mutagenesis, or a combination of both, and are described in the literature as well as in patents. Our hypothesis is that this material could be subjected to a machine learning approach in order to generate new variants with similar properties. We therefore compiled 1323 Fc variants affecting the affinity for FcRn, which were disclosed in twenty patents. These data were used to train several algorithms, with two different models, in order to predict the affinity for FcRn of new randomly generated Fc variants. To determine which algorithm was the most robust, we first assessed the correlation between measured and predicted affinity in a 10-fold cross-validation test. We then generated variants by in silico random mutagenesis and compared the prediction made by the different algorithms. As a final validation, we produced variants, not described in any patents, and compared the predicted affinity with the experimental binding affinities measured by surface plasmon resonance (SPR). The best mean absolute error (MAE) between predicted and experimental values was obtained with a support vector regressor (SVR) using six features and trained on 1251 examples. With this setting, the error on the log(KD) was less than 0.17. The obtained results show that such an approach could be used to find new variants with better half-life properties that are different from those already extensively used in therapeutic antibody development.

Comparative Thermodynamics of the Reversible Self-Association of Therapeutic mAbs Reveal Opposing Roles for Linked Proton- and Ion-Binding Events.

Reversible self-association (RSA) has long been a concern in therapeutic monoclonal antibody (mAb) development. Because RSA typically occurs at high mAb concentrations, accurate assessment of the underlying interaction parameters requires explicitly addressing hydrodynamic and thermodynamic nonideality. We previously examined the thermodynamics of RSA for two mAbs, C and E, in phosphate buffered saline (PBS). Here we continue to explore the mechanistic aspects of RSA by examining the thermodynamics of both mAbs under reduced pH and salt conditions. Dynamic light scattering and sedimentation velocity (SV) studies were conducted for both mAbs at multiple protein concentrations and temperatures, with the SV data analyzed via global fitting to determine best-fit models, interaction energetics, and nonideality contributions. We find that mAb C self-associates isodesmically irrespective of temperature, and that association is enthalpically driven but entropically penalized. Conversely, mAb E self-associates cooperatively and via a monomer-dimer-tetramer-hexamer reaction pathway. Moreover, all mAb E reactions are entropically driven and enthalpically modest or minimal. The thermodynamics for mAb C self-association are classically seen as originating from van der Waals interactions and hydrogen bonding. However, relative to the energetics we determined in PBS, self-association must also be linked to proton release and/or ion uptake events. For mAb E, the thermodynamics implicate electrostatic interactions. Furthermore, self-association is instead linked to proton uptake and/or ion release, and primarily by tetramers and hexamers. Finally, although the origins of mAb E cooperativity remain unclear, ring formation remains a possibility whereas linear polymerization reactions can be eliminated.

Polyspecificity - An emerging trend in the development of clinical antibodies.

The essence of the growth and development of therapeutic conventional monoclonal antibodies (MAbs) for the treatment of various disorders is the aptitude of MAbs to precisely bind a target antigen and neutralise or promote its activity. However, the conventional antibodies are monoclonal i.e., both paratopes bind to the same epitope. But most of the pathophysiological conditions are multifaceted, hence targeting/blocking/inhibition of more than one epitope/antigen is more promising than one epitope/antigen. Polyspecific antibodies (PsAbs) have the potential to concurrently bind to more than one target and are the next-generation antibodies that augment efficacy in both clinical and non-clinical contexts. Thus, the trend of engineering and developing various formats of PsAbs is emerging. In this review, we have briefly discussed the importance of antibody polyspecificity and PsAbs approved for clinical use. Subsequently, we have discussed the role of TNF-α and IL-23 in inflammatory diseases and stressed the need for developing anti-TNF-α and anti-IL-23 bispecific antibodies.

Fibroblast, Epithelial and Endothelial Cell-Derived Human Cytomegalovirus Strains Display Distinct Neutralizing Antibody Responses and Varying Levels of gH/gL Complexes.

In sequential sera from pregnant women with HCMV primary infection (PI), the serum neutralizing activity is higher against virions produced in epithelial and endothelial cells than in fibroblasts. Immunoblotting shows that the pentamer complex/trimer complex (PC/TC) ratio varies according to the producer cell culture type used for the virus preparation to be employed in the neutralizing antibody (NAb) assay, and is lower in fibroblasts and higher in epithelial, and especially endothelial cells. The blocking activity of TC- and PC-specific inhibitors varies according to the PC/TC ratio of virus preparations. The rapid reversion of the virus phenotype following its back passage to the original cell culture (fibroblasts) potentially argues in favor of a producer cell effect on virus phenotype. However, the role of genetic factors cannot be overlooked. In addition to the producer cell type, the PC/TC ratio may differ in single HCMV strains. In conclusion, the NAb activity not only varies with different HCMV strains, but is a dynamic parameter changing according to virus strain, type of target and producer cells, and number of cell culture passages. These findings may have some important implications for the development of both therapeutic antibodies and subunit vaccines.

Two-Dimensional Capillary Zone Electrophoresis-Mass Spectrometry: Intact mAb Charge Variant Separation Followed by Peptide Level Analysis Using In-Capillary Digestion.

Characterization of charge heterogeneity is an essential pillar for pharmaceutical development and quality control of therapeutic monoclonal antibodies (mAbs). The highly selective and commonly applied capillary zone electrophoresis (CZE) method containing high amounts of ε-aminocaproic acid (EACA) provides a detailed and robust charge heterogeneity profile of intact mAb variants. Nevertheless, the exact location of protein modifications within these charge profiles remains ambiguous. Electrospray ionization mass spectrometry (ESI-MS) is a promising tool for this purpose; however, EACA is incompatible with electrospray. In this context, we present a two-dimensional CZE-CZE-MS system to combine efficient charge variant separation of intact mAbs with subsequent peptide analysis after in-capillary digestion of selected charge variants. The first dimension is based on a generic CZE(EACA) method in a fused silica capillary. In the second dimension, a neutral-coated capillary is used for in-capillary reduction and digestion with Tris(2-carboxyethyl)phosphine (TCEP) and pepsin, followed by CZE separation and MS/MS-characterization of the resulting peptides. The setup is demonstrated using stressed and nonstressed mAbs where peaks of basic, main, and acidic variants were transferred in a heart-cut fashion, digested, and characterized on the peptide level. Sequence coverages of more than 90% were obtained for heavy chain (HC) and light chain (LC) for four different mAbs, including low-abundance variants (<2% of the main peak). Frequently observed modifications (deamidation, oxidation, etc.) could be detected and localized. This study demonstrates a proof-of-concept for identification and localization of protein modifications from CZE charge heterogeneity profiles and, in this way, is expected to support the development and quality control testing of protein pharmaceuticals.

Antibody Fc-chimerism and effector functions: When IgG takes advantage of IgA.

Recent advances in the development of therapeutic antibodies (Abs) have greatly improved the treatment of otherwise drug-resistant cancers and autoimmune diseases. Antibody activities are mediated by both their Fab and the Fc. However, therapeutic Abs base their protective mechanisms on Fc-mediated effector functions resulting in the activation of innate immune cells by FcRs. Therefore, Fc-bioengineering has been widely used to maximise the efficacy and convenience of therapeutic antibodies. Today, IgG remains the only commercially available therapeutic Abs, at the expense of other isotypes. Indeed, production, sampling, analysis and related in vivo studies are easier to perform with IgG than with IgA due to well-developed tools. However, interest in IgA is growing, despite a shorter serum half-life and a more difficult sampling and purification methods than IgG. Indeed, the paradigm that the effector functions of IgG surpass those of IgA has been experimentally challenged. Firstly, IgA has been shown to bind to its Fc receptor (FcR) on effector cells of innate immunity with greater efficiency than IgG, resulting in more robust IgA-mediated effector functions in vitro and better survival of treated animals. In addition, the two isotypes have been shown to act synergistically. From these results, new therapeutic formats of Abs are currently emerging, in particular chimeric Abs containing two tandemly expressed Fc, one from IgG (Fcγ) and one from IgA (Fcα). By binding both FcγR and FcαR on effector cells, these new chimeras showed improved effector functions in vitro that were translated in vivo. Furthermore, these chimeras retain an IgG-like half-life in the blood, which could improve Ab-based therapies, including in AIDS. This review provides the rationale, based on the biology of IgA and IgG, for the development of Fcγ and Fcα chimeras as therapeutic Abs, offering promising opportunities for HIV-1 infected patients. We will first describe the main features of the IgA- and IgG-specific Fc-mediated signalling pathways and their respective functional differences. We will then summarise the very promising results on Fcγ and Fcα containing chimeras in cancer treatment. Finally, we will discuss the impact of Fcα-Fcγ chimerism in prevention/treatment strategies against infectious diseases such as HIV-1.

Structures of Cancer Antigen Mesothelin and Its Complexes with Therapeutic Antibodies.

The structures of full-length mesothelin and its complexes with antibodies reported here are the first to be determined experimentally, providing atomic models for structural organization of this protein and its interactions with antibodies. It offers insights into the function of mesothelin and guidance for further development of therapeutic antibodies.

Membrane binding is involved in the action of broadly neutralizing influenza antibodies.

Influenza poses a major health issue globally. Characterized by annual epidemics with mild to severe symptoms and even a significant number of deaths, the virus has historically resulted in substantial economic and societal problems in the world. Neutralizing antibodies that bind to the membrane-proximal region of hemagglutinin (HA) provide considerable protection against the infection by blocking the fusion of virus to the host cell. Here, using an array of advanced modeling and simulation techniques employed to a very large molecular assembly, for the first time, we develop a high-resolution structural model of full-length, antibody-bound HA in a native viral membrane and describe key molecular interactions that determine the binding affinity of the antibody to the viral membrane. The model allowed us to identify key residues in the antibody that interact with and bind to the membrane. Guided by the simulation predictions, the virus neutralization activity of the antibody was evaluated by mutagenesis experiments and infectivity assays. In alignment with the simulation results, the experiments show that mutating the predicted basic residues located away from the antigen binding site in the antibody decreases the neutralizing activity by up to an order of magnitude. Based on the results, we propose that the direct interaction of membrane-proximal antibodies with the viral membrane plays a role in their neutralizing activity against the virus. Introducing additional basic side chains enhancing phospholipid-binding is expected to further stabilize the HA-antibody. Given the rapid evolution of the influenza virus, the developed model provides a framework for rational design and development of more effective therapeutic antibodies.

Development of machine learning models for prediction of antibody non-specificity.

For the development of therapeutic antibodies, it is acknowledged that optimal target binding affinity should be accompanied by high specificity. In recent years, there has been an increased focus on lowering non-specific binding during early-stage drug development. In particular, non-specificity has been recognised as a root-cause for failure in numerous antibody-based drug programs due to unexpected pharmacokinetics and elevated toxicity. Hence, there is a need for predictive tools for flagging and deselecting non-specific antibodies at an early stage to mitigate failure at clinical late-stage development. From a computational design perspective, prediction has remained a challenge as non-specificity is currently a poorly understood biophysical phenomena, typically involving weak interactions to various, unrelated targets and surfaces. In this work, we describe the development of sequence-based machine learning (ML) models for prediction of antibody non-specificity. A dataset of >1000 antibody sequences paired with their respective non-specificity flag, defined by ELISA using a panel of common antigens, were retrieved from public sources (Jain et al., 2017; Boughter et al., 2020). The sequences were embedded using state-of-the-art protein language models for representation of their physiochemical properties, whereafter the vectorized embeddings were served as features for training of binary classifiers for non-specificity flags. A total of 12 different antibody domain-specific models were generated. Highest predictability of non-specificity was obtained for ML algorithms trained on the heavy-chain variable domain (VH), and the complementarity determining regions (CDRs), and resulted in a mean accuracy of 73% across 3-, 5-, and 10-fold cross-validations. The classification probability of non-specificity followed an uptrend with the experimental data (non-specificity flags), thereby mimicking regression behaviour. Altogether, our work demonstrates feasibility for prediction of non-specific binding from the antibody sequence, and that the non-specificity primarily stems from the VH domain, with main contributions from the heavy-chain CDRs.

Recombinant Soluble Henipavirus Glycoprotein Preparation.

Henipaviruses possess two envelope glycoproteins, the attachment (G) and the fusion (F) proteins that mediate cellular entry and are the major targets of virus-neutralizing antibody responses. Recombinant expression technologies have been used to produce soluble G and F proteins (sG and sF) that retain native-like oligomeric conformations and epitopes, which are advantageous for the development and characterization of vaccines and antiviral antibody therapeutics. In addition to Hendra virus and Nipah virus tetrameric sG and trimeric sF production, we also describe the expression and purification of Cedar virus tetrameric sG and Ghana virus trimeric sF glycoproteins. These henipavirus glycoproteins were also used as immunizing antigens to generate monoclonal antibodies, and binding was demonstrated with a pan-henipavirus multiplex microsphere immunoassay.