mAb Binding Research Articles

The mechanistic basis of protection by non-neutralizing anti-alphavirus antibodies.

SUMMARYAlthough neutralizing monoclonal antibodies (mAbs) against epitopes within the alphavirus E2 protein can protect against infection, the functional significance of non-neutralizing mAbs is poorly understood. Here, we evaluate the activity of 13 non-neutralizing mAbs against Mayaro virus (MAYV), an emerging arthritogenic alphavirus. These mAbs bind to the MAYV virion and surface of infected cells but fail to neutralize infection in cell culture. Mapping studies identify six mAb binding groups that localize to discrete epitopes within or adjacent to the A domain of the E2 glycoprotein. Remarkably, passive transfer of non-neutralizing mAbs protects against MAYV infection and disease in mice, and their efficacy requires Fc effector functions. Monocytes mediate the protection of non-neutralizing mAbs in vivo, as Fcγ-receptor-expressing myeloid cells facilitate the binding, uptake, and clearance of MAYV without antibody-dependent enhancement of infection. Humoral protection against alphaviruses likely reflects contributions from non-neutralizing antibodies through Fc-dependent mechanisms that accelerate viral clearance.

Rapid and efficient generation of T-cell receptor-like antibodies using chip-based single-cell analysis.

Generation of TCR-like monoclonal antibodies using conventional methods is markedly laborious and inefficient. We have proposed improvements of ISAAC (chip-based Ab-secreting cell [ASC] screening method), allows comprehensive analysis of ASCs at the single-cell level to obtain TCR-like antibodies; blocking procedure enables us to avoid the detection of non-TCR-like antibodies.

Mutations in the Hemagglutinin Stalk Domain Do Not Permit Escape from a Protective, Stalk-Based Vaccine-Induced Immune Response in the Mouse Model.

ABSTRACTCurrent seasonal influenza virus vaccines target regions of the hemagglutinin (HA) head domain that undergo constant antigenic change, forcing the painstaking annual reformulation of vaccines. The development of broadly protective or universal influenza virus vaccines that induce cross-reactive, protective immune responses could circumvent the need to reformulate current seasonal vaccines. Many of these vaccine candidates target the HA stalk domain, which displays epitopes conserved within and across influenza virus subtypes, including those with pandemic potential. While HA head-mediated antigenic drift is well understood, the potential for antigenic drift in the stalk domain is understudied. Using a panel of HA stalk-specific monoclonal antibodies (MAbs), we applied selection pressure to the stalk domain of A/Netherlands/602/2009 (pdmH1N1) to determine fitness and phenotypes of escape mutant viruses (EMVs). We found that HA stalk MAbs with lower cross-reactivity caused single HA stalk escape mutations, whereas MAbs with broader cross-reactivity forced multiple mutations in the HA. Each escape mutant virus greatly decreased mAb neutralizing activity, but escape mutations did not always ablate MAb binding or Fc-Fc receptor-based effector functions. Escape mutant viruses were not attenuated in vitro but showed attenuation in an in vivo mouse model. Importantly, mice vaccinated with a chimeric HA universal vaccine candidate were protected from lethal challenge with EMVs despite these challenge viruses containing escape mutations in the stalk domain. Our study indicates that while the HA stalk domain can mutate under strong MAb selection pressure, mutant viruses may have attenuated phenotypes and do not evade a polyclonal, stalk-based vaccine-induced response.

Sensitive Immunoassay Detection of Plasmodium Lactate Dehydrogenase by Inductively Coupled Plasma Mass Spectrometry.

Rapid, reliable, and sensitive detection of Plasmodium infection is central to malaria control and elimination. Many Malaria Rapid Diagnostic Tests (RDTs) developed for this purpose depend upon immunoassays that can be improved by advances in bound antibody sensor technology. In a previous study, immuno-polymerase chain reaction (PCR) was shown to provide highly sensitive detection of Plasmodium falciparum lactate dehydrogenase (PfLDH) in monoclonal antibody (mAb) sandwich assays. Here, we show comparably high immunoassay sensitivity by inductively coupled plasma mass spectrometry (ICP-MS) detection of gold nanoparticles (AuNPs). Following capture of PfLDH with the primary mAb and binding of the AuNP-labeled detection mAb, ICP-MS signals from the AuNPs provided quantitative measures of recombinant PfLDH test dilutions and P. falciparum-infected erythrocytes. A detection limit of 1.5 pg/mL was achieved with the PfLDH protein. Parasitemia in cultures of P. falciparum-infected erythrocytes could be detected to a lower limit of 1.6 parasite/μl (p/μl) for early ring-stage forms and 0.3 p/μl for mixed stages including mature trophozoites and schizont-stages. These results show that ICP-MS detection of AuNPs can support highly sensitive and accurate detection of Plasmodium infection.

Anti-GPR56 monoclonal antibody potentiates GPR56-mediated Src-Fak signaling to modulate cell adhesion

GPR56 is a member of the adhesion G-protein-coupled receptor family shown to play important roles in cell adhesion, brain development, immune function, and tumorigenesis. GPR56 is highly upregulated in colorectal cancer and correlates with poor prognosis. Several studies have shown GPR56 couples to the Gα12/13 class of heterotrimeric G-proteins to promote RhoA activation. However, due to its structural complexity and lack of a high-affinity receptor-specific ligand, the complete GPR56 signaling mechanism remains largely unknown. To delineate the activation mechanism and intracellular signaling functions of GPR56, we generated a monoclonal antibody (mAb) that binds with high affinity and specificity to the extracellular domain (ECD). Using deletion mutants, we mapped the mAb binding site to the GAIN domain, which mediates membrane-proximal autoproteolytic cleavage of the ECD. We showed that GPR56 overexpression in 293T cells leads to increased phosphorylation of Src, Fak, and paxillin adhesion proteins and activation of the Gα12/13-RhoA-mediated serum response factor (SRF) pathway. Treatment with the mAb potentiated Src-Fak phosphorylation, RhoA–SRF signaling, and cell adhesion. Consistently, GPR56 knockdown in colorectal cancer cells decreased Src–Fak pathway phosphorylation and cell adhesion. Interestingly, GPR56-mediated activation of Src–Fak phosphorylation occurred independent of RhoA, yet mAb-induced potentiation of RhoA–SRF signaling was Src-dependent. Furthermore, we show that the C-terminal portion of the Serine–Threonine–Proline-rich (STP) region, adjacent to the GAIN domain, was required for Src–Fak activation. However, autoproteolytic cleavage of the ECD was dispensable. These data support a new ECD-dependent mechanism by which GPR56 functions to regulate adhesion through activation of Src–Fak signaling.

Understanding the mechanism of HIV-1 protease inhibition by monoclonal antibodies

HIV-1 protease is an essential enzyme in the life cycle of human immunodeficiency virus (HIV) and hence is one of the most important targets for antiviral drug design. Although there are ten FDA approved drugs against HIV protease (PR), their long term usage elicits mutations leading to drug resistance. As a result, novel therapeutic approaches are being explored including synthetic antibodies. Recently, a murine monoclonal antibody, mAB1696 (mAB) was reported to inhibit PR by preventing dimerization. Crystallographic data could reveal only six protease residues that interact with mAB. The present study employs a range of computational techniques, starting from protein-protein docking to all-atomic molecular dynamics simulations to generate plausible 3D structures of PR-mAB complex. Results show that mAB interacts very strongly with several PR dimer interface residues, such as Gln7, Arg8 (N-terminal), Cys95, Leu97 (C-terminal), Thr26, Gly27 (active site), Gly49, Ile50 (flap), apart from its interactions with the PR epitope region, Pro1-Trp6 (N-terminal). These observations support the hypothesis that binding of mAB prevents the dimerization of PR. The interactions and binding conformations identified in this study could form the basis for designing allosteric inhibitors preventing the dimerization of HIV-1 Protease.

Ligand binding to a humanized anti-cocaine mAb measured by dye absorption spectroscopy

Here we demonstrate that the antigen binding function of a humanized anti-cocaine mAb (h2E2) can be directly and easily determined using simple and inexpensive absorption spectroscopy and dyes commonly used for differential scanning fluorimetry, such as DASPMI and SYPRO Orange. Therapeutic monoclonal antibodies are commonly formulated in buffers which can interfere with necessary functional assays, containing additives and excipients such as mild detergents. Using the undiluted therapeutic product h2E2 mAb in its formulation buffer containing 0.01% polysorbate 80, the number of antigen/cocaine binding sites can be determined by the increase in absorbance (for DASPMI dye) or by the decrease in absorbance maximum wavelength (for SYPRO Orange dye), confirming proper function of the therapeutic mAb product. This ligand-induced visible dye absorption change can also be used to qualitatively evaluate the relative affinities of various metabolites of cocaine. These results are confirmed and extended by binding data obtained in the same formulation buffer using intrinsic tyrosine and tryptophan fluorescence quenching by cocaine, as well as by differential scanning fluorimetry. Interestingly, the binding of the cocaine metabolite norcocaine was demonstrated to be differentially affected by the pH 6 formulation buffer used for this mAb, presumably due to the differential ionizability of the demethylated norcocaine tropane ring nitrogen. This simple, direct, and inexpensive technique should prove useful for evaluation of other small molecule binding mAbs directly in their formulation buffers containing detergent, allowing rapid functional assessment of the produced therapeutic proteins.

Affinity-Restricted Memory B Cells Dominate Recall Responses to Heterologous Flaviviruses

Memory B cells (MBCs) can respond to heterologous antigens either by molding new specificities through secondary germinal centers (GCs) or by selecting preexisting clones without further affinity maturation. To distinguish these mechanisms in flavivirus infections and immunizations, we studied recall responses to envelope protein domain III (DIII). Conditional deletion of activation-induced cytidine deaminase (AID) between heterologous challenges of West Nile, Japanese encephalitis, Zika, and dengue viruses did not affect recall responses. DIII-specific MBCs were contained mostly within the plasma-cell-biased CD80+ subset, and few GCs arose following heterologous boosters, demonstrating that recall responses are confined by preexisting clonal diversity. Measurement of monoclonal antibody (mAb) binding affinity to DIII proteins, timed AID deletion, single-cell RNA sequencing, and lineage tracing experiments point to selection of relatively low-affinity MBCs as a mechanism to promote diversity. Engineering immunogens to avoid this MBC diversity may facilitate flavivirus-type-specific vaccines with minimized potential for infection enhancement.

Tackling COVID19 by Exploiting Pre-existing Cross-Reacting Spike-Specific Immunity

Tackling COVID19 by Exploiting Pre-existing Cross-Reacting Spike-Specific Immunity

A stabilized glycomimetic conjugate vaccine inducing protective antibodies against Neisseria meningitidis serogroup A

Neisseria meningitidis serogroup A capsular polysaccharide (MenA CPS) consists of (1 → 6)-2-acetamido-2-deoxy-α-D-mannopyranosyl phosphate repeating units, O-acetylated at position C3 or C4. Glycomimetics appear attractive to overcome the CPS intrinsic lability in physiological media, due to cleavage of the phosphodiester bridge, and to develop a stable vaccine with longer shelf life in liquid formulation. Here, we generate a series of non-acetylated carbaMenA oligomers which are proven more stable than the CPS. An octamer (DP8) inhibits the binding of a MenA specific bactericidal mAb and polyclonal serum to the CPS, and is selected for further in vivo testing. However, its CRM197 conjugate raises murine antibodies towards the non-acetylated CPS backbone, but not the natural acetylated form. Accordingly, random O-acetylation of the DP8 is performed, resulting in a structure (Ac-carbaMenA) showing improved inhibition of anti-MenA CPS antibody binding and, after conjugation to CRM197, eliciting anti-MenA protective murine antibodies, comparably to the vaccine benchmark.

Abstract 3151: Active immunization with PD1-derived mimotope-Combination immunotherapy against Her-2/neu-expressing tumors

Abstract Application of monoclonal antibodies (mAbs) as immune checkpoint inhibitors (ICIs) has demonstrated a tremendous effect in cancer immunotherapy. However, high costs for and frequent applications of mono- or combination-therapies or possible development of resistance justify modifications of this approach. In this line, active immunization with mimotopes (B cell peptides) of ICIs, activating the patients' own immune system, rather than application of the corresponding therapeutic mAbs, may provide advantages over the costly treatment of the mAbs. Overlapping peptides spanning the extracellular domains of human PD1 (hPD1) were used to identify hPD1-derived mimotope, using the therapeutic mAb Nivolumab as a proof of concept. Additionally, for in vivo evaluation in a tumor mouse model, a mouse PD1 (mPD1)-derived mimotope was identified using an anti-mPD1 mAb with mPD1/mPDL-1 blocking capacity. The identified mimotopes were characterized by in vitro assays, including a reporter cell-based assay, and their anti-tumor effect was evaluated in a syngeneic mouse model involving grafting with human Her-2/neu-expressing tumors. The PD1-mimotopes were shown to specifically inhibit the binding of the corresponding mAbs, and also the mAbs capacity in blocking the respective PD1-PDL1 interactions. Applying the syngeneic tumor mouse model, a significant tumor growth reduction following active immunization with the mPD1-mimotope was shown. Importantly, combined vaccination with the mimotope and a multiple B-cell epitope Her-2/neu vaccine potentiated the anti-tumor effect in the syngeneic mice. Tumor growth reduction in the mice immunized with the mimotope was associated with a significant increase of the apoptotic (CC3) and significant reduction of proliferation (Ki67) markers, as evaluated by immunohistochemistry (IHC) staining. However, the tumor growth reduction following active immunization with the cancer vaccine (alone or in combination with the checkpoint mimotope) showed a different IHC picture, namely highly increased levels of CC3 without reduction of proliferation (Ki67 levels) in the tumor cells. In depth studies on the underlying mechanisms of tumor growth reduction are currently ongoing. Our results suggest active immunization with B cell mimotopes of ICIs as either monovalent vaccine or a combination therapy with tumor specific vaccines to enhance the anti-tumor efficacy. Such strategies may potentially lead to multipurpose treatment regimens adapted to the type, stage and progression phase of the various tumors. Citation Format: Joshua Tobias, Claire Battin, Annika De Sousa Linhares, Michael Lebens, Karin Baier, Katharina Ambroz, Mirjana Drinić, Sandra Högler, Aleksandra Inic-Kanada, Erika Garner-Spitzer, Matthias Preusser, Lukas Kenner, Michael Kundi, Christoph C. Zielinski, Peter Steinberger, Ursula Wiedermann. Active immunization with PD1-derived mimotope-Combination immunotherapy against Her-2/neu-expressing tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3151.

A novel therapeutic antibody screening method using bacterial high-content imaging reveals functional antibody binding phenotypes of Escherichia coli ST131

The increase of antimicrobial resistance (AMR), and lack of new classes of licensed antimicrobials, have made alternative treatment options for AMR pathogens increasingly attractive. Recent studies have demonstrated anti-bacterial efficacy of a humanised monoclonal antibody (mAb) targeting the O25b O-antigen of Escherichia coli ST131. To evaluate the phenotypic effects of antibody binding to diverse clinical E. coli ST131 O25b bacterial isolates in high-throughput, we designed a novel mAb screening method using high-content imaging (HCI) and image-based morphological profiling to screen a mAb targeting the O25b O-antigen. Screening the antibody against a panel of 86 clinical E. coli ST131 O25:H4 isolates revealed 4 binding phenotypes: no binding (18.60%), weak binding (4.65%), strong binding (69.77%) and strong agglutinating binding (6.98%). Impaired antibody binding could be explained by the presence of insertion sequences or mutations in O-antigen or lipopolysaccharide core biosynthesis genes, affecting the amount, structure or chain length of the O-antigen. The agglutinating binding phenotype was linked with lower O-antigen density, enhanced antibody-mediated phagocytosis and increased serum susceptibly. This study highlights the need to screen candidate mAbs against large panels of clinically relevant isolates, and that HCI can be used to evaluate mAb binding affinity and potential functional efficacy against AMR bacteria.

Monoclonal antibodies for the S2 subunit of spike of SARS-CoV-1 cross-react with the newly-emerged SARS-CoV-2.

BackgroundA novel coronavirus, SARS-CoV-2, which emerged at the end of 2019 and causes COVID-19, has resulted in worldwide human infections. While genetically distinct, SARS-CoV-1, the aetiological agent responsible for an outbreak of severe acute respiratory syndrome (SARS) in 2002–2003, utilises the same host cell receptor as SARS-CoV-2 for entry: angiotensin-converting enzyme 2 (ACE2). Parts of the SARS-CoV-1 spike glycoprotein (S protein), which interacts with ACE2, appear conserved in SARS-CoV-2.AimThe cross-reactivity with SARS-CoV-2 of monoclonal antibodies (mAbs) previously generated against the S protein of SARS-CoV-1 was assessed.MethodsThe SARS-CoV-2 S protein sequence was aligned to those of SARS-CoV-1, Middle East respiratory syndrome (MERS) and common-cold coronaviruses. Abilities of mAbs generated against SARS-CoV-1 S protein to bind SARS-CoV-2 or its S protein were tested with SARS-CoV-2 infected cells as well as cells expressing either the full length protein or a fragment of its S2 subunit. Quantitative ELISA was also performed to compare binding of mAbs to recombinant S protein.ResultsAn immunogenic domain in the S2 subunit of SARS-CoV-1 S protein is highly conserved in SARS-CoV-2 but not in MERS and human common-cold coronaviruses. Four murine mAbs raised against this immunogenic fragment could recognise SARS-CoV-2 S protein expressed in mammalian cell lines. In particular, mAb 1A9 was demonstrated to detect S protein in SARS-CoV-2-infected cells and is suitable for use in a sandwich ELISA format.ConclusionThe cross-reactive mAbs may serve as useful tools for SARS-CoV-2 research and for the development of diagnostic assays for COVID-19.

Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

The Myo/Nog cell lineage was discovered in the chick embryo and is also present in adult mammalian tissues. The cells are named for their expression of mRNA for the skeletal muscle specific transcription factor MyoD and bone morphogenetic protein inhibitor Noggin. A third marker for Myo/Nog cells is the cell surface molecule recognized by the G8 monoclonal antibody (mAb). G8 has been used to detect, track, isolate and kill Myo/Nog cells. In this study, we screened a membrane proteome array for the target of the G8 mAb. The array consisted of >5,000 molecules, each synthesized in their native confirmation with appropriate post-translational modifications in a single clone of HEK-293T cells. G8 mAb binding to the clone expressing brain-specific angiogenesis inhibitor 1 (BAI1) was detected by flow cytometry, re-verified by sequencing and validated by transfection with the plasmid construct for BAI1. Further validation of the G8 target was provided by enzyme-linked immunosorbent assay. The G8 epitope was identified by screening a high-throughput, site directed mutagenesis library designed to cover 95–100% of the 954 amino acids of the extracellular domain of the BAI1 protein. The G8 mAb binds within the third thrombospondin repeat of the extracellular domain of human BAI1. Immunofluorescence localization experiments revealed that G8 and a commercially available BAI1 mAb co-localize to the subpopulation of Myo/Nog cells in the skin, eyes and brain. Expression of the multi-functional BAI1 protein in Myo/Nog cells introduces new possibilities for the roles of Myo/Nog cells in normal and diseased tissues.

Targeted inhibition of activated protein C by a non-active-site inhibitory antibody to treat hemophilia

Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Based on the hypothesis that specific inhibition of APC’s anticoagulant but not its cytoprotective activity can be beneficial for hemophilia therapy, 2 types of inhibitory monoclonal antibodies (mAbs) are tested: A type I active-site binding mAb and a type II mAb binding to an exosite on APC (required for anticoagulant activity) as shown by X-ray crystallography. Both mAbs increase thrombin generation and promote plasma clotting. Type I blocks all APC activities, whereas type II preserves APC’s cytoprotective function. In normal monkeys, type I causes many adverse effects including animal death. In contrast, type II is well-tolerated in normal monkeys and shows both acute and prophylactic dose-dependent efficacy in hemophilic monkeys. Our data show that the type II mAb can specifically inhibit APC’s anticoagulant function without compromising its cytoprotective function and offers superior therapeutic opportunities for hemophilia.

Remodeling of the Histoplasma Capsulatum Membrane Induced by Monoclonal Antibodies.

Antibodies play a central role in host immunity by directly inactivating or recognizing an invading pathogen to enhance different immune responses to combat the invader. However, the cellular responses of pathogens to the presence of antibodies are not well-characterized. Here, we used different mass spectrometry techniques to study the cellular responses of the pathogenic fungus Histoplasma capsulatum to monoclonal antibodies (mAb) against HSP60, the surface protein involved in infection. A proteomic analysis of H. capsulatum yeast cells revealed that mAb binding regulates a variety of metabolic and signaling pathways, including fatty acid metabolism, sterol metabolism, MAPK signaling and ubiquitin-mediated proteolysis. The regulation of the fatty acid metabolism was accompanied by increases in the level of polyunsaturated fatty acids, which further augmented the degree of unsaturated lipids in H. capsulatum’s membranes and energy storage lipids, such as triacylglycerols, phosphatidylcholines, phosphatidylethanolamines and phosphatidylinositols. MAb treatment also regulated sterol metabolism by increasing the levels of cholesterol and ergosterol in the cells. We also showed that global changes in the lipid profiles resulted in an increased susceptibility of H. capsulatum to the ergosterol-targeting drug amphotericin B. Overall, our data showed that mAb induction of global changes in the composition of H. capsulatum membranes can potentially impact antifungal treatment during histoplasmosis.

Depatuxizumab Mafodotin (ABT-414)-induced Glioblastoma Cell Death Requires EGFR Overexpression, but not EGFRY1068 Phosphorylation.

Glioblastomas commonly (40%) exhibit epidermal growth factor receptor (EGFR) amplification; half of these tumors carry the EGFRvIII deletion variant characterized by an in-frame deletion of exons 2-7, resulting in constitutive EGFR activation. Although EGFR tyrosine kinase inhibitors had only modest effects in glioblastoma, novel therapeutic agents targeting amplified EGFR or EGFRvIII continue to be developed.Depatuxizumab mafodotin (ABT-414) is an EGFR-targeting antibody-drug conjugate consisting of the mAb 806 and a toxic payload, monomethyl auristatin F. Because glioma cell lines and patient-derived glioma-initiating cell models expressed too little EGFR in vitro to be ABT-414-sensitive, we generated glioma sublines overexpressing EGFR or EGFRvIII to explore determinants of ABT-414-induced cell death.Overexpression of EGFRvIII induces sensitization to ABT-414 more readily than overexpression of EGFR in vitro and in vivo Exposure to ABT-414 in vivo eliminated EGFRvIII-expressing tumor cells, and recurrent tumors were devoid of EGFRvIII expression. There is no bystander killing of cells devoid of EGFR expression. Surprisingly, either exposure to EGF or to EGFR tyrosin kinase inhibitors reduce EGFR protein levels and are thus not strategies to promote ABT-414-induced cell killing. Furthermore, glioma cells overexpressing kinase-dead EGFR or EGFRvIII retain binding of mAb 806 and sensitivity to ABT-414, allowing to dissociate EGFR phosphorylation from the emergence of the "active" EGFR conformation required for ABT-414 binding.The combination of EGFR-targeting antibody-drug conjugates with EGFR tyrosine kinase inhibitors carries a high risk of failure. Promoting EGFR expression rather than phosphorylation should result in glioblastoma cell sensitization to ABT-414.

Neutralizing monoclonal antibodies against Venezuelan equine encephalitis virus envelope glycoprotein E2 protect from lethal encephalitis

Abstract Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne New World alphavirus responsible for severe morbidity and mortality in humans and equines. The development of therapeutics against VEEV is critical due to its potential for outbreak and aerosolization as a bioterrorist agent. However, effective and widely accessible antiviral agents for VEEV do not exist. Humoral immunity is an important defense against many alphavirus infections, and we thus generated a panel of 17 highly neutralizing murine VEEV-specific monoclonal antibodies (mAbs) using the VEEV vaccine TC-83 and attenuated chimeric SINV-VEEV Trinidad donkey (TRD) subtype IAB. Nine of these mAbs demonstrate 50% effective inhibitory concentrations (EC50) values below 100 ng/ml when tested against enzootic subtype ID and epizootic VEEV subtypes IAB and IC virion envelope glycoproteins. To define specific viral epitopes important for mAb binding, mapping studies including competition assays, HDX, alanine-scanning mutagenesis, and viral escape mutants are ongoing. Preliminary data suggests the epitopes for these mAbs are found in both the A and B domains of the surface virion envelope 2 glycoprotein. Additionally, several of these mAbs confer prophylactic and therapeutic protection in mice aerosol challenged with virulent TRD VEEV. Mechanism of action studies are underway to define the steps in the virus life cycle (viral attachment, entry, fusion, and egress) at which our most potent protective antibodies act. Identification of specific epitopes targeted by these protective mAbs will better inform the development of targeted immunotherapeutics and vaccines against VEEV and related encephalitic alphaviruses.

MRNA Display Discovery of a Novel Programmed Death Ligand 1 (PD-L1) Binding Peptide (a Peptide Ligand for PD-L1).

Programmed death ligand 1 (PD-L1) is a critical immune checkpoint ligand whose overexpression on tumor cells provides a mechanism of escape from immune surveillance. The interaction between PD-L1 and PD-1 on T cell lymphocytes suppresses both T cell activation and effector function and is engaged by cancers to dampen antitumor immunity. Here, we used mRNA display to engineer an 18-residue linear peptide that binds to human PD-L1. This peptide, which we term SPAM (signal peptide-based affinity maturated ligand), is nonhomologous to known PD-L1 binding peptides and mAbs, with dissociation constants (KD) of 119 and 67 nM for unglycosylated and glycosylated human PD-L1, respectively. The SPAM peptide is highly selective for human PD-L1 and shows no significant binding to either mouse PD-L1 or human PD-L2. Competition binding assays indicate that the SPAM peptide binding site overlaps with the binding site of PD-1 as well as therapeutic anti-PD-L1 antibodies. Taken together, these results suggest that the SPAM peptide specifically binds to human PD-L1 and could potentially serve as a PD-L1 affinity agent and PD-L1/PD-1 pathway modulator.

Non-neutralizing Antibodies from a Marburg Infection Survivor Mediate Protection by Fc-Effector Functions and by Enhancing Efficacy of Other Antibodies

Marburg virus (MARV) and Ebola virus (EBOV) belong to the family Filoviridae. MARV causes severe disease in humans with high fatality. We previously isolated a large panel of monoclonal antibodies (mAbs) from B cells of a human survivor with previous naturally acquired MARV infection. Here, we characterized functional properties of these mAbs and identified non-neutralizing mAbs targeting the glycoprotein (GP) 2 portion of the mucin-like domain (MLD) of MARV GP, termed the wing region. One mAb targeting the GP2 wing, MR228, showed therapeutic protection in mice and guinea pigs infected with MARV. The protection was mediated by the Fc fragment functions of MR228. Binding of another GP2 wing-specific non-neutralizing mAb, MR235, to MARV GP increased accessibility of epitopes in the receptor-binding site (RBS) for neutralizing mAbs, resulting in enhanced virus neutralization by these mAbs. These findings highlight an important role for non-neutralizing mAbs during natural human MARV infection.