Computationally restoring the potency of a clinical antibody against Omicron.

The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs1-3 and revealed how quickly viral escape can curtail effective options4,5. When the SARS-CoV-2 Omicron variant emerged in 2021, many antibody drug products lost potency, including Evusheld and its constituent, cilgavimab4-6. Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination4 and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign and renew the efficacy of COV2-2130 against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and subsequent variants of concern, and provides protection in vivo against the strains tested: WA1/2020, BA.1.1 and BA.5. Deep mutational scanning of tens of thousands of pseudovirus variants reveals that 2130-1-0114-112 improves broad potency without increasing escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Our computational approach does not require experimental iterations or pre-existing binding data, thus enabling rapid response strategies to address escape variants or lessen escape vulnerabilities.

Computationally restoring the potency of a clinical antibody against SARS-CoV-2 Omicron subvariants.

The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs1-3, but also revealed how quickly viral escape can curtail effective options4,5. With the emergence of the SARS-CoV-2 Omicron variant in late 2021, many clinically used antibody drug products lost potency, including Evusheld™ and its constituent, cilgavimab4,6. Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination4 and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies with a known clinical profile to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign COV2-2130 to rescue in vivo efficacy against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the contemporaneously dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and many variants of concern that subsequently emerged, and provides protection in vivo against the strains tested, WA1/2020, BA.1.1, and BA.5. Deep mutational scanning of tens of thousands pseudovirus variants reveals 2130-1-0114-112 improves broad potency without incurring additional escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Because our approach is computationally driven, not requiring experimental iterations or pre-existing binding data, it could enable rapid response strategies to address escape variants or pre-emptively mitigate escape vulnerabilities.

Healthy humans can be a source of antibodies countering COVID-19.

Here, we describe the isolation of 18 unique anti SARS-CoV-2 human single-chain antibodies from an antibody library derived from healthy donors. The selection used a combination of phage and yeast display technologies and included counter-selection strategies meant to direct the selection of the receptor-binding motif (RBM) of SARS-CoV-2 spike protein's receptor binding domain (RBD2). Selected antibodies were characterized in various formats including IgG, using flow cytometry, ELISA, high throughput SPR, and fluorescence microscopy. We report antibodies' RBD2 recognition specificity, binding affinity, and epitope diversity, as well as ability to block RBD2 binding to the human receptor angiotensin-converting enzyme 2 (ACE2) and to neutralize authentic SARS-CoV-2 virus infection in vitro. We present evidence supporting that: 1) most of our antibodies (16 out of 18) selectively recognize RBD2; 2) the best performing 8 antibodies target eight different epitopes of RBD2; 3) one of the pairs tested in sandwich assays detects RBD2 with sub-picomolar sensitivity; and 4) two antibody pairs inhibit SARS-CoV-2 infection at low nanomolar half neutralization titers. Based on these results, we conclude that our antibodies have high potential for therapeutic and diagnostic applications. Importantly, our results indicate that readily available non immune (naïve) antibody libraries obtained from healthy donors can be used to select high-quality monoclonal antibodies, bypassing the need for blood of infected patients, and offering a widely accessible and low-cost alternative to more sophisticated and expensive antibody selection approaches (e.g. single B cell analysis and natural evolution in humanized mice).

Construction, characterization and crystal structure of a fluorescent single-chain Fv chimera.

In vitro display technologies based on phage and yeast have a successful history of selecting single-chain variable fragment (scFv) antibodies against various targets. However, single-chain antibodies are often unstable and poorly expressed in Escherichia coli. Here, we explore the feasibility of converting scFv antibodies to an intrinsically fluorescent format by inserting the monomeric, stable fluorescent protein named thermal green, between the light- and heavy-chain variable regions. Our results show that the scTGP format maintains the affinity and specificity of the antibodies, improves expression levels, allows one-step fluorescent assay for detection of binding and is a suitable reagent for epitope binning. We also report the crystal structure of an scTGP construct that recognizes phosphorylated tyrosine on FcεR1 receptor of the allergy pathway.

Development of Anti-Yersinia pestis Human Antibodies with Features Required for Diagnostic and Therapeutic Applications.

BACKGROUND: Yersinia pestis is a category A infective agent that causes bubonic, septicemic, and pneumonic plague. Notably, the acquisition of antimicrobial or multidrug resistance through natural or purposed means qualifies Y. pestis as a potential biothreat agent. Therefore, high-quality antibodies designed for accurate and sensitive Y. pestis diagnostics, and therapeutics potentiating or replacing traditional antibiotics are of utmost need for national security and public health preparedness. METHODS: Here, we describe a set of human monoclonal immunoglobulins (IgG1s) targeting Y. pestis fraction 1 (F1) antigen, previously derived from in vitro evolution of a phage-display library of single-chain antibodies (scFv). We extensively characterized these antibodies and their effect on bacterial and mammalian cells via: ELISA, flow cytometry, mass spectrometry, spectroscopy, and various metabolic assays. RESULTS: Two of our anti-F1 IgG (αF1Ig 2 and αF1Ig 8) stood out for high production yield, specificity, and stability. These two antibodies were additionally attractive in that they displayed picomolar affinity, did not compete when binding Y. pestis, and retained immunoreactivity upon chemical derivatization. Most importantly, these antibodies detected <1,000 Y. pestis cells in sandwich ELISA, did not harm respiratory epithelial cells, induced Y. pestis agglutination at low concentration (350 nM), and caused apparent reduction in cell growth when radiolabeled at a nonagglutinating concentration (34 nM). CONCLUSION: These antibodies are amenable to the development of accurate and sensitive diagnostics and immuno/radioimmunotherapeutics.

Selection and verification of antibodies against the cytoplasmic domain of M2 of influenza, a transmembrane protein.

Interactions between the cytoplasmic domains of viral transmembrane proteins and host machinery often determine the outcome of viral infection. The M2 protein of influenza A has been identified as a key player in autophagy-mediated viral replication. Here, we describe the engineering and validation of an antibody specific for the cytoplasmic domain of the M2 protein. Through phage and yeast display selection techniques, we obtained an antibody that recognizes: 1) the M2 cytoplasmic domain purified from bacterial inclusion bodies and refolded, 2) full-length M2 recombinant protein expressed in mammalian cells, and 3) native M2 protein in influenza A infected cells. This antibody can serve as a molecular tool to enhance our knowledge of protein-protein interactions between influenza A virus and the host cell machinery. We anticipate the methods described herein will further the development of antibodies specific to the cytoplasmic domains of transmembrane proteins.

Analytics for Investigation of Disease Outbreaks: Web-Based Analytics Facilitating Situational Awareness in Unfolding Disease Outbreaks.

BACKGROUND: Information from historical infectious disease outbreaks provides real-world data about outbreaks and their impacts on affected populations. These data can be used to develop a picture of an unfolding outbreak in its early stages, when incoming information is sparse and isolated, to identify effective control measures and guide their implementation. OBJECTIVE: This study aimed to develop a publicly accessible Web-based visual analytic called Analytics for the Investigation of Disease Outbreaks (AIDO) that uses historical disease outbreak information for decision support and situational awareness of an unfolding outbreak. METHODS: We developed an algorithm to allow the matching of unfolding outbreak data to a representative library of historical outbreaks. This process provides epidemiological clues that facilitate a user's understanding of an unfolding outbreak and facilitates informed decisions about mitigation actions. Disease-specific properties to build a complete picture of the unfolding event were identified through a data-driven approach. A method of analogs approach was used to develop a short-term forecasting feature in the analytic. The 4 major steps involved in developing this tool were (1) collection of historic outbreak data and preparation of the representative library, (2) development of AIDO algorithms, (3) development of user interface and associated visuals, and (4) verification and validation. RESULTS: The tool currently includes representative historical outbreaks for 39 infectious diseases with over 600 diverse outbreaks. We identified 27 different properties categorized into 3 broad domains (population, location, and disease) that were used to evaluate outbreaks across all diseases for their effect on case count and duration of an outbreak. Statistical analyses revealed disease-specific properties from this set that were included in the disease-specific similarity algorithm. Although there were some similarities across diseases, we found that statistically important properties tend to vary, even between similar diseases. This may be because of our emphasis on including diverse representative outbreak presentations in our libraries. AIDO algorithm evaluations (similarity algorithm and short-term forecasting) were conducted using 4 case studies and we have shown details for the Q fever outbreak in Bilbao, Spain (2014), using data from the early stages of the outbreak. Using data from only the initial 2 weeks, AIDO identified historical outbreaks that were very similar in terms of their epidemiological picture (case count, duration, source of exposure, and urban setting). The short-term forecasting algorithm accurately predicted case count and duration for the unfolding outbreak. CONCLUSIONS: AIDO is a decision support tool that facilitates increased situational awareness during an unfolding outbreak and enables informed decisions on mitigation strategies. AIDO analytics are available to epidemiologists across the globe with access to internet, at no cost. In this study, we presented a new approach to applying historical outbreak data to provide actionable information during the early stages of an unfolding infectious disease outbreak.

Conjugation of Amphiphilic Proteins to Hydrophobic Ligands in Organic Solvent.

Protein-ligand conjugations are usually carried out in aqueous media in order to mimic the environment within which the conjugates will be used. In this work, we focus on the conjugation of amphiphilic variants of elastin-like polypeptide (ELP), short elastin (sEL), to poorly water-soluble compounds like OPPVs ( p-phenylenevinylene oligomers), triarylamines, and polypyridine-metal complexes. These conjugations are problematic when carried out in aqueous phase because hydrophobic ligands tend to avoid exposure to water, which in turn causes the ligand to self-aggregate and/or interact noncovalently with hydrophobic regions of the amphiphile. Ultimately, this behavior leads to low conjugation efficiency and contamination with strong noncovalent "conjugates". After exploring the solubility of sEL in various organic solvents, we have established an efficient conjugation methodology for obtaining covalent conjugates virtually free of contaminating noncovalent complexes. When conjugating carboxylated ligands to the amphiphile amines, we demonstrate that even when only one amine (the N-terminus) is present, its derivatization is 98% efficient. When conjugating amine moieties to the amphiphile carboxyls (a problematic configuration), protein multimerization is avoided, 98-100% of the protein is conjugated, and the unreacted ligand is recovered in pure form. Our syntheses occur in "one pot", and our purification procedure is a simple workup utilizing a combination of water and organic solvent extractions. This conjugation methodology might provide a solution to problems arising from solubility mismatch of protein and ligand, and it is likely to be widely applied for modification of recombinant amphiphiles used for drug delivery (PEG-antibodies, polymer-enzymes, food proteins), cell adhesion (collagen, hydrophobins), synthesis of nanostructures (peptides), and engineering of biocompatible optoelectronics (biological polymers), to cite a few.

An extensible framework and database of infectious disease for biosurveillance.

Biosurveillance, a relatively young field, has recently increased in importance because of increasing emphasis on global health. Databases and tools describing particular subsets of disease are becoming increasingly common in the field. Here, we present an infectious disease database that includes diseases of biosurveillance relevance and an extensible framework for the easy expansion of the database.

Development of phage-based single chain Fv antibody reagents for detection of Yersinia pestis.

BACKGROUND: Most Yersinia pestis strains are known to express a capsule-like antigen, fraction 1 (F1)(.) F1 is encoded by the caf1 gene located on the large 100-kb pFra plasmid, which is found in Y. pestis but not in closely related species such as Yersinia enterocolytica and Yersinia pseudotuberculosis. In order to find antibodies specifically binding to Y. pestis we screened a large single chain Fv antibody fragment (scFv) phage display library using purified F1 antigen as a selection target. Different forms of the selected antibodies were used to establish assays for recombinant F1 antigen and Y. pestis detection. METHODS: Phage antibody panning was performed against F1 in an automated fashion using the Kingfisher magnetic bead system. Selected scFvs were screened for F1-binding specificity by one-step alkaline phosphatase enzyme linked immunosorbant assay (ELISA), and assayed for binding to recombinant antigen and/or Y. pestis by flow cytometry and whole-cell ELISA. RESULTS: Seven of the eight selected scFvs were shown to specifically bind both recombinant F1 and a panel of F1-positive Yersinia cells. The majority of the soluble scFvs were found to be difficult to purify, unstable and prone to cross-reactivity with F1-negative Yersinia strains, whereas phage displayed scFvs were found to be easy to purify/label and remarkably stable. Furthermore direct fluorescent labeling of phage displaying scFv allowed for an easy one-step flow cytometry assay. Slight cross-reactivity was observed when fixed cells were used in ELISA. CONCLUSIONS: Our high throughput methods of selection and screening allowed for time and cost effective discovery of seven scFvs specifically binding Y. pestis F1 antigen. We describe implementation of different methods for phage-based immunoassay. Based on the success of these methods and the proven stability of phage, we indicate that the use of phage-displayed, rather than phage-free proteins, might generally overcome the shortcomings of scFv antibodies.

Targeting heat shock proteins on cancer cells: selection, characterization, and cell-penetrating properties of a peptidic GRP78 ligand.

Peptidic ligands can be used for specific cell targeting and the delivery of payloads into the target cell. Here we describe the screening of a pool of cyclic peptide phage display libraries using whole-cell panning against human melanoma cell line Me6652/4. This strategy resulted in the selection of the cyclic 13-mer Pep42, CTVALPGGYVRVC, which showed preferential internalization into melanoma cell line Me6652/4 versus the reference cell line Me6652/56. This translocation is a receptor-mediated process that does not require electrostatic interactions nor does it involve transfer to the lysosomal compartment. The cellular receptor for Pep42 was identified as the surface membrane form of glucose-regulated protein 78 (GRP78), a member of the heat shock protein family and a marker on malignant cancer cells. The cellular uptake and intracellular trafficking of Pep42-Quantum Dot conjugates was monitored by confocal laser microscopy, and colocalization within the endoplasmic reticulum was observed. The uptake of Pep42 could be blocked by a monoclonal antibody against the identified receptor. Furthermore, Pep42 was shown to target specifically GRP78-expressing cancer cells. The in vitro cytotoxicity of a Pep42-Taxol conjugate was evaluated by flow cytometry wherein the conjugate was shown to induce apoptosis and was more effective in promoting programmed cell death in Me6652/4 cells. In summary, the data presented suggest that cyclic peptide Pep42 might be a powerful tool in the construction of drug conjugates designed to selectively kill malignant cancer cells.

A contiguous stretch of methionine residues mediates the energy-dependent internalization mechanism of a cell-penetrating peptide.

Recently we characterized an unusual switch in the internalization mechanism of the monomeric and dimeric forms of the cell-penetrating peptide RDLWEMMMVSLACQY. Here, we observed both energy-dependent and energy-independent modes of peptide uptake by the target B-lymphocytes WI-L2-729HF2, suggesting that higher-order structure might modulate the action of this novel cell-penetrating peptide. In the present work, we propose a possible internalization mechanism for the dimeric peptide which involves an initial interaction with the cell membrane, followed by an energy-dependent internalization process which requires the contiguous Met(6-8) sequence.

A dimerization "switch" in the internalization mechanism of a cell-penetrating peptide.

The internalization mechanism of a cell-penetrating peptide has been explored through combinatorial selection of a phage-displayed peptide dimer library, chemical synthesis, and biophysical characterization. Both energy-dependent and energy-independent modes for peptide uptake by the target mammalian cells were observed, suggesting a role for higher-order structure in modulating the action of this novel cell-penetrating peptide.

Combinatorial antibody libraries from cancer patients yield ligand-mimetic Arg-Gly-Asp-containing immunoglobulins that inhibit breast cancer metastasis.

Combinatorial antibody libraries have the potential to display the entire immunological record of an individual, allowing one to detect and recover any antibody ever made, irrespective of whether it is currently being produced. We have termed this the "fossil record" of an individual's antibody response. To determine whether cancer patients have ever made antibodies with disease-fighting potential, we screened combinatorial antibody libraries from cancer patients for immunoglobulins that can identify metastatic tumor cells. This strategy yielded human antibodies specific for the activated conformation of the adhesion receptor integrin alphavbeta3 that is associated with a metastatic phenotype. In a remarkable example of convergent evolution, two of these antibodies were shown to contain the Arg-Gly-Asp integrin recognition motif of the natural ligand within the third complementarity-determining region of the heavy chain. These antibodies interfered with lung colonization by human breast cancer cells in a mouse model and inhibited existing metastatic disease. Our data imply that, at least at some time, these antibodies were part of a patient's surveillance system against metastatic cells, targeting the activated conformer of integrin alphavbeta3 and disrupting its functions. The ligand-mimetic nature of these antibodies, combined with specificity for a single receptor, is unique in the integrin-ligand repertoire. The convergent evolution of critical sequences in antibodies and other ligands that bind to the same target means that the immune response has sufficient power to find a best chemical solution for the optimization of binding energy, even though antibodies evolve in real time, as compared with billions of years for the natural ligand.

Kinetic analysis of Escherichia coli 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase, wild type and mutants, reveals roles of active site amino acids.

Escherichia coli 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (YgbP or IspD) catalyzes the conversion of 2-C-methyl-D-erythritol 4-phosphate (MEP) and cytidine triphosphate (CTP) to 4-diphosphocytidyl-2-C-methylerythritol (CDPME). Pulse chase experiments established that the reaction involves an ordered sequential mechanism with mandatory initial binding of CTP. On the basis of analysis of the previously reported crystal structures of apo-YgbP as well as YgbP complexed with both CTP.Mg(2+) and CDPME.Mg(2+) [Richard, S. B., Bowman, M. E., Kwiatkowski, W., Kang, I., Chow, C., Lillo, A. M., Cane, D. E., and Noel, J. P. (2001) Nat. Struct. Biol. 8, 641-648], a group of active site residues were selected for site-directed mutagenesis and steady-state kinetic analysis. Both Lys27 and Lys213 were shown to be essential to catalytic activity, consistent with their proposed role in stabilization of a pentacoordinate phosphate transition state resulting from in-line attack of the MEP phosphate on the alpha-phosphate of CTP. In addition, Thr140, Arg109, Asp106, and Thr165 were all shown to play critical roles in the binding and proper orientation of the MEP substrate.

A human single-chain antibody specific for integrin alpha3beta1 capable of cell internalization and delivery of antitumor agents.

Selective antitumor chemotherapy can be achieved by using antibody-drug conjugates that recognize surface proteins upregulated in cancer cells. One such receptor is integrin alpha3beta1, which is overexpressed on malignant melanoma, prostate carcinoma, and glioma cells. We previously identified a human single-chain Fv antibody (scFv), denoted Pan10, specific for integrin alpha3beta1 that is internalized by human pancreatic cancer cells. Herein, we describe the chemical introduction of reactive thiol groups onto Pan10, the specific conjugation of the modified scFv to maleimide-derivatized analogs of the potent cytotoxic agent duocarmycin SA, and the properties of the resultant conjugates. Our findings provide evidence that Pan10-drug conjugates maintain the internalizing capacity of the parent scFv and are cytotoxic at nanomolar concentrations. Our Pan10-drug conjugates may be promising candidates for targeted chemotherapy of malignant diseases associated with overexpression of integrin alpha3beta1.

Functional expression and characterization of EryA, the erythritol kinase of Brucella abortus, and enzymatic synthesis of L-erythritol-4-phosphate.

The eryA gene of the bacterial pathogen Brucella abortus has been functionally expressed in Escherichia coli. The resultant EryA was shown to catalyze the ATP-dependent conversion of erythritol to L-erythritol-4-phosphate (L-E4P). The steady state kinetic parameters of this reaction were determined and the enzyme was used to prepare L-E4P which was shown to be a weak inhibitor of 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (YgbP).

Structure and mechanism of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase. An enzyme in the mevalonate-independent isoprenoid biosynthetic pathway.

The enzyme 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP) synthase catalyzes the conversion of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-ME2P) to MECDP, a highly unusual cyclodiphosphate-containing intermediate on the mevalonate-independent pathway to isopentenyl diphosphate and dimethylallyl diphosphate. We now report two x-ray crystal structures of MECDP synthase refined to 2.8-A resolution. The first structure contains a bound Mn(2+) cation, and the second structure contains CMP, MECDP, and Mn(2+). The protein adopts a homotrimeric quaternary structure built around a central hydrophobic cavity and three externally facing active sites. Each of these active sites is located between two adjacent monomers. A tetrahedrally arranged transition metal binding site, potentially occupied by Mn(2+), sits at the base of the active site cleft. A phosphate oxygen of MECDP and the side chains of Asp(8), His(10), and His(42) occupy the metal ion coordination sphere. These structures reveal for the first time the structural determinants underlying substrate, product, and Mn(2+) recognition and the likely catalytic mechanism accompanying the biosynthesis of the cyclodiphosphate-containing isoprenoid precursor, MECDP.