PET imaging of Aspergillus infection using Zirconium-89 labeled anti-ß-glucan antibody fragments.

PURPOSE: Invasive fungal diseases, such as pulmonary aspergillosis, are common life-threatening infections in immunocompromised patients and effective treatment is often hampered by delays in timely and specific diagnosis. Fungal-specific molecular imaging ligands can provide non-invasive readouts of deep-seated fungal pathologies. In this study, the utility of antibodies and antibody fragments (Fab) targeting ß-glucans in the fungal cell wall to detect Aspergillus infections was evaluated both in vitro and in preclinical mouse models. METHODS: The binding characteristics of two commercially available ß-glucan antibody clones and their respective antigen-binding Fabs were tested using biolayer interferometry (BLI) assays and immunofluorescence staining. In vivo binding of the Zirconium-89 labeled antibodies/Fabs to fungal pathogens was then evaluated using PET/CT imaging in mouse models of fungal infection, bacterial infection and sterile inflammation. RESULTS: One of the evaluated antibodies (HA-ßG-Ab) and its Fab (HA-ßG-Fab) bound to ß-glucans with high affinity (KD = 0.056 & 21.5 nM respectively). Binding to the fungal cell wall was validated by immunofluorescence staining and in vitro binding assays. ImmunoPET imaging with intact antibodies however showed slow clearance and high background signal as well as nonspecific accumulation in sites of infection/inflammation. Conversely, specific binding of [89Zr]Zr-DFO-HA-ßG-Fab to sites of fungal infection was observed when compared to the isotype control Fab and was significantly higher in fungal infection than in bacterial infection or sterile inflammation. CONCLUSIONS: [89Zr]Zr-DFO-HA-ßG-Fab can be used to detect fungal infections in vivo. Targeting distinct components of the fungal cell wall is a viable approach to developing fungal-specific PET tracers.

Chemogenetic Control of Nanobodies.

We introduce an engineered nanobody whose affinity to green fluorescent protein (GFP) can be switched on and off with small molecules. By controlling the cellular localization of GFP fusion proteins, the engineered nanobody allows interrogation of their roles in basic biological processes, an approach that should be applicable to numerous previously described GFP fusions. We also outline how the binding affinities of other nanobodies can be controlled by small molecules.

Framework-Directed Amino-Acid Insertions Generated over 55-Fold Affinity-Matured Antibody Fragments That Enabled Sensitive Luminescent Immunoassays of Cortisol.

We generated three single-chain Fv fragments (scFvs) specific to cortisol according to our original affinity-maturation strategy and verified their utility in developing immunoassays. These scFv mutants (m-scFvs) had insertion of one, four, or six amino acid(s) in the framework region 1 of the VH-domain and showed >55-fold higher affinity (Ka, 2.0 - 2.2 × 1010 M-1) than the unmodified scFv (wt-scFv). Each m-scFv was fused with NanoLuc luciferase (NLuc) for the use in enzyme-linked immunosorbent assays (ELISAs). In these ELISA, the m-scFv-NLuc fusions were competitively reacted with immobilized cortisol residues and cortisol standards, and then the bound NLuc activity was monitored luminometrically. The luminescent ELISAs generated dose-response curves with extremely low midpoints (approx. 3 pg/assay) and were >150-fold more sensitive than the colorimetric ELISAs using wt-scFv and >8000-fold more sensitive than the ELISA using the parental native antibody. The luminescent ELISAs showed acceptable cross-reactivity patterns with related steroids, and the determination of control sera afforded cortisol levels in the reference range with satisfactory parallelism.

Emerging Paradigm of Intracellular Targeting of G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) recognize a diverse array of extracellular stimuli, and they mediate a broad repertoire of signaling events involved in human physiology. Although the major effort on targeting GPCRs has typically been focused on their extracellular surface, a series of recent developments now unfold the possibility of targeting them from the intracellular side as well. Allosteric modulators binding to the cytoplasmic surface of GPCRs have now been described, and their structural mechanisms are elucidated by high-resolution crystal structures. Furthermore, pepducins, aptamers, and intrabodies targeting the intracellular face of GPCRs have also been successfully utilized to modulate receptor signaling. Moreover, small molecule compounds, aptamers, and synthetic intrabodies targeting ß-arrestins have also been discovered to modulate GPCR endocytosis and signaling. Here, we discuss the emerging paradigm of intracellular targeting of GPCRs, and outline the current challenges, potential opportunities, and future outlook in this particular area of GPCR biology.

GlycoTAIL and FlexiTAIL as Half-Life Extension Modules for Recombinant Antibody Fragments.

Many therapeutic proteins are small in size and are rapidly cleared from circulation. Consequently, half-life extension strategies have emerged to improve pharmacokinetic properties, including fusion or binding to long-lasting serum proteins, chemical modifications with hydrophilic polymers such as PEGylation, or, more recently, fusion to PEG mimetic polypeptides. In the present study, two different PEG mimetic approaches, the GlycoTAIL and the FlexiTAIL, were applied to increase the hydrodynamic radius of antibody fragments of different sizes and valencies, including scFv, diabody, and scFv-EHD2 fusion proteins. The GlycoTAIL and FlexiTAIL sequences of varying lengths are composed of aliphatic and hydrophilic residues, with the GlycoTAIL furthermore comprising N-glycosylation sites. All modified proteins could be produced in a mammalian expression system without reducing stability and antigen binding, and all modified proteins exhibited a prolonged half-life and increased drug disposition in mice. The strongest effects were observed for proteins comprising a FlexiTAIL of 248 residues. Thus, the GlycoTAIL and FlexiTAIL sequences represent a flexible and modular system to improve the pharmacokinetic properties of proteins.

Site-Specific Antibody Fragment Conjugates for Reversible Staining in Fluorescence Microscopy.

Antibody conjugates have taken a great leap forward as tools in basic and applied molecular life sciences that was enabled by the development of chemoselective reactions for the site-specific modification of proteins. Antibody-oligonucleotide conjugates combine the antibody's target specificity with the reversible, sequence-encoded binding properties of oligonucleotides like DNAs or peptide nucleic acids (PNAs), allowing sequential imaging of large numbers of targets in a single specimen. In this report, we use the Tub-tag® technology in combination with Cu-catalyzed azide-alkyne cycloaddition for the site-specific conjugation of single DNA and PNA strands to an eGFP-binding nanobody. We show binding of the conjugate to recombinant eGFP and subsequent sequence-specific annealing of fluorescently labelled imager strands. Furthermore, we reversibly stain eGFP-tagged proteins in human cells, thus demonstrating the suitability of our conjugation strategy to generate antibody-oligonucleotides for reversible immunofluorescence imaging.

Sensitivity and reproducibility enhancement in enzyme immunosorbent assays based on half fragment antibodies.

The free sulfhydryl groups of the hinge region of monovalent antibody fragments (rIgG) allow the orientation of rIgG on functionalized surfaces in immunosensors. To evaluate the contribution of reduction and orientation on signal enhancement we compared the performance of whole antibodies and their rIgG in ELISA performed on polystyrene or maleimide-functionalized microplates. Monoclonal anti-horseradish peroxidase (anti-HRP) and monoclonal anti-fPSA antibodies (1 mg/mL) were reduced with 2-mercaptoethylamine (53 mM). Western blot confirmed the presence of rIgG as a band at 75 kDa, detectable only by anti-heavy chain but not by anti-light chain antibodies, suggesting a possible folding rearrangement. Using anti-HRP we confirmed the retention of the antigen binding capacity of rIgG. Moreover, we observed a signal enhancement for rIgG even if randomly absorbed on polystyrene [linear regression slope (95%CI): rIgG 0.524 (0.434-0.614), IgG 0.370 (0.430-0.399); P = 0.0016] suggesting that chemical reduction might affect the antigen binding capacity of antibodies. ELISA with anti-fPSA rIgG coated on polystyrene confirmed these observations. Oriented anti-fPSA rIgG on a maleimide surface showed comparable signals to the assay performed on polystyrene for each analyzed concentration of antigen (PANOVA = 0.1980), anyway, with a significant improvement of the repeatability likely providing a more homogeneous capturing surface (SD rIgGmaleimide-rIgGpolystirene: fPSA 0.725 ng/mL:0.74-2.89; 1.45 ng/mL:1.56-8.69; 3.625 ng/mL:3.52-15.03; 7.25 ng/mL:7.78-18.44).

Expression of soluble recombinant human matrix metalloproteinase 9 and generation of its monoclonal antibody.

We have successfully produced a recombinant human matrix metalloproteinase 9 (hMMP9) antigen with high yield and purity and used it to generate a hybridoma cell-culture-based monoclonal anti-hMMP9 antibody. We selected the most effective antibody for binding antigens and successfully identified its nucleotide sequence. The entire antigen and antibody developmental procedures described herein can be a practical approach for producing large amounts of monoclonal antibodies against hMMP9 and other antigens of interest. Additionally, the nucleotide sequence information of the anti-hMMP9 monoclonal antibody revealed herein will be useful for the generation of recombinant antibodies or antibody fragments against hMMP9.

Anti-CTLA-4 therapy requires an Fc domain for efficacy.

Ipilimumab, a monoclonal antibody that recognizes cytotoxic T lymphocyte antigen (CTLA)-4, was the first approved "checkpoint"-blocking anticancer therapy. In mouse tumor models, the response to antibodies against CTLA-4 depends entirely on expression of the Fcγ receptor (FcγR), which may facilitate antibody-dependent cellular phagocytosis, but the contribution of simple CTLA-4 blockade remains unknown. To understand the role of CTLA-4 blockade in the complete absence of Fc-dependent functions, we developed H11, a high-affinity alpaca heavy chain-only antibody fragment (VHH) against CTLA-4. The VHH H11 lacks an Fc portion, binds monovalently to CTLA-4, and inhibits interactions between CTLA-4 and its ligand by occluding the ligand-binding motif on CTLA-4 as shown crystallographically. We used H11 to visualize CTLA-4 expression in vivo using whole-animal immuno-PET, finding that surface-accessible CTLA-4 is largely confined to the tumor microenvironment. Despite this, H11-mediated CTLA-4 blockade has minimal effects on antitumor responses. Installation of the murine IgG2a constant region on H11 dramatically enhances its antitumor response. Coadministration of the monovalent H11 VHH blocks the efficacy of a full-sized therapeutic antibody. We were thus able to demonstrate that CTLA-4-binding antibodies require an Fc domain for antitumor effect.

Human Antibody Domains and Fragments Targeting Neutrophil Elastase as Candidate Therapeutics for Cancer and Inflammation-Related Diseases.

Neutrophil elastase (NE) is a serine protease released during neutrophil maturation. High levels of NE are related to lung tissue damage and poor prognosis in cancer; thus, NE is a potential target for therapeutic immunotherapy for multiple lung diseases and cancers. Here, we isolate and characterize two high-affinity, specific, and noncompetitive anti-NE antibodies Fab 1C10 and VH 1D1.43 from two large phage-displayed human Fab and VH libraries. After fusion with human IgG1 Fc, both of them (VH-Fc 1D1.43 and IgG1 1C10) inhibit NE enzymatic activity with VH-Fc 1D1.43 showing comparable inhibitory effects to that of the small molecule NE inhibitor SPCK and IgG1 1C10 exhibiting even higher (2.6-fold) activity than SPCK. Their epitopes, as mapped by peptide arrays combined with structural modeling, indicate different mechanisms for blocking NE activity. Both VH-Fc and IgG1 antibodies block NE uptake by cancer cells and fibroblast differentiation. VH-Fc 1D1.43 and IgG1 1C10 are promising for the antibody-based immunotherapy of cancer and inflammatory diseases.

An engineered antibody fragment targeting mutant ß-catenin via major histocompatibility complex I neoantigen presentation.

Mutations in CTNNB1, the gene encoding ß-catenin, are common in colon and liver cancers, the most frequent mutation affecting Ser-45 in ß-catenin. Peptides derived from WT ß-catenin have previously been shown to be presented on the cell surface as part of major histocompatibility complex (MHC) class I, suggesting an opportunity for targeting this common driver gene mutation with antibody-based therapies. Here, crystal structures of both the WT and S45F mutant peptide bound to HLA-A*03:01 at 2.20 and 2.45 Å resolutions, respectively, confirmed the accessibility of the phenylalanine residue for antibody recognition. Phage display was then used to identify single-chain variable fragment clones that selectively bind the S45F mutant peptide presented in HLA-A*03:01 and have minimal WT or other off-target binding. Following the initial characterization of five clones, we selected a single clone, E10, for further investigation. We developed a computational model of the binding of E10 to the mutant peptide-bound HLA-A3, incorporating data from affinity maturation as initial validation. In the future, our model may be used to design clones with maintained specificity and higher affinity. Such derivatives could be adapted into either cell-based (CAR-T) or protein-based (bispecific T-cell engagers) therapies to target cancer cells harboring the S45F mutation in CTNNB1.

Complexation with a Cognate Antibody Fragment Facilitates Affinity Measurements of Fluorescein-Linked Small Molecule Ligands.

The availability of reliable methods for the characterization of the binding of small molecule ligands to protein targets is crucially important for drug discovery. We have adapted a method, routinely used for the characterization of monoclonal antibodies (enzyme-linked immunosorbent assay, or "ELISA"), to small molecule ligands, using fluorescein conjugates and antifluorescein antibodies as detection reagents. The new small molecule-ELISA methodology was tested using a panel of binders specific to carbonic anhydrase II, with dissociation constants ranging between 6 µM and 14 nM. An excellent agreement was found between ELISA measurements and fluorescence polarization results. The methodology was also extended to BIAcore measurements and implemented for ligands coupled to oligonucleotides. Small molecule-ELISA procedures are particularly useful in the context of DNA-encoded libraries, for which hit validation procedures need to be performed on dozens of candidate molecules and hit compounds can be conveniently resynthesized on DNA.

Isolation and characterization of antibody fragments selective for human FTD brain derived TDP-43 variants.

BACKGROUND: Frontotemporal dementia (FTD) is the second leading cause of early onset dementia following Alzheimer's disease. It involves atrophy of the frontal and temporal regions of the brain affecting language, memory, and behavior. Transactive response DNA-binding protein 43 (TDP-43) pathology is found in most FTD and ALS cases. It plays a role in transcription, translation and serves as a shuttle between the nucleus and cytoplasm. Prior to its aggregation, TDP-43 exists as polyubiquitinated, hyperphosphorylated C-terminal fragments that correlate well with FTD disease progression. Because of the importance of TDP-43 in these diseases, reagents that can selectively recognize specific toxic TDP variants associated with onset and progression of FTD can be effective diagnostic and therapeutic tools. RESULTS: We utilized a novel atomic force microscopy (AFM) based biopanning protocol to isolate single chain variable fragments (scFvs) from a phage display library that selectively bind TDP variants present in human FTD but not cognitively normal age matched brain tissue. We then used the scFvs (FTD-TDP1 through 5) to probe post-mortem brain tissue and sera samples for the presence of FTD related TDP variants. The scFvs readily selected the FTD tissue and sera samples over age matched controls. The scFvs were used in immunohistochemical analysis of FTD and control brain slices where the reagents showed strong staining with TDP in FTD brain tissue slice. FTD-TDP1, FTD-TDP2, FTD-TDP4 and FTD-TDP5 all protected neuronal cells against FTD TDP induced toxicity suggesting potential therapeutic value. CONCLUSIONS: These results show existence of different disease specific TDP variants in FTD individuals. We have identified a panel of scFvs capable of recognizing these disease specific TDP variants in postmortem FTD tissue and sera samples over age matched controls and can thus serve as a biomarker tool.

Continuous directed evolution of proteins with improved soluble expression.

We report the development of soluble expression phage-assisted continuous evolution (SE-PACE), a system for rapidly evolving proteins with increased soluble expression. Through use of a PACE-compatible AND gate that uses a split-intein pIII, SE-PACE enables two simultaneous positive selections to evolve proteins with improved expression while maintaining their desired activities. In as little as three days, SE-PACE evolved several antibody fragments with >5-fold improvement in expression yield while retaining binding activity. We also developed an activity-independent form of SE-PACE to correct folding-defective variants of maltose-binding protein (MBP) and to evolve variants of the eukaryotic cytidine deaminase APOBEC1 with improved expression properties. These evolved APOBEC1 variants were found to improve the expression and apparent activity of Cas9-derived base editors when used in place of the wild-type cytidine deaminase. Together, these results suggest that SE-PACE can be applied to a wide variety of proteins to rapidly improve their soluble expression.

Optimizing antibody affinity and stability by the automated design of the variable light-heavy chain interfaces.

Antibodies developed for research and clinical applications may exhibit suboptimal stability, expressibility, or affinity. Existing optimization strategies focus on surface mutations, whereas natural affinity maturation also introduces mutations in the antibody core, simultaneously improving stability and affinity. To systematically map the mutational tolerance of an antibody variable fragment (Fv), we performed yeast display and applied deep mutational scanning to an anti-lysozyme antibody and found that many of the affinity-enhancing mutations clustered at the variable light-heavy chain interface, within the antibody core. Rosetta design combined enhancing mutations, yielding a variant with tenfold higher affinity and substantially improved stability. To make this approach broadly accessible, we developed AbLIFT, an automated web server that designs multipoint core mutations to improve contacts between specific Fv light and heavy chains (http://AbLIFT.weizmann.ac.il). We applied AbLIFT to two unrelated antibodies targeting the human antigens VEGF and QSOX1. Strikingly, the designs improved stability, affinity, and expression yields. The results provide proof-of-principle for bypassing laborious cycles of antibody engineering through automated computational affinity and stability design.

Principles for computational design of binding antibodies.

Natural proteins must both fold into a stable conformation and exert their molecular function. To date, computational design has successfully produced stable and atomically accurate proteins by using so-called "ideal" folds rich in regular secondary structures and almost devoid of loops and destabilizing elements, such as cavities. Molecular function, such as binding and catalysis, however, often demands nonideal features, including large and irregular loops and buried polar interaction networks, which have remained challenging for fold design. Through five design/experiment cycles, we learned principles for designing stable and functional antibody variable fragments (Fvs). Specifically, we (i) used sequence-design constraints derived from antibody multiple-sequence alignments, and (ii) during backbone design, maintained stabilizing interactions observed in natural antibodies between the framework and loops of complementarity-determining regions (CDRs) 1 and 2. Designed Fvs bound their ligands with midnanomolar affinities and were as stable as natural antibodies, despite having >30 mutations from mammalian antibody germlines. Furthermore, crystallographic analysis demonstrated atomic accuracy throughout the framework and in four of six CDRs in one design and atomic accuracy in the entire Fv in another. The principles we learned are general, and can be implemented to design other nonideal folds, generating stable, specific, and precise antibodies and enzymes.

Wavelength-Dependent Fluorescent Immunosensors via Incorporation of Polarity Indicators near the Binding Interface of Antibody Fragments.

Herein, we describe a fluorescent immunosensor designed by incorporating an unnatural amino acid fluorophore into the binding site of an EGFR-specific antibody fragment, resulting in quantifiable EGFR-dependent changes in peak fluorescence emission wavelength. To date, immunosensor design strategies have relied on binding-induced changes in fluorescence intensity that are prone to excitation source fluctuations and sample-dependent noise. In this study, we used a rational design approach to incorporate a polarity indicator (Anap) into specific positions of an anti-EGFR single chain antibody to generate an emission wavelength-dependent immunosensor. We found that when incorporated within the topological neighborhood of the antigen binding interface, the Anap emission wavelength is blue-shifted by EGFR-binding in a titratable manner, up to 20 nm, with nanomolar detection limits. This approach could be applicable to other antibody/antigen combinations for integration into a wide range of assay platforms (including homogeneous, solid-phase assay, or microfluidic assays) for one-step protein quantification.

Dual Site-Specific Labeling of an Antibody Fragment through Sortase A and π-Clamp Conjugation.

The functionalization of proteins with different cargo molecules is highly desirable for a broad range of applications. However, the reproducible production of defined conjugates with multiple functionalities is a significant challenge. Herein, we report the dual site-specific labeling of an antibody fragment, utilizing the orthogonal Sortase A and π-clamp conjugation methods, and demonstrate that binding of the antibody fragment to its target receptor is retained after dual labeling.

Characterization of therapeutic antibody fragmentation using automated capillary western blotting as an orthogonal analytical technique.

Fragmentation in protein-based molecules continues to be a challenge during manufacturing and storage, and requires an appropriate control strategy to ensure purity and integrity of the drug product. Electrophoretic and chromatographic methods are commonly used for monitoring the fragments. However, size-exclusion chromatography often suffers from low resolution of low molecular weight fragments. Electrophoretic methods like CE-SDS are not compatible with enriching fragments for additional characterization tests such as MS. These limitations may result in inadequate control strategy for monitoring and characterizing fragments for protein-based molecules. Capillary western blotting was used in this study as an orthogonal method for characterization of fragments in an IgG1 antibody under reduced conditions. To achieve a comprehensive mapping of various fragments generated by thermal stress, capillary western profiles were generated using recognition antibodies for IgG kappa (κ) light chain, Fc, and Fab regions that enabled unambiguous fragment identification. Additionally, three different enzymatic digestion methods (IdeS, PNGase F, and IgdE) were applied coupled with capillary western blotting for clip identifications. Finally, complementary data collected using traditional chromatographic and electrophoretic methods allowed to establish a comparison of analytical profiles with an added benefit of fragment identification offered by capillary western profiling. In addition to various Fc and Fab-related low molecular weight fragments, a non-reducible thio-ether linked 75 kDa HL fragment was also identified.

Complementary Design for Pairing between Two Types of Nanoparticles Mediated by a Bispecific Antibody: Bottom-Up Formation of Porous Materials from Nanoparticles.

Recent advances in biotechnology have enabled the generation of antibodies with high affinity for the surfaces of specific inorganic materials. Herein, we report the synthesis of functional materials from multiple nanomaterials by using a small bispecific antibody recombinantly constructed from gold-binding and ZnO-binding antibody fragments. The bispecific antibody-mediated spontaneous linkage of gold and ZnO nanoparticles forms a binary gold-ZnO nanoparticle composite membrane. The relatively low melting point of the gold nanoparticles and the solubility of ZnO in dilute acidic solution then allowed for the bottom-up synthesis of a nanoporous gold membrane by means of a low-energy, low-environmental-load protocol. The nanoporous gold membrane showed high catalytic activity for the reduction of p-nitrophenol to p-aminophenol by sodium borohydride. Here, we show the potential utility of nanoparticle pairing mediated by bispecific antibodies for the bottom-up construction of nanostructured materials from multiple nanomaterials.