Offsetting Low-Affinity Carbohydrate Binding with Covalency to Engage Sugar-Specific Proteins for Tumor-Immune Proximity Induction.

Carbohydrate-binding receptors are often used by the innate immune system to potentiate inflammation, target endocytosis/destruction, and adaptive immunity (e.g., CD206, DC-SIGN, MBL, and anticarbohydrate antibodies). To access this class of receptors for cancer immunotherapy, a growing repertoire of bifunctional proximity-inducing therapeutics use high-avidity multivalent carbohydrate binding domains to offset the intrinsically low affinity associated with monomeric carbohydrate-protein binding interactions (Kd ≈ 10-3-10-6 M). For applications aimed at recruiting anticarbohydrate antibodies to tumor cells, large synthetic scaffolds are used that contain both a tumor-binding domain (TBD) and a multivalent antibody-binding domain (ABD) comprising multiple l-rhamnose monosaccharides. This allows for stable bridging between tumor cells and antibodies, which activates tumoricidal immune function. Problematically, such multivalent macromolecules can face limitations including synthetic and/or structural complexity and the potential for off-target immune engagement. We envisioned that small bifunctional "proximity-inducing" molecules containing a low-affinity monovalent ABD could efficiently engage carbohydrate-binding receptors for tumor-immune proximity by coupling weak binding with covalent engagement. Typical covalent drugs and electrophilic chimeras use high-affinity ligands to promote the fast covalent engagement of target proteins (i.e., large kinact/KI), driven by a favorably small KI for binding. We hypothesized the much less favorable KI associated with carbohydrate-protein binding interactions can be offset by a favorably large kinact for the covalent labeling step. In the current study, we test this hypothesis in the context of a model system that uses rhamnose-specific antibodies to induce tumor-immune proximity and tumoricidal function. We discovered that synthetic chimeric molecules capable of preorganizing an optimal electrophile (i.e., SuFEx vs activated ester) for protein engagement can rapidly covalently engage natural sources of antirhamnose antibody using only a single low-affinity rhamnose monosaccharide ABD. Strikingly, we observe chimeric molecules lacking an electrophile, which can only noncovalently bind the antibody, completely lack tumoricidal function. This is in stark contrast to previous work targeting small molecule hapten and peptide-specific antibodies. Our findings underscore the utility of covalency as a strategy to engage low-affinity carbohydrate-specific proteins for tumor-immune proximity induction.

Myeloid-specific deletion of activating transcription factor 6 alpha increases CD11b+ macrophage subpopulations and aggravates lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic interstitial lung disease of unknown etiology. The accumulation of macrophages is associated with disease pathogenesis. The unfolded protein response (UPR) has been linked to macrophage activation in pulmonary fibrosis. To date, the impact of activating transcription factor 6 alpha (ATF6α), one of the UPR mediators, on the composition and function of pulmonary macrophage subpopulations during lung injury and fibrogenesis is not fully understood. We began by examining the expression of Atf6α in IPF patients' lung single-cell RNA sequencing dataset, archived surgical lung specimens, and CD14+ circulating monocytes. To assess the impact of ATF6α on pulmonary macrophage composition and pro-fibrotic function during tissue remodeling, we conducted an in vivo myeloid-specific deletion of Atf6α. Flow cytometric assessments of pulmonary macrophages were carried out in C57BL/6 and myeloid specific ATF6α-deficient mice in the context of bleomycin-induced lung injury. Our results demonstrated that Atf6α mRNA was expressed in pro-fibrotic macrophages found in the lung of a patient with IPF and in CD14+ circulating monocytes obtained from blood of a patient with IPF. After bleomycin administration, the myeloid-specific deletion of Atf6α altered the pulmonary macrophage composition, expanding CD11b+ subpopulations with dual polarized CD38+ CD206+ expressing macrophages. Compositional changes were associated with an aggravation of fibrogenesis including increased myofibroblast and collagen deposition. A further mechanistic ex vivo investigation revealed that ATF6α was required for CHOP induction and the death of bone marrow-derived macrophages. Overall, our findings suggest a detrimental role for the ATF6α-deficient CD11b+ macrophages which had altered function during lung injury and fibrosis.

Therapeutic strategies targeting pro-fibrotic macrophages in interstitial lung disease.

Idiopathic pulmonary fibrosis (IPF) is the representative phenotype of interstitial lung disease where severe scarring develops in the lung interstitium. Although antifibrotic treatments are available and have been shown to slow the progression of IPF, improved therapeutic options are still needed. Recent data indicate that macrophages play essential pro-fibrotic roles in the pathogenesis of pulmonary fibrosis. Historically, macrophages have been classified into two functional subtypes, "M1″ and "M2," and it is well described that "M2″ or "alternatively activated" macrophages contribute to fibrosis via the production of fibrotic mediators, such as TGF-ß, CTGF, and CCL18. However, highly plastic macrophages may possess distinct functions and phenotypes in the fibrotic lung environment. Thus, M2-like macrophages in vitro and pro-fibrotic macrophages in vivo are not completely identical cell populations. Recent developments in transcriptome analysis, including single-cell RNA sequencing, have attempted to depict more detailed phenotypic characteristics of pro-fibrotic macrophages. This review will outline the role and characterization of pro-fibrotic macrophages in fibrotic lung diseases and discuss the possibility of treating lung fibrosis by preventing or reprogramming the polarity of macrophages. We also utilized a systematic approach to review the literature and identify novel and promising therapeutic agents that follow this treatment strategy.

A covalent opsonization approach to enhance synthetic immunity against viral escape variants.

The sensitivity of therapeutic antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral "escape" mutations has inspired efforts to develop treatment strategies that are still effective in the face of rapidly mutating viral surface proteins. Here, we demonstrate a chemical strategy that enforces viral opsonization by natural serum antibodies. This strategy uses chimeric molecules that we call covalent viral opsonizers, which covalently label viral surface proteins, with synthetic antibody-binding ligands. As a proof of concept, we develop covalent viral opsonizers that covalently label the spike protein on SARS-CoV-2 using a "mutation-proof" small-molecule-binding ligand for anti-dinitrophenyl serum antibodies. In model assays, we observe that covalent viral opsonizers can rapidly and selectively covalently label the receptor-binding domain of both native and mutant spike proteins, leading to antibody opsonization. Opsonization mediated by this strategy is able to efficiently block the key binding domain interactions, in contrast to non-covalent analogs. We also show that covalent viral opsonizers enact targeted anti-viral phagocytotic immune function. This strategy has potential general utility for the rapid deployment of anti-viral synthetic immunotherapeutics at the onset of a new pandemic to reinforce vaccination and antibody engineering efforts.

Tunable Multivalent Platform for Immune Recruitment to Lower Antigen Expressing Cancers.

Chemical immunotherapeutic strategies including Antibody Recruiting Molecules (ARMs - bivalent small molecules containing an antibody-binding domain (ABD) and a target-binding domain (TBD)) direct immune-mediated clearance of diseased cells. Anti-cancer ARM function relies on high tumor antigen valency, limiting function against lower antigen expressing tumors. To address this limitation, we report a tunable multivalent immune recruitment (MIR) platform to amplify/stabilize antibody recruitment to cells with lower antigen valencies. An initial set of polymeric ARMs (pARMs) were synthesized and screened to evaluate ABD/TBD copy number, ratio, and steric occlusion on specific immune induction. Most pARMs demonstrated simultaneous high avidity binding to anti-dinitrophenyl antibodies and prostate-specific membrane antigens on prostate cancer. Optimized pARMs mediated enhanced anti-cancer immune function against lower antigen expressing target cells compared to an analogous ARM.

Covalent Immune Proximity-Induction Strategy Using SuFEx-Engineered Bifunctional Viral Peptides.

Covalent antibody recruiting molecules (cARMs) constitute a proximity-inducing chemical strategy to modulate the recognition and elimination of cancer cells by the immune system. Recognition is achieved through synthetic bifunctional molecules that use covalency to stably bridge endogenous hapten-specific antibodies like anti-dinitrophenyl (anti-DNP), with tumor antigens on cancer cell surfaces. To recruit these antibodies, cARMs are equipped with the native hapten-binding molecule. The majority of cancer-killing immune machinery, however, recognizes epitopes on protein ligands and not small molecule haptens (e.g., Fc receptors, pathogen-specific antibodies). To access this broader class of immune machinery for recruitment, we developed a covalent immune proximity-inducing strategy. This strategy uses synthetic bifunctional electrophilic peptides derived from the native protein ligand. These bifunctional peptides are engineered to contain both a tumor-targeting molecule and a sulfonyl (VI) fluoride exchange (SuFEx) electrophile. As a proof of concept, we synthesized bifunctional electrophilic peptides derived from glycoprotein D (gD) on herpes simplex virus (HSV), to recruit gD-specific serum anti-HSV antibodies to cancer cells expressing the prostate-specific membrane antigen (PSMA). We demonstrate that serum anti-HSV antibodies can be selectively and irreversibly targeted by these electrophilic peptides and that the reaction rate can be uniquely enhanced by tuning SuFEx chemistry without a loss in selectivity. In cellular assays, electrophilic peptides demonstrated enhanced anti-tumor immunotherapeutic efficacy compared to analogous peptides lacking electrophilic functionality. This enhanced efficacy was especially prominent in the context of (a) natural anti-HSV antibodies isolated from human serum and (b) harder to treat tumor cells associated with lower PSMA expression levels. Overall, we demonstrate a new covalent peptide-based approach to immune proximity induction and reveal the potential utility of anti-viral antibodies in synthetic tumor immunotherapy.

Development of a B-cell maturation antigen-specific T-cell antigen coupler receptor for multiple myeloma.

BACKGROUND AIMS: T cells engineered with synthetic receptors have delivered powerful therapeutic results for patients with relapsed/refractory hematologic malignancies. The authors have recently described the T-cell antigen coupler (TAC) receptor, which co-opts the endogenous T-cell receptor (TCR) and activates engineered T cells in an HLA-independent manner. Here the authors describe the evolution of a next-generation TAC receptor with a focus on developing a TAC-engineered T cell for multiple myeloma. METHODS: To optimize the TAC scaffold, the authors employed a bona fide antigen-binding domain derived from the B-cell maturation antigen-specific monoclonal antibody C11D5.3, which has been used successfully in the clinic. The authors first tested humanized versions of the UCHT1 domain, which is used by the TAC to co-opt the TCR. The authors further discovered that the signal peptide affected surface expression of the TAC receptor. Higher density of the TAC receptor enhanced target binding in vitro, which translated into higher levels of Lck at the immunological synapse and stronger proliferation when only receptor-ligand interactions were present. RESULTS: The authors observed that the humanized UCHT1 improved surface expression and in vivo efficacy. Using TAC T cells derived from both healthy donors and multiple myeloma patients, the authors determined that despite the influence of receptor density on early activation events and effector function, receptor density did not impact late effector functions in vitro, nor did the receptor density affect in vivo efficacy. CONCLUSIONS: The modifications to the TAC scaffold described herein represent an important step in the evolution of this technology, which tolerates a range of expression levels without impacting therapeutic efficacy.

A Versatile Platform for the Development of Radiolabeled Antibody-Recruiting Small Molecules.

Building on clinical case reports of the abscopal effect, there has been considerable interest in the synergistic effects of radiation and immunotherapies for the treatment of cancer. Here, the first radiolabeled antibody-recruiting small molecule that can chelate a variety of cytotoxic radionuclides is described. The platform consists of a tunable antibody-binding domain against a serum antibody of interest (e.g., dinitrophenyl hapten) to recruit endogenous antibodies that activate effector cell function, a chelate capable of binding diagnostic and therapeutic radiometals, and a tetrazine for bioorthogonal coupling with trans-cyclooctene-modified targeting vectors. The dinitrophenyl-tetrazine ligand was shown to both affect dose-dependent antibody recruitment and immune cell function (phagocytosis) in vitro, and the bisphosphonate 177Lu-complex was shown to accumulate at sites of calcium accretion in vivo, which was achieved using both active and pretargeting strategies.

Cell-specific drug targeting in the lung.

Non-targeted drug delivery systems have several limitations including the decreased bioavailability of the drug, poor stability and rapid clearance in addition to off-target distribution. Cell-specific targeted delivery approaches promise to overcome some of these limitations and enhance therapeutic selectivity. In this review, we aim to discuss cell-specific targeted approachesin the lung at the biochemical and molecular levels. These approaches include;a) directly administered small molecule drugs with intracellular action; b) targeted biologics and synthetic hybrids with extracellular action; c) site activateddrugs; and d) delivery systems.We discuss the pharmaceutical and biochemical parameters that govern the fate of drug molecules at delivery sites while presenting an overview of relevant literature surrounding this area of research and current advancements.

Covalent Stabilization of Antibody Recruitment Enhances Immune Recognition of Cancer Targets.

Antibody recruiting molecules (ARMs) represent an important class of "proximity-inducing" chemical tools with therapeutic potential. ARMs function by simultaneously binding to a hapten-specific serum antibody (Ab) (e.g., anti-dinitrophenyl (DNP)) and a cancer cell surface protein, enforcing their proximity. ARM anticancer efficacy depends on the formation of ARM:Ab complexes on the cancer cell surface, which activate immune cell recognition and elimination of the cancer cell. Problematically, ARM function in human patients may be limited by conditions that drive the dissociation of ARM:Ab complexes, namely, intrinsically low binding affinity and/or low concentrations of anti-hapten antibodies in human serum. To address this potential limitation, we previously developed a covalent ARM (cARM) chemical tool that eliminates the ARM:antibody equilibrium through a covalent linkage. In the current study, we set out to determine to what extent maximizing the stability of ARM:antibody complexes via cARMs enhances target immune recognition. We observe cARMs significantly increase target immune recognition relative to ARMs across a range of therapeutically relevant antibody concentrations. These results demonstrate that ARM therapeutic function can be dramatically enhanced by increasing the kinetic stability of ARM:antibody complexes localized on cancer cells. Our findings suggest that a) high titres/concentrations of target antibody in human serum are not neccessary and b) saturative antibody recruitment to cancer cells not sufficient, to achieve maximal ARM therapeutic function.

Methods to Validate Binding and Kinetics of "Proximity-Inducing" Covalent Immune-Recruiting Molecules.

The emergence of covalent inhibitors and chemoproteomic probes in translational chemical biology research requires the development of robust biophysical and analytical methods to characterize their complex interactions with target biomolecules. Importantly, these methods must efficiently assess target selectivity and accurately discern noncovalent binding from the formation of resultant covalent adducts. One recently reported covalent chemical tool used in tumor immune oncology, covalent immune recruiters (CIRs), increases the proximity of immune cells and cancer cells, promoting immune recognition and response. Herein we describe biolayer interferometry (BLI) biosensor, flow cytometry, and solution fluorescence-based assay approaches to characterize CIR:antibody binding and CIR-antibody covalent-labeling kinetics. BLI technology, akin to surface plasmon resonance, provides the unique opportunity to investigate molecular binding and labeling kinetics both on a solid surface (Basic Protocol 1) and in solution (Alternate Protocol 1). Here, recruitment of mass-containing proteins to the BLI probe via CIR is measured with high sensitivity and is used as a readout of CIR labeling activity. Further, CIR technology is used to label antibodies with a fluorescent handle. In this system, labeling is monitored via SDS-PAGE with a fluorescence gel imager, where increased fluorescence intensity of a sample reflects increased labeling (Basic Protocol 2). Analysis of CIR:antibody target-specific immune activation is demonstrated with a flow cytometry-based antibody-dependent cellular phagocytosis (ADCP) assay (Basic Protocol 3). This ADCP protocol may be further used to discern CIR:antibody binding from covalent adduct formation (Alternate Protocol 3). For the protocols described, each method may be used to analyze characteristics of any covalent-tagging or antibody-recruiting small molecule or protein-based technology. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Determining "on-probe" reaction kinetics of CIR1/CIR4 via biolayer interferometry with Octet RED96 Alternate Protocol 1: Determining "in-solution" reaction kinetics of prostate-specific membrane antigen targeting CIR (CIR3) via biolayer interferometry with Octet RED96 Basic Protocol 2: Reaction kinetics of covalently labeled antibodies via fluorescence SDS-PAGE Basic Protocol 3: Small molecule-directed antibody-dependent cellular phagocytosis on live human cells measured via flow cytometry Alternate Protocol 2: Kinetic analysis of CIR3:antibody labeling via antibody-dependent cellular phagocytosis on flow cytometry Support Protocol 1: Activation of U937 monocytes with interferon γ Support Protocol 2: Labeling streptavidin beads with biotinylated prostate-specific membrane antigen receptor.

Covalent Immune Recruiters: Tools to Gain Chemical Control Over Immune Recognition.

Unprecedented progress made in the treatment of cancer using the body's own immune system has encouraged the development of synthetic molecule based immunotherapeutics. An emerging class of these compounds, called Antibody Recruiting Molecules (ARMs) or Antibody Engagers (AEs), functions by reversibly binding antibodies naturally present in human serum and recruiting these to cancer cells. The recruited antibodies then engage immune cells to form quaternary complexes that drive cancer erradication. Despite their promise, the requirement to form quaternary complexes governed by multiple equilibria complicates an understanding of their in vivo efficacy. Particularly problematic are low endogenous serum antibody concentrations and rapid clearance of AEs from circulation. Here we describe a new class of trifunctional chemical tools we call covalent immune recruiters (CIRs). CIRs covalently label specific serum antibodies in a selective manner with a target protein binding ligand. CIRs thereby exert well-defined control over antibody recruitment and simplify quaternary complex equilibium, enabling probing of the resultant effects on immune recognition. We demonstrate CIRs can selectively covalently label anti-DNP IgG, a natural human antibody, directly in human serum to drive efficient immune cell recognition of targets. We expect CIRs will be useful tools to probe how quaternary complex stability impacts the immune recognition of cancer in vivo, revealing new design principles to guide the development of future AEs.

Improved Efficacy of Antibody Cancer Immunotherapeutics through Local and Sustained Delivery.

Antibodies are a growing class of cancer immunotherapeutics that facilitate immune-cell-mediated killing of tumors. However, the efficacy and safety of immunotherapeutics are limited by transport barriers and poor tumor uptake, which lead to high systemic concentrations and potentially fatal side effects. To increase tumor antibody immunotherapeutic concentrations while decreasing systemic concentrations, local delivery vehicles for sustained antibody release are being developed. The focus of this review is to define the material properties required for implantable controlled antibody delivery and highlight the controlled-release strategies that are applicable to antibody immunotherapeutics.

Re-engineering the Immune Response to Metastatic Cancer: Antibody-Recruiting Small Molecules Targeting the Urokinase Receptor.

Developing selective strategies to treat metastatic cancers remains a significant challenge. Herein, we report the first antibody-recruiting small molecule (ARM) that is capable of recognizing the urokinase-type plasminogen activator receptor (uPAR), a uniquely overexpressed cancer cell-surface marker, and facilitating the immune-mediated destruction of cancer cells. A co-crystal structure of the ARM-U2/uPAR complex was obtained, representing the first crystal structure of uPAR complexed with a non-peptide ligand. Finally, we demonstrated that ARM-U2 substantially suppresses tumor growth in vivo with no evidence of weight loss, unlike the standard-of-care agent doxorubicin. This work underscores the promise of antibody-recruiting molecules as immunotherapeutics for treating cancer.

Peptide-glycosaminoglycan cluster formation involving cell penetrating peptides.

Glycosaminoglycans (GAGs) affect the efficiency of cellular uptake of a wide range of cell penetrating peptides (CPPs). GAGs have been proposed to cluster with CPPs at the cell surface before uptake but little is known about the formation or stability of CPP-GAG clusters. Here we apply a combination of heparin affinity chromatography, dynamic light scattering, and fluorescence spectroscopy to characterize the formation, stability, and size of the clusters formed between CPPs and heparin. Under conditions similar to those used in cell uptake experiments the CPP, penetratin (Antp), was observed to form significantly more stable clusters with heparin than the CPP TAT, despite TAT showing a comparable affinity for heparin. This difference in cluster stability may explain the origins of the preferred cell uptake pathways followed by Antp and TAT, and may be an important parameter for optimizing the efficiency of designed CPP delivery vectors.

Fluorescence resonance energy transfer in gaseous, mass-selected polyproline peptides.

Despite the many successes of mass spectrometry in the analysis of biological samples, the need to better understand the correlation between condensed-phase properties and those of electrospray species remains. In particular, the link between structures in the condensed phase and in the gaseous environment of the mass spectrometer is still elusive. Here, we show that fluorescence resonance energy transfer (FRET) can be used to probe the conformations of gaseous biopolymers which are formed by electrospray ionization (ESI) and manipulated in a quadrupole ion trap mass spectrometer. A rhodamine dye pair suitable for gas-phase FRET is characterized. Both steady state spectra and lifetime measurements are used to monitor energy transfer in a series of dye-labeled polyproline-based peptides. FRET efficiency is explored as a function of peptide chain length and charge state. For the peptide with eight proline repeats, virtually complete energy transfer is observed. For the peptide with 14 proline repeats, energy transfer decreases as the charge state increases, consistent with Coulomb repulsion induced elongation of the peptide backbone. FRET measurements of the longest peptide examined, which has 20 proline repeats, indicates that the peptide adopts a bent configuration. Evidence for multiple conformations present within the ensemble of trapped ions is provided by fluorescence lifetime measurements. Gas-phase FRET measurements promise to be a new route to probe the conformations of large gaseous ions.

Importance of the spatial display of charged residues in heparin-peptide interactions.

Many studies have examined consensus sequences required for protein-glycosaminoglycan interactions. Through the synthesis of helical heparin binding peptides, this study probes the relationship between spatial arrangement of positive charge and heparin binding affinity. Peptides with a linear distribution of positive charge along one face of the alpha-helix had the highest affinity for heparin. Moving the basic residues away from a single face resulted in drastic changes in heparin binding affinity of up to three orders of magnitude. These findings demonstrate that amino acid sequences, different from the known heparin binding consensus sequences, will form high affinity protein-heparin binding interactions when the charged residues are aligned linearly.