Self-Immolative Carbamate Linkers for CD19-Budesonide Antibody-Drug Conjugates.

Antibody-drug conjugates consist of potent small-molecule payloads linked to a targeting antibody. Payloads must possess a viable functional group by which a linker for conjugation can be attached. Linker-attachment options remain limited for the connection to payloads via hydroxyl groups. A releasing group based on 2-aminopyridine was developed to enable stable attachment of para-aminobenzyl carbamate (PABC) linkers to the C21-hydroxyl group of budesonide, a glucocorticoid receptor agonist. Payload release involves a cascade of two self-immolative events that are initiated by the protease-mediated cleavage of the dipeptide-PABC bond. Budesonide release rates were determined for a series of payload-linker intermediates in buffered solution at pH 7.4 and 5.4, leading to the identification of 2-aminopyridine as the preferred releasing group. Addition of a poly(ethylene glycol) group improved linker hydrophilicity, thereby providing CD19-budesonide ADCs with suitable properties. ADC23 demonstrated targeted delivery of budesonide to CD19-expressing cells and inhibited B-cell activation in mice.

Discovery of ABBV-154, an anti-TNF Glucocorticoid Receptor Modulator Immunology Antibody-Drug Conjugate (iADC).

Stable attachment of drug-linkers to the antibody is a critical requirement, and for maleimide conjugation to cysteine, it is achieved by ring hydrolysis of the succinimide ring. During ADC profiling in our in-house property screening funnel, we discovered that the succinimide ring open form is in equilibrium with the ring closed succinimide. Bromoacetamide (BrAc) was identified as the optimal replacement, as it affords stable attachment of the drug-linker to the antibody while completely removing the undesired ring open-closed equilibrium. Additionally, BrAc also offers multiple benefits over maleimide, especially with respect to homogeneity of the ADC structure. In combination with a short, hydrophilic linker and phosphate prodrug on the payload, this afforded a stable ADC (ABBV-154) with the desired properties to enable long-term stability to facilitate subcutaneous self-administration.

Optimization of Drug-Linker to Enable Long-term Storage of Antibody-Drug Conjugate for Subcutaneous Dosing.

To facilitate subcutaneous dosing, biotherapeutics need to exhibit properties that enable high-concentration formulation and long-term stability in the formulation buffer. For antibody-drug conjugates (ADCs), the introduction of drug-linkers can lead to increased hydrophobicity and higher levels of aggregation, which are both detrimental to the properties required for subcutaneous dosing. Herein we show how the physicochemical properties of ADCs could be controlled through the drug-linker chemistry in combination with prodrug chemistry of the payload, and how optimization of these combinations could afford ADCs with significantly improved solution stability. Key to achieving this optimization is the use of an accelerated stress test performed in a minimal formulation buffer.

Suppression of local inflammation via galectin-anchored indoleamine 2,3-dioxygenase.

The treatment of chronic inflammation with systemically administered anti-inflammatory treatments is associated with moderate-to-severe side effects, and the efficacy of locally administered drugs is short-lived. Here we show that inflammation can be locally suppressed by a fusion protein of the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO) and galectin-3 (Gal3). Gal3 anchors IDO to tissue, limiting the diffusion of IDO-Gal3 away from the injection site. In rodent models of endotoxin-induced inflammation, psoriasis, periodontal disease and osteoarthritis, the fusion protein remained in the inflamed tissues and joints for about 1 week after injection, and the amelioration of local inflammation, disease progression and inflammatory pain in the animals were concomitant with homoeostatic preservation of the tissues and with the absence of global immune suppression. IDO-Gal3 may serve as an immunomodulatory enzyme for the control of focal inflammation in other inflammatory conditions.

Discovery of ABBV-3373, an Anti-TNF Glucocorticoid Receptor Modulator Immunology Antibody Drug Conjugate.

Using a convergent synthetic route to enable multiple points of diversity, a series of glucocorticoid receptor modulators (GRM) were profiled for potency, selectivity, and drug-like properties in vitro. Despite covering a large range of diversity, profiling the nonconjugated small molecule was suboptimal and they were conjugated to a mouse antitumor necrosis factor (TNF) antibody using the MP-Ala-Ala linker. Screening of the resulting antibody drug conjugates (ADCs) provided a better assessment of efficacy and physical properties, reinforcing the need to conduct structure-activity relationship studies on the complete ADC. DAR4 ADCs were screened in an acute mouse contact hypersensitivity model measuring biomarkers to ensure a sufficient therapeutic window. In a chronic mouse arthritis model, mouse anti-TNF GRM ADCs were efficacious after a single dose of 10 mg/kg i.p. for over 30 days. Data on the unconjugated payloads and mouse surrogate anti-TNF ADCs identified payload 17 which was conjugated to a human anti-TNF antibody and advanced to the clinic as ABBV-3373.

Site-Specific Cross-Linking of Galectin-1 Homodimers via Poly(ethylene glycol) Bismaleimide.

INTRODUCTION: The promise of the natural immunoregulator, Galectin-1 (Gal1), as an immunomodulatory therapeutic is challenged by its unstable homodimeric conformation. Previously, a Gal1 homodimer stabilized via covalent poly(ethylene glycol) diacrylate (PEGDA) cross-linking demonstrated higher activity relative to the non-covalent homodimer. METHODS: Here, we report Gal1 homodimers formed using an alternative thiol-Michael addition linker chemistry. RESULTS: Poly(ethylene glycol) bismaleimide (PEGbisMal) reacted with Gal1 at multiple sites with greater efficiency than PEGDA. However, multiple PEGbisMal molecules were conjugated to Gal1 C130, a Gal1 mutant with one surface cysteine (cys-130) and two cysteines thought to be buried in the solvent-inaccessible protein core (cys-42 and cys-60). Site-directed mutagenesis demonstrated that cys-60 was the site at which additional PEGbisMal molecules were conjugated onto Gal1 C130. Compared to WT-Gal1, Gal1 C130 had low activity for inducing Jurkat T cell death, characterized by phosphatidylserine exposure and membrane permeability. PEG cross-linking could restore the function of Gal1 C130, such that at high concentrations Gal1 C130 cross-linked by PEGbisMal had higher activity than both WT-Gal1 and Gal1 C130 cross-linked by PEGDA. Mutating cys-42 and cys-60 to serines in Gal1 C130 did not affect the cell death signaling activity of the Gal1 C130 homodimer cross-linked by PEGbisMal. PEGylated Gal1 C130 variants also eliminated the need for a reducing agent, such as dithiothreitol, which is required to maintain WT-Gal1 signaling activity. CONCLUSION: Collectively, these data demonstrate that thiol-Michael addition bioconjugation leads to a PEG-cross-linked Gal1 homodimer with improved extracellular signaling activity that does not require a reducing environment to be functional.

Intracellular immune sensing promotes inflammation via gasdermin D-driven release of a lectin alarmin.

Inflammatory caspase sensing of cytosolic lipopolysaccharide (LPS) triggers pyroptosis and the concurrent release of damage-associated molecular patterns (DAMPs). Collectively, DAMPs are key determinants that shape the aftermath of inflammatory cell death. However, the identity and function of the individual DAMPs released are poorly defined. Our proteomics study revealed that cytosolic LPS sensing triggered the release of galectin-1, a ß-galactoside-binding lectin. Galectin-1 release is a common feature of inflammatory cell death, including necroptosis. In vivo studies using galectin-1-deficient mice, recombinant galectin-1 and galectin-1-neutralizing antibody showed that galectin-1 promotes inflammation and plays a detrimental role in LPS-induced lethality. Mechanistically, galectin-1 inhibition of CD45 (Ptprc) underlies its unfavorable role in endotoxin shock. Finally, we found increased galectin-1 in sera from human patients with sepsis. Overall, we uncovered galectin-1 as a bona fide DAMP released as a consequence of cytosolic LPS sensing, identifying a new outcome of inflammatory cell death.

The CD28 Transmembrane Domain Contains an Essential Dimerization Motif.

CD28 plays a critical role in regulating immune responses both by enhancing effector T cell activation and differentiation and controlling the development and function of regulatory T cells. CD28 is expressed at the cell surface as a disulfide linked homodimer that is thought to bind ligand monovalently. How ligand binding triggers CD28 to induce intracellular signaling as well as the proximal signaling pathways that are induced are not well-understood. In addition, recent data suggest inside-out signaling initiated by the T cell antigen receptor can enhance CD28 ligand binding, possibly by inducing a rearrangement of the CD28 dimer interface to allow for bivalent binding. To understand how possible conformational changes during ligand-induced receptor triggering and inside-out signaling are mediated, we examined the CD28 transmembrane domain. We identified an evolutionarily conserved YxxxxT motif that is shared with CTLA-4 and resembles the transmembrane dimerization motif within CD3ζ. We show that the CD28 transmembrane domain can drive protein dimerization in a bacterial expression system at levels equivalent to the well-known glycophorin A transmembrane dimerization motif. In addition, ectopic expression of the CD28 transmembrane domain into monomeric human CD25 can drive dimerization in murine T cells as detected by an increase in FRET by flow cytometry. Mutation of the polar YxxxxT motif to hydrophobic leucine residues (Y145L/T150L) attenuated CD28 transmembrane mediated dimerization in both the bacterial and mammalian assays. Introduction of the Y145L/T150L mutation of the CD28 transmembrane dimerization motif into the endogenous CD28 locus by CRISPR resulted in a dramatic loss in CD28 cell surface expression. These data suggest that under physiological conditions the YxxxxT dimerization motif within the CD28 transmembrane domain plays a critical role in the assembly and/or expression of stable CD28 dimers at the cell surface.

A chimeric, multivalent assembly of galectin-1 and galectin-3 with enhanced extracellular activity.

Galectins are attractive therapeutic candidates to control aberrant immune system activation because they can alter the phenotype and function of various innate and adaptive immune cells. However, use of exogenous galectin-1 ("G1") and galectin-3 ("G3") as immunomodulators is challenged by their high dosing requirements and dynamic quaternary structures. Here we report a chimeric assembly of G1 and G3 with enhanced extracellular activity ("G1/G3 Zipper"), which was created by recombinant fusion of G1 and G3 via a peptide linker that forms a two-stranded α-helical coiled-coil. G1/G3 Zipper had higher apparent binding affinity for immobilized lactose and a lower concentration threshold for inducing soluble glycoprotein crosslinking than G1, a recombinant fusion of G1 and G3 with a flexible peptide linker ("G1/G3"), or a recently reported stable G1 dimer crosslinked by poly(ethylene glycol) diacrylate ("G1-PEG-G1"). As a result, G1/G3 Zipper was more effective at inducing Jurkat T cell apoptosis in media containing serum, and was the only variant that could induce apoptosis at low concentrations under serum-free conditions. The monomeric G1/G3 fusion protein lacked extracellular activity under all conditions tested, suggesting that the enhanced activity of G1/G3 Zipper was due to its quaternary structure and increased carbohydrate-recognition domain valency. Thus, combining G1 and G3 into a non-native chimeric assembly provides a new candidate therapeutic with greater immunomodulatory potency than the wild-type proteins and previously reported engineered variants.

A Synthetic Tetramer of Galectin-1 and Galectin-3 Amplifies Pro-apoptotic Signaling by Integrating the Activity of Both Galectins.

Galectin-1 (G1) and galectin-3 (G3) are carbohydrate-binding proteins that can signal apoptosis in T cells. We recently reported that a synthetic tetramer with two G1 and two G3 domains ("G1/G3 Zipper") induces Jurkat T cell death more potently than G1. The pro-apoptotic signaling pathway of G1/G3 Zipper was not elucidated, but we hypothesized based on prior work that the G1 domains acted as the signaling units, while the G3 domains served as anchors that increase glycan-binding affinity. To test this, here we studied the involvement of different cell membrane glycoproteins and intracellular mediators in pro-apoptotic signaling via G1/G3 Zipper, G1, and G3. G1/G3 Zipper induced Jurkat T cell death more potently than G1 and G3 alone or in combination. G1/G3 Zipper, G1, and G3 increased caspase-8 activity, yet only G1 and G3 depended on it to induce cell death. G3 increased caspase-3 activity more than G1/G3 Zipper and G1, while all three galectin variants required it to induce cell death. JNK activation had similar roles downstream of G1/G3 Zipper, G1, and G3, whereas ERK had differing roles. CD45 was essential for G1 activity, and was involved in signaling via G1/G3 Zipper and G3. CD7 inhibited G1/G3 Zipper activity at low galectin concentrations but not at high galectin concentrations. In contrast, CD7 was necessary for G1 and G3 signaling at low galectin concentration but antagonistic at high galectin concentrations. Collectively, these observations suggest that G1/G3 Zipper amplifies pro-apoptotic signaling through the integrated activity of both the G1 and G3 domains.

Locally anchoring enzymes to tissues via extracellular glycan recognition.

Success of enzymes as drugs requires that they persist within target tissues over therapeutically effective time frames. Here we report a general strategy to anchor enzymes at injection sites via fusion to galectin-3 (G3), a carbohydrate-binding protein. Fusing G3 to luciferase extended bioluminescence in subcutaneous tissue to ~7 days, whereas unmodified luciferase was undetectable within hours. Engineering G3-luciferase fusions to self-assemble into a trimeric architecture extended bioluminescence in subcutaneous tissue to 14 days, and intramuscularly to 3 days. The longer local half-life of the trimeric assembly was likely due to its higher carbohydrate-binding affinity compared to the monomeric fusion. G3 fusions and trimeric assemblies lacked extracellular signaling activity of wild-type G3 and did not accumulate in blood after subcutaneous injection, suggesting low potential for deleterious off-site effects. G3-mediated anchoring to common tissue glycans is expected to be broadly applicable for improving local pharmacokinetics of various existing and emerging enzyme drugs.

Engineering Reactive Oxygen Species-Resistant Galectin-1 Dimers with Enhanced Lectin Activity.

Galectin-1 is an immunomodulatory carbohydrate-binding protein with demonstrated efficacy in various preclinical models. However, its potential for clinical use is challenged by two features of the protein. First, galectin-1 (Gal-1) can be inactivated in oxidative environments, such as sites of inflammation, via covalent cross-linking of surface-exposed cysteine residues. Second, the active conformation of galectin-1 is a noncovalent homodimer with a micromolar dissociation constant. Together, these features necessitate frequent administration of high doses of galectin-1 for therapeutic efficacy. To address this challenge, we report an engineered dimeric variant of Gal-1 that is resistant to oxidative inactivation. Specifically, to prevent oxidative inactivation we mutated 3 of 4 surface cysteine residues to serine residues on Gal-1 ("Tri Gal-1"), and then cross-linked two Tri Gal-1 molecules with poly(ethylene glycol) diacrylate to create a stable homodimer ("Tri-PEG-Tri"). Our data demonstrate that cysteine-to-serine galectin-1 mutants retain the carbohydrate-binding properties and pro-apoptotic activity of wild-type Gal-1. Mutants lacking all surface cysteine residues are completely resistant to covalent cross-linking in oxidative environments. At sufficient polymer:protein ratios, poly(ethylene glycol) diacrylate reacts with the surface cysteine on two Tri Gal-1 molecules to form Tri-PEG-Tri. The effective dose of Tri-PEG-Tri is more than an order of magnitude lower than that of non-PEGylated Gal-1. Together, these data demonstrate reactive oxygen species (ROS)-resistant Tri-PEG-Tri dimers with enhanced lectin activity that may be broadly useful for improving the therapeutic efficacy of Gal-1 in immune modulation, transplant tolerance, and treatment of chronic inflammation.

Glycomaterials for immunomodulation, immunotherapy, and infection prophylaxis.

Synthetic materials that can engage the innate and adaptive immune systems are receiving increasing interest to confer protection against onset of future disease, such as pathogen infection, as well as to treat established diseases, such as autoimmunity and cancer. Carbohydrates are integral to various immune-related processes, including inflammation, adaptive memory, and tolerance, through both their non-covalent recognition of carbohydrate-binding proteins and as chemical signatures that distinguish self from non-self. Harnessing the biological activity of carbohydrates, however, was long hindered by the lack of a 'sugar code' defining their structure-function relationships, the difficulty of carbohydrate synthesis, and the weak binding affinity of monovalent carbohydrates for proteins or other biomolecules. The advent of new glycan synthesis approaches, combined with increased understanding of the role of multivalent carbohydrate clusters in immunology, has spurred significant recent growth in the development of multivalent synthetic materials modified with carbohydrates (i.e."glycomaterials") to activate, temper, or inhibit specific immunological processes. In this review, we highlight recent advances in glycomaterials that can inhibit T cell apoptosis, establish antigen-specific tolerance, suppress inflammation, or inhibit viral entry into host cells via non-covalent recognition of carbohydrate-binding proteins. In addition, we survey glycomaterials that can act as vaccines for adaptive immunological recognition and memory of carbohydrate antigens, with a particular emphasis on vaccines against tumor-associated carbohydrate antigens as cancer immunotherapies. In total, these examples demonstrate the enormous potential of glycomaterials to prevent or treat diverse diseases by engaging specific immunological processes.

Microgels with tunable affinity-controlled protein release via desolvation of self-assembled peptide nanofibers.

With a growing number of bioactive protein drugs approved for clinical use each year, there is increasing need for vehicles for localized protein delivery to reduce administered doses, prevent off-target activity, and maintain protein bioactivity. Ideal protein delivery vehicles provide high encapsulation efficiency of bioactive drug, enable fine-tuning of protein release profiles, are biocompatible, and can be administered via minimally-invasive routes. Here we developed an approach to create micron-sized hydrated gels (i.e."microgels") for protein delivery that fulfill these requirements via desolvation of self-assembled ß-sheet peptide nanofibers. Specifically, aqueous solutions of peptide nanofibers were diluted under stirring conditions in a "desolvating agent", such as ethanol, which is miscible with water but poorly solvates peptides. The desolvating agent induced nanofiber physical crosslinking into microgels that retained ß-sheet secondary structure and were stable in aqueous solutions. Microgels did not activate dendritic cells in vitro, suggesting they are biocompatible. Peptide nanofibers and proteins having similar non-solvent immiscibility properties were co-desolvated to produce protein-loaded microgels with loading efficiencies of ∼85%. Encapsulated bioactive proteins rapidly diffused into bulk aqueous media, as expected for hydrated gels. Modifying peptide nanofibers with a protein-binding ligand provided tunable affinity-controlled protein release. Biocompatible microgels formed via desolvation of self-assembled peptide nanofibers are therefore likely to be broadly useful as vehicles for localized delivery of bioactive proteins, as well as other therapeutic molecules.

T cell receptor signaling can directly enhance the avidity of CD28 ligand binding.

T cell activation takes place in the context of a spatial and kinetic reorganization of cell surface proteins and signaling molecules at the contact site with an antigen presenting cell, termed the immunological synapse. Coordination of the activation, recruitment, and signaling from T cell receptor (TCR) in conjunction with adhesion and costimulatory receptors regulates both the initiation and duration of signaling that is required for T cell activation. The costimulatory receptor, CD28, is an essential signaling molecule that determines the quality and quantity of T cell immune responses. Although the functional consequences of CD28 engagement are well described, the molecular mechanisms that regulate CD28 function are largely unknown. Using a micropipet adhesion frequency assay, we show that TCR signaling enhances the direct binding between CD28 and its ligand, CD80. Although CD28 is expressed as a homodimer, soluble recombinant CD28 can only bind ligand monovalently. Our data suggest that the increase in CD28-CD28 binding is mediated through a change in CD28 valency. Molecular dynamic simulations and in vitro mutagenesis indicate that mutations at the base of the CD28 homodimer interface, distal to the ligand-binding site, can induce a change in the orientation of the dimer that allows for bivalent ligand binding. When expressed in T cells, this mutation allows for high avidity CD28-CD80 interactions without TCR signaling. Molecular dynamic simulations also suggest that wild type CD28 can stably adopt a bivalent conformation. These results support a model whereby inside-out signaling from the TCR can enhance CD28 ligand interactions by inducing a change in the CD28 dimer interface to allow for bivalent ligand binding and ultimately the transduction of CD28 costimulatory signals that are required for T cell activation.