A distinct topology of BTN3A IgV and B30.2 domains controlled by juxtamembrane regions favors optimal human γδ T cell phosphoantigen sensing.

Butyrophilin (BTN)-3A and BTN2A1 molecules control the activation of human Vγ9Vδ2 T cells during T cell receptor (TCR)-mediated sensing of phosphoantigens (PAg) derived from microbes and tumors. However, the molecular rules governing PAg sensing remain largely unknown. Here, we establish three mechanistic principles of PAg-mediated γδ T cell activation. First, in humans, following PAg binding to the intracellular BTN3A1-B30.2 domain, Vγ9Vδ2 TCR triggering involves the extracellular V-domain of BTN3A2/BTN3A3. Moreover, the localization of both protein domains on different chains of the BTN3A homo-or heteromers is essential for efficient PAg-mediated activation. Second, the formation of BTN3A homo-or heteromers, which differ in intracellular trafficking and conformation, is controlled by molecular interactions between the juxtamembrane regions of the BTN3A chains. Finally, the ability of PAg not simply to bind BTN3A-B30.2, but to promote its subsequent interaction with the BTN2A1-B30.2 domain, is essential for T-cell activation. Defining these determinants of cooperation and the division of labor in BTN proteins improves our understanding of PAg sensing and elucidates a mode of action that may apply to other BTN family members.

Diester Prodrugs of a Phosphonate Butyrophilin Ligand Display Improved Cell Potency, Plasma Stability, and Payload Internalization.

Activation of Vγ9Vδ2 T cells with butyrophilin 3A1 (BTN3A1) agonists such as (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) has the potential to boost the immune response. Because HMBPP is highly charged and metabolically unstable, prodrugs may be needed to overcome these liabilities, but the prodrugs themselves may be limited by slow payload release or low plasma stability. To identify effective prodrug forms of a phosphonate agonist of BTN3A1, we have prepared a set of diesters bearing one aryl and one acyloxymethyl group. The compounds were evaluated for their ability to stimulate Vγ9Vδ2 T cell proliferation, increase production of interferon γ, resist plasma metabolism, and internalize into leukemia cells. These bioassays have revealed that varied aryl and acyloxymethyl groups can decouple plasma and cellular metabolism and have a significant impact on bioactivity (>200-fold range) and stability (>10 fold range), including some with subnanomolar potency. Our findings increase the understanding of the structure-activity relationships of mixed aryl/acyloxymethyl phosphonate prodrugs.

Mutations to the BTN2A1 Linker Region Impact Its Homodimerization and Its Cytoplasmic Interaction with Phospho-Antigen-Bound BTN3A1.

Intracellular binding of small-molecule phospho-Ags to the HMBPP receptor complex in infected cells leads to extracellular detection by T cells expressing the Vγ9Vδ2 TCR, a noncanonical method of Ag detection. The butyrophilin proteins BTN2A1 and BTN3A1 are part of the complex; however, their precise roles are unclear. We suspected that BTN2A1 and BTN3A1 form a tetrameric (dimer of dimers) structure, and we wanted to probe the importance of the BTN2A1 homodimer. We analyzed mutations to human BTN2A1, using internal domain or full-length BTN2A1 constructs, expressed in Escherichia coli or human K562 cells, that might disrupt its structure and/or function. Although BTN2A1 is a disulfide-linked homodimer, mutation of cysteine residues C247 and C265 did not affect the ability to stimulate T cell IFN-γ production by ELISA. Two mutations of the juxtamembrane region (at EKE282) failed to impact BTN2A1 function. In contrast, single point mutations (L318G and L325G) near the BTN2A1 B30.2 domain blocked phospho-Ag response. Size exclusion chromatography and nuclear magnetic resonance (NMR) experiments showed that the isolated BTN2A1 B30.2 domain is a homodimer, even in the absence of its extracellular and transmembrane region. [31P]-NMR experiments confirmed that HMBPP binds to BTN3A1 but not BTN2A1, and binding abrogates signals from both phosphorus atoms. Furthermore, the BTN2A1 L325G mutation but not the L318G mutation prevents both homodimerization of BTN2A1 internal domain constructs in size exclusion chromatography (and NMR) experiments and their binding to HMBPP-bound BTN3A1 in isothermal titration calorimetry experiments. Together, these findings support the importance of homodimerization within the BTN2A1 internal domain for phospho-Ag detection.

Division of labor and cooperation between different butyrophilin proteins controls phosphoantigen-mediated activation of human γδ T cells.

Butyrophilin (BTN)-3A and BTN2A1 molecules control TCR-mediated activation of human Vγ9Vδ2 T-cells triggered by phosphoantigens (PAg) from microbes and tumors, but the molecular rules governing antigen sensing are unknown. Here we establish three mechanistic principles of PAg-action. Firstly, in humans, following PAg binding to the BTN3A1-B30.2 domain, Vγ9Vδ2 TCR triggering involves the V-domain of BTN3A2/BTN3A3. Moreover, PAg/B30.2 interaction, and the critical γδ-T-cell-activating V-domain, localize to different molecules. Secondly, this distinct topology as well as intracellular trafficking and conformation of BTN3A heteromers or ancestral-like BTN3A homomers are controlled by molecular interactions of the BTN3 juxtamembrane region. Finally, the ability of PAg not simply to bind BTN3A-B30.2, but to promote its subsequent interaction with the BTN2A1-B30.2 domain, is essential for T-cell activation. Defining these determinants of cooperation and division of labor in BTN proteins deepens understanding of PAg sensing and elucidates a mode of action potentially applicable to other BTN/BTNL family members.

Generation of effector Vγ9Vδ2 T cells and evaluation of their response to phosphoantigen-loaded cells.

Vγ9Vδ2 T cells are non-canonical T cells that use their T cell receptor to detect phosphoantigens bound to the internal domain of the HMBPP receptor (butyrophilin 3/2A1 complex). This protocol describes the expansion and purification of human effector Vγ9Vδ2 T cells from human buffy coat and describes how to assess their activation by antigen-containing target cells. While specifically focused on cytokine production, this protocol can be readily adapted to evaluate other effector functions of activated Vγ9Vδ2 T cells. For complete details on the use and execution of this protocol, please refer to Hsiao et al. (2022) and Hsiao and Wiemer (2018).

New Technologies Bloom Together for Bettering Cancer Drug Conjugates.

Drug conjugates, including antibody-drug conjugates, are a step toward realizing Paul Ehrlich's idea from over 100 years ago of a "magic bullet" for cancer treatment. Through balancing selective targeting molecules with highly potent payloads, drug conjugates can target specific tumor microenvironments and kill tumor cells. A drug conjugate consists of three parts: a targeting agent, a linker, and a payload. In some conjugates, monoclonal antibodies act as the targeting agent, but new strategies for targeting include antibody derivatives, peptides, and even small molecules. Linkers are responsible for connecting the payload to the targeting agent. Payloads impact vital cellular processes to kill tumor cells. At present, there are 12 antibody-drug conjugates on the market for different types of cancers. Research on drug conjugates is increasing year by year to solve problems encountered in conjugate design, such as tumor heterogeneity, poor circulation, low drug loading, low tumor uptake, and heterogenous expression of target antigens. This review highlights some important preclinical research on drug conjugates in recent years. We focus on three significant areas: improvement of antibody-drug conjugates, identification of new conjugate targets, and development of new types of drug conjugates, including nanotechnology. We close by highlighting the critical barriers to clinical translation and the open questions going forward. SIGNIFICANCE STATEMENT: The development of anticancer drug conjugates is now focused in three broad areas: improvements to existing antibody drug conjugates, identification of new targets, and development of new conjugate forms. This article focuses on the exciting preclinical studies in these three areas and advances in the technology that improves preclinical development.

Efficiency of bis-amidate phosphonate prodrugs.

Bis-amidate derivatives have been viewed as attractive phosphonate prodrug forms because of their straightforward synthesis, lack of phosphorus stereochemistry, plasma stability and nontoxic amino acid metabolites. However, the efficiency of bis-amidate prodrug forms is unclear, as prior studies on this class of prodrugs have not evaluated their activation kinetics. Here, we synthetized a small panel of bis-amidate prodrugs of butyrophilin ligands as potential immunotherapy agents. These compounds were examined relative to other prodrug forms delivering the same payload for their stability in plasma and cell lysate, their ability to stimulate T cell proliferation in human PBMCs, and their activation kinetics in a leukemia co-culture model of T cell cytokine production. The bis-amidate prodrugs demonstrate high plasma stability and improved cellular phosphoantigen activity relative to the free phosphonic acid. However, the efficiency of bis-amidate activation is low relative to other prodrugs that contain at least one ester such as aryl-amidate, aryl-acyloxyalkyl ester, and bis-acyloxyalkyl ester forms. Therefore, bis-amidate prodrugs do not drive rapid cellular payload accumulation and they would be more useful for payloads in which slower, sustained-release kinetics are preferred.

Synthesis and Metabolism of BTN3A1 Ligands: Studies on Diene Modifications to the Phosphoantigen Scaffold.

Phosphoantigens (pAgs) are small organophosphorus compounds such as (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) that trigger an immune response. These molecules bind to butyrophilin 3A1 (part of the HMBPP receptor) and activate Vγ9Vδ2 T cells. To explore the structure-activity relationships underlying this process, we evaluated a series of novel diene analogs of HMBPP. Here we report that prodrug forms of [(1E)-4-methylpenta-1,3-dien-1-yl] phosphonic acid that lack the allylic alcohol of HMBPP but instead contained a diene scaffold exhibit mid-nanomolar potency for the activation of Vγ9Vδ2 T cells. The compounds also trigger the production of T-cell interferon γ upon exposure to loaded K562 cells. Although both the allylic alcohol and the diene scaffold boost pAg activity, the combination of the two decreases the activity and results in glutathione conjugation. Together, these data show that the diene scaffold results in intermediate pAgs that may have implications for the mechanisms regulating the HMBPP receptor.

Ligand-induced interactions between butyrophilin 2A1 and 3A1 internal domains in the HMBPP receptor complex.

The ligand-bound (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) receptor (BTN3A1 and BTN2A1) is detectable by the T cell receptor (TCR) of Vγ9Vδ2 T cells. Although BTN3A1 binds to phosphoantigens (pAgs), the mechanisms resulting in receptor activation are not clear. We used CRISPR-Cas9, ELISA, nano-bioluminescence resonance energy transfer (BRET), and isothermal titration calorimetry (ITC) to evaluate the role of BTN2A1. Depletion of BTN2A1 and rescue experiments demonstrate that its internal domain is essential for pAg detection. Internal hetero-BRET signals are observed between BTN2A1 and BTN3A1 that are increased by pAg. ITC detects a direct interaction between the intracellular domains of BTN3A1 and BTN2A1 only in the presence of pAg. This interaction is abrogated by removal of the BTN2A1 juxtamembrane (JM) region but not by removal of the BTN3A1 JM region. Regional mutations between BTN2A1 316-326 clearly affect the interferon γ (IFNγ) response and hetero-BRET signal. Mutations to amino acids L318, W320, and L325 indicate that these amino acids are crucial. This study demonstrates a pAg-inducible interaction between BTN2A1 and BTN3A1 internal domains.

Stepping forward in antibody-drug conjugate development.

Antibody-drug conjugates (ADCs) are cancer therapeutic agents comprised of an antibody, a linker and a small-molecule payload. ADCs use the specificity of the antibody to target the toxic payload to tumor cells. After intravenous administration, ADCs enter circulation, distribute to tumor tissues and bind to the tumor surface antigen. The antigen then undergoes endocytosis to internalize the ADC into tumor cells, where it is transported to lysosomes to release the payload. The released toxic payloads can induce apoptosis through DNA damage or microtubule inhibition and can kill surrounding cancer cells through the bystander effect. The first ADC drug was approved by the United States Food and Drug Administration (FDA) in 2000, but the following decade saw no new approved ADC drugs. From 2011 to 2018, four ADC drugs were approved, while in 2019 and 2020 five more ADCs entered the market. This demonstrates an increasing trend for the clinical development of ADCs. This review summarizes the recent clinical research, with a specific focus on how the in vivo processing of ADCs influences their design. We aim to provide comprehensive information about current ADCs to facilitate future development.

Generation of Single-Chain Variable Fragment (scFv) Libraries for Use in Phage Display.

Phage display is a powerful platform for the discovery of novel biologics with high binding affinities to a specific target protein. Here, we describe methods to construct a phage display library containing diverse single-chain variable antibody fragments (scFvs). Specifically, updated methods for polymerase chain reaction (PCR) amplification and fusion of human antibody genes, their ligation into the pComb3X vector for transformation into 5αF'Iq competent bacterial cells, and their expression in M13KO7 helper phage are presented. Additionally, we describe how to amplify and quantify the phage library and to prepare it in various formats for short- and long-term storage. © 2021 Wiley Periodicals LLC. Basic Protocol 1: First-round polymerase chain reaction (PCR) for isolation of antibody fragments Basic Protocol 2: Ethanol precipitation and pooling of fragment DNA Basic Protocol 3: Second-round polymerase chain reaction with splicing by overlap extension (SOE) for antibody fragment fusion Basic Protocol 4: Restriction digestion of individual scFv constructs and pComb3XSS vector Basic Protocol 5: Directional ligation of the scFv constructs and pComb3X backbone Basic Protocol 6: Transformation of pComb-scFv plasmids into 5αF'Iq competent cells Basic Protocol 7: Collection of bacteria containing the scFv library Basic Protocol 8: Preparation of bacterial glycerol stock Basic Protocol 9: Preparation of phage library glycerol stock Basic Protocol 10: Preparation of plasmid DNA stock Basic Protocol 11: Amplification of M13KO7 helper phage Basic Protocol 12: Phage titer by plate assay Alternate Protocol: One-plate phage plaque assay.

Incorporation of a FRET pair within a phosphonate diester.

Cell-cleavable protecting groups are an effective tactic for construction of biological probes because such compounds can improve problems with instability, solubility, and cellular uptake. Incorporation of fluorescent groups in the protecting groups may afford useful probes of cellular functions, especially for payloads containing phosphonates that would be highly charged if not protected, but little is known about the steric or electronic factors that impede release of the payload. In this report we present a strategy for the synthesis of a coumarin fluorophore and a 4-((4-(dimethylamino)phenyl)diazenyl)benzoic acid (DABCYL) ester chromophore incorporated as a FRET pair within a single phosphonate. Such compounds were designed to deliver a BTN3A1 ligand payload to its intracellular receptor. Both final products and some synthetic intermediates were evaluated for their ability to undergo metabolic activation in γδ T cell functional assays, and for their photophysical properties by spectrophotometry. One phosphonate bearing a DABCYL acyloxyester and a novel tyramine-linked coumarin fluorophore exhibited strong, rapid, and potent cellular activity for γδ T cell stimulation and also showed FRET interactions. This strategy demonstrates that bioactivatable phosphonates containing FRET pairs can be utilized to develop probes to monitor cellular uptake of otherwise charged payloads.

Synthesis and Metabolism of BTN3A1 Ligands: Studies on Modifications of the Allylic Alcohol.

(E)-4-Hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) and its phosphonate analogs are potent phosphoantigens. HMBPP contains an (E)-allylic alcohol which interacts with the molecular target BTN3A1 giving an antigenic signal to activate Vγ9Vδ2 T cells. As probes of BTN3A1 function, we prepared prodrug derivatives of the HMBPP analog C-HMBP that lack the (E)-allylic alcohol or have modified it to an aldehyde or aldoxime and evaluated their biological activity. Removal of the alcohol completely abrogates phosphoantigenicity in these compounds while the aldoxime modification decreases potency relative to the (E)-allylic alcohol form. However, homoprenyl derivatives oxidized to an aldehyde stimulate Vγ9Vδ2 T cells at nanomolar concentrations. Selection of phosphonate protecting groups (i.e., prodrug forms) impacts the potency of phosphoantigen aldehydes, with mixed aryl acyloxyalkyl forms exhibiting superior activity relative to aryl amidate forms. The activity correlates with the cellular reduction of the aldehyde to the alcohol form. Thus, the functionality on this ligand framework can be altered concurrently with phosphonate protection to promote cellular transformation to highly potent phosphoantigens.

Potent double prodrug forms of synthetic phosphoantigens.

Phosphoantigens are ligands of BTN3A1 that stimulate anti-cancer functions of γδ T cells, yet the potency of natural phosphoantigens is limited by low cell permeability and low metabolic stability. Derivatives of BTN3A1 ligand prodrugs were synthesized that contain an acetate-protected allylic alcohol and act as doubly protected prodrugs. A novel set of phosphonates, phosphoramidates, and phosphonamidates has been prepared through a new route that simplifies synthesis and postpones the point of divergence into different prodrug forms. One of the new prodrugs, compound 11, potently stimulates γδ T cell proliferation (72 h EC50 = 0.12 nM) and interferon γ response to loaded leukemia cells (4 h EC50 = 19 nM). This phosphonamidate form was > 900x more potent than the corresponding phosphoramidate, and the phosphonamidate form was also significantly more stable in plasma following acetate hydrolysis. Therefore, prodrug modification of phosphonate butyrophilin ligands at the allylic alcohol can both facilitate chemical synthesis and improve potency of γδ T cell stimulation.

Metabolic Efficacy of Phosphate Prodrugs and the Remdesivir Paradigm.

Drugs that contain phosphates (and phosphonates or phosphinates) have intrinsic absorption issues and are therefore often delivered in prodrug forms to promote their uptake. Effective prodrug forms distribute their payload to the site of the intended target and release it efficiently with minimal byproduct toxicity. The ability to balance unwanted payload release during transit with desired release at the site of action is critical to prodrug efficacy. Despite decades of research on prodrug forms, choosing the ideal prodrug form remains a challenge which is often solved empirically. The recent emergency use authorization of the antiviral remdesivir for COVID-19 exemplifies a new approach for delivery of phosphate prodrugs by parenteral dosing, which minimizes payload release during transit and maximizes tissue payload distribution. This review focuses on the role of metabolic activation in efficacy during oral and parenteral dosing of phosphate, phosphonate, and phosphinate prodrugs. Through examining prior structure-activity studies on prodrug forms and the choices that led to development of remdesivir and other clinical drugs and drug candidates, a better understanding of their ability to distribute to the planned site of action, such as the liver, plasma, PBMCs, or peripheral tissues, can be gained. The structure-activity relationships described here will facilitate the rational design of future prodrugs.

Structure-Activity Relationships of Butyrophilin 3 Ligands.

Phosphoantigens (pAgs) are small phosphorus-containing molecules that stimulate Vγ9Vδ2 T cells with sub-nanomolar cellular potency. Recent work has revealed that these compounds work through binding to the transmembrane immunoglobulin butyrophilin 3A1 (BTN3A1) within its intracellular B30.2 domain. Engagement of BTN3A1 is critical to the formation of an immune synapse between cells that contain pAgs and the Vγ9Vδ2 T cells. This minireview summarizes the structure-activity relationships of pAgs and their implications to the mechanisms of butyrophilin 3 activation leading to Vγ9Vδ2 T cell response.

Regulation of the Notch-ATM-abl axis by geranylgeranyl diphosphate synthase inhibition.

Notch proteins drive oncogenesis of many cancers, most prominently T-cell acute lymphoblastic leukemia (T-ALL). Because geranylgeranylated Rab proteins regulate Notch processing, we hypothesized that inhibition of geranylgeranyl diphosphate synthase (GGDPS) would impair Notch processing and reduce viability of T-ALL cells that express Notch. Here, we show that GGDPS inhibition reduces Notch1 expression and impairs the proliferation of T-ALL cells. GGDPS inhibition also reduces Rab7 membrane association and depletes Notch1 mRNA. GGDPS inhibition increases phosphorylation of histone H2A.X, and inhibitors of ataxia telangiectasia-mutated kinase (ATM) mitigate GGDPS inhibitor-induced apoptosis. GGDPS inhibition also influences c-abl activity downstream of caspases, and inhibitors of these enzymes prevent GGDPS inhibitor-induced apoptosis. Surprisingly, induction of apoptosis by GGDPS inhibition is reduced by co-treatment with γ-secretase inhibitors. While inhibitors of γ-secretase deplete one specific form of the Notch1 intracellular domain (NICD), they also increase Notch1 mRNA expression and increase alternate forms of Notch1 protein expression in cells treated with a GGDPS inhibitor. Furthermore, inhibitors of γ-secretase and ATM increase Notch1 mRNA stability independent of GGDPS inhibition. These results provide a model by which T-ALL cells use Notch1 to avoid DNA-damage-induced apoptosis, and can be overcome by inhibition of GGDPS through effects on Notch1 expression and its subsequent response.

Toward Broad Spectrum Dihydrofolate Reductase Inhibitors Targeting Trimethoprim Resistant Enzymes Identified in Clinical Isolates of Methicillin Resistant Staphylococcus aureus.

The spread of plasmid borne resistance enzymes in clinical Staphylococcus aureus isolates is rendering trimethoprim and iclaprim, both inhibitors of dihydrofolate reductase (DHFR), ineffective. Continued exploitation of these targets will require compounds that can broadly inhibit these resistance-conferring isoforms. Using a structure-based approach, we have developed a novel class of ionized nonclassical antifolates (INCAs) that capture the molecular interactions that have been exclusive to classical antifolates. These modifications allow for a greatly expanded spectrum of activity across these pathogenic DHFR isoforms, while maintaining the ability to penetrate the bacterial cell wall. Using biochemical, structural, and computational methods, we are able to optimize these inhibitors to the conserved active sites of the endogenous and trimethoprim resistant DHFR enzymes. Here, we report a series of INCA compounds that exhibit low nanomolar enzymatic activity and potent cellular activity with human selectivity against a panel of clinically relevant TMP resistant (TMPR) and methicillin resistant Staphylococcus aureus (MRSA) isolates.

A luciferase lysis assay reveals in vivo malignant cell sensitization by phosphoantigen prodrugs.

Human Vγ9Vδ2 T cells respond to small phosphorus-containing compounds, often called phosphoantigens, which are now known to be intracellular ligands of the immune receptor butyrophilin 3A1 (BTN3A1). In order to compare the efficiency of butyrophilin ligands, we developed a luciferase-based lysis assay that measures the direct cytolysis by Vγ9Vδ2 T cells of luciferase-expressing K562 leukemia cells sensitized by phosphoantigen prodrugs. Our results show that the luciferase-based lysis assay allows in vitro and in vivo assessment of phosphoantigen activity in a way that does not require the extensive processing of flow cytometry or ELISA based approaches. In cellular assays, the structure activity relationships of phosphoantigen prodrugs correlate with ELISA-based activation assays, though phosphoantigen induced target cell lysis occurs at lower concentrations relative to T cell interferon γ production measured by ELISA. In mice dosed with phosphoantigens, a racemic aryl phosphonamidate prodrug, methyl 2-[[[(E)-5-hydroxy-4-methyl-pent-3-enyl]-(1-naphthyloxy)phosphoryl]amino]acetate (1-Nap/GlyOMe C-HMBP, 5), sensitized subcutaneous K562 tumors within minutes, and this effect was maintained at least four hours after treatment. In vivo activity of compound 5 was stronger than that of an equivalent dose of zoledronate. This luciferase lysis assay can be used for evaluation of phosphoantigens due to its time efficiency, high sensitivity, and in vivo compatibility and demonstrates rapid in vitro and in vivo sensitization of tumor cells by phosphoantigen prodrugs.

Synthesis and Bioactivity of the Alanyl Phosphonamidate Stereoisomers Derived from a Butyrophilin Ligand.

Aryloxy phosphonamidate derivatives of a butyrophilin 3A1 ligand are stimulants of Vγ9 Vδ2 T cells. However, when bonded to an aryl ester and an amine, the phosphorus is stereogenic, and past compounds were studied as racemates. To determine the impact of stereochemistry on the activity, we now have prepared phosphonate derivatives of l- and d-alanine ethyl ester, separated the diastereomers, and evaluated their biological activity as single stereoisomers. The results demonstrate that phosphonamidates substituted with l-alanine stimulate Vγ9 Vδ2 T cells at lower concentrations than the racemic glycine counterpart, while those derived from d-alanine require higher concentrations. All four diastereomers are more active than charged phosphoantigens such as HMBPP. Surprisingly, only a 2-fold difference was observed between the l-alanine phosphorus isomers, with the R P isomer more potent. This suggests that the small phosphoantigen scaffold reduces but does not eliminate dependence upon phosphorus stereochemistry for cellular activity.