Enhanced Plasma Stability and Potency of Aryl/Acyloxy Prodrugs of a BTN3A1 Ligand.

While ester-based phosphonate prodrugs excel at delivering payloads into cells, their instability in plasma is a hurdle for their advancement. Here, we synthesized new aryl/acyloxy prodrugs of a phosphonate BTN3A1 ligand. We evaluated their phosphoantigen potency by flow cytometry and ELISA and their plasma and cellular metabolism by LC-MS.

Synthesis and evaluation of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate analogs as competitive partial agonists of butyrophilin 3A1.

Phosphoantigens (pAgs) induce conformational changes after binding to the intracellular region of BTN3A1 which result in its clustering with BTN2A1, forming an activating ligand for the Vγ9Vδ2 T cell receptor. Here, we designed a small panel of bulky analogs of the prototypical pAg (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) that contain an aromatic ring attached to the C-3 position in place of methyl group. These compounds bind with high affinity to BTN3A1 but fail to fully support its interaction with BTN2A1 and only partially trigger T cell activation relative to HMBPP.

Antibody-Drug Conjugates in the Pipeline for Treatment of Melanoma: Target and Pharmacokinetic Considerations.

Melanoma is an aggressive, rapidly developing form of skin cancer that affects about 22 per 100,000 individuals. Treatment options for melanoma patients are limited and typically involve surgical excision of moles and chemotherapy. Survival has been improved in recent years through targeted small molecule inhibitors and antibody-based immunotherapies.

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.

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 ()-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.

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.

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.

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.

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.

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.

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

Phosphoantigens (pAgs) are small organophosphorus compounds such as ()-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.

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.

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'I competent bacterial cells, and their expression in M13KO7 helper phage are presented.

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.

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.

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

()-4-Hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) and its phosphonate analogs are potent phosphoantigens. HMBPP contains an ()-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 ()-allylic alcohol or have modified it to an aldehyde or aldoxime and evaluated their biological activity.

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.

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.

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.

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.

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.

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

The spread of plasmid borne resistance enzymes in clinical 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.

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.

Probing the Ligand-Binding Pocket of BTN3A1.

Small-molecule phosphoantigens such as ()-4-hydroxy-3-methyl-but-2-enyl diphosphate stimulate human Vγ9Vδ2 T cells after binding to the intracellular B30.2 domain of the immune receptor butyrophilin 3 isoform A1 (BTN3A1). To understand the ligand-target interaction in greater detail, we performed molecular docking.

Stability and Efficiency of Mixed Aryl Phosphonate Prodrugs.

A set of phosphonate prodrugs of a butyrophilin ligand was synthesized and evaluated for plasma stability and cellular activity. The mixed aryl acyloxy esters were prepared either via a standard sequence through the phosphonic acid chloride, or through the more recently reported, and more facile, triflate activation. In the best of cases, this class of prodrugs shows cellular potency similar to that of bis-acyloxyalkyl phosphonate prodrugs and plasma stability similar to that of aryl phosphonamidates.