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.

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.

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.

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.

The butyrophilin 3A1 intracellular domain undergoes a conformational change involving the juxtamembrane region.

Small isoprenoid diphosphates, such as (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), are ligands of the internal domain of BTN3A1. Ligand binding in target cells promotes activation of Vγ9Vδ2 T cells. We demonstrate by small-angle X-ray scattering (SAXS) that HMBPP binding to the internal domain of BTN3A1 induces a conformational change in the position of the B30.2 domain relative to the juxtamembrane (JM) region. To better understand the molecular details of this conformational rearrangement, NMR spectroscopy was used to discover that the JM region interacts with HMBPP, specifically at the diphosphate. The spectral location of the affected amide peaks, partial NMR assignments, and JM mutants (ST296AA or T304A) investigated, confirm that the backbone amide of at least one Thr (Thr304), adjacent to conserved Ser, comes close to the HMBPP diphosphate, whereas double mutation of nonconserved residues (Ser/Thr296/297) may perturb the local fold. Cellular mutation of either of the identified Thr residues reduces the activation of Vγ9Vδ2 T cells by HMBPP, zoledronate, and POM2-C-HMBP, but not by a partial agonist BTN3 antibody. Taken together, our results show that ligand binding to BTN3A1 induces a conformational change within the intracellular domain that involves the JM region and is required for full activation.-Nguyen, K., Li, J., Puthenveetil, R., Lin, X., Poe, M. M., Hsiao, C.-H. C., Vinogradova, O., Wiemer, A. J. The butyrophilin 3A1 intracellular domain undergoes a conformational change involving the juxtamembrane region.

HMBPP Analog Prodrugs Bypass Energy-Dependent Uptake To Promote Efficient BTN3A1-Mediated Malignant Cell Lysis by Vγ9Vδ2 T Lymphocyte Effectors.

Vγ9Vδ2 effector T cells lyse cells in response to phosphorus-containing small molecules, providing primates a unique route to remove infected or malignant cells. Yet, the triggering mechanisms remain ill defined. We examined lysis mediated by human Vγ9Vδ2 effector T cells in response to the naturally occurring (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) or a synthetic cell-permeable prodrug, bis (pivaloyloxymethyl) (E)-4-hydroxy-3-methyl-but-2-enyl phosphonate. CD27(+)/CD45RA(-) Th1-like effector cells killed K562 target cells through a mechanism that could be enhanced by either compound or TCR Ab and blocked by Src inhibition or butyrophilin 3 isoform A1 (BTN3A1) disruption. Pretreatment at 4 °: C decreased HMBPP-induced lysis but did not reduce lysis induced by bis (pivaloyloxymethyl) (E)-4-hydroxy-3-methyl-but-2-enyl phosphonate. Together, our results show that internalization of HMBPP into target cells is required for BTN3A1-dependent lysis by Vγ9Vδ2 effector T cells. The enhanced activity of the prodrug analog is due to its ability to bypass the pathways required for entry of HMBPP. These findings support an inside-out model of T cell triggering driven by small-molecule induction of BTN3A1.

Molecular mechanisms linking geranylgeranyl diphosphate synthase to cell survival and proliferation.

Geranylgeranyl diphosphate is a 20-carbon isoprenoid phospholipid whose lipid moiety can be post-translationally incorporated into proteins to promote membrane association. The process of geranylgeranylation has been implicated in anti-proliferative effects of clinical agents that inhibit enzymes of the mevalonate pathway (i.e. statins and nitrogenous bisphosphonates) as well as experimental agents that deplete geranylgeranyl diphosphate. Inhibitors of geranylgeranyl diphosphate synthase are an attractive way to block geranylgeranylation because they possess a calcium-chelating substructure to allow localization to bone and take advantage of a unique position of the enzyme within the biosynthetic pathway. Here, we describe recent advances in geranylgeranyl diphosphate synthase expression and inhibitor development with a particular focus on the molecular mechanisms that link geranylgeranyl diphosphate to cell proliferation via geranylgeranylated small GTPases.

Evaluation of a 7-Methoxycoumarin-3-carboxylic Acid Ester Derivative as a Fluorescent, Cell-Cleavable, Phosphonate Protecting Group.

Cell-cleavable protecting groups often enhance cellular delivery of species that are charged at physiological pH. Although several phosphonate protecting groups have achieved clinical success, it remains difficult to use these prodrugs in live cells to clarify biological mechanisms. Here, we present a strategy that uses a 7-methoxycoumarin-3-carboxylic acid ester as a fluorescent protecting group. This strategy was applied to synthesis of an (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) analogue to assess cellular uptake and human Vγ9Vδ2 T cell activation. The fluorescent ester displayed low cellular toxicity (IC50 >100 µm) and strong T cell activation (EC50 =0.018 µm) relative to the unprotected anion (EC50 =23 µm). The coumarin-derived analogue allowed no-wash analysis of biological deprotection, which revealed rapid internalization of the prodrug. These results demonstrate that fluorescent groups can be applied both as functional drug delivery tools and useful biological probes of drug uptake.

Novel α-substituted tropolones promote potent and selective caspase-dependent leukemia cell apoptosis.

Tropolones, such as ß-thujaplicin, are small lead-like natural products that possess a variety of biological activities. While the ß-substituted natural products and their synthetic analogs are potent inhibitors of human cancer cell growth, less is known about their α-substituted counterparts. Recently, we synthesized a series of α-substituted tropolones including 2-hydroxy-7-(naphthalen-2-yl)cyclohepta-2,4,6-trien-1-one (α-naphthyl tropolone). Here, we evaluate the antiproliferative mechanisms of α-naphthyl tropolone and the related α-benzodioxinyl analog. The α-substituted tropolones inhibit growth of lymphocytic leukemia cells, but not healthy blood cells, with nanomolar potency. Treatment of leukemia cell lines with the tropolone dose-dependently induces apoptosis as judged by staining with annexin V and propidium iodide and Western blot analysis of cleaved caspase 3 and 7. Moreover, pre-treatment of cells with the caspase inhibitor Z-VAD-FMK inhibited the apoptotic effects of the tropolone in two lymphocytic lines. Caspase inhibition also blocked elevated histone acetylation caused by the tropolone, indicating that its effects on histone acetylation are potentiated by caspases. In contrast, α-naphthyl tropolone upregulated p53 expression and phosphorylation of Akt and mTOR in a manner that was not rescued by caspase inhibition. The effects of tropolone were blocked by co-incubation with high levels of free extracellular iron but not by pre-loading with iron. Additionally, dose and time dependent reduction in ex vivo viability of cells from leukemia patients was observed. Taken together, we demonstrate that α-substituted tropolones upregulate DNA damage repair pathways leading to caspase-dependent apoptosis in malignant lymphocytes.

Synthesis and biological evaluation of santacruzamate A analogues for anti-proliferative and immunomodulatory activity.

Santacruzamate A (SCA) is a natural product isolated from a Panamanian marine cyanobacterium, previously reported to have potent and selective histone deacetylase (HDAC) activity. To optimize the enzymatic and cellular activity, 40 SCA analogues were synthesized in a systematic exploration of the zinc-binding group (ZBG), cap terminus, and linker region. Two cap group analogues inhibited proliferation of MCF-7 breast cancer cells, with analogous increased degranulation of cytotoxic T cells (CTLs), while one cap group analogue reduced CTL degranulation, indicative of suppression of the immune response. Additional testing of these analogues resulted in reevaluation of the previously reported SCA mechanism of action. These analogues and the resulting structure-activity relationships will be of interest for future studies on cell proliferation and immune modulation.

Prodrugs of phosphonates and phosphates: crossing the membrane barrier.

A substantial portion of metabolism involves transformation of phosphate esters, including pathways leading to nucleotides and oligonucleotides, carbohydrates, isoprenoids and steroids, and phosphorylated proteins. Because the natural substrates bear one or more negative charges, drugs that target these enzymes generally must be charged as well, but small charged molecules can have difficulty traversing the cell membrane by means other than endocytosis. The resulting dichotomy has stimulated a great deal of effort to develop effective prodrugs, compounds that carry little or no charge to enable them to transit biological membranes, but able to release the parent drug once inside the target cell. This chapter presents recent studies on advances in prodrug forms, along with representative examples of their application to marketed and developmental drugs.

Cyclopropene cycloadditions with annulated furans: total synthesis of (+)- and (-)-frondosin B and (+)-frondosin A.

The asymmetric total syntheses of the natural products (+)- and (-)-frondosin B and (+)-frondosin A are reported based on a diastereoselective cycloaddition between tetrabromocyclopropene and an annulated furan to provide a highly functionalized common building block. The bridged bicyclic intermediate could be stereo- and chemoselectively manipulated to produce the two structurally distinct members of the frondosins. Both syntheses feature regioselective palladium-coupling reactions and an unprecedented phosphine-mediated ether bridge cleavage. Surprisingly, the planned enantioselective synthesis of frondosin B led to the opposite epimer of the natural product, suggesting an unusual late stage stereoinversion at C8. Frondosin A, but not frondosin B, was shown to have selective antiproliferative activity against several B-cell lines.

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. However, the long-term side effects that arise from taking chemotherapies can negatively impact the lives of patients because they lack specificity and impact healthy cells along with the cancer cells. Antibody-drug conjugates are a promising new class of drugs for the treatment of melanoma. This review focuses on the development of antibody-drug conjugates for melanoma and discusses the existing clinical trials of antibody-drug conjugates and their use as a melanoma treatment. So far, the antibody-drug conjugates have struggled from efficacy problems, with modest effects at best, leading many to be discontinued for melanoma. At the same time, conjugates such as AMT-253, targeting melanoma cell adhesion molecule, and mecbotamab vedotin  targeting AXL receptor tyrosine kinase, are among the most exciting for melanoma treatment in the future.

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. These compounds displayed low nanomolar to high picomolar potency. Addition of a p-isopropyl group to the phenyl substituent and use of cyclohexyl or p-methoxybenzyl groups as the acyloxy substituent significantly increased human, but not mouse or rat, plasma stability without negatively impacting potency. Combinations of these prodrug moieties further improved stability, with the best combination achieving a half-life of over 12 h in human plasma, a marked improvement on prior compounds. In contrast, oxane analogs improved water solubility and cellular payload delivery but remained unstable in human plasma. The studies suggest that certain ester-based phosphonate prodrugs quickly deliver active payloads inside cells and show substantial stability in human plasma.

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. Furthermore, they can compete with HMBPP for cellular binding to BTN3A1 and reduce the cellular response to HMBPP, a classic partial agonist phenotype. Trifluoromethyl analog 6e was the weakest agonist but the strongest inhibitor of HMBPP ELISA response. Our study provides a rationale for the mode of action of pAg-induced γδ T cell activation and provides insights into other naturally occurring BTN proteins and their respective ligands.

A live imaging cell motility screen identifies prostaglandin E2 as a T cell stop signal antagonist.

The T cell migration stop signal is a central step in T cell activation and inflammation; however, its regulatory mechanisms remain largely unknown. Using a live-cell, imaging-based, high-throughput screen, we identified the PG, PGE(2), as a T cell stop signal antagonist. Src kinase inhibitors, microtubule inhibitors, and PGE(2) prevented the T cell stop signal, and impaired T cell-APC conjugation and T cell proliferation induced by primary human allogeneic dendritic cells. However, Src inhibition, but not PGE(2) or microtubule inhibition, impaired TCR-induced ZAP-70 signaling, demonstrating that T cell stop signal antagonists can function either upstream or downstream of proximal TCR signaling. Moreover, we found that PGE(2) abrogated TCR-induced activation of the small GTPase Rap1, suggesting that PGE(2) may modulate T cell adhesion and stopping through Rap1. These results identify a novel role for PGs in preventing T cell stop signals and limiting T cell activation induced by dendritic cells.

Contact-dependent T cell activation and T cell stopping require talin1.

T cell-APC contact initiates T cell activation and is maintained by the integrin LFA-1. Talin1, an LFA-1 regulator, localizes to the immune synapse (IS) with unknown roles in T cell activation. In this study, we show that talin1-deficient T cells have defects in contact-dependent T cell stopping and proliferation. Although talin1-deficient T cells did not form stable interactions with APCs, transient contacts were sufficient to induce signaling. In contrast to prior models, LFA-1 polarized to T cell-APC contacts in talin1-deficient T cells, but vinculin and F-actin polarization at the IS was impaired. These results indicate that T cell proliferation requires sustained, talin1-mediated T cell-APC interactions and that talin1 is necessary for F-actin polarization and the stability of the IS.

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.

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.