Design and Evaluation of Phosphonamidate-Linked Exatecan Constructs for Highly Loaded, Stable, and Efficacious Antibody-Drug Conjugates.

Topoisomerase I (TOP1) Inhibitors constitute an emerging payload class to engineer antibody-drug conjugates (ADC) as next-generation biopharmaceutical for cancer treatment. Existing ADCs are using camptothecin payloads with lower potency and suffer from limited stability in circulation. With this study, we introduce a novel camptothecin-based linker-payload platform based on the highly potent camptothecin derivative exatecan. First, we describe general challenges that arise from the hydrophobic combination of exatecan and established dipeptidyl p-aminobenzyl-carbamate (PAB) cleavage sites such as reduced antibody conjugation yields and ADC aggregation. After evaluating several linker-payload structures, we identified ethynyl-phosphonamidates in combination with a discrete PEG24 chain to compensate for the hydrophobic PAB-exatecan moiety. Furthermore, we demonstrate that the identified linker-payload structure enables the construction of highly loaded DAR8 ADCs with excellent solubility properties. Head-to-head comparison with Enhertu, an approved camptothecin-based ADC, revealed improved target-mediated killing of tumor cells, excellent bystander killing, drastically improved linker stability in vitro and in vivo and superior in vivo efficacy over four tested dose levels in a xenograft model. Moreover, we show that ADCs based on the novel exatecan linker-payload platform exhibit antibody-like pharmacokinetic properties, even when the ADCs are highly loaded with eight drug molecules per antibody. This ADC platform constitutes a new and general solution to deliver TOP1 inhibitors with highest efficiency to the site of the tumor, independent of the antibody and its target, and is thereby broadly applicable to various cancer indications.

Biomolecular condensate drives polymerization and bundling of the bacterial tubulin FtsZ to regulate cell division.

Cell division is spatiotemporally precisely regulated, but the underlying mechanisms are incompletely understood. In the social bacterium Myxococcus xanthus, the PomX/PomY/PomZ proteins form a single megadalton-sized complex that directly positions and stimulates cytokinetic ring formation by the tubulin homolog FtsZ. Here, we study the structure and mechanism of this complex in vitro and in vivo. We demonstrate that PomY forms liquid-like biomolecular condensates by phase separation, while PomX self-assembles into filaments generating a single large cellular structure. The PomX structure enriches PomY, thereby guaranteeing the formation of precisely one PomY condensate per cell through surface-assisted condensation. In vitro, PomY condensates selectively enrich FtsZ and nucleate GTP-dependent FtsZ polymerization and bundle FtsZ filaments, suggesting a cell division site positioning mechanism in which the single PomY condensate enriches FtsZ to guide FtsZ-ring formation and division. This mechanism shares features with microtubule nucleation by biomolecular condensates in eukaryotes, supporting this mechanism's ancient origin.

Compact hydrophilic electrophiles enable highly efficacious high DAR ADCs with excellent in vivo PK profile.

The recent success of antibody-drug conjugates (ADC), exemplified by seven new FDA-approvals within three years, has led to increased attention for antibody based targeted therapeutics and fueled efforts to develop new drug-linker technologies for improved next generation ADCs. We present a highly efficient phosphonamidate-based conjugation handle that combines a discrete hydrophilic PEG-substituent, an established linker-payload and a cysteine-selective electrophile in one compact building block. This reactive entity provides homogeneous ADCs with a high drug-to-antibody ratio (DAR) of 8 in a one-pot reduction and alkylation protocol from non-engineered antibodies. The compact branched PEG-architecture introduces hydrophilicity without increasing the distance between antibody and payload, allowing the generation of the first homogeneous DAR 8 ADC from VC-PAB-MMAE without increased in vivo clearance rates. This high DAR ADC exhibits excellent in vivo stability and increased antitumor activity in tumour xenograft models relative to the established FDA approved VC-PAB-MMAE ADC Adcetris, clearly showing the benefit of the phosphonamidate based building-blocks as a general tool for the efficient and stable antibody-based delivery of highly hydrophobic linker-payload systems.

Targeting FLT3 with a new-generation antibody-drug conjugate in combination with kinase inhibitors for treatment of AML.

Fms-like tyrosine kinase 3 (FLT3) is often overexpressed or constitutively activated by internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations in acute myeloid leukemia (AML). Despite the use of receptor tyrosine kinase inhibitors (TKI) in FLT3-ITD-positive AML, the prognosis of patients is still poor, and further improvement of therapy is required. Targeting FLT3 independent of mutations by antibody-drug conjugates (ADCs) is a promising strategy for AML therapy. Here, we report the development and preclinical characterization of a novel FLT3-targeting ADC, 20D9-ADC, which was generated by applying the innovative P5 conjugation technology. In vitro, 20D9-ADC mediated potent cytotoxicity to Ba/F3 cells expressing transgenic FLT3 or FLT3-ITD, to AML cell lines, and to FLT3-ITD-positive patient-derived xenograft AML cells. In vivo, 20D9-ADC treatment led to a significant tumor reduction and even durable complete remission in AML xenograft models. Furthermore, 20D9-ADC demonstrated no severe hematotoxicity in in vitro colony formation assays using concentrations that were cytotoxic in AML cell line treatment. The combination of 20D9-ADC with the TKI midostaurin showed strong synergy in vitro and in vivo, leading to reduction of aggressive AML cells below the detection limit. Our data indicate that targeting FLT3 with an advanced new-generation ADC is a promising and potent antileukemic strategy, especially when combined with FLT3-TKI in FLT3-ITD-positive AML.

The footprint of human-induced climate change on heat-related deaths in the summer of 2022 in Switzerland.

Human-induced climate change is leading to an increase in the intensity and frequency of extreme weather events, which are severely affecting the health of the population. The exceptional heat during the summer of 2022 in Europe is an example, with record-breaking temperatures only below the infamous 2003 summer. High ambient temperatures are associated with many health outcomes, including premature mortality. However, there is limited quantitative evidence on the contribution of anthropogenic activities to the substantial heat-related mortality observed in recent times. Here we combined methods in climate epidemiology and attribution to quantify the heat-related mortality burden attributed to human-induced climate change in Switzerland during the summer of 2022. We first estimated heat-mortality association in each canton and age/sex population between 1990 and 2017 in a two-stage time-series analysis. We then calculated the mortality attributed to heat in the summer of 2022 using observed mortality, and compared it with the hypothetical heat-related burden that would have occurred in absence of human-induced climate change. This counterfactual scenario was derived by regressing the Swiss average temperature against global mean temperature in both observations and CMIP6 models. We estimate 623 deaths [95% empirical confidence interval (95% eCI): 151-1068] due to heat between June and August 2022, corresponding to 3.5% of all-cause mortality. More importantly, we find that 60% of this burden (370 deaths [95% eCI: 133-644]) could have been avoided in absence of human-induced climate change. Older women were affected the most, as well as populations in western and southern Switzerland and more urbanized areas. Our findings demonstrate that human-induced climate change was a relevant driver of the exceptional excess health burden observed in the 2022 summer in Switzerland.

Drought self-propagation in drylands due to land-atmosphere feedbacks.

Reduced evaporation due to dry soils can affect the land surface energy balance, with implications for local and downwind precipitation. When evaporation is constrained by soil moisture, the atmospheric supply of water is depleted, and this deficit may propagate in time and space. This mechanism could theoretically result in the self-propagation of droughts, but the extent to which this process occurs is unknown. Here we isolate the influence of soil moisture drought on downwind precipitation using Lagrangian moisture tracking constrained by observations from the 40 largest recent droughts worldwide. We show that dryland droughts are particularly prone to self-propagating, because evaporation tends to respond strongly to enhanced soil water stress. In drylands precipitation can decline by more than 15% due to upwind drought in during a single event, and up to 30% during individual months. In light of projected widespread reductions in water availability, this feedback may further exacerbate future droughts.

Three PilZ Domain Proteins, PlpA, PixA, and PixB, Have Distinct Functions in Regulation of Motility and Development in Myxococcus xanthus.

In bacteria, the nucleotide-based second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) binds to effectors to generate outputs in response to changes in the environment. In Myxococcus xanthus, c-di-GMP regulates type IV pilus-dependent motility and the starvation-induced developmental program that results in formation of spore-filled fruiting bodies; however, little is known about the effectors that bind c-di-GMP. Here, we systematically inactivated all 24 genes encoding PilZ domain-containing proteins, which are among the most common c-di-GMP effectors. We confirm that the stand-alone PilZ domain protein PlpA is important for regulation of motility independently of the Frz chemosensory system and that Pkn1, which is composed of a Ser/Thr kinase domain and a PilZ domain, is specifically important for development. Moreover, we identify two PilZ domain proteins that have distinct functions in regulating motility and development. PixB, which is composed of two PilZ domains and an acetyltransferase domain, binds c-di-GMP in vitro and regulates type IV pilus-dependent and gliding motility in a Frz-dependent manner as well as development. The acetyltransferase domain is required and sufficient for function during growth, while all three domains and c-di-GMP binding are essential for PixB function during development. PixA is a response regulator composed of a PilZ domain and a receiver domain, binds c-di-GMP in vitro, and regulates motility independently of the Frz system, likely by setting up the polarity of the two motility systems. Our results support a model whereby PlpA, PixA, and PixB act in independent pathways and have distinct functions in regulation of motility. IMPORTANCE c-di-GMP signaling controls bacterial motility in many bacterial species by binding to downstream effector proteins. Here, we identify two PilZ domain-containing proteins in Myxococcus xanthus that bind c-di-GMP. We show that PixB, which contains two PilZ domains and an acetyltransferase domain, acts in a manner that depends on the Frz chemosensory system to regulate motility via the acetyltransferase domain, while the intact protein and c-di-GMP binding are essential for PixB to support development. In contrast, PixA acts in a Frz-independent manner to regulate motility. Taking our results together with previous observations, we conclude that PilZ domain proteins and c-di-GMP act in multiple independent pathways to regulate motility and development in M. xanthus.

PomX, a ParA/MinD ATPase activating protein, is a triple regulator of cell division in Myxococcus xanthus.

Cell division site positioning is precisely regulated but the underlying mechanisms are incompletely understood. In the social bacterium Myxococcus xanthus, the ~15 MDa tripartite PomX/Y/Z complex associates with and translocates across the nucleoid in a PomZ ATPase-dependent manner to directly position and stimulate formation of the cytokinetic FtsZ-ring at midcell, and then undergoes fission during division. Here, we demonstrate that PomX consists of two functionally distinct domains and has three functions. The N-terminal domain stimulates ATPase activity of the ParA/MinD ATPase PomZ. The C-terminal domain interacts with PomY and forms polymers, which serve as a scaffold for PomX/Y/Z complex formation. Moreover, the PomX/PomZ interaction is important for fission of the PomX/Y/Z complex. These observations together with previous work support that the architecturally diverse ATPase activating proteins of ParA/MinD ATPases are highly modular and use the same mechanism to activate their cognate ATPase via a short positively charged N-terminal extension.

SMC and the bactofilin/PadC scaffold have distinct yet redundant functions in chromosome segregation and organization in Myxococcus xanthus.

In bacteria, ParABS systems and structural maintenance of chromosome (SMC) condensin-like complexes are important for chromosome segregation and organization. The rod-shaped Myxococcus xanthus cells have a unique chromosome arrangement in which a scaffold composed of the BacNOP bactofilins and PadC positions the essential ParB∙parS segregation complexes and the DNA segregation ATPase ParA in the subpolar regions. We identify the Smc and ScpAB subunits of the SMC complex in M. xanthus and demonstrate that SMC is conditionally essential, with Δsmc or ΔscpAB mutants being temperature sensitive. Inactivation of SMC caused defects in chromosome segregation and organization. Lack of the BacNOP/PadC scaffold also caused chromosome segregation defects but this scaffold is not essential for viability. Inactivation of SMC was synthetic lethal with lack of the BacNOP/PadC scaffold. Lack of SMC interfered with formation of the BacNOP/PadC scaffold while lack of this scaffold did not interfere with chromosome association by SMC. Altogether, our data support that three systems function together to enable chromosome segregation in M. xanthus. ParABS constitutes the basic and essential machinery. SMC and the BacNOP/PadC scaffold have different yet redundant roles in chromosome segregation with SMC supporting individualization of daughter chromosomes and BacNOP/PadC making the ParABS system operate more robustly.

Atmospheric heat and moisture transport to energy- and water-limited ecosystems.

The land biosphere is a crucial component of the Earth system that interacts with the atmosphere in a complex manner through manifold feedback processes. These relationships are bidirectional, as climate affects our terrestrial ecosystems, which, in turn, influence climate. Great progress has been made in understanding the local interactions between the terrestrial biosphere and climate, but influences from remote regions through energy and water influxes to downwind ecosystems remain less explored. Using a Lagrangian trajectory model driven by atmospheric reanalysis data, we show how heat and moisture advection affect gross carbon production at interannual scales and in different ecoregions across the globe. For water-limited regions, results show a detrimental effect on ecosystem productivity during periods of enhanced heat and reduced moisture advection. These periods are typically associated with winds that disproportionately come from continental source regions, as well as positive sensible heat flux and negative latent heat flux anomalies in those upwind locations. Our results underline the vulnerability of ecosystems to the occurrence of upwind climatic extremes and highlight the importance of the latter for the spatiotemporal propagation of ecosystem disturbances.

N-Hydroxysuccinimide-Modified Ethynylphosphonamidates Enable the Synthesis of Configurationally Defined Protein Conjugates.

Herein, the application of N-hydroxysuccinimide-modified phosphonamidate building blocks for the incorporation of cysteine-selective ethynylphosphonamidates into lysine residues of proteins, followed by thiol addition with small molecules and proteins, is reported. It is demonstrated that the building blocks significantly lower undesired homo-crosslinking side products that can occur with commonly applied succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) under physiological pH. The previously demonstrated stability of the phosphonamidate moiety additionally solves the problem of premature maleimide hydrolysis, which can hamper the efficiency of subsequent thiol addition. Furthermore, a method to separate the phosphonamidate enantiomers to be able to synthesize protein conjugates in a defined configuration has been developed. Finally, the building blocks are applied to the construction of functional antibody-drug conjugates, analogously to FDA-approved, SMCC-linked Kadcyla, and to the synthesis of a functional antibody-protein conjugate.

One-Step Fluorescent Protein Labeling by Tubulin Tyrosine Ligase.

Tub-tag labeling, a novel chemoenzymatic protein functionalization method, facilitates one-step fluorescent labeling of functional biomolecules. The enzyme tubulin tyrosine ligase incorporates coumarin-amino acids to the terminal carboxylic acid of proteins containing a short peptidic recognition sequence called Tub-tag. Here we describe the one-step Tub-tag protein modification protocol in detail and explain its utilization to generate fluorescently labeled proteins for advanced applications in imaging and diagnostics.

Ethynylphosphonamidates for the Rapid and Cysteine-Selective Generation of Efficacious Antibody-Drug Conjugates.

Requirements for novel bioconjugation reactions for the synthesis of antibody-drug conjugates (ADCs) are exceptionally high, since conjugation selectivity as well as the stability and hydrophobicity of linkers and payloads drastically influence the performance and safety profile of the final product. We report Cys-selective ethynylphosphonamidates as new reagents for the rapid generation of efficacious ADCs from native non-engineered monoclonal antibodies through a simple one-pot reduction and alkylation. Ethynylphosphonamidates can be easily substituted with hydrophilic residues, giving rise to electrophilic labeling reagents with tunable solubility properties. We demonstrate that ethynylphosphonamidate-linked ADCs have excellent properties for next-generation antibody therapeutics in terms of serum stability and in vivo antitumor activity.

Tubulin Tyrosine Ligase-Mediated Modification of Proteins.

Tubulin tyrosine ligase (TTL) catalyzes the addition of tyrosine derivatives to the C-terminal carboxylic acid of proteins. The enzyme binds to a 14-amino acid recognition sequence, termed Tub-tag, and allows for the introduction of tyrosine derivatives that carry a unique chemical handle. These handles enable subsequent bioorthogonal reactions with a great variety of probes or effector molecules. Clearly, this two-step chemoenzymatic approach, facilitates the site-specific functionalization of proteins. Furthermore, due to its broad substrate tolerance, tubulin tyrosine ligase also enables an enzymatic one-step modification. For example, a coumarin amino acid was utilized to generate fluorescently labeled proteins for advanced applications in imaging and diagnostics. Here we describe the modification of proteins using TTL in detail via a one-step as well as two-step procedure and highlight its practicability for applications in imaging, diagnostics, and cell biology.

TuPPL: Tub-tag mediated C-terminal protein-protein-ligation using complementary click-chemistry handles.

We introduce a chemoenzymatic strategy for straightforward in vitro generation of C-terminally linked fusion proteins. Tubulin tyrosine ligase is used for the incorporation of complementary click chemistry handles facilitating subsequent formation of functional bispecific antibody-fragments. This simple strategy may serve as central conjugation hub for a modular protein ligation platform.

Cysteine-Selective Phosphonamidate Electrophiles for Modular Protein Bioconjugations.

We describe a new technique in protein synthesis that extends the existing repertoire of methods for protein modification: A chemoselective reaction that induces reactivity for a subsequent bioconjugation. An azide-modified building block reacts first with an ethynylphosphonite through a Staudinger-phosphonite reaction (SPhR) to give an ethynylphosphonamidate. The resulting electron-deficient triple bond subsequently undergoes a cysteine-selective reaction with proteins or antibodies. We demonstrate that ethynylphosphonamidates display excellent cysteine-selective reactivity combined with superior stability of the thiol adducts, when compared to classical maleimide linkages. This turns our technique into a versatile and powerful tool for the facile construction of stable functional protein conjugates.

FLEXamers: A Double Tag for Universal Generation of Versatile Peptide-MHC Multimers.

Peptide-MHC (pMHC) multimers have become a valuable tool for immunological research, clinical immune monitoring, and immunotherapeutic applications. Biotinylated tetramers, reversible Streptamers, or dye-conjugated pMHC multimers are distinct pMHC reagents tailored for T cell identification, traceless T cell isolation, or TCR characterization, respectively. The specific applicability of each pMHC-based reagent is made possible either through conjugation of probes or reversible multimerization in separate production processes, which is laborious, time-consuming, and prone to variability between the different types of pMHC reagents. This prohibits broad implementation of different types of pMHC reagents as a standard toolbox in routine clinical immune monitoring and immunotherapy. In this article, we describe a novel method for fast and standardized generation of any pMHC multimer reagent from a single precursor ("FLEXamer"). FLEXamers unite reversible multimerization and versatile probe conjugation through a novel double tag (Strep-tag for reversibility and Tub-tag for versatile probe conjugation). We demonstrate that FLEXamers can substitute conventional pMHC reagents in all state-of-the-art applications, considerably accelerating and standardizing production without sacrificing functional performance. Although FLEXamers significantly aid the applicability of pMHC-based reagents in routine workflows, the double tag also provides a universal tool for the investigation of transient molecular interactions in general.

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus.

Fluorescence live-cell imaging of bacterial cells is a key method in the analysis of the spatial and temporal dynamics of proteins and chromosomes underlying central cell cycle events. However, imaging of these molecules in slow-growing bacteria represents a challenge due to photobleaching of fluorophores and phototoxicity during image acquisition. Here, we describe a simple protocol to circumvent these limitations in the case of Myxococcus xanthus (which has a generation time of 4 - 6 h). To this end, M. xanthus cells are grown on a thick nutrient-containing agar pad in a temperature-controlled humid environment. Under these conditions, we determine the doubling time of individual cells by following the growth of single cells. Moreover, key cellular processes such as chromosome segregation and cell division can be imaged by fluorescence live-cell imaging of cells containing relevant fluorescently labeled marker proteins such as ParB-YFP, FtsZ-GFP, and mCherry-PomX over multiple cell cycles. Subsequently, the acquired images are processed to generate montages and/or movies.

Tub-Tag Labeling; Chemoenzymatic Incorporation of Unnatural Amino Acids.

Tub-tag labeling is a chemoenzymatic method that enables the site-specific labeling of proteins. Here, the natural enzyme tubulin tyrosine ligase incorporates noncanonical tyrosine derivatives to the terminal carboxylic acid of proteins containing a 14-amino acid recognition sequence called Tub-tag. The tyrosine derivative carries a unique chemical reporter allowing for a subsequent bioorthogonal modification of proteins with a great variety of probes. Here, we describe the Tub-tag protein modification protocol in detail and explain its utilization to generate labeled proteins for advanced applications in cell biology, imaging, and diagnostics.

Nanobodies: Chemical Functionalization Strategies and Intracellular Applications.

Nanobodies can be seen as next-generation tools for the recognition and modulation of antigens that are inaccessible to conventional antibodies. Due to their compact structure and high stability, nanobodies see frequent usage in basic research, and their chemical functionalization opens the way towards promising diagnostic and therapeutic applications. In this Review, central aspects of nanobody functionalization are presented, together with selected applications. While early conjugation strategies relied on the random modification of natural amino acids, more recent studies have focused on the site-specific attachment of functional moieties. Such techniques include chemoenzymatic approaches, expressed protein ligation, and amber suppression in combination with bioorthogonal modification strategies. Recent applications range from sophisticated imaging and mass spectrometry to the delivery of nanobodies into living cells for the visualization and manipulation of intracellular antigens.