Potent antitumor activity of anti-HER2 antibody-topoisomerase I inhibitor conjugate based on self-immolative dendritic dimeric-linker.

Antibody-drug conjugates (ADCs) are a rapidly expanding class of anticancer therapeutics, with 14 ADCs already approved worldwide. We developed unique linker technologies for the bioconjugation of drug molecules with controlled-release applications. We synthesized cathepsin-cleavable ADCs using a dimeric prodrug system based on a self-immolative dendritic scaffold, resulting in a high drug-antibody ratio (DAR) with the potential to reach 16 payloads due to its dendritic structure, increased stability in the circulation and efficient release profile of a highly cytotoxic payload at the targeted site. Using our novel cleavable linker technologies, we conjugated the anti-human epidermal growth factor receptor 2 (anti-HER2) antibody, trastuzumab, with topoisomerase I inhibitors, exatecan or belotecan. The newly synthesized ADCs were tested in vitro on mammary carcinoma cells overexpressing human HER2, demonstrating a substantial inhibitory effect on the proliferation of HER2-positive cells. Importantly, a single dose of our trastuzumab-based ADCs administered in vivo to mice bearing HER2-positive tumors, showed a dose-dependent inhibition of tumor growth and survival benefit, with the most potent antitumor effects observed at 10 mg/kg, which resulted in complete tumor regression and survival of 100% of the mice. Overall, our novel dendritic technologies using the protease-cleavable Val-Cit linker present an opportunity for the development of highly selective and potent controlled-released therapeutic payloads. This strategy could potentially lead to the development of novel and effective ADC technologies for patients diagnosed with HER2-positive cancers. Moreover, our proposed ADC linker technology can be implemented in additional medical conditions such as other malignancies as well as autoimmune diseases that overexpress targets, other than HER2.

Generation and Characterization of Iduronidase-Cleavable ADCs.

A crucial design feature for the therapeutic success of antibody-drug conjugates (ADCs) is the linker that connects the antibody with the drug. Linkers must be stable in circulation and efficiently release the drug inside the target cell, thereby having a fundamental impact on ADC pharmacokinetics and efficacy. The variety of enzymatically cleavable linkers applied in ADCs is limited, and some are believed to be associated with unwanted side effects due to the expression of cleavage-mediating enzymes in nonmalignant cells. Based on a bioinformatic screen of lysosomal enzymes, we identified α-l-iduronidase (IduA) as an interesting candidate for ADC linker cleavage because of its low expression in normal tissues and its overexpression in several tumor types. In the present study, we report a novel IduA-cleavable ADC linker using exatecan and duocarmycin as payloads. We showed the functionality of our linker system in cleavage assays using recombinant IduA or cell lysates and compared it to established ADC linkers. Subsequently, we coupled iduronide-exatecan via interchain cysteines or iduronide-duocarmycin via microbial transglutaminase (mTG) to an anti-CEACAM5 (aCEA5) antibody. The generated iduronide-exatecan ADC showed high serum stability and similar target-dependent tumor cell killing in the subnanomolar range but reduced toxicity on nonmalignant cells compared to an analogous cathepsin B-activatable valine-citrulline-exatecan ADC. Finally, in vivo antitumor activity could be demonstrated for an IduA-cleavable duocarmycin ADC. The presented results emphasize the potential of iduronide linkers for ADC development and represent a tool for further balancing out tumor selectivity and safety.

AI/ML combined with next-generation sequencing of VHH immune repertoires enables the rapid identification of de novo humanized and sequence-optimized single domain antibodies: a prospective case study.

Introduction: In this study, we demonstrate the feasibility of yeast surface display (YSD) and nextgeneration sequencing (NGS) in combination with artificial intelligence and machine learning methods (AI/ML) for the identification of de novo humanized single domain antibodies (sdAbs) with favorable early developability profiles. Methods: The display library was derived from a novel approach, in which VHH-based CDR3 regions obtained from a llama (Lama glama), immunized against NKp46, were grafted onto a humanized VHH backbone library that was diversified in CDR1 and CDR2. Following NGS analysis of sequence pools from two rounds of fluorescence-activated cell sorting we focused on four sequence clusters based on NGS frequency and enrichment analysis as well as in silico developability assessment. For each cluster, long short-term memory (LSTM) based deep generative models were trained and used for the in silico sampling of new sequences. Sequences were subjected to sequence- and structure-based in silico developability assessment to select a set of less than 10 sequences per cluster for production. Results: As demonstrated by binding kinetics and early developability assessment, this procedure represents a general strategy for the rapid and efficient design of potent and automatically humanized sdAb hits from screening selections with favorable early developability profiles.

A library approach for the de novo high-throughput isolation of humanized VHH domains with favorable developability properties following camelid immunization.

In this study, we generated a novel library approach for high throughput de novo identification of humanized single-domain antibodies following camelid immunization. To achieve this, VHH-derived complementarity-determining regions-3 (CDR3s) obtained from an immunized llama (Lama glama) were grafted onto humanized VHH backbones comprising moderately sequence-diversified CDR1 and CDR2 regions similar to natural immunized and naïve antibody repertoires. Importantly, these CDRs were tailored toward favorable in silico developability properties, by considering human-likeness as well as excluding potential sequence liabilities and predicted immunogenic motifs. Target-specific humanized single-domain antibodies (sdAbs) were readily obtained by yeast surface display. We demonstrate that, by exploiting this approach, high affinity sdAbs with an optimized in silico developability profile can be generated. These sdAbs display favorable biophysical, biochemical, and functional attributes and do not require any further sequence optimization. This approach is generally applicable to any antigen upon camelid immunization and has the potential to significantly accelerate candidate selection and reduce risks and attrition rates in sdAb development.

Population-level antagonism between FGF and BMP signaling steers mesoderm differentiation in embryonic stem cells.

The mesodermal precursor populations for different internal organ systems are specified during gastrulation by the combined activity of extracellular signaling systems such as BMP, Wnt, Nodal and FGF. The BMP, Wnt and Nodal signaling requirements for the differentiation of specific mesoderm subtypes in mammals have been mapped in detail, but how FGF shapes mesodermal cell type diversity is not precisely known. It is also not clear how FGF signaling integrates with the activity of other signaling systems involved in mesoderm differentiation. Here, we address these questions by analyzing the effects of targeted signaling manipulations in differentiating stem cell populations at single-cell resolution. We identify opposing functions of BMP and FGF, and map FGF-dependent and -independent mesodermal lineages. Stimulation with exogenous FGF boosts the expression of endogenous Fgf genes while repressing Bmp ligand genes. This positive autoregulation of FGF signaling, coupled with the repression of BMP signaling, may contribute to the specification of reproducible and coherent cohorts of cells with the same identity via a community effect, both in the embryo and in synthetic embryo-like systems.

Intermittent ERK oscillations downstream of FGF in mouse embryonic stem cells.

Signal transduction networks generate characteristic dynamic activities to process extracellular signals and guide cell fate decisions such as to divide or differentiate. The differentiation of pluripotent cells is controlled by FGF/ERK signaling. However, only a few studies have addressed the dynamic activity of the FGF/ERK signaling network in pluripotent cells at high time resolution. Here, we use live cell sensors in wild-type and Fgf4-mutant mouse embryonic stem cells to measure dynamic ERK activity in single cells, for defined ligand concentrations and differentiation states. These sensors reveal pulses of ERK activity. Pulsing patterns are heterogeneous between individual cells. Consecutive pulse sequences occur more frequently than expected from simple stochastic models. Sequences become more prevalent with higher ligand concentration, but are rarer in more differentiated cells. Our results suggest that FGF/ERK signaling operates in the vicinity of a transition point between oscillatory and non-oscillatory dynamics in embryonic stem cells. The resulting heterogeneous dynamic signaling activities add a new dimension to cellular heterogeneity that may be linked to divergent fate decisions in stem cell cultures.

EGFR binding Fc domain-drug conjugates: stable and highly potent cytotoxic molecules mediate selective cell killing.

The exposition of cancer cells to cytotoxic doses of payload is fundamental for the therapeutic efficacy of antibody drug conjugates (ADCs) in solid cancers. To maximize payload exposure, tissue penetration can be increased by utilizing smaller-sized drug conjugates which distribute deeper into the tumor. Our group recently explored small human epidermal growth factor receptor 2 (HER2) targeting Fc antigen binding fragments (Fcabs) for ADC applications in a feasibility study. Here, we expand this concept using epidermal growth factor receptor (EGFR) targeting Fcabs for the generation of site-specific auristatin-based drug conjugates. In contrast to HER2-targeting Fcabs, we identified novel conjugation sites in the EGFR-targeting Fcab scaffold that allowed for higher DAR enzymatic conjugation. We demonstrate feasibility of resultant EGFR-targeting Fcab-drug conjugates that retain binding to half-life prolonging neonatal Fc receptor (FcRn) and EGFR and show high serum stability as well as target receptor mediated cell killing at sub-nanomolar concentrations. Our results emphasize the applicability of the Fcab format for the generation of drug conjugates designed for increased penetration of solid tumors and potential FcRn-driven antibody-like pharmacokinetics.

Cell-cell communication through FGF4 generates and maintains robust proportions of differentiated cell types in embryonic stem cells.

During embryonic development and tissue homeostasis, reproducible proportions of differentiated cell types are specified from populations of multipotent precursor cells. Molecular mechanisms that enable both robust cell-type proportioning despite variable initial conditions in the precursor cells, and the re-establishment of these proportions upon perturbations in a developing tissue remain to be characterized. Here, we report that the differentiation of robust proportions of epiblast-like and primitive endoderm-like cells in mouse embryonic stem cell cultures emerges at the population level through cell-cell communication via a short-range fibroblast growth factor 4 (FGF4) signal. We characterize the molecular and dynamical properties of the communication mechanism and show how it controls both robust cell-type proportioning from a wide range of experimentally controlled initial conditions, as well as the autonomous re-establishment of these proportions following the isolation of one cell type. The generation and maintenance of reproducible proportions of discrete cell types is a new function for FGF signaling that might operate in a range of developing tissues.

Effect of Conjugation Site and Technique on the Stability and Pharmacokinetics of Antibody-Drug Conjugates.

Appropriate selection of conjugation sites and conjugation technologies is now widely accepted as crucial for the success of antibody-drug conjugates (ADCs). Herein, we present ADCs conjugated by different conjugation methods to different conjugation positions being systematically characterized by multiple in vitro assays as well as in vivo pharmacokinetic (PK) analyses in transgenic Tg276 mice. Conjugation to cysteines, genetically introduced at positions N325, L328, S239, D265, and S442, was compared to enzymatic conjugation via microbial transglutaminase (mTG) either to C-terminal light (LC) or heavy chain (HC) recognition motifs or to endogenous position Q295 of a native antibody. All conjugations yielded homogeneous DAR 2 ADCs with similar hydrophobicity, thermal stability, human neonatal Fc receptor (huFcRn) binding, and serum stability properties, but with pronounced differences in their PK profiles. mTG-conjugated ADC variants conjugated either to Q295 or to LC recognition motifs showed superior PK behavior. Within the panel of engineered cysteine variants L328 showed a similar PK profile compared to previously described S239 but superior PK compared to S442, D265, and N325. While all positions were first tested with trastuzumab, L328 and mTG LC were further evaluated with additional antibody scaffolds derived from clinically evaluated monoclonal antibodies (mAb). Based on PK analyses, this study confirms the newly described position L328 as favorable site for cysteine conjugation, comparable to the well-established engineered cysteine position S239, and emphasizes the favorable position Q295 of native antibodies and the tagged LC antibody variant for enzymatic conjugations via mTG. In addition, hemizygous Tg276 mice are evaluated as an adequate model for ADC pharmacokinetics, facilitating the selection of suitable ADC candidates early in the drug discovery process.

Generation and Biological Evaluation of Fc Antigen Binding Fragment-Drug Conjugates as a Novel Antibody-Based Format for Targeted Drug Delivery.

Fragment crystallizable (Fc) antigen binding fragments (Fcabs) represent a novel antibody format comprising a homodimeric Fc region with an engineered antigen binding site. In contrast to their full-length antibody offspring, Fcabs combine Fc-domain-mediated and antigen binding functions at only one-third of the size. Their reduced size is accompanied by elevated tissue penetration capabilities, which is an attractive feature for the treatment of solid tumors. In the present study, we explored for the first time Fcabs as a novel scaffold for antibody-drug conjugates (ADCs). As model, various HER2-targeting Fcab variants coupled to a pH-sensitive dye were used in internalization experiments. A selective binding on HER2-expressing tumor cells and receptor-mediated endocytosis could be confirmed for selected variants, indicating that these Fcabs meet the basic prerequisite for an ADC approach. Subsequently, Fcabs were site-specifically coupled to cytotoxic monomethyl auristatin E yielding homogeneous conjugates. The conjugates retained HER2 and FcRn binding behavior of the parent Fcabs, showed a selective in vitro cell killing and conjugation site-dependent serum stability. Moreover, Fcab conjugates showed elevated penetration in a spheroid model, compared to their full-length antibody and Trastuzumab counterparts. Altogether, the presented results emphasize the potential of Fcabs as a novel scaffold for targeted drug delivery in solid cancers and pave the way for future in vivo translation.

Robustness and timing of cellular differentiation through population-based symmetry breaking.

During mammalian development and homeostasis, cells often transition from a multilineage primed state to one of several differentiated cell types that are marked by the expression of mutually exclusive genetic markers. These observations have been classically explained by single-cell multistability as the dynamical basis of differentiation, where robust cell-type proportioning relies on pre-existing cell-to-cell differences. We propose a conceptually different dynamical mechanism in which cell types emerge and are maintained collectively by cell-cell communication as a novel inhomogeneous state of the coupled system. Differentiation can be triggered by cell number increase as the population grows in size, through organisation of the initial homogeneous population before the symmetry-breaking bifurcation point. Robust proportioning and reliable recovery of the differentiated cell types following a perturbation is an inherent feature of the inhomogeneous state that is collectively maintained. This dynamical mechanism is valid for systems with steady-state or oscillatory single-cell dynamics. Therefore, our results suggest that timing and subsequent differentiation in robust cell-type proportions can emerge from the cooperative behaviour of growing cell populations during development.

Live Visualization of ERK Activity in the Mouse Blastocyst Reveals Lineage-Specific Signaling Dynamics.

FGF/ERK signaling is crucial for the patterning and proliferation of cell lineages that comprise the mouse blastocyst. However, ERK signaling dynamics have never been directly visualized in live embryos. To address whether differential signaling is associated with particular cell fates and states, we generated a targeted mouse line expressing an ERK-kinase translocation reporter (KTR) that enables live quantification of ERK activity at single-cell resolution. 3D time-lapse imaging of this biosensor in embryos revealed spatially graded ERK activity in the trophectoderm prior to overt polar versus mural differentiation. Within the inner cell mass (ICM), all cells relayed FGF/ERK signals with varying durations and magnitude. Primitive endoderm cells displayed higher overall levels of ERK activity, while pluripotent epiblast cells exhibited lower basal activity with sporadic pulses. These results constitute a direct visualization of signaling events during mammalian pre-implantation development and reveal the existence of spatial and temporal lineage-specific dynamics.

De novo structure determination of butadiene by isotope-resolved rotational Raman spectroscopy.

Isotope-selective rotational spectroscopy allows to calculate molecular structures independent of assumptions or theoretical predictions. Here, we present the first de novo structure determination based on mass-correlated rotational Raman spectroscopy, analyzing the carbon atom positions of butadiene. Mass correlation allowed us to analyze signals of rare 13C isotopologues at natural abundance, without interference from the main isotopologue signals. Fitted rotational constants and structural parameters confirm literature data from rovibrational spectroscopy of synthetic isotopologues and electron diffraction experiments.

Site-Specific Conjugation of Native Antibodies Using Engineered Microbial Transglutaminases.

Site-specific bioconjugation technologies are frequently employed to generate homogeneous antibody-drug conjugates (ADCs) and are generally considered superior to stochastic approaches like lysine coupling. However, most of the technologies developed so far require undesired manipulation of the antibody sequence or its glycan structures. Herein, we report the successful engineering of microbial transglutaminase enabling efficient, site-specific conjugation of drug-linker constructs to position HC-Q295 of native, fully glycosylated IgG-type antibodies. ADCs generated via this approach demonstrate excellent stability in vitro as well as strong efficacy in vitro and in vivo. As it employs different drug-linker structures and several native antibodies, our study additionally proves the broad applicability of this approach.

Selection and Characterization of Anti-idiotypic Shark Antibody Domains.

The antibody repertoire of cartilaginous fish comprises an additional heavy-chain-only antibody isotype that is referred to as IgNAR (immunoglobulin novel antigen receptor). Its antigen-binding site consists of one single domain (vNAR) that is reportedly able to engage a respective antigen with affinities similar to those achieved by conventional antibodies. While vNAR domains offer a reduced size, which is often favorable for applications in a therapeutic as well as a biotechnological setup, they also exhibit a high physicochemical stability. Together with their ability to target difficult-to-address antigens such as virus particles or toxins, these shark-derived antibody domains seem to be predestined as tools for biotechnological and diagnostic applications. In the following chapter, we will describe the isolation of anti-idiotypic vNAR domains targeting monoclonal antibody paratopes from semi-synthetic, yeast-displayed libraries. Anti-idiotypic vNAR variants could be employed for the characterization of antibody-based therapeutics (such as antibody-drug conjugates) or as positive controls in immunogenicity assays. Peculiarly, when using semi-synthetic vNAR libraries, we found that it is not necessary to deplete the libraries using unrelated antibody targets, which enables a fast and facile screening procedure that exclusively delivers anti-idiotypic binders.

Isolation of Tailor-Made Antibody Fragments from Yeast-Displayed B-Cell Receptor Repertoires by Multiparameter Fluorescence-Activated Cell Sorting.

In the past decades, monoclonal antibodies have made an unprecedented transformation from research tools to a rapidly growing class of therapeutics. Advancements in the yeast surface display platform enable the selection of favorable mouse or human antibody variants from large B-cell receptor (BCR) gene repertoires that are derived from immunized normal or transgenic animals. Application of high-throughput fluorescence-activated cell sorting (FACS) screening along with well-chosen selection settings can be utilized to identify variants with desired affinities and predefined epitope binding properties. In the following chapter, we describe in detail a multiparameter screening protocol for the selection of antibody variants from yeast libraries generated from BCR gene repertoires from immunized transgenic rats. The procedure provides guidance for the selection of antigen-specific, high-affinity binding, and species cross-reactive human antibodies with a broad epitope coverage. Essentially, this can accelerate target-specific antibody characterization as multiple desirable antibody features can be easily integrated into the selection procedure. In addition, we provide information on how to validate binding behavior of selected candidates after expression as soluble, full-length IgG molecules.

Isolation of Anti-Hapten Antibodies by Fluorescence-Activated Cell Sorting of Yeast-Displayed B-Cell Receptor Gene Repertoires.

Anti-hapten antibodies are widely used as specific immunochemical detection tools in a variety of clinical and environmental analyses. The sensitivity, however, is limited due to the resulting antibody affinities to the haptens which, in turn, leads to a high demand for specific affinity reagents. A well-established path for the generation of high-affinity antibodies is the immunization of animals with the target antigen. However, the generation of anti-hapten antibodies via immunization remains challenging as small molecule haptens usually possess low immunogenicity and, therefore, must be coupled to an immunogenic and high molecular weight carrier to provoke an immune response.Consequently, antibodies are primarily raised against the carrier molecule or structural features of the hapten-linker fused to the carrier protein. This turns the generation of antibodies which bind exclusively to the hapten structure into a search for the needle in a haystack. In the following chapter, we describe how yeast surface display and high-throughput fluorescence-activated cell sorting can be used to isolate anti-hapten antibodies from a large, yeast-displayed B-cell receptor gene library derived from immunized animals. For this, we describe in detail the preparation of protein-hapten conjugates, the immunization procedure, and the subsequent screening process. Moreover, we provide a simple flow cytometry protocol that allows for a rapid analysis of the enriched clones toward free hapten binding.

Impact of Accidental Hypothermia on Pulmonary Complications in Multiply Injured Patients With Blunt Chest Trauma - A Matched-pair Analysis.

BACKGROUND/AIM: Blunt chest trauma is one of the major injuries in multiply injured patients and is associated with an increased risk of acute respiratory distress syndrome (ARDS) and ventilator-associated pneumonia (VAP). Accidental hypothermia is a common accompaniment of multiply injured patients. The objective of this study was to analyze the influence of accidental hypothermia on pulmonary complications in multiply injured patients with blunt chest trauma. PATIENTS AND METHODS: Multiply injured patients [injury severity score (ISS) ≥16] with severe blunt chest trauma [abbreviated injury scale of the chest (AISchest) ≥3] were analyzed. Hypothermia was defined as body core temperature <35°C. The primary endpoint was the development of ARDS and VAP. Propensity score matching was performed. RESULTS: Data were analyzed for 238 patients, with a median ISS of 26 (interquartile range=12). A total of 67 patients (28%) were hypothermic on admission. Hypothermic patients were injured more severely (median ISS 34 vs. 24, p<0.001) and had a higher transfusion requirement (p<0.001). Their mortality rate was consequently increased (10% vs. 1%, p=0.002); After propensity score matching, the mortality rate was still higher (10% vs. 2%, p=0.046). However, hypothermia was not an independent predictor of mortality. Hypothermic patients had to be ventilated longer (p=0.02). However, there were no differences in occurrence of ARDS and VAP. Hypothermia was not identified as an independent predictor of ARDS and VAP. CONCLUSION: Among multiply injured patients with severe blunt chest trauma, accidental hypothermia is not an independent predictor of ARDS and VAP and is more likely to be an accompaniment of injury severity and hemorrhage.

Mouse ICM Organoids Reveal Three-Dimensional Cell Fate Clustering.

During mammalian preimplantation, cells of the inner cell mass (ICM) adopt either an embryonic or an extraembryonic fate. This process is tightly regulated in space and time and has been studied previously in mouse embryos and embryonic stem cell models. Current research suggests that cell fates are arranged in a salt-and-pepper pattern of random cell positioning or a spatially alternating pattern. However, the details of the three-dimensional patterns of cell fate specification have not been investigated in the embryo nor in in vitro systems. We developed ICM organoids as a, to our knowledge, novel three-dimensional in vitro stem cell system to model mechanisms of fate decisions that occur in the ICM. ICM organoids show similarities to the in vivo system that arise regardless of the differences in geometry and total cell number. Inspecting ICM organoids and mouse embryos, we describe a so far unknown local clustering of cells with identical fates in both systems. These findings are based on the three-dimensional quantitative analysis of spatiotemporal patterns of NANOG and GATA6 expression in combination with computational rule-based modeling. The pattern identified by our analysis is distinct from the current view of a salt-and-pepper pattern. Our investigation of the spatial distributions both in vivo and in vitro dissects the contributions of the different parts of the embryo to cell fate specifications. In perspective, our combination of quantitative in vivo and in vitro analyses can be extended to other mammalian organisms and thus creates a powerful approach to study embryogenesis.

Construction of Histidine-Enriched Shark IgNAR Variable Domain Antibody Libraries for the Isolation of pH-Sensitive vNAR Fragments.

The adaptive immune system of sharks comprises a heavy chain-only antibody isotype, referred to as immunoglobulin new antigen receptor (IgNAR). Antigen binding in case of IgNAR antibodies is mediated by a single variable domain (vNAR). Due to their inherent beneficial biophysical properties, such as small size and high thermal stability combined with a high specificity and affinity to their target antigens, vNAR domains emerged as promising tools for biotechnological and biomedical applications. Herein, we present detailed protocols for the engineering of pH-sensitivity into IgNAR V domains by constructing histidine-enriched and CDR3-diversified semisynthetic antibody libraries which can then be screened upon using yeast surface display. Protonation or deprotonation of incorporated histidine residues at different pH values results in structural transitions caused by altered electrostatic interactions. These interactions account for an altered binding behavior toward the target antigen. In the following protocol, we describe the generation of a semisynthetic vNAR master library that comprises two histidine residues on average in the 12-residue CDR3 loop. Moreover, once a pH-dependent vNAR population toward the target antigen is identified, this population can further be optimized in terms of affinity and pH sensitivity upon conducting a CDR1-mediated affinity maturation.