Respiratory syncytial virus-approved mAb Palivizumab as ligand for anti-idiotype nanobody-based synthetic cytokine receptors.

Synthetic cytokine receptors can modulate cellular functions based on an artificial ligand to avoid off-target and/or unspecific effects. However, ligands that can modulate receptor activity so far have not been used clinically because of unknown toxicity and immunity against the ligands. Here, we developed a fully synthetic cytokine/cytokine receptor pair based on the antigen-binding domain of the respiratory syncytial virus-approved mAb Palivizumab as a synthetic cytokine and a set of anti-idiotype nanobodies (AIPVHH) as synthetic receptors. Importantly, Palivizumab is neither cross-reactive with human proteins nor immunogenic. For the synthetic receptors, AIPVHH were fused to the activating interleukin-6 cytokine receptor gp130 and the apoptosis-inducing receptor Fas. We found that the synthetic cytokine receptor AIPVHHgp130 was efficiently activated by dimeric Palivizumab single-chain variable fragments. In summary, we created an in vitro nonimmunogenic full-synthetic cytokine/cytokine receptor pair as a proof of concept for future in vivo therapeutic strategies utilizing nonphysiological targets during immunotherapy.

Better safe than sorry: dual targeting antibodies for cancer immunotherapy.

Antibody-based therapies are revolutionizing cancer treatment and experience a steady increase from preclinical and clinical pipelines to market share. While the clinical success of monoclonal antibodies is frequently limited by low response rates, treatment resistance and various other factors, multispecific antibodies open up new prospects by addressing tumor complexity as well as immune response actuation potently improving safety and efficacy. Novel antibody approaches involve simultaneous binding of two antigens on one cell implying increased specificity and reduced tumor escape for dual tumor-associated antigen targeting and enhanced and durable cytotoxic effects for dual immune cell-related antigen targeting. This article reviews antibody and cell-based therapeutics for oncology with intrinsic dual targeting of either tumor cells or immune cells. As revealed in various preclinical studies and clinical trials, dual targeting molecules are promising candidates constituting the next generation of antibody drugs for fighting cancer.

Bovine ultralong CDR-H3 derived knob paratopes elicit potent TNF-α neutralization and enable the generation of novel adalimumab-based antibody architectures with augmented features.

In this work we have generated cattle-derived chimeric ultralong CDR-H3 antibodies targeting tumor necrosis factor α (TNF-α) via immunization and yeast surface display. We identified one particular ultralong CDR-H3 paratope that potently neutralized TNF-α. Interestingly, grafting of the knob architecture onto a peripheral loop of the CH3 domain of the Fc part of an IgG1 resulted in the generation of a TNF-α neutralizing Fc (Fcknob) that did not show any potency loss compared with the parental chimeric IgG format. Eventually, grafting this knob onto the CH3 region of adalimumab enabled the engineering of a novel TNF-α targeting antibody architecture displaying augmented TNF-α inhibition.

Welding PROxAb Shuttles: A Modular Approach for Generating Bispecific Antibodies via Site-Specific Protein-Protein Conjugation.

Targeted protein degradation is an innovative therapeutic strategy to selectively eliminate disease-causing proteins. Exemplified by proteolysis-targeting chimeras (PROTACs), they have shown promise in overcoming drug resistance and targeting previously undruggable proteins. However, PROTACs face challenges, such as low oral bioavailability and limited selectivity. The recently published PROxAb Shuttle technology offers a solution enabling the targeted delivery of PROTACs using antibodies fused with PROTAC-binding domains derived from camelid single-domain antibodies (VHHs). Here, a modular approach to quickly generate PROxAb Shuttles by enzymatically coupling PROTAC-binding VHHs to off-the-shelf antibodies was developed. The resulting conjugates retained their target binding and internalization properties, and incubation with BRD4-targeting PROTACs resulted in formation of defined PROxAb-PROTAC complexes. These complexes selectively induced degradation of the BRD4 protein, resulting in cytotoxicity specifically to cells expressing the antibody's target. The chemoenzymatic approach described herein provides a versatile and efficient solution for generating antibody-VHH conjugates for targeted protein degradation applications, but it could also be used to combine antibodies and VHH binders to generate bispecific antibodies for further applications.

Potent Apoptosis Induction by a Novel Trispecific B7-H3xCD16xTIGIT 2+1 Common Light Chain Natural Killer Cell Engager.

Valued for their ability to rapidly kill multiple tumor cells in succession as well as their favorable safety profile, NK cells are of increasing interest in the field of immunotherapy. As their cytotoxic activity is controlled by a complex network of activating and inhibiting receptors, they offer a wide range of possible antigens to modulate their function by antibodies. In this work, we utilized our established common light chain (cLC)-based yeast surface display (YSD) screening procedure to isolate novel B7-H3 and TIGIT binding monoclonal antibodies. The chicken-derived antibodies showed single- to low-double-digit nanomolar affinities and were combined with a previously published CD16-binding Fab in a 2+1 format to generate a potent NK engaging molecule. In a straightforward, easily adjustable apoptosis assay, the construct B7-H3xCD16xTIGIT showed potent apoptosis induction in cancer cells. These results showcase the potential of the TIGIT NK checkpoint in combination with activating receptors to achieve increased cytotoxic activity.

Novel amino-Li resin for water-based solid-phase peptide synthesis.

We report the first application of a novel amino-Li resin to water-based solid-phase peptide synthesis (SPPS) applying the Smoc-protecting group approach. We demonstrated that it is a suitable support for the sustainable water-based alternative to a classical SPPS approach. The resin possesses good swelling properties in aqueous milieu, provides significant coupling sites, and may be applicable to the synthesis of difficult sequences and aggregation-prone peptides.

Affinity Maturation of B7-H6 Translates into Enhanced NK Cell-Mediated Tumor Cell Lysis and Improved Proinflammatory Cytokine Release of Bispecific Immunoligands via NKp30 Engagement.

Activating NK cell receptors represent promising target structures to elicit potent antitumor immune responses. In this study, novel immunoligands were generated that bridge the activating NK cell receptor NKp30 on NK cells with epidermal growth factor receptor (EGFR) on tumor cells in a bispecific IgG-like format based on affinity-optimized versions of B7-H6 and the Fab arm derived from cetuximab. To enhance NKp30 binding, the solitary N-terminal IgV domain of B7-H6 (ΔB7-H6) was affinity matured by an evolutionary library approach combined with yeast surface display. Biochemical and functional characterization of 36 of these novel ΔB7-H6-derived NK cell engagers revealed an up to 45-fold-enhanced affinity for NKp30 and significantly improved NK cell-mediated, EGFR-dependent killing of tumor cells compared with the NK cell engager based on the wild-type ΔB7-H6 domain. In this regard, potencies (EC50 killing) of the best immunoligands were substantially improved by up to 87-fold. Moreover, release of IFN-γ and TNF-α was significantly increased. Importantly, equipment of the ΔB7-H6-based NK cell engagers with a human IgG1 Fc part competent in Fc receptor binding resulted in an almost 10-fold superior killing of EGFR-overexpressing tumor cells compared with molecules either triggering FcγRIIIa or NKp30. Additionally, INF-γ and TNF-α release was increased compared with molecules solely triggering FcγRIIIa, including the clinically approved Ab cetuximab. Thus, incorporating affinity-matured ligands for NK cell-activating receptors might represent an effective strategy for the generation of potent novel therapeutic agents with unique effector functions in cancer immunotherapy.

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.

Lateral Dermal Penetration Is Dependent on the Lipophilicity of Active Ingredients.

INTRODUCTION: With its large surface area, skin facilitates a topical administration of active ingredients, and thus percutaneous delivery to a specific target site. Due to its high barrier function and different diffusion characteristics, skin governs the efficacy of these active ingredients and a bioavailability in the epidermal and dermal tissue. OBJECTIVE: In order to characterize the vertical and lateral movement of molecules into and inside the skin, the diffusivity of active ingredients with different physicochemical properties and their penetration ability in different dermal skin layers was investigated. METHODS: A novel lateral dermal microdialysis (MD) penetration setup was used to compare the diffusion characteristics of active ingredients into superficial and deep-implanted MD membranes in porcine skin. The corresponding membrane depth was determined via ultrasound and the active ingredients concentration via high-pressure liquid chromatography measurement. RESULTS: The depth depended penetration of superficial and deep-implanted MD membranes and the quantitative diffusivity of two active ingredients was compared. An experimental lateral MD setup was used to determine the influence of percutaneous skin penetration characteristics of an active ingredient with different lipophilic and hydrophilic characteristics. Therefore, hydrophilic caffeine and lipophilic LIP1, which have an identical molecular weight but different lipophilic characteristics, were tested for their penetration ability inside a propylene glycol and oleic acid formulation. CONCLUSION: The vertical and lateral penetration movement of caffeine was found to exceed that of LIP1 through the hydrophilic dermal environment. The findings of this study show that the lipophilicity of active ingredients influences the penetration movement and that skin enables a conical increasing lateral diffusivity and transdermal delivery.

Targeted Phagocytosis Induction for Cancer Immunotherapy via Bispecific MerTK-Engaging Antibodies.

The Tyro, Axl, and MerTK receptors (TAMRs) play a significant role in the clearance of apoptotic cells. In this work, the spotlight was set on MerTK, as it is one of the prominent TAMRs expressed on the surface of macrophages and dendritic cells. MerTK-specific antibodies were previously isolated from a transgenic rat-derived immune library with suitable biophysical properties. Further characterisation resulted in an agonistic MerTK antibody that led to phospho AKT activation in a dose-dependent manner. In this proof-of-concept study, a MerTK-specific antibody, MerK28, was combined with tandem, biparatopic EGFR-binding VHH camelid antibody domains (7D9G) in different architectures to generate bispecific antibodies with the capacity to bind EGFR and MerTK simultaneously. The bispecific molecules exhibited appropriate binding properties with regard to both targets in their soluble forms as well as to cells, which resulted in the engagement of macrophage-like THP-1 cells with epidermoid carcinoma A431 cells. Furthermore, targeted phagocytosis in co-culture experiments was observed only with the bispecific variants and not the parental MerTK-binding antibody. This work paves the way for the generation of bispecific macrophage-engaging antibodies for targeted phagocytosis harnessing the immune-modulating roles of MerTK in immunotherapy.

Sactipeptide Engineering by Probing the Substrate Tolerance of a Thioether-Bond-Forming Sactisynthase.

Sactipeptides are ribosomally synthesized peptides containing a unique sulfur to α-carbon crosslink. Catalyzed by sactisynthases, this thioether pattern endows sactipeptides with enhanced structural, thermal, and proteolytic stability, which makes them attractive scaffolds for the development of novel biotherapeutics. Herein, we report the in-depth study on the substrate tolerance of the sactisynthase AlbA to catalyze the formation of thioether bridges in sactipeptides. We identified a possible modification site within the sactipeptide subtilosin A allowing for peptide engineering without compromising formation of thioether bridges. A panel of natural and hybrid sactipeptides was produced to study the AlbA-mediated formation of thioether bridges, which were identified mass-spectrometrically. In a proof-of-principle study, we re-engineered subtilosin A to a thioether-bridged, specific streptavidin targeting peptide, opening the door for the functional engineering of sactipeptides.

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.

Use of 5-Thio-L-Fucose to modulate binding affinity of therapeutic proteins.

The reduction of antibody core-fucosylation is known to enhance antibody-dependent cellular cytotoxicity (ADCC). In this study, 5-Thio-l-Fucose (ThioFuc) was investigated as a media and feed supplement for modulating the fucosylation profile of therapeutic proteins and, thereby, improving the resulting effector functions. Glycan analysis of five different therapeutic proteins produced by a diverse set of Chinese hamster ovary cell lines demonstrated a clone dependent impact of ThioFuc treatment. Using rituximab as a model, an efficient dose- and time-dependent reduction of core-fucosylation up to a minimum of 5% were obtained by ThioFuc. Besides a concomitant increase in the afucosylation level up to 48%, data also revealed up to 47% incorporation of ThioFuc in place of core-fucosylation. In accordance with the glycan data, antibodies produced in the presence of ThioFuc revealed an enhanced FcγRIIIa binding up to 7.7-fold. Furthermore, modified antibodies subjected to a cell-based ADCC reporter bioassay proved to exert both a 1.5-fold enhanced ADCC efficacy and 2.6-fold enhancement in potency in comparison to their native counterparts-both of which contribute to an improvement in the ADCC activity. In conclusion, ThioFuc is a potent fucose derivative with potential applications in drug development processes.

Toward Fabrication of Bioactive Papers: Covalent Immobilization of Peptides and Proteins.

Herein, we report a novel two-step method for the covalent, site-directed, and efficient immobilization of proteins on lab-made paper sheets. First, paper fibers were modified with a peptidic anchor comprising enzyme recognition motifs. Four different conjugation strategies for peptide immobilization were evaluated with respect to reproducibility and fiber loading efficiency. After manufacturing of the peptide-preconditioned paper, oriented conjugation of the model protein tGFP containing a C-terminal recognition sequence for either sortase A or microbial transglutaminase was assessed semiquantitatively by fluorescence measurement and inspected by confocal laser scanning microscopy (CLSM). The two enzymes utilized for protein conjugation used the same oligoglycine peptide anchor, and both proved to be suitable for controlled oriented linkage of substrate proteins at physiological conditions.

Multivalent dextran hybrids for efficient cytosolic delivery of biomolecular cargoes.

The development of novel biotherapeutics based on peptides and proteins is often limited to extracellular targets, because these molecules are not able to reach the cytosol. In recent years, several approaches were proposed to overcome this limitation. A plethora of cell-penetrating peptides (CPPs) was developed for cytoplasmic delivery of cell-impermeable cargo molecules. For many CPPs, multimerization or multicopy arrangement on a scaffold resulted in improved delivery but also higher cytotoxicity. Recently, we introduced dextran as multivalent, hydrophilic polysaccharide scaffold for multimerization of cell-targeting cargoes. Here, we investigated covalent conjugation of a CPP to dextran in multiple copies and assessed the ability of resulted molecular hybrid to enter the cytoplasm of mammalian cells without largely compromising cell viability. As a CPP, we used a novel, low-toxic cationic amphiphilic peptide L17E derived from M-lycotoxin. Here, we show that cell-penetrating properties of L17E are retained upon multivalent covalent linkage to dextran. Dextran-L17E efficiently mediated cytoplasmic translocation of an attached functional peptide and a peptide nucleic acid (PNA). Moreover, a synthetic route was established to mask the lysine side chains of L17E with a photolabile protecting group thus opening avenues for light-triggered activation of cellular uptake.

Dissecting capture and twisting of aureolysin and pseudolysin: functional amino acids of the Dispase autolysis-inducing protein.

The Dispase autolysis-inducing protein (DAIP) from Streptomyces mobaraensis attracts M4 metalloproteases, which results in inhibition and autolysis of bacillolysin (BL) and thermolysin (TL). The present study shows that aureolysin (AL) from Staphylococcus aureus and pseudolysin (LasB) from Pseudomonas aeruginosa are likewise impaired by DAIP. Complete inhibition occurred when DAIP significantly exceeded the amount of the target protease. At low DAIP concentrations, AL and BL performed autolysis, while LasB and TL degradation required reductants or detergents that break intramolecular disulfide bonds or change the protein structure. Site directed mutagenesis of DAIP and removal of an exposed protein loop either influenced binding or inhibition of AL and TL but had no effect on LasB and BL. The Y170A and Δ239-248 variants had completely lost affinity for TL and AL. The exchange of Asn-275 also impaired the interaction of DAIP with AL. In contrast, DAIP Phe-297 substitution abolished inhibition and autolysis of both target proteases but still allowed complex formation. Our results give rise to the conclusion that other, yet unknown DAIP amino acids inactivate LasB and BL. Obviously, various bacteria in the same habitat caused Streptomyces mobaraensis to continuously optimize DAIP in inactivating the tackling metalloproteases.

A tightly regulated and adjustable CRISPR-dCas9 based AND gate in yeast.

The robust and precise on and off switching of one or more genes of interest, followed by expression or repression is essential for many biological circuits as well as for industrial applications. However, many regulated systems published to date influence the viability of the host cell, show high basal expression or enable only the overexpression of the target gene without the possibility of fine regulation. Herein, we describe an AND gate designed to overcome these limitations by combining the advantages of three well established systems, namely the scaffold RNA CRISPR/dCas9 platform that is controlled by Gal10 as a natural and by LexA-ER-AD as heterologous transcription factor. We hence developed a predictable and modular, versatile expression control system. The selection of a reporter gene set up combining a gene of interest (GOI) with a fluorophore by the ribosomal skipping T2A sequence allows to adapt the system to any gene of interest without losing reporter function. In order to obtain a better understanding of the underlying principles and the functioning of our system, we backed our experimental findings with the development of a mathematical model and single-cell analysis.

Comparison of Membrane Depth Determination Techniques for Active Ingredient Skin Penetration Studies Using Microdialysis.

INTRODUCTION: The skin is a major physical barrier to the environment, and thus, percutaneous delivery of active ingredients to the dermal target site faces a unique set of hurdles. The efficacy of these active ingredients is governed by their release into the underlying epidermal and dermal tissue, especially when administered topically. OBJECTIVE: The aim of this study was to understand if different physicochemical properties influence the skin penetration of active ingredients and the depth to which they penetrate into the dermis. METHODS: A microdialysis (MD) setup was used to compare the percutaneous penetration in superficial and deep implanted MD membranes in porcine skin. The precise MD membrane depth was determined using histological sectioning paired with microscopy, ultrasound, and a novel computed tomographic approach. RESULTS: In study A, the measured depth of the superficial and deep implanted MD membranes was compared using histological sectioning, ultrasound, and computed tomography. Experimental determination of the depth up to which penetration occurs was found to be crucial to percutaneous penetration studies. In study B, the lipophilic differences of the active ingredients and its influences on the penetration was tested using hydrophilic caffeine and lipophilic LIP1 as model compounds, which have an identical molecular weight with different lipophilic characteristics. It is assumed that the lipophilic characteristics of active ingredients influence their penetration and thus governs the concentration of these molecules reaching their target site. CONCLUSION: The transdermal penetration of caffeine was found to exceed that of LIP1 through the hydrophilic environment of the dermis. Thus, the findings of this study show that the precise MD dermis localization and the physicochemical properties, such as lipophilicity, influence the penetration rate of active ingredients and lay the foundation for creating optimized transdermal delivery systems.

Vom Huhn abgeleitete Antikörper für Diagnostik und Immuntherapie.

Due to the large evolutionary distance between birds (Aves) und humans, immunization of chickens with human proteins results in a strong response of the bird's adaptive immune system to proteins of mammalian origin. Additionally, chicken-derived antibodies display less undesired cross-reactivity in analytical setups than conventional rodent-derived antibodies. Due to these features as well as the facile amplification of antibody-coding genes, chicken-derived antibodies emerged as promising molecules for the immunotherapy and various biotechnological applications.

Recent progress in transglutaminase-mediated assembly of antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are hybrid molecules intended to overcome the drawbacks of conventional small molecule chemotherapy and therapeutic antibodies by merging beneficial characteristics of both molecule classes to develop more efficient and patient-friendly options for cancer treatment. During the last decades a versatile bioconjugation toolbox that comprises numerous chemical and enzymatic technologies have been developed to covalently attach a cytotoxic cargo to a tumor-targeting antibody. Microbial transglutaminase (mTG) that catalyzes isopeptide bond formation between proteinaceous or peptidic glutamines and lysines, provides many favorable properties that are beneficial for the manufacturing of these conjugates. However, to efficiently utilize the enzyme for the constructions of ADCs, different drawbacks had to be overcome that originate from the enzyme's insufficiently understood substrate specificity. Within this review, pioneering methodologies, recent achievements and remaining limitations of mTG-assisted assembly of ADCs will be highlighted.