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.

TRAIL-Inspired Multivalent Dextran Conjugates Efficiently Induce Apoptosis upon DR5 Receptor Clustering.

Triggering apoptosis of tumor cells has been in focus of cancer-inspired research since decades. As clustering of death receptor 5 (DR5), which is overexpressed on various cancer cells, leads to formation of the death-inducing signaling cascade (DISC), DR5 has recently become a promising target for tumor treatment. Herein, we demonstrate that covalent multimerization of a death receptor targeting peptide (DR5TP) on a dextran scaffold generates potent apoptosis-inducing conjugates (EC50 =2-20 nm). A higher conformational flexibility compared to reported DR5TP multimerization approaches, introduced by the polysaccharide framework compensates the reported need for the defined ligand orientation that was considered as essential prerequisite for effective receptor clustering and apoptosis induction. Enzyme-catalyzed ligation of a hydrophilic dextran conjugate bearing multiple DR5-targeting sites to a human fragment crystallizable (Fc) receptor did not affect the potency (EC50 =2-7 nm), providing an option for improved in vivo half-life and prospective conjugation to an antibody of interest in view of bispecific tumor targeting.

Light-Controlled Chemoenzymatic Immobilization of Proteins towards Engineering of Bioactive Papers.

Efficient and reliable methods for the generation of bioactive papers are of growing interest in relation to point-of-care testing devices that do not require extensive analytical equipment. Herein, we report the immobilization of functional proteins on paper fibers using a modular chemoenzymatic approach. The synthetic strategy relies on a combination of highly efficient spatially controllable photo-triggered cycloaddition followed by site-specific sortase A-catalyzed transamidation. This site-directed and regiospecific method has allowed unidirectional and covalent immobilization of several proteins displaying different functional properties, with ramifications for application in paper-based diagnostics.

Directed Evolution of a Bond-Forming Enzyme: Ultrahigh-Throughput Screening of Microbial Transglutaminase Using Yeast Surface Display.

Microbial transglutaminase from Streptomyces mobaraensis (mTG) has emerged as a useful biotechnological tool due to its ability to crosslink a side chain of glutamine and primary amines. To date, the substrate specificity of mTG is not fully understood, which poses an obvious challenge when mTG is used to address novel targets. To that end, a viable strategy providing an access to tailor-made transglutaminases is required. This work reports an ultrahigh-throughput screening approach based on yeast surface display and fluorescence-activated cell sorting (FACS) that enabled the evolution of microbial transglutaminase towards enhanced activity. Five rounds of FACS screening followed by recombinant expression of the most potent variants in E. coli yielded variants that possessed, compared to the wild type enzyme, improved enzymatic performance and labeling behavior upon conjugation with an engineered therapeutic anti-HER2 antibody. This robust and generally applicable platform enables tailoring of the catalytic efficiency of mTG.

A Two-Step Golden Gate Cloning Procedure for the Generation of Natively Paired YSD Fab Libraries.

In vitro antibody display libraries have emerged as powerful tools for a streamlined discovery of novel antibody binders. While in vivo antibody repertoires are matured and selected as a specific pair of variable heavy and light chains (VH and VL) with optimal specificity and affinity, during the recombinant generation of in vitro libraries, the native sequence pairing is not maintained. Here we describe a cloning method that combines the flexibility and versatility of in vitro antibody display with the advantages of natively paired VH-VL antibodies. In this regard, VH-VL amplicons are cloned via a two-step Golden Gate cloning procedure, allowing the display of Fab fragments on yeast cells.

An integrated mammalian library approach for optimization and enhanced microfluidics-assisted antibody hit discovery.

Recent years have seen the development of a variety of mammalian library approaches for display and secretion mode. Advantages include library approaches for engineering, preservation of precious immune repertoires and their repeated interrogation, as well as screening in final therapeutic format and host. Mammalian display approaches for antibody optimization exploit these advantages, necessitating the generation of large libraries but in turn enabling early screening for both manufacturability and target specificity. For suitable libraries, high antibody integration rates and resulting monoclonality need to be balanced - we present a solution for sufficient transmutability and acceptable monoclonality by applying an optimized ratio of coding to non-coding lentivirus. The recent advent of microfluidic-assisted hit discovery represents a perfect match to mammalian libraries in secretion mode, as the lower throughput fits well with the facile generation of libraries comprising a few million functional clones. In the presented work, Chinese Hamster Ovary cells were engineered to both express the target of interest and secrete antibodies in relevant formats, and specific clones were strongly enriched by high throughput screening for autocrine cellular binding. The powerful combination of mammalian secretion libraries and microfluidics-assisted hit discovery could reduce attrition rates and increase the probability to identify the best possible therapeutic antibody hits faster.

Isolation of Antigen-Specific Unconventional Bovine Ultra-Long CDR3H Antibodies Using Cattle Immunization in Combination with Yeast Surface Display.

Cattle are known for their repertoire of antibodies harboring extremely long CDR3H regions that form extensive "knob on stalk" cysteine-rich structures. The compact knob domain allows for the recognition of epitopes potentially not accessible to classical antibodies. To effectively access the potential of bovine-derived antigen-specific ultra-long CDR3 antibodies, a straightforward and effective high-throughput method based on yeast surface display and fluorescence-activated cell sorting is described.

One-Pot Droplet RT-OE-PCR for the Generation of Natively Paired Antibody Immune Libraries.

Classical yeast surface display (YSD) antibody immune libraries are generated by a separate amplification of heavy- and light-chain antibody variable regions (VH and VL, respectively) and subsequent random recombination during the molecular cloning procedure. However, each B cell receptor comprises a unique VH-VL combination, which has been selected and affinity matured in vivo for optimal stability and antigen binding. Thus, the native variable chain pairing is important for the functioning and biophysical properties of the respective antibody. Herein, we present a method for the amplification of cognate VH-VL sequences, compatible with both next-generation sequencing (NGS) and YSD library cloning. We employ a single B cell encapsulation in water-in-oil droplets, followed by a one-pot reverse transcription overlap extension PCR (RT-OE-PCR), resulting in a paired VH-VL repertoire from more than a million B cells in a single day.

Cattle-derived knob paratopes grafted onto peripheral loops of the IgG1 Fc region enable the generation of a novel symmetric bispecific antibody format.

In this work we present a novel symmetric bispecific antibody format based on engraftments of cattle-derived knob paratopes onto peripheral loops of the IgG1 Fc region. For this, knob architectures obtained from bovine ultralong CDR-H3 antibodies were inserted into the AB loop or EF loop of the CH3 domain, enabling the introduction of an artificial binding specificity into an IgG molecule. We demonstrate that inserted knob domains largely retain their binding affinities, resulting into bispecific antibody derivatives versatile for effector cell redirection. Essentially, generated bispecifics demonstrated adequate biophysical properties and were not compromised in their Fc mediated functionalities such as FcRn or FcγRIIIa binding.

Generation and engineering of potent single domain antibody-based bispecific IL-18 mimetics resistant to IL-18BP decoy receptor inhibition.

Here, we generated bispecific antibody (bsAb) derivatives that mimic the function of interleukin (IL)-18 based on single domain antibodies (sdAbs) specific to IL-18 Rα and IL-18 Rß. For this, camelids were immunized, followed by yeast surface display (YSD)-enabled discovery of VHHs targeting the individual receptor subunits. Upon reformatting into a strictly monovalent (1 + 1) bispecific sdAb architecture, several bsAbs triggered dose-dependent IL-18 R downstream signaling on IL-18 reporter cells, as well as IFN-γ release by peripheral blood mononuclear cells in the presence of low-dose IL-12. However, compared with IL-18, potencies and efficacies were considerably attenuated. By engineering paratope valencies and the spatial orientation of individual paratopes within the overall design architecture, we were able to generate IL-18 mimetics displaying significantly augmented functionalities, resulting in bispecific cytokine mimetics that were more potent than IL-18 in triggering proinflammatory cytokine release. Furthermore, generated IL-18 mimetics were unaffected from inhibition by IL-18 binding protein decoy receptor. Essentially, we demonstrate that this strategy enables the generation of IL-18 mimetics with tailor-made cytokine functionalities.

The influence of L-proline and fulvic acid on oxidative stress and semen quality of buffalo bull semen following cryopreservation.

BACKGROUND: This study investigates the effects of cryopreservation and supplementation of Azeri water buffalo's semen with proline (Lp) and fulvic acid (FA). OBJECTIVES: Therefore, this study aimed to assess motility parameters, sperm viability, oxidative stress parameters, and DNA damage to detect the optimum concentrations of Lp and FA for buffalo semen cryopreservation. METHODS: Thirty semen samples of three buffalo bulls were diluted in Tris-egg yolk extender and divided into 12 equal groups including control (C), Lp-10, Lp-20, Lp-40, Lp-60, Lp-80 (containing 10, 20, 40, 60, 80 mM L-proline, respectively), FA-0.2, FA-0.5, FA-0.8, FA-1.1, FA-1.4 and FA-1.7 (containing 0.2%, 0.5%, 0.8%, 1.1%, 1.4% and 1.7% fulvic acid, respectively). RESULTS: The velocity parameters, TM and PM were improved by FA-1.7, FA-1.4, Lp-40 and Lp-60 groups compared to the C group but no significant difference was found regarding the amplitude of lateral head displacement and straightness compared to the control groups. The percentage of sperm viability and PMF were increased by FA-1.7, FA-1.4, FA-1.1, Lp-40 and Lp-60 groups compared to C group, while in terms of sperm DNA damage FA-1.7, FA-1.4, FA-1.1, Lp-10, Lp-20, Lp-40 and Lp-60 groups showed better results compared to C group. The results also showed that FA-1.7, FA-1.4, FA-1.1, Lp-20, Lp-40 and Lp-60 groups could improve TAC, SOD, GSH and decrease MDA levels. Also, FA-1.7, FA-1.4, Lp-20 and Lp-40 groups could improve GPx levels but just FA-1.7, and Lp-40 groups could improve CAT levels compared to C group. CONCLUSIONS: Thus, it can be concluded that L-proline and fulvic acid supplementations can improve the quality parameters of post-thawed buffalo bull semen.

Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities.

Controlled Fab arm exchange (cFAE) has proven to be a generic and versatile technology for the efficient generation of IgG-like bispecific antibodies (DuoBodies or DBs), with several in clinical development and one product, amivantamab, approved by the Food and Drug Administration. In this study, we expand the cFAE-toolbox by incorporating VHH-modules at the C-termini of DB-IgGs, termed DB-VHHs. This approach enables the combinatorial generation of tri- and tetraspecific molecules with flexible valencies in a straightforward fashion. Using cFAE, a variety of multispecific molecules was produced and assessed for manufacturability and physicochemical characteristics. In addition, we were able to generate DB-VHHs that efficiently triggered natural killer cell mediated lysis of tumor cells, demonstrating the utility of this format for potential therapeutic applications.

Isolation of Common Light Chain Antibodies from Immunized Chickens Using Yeast Biopanning and Fluorescence-Activated Cell Sorting.

The phylogenetic distance between chickens and humans accounts for a strong immune response and a broader epitope coverage compared to rodent immunization approaches. Here the authors report the isolation of common light chain (cLC)-based chicken monoclonal antibodies from an anti-epidermal growth factor receptor (EGFR) immune library utilizing yeast surface display in combination with yeast biopanning and fluorescence-activated cell sorting (FACS). For the selection of high-affinity antibodies, a yeast cell library presenting cLC-comprising fragment antigen binding (Fab) fragments is panned against hEGFR-overexpressing A431 cells. The resulting cell-cell-complexes are sorted by FACS resulting in gradual enrichment of EGFR-binding Fabs in three sorting rounds. The isolated antibodies share the same light chain and show high specificity for EGFR, resulting in selective binding to A431 cells with notable EC50 values. All identified antibodies show very good aggregation propensity profiles and thermostabilities. Additionally, epitope binning demonstrates that these cLC antibodies cover a broad epitope space. Isolation of antibodies from immunized chickens by yeast cell biopanning makes an addition to the repertoire of methods for antibody library screening, paving the way for the generation of cLC-based bispecific antibodies against native mammalian receptors.

Protease-Activation of Fc-Masked Therapeutic Antibodies to Alleviate Off-Tumor Cytotoxicity.

The interaction of the Fc region of therapeutic antibodies and antibody-drug conjugates with Fcγ receptors (FcγRs) can lead to unpredictable and severe side effects. Over the last decades several strategies have been developed to overcome this drawback, including extensive Fc- and glycoengineering and antibody isotype switching. However, these approaches result in permanently Fc-silenced antibody derivates which partially or completely lack antibody-mediated effector functions. Nevertheless, for a majority of antibody-based drugs, Fc-mediated effector functions, like antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) as well as complement-dependent cytotoxicity (CDC), represent the most substantial modes of action. We argued that a new strategy combining the beneficial properties of Fc-silencing and controlled activation of effector functions can pave the way to potent antibody therapeutics, reducing the FcγRs-mediated off-target toxicity. We present a novel Fc-tamed antibody format, where the FcγR-binding sites of antibodies are blocked by anti-isotypic masking units, hindering the association of FcγR and complement component 1 (c1q) to the Fc domain. The masking units were genetically fused to trastuzumab, including a protease-addressable peptide-liker. Our Fc-tamed antibodies demonstrated completely abolished interaction to soluble high-affinity Fcγ-Receptor I and c1q. In reporter cell-based ADCC assays, our Fc-tamed antibodies exhibited a 2,700 to 7,100-fold reduction in activation, compared to trastuzumab. Upon demasking by a tumor-associated protease, the Fc-activated antibodies demonstrated restored FcγR-binding, c1q-binding and the ability to induce potent ADCC activation. Furthermore, cell killing assays using donor-derived NK cells were performed to validate the functionality of the Fc-tamed antibody variants. To our knowledge, this approach represents the first non-permanently Fc-silenced antibody, which can be re-activated by a tumor-associated protease, eventually extending the field of novel antibody formats.

Enhancing the Pharmacokinetics and Antitumor Activity of an α-Amanitin-Based Small-Molecule Drug Conjugate via Conjugation with an Fc Domain.

Herein we describe the design and biological evaluation of a novel antitumor therapeutic platform that combines the most favorable properties of small-molecule drug conjugates (SMDCs) and antibody drug conjugates (ADCs). Although the small size of SMDCs, compared to ADCs, is an appealing feature for their application in the treatment of solid tumors, SMDCs usually suffer from poor pharmacokinetics, which severely limits their therapeutic efficacy. To overcome this limitation, in this proof-of-concept study we grafted an α-amanitin-based SMDC that targets prostate cancer cells onto an immunoglobulin Fc domain via a two-step "program and arm" chemoenzymatic strategy. We demonstrated the superior pharmacokinetic properties and therapeutic efficacy of the resulting Fc-SMDC over the SMDC in a prostate cancer xenograft mouse model. This approach may provide a general strategy toward effective antitumor therapeutics combining small size with pharmacokinetic properties close to those of an ADC.

Milking the Cow: Cattle-Derived Chimeric Ultralong CDR-H3 Antibodies and Their Engineered CDR-H3-Only Knobbody Counterparts Targeting Epidermal Growth Factor Receptor Elicit Potent NK Cell-Mediated Cytotoxicity.

In this work, we have generated epidermal growth factor receptor (EGFR)-specific cattle-derived ultralong CDR-H3 antibodies by combining cattle immunization with yeast surface display. After immunization, ultralong CDR-H3 regions were specifically amplified and grafted onto an IGHV1-7 scaffold by homologous recombination to facilitate Fab display. Antigen-specific clones were readily obtained by fluorescence-activated cell sorting (FACS) and reformatted as chimeric antibodies. Binning experiments revealed epitope targeting of domains I, II, and IV of EGFR with none of the generated binders competing with Cetuximab, Matuzumab, or EGF for binding to EGFR. Cattle-derived chimeric antibodies were potent in inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against EGFR-overexpressing tumor cells with potencies (EC50 killing) in the picomolar range. Moreover, most of the antibodies were able to significantly inhibit EGFR-mediated downstream signaling. Furthermore, we demonstrate that a minor fraction of CDR-H3 knobs derived from generated antibodies was capable of independently functioning as a paratope facilitating EGFR binding when grafted onto the Fc part of human IgG1. Besides slightly to moderately diminished capacities, these engineered Knobbodies largely retained main properties of their parental antibodies such as cellular binding and triggering of ADCC. Hence, Knobbodies might emerge as promising tools for biotechnological applications upon further optimization.

FACS-Based Functional Protein Screening via Microfluidic Co-encapsulation of Yeast Secretor and Mammalian Reporter Cells.

In this study, we present a straightforward approach for functional cell-based screening by co-encapsulation of secretor yeast cells and reporter mammalian cells in millions of individual agarose-containing microdroplets. Our system is compatible with ultra-high-throughput selection utilizing standard fluorescence-activated cell sorters (FACS) without need of extensive adaptation and optimization. In a model study we co-encapsulated murine interleukin 3 (mIL-3)-secreting S. cerevisiae cells with murine Ba/F3 reporter cells, which express green fluorescent protein (GFP) upon stimulation with mIL-3, and could observe specific and robust induction of fluorescence signal compared to a control with yeast cells secreting a non-functional mIL-3 mutant. We demonstrate the successful enrichment of activating mIL-3 wt-secreting yeast cells from a 1:10,000 dilution in cells expressing the inactive cytokine variant by two consecutive cycles of co-encapsulation and FACS. This indicates the suitability of the presented strategy for functional screening of high-diversity yeast-based libraries and demonstrates its potential for the efficient isolation of clones secreting bioactive recombinant proteins.

Dextramabs: A Novel Format of Antibody-Drug Conjugates Featuring a Multivalent Polysaccharide Scaffold.

Antibody-drug conjugates (ADCs) are multicomponent biomolecules that have emerged as a powerful tool for targeted tumor therapy. Combining specific binding of an immunoglobulin with toxic properties of a payload, they however often suffer from poor hydrophilicity when loaded with a high amount of toxins. To address these issues simultaneously, we developed dextramabs, a novel class of hybrid antibody-drug conjugates. In these architectures, the therapeutic antibody trastuzumab is equipped with a multivalent dextran polysaccharide that enables efficient loading with a potent toxin in a controllable fashion. Our modular chemoenzymatic approach provides an access to synthetic dextramabs bearing monomethyl auristatin as releasable cytotoxic cargo. They possess high drug-to-antibody ratios, remarkable hydrophilicity, and high toxicity in vitro.

Yeast Surface Display in Combination with Fluorescence-activated Cell Sorting Enables the Rapid Isolation of Antibody Fragments Derived from Immunized Chickens.

Yeast surface display emerged as a viable tool for the generation of human and murine monoclonal antibodies. This platform technology enables the careful definition of selection conditions, the potential for high-throughput screening, as well as the isolation of antibodies recognizing predefined epitopes. In this study, the applicability of yeast surface display in combination with fluorescence-activated cell sorting (FACS) for the isolation of antigen-specific chicken-derived antibodies is demonstrated. To this end, yeast-displayed recombinant antibody libraries from splenic mRNA of chickens immunized with epidermal growth factor receptor (EGFR) and human chorionic gonadotropin (hCG) were constructed as single chain variable fragments (scFv) by overlap extension polymerase chain reaction. A large number of antigen binding scFvs were readily isolated in a convenient screening process. Target-specific scFv-Fc molecules were produced as soluble proteins and more extensively characterized by confirming specificity, thermostability and high affinity. Essentially, we demonstrated the biotechnological applicability of binders directed against both antigens via specific cellular binding for EGFR and in the context of a lateral flow test by utilizing hCG-binding scFvs as capturing antibodies for pregnancy detection. Altogether, the described strategy using yeast surface display expands the repertoire of display methods for the isolation of antibodies resulting from chicken immunization campaigns.

Semi-synthetic vNAR libraries screened against therapeutic antibodies primarily deliver anti-idiotypic binders.

Anti-idiotypic binders which specifically recognize the variable region of monoclonal antibodies have proven to be robust tools for pharmacokinetic studies of antibody therapeutics and for the development of cancer vaccines. In the present investigation, we focused on the identification of anti-idiotypic, shark-derived IgNAR antibody variable domains (vNARs) targeting the therapeutic antibodies matuzumab and cetuximab for the purpose of developing specific capturing ligands. Using yeast surface display and semi-synthetic, CDR3-randomized libraries, we identified several highly specific binders targeting both therapeutic antibodies in their corresponding variable region, without applying any counter selections during screening. Importantly, anti-idiotypic vNAR binders were not cross-reactive towards cetuximab or matuzumab, respectively, and comprised good target recognition in the presence of human and mouse serum. When coupled to magnetic beads, anti-idiotypic vNAR variants could be used as efficient capturing tools. Moreover, a two-step procedure involving vNAR-functionalized beads was employed for the enrichment of potentially bispecific cetuximab × matuzumab antibody constructs. In conclusion, semi-synthetic and CDR3-randomized vNAR libraries in combination with yeast display enable the fast and facile identification of anti-idiotypic vNAR domains targeting monoclonal antibodies primarily in an anti-idiotypic manner.