In this study, we present a strategy to uncouple tumor necrosis factor (TNF)-like cell death induction from TNFR2 agonism in a tumor-targeted fashion. Single-domain antibodies (sdAbs) targeting TNFR1 were generated by combining camelid immunization with yeast surface display. Reformatting of resulting paratopes as bispecific antibodies (bsAbs) in a 2 + 2 manner by employing an sdAb-based paratope targeting HER2 revealed the identification of an immunocytokine-like bsAb, referred to as immunocytokine mimetic (ICM), which triggered TNF-like tumor cell death of HER2-overexpressing cancer cells as well as robust caspase-1,-3, and-8 activation in a cis-targeted manner. By modulating the valency of the TNFR1-directed sdAb, killing capacities as well as caspase activities of HER2-targeted ICMs were significantly augmented, eventually resulting in enhanced cell death induction when compared with TNF. Moreover, HER2-targeted TNFR1 ICMs also displayed a beneficial, i.e., substantially reduced profile in inducing unconditional pro-inflammatory cytokine release from peripheral blood mononuclear cells (PBMCs).

In this work, we developed bispecific antibody (bsAb)-derived surrogate agonists which mimic the function of IL-21 by targeting the IL-21 receptor composed of IL-21 R (CD360) and IL-2 Rγ (CD132). For this, antigen-specific VHHs (variable domains of the heavy chain of heavy-chain-only antibodies) were obtained by immunization of camelids and isolated using yeast surface display. Combinatorial reformatting of IL-21 R-specific single‑domain antibodies (sdAbs) and IL-2 Rγ-targeting paratopes into a monovalent bispecific antibody architecture enabled the identification of IL-21 mimetics displaying attenuated capacities in triggering STAT3 phosphorylation compared to the wild-type cytokine as demonstrated in NK-92 cells as well as peripheral blood mononuclear cells (PBMCs). Moreover, by applying different protein engineering strategies, we demonstrate that agonism capacities of the generated IL-21 mimetics, such as pSTAT3 induction or Granzyme B expression of cytotoxic T cells, can be significantly optimized. For this, framework mutations were introduced to engineer VHH:VHH interactions within the bispecific sdAb-Fc fusion geometry for a more rigid receptor targeting. Furthermore, we show that antibody format engineering, in which the VHHs were arranged in an IgG-like scaffold that replaces the conventional IgG VH and VL domains with the corresponding VHHs, combined with rigidifying mutations, enables IL-21 R agonism comparable to the wild-type cytokine. Taken together, these findings show that IL-21 receptor agonism can be substantially optimized by adapting the spatial orientation of paratopes targeting both receptor subunits via forced dimerization, without altering paratope valencies.

Allosteric regulation provides a powerful framework for modulating receptor signaling in both physiological and therapeutic contexts. The epidermal growth factor receptor (EGFR), a receptor tyrosine kinase frequently dysregulated in cancer, undergoes activation through conformational transitions that couple extracellular ligand binding to intracellular kinase signaling. Here, we explore how camelid derived VHH (variable domain of the heavy chain of a heavy chain-only)-antibodies can exploit this allosteric architecture to inhibit EGFR function. Using a panel of single domain monospecific and biparatopic antibodies, targeting non-overlapping EGFR epitopes, we combined experimental assays with structure-based modeling to dissect their effects on EGFR signaling and internalization. AlphaFold3-predicted EGFR-antibody complexes were analyzed using the Structure-Based Statistical Mechanical Model of Allostery (SBSMMA) to compute residue-level allosteric modulations induced upon binding. The resulting profiles revealed that only a subset of epitope combinations produced long-range allosteric responses reaching the juxtamembrane segment and the kinase domain. These patterns correlated with effective inhibition of downstream ERK and AKT signaling in cellular assays. In contrast, some constructs with high internalization capacity induced minimal allosteric propagation and weak signaling suppression, indicating a mechanistic decoupling of receptor uptake from conformational regulation. Together, these results define distinct allosteric modes of EGFR modulation by VHH-antibodies and show how computational modeling based on energetic propagation can complement experimental screening to guide the design of next-generation allosteric biologics.

Immunoglobulin (Ig) A has attracted interest as a proposed therapeutic agent due to its ability to engage cell groups differently compared to an IgG scaffold and elicit tumor eradication. Further, its multimeric forms enable increased flexibility in the design of available paratopes. The latter is particularly advantageous for bi- and multispecific antibody formats, which are unparalleled in their enhanced selectivity and unique biological functions. We engineered bispecific heterodimeric IgA-based antibodies using the strand-exchanged engineered domain (SEED) technology, which relies on intertwined segments of IgA and IgG in the CH3 domain, and applied mutagenesis to introduce two additional binding sites to enable the interaction of IgA-Fc with the myeloid cell-activating receptor CD89 (FcαR). These antibodies exhibited good biophysical properties and thermostability similar to the parental SEED molecule. Binding capacity to both antigens recognized by variable domains, epidermal growth factor receptor (EGFR) and receptor tyrosine kinase like orphan receptor 1 (ROR1), was not impaired, and in contrast to the original SEED-IgA, trispecific mutants could bind to CD89-expressing cells, mediate tumor cell-effector cell clustering, and induce neutrophil-mediated specific lysis of tumor cells. Trispecific design was applicable to both SEED-IgA1 and -IgA2 scaffolds. Interestingly, HEK-expressed mutants featured a CH2-linked N-glycan pattern more similar to wild-type IgA, with reduced core fucosylation in comparison with IgA-SEED. Collectively, the presented format combines the mobilization of CD89-positive effector cells with the flexibility of incorporating antigen specificities of choice into the variable domains, and thus is a promising basis for biochemically stable multispecific IgA with high therapeutic potential.

In this work, we have generated bispecific interleukin (IL)-12 surrogate agonists based on camelid-derived single-domain antibodies (sdAbs) targeting the IL-12 receptor (IL-12R) subunits IL-12Rβ1 and IL-12Rβ2. Following immunization and antibody display-based paratope isolation, respective sdAbs were combinatorially reformatted into a monovalent bispecific architecture by grafting resulting paratopes onto the hinge region of a heterodimeric Fc region. Functional characterization using NK-92 cells enabled the identification of multiple different sdAb-based bispecifics displaying divergent IL-12R agonism capacities as analyzed by STAT4 phosphorylation. Further investigations by harnessing peripheral blood mononuclear cells (PBMCs) from healthy donors revealed attenuated pSTAT4 activation compared to recombinant human (rh) wild-type IL-12 regarding both natural killer (NK)-cell and T-cell activation but robust IL-12R agonism on stimulated T cells. While several sdAb-based IL-12 mimetics were nearly inactive on NK cells as well as T cells obtained from PBMCs, they elicited significant STAT4 phosphorylation and interferon (IFN)-γ release on stimulated T cells as well as an IL-12-like transcriptional signature. Furthermore, we demonstrate that the activity of receptor agonism of generated bispecific IL-12 mimetics can also be biased towards stimulated T cells by changing the spatial orientation of the individual sdAbs within the molecular design architecture. Taken together, we present an alternative strategy to generate IL-12-like biologics with tailor-made characteristics.

In this work, we report the discovery and engineering of allosteric variable domains of the heavy chain (VHHs) derived from camelid immunization targeting NKp30, an activating receptor on natural killer (NK) cells. The aim was to enhance NK cell-mediated killing capacities by identifying VHHs that do not compete with the natural ligand of NKp30:B7-H6, thereby maximizing the recognition of B7-H6+ tumor cells. By relying on the DuoBody technology, bispecific therapeutic antibodies were engineered, creating a panel of bispecific antibodies against NKp30xEGFR (cetuximab moiety) or NKp30xHER2 (trastuzumab moiety), called natural killer cell engagers (NKCEs). These NKCEs were assessed for their killing capacities on B7-H6-expressing tumor cells. The results demonstrated an enhancement in NK killing capacities for both EGFR-expressing (HeLa) and HER2-expressing (SK-BR-3) cells, indicating the significance of the natural NKp30/B7-H6 axis in tumor recognition by the immune system. Notably, engineering NKCEs to allow natural recognition of B7-H6 was found to be more effective in promoting NKCE-mediated killing of B7-H6+ tumor cells via enhancement of cytokine release. This study highlights the potential of an enhanced-targeting approach, wherein tumor cell surface antigens are targeted while still enabling the natural recognition of the activating ligand (B7-H6) by the immune cells.

In this work, we report the discovery of potent anti-epidermal growth factor receptor (EGFR) allosteric heavy-chain antibodies by combining camelid immunization and fluorescence-activated cell sorting (FACS). After immunization and yeast surface display library construction, allosteric clones were obtained by introducing the labeled EGF Fc fusion protein as an additional criterion for FACS. This sorting method enabled the identification of 11 heavy-chain antibodies that did not compete with the orthosteric ligand EGF for the binding to EGFR. These antibodies bind to a triple-negative breast cancer cell line expressing EGFR with affinities in the picomolar to nanomolar range. Those camelid-derived antibodies also exhibit interesting properties by modulating EGFR affinity for EGF. Moreover, they are also able to inhibit EGF-induced downstream signaling pathways. In particular, we identified one clone that is more potent than the approved blocking antibody cetuximab in inhibiting both PI3K/AKT and MAPK/ERK pathways. Our results suggest that allosteric antibodies may be potential new modalities for therapeutics.

Allostery represents a fundamental mechanism in protein regulation, enabling modulation of protein function from sites distal to the active site. While traditionally explored in the context of small molecules, allosteric modulation is gaining traction as a main mode of action in the realm of antibodies, which offer enhanced specificity and reduced toxicity. This review delves into the rapidly growing field of allosteric antibodies, highlighting recent therapeutic advancements and novel druggability avenues. We also explore the potential of these antibodies as innovative tools in drug discovery and discuss contemporary strategies for designing novel allosteric antibodies, leveraging state-of-the-art computational approaches.

In this work, we report the development of a platform for the early selection of non-competitive antibody-fragments against cell surface receptors that do not compete for binding of their natural ligand. For the isolation of such subtype of blocking antibody-fragments, we applied special fluorescence-activated cell sorting strategies for antibody fragments isolation from yeast surface display libraries. Given that most of the monoclonal antibodies approved on the market are blocking ligand-receptor interactions often leading to resistance and/or side effects, targeting allosteric sites represents a promising mechanism of action to open new avenues for treatment. To directly identify these antibody-fragments during library screening, we employed immune libraries targeting the epidermal growth factor receptor as proof of concept. Incorporating a labeled orthosteric ligand during library sorting enables the early selection of non-competitive binders and introduces an additional criterion to refine the selection of candidates exhibiting noteworthy properties. Furthermore, after sequencing, more candidates were identified compared to classical sorting based solely on target binding. Hence, this platform can significantly improve the drug discovery process by the early selection of more candidates with desired properties.

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.

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 Ra and IL-18 R ss. 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-. 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.

BACKGROUND:Site-specific coupling of toxin entities to antibodies has become a popular method of synthesis of antibody-drug conjugates (ADCs), as it leads to a homogenous product and allows a free choice of a convenient site for conjugation.METHODS:We introduced a short motif, containing a single cysteine surrounded by aromatic residues, into the N-terminal FG-loop of the CH2 domain of two model antibodies, cetuximab and trastuzumab. The extent of conjugation with toxic payload was examined with hydrophobic interaction chromatography and mass spectrometry and the activity of resulting conjugates was tested on antigen-overexpressing cell lines.RESULTS:Antibody mutants were amenable for rapid coupling with maleimide-based linker endowed toxin payload and the modifications did not impair their reactivity with target cell lines or negatively impact their biophysical properties. Without any previous reduction, up to 50% of the antibody preparation was found to be coupled with two toxins per molecule. After the isolation of this fraction with preparative hydrophobic interaction chromatography, the ADC could elicit a potent cytotoxic effect on the target cell lines.CONCLUSION:By fine-tuning the microenvironment of the reactive cysteine residue, this strategy offers a simplified protocol for production of site-selectively coupled ADCs.GENERAL SIGNIFICANCE:Our unique approach allows the generation of therapeutic ADCs with controlled chemical composition, which facilitates the optimization of their pharmacological activity. This strategy for directional coupling could in the future simplify the construction of ADCs with double payloads ("dual warheads") introduced with orthogonal techniques.

A library of glycoforms of human interleukin 6 (IL-6) comprising complex and mannosidic N-glycans was generated by semisynthesis. The three segments were connected by sequential native chemical ligation followed by two-step refolding. The central glycopeptide segments were assembled by pseudoproline-assisted Lansbury aspartylation and subsequent enzymatic elongation of complex N-glycans. Nine IL-6 glycoforms were synthesized, seven of which were evaluated for in vivo plasma clearance in rats and compared to non-glycosylated recombinant IL-6 from E. coli. Each IL-6 glycoform was tested in three animals and reproducibly showed individual serum clearances depending on the structure of the N-glycan. The clearance rates were atypical, since the 2,6-sialylated glycoforms of IL-6 cleared faster than the corresponding asialo IL-6 with terminal galactoses. Compared to non-glycosylated IL-6 the plasma clearance of IL-6 glycoforms was delayed in the presence of larger and multibranched N-glycans in most cases

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.

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.

Bispecific antibodies comprise extremely diverse architectures enabling complex modes of action, such as effector cell recruitment or conditional target modulation via dual targeting, not conveyed by monospecific antibodies. In recent years, research on bispecific therapeutics has substantially grown. However, evaluation of binding moiety combinations often leads to undesired prolonged development times. While high throughput screening for small molecules and classical antibodies has evolved into a mature discipline in the pharmaceutical industry, dual-targeting antibody screening methodologies lack the ability to fully evaluate the tremendous number of possible combinations and cover only a limited portion of the combinatorial screening space. Here, we propose a novel combinatorial screening approach for bispecific IgG-like antibodies to extenuate screening limitations in industrial scale, expanding the limiting screening space. Harnessing the ability of a protein trans-splicing reaction by the split intein Npu DnaE, antibody fragments were reconstituted within the hinge region in vitro. This method allows for fully automated, rapid one-pot antibody reconstitution, providing biological activity in several biochemical and functional assays. The technology presented here is suitable for automated functional and combinatorial high throughput screening of bispecific antibodies.

The classical yeast surface display (YSD)-based antibody hit discovery relies on the enrichment of candidates from large antibody fragment libraries by fluorescence-activated cell sorting (FACS), followed by sequencing of the remaining diversity. The sequences of hit candidates are then transferred into plasmids, which allow their expression as full-length IgG in mammalian cells. After production and downstream processing, antibodies are further qualified in biochemical and functional assays. This multistep process has been proven to be successful in academia and has also found its way to the biopharmaceutical industry. However, during the recent decade, several groups tried to simplify this process by enabling either switchable or simultaneous secretion and display of antibodies or antibody fragments by yeast cells. Therefore, functional testing of candidates can be accomplished immediately during the screening process, while eliminating the need for tedious sequence reformatting and production host change. In this protocol, we give guidance for the establishment of a full-length antibody display system on yeast cells, which permits a simple switch to soluble antibody secretion.

In addition to conventional hetero-tetrameric antibodies, the adaptive immune repertoire of camelids comprises the so-called heavy chain-only antibodies devoid of light chains. Consequently, antigen binding is mediated solely by the variable domain of the heavy chain, referred to as VHH. In recent years, these single-domain moieties emerged as promising tools for biotechnological and biomedical applications. In this chapter, we describe the generation of VHH antibody yeast surface display libraries from immunized Alpacas and Lamas as well as the facile isolation of antigen-specific molecules in a convenient fluorescence-activated cell sorting (FACS)-based selection process.