Screening & Hit-to-Lead

Science Spyglass Application of VHH in drug discovery Antibodies represent a major class of biologic therapeutics and, since the advent of recombinant monoclonal antibody (mAb) production in mammalian cells, have transformed the treatment of numerous diseases, particularly cancer.VHH, also known as single-domain antibodies, or Nanobodies® (Nanobody® is a registered trademark of Ablynx N.V.), are the antigen-binding domains of heavy-chain-only antibodies naturally found in camelids. Since their discovery in the 1990s, VHH have attracted considerable interest as an alternative to conventional antibodies due to their small size, high stability, ease of recombinant production and high affinity and specificity for their targets. These properties have supported their development for therapeutic and diagnostic applications. In 2019, the FDA approved caplacizumab, the first VHH-based drug for the treatment of a rare blood disease. That was followed a few years later by ozoralizumab, envafolimab and the VHH-CAR-T therapy ciltacabtagene autoleucel, for the treatment of rheumatoid arthritis, solid tumors and blood cancers, respectively. As of 2025, over 35 VHH-based therapeutics have entered clinical trials, with more foreseen for the near future, suggesting that the market of VHH drugs will likely grow significantly over the next years. By joining forces with Isogenica Ltd, a Cambridge (UK)-based company specializing in VHH discovery, Axxam aims to broaden the services offered to clients by integrating the power of Isogenica’s VHH library, the largest in the market, with Axxam’s portfolio of HTS-ready functional assays and newly developed high-throughput VHH production platform, enabling the automated production, purification and functional characterization of hundreds of VHH. Case study: VHH discovery, automated production and testing against CDK2/CycE1 complex To test the integration of both companies’ services, a joint case study was conducted using CDK2 as a target for VHH selections, production, and testing in biochemical functional assays. CDK2/CycE1 is an important regulator of G1-S cell cycle pathway (Chu et al., 2021), and its impaired function is linked to a variety of cancers, such as ovarian, breast and endometrial tumours. Most notably, overexpression of CycE1 is linked to increased resistance in standard-of-care therapies (Tshemiak et al., 2017; Nakayama et al., 2010; Turner et al., 2019). CDK2 has been considered an important drug target for years and several SMC inhibitors have been developed. However, first-generation inhibitors, like dinaciclib, are not selective against CDK2 and cause important side effects or are characterized by acute toxicity. In recent years, selective CDK2 inhibitors are being studied and are currently in Phase I/II clinical trials, like Pfizer’s PF-07104091 or INX-315 by Incyclix Bio (Yap et al., 2023; Dietrich et al., 2024). However, tumors can develop resistance mechanisms to CDK2 inhibitors, such as by upregulating CycD1 and triggering the CDK4/6 pathway, de facto bypassing the effect of the inhibitor, or by up-regulating CDK2 expression levels so that a higher concentration of inhibitor must be used, thereby increasing the chance of acute toxicity (Taylor-Harding et al., 2014; Gorski et al., 2020). No inhibitors are currently reported that specifically target CDK2/CycE1 complex formation. New modalities to selectively target CDK2 and/or to impair CDK2/CycE1 complex formation in CycE1-overexpressing cancers are needed to overcome such hurdles, and VHH antibodies constitute a valid alternative to SMC inhibitors, given that the intracellular delivery of VHH, such as in the intrabody format (De Genst et al., 2022), is being explored as the next frontier of biological therapeutics. To date, no VHH targeting CDK2 or its complex with CycE1 are reported. The Axxam-Isogenica joint case study constitutes the first attempt to identify and characterize VHH targeting CDK2 activity and its interaction with CycE1. VHH selection campaign against pre-activated CDK2 A high-quality antigen is the cornerstone of any good antibody discovery campaign. Pre-activated CDK2 was expressed and purified by Axxam, undergoing essential QC steps before biotinylation at Isogenica to enable immobilization during selections and screening. Since any biochemical change has the potential to disrupt the antigen structure and impact the antibody selection process, biotinylated CDK2 was re-validated to ensure the antigen had retained its functionality during the biotinylation process. This ensured that the discovered VHH antibodies would be as physiologically relevant as possible. VHH selections were carried out against CDK2 using Isogenica’s LlamdA® and VHHantage™ synthetic libraries under a range of parallel phage display conditions. During this process, the libraries were exposed to CDK2, and hits were enriched through immobilization using the biotin tag. The campaign included both total and specific elution strategies in order to target a range of epitopes. Specific hits were identified by ELISA and sent for DNA sequencing to match the antibody amino acid sequences to the binding data. Figure 1: Selection using VHH antibody libraries. Unlike small molecule libraries where each compound is present in an individual well, Isogenica’s VHH antibody libraries are encoded into DNA, with billions of different VHH sequences in a single tube. The best binders to CDK2 were identified by cycles of binding, washing, and enriching under different parallel conditions, and the identity of each hit is determined by DNA sequencing at the end of the process. Through the selection process, 192 VHH antibodies were identified for testing. These were purified according to Isogenica’s optimised bead-based expression processes, which routinely handle 200-300 samples per day in order to generate sufficient protein for analytical size exclusion chromatography (aSEC), ELISA, cell binding, and other early antibody assays, typically in 96 well format. Since Axxam’s HTS assays take place in 384-1536 format, smaller quantities of protein are needed, and the potential to screen more samples is considerably higher. Then, the challenge became the miniaturisation of both the expression and purification processes. High-Throughput automated VHH purification Whilst VHH selections can be tailored to targeting specific epitopes, address cross-reactivity or bias towards high affinities, addressing functionality during selections is still a limiting factor. In order to overcome that, increasing the number and diversity of hits to be screened in functional assays presents as a reliable strategy. Nevertheless, this leads to the need for production and characterization of higher numbers of VHH in the subsequent phases, constituting a significant technical burden as thousands of VHH have to

Science Spyglass Unlocking the Power of Thermal Shift Assays for Early-Stage Drug Discovery Thermal Shift Assays (TSA) provide valuable insights into protein stability, ligand interactions, and quality control, making them indispensable for drug development and protein engineering. Key Takeaways TSA supports protein stability and ligand binding studies Compatible with high-throughput screening workflows Enables efficient hit validation and protein characterization Seamlessly integrates with our biochemical assay capabilities Consult our TSA specialists Explore our full biochemical assay capabilities Why it matters in Drug Discovery? Thermal Shift Assays are widely used in early-stage drug discovery because they combine low sample consumption, scalability, and rapid experimental execution. They provide a fast and cost-effective way to assess protein stability and ligand binding, supporting informed decision-making from hit identification through lead optimization. Key applications include: Hit identification by screening compound libraries for molecules that stabilize or destabilize the target protein Protein characterization by assessing thermal stability and mutation effects Ligand engagement studies to confirm protein–ligand interactions Affinity and inhibition analysis, including estimation of binding parameters (Kd) and inhibitor potency Quality control for evaluating protein stability and formulation consistency Additionally, TSA enables the ranking of compounds based on their affinity to the target protein, providing valuable insights into their binding properties. By using TSA, researchers can confidently evaluate compounds and identify those with a higher or lower affinity to the target protein, aiding in drug discovery and optimization processes. How can Thermal Shift Assays (TSA) support early-stage drug discovery? Thermal Shift Assays (TSA) are a versatile tool that can support multiple stages of early drug discovery. They are particularly recommended for:   Primary screening of small, focused, or unbiased compound libraries, where TSA enables the rapid identification of promising compounds for further investigation. Secondary assays for hit validation and target engagement, providing additional evidence of direct interactions between compounds and target proteins to further validate potential hits. Characterizing lead compounds during the hit-to-lead phase, providing crucial information on their stability and binding properties to support the selection of the most viable candidates for further development. For the identification of proteolysis targeting chimera (PROTACTM) scaffold molecules. The interactions between PROTACs and target proteins can be assessed by utilizing TSA, facilitating the development of effective protein-degradation strategies. Characterization of protein variants and mutants, enabling researchers to study how mutations affect protein stability and ligand interactions. In addition to these applications, TSA offers several practical advantages over traditional protein stability methods. It is simple, rapid, and cost-effective, requires relatively small amounts of protein, and can be easily miniaturized to 384-well plate formats, making it suitable for medium- and high-throughput screening workflows. What are Thermal Shift Assays? TSA is a highly parallelizable, cost-effective technique for studying protein stability and protein binders. It measures the thermal denaturation of a target protein by monitoring the fluorescence intensity of a dye that binds to exposed hydrophobic regions of the protein during heating. As the protein unfolds, the exposed hydrophobic regions increase, leading to an increase in fluorescence signal. Compounds, such as inhibitors or in general modulators, stabilize the three-dimensional conformation of the target protein and as a consequence the temperature needed to denature the protein is higher (see purple curve) The figure shows fluorescence intensity curves (top panel). The blue curve is the target protein with the dye; the other traces correspond to the addition of increasing concentration of a binder compound that causes a shift of the melting temperature of the protein complex. The melting temperature can be calculated as the inflection point of the fluorescence intensities or as the first derivative of the curves (peaks in the bottom panel). TSA and differential scanning fluorimetry (DSF) can generally be used interchangeably, a primary distinguishing feature between the two lies in their denaturation methods. TSA specifically refers to the denaturation of a target protein triggered solely by temperature intensification. On the other hand, DSF encompasses techniques that employ various physicochemical agents, including but not limited to temperature intensification, in order to induce denaturation in the target protein. In this article, our focus will be directed towards exploring the specific aspects of TSA. What are the key steps in a Thermal Shift Assay (TSA) experiment? The TSA methodology involves a series of simple steps that make it suitable for routine laboratory use. The following is a brief outline of the key steps involved in performing a TSA experiment: Protein sample preparation: purify the protein of interest ≥ 90% using established protocols, ensuring its quality and concentration. Fluorescent dye binding: add a fluorescent dye, such as SYPROTM Orange (a gold standard dye), to the protein solution. The dye selectively binds to hydrophobic regions exposed during protein unfolding. Thermal gradient generation: set up a thermal gradient by using a thermocycler. Typically, the temperature is increased in small increments, allowing for gradual protein denaturation. Fluorescence detection: monitor the fluorescence intensity of the dye at each temperature increment. The increase in fluorescence corresponds to protein denaturation. Data analysis: analyze the thermal-shift data to determine the protein melting temperature (Tm), which represents the temperature at which 50% of the protein is unfolded. Tm provides insights into protein stability and ligand interactions. Explore TSA with our experts Axxam’s Thermal Shift Assays capabilities Axxam provides Thermal Shift Assay (TSA), also known as Differential Scanning Fluorimetry (DSF), services for early-stage drug discovery. These assays can be seamlessly integrated into scalable drug discovery workflows to accelerate hit identification, compound evaluation, protein characterization, and decision-making. TSA instrument platform Our TSA experiments are conducted using advanced thermocyclers that allow for the creation of precise temperature gradients ranging from 20 to 100 °C. These instruments are equipped with the capability to accurately read the fluorescence emission of SYPROTM Orange dye, supplying reliable data for your TSA analysis. Screening compatibility For projects requiring higher throughput, we have the capability to run TSA in a 384-well plate format. This screening choice allows for efficient analysis of a larger number of samples. Moreover, we have integrated suitable instruments into our automated screening station, ensuring streamlined processes and precise data acquisition.

Science Spyglass Why is shRNA better than siRNA for gene knock down in screening campaigns? One of the first challenges to face in the drug discovery process, is the generation of reliable in-vitro models that mimic the pathological conditions of the disease being studied. Many diseases are caused by the loss of function of a gene, with consequences on downstream pathways and detrimental effects on cell functions. Different approaches can be used in-vitro to obtain a loss of function model at the DNA, RNA or protein level. We are not new to the use of RNA interference for silencing the mRNA transcripts of such genes, having worked for many years with small interfering RNA (siRNA) transfection. Despite allowing for a great flexibility and ease of use, this approach poses some issues in terms of throughput and variability, when screening large compound libraries to find molecules able to revert the pathological conditions into healthy ones, mainly due to the need to transfect many batches of cells during the screening process. An alternative to siRNA transfection, well-suited for high-throughput screening (HTS) and hit-to-lead campaigns, is the development of assays based on inducible short hairpin RNA (shRNA) expression, to knock down the gene of interest. How does it work? In our experimental model, we treat the cells with lentiviral particles encoding for inducible shRNA directed against the gene of interest, in order to generate a stable cell line able to mimic the pathological condition, upon gene downregulation triggered by doxycycline. What are the advantages? Compared to siRNA transfection, that is a transient approach, the shRNA viral transduction generates a stable cell line, and the gene downregulation can be sustained over time by treatment with doxycycline, making this approach very suitable for HTS campaigns and hit-to-lead programs. Indeed, the entire process is particularly fit for automation as it requires fewer handling steps. One additional important advantage of this approach is that the shRNA induction with doxycycline is dose- dependent (the possibility to modulate the expression is especially important when studying essential and lethal genes) and time-dependent (reversible upon withdrawal of doxycycline). When to apply this approach? Independently of the therapeutic area, this approach is particularly meaningful when studying diseases characterized by a depletion or mutation of a “disease causing gene” (loss of function) with the final aim of identifying small molecules that revert such pathological conditions to the normal state. The experimental disease-related model can be used also to study “downstream genes” regulated or at least influenced by the disease causing gene that will be knock down by specific shRNA. A case study:focus on a disease caused by the mis-splicing of a target gene We developed a disease model transducing shRNA expressing lentivirus into a human neuroblastoma cell line, to silence a splicing factor (the disease-causing gene), whose downregulation causes a mis-splicing in a target gene (downstream gene). The downstream gene was measured by multiplex TaqManTM q-PCR in dose response, quantifying normal and aberrant slice variants at the transcriptional level. In addition, the downstream gene was monitored at the protein level with Nano-Glo® HiBiT detection system by Promega. Related content Drugging the RNA world at cellular and subcellular levels Webinar available on demand Contact us Back