Introduction

In the pursuit of new therapeutics, discovery teams encounter a huge variety of target proteins – from simple soluble enzymes to complex multi-pass receptors. Many of these targets are deemed “difficult” or even “undruggable” for traditional antibody discovery methods. Isogenica has built its platform to embrace this breadth of targets.

Using established phage display methods, Isogenica’s CIS Display™ in vitro selection technology and the world’s largest synthetic VHH libraries, we can tackle these kinds of targets. Whether the antigen is membrane-bound, intracellular, toxic, or part of a large complex, our platforms provide the flexibility, speed, and control needed to find specific, high-affinity binders.

If you are unfamiliar with VHHs: they are the antigen-binding domains of camelid heavy-chain antibodies and combine small size (~15 kDa), robustness, and high affinity, offering unique advantages over conventional antibodies. Often called “Nanobodies”, they tolerate harsh pH and temperature conditions, remain soluble, and can be expressed efficiently in microbial systems. Thanks to their long and flexible CDR3 loops, VHHs can access recessed or conformational epitopes that bulkier antibodies cannot reach, including enzyme active sites and receptor clefts. Their modularity allows straightforward formatting into multivalent, bispecific, or payload-conjugated constructs, while half-life can be tuned by Fc or albumin fusions.

In this article, we will explore the classes of proteins we work with and the antigen formats and binder scaffolds that enable success against the various targets​.

Trends in antibody discovery targets

Isogenica’s technology was built to handle a broad spectrum of antigen types. Here are some of the typical target categories and how they fit into in vitro discovery approaches:

Soluble proteins

This category includes typical extracellular proteins, like cytokines and enzymes, that are readily produced in recombinant form. Soluble antigens are the most straightforward to work with, and our libraries routinely yield high-affinity VHH antibodies against them. With well-behaved soluble proteins, standard panning techniques often suffice. However, we still leverage our large library size to explore vast binder diversity beyond what animal immunization might achieve. The result is fast identification of specific binders, even for soluble targets that might have highly conserved regions or subtle epitopes. For discovery teams, this means rapid routes to specific binders, and improved sequence diversity compared with previously patented sequences on more popular targets.

Membrane proteins

Membrane proteins — such as GPCRs and ion channels — are notorious challenges in drug discovery. They are difficult to express and often unstable outside their lipid bilayer context. In partnership with membrane protein experts, we use high integrity nanodisc embedded antigens. This ensures the protein’s structure is maintained for selection, while biotinylation of the nanodisc polymer enables efficient capture and counter-selection. In many cases, stably transfected cell lines can also provide a valuable addition to the discovery toolbox, providing orthogonal validation of antigen confirmation.

Highly conserved proteins

Many therapeutically relevant targets are highly conserved across species, which makes them unsuitable for traditional animal immunization. Conventional immune systems recognize them as “self”, producing little or no antibody response.
Because Isogenica’s discovery platforms operate entirely in vitro, such conserved proteins can still be explored effectively using synthetic libraries.

A recent example is a campaign led by AstraZeneca using phage display against phospholamban, a cardiac regulatory protein highly conserved across species. In this study, the whole protein was not used as antigen; instead, different peptides derived from phospholamban were selected to represent key regions of interest. This approach enabled the successful isolation of VHH antibodies that could distinguish between phosphorylation states of the protein — something that would be extremely difficult to achieve through immunization.

Toxic antigens

Toxic antigens like bacterial toxins or potent signaling molecules pose obvious problems for animal immunization – they can harm the host or evoke no immune response due to toxicity. Isogenica’s in vitro discovery platforms are unaffected by these issues as the antigen is never present alongside cells. This would be more challenging for yeast and mammalian display platforms which could be negatively impacted by the presence of the toxin.

Complex and multimeric proteins

Complex or multimeric proteins—such as oligomeric enzymes, viral particles, or multi-subunit assemblies—pose unique challenges for antibody discovery. Their native conformation is often fragile, and maintaining it is essential for generating binders that recognize the functional, assembled form. Producing sufficient quantities of these complexes for immunization can also be prohibitively expensive. Moreover, they may lose stability or change conformation outside their native formulation, making traditional immunization unreliable.

By contrast, in vitro discovery platforms allow selections to take place under stable and precisely controlled conditions. This ensures the structural integrity of complex targets and supports the identification of antibodies against their native, biologically relevant conformations, or home in on particular subunints through effective deselection methods.

By covering all these protein classes, we ensure that clients – whether working on a soluble enzyme or a 7-transmembrane receptor – can bring their target to us with confidence that we have a solution. The next section discusses the antigen formats and how we tailor the presentation of these targets to maximize success.

A broad range of antigen classes supported

Just as important as the type of protein is the format in which the antigen is presented. Choosing the right format makes the difference between a failed campaign and a fruitful one. Isogenica supports a wide array of antigen formats:

Purified recombinant proteins

Most projects use purified recombinant proteins — typically produced in bacterial, insect, or mammalian systems — as at least one of the antigens. We can work with tagged or untagged proteins and can immobilize them on solid supports for panning. When a target is easily expressed and stable in isolation, a purified protein provides a straightforward path. Even for large multimeric proteins, if they can be isolated in native form, we will use them directly. High purity and proper folding are essential — even the most advanced discovery platform cannot overcome a poor-quality antigen. Accordingly, we advise on antigen preparation and sometimes co-develop the expression strategy with our partners to ensure the protein is in the best possible form for discovery campaigns.

Peptides and peptide conjugates

Sometimes the desired epitope is a specific peptide sequence, such as a short region of a protein or a disease-specific neo-epitope. Peptides (≈10–20 amino acids) are an attractive way to target a very precise site or to work around expression issues with a full protein. However, peptides are infamously difficult targets: their small size and flexibility often lead to poor immune responses and difficulty finding high-affinity binders.

Our team has developed strategies to succeed in anti-peptide campaigns. In one case, a client needed antibodies to a tiny peptide that differed by only a few amino acids from other proteins. Previous approaches had failed to find something specific. By tweaking our panning conditions (stringent blocking, strategic deselection etc.), we isolated multiple VHH antibodies highly specific to that target peptide. With the right adjustments, even “impossible” peptide targets can be conquered.

Membrane proteins

For membrane proteins, a soluble recombinant format may not exist or may not present the native structure. In these cases, we offer multiple options to present the target in a membrane-like context:

• Lipid nanodiscs – they are nanoscale lipid bilayers encircled by scaffold proteins or polymers. Nanodiscs stabilize membrane proteins in a near-native environment, keeping them soluble yet properly folded. Our partner-assisted nanodisc preparations have yielded high-quality GPCR and ion channel antigens, maintaining conformation and function for meaningful binder selection.

• Virus-like particles (VLPs) – these self-assembling, non-infectious particles mimic the native membrane topology of viral proteins. They provide a multivalent and correctly folded display of antigens, enabling the selection of antibodies that recognize conformational epitopes relevant to infection or vaccine development.
  

• Whole cells (cell-based antigens) – using intact cells expressing the target protein is an established method to facilitate binder selection under fully native conditions. This approach preserves post-translational modifications, membrane orientation, and local microenvironments. In these selections, ensuring good cell surface expression of the target is important when choosing the right cell lines.
  

Our philosophy is to choose the antigen format (or formats!) that best maintains the target’s native properties and maximizes the chances of finding the desired antibody. By being agnostic to antigen format – working with anything from a small peptides to a full membrane protein on a cell – we give each campaign multiple shots on goal.

How in vitro discovery unlocks difficult targets

In vitro discovery platforms like Isogenica’s vast libraries confer several advantages over traditional in vivo (animal-based) antibody generation, which directly address the challenges of difficult targets:

No dependence on an immune response

Conventional hybridoma or immune library methods rely on an animal host, which limits which targets can be addressed. Conserved human proteins may be ignored as “self”, toxic proteins can harm the host, and unstable antigens can either degrade in the animal’s circulation, or be prohibitively costly to produce in the required quantities. In contrast, in vitro synthetic libraries bypass these constraints: any antigen can be presented and selections are carried out entirely at the lab bench. This enables discovery without safety risks to host species, expanding the universe of viable targets – constrained only by what can be biochemically prepared and screened (1,2).

Precise control over selection conditions

In vitro selection is a highly tunable process. We can modify binding and washing conditions, add competitors or decoys, adjust antigen concentration, and much more. This way, we can steer the outcome toward the binders we want. This control is a luxury that immunization does not offer since an animal’s immune system is a “black box” we cannot precisely direct.

Isogenica takes full advantage of selection control to tackle tricky targets. We routinely pan using different stringencies (salt, detergent, washing, temperature) to enrich the most robust or kinetically favourable binders. In the earlier peptide example, the ability to customize blocking and elution stringency was critical – as our scientists noted, “the luxury of a fully synthetic system is the control you can apply to selection conditions”. Besides, being able to experiment with selection parameters in parallel gives us a better shot at success when standard approaches fail. This tunability means antigen instability or expression issues do not halt progress

Massive library diversity and unbiased discovery

Isogenica’s synthetic libraries are extremely large (up to 10¹³ unique VHHs in LlamdA®), far exceeding the diversity one can practically sample by immunization. This scale matters. It means that even if the perfect binder for a rare epitope exists only as 1 in a trillion clones, it could very well be present in our library and retrievable by panning. Additionally, synthetic libraries are unbiased by immunodominance. This enables the selection of antibodies for hidden or transient epitopes that natural responses might miss. For example, in the AstraZeneca phospholamban intrabody campaign, VHHs were isolated that distinguished phosphorylation-dependent conformations. By using both active- and inactive-state antigens during panning, state-selective binders can be enriched. The combination of vast library diversity and guided selection allows isolation of antibodies against minor or conformationally dynamic epitopes, which is critical for therapeutic programs requiring high specificity.

Speed and scalability

In vivo immunization campaigns can take months and usually focus on one or two targets at a time, with the quality of immune responses varying wildly between individual animals. By contrast, in vitro antibody discovery can deliver binder candidates within weeks. The process is automatable and parallelizable, allowing multiple simultaneous panning strategies (e.g., testing a complex multi-subunit target in a variety of buffers) to quickly identify the most productive route. Skipping animal immunization removes a major bottleneck, so discovery begins as soon as the antigen is ready.

In summary, our in vitro discovery platform, powered by the world’s largest synthetic libraries, not only broadens the targets we can pursue (by sidestepping biological limitations), but also gives us dials to adjust that greatly improve the odds of success on challenging campaigns. Difficult targets often require this kind of extra finesse and additional diversity. It is the combination of diversity, control, and adaptability that makes previously “undruggable” targets now very much druggable in our hands.

Conclusion: Challenging Targets, Meet Your Match

Isogenica’s synthetic VHH libraries demonstrate that even the hardest targets can be addressed effectively. Before any panning begins, we place strong emphasis on antigen quality control. By that way, we verify integrity, conformation, and functionality, using project-specific controls to ensure each target is presented in a physiologically relevant state. By combining this rigorous QC with ultra-diverse synthetic libraries and technologies that preserve native conformations, then we deliver drug-quality VHH antibodies and peptides against proteins once considered intractable — from soluble enzymes and conserved intracellular proteins to GPCRs stabilized in nanodiscs and other complex antigens.

For biotech and pharma teams, the key message is: difficult does not mean impossible. With flexible antigen support, rigorous screening, and rapid timelines, discovery campaigns can move forward with greater confidence.

If you are wondering how we can help your drug project move forward, book a discovery call and let’s start talking.

References

[1] Isogenica (2024). The importance of library size in antibody discovery. Available from: https://isogenica.com/library_size/

[2] Chan CEZ, Chan AHY, Lim APC, Hanson BJ (2011). Comparison of the efficiency of antibody selection from semi-synthetic scFv and non-immune Fab phage display libraries against protein targets for rapid development of diagnostic immunoassays. J Immunol Methods, 373(1): 79–88.