Choosing the right display technology is a critical decision in therapeutic and diagnostic discovery procedures. This article compares CIS Display™ with Phage Display, focusing on their underlying techniques, important benefits, and suitable applications. This blog intends to help protein engineers, discovery scientists, and pharmaceutical development teams choose the most successful method for their antibody, peptide, and scaffold protein discovery goals by highlighting the strengths and limits of each platform.

Introduction

Broadly speaking, in vitro library display technologies expose protein fragments such as stretches of VHH or peptides to a desired antigen target with the aim of finding the best binders. While all in vitro technologies share certain characteristics and advantages over in vivo immunization-based discovery, choosing the right display technology for each project depends on project requirements, resource constraints, and desired throughput. In the rapidly changing world of antibody, peptide, and scaffold protein discovery, the ability to choose the best screening platform for the job can improve selection fidelity, accelerate timelines, and enhance downstream candidate success [1]. Among the options available are [2]:

• Phage Display, an established approach based on bacteriophage expression systems.
• CIS Display™, a next-generation, completely in vitro platform with ultra-large library screening capabilities.

Both techniques offer unique advantages and disadvantages. This article will compare CIS Display™ and Phage Display, looking at their methodology, essential features, and best applications to enable protein engineers, discovery scientists, and pharmaceutical development teams to make informed decisions.

How the Technologies Work

Phage Display: Mimicking Natural Selection

George P. Smith pioneered phage display in 1985 for peptide display, and scientists such as Sir Greg Winter and John McCafferty made substantial advances in antibody development [3-5]. It replicates the natural selection process of the human immune system by iteratively choosing and amplifying high-affinity binders from a variety of libraries in a controlled, in vitro environment [6].

Phages display a diverse library of antibody or protein variants on their surface. Researchers gradually enrich for particular binders to a selected target by performing a sequence of biopanning stages that include target binding, deselection, washing, elution, and amplification of desired phages, in bacteria [7]. Libraries containing up to 10¹⁰ variants may be screened, with each selection cycle lasting several days [8,9].

The upper limit of panning arms depends on the capacity of the research team, but generally reaches about 6; which is sufficient for most programme requirements. Those criteria might include desired behaviors such as pH dependent binding, exclusion of isoforms, salinity or temperature ranges, species cross reactivity or either mimicking or avoiding features of existing controls.

Yield & Fidelity

In Phage Display, yield is intrinsically limited by the effectiveness of bacterial transformation and phage propagation, which might introduce bias and result in under-representation of some clones, particularly those that are poorly expressed or toxic in E. coli. This might reduce the variety of the selected pool in subsequent rounds. In contrast, CIS Display™ maintains high library fidelity during selection rounds because it completely avoids cellular transformation. Its in vitro approach better retains uncommon and difficult sequences, ensuring that ultra-large, diverse libraries are well-represented even under harsh selection circumstances.

CIS Display™: Ultra-Large, Cell-Free Selection

CIS Display™, a form of in vitro display, is a completely cell-free system that connects protein variants to their encoding DNA via a covalent bond between a DNA-binding protein called RepA fused to the nascent peptide [10]. This technique eliminates the necessity for bacterial transformation, allowing for selection from libraries with over 10¹² variants, which is larger than typical Phage Display libraries.

Its short selection cycles (typically under 48 hours) and compatibility with high-throughput, automated workflows make it an increasingly attractive alternative for early-stage hit identification and quick panning condition adjustment [12].

Because CIS Display™ is a cell-free platform, it is easily conducive to automation; expanding the number of panning arms to 48 or even 96 different conditions in parallel. This type of high-throughput panning is not required for every project, but offers the ability to explore many antigen types and conditions rapidly and in parallel which is especially useful for difficult target types or demanding binder profiles. Layering on NGS sequencing to these many panning conditions further expands the value of the panning exercises, allowing for deep sequence analysis and identification of ultra rare binders.

Side-by-Side Platform Comparison (Panel 2)

* CIS Display™ was only created in 2004 compared to 1985 for Phage. Currently, 3 molecules are in clinical trials.

Binding Affinity Progression Across Selection Rounds: CIS Display™ vs. Phage Display 

One of the most important criteria in any display-based discovery tool is how well selection rounds enrich for high-affinity binders. Both Phage Display and CIS Display™ provide iterative selection (or “biopanning”) cycles, however, the dynamics and advancement profiles differ owing to platform mechanics and stringency management.

Typical Affinity Progression in Phage Display

In Phage Display, each selection cycle consists of infecting E. coli, multiplying the phage, and selecting for binding clones. Conditions can be altered through the rounds to direct outcomes such as towards higher affinities, species cross-reactivity, or isoform selectivity:

• Round 1: Capture a broad spectrum of binders, including rare and weakly interacting clones
• Round 2–3: Rapid elimination of non-specific binders and enrichment of mid-affinity (50–200 nM)
• Round 4–5: Select high-affinity clones (<10 nM) by drastically lowering target concentration or introducing off-rate selection

However, transformation efficiency and library size constraints (up to 10¹⁰ variations) can limit the diversity of high-affinity binders.

Typical Affinity Progression in CIS Display™

CIS Display™ uses its huge, cell-free libraries (>10¹⁴ variations) and instantaneous control over selection stringency (e.g., lowering target concentration, increasing wash strength) to enable faster affinity maturation:

• Round 1: Enrichment of initial binders with low-to-moderate affinity (typically 100–500 nM range)
• Round 2-3: Progressive enrichment of tighter binders (10–100 nM range)
• Round 4-5: Selection of high-affinity clones (<10 nM), especially when combined with off-rate selection or competitive elution

Because of the absence of transformation bias and quicker iteration periods (less than 48 hours per round), Phage Display can often identify high-affinity binders in 2-3 rounds, whereas CIS Display™ may need 4 or 5 rounds due to the larger initial library size.

Takeaway

Phage Display’s flexibility and biological relevance make it an efficient platform for identifying early-stage hits, particularly when targeting complex, membrane-associated, or extracellular targets in physiologically relevant circumstances. Its ability to perform repeated rounds of enrichment and functional screening allows for the identification of moderate-affinity binders, which may subsequently be further optimized. Phage Display remains a proven, reliable platform of antibody and peptide discovery often representing the right balance of throughput and complexity.

CIS Display™ ‘s quick, scalable, cell-free selection capabilities offer strategic advantages in antigen triage and late-stage affinity maturation, allowing for accurate off-rate selection and rapid enrichment of high-affinity variants from targeted libraries. This makes it especially useful for establishing successful antigen conditions and refining candidate pools once initial functional binders have been found and the sequence space has been appropriately constrained. The high panning arm capacity of CIS Display™ also lends itself naturally to NGS analysis and training of algorithms, including the parallel generation of negative datasets.

When to Use Each Platform

While both approaches allow the identification of high-affinity binders, the right choice is based on the unique project objectives.

• Phage Display is still the gold standard for guided panning and downstream validation. Its capacity to show antibody fragments on phages enables functional experiments in physiologically realistic situations, making it ideal for selections involving fragile or difficult-to-immobilize targets.
• CIS Display™ excels at quick, high-throughput discovery, especially for unstable targets and rare scaffolds. It’s also recommended for large, exploratory library screenings, antigen triage, and rapid iterations easily flowing into NGS analysis and AI/ML training and learning cycles.

Complementary Use in Discovery Workflows

Instead of considering these platforms as incompatible, many discovery platforms intentionally combine them to speed up and optimize hit detection [15].

Case Study: Hybrid Workflow for EphA2-Targeting Abdurins

In a study by Ullman et al., researchers combined Phage Display and CIS Display™ to discover and optimize high-affinity binders targeting the cancer-associated receptor EphA2 [16].

Workflow Summary:

• Phage Display: A large Abdurin scaffold library was presented on the M13 phage and tested for recombinant EphA2. Three rounds of biopanning resulted in the isolation of several early binders.
• CIS Display™: The most effective phage-derived clones were used to create concentrated affinity maturation libraries. These were screened by CIS Display™ under progressively stringent conditions, resulting in the identification of low nanomolar binders.
• Validation: Top candidates were evaluated in mammalian cells and a prostate cancer xenograft model, with radiolabelled Abdurins showing selective tumor uptake in PET/CT imaging.

Result:

The hybrid methodology produced affinity-matured, high-specificity binders. Furthermore, because of their tiny size, Abdurins entered tumors more effectively than larger monoclonal antibodies. In addition, the lengthy half-life of the Abdurins slowed removal from the animals, resulting in considerable Abdurin buildup in the tumors after 48 hours.

Takeaway:

This example demonstrates how integrating CIS Display™ ‘s stringent, cell-free selection with Phage Display’s comprehensive screening platform speeds up discovery while boosting candidate quality for therapeutic and diagnostic applications.

Conclusions

Both CIS Display™ and Phage Display are essential tools in the discovery scientist’s arsenal, with each providing distinct benefits targeted to particular phases of therapeutic and diagnostic development workflows. Researchers may accelerate hit detection, enhance binder selection results, and ultimately support more efficient progression of validated candidates into clinical development. 

References

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[5] Winter, G., Griffiths, A. D., Hawkins, R. E., & Hoogenboom, H. R. (1994). Making antibodies by phage display technology. Annual Review of Immunology, 12, 433–455. https://doi.org/10.1146/annurev.iy.12.040194.002245

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[12] Collier, A. (2018). Isogenica licenses CIS Display technology to Aro Biotherapeutics for Centyrin-based biopharmaceutical development. Technology Networks. Retrieved from https://www.technologynetworks.com/tn/go/lc/further-information-306705?utm_source=306704&utm_medium=pdf&utm_campaign=pdf_lead_conversion

[13] Valldorf, B., Hinz, S. C., Russo, G., Pekar, L., Mohr, L., Klemm, J., Doerner, A., Krah, S., Hust, M., & Zielonka, S. (2022). Antibody display technologies: Selecting the cream of the crop. Biological Chemistry, 403(5–6), 455–477. https://doi.org/10.1515/hsz-2020-0377

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