Immune checkpoint discovery is not an affinity-only problem. A phage-displayed peptide or antibody fragment may bind PD-1, PD-L1, or another checkpoint target without disrupting the receptor-ligand interaction, and a competitor may still fail to change downstream signaling in the relevant cell context. Creative Biolabs builds Functional Phage Display Screening programs around the functional direction you need to evaluate, with binding, competition, signaling, and cell behavior treated as distinct evidence levels.
We can begin from a known checkpoint receptor or ligand, a candidate emerging from target-discovery work, or a cell-surface system in which membrane context is important. Selection can use purified domains, target-positive cells, related receptors, target-negative backgrounds, or staged combinations of these materials. After enrichment, individual candidates are tested in the assay that answers the next question—direct recognition, natural-ligand competition, receptor-proximal signaling, or a more contextual immune-cell phenotype.
This evidence-based progression is especially important for newer checkpoint axes where the biology is still being defined. We do not reduce immune modulation to one cytokine or assume that a strong engineered reporter response will translate directly to primary-cell behavior. Instead, we help you build a candidate set whose activity is traceable to the target system and whose limitations are clear before deeper mechanistic work begins.
Fig.1 Therapeutic strategies for targeting immune checkpoints.1
Define the Checkpoint Axis and the Functional Direction
Checkpoint pathways differ in receptor family, ligand geometry, expression pattern, signaling mechanism, and cell type. The first design question is therefore not simply which library to screen; it is what you want the candidate to do. A blocking program, agonist-like program, ligand-displacement study, target-selective binder campaign, and novel-axis hypothesis all require different controls and different evidence at handoff.
Objective
Primary Question
Evidence Needed to Advance
Checkpoint binder discovery
Does the candidate recognize the intended receptor or ligand?
Independent binding plus specificity against target-negative or related molecules.
Ligand blocking or competition
Does the candidate perturb the defined receptor-ligand interaction?
Competition assay under documented target/ligand conditions.
Agonist/antagonist-like modulation
Does the candidate change receptor-linked signaling in the intended direction?
Target-dependent reporter or pathway assay with reference and negative controls.
Immune-cell functional modulation
Does the candidate change an interpretable cellular response?
A panel such as activation, proliferation, cytokines, degranulation/cytotoxicity, and viability as appropriate.
Novel checkpoint-axis discovery
Is a proposed interaction functionally meaningful?
Binding/competition plus pathway perturbation or other evidence linking the response to the candidate axis.
Document the receptor/ligand, species or isoform, desired modulation, target presentation, and evidence needed at handoff.
02
Select the Library and Screening Format
Use purified domains for controlled selection or cells when membrane context matters; add counterselection and cross-format confirmation as needed.
03
Enrich and Identify Candidate Families
Use selection, counterselection, and clone sequencing or NGS to identify recurring candidate families.
04
Confirm Binding and Competition
Retest individual candidates for direct binding, then measure natural or reference ligand competition when interaction perturbation is relevant.
05
Evaluate Functional Activity
Use target-dependent reporter or signaling assays, adding immune-cell, co-culture, or mechanistic follow-up only when needed for the research decision.
Build the Functional Assay Around Target Dependence
Reporter and cell-functional assays are designed around target dependence rather than a single universal readout. We confirm the relevant receptor/ligand context and use target-negative or ligand-negative conditions, reference controls, and viability measurements where appropriate so a signal can be linked to the intended checkpoint axis.
Primary immune cells or tumor/immune co-cultures can add context but also introduce donor, activation-state, target-density, cytokine-feedback, and cell-health variability. Depending on the checkpoint, activation markers, proliferation, cytokines, degranulation/cytotoxicity, and target-cell viability can be combined to define the phenotype more clearly.
You do not need to have the full checkpoint assay cascade finalized before contacting us. Start with the receptor/ligand axis, the functional direction you want to test, and any target or cell materials already available; we can help define the most informative screening, counterselection, and follow-up sequence.
Project Inputs
What to Share
Checkpoint receptor or ligand and species/isoform
Target presentation or cell model
Desired blocking, activating, or binding objective
Reference ligand or modulator, if available
Related checkpoints or negative materials
Preferred library format
Existing reporter/cell assay and desired validation depth
Potential Project Outputs
What You May Receive
Enriched pools and sequence families
Clone-level target binding data
Receptor-ligand competition results
Reporter concentration-response data
Non-phage/reformatted candidate confirmation
Selected immune-cell or co-culture results
Prioritized candidates and follow-up recommendations
Each output is reported according to the system tested. High affinity, one-point blocking, an isolated cytokine change, or an engineered reporter result is not treated as proof of a complete checkpoint mechanism.
Fig.2 Screening of functional scFv antibodies using a BLI-based PD-1/PD-L1 binding inhibition assay.2
Choi and colleagues integrated a BLI-based PD-1/PD-L1 inhibition assay into phage-display antibody screening so candidate scFvs could be evaluated for interaction perturbation before full IgG reformatting. This adds a functional selection layer beyond ELISA or cell-surface binding and helps prioritize clones according to the checkpoint interaction they alter.
For checkpoint projects, the transferable principle is straightforward: binding identifies a candidate, competition tests whether the receptor-ligand interaction is perturbed, and target-dependent reporter or cell assays establish functional direction when that evidence is required.
Build the Checkpoint Screen Around the Function You Need to Demonstrate
Share your checkpoint receptor/ligand pair, target format, desired functional direction, and the information already available. Creative Biolabs can help define a research-use path from phage enrichment to binding, competition, signaling, and cell-function evidence without collapsing those stages into one claim.
Does binding to an immune checkpoint prove functional modulation?
No. Binding shows that the candidate recognizes the target under the tested conditions. A modulation claim requires evidence that the candidate changes a relevant molecular interaction or target-dependent signaling response. For receptor-ligand checkpoints, competition can establish interaction perturbation, while reporter or cellular assays are needed to determine whether the effect has a functional direction.
Can primary human immune cells be included in the workflow?
They can be considered when sourcing, assay design, biosafety, and project scope are appropriate. Primary cells add context but also introduce donor-to-donor variability, activation-state differences, and sensitivity to culture conditions. We therefore define controls and readout panels carefully and avoid treating one donor, one cytokine, or one endpoint as a universal checkpoint response.
Which controls are most important for a checkpoint reporter assay?
Useful controls typically include a target-negative or ligand-negative context where feasible, vehicle or isotype/nonbinding controls, confirmation of target expression, a reference modulator when one is available, and an independent viability or cell-health measurement. The exact control set depends on the checkpoint axis and reporter architecture; the goal is to demonstrate that the response follows the intended target system.
Can phage display be used for novel checkpoint targets with limited reference biology?
Yes, but the evidence must be staged carefully. We can screen a defined receptor, ligand, domain, or cell-surface context and then test direct binding and interaction perturbation. When benchmark agonists or antagonists are unavailable, genetic or antibody-based pathway controls can be especially useful. The output is treated as a research hypothesis until target-dependent functional evidence is established.
Should candidates be tested as soluble peptides or reformatted antibodies?
Yes when the intended downstream format differs from the phage particle. Multivalent display can increase apparent affinity, cluster receptors, or alter uptake and signaling. We repeat decisive binding, competition, and functional experiments with the soluble peptide, antibody fragment, IgG, or other intended format so the observed modulation can be attributed to the candidate itself.
How do you decide whether to use purified protein or cells for checkpoint selection?
Purified targets provide strong control over the molecular interaction and counterselection, while cells preserve membrane presentation, receptor density, glycosylation, and neighboring surface molecules. We choose the starting format according to what must be preserved during discovery and often combine platforms when each step answers a different question rather than assuming one format is universally superior.
References
Zamani, Mohammad Reza, and Pavel Šácha. "Immune checkpoint inhibitors in cancer therapy: what lies beyond monoclonal antibodies?." Medical Oncology 42.7 (2025): 273. Distributed under Open Access license CC BY 4.0, without modification. https://doi.org/10.1007/s12032-025-02822-1
Choi, et al. BLI-Based Functional Assay in Phage Display Benefits the Development of a PD-L1-Targeting Therapeutic Antibody. Viruses 12.6 (2020): 684. Distributed under Open Access license CC BY 4.0, without modification. https://doi.org/10.3390/v12060684
Please kindly note that our services can only be used to support research purposes (Not for clinical use).
Creative Biolabs is a globally recognized phage company. Creative Biolabs is committed to providing researchers with the most reliable service and the most competitive price.
