Anti-Human PD-L1 (CD274) [Clone 29E.2A3] — Purified in vivo PLATINUM™ Functional Grade

Clone 29E.2A3 is most commonly used in mice for in vivo blockade of human PD-L1 signaling to study immune checkpoint inhibition and tumor immunology. This approach helps researchers analyze the functional role of PD-L1 in tumor immune escape, T cell regulation, and responses to immunotherapy, especially in the context of humanized mouse models expressing human PD-L1.

Typical in vivo applications in mice include:

• Blocking PD-L1 signaling: Clone 29E.2A3 specifically targets human PD-L1 (CD274), and in mice, it is used to block the interaction between PD-L1 and its receptor PD-1, thereby preventing immunosuppressive signaling. This is essential in preclinical studies of immune checkpoint blockade, often mimicking clinical anti-PD-L1 therapies.
• Tumor immunology studies: The antibody helps analyze how inhibition of PD-L1 affects tumor growth, immune cell infiltration, and anti-tumor immune responses. These studies are especially relevant in humanized mouse models with human tumors and human immune cells.
• Functional immune assays: Researchers use clone 29E.2A3 to investigate the effect of PD-L1 blockade on T cell activation, cytokine production, and modulation of immune tolerance.
• Assessment of immunotherapy combinations: The antibody is also used in combination with other agents to study synergistic effects in anti-tumor immune responses or to explore toxicity and efficacy of combinatorial treatments.

Key contextual points:

• 29E.2A3 binds to an epitope within the PD-L1–CD80 binding region, which is crucial for disrupting PD-L1-mediated inhibitory signaling.
• It does not cross-react with mouse PD-L1, so studies utilize humanized models (expressing human PD-L1) or grafts of human tumor cells in mice.
• Formats of 29E.2A3 intended for in vivo use are azide-free and low-endotoxin to ensure safe administration in functional assays.
• In vivo applications almost exclusively focus on blockade; other uses (e.g., immunohistochemistry, flow cytometry) are primarily for ex vivo or in vitro analyses.

In summary, clone 29E.2A3 is a tool of choice in mouse models—particularly humanized— for studying the immunobiology of human PD-L1, testing immune checkpoint blockade efficacy, and elucidating mechanisms of antitumor immunity.

Commonly used antibodies or proteins paired with 29E.2A3 (an anti-human PD-L1/CD274 antibody) in the literature include several secondary detection antibodies, immune checkpoint markers, and lineage-specific immune markers. These are often utilized for multi-color flow cytometry, blocking assays, and immunofluorescence.

Key commonly used antibodies or proteins with 29E.2A3:

• Secondary Antibodies:

  • Goat anti-mouse (GAM), typically conjugated with fluorophores such as APC (Allophycocyanin), is frequently used as a secondary antibody with 29E.2A3 when the latter is not directly conjugated for flow cytometry detection.
  • Directly conjugated 29E.2A3 is also available (e.g., with PerCP/Cy5.5, Alexa Fluor 700, APC, etc.) for multi-color analysis, avoiding the need for secondary detection.

• Immune Checkpoint and Activation Markers:

  • PD-1 (CD279): The natural receptor for PD-L1, often measured alongside PD-L1 in immune profiling.
  • CTLA-4 (CD152): Another key checkpoint protein often co-analyzed with PD-L1 and PD-1 to assess immune regulatory pathways.
  • CD28: A T cell activation molecule sometimes included in immunophenotyping panels with PD-L1.

• Other Lineage/Activation Markers (for multiparametric analysis of immune cell subsets):

  • CD3, CD4, CD8: General T cell markers frequently stained in combination with PD-L1 to characterize PD-L1 expression on specific T cell subsets.
  • CD14, CD19, CD56: These may be included to identify monocytes/macrophages, B cells, or NK cells, respectively, alongside PD-L1 in broader immune profiling.

• Functional and Blocking Assays:

  • PD-1-Ig fusion protein: Used as a reagent to test the blocking capability of 29E.2A3 for PD-1/PD-L1 interactions in functional studies.

• Fluorophore-conjugated Antibodies:

  • Antibodies conjugated to fluorophores such as APC, PerCP/Cy5.5, Alexa Fluor 700, and others are used for multicolor panels in flow cytometry with 29E.2A3.
  • Note: There is a known technical interaction between 29E.2A3 and Alexa Fluor 700-conjugated antibodies, resolved by sequential staining.

Summary table of commonly used antibodies/proteins with 29E.2A3:

In summary, 29E.2A3 is commonly used with secondary detection reagents and various immune markers—particularly those related to T cell checkpoints (PD-1, CTLA-4) and lineage markers—in both phenotype and functional studies.

Clone 29E.2A3, an anti-human PD-L1 (CD274/B7-H1) antibody, has generated significant findings across multiple areas of immunology research. This widely-used antibody clone has been instrumental in advancing our understanding of PD-L1 biology, but has also revealed important technical considerations for its use.

Functional Characterization and Applications

Clone 29E.2A3 recognizes an epitope on PD-L1 within the PD-L1-CD80 binding region. This positioning makes it particularly valuable for functional studies, as it has demonstrated blocking capabilities in multiple experimental contexts. The antibody has been successfully employed in various applications including flow cytometry, immunohistochemical staining of acetone-fixed frozen sections, and blocking assays. However, it notably does not work in Western blot applications.

The scientific literature includes several key studies utilizing this clone. Research by Brown et al. (2003) employed 29E.2A3 for flow cytometry, immunohistochemistry, and blocking experiments. Subsequent studies by Radziewicz et al. (2007) and Nakamoto et al. (2009) used the antibody in blocking assays to investigate PD-L1 function. More recent work by Barsoum et al. (2014) and Mahoney et al. (2015) continued to leverage this clone for cancer research applications.

Critical Discovery of Fluorophore Cross-Reactivity

A particularly important finding emerged regarding an unexpected interaction between clone 29E.2A3 and the Alexa Fluor 700 (AF700) fluorophore. Research demonstrated that this antibody clone can bind directly to AF700 fluorescent conjugates during multi-color immunofluorescent staining. This discovery came from careful validation studies examining PD-L1 expression on neutrophils.

When investigating PD-L1 expression on neutrophils from healthy volunteers and COPD patients, researchers initially observed increased PD-L1 detection when using AF700-conjugated anti-CD16 antibodies alongside clone 29E.2A3. However, this turned out to be an artifact. Co-staining experiments with AF700-conjugated IgG1κ and clone 29E.2A3 revealed a reduction in detected PD-L1 expression, indicating that the PD-L1 antibody was binding directly to the AF700 fluorochrome rather than detecting genuine biological PD-L1 expression.

This was confirmed by comparing clone 29E.2A3 with an alternative PD-L1 clone (130,021 from R&D Systems). While clone 29E.2A3 produced false-positive PD-L1 detection in combination with AF700-conjugated anti-CD16, clone 130,021 did not reproduce this effect. The interaction was further supported by small decreases in the detected mean fluorescence intensity (MFI) of AF700 in the presence of clone 29E.2A3, likely due to disruption of the fluorescence signal from direct antibody binding to the fluorophore.

Practical Implications and Recommendations

To address the fluorophore binding issue, researchers can use sequential staining protocols—staining with the 29E.2A3 antibody first, followed by the AF700 conjugate of interest. This workflow resolves the interaction and prevents false-positive results.

The discovery of this fluorophore cross-reactivity represents a novel mode of antibody cross-reactivity that extends beyond typical antigen-antibody interactions. This finding emphasizes the critical importance of rigorous antibody validation in multi-color flow cytometry panels, particularly when using new combinations of clones, fluorophores, and binding targets. The use of appropriate controls, such as fluorescence minus one (FMO) controls, is essential for identifying false-positive results.

Expression Pattern Studies

Clone 29E.2A3 has been extensively used to characterize PD-L1 expression patterns across various cell types and disease states. Studies have analyzed PD-L1 expression in 19 human tumor cell lines using this clone, categorizing surface expression as low, medium, or high. PD-L1 is expressed constitutively on macrophages and dendritic cells, and can be induced on activated T cells, B cells, endothelial cells, and epithelial cells in response to interferons alpha, beta, and gamma. Research has also found abundant PD-L1 expression on many murine and human cancers, with further upregulation possible upon IFN-gamma stimulation.

These collective findings establish clone 29E.2A3 as both a valuable research tool and a cautionary example of the need for thorough antibody validation in immunological research.

Overview

The clone 29E.2A3 specifically targets human PD-L1 (CD274, B7-H1) and is primarily used for in vitro assays, epitope mapping, and detecting human PD-L1 expression in research applications. There is no established or standardized dosing regimen for clone 29E.2A3 in mouse models reported in the literature, as its direct therapeutic use is rare—most in vivo studies using PD-L1 blockade in mice employ anti-mouse PD-L1 clones (such as 10F.9G2).

Current Understanding

• Standard Mouse PD-L1 Clones: For blocking mouse PD-L1, clones like 10F.9G2 are commonly used, with typical dosing regimens of 100–250 μg per mouse, administered intraperitoneally 2–3 times per week. This reflects species-specific antibody targeting—mouse PD-L1 blockade requires mouse-specific antibodies for functional blockade.
• 29E.2A3 Function: Clone 29E.2A3 is an anti-human PD-L1 antibody, not anti-mouse, so it cannot functionally block mouse PD-L1 signaling in standard syngeneic tumor models. Its use in vivo is generally for epitope detection or passive assessment of human PD-L1 expression in xenograft models using human tumor lines, not for immune checkpoint blockade.
• Humanized Mouse Models: In models where human immune cells (humanized immune system, HIS-mice) and/or human tumor cells are engrafted, the focus is on human immune checkpoint molecules. However, even in these advanced models, precise dosing regimens for 29E.2A3 are not reported in the literature. If used in such contexts, dosing would likely be extrapolated from clinically relevant anti-human PD-L1 antibodies (e.g., atezolizumab), but no peer-reviewed data specifically guide 29E.2A3 dosing in mice.

Summary Table

Key Points

• Clone 29E.2A3 is not a standard tool for immunotherapy in conventional mouse models due to its target specificity (human PD-L1, not mouse).
• No published, peer-reviewed dosing regimens for 29E.2A3 in any mouse model are available, as its principal applications are in vitro and epitope detection.
• In humanized models, if 29E.2A3 were used to target human PD-L1 on xenografts or human immune cells, dosing would need to be empirically determined and would not follow the conventions established for mouse-specific antibodies.
• For functional PD-L1 blockade in mice, always use species-matched antibodies (e.g., 10F.9G2 for mouse PD-L1).

Conclusion

Dosing regimens for clone 29E.2A3 are not established across mouse models and, given its specificity for human PD-L1, it is not used for immune checkpoint blockade in standard mouse studies. Researchers should use species-matched antibodies for functional studies and consult product-specific resources for any intended in vivo use of 29E.2A3 in specialized humanized systems.