Anti-Mouse CD279 (PD-1) [Clone 29F.1A12] — Purified in vivo GOLD™ Functional Grade

Clone 29F.1A12 is a rat anti-mouse monoclonal antibody widely used in mice for in vivo immune checkpoint blockade by targeting and blocking the PD-1 receptor, thereby preventing its interaction with its ligands (PD-L1 and PD-L2).

Key in vivo applications include:

• Cancer immunotherapy research: Used to block PD-1 signaling, 29F.1A12 enhances anti-tumor immune responses in mouse cancer models by promoting T cell activation and restoring anti-tumor immunity. Its efficacy has been evaluated alone or in combination with other immunotherapies to study synergy and mechanisms of checkpoint blockade.
• Mechanistic studies of T cell regulation: Utilized to dissect the role of PD-1/PD-L1 interactions in T cell exhaustion, tolerance, and activation, especially relevant in tumor-bearing and chronic infection models.
• Comparative immunotherapy studies: Employed to evaluate the relative efficacy of different anti-PD-1 clones (e.g., compared versus RMP1-14 or J43) and to model clinical anti-PD-1 therapeutic antibodies, given its high PD-1 blocking affinity.
• Genetic mouse models: Specifically applied to explore PD-1 blockade effects in context of unique mutations or cancer syndromes; for example, in murine DNA polymerase mutator models, 29F.1A12 was used to study cancer onset and survival, with variable efficacy depending on the genetic context.

Additional relevant details:

• Isotype: Rat IgG2a.
• Does not deplete PD-1+ cells: Acts by blocking receptor-ligand interaction rather than by cell depletion.
• Other use cases: While primarily for in vivo functional blockade, 29F.1A12 is also used in assays such as flow cytometry and immunohistochemistry to detect PD-1.

In summary, 29F.1A12 is most commonly used in live mouse studies for immune checkpoint blockade, chiefly in cancer immunotherapy research, mechanistic T cell studies, and comparative immunotherapy modeling, with proven utility in a broad array of mouse disease models.

Commonly Used Antibodies and Proteins with 29F.1A12

29F.1A12 is a widely utilized rat anti-mouse PD-1 (CD279) monoclonal antibody, frequently employed in both in vitro and in vivo research to block the PD-1/PD-L1 pathway in mouse models. In the literature, several other antibodies and proteins are commonly paired with 29F.1A12 for comprehensive pathway analysis, combination therapies, or to serve as positive/negative controls.

Frequently Used Anti-PD-1 Antibodies

• RMP1-14: Another rat anti-mouse PD-1 clone, often compared directly with 29F.1A12. While both are used for in vivo PD-1 blockade, 29F.1A12 generally exhibits higher binding affinity and more potent blocking activity at lower concentrations.
• RMP1-30: Also a rat anti-mouse PD-1 clone, used for flow cytometry and sometimes in combination with 29F.1A12 to confirm PD-1 expression and specificity in co-staining experiments.
• J43: A hamster anti-mouse PD-1 antibody, sometimes used as an alternative for PD-1 blockade in mouse models, though less frequently paired with 29F.1A12 in the same studies.

Frequently Used Anti-PD-L1 Antibodies

• 10F.9G2: A rat anti-mouse PD-L1 (B7-H1) monoclonal antibody, commonly used alongside 29F.1A12 to model complete blockade of the PD-1/PD-L1 axis in both in vitro and in vivo settings.
• MIH6: Another rat anti-mouse PD-L1 antibody, nearly equivalent to 10F.9G2 in blocking activity and often used interchangeably in combination with 29F.1A12 for comprehensive pathway inhibition.

Functional Proteins and Disease Models

• Recombinant PD-L1: Used in functional assays to directly assess the blocking efficacy of 29F.1A12 on the interaction between PD-1 and its ligands.
• Cytokines (e.g., IL-2): Sometimes measured as downstream readouts of T-cell activation in studies using 29F.1A12 to demonstrate functional blockade of PD-1 signaling.
• Anti-CD28 antibodies: Occasional use in combination to provide co-stimulation and better model T-cell activation in vitro.

Summary Table

Key Takeaways

• 29F.1A12 is often used in combination with other anti-PD-1 clones (RMP1-14, RMP1-30, J43) or anti-PD-L1 clones (10F.9G2, MIH6) to fully interrogate the PD-1/PD-L1 pathway, validate specificity, and model combination immunotherapies.
• Recombinant PD-L1 protein is sometimes employed in functional assays to directly measure blockade efficacy.
• Choice of antibody pair depends on the experimental question, with 29F.1A12 favored for its high affinity and strong blocking activity, especially when modeling human therapeutic responses in mice.

The 29F.1A12 monoclonal antibody clone targeting mouse PD-1 (CD279) has generated several important findings across multiple studies, establishing it as a critical tool in immunology and cancer research.

Blocking Activity and Ligand Interaction

The most significant characteristic of 29F.1A12 is its exceptional blocking capability. This clone functions as a highly effective blocking antibody that prevents PD-1 from interacting with its ligand PD-L1. The blocking activity is so comprehensive that 29F.1A12 completely prevents PD-1 detection by nearly all other antibody clones when used in competition assays. Among four tested anti-PD-1 clones, 29F.1A12 stands out as the most effective blocking antibody, completely displacing PD-L1-Fc binding at higher concentrations. At concentrations as low as 50 ng/ml, it can reduce PD-L1-Fc binding to PD-1-expressing cells.

Binding Specificity and Cell Recognition

Studies have confirmed that 29F.1A12 recognizes surface PD-1 protein on live cells with high specificity. The clone successfully detects PD-1 on live B16-F10 melanoma cells and activated wild-type T-cells. When researchers FACS-purified PD-1+ versus PD-1- subpopulations, they found that Pdcd1 gene expression levels were more than 19-fold enriched in PD-1+ melanoma cell fractions and 6-fold enriched in T-cell isolates.

The antibody demonstrates dual recognition patterns when co-stained with another clone (RMP1-30), showing dual positivity by nearly all PD-1 antibody-reactive wild-type melanoma cells (90.8%) and PD-1 overexpressing B16-F10 cells (96.7%). Similarly, overlapping subpopulations of unactivated (57.2%) and activated wild-type T-cells (48.1%) were dually bound by both antibodies.

Culture Condition Effects

A notable finding is that 29F.1A12 reactivity varies significantly based on culture conditions. Both 29F.1A12 and RMP1-30 showed more than 3-fold increased reactivity to live wild-type B16-F10 cells in three-dimensional (3D) versus two-dimensional (2D) cultures. This aligns with the observation that the 29F.1A12 PD-1 blocking antibody inhibits B16-F10 melanoma growth in 3D tumor spheroid cultures but not in standard 2D cultures.

Cross-Reactivity with Dying Cells

An important caveat identified in the literature is that 29F.1A12 shows some cross-reactivity with dying cells. While the clone maintains PD-1 specificity for live cells, it demonstrates increased reactivity with fixable viability dye-positive (FVD+) cells, indicating reactivity with dead or dying cells. Researchers observed that PD-1 staining with 29F.1A12 uncovered a forward scatter-height (FSC-H) low population predominantly positive for PD-1, which mainly contained dead cells.

Functional Applications

The 29F.1A12 antibody has been validated for blocking PD-1 binding to its ligands in vivo, similar to other therapeutic clones like RMP1-14 and J43. This blocking capability has proven valuable in studying cancer immunotherapy, as PD-L1 overexpression in tumors increases resistance to CD8 T cell-mediated lysis, and blocking the PD-1/PD-L1 interaction can transiently arrest tumor growth in mouse models of melanoma.

Interestingly, while 29F.1A12 is primarily known as a blocking antibody, weak agonist activity was also detected with this clone, revealing additional functional complexity beyond its primary blocking mechanism.

Dosing regimens for clone 29F.1A12 (anti-PD-1 antibody) in mouse models are most commonly 100–200 μg per mouse administered intraperitoneally every 3 days for three doses, but regimens are tailored based on mouse strain, tumor model, and experimental goals.

Key dosing variations across mouse models:

• Standard regimen: 100–200 μg per mouse, intraperitoneal (IP), every 3 days for three doses.
• Alternative schedules: Some experiments adopt biweekly (twice per week) dosing or alter intervals (every 3–4 days, or weekly) depending on the desired duration of PD-1 blockade, tumor kinetics, or mouse strain sensitivities.
• Dose adjustments: Lower doses (e.g., 50 μg every 3 days for four doses) or higher doses (up to 7.5 mg/kg, approx. 150–300 μg/mouse, twice weekly) are used based on antibody affinity, mouse weight, or to test efficacy thresholds.
• Dosing in BALB/c vs. C57BL/6: While the typical range remains 100–200 μg/mouse, publications note minor variations by genetic background. For example, some tumor models in BALB/c mice use up to 3-week intervals or different booster schedules.

Factors influencing dosing variation include:

• Mouse strain and weight (BALB/c vs. C57BL/6 may differ slightly)
• Tumor model aggressiveness and burden
• Immune status (naïve vs. tumor-bearing)
• Affinity of antibody constructs used
• Combination with other immunotherapies

For most studies, 100–200 μg per mouse, IP, every 3 days for 3 doses remains the reference regimen, but deviations are common based on experimental needs and mouse model specifics.