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

Clone 29F.1A12 is a rat anti-mouse PD-1 monoclonal antibody widely used in in vivo mouse studies to block the interaction between PD-1 and its ligands, typically as a tool to study immune checkpoint blockade therapy.

In these studies, 29F.1A12 is used to:

• Enhance anti-tumor immune responses and investigate the role of PD-1 signaling in various cancer and chronic infection mouse models.
• Serve as a functional blocking antibody that interrupts PD-1-mediated inhibitory signaling on T cells, thereby promoting T cell activity against tumors.
• Be combined with other immunotherapies to assess combinatorial anti-cancer effects or mechanisms underlying checkpoint blockade.
• Characterize PD-1 expression by flow cytometry or immunohistochemistry in mouse tissues or immune cell subsets, demonstrating specificity for mouse PD-1 both on T cells and, in specific contexts, on tumor cells.

Typical experimental protocols include:

• Intraperitoneal or intravenous injection of the antibody at defined doses and schedules, e.g., biweekly, in models such as melanoma or genetically engineered cancer-prone mice.
• Measuring outcomes such as tumor growth delay, survival extension, T cell activation, and changes in tumor immune infiltration.
• Detecting PD-1 surface protein on live T cells or tumor cells via flow cytometry, with the 29F.1A12 clone having defined specificity for PD-1 (and showing high-intensity staining compared to other clones).

Considerations:

• The 29F.1A12 clone has been rigorously characterized for in vivo use, offering proven efficacy in diverse disease models.
• It is also used to dissect mechanistic aspects of PD-1 blockade, such as which immune subsets are affected and how blocking PD-1 alters disease progression.
• Notably, there are documented differences in efficacy between anti-PD-1 clones across mouse models; for example, 29F.1A12 showed variable effects in DNA polymerase mutator syndromes and may perform differently than other commonly used clones like RMP1-14 in some models.

In summary, clone 29F.1A12 is a gold-standard tool for in vivo PD-1 blockade in mice, supporting functional studies of immune modulation, cancer immunotherapy, and basic T-cell biology.

Commonly Used Antibodies and Proteins Co-Used with 29F.1A12 in Literature

29F.1A12 is a monoclonal antibody targeting mouse PD-1, commonly used in research to block the PD-1/PD-L1 pathway and study its role in immune regulation and cancer biology. Several other antibodies and proteins are frequently used alongside 29F.1A12 in experimental setups, especially in flow cytometry, in vitro blocking assays, and co-staining experiments.

Common Companion Antibodies

Co-Staining with Other PD-1 Antibodies

• RMP1-30: Frequently used for flow cytometric analysis of PD-1 surface expression, often in co-staining experiments with 29F.1A12 to validate PD-1 expression on various cell types such as melanoma cells and T cells. The two antibodies show overlapping positivity, indicating they recognize similar PD-1 epitopes or populations.
• RMP1-14: Another anti-mouse PD-1 antibody, sometimes used in parallel with 29F.1A12, especially for comparison of blocking efficacy and binding affinity. 29F.1A12 generally demonstrates higher affinity and more effective blockade of PD-1/PD-L1 interactions compared to RMP1-14.
• 384-35: A fully murinized anti-mouse PD-1 antibody, commercially available and sometimes mentioned in the context of PD-1 research, though less commonly cited in direct comparison with 29F.1A12.

Blocking PD-L1 Interactions

• Recombinant PD-L1 (rPD-L1): Used in functional assays to test the blocking ability of 29F.1A12. Flow cytometry experiments often involve adding recombinant PD-L1 to cells, then measuring binding in the presence or absence of 29F.1A12 to validate PD-1 blockade.
• PD-L2: Sometimes included in assays to test the specificity of PD-1 blockade, although major studies primarily focus on PD-L1.

Co-Use with Anti-PD-L1 Antibodies

• 10F.9G2 and MIH6: These are anti-mouse PD-L1 antibodies used to study the PD-1/PD-L1 pathway. While not directly co-stained with 29F.1A12, they are used in parallel studies for pathway blockade and to model the effects of human PD-1 therapeutics in mouse systems.
• Functional Blockade Assays: Experiments often involve combining PD-1 (29F.1A12, RMP1-14, RMP1-30) and PD-L1 (10F.9G2, MIH6) antibodies to assess their ability to reverse PD-1-mediated inhibition of TCR/CD28 signaling.

Typical Experimental Approaches

Summary

In the literature, 29F.1A12 is most frequently used alongside other anti-PD-1 antibodies like RMP1-30 (for co-staining and validation) and RMP1-14 (for comparative blocking studies). Functional experiments often include recombinant PD-L1 to assess blockade efficacy, while parallel studies may use anti-PD-L1 antibodies such as 10F.9G2 and MIH6 to model comprehensive pathway inhibition. These combinations are standard in preclinical research aiming to dissect the PD-1/PD-L1 axis and evaluate potential therapeutic interventions.

The 29F.1A12 anti-PD-1 antibody clone has emerged as a significant research tool in immunotherapy studies, with several key findings documented across scientific literature.

Specificity and Detection Properties

The 29F.1A12 clone demonstrates robust specificity for mouse PD-1, recognizing a specific epitope on the PD-1 protein. Flow cytometric analyses have shown that this clone effectively detects PD-1 surface protein expression on live murine cells, with particularly high reactivity observed on activated T-cells (52.2 ± 7.9%) compared to unactivated T-cells (6.0 ± 1.5%). Importantly, the clone shows minimal cross-reactivity with PD-1 knockout cells, confirming its specificity for live cells while showing lesser specificity for dead cells.

Functional Blocking Activity

A critical finding is that 29F.1A12 functions as a blocking antibody that prevents PD-1 from interacting with its ligand PD-L1. This blocking capability is so comprehensive that it completely prevents PD-1 detection by nearly all other antibody clones when used in competition assays. The antibody's blocking function translates into therapeutic potential, as it can inhibit B16-F10 melanoma growth in three-dimensional tumor spheroid cultures, though notably not in standard two-dimensional cultures.

Clone Comparison and Overlap

When compared to other anti-PD-1 clones like RMP1-30, the 29F.1A12 antibody shows the brightest staining intensity among tested clones. Co-staining experiments reveal that 29F.1A12 and RMP1-30 recognize overlapping cell subpopulations, with dual positivity observed in nearly all PD-1-reactive melanoma cells (90.8-96.7%) and T-cells (48.1-57.2%).

Culture Condition Sensitivity

A notable finding is that both 29F.1A12 and RMP1-30 reactivity increases more than 3-fold in three-dimensional versus two-dimensional culture conditions. This suggests that PD-1 expression levels vary significantly depending on the cellular environment, which has important implications for experimental design and therapeutic applications.

Variable Therapeutic Efficacy

In preclinical studies, 29F.1A12 has shown differential efficacy across genetic contexts. While the clone demonstrated initial benefits in delaying cancer onset and improving survival in certain mouse models (such as Pole L424V mutant mice), it showed variable or limited effects in other genetic backgrounds compared to alternative clones like RMP-14. This variability highlights the importance of clone selection based on specific experimental contexts and genetic models.

Technical Considerations

An important caveat identified in the literature is that PD-1-specific blocking antibodies like 29F.1A12 can deplete PD-1+ T cells, presenting a potential confounding variable in preclinical immunotherapy experiments. This depletion effect must be considered when interpreting experimental results and designing studies.

The collective findings establish 29F.1A12 as a highly specific, functionally active anti-PD-1 antibody with strong blocking capabilities, but researchers should carefully consider its variable efficacy across different experimental contexts and potential for T-cell depletion when designing studies.

Dosing regimens of clone 29F.1A12 (anti-mouse PD-1 monoclonal antibody) in mouse models typically use doses between 100–200??g per mouse given by intraperitoneal injection, but frequency and total duration can vary depending on study design, mouse strain, disease model, and experimental goals.

Key details by context:

• Standard Dosing Range: Most studies recommend 100–200??g per mouse (~5?mg/kg) via intraperitoneal injection.
• Typical Schedule: Common regimens involve administration three times at three-day intervals, though some protocols extend to dosing every 3–4 days for several weeks.
• Biweekly Regimens: Some experiments, especially those in genetically engineered models like Pold1 and Pole mutant mice, use biweekly (twice weekly) treatments.
• Low Dose and Frequency Variations: Efficacy studies have employed regimens as low as 50??g every three days for four doses or 100??g every seven days for three doses in MC38 tumor-bearing mice, showing that both lower dose and less frequent dosing can reduce efficacy in some models.

• Route of Administration: Usually intraperitoneal injection; rarely intravenous or subcutaneous, depending on specific requirements.
• Combination Therapies: 29F.1A12 is sometimes combined with other checkpoint inhibitors or chemotherapy to assess synergistic effects.

Variation across models:

• The frequency and total dose may be adapted to tumor growth kinetics, immune competence, or study length. Higher frequency or prolonged dosing is used in aggressive or chronic disease models to maintain blockade.
• Genetic background of the mouse and immune status (immunocompetent vs. immunodeficient) can drive further adjustments to ensure efficacy and avoid toxicity.

Efficacy Considerations:

• Both the total dose and dosing interval impact how well PD-1 is saturated in the tumor microenvironment and, thus, therapeutic outcomes.
• Some genetically engineered tumor models may respond differently to the same regimen, requiring protocol customization.

In summary, clone 29F.1A12 is typically administered at 100–200??g per mouse intraperitoneally every 3–4 days, with variations according to the mouse model, tumor type, and experimental goal. Researchers should tailor regimens to their specific system, referencing published protocols for the closest matching model.