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

The 29F.1A12 clone is a rat anti-mouse PD-1 monoclonal antibody widely used in preclinical in vivo mouse studies to investigate immune checkpoint blockade and cancer immunotherapy mechanisms.

Primary Applications in Cancer Models

The 29F.1A12 antibody functions as a blocking antibody that prevents PD-1 from interacting with its ligand PD-L1, thereby enhancing anti-tumor immune responses. In melanoma studies, this clone has been shown to transiently arrest tumor growth when used alone or in combination with anti-PD-L1 antibodies. The antibody effectively promotes anti-tumor immunity by releasing the brake on T cell activation that normally occurs through the PD-1/PD-L1 pathway.

Specific Research Applications

Cancer Immunotherapy Studies: The 29F.1A12 clone has been employed in various cancer models to investigate the role of PD-1 in tumor immune evasion. It has demonstrated the ability to enhance anti-tumor immune responses and is particularly suitable for studying combination therapy approaches with other immunotherapeutic agents.

DNA Polymerase Mutator Studies: In research examining DNA polymerase mutator syndromes, 29F.1A12 showed variable efficacy across different genetic contexts. While it provided initial delays in cancer onset and improved survival in Pole L424V mutant mice, it showed less significant improvements compared to other PD-1 antibody clones like RMP-14 in Pold1 mutation models.

Mechanistic Research: The antibody is used to investigate the mechanistic aspects of PD-1 blockade, including studying how checkpoint inhibition affects T cell function and tumor microenvironment dynamics.

Technical Characteristics for In Vivo Use

The recombinant version of 29F.1A12 is produced as a mouse IgG2c antibody with kappa light chains in mammalian cells, achieving greater than 95% purity. It's formulated as a 0.2 ?M filtered solution in PBS and is specifically graded for in vivo use, ensuring low endotoxin levels critical for accurate immune studies.

The antibody demonstrates stronger binding affinity to mouse PD-1 compared to other commonly used clones like RMP1-14. However, researchers should note that 29F.1A12 can deplete PD-1+ T cells, which represents an important variable to consider in experimental design.

Flow Cytometry and Detection Applications

Beyond therapeutic applications, 29F.1A12 serves as an excellent detection reagent for flow cytometry studies. It shows the brightest staining intensity among PD-1 antibody clones and can completely prevent PD-1 detection by other antibody clones when used as a blocking agent. The antibody specifically recognizes surface PD-1 protein on live cells, including melanoma cells and activated T cells, with minimal cross-reactivity to dead cells.

Other commonly used antibodies or proteins used with 29F.1A12 in the literature include RMP1-30 (another anti-PD-1 clone), RMP1-14 (anti-PD-1), and anti-PD-L1 antibodies such as 10F.9G2 and MIH6.

Key combinations and usage patterns:

• RMP1-30: Frequently used alongside 29F.1A12 for co-staining and confirming PD-1 expression; both recognize overlapping PD-1-positive cell populations in murine models.
• RMP1-14: Another anti-PD-1 antibody, commonly compared in efficacy and used in experimental panels to evaluate PD-1 blockade either alone or in combination.
• 10F.9G2 and MIH6 (anti-PD-L1 antibodies): Used in parallel or combination with 29F.1A12 to study the PD-1/PD-L1 interaction pathway, blockade experiments, and for comparing blocking efficiency in mouse models.
• Recombinant PD-L1 (rPD-L1): Utilized in binding and blocking assays to measure PD-1 interaction and antibody efficacy on target cells, often in combination with 29F.1A12.

In summary, combinations commonly used with 29F.1A12 target:

• PD-1 (using other anti-PD-1 clones like RMP1-30 and RMP1-14)
• PD-L1 (using antibodies such as 10F.9G2 and MIH6)
• Recombinant protein ligands like rPD-L1 for functional and blocking assays

These combinations provide comprehensive assessment of the PD-1/PD-L1 axis and enhance experimental robustness in immunological and cancer research contexts.

Key Findings from Clone 29F.1A12 Citations in Scientific Literature

Specificity and Recognition of PD-1

• PD-1 Detection: Clone 29F.1A12 is a monoclonal antibody specifically recognizing an epitope on mouse PD-1 protein. It is highly effective for detecting PD-1 surface expression by flow cytometry, with particularly strong reactivity on live murine cells such as T-cells and melanoma cells, but much less so on dead cells.
• Gene Expression Correlation: Flow cytometry-sorted PD-1-positive (PD-1+) B16-F10 melanoma cells and activated T-cells, specifically identified using 29F.1A12, showed markedly higher Pdcd1 (PD-1 gene) mRNA levels compared to PD-1-negative cells, providing a clear link between protein detection and gene expression.
• Overlapping Recognition: When co-stained with another PD-1 antibody clone (RMP1-30), 29F.1A12 showed nearly complete overlap in recognizing PD-1+ cell populations, further confirming its specificity for PD-1.

Functional Characteristics

• Blockade of PD-1/PD-L1 Interaction: The 29F.1A12 clone is a "blocking" antibody that prevents PD-1 from interacting with its ligand PD-L1, thereby serving as an immune checkpoint inhibitor.
• Epitope Competition: In blocking studies, 29F.1A12 completely prevented detection of PD-1 by nearly all other antibody clones, indicating its strong masking of the targeted epitope.
• Staining Intensity: Compared to other anti-PD-1 clones (RMP1-14, J43, G4, RMP1-30), 29F.1A12 consistently yields the brightest staining, suggesting a high avidity for PD-1.

Preclinical Therapeutic Effects

• Differential Efficacy: The efficacy of 29F.1A12 varies by genetic context. In mouse models with specific DNA polymerase mutations, 29F.1A12 improved survival and delayed cancer onset in some (e.g., Pole L424V mutant mice) but not in others (e.g., Pold1 D400A mice), unlike some other PD-1 antibody clones.
• Tumor Microenvironment Dependence: Flow cytometric reactivity of 29F.1A12 (and RMP1-30) on B16-F10 cells was more than 3-fold higher in three-dimensional (3D) tumor spheroids than in standard two-dimensional (2D) cultures, suggesting that PD-1 expression and antibody binding are influenced by the tumor microenvironment.
• In Vivo Applications: 29F.1A12 is validated for in vivo use and has been employed in functional assays, including immune checkpoint blockade studies.

Summary Table: Key Properties of 29F.1A12

Conclusions

Clone 29F.1A12 is a well-characterized, high-avidity, blocking anti-PD-1 antibody for mouse models, suitable for both detection and functional studies of PD-1 in immune regulation and cancer immunotherapy. Its activity is highly context-dependent, with robust in vivo and ex vivo validation, but variable therapeutic outcomes depending on the genetic background of the host.

Dosing regimens of clone 29F.1A12 (anti-mouse PD-1 antibody) in mouse models commonly range from 100–200??g per mouse per injection, administered intraperitoneally at 3-day intervals for three doses, but can vary based on the disease model, genetic background, and study objectives.

• Standard regimen: 100–200??g per mouse (~5?mg/kg), intraperitoneal injection, three doses spaced by three days.
• Alternative regimens: In some studies, lower doses (e.g., 50??g every three days for four doses) or higher single doses (up to 135??g) have been tested to assess saturation and efficacy in tumor models. Dosing interval may also be extended to 100??g every seven days for three doses in some experiments.

Experimental variations:

• In DNA polymerase mutator syndrome mouse models (e.g., Pole and Pold1 mutants), biweekly dosing (every two weeks) has been used, usually in comparison to other anti-PD-1 clones like RMP-14.
• Dosing efficacy and survival outcomes may vary across different genetically engineered models; for instance, some regimens improved survival in Pole L424V mutant, but not in Pold1 D400A model.
• Higher-affinity antibodies may allow for decreased dosing amounts or extended intervals, but 29F.1A12 sits near the threshold where further affinity increases do not improve efficacy.

Key considerations:

• The route is consistently intraperitoneal.
• Regimens may adjust based on tumor type, immune context, and antibody affinity.
• Many protocols apply the antibody in combination treatments or with other checkpoint inhibitors.

In summary: The typical dose of clone 29F.1A12 across mouse models is 100–200??g per mouse by intraperitoneal injection every 3 days for three doses, but variations exist (biweekly dosing, different intervals, or lower/higher doses) depending on the experimental requirements and genetic background of the mice.