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

In Vivo Applications of Clone 29F.1A12 in Mice

Clone 29F.1A12 is a rat monoclonal antibody raised against mouse programmed death-1 (PD-1), also known as CD279. This clone is widely utilized in preclinical mouse models for immune checkpoint blockade studies, especially in the context of cancer immunotherapy.

Major In Vivo Uses

• Immune Checkpoint Blockade in Tumor Models: 29F.1A12 is extensively employed to investigate the effects of PD-1 pathway inhibition on tumor growth and immune response. It is used to block the interaction between PD-1 and its ligands (PD-L1/PD-L2), thereby enhancing anti-tumor immune responses in vivo.
• Cancer Immunotherapy Studies: The antibody is applied in various mouse cancer models to assess the therapeutic potential of PD-1 blockade, either alone or in combination with other immunotherapies. Its use has been documented in delaying tumor onset and extending survival in certain genetic contexts, although efficacy can vary across different models.
• Mechanistic Studies of PD-1 Signaling: Researchers use 29F.1A12 to dissect the role of PD-1 in immune regulation, including its impact on T-cell activation, exhaustion, and tolerance in vivo. The clone is also suitable for studying combinatorial effects with other checkpoint inhibitors or conventional therapies.
• Preclinical Evaluation of Immune Responses: The antibody is used to evaluate how PD-1 blockade affects immune cell populations, cytokine profiles, and overall host defense mechanisms in mouse models of cancer, infection, and autoimmunity.

Technical Considerations

• High PD-1 Blocking Affinity: 29F.1A12 is reported to have approximately 100-fold higher PD-1 blocking affinity than other common clones (e.g., RMP1-14), making it particularly effective for in vivo studies where robust blockade is desired.
• Non-Depleting: Like other anti-PD-1 antibodies used in mice, 29F.1A12 functions by sterically blocking PD-1/PD-L1 interactions rather than depleting PD-1-expressing cells.
• Formulation and Purity: The antibody is available in highly purified, low-endotoxin formulations specifically optimized for in vivo use, ensuring minimal off-target effects and maximal reproducibility.
• Administration: Typically administered via intraperitoneal injection in preclinical studies, with dosing regimens optimized for the specific experimental model.

Comparative Context

29F.1A12 is one of the three most commonly used anti-mouse PD-1 clones for in vivo research, alongside RMP1-14 and J43. The choice among these clones depends on the specific research question, desired affinity, and compatibility with the experimental setup.

Summary Table: Key Features of 29F.1A12 In Vivo Use

Conclusion

Clone 29F.1A12 is a cornerstone tool for in vivo PD-1 blockade studies in mice, especially in cancer immunotherapy research. Its high blocking affinity, specificity, and suitability for combinatorial approaches make it a preferred choice for mechanistic and therapeutic investigations in preclinical models.

The anti-PD-1 antibody 29F.1A12 is commonly used in conjunction with several other antibodies or proteins in the literature, especially those targeting the PD-1/PD-L1 axis. Frequently paired and comparative antibodies include:

• Anti-PD-1 (RMP1-30): Used for co-staining, validation, and comparative studies with 29F.1A12, due to recognition of overlapping subpopulations and differences in blocking efficacy.
• Anti-PD-1 (RMP1-14): Utilized for comparative blocking studies, with noted differences in binding and blocking efficiency compared to 29F.1A12.
• Anti-PD-1 (J43): Another clone regularly used in PD-1 research alongside or as an alternative to 29F.1A12, particularly in immunotherapy and functional assays.
• Anti-PD-L1 (10F.9G2 and MIH6): These antibodies are often employed with 29F.1A12 to assess comprehensive pathway inhibition and to study the functional blockade of the PD-1/PD-L1 interaction in vivo.

Additional proteins and controls commonly used include:

• Recombinant PD-L1: To assess the efficacy of blockade by 29F.1A12 in functional experiments.
• Isotype controls (e.g., rat IgG2b, anti-keyhole limpet hemocyanin and anti-trinitrophenol): Used for specificity and background control in immunological studies.
• Other checkpoint modulators: In combination therapy mouse models, 29F.1A12 is sometimes used with antibodies targeting other immune pathways (such as anti-CD28), to study combinatorial effects and mechanistic aspects of immune blockade.

These combinations allow researchers to analyze:

• Specificity of PD-1 detection
• PD-1/PD-L1 blockade efficacy
• Comprehensive immune checkpoint dynamics
• Functional outcomes in cancer and chronic infection models

In summary, 29F.1A12 is routinely used with anti-PD-1 clones like RMP1-30, RMP1-14, J43, and anti-PD-L1 clones like 10F.9G2, MIH6, as well as recombinant PD-L1 and various isotype controls for rigorous experimental design in immunology and preclinical cancer research.

Clone 29F.1A12 is a widely used monoclonal antibody specific for mouse PD-1 (CD279), and its citations in scientific literature highlight several key findings relevant to both mechanistic immunology and preclinical therapy development:

• Potent PD-1/PD-L1 Pathway Blockade: 29F.1A12 is characterized as a highly effective blocking antibody—it prevents PD-1 from interacting with its ligand PD-L1, thereby inhibiting PD-1–mediated immune suppression. This makes it extensively used for studying immune checkpoint blockade in vivo and in vitro.

• Detection and Specificity:

  • 29F.1A12 is validated to detect surface PD-1 protein on live murine T cells and certain tumor cell lines (e.g., B16-F10 melanoma).
  • The clone demonstrates brightest staining intensity among several PD-1–targeting clones, making it a preferred option for flow cytometry-based detection of PD-1 expression on murine cells.
  • When used in competition assays, 29F.1A12’s blocking is so effective that it can completely prevent PD-1 detection by most other anti-PD-1 clones—an essential consideration for experimental design. Researchers must sequence antibody incubations carefully to avoid false negatives.

• Cross-reactivity and Artifacts:

  • There is documented cross-reactivity with a nuclear antigen that becomes exposed in dead or dying cells, which can cause false positive signals if viability dyes or careful gating are not employed.
  • Literature notes a preference for live-cell assays due to this artifact, and researchers are advised to interpret results in light of potential non-specific staining on non-viable cells.

• Therapeutic and Biological Activity:

  • 29F.1A12 has been used to demonstrate that immune checkpoint blockade can delay tumor growth and extend survival in certain mouse cancer models, though efficacy can vary depending on genetic background and model system (e.g., Pold1, Pole mutant mice).
  • Comparative studies indicate the blocking activity of 29F.1A12 surpasses other clones like RMP1-30, but activity may differ from RMP1-14 and J43 depending on context.

• Experimental Considerations:

  • Co-staining with 29F.1A12 and other PD-1 antibodies (like RMP1-30) shows that they recognize overlapping cell populations, supporting its specificity for PD-1.
  • 29F.1A12 binding and detection increase in three-dimensional (3D) tumor spheroid cultures compared to two-dimensional conditions, reflecting context-dependent PD-1 expression.
  • Some studies report that certain PD-1 clones, including 29F.1A12, can deplete PD-1+ T cells in vivo, which could confound interpretations in preclinical immunotherapy experiments focusing on T cell responses.

Summary Table: Key Properties of 29F.1A12

Researchers using clone 29F.1A12 should consider its potent blocking ability, strong detection of live-cell PD-1, cross-reactivity concerns with dead cells, and context-dependent therapeutic effects in preclinical models. Proper experimental controls and awareness of clone interactions are essential for accurate data interpretation.

Dosing regimens of clone 29F.1A12 (anti-mouse PD-1 antibody) in mouse models commonly range from 100–200 μg per mouse via intraperitoneal (i.p.) injection every 3 days for three doses, but both the dose and schedule are frequently adjusted based on model and experimental goals. In some studies, doses as low as 10 μg or as high as 7.5 mg/kg are used, and the injection interval can be varied (twice weekly, every 3–4 days, or weekly).

Key variations in dosing regimens include:

• Standard regimen: 100–200 μg per mouse, i.p., every 3 days, typically for three doses.
• Alternative by body weight: 2.5–7.5 mg/kg, i.p., administered twice a week (especially when precise dosing by mass is required).
• Adjusted frequency/intervals: Sometimes biweekly (twice weekly), or weekly doses (e.g., 100 μg once every 7 days for three doses), depending on the study design and tumor model.
• Lower or higher doses: Studies may decrease the dose (e.g., 50 μg or even 10 μg every three days) to investigate pharmacodynamics and effects on receptor saturation, or increase the dose for larger mice or different experimental endpoints.
• Combination therapy and model-specific adjustments: Dosing may be adjusted when combined with other agents (e.g., anti-CTLA-4) or according to the mouse strain or tumor burden.

Applications covered by these regimens include:

• Cancer immunotherapy (syngeneic tumor models, e.g., MC38, B16 melanoma)
• Reinvigorating exhausted T cells in chronic infection models
• Testing combination immunotherapies and mechanistic studies of immune checkpoint blockade.

Summary Table: Dosing Regimen Variations for 29F.1A12

In summary, clone 29F.1A12 is administered using a flexible dosing strategy with a core range of 100–200 μg/mouse i.p. every 3 days, but regimens vary with the mouse model, experimental objective, and combination therapies.