Anti-Mouse CD279 (PD-1) [Clone RMP1-14] — Purified in vivo GOLD™ Functional Grade

Use of RMP1-14 Clone in In Vivo Mouse Studies

RMP1-14 is a rat monoclonal antibody that specifically targets the mouse PD-1 (Programmed Cell Death Protein 1, CD279), a key immune checkpoint receptor expressed on activated T and B cells. The antibody is widely used in pre-clinical research to investigate the role of PD-1 blockade in immune regulation, especially in cancer immunotherapy.

Mechanism of Action

• Blockade of PD-1/PD-L1 Interaction: RMP1-14 binds to the extracellular domain of mouse PD-1, effectively blocking its interaction with PD-L1 (and PD-L2), thereby preventing the inhibitory signal that suppresses T cell activation and cytokine release.
• Enhanced Immune Response: By blocking PD-1, RMP1-14 enhances T cell activity, leading to improved anti-tumor immune responses in murine cancer models.

Experimental Design Considerations

• Animal Models: RMP1-14 is typically used in syngeneic mouse tumor models, such as MC38 colon carcinoma or B16 melanoma, which are well-established for evaluating PD-1 blockade efficacy.
• Dosing: Common dosing regimens range from 200–500 ?g per injection, administered intraperitoneally every 3–4 days. Dosing may be adjusted based on tumor model, animal strain, and study objectives.
• Administration: The antibody is usually delivered via intraperitoneal injection.
• Monitoring: Researchers must monitor for potential adverse effects, including autoimmunity or toxicity, which can occur with PD-1 blockade. Dose adjustment or discontinuation may be necessary if severe side effects are observed.

Research Applications

• Therapeutic Efficacy: RMP1-14 has demonstrated efficacy in enhancing anti-tumor immunity across multiple cancer models, making it a standard tool for in vivo PD-1 blockade studies in mice.
• Mechanistic Studies: Beyond therapy, it is used to elucidate the role of PD-1 in immune tolerance, autoimmunity, and chronic infections.
• Publication Record: RMP1-14 has an extensive publication record for in vivo PD-1 blocking, though its utility is generally limited to in vivo applications (unlike some other clones, which are also used for in vitro assays).

Technical Details

• Host/Isotype: Rat IgG2a kappa.
• Specificity: Recognizes an epitope on mouse PD-1; does not cross-react with human PD-1.
• Quality: High purity (>95% monomer), ultra-low endotoxin levels (<0.5 EU/mg), ensuring suitability for in vivo use.

Comparison with Other Clones

Summary

RMP1-14 is a robust and well-characterized tool for in vivo PD-1 blockade studies in mice, primarily used to model checkpoint immunotherapy in syngeneic tumor systems. Its strong publication record, established dosing protocols, and specificity for mouse PD-1 make it a staple in pre-clinical immuno-oncology research. However, selection of antibody clone should be guided by specific experimental needs and published precedents in the relevant mouse model.

The correct storage temperature for sterile packaged clone RMP1-14 depends on the intended storage duration:

• Short-term storage (up to 2–4 weeks): Store at 2–8°C (refrigerator temperature).
• Long-term storage: Store at -20°C or lower (preferably -70°C to -80°C) for up to 12 months or longer. Avoid repeated freeze-thaw cycles.

Most suppliers recommend storing the antibody undiluted and in the dark at the temperature appropriate for the storage duration. For storage periods beyond one month, aliquoting and freezing at -70°C or -80°C is advised to maintain stability.
Do not freeze the antibody if the instructions specify "do not freeze," as this may apply to some formulations or ready-to-use products.

Summary Table:

Based on the research literature, several antibodies and proteins are commonly used alongside RMP1-14 in various experimental contexts. These can be broadly categorized into complementary antibodies, comparative antibodies, and control reagents.

Complementary Anti-PD-L1 Antibodies

The most frequently used antibody in combination with RMP1-14 is 10F.9G2, which targets PD-L1 (B7-H1). This combination is particularly valuable because while RMP1-14 blocks PD-1 on T cells, 10F.9G2 blocks PD-L1 on tumor cells and antigen-presenting cells, providing complementary checkpoint inhibition from both sides of the PD-1/PD-L1 axis.

Other Anti-PD-1 Clones for Comparison

Several other anti-PD-1 antibodies are routinely used alongside RMP1-14 in comparative studies:

29F.1A12 is perhaps the most important comparator, as it demonstrates significantly higher avidity than RMP1-14. Research shows that 29F.1A12 blocks PD-1/PD-L1 interactions with an IC?? of 0.036 ?g/mL compared to RMP1-14's IC?? of 3.23 ?g/mL. Studies indicate that experiments with 29F.1A12 most closely model human PD-1 therapeutic antibodies due to its high affinity and blocking ability.

J43 represents another commonly used anti-PD-1 clone that appears in comparative efficacy studies with RMP1-14. RMP1-30 serves as an additional comparator, showing intermediate avidity between 29F.1A12 and RMP1-14, and is particularly useful for staining experiments when therapeutic antibodies are bound to PD-1.

Costimulatory and Control Antibodies

Anti-human CD28 (Clone 9.3) is frequently used in functional assays to study T cell receptor and CD28 signaling pathways in conjunction with PD-1 blockade. This combination helps researchers understand how PD-1 inhibition affects T cell activation and proliferation.

Isotype control antibodies are essential components of RMP1-14 studies, including rat IgG2b isotype control (anti-keyhole limpet hemocyanin, Clone LTF-2) and rat IgG2a isotype control (anti-trinitrophenol, Clone 2A3). These controls help distinguish specific antibody effects from non-specific binding.

Ligand Proteins for Binding Studies

PD-L1 and PD-L2 Fc fusion proteins are commonly employed in blocking assays to test RMP1-14's ability to prevent ligand binding to PD-1. These recombinant proteins are crucial for understanding the mechanistic basis of RMP1-14's therapeutic activity and for comparing its blocking capacity to other anti-PD-1 antibodies.

The selection of companion antibodies often depends on the specific research question, with combination therapy studies requiring careful consideration of each antibody's blocking potency to avoid misestimating therapeutic effects.

Clone RMP1-14 has emerged as a pivotal research tool in cancer immunotherapy, with scientific literature revealing several key findings that demonstrate its efficacy and versatility in preclinical studies.

Mechanism and Development

RMP1-14 was developed using hybridoma technology by immunizing rats with mouse PD-1 protein and fusing rat B cells with myeloma cells. The clone was specifically selected for its high affinity and specificity for mouse PD-1, as well as its potent blocking activity in functional assays. This rat IgG2a isotype antibody binds to the extracellular domain of mouse PD-1 and effectively blocks the binding of both PD-L1 and PD-L2 to PD-1, preventing the inhibitory signaling that dampens T cell activation.

Efficacy in Tumor Models

Superior Anti-Tumor Activity

Multiple studies have demonstrated RMP1-14's exceptional performance across diverse cancer models. The antibody has shown remarkable tumor suppression rates, with studies reporting up to 82% tumor reduction in mesothelioma models when used in combination therapies. In melanoma models, RMP1-14 achieved 80% tumor suppression when combined with photodynamic therapy (PDT).

Comparative Performance

When compared to other anti-PD-1 clones, RMP1-14 has consistently demonstrated superior efficacy. In a comparative study by Wei et al. (2018), RMP1-14 showed greater tumor growth inhibition and prolonged survival compared to the J43 clone in a mouse model of colon carcinoma. This enhanced efficacy was attributed to RMP1-14's higher binding affinity and stronger effector functions.

Immune System Enhancement

T-Cell Response Amplification

Research has revealed that RMP1-14 significantly enhances immune cell function. A 2024 study by Anto and Rudd found that treatment with RMP1-14 increases T-cell proliferation by 45%, while simultaneously enhancing cytokine responses, leading to better tumor control in melanoma models. The antibody restores T-cell activation by blocking the PD-1 pathway, which is crucial in immune suppression within tumors.

Optimized Specificity

RMP1-14 offers exceptional specificity to murine PD-1, allowing for targeted immune checkpoint studies with minimal off-target effects. This precision makes it particularly valuable for modeling human-like immune responses in animal studies without affecting unrelated pathways.

Combination Therapy Success

Enhanced Therapeutic Outcomes

The literature consistently shows that RMP1-14 performs exceptionally well in combination therapies. Lou et al. (2021) demonstrated that combining RMP1-14 with photodynamic therapy achieved tumor reduction rates of up to 80% in mesothelioma models. Similarly, combination with NOS inhibitors in breast cancer models yielded 75% tumor suppression rates.

Diagnostic and Biomarker Applications

Biomarker Discovery

Beyond therapeutic applications, RMP1-14 has enabled significant breakthroughs in biomarker discovery for PD-1/PD-L1 pathways. Suzuki et al. (2023) described how RMP1-14 helps identify immune-resistant tumor variants, establishing its use in diagnostics and patient stratification for PD-1-targeted therapies.

Research Reliability and Accessibility

Established Protocols

RMP1-14 benefits from well-established dosing and administration protocols that have been extensively validated and optimized across different tumor models and research settings. This extensive validation provides researchers with a solid foundation for designing studies, reducing the need for extensive optimization and troubleshooting.

Binding Characteristics

Comparative binding studies by Butte et al. (2007) found that RMP1-14 had the highest binding affinity and most potent blocking activity among mouse-specific anti-PD-1 clones. These superior binding characteristics contribute to its effectiveness in blocking PD-1 signaling and enhancing anti-tumor immune responses.

The collective findings from scientific literature establish RMP1-14 as a highly effective, specific, and reliable tool for cancer immunotherapy research, with proven efficacy across multiple tumor types and exceptional performance in combination therapeutic approaches.