Anti-Mouse CD223 (LAG-3) [Clone C9B7W] — Purified in vivo GOLD™ Functional Grade

In Vivo Applications of Clone C9B7W in Mice

Clone C9B7W is a widely used monoclonal antibody that targets mouse LAG-3 (CD223), an inhibitory immune checkpoint receptor expressed on activated T lymphocytes (including both CD4+ and CD8+ T cells), natural killer (NK) cells, and regulatory T (Treg) cells. While C9B7W recognizes an epitope in the D2 domain of LAG-3 and does not block binding of LAG-3 to MHC class II, it is a functional antagonist with several established in vivo applications in murine models.

Functional Blockade of LAG-3 Signaling

• Tumor Immunotherapy Research: C9B7W is most frequently employed in vivo to block LAG-3-mediated immune suppression, particularly in studies of cancer immunotherapy. By interrupting LAG-3 signaling, C9B7W can enhance anti-tumor T cell responses, often in combination with other checkpoint inhibitors (e.g., anti-PD-1, anti-CTLA-4).
• Inhibition of T Regulatory Cell Suppressor Function: C9B7W has been shown to block the suppressor function of Treg cells in vivo, which can augment effector T cell activity and promote immune activation.
• Synergy with Other Therapies: C9B7W is used to study potential synergistic effects with other immunotherapies, such as vaccines or additional checkpoint inhibitors, to enhance immune responses against tumors or pathogens.

Mechanistic Insights

• Not a Ligand-Blocker: Unlike some other anti-LAG-3 antibodies, C9B7W does not block the binding of LAG-3 to MHC class II molecules. Instead, it is thought to induce conformational changes in LAG-3 that attenuate its inhibitory function, possibly by interfering with other downstream signaling pathways or co-receptor interactions.
• Impact on NK Cell Function: Studies in LAG-3-deficient mice suggest that LAG-3 contributes to NK cell-mediated tumor cell killing. While direct in vivo data with C9B7W in this context are limited, the antibody’s blockade of LAG-3 could theoretically affect NK cell activity.

Additional Research Applications

• Basic Immunology: C9B7W is used to investigate the role of LAG-3 in T cell homeostasis, activation, and exhaustion, as well as in models of autoimmunity and chronic infection.
• Combination Studies: Researchers employ C9B7W in vivo to dissect the contributions of LAG-3 relative to other immune checkpoints in complex immunological settings.

Summary Table: Key In Vivo Uses of C9B7W in Mice

Conclusion

Clone C9B7W is a cornerstone tool in murine immunology, especially for in vivo studies aiming to block LAG-3-mediated immune suppression in cancer immunotherapy, to interrogate Treg function, and to explore LAG-3’s role in immune regulation. Its unique mechanism—functional blockade without preventing MHC class II binding—makes it particularly valuable for mechanistic studies and combination therapies.

The antibody C9B7W is most commonly used to detect or functionally block mouse LAG-3 (CD223) in immunology research, particularly in the study of immune regulation and checkpoint blockade. In the literature, it is routinely paired with other antibodies or proteins to characterize immune cell populations or analyze functional pathways.

Other commonly used antibodies or proteins with C9B7W include:

• Anti-CD3: Used to activate T cells in vitro, commonly applied with C9B7W to study LAG-3 function in activated splenocytes.
• Anti-CD28: Often paired with anti-CD3 for co-stimulation in T cell activation assays alongside C9B7W.
• Polyclonal anti-mouse CD223: Used as a detection antibody in ELISA, complementing C9B7W as the capture antibody for LAG-3 protein quantification.
• Anti-PD-1 (Programmed cell death protein 1): Frequently combined with C9B7W for dual checkpoint blockade experiments investigating T cell responses and cancer immunotherapy.
• MHC Class II Tetramers: Utilized to study the binding of LAG-3 to MHC-II; for instance, OVA-MHC-II tetramers are used to assess whether C9B7W can block this interaction on cells.
• Anti-CD4, Anti-CD8: Applied in flow cytometry panels to delineate T cell subpopulations expressing LAG-3 detected by C9B7W.
• CD49b: Co-staining with C9B7W and anti-CD49b is used to identify Tr1 regulatory T cells in both mice and humans.

Summary Table

These combinations facilitate in-depth investigation into T cell activation, regulatory function, immune checkpoint interactions, and cell phenotyping. Selection depends on the experimental design, such as immunophenotyping (flow cytometry), functional blockade (in vitro/in vivo), or protein quantification (ELISA).

If you are seeking a specific panel for flow cytometry or in vivo blockade studies, anti-CD3, anti-CD28, anti-CD4, anti-CD8, anti-PD-1, and MHC-II tetramers represent the most widely reported partners for C9B7W in the literature.

Key findings from citations referencing clone C9B7W in scientific literature include:

1. Target Specificity: C9B7W is a rat monoclonal antibody that specifically targets the D2 domain of mouse LAG-3 (CD223), a 70 kDa activation-induced cell surface molecule expressed on activated T cells and a subset of natural killer cells.

2. Functional Impact: Although C9B7W is reported to block the in vitro function of murine LAG-3, it does not block the binding of LAG-3 to MHC class II molecules directly. Instead, it is thought to induce conformational changes that attenuate LAG-3 function.

3. Dimerization and Function: Recent studies suggest that C9B7W disrupts LAG-3 dimerization, which is crucial for its function. This disruption affects LAG-3's ability to engage with ligands like MHCII and FGL1.

4. Applications: C9B7W has been used in various applications, including flow cytometry, immunoprecipitation, and ELISA. It is also used in functional assays to study T cell regulation and in vivo studies to examine its anti-tumor activity.

Overall, C9B7W is a valuable tool for studying LAG-3 function and its role in immune regulation, particularly in the context of T cell activation and inhibition.

Dosing regimens for clone C9B7W (anti-mouse LAG-3/CD223) vary significantly depending on the specific mouse model, the experimental context, and the therapeutic goals of the study. While the antibody is widely used for both in vitro functional assays and in vivo blockade of LAG-3, there is no single universal regimen; researchers tailor the dose, frequency, and route based on their model and endpoint.

Key details on dosing variation:

• Dose Amounts: Studies commonly use doses ranging from 100–500 μg per mouse per injection. Some protocols use up to 10 mg/kg, but most preclinical immuno-oncology models use flat doses on the order of several hundred micrograms per dose per mouse.
• Frequency: Typical regimens involve dosing every 3–7 days (e.g., every 3 days, twice weekly, or every 4 days).
• Route: The most common route is intraperitoneal (i.p.) injection, though intravenous (i.v.) administration is occasionally used depending on the study design.
• Mouse Models:
  • In syngeneic tumor models (e.g., MC38, B16), dosing typically begins around the time of tumor inoculation or when tumors reach a specified size, and continues for 2–4 weeks.
  • In autoimmune or infection models, regimens may be adjusted to match disease course and desired immunomodulation window.
• Combination Therapy: When C9B7W is combined with other immunotherapies such as anti-PD-1, dosing intervals and amounts may be coordinated or staggered to optimize efficacy and minimize immunogenicity.

The following table summarizes regimen aspects as reported in product datasheets and literature:

Combination and Scheduling:

• In immunotherapy combination studies (e.g., with anti-PD-1), C9B7W is usually given on a similar schedule as the partnering antibody, but the sequence may be adjusted depending on the protocol.
• Some studies generate a mouse IgG1 variant of C9B7W to reduce anti-drug antibody effects—dosing remains similar.

Summary:
Exact C9B7W regimens must be determined by protocol specifics, but most published and commercial sources recommend starting within the range indicated above, with adjustment based on pilot results, disease model, and combination strategies. Always consult contemporary literature or datasheets for regimen optimization in context.