Anti-Mouse CTLA-4 [Clone 9D9] — Purified in vivo GOLD™ Functional Grade

Clone 9D9 is a monoclonal antibody targeting mouse CTLA-4 (CD152) and is widely used in in vivo mouse studies as a surrogate for CTLA-4 checkpoint blockade, especially in cancer immunotherapy models.

• Mechanism: In vivo, 9D9 functions primarily by depleting intratumoral regulatory T cells (Tregs) and neutralizing CTLA-4, which enhances anti-tumor immune responses. Its mouse IgG2b Fc enables effective Treg depletion within the tumor microenvironment, a property leveraged to improve outcomes in immunotherapy research.

• Standard use: 9D9 is administered via intraperitoneal injection (sometimes intratumoral), typically at doses between 100–250??g per mouse every 3 days. The antibody is effective as monotherapy and in combination approaches across various tumor models, including melanoma and colon cancer.

• Experimental outcomes: Studies show that 9D9 increases lymphocyte and CD8+ T cell infiltration into tumors, boosts activation markers on T cells (CD44, CD69, PD1), reduces the proportion of regulatory T cells (CD4+/CD25+/FoxP3+), and induces tumor regression.

• Additional notes: 9D9 binds an epitope on mouse CTLA-4 that is distinct from epitopes of human therapeutic antibodies (e.g., ipilimumab) and demonstrates binding properties that may differ under acidic physiological conditions.

Applications in in vivo mouse studies:

• Cancer immunotherapy and checkpoint blockade
• Mechanistic studies of Treg depletion and immune activation within tumors
• Surrogate testing for potential human therapeutics

Researchers choose 9D9 when precise depletion of intratumoral Tregs and robust CTLA-4 blockade are necessary to study immune responses in preclinical mouse cancer models.

The correct storage temperature for sterile packaged clone 9D9 is 2–8?°C for short-term storage (up to one month), and -20 to -70?°C for long-term storage (up to 12 months) as supplied.

For most research purposes:

• Up to 1 month: Store at 2–8?°C (refrigerator). Do not freeze during this period if you plan short-term use.
• Up to 12 months: For extended storage, keep at -20 to -70?°C (manual defrost freezer) and avoid repeated freeze-thaw cycles.

These guidelines presume the antibody is supplied without glycerol or other stabilizers; always consult the product datasheet for formulation-specific instructions. The product is typically shipped cold (with ice pack) and should be refrigerated immediately upon receipt for short-term, or frozen for long-term storage.

In the literature, the 9D9 antibody is most commonly used for targeting mouse CTLA-4 (CD152) as a surrogate for CTLA-4 blockade studies in murine cancer models. Other antibodies and proteins frequently used together with 9D9, for comparison or combination therapy, include:

• Anti-CTLA-4 antibodies targeting human CTLA-4, such as:

  • Ipilimumab
  • Tremelimumab

  These are typically referenced for comparison because 9D9 targets a different epitope on CTLA-4 and has different biophysical properties from these clinically used antibody drugs. Engineered variants of ipilimumab that bind both human and mouse CTLA-4 are also used to facilitate translational studies.

• Regulatory T cell (Treg) depletion antibodies or constructs, such as:

  • b1s1e2-Fc, a nonantagonistic CTLA-4 binder

  This protein is sometimes administered alongside 9D9 in studies examining mechanisms related to tumor immune microenvironments and Treg modulation.

• General checkpoint blockade controls:

  • Other checkpoint inhibitors or immune modulators may be administered either alone or in combination with 9D9 to assess synergy or mechanistic interactions. However, the specific antibodies vary by study design and are less frequently mentioned in the context of direct comparison to 9D9 than ipilimumab, tremelimumab, or engineered cross-reactive variants.

• Isotype controls and framework-modified anti-CTLA-4 constructs:

  • Modified forms of 9D9 (e.g., for DNA-encoded mAb delivery) and isotype control antibodies are used to ensure specificity of immune effects in research settings.

In summary, ipilimumab, tremelimumab, and their engineered variants (for murine cross-reactivity), as well as proteins affecting Treg cell populations, are among the most frequently used antibodies or proteins with 9D9 in published research, serving roles in both mechanistic comparison and combination therapy studies.

The key findings from scientific literature citing clone 9D9—a mouse anti-mouse CTLA-4 monoclonal antibody—center on its role as a surrogate for immune checkpoint blockade studies, its anti-tumor efficacy, unique binding properties, and mechanisms of T cell activation in preclinical models.

Major findings include:

• Potent anti-tumor activity in mouse models: 9D9 demonstrates significant anti-tumor effects, including enhanced lymphocyte infiltration into tumors, activation of CD8+ T cells, reduction of immunosuppressive regulatory T cells, and high rates of tumor clearance in therapeutic settings.
• Enhanced T cell priming and activation: Treatment with 9D9 increases effector T cell proliferation and activation markers in tumor-draining lymph nodes, showing that CTLA-4 antagonism is required for optimal T cell priming and maximum antitumor efficacy of anti-CTLA-4 therapies.
• Unique epitope specificity and pH-dependent binding: Structural analysis reveals that 9D9 binds a distinct epitope on mouse CTLA-4 compared to clinically used antibodies (ipilimumab and tremelimumab). Additionally, 9D9 loses binding under acidic conditions (unlike ipilimumab), which has implications for mechanistic studies and antibody engineering.
• Frequently used as a surrogate in mouse research: Due to limited cross-reactivity of clinical CTLA-4 antibodies with mouse proteins, 9D9 is a preferred surrogate for studying immune checkpoint blockade in mouse cancer models.

Additional insights:

• Modifications can improve 9D9 expression: Engineering the DNA sequence encoding 9D9 can substantially increase antibody yield without affecting target binding, facilitating mechanistic studies and comparative efficacy analyses.
• Biophysical differences from therapeutics: The distinctive binding characteristics of 9D9 compared to therapeutic antibodies highlight challenges in preclinical-to-clinical translatability and motivate the development of cross-reactive engineered variants.

In summary:9D9 is a well-established tool for preclinical CTLA-4 blockade research, showing strong efficacy in activating anti-tumor immune responses and possessing unique structural and biophysical properties that are important for interpreting preclinical results and developing translatable antibody therapeutics.