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

Clone 9D9, a monoclonal antibody targeting mouse CTLA-4 (CD152), is commonly used in in vivo applications for cancer immunotherapy research in mice. Here are some of its primary applications:

1. Cancer Immunotherapy: Clone 9D9 is used to block CTLA-4, a checkpoint molecule that inhibits T-cell activation. By blocking CTLA-4, it enhances anti-tumor immunity, often in combination with other immunotherapies like anti-PD-1/PD-L1.

2. Tumor Microenvironment Studies: It is used to study the effects of CTLA-4 blockade on the tumor microenvironment, particularly in increasing T-cell infiltration and reducing regulatory T cells (Tregs).

3. Tumor Growth Control: Clone 9D9 can control tumor growth and induce tumor clearance in mouse models, making it a valuable tool for therapeutic studies.

4. Treg Depletion: Its ability to deplete intratumoral Tregs enhances anti-tumor responses by promoting a favorable immune environment.

For in vivo studies, clone 9D9 is typically administered via intraperitoneal injection, with a recommended dose range of 100-250 μg per mouse, and dosing is usually performed every 3 days.

In the literature, 9D9, a monoclonal antibody specific to mouse CTLA-4, is often used alone for CTLA-4 checkpoint blockade studies. However, it is compared or mentioned alongside other CTLA-4 targeting antibodies, such as:

• Ipilimumab: A therapeutic antibody targeting human CTLA-4, used in clinical settings.
• Tremelimumab: Another anti-human CTLA-4 antibody, also used in clinical contexts.

For broader immune checkpoint studies, while not specifically paired with 9D9, other antibodies targeting different pathways, such as PD-1/PD-L1, are commonly used in cancer immunotherapy research.

There is limited direct mention of other specific antibodies or proteins used in combination with 9D9 in the literature, but it is often used as a surrogate in mouse models to study CTLA-4 blockade mechanisms.

Clone 9D9, an anti-mouse CTLA-4 antibody, has emerged as a pivotal tool in cancer immunotherapy research, with scientific literature revealing its multifaceted mechanisms and therapeutic potential. This antibody operates through dual mechanisms that distinguish it from other CTLA-4-targeting agents, making it particularly valuable for preclinical studies.

Dual Mechanism of Action

Clone 9D9 functions through two primary mechanisms that work synergistically to enhance anti-tumor immunity. The antibody blocks CTLA-4, a negative regulator of T cell activation, while simultaneously depleting intratumoral regulatory T cells (Tregs). This dual action sets it apart from other anti-CTLA-4 clones and contributes to its robust therapeutic efficacy.

The CTLA-4 blockade component works by preventing negative signaling and reducing B7 transendocytosis, which enhances T cell priming in tumor-draining lymph nodes. Within just 3 days of treatment, 9D9 increases the percentage of CD4+ and CD8+ effector T cells expressing proliferation marker Ki67 and activation marker PD-1 in these lymph nodes.

Impact on Tumor Microenvironment

Studies demonstrate that 9D9 profoundly alters the tumor microenvironment in ways that favor anti-tumor immunity. The antibody increases global lymphocyte infiltration (CD3+ cells) as well as CD8+ T cell infiltration specifically. These infiltrating CD8+ T cells display heightened activation, expressing elevated levels of CD44, CD69, and PD1.

Critically, tumors treated with 9D9 exhibit a significantly lower proportion of regulatory T cells (CD4+/CD25+/FoxP3+), reflecting the antibody's Treg-depleting capacity. This reduction in immunosuppressive Tregs within the tumor creates a more permissive environment for effector T cell function.

Therapeutic Efficacy

The therapeutic potential of 9D9 has been validated across multiple tumor models and treatment settings. In the CT26 tumor model, 9D9 demonstrated effectiveness in a therapeutic setting when administration began 3 days after tumor implantation, inducing tumor clearance in 8 out of 10 mice. The antibody has proven versatile across different mouse strains and tumor types, consistently showing tumor regression capabilities.

However, research indicates that both CTLA-4 antagonism and intratumoral Treg depletion are essential for maximum efficacy. Studies comparing 9D9 to modified versions with silenced Fc regions revealed that antagonism alone is insufficient—the Fc-silenced version resulted in survival similar to control mice. Conversely, constructs that depleted Tregs but lacked antagonistic properties also proved less effective than wild-type 9D9, with only the full 9D9 showing signs of enhanced T cell activation.

Limitations and Treg Expansion

Despite its efficacy, 9D9 cured less than 50% of mice in some studies, a limitation attributed to nonspecific T cell priming in tumor-draining lymph nodes. An important consequence of CTLA-4 antagonism is that nodal Tregs, which express lower levels of CTLA-4 than intratumoral Tregs, experience enhanced costimulation and subsequent expansion rather than depletion. This proliferative Treg response may allow immunosuppressive functions to dominate the anti-tumor immune response, potentially limiting overall therapeutic benefit.

DNA-Encoded Antibody Platform

Recent innovations have explored delivering 9D9 through synthetic DNA-encoded monoclonal antibodies (DMAbs). Initial designs produced relatively low antibody levels (~660ng/mL in vitro and ~1.2μg/mL in serum), but framework modifications improved expression nearly 10-fold without altering binding to mouse CTLA-4 protein. The optimized DMAb version (mod #4) achieved serum levels of approximately 7.9μg/mL, representing over 6-fold improvement while maintaining similar IC50 values (36.105–44.25ng/mL) compared to recombinant 9D9.

These findings establish clone 9D9 as a cornerstone tool in mouse cancer immunotherapy research, valued for its ability to simultaneously block CTLA-4 signaling and deplete intratumoral Tregs, though its therapeutic ceiling remains constrained by nodal Treg expansion dynamics.

Dosing regimens of clone 9D9 in mouse models generally use 100–250 μg per mouse, administered intraperitoneally (i.p.) every 3 days, but can vary depending on the specific study design, application, and sometimes route of administration.

Key points on variation across different mouse models:

• Dose Range: Most studies use doses between 100–250 μg per mouse.
• Route of Administration: The intraperitoneal (i.p.) injection is standard, but some settings test intratumoral injection especially in tumor models.
• Frequency: The typical schedule is injection every 3 days, though clinical model needs or combination therapy designs may adjust this interval.
• Mouse Strain: Dosing amount and frequency are generally similar among common laboratory strains (e.g., C57BL/6, BALB/c, CD1), with modifications sometimes based on tumor burden, immune status, or body weight.
• Model Specific Differences:
  • In syngeneic tumor models (like CT26, MC38, B16), dosing usually sticks to 100–250 μg, i.p., every 3 days, matching the standard application for checkpoint blockade immunotherapy.
  • For antibody gene delivery studies (as DNA-encoded monoclonal antibody, DMAb), dosing references the DNA construct (e.g., 100 μg DNA via IM-EP), with resulting serum levels reflecting expression rather than direct antibody injection.
• Antibody Format: Studies using mIgG1 backbone for 9D9 may report altered pharmacokinetics (e.g., longer half-life, leading to possible accumulation), potentially justifying model-specific dose adjustment.
• Combinatorial Studies: When combined with other therapies (e.g., anti-PD-1/PD-L1 antibodies, radiotherapy, or chemotherapy), 9D9 dosing is usually unchanged, but frequency or duration of treatment might be optimized based on therapeutic synergy or toxicity observed in that particular mouse model.

Summary Table: Clone 9D9 Dosing Regimens in Mouse Models

• i.p. = intraperitoneal, i.t. = intratumoral, IM-EP = intramuscular electroporation
• Dose and regimen can depend on tumor size, mouse strain, and concurrent therapies

In summary: While the core regimen for clone 9D9 across mouse models remains 100–250 μg i.p. every 3 days, model-specific adjustments (e.g., route, formulation, or frequency) are made based on experimental needs, such as pharmacokinetic profiling, gene delivery, or combination immunotherapy.