Anti-Mouse CD152 (CTLA-4) [Clone 9H10] — Purified in vivo GOLD™ Functional Grade

Clone 9H10 is a widely used Syrian hamster IgG monoclonal antibody that targets mouse CTLA-4 (CD152) and has been extensively employed in preclinical cancer immunotherapy studies. This antibody functions through multiple mechanisms that make it particularly valuable for in vivo research applications.

Primary Mechanisms of Action

Clone 9H10 operates by blocking CTLA-4 binding to its ligands, which allows for enhanced CD28 binding and promotes T cell co-stimulation. This mechanism is crucial for unleashing the immune system's ability to mount stronger anti-tumor responses. Additionally, the antibody demonstrates the ability to deplete intra-tumoral regulatory T cells (Tregs), which are immunosuppressive cells that often limit effective anti-cancer immunity.

Research Applications and Efficacy

In mouse melanoma models, clone 9H10 has shown slightly stronger Treg depletion efficacy compared to the 9D9 clone, making it particularly effective for studies investigating tumor immunity. The antibody has been instrumental in demonstrating the potential of CTLA-4 blockade in enhancing anti-tumor immunity, with early studies establishing its efficacy in various cancer models.

Functional Characteristics

Clone 9H10 is formulated to neutralize CTLA-4 both in vitro and in vivo, providing researchers with flexibility in experimental design. The antibody is available in high-purity formats (>95% determined by SDS-PAGE) and is rigorously tested to ensure it is murine pathogen-free with endotoxin levels below 1EU/mg. This quality control is essential for in vivo studies where contamination could confound experimental results.

Experimental Considerations

For in vivo applications, clone 9H10 requires specific handling protocols, including the use of InVivoPure™ pH 7.0 Dilution Buffer and appropriate isotype controls (polyclonal Syrian hamster IgG). The antibody is available in multiple sizes ranging from 5mg to 100mg and more, accommodating various study scales and dosing requirements. Researchers have successfully used this clone in both monotherapy approaches and combination studies with other immune checkpoint inhibitors or conventional cancer treatments.

In the literature, the antibody 9H10 is used in two distinct contexts: one targeting the influenza hemagglutinin stalk and another targeting CTLA-4 (Cytotoxic T-lymphocyte-associated protein 4).

Influenza Hemagglutinin Context

In studies related to influenza, antibodies like 9H10 that target the hemagglutinin stalk are often compared or used alongside other broadly neutralizing antibodies such as CR8020 and CR8043. These antibodies are known for their ability to neutralize a wide range of influenza A viruses by targeting the conserved stalk region of the hemagglutinin protein.

CTLA-4 Context

In immunology, particularly in the context of CTLA-4, 9H10 is frequently used in conjunction with other anti-CTLA-4 antibodies like 9D9 and UC10-4F10-11. These antibodies are used for various applications, including Western blot, flow cytometry, and in vivo studies, particularly for blocking CTLA-4 function and depleting regulatory T cells (Tregs).

Some of the other proteins or antibodies commonly used with 9H10 in the CTLA-4 context include:

• CD28: This protein is involved in T cell co-stimulation, and its function is indirectly promoted when CTLA-4 binding is blocked by antibodies like 9H10, allowing CD28 to interact more effectively with its ligands.
• B7-1 and B7-2: These are the ligands for both CTLA-4 and CD28. Understanding their interaction is crucial in studies involving CTLA-4 blocking antibodies like 9H10.

In the context of influenza, other proteins or components might involve the hemagglutinin itself, NA proteins, or other viral components critical for viral replication and infection processes.

Clone 9H10 is a well-characterized antibody targeting the immune checkpoint protein CTLA-4, and key findings from its citations in scientific literature emphasize its mechanism of action, efficacy, and applications in immuno-oncology.

• Mechanism and Efficacy:

  • 9H10 is a Syrian hamster IgG that binds mouse CTLA-4, blocking its interaction with ligands and promoting T cell co-stimulation, allowing for more robust activation of effector T cells.
  • Notably, 9H10 also depletes intra-tumoral regulatory T cells (Tregs) via antibody-dependent cellular cytotoxicity (ADCC), improving antitumor memory responses. Its efficacy in Treg depletion is reported to be slightly stronger than that of the 9D9 clone in mouse melanoma models.
  • Upon tumor rechallenge, mice treated with 9H10 showed minimal tumor growth, indicating a durable memory response, superior to anti-PD-1 antibodies and other anti-CTLA-4 clones evaluated. This enhanced memory depends on T-cell intrinsic effects rather than solely on Treg depletion.
  • 9H10 treatment has led to significantly longer survival in mouse CT26 colon carcinoma models, demonstrating its antitumor potency.

• Comparisons and Structural Considerations:

  • Compared to other anti-mouse CTLA-4 antibodies (such as 9D9 and UC10-4F10), 9H10 is distinguished by its host species, isotype, and both in vitro and in vivo blocking capacities.
  • Chimeric and novel antibodies have been structurally compared to 9H10, with new formats demonstrating higher in vitro blocking affinities but highlighting the importance of the Fc domain for in vivo efficacy, which 9H10 possesses.

• Applications:

  • 9H10 is widely used for CTLA-4 detection in Western blot assays, and its ability to deplete Tregs is crucial for studying immunotherapy mechanisms.
  • It serves as a standard or control antibody in preclinical models to compare newer antibody formats, epitope vaccines, or combination therapies.

• Key Insights:

  • Intra-tumoral Treg depletion by 9H10 underpins its unique contribution to antitumor memory and survival benefits compared to other clones and checkpoint inhibitors.
  • The Fc region and ability to mediate ADCC are critical for its in vivo activity, setting it apart from antibodies lacking this feature.
  • 9H10’s robust antitumor effects inform ongoing antibody engineering to improve immunotherapeutic strategies.

In summary, clone 9H10’s main scientific impact is as a potent anti-CTLA-4 antibody that promotes T cell activation, depletes intra-tumoral Tregs, enables stronger and longer-term antitumor immune memory, and serves as a benchmark in preclinical immunotherapy studies.

The dosing regimens of clone 9H10 (anti-mouse CTLA-4 antibody) vary by dose, route, schedule, and mouse model, but standard approaches have emerged for preclinical studies:

• Standard Dose Range: Commonly reported doses are 100–200??g per mouse, typically delivered via intraperitoneal injection every ~3 days in cancer immunotherapy models. Some studies use weight-based dosing, ranging from 0.625 to 10?mg/kg, administered intravenously every 3 days for 3 doses.

• Variation by Model and Protocol:

  • In the D2F2 mammary tumor model, 200??g/dose of 9H10 was given, with frequency unspecified but likely matching the every-3-day interval typical for such studies.
  • In a syngeneic tumor model validated across studies, dosages ranged from 0.625 to 10?mg/kg IV every 3 days × 3, with tumor volume tracked to assess efficacy and tolerability.
  • In combination immunotherapy (such as with anti-OX40), much lower doses (e.g., 10–30??g per injection, given biweekly or at similar frequencies) have shown significant efficacy with minimal toxicity in certain models like MC38 and A20 tumors.

• Toxicity and Tolerance:

  • Higher doses (e.g., repeated 30??g injections in combination regimens) carried increased risk of toxicity, particularly in sensitive models (deaths occurred in the MC38 model at the highest repeated dose). Lower doses (10??g) produced strong anti-tumor effects with minimal observed systemic toxicity.

• Route and Frequency:

  • Most protocols use intraperitoneal (IP) or intravenous (IV) routes, with dosing every 3 days being a common schedule.
  • Intra-tumoral injections have been explored but are less common and more protocol-specific.

• Functional differences:

  • Clone 9H10 is not only a CTLA-4 blocker but also depletes intra-tumoral regulatory T cells (Tregs), which can impact the required dose and expected toxicity depending on the tumor microenvironment and mouse strain.

Summary Table: Clone 9H10 Dosing Regimens in Mouse Models

In summary, dose adjustments are made primarily based on mouse model, tumor type, combination partners, and desired toxicity profile. Doses can range from as low as 10??g (in combination settings for minimal toxicity) to 10?mg/kg for single-agent pharmacology studies, but 100–200??g every 3 days, intraperitoneally, is the most common monotherapy protocol.