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

Use of Clone 9D9 in In Vivo Mouse Studies

Clone 9D9 is a monoclonal antibody specific to mouse CTLA-4 (CD152), widely used as a surrogate for CTLA-4 checkpoint blockade in preclinical cancer immunotherapy studies. Its primary applications and mechanisms are summarized below.

Key Mechanisms

• CTLA-4 Blockade: 9D9 neutralizes CTLA-4, a negative regulator of T cell activation, thereby enhancing anti-tumor immune responses.
• Treg Depletion: Due to its mouse IgG2b Fc region, 9D9 can also deplete regulatory T cells (Tregs) within tumors, further promoting anti-tumor immunity.
• Immune Modulation: Treatment with 9D9 leads to increased infiltration of CD3+ and CD8+ T cells into tumors, with CD8+ T cells showing elevated activation markers (CD44, CD69, PD-1), indicating a potent immune stimulatory effect.

Experimental Protocols

• Dosing: The standard dose range is 100–250 ?g per mouse, administered via intraperitoneal injection, though intratumoral delivery has also been tested. Dosing is typically repeated every 3 days.
• Models: 9D9 has been employed in various tumor models, including melanoma and colon cancer, both as monotherapy and in combination with other immunotherapies (e.g., anti-PD-1/PD-L1).
• Efficacy: In therapeutic settings (e.g., CT26 colon carcinoma), 9D9 administration starting a few days after tumor implantation can lead to significant tumor control and even clearance in a majority of treated mice.
• DNA-Encoded Antibody Delivery: Advanced platforms deliver 9D9 as a DNA-encoded monoclonal antibody (DMAb), enabling in vivo production of the antibody after intramuscular electroporation. This approach maintains functional activity, including tumor infiltration by activated T cells and Treg depletion.

Comparative Context

• Surrogate for Human Therapeutics: 9D9 is commonly used as a mouse-specific surrogate in preclinical studies because most human anti-CTLA-4 antibodies (e.g., ipilimumab) do not cross-react with mouse CTLA-4.
• Epitope Differences: The epitope recognized by 9D9 on mouse CTLA-4 is distinct from those targeted by clinical antibodies like ipilimumab, which may limit direct translation of findings to human therapies.

Summary Table: 9D9 in Mouse In Vivo Studies

Conclusion

Clone 9D9 is a cornerstone tool in mouse cancer immunotherapy research, valued for its dual action of CTLA-4 blockade and intratumoral Treg depletion. It is administered systemically or via advanced DNA delivery platforms, with dosing and scheduling optimized for maximal immune activation and tumor control. However, researchers should be aware of its distinct epitope and potential differences from human therapies when interpreting results.

In the literature, the antibody 9D9 is often used in the context of CTLA-4 checkpoint blockade studies, particularly in mouse models. It is frequently compared or used alongside other therapeutic CTLA-4 antibodies like:

• Ipilimumab: This is a well-known anti-CTLA-4 antibody used in human cancer treatment, known for its pH-independent binding to human CTLA-4.
• Tremelimumab: Another anti-CTLA-4 antibody, which, like ipilimumab, targets a different epitope on CTLA-4 compared to 9D9.
• Nonantagonistic CTLA-4 binders (e.g., b1s1e2-Fc): These are used in studies to compare the effects of antagonistic versus nonantagonistic CTLA-4 interactions in mouse tumor models.

In addition to these CTLA-4 targeting agents, other antibodies or proteins may be used in the broader context of immune checkpoint studies, including those targeting PD-1/PD-L1 pathways, but they are not specifically paired with 9D9 in the available literature.

Key Findings from Clone 9D9 in Scientific Literature

Structure and Binding Properties

• Clone 9D9 is a mouse monoclonal antibody that specifically binds mouse CTLA-4 (CD152).
• Crystallographic analysis revealed that 9D9 binds to an epitope on mouse CTLA-4 distinct from that targeted by human therapeutic antibodies like ipilimumab and tremelimumab, indicating significant biophysical differences between surrogate and clinical anti-CTLA-4 antibodies.
• Unlike ipilimumab, which binds human CTLA-4 in a pH-independent manner, 9D9 loses binding to mouse CTLA-4 at physiologically relevant acidic pH, highlighting a functional difference that may affect its behavior in certain tumor microenvironments.

In Vitro and In Vivo Expression

• DNA-encoded monoclonal antibody (DMAb) platforms using the 9D9 sequence demonstrated that initial antibody expression was low both in vitro and in vivo, but engineering modifications to the heavy chain sequence significantly boosted production—up to a 10-fold increase in vitro and over 6-fold in mouse serum.
• Framework modifications improved expression without altering the antibody's binding affinity for mouse CTLA-4, as confirmed by similar IC50 values before and after modification.

Functional Activity in Preclinical Models

• 9D9 exhibits potent immune stimulatory effects: In mouse tumor models, 9D9 DMAb treatment led to increased infiltration of CD3+ and CD8+ T cells into tumors, with CD8+ T cells showing higher expression of activation markers (CD44, CD69, PD-1).
• Depletion of regulatory T cells (Tregs): Tumors treated with 9D9 had a significantly lower proportion of CD4+/CD25+/FoxP3+ Tregs, consistent with its known mechanism of depleting intra-tumoral Tregs in vivo.
• Anti-tumor efficacy: In therapeutic settings (e.g., CT26 tumor model), 9D9 DMAb administration resulted in high antibody expression and effective tumor control, inducing tumor clearance in the majority of treated mice.

Comparative Notes with Other Anti-CTLA-4 Clones

• Distinct mechanisms: While 9D9 primarily depletes Tregs, other clones like 9H10 block CTLA-4 binding to its ligands, promoting T cell co-stimulation, and also deplete Tregs, sometimes with slightly stronger efficacy in certain models.
• 9D9 is widely used as a surrogate in preclinical studies due to its ability to bind mouse CTLA-4, but its epitope and pH sensitivity differences from human therapeutics should be considered when interpreting translatability to clinical settings.

Summary Table: Functional Comparison of Anti-Mouse CTLA-4 Clones

Expert Perspective

Clone 9D9 is a critical tool for studying CTLA-4 blockade in mice, with well-documented effects on T cell activation and Treg depletion. However, its structural and biophysical differences from clinical anti-CTLA-4 antibodies underscore the importance of careful interpretation when extrapolating mouse data to human therapy. Engineering strategies have successfully enhanced its expression for advanced delivery platforms, further cementing its utility in preclinical immunology and oncology research.

Dosing regimens of clone 9D9 (anti-CTLA-4) in mouse models commonly use doses ranging from 100–250??g per mouse, administered intraperitoneally (i.p.) every 3 days. The route and specific dose can vary depending on the application and experimental design.

Key regimen details:

• Dose range: 100–250??g per mouse.
• Route: Most frequently intraperitoneal injection, though intratumoral injection has also been tested in some studies.
• Frequency: Every 3 days is typical for cancer immunotherapy and Treg depletion applications in tumor models.

Model-specific regimen adaptations:

• Standard syngeneic tumor models: In studies using models such as CT26 (colon carcinoma) and others in C57BL/6 or BALB/c mice, dosing at 100–250??g i.p. every 3 days is routine. This regimen is chosen to achieve sufficient target engagement for CTLA-4 blockade and maximize anti-tumor effects.
• Intratumoral administration: Some studies explore direct delivery into the tumor, potentially enabling a lower systemic exposure but higher local concentration. The precise dose for intratumoral administration may be adjusted and is determined empirically, but still within the 100–250??g per mouse range.
• DNA-encoded delivery (DMAb): In engineered mouse models with DNA-encoded monoclonal antibody strategy, a single administration of 100??g plasmid DNA delivered intramuscularly via electroporation has been tested, resulting in sustained antibody expression and measurable serum levels of 9D9. This approach is not a typical direct antibody dosing regimen but provides a continuous in vivo supply, rather than repeated bolus doses.

Variation across mouse strains and tumor models:

• There is no evidence that major protocol differences are used between common immunocompetent strains (e.g., BALB/c vs. C57BL/6); the same dosing schedules and ranges are reported effective across these backgrounds.
• Adjustments in dosing are primarily determined by experimental goals (e.g., maximizing Treg depletion vs. balancing efficacy and toxicity), mode of antibody delivery, and the need to combine 9D9 with other immunotherapies.

When employing 9D9 in new settings, dose titration within the 100–250??g per mouse every 3 days range (i.p.), or following previously published regimens for comparable models, is recommended unless specific pharmacodynamic or toxicity data warrant further optimization.