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

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

Product No.: C2856

[product_table name="All Top" skus="C2856"]

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Clone
9D9
Target
CTLA-4
Formats AvailableView All
Product Type
Monoclonal Antibody
Alternate Names
CD152, Cytotoxic T Lymphocyte-Associated Antigen-4, Ly-56
Isotype
Mouse IgG2b
Applications
FA
,
in vivo
,
WB

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Data

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Antibody Details

Product Details

Reactive Species
Mouse
Host Species
Mouse
Recommended Isotype Controls
Recommended Dilution Buffer
Immunogen
Not Available
Product Concentration
≥ 5.0 mg/ml
Endotoxin Level
<0.5 EU/mg as determined by the LAL method
Purity
≥98% monomer by analytical SEC
>95% by SDS Page
Formulation
This monoclonal antibody is aseptically packaged and formulated in 0.01 M phosphate buffered saline (150 mM NaCl) PBS pH 7.2 - 7.4 with no carrier protein, potassium, calcium or preservatives added. Due to inherent biochemical properties of antibodies, certain products may be prone to precipitation over time. Precipitation may be removed by aseptic centrifugation and/or filtration.
Product Preparation
Functional grade preclinical antibodies are manufactured in an animal free facility using in vitro cell culture techniques and are purified by a multi-step process including the use of protein A or G to assure extremely low levels of endotoxins, leachable protein A or aggregates.
Pathogen Testing
To protect mouse colonies from infection by pathogens and to assure that experimental preclinical data is not affected by such pathogens, all of Leinco’s Purified Functional PLATINUM™ antibodies are tested and guaranteed to be negative for all pathogens in the IDEXX IMPACT I Mouse Profile.
Storage and Handling
Functional grade preclinical antibodies may be stored sterile as received at 2-8°C for up to one month. For longer term storage, aseptically aliquot in working volumes without diluting and store at ≤ -70°C. Avoid Repeated Freeze Thaw Cycles.
Country of Origin
USA
Shipping
Next Day 2-8°C
Each investigator should determine their own optimal working dilution for specific applications. See directions on lot specific datasheets, as information may periodically change.

Description

Description

Specificity
Clone 9D9 recognizes an epitope on mouse CTLA-4.
Background
CTLA-4 is a 33 kD member of the Ig superfamily similar to CD28 in amino acid sequence, structure, and genomic organization. CTLA-4 is a protein receptor that functions as an immune checkpoint and downregulates immune responses. It is involved in the development of protective immunity and thymocyte regulation, in addition to the induction and maintenance of immunological tolerance. CTLA-4 has therapeutic potential both as an agonist to reduce immune activity, and an antagonist to increase immune activity.
Antigen Distribution
CTLA-4 is expressed on activated T and B lymphocytes.
Ligand/Receptor
CD80 (B7.1), CD86 (B7.2)
Function
Negative regulator of T cell activation
NCBI Gene Bank ID
Research Area
Immunology
.
Inhibitory Molecules

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Common In Vivo Applications of Clone 9D9 in Mice

Clone 9D9 is a monoclonal antibody targeting mouse CTLA-4 (CD152), a key immune checkpoint that negatively regulates T cell activation. Its in vivo applications are primarily in the field of cancer immunotherapy, tumor microenvironment research, and mechanistic immunology studies in mice.

Cancer Immunotherapy

  • Monotherapy and Combination Therapy: 9D9 is widely used to block CTLA-4, thereby enhancing T cell activation and anti-tumor immunity. It has shown efficacy as both a standalone treatment and in combination with other immunotherapies, such as anti-PD-1/PD-L1 agents, mirroring the clinical use of drugs like ipilimumab.
  • Tumor Models: The antibody has been tested in various tumor models, including melanoma and colon cancer, where it demonstrates significant tumor growth control and, in some settings, complete tumor clearance.
  • Dosing: Typical in vivo dosing is 100–250 µg per mouse, administered intraperitoneally every 3 days. Intratumoral injection has also been explored.

Tumor Microenvironment Studies

  • Immune Cell Infiltration: 9D9 treatment increases global lymphocyte and specifically CD8+ T cell infiltration into tumors, along with upregulation of T cell activation markers (CD44, CD69, PD-1).
  • Regulatory T Cell (Treg) Modulation: Due to its mouse IgG2b isotype, 9D9 can deplete intratumoral Tregs, further enhancing anti-tumor immunity. Studies show a significant reduction in the proportion of CD4+CD25+FoxP3+ Tregs within treated tumors.
  • Mechanistic Insights: By altering the balance of effector and regulatory T cells, 9D9 helps elucidate the role of CTLA-4 in immune suppression within the tumor microenvironment.

Alternative Delivery Methods

  • DNA-Encoded Antibody (DMAb) Platforms: 9D9 has been delivered via DNA plasmids, enabling in vivo production of the antibody after intramuscular electroporation. This approach achieves therapeutic levels of antibody in the serum and demonstrates efficacy in controlling tumor growth, offering a versatile platform for antibody delivery in preclinical studies.

Additional Applications

  • Functional Assays: Beyond tumor models, 9D9 is used in assays to study biological pathways affected by CTLA-4, including pharmacokinetic (PK) and anti-drug antibody (ADA) assays.
  • Tool for Immunological Research: As a surrogate for human CTLA-4 blockade, 9D9 is instrumental in preclinical studies aiming to understand checkpoint inhibition and optimize combination regimens.

Summary Table: Key In Vivo Applications of 9D9

Application AreaDetailsCitation
Cancer ImmunotherapyMonotherapy/combination, tumor growth control, T cell activation
Tumor MicroenvironmentEnhanced T cell infiltration, Treg depletion, immune activation markers
Delivery MethodsProtein injection, DNA-encoded antibody (DMAb) platforms
Mechanistic StudiesRole of CTLA-4 in immune suppression, combination therapy mechanisms
Functional AssaysPK/ADA assays, pathway analysis

Conclusion

Clone 9D9 is a cornerstone tool in mouse immunology and cancer research, primarily for studying CTLA-4 blockade, enhancing anti-tumor immunity, modulating the tumor microenvironment, and testing novel antibody delivery platforms. Its ability to deplete Tregs and promote CD8+ T cell activity makes it especially valuable for understanding and improving cancer immunotherapy strategies.

In the literature, antibodies or proteins commonly used alongside the 9D9 monoclonal antibody, which targets mouse CTLA-4, include:

  • Ipilimumab and Tremelimumab: These are human CTLA-4 targeting antibodies often referenced in studies for comparison or combination therapy with 9D9.
  • PD-1/PD-L1 Pathway Targeting Antibodies: While not specifically paired with 9D9, these are used in broader immune checkpoint studies, including combination therapies with anti-CTLA-4 antibodies like ipilimumab to enhance antitumor responses.
  • Other anti-mouse CTLA-4 clones: Such as 9H10 and 4F10, which are also used in mouse models for CTLA-4 blockade studies.

These antibodies and proteins are used in various research contexts, including cancer immunotherapy models, to better understand the mechanisms of CTLA-4 blockade and to develop more effective treatments.

Clone 9D9 is a widely used anti-mouse CTLA-4 monoclonal antibody with significant impact in immuno-oncology research. Key findings from scientific literature on 9D9 reveal that:

  • 9D9 promotes anti-tumor immune responses by increasing lymphocyte and CD8+ T cell infiltration into tumors, elevating activation markers (CD44, CD69, PD1) on T cells, and reducing the proportion of regulatory T cells (Tregs; CD4+/CD25+/FoxP3+), resulting in tumor regression.

  • The therapeutic efficacy of 9D9 depends on its ability to both antagonize CTLA-4 signaling and deplete intra-tumoral Tregs. Studies directly comparing 9D9 with non-antagonistic constructs show that while both can deplete Tregs, only 9D9 enhances T cell activation, T cell priming in tumor-draining lymph nodes, and achieves higher rates of long-term tumor cures in mouse models.

  • Expression optimization in DNA-encoded monoclonal antibody systems has been achieved for 9D9, significantly increasing antibody yield in vitro and in vivo without compromising CTLA-4 binding specificity or efficacy.

  • Mechanistic insights demonstrate that 9D9’s antitumor activity is not solely due to Treg depletion; CTLA-4 antagonism is required for optimal T cell priming and maximum survival benefit. When 9D9’s Fc region is silenced, it loses therapeutic efficacy, confirming the necessity of both Treg depletion and checkpoint blockade.

  • Comparative studies have found differences among anti-CTLA-4 clones (e.g., 9D9, 9H10, UC10-4F10-11), with 9D9 excelling in intratumoral Treg depletion and immune activation.

In summary, clone 9D9’s unique capability to combine CTLA-4 antagonism with effective Treg depletion leads to enhanced T cell activation and robust antitumor immunity, supporting its use in studies of immune checkpoint blockade and cancer immunotherapy.

Dosing regimens of clone 9D9 (anti-CTLA-4) show remarkable consistency across different mouse models, though specific applications have introduced some variations in administration routes and frequencies.

The standard dosing approach for clone 9D9 uses 100-250 μg per mouse administered via intraperitoneal injection every 3 days. This range has become the established protocol across most cancer immunotherapy studies. The specific dose within this range often depends on the experimental objectives and tumor model being studied.

Route of Administration

While intraperitoneal injection represents the primary delivery method, clone 9D9 has also been tested via intratumoral administration in certain experimental designs. This flexibility in delivery routes reflects the antibody's versatility in different research contexts, though intraperitoneal remains the most commonly reported approach.

Timing and Schedule

The every-3-day dosing schedule appears consistently across various mouse models. This frequency has been optimized to maintain therapeutic antibody levels while allowing for effective checkpoint blockade. In some protocols, treatment begins as early as 3 days after tumor implantation, demonstrating the antibody's utility in early-stage disease models.

Combination Therapy Contexts

Clone 9D9 is frequently employed in combination therapy studies, where it's paired with anti-PD-1 or anti-PD-L1 antibodies. In these combination regimens, the dosing typically remains within the standard 100-250 μg range. Multiple-dose pharmacokinetic studies have shown that when administered at 1-10 mg/kg twice weekly, clone 9D9 demonstrates a notably long terminal half-life, approximately five-fold longer than some comparator antibodies, leading to greater than two-fold accumulation over repeated dosing cycles.

Unique Functional Properties

An important consideration for clone 9D9 across different models is its dual mechanism of action. Due to its mouse IgG2b Fc domain, this clone not only blocks CTLA-4 but also depletes intratumoral regulatory T cells (Tregs), which enhances its anti-tumor immunity effects. This property distinguishes it from other anti-CTLA-4 clones and may influence dosing decisions in specific experimental contexts.

References & Citations

FA
in vivo Protocol
General Western Blot Protocol

Certificate of Analysis

Formats Available

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Disclaimer AlertProducts are for research use only. Not for use in diagnostic or therapeutic procedures.