Anti-Mouse CD40 [Clone FGK4.5] — Purified in vivo PLATINUM™ Functional Grade

Clone FGK4.5 is an agonistic anti-mouse CD40 monoclonal antibody that has several important in vivo applications in mouse research models, primarily focused on immune system activation and modulation.

Primary Applications

The FGK4.5 antibody is widely used in in vivo mouse studies to activate CD40-expressing antigen-presenting cells (APCs), including B cells, dendritic cells, and macrophages. As an agonistic antibody, it mimics the natural CD40-CD154 interaction, triggering downstream immune responses without requiring the presence of CD154 ligand.

Immune Cell Activation Studies

In vivo administration of FGK4.5 leads to significant changes in immune cell populations. Studies have demonstrated that treatment with this antibody causes a substantial increase in the total number of CD8+ T cells, B220+ B cells, and CD11c+ dendritic cells in the spleen. Additionally, it promotes cell proliferation, with measurable increases in DNA synthesis among splenic B cells and T cells. This makes FGK4.5 particularly valuable for researchers studying immune cell expansion and activation dynamics.

Cancer Immunotherapy Research

Agonistic CD40 monoclonal antibodies like FGK4.5 have shown promise in anti-tumor research by activating APCs and promoting anti-tumor T cell responses. The ability to enhance immune activation against tumors makes this clone an important tool for investigating cancer immunotherapy approaches and understanding how APC activation can be leveraged for therapeutic benefit.

Blocking CD40/CD154 Interactions

Beyond its agonistic properties, FGK4.5 can be used to inhibit CD40/CD154 interaction both in vitro and in vivo. This dual functionality allows researchers to study the consequences of blocking this critical co-stimulatory pathway, which is relevant for understanding autoimmune conditions and transplantation immunity.

The antibody is typically administered at doses around 500 μg per injection, with treatment regimens varying based on experimental design.

Commonly Used Antibodies and Proteins with FGK4.5 in the Literature

FGK4.5 (clone name: FGK4.5 or FGK45) is a well-known agonistic anti-mouse CD40 monoclonal antibody commonly used to study immune activation, particularly in murine models. In research, FGK4.5 is often used in combination with other antibodies and proteins to assess immune cell activation, co-stimulation, and modulation of immune responses.

Detection of Immune Activation and Co-Stimulation

When FGK4.5 is used to activate CD40-expressing antigen-presenting cells (APCs), researchers commonly measure downstream activation markers associated with immune cell stimulation. Some of the most frequently co-analyzed antibodies and proteins include:

• MHC class II (I-A, I-E): To assess antigen presentation capacity and APC activation.
• MHC class I (H-2D^b^): To evaluate cross-presentation and MHC I upregulation.
• CD80 (B7-1): A key co-stimulatory molecule upregulated upon activation.
• CD86 (B7-2): Another critical co-stimulatory molecule, upregulated in activated APCs.
• CD70: A marker that can also be upregulated with APC activation.

These markers are typically detected using flow cytometry or other immunoassays to confirm that FGK4.5 treatment elicits a robust activation of B cells, dendritic cells, and macrophages.

Functional Manipulation and Competitive Inhibition

FGK4.5 is also used in assays designed to block or compete with the natural ligand, CD154 (CD40L). For example, Flag-tagged soluble CD154 (sCD154) is used in competitive binding assays to demonstrate that FGK4.5 can inhibit the interaction between CD40 and its ligand. The ability of FGK4.5 to compete with sCD154 for binding to CD40 is a common experimental endpoint, often compared to other anti-CD40 antibodies.

In Vivo and Combination Studies

In in vivo studies, FGK4.5 is sometimes used in combination with other therapeutic antibodies, such as CTLA-4 Ig, to investigate synergistic effects on immune modulation. Additionally, other anti-mouse CD40 monoclonal antibodies like 1C10 and 3/23 are frequently referenced alongside FGK4.5, especially in studies comparing the potency or mechanism of different CD40-targeting antibodies.

Other Relevant Proteins

• Natural killer (NK) cell activation: FGK4.5 is sometimes used to study indirect activation of NK cells, which can have antitumor and antimetastatic effects.
• Functional grade and low endotoxin preparations: These are important for minimizing confounding effects in in vivo studies.

Summary Table

Key Points

• FGK4.5 is most commonly used with antibodies against MHC II, MHC I, CD80, CD86, and CD70 to assess APC activation and co-stimulation.
• Competitive assays with CD154 are used to evaluate the mechanistic basis of FGK4.5’s agonistic activity.
• In vivo, FGK4.5 is often compared to other anti-CD40 mAbs (e.g., 1C10, 3/23), and is sometimes combined with other immunomodulators like CTLA-4 Ig.
• Functional and low endotoxin preparations are critical for in vivo research to minimize non-specific immune activation.

This combination of tools and markers allows researchers to dissect the role of CD40 signaling in immune activation, tolerance, and therapeutic contexts.

Clone FGK4.5 is a functionally active, agonistic anti-mouse CD40 monoclonal antibody that has been widely used to study immune activation, particularly of antigen-presenting cells (APCs) such as dendritic cells, B cells, and macrophages. Key findings from scientific literature incorporating citations of clone FGK4.5 include:

• Potent agonist of CD40: FGK4.5 robustly activates CD40-expressing APCs both in vitro and in vivo, promoting upregulation of activation markers (e.g., MHC II, CD86, CD80) on B cells and macrophages.
• Dependence on FcγRIIB receptor: The agonistic activity and immune activation induced by FGK4.5 in vivo require engagement of the murine inhibitory Fcγ receptor, FcγRIIB. This receptor dependency is necessary for optimal upregulation of activation markers on B cells and macrophages in mice.
• Macrophage polarization and metabolic effects: FGK45 (the same as FGK4.5) reprograms tumor-associated macrophages (TAMs) to an anti-tumoral state. Mechanistically, it drives re-education of TAMs towards a pro-inflammatory phenotype via a metabolic switch involving glucose-independent, fatty acid oxidation (FAO)-dependent lactate production and altered glutamine metabolism, which are essential for CD40-driven macrophage polarization and anti-tumor activity.
• Induction of hemophagocytic syndrome: Administration of FGK4.5 can induce the full clinical spectrum of hemophagocytic syndrome in mice, thereby selectively activating macrophages and modeling relevant immune pathologies.
• Functional studies in mouse models: FGK4.5 has been instrumental in demonstrating the essential role of CD40-CD154 interactions in a range of immune responses, including B cell activation, germinal center formation, immunoglobulin class switching, and the development of memory B cells.
• Synergistic effect in combination immunotherapy: FGK4.5 has been used in studies showing synergy with other immune modulators, such as CTLA-4 blockade, in enhancing immune responses in transplantation and tumor models.

Summary Table of Key Findings

In summary, FGK4.5 is a central tool in mouse immunology, particularly for dissecting pathways of immune activation, APC function, macrophage polarization, and as a preclinical model for agonist CD40-based therapies.

Dosing regimens for clone FGK4.5 (anti-mouse CD40 agonist antibody) vary widely based on mouse strain, disease model, administration route, and experimental objectives, with reported doses ranging from about 24 μg to 5 mg/kg per mouse per injection.

Key reported regimens in literature:

• Cancer models (e.g., pancreatic tumor; Pan02)

  • 3 mg/kg, administered on days 7, 14, and 21 (intraperitoneal injection), assessed on day 22 for effects on tumor microenvironment.

• Oncolytic virus/virotherapy combination

  • 24 μg per mouse, intratumoral injection (i.t.), typically a single dose within a treatment schedule.

• Lymphoma model (A20 lymphoma)

  • 100 μg per mouse, intraperitoneal (i.p.) injection, used for transient activation of B cells and APCs.

• Single high-dose immunomodulation (C57BL/6 mice)

  • 500 μg per mouse, single i.p. injection.

• Cross-species potency comparison

  • Mouse studies often use 5 mg/kg dose (i.p.), which is much higher than human-compatible anti-CD40 doses due to lower potency and crosslinking requirements.

Additional context and practice:

• Dosing frequency: Reported schedules range from single injections to repeated administration every 3–7 days, depending on experimental needs and disease progression.
• Administration route: Intraperitoneal is standard for systemic activation; intratumoral is used for localized effects, especially in tumor or virotherapy models.
• Dose rationale: Lower doses (10–100 μg) are used for activation or costimulation studies; higher doses (mg/kg scale) are needed for robust agonistic effects or when mimicking clinical potency.
• Optimization: The manufacturer recommends titration for optimal performance and cellular activation in each experiment, as exact dose requirements can vary with mouse strain, disease context, and desired immunological outcome.

Summary Table: FGK4.5 Dosing Examples

For any specific model or application, it is essential to consult primary literature or manufacturers' dosing guides and to empirically determine optimal dosing through pilot titration. Variability in dosing reflects differences in disease mechanisms, antibody potency, administration method, and desired immunomodulatory outcomes.