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

Clone FGK4.5 (also known as FGK45) is an agonistic rat anti-mouse CD40 monoclonal antibody with several important in vivo applications in murine research. This antibody functions by binding to and activating CD40, a transmembrane glycoprotein expressed on antigen-presenting cells.

Activation of Antigen-Presenting Cells

The primary in vivo application of FGK4.5 is the activation of CD40-expressing antigen-presenting cells (APCs), including B cells, dendritic cells, and macrophages. When administered in vivo, this agonistic antibody mimics the natural interaction between CD40 and its ligand CD154, triggering costimulatory signals that activate these immune cells.

Immune Cell Expansion and Proliferation

In vivo administration of FGK4.5 leads to measurable changes in splenic immune cell populations. Studies have shown that mice treated with FGK4.5 (typically at doses of 500 μg given on days 0, 2, 4, and 6) exhibit significant increases in the total numbers of CD8+ T cells, B220+ B cells, and CD11c+ dendritic cells in the spleen. The antibody also stimulates DNA synthesis and proliferation in these cell populations, as demonstrated by BrdU incorporation assays.

Cancer Immunotherapy Research

Agonistic CD40 monoclonal antibodies like FGK4.5 have been shown to activate APCs and promote anti-tumor T cell responses. This makes the clone particularly valuable for investigating CD40 as a potential target for cancer immunotherapy, as CD40 activation can enhance immune surveillance against tumors.

CD40/CD154 Interaction Studies

Beyond its agonistic properties, FGK4.5 can be used to inhibit the interaction between CD40 and its natural ligand CD154 both in vitro and in vivo. This blocking capability is useful for studying the role of CD40-CD154 signaling in various immune processes, including autoimmune diseases and transplant rejection.

Dendritic Cell Maturation Studies

The antibody is employed in research investigating dendritic cell maturation, an important process for initiating adaptive immune responses. By activating CD40 on dendritic cells, FGK4.5 helps researchers understand how these cells develop their antigen-presenting capabilities.

Commonly used antibodies or proteins studied alongside FGK4.5 (an agonistic anti-mouse CD40 monoclonal antibody) include MHC class II, MHC class I (H-2D^b^), CD80, and CD86, which are key immune activation and co-stimulatory markers.

Essential context:

• These molecules are typically measured by flow cytometry to assess activation states of splenic B cells and macrophages following FGK4.5 treatment in mouse models.
• The upregulation of MHC class II, MHC class I, CD80, and CD86 is used as a surrogate for immune activation induced by CD40 stimulation.

Additional relevant proteins/antibodies:

• Soluble CD154 (CD40L): Used in competition and inhibition assays to characterize blockade or agonism of CD40-CD40L interactions, as shown by testing FGK4.5 alongside 7E1-G1 and 7E1-G2b, with detection via anti-FLAG-tagged antibodies.
• Isotype control antibodies: Often used as baseline controls for evaluating the specific effects of FGK4.5 in vivo.
• Other agonistic anti-mouse CD40 antibodies (such as 1C10 and 3/23) are sometimes used in comparative studies with FGK4.5 to examine Fc receptor dependence and potency.
• FcγRIIB receptor knockout mice and associated detection reagents are commonly included to evaluate the role of Fc receptor engagement in the agonistic activity of FGK4.5.

Summary of typical combinations in the literature:

These are the most commonly cited antibodies and proteins used with FGK4.5 in immunology and oncology research employing mouse models.

Clone FGK4.5 (also known as FGK45) is a widely used agonistic anti-mouse CD40 monoclonal antibody that plays a critical role in activating antigen-presenting cells and modulating immune responses in mice. Key findings from scientific literature on FGK4.5 cite the following:

• Potent Agonist of CD40: FGK4.5 robustly activates CD40-expressing antigen-presenting cells—including dendritic cells, B cells, and macrophages—in both in vitro and in vivo models.
• Immune Cell Activation and Cancer Immunotherapy: It mediates a wide array of immune and inflammatory responses, such as T cell-dependent immunoglobulin class switching, germinal center formation, memory B cell development, and reeducation of tumor-associated macrophages toward an anti-tumor phenotype.
• Metabolic Rewiring in Macrophages: FGK4.5 induces changes in macrophage metabolism, such as histone acetylation and the utilization of fatty acid/glutamine pathways, which are necessary for pro-inflammatory macrophage polarization and anti-tumor responses.
• Fc Receptor (FcR) Crosslinking Requirement: FGK4.5's in vivo potency depends on Fcγ receptor (FcγRIIB)-mediated crosslinking for full agonistic activity. It's significantly less potent than some clinical human antibodies and requires higher dosing in preclinical mouse models.
• Induction of Hemophagocytic Syndrome: In mice, selective macrophage activation with FGK4.5 can induce hemophagocytic syndrome, recapitulating many features of this clinical entity.
• Combination Therapy Potential: FGK4.5 has shown synergism with other immunotherapies, such as checkpoint blockade (e.g., anti-PD-1) and CTLA-4 Ig, enhancing anti-tumor efficacy and modulating immune cell function.

Supportive detail from early and recent citations highlights that FGK4.5:

• Mimics the physiological interaction of CD40 and CD154 (CD40L), acting as a powerful experimental tool in immunology, autoimmunity, and cancer research.
• Has been instrumental in dissecting CD40's role in antigen presentation, B cell function, and dendritic cell cytokine production, including the differential induction of IL-10 and IL-12 in dendritic cells.

In summary, FGK4.5 is established as a powerful tool for the activation and study of mouse CD40 signaling, immune modulation, and therapeutic strategies targeting the CD40 pathway in preclinical research.

Dosing regimens of clone FGK4.5 (an agonistic anti-CD40 antibody) vary considerably across mouse models depending on the application, tumor type, administration route, and experimental design. Reported doses span from 24 μg/mouse via intratumoral injection to 3–5 mg/kg via systemic administration, with dosing frequency and route chosen according to the model's goals.

Key dosing regimens reported in peer-reviewed studies:

• High systemic dose for tumor models:
  In an orthotopic pancreatic cancer model (Pan02), mice received 3 mg/kg of FGK4.5 via systemic administration on days 7, 14, and 21, with assessment on day 22. This regimen is suitable for robust immune activation and modulation of the tumor microenvironment.

• Low intratumoral dose for virotherapy studies:
  In studies combining CD40 agonists with oncolytic virus therapy, 24 μg FGK4.5/mouse was administered intratumorally in combination with virotherapy, designed to provide localized immune stimulation with reduced systemic exposure.

• Intermediate systemic dose for lymphoma model:
  In the A20 B cell lymphoma mouse model, mice received 100 μg FGK4.5 per mouse intraperitoneally (i.p.) to activate antigen-presenting cells (APCs) and stimulate immune responses. This dose elicits transient B cell activation and is a common starting point for in vivo studies.

• Cross-species dose comparison and potency:
  Some studies indicate doses up to 5 mg/kg may be needed for potent activation in mice, reflecting differences in potency compared to human CD40 agonists.

General principles for regimen selection:

• Route of administration varies:
  • Intraperitoneal (i.p.): Common for systemic immune activation and in cancer models.
  • Intratumoral (i.t.): Used for localized action in tumor or virotherapy studies, allowing lower doses.
• Frequency:
  • Most regimens involve dosing every 3–7 days, with some studies using single or repeated doses, tailored to model kinetics and antibody half-life.
• Dose selection:
  • Low-dose regimens (10–100 μg/mouse) tend to be used for localized or short-term studies.
  • High-dose regimens (1–5 mg/kg) are used for systemic immune activation or when cross-linking potency demands higher levels.

Variations by model/application:

• Tumor models (e.g., Pan02, A20, solid tumors): Doses range from 100 μg–5 mg/kg, typically administered i.p. or intravenously. Schedules may be once, every few days, or weekly depending on tumor growth kinetics and study endpoints.
• Virotherapy and localized tumor studies: Lower intratumoral doses (24–100 μg) dominate, given the desire for localized, rather than systemic, immune activation.
• Autoimmunity and immunology research: Lower doses are sometimes used to dissect immune regulation without overwhelming systemic activation.

Summary Table: FGK4.5 Dosing Regimens in Mouse Models

In summary:

• The most common doses for FGK4.5 in mice range from 100 μg to 5 mg/kg.
• Route (intraperitoneal vs. intratumoral) and frequency (single vs. repeated dosing) are adapted to model requirements.
• For robust systemic effects, higher doses are chosen, while intratumoral regimens permit lower, localized doses.

If you have a specific mouse disease model or immune context, dose and schedule should be carefully titrated with preliminary pilot studies, guided by published protocols in comparable models.