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

Clone FGK4.5 is a rat monoclonal antibody that acts as an agonist of mouse CD40 and is widely used in in vivo mouse studies to activate immune cells, particularly antigen-presenting cells (APCs) such as B cells, dendritic cells, and macrophages. In these studies, FGK4.5 is typically administered intraperitoneally (i.p.) at doses around 100 ?g per mouse to induce robust activation and upregulation of activation markers (such as MHC class II, CD80, CD86) on the target cells.

Key details on its use in in vivo mouse studies:

• Function: FGK4.5 is an agonistic anti-CD40 antibody that mimics the effects of the physiological ligand CD154 (CD40L), leading to potent activation of CD40 on APCs. This results in upregulation of costimulatory molecules and enhanced immune activation.
• Administration: Standard practice involves intraperitoneal injection of FGK4.5 (commonly at 100 ?g per mouse), which effectively activates immune cells in wild-type mice. The antibody is provided as a purified, unconjugated reagent, usually at concentrations suitable for both in vivo and in vitro experiments.
• Mechanism Dependence: Full in vivo agonistic activity of FGK4.5 relies on engagement with the Fc?RIIB receptor. Mice deficient in Fc?RIIB show reduced activation following FGK4.5 administration, especially for upregulation of certain costimulatory markers on B cells and macrophages, indicating that Fc receptor-mediated crosslinking is important for optimal immune stimulation.
• Applications: FGK4.5 is used in:
  • Studies of immune modulation, costimulatory signaling, and immunotherapy
  • Experimental models involving cancer, autoimmune disease, and vaccine adjuvanticity
  • Exploration of cellular and molecular mechanisms in B cell activation, class switching, and T cell help.
• Additional Uses: While primarily employed as an agonist, FGK4.5 can also interfere with the CD40/CD154 interaction in some settings.

In summary, FGK4.5 is a critical tool for experimental activation of the CD40 pathway in mice, with its in vivo bioactivity highly dependent on Fc?RIIB-mediated crosslinking, underpinning its use in a range of immunological research settings.

The correct storage temperature for sterile packaged clone FGK4.5 (anti-mouse CD40 monoclonal antibody) depends on the intended storage duration:

• Short-term storage (up to 1 month): 2°C to 8°C (refrigerated).
• Long-term storage: -20°C to -70°C (manual defrost freezer, avoid repeated freeze-thaw cycles).

Store the antibody as supplied, undiluted, and protected from light if it is conjugated. Do not freeze if only storing short-term at 2–8°C. If storage is anticipated beyond one month, aliquot and freeze at -20°C or below.

Based on the available research literature, several antibodies and proteins are commonly used alongside FGK4.5 (also known as FGK45) in experimental studies.

Other Anti-CD40 Monoclonal Antibodies

The most frequently co-referenced antibodies with FGK4.5 are other anti-mouse CD40 monoclonal antibodies, particularly 1C10 and 3/23. These antibodies share similar functional characteristics with FGK4.5, as they all require Fc?RIIB receptor engagement for optimal agonistic activity in vivo. Researchers often use these antibodies in comparative studies to evaluate different approaches to CD40 targeting.

Another notable anti-CD40 antibody used in conjunction with FGK4.5 is the 7E1 antibody system, which includes variants 7E1-G1 and 7E1-G2b. These antibodies have been studied alongside FGK4.5 to compare their competitive binding capabilities with CD154, showing distinct binding characteristics with different EC50 values.

CD40 Ligand and Related Proteins

Soluble CD154 (sCD154), the natural ligand for CD40, is frequently used in studies with FGK4.5. Researchers use Flag-tagged soluble CD154 fusion proteins to assess competitive binding interactions and to understand how different anti-CD40 antibodies compete with the natural ligand for receptor binding. The CD40-CD154 interaction is fundamental to the biological pathway that FGK4.5 targets.

Immune Checkpoint and Costimulatory Molecules

Studies involving FGK4.5 often examine its effects on various activation markers and costimulatory molecules. These include MHC class I and II molecules, CD86, and CD80, which serve as readouts for immune cell activation following FGK4.5 treatment. Additionally, CTLA-4 Ig has been used in combination studies with anti-CD40 antibodies, suggesting potential synergistic therapeutic applications.

Human CD40 Targeting Agents

For translational research purposes, CP-870,893, a human anti-CD40 monoclonal antibody, is often discussed alongside FGK4.5 studies. While CP-870,893 targets human CD40 and FGK4.5 targets mouse CD40, researchers use both to understand species-specific differences in CD40 signaling and to bridge preclinical mouse studies with clinical applications.

The literature shows that FGK4.5 is predominantly used in studies focused on immune activation, B cell biology, and cancer immunotherapy research, where these companion antibodies and proteins help researchers understand the broader immunological context of CD40 signaling.

Key findings from scientific literature involving clone FGK4.5 (also referred to as FGK45) center on its role as a monoclonal antibody that activates mouse CD40 and its broad immunological and metabolic effects in vitro and in vivo.

• Immune activation: FGK4.5/FGK45 binds specifically to mouse CD40, a receptor on antigen-presenting cells such as dendritic cells, B cells, and macrophages. This interaction is essential for immune processes such as immunoglobulin class switching, formation of germinal centers, and development of memory B cells. FGK45 is widely used to study or induce activation of these cells both in vitro and in vivo.

• Pro-inflammatory and metabolic reprogramming: Recent literature demonstrates that FGK45 signaling not only increases lipid uptake and mitochondrial activity but also rewires cellular metabolism. Specifically, FGK45 stimulation in bone marrow-derived macrophages (BMDMs) enhances fatty acid oxidation, promotes lactate production through the enzyme LDHA, and leads to histone acetylation (notably H3K27ac), driving pro-inflammatory gene expression. Blocking key metabolic enzymes (ACLY or LDHA) abrogates these effects, underlining their necessity for CD40-driven macrophage polarization.

• Cancer immunotherapy implications: FGK45 antibody activation of CD40 augments immune cell activity, improving responses to immune checkpoint blockade therapies (e.g., anti-PD-1) in murine tumor models, such as intrahepatic cholangiocarcinoma. The efficacy depends on intact metabolic pathways enabling pro-inflammatory programming of tumor-associated macrophages.

• B cell proliferation: FGK45 also induces robust proliferation of mouse B cells, further highlighting its utility in dissecting B cell activation mechanisms.

• Competitive binding and epitope specificity: FGK45 blocks binding by other CD40 antibodies (such as HM40-3), confirming its epitope specificity and functional relevance for CD40 activation studies.

In summary, clone FGK4.5/FGK45 is a principal experimental tool for activating CD40 in mice, providing insights into immune cell activation, metabolic rewiring, and therapeutic strategies targeting the CD40 pathway in immunology and oncology.