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| Targets |
Belatacept targets the costimulatory molecules CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells (APCs). It binds to CD80 with an IC50 of 0.009 ug/mL and to CD86 with an IC50 of 0.102 ug/mL. By binding to these ligands, Belatacept blocks the CD28-mediated costimulatory signal required for full T-cell activation. Without this second signal, T cells become anergic (unresponsive), leading to immune suppression and reduced rejection of transplanted organs. It is a selective T-cell costimulation blocker.
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| ln Vitro |
In a dose-dependent manner, belatacept (0–5 mg/mL, 1 hour) inhibits T cell proliferation [2]. After allogeneic stimulation, belatacept (500 ng/mL, 7 days) increases effector memory T cell dominance [2]. Isolated effector memory T cell differentiation and allogeneic IFNγ production were unaffected by belatacept (100, 500 ng/mL, 7 days) [2]. The differentiation of B cells dependent on follicular T cells is not inhibited by belatacept (10 μg/mL, 1 hour) [4]. T cell-independent reductions in plasmablast differentiation, Ig production, and the major transcription factor Blimp-1 are observed with belatacept (40 μg/mL, 10 days) [5]. When administered at a dose of 40 μg/mL for 30 minutes, belatacept decreases the expression of CD86 and activates the STAT3 transcription factor in stimulated B cells [5].
In vitro, Belatacept potently inhibits T-cell activation and proliferation. In mixed lymphocyte reaction (MLR) assays, Belatacept blocks the proliferation of alloreactive T cells in response to donor antigens. It suppresses the production of T-cell cytokines, including interleukin-2 (IL-2), interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha). The compound also inhibits the differentiation of naïve T cells into effector T cells. These in vitro activities confirm its mechanism of action as a costimulation blocker and support its use as an immunosuppressive agent. |
| ln Vivo |
Belatacept (ip, 60 mg/kg) suppresses antibody-mediated rejection (ABMR) and, when paired with BTLA (B and T lymphocyte attenuator) overexpression treatment, can suppress acute rejection [3]. In monkeys given sheep red blood cells as an immunization, belatacept (20 mg/kg administered intravenously) exhibited immunosuppressive effects [6].
In vivo, Belatacept has demonstrated efficacy in preventing allograft rejection in animal models of solid organ transplantation. In preclinical studies, Belatacept prolonged graft survival in kidney, heart, and islet transplantation models. In clinical trials, Belatacept was shown to be non-inferior to cyclosporine for the prevention of acute rejection in kidney transplant recipients, with superior renal function and improved cardiovascular and metabolic risk profiles. It is approved for use in Epstein-Barr virus (EBV)-seropositive kidney transplant recipients as part of an immunosuppressive regimen. |
| Enzyme Assay |
For in vitro binding assays, a standard protocol uses a competitive ELISA. Recombinant human CD80 or CD86 is coated onto a 96-well plate. Belatacept is added at varying concentrations (e.g., 0.001-100 ug/mL) in the presence or absence of a fixed concentration of a labeled CD28-Fc fusion protein. After incubation and washing, bound Belatacept is detected using an anti-human IgG-Fc antibody conjugated to HRP. The IC50 for inhibition of CD28 binding is calculated from the dose-response curve. Alternatively, a surface plasmon resonance (SPR) assay can be used to measure binding kinetics.
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| Cell Assay |
Cell viability assay [2]
Cell Types: PBMC from healthy volunteers Tested Concentrations: 0-5 mg/mL Incubation Duration: 1 hour Experimental Results: Inhibited T cell proliferation with an IC50 value of 215 ng/mL, and residual T cell proliferation (± 30%) persisted at high doses. Western Blot Analysis [5] Cell Types: CD40L and IL-21 stimulated B cells Tested Concentrations: 40 μg/mL Incubation Duration: 15, 30 min Experimental Results: Increased STAT signaling as determined by increased STAT3 phosphorylation. For in vitro cell-based assays, a mixed lymphocyte reaction (MLR) is used. Human peripheral blood mononuclear cells (PBMCs) are isolated from two HLA-mismatched donors. Stimulator cells (PBMCs) are irradiated to prevent proliferation. Responder cells (PBMCs) are co-cultured with irradiated stimulator cells in 96-well plates. Belatacept is added at varying concentrations (e.g., 0.1-100 ug/mL). After 5-7 days, T-cell proliferation is measured by 3H-thymidine incorporation or by CFSE dilution using flow cytometry. Cytokine production (IL-2, IFN-gamma) in the supernatant is measured by ELISA. The IC50 for inhibition of proliferation is calculated. |
| Animal Protocol |
Animal/Disease Models: Rat orthotopic renal transplantation acute rejection model [3]
Doses: 60 mg/kg Route of Administration: intraperitoneal (ip) injection, after transplantation and 4 days after transplantation. Experimental Results: Inhibition of creatinine increase after renal transplantation (combined with BTLA overexpression treatment). There was diminished C4d in graft IF staining, CD138 infiltration, and DSA production. Animal/Disease Models: immunize rhesus monkeys with sheep red blood cells [6] Doses: 10 mg/kg intraoperatively, day 4 (15 mg/kg) and postoperative days 14, 28, 42, 56, and 70 (20 mg/kg ). Route of Administration: intravenous (iv) (iv)injection Experimental Results: resulted in a 50% reduction in the peak anti-SRBC response. Prolonged renal allograft survival and synergy with conventional immunosuppression. For in vivo animal studies, a rat or mouse model of allogeneic kidney or heart transplantation is used. Recipient animals receive a kidney or heart transplant from a donor of a different strain. Belatacept is administered intravenously at a dose of 1-10 mg/kg on days 0, 1, 4, and 7 after transplantation. Control animals receive vehicle or a control immunosuppressive agent (e.g., cyclosporine). Graft survival is monitored daily, and rejection is confirmed by histology at the time of graft failure. Blood samples are collected for measurement of drug concentration and markers of immune activation. |
| ADME/Pharmacokinetics |
Belatacept is administered intravenously and has a dose-dependent pharmacokinetic profile. The terminal half-life is approximately 8-10 days in humans. Peak serum concentrations are reached at the end of the intravenous infusion. The volume of distribution at steady state is approximately 0.1 L/kg, primarily confined to the vascular space. Metabolism is via proteolytic degradation. Belatacept is a large fusion protein (approximately 90 kDa) and is not cleared renally. The recommended dosing regimen for kidney transplant recipients is a weight-based dose given monthly after the initial loading phase.
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| Toxicity/Toxicokinetics |
Belatacept has a black box warning for an increased risk of post-transplant lymphoproliferative disorder (PTLD), particularly involving the central nervous system (CNS), and for the risk of progressive multifocal leukoencephalopathy (PML). The FDA label includes a boxed warning for these serious risks. Common adverse reactions include urinary tract infection, hypertension, pyrexia, diarrhea, headache, and anemia. It is contraindicated in EBV-seronegative patients due to the increased risk of PTLD. Use in liver transplant patients is not recommended due to an increased risk of graft loss and death. Belatacept is a potent immunosuppressant and should be prescribed by experienced physicians.
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| References |
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| Additional Infomation |
Belatacept is a FDA-approved immunosuppressive drug for the prophylaxis of organ rejection in adult kidney transplant recipients. It is marketed under the trade name Nulojix. Belatacept was derived from abatacept (Orencia) by introducing two amino acid substitutions (L104E and A29Y) in the CTLA-4 ligand-binding domain, which increase its avidity for CD80 and CD86 by approximately 10-fold. Unlike calcineurin inhibitors (e.g., cyclosporine, tacrolimus), Belatacept does not cause nephrotoxicity, a major advantage for long-term renal transplant function. The compound is for research use only in vitro; clinical use requires medical supervision.
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| Molecular Formula |
0
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| Molecular Weight |
0
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| CAS # |
706808-37-9
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| Appearance |
White to off-white solid powder
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
H2O : ~50 mg/mL
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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