生物活性

Theneutralization activity (IC50) of LymphoStat-B in the murine splenocyteproliferation assay is 0.02 nM.[1]

LymphoStat-Bbound to BLyS with an EC50 value of 0.024 nM. The binding of LymphoStat-B wasreadily inhibited with soluble BLyS, with an IC50 value of 8.5 nM.[1]

hebinding inhibition of LymphoStat-B[1]

体外研究

体内研究

激酶实验

Binding to BLyS in solid phase (directenzyme-linked immunosorbent assay [ELISA])[1]

ELISA plates (Immulon-II) were coated with1μg/ml of streptavidin in PBS. After washing in PBS and blocking with3% bovine serum albumin (BSA), 100 μl of biotinylated BLyS solutionin PBST (1μg/ml in PBS, 0.1% Tween 20, 0.1% BSA) was added to each well, andthe wells were incubated for 1 hour at room temperature. After washing, 100 μlof serial dilutions of antibodies (66–6.6 X 10^–6 nM) in diluentbuffer (PBS, 0.1% Tween 20, 0.1% BSA) was dispensed into individual wells ofBLyS-coated plates. The plates were incubated for 2 hours at room temperature,washed 4 times with PBST, and then 100μl of peroxidase-conjugated goatanti-human IgG (1 μg/ml in diluent buffer) was added to each well. Plates wereincubated for 1 hour at room temperature, washed 4 times with PBST, anddeveloped with tetramethylbenzidine substrate. Absorption at 450 nm wasmeasured with a Spectra- Max 3000 instrument.

Bindingto BLyS in solution (competition ELISA)

Serial dilutions of BLyS (10³–10^–4 nM) were prepared in diluent buffer, and 50-μl aliquots ofeach dilution were distributed to individual wells of BLyS-coated plates(prepared as described above). Antibodies were diluted to the 50% maximum responseconcentration (EC50), and 50 μl of the resultant dilution was added to the wells alreadycontaining BLyS. The plates were further processed as described above.

细胞实验

Cells[2]

Peripheral blood mononuclear cells (PBMCs)from CLL patients were obtained at time of diagnosis prior to therapy. PolyclonalNK cells (pNKCs) were generated using K562-41BBL-IL15 feeder cells. Resting NKcells and monocytes were purified by MACS isolation. Purity of cellpreparations was confirmed by fluorescence-activated cell sorting (FACS) andalways above 90%.

Metabolicactivity

Primary CLL cells were plated at 2 × 10⁶ per well and metabolic activity was measured after 72 h using the CellProliferation Reagent WST-1 set from Roche according to the manufacturer’sinstructions. Results are shown as means of triplicate measurements with s.e.m.

NK-cellactivation, degranulation and cytotoxicity

Upregulation of CD69 and CD107a as markersfor NK-cell activation and degranulation, respectively, was analyzed by FACS.Direct cytotoxicity and ADCC of NK cells against CLL cells were analyzed by 2 hBATDA Europium assays.

动物实验

Neutralizing activity of LymphoStat-B antibodyin mice[1]

On day 1, female BALB/c mice (ages 8–9 weeks)were injected intravenously with diluent (PBS) or with LymphoStat-B or controlhuman monoclonal IgG1 antibody at doses of 0.05, 0.15, 0.5, 1.5, or 5 mg/kg.Recombinant human BLyS at 0.3 mg/kg or BLyS vehicle (12.5 mM citrate, 125 Mm NaCl,pH 6) was injected subcutaneously 1 hour after antibody injection. On day 3,mice received a second dose of antibody (1 hour before BLyS injection). BLySwas injected subcutaneously once a day for 4 consecutive days. On day 5, allmice were killed 24 hours after the last BLyS injection. Sera were analyzed forIgA content by ELISA, wet spleen weights were recorded, and splenocytes wereanalyzed by flow cytometry using phycoerythrin (PE)–conjugated anti–B220 (anti-CD45R) and fluorescein isothiocyanate (FITC)–conjugated anti-ThB (anti-Ly6D) asmarkers.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Baker KP, E. B., Main SH, Choi GH, Wager RE, Halpern WG, Lappin PB, Riccobene T, Abramian D, Sekut L, Sturm B, Poortman C, Minter RR, Dobson CL, Williams E, Carmen S, Smith R, Roschke V, Hilbert DM, Vaughan TJ, Albert VR, Generation and characterization of LymphoStat-B, a human monoclonal antibody that antagonizes the bioactivities of B lymphocyte stimulator. Arthritis Rheum 2003, 48 (11), 3253-65.
[2] Wild, J.; Schmiedel, B. J.; Maurer, A.; Raab, S.; Prokop, L.; Stevanovic, S.; Dorfel, D.; Schneider, P.; Salih, H. R., Neutralization of (NK-cell-derived) B-cell activating factor by Belimumab restores sensitivity of chronic lymphoid leukemia cells to direct and Rituximab-induced NK lysis. Leukemia 2015, 29 (8), 1676-83.

体内溶解度

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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