生物活性

Trastuzumab, an anti- ERBB2 (HER2 or HER-2/neu) humanized monoclonal antibody, is used to treat “HER2-positive” tumors.. FcγRs binding affinity of trastuzumab[1]

体外研究

体内研究

激酶实验

Measurement of FcγRs binding assays[1]

Macrophages were stained with rabbitanti-mouse FcγRIV and followed by Alexa Fluor 488–anti-rabbit IgG. After staining,the cells were detected using the guava easyCyte HT instrument. Bindingconstant (Kd) of trastuzumab to human FcγRIII incomparison with murine FcgRIV was determined using the surface plasmonresonance method with a Biacore T-100 instrument. Binding of mouse FcγRswith trastuzumab was also determined by ELISA. Briefly, FcγRproteins (2mg/ml) were coated on a MaxiSorp plate, and binding of trastuzumabwas detected using an F(ab’)₂ fragment of goat anti-human F(ab’)₂ Ab conjugated with alkaline phosphatase using the 4-methylumbelliferylphosphate substrate.Fluorescence intensity was read using a plate reader. 

Recombinant proteins consisting of theextracellular domains of human FcγRs fused with glutathioneS-transferase were prepared. There are two genotypes in FcγRIIIA,FcγRIIIA-Val158 and FcγRIIIA-Phe158. In the present study, FcγRIIIA-Val158was used since its binding affinity to Fc is higher than FcγRIIIA-Phe158.Microtiter plates were coated with 4 μg/ml of the FcγR proteinsin PBS at 4^oC overnight and blocked with 25mM Tris HCl (pH 7.4)containing 0.14M NaCl, 0.5% BSA, 2mM EDTA and 0.05% Tween 20 (ELISA buffer) at37^oC for 2 h. TRAs were serially diluted in ELISA buffer anddispensed into individual wells of the FcγR-coatedplates, which were then incubated at 37^oC for 2 h. The plates werewashed three times with ELISA buffer, and then 0.25μg/mlhorseradish peroxidase (HRP)-conjugated goat antihuman IgG (H+L)affinity-purified F(ab’)₂ in ELISA buffer was dispensed into thewells, and the plates were incubated at 37^oC for 1 h. After washingagain in the same manner, 0.1 ml of 0.4% o-phenylenediamine dihydrochloride and0.01% hydrogen peroxide in 0.1M citrate buffer (pH 5.0) were added to thewells, and the plates were allowed to stand at room temperature for 20 min. Absorbanceat 450nm was measured by using a microplate reader.

细胞实验

Cell lines and cell culture conditions[3]

The cell lines AGS, Hs746T, KATOIII, LMSU,MKN1, MKN28, MKN45, GSU, H111TC, HGC-27 and MKN7 were obtained. GSU, H111TC andMKN7 cells were grown in RPMI-1640 medium, and HGC-27 cells were cultured inEagle’s minimum essential medium (MEM). Both media were supplemented with 10%foetal bovine serum Sera Plus and penicillin–streptomycin (100 internationalunits (IU)/ml, 100μg/ml); in addition, RPMI-1640 was supplemented with 2mM L-glutamine.

WST‑1 cell proliferationassay

To assess the effects of the treatments oncell proliferation, the WST-1 cell proliferation assay was used. All samples wereanalysed in triplicate. Cells were seeded at densities between 0.5 × 10³ and 5 × 10³ cells per well in 80μl culture medium and allowed tosettle for 24 h. The following day, trastuzumab was added to a final volume of100μl per well. The isotype control was added to a final concentration of40μg/ml for trastuzumab. Due to the high volume needed, we investigated theeffect of the isotype solvent as well. The applied volume for the trastuzumabsolvent corresponded to 40μg/ml trastuzumab. Assays with trastuzumab wereincubated for 72 h. After this incubation period, pre-warmed WST-1 reagent wasadded. The absorbance of the samples was measured after an incubation periodbetween 30 min and 2 h, depending on the cell line. An Asys Expert Plusmicroplate reader was used for measurements.

动物实验

Animals[4]

Five week-old female BALB/c nude mice were usedin the study.

Invivo therapy study

BT-474 cells (5 x 10⁶ per mouse)were inoculated subcutaneously into the right flank of female BALB/c nude mice.When tumor volumes reached an average of about 150 mm³, the micewere randomly divided into 4 groups of 6 mice each. Mice were intravenously injectedwith control human IgG, trastuzumab (20 mg/kg), nimotuzumab (20 mg/kg) ortrastuzumab (20 mg/kg) plus nimotuzumab (20 mg/kg) twice weekly. Tumors weremeasured with digital calipers, and tumor volumes were calculated by the formula:volume = [length x (width) ²]/2

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] Shi, Y.; Fan, X.; Deng, H.; Brezski, R. J.; Rycyzyn, M.; Jordan, R. E.; Strohl, W. R.; Zou, Q.; Zhang, N.; An, Z., Trastuzumab triggers phagocytic killing of high HER2 cancer cells in vitro and in vivo by interaction with Fcgamma receptors on macrophages. J Immunol 2015, 194 (9), 4379-86.
[2] Suzuki, M.; Yamanoi, A.; Machino, Y.; Ootsubo, M.; Izawa, K.; Kohroki, J.; Masuho, Y., Effect of trastuzumab interchain disulfide bond cleavage on Fcgamma receptor binding and antibody-dependent tumour cell phagocytosis. J Biochem 2016, 159 (1), 67-76.
[3] Kneissl, J.; Hartmann, A.; Pfarr, N.; Erlmeier, F.; Lorber, T.; Keller, S.; Zwingenberger, G.; Weichert, W.; Luber, B., Influence of the HER receptor ligand system on sensitivity to cetuximab and trastuzumab in gastric cancer cell lines. J Cancer Res Clin Oncol 2017, 143 (4), 573-600.
[4] Yang, Y.; Guo, R.; Tian, X.; Zhang, Z.; Zhang, P.; Li, C.; Feng, Z., Synergistic anti-tumor activity of Nimotuzumab in combination with Trastuzumab in HER2-positive breast cancer. Biochem Biophys Res Commun 2017, 489 (4), 523-527.

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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