生物活性

Muromonab CD3 (OKT3), the murine anti-CD3monoclonal antibody of the IgG-2a class, used as a powerful immunosuppressiveagent in solid organ transplantation and the first-line treatment of acuteallograft rejection.

体外研究

体内研究

激酶实验

细胞实验

Cells[1]

Responder lymphocytes were mononuclearcells prepared from the peripheral blood of adult, healthy donors. Buffy coatcells were centrifuged through a density gradient and mononuclear cells wererecovered from the density interface and washed in medium. These cells wereused as responder cells in mixed lymphocyte culture (MLC) without furtherfractionation. Stimulator cells were799CP71, an Epstein-Barr virus-transformedhuman B cell line. They were grown in RPMI-1640 supplemented with 20% pooledhuman plasma, 2mM glutamine, penicillin (100units/ml) and streptomycin (100μg/ml),10mM Hepes and 50μM 2-mercaptoethanol.

Generationof CTLs

Mononuclear cells (1 x 10⁷) and1 x10⁵ mitomycin C-treated 799CP71 cells (responder/stimulator cellratio, 100:1) were incubated in 5ml of medium (RPMI-1640 supplemented with 20%heat-inactivated autologous plasma, glutamine, penicillin, streptomycin,2-mercaptoethanol, and Hepes) in upright culture flasks maintained in a 95%air/5% CO₂ atmosphere for 6 days at 37°C. After 2 and 4 days ofculture, 2.5ml of fresh medium was added to the flasks. For mitomycin Ctreatment, 799CP71 cells- were incubated at 1 x 10⁷/ml withmitomycin C (50μg/ml) for 30 min at 37°C. Normal mononuclear cells were also used asstimulator cells in MLC. Culturing procedure and mitomycin C treatment were thesame as with 799CP71 tumor cells except that 1 x 10⁷ stimulatorcells were used (responder/stimulator cell ratio, 1:1).

Assayfor CTL-Mediated Cytolysis

799CP71-cells were used as target cells;they were labeled with Na₂⁵¹CrO₄. Cells (1 x10⁷) were incubated in 0.5 ml of medium containing 250μCi(1Ci= 3.7 x10¹⁰ becquerels) of ⁵¹Cr for 60min at 37^oC.In the cytotoxicity assays, 1 x 10⁴ target cells were mixed witheffector cells at desired ratios in a total volume of 200μlof medium in round-bottom wells of microtiter plates and were incubated at 37°Cfor 4hr. The medium contained 10% pooled human plasma unless otherwisespecified. The ⁵¹Cr released to the medium by the lysed target cellswas determined in a gamma scintillation counter. Percent specific release wasdefined as 100 (ER-SR)/ (MR-SR), in which ER (experimental release) and SR(spontaneous release) were ⁵¹Cr released in the presence andabsence, respectively, of immune effector cells and MR (maximal release) was ⁵¹Crreleased from target cells after they were frozen and thawed three times.

OKT3-InducedMitogenesis

Mononuclear cells (2 x 10⁵) wereincubated with serially diluted OKT3 antibody in a total volume of 200μlof medium in flat-bottom wells of microtiter plates. The medium contained 10%fetal calf serum or autologous plasma as specified. After approximately 65hr ofculture at 37^oC, 0.5μCi of [³H] thymidine in 10μl of mediumwas added to the wells and the cultures were incubated for an additional 6 hr.Cells were harvested and washed by using an automatic cell harvester. The incorporated ³H trapped in the filter was determined by using liquidscintillation counting.

动物实验

Xenografts[2]

Mice were placed on doxycycline chow priorto and immediately following conditioning or BM aspiration. Conditioningconsisted of IP injection of busulfan (BU) 24 hours prior to IV injection ofcells. BU powder was freshly dissolved in dimethylsulfoxide to a stockconcentration of 30 mg/mL and temporarily kept at room temperature. Immediatelybefore injection, this stock solution was diluted 10-fold in PBS. Conditioning(30mg/kg SCID mice and 40mg/kg RAG mice, IP) is occurred 24 hours prior to IVinjection of cells. Alternatively, mice received 250 cGy of total bodyirradiation 4 hours prior to cell injection. For secondary transplants, bones fromprimary xenografted mice were crushed and filtered. Then 10 to 15 million totalcells were infused into BU-conditioned recipients.

Cytotoxicantibodies

OKT3, UCHT1 and ATG (Thymoglobulin) were dilutedto 1mg/mL solutions in sterile PBS containing 2% FBS. These dilutions werestored at 4°C until use. Antibodies were injected IP (1-10mg/kg) or incubatedwith cells in suspension on ice for ~30 minutes at a concentration of1mL/1million WBC (1mg/million) just prior to IV injection.

SRCfrequency determination by limiting dilution

Conditioned mice received 5-fold dilutionsbetween 5x10⁶ and 8x10³ total UBC-WBCs by IV delivery.Ten weeks after engraftment, BM aspirates were evaluated for multi-lineageengraftment. The number of CD341 cells injected was calculated by staining analiquot of the cell suspension with CD34-APC at the time of injection. SRCfrequency was determined using the Extreme Limiting Dilution Analysisapplication.

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] Chang TW, K. P., Gingras SP, Goldstein G, Does OKT-3 monoclonal antibody react with an antigen-recognition structure on human T cells? Proc Natl Acad Sci U S A 1981, 78 (3), 1805-8.
[2] Wunderlich, M.; Brooks, R. A.; Panchal, R.; Rhyasen, G. W.; Danet-Desnoyers, G.; Mulloy, J. C., OKT3 prevents xenogeneic GVHD and allows reliable xenograft initiation from unfractionated human hematopoietic tissues. Blood 2014, 123 (24), e134-44.

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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