~（99）Tc~m-MAG_3-K237的制备、鉴定及其动物体内实验研究
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摘要
研究背景及目的
血管生成与恶性肿瘤的生长及转移密切相关,肿瘤直径大于2 mm时,通过新生血管输送足够的养分和排除代谢废物,并为转移提供便利。
血管生成需多种因子的参与,其中血管内皮生长因子(vascular endothelial growth factor,VEGF)信号通路是主要的限速环节,且VEGFR-2(KDR)是发挥血管生成生物学作用的主要受体类型,其在正常组织和良性肿瘤组织不表达或低表达,而在恶性肿瘤组织中呈高水平表达。针对多种恶性肿瘤的免疫组化研究显示,KDR主要表达于新生血管内皮细胞,某些肿瘤细胞本身也高表达该受体。
在胞膜受体高表达的肿瘤中,选择性受体靶向的放射性多肽已成为一类重要的分子显像和治疗放射性药物。这种被发射γ射线的核素标记多肽可与受体结合,进而达到无创探测表达有该受体的肿瘤的目的。
近年来,噬菌体展示技术及核糖体展示技术的出现为寻找VEGFR特异性结合物提供了一个新的途径。该技术用已知受体在高通量噬菌体肽库或核糖体肽库中进行筛选,可保证筛选出的多肽与受体高特异性及高亲和力结合。K237即是应用该技术筛选出的十二肽,研究证实,K237可阻断VEGF与KDR的结合,抑制VEGF介导的人脐静脉内皮细胞(HUVEC)增殖及绒毛膜尿囊膜的血管生成,并可抑制SCID小鼠BICR-H1系乳腺癌细胞的生长和转移。
本实验研究通过对K237进行99Tcm标记,探索其作为KDR受体导向的恶性实体瘤分子显像剂的可行性,并为日后开展放射性核素内照射治疗研究提供实验基础。方法
1.~(99)Tc~m-MAG_3-K237的制备:采用酒石酸亚锡还原法制备~(99)Tc~m-MAG_3-K237, 3MM色谱纸层析测定放射化学纯度,应用正交试验设计方法筛选最佳标记条件。
2.~(99)Tc~m-MAG_3-K23的体外稳定性鉴定:通过室温稳定性、半胱氨酸置换和三氯乙酸蛋白沉淀等体外稳定性实验,评价~(99)Tc~m-MAG_3-K237的理化性质。
3.~(99)Tc~m-MAG_3-K237在健康家兔体内的示踪动力学分析:取6只日本雄性大耳兔,经耳缘静脉注射37 MBq ~(99)Tc~m-MAG_3-K237溶液,然后分别于1.5、3、5、10、30、60、120、240、480 min从对侧耳缘静脉取血,称重并测量放射性计数,结果经测量效率校正,计算放射性浓度(KBq/L)。运用药代动力学分析软件DAS 2.1.1版判断最佳房室模型,并得出药代动力学参数。
4 . ~(99)Tc~m-MAG_3-K237的健康动物体内分布及显像研究:将7.4 MBq ~(99)Tc~m-MAG_3-K237溶液稀释至500 ml,分别取1 ml加入至5只放免试管中,将测得的放射性计数(cpm)的均数乘以500,即得到参考源的放射性计数。取35只成年昆明小鼠,按随机数字表法分为7组,每组5只。经尾静脉注射7.4 MBq ~(99)Tc~m-MAG_3-K237溶液,分别于1、5、10、30、60、120、240 min摘眼球法取血,然后将小鼠以颈椎脱臼法处死,收集心、肺、肝、肾、肠、肌肉、骨、脑,分别称重并测量放射性计数,经参考源校正后计算每克组织百分注射剂量率(%ID/g)。家兔固定于木制板上,注射~(99)Tc~m-MAG_3-K237溶液37 MBq后立即以1帧/min采集60 min,并于90、120、180、240 min预置计时2 min各采集图像1帧,观察各器官的放射性分布动态变化。
5.~(99)Tc~m-MAG_3-K237的荷HepG2裸鼠体内分布研究:将7.4 MBq ~(99)Tc~m-MAG_3-K237溶液稀释至500 ml,分别取1 ml加入至5只放免试管中,将测得的放射性计数(cpm)的均数乘以500,即得到参考源的放射性计数。取15只荷HepG2裸鼠,按随机数字表法分为5组。经尾静脉注射7.4 MBq ~(99)Tc~m-MAG_3-K237溶液,分别于10、30、60、120、240 min通过摘除眼球采集血液,然后将小鼠以颈椎脱臼法处死,收集心、肺、肝、肾、肠、肌肉、骨、脑、肿瘤,分别称重并测量放射性计数(cpm),经参考源校正后计算每克组织百分注射剂量率(%ID/g),并计算各时相的T/NT比值。
6.~(99)Tc~m-MAG_3-K237荷瘤裸鼠显像及竞争结合显像研究:取荷HepG2裸鼠固定于木制板上,尾静脉注射~(99)Tc~m-MAG_3-K237溶液17.5 MBq后分别于30、60、90、120、180、240 min各预置计时2 min采集图像1帧,观察肿瘤组织放射性分布动态变化。取另一只荷瘤裸鼠,尾静脉注射混有0.5 mg MAG_3-K237的~(99)Tc~m-MAG_3-K237溶液17.5 MBq,观察MAG_3-K237对肿瘤显像的抑制作用。
结果
1.~(99)Tc~m-MAG_3-K237的制备:99Tcm标记MAG_3-K237(20μg)的最佳反应条件为: 0.25 mol/L醋酸铵缓冲液(pH 5.2)、16μg酒石酸钾钠、4μg酒石酸亚锡、37 MBq 99TcmO4-,总反应体积182μl,充氮、密封条件下于沸水浴中加热30 min。Whatmann 3MM纸层析,γ计数器测量标记混合物各放射性组分在生理盐水、氨水及丙酮3种流动相中的比移值(Rf值)。本方法制备的~(99)Tc~m-MAG_3-K237的标记率为(97.98±0.76)%,直接法计算其比活度为(3.54±0.03)TBq/mmol。
2.~(99)Tc~m-MAG_3-K23的体外稳定性:标记物室温下放置8 h,标记率仍大于95%,为(96.15±0.37)%;不同浓度半胱氨酸置换实验,~(99)Tc~m-MAG_3-K237中未结合99Tcm的变化均小于1%;三氯乙酸沉淀实验显示~(99)Tc~m-MAG_3-K237与血清蛋白无明显结合。
3.~(99)Tc~m-MAG_3-K237在健康家兔体内的示踪动力学分析:标记多肽的家兔体内动力学过程符合权重为1/c2的三室模型,t1/2α为4.00±3.53 min,t1/2β为24.48±9.84 min,t1/2γ为852.24±444.00 min,清除率(CL)为1.83±0.41 ml/min。
4.~(99)Tc~m-MAG_3-K237在健康动物体内的生物分布及显像:标记物在健康小鼠体内清除迅速,肾脏、肝脏及肠道分布较多,但肝脏30 min后分布很少。
5.~(99)Tc~m-MAG_3-K237荷瘤裸鼠体内分布及显像:荷HepG2裸鼠体内分布显示,10 min时相肿瘤组织摄取放射性最高,为(0.90±0.18) (%ID/g);肿瘤与对侧肌肉放射性比值(T/NT)1 h达最高值,为2.19±0.22;肾脏、肝脏及肠道放射性分布较多。荷HepG2裸鼠SPECT显像中,肿瘤1 h显影最清晰,MAG_3-K237阻断后,肿瘤影像明显减淡。
结论
1.本研究制备的~(99)Tc~m-MAG_3-K237标记率>95%,勿需分离纯化直接应用,具有良好的体内外稳定性。
2.~(99)Tc~m-MAG_3-K237在健康动物体内具有良好的体内分布及示踪动力学特性,血液清除快,通过肾脏及肝脏双重途径排泄。
3.~(99)Tc~m-MAG_3-K237特异性浓聚于荷瘤裸鼠肿瘤组织,肿瘤显像清楚,且T/NT比值较高,是一具有潜在应用价值的肿瘤KDR分子显像剂。
Background and objectives:
Angiogenesis is closely related to the growth and metastasis of malignant tumor. Tumors with diameter more than 2 mm derive nutrition, exclude metabolic waste and facilitate metastasis through new blood vessel.
The process of angiogenesis includes participation of many factors, among which vascular endothelial growth factor(VEGF)and KDR signal transduction pathway is the most important. In normal and benign tumor tissues, KDR is expressed at low level, whereas malignant tumor tissues express KDR at high level. Some immunohistochemistry studies on malignant tumors revealed that KDR was mainly expressed in the new blood vessel cell and some kind of tumor cell itself.
In the tumors highly expressing some receptor, selective receptor targeting radioactive peptide has become an important kind of molecular imaging and treatment drug. The peptide labeled by nuclide emittingγ-ray is able to combine with its receptor, and then is able to noninvasively explore malignant tumor in vivo.
In recent years, phage display technique and ribosome display technique have become two kinds of ways to find VEGFR specific bond. It can guarantee high chemical affinity between the peptide and its receptor. K237 was isolated from a phage-displayed peptide library, which could bind to KDR with high affinity and specificity. By interfering with VEGF-KDR interaction, the peptide K237 inhibited proliferation of cultured primary human umbilical vein endothelial cells. K237 also exerted an anti-angiogenesis activity in vivo in chick embryo chorioallantoic membrane and inhibited the growth and metastasis of breast cancer in SCID mice.
This study focused on the labeling of K237 with 99Tcm and feasibility of the labeled peptide as a molecular imaging agent mediated by KDR receptor in solid malignant tumors,which would lay an experimental foundation for the nuclide-therapy study in the future.
Methods:
1.Preparation of ~(99)Tc~m-MAG_3-K237:~(99)Tc~m-MAG_3-K237 was prepared by way of reduction with stannous tartrate. RCP analysis of ~(99)Tc~m-MAG_3-K237 was demonstrated by Whatman 3MM paper chromatographic system. Orthogonal design was also applied to detect the optimized labeling condition.
2.In vitro stability identification of ~(99)Tc~m-MAG_3-K237: Evaluate the stability of ~(99)Tc~m-MAG_3-K237 in vitro through a series of experiment such as stability at room temperature, displacement by Cysteine and protein precipitation by trichloroacetic acid.
3.Tracer kinetics analysis in vivo of ~(99)Tc~m-MAG_3-K237 in healthy rabbits: Each of six rabbits was injected 37 MBq ~(99)Tc~m-MAG_3-K237, then draw blood from ear vein at the following time: 1.5, 3, 5, 10, 30, 60, 120, 240, 480 min. These blood sample was weighed and measured byγ-immuno-counter, and then we calculated the radioactive concentration of every sample after correction by measurement efficiency ofγ-immuno-counter. At last, we evaluated the best compartmental model and parameter by virtue of pharmacokinetics analytical software.
4.Body distribution and imaging study of ~(99)Tc~m-MAG_3-K237 in vivo in healthy animals: 35 adult Kunming mice were involved in the study of tissue distribution, which were divided to 7 groups by random digits table. 7.4 MBq ~(99)Tc~m-MAG_3-K237 was administrated via caudal vein of the mice. At 1, 5, 10, 30, 60, 120, 240 min after injection, mice were sacrificed and the blood, heart, lungs, liver, kidneys, intestines, muscles, bone and brain were excised, weighed and counted for radioactivity by a gamma counter. And then we calculated the percentage of the injected radioactive dose per gram of tissue wet weigh (%ID/g) after correction by reference source. In the study of imaging in healthy animals, we injected 37 MBq ~(99)Tc~m-MAG_3-K237 into ear vein of rabbits. The dynamic imaging was observed by the scintigraphy with SPECT for 60 minutes and then static imaging at 90, 120, 180 and 240 min.
5.Tissue distribution study of ~(99)Tc~m-MAG_3-K237 in vivo in tumor bearing nude mice: In the study of tissue distribution, 15 tumor bearing nude mice were divided to 5 groups. 7.4 MBq ~(99)Tc~m-MAG_3-K237 was administrated into the caudal vein of each mice, and then we sacrificed them and collected their blood, heart, lungs, liver, kidneys, intestines, muscles, bone, brain and tumor tissues at 10, 30, 60, 120 and 240 min. At last, we calculated the percentage of the injected radioactive dose per gram of tissue wet weigh (%ID/g) and tumor/non-tumor ratio(T/NT) at various time.
6.SPECT imaging and competitive imaging by MAG_3-K237: In the study of imaging, 17.5 MBq ~(99)Tc~m-MAG_3-K237 was injected into the caudal vein of the mice. The dynamic distribution was observed by the scintigraphy with SPECT at 30, 60, 90, 120, 180 and 240 min. In the study of competitive imaging, mixture of 17.5 MBq ~(99)Tc~m-MAG_3-K237 and 0.5 mg MAG_3-K237 was administrated into the caudal vein of the mice, then competitive imaging inhibited by MAG_3-K237 was observed.
Results:
1.Preparation of ~(99)Tc~m-MAG_3-K237: ~(99)Tc~m-MAG_3-K237 could be successfully prepared following the steps below: added 20μg MAG_3-K237, 50μl 0.25mol/L ammonium acetate buffer solution(pH 5.2), 16μg potassium sodium tartrate, 4μg stannous tartrate, 37 MBq 99TcmO4- to the cell frozen tube, sealed the tube and boiled it under boiling water for 30 minutes. The RCP of the labeled peptide was (97.98±0.76)%, and its specific activity was (3.54±0.03)TBq/mmol.
2.Identification of stability of ~(99)Tc~m-MAG_3-K237 in vitro: after 8 h, the RCP of ~(99)Tc~m-MAG_3-K237 was (96.15±0.37)%; Cysteine replacement rate was less than 1% after adding cysteine of various concentration; trichloroacetic acid precipitation test revealed the labeled peptide couldn’t combine with serum protein.
3.Tracer kinetics analysis in vivo of ~(99)Tc~m-MAG_3-K237 in healthy rabbits: tracer kinetics process in vivo of ~(99)Tc~m-MAG_3-K237 in healthy rabbits followed three compartment model weighed by 1/c2 and the main parameters t1/2α, , t1/2β, t1/2γand CL were 4.00±3.53 min, 24.48±9.84 min, 852.24±444.00 min and 1.83±0.41 ml/min respectively.
4.Tissue distribution and imaging study of ~(99)Tc~m-MAG_3-K237 in vivo in healthy animals: ~(99)Tc~m-MAG_3-K237 was cleared quickly from the mice, it mainly distributed in the kidneys, liver and intesteins, but the distribution in liver was little after 30 min.
5.Tissue distribution and imaging study of ~(99)Tc~m-MAG_3-K237 in vivo in tumor bearing nude mice: In the study of tissue distribution, %ID/g of tumor was highest ,(0.90±0.18) at 10 min, T/NT ratio was highest, 2.19±0.22 at 1 h, and the labeled peptide was mainly distributed in kidneys, liver and intestines. In the study of imaging, the tumor was clearest at 1 h, and the imaging was much less clear after blocked by MAG_3-K237.
Conclusions:
1.~(99)Tc~m-MAG_3-K237 was successfully prepared with high RCP(>95%) and perfect stability in vitro and in vivo.
2.~(99)Tc~m-MAG_3-K237 had satisfactory characteristics in tracer kinetics and tissue distribution and was cleared quickly from blood and excreted quickly from kidneys and liver.
3.~(99)Tc~m-MAG_3-K237 could concentrate in the tumor tissue with specificity in tumor bearing nude mice, which showed its bright future in the field of molecular imaging in KDR positive tumor.
血管生成与恶性肿瘤的生长及转移密切相关,肿瘤直径大于2 mm时,通过新生血管输送足够的养分和排除代谢废物,并为转移提供便利。
血管生成需多种因子的参与,其中血管内皮生长因子(vascular endothelial growth factor,VEGF)信号通路是主要的限速环节,且VEGFR-2(KDR)是发挥血管生成生物学作用的主要受体类型,其在正常组织和良性肿瘤组织不表达或低表达,而在恶性肿瘤组织中呈高水平表达。针对多种恶性肿瘤的免疫组化研究显示,KDR主要表达于新生血管内皮细胞,某些肿瘤细胞本身也高表达该受体。
在胞膜受体高表达的肿瘤中,选择性受体靶向的放射性多肽已成为一类重要的分子显像和治疗放射性药物。这种被发射γ射线的核素标记多肽可与受体结合,进而达到无创探测表达有该受体的肿瘤的目的。
近年来,噬菌体展示技术及核糖体展示技术的出现为寻找VEGFR特异性结合物提供了一个新的途径。该技术用已知受体在高通量噬菌体肽库或核糖体肽库中进行筛选,可保证筛选出的多肽与受体高特异性及高亲和力结合。K237即是应用该技术筛选出的十二肽,研究证实,K237可阻断VEGF与KDR的结合,抑制VEGF介导的人脐静脉内皮细胞(HUVEC)增殖及绒毛膜尿囊膜的血管生成,并可抑制SCID小鼠BICR-H1系乳腺癌细胞的生长和转移。
本实验研究通过对K237进行99Tcm标记,探索其作为KDR受体导向的恶性实体瘤分子显像剂的可行性,并为日后开展放射性核素内照射治疗研究提供实验基础。方法
1.~(99)Tc~m-MAG_3-K237的制备:采用酒石酸亚锡还原法制备~(99)Tc~m-MAG_3-K237, 3MM色谱纸层析测定放射化学纯度,应用正交试验设计方法筛选最佳标记条件。
2.~(99)Tc~m-MAG_3-K23的体外稳定性鉴定:通过室温稳定性、半胱氨酸置换和三氯乙酸蛋白沉淀等体外稳定性实验,评价~(99)Tc~m-MAG_3-K237的理化性质。
3.~(99)Tc~m-MAG_3-K237在健康家兔体内的示踪动力学分析:取6只日本雄性大耳兔,经耳缘静脉注射37 MBq ~(99)Tc~m-MAG_3-K237溶液,然后分别于1.5、3、5、10、30、60、120、240、480 min从对侧耳缘静脉取血,称重并测量放射性计数,结果经测量效率校正,计算放射性浓度(KBq/L)。运用药代动力学分析软件DAS 2.1.1版判断最佳房室模型,并得出药代动力学参数。
4 . ~(99)Tc~m-MAG_3-K237的健康动物体内分布及显像研究:将7.4 MBq ~(99)Tc~m-MAG_3-K237溶液稀释至500 ml,分别取1 ml加入至5只放免试管中,将测得的放射性计数(cpm)的均数乘以500,即得到参考源的放射性计数。取35只成年昆明小鼠,按随机数字表法分为7组,每组5只。经尾静脉注射7.4 MBq ~(99)Tc~m-MAG_3-K237溶液,分别于1、5、10、30、60、120、240 min摘眼球法取血,然后将小鼠以颈椎脱臼法处死,收集心、肺、肝、肾、肠、肌肉、骨、脑,分别称重并测量放射性计数,经参考源校正后计算每克组织百分注射剂量率(%ID/g)。家兔固定于木制板上,注射~(99)Tc~m-MAG_3-K237溶液37 MBq后立即以1帧/min采集60 min,并于90、120、180、240 min预置计时2 min各采集图像1帧,观察各器官的放射性分布动态变化。
5.~(99)Tc~m-MAG_3-K237的荷HepG2裸鼠体内分布研究:将7.4 MBq ~(99)Tc~m-MAG_3-K237溶液稀释至500 ml,分别取1 ml加入至5只放免试管中,将测得的放射性计数(cpm)的均数乘以500,即得到参考源的放射性计数。取15只荷HepG2裸鼠,按随机数字表法分为5组。经尾静脉注射7.4 MBq ~(99)Tc~m-MAG_3-K237溶液,分别于10、30、60、120、240 min通过摘除眼球采集血液,然后将小鼠以颈椎脱臼法处死,收集心、肺、肝、肾、肠、肌肉、骨、脑、肿瘤,分别称重并测量放射性计数(cpm),经参考源校正后计算每克组织百分注射剂量率(%ID/g),并计算各时相的T/NT比值。
6.~(99)Tc~m-MAG_3-K237荷瘤裸鼠显像及竞争结合显像研究:取荷HepG2裸鼠固定于木制板上,尾静脉注射~(99)Tc~m-MAG_3-K237溶液17.5 MBq后分别于30、60、90、120、180、240 min各预置计时2 min采集图像1帧,观察肿瘤组织放射性分布动态变化。取另一只荷瘤裸鼠,尾静脉注射混有0.5 mg MAG_3-K237的~(99)Tc~m-MAG_3-K237溶液17.5 MBq,观察MAG_3-K237对肿瘤显像的抑制作用。
结果
1.~(99)Tc~m-MAG_3-K237的制备:99Tcm标记MAG_3-K237(20μg)的最佳反应条件为: 0.25 mol/L醋酸铵缓冲液(pH 5.2)、16μg酒石酸钾钠、4μg酒石酸亚锡、37 MBq 99TcmO4-,总反应体积182μl,充氮、密封条件下于沸水浴中加热30 min。Whatmann 3MM纸层析,γ计数器测量标记混合物各放射性组分在生理盐水、氨水及丙酮3种流动相中的比移值(Rf值)。本方法制备的~(99)Tc~m-MAG_3-K237的标记率为(97.98±0.76)%,直接法计算其比活度为(3.54±0.03)TBq/mmol。
2.~(99)Tc~m-MAG_3-K23的体外稳定性:标记物室温下放置8 h,标记率仍大于95%,为(96.15±0.37)%;不同浓度半胱氨酸置换实验,~(99)Tc~m-MAG_3-K237中未结合99Tcm的变化均小于1%;三氯乙酸沉淀实验显示~(99)Tc~m-MAG_3-K237与血清蛋白无明显结合。
3.~(99)Tc~m-MAG_3-K237在健康家兔体内的示踪动力学分析:标记多肽的家兔体内动力学过程符合权重为1/c2的三室模型,t1/2α为4.00±3.53 min,t1/2β为24.48±9.84 min,t1/2γ为852.24±444.00 min,清除率(CL)为1.83±0.41 ml/min。
4.~(99)Tc~m-MAG_3-K237在健康动物体内的生物分布及显像:标记物在健康小鼠体内清除迅速,肾脏、肝脏及肠道分布较多,但肝脏30 min后分布很少。
5.~(99)Tc~m-MAG_3-K237荷瘤裸鼠体内分布及显像:荷HepG2裸鼠体内分布显示,10 min时相肿瘤组织摄取放射性最高,为(0.90±0.18) (%ID/g);肿瘤与对侧肌肉放射性比值(T/NT)1 h达最高值,为2.19±0.22;肾脏、肝脏及肠道放射性分布较多。荷HepG2裸鼠SPECT显像中,肿瘤1 h显影最清晰,MAG_3-K237阻断后,肿瘤影像明显减淡。
结论
1.本研究制备的~(99)Tc~m-MAG_3-K237标记率>95%,勿需分离纯化直接应用,具有良好的体内外稳定性。
2.~(99)Tc~m-MAG_3-K237在健康动物体内具有良好的体内分布及示踪动力学特性,血液清除快,通过肾脏及肝脏双重途径排泄。
3.~(99)Tc~m-MAG_3-K237特异性浓聚于荷瘤裸鼠肿瘤组织,肿瘤显像清楚,且T/NT比值较高,是一具有潜在应用价值的肿瘤KDR分子显像剂。
Background and objectives:
Angiogenesis is closely related to the growth and metastasis of malignant tumor. Tumors with diameter more than 2 mm derive nutrition, exclude metabolic waste and facilitate metastasis through new blood vessel.
The process of angiogenesis includes participation of many factors, among which vascular endothelial growth factor(VEGF)and KDR signal transduction pathway is the most important. In normal and benign tumor tissues, KDR is expressed at low level, whereas malignant tumor tissues express KDR at high level. Some immunohistochemistry studies on malignant tumors revealed that KDR was mainly expressed in the new blood vessel cell and some kind of tumor cell itself.
In the tumors highly expressing some receptor, selective receptor targeting radioactive peptide has become an important kind of molecular imaging and treatment drug. The peptide labeled by nuclide emittingγ-ray is able to combine with its receptor, and then is able to noninvasively explore malignant tumor in vivo.
In recent years, phage display technique and ribosome display technique have become two kinds of ways to find VEGFR specific bond. It can guarantee high chemical affinity between the peptide and its receptor. K237 was isolated from a phage-displayed peptide library, which could bind to KDR with high affinity and specificity. By interfering with VEGF-KDR interaction, the peptide K237 inhibited proliferation of cultured primary human umbilical vein endothelial cells. K237 also exerted an anti-angiogenesis activity in vivo in chick embryo chorioallantoic membrane and inhibited the growth and metastasis of breast cancer in SCID mice.
This study focused on the labeling of K237 with 99Tcm and feasibility of the labeled peptide as a molecular imaging agent mediated by KDR receptor in solid malignant tumors,which would lay an experimental foundation for the nuclide-therapy study in the future.
Methods:
1.Preparation of ~(99)Tc~m-MAG_3-K237:~(99)Tc~m-MAG_3-K237 was prepared by way of reduction with stannous tartrate. RCP analysis of ~(99)Tc~m-MAG_3-K237 was demonstrated by Whatman 3MM paper chromatographic system. Orthogonal design was also applied to detect the optimized labeling condition.
2.In vitro stability identification of ~(99)Tc~m-MAG_3-K237: Evaluate the stability of ~(99)Tc~m-MAG_3-K237 in vitro through a series of experiment such as stability at room temperature, displacement by Cysteine and protein precipitation by trichloroacetic acid.
3.Tracer kinetics analysis in vivo of ~(99)Tc~m-MAG_3-K237 in healthy rabbits: Each of six rabbits was injected 37 MBq ~(99)Tc~m-MAG_3-K237, then draw blood from ear vein at the following time: 1.5, 3, 5, 10, 30, 60, 120, 240, 480 min. These blood sample was weighed and measured byγ-immuno-counter, and then we calculated the radioactive concentration of every sample after correction by measurement efficiency ofγ-immuno-counter. At last, we evaluated the best compartmental model and parameter by virtue of pharmacokinetics analytical software.
4.Body distribution and imaging study of ~(99)Tc~m-MAG_3-K237 in vivo in healthy animals: 35 adult Kunming mice were involved in the study of tissue distribution, which were divided to 7 groups by random digits table. 7.4 MBq ~(99)Tc~m-MAG_3-K237 was administrated via caudal vein of the mice. At 1, 5, 10, 30, 60, 120, 240 min after injection, mice were sacrificed and the blood, heart, lungs, liver, kidneys, intestines, muscles, bone and brain were excised, weighed and counted for radioactivity by a gamma counter. And then we calculated the percentage of the injected radioactive dose per gram of tissue wet weigh (%ID/g) after correction by reference source. In the study of imaging in healthy animals, we injected 37 MBq ~(99)Tc~m-MAG_3-K237 into ear vein of rabbits. The dynamic imaging was observed by the scintigraphy with SPECT for 60 minutes and then static imaging at 90, 120, 180 and 240 min.
5.Tissue distribution study of ~(99)Tc~m-MAG_3-K237 in vivo in tumor bearing nude mice: In the study of tissue distribution, 15 tumor bearing nude mice were divided to 5 groups. 7.4 MBq ~(99)Tc~m-MAG_3-K237 was administrated into the caudal vein of each mice, and then we sacrificed them and collected their blood, heart, lungs, liver, kidneys, intestines, muscles, bone, brain and tumor tissues at 10, 30, 60, 120 and 240 min. At last, we calculated the percentage of the injected radioactive dose per gram of tissue wet weigh (%ID/g) and tumor/non-tumor ratio(T/NT) at various time.
6.SPECT imaging and competitive imaging by MAG_3-K237: In the study of imaging, 17.5 MBq ~(99)Tc~m-MAG_3-K237 was injected into the caudal vein of the mice. The dynamic distribution was observed by the scintigraphy with SPECT at 30, 60, 90, 120, 180 and 240 min. In the study of competitive imaging, mixture of 17.5 MBq ~(99)Tc~m-MAG_3-K237 and 0.5 mg MAG_3-K237 was administrated into the caudal vein of the mice, then competitive imaging inhibited by MAG_3-K237 was observed.
Results:
1.Preparation of ~(99)Tc~m-MAG_3-K237: ~(99)Tc~m-MAG_3-K237 could be successfully prepared following the steps below: added 20μg MAG_3-K237, 50μl 0.25mol/L ammonium acetate buffer solution(pH 5.2), 16μg potassium sodium tartrate, 4μg stannous tartrate, 37 MBq 99TcmO4- to the cell frozen tube, sealed the tube and boiled it under boiling water for 30 minutes. The RCP of the labeled peptide was (97.98±0.76)%, and its specific activity was (3.54±0.03)TBq/mmol.
2.Identification of stability of ~(99)Tc~m-MAG_3-K237 in vitro: after 8 h, the RCP of ~(99)Tc~m-MAG_3-K237 was (96.15±0.37)%; Cysteine replacement rate was less than 1% after adding cysteine of various concentration; trichloroacetic acid precipitation test revealed the labeled peptide couldn’t combine with serum protein.
3.Tracer kinetics analysis in vivo of ~(99)Tc~m-MAG_3-K237 in healthy rabbits: tracer kinetics process in vivo of ~(99)Tc~m-MAG_3-K237 in healthy rabbits followed three compartment model weighed by 1/c2 and the main parameters t1/2α, , t1/2β, t1/2γand CL were 4.00±3.53 min, 24.48±9.84 min, 852.24±444.00 min and 1.83±0.41 ml/min respectively.
4.Tissue distribution and imaging study of ~(99)Tc~m-MAG_3-K237 in vivo in healthy animals: ~(99)Tc~m-MAG_3-K237 was cleared quickly from the mice, it mainly distributed in the kidneys, liver and intesteins, but the distribution in liver was little after 30 min.
5.Tissue distribution and imaging study of ~(99)Tc~m-MAG_3-K237 in vivo in tumor bearing nude mice: In the study of tissue distribution, %ID/g of tumor was highest ,(0.90±0.18) at 10 min, T/NT ratio was highest, 2.19±0.22 at 1 h, and the labeled peptide was mainly distributed in kidneys, liver and intestines. In the study of imaging, the tumor was clearest at 1 h, and the imaging was much less clear after blocked by MAG_3-K237.
Conclusions:
1.~(99)Tc~m-MAG_3-K237 was successfully prepared with high RCP(>95%) and perfect stability in vitro and in vivo.
2.~(99)Tc~m-MAG_3-K237 had satisfactory characteristics in tracer kinetics and tissue distribution and was cleared quickly from blood and excreted quickly from kidneys and liver.
3.~(99)Tc~m-MAG_3-K237 could concentrate in the tumor tissue with specificity in tumor bearing nude mice, which showed its bright future in the field of molecular imaging in KDR positive tumor.
引文
1. Hicklin DJ, Ellis LM. Role of the Vascular Endothelial Growth Factor Pathway in Tumor Growth and Angiogenesis. Journal of Clinical Oncology, 2005, 23(5): 1011-1027.
2. Napoleone Ferrara . Vascular Endothelial Growth Factor: Basic Science and Clinical Progress . Endocrine Reviews,2004,25 (4): 581-611
3. Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med, 1971, 285(21): 1182-1186.
4. Masabumi S. Differential Roles of Vascular Endothelial Growth Factor Receptor-1 and Receptor-2 in Angiogenesis. Journal of Biochemistry and Molecular Biology, 2006, 39(5) : 469-478.
5. Li YJ, Wen G, Wang Q. Expression of vascular endothelial growth factor and its receptor Flk-1/KDR in benign and malignant breast neoplasms. Nan Fang Yi Ke Da Xue Xue Bao.2009, 29(1) : 70-71, 74.
6. Liu L, Zhu D, Gao R, Guo H. Expression of vascular endothelial growth factor, receptor KDR and p53 protein in transitional cell carcinoma of the bladder. Urol Int, 2008, 81(1) : 72-76.
7. Giatromanolaki A, Koukourakis MI, Sivridis E, Chlouverakis G, Vourvouhaki E, Turley H, Harris AL, Gatter KC. Activated VEGFR2/KDR pathway in tumour cells and tumour associated vessels of colorectal cancer . Eur J Clin Invest, 2007 , 37(11): 878-886.
8. An SJ, Nie Q, Chen ZH, Lin QX, Wang Z, Xie Z, Chen SL, Huang Y, Zhang AY, Yan JF, Wu HS, Lin JY, Li R, Zhang XC, Guo AL, Mok TS, Wu YL . KDR expression is associated with the stage and cigarette smoking of the patients with lung cancer . J Cancer Res Clin Oncol , 2007 , 133(9): 635-642
9. Inan S, Vatansever S, Celik-Ozenci C, Sanci M, Dicle N, Demir R . Immunolocalizations of VEGF, its receptors flt-1, KDR and TGF-beta's in epithelial ovarian tumors . Histol Histopathol , 2006 , 21(10): 1055-1064.
10. Ozdemir F, Akdogan R, Aydin F, Reis A, Kavgaci H, Gul S, Akdogan E . The effects of VEGF and VEGFR-2 on survival in patients with gastric cancer . J Exp Clin CancerRes , 2006 , 25(1): 83-88
11. Damiano V, Melisi D, Bianco C, Raben D, Caputo R, Fontanini G, Bianco R, Ryan A, Bianco AR, De Placido S, Ciardiello F, Tortora G . Cooperative antitumor effect of multitargeted kinase inhibitor ZD6474 and ionizing radiation in glioblastoma . Clin Cancer Res , 2005 , 11(15): 5639-5644.
12. Lei HT, An P, Song SM etc . A Novel Peptide Isolated from a Phage Display Library Inhibits Tumor Growth and Metastasis by Blocking the Binding of Vascular Endothelial Growth Factor to Its Kinase Domain Receptor. The Journal of Biological Chemitry , 2002 , 277(45): 43137-43142.
13. Liu Z, Wu K. Peptides homing to tumor vasculature: imaging and therapeutics for cancer. Recent Pat Anticancer Drug Discov. 2008, 3(3):202-8.
14. Sosabowsky J, Melendez-Alafort L, Mather S. Radiolabelling of peptides for diagnosis and therapy of non-oncological diseases. Q J Nucl Med. 2003, 47(4):223-37.
15.兰晓莉.两种99Tcm标记双功能螯合剂: NHS-MAG3和HYNIC.国外医学·放射医学核医学分册, 2005, 29(1) : 15-18
16. Winnard Jr P, Chang F, Rusckowski M, Mardirossian G, Hnatowich DJ. Preparation and use of NHS-MAG3 for technetium-99m labelingof DNA. Nucl Med Biol. 1997, 24(5):425–32.
17. Márquez M, Westlin JE, Nilsson S, Holmberg AR. 99mTc-dextran-antibody conjugates. Labelling procedures. Acta Oncol. 1996, 35(4):489-92.
18. Itoh K. 99mTc-MAG3: review of pharmacokinetics, clinical application to renal diseases and quantification of renal function. Ann Nucl Med. 2001, 15(3):179-90.
1. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell,1996,86(3): 353-64.
2. Napoleone Ferrara . Vascular Endothelial Growth Factor: Basic Science and Clinical Progress . Endocrine Reviews,2004, 25 (4): 581-611
3. Tischer E, Mitchell R, Hartman T, et al. The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem,1991,266(18):11947-54.
4. Cho NK, Keyes L, Johnson E,et al. Developmental control of blood cell migration by the Drosophila VEGF pathway. Cell,2002, 108(6):865-76.
5. Dor Y, Porat R, Keshet E. Vascular endothelial growth factor and vascular adjustments to perturbations in oxygen homeostasis. Am J Physiol Cell Physiol , 2001 ,280(6):C1367-74.
6. Terman BI, Carrion ME, Kovacs E, et al. Identification of a new endothelial cell growth factor receptor tyrosine kinase. Oncogene,1991,6(9):1677-83.
7. Fong GH, Zhang L, Bryce DM, et al. Increased hemangioblast commitment, not vascular disorganization, is the primary defect in flt-1 knock-out mice. Development,1999,126(13):3015-25.
8. Hiratsuka S, Maru Y, Okada A, et al. Involvement of Flt-1 tyrosine kinase (vascular endothelial growth factor receptor-1) in pathological angiogenesis. Cancer Res,2001,61(3):1207-13.
9. Hiratsuka S, Minowa O, Kuno J, et al. Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice. Proc Natl Acad Sci U S A,1998,95(16):9349-54.
10. Waltenberger, J, Claesson-Welsh, L, Siegbahn, A, et al. Different signal transduction properties of KDR and Flt1, two receptors for Vascular Endothelial Growth Factor. J Biol Chem,1994,269(43):26988-95.
11. Shalaby, F., Rossant, J., Yamaguchi, T. P., et al. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature,1995,376(6535):62-6.
12. Carmeliet, P., Moons, L., Luttun, A., et al. Synergism between vascular endothelialgrowth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. Nat Med,2001,7(5):575-83.
13. Tanigawa N , Amaya H, M atsumura M,et al. Correlation between expression of vascular endothelial growgh factor and tumor vascularity and patient outcome in human gastric carcinoma. J Clin Oncol,1997,15(2):826-32.
14.宋述梅;曾莉;吴健等. VEGF受体KDR胞外Ⅴ～Ⅶ区的克隆、单抗制备及KDR在不同来源肿瘤组织中的表达.中华肿瘤杂志,1999年第2期No.21999
15. Li S, Peck-Radosavljevic M, Koller E, et al. Characterization of (123)I-vascular endothelial growth factor-binding sites expressed on human tumour cells: possible implication for tumour scintigraphy. Int J Cancer,2001,91(6):789-96.
16. Cornelissen B, Oltenfreiter R, Kersemans V, et al. In vitro and in vivo evaluation of [123I]-VEGF165 as a potential tumor marker. Nucl Med Biol,2005,32(5):431-6.
17. Yoshimoto M, Kinuya S, Kawashima A, et al. Radioiodinated VEGF to image tumor angiogenesis in a LS180 tumor xenograft model. Nucl Med Biol,2006,33(8):963-9..
18. Backer MV, Levashova Z, Patel V, et al. Molecular imaging of VEGF receptors in angiogenic vasculature with single-chain VEGF-based probes. Nat Med,2007,13(4):504-9.
19. Qin ZX, Li QW, Liu GY, et al. Imaging targeted at tumor with (188)Re-labeled VEGF(189) exon 6-encoded peptide and effects of the transfecting truncated KDR gene in tumor-bearing nude mice. Nucl Med Biol,2009,36(5):535-43.
20. Chan C, Sandhu J, Guha A, et al. A human transferrin-vascular endothelial growth factor (hnTf-VEGF) fusion protein containing an integrated binding site for (111)In for imaging tumor angiogenesis. J Nucl Med,2005,46(10):1745-52.
21. Backer MV, Levashova Z, Patel V, et al. Molecular imaging of VEGF receptors in angiogenic vasculature with single-chain VEGF-based probes. Nat Med,2007,13(4):504-9.
22. Schenone S, Bondavalli F, Botta M. Antiangiogenic agents: an update on small molecule VEGFR inhibitors. Curr Med Chem,2007,14(23):2495-516.
23. Hetian L, Ping A, Shumei S, et al.A novel peptide isolated from a phage display library inhibits tumor growth and metastasis by blocking the binding of vascular endothelialgrowth factor to its kinase domain receptor. J Biol Chem,2002,77(45):43137-42.
24. Jayson GC, Zweit J, Jackson A, et al. Molecular imaging and biological evaluation of HuMV833 anti-VEGF antibody: implications for trial design of antiangiogenic antibodies. J Natl Cancer Inst,2002,94(19):1484-93.
25. Nagengast WB, de Vries EG, Hospers GA, et al. In vivo VEGF imaging with radiolabeled bevacizumab in a human ovarian tumor xenograft. J Nucl Med,2007,48(8):1313-9.
26. Cai W, Chen K, Mohamedali KA, et al. PET of vascular endothelial growth factor receptor expression. J Nucl Med,2006,47(12):2048-56.
27. Wang H, Cai W, Chen K, et al. A new PET tracer specific for vascular endothelial growth factor receptor 2. Eur J Nucl Med Mol Imaging,2007,34(12):2001-10.
2. Napoleone Ferrara . Vascular Endothelial Growth Factor: Basic Science and Clinical Progress . Endocrine Reviews,2004,25 (4): 581-611
3. Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med, 1971, 285(21): 1182-1186.
4. Masabumi S. Differential Roles of Vascular Endothelial Growth Factor Receptor-1 and Receptor-2 in Angiogenesis. Journal of Biochemistry and Molecular Biology, 2006, 39(5) : 469-478.
5. Li YJ, Wen G, Wang Q. Expression of vascular endothelial growth factor and its receptor Flk-1/KDR in benign and malignant breast neoplasms. Nan Fang Yi Ke Da Xue Xue Bao.2009, 29(1) : 70-71, 74.
6. Liu L, Zhu D, Gao R, Guo H. Expression of vascular endothelial growth factor, receptor KDR and p53 protein in transitional cell carcinoma of the bladder. Urol Int, 2008, 81(1) : 72-76.
7. Giatromanolaki A, Koukourakis MI, Sivridis E, Chlouverakis G, Vourvouhaki E, Turley H, Harris AL, Gatter KC. Activated VEGFR2/KDR pathway in tumour cells and tumour associated vessels of colorectal cancer . Eur J Clin Invest, 2007 , 37(11): 878-886.
8. An SJ, Nie Q, Chen ZH, Lin QX, Wang Z, Xie Z, Chen SL, Huang Y, Zhang AY, Yan JF, Wu HS, Lin JY, Li R, Zhang XC, Guo AL, Mok TS, Wu YL . KDR expression is associated with the stage and cigarette smoking of the patients with lung cancer . J Cancer Res Clin Oncol , 2007 , 133(9): 635-642
9. Inan S, Vatansever S, Celik-Ozenci C, Sanci M, Dicle N, Demir R . Immunolocalizations of VEGF, its receptors flt-1, KDR and TGF-beta's in epithelial ovarian tumors . Histol Histopathol , 2006 , 21(10): 1055-1064.
10. Ozdemir F, Akdogan R, Aydin F, Reis A, Kavgaci H, Gul S, Akdogan E . The effects of VEGF and VEGFR-2 on survival in patients with gastric cancer . J Exp Clin CancerRes , 2006 , 25(1): 83-88
11. Damiano V, Melisi D, Bianco C, Raben D, Caputo R, Fontanini G, Bianco R, Ryan A, Bianco AR, De Placido S, Ciardiello F, Tortora G . Cooperative antitumor effect of multitargeted kinase inhibitor ZD6474 and ionizing radiation in glioblastoma . Clin Cancer Res , 2005 , 11(15): 5639-5644.
12. Lei HT, An P, Song SM etc . A Novel Peptide Isolated from a Phage Display Library Inhibits Tumor Growth and Metastasis by Blocking the Binding of Vascular Endothelial Growth Factor to Its Kinase Domain Receptor. The Journal of Biological Chemitry , 2002 , 277(45): 43137-43142.
13. Liu Z, Wu K. Peptides homing to tumor vasculature: imaging and therapeutics for cancer. Recent Pat Anticancer Drug Discov. 2008, 3(3):202-8.
14. Sosabowsky J, Melendez-Alafort L, Mather S. Radiolabelling of peptides for diagnosis and therapy of non-oncological diseases. Q J Nucl Med. 2003, 47(4):223-37.
15.兰晓莉.两种99Tcm标记双功能螯合剂: NHS-MAG3和HYNIC.国外医学·放射医学核医学分册, 2005, 29(1) : 15-18
16. Winnard Jr P, Chang F, Rusckowski M, Mardirossian G, Hnatowich DJ. Preparation and use of NHS-MAG3 for technetium-99m labelingof DNA. Nucl Med Biol. 1997, 24(5):425–32.
17. Márquez M, Westlin JE, Nilsson S, Holmberg AR. 99mTc-dextran-antibody conjugates. Labelling procedures. Acta Oncol. 1996, 35(4):489-92.
18. Itoh K. 99mTc-MAG3: review of pharmacokinetics, clinical application to renal diseases and quantification of renal function. Ann Nucl Med. 2001, 15(3):179-90.
1. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell,1996,86(3): 353-64.
2. Napoleone Ferrara . Vascular Endothelial Growth Factor: Basic Science and Clinical Progress . Endocrine Reviews,2004, 25 (4): 581-611
3. Tischer E, Mitchell R, Hartman T, et al. The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem,1991,266(18):11947-54.
4. Cho NK, Keyes L, Johnson E,et al. Developmental control of blood cell migration by the Drosophila VEGF pathway. Cell,2002, 108(6):865-76.
5. Dor Y, Porat R, Keshet E. Vascular endothelial growth factor and vascular adjustments to perturbations in oxygen homeostasis. Am J Physiol Cell Physiol , 2001 ,280(6):C1367-74.
6. Terman BI, Carrion ME, Kovacs E, et al. Identification of a new endothelial cell growth factor receptor tyrosine kinase. Oncogene,1991,6(9):1677-83.
7. Fong GH, Zhang L, Bryce DM, et al. Increased hemangioblast commitment, not vascular disorganization, is the primary defect in flt-1 knock-out mice. Development,1999,126(13):3015-25.
8. Hiratsuka S, Maru Y, Okada A, et al. Involvement of Flt-1 tyrosine kinase (vascular endothelial growth factor receptor-1) in pathological angiogenesis. Cancer Res,2001,61(3):1207-13.
9. Hiratsuka S, Minowa O, Kuno J, et al. Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice. Proc Natl Acad Sci U S A,1998,95(16):9349-54.
10. Waltenberger, J, Claesson-Welsh, L, Siegbahn, A, et al. Different signal transduction properties of KDR and Flt1, two receptors for Vascular Endothelial Growth Factor. J Biol Chem,1994,269(43):26988-95.
11. Shalaby, F., Rossant, J., Yamaguchi, T. P., et al. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature,1995,376(6535):62-6.
12. Carmeliet, P., Moons, L., Luttun, A., et al. Synergism between vascular endothelialgrowth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. Nat Med,2001,7(5):575-83.
13. Tanigawa N , Amaya H, M atsumura M,et al. Correlation between expression of vascular endothelial growgh factor and tumor vascularity and patient outcome in human gastric carcinoma. J Clin Oncol,1997,15(2):826-32.
14.宋述梅;曾莉;吴健等. VEGF受体KDR胞外Ⅴ～Ⅶ区的克隆、单抗制备及KDR在不同来源肿瘤组织中的表达.中华肿瘤杂志,1999年第2期No.21999
15. Li S, Peck-Radosavljevic M, Koller E, et al. Characterization of (123)I-vascular endothelial growth factor-binding sites expressed on human tumour cells: possible implication for tumour scintigraphy. Int J Cancer,2001,91(6):789-96.
16. Cornelissen B, Oltenfreiter R, Kersemans V, et al. In vitro and in vivo evaluation of [123I]-VEGF165 as a potential tumor marker. Nucl Med Biol,2005,32(5):431-6.
17. Yoshimoto M, Kinuya S, Kawashima A, et al. Radioiodinated VEGF to image tumor angiogenesis in a LS180 tumor xenograft model. Nucl Med Biol,2006,33(8):963-9..
18. Backer MV, Levashova Z, Patel V, et al. Molecular imaging of VEGF receptors in angiogenic vasculature with single-chain VEGF-based probes. Nat Med,2007,13(4):504-9.
19. Qin ZX, Li QW, Liu GY, et al. Imaging targeted at tumor with (188)Re-labeled VEGF(189) exon 6-encoded peptide and effects of the transfecting truncated KDR gene in tumor-bearing nude mice. Nucl Med Biol,2009,36(5):535-43.
20. Chan C, Sandhu J, Guha A, et al. A human transferrin-vascular endothelial growth factor (hnTf-VEGF) fusion protein containing an integrated binding site for (111)In for imaging tumor angiogenesis. J Nucl Med,2005,46(10):1745-52.
21. Backer MV, Levashova Z, Patel V, et al. Molecular imaging of VEGF receptors in angiogenic vasculature with single-chain VEGF-based probes. Nat Med,2007,13(4):504-9.
22. Schenone S, Bondavalli F, Botta M. Antiangiogenic agents: an update on small molecule VEGFR inhibitors. Curr Med Chem,2007,14(23):2495-516.
23. Hetian L, Ping A, Shumei S, et al.A novel peptide isolated from a phage display library inhibits tumor growth and metastasis by blocking the binding of vascular endothelialgrowth factor to its kinase domain receptor. J Biol Chem,2002,77(45):43137-42.
24. Jayson GC, Zweit J, Jackson A, et al. Molecular imaging and biological evaluation of HuMV833 anti-VEGF antibody: implications for trial design of antiangiogenic antibodies. J Natl Cancer Inst,2002,94(19):1484-93.
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