In vitro characterization of radiolabeled monoclonal antibodies specific for the extracellular domain of prostate-specific membrane antigen.
In vitro characterization of radiolabeled monoclonal antibodies specific for the extracellular domain of prostate-specific membrane antigen.
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发表时间：
2000-09
期刊：
影响因子：
11.2
通讯作者：
P. Smith-Jones;Shankar Vallabahajosula;S. Goldsmith;Vincent Navarro;Catherine J. Hunter;D. Bastidas;N. Bander
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文献类型：
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作者：
P. Smith-Jones;Shankar Vallabahajosula;S. Goldsmith;Vincent Navarro;Catherine J. Hunter;D. Bastidas;N. Bander
前列腺特异性膜抗原（PSMA）是一种特性良好的细胞表面抗原，几乎在所有前列腺癌（PCas）中表达。PSMA已经成功地在体内用（111）in -labeled 7E11单克隆抗体（mAb; ProstaScint; Cytogen, Princeton， NJ）靶向，该抗体结合PSMA的细胞内表位。本研究报道了最近开发的三种结合PSMA细胞外结构域（PSMAext）的单克隆抗体的体外特性。小鼠单克隆抗体J415、J533、J591和7E11用131I放射性标记，并在LNCaP细胞底物的竞争和饱和结合研究中进行了评估。J415和J591与1,4,7,10-四氮杂环十二烷-N，N‘，N’，N' -四乙酸偶联，标记为（111）In。在活的LNCaP细胞中评估这些抗体的摄取和细胞加工。所有四种单抗都可以用131I标记，特异性活性高达350 MBq/mg，没有或几乎没有明显的免疫反应性损失。竞争分析显示J415和J591在与PSMAext抗原结合方面存在竞争。J533结合到靠近J591结合表位的区域，但J533不干扰J415与PSMA的结合。mAb 7E11不抑制J415、J533或J591的结合（反之亦然），这与早期的研究一致，即这些后一种mAb结合PSMAext，而7E11结合PSMA的细胞内结构域。饱和结合实验表明，J415和J591的结合亲和力相近（Kd值分别为1.76和1.83 nM），而J533的结合亲和力较低（Kd值为18 nM）。在平行研究中，所有四种单克隆抗体结合到通透化细胞表达的PSMA位点的数量相似（1,000,000-1,300,000个位点/细胞）。在用活的LNCaP细胞进行的平行研究中，J415、J533和J591结合到相似数量的PSMA位点（即60 -80万个位点/细胞），而7E11只结合到可用PSMA位点的一个亚群（95000个位点/细胞）。当细胞被胰蛋白酶化和悬浮时，产生的5-7%的通透性细胞亚群可以解释7E11与活细胞的明显结合。多达5种DOTA螯合物可以与J415或J591结合而不影响免疫反应性。131I-和（111）In标记抗体的细胞摄取和代谢过程的比较表明，131I从细胞中快速消除，（111）In保留率高。所有四种单克隆抗体都能识别并结合由破裂的LNCaP细胞（即暴露的PSMA的胞内和胞外结构域）表达的相似数量的PSMA。与J415和J591相比，J533的结合亲和力较低。J415和J591都能识别并结合完整LNCaP表达的相同数量的psma。相比之下，7E11结合到完整LNCaP细胞表达的较少位点（即PSMA暴露的胞外结构域）。J415和J591都是有前景的单克隆抗体，用于诊断和治疗金属放射性核素靶向活的表达psma的组织。
Prostate-specific membrane antigen (PSMA) is a well-characterized cell surface antigen expressed by virtually all prostate cancers (PCas). PSMA has been successfully targeted in vivo with (111)In-labeled 7E11 monoclonal antibody (mAb; ProstaScint; Cytogen, Princeton, NJ), which binds to an intracellular epitope of PSMA. This work reports the in vitro characterization of three recently developed mAbs that bind the extracellular domain of PSMA (PSMAext). Murine mAbs J415, J533, J591, and 7E11 were radiolabeled with 131I and evaluated in competitive and saturation binding studies with substrates derived from LNCaP cells. J415 and J591 were conjugated to 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid labeled with (111)In. The uptake and cellular processing of these antibodies were evaluated in viable LNCaP cells. All four mAbs could be labeled with 131I up to a specific activity of 350 MBq/mg with no or little apparent loss of immunoreactivity. Competition assays revealed that J415 and J591 compete for binding to PSMAext antigen. J533 bound to a region close to the J591 binding epitope, but J533 did not interfere with J415 binding to PSMA. mAb 7E11 did not inhibit the binding of J415, J533, or J591 (or vice versa), consistent with earlier work that these latter mAbs bind PSMAext whereas 7E11 binds the intracellular domain of PSMA. Saturation binding studies demonstrated that J415 and J591 bound with a similar affinity (Kds 1.76 and 1.83 nM), whereas J533 had a lower affinity (Kd, 18 nM). In parallel studies, all four mAbs bound to a similar number of PSMA sites expressed by permeabilized cells (1,000,000-1,300,000 sites/cell). In parallel studies performed with viable LNCaP cells, J415, J533, and J591 bound to a similar number of PSMA sites (i.e., 600,000-800,000 sites/cell), whereas 7E11 bound only to a subpopulation of the available PSMA sites (95,000 sites/cell). This apparent binding of 7E11 to viable cells can be accounted for by a 5-7% subpopulation of permeabilized cells produced when the cells were trypsinized and suspended. Up to five DOTA chelates could be bound to either J415 or J591 without compromising immunoreactivity. A comparison of the cellular uptake and metabolic processing of the 131I- and (111)In-labeled antibodies showed a rapid elimination of 131I from the cell and a high retention of (111)In. All four mAbs recognized and bound to similar numbers of PSMAs expressed by ruptured LNCaP cells (i.e., the exposed intracellular and extracellular domains of PSMA). By comparison to J415 and J591, J533 had a lower binding affinity. Both J415 and J591 recognized and bound to the same high number of PSMAs expressed by intact LNCaP. By contrast, 7E11 bound to fewer sites expressed by intact LNCaP cells (i.e., the exposed extracellular domain of PSMA). Both J415 and J591 are promising mAbs for the targeting of viable PSMA-expressing tissue with diagnostic and therapeutic metallic radionuclides.