Induction of antibodies against prostate-specific membrane antigen (PSMA) by vaccination with a PSMA DNA vector.
Induction of antibodies against prostate-specific membrane antigen (PSMA) by vaccination with a PSMA DNA vector.
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通过接种 PSMA DNA 载体来诱导针对前列腺特异性膜抗原 (PSMA) 的抗体。
DOI：
10.1016/s0302-2838(02)00211-7
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发表时间：
2002
期刊：
影响因子：
23.4
通讯作者：
Haas,GabrielP
中科院分区：
文献类型：
--
作者：
Kuratsukuri,Katsuyuki;Wang,ChingY;Sone,Tomomichi;Nishisaka,Nobuyasu;Jones,RichardF;Haas,GabrielP
简介和目的前列腺特异性膜抗原 (PSMA) 是一种 750 个氨基酸的表面蛋白，主要在前列腺上皮中表达，在前列腺癌中表达上调 10 倍。因此，它是免疫治疗的一个有吸引力的靶点。然而，大多数报道的 PSMA 抗体明显识别胞外结构域残基 43-570 区域中的表位，并且在结合后通过内化迅速从细胞表面去除。这可能会限制它们介导 Fc 依赖性细胞毒性的能力。在本研究中，我们构建了一个 DNA 表达载体 pV/TM–PSMc，其中该区域从全长 PSMA cDNA 中删除。给小鼠接种 pV/TM–PSMc DNA 疫苗，以确定该 C 末端区域是否可以诱导针对 PSMA 阳性人前列腺癌细胞的体液反应。方法使用基于聚合酶链反应 (PCR) 的技术从人 PSMA cDNA 编码区删除密码子 50–570，从而将 C 末端 (PSMc) 连接到 N 末端细胞质/跨膜结构域 (TM)。该截短产物 TM–PSMc 被克隆到载体 pNGVL3 (pV) 中。所得载体 pV/TM–PSMc 通过 DNA 测序、使用逆转录酶 (RT)–PCR 进行转录本的表达研究以及使用 PSMA 单克隆抗体 (mAb) 7E11.C5 进行免疫组织化学 (IHC) 染色进行了确认。 BALB/c小鼠胫骨前肌注射四次，每两周一次，每次注射100μg载体DNA。最后一次注射一周后，抽血制备血清。通过 LNCaP（一种 PSMA 阳性人类前列腺癌系）的 IHC 染色来测定血清中的抗 PSMA 抗体。通过 RT-PCR 测定 TM-PSMc 转录物来测定接种疫苗的肌肉细胞中的表达。结果用 mAb 7E11.C5 进行 IHC 反应，转染 pV/TM-PSMc 的 NIH3T3 细胞染色呈阳性。向小鼠肌内 (i.m.) 注射 100μg pV/TM–PSMc 载体 DNA 48 小时后，通过 RT–PCR 分析可在肌肉 RNA 中检测到 TM–PSMc 转录本。来自接种 pV/TM–PSMc-DNA 疫苗的小鼠的抗血清，以 1:20 的稀释度，对活的和固定的 LNCaP 细胞进行强烈的 IHC 染色。结论这些结果表明，肌内注射诱导了抗 PSMA 体液反应。给小鼠注射 pV/TM–PSMc DNA。抗血清中的抗体针对人类前列腺癌细胞表达的天然 PSMA 的细胞外表位。使用 pV/TM–PSMc 等 DNA 表达载体进行疫苗接种可能为治疗前列腺癌提供免疫治疗方法。
Introduction and ObjectivesProstate-specific membrane antigen (PSMA) is a 750 amino acid surface protein expressed primarily in prostate epithelium, and is upregulated 10-fold in prostate cancer. It is therefore an attractive target for immunotherapy. However, most reported antibodies to PSMA apparently recognize epitopes in the residue 43–570 region of the extracellular domain, and upon binding are rapidly removed from the cell surface by internalization. This would potentially limit their ability to mediate Fc-dependent cytoxicity. In this study, we constructed a DNA expression vector, pV/TM–PSMc, in which this region was deleted from full-length PSMA cDNA. Mice were vaccinated with pV/TM–PSMc DNA to determine whether humoral responses directed against PSMA-positive human prostate cancer cells could be induced by this C-terminal region.MethodsPolymerase chain reaction (PCR)-based techniques were used to delete codons 50–570 from the coding region of human PSMA cDNA, thereby joining the C-terminal end (PSMc) to the N-terminal cytoplasmic/transmembrane domain (TM). This truncated product, TM–PSMc, was cloned into the vector pNGVL3 (pV). The resulting vector, pV/TM–PSMc, was confirmed by DNA sequencing, and by expression studies using reverse transcriptase (RT)–PCR for transcripts and immunohistochemical (IHC) staining with the PSMA monoclonal antibody (mAb) 7E11.C5. BALB/c mice were injected in the tibialis anterior muscle four times, at biweekly intervals, with 100μg vector DNA per injection. One week after the last injection, blood was drawn for serum preparation. The serum was assayed for antibodies against PSMA by IHC staining of LNCaP, a PSMA-positive human prostate cancer line. Expression in vaccinated muscle cells was determined by RT–PCR assay for TM–PSMc transcripts.ResultsNIH3T3 cells transfected with pV/TM–PSMc stained positively by IHC reaction with mAb 7E11.C5. 48h after one intramuscular (i.m.) injection of mice with 100μg pV/TM–PSMc vector DNA, TM–PSMc transcripts were detectable in muscle RNA by RT–PCR analysis. Anti-serum from pV/TM–PSMc-DNA vaccinated mice, at a dilution of 1:20, intensely IHC-stained both live and fixed LNCaP cells.ConclusionsThese results demonstrate that anti-PSMA humoral responses were induced by i.m. injection of mice with pV/TM–PSMc DNA. Antibodies in the anti-serum were directed against extracellular epitopes of native PSMA expressed by human prostate cancer cells. Vaccination with DNA expression vectors such as pV/TM–PSMc may provide an immunotherapeutic approach for the treatment of prostate cancer.