前列腺癌噬菌体抗体库的构建及抗特异性膜抗原抗体的筛选

目的建立国人前列腺癌噬菌体Fab抗体片段库，筛选、鉴定抗前列腺特异性膜抗原（PSMA）的人源Fab抗体可变区的编码基因，为前列腺癌的诊断与基因治疗研究开辟新途径。方法20例前列腺癌患者外周血分离淋巴细胞，提取其总RNA，经RTPCR及半套式扩增得到免疫球蛋白全部轻链和重链Fd段基因，克隆进载体pComb3，并电转化大肠杆菌XLl-Blue，构建前列腺癌噬菌体Fab抗体片段库。采用噬菌体表面展示技术，以PSMA原核表达重组子pET30a（＋）-PSMA诱导表达并纯化后的PSMA为固相抗原，从噬菌体Fab抗体库中经过“吸附洗脱扩增”筛选过程，获得抗原结合活性和特异性较强的PSMA人源Fab可变区抗体基因片段的阳性克隆，并进行免疫检测及序列测定。结果成功构建前列腺癌噬菌体抗体Fab片段库，其轻链及重链Fd段与载体DNA的重组率分别为87％、79％，库容为2．32×10^7，滴度为8．7×10^13pfu／ml。筛选得到Fab抗体克隆的Fd段基因核苷酸序列为696bp，编码由232个氨基酸残基组成的多肽，与人免疫球蛋白γ链恒定区的同源性为98％；轻链κ基因核苷酸序列为630bp，编码由210个氨基酸残基组成的多肽，与κ链恒定区同源性为93％。结论成功构建了前列腺癌噬菌体抗体库，并获得了PSMA人Fab抗体可变区编码基因克隆，该克隆抗体基因具有典型的免疫球蛋白轻链和重链可变区的结构特点，基因编码产物具有与PSMA反应的免疫学活性和特异性。     

抗人膀胱癌单克隆抗体重,轻链可变区基因体外扩增,克隆及序列分析

应用人工合成的两套引物，从分泌抗人膀胱癌特异性单抗的杂交瘤细胞ＢＤＩ－１中提取总ＤＮＡ。以之作模板，经ＰＣＲ法扩增出重、轻链可变区基因片段。将扩增产物分别插入ＰＵＣ１９质粒，筛选出阳性克隆，用链终止法进行ＤＮＡ序列测定。结果表明：重链可变区基因全长３６６ｂｐ，编码１２２个氨基酸，属于小鼠重链可变区的ＳｕｂｇｒｏｕｐＩＩ，由种系基因中的ＶＨ与Ｄ基因的Ｄｓｐ２．２及ＪＨ４基因重排而成；轻链可变区基因全长３２４ｂｐ，编码１０８个氨基酸，属于小鼠轻链可变区的ＳｕｂｇｒｏｕｐＩＶ，由种系基因中的Ｖκ与Ｊκ４基因重排而成。与已发表的抗体可变区基因序列比较，证明它们是功能性的可变区基因     

胶质瘤单抗SZ-38可变区基因的克隆和序列分析

目的　克隆高特性、高亲和力的胶质瘤靶向载体基因,为制备重组免疫毒素奠定基础。方法　从胶质瘤单抗杂交瘤细胞 Ｓ Ｚ３８ 提取总 Ｒ Ｎ Ａ, Ｒ Ｔ Ｐ Ｃ Ｒ 扩增抗体的重链及轻链可变区基因,并测序分析。结果　重链可变区全长３０６ ｂｐ ,编码１０２ 个氨基酸;轻链可变区全长３１２ ｂｐ ,编码１０４ 个氨基酸,分属重链Ⅲ（ Ｄ） 亚组和 Ｋａｐｐａ 链Ⅵ亚组。结论　新克隆的可变区基因符合功能重排的鼠抗体可变区基因特征,可用于重组免疫毒素的制备。     

肝癌特异性噬菌体多肽的筛选和初步鉴定

目的 利用噬菌体展示肽库筛选与肝癌HepG2细胞特异性结合的多肽,为筛选及明确新的肝癌早期诊断和治疗标志物打下基础.方法 以肝癌细胞HepG2为靶细胞,LO-2为吸附细胞,在37℃条件下对噬菌体随机12肽库进行多轮减性筛选,挑取单克隆扩增并鉴定.利用ELISA初步鉴定克隆亲和力,测定阳性克隆DNA测序并进行同源性及氨基酸分析.结果 经过3轮减性筛选发现,随机挑选的30个单克隆中,其中ZS-9对HepG2具有较高亲和力,氨基酸测序结果表明,该序列与美国国立生物技术信息中心(NCBI)GenBankDNA序列数据库和Swiss-Prot蛋白数据库中的已知基因和蛋白无同源性,而且,国内外文献均未见报道,表明笔者筛选到一新的肝癌相关抗原的配体.结论 利用噬菌体随机12肽库成功筛选到与肝癌细胞HepG2具有较高亲和力的多肽,为筛选鉴定新的肝癌特异的标志物奠定工作基础,也为肝癌的早期诊断和靶向治疗进一步研发确定了靶标.     

利用噬菌体随机七肽库技术筛选人膀胱癌相关表面标志物Her2、Survivin表位模拟肽

目的 利用噬菌体随机七肽库技术筛选人膀胱癌相关表面标志物Her2、Survivin表位模拟肽.方法 分别以Her2、Survivin多克隆抗体为固相筛选分子,对噬菌体随机七肽库进行3轮生物淘洗,随机挑选单克隆噬菌体进行酶联免疫吸附试验(ELISA)分析检测其结合力,竞争抑制实验鉴定其阳性克隆,对结合力较好的噬菌体提取DNA并测序,寻找比较保守的核心序列,合成多肽,最后鉴定合成多肽的结合力.结果经噬菌体随机七肽库3轮淘洗后,特异性噬菌体模拟肽得到富集,分别挑选Her2、Survivin20个克隆,经DNA测序分析,Her2较保守的序列为ACT ACG GCT GAG AAT AAT GAG,Survivin为TCT CCT CCT CAT CTT TCG CAG,合成多肽,Her2为Thr-Thr-Ala-Glu-Asn-Asn-Glu(TTAENNE),Survivin为Ser-Pro-Pro-His-Leu-Ser-Gln(SPPHLSQ),ELISA分析具有良好结合力.结论 利用噬菌体随机七肽库成功筛选到人膀胱癌相关表面标志物Her2、Survivin表位模拟肽,从而为今后膀胱癌多肽疫苗的研制提供良好的基础和条件.     

抗人膀胱癌基因工程单链抗体的制备研究

应用ＰＣＲ方法，从分泌鼠抗人膀胱癌抗体的杂交瘤细胞中扩增抗体重、轻链可变区基因，经ＤＮＡ接头连接后与噬菌粒ｐＣＡＮＴＡＢ５重组，转化大肠杆菌后，通过亲和筛选、再转染、克隆及鉴定，以此获得抗人膀胱癌噬菌体单链抗体。     

抗膀胱癌单克隆抗体重链可变区基因扩增克隆及序列测定

从分泌人膀胱癌特异性单克隆抗体的杂交癌细胞ＢＤＩ－１中提取总ＤＮＡ，应用人工合成的引物经聚合酶链反应（ＰＣＲ）扩增出重链可变区基因片段，将扩增产物插入ＰＵＣ１９质粒，筛选出阳性克隆，用链终止法进行ＤＮＡ序列测定分析，结果证实抗体重链可变区基因片段全长由３６６个碱基组成。     

人前列腺特异性膜抗原cDNA的克隆、原核表达、鉴定和抗原初步纯化

目的　克隆中国人前列腺特异性膜抗原 (PSMA)cDNA全长 ,构建PSMA原核表达重组子。诱导PSMA原核表达 ,并对表达产物进行初步纯化。　方法　从前列腺癌患者组织中提取总RNA ,利用PSMAcDNA中的EcoRⅠ位点 ,将PSMA分为两段分别作RT PCR ,分别连接到pET 30a中 ,从而得到完整PSMAcDNA的原核表达重组子pET 30a PSMA ,并进行酶切鉴定与序列测定。确定序列正确后 ,利用IPTG诱导重组子表达 ,采用Ni NTAAgarose螯合层析柱对表达产物初步纯化 ,SDS PAGE和Western Blotting对表达产物和纯化产物进行鉴定。　结果　成功克隆到序列完全正确的人源PSMAcDNA ,诱导表达并初步纯化出PSMA蛋白 ,该蛋白具有较好的抗原性和特异性。　结论　此项研究为PSMA功能的研究及前列腺癌噬菌体抗体库的筛选提供了实验依据     

噬菌体展示的肽库中N-甲基-D-天门冬氨酸受体2B亚基模拟抗原表位的筛选

目的 从噬菌体展示的随机十二肽库中筛选N-甲基-D-天门冬氨酸受体（NMDAR）2B亚基模拟抗原表位。方法以NMDAR2B单克隆抗体为配基，免疫亲和筛选以融合蛋白形式表达在丝状噬菌体M13外壳蛋白Ⅲ上的随机十二肽。经过3轮筛选，从第3轮洗脱物中随机挑选12个单克隆噬菌体扩增后进行ELISA鉴定，用酶标仪测定450nm处的吸光值（A450）。对这12个单克隆噬菌体分别进行扩增、纯化，并对DNA测序，以确定插入十二肽的氨基酸序列。通过细胞竞争ELISA法分析阳性单克隆噬菌体对NMDAR2B天然抗原表位与其特异性抗体结合的竞争性抑制。结果经过3轮筛选后能与NMDAR2B单克隆抗体特异性结合的噬菌体得到了有效富积，12个单克隆噬菌体中有9个单克隆噬菌体的A450高于其它3个。DNA测序结果表明这9个单克隆噬菌体表达了一个共同氨基酸序列：SHPPVMPWPTST，将其命名为阳性克隆噬菌体，该阳性克隆噬菌体可以竞争性抑制细胞表面天然抗原与特异性抗体的结合，抑制率（45土3）％，具有抗原模拟性。结论从噬菌体展示的随机十二肽库中成功筛选到了能与NMDAR2B特异性抗体结合的短肽，该肽模拟了天然抗原的某个表位。     

胶质瘤单链抗体的基因构建及表达

单链抗体 (ＳｃＦｖ)因其能较好地保持对抗原亲和活性 ,分子量小 ,穿透力强 ,抗原性弱 ,且易与效应分子相拼接 ,是构建免疫毒素和双功能抗体的较理想元件。我们在已克隆抗胶质瘤单抗ＳＺ39重链可变区 (ＶＨ)和轻链可变区(ＶＬ)基因的基础上 ,构建了抗人脑胶质瘤ＳｃＦｖ基因 ,并克隆于表达载体ｐＧＥＸ 5Ｘ 1,在大肠杆菌中诱导表达 ,现将结果报道如下。一、材料和方法1.材料 :(1)菌种与质粒载体 :ｐＧＥＭ Ｔ 39ＶＨ、ｐＧＥＭ Ｔ 39ＶＬ为含有ＶＨ、ＶＬ基因的克隆质粒 ,由本室构建。ｐＳＷ1 ＳｃＦｖ为含有一编码疏水性多肽接头 (Ｇｌｙ4 …     

Anti-prostate specific membrane antigen designer T cells for prostate cancer therapy

BACKGROUND: Designer T cells are T lymphocytes engineered toward specific antibody-type membrane antigens through chimeric immunoglobulin-T-cell receptor (IgTCR) genes that have been used for adoptive cellular immunotherapy. We have extended this approach to prostate specific membrane antigen (PSMA) as a means to attack prostate cancer. METHODS: A chimeric anti-PSMA IgTCR gene was constructed based on an anti-PSMA monoclonal antibody, 3D8. Both T-cell lines and primary cultured human T lymphocytes were transduced with the chimeric anti-PSMA IgTCR construct and were analyzed for IgTCR expression, specific activation by PSMA, cytotoxicity against PSMA-expressing tumor cells in vitro, and retardation of tumor growth in an animal model. RESULTS: The IgTCR was incorporated into the TCR-CD3 complex and formed a functional chimeric complex. The IgTCR-modified T cells were specifically activated through the chimeric receptor with PSMA as measured by IL-2 production and increased CD25 expression and specifically lysed the PSMA-expressing prostate cancer cells in vitro as well as retarded tumor growth in an animal model. CONCLUSIONS: The anti-PSMA designer T cells exhibit an antibody-type specificity that can recognize PSMA expressing tumor cells in a MHC-independent fashion, resulting in T-cell activation, target cell lysis in vitro and inhibition of tumor growth in vivo.     

Further investigation of the epitope recognized by the new monoclonal antibody 2C9

OBJECTIVE: We established a new monoclonal antibody (2C9) that reacted with prostate tissue. The immunohistochemical reactivity of this antibody is similar to anti-prostate-specific membrane antigen (PSMA). Herein, we report the antigenic determinant of 2C9 antibody. METHODS: The reactivity of the antibody was characterized by immunohistochemical staining and the antigen target was characterized by amino acid sequencing after immuno-affinity purification from an LNCaP cell lysate and cloning of a cDNA using a mammalian expression cDNA cloning system. RESULTS: The amino acid and nucleotide sequences for the antigen molecule recognized with 2C9 monoclonal antibody demonstrated identity with PSMA. CONCLUSION: The target molecule of the 2C9 monoclonal antibody is PSMA, pointing to future diagnostic and therapeutic applications.     

A new generation of monoclonal and recombinant antibodies against cell-adherent prostate specific membrane antigen for diagnostic and therapeutic targeting of prostate cancer

BACKGROUND: Prostate-specific membrane antigen (PSMA) is an excellent candidate for targeting prostate cancer by virtue of its restricted expression on prostatic epithelial cells and its upregulation on prostatic carcinoma cells. PSMA is expressed on the cell surface displaying a specific three-dimensional structure. Therefore, only antibodies with a high cell binding activity will have an important impact on antibody-based imaging and therapy. METHODS: Monoclonal antibodies (mAbs) and single chain antibody fragments (scFvs) were prepared from spleen cells of mice that had been immunized either with purified PSMA or a cell lysate of prostate cancer LNCaP cells containing native PSMA. mAbs and scFvs were screened for reactivity with purified PSMA and binding to PSMA-expressing LNCaP cells. RESULTS: From mice immunized with purified PSMA, we obtained three mAbs (K7, K12, D20) and four scFvs (G0, G1, G2, G4), which were highly reactive with the isolated antigen, but showed weak or no reaction with viable LNCaP cells. From mice immunized with unpurified LNCaP lysate, we obtained three mAbs (3/E7, 3/F11, 3/A12), and one scFv (A5), which were reactive with purified PSMA, also showing a strong and specific binding to viable LNCaP cells and PSMA-transfected cells. CONCLUSIONS: Our results suggest that only the mAbs and scFvs, that were elicited with unpurified LNCaP lysate and not with purified PSMA will be useful agents for diagnostic imaging and therapeutic applications of prostate cancer.     

Cell-Surface labeling and internalization by a fluorescent inhibitor of prostate-specific membrane antigen

BACKGROUND: [corrected] Prostate-specific membrane antigen (PSMA) remains an attractive target for imaging and therapeutic applications for prostate cancer. Recent efforts have been made to conjugate inhibitors of PSMA with imaging agents. Compared to antibodies, small-molecule inhibitors of PSMA possess apparent advantages for in vivo applications. To date, there are no reports on the cellular fate of such constructs once bound the extracellular domain of PSMA. The present study was focused on precisely defining the binding specificity, time-dependent internalization, cellular localization, and retention of inhibitor conjugates targeted to PSMA on LNCaP cells. A novel fluorescent inhibitor was prepared as a model to examine these processes. METHODS: Fluorescence microscopy of LNCaP and PC-3 cell lines was used to monitor the specificity, time-dependent internalization, cellular localization, and retention of a fluorescent PSMA inhibitor. RESULTS: Fluorescent inhibitor 2 was found to be a potent inhibitor (IC50 = 0.35 nM) of purified PSMA. Its high affinity for PSMA on living cells was confirmed by antibody blocking and competitive binding experiments. Specificity for LNCaP cells was demonstrated as no labeling by 2 was observed for negative control PC-3 cells. Internalization of 2 by viable LNCaP cells was detected after 30 min incubation at 37 degrees C, followed by accumulation in the perinuclear endosomes. It was noted that internalized fluorescent inhibitor can be retained within endosomes for up to 150 min without loss of signal. CONCLUSIONS: Our results suggest that potent, small-molecule inhibitors of PSMA can be utilized as carriers for targeted delivery for prostate cancer for future imaging and therapeutic applications.     

前列腺特异性膜抗原在前列腺癌诊治中的研究进展

近年来,前列腺癌特异性的分子标志物——前列腺特异性膜抗原(PSMA)已成为前列腺癌临床研究中的热点之一。PSMA在前列腺癌的早期诊断、基因治疗、预后评估中所起的作用变得越来越重要。本文就PSMA蛋白的结构、功能、表达特点、基因表达以及基于PSMA的前列腺癌放射免疫显像、DNA疫苗、自杀基因治疗的相关研究进展及其在前列腺癌诊治中的作用进行了综述。     

Specific Fab fragments recovered by phage display technique recognizing human spermatozoa

Human hybridoma cell lines are often unstable and loose ability for antibody production. Sometimes, they show low and varying levels of heavy and light chains synthesis. Therefore it is reasonable to preserve generated specificities of light and heavy chains by cloning them to phagemid vector and creating phage display library. The aim of this study was to construct phage display library of Fab fragments recognizing sperm surface antigens. The source of mRNA constituted seven hybridoma cell lines producing antisperm antibodies which was proved by ELISA, and agglutination test as well as by inhibition of sperm to penetrate hamster oocytes. Fragments of cDNA encoding κ/λ and γ chains were cloned into pComb3HSS phagemid vector and amplified in XL-1Blue. The library was panned against whole unfixed sperm cells. Three positive clones selected after fourth round of panning showed heavy chain belonging to VH4 family, two of them (G28, K61) possessed lambda chain from VL2 family and one (H43) kappa chain from VK1 family. As these Fabs revealed similarities to antibodies against some proteins involved in sperm motility and cell fusion it can be suggested that these Fabs may be a cause of infertility. Finally, we proved that it is feasible to preserve specificities produced by human hybridomas using phage display technique and we recovered some Fabs which may be of diagnostic and research value, and may also have some value for contraceptive vaccine.     

Imaging with radiolabelled monoclonal antibody (MUJ591) to prostate-specific membrane antigen in staging of clinically localized prostatic carcinoma: comparison with clinical, surgical and histological staging

OBJECTIVE: To evaluate the reliability of prostate scintigraphy using a radiolabelled antibody (MUJ591) raised against the external domain of prostate-specific membrane antigen (PSMA) in the staging of early prostate cancer. PATIENTS AND METHODS: This was a prospective study of 16 patients who had radical retropubic prostatectomies (median PSA 9.75 ng/mL). All patients underwent PSMA imaging using MUJ591 radiolabelled with (99m)Tc using a photo-reduction technique. RESULTS: The findings of prostate imaging and histology were identical in seven patients. Scans showed understaging and overstaging in six and three patients, respectively. CONCLUSIONS: PSMA scintigraphy using (99m)Tc-labelled MUJ591 identifies the presence of prostate cancer, but is not sensitive in delineating micro-invasion of the capsule, seminal vesicles or bladder neck. As in other studies it seems to be useful in detecting prostate bed recurrence and distant micrometastasis.     

Effect of carbohydrate moieties on the folate hydrolysis activity of the prostate specific membrane antigen

BACKGROUND: Prostate specific membrane antigen or PSMA has been recognized as one of the important cellular markers for prostate cancer, the expression of which is enhanced many fold in prostate cancer and other tumor neovasculature. PSMA is a type II membrane glycoprotein with a short cytoplasmic N-terminal region, a transmembrane domain, and a 701 amino acid extracellular portion with 10 potential N-linked glycosylation sites. PSMA is a folate hydrolase, which cleaves terminal glutamates from poly- and gamma-glutamated folates; and NAALADase, which hydrolyses alpha-glutamate-linked dipeptide, N-acetyl-aspartyl-glutamate (NAAG) and is a glutamate carboxypeptidase. METHODS: In our study we have used various enzymes or site directed mutagenesis to remove sugar molecules from PSMA protein and studied its folate hydrolase function. We have performed a biochemical characterization of N-linked glycosylation of the various mutant proteins. RESULTS: PSMA protein expressed in different prostate cancer cell lines is differentially glycosylated. Removal of sugar residues either enzymatically or by mutagenesis abolishes the enzyme activity of PSMA protein completely. CONCLUSION: N-linked carbohydrate structures are important for the folate hydrolase function of the protein. Removal of sugars partially or completely causes PSMA to be enzymatically inactive, improperly folded, resulting in increased rate of degradation.     

Prostate specific membrane antigen (PSMA) is a tissue-specific target for adenoviral transduction of prostate cancer in vitro

BACKGROUND: Adenovirus binds to the coxsackievirus and adenovirus receptor (CAR) as a first step in the process of cellular infection. This dependence on CAR potentially limits the use of adenovirus in gene therapy, since CAR is expressed in many tissues of the body, and expression of CAR may be low or lost upon progression of certain tumors. These limitations may be overcome by transductional targeting of adenovirus towards other cell surface molecules. We have evaluated the pantumoral epithelial cell adhesion molecule (EpCAM) and prostate specific membrane antigen (PSMA) as possible targets for adenoviral transduction of prostate cancer cells. METHODS: Bispecific antibodies, constructed as conjugates between an anti-adenovirus fiber knob Fab' fragment and anti-EpCAM or anti-PSMA monoclonal antibodies, were incubated with an eGFP-expressing adenovirus to retarget this vector. A cell panel, that includes two prostate cancer cell lines and four non-prostate control lines, were infected with serial dilutions of the retargeted vector and specificity of infection was determined. RESULTS: Receptor-specific transduction was obtained for both EpCAM and PSMA. PSMA-retargeting was shown to be selective for the prostate cancer cell lines. CONCLUSIONS: PSMA serves as a tissue-specific target for adenoviral vectors and may be applicable for gene therapeutical treatment of prostate cancer.