Active immunotherapy with anti-idiotypic antibody for patients with nasopharyngeal carcinoma (NPC)

Two anti-idiotypic monoclonal antibodies (Ab2), designated 2H4 and 5D3, against two antitumor antibodies Ab1 (FC2 and HNL5) that recognize nasopharyngeal carcinoma (NPC) associated antigen were generated. They could substitute NPC antigen to induce humoral and cellular immune response against NPC cells in syngeneic mice. Nineteen patients with NPC at stage IV were chosen for active immunotherapy. They were treated with aluminum hydroxide-precipitated Ab2 2H4 or 5D3 accompanying radiotherapy. None of the immunization of anti-idiotypic monoclonal antibody (mAb) was associated with toxicity or allergies reactions. Nine patients with radiotherapy alone served as control. Both anti-anti-idiotypic antibodies (Ab3) and anti-NPC antibodies (Ab1') were increased and human anti-mouse Ig antibodies (HAMA) occurred in nineteen patients of the experimental group; whereas the levels of Ab1' did not rise in the control group. Serum IL-2, IFN-gamma, and TNF-alpha levels were increased in most patients in the experimental group, while in the control group, there were no differences of Ab1' and cytokine level between pretherapy and posttherapy. In addition, IL-2 mRNA expression in peripheral blood mononuclear cells (PBMC) of NPC patients was closely related to serum IL-2 (r = +0.8829) by in situ hybridization. Therefore, mouse anti-idiotypic antibodies 2H4 and 5D3 are safe for active immunotherapy and might enhance humoral and/or cellular immunity of NPC patients receiving radiotherapy.     

小细胞肺癌抗独特型疫苗功能的试验研究

目的探讨小细胞肺癌(SCLC)抗独特型抗体3F6和其单链抗体(3F6ScFv)诱导体液和细胞免疫应答的能力,以证明其作为抗SCLC疫苗的可行性。方法3F6和3F6ScFv(Ab2)免疫BALB/c小鼠获得抗血清,以ELISA和Westernblot方法分别检测抗血清中Ab3结合SCLC细胞膜表面特异抗原(NCI H128抗原)的能力,用竞争Westernblot检测Ab3与2F7竞争结合NCI H128抗原的能力。以迟发型超敏反应(DTH反应)和小鼠脾脏淋巴细胞增殖实验检测3F6及3F6ScFv诱发细胞免疫应答的潜能。结果ELISA和Westernblot方法均证明,3F6和3F6ScFv免疫同系小鼠所产生的Ab3能特异的与NCI H128抗原相结合,与对照血清相比,差异有统计学意义(P<0.001),且有很强的与2F7(Ab1)竞争结合靶抗原的能力。在DTH反应中,3F6和3F6ScFv所致小鼠足垫肿胀的程度均明显高于对照组(P<0.001)。小鼠脾脏淋巴细胞增殖反应试验证明,用3F6和3F6ScFv免疫的小鼠脾脏淋巴细胞对靶细胞的再次刺激有明显的增殖反应,与阴性肿瘤细胞对照组和阴性抗体对照组相比,差异有统计学意义(P<0.05)。结论抗独特型抗体3F6和3F6ScFv均有模拟SCLC细胞膜表面特异抗原的能力,成功诱导了相应的体液和细胞免疫应答,可作为抗SCLC疫苗进行深入研究。     

鼻咽癌人源抗独特型单链抗体的制备及筛选

背景与目的:抗独特型抗体作为肿瘤抗原替代物可用于肿瘤治疗,这已在临床试验中得到证实。但由于目前所使用的抗独特型抗体多为鼠源性,用于人体可产生人抗鼠抗体反应,从而影响疗效。本实验拟构建噬菌体人源抗独特型抗体库,并从中筛选出能模拟鼻咽癌相关抗原的β型抗独特型单链抗体scFv(Ab2βscFv),以解决鼠源性抗独特型抗体用于临床所产生的人抗鼠抗体反应。方法:体外致敏并用EB病毒(Epstein-Barrvirus,EBV)转化鼻咽癌患者的外周血单个核细胞(peripheralbloodmononuclearcell,PBMC),用RT-PCR分别扩增VH和VL基因并连接成scFv基因,将scFv基因与载体fUSE5连接后,转化大肠杆菌MC1061,构建噬菌体呈现型scFv库。在用单抗FC2对文库进行4轮筛选后,用SandwichELISA和结合抑制法从中筛选出β型Ab2scFv。结果:用单抗FC2体外致敏并经EBV转化的10例鼻咽癌患者的PBMC中,8例有鼻咽癌抗独特型抗体产生。经PCR分别扩增出5种VH(γ、μ)和7种VL(κ、λ)基因,经连接组成14种scFv基因。在与载体连接后,导入大肠杆菌MC1061,得到库容为1.5×108的初级噬菌体抗独特型抗体库。经富集筛选后,从中随机挑取270个克隆进行ELISA筛选,得到91个Ab2scFv单克隆,阳性率为33.7%。再用结合抑制法从中初步筛选出5个可能为β型的Ab2scFv。结论:联     

Monoclonal anti-idiotypic antibody functionally mimics the human gastrointestinal carcinoma epitope GA733

Anti-idiotypic antibodies (Ab2) that bind to the antigen-combining region of anti-tumor antibodies (Ab1) may functionally, and even structurally, mimic tumor antigen. We have previously demonstrated that polyclonal goat Ab2 directed against anti-human gastrointestinal carcinoma Ab1 GA733 induces anti-anti-idiotypic antibodies (Ab3) in animals that are Ab1-like in their binding specificity and idiotope expression. To obtain more defined Ab2 vaccines with potentially increased specificity and efficacy, a monoclonal Ab2 (FG1) was produced against Ab1 GA733 in rats. The monoclonal Ab2 FG1, similar to the polyclonal Ab2 described previously, induced Ab3 in rabbits that were Ab1-like in their idiotope expression and binding specificity to tumor cells and antigen. Antigen-specific Ab3 induced by Ab2 FG1 were easily detected in unprocessed rabbit sera, whereas the demonstration of such Ab3 after polyclonal Ab2 immunization required purification of the Ab3 from the rabbit sera. In addition, Ab2 FG1 induced antigen-specific humoral and cellular immunity in mice. Murine Ab3 bound specifically to antigen-positive tumor cells. Ab2-immunized mice showed antigen-specific delayed-type hypersensitivity (DTH) reaction, and cultured splenocytes from the immune mice demonstrated specific proliferation and cytokine (interferon-γ and interleukin-4) secretion upon stimulation with GA733 antigen. However, immune mice were not protected against a challenge with syngeneic GA733 antigen-expressing colon carcinoma cells. © 1996 Wiley-Liss, Inc.     

Murine monoclonal anti-idiotypic antibody as a surrogate antigen for human Her-2/neu

The anti-idiotypic (Id) monoclonal antibody (MAb) 520C9-6b (IgG1k), raised in syngenic mice against the murine anti-Her2/neu MAb 520C9 (Ab1), functionally mimics a human Her2/neu epitope and serves as a surrogate for the protein antigen. Immunization of allogeneic C57BL/6 mice and rabbits with 520C9-6b (Ab2) induced anti-Her2/neu-specific antibodies that react with antigen-positive SKBr3 cells by ELISA and FACS analysis. The immune sera inhibited binding between Ab1 and Ab2 and vice versa (binding of Ab2 to Ab1), indicating that it was a true anti-anti-Id (Ab3) antibody. The Ab3 sera or purified Ab3 specifically lysed Her2/neu-positive SKBr3 cells, but no significant lysis was observed in antigen-negative LS174T cells in an antibody-dependent cellular cytotoxicity assay. An Id-specific cellular immune response was also demonstrated in an in vitro lymphocyte proliferation assay. Furthermore, a panel of tumor tissues and tumor cells was screened for the presence of the Her2/neu epitope by its reactivity with Ab1 and Ab3 using immunohistochemistry and FACS analysis. Identical results were obtained using either Ab1 or Ab3 (Ab1'). The data indicated that anti-Id 520C9-6b can induce Her2/neu-specific antibody in experimental animals and may serve as a potential network antigen for the treatment of patients bearing Her2/neu-positive tumors.     

The Epstein-Barr Virus (EBV) major envelope glycoprotein gp350/220-specific antibody reactivities in the sera of patients with different EBV-associated diseases

gp350 of Epstein-Barr virus (EBV) induces a strong immune response in EBV-infected individuals, but relatively little is known about the clinical relevance of this response in patients with different EBV-associated malignancies and other diseases. Using our gp350-expressing cell clones, we studied gp350-specific humoral immune responses in the sera of individuals with nasopharyngeal carcinoma (NPC), chronic symptomatic EBV infection (CEI), Hodgkin's disease (HD), acute infectious mononucleosis (IM) and healthy EBV-seropositive individuals (HI). The titres of antibody-dependent cellular cytotoxicity (ADCC) antibodies were highest in HI followed by CEI, HD and NPC. EBV-neutralizing (NA) and gp350-specific IgG antibody profiles in these conditions were: CEI > HI > NPC > HD, whereas IgA titres were the highest in NPC sera followed by CEI and HD. The sera from IM patients were found to be negative for gp350-specific ADCC and IgA activities. Sera from HI were also negative for gp350-specific IgA. A significant positive correlation was found between serum gp350 IgA and viral capsid antigen IgA and a significant negative one between IgM and ADCC titres. High IgA titres were also found in CEI and EBV-genome positive HD in addition to NPC. Importantly, gp350-specific IgA titres were of prognostic value in NPC patients. Our data provide new insights about the clinical relevance of gp350-specific immune responses in these diseases. Int. J. Cancer (Pred. Oncol.) 79:481–486, 1998.© 1998 Wiley-Liss, Inc.     

Anti-idiotype vaccine against Hodgkin's lymphoma: induction of B- and T-cell immunity across species barriers against CD30 antigen by murine monoclonal internal image antibodies.

Murine monoclonal antibody HRS-4 (Ab1), which defines the cell-bound and soluble CD30 antigen associated with Hodgkin's lymphoma, was used to generate monoclonal anti-idiotypic antibodies (Ab2) in syngeneic BALB/c mice. Murine monoclonal Ab2 14G9 and Ab2 9G10 directed against HRS-4 were shown to be anti-idiotypic Ab2 beta carrying the internal image of the CD30 antigen. These antibodies bound specifically to HRS-4 and effectively inhibited binding of HRS-4 to a CD30 antigen preparation at concentrations as low as 50 ng/ml. KLH-coupled Ab2 beta 14G9 and 9G10 induced in BALB/c mice and New Zealand white rabbits a specific polyclonal humoral response against the 120 kDa band of the CD30 antigen. Moreover, BALB/c mice immunized i.p. with KLH-coupled 14G9 and 9G10 exhibited a statistically significant (p less than 0.01) delayed-type hypersensitivity reaction against CD30 expressing Hodgkin-derived L540-cells. We conclude from these data that Ab2 beta 14G9 and 9G10, mimicking structures of the nominal CD30 antigen, are capable of inducing a CD30-specific T-cell- and B-cell-mediated immune response in mice and even across species barriers in rabbits. These CD30 anti-id antibodies may hold promise for use as vaccines against CD30-antigen-expressing lymphomas.     

鼻咽癌患者血清抗体相关抗原优势表位的筛选

背景与目的：EB病毒与鼻咽癌(nasopharyngeal carcinoma,NPC)密切相关。可应用多种血清学方法检测鼻咽癌患者血清EBV抗体,本研究利用鼻咽癌患者血清中纯化的EB病毒相关多抗,从噬菌体展示的随机肽库中筛选相关的优势抗原表位,在表位水平为鼻咽癌患者血清抗体的检测寻找新的检测抗原。方法：以B95-8细胞EBV蛋白为抗原,从鼻咽癌患者血清中洗脱EB病毒相关多抗,对噬菌体展示的随机12肽库进行三轮生物淘洗。用夹心ELISA法和竞争抑制实验筛选阳性克隆,对这些阳性克隆进行测序,并将其展示的外源多肽与EB病毒蛋白的抗原性区域进行同源序列比对分析。结果：从第三轮的淘洗洗脱液中随机挑取64个噬菌体克隆,用夹心ELISA法筛选到25个阳性克隆,阳性率为39．06％,选取其中13个克隆进行竞争ELISA实验,有11个克隆出现抑制现象,抑制率在18．09％～65．94％之问。选取吸光度(A)值和抑制率均较高的5个克隆为阳性克隆,它们所展示的外源多肽序列分别为-A-T-S-H-L-H-V-R-L-P-W-T-(d15／d18)、-G-S-T-H-K-H-N-H-F-N-K-T-(d19)、-K-P-I-H-E-H-P-H-R-F-K-S-(e8)、-H-T-H-K一1-K-I-P-LP-I-Q-(e23)。这些多肽序列分别与EB病毒膜蛋白(d15／d18)、EB病毒胸苷激酶(d19)、EB病毒主要壳蛋白(e8,e23)抗原区域的氨基酸存在序列相似性。结论：利用鼻咽癌患者血清中的特异多抗可以从噬菌体展示的随机肽库中筛选到EB病毒相关抗原表位。     

Isolation and characterisation of a human anti-idiotypic scFv used as a surrogate tumour antigen to elicit an anti-HER-2/neu humoral response in mice

HER-2/neu is a tumour antigen that is overexpressed in human breast tumours. Among the vaccine strategies developed to overcome immune tolerance to self-proteins, vaccination with anti-idiotypic (anti-Id) antibodies has been described as a promising approach for treatment of several malignant diseases. To develop an active immunotherapy for cancer patients positive for HER-2/neu, we investigated immunisation with human anti-Id single-chain fragments (scFv) mimicking the conformation of HER-2/neu protein to induce a humoral response in mice. We selected by phage display two human anti-Id scFv (Ab2beta) directed against trastuzumab F(ab')2 fragments (Ab1), a humanised anti-HER-2/neu monoclonal antibody. Using competitive ELISA and Biacore biosensor analysis, we showed that anti-Id scFv 40 and scFv 69 could inhibit HER-2/neu binding to trastuzumab. Following vaccination of BALB/c mice with the soluble or phage-displayed scFv, Ab3 polyclonal antibodies, and among them Ab1' antibodies able to bind HER-2/neu, were detected in the sera of the immunised mice. These results demonstrate that the human anti-Id scFv could act as a surrogate antigen for HER-2/neu. The present study strongly suggests that the novel 30 kDa human mini-antibody could be used as an anti-idiotype-based vaccine formulation to induce an effective humoral response in patients bearing HER-2/neu-positive tumours.     

Construction and selection of human anti-idiotypic antibody single chain variable fragments or CDR3 fragments of nasopharyngeal carcinoma

Peripheral blood mononuclear cells (PBMCs) of patients with NPC were immunized in vitro by anti-NPC monoclonal antibody FC2 and transformed by Epstein-Barr virus (EBV). Detection showed that of 10 NPC patients, 8 patients' B cells immunized by FC2 and transformed by EBV produced anti-idiotypic antibodies to NPC. Five types of VH genes and 7 types of VL genes were obtained by RT-PCR amplification and then connected with (Gly4Ser)3 linker to form 14 types of scFv genes. ScFv genes digested with Sfi I were cloned into vector fUSE5 and transformed into E. coli MC 1061. Phage anti-idiotypic antibody library with 1.5 x 10(8) clones was obtained. After four rounds of panning, 270 phage clones were selected randomly and 91 FC2-positive clones were obtained by Sandwich ELISA, the positive ratio was 33.7%. 5 clones (D83, E92, G22, I50, I54), which might display beta type Ab2 scFv, were selected by binding inhibition test. These 5 phage anti-idiotypic antibodies were further analyzed by DNA sequencing. The VDJ regions of G22, I50, I54 belonged to VH4-39-D4-11-JH3-linker-V1-19-JL2, VH4-4-D4-11-JH6 and VH4-31-D4-11-JH6, respectively. E92 had the same VDJ regions with G22; D83 had the same VDJ regions with 150. So, a strategy for preparing and selecting beta type Ab2 scFv or CDR by means of immunization in vitro, EBV transformation and phage display technique is feasible, which paves a way for preparing cancer vaccine using beta type Ab2 scFv.     

Development of an enzyme-linked immunosorbent assay (ELISA) for detecting IgA antibodies to the Epstein-Barr virus

A 3-step enzyme-linked immunosorbent assay (ELISA) was developed for detecting IgA antibodies to purified Epstein-Barr virus (EBV) polypeptides. The 3-step procedure included the use of a mouse anti-human IgA monoclonal antibody (MAb) to amplify the IgA reaction. The 2 major EBV proteins used in this assay were the 125-kDa component (gp125) associated with the viral capsid antigen (VCA) complex and a major glycoprotein (gp250/200) associated with the membrane antigen (MA) complex. Eighty-two sera were tested on ELISA plates containing either both of the glycoproteins or each one separately. These included 45 IgA antibody-positive sera from patients with nasopharyngeal carcinoma (NPC). With these sera, there was a good correlation, both qualitatively and quantitatively, between results with the immunofluorescence (IF) and ELISA procedures. Although most IgA antibody-positive sera contained antibodies reactive with both gp125 and gp250/200, a number of sera contained antibodies reactive with one of the glycoproteins but not with both. The data indicated that both of these glycoproteins should be used in assays for detecting IgA antibodies to EBV, to avoid false-negative results. This assay should be useful for screening large populations for IgA antibodies to EBV and also possibly for monitoring disease course in patients with NPC.     

Serum and salivary IgA antibodies against a defined epitope of the Epstein-Barr virus nuclear antigen (EBNA) are elevated in nasopharyngeal carcinoma.

The Epstein-Barr virus nuclear antigen I (EBNA I) is the only latent EBV antigen consistently expressed in malignant tissues of the nasopharynx. A 20-amino-acid synthetic peptide, p107 contains a major epitope of EBNA I. We tested sera from 210 patients with nasopharyngeal carcinoma (NPC) and from 128 normal individuals (NHS) for IgA antibodies to p107 using an enzyme-linked immunosorbent assay (ELISA). Whereas 191/210 (91%) of NPC patients had IgA antibodies to p107, only 17/128 (13.3%) of NHS had such antibodies and only 6/57 (10.5%) of sera from patients with malignancies other than NPC had IgA-p107 reactivity. Thirty-nine salivary samples from 46 NPC patients (84.8%) also contained IgA-p107 antibodies whereas only 3/42 (7.1%) of normal saliva samples were IgA-p107 positive. The results suggest that IgA antibodies to EBNA I may become a useful, easily measurable, marker for NPC.     

IL-23促进人肺腺癌A549细胞的迁移和侵袭

背景与目的前炎症因子白细胞介素23(interleukin 23,IL-23)是与慢性炎症和肿瘤微环境相关的一种重要的细胞因子,同时IL-23受体在结直肠癌、肺癌、口腔鳞癌等肿瘤细胞中有表达。本研究旨在探讨IL-23能否促进人肺腺癌细胞A549的迁移和侵袭并探讨其机制。方法用划痕试验和Transwell小室法测定IL-23对A549的迁移和侵袭的影响,用Real-time PCR和ELISA检测IL-23对基质金属蛋白酶9(matrix metalloproteinase 9,MMP-9)的mRNA和蛋白表达的影响,通过IL-23中和抗体阻断IL-23的作用,进一步证实IL-23对A549迁移和侵袭的影响。结果 IL-23明显增加了A549细胞的迁移和侵袭能力;同时IL-23能提高A549细胞MMP-9的mRNA表达和其培养上清中MMP-9的蛋白表达,IL-23中和抗体能有效地阻断IL-23对A549的迁移和侵袭的作用。结论 IL-23可刺激A549细胞表达MMP-9,从而促进A549细胞的迁移和侵袭。     

Induction of interleukin-2, natural killer cell activity and anti-melanoma antibodies resulting from immunization of mice with formalinized extracellular antigens (FECA) of murine melanoma.

Extracellular products from melanoma cells may play an important role in the pathogenesis of metastatic melanoma. Studies were designed to evaluate the effect of vaccination with formalinized extracellular antigens (FECA) of murine melanoma cells (MMM B16-F10) on survival and immune response of C57BL/6 mice. The cellular immune response was evaluated by assessing interleukin-2 (IL-2) production and natural killer cell activity, whereas the humoral immune response was examined by measuring the production of specific antibodies to extracellular antigens (ECA). IL-2 production by the splenocytes from immunized animals was significantly higher (4.7 U/ml and 3.7 U/ml) than that of controls (1.38 U/ml). The splenocytes from immunized mice revealed significantly higher natural killer cell activity. Similarly, immunized animals responded by producing specific antibodies against the extracellular melanoma antigens as detected by ELISA. The peak production of antibodies against ECA was observed on the 21st day post-immunization. These results suggest that FECA are immunogenic and may enhance active cellular and humoral anti-melanoma immunity.     

Tumor-antigen-specific humoral immune response of animals to anti-idiotypic antibodies and comparative serological analysis of patients with small-cell lung carcinoma.

We have previously developed 3 monoclonal anti-idiotypic antibodies (Ab2) of LOU rat origin directed against the binding site of the murine monoclonal IgM LAM8, which recognizes the small-cell lung carcinoma (SCLC)-associated sialoglycoprotein antigen sGP 90-135. The aim of this study was to compare the efficiencies of these 3 Ab2, designated LY8-229, LX8-531 and LX8-632, to induce antigen-specific immunity in different animal species without prior exposure of the recipients to the nominal antigen, and thereby possibly select an Ab2 candidate for active immunotherapy against SCLC in patients. The feasibility of this approach was further evaluated by a serological analysis of patients with SCLC compared with healthy individuals, in whom the spontaneous antibody reactivities against SCLC cell lines and Ab2 were tested. LY8-229 was shown to be the most effective Ab2 in inducing antigen-specific antibodies in BALB/c mice, CBA/J/Zur mice and one NZW rabbit. Furthermore, LY8-229 was the only Ab2 against which significantly elevated idiotype-specific antibody reactivities existed in sera of patients with SCLC. These reactivities correlated positively with binding to antigen-positive tumor cells. Our findings suggest that LY8-229 represents in its reactivity pattern the nominal SCLC antigen in humans also, and therefore may be of diagnostic and possibly therapeutic relevance for patients with SCLC.     

Intrasplenic vaccination against experimental melanoma

The immunodominant component of a formalinized extracellular antigen (fECA) vaccine prepared from B16 F10 melanoma cells is the melanoma-associated antigen B700. We now demonstrate that a single prophylactic intrasplenic inoculation of B700 antigen (1-10 mu g) stimulates the production of antibodies which have antiproliferative effects on B16 F10 melanoma cells in vitro. In addition, potential cytotoxic effects of splenocytes from B700 antigen inoculated mice were evaluated for two cellular immune effector functions, natural killer (NK) cell activity and lymphokine activated killer (LAK) cell activity; both activities were increased following B700 antigen inoculation. Intrasplenic injection of B700 antigen elicited an increase in the expression of the CD25 surface antigen (IL-2 R alpha) by T lymphocytes and up-regulated the expression of IL-2 R alpha mRNA. Thus both humoral and cellular cytotoxic immune responses might play roles in the decreased growth of primary tumors in B700 antigen inoculated mice and in the higher survival rate in this group of animals.     

Interleukin-10 is a growth factor for human melanoma cells and down-regulates HLA class-I,HLA class-II and ICAM-1 molecules

IL-10 is a cytokine which shows various effects including inhibition of T-cell proliferation or HLA-dependent antigen presentation. In this study, we analysed the effects of exogenous or autocrine IL-10 on proliferation and expression of immunocritical surface molecules. Fourteen cultures of human melanoma cells were established from primary melanomas, locoregional lymph-node or distant metastases. In 5 melanoma cell cultures, proliferation in the presence of IL-10, anti-IL-10 antibodies (Ab) or control Ab was assessed with colorimetric and [³H]thymidine uptake assays. Flow cytometric analysis was used to quantify the expression of human leukocyte antigen (HLA) class-I, HLA class-II and intercellular adhesion molecule (ICAM)-I and the IL-10 receptor (IL-10R). IL-10 production of melanoma cells was documented by RT-PCR and IL-10 protein was detected in the supernatants by means of ELISA. IL-10 enhanced proliferation and prolonged survival of melanoma cells in 5 out of 5 cultures. Anti-IL-10 Ab decreased proliferation. IL-10R expression was found in 12 out of 14 (86%) melanoma cell cultures. The expression of HLA-I, HLA-II and ICAM-I on all melanoma cells that were positive for IL-10R showed a reduction of 10-60% by IL-10, whereas the surface levels of HLA-I, HLA-II and ICAM-I in 5 out of 5 cell cultures revealed an increase of 10-170% by anti-IL-10 Ab. These findings suggest that IL-10 is an autocrine growth factor with significant impact on immunocritical molecules in melanoma. IL-10 effects have to be considered when planning therapeutic immunointerventions in melanoma patients. Int. J. Cancer 71:630-637, 1997. © 1997 Wiley-Liss, Inc.     

Immunological response of nasopharyngeal carcinoma patients to the Epstein-Barr-virus-coded thymidine kinase expressed in Escherichia coli

A bacterial expression system which produces large amounts of the Epstein-Barr-virus-coded thymidine kinase has been developed and used to produce protein for Western blot analysis of a number of human antisera. Interestingly, only sera from nasopharyngeal carcinoma (NPC) patients had any detectable IgA antibody which reacted with the EBV TK. These findings provided the basis for ELISA tests using a crude lysate of the E. coli cells expressing the EBV TK as target antigen. Sera from NPC patients showed high levels of IgA reactive antibodies in this test while other sera did not.     

Increase of auto anti-phosphatidylinositol antibodies in plasma of female rats during the appearance of DMBA-induced malignant mammary tumors

Using a lipid-adapted ELISA, antiphosphatidylinositol (PtdIns) antibodies (Ab) have been found in sera of cancer patients. With the same procedure, we have checked their possible raising in plasma of female rats during the appearance of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors. Twenty days after DMBA administration, the mean anti-PtdIns Ab level was already higher than that of control rats (oil). Immunochemical analysis of the anti-PtdIns Ab site showed a rather high Ab avidity (8 x 10(-9) M, at half-displacement) and high specificity for glyceryl phosphate inositol residues. Other phospholipids (PL) were not recognized by the anti-PtdIns Ab induced by malignant cell transformation.     

Network of idiotypic and anti-idiotypic antibodies related to the ovarian carcinoma-associated folate binding protein.

The present paper describes the generation and characterization of anti-idiotypic monoclonal antibodies (Ab2 MAbs) produced against the MOv19 MAb (Ab1 MAb), which shows a restricted reactivity with human ovarian carcinoma. The Ab2 MAbs were produced by immunizing mice with MOv19 conjugated to syngeneic mouse red blood cells (MRBC). After the somatic hybridization, five Ab2 MAbs, designated Id19.1, Id19.2, Id19.3, Id19.4 and Id19.5, were selected by a sandwich assay on the basis of their specific reactivity to the MOv19 MAb, but not to the isotype-matched MAb used as a control. A complete cross-inhibition between these 5 MAbs was observed, indicating that they recognize overlapping idiotopes on MOv19. The Ab2/antigen relationship of two Ab2 MAbs (Id19.1 and Id19.2) was investigated by competition experiments on a relevant tumor target cell line. We showed that both Ab2 MAbs were able to interfere with the antigen binding capacity of 125I-MOv19. Moreover, Id19.1 was able partially to inhibit the binding of the purified radiolabelled antigen recognized by the MOv19 MAb (125I-CaMOv19) on insolubilized MOv19. To investigate further whether the Ab2 MAb is the "internal image" of CaMOv19, Id19.1 was used as immunogen with the aim to induce an anti-CaMOv19 response. The antisera tested by indirect solid-phase radioimmunoassay (RIA) on the Ab2 MAb and the irrelevant isotype-matched control MAb revealed an anti-anti-idiotypic response (Ab3). However, no Ab3 antibodies with Ab1-like specificity (Ab1') were found in the immune sera, as assessed by testing the antisera both in indirect immunofluorescence (IF) and solid-phase RIA on CaMOv19-positive target cell lines.