PRODUCTION OF PHAGE-DISPLAYED ANTI-IDIOTYPIC ANTIBODY SINGLE CHAIN VARIABLE FRAGMENTS TO MG7 MONOCLONAL ANTIBODY DIRECTED AGAINST GASTRIC CARCINOMA

Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti - Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer.Methods. Balb/c mice were immunized i. p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL) genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TGI. The transformants were infected with M13K07 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA.Results. The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed β or γ type anti-Id ScFv.Conclusion. The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.

Isolation and Characterization of Recombinant Variable Domain of Heavy Chain Anti-idiotypic Antibodies Specific to Aflatoxin B_1

Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain （VHH）. The recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. In particular, they might be very useful for molecular mimicry. The present study demonstrated an alpaca immunized with the F（ab＇）z fragment of anti-aflatoxin B1 mAb and developed an important anti-idiotypic （anti-ld） responses. Antigen-specific elution method was used for panning private anti-ld VHHs from the constructed alpaca VHH library. The selected VHHs were expressed, renatured, purified, and then identified by a competitive enzyme-linked immunosorbent assay （ELISA）. Our findings indicated that the VHH would be an alternative tool for haptens mimicry studies.

BASIC STUDY AND PHASE I CLINIC TRIAL OF ANTI-IDIOTYPIC ANTIBODY MIMICKING NASOPHARYNGEAL CARCINOMA CELL ANTIGEN

An anti-idiotypic monoclonal antibody 2A9 (Ab2)was prepared, which mimicked the nasopharynegealcarcinoma (NPC) cell antigen defined by anti-NPC McAbFel. The abilities of 2A9's inducing humoral and cellularimmunity against NPC cell antigen were studied insyngeneic mice by inducing anti-Ab2 sera (Ab3) anddelayed-type hypersensitivity. Two periods of phasc Ⅰclinical trials were carried out, stage Ⅳ NPC patientsreceiving radiotherapy were chosen. Human anti-mouseantibodies (HAMA), anti-anti-idiotypic antibodies (Ab3),and anti-NPC cell antibodies (Ab1') were detected byELISA. TNF-α,IL-2, IFN-γ levels in sera were determinedby ELISA Kits. IL-2 mRNA expression in peripheralblood mononuclear cells (PBMC) were shown by in situhybridization. The results showed that 2A9 was safe inapplying on NPC patients and induced some humoraland/or cellular immunc responses.

PREPARATION OF ANTI-IDIOTYPIC ANTIBODIES SPECIFIC FOR ANTI-HEL AND ANALYSIS OF THEIR FUNCTIONAL MIMICRY

Objective. This study is to investigate the functional mimicry by using antiidiotypic antibodies of enzymes. Methods.Monoclonal antiidiotypic antibodies against antiHEL(hen eggwhite lysozyme, HEL) antibodies were obtained by fusion of Sp2/0 myeloma cells with spleen cells of syngeneic mice immunized with monoclonal antiHEL antibodies against HELs different antigenic epitopes. Then bacteriolysis of the antiidiotypic antibodies were observed. Results.Eight hybridomas strains secreting antiidiotypic antibodies were selected and characterized. It was shown that two of eight antiidiotypic antibodies secreted by two hybridomas(1A 10 C 9 and 2A 11 C 1B 3) could mimic HEL catalytic activity to lyse Micrococcus lysodeikticus and that the catalytic effect of mixed antiidiotypic antibodies of 1A 10 C 9 and 2A 11 C 1B 3 was stronger than that of one of them, but less than HEL. Conclusion. The results demonstrated that the antiidiotypic antibodies that could mimic enzyme activity existed in the idiotype network during antienzymatic immune response.

抗HBsAg和抗RBC双特异minibody载体的构建及表达

目的 :用抗HBsAg和抗RBC单链抗体构建双特异minibody抗体模型。方法 :将人IgG1CH3 经点突变方式 ,在CH3界面由大分子氨基酸代替小分子氨基酸形成“knob”(T36 6W) (杵状结构 ) ,另一个CH3 分子由 3个小分子氨基酸代替 3个大分子氨基酸形成“hole”(T36 6S :L36 8A :Y40 7V) (臼状结构 ) ,并在此基础上在适当位置引入半胱氨酸残基形成二硫键 (S35 4C :T36 6W /Y34 9C :T36 6S :L36 8A :Y40 7V)。然后将“knob”和“hole”分别接连于抗HBsAg和抗RBC单链抗体基因 3’端 ,并将两基因组装于同一表达载体中 ,在大肠杆菌中分泌表达。结果 :依CH3 界面不同共构建 3种不同的表达载体 ,分别带有①野生型CH3 ,②杵臼结构 (T36 6W/T36 6S :L36 8A :Y40 7V)突变型CH3 ,③杵臼结构加二硫键 (S35 4C :T36 6W/Y34 9C :T36 6S :L36 8A :Y40 7V)突变型CH3 。大肠杆菌分泌表达后 ,ELISA法检测抗HBsAg和抗RBC活性 ,3种表达上清均有相同的抗HBsAg和抗RBC活性 ;两种带有突变型CH3 的minibody用血球凝集法可检测到抗HBsAg和抗RBC双特异活性。结论 :CH3 界面经适当改造可促进异源二聚体形成 ,并在大肠杆菌获得功能性表达 ,得到双特异minibody。

Enzyme-linked Immunosorbent Assay for Detection of Anti-idiotype Antibodies to Antibodies to Ligand of Nicotinic Acetylcholine Receptor in Sera of Patients with Myasthenia Gravis

Anti-bungarotoxin anti-serum,which has the internal image of nicotinicacetylcholine receptor,was used as a tool to measure anti-idiotypic antibodies toantibodies to Iigand of nicotinic acctylcholine receptor in scra from 81 patients withmyasthenia gravis.Enzyme-linked immunosorbcnt assay was adopted.Thc positive ratewas 46.9%(38/81).The specific cross inhibitory test with nicotinic acetylcholinereceptor was positive.Anti-idiotype antibodies to antibodies to ligand of nicotinicacetylcholine receptor in sera of different types of myasthenia gravis patients classified ac-cording to modified Osserman’s standard and myasthenia gravis patients with or withoutthymoma were comparcd in this study and the role of anti-idiotype antibodies toantibodies to Iigand of nicotinic acctylcholinc receptor in the immunity of myasthcniagravis and the possibility of thcrapeutic use of anti-idiotype antibodies arc discussed.

卵巢癌抗独特型微抗体主动免疫治疗卵巢癌动物实验

目的用模拟人卵巢癌抗原并有满意免疫原性的抗独特型微抗体进行临床前动物实验研究。方法将已构建的抗独特型微抗体融合基因在大肠杆菌进行表达,用Westernblot、竞争抑制ELISA进行微抗体活性测定。20只人淋巴细胞免疫功能重建的荷卵巢癌腹腔瘤SCID小鼠分2组,10只用微抗体免疫后取血采用间接ELISA检测Ab3。观察小鼠腹腔积液生成时间、生存期,取脾做流式细胞分析CD4+、CD8+。结果在宿主大肠杆菌BL21(DE3)成功诱导表达出微抗体,Westernblot结果表明微抗体可同时与COC166-9(6B11的一抗)及羊抗人的IgG1反应。竞争抑制ELISA表明微抗体可模拟原始抗原与一抗结合。微抗体免疫小鼠后可诱导产生Ab3,在末次免疫14d时最高,持续6周降至对照组水平;CD4+/CD8+在末次免疫13d达最高值。对照组和微抗体治疗组小鼠分别在(37.7±5.5)d和(48.6±14.3)d长出血性腹腔积液(P=0.04);两组小鼠生存期分别为(42.5±1.8)d和(59.4±16.8)d(P=0.011)。结论微抗体保留了6B11scFv和人IgG的双重免疫学活性,达到了部分人源化的目的,并有满意的免疫原性,可诱导小鼠产生特异性体液免疫反应,抑制荷瘤小鼠腹腔积液的生成,延长生存期,或许将来可作为卵巢癌疫苗用于临床。

卵巢癌抗独特型微抗体疫苗诱导BALB/c小鼠体内抗肿瘤免疫应答的实验研究

背景与目的6B11抗独特型微抗体由模拟人卵巢癌抗原的抗独特型单链抗体(6B11ScFv)融合人IgG1铰链区和CH3区所构成,它具有6B11ScFv和人IgGFc的双重免疫学活性。本研究观察6B11抗独特型微抗体在BALB/c小鼠体内诱导抗肿瘤免疫反应情况,探讨其作为卵巢癌疫苗的可能性。方法用卵巢癌6B11抗独特型微抗体免疫BALB/c小鼠。采用间接ELISA、竞争抑制实验和流式细胞术检测免疫鼠血清。结果6B11抗独特型微抗体免疫小鼠后,在不用佐剂的情况下可诱导小鼠产生较高的Ab3,末次免疫后30天仍持续在较高的水平。分别在末次免疫后4天、14天、24天和30天可刺激小鼠脾脏淋巴细胞CD4+T细胞和CD8+T细胞明显升高。结论6B11抗独特型微抗体可诱导机体产生特异体液免疫和细胞免疫反应,这为抗独特型微抗体疫苗的临床应用提供了一定的实验依据。

卵巢癌特异性免疫细胞治疗的体内外实验研究

背景与目的:卵巢癌抗独特型微抗体(6B11mini)是部分人源化的抗独特型卵巢癌疫苗,体内外实验研究证明,它可以诱导出特异的抗卵巢癌体液免疫和细胞免疫。本研究评价将6B11mini作为抗原,采用免疫细胞的方法处理卵巢癌时的安全性和有效性。方法:用纯化的6B11mini负载树突细胞(dendritic cell,DC),与外周血单个核细胞(peripheral blood mononucleocyte,PBMNC)混合培养,在多种细胞因子共同作用下获得效应细胞6B11-OCIK。通过软琼脂克隆形成实验及接种裸鼠皮下瘤实验,观察6B11-OCIK在体内和体外的成瘤能力;将6B11-OCIK静脉注射BALB/c小鼠,观察急性毒性反应;体外Cr释放实验检测6B11-OCIK对靶细胞的杀伤作用。51用人卵巢癌细胞株SKOV3构建严重联合免疫缺陷(severe combined immune deficieney,SCID)小鼠卵巢癌移植瘤模型,注射6B11-OCIK,并以CIK、PBMNC细胞以及生理盐水作为对照组,分别观察各组肿瘤生长情况。结果:软琼脂培养14d,卵巢癌SKOV3细胞克隆形成良好,克隆形成率50%;皮下接种裸鼠后14d,阳性对照的宫颈癌HeLa细胞组全部成瘤,6B11-OCIK、CIK、WI-38组和新鲜PBMNC组持续观察13周没有成瘤。6B11-OCIK静脉注射BALB/c小鼠,30min内各剂量实验组和生理盐水对照组动物都无任何明显的不良反应;输注后第13天处死小鼠,解剖小鼠观察其各主要脏器无明显异常。在体外杀伤实验中6B11-OCIK对抗原阳性的肿瘤细胞具有特异性杀伤作用,并且与MHC限制性相关;在荷瘤SCID小鼠中6B11-OCIK治疗组肿瘤重量与生理盐水对照组相比差异有统计学意义(P=0.023),与CIK组、新鲜单采组相比差异无统计学意义(P=0.540,P=0.285)。结论:6B11-OCIK在动物体内应用时符合安全性指标,并对卵巢癌的生长有一定的抑制作用。

原核表达系统pET28a(＋)中抗CD20微抗体的构建和表达

目的:改造Rituximab,构建包含VL、VH和CH3的抗CD20微抗体minibody,并探索诱导其可溶性表达的最佳方法.方法:首先通过overlap PCR将Rituximab的VL和VH通过Linker连接在一起,成为单链抗体ScFv,再将ScFv和人源IgG1 CH3通过人源IgG1铰链区连成minibody的cDNA.将其克隆至表达载体pET28a(+)中,并在大肠杆菌中用IPTG诱导表达.结果:SDS-PAGE和Western blot结果表明,抗CD20的微抗体基因表达产物分子量约41.88 kDa,在20℃、1.0 mmol/L IPTG诱导24 h时minibody的可溶性表达量最大,同时还有包涵体形式的蛋白.可溶性抗体蛋白通过镍柱纯化,纯度达到90.25%,包涵体经过变、复性获得了高纯度的抗体蛋白.重组蛋白minibody可特异性地被兔抗人IgG1单克隆抗体识别,并且可特异性结合靶抗原CD20.结论:抗CD20微抗体minibody基因在此原核表达系统中成功表达,为其生物学功能和更好地针对B淋巴系统恶性肿瘤的靶向治疗奠定了基础.

A clinical trial of active immunotherapy with anti-idiotypic vaccine in nasopharyngeal carcinoma patients

OBJECTIVE: To investigate the effect of active immunotherapy with anti-idiotypic vaccine in patients with nasopharyngeal carcinoma (NPC). METHODS: Anti-idiotypic antibodies (2H4/5D3) bearing the internal image of the NPC antigen were used in active immunotherapy in NPC patients receiving radiotherapy. Antibodies and cytokine levels in patient sera were determined using ELISA before and after active immunotherapy. IL-2 mRNA expression in the peripheral blood mononuclear cells (PBMC) was measured by in situ hybridization. RESULTS: Nineteen patients with NPC at stage IV were treated with alum-precipitated 2H4 or 5D3. Neither hypersensitivity nor adverse side effects were observed. The levels of anti-anti-idiotypic antibodies (Ab3) and anti-NPC antibodies (Ab1') were increased. Human anti-mouse antibodies (HAMA) were seen in 19 patients of the experimental group; the levels of Ab1' did not increase in the control group. Serum IL-2, IFN-gamma and TNF-alpha levels were increased in most patients in the experimental group, while no differences were observed in Ab1' and cytokine levels between pre- and post-therapy in the control group. In addition, IL-2 mRNA expression in PBMCs from NPC patients was closely related to serum IL-2 (r = + 0.8829) levels by in situ hybridization. CONCLUSIONS: Anti-idiotype vaccine is safe for clinical active immunotherapy. Anti-idiotypic vaccine might be able to enhance humoral and/or cellular immunity in NPC patients receiving radiotherapy.

Preparation of Anti-Idiotypic Antibody against Avian Influenza Virus Subtype H9

To generate monoclonal anti-idiotypic antibodies(mAb2)against avian influenza virus subtype H9(H9 AⅣ), BALB/c mice were immunized with purified chicken anti-H9-AⅣ IgG and the splenocytes of immunized mice were fused with myeloma cells NS-1.Hybridoma cells were screened by indirect enzyme-linked immunosorbent assays with both chicken and rabbit anti-H9-AⅣ IgG as coating antigens.One hybridoma cell clone secreting monoclonal antibody against idiotypes shared by both chicken and rabbit anti-H9-AⅣ IgG was established.Experiments demonstrated the mAb2 was able to inhibit the binding of hemagglutinin to anti-H9-AⅣ IgG and to induce chickens to generate hemagglutination inhibition antibodies,indicating this anti-species-sharing-idiotypic antibody bore the internal image of hemagglutinin on avian influenza virus.Cellular & Molecular Immunology.2005;2(2): 155-157.

ANTI-IDIOTYPIC MONOCLONAL ANTIBODIES AGAINST ANTI-OVARIAN CARCINOMA MONOCLONAL ANTIBODY COC166-9 GENERATION AND APPLICATION

Anri-idiotypic monoclonal antibody (Mab Ab2 ) by MAb COC166-9 against ovarian serous papillary adenocarcinoma was prepared. Hybridomas of Ab2 screened by sandwich ELISA and immunocompetitive inhibition tests were procured and named as 6B11 and 1H12. The number of their chromosomes were 93 and 91, and DNA analysis also proved the characteristics of hybridomas. These Ab2s could induce delayed type hypersensitivity (DTH), the cellular immune response. The results of the immune reaction of 6B11 with SKOV3 (ovarian carcinoma cell line) were similar to OC166-9 (Ag), the positive control, while 1H12 was weaker. Anti-and-idiotypic antibody (Ab3) was also raised by 6B11 and 1H12 respectively. They all showed positive immunohistochemical stainings with ovarian serous adenocarcinoma tissue sections and immunocytochemical stainings with SKOV3 cells as was shown by COC166-9. In the antibody dependent cell mediated cytotoxicity (ADCC) tests, they showed no differences against SKOV3 as compared with COC166-9. We anticipate that 6B11 and 1H12 may be used as vaccines against ovarian carcinoma and may provide a clue for its prevention and treatment.

Humoral immune responses induced by anti-idiotypic antibody fusion protein of 6B11scFv/hGM-CSF in BALB/c mice

Background We have previously developed and characterized a monoclonal anti-idiotype antibody, designated 6B 11, which mimics an ovarian carcinoma associated antigen OC166-9 and whose corresponding monoclonal antibody is COC166-9 （Abl）. In this study, we evaluate the humoral immune responses induced by the fusion protein 6B11 single-chain variable fragment （scFv）/human granulocyte macrophage colony-stimulating factor （hGM-CSF） and 6B 1 lscFv in BALB/c mice. Methods The fusion protein 6B 11 scFv/hGM-CSF was constructed by fusing a recombinant single-chain variable fragment of 6B11scFv to GM-CSE BALB/c mice were administrated by 6B11scFv/hGM-CSF and 6B11scFv, respectively. Results The fusion protein 6B11scFv/hGM-CSF retained binding to the anti-mouse F（ab）2＇ and was also biologically active as measured by proliferation of human GM-CSF dependent cell TF1 in vitro. After immunization with the 6B11scFv/hGM-CSF and 6BllScFv, BALB/c mice showed significantly enhanced Ab3 antibody responses to 6B11 scFv/hGM-CSF compared with the 6B11 scFv alone. The level of Ab3 was the highest after the first week and maintained for five weeks after the last immunization. Another booster was given when the Ab3 titer descended, and it would reach to the high level in a week. Conclusion The fusion protein 6B11scFv/hGM-CSF can induce humoral immunity against ovarian carcinoma in vivo. We also provide the theoretical foundation for the application of the fusion protein 6B11 scFv/hGM-CSF for active immunotherapy of ovarian cancer.

Nanoparticles as a vaccine adjuvant of anti-idiotypic antibody against schistosomiasis

Background The development of new adjuvants for human use has been the focus of attention. This study’s aim is to explore the possibility of using nanoparticle Ca nanoparticles (CA) as a vaccine adjuvant of anti-idiotypic antibody NP30 against schistosomiasis and its protective mechanisms. Methods Nanoparticle CA-NP30 conjugate (CA-NP30) was fabricated. BALB/c mice were immunized actively with CA-NP30 to evaluate its effects of protective immunity on mice. The serum levels of specific IgG,IgG1 and IgG2a antibodies against NP30 and the concentrations of IFN-γ and IL-4 in supernatant of splenocytes were determined via ELISA. Results Nanoparticle CA could enhance significantly the protective immunity of NP30 against infection of Schistosoma japonicum and the worm reduction rose from 36.0% (NP30 alone) to 52.6%. The serum levels of specific IgG,IgG1 and IgG2a antibodies against NP30 increased remarkably,as compared with those of the group immunized with NP30 alone. The concentration of IFN-γ in supernatant of splenocyte was drastically elevated [the groups immunized with CA-NP30 and NP30 alone were (493.80±400.74) pg/ml and (39.03±39.58) pg/ml,respectively],but the concentration of IL-4 showed no significant difference from that of NP30 alone [(27.94±9.84) pg/ml vs (27.28±14.44) pg/ml]. Conclusions Nanoparticle CA could act as a vaccine adjuvant of anti-idiotypic antibody NP30 against schistosomiasis. The mechanism could be that CA-NP30 enhances humoral and cellular immune responses in mice.

Induction of Human Anti-Human Antibody Responses (Ab2) after Application of a Humanized Lewis Y Carbohydrate Specific Antibody (Ab1): Connection of Prolonged Disease Stabilization with Ab3 Induction?

Purpose: Detailed analysis of a patient with epithelial Lewis Y (LeY) positive cancer who received twice 50 mg of the humanized Lewis Y carbohydrate specific mAb IGN311 and developed a clinically significant human anti-human antibody (HAHA) response (Ab2). Results: Clinical stabilization of the disease was assigned to in this patient. The HAHA response consisted mainly of IgG1 and was found to be directed against the IGN311 binding site. Consistent with the induction of the HAHA response, CDC activity against Lewis Y positive target cells was completely abolished at day 8 and could not be restored by the second 50 mg infusion indicating complete neutralization of applied IGN311. The ADCC reactivity was also significantly reduced and anti-anti idiotype-specific antibodies (Ab3) were detectable at day 65. Conclusions: Induction of Ab3 antibodies should be considered as an additional factor influencing the efficacy of humanized antibodies. In this context, the potential threat of induced HAHA responses against therapeutic mAbs might have to be reconsidered because they might actually have also beneficial immunological long-term effects leading to an active immunization component induced by therapeutic antibodies.

Carbohydrate-associated epitope-based anti-cancer drugs and vaccines

RP215 is one of the three thousand monoclonal antibodies (Mabs) which were generated against the OC-3-VGH ovarian cancer cell line. RP215 was shown to react with a carbohydrate-associated epitope located specifically on glycoproteins, known as CA215, from cancer cells. Further molecular analysis by matrix adsorption laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed that CA215 consists mainly of immunoglobulin super-family (IgSF) proteins, including immunoglobulins, T-cell receptors, and cell adhesion molecules, as well as several other unrelated proteins. Peptide mappings and glycoanalysis were performed with CA215 and revealed high-mannose and complex bisecting structures with terminal sialic acid in N-glycans. As many as ten O-glycans, which are structurally similar to those of mucins, were also identified. In addition, two additional O-linked glycans were exclusively detected in cancerous immunoglobulins but not in normal B cell-derived immunoglobulins. Immunizations of mice with purified CA215 resulted in the predominant generation of RP215-related Mabs, indicating the immunodominance of this carbohydrate-associated epitope. Anti-idiotype (anti-id) Mabs of RP215, which were generated in the rat, were shown to contain the internal images of the carbohydrate-associated epitope. Following immunizations of these anti-id Mabs in mice, the resulting anti-anti-id (Ab3) responses in mice were found to be immunologically similar to that of RP215. Judging from these observations, anti-id Mabs, which carry the internal image of the RP215-specific epitope, may be suitable candidates for anticancer vaccine development in humans.

Immunomodulation of Human Carcinogenesis by the Blood Serum Antibodies against Benzo[a]pyrene, Estradiol and Progesterone

It was supposed that lung and breast cancer risks significantly increased when the levels of serum immunoglobulins A antibodies against benzo[a]pyrene and estradiol increased together, but did not separately. However, the cancer risks dramatically decreased when the levels of immunoglobulins A against progesterone elevated separately or together with immunoglobulins A against benzo[a]pyrene and estradiol. So, immunoglobulins A against benzo[a]pyrene and immunoglobulins A against estradiol acted as co-initiator and co-promoter in developing cancer scenario, but immunoglobulins A against progesterone acted along or conjointly with immunoglobulins A against benzo[a]pyrene and estradiol as strongly inhibitor in human carcinogenesis. Also it was suggested the precise mechanism of carcinogenesis modulation using anti-idiotypic antibodies against estradiol and progesterone through their membrane steroid receptors.

Construction and Expression of a Single Chain Antibody Mimicing Human Ovarian Cancer Antigen CA125

One concept for immune therapy of cancer involves induction of antigen mimic antibodies to trigger the immune response against tumor cells. Anti-idiotypic antibodies directed against the antigen-binding site of antibodies specific for tumor antigen may functionally and even structurally mimic antigen and induce anti-anti-idiotypic immune response. Monoclonal antibody W J02 is one of such anti-idiotypic antibodies, which contains internal image of CA125. In order to improve the immunospecificity of mAb WJ02, we constructed a single chain of mAb WJ02 in Vl-linker-Vh orientation. The scFv-WJ02, could be expressed and secreted in the recombinant Pichia pastoris system. The secreted scFv protein with a molecular weight of 30 kD retained the biological activity of mAb WJ02, which was proved by a direct binding assay and inhibition experiment. Our results indicated that the scFv-WJ02 could be used as a possible tool for idiotypic therapy against ovarian cancer, which might enhance the possibility of eliminating nonspecific responses induced by mAb WJ02.

Characterization and evaluation of a novel anti-MUC-1 monoclonal antibody: induction of the idiotypic network in experimental mice

Objective To investigate the anti-idiotypic effect induced by a monoclonal antibody. Methods A conventional fusion method was used to obtain hybridoma cells produing monoclonal antibody, which were detected by flow cytometry. ELISA were used to detect the humoral response induced by the antibody in mice. Cytotoxic and proliferation experiments were used to detect the cellular response induced by the antibody in mice. Results CS20 is a MUC-1 specific monoclonal antibody that strongly reacts with MUC-1 antigen expressed on the cell surface of breast cancer cells. The antibody could not kill tumor cells directly through complement-dependant cytotoxicity or antibody-dependant cell-mediated cytotoxicity. However, after 6 administrations of mAb CS20-KLH (keyhole limpet hemocyanin) conjugated to BALB/c mice (n=5) at a dose of 50 μg/mouse, anti-idiotypic antibodies and anti-anti-idiotypic antibodies were induced. T cells derived from CS20-KLH-immunized mice responded to mAb CS20, indicating the existence of idiotype-specific T cells. Conclusion These data indicated the possibility of using MUC-1 specific antibody for active immunotherapy of breast cancer.