嵌合抗原受体T细胞技术及其临床应用

嵌合抗原受体 T 细胞（CAR-T）疗法是利用基因改造技术,使 T 细胞表达肿瘤特异性嵌合抗原受体,以抗原依赖、非主要组织相容性复合体（MHC）限制的方式结合肿瘤抗原,肿瘤相关抗原（TAA）的单链抗体（scFv）和 T 细胞的活化序列在体外进行基因重组,形成重组质粒。在体外通过转染技术,转染经纯化与大规模扩增后的 T 细胞,启动并活化特异性杀伤肿瘤反应,其临床细胞治疗的应用显示出高效性,在白血病、淋巴瘤、黑色素瘤等恶性肿瘤治疗中均显示出良好的抗肿瘤效应,使得 CAR-T 技术成为主流细胞治疗手段。     

嵌合抗原受体修饰的免疫细胞及其在肿瘤免疫治疗中的应用

嵌合抗原受体修饰的T(chimeric antigen receptor-modified T,CAR-T)细胞是指通过基因修饰技术将带有特异性抗原识别结构域及T细胞激活信号的遗传物质转入T细胞并表达,使T细胞能直接与肿瘤细胞表面的特异性抗原相结合而被激活的细胞疗法.近年来,CAR-T细胞在血液病治疗中取得了显著的效果...     

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.     

Immunotherapy with monoclonal anti-idiotypic antibodies: tumour reduction and lymphokine production

A patient with a B-cell chronic lymphocytic leukaemia was treated with a murine IgG1 monoclonal anti-idiotypic antibody (MoAb anti-id). After a total of 773.2 mg MoAb anti-id had been administered with a maximum daily dose of 83.2 mg, 90% tumour reduction was established within 2 weeks. Although in vitro the tumour cells were stimulated by MoAb anti-id no signs of tumour cell activation were observed in vivo during therapy with MoAb anti-id. The rapid tumour reduction suggests that the proliferation-enhancing property of anti-id is not a contra-indication for immunotherapy. The FcR II receptors on the patients monocytes could interact with the IgG1 monoclonal used. MoAb anti-id administration induced strongly decreased platelet counts, dependent on the amount of serum idiotype that had to be cleared. Antibody administration activated the macrophage/monocyte system, reflected in the neopterin profile, resulting in TNF alpha production and simultaneously strong reductions of circulating tumour cells. The partial remission lasted 3 months, then the tumour reappeared. These data show that a straightforward therapy with MoAb anti-id, in itself, has a strong potential, but is not sufficient to eradicate the tumour permanently. Further study will be needed to improve the clinical results with this kind of therapy.     

抗CEA抗体可变区基因的克隆及其嵌合抗体的表达

目的 在真核细胞中表达抗癌胚抗原（carcinoembryonic antigen,CEA)人－鼠嵌合抗体。方法 从分泌抗CEA鼠单抗C50的杂交瘤细胞扩增、克隆可变区基因。测序鉴定后连入真核表达载体,导入二氢叶酸还原酶缺陷型中国仓鼠卵巢（dihydrofolate reductase-deficient Chinese hamster ovary CHO-dhfr）细胞进行表达。采用间接和竞争抑制ELISA检测表达产物的人源性和抗原结合特异性。结果 序列测定初步确定所克隆的是功能性抗体可变区基因。在转染细胞上清中测到抗CEA嵌合抗体的表达。ELISA试验证实嵌合抗体具有人的恒定区,并与原鼠单抗C50有相同的抗原结合特异性。结论 成功地在真核细胞中表达了抗CEA人－鼠嵌合抗体。     

HER2抗独特型单克隆抗体的制备和初步研究

目的:研制HER2抗独特型单克隆抗体,为进一步深入研究乳腺癌抗独特型抗体疫苗奠定基础。方法:用人HER2蛋白免疫家兔,获得特异性兔抗HER2抗体。再用兔抗HER2抗体免疫Balb/c小鼠,采用杂交瘤技术制备HER2抗独特型单克隆抗体。并筛选出β型HER2抗独特型单克隆抗体。结果:ELISA检测结果表明,获得的兔抗HER2抗体能特异性地与HER2蛋白结合,其OD值随HER2的浓度呈正线性关系。用兔抗HER2抗体免疫小鼠获得一株稳定分泌HER2抗独特型单克隆抗体的杂交瘤细胞1F5,其分泌的单克隆抗体能特异性的和兔抗HER2多克隆抗体结合,并与HER2产生竞争抑制作用。用1F5抗独特型单克隆抗体免疫家兔得到的抗血清能和HER2特异性的结合。该抗体亚型为IgG3,未浓缩腹水的效价为1∶1.02×104。结论:抗独特型单克隆抗体为β型HER2抗独特型抗体,有可能成为乳腺癌抗独特型单克隆抗体疫苗。     

Recombinant vaccinia viruses expressing immunoglobulin variable regions efficiently and selectively protect mice against tumoral B-cell growth.

The variable regions of the immunoglobulin (Ig) expressed on the surface of a malignant B cell can be considered tumor-specific antigens and, as such, could be targets for immunotherapeutic approaches. However, because until now the immunization procedures have been complex and have given only partial protection, it was necessary to find new methods of immunotherapy. Here, we present a successful method of vaccination against B-cell tumors in a murine model. We produced recombinant vaccinia viruses (rVV) expressing the heavy and the light chain of surface Ig of a patient's malignant B cells and we tested the ability of these rVV to protect immunized mice against tumor growth of transfectomas producing the same human Ig. The protection of the mice was complete and specific to the variable region of the immunizing heavy chain although specific lymphoproliferative and cytotoxic responses were not detectable in vitro. The protection was strictly dependent on the presence of CD4 T cells and asialo GM1+ cells. Furthermore, tumor protection clearly required gamma-interferon and was partially inhibited by blocking the Fas-Fas ligand interaction. We also show, in a murine syngeneic model, that rVV expressing a poorly mutated Ig protects against the growth of Ig-producing tumor.     

高感染力嵌合型腺病毒载体的构建及其感染力的流式细胞术测定

目的构建1种35型腺病毒(Ad35)Fiber替代5型腺病毒(Ad5)Fiber的嵌合型腺病毒载体,探索该腺病毒载体感染肿瘤细胞的效率。方法PCR扩增Ad35腺病毒Fiber序列,插入并置换Ad5的Fiber序列,构建Ad5-F35嵌合型腺病毒载体,使之携带绿色荧光蛋白报告基因EGFP;以MOI=5的Ad5-F35EGFP病毒感染度感染淋巴瘤细胞raji和胃癌细胞SGC-7901,流式细胞术检测EGFP表达,借以判断病毒感染细胞的能力。结果成功构建携带EGFP的嵌合型腺病毒载体pAd5-F35EGFP,并在293细胞中重组出Ad5-F35EGFP嵌合型腺病毒,扩增纯化后病毒滴度达到3.4×109pfu/ml;流式细胞仪检测发现,Ad5-F35EGFP在淋巴瘤细胞raji和胃癌细胞SGC-7901内表达EGFP的强度明显比传统的Ad5-EGFP提高。结论Ad5-F35嵌合型腺病毒感染肿瘤细胞的能力明显增强,为提高腺病毒载体转染抗肿瘤基因的效率、改进肿瘤基因治疗的临床疗效奠定了基础。     

鼻咽癌抗独特型疫苗的制备及临床研究

目的：制备具有内影像特征的抗独特型抗体,用于鼻咽癌病人的主动免疫治疗以探讨其抗肿瘤效应。方法：用杂交瘤技术制备针对Ａｂ１可变区的抗独特型单抗（Ａｂ２）,免疫同系动物诱导产生免疫应答并用氢氧化铝凝胶沉淀法制成抗独物型疫苗,对１９例晚期鼻咽癌放疗病人作主动免疫治疗,９例放疗加生理盐水注射为对照组,用ＥＬＩＳＡ检测治疗前后病人血清抗体和细胞因子水平。用原位Ｎｏｒｔｈｅｒｎ杂交检测外周血单个核细胞（ＰＢＭ     

Monoclonal anti-idiotypic antibodies to human melanoma-associated proteoglycan antigen: generation and characterization of anti-idiotype antibodies.

We have characterized a number of monoclonal anti-idiotype antibodies (mAb2s) made against a monoclonal antitumor antibody, MEM136. The monoclonal antibody 1 (mAb1) MEM136 recognizes an epitope on human melanoma-associated proteoglycan and blocks melanoma cell interaction with basement membrane components in vitro. The anti-idiotype antibodies (Ab2s) made against MEM136 each cross-inhibited, to varying degrees, their binding to MEM136. Thus, the mAb2s recognized overlapping idiotopes on MEM136. In an attempt to identify potential internal image candidates we set up a cell migration inhibition assay. In this assay, migration of melanoma-associated proteoglycan-positive Colo38 cells was determined through a membrane barrier impregnated with Matrigel, which is composed of extracellular matrix components, i.e., collagen type IV, heparan sulfate, and laminin. Interestingly, only Ab2s IM06 and IM32 inhibited melanoma cell migration. Additional studies indicate that of eight mAb2s tested, only IM32 and IM06 induced anti-MPG responses in rabbits. The possibility that IM32 and IM06 bear images of melanoma cell surface-associated proteoglycan epitopes is discussed.     

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     

Consumption of monoclonal anti-idiotypic antibody by neoplastic B lymphocytes: a guide for immunotherapy

A quantitative analysis in vitro of events which might occur on administration of mouse monoclonal anti-idiotypic antibody to a recipient with a B cell neoplasm has been made. The L2C leukaemic cells of guinea pigs, which closely resemble those of human lymphoma in expression and metabolism of immunoglobulin have been used as a model. Exposure of neoplastic B cells to antibody results in rapid binding of approximately 420,000 molecules of antibody per cell at saturation, and the amount consumed does not increase markedly over the next 4 h of exposure at 37 degrees C. This is in spite of the fact that secretion of idiotypic IgM continues unaffected by the presence of antibody, and reflects the fact that the amount of IgM secreted during this period is low compared to the amount displayed on the cell surface. If cells undergo lysis, however, the antibody consumed is approximately doubled: thus a recipient with an estimated tumour load of 10(12) cells would require 200 mg of monoclonal anti-idiotype for binding to surface and intracellular antigen. The effect of the soluble idiotypic IgM found in serum on the ability of antibody to bind target cells has been examined by means of the fluorescence activated cell sorter. Access of antibody to the cells is efficiently blocked by competing idiotypic IgM in the fluid phase, with no indication of preferential binding to cell surface idiotype. Immunotherapeutic doses should be designed therefore to overcome this additional antigenic load in secreting tumours, which form the majority of B cell neoplasms.     

Impaired lymphocyte responses and their restoration in oral cancer patients expressing distinct TCR variable region

We have analyzed TCR Vbeta gene usage and clonality of T cells in the peripheral blood of patients with oral cancer and healthy individuals. A large repertoire of clonal TCR Vbeta was observed in the peripheral blood of cancer patients, and this clonal expansion was dominantly represented in the CD8+ T cells. A marked decrease in the lymphocyte proliferative responses to mitogen and anti-CD3 MAb and spontaneous apoptosis was observed in lymphocytes. Appropriate co-stimulation of lymphocytes (anti-TCR Vbeta MAb and anti-CD28 MAb) restored the lymphocyte proliferative responses and CD3-zeta chain expression in these patients.     

Cancer patients' perceptions of their disease and its treatment

One hundred cancer patients undergoing active treatment were interviewed to determine how they perceived their illness and how their perceptions compared with those of their attending physicians. Ninety-eight patients recognized that they had cancer and 87 correctly identified the tumour type. Sixty-four of 67 patients with local or regional disease were aware of this, but 11 of 33 patients with metastatic disease incorrectly believed that the cancer was localized. Five of 52 patients being treated for cure thought they were being treated palliatively, and 16 of 48 patients receiving palliative treatment believed that the doctor's aim was to cure them. Forty of these 48 patients significantly overestimated the probability that the treatment would prolong their lives. Patients with little secondary education were significantly more likely to underestimate the seriousness of their condition. Interactions between doctor and patients were not observed directly and it was therefore not possible to determine whether patients' inaccurate views of their illness were due to suboptimal communication or denial. Doctors frequently failed to recognize their patients' misconceptions. In only one of the 16 cases in which a patient, who was being treated palliatively, believed that the treatment was curative did the doctor recognize that this misunderstanding existed.     

Relationship between antigenicity and pathogenicity for Burkholderia pseudomallei and Burkholderia mallei revealed by a large panel of mouse MAbs

Burkholderia pseudomallei and Burkholderia mallei are two closely related gram-negative bacterial species classified by the CDC as category B biowarfare agents. To develop monoclonal antibodies (MAbs) that can recognize as many different strains and/or clinical isolates of these two pathogens as possible, we immunized mice with heat-killed bacterial whole cells and membrane preparations from multiple strains and/or clinical isolates of B. pseudomallei and B. mallei. More than 100 different hybridoma clones that produced MAbs strongly reacting to B. pseudomallei and/or B. mallei have been developed. These MAbs were categorized into eight different groups according to their reaction specificity against different species of Burkholderia bacteria as well as the different nature of target antigens (LPS, capsule polysaccharides, proteins, and glycoproteins) on the bacteria they recognized. Characterization of this large panel of MAbs has demonstrated an interesting pattern of various antigenic epitopes shared by the different species of Burkholderia bacteria. More importantly, this study has revealed a pathogenicity-linked antigen epitope(s) on capsule-like polysaccharides found only in the pathogenic species of Burkholderia bacteria and a Burkholderia-specific antigen epitope(s) that did not exist in other gram-negative bacterial species. Our MAbs should prove to be highly valuable in the development of detection, diagnosis, and therapeutic applications against B. mallei and B. pseudomallei infections.     

Cleavage of Fyn and Lyn in their N-terminal unique regions during induction of apoptosis: a new mechanism for Src kinase regulation

The members of the Src kinase family are expressed in a wide variety of tissues, but some of them such as Blk, Hck, Fgr, Lck and Lyn are found primarily in hematopoietic cells. In the present study, we have undertaken experiments to test whether Src kinase cleavage and relocation is a general mechanism during induction of apoptosis. Our results indicate that Fyn and Lyn are efficiently cleaved in their unique region in hematopoietic cells undergoing apoptosis. Fyn cleavage occurred in Fas-stimulated Jurkat T cells but Fyn and Lyn were also processed in the SKW6.4 B cell line. Inhibition of caspases by Z-VAD-fmk or Ac-DEVD-CHO totally prevented Fyn and Lyn cleavage in both intact cells and in vitro. Fyn and Lyn but not Lck, Src and Hck were processed in vitro by human recombinant caspase 3 and by cellular extracts prepared from Fas-stimulated cells. Single mutation of Asp 19 or Asp 18 in the unique N-terminal domains of Fyn and Lyn respectively abolished their cleavage and relocation into the cytoplasm of apoptotic cells. When immunoprecipitated from COS cells N-terminal deleted Src kinases exhibited increased enzymatic kinase activity toward enolase. Thus, cleavage of Fyn and Lyn during induction of apoptosis represents a new mechanism for the regulation of Src kinases that may have important functional and physiological consequences.