抗EGFRvⅢ噬菌体抗体库的构建和筛选

目的:应用噬菌体展示技术筛选针对表皮生长因子受体突变体Ш(epidermal growth factorreceptor variant typeⅢ,EGFRvIII)的单链抗体(single chain Fv,scFv)。方法:利用原核表达纯化的人EGFRvIIIex蛋白和高表达EGFRvIIIex的小鼠成纤维细胞系NIH3T3免疫小鼠,扩增VH和VL片段并拼装成scFv基因,连接至噬菌粒pCANTAB5E,电击转化Hpd3cells,构建噬菌体单链抗体库,并进行3轮富集筛选。在第4轮筛选时,采用了降低抗原浓度的方法。然后将筛选得到的阳性克隆测序分析,转化E.coliHB2151,IPTG诱导可溶性scFv的表达。结果:构建了库容为7.9×107的噬菌体单链抗体库。经过第4轮低浓度抗原筛选,得到了较高亲和力的克隆。取单个阳性克隆测序分析结果表明,该抗EGFRvIII scFv基因序列长807bp,编码268个氨基酸。IPTG诱导后表达的可溶性scFv可分别与纯化的EGFRvⅢex抗原以及细胞表面的EGFRvⅢex结合。结论:利用噬菌体抗体库筛选得到了高亲和力的抗EGFRvⅢ scFv,为开发针对EGFRvⅢ...     

噬菌体抗体库的构建及抗乳腺癌细胞单链抗体的筛选

构建抗人乳腺癌细胞MCF 7的噬菌体单链抗体库 ,从中筛选MCF 7细胞特异性单链抗体。用MCF-7细胞免疫BALB C小鼠 ,取脾脏 ,提取总RNA ,用RT-PCR技术扩增小鼠抗体重链 (V_H)和轻链 (V_L)可变区基因 ,经重叠PCR(SOE-PCR) ,在体外将V_H和V_{L连接成单链抗体 (scFv)基因 ,并克隆到噬菌粒载体pCANTAB5E中 ,电转化至大肠杆菌TG1,经辅助噬菌体超感染 ,构建噬菌体单链抗体库。从该抗体库中筛选特异性识别MCF-7细胞的噬菌体单链抗体 ,将表面展示单链抗体的单克隆噬菌体转化大肠杆菌TOP10进行可溶性表达。成功地构建了库容为12×10⁶ 的抗MCF-7乳腺癌细胞的单链抗体库 ,初步筛选到了与MCF 7细胞特异性结合的scFv,Westernblot检测表明 ,在大肠杆菌TOP10中实现了单链抗体可溶性表达}     

Differences in antibody repertoires for (4-hydroxy-3-nitrophenyl)acetyl (NP) in splenic vs immature bone marrow precursor cells

To evaluate the contribution of environmental regulatory mechanisms in fashioning the primary B cell repertoire, we have compared the repertoire of (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific primary splenic B cells with that of precursor cells present as surface immunoglobulin-negative (sIg-) cells in adult bone marrow of C.B20 (Ighb) mice. Previous analyses using a variety of antigens have led to the conclusion that the antibody repertoire expressed in the spleen is similar to that expressed in newly generated B cell precursors with respect to both repertoire diversity and the representation of various predominant clonotypes. However, in the response to NP of C.B20 precursor cells, two marked disparities have been identified between the repertoire of sIg- bone marrow cells vs splenic precursor cells. The first concerns precursor cells that give rise to lambda-bearing NP-specific antibodies with heteroclitic fine specificity. Such antibodies normally dominate the primary response of Ighb mice; however, the representation of precursor cells giving rise to lambda-bearing antibodies is disproportionately low in the sIg- bone marrow cell population of C.B20 mice. Thus, during the maturation of these cells, post-sIg receptor expression, there is an apparent increase in the proportionate representation of lambda-bearing NP-specific cells. The second disparity concerns precursor cells whose antibody products bear kappa-light chains and exhibit high affinity and homoclitic binding for the NP haptenic determinant. Such precursor cells are poorly represented in the spleen, but represent a sizeable proportion of the sIg- NP-specific precursor cell population. Thus, there seems to be a selective elimination of high affinity, kappa-homoclitic anti-NP antibody-bearing cells as they acquire their sIg receptors. The elimination of this cell population could partially account for the dominance of lambda-heteroclitic antibodies in the serum responses to NP of C.B20 mice.     

In aged mice,low surrogate light chain promotes pro‐B‐cell apoptotic resistance,compromises the PreBCR checkpoint,and favors generation of autoreactive,phosphorylcholine‐specific B cells

In aged mice, new B‐cell development is diminished and the antibody repertoire becomes more autoreactive. Our studies suggest that (i) apoptosis contributes to reduced B lymphopoiesis in old age and preferentially eliminates those B‐cell precursors with higher levels of the surrogate light chain (SLC) proteins (λ5/VpreB) and (ii) λ5^low B‐cell precursors generate new B cells which show increased reactivity to the self‐antigen/bacterial antigen phosphorylcholine (PC). Pro‐B cells in old bone marrow as well as pro‐B cells from young adult λ5‐deficient mice are resistant to cytokine‐induced apoptosis (TNFα; TGFβ), indicating that low λ5 expression in pro‐B cells is sufficient to cause increased survival. Transfer of TNFα‐producing ‘age‐associated B cells’ (ABC; CD21/35^? CD23^?) or follicular (FO) B cells from aged mice into RAG‐2 KO recipients led to preferential loss of λ5^high pro‐B cells, but retention of λ5^low, apoptosis‐resistant pro‐B cells. In old mice, there is increased reactivity to PC in both immature bone marrow B cells and mature splenic FO B cells. In young mice, absence of λ5 expression led to a similar increase in PC reactivity among bone marrow and splenic B cells. We propose that in old age, increased apoptosis, mediated in part by TNFα‐producing B cells, results in preferential loss of SLC^high pro‐B cells within the bone marrow. Further B‐cell development then occurs via an ‘SLC^low’ pathway that not only impairs B‐cell generation, but promotes autoreactivity within the naïve antibody repertoires in the bone marrow and periphery.     

抗松材线虫纤维素酶单链抗体库的构建及筛选

构建鼠源性松材线虫纤维素酶（Bursaphelenchus xylophilus cellulase, BXC）的噬菌体单链抗体库,从中筛选特异性BXC的单链抗体。以BXC为抗原免疫BALB/C小鼠,从脾脏提取总RNA,用RT-PCR技术扩增小鼠抗体重链（V_H）和轻链(V_L)可变区基因。经重叠PCR(SOE-PCR)在体外将V_H和V_L连接成单链抗体(scFv)基因,并克隆到噬菌粒载体pCANTAB5E中,电转化至大肠杆菌TG1,经辅助噬菌体超感染,成功构建了库容为5×10⁴的Anti-BXC单链抗体库,并从该抗体库中初步筛选到了特异性识别BXC的噬菌体单链抗体scFv。将表面展示单链抗体的单克隆噬菌体转化大肠杆菌HB2151进行可溶性表达,SDS-PAGE及Western blot分析结果显示,可溶性scFv获得表达,且与BXC具有结合活性,为松材线虫的检验检疫以及病理学研究奠定了基础。     

Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing

High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.     

Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing

High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.     

The frequency and fine specificity of B cells responsive to (4-hydroxy-3-nitrophenyl)acetyl in aged mice

The B-cell response to NP-Hy of two murine strains has been analyzed in order to evaluate the affects of aging on B-cell repertoire expression. The results indicate that for both BALB/c mice (Igha) and B10.D2 mice (Ighb) the frequency of (4-hydroxy-3-nitrophenyl)acetyl (NP)-responsive splenic B cells is approximately twofold lower, on a per B cell basis, in aged mice as compared to young adults. However, as with previous assessments of the response to DNP-Hy in aged mice, the frequency of newly generated surface immunoglobulin negative bone marrow precursor cells specific for NP in aged BALB/c mice is the same as in young mice. The decrease in frequency of responsive splenic B cells is not accompanied by a measurable decrease in repertoire diversity or changes in clonotype distribution as assessed by representation of kappa vs lambda light chain-bearing antibodies, binding of monoclonal antibodies to a panel of analogues of NP, and the proportionate representation of B10.D2 monoclonal antibodies that bear NPb idiotypic determinants. By these criteria it appears that down-regulation of B cells as they mature and emerge from the bone marrow of aged mice is pan-specific and does not disproportionately affect B cells of a predominant clonotype family. Consistent with other investigations which have demonstrated differences in secreted antibodies of immunized aged vs young mice, we have observed that 4 weeks after immunization of B10.D2 mice with NP-BSA, the frequency of newly generated secondary B cells is lower in aged than in young mice and the generation of lambda-bearing secondary precursor cells is particularly low. Thus, clonotype-specific down-regulation may play a role in shaping the B-cell repertoire subsequent to immunization of aged mice.     

Monoclonal anti-Id antibodies react with varying proportions of human B lineage cells

Monoclonal antibodies to idiotypic determinants are being used with increasing frequency for analysis and treatment of B cell malignancies. In the present study we have compared the idiotypic specificities of a panel of 39 mouse monoclonal anti-idiotype (anti-Id) antibodies developed against 16 monoclonal human immunoglobulins (Ig). The Id cross-reactivities of these antibodies with Ig products of normal and abnormal B cells were examined by immunofluorescence and immunochemical methods. The reactivity patterns of these anti-Id antibodies with a normal population of plasma cells were highly variable in the immunofluorescence assay. Six were reactive with 2 to 10% of normal plasma cells, 30 with 0.1 to 2% of plasma cells, and three with less than 0.1% of plasma cells from blood, bone marrow, spleen, or tonsils. These reactivity patterns were relatively consistent among samples from 23 Caucasian, black, and Oriental adults. Although the reactivities of most anti-Id antibodies in the panel were not restricted to a particular Ig isotype, several were preferentially reactive with a particular heavy or light chain isotype: one IgM-, two IgA-, two kappa-, and three lambda-restricted antibodies. The immunofluorescence data was confirmed by biosynthetic analysis of Id+ molecules produced by a normal plasma cell population. When the reactivity of this panel of anti-Id antibodies with nonhomologous B cell neoplasms was examined, seven of 30 myelomas or leukemia-derived products and one of nine B cell leukemias or lymphomas without paraproteins were found to be cross-reactive with one or two of the anti-Id antibodies. Although clearly significant, the cross-reactivity between the Id of these paraproteins appeared to be of lower affinity than the reactivity of the homologous Id with their respective anti-Id antibodies. The results reveal a remarkable diversity in the specificities of monoclonal antibodies classified by conventional criteria as anti-Id antibodies, and indicate the potential usefulness of a panel of antibodies for analyzing clonal diversity in normal and abnormal B cell development.     

A proteomics approach for the identification and cloning of monoclonal antibodies from serum

We describe a proteomics approach that identifies antigen-specific antibody sequences directly from circulating polyclonal antibodies in the serum of an immunized animal. The approach involves affinity purification of antibodies with high specific activity and then analyzing digested antibody fractions by nano-flow liquid chromatography coupled to tandem mass spectrometry. High-confidence peptide spectral matches of antibody variable regions are obtained by searching a reference database created by next-generation DNA sequencing of the B-cell immunoglobulin repertoire of the immunized animal. Finally, heavy and light chain sequences are paired and expressed as recombinant monoclonal antibodies. Using this technology, we isolated monoclonal antibodies for five antigens from the sera of immunized rabbits and mice. The antigen-specific activities of the monoclonal antibodies recapitulate or surpass those of the original affinity-purified polyclonal antibodies. This technology may aid the discovery and development of vaccines and antibody therapeutics, and help us gain a deeper understanding of the humoral response.     

In vitro immunization can elicit the expansion of diverse repertoire of B cells from peripheral blood mononuclear cells

We previously developed an in vitro immunization (IVI) protocol of human peripheral blood mononuclear cells (PBMC) for generating antigen-specific human antibodies. In order to clarify whether IVI protocolinduces antigen-specific B cell responses in PBMC, we analyzed family gene usage and sequence of the variable region gene of immunoglobulin heavy chain (VH gene) of the antibody produced from the in vitro immunized PBMC. Sequence homology analyses of VH gene demonstrated that a larger repertoire of B cells can be sensitized with mite-extract than with cholera toxin B subunit and rice allergen. Further, antigen-specific B cells were efficiently expanded by using CpG oligodeoxynucleotide as adjuvant. These results suggest that appropriate combination of sensitizing antigen and adjuvant is primarily important for expansion of antigen-specific B cells in IVI protocol.     

Human Immunoglobulin Repertoires against Tetanus Toxoid Contain a Large and Diverse Fraction of High-Affinity Promiscuous VH Genes

To study the contribution of antibody light (L) chains to the diversity and binding properties of immune repertoires, a phage display repertoire was constructed from a single human antibody L chain and a large collection of antibody heavy (H) chains harvested from the blood of two human donors immunized with tetanus toxoid (TT) vaccine. After selection for binding to TT, 129 unique antibodies representing 53 variable immunoglobulin H chain (V_H) gene rearrangements were isolated. This panel of anti-TT antibodies restricted to a single variable immunoglobulin L chain (V_L) could be organized into 17 groups binding non-competing epitopes on the TT molecule. Comparison of the V_H regions in this V_L-restricted panel with a previously published repertoire of anti-TT V_H regions with cognate V_H-V_L pairing showed a very similar distribution of V_H, D_H and J_H gene segment utilization and length of the complementarity-determining region 3 of the H chain. Surface plasmon resonance analysis of the single-V_L anti-TT repertoire unveiled a range of affinities, with a median monovalent affinity of 2 nM. When the single-V_L anti-TT V_H repertoire was combined with a collection of naïve V_L regions and again selected for binding to TT, many of the V_H genes were recovered in combination with a diversity of V_L regions. The affinities of a panel of antibodies consisting of a single promiscuous anti-TT V_H combined with 15 diverse V_L chains were determined and found to be identical to each other and to the original isolate restricted to a single-V_L chain. Based on previous estimates of the clonal size of the human anti-TT repertoire, we conclude that up to 25% of human anti-TT-encoding V_H regions from an immunized repertoire have promiscuous features. These V_H regions readily combine with a single antibody L chain to result in a large panel of anti-TT antibodies that conserve the expected epitope diversity, V_H region diversity and affinity of a natural repertoire.     

Hypogammaglobulinemia in BLT Humanized Mice – An Animal Model of Primary Antibody Deficiency

Primary antibody deficiencies present clinically as reduced or absent plasma antibodies without another identified disorder that could explain the low immunoglobulin levels. Bone marrow-liver-thymus (BLT) humanized mice also exhibit primary antibody deficiency or hypogammaglobulinemia. Comprehensive characterization of B cell development and differentiation in BLT mice revealed other key parallels with primary immunodeficiency patients. We found that B cell ontogeny was normal in the bone marrow of BLT mice but observed an absence of switched memory B cells in the periphery. PC-KLH immunizations led to the presence of switched memory B cells in immunized BLT mice although plasma cells producing PC- or KLH- specific IgG were not detected in tissues. Overall, we have identified the following parallels between the humoral immune systems of primary antibody deficiency patients and those in BLT mice that make this in vivo model a robust and translational experimental platform for gaining a greater understanding of this heterogeneous array of humoral immunodeficiency disorders in humans: (i) hypogammaglobulinemia; (ii) normal B cell ontogeny in bone marrow; and (iii) poor antigen-specific IgG response to immunization. Furthermore, the development of strategies to overcome these humoral immune aberrations in BLT mice may in turn provide insights into the pathogenesis of some primary antibody deficiency patients which could lead to novel clinical interventions for improved humoral immune function.     

基于单个B细胞抗体基因扩增技术筛选马IgG1单克隆抗体*

在马的免疫学研究领域中,由于目前市场上缺乏商业化的马IgG单克隆抗体,使得对马的B细胞研究受到很大阻碍,IgG是B细胞受体(BCR)的重要构成成分,与B细胞分化成熟相关。为了获得马IgG特异性单克隆抗体,利用单个B细胞扩增技术进行抗体筛选。首先,将马IgG蛋白(EqIgG1-C)密码子优化后合成到真核表达载体pcDNA3.4上,纯化出抗原蛋白。随后,使用蛋白质免疫小鼠,分离脾细胞后利用流式细胞术分离特异性单个B细胞,扩增出抗体重链和轻链的可变区基因,用overlapping PCR方法扩增出线性化的完整抗体,并进行鉴定。结果从80个B细胞中获得了27株特异性重组单克隆抗体,并挑选出3株线性结合活性最强的抗体基因构建到表达载体上,共转染Expi293F^TM细胞后表达纯化,经过ELISA和Western blot验证,显示获得的抗体可以和EqIgG1-C蛋白有良好的结合作用。使用该方法可以省时高效的获得特异性抗体,为马的免疫学研究提供了重要研究工具,为鼠单克隆抗体筛选提供了技术拓展。     

Monoclonal antibodies to HLA-A,B, and Ia-like antigens inhibit colony formation by human myeloid progenitor cells

Hybridomas derived from the fusion of murine myeloma cells with splenocytes from mice immunized with human cultured lymphoid cells secreted monoclonal antibodies to human cell surface antigens. Serologic and immunochemical assays showed that 4 monoclonal antibodies (Ab Q2/47, Q2/61, Q2/70, Q2/80) recognize framework determinants of Ia-like antigens and 1 monoclonal antibody (Ab Q1/28) reacts with determinants expressed on the heavy chain of HLA-A,B antigens. Both anti-HLA-A,B and anti-Ia-like antigen monoclonal antibodies caused complement-dependent inhibition of granulocyte-macrophage colony formation by human bone marrow grown in soft agar. Mixing experiments excluded the possibility of an indirect effect on progenitor cells by lysis of auxiliary cells. These results indicate that human myeloid progenitor cells express HLA-A,B and Ia-like antigens.     

Generation of functional scFv intrabodies for triggering anti-tumor immunity

Intracellular expression of recombinant antibodies (intrabodies) allows to interfere with the functions of oncogenic or viral molecules expressed in different cell compartments and has therefore a vast clinical potential in therapy. Although the use of phage-display libraries has made it possible to select Fab or single chain Fv (scFv) antibody fragments usable for intracellular targeting, a major source of recombinant antibodies for therapeutic use still remains hybridoma B cells producing well-characterized monoclonal antibodies (mAbs). However, the cloning and the intracellular expression of antibody fragments derived from mAbs can be markedly hampered by a number of technical difficulties that include failure of cloning functional variable regions as well as lack of binding of the antibody fragments to the targeted molecule in an intracellular environment. We discuss herein various molecular methods that have been developed to generate functional recombinant antibody fragments usable as anti-tumor triggering agents when expressed in tumor cells. Such antibodies can neutralize or modify the activity of oncogenic molecules when addressed in specific subcellular compartments and/or they can be used to trigger anti-tumor immunity when expressed on tumor cell surface.     

噬菌体呈现技术制备结肠癌抗独特型抗体

 以纯化的鼠抗人结肠癌细胞单克隆抗体 (简称单抗 )MC5与钥孔血蓝素 (KLH)的交联物经腹腔免疫Balb c小鼠 ,取脾分离mRNA .RT PCR分别扩增抗体重、轻链可变区基因片段 (VH 和VLcD NA ,大小分别约为 340bp和 32 0bp) ,二者经linkerDNA连接形成ScFv(singlechainvariablefragment)DNA(约 75 0bp) .将ScFvDNA与噬菌粒载体pCANTAB5E的连接产物转化于大肠杆菌TG1,经辅助噬菌体M13KO7感染后 ,获得重组噬菌体抗体ScFv文库 .以单抗MC5对ScFv文库进行 4轮亲和筛选后 ,随机挑取 80个克隆经酶联免疫吸附实验 (ELISA)筛选出 2 2个呈现ScFv形式抗独特型抗体(抗 IdScFv)的噬菌体单克隆 .竞争抑制实验表明 ,在 2 2个阳性克隆中有 4个克隆所呈现的抗 IdScFv属β或γ型 .针对单抗MC5的噬菌体呈现型抗 IdScFv的制备 ,为筛选新的结肠癌重组抗 Id瘤苗候选分子奠定了基础