Immunoglobulin repertoire of B lymphocytes infiltrating breast medullary carcinoma

Tumor specific peptides recognized by T lymphocytes infiltrating solid tumors, as well as the corresponding T cell receptor (TcR) repertoire usage, have been extensively investigated. By contrast, tumor infiltrating B cells and their immunoglobulin (Ig) repertoire have been studied only in a limited number of tumors. The objective of the present study was to determine, whether DNA sequence analysis of the expressed immunoglobulin variable regions of B cells that infiltrate breast cancer, could be used to reveal a potential specific tumor binding capacity of the antibodies. To answer this question, about 200 expressed Ig heavy (VH) and light chain variable gene (VL) regions were cloned, sequenced and comparatively analysed from a typical medullary beast carcinoma (MBC), where the massive B and plasma cell infiltration correlates with favourable prognosis despite of its high grade. The tumor infiltrating B cell Ig heavy and light chain sequences could be classified into clusters, families and subgroups, based on the identity level to germline, showing a pattern of oligoclonality. Some overrepresented clusters could be determined. In the course of a detailed analysis and search in Blastn database, a number of VH and VL sequences showed more than 99% homology to DNA sequences of Ig VH region, with proved tumor antigen binding capacity. Our data suggest, that potential tumor binder Ig VH and VL sequences might be selected using a detailed immunoglobulin variable region analysis. This new approach might have a benefit for further antibody engineering, as difficulties in search for tumor binders by phage library selection might be reduced and the time for selection shortened.     

具有GPX活性的单克隆抗体可变区基因的克隆和序列分析

目的利用分泌具有GPX活性的单克隆抗体(mAb)的杂交瘤细胞3G5,克隆其mAb体的可变区基因。方法提取杂交瘤细胞的总RNA ,分离mRNA ,反转录合成cDNA。经PCR扩增VH 基因和VL 基因 ,将VH 基因和VL基因与载体 pGEM T连接后 ,进行酶切鉴定和序列分析。结果构建了2个分别含有VH 和VL 基因的重组质粒。序列分析表明 ,VH 和VL 分别属于mouscheavychainsubgroupIII和mouselightchainsubgroupV亚群 ,长度为372和324bp ,编码124和108个氨基酸 ,在高变区分别有4和2个丝氨酸。结论克隆的VH 和VL 基因符合功能性重排的鼠抗体可变区基因特征 ,为将来制备具有GPX活性的单链抗体提供了可靠的基因材料。     

抗人CD154单抗轻重链可变区基因克隆与序列分析

目的:克隆抗人CD154抗体轻重链可变区基因,并分析其核苷酸序列,为基因工程抗体的构建奠定基础。方法:从分泌能抑制免疫反应的抗人CD154单克隆抗体杂交瘤细胞株 7E8中提取总RNA,合成cDNA第一链后,经PCR扩增获得抗人CD154单抗轻链可变区 (VL)和重链可变区 (VH)基因,分别克隆入pUC18载体,并进行序列分析。结果:①抗体的轻链可变区基因全长为 341bp,编码113个氨基酸,归属于Ig的Vκ2基因,氨基酸序列分析结果显示轻链可变区含有明确的 4个骨架区和 3个抗原决定簇互补区,在第 2 3位和第 93位氨基酸为半胱氨酸,是与抗体二硫键形成有关的两个特征性氨基酸;②抗体的重链可变区基因全长为 354bp,编码118个氨基酸,归属于小鼠IgVH基因,D、J区基因分别属于DSP2.9和JH2,氨基酸序列分析结果显示,重链可变区含有明确的 4个骨架区和 3个抗原决定簇互补区,在第 2 3和第 97位氨基酸为半胱氨酸,是与抗体二硫键形成有关的两个特征性氨基酸。结论:经核苷酸序列分析证明所克隆的基因分别为抗体的轻、重链可变区基因.     

抗肿瘤坏死因子相关凋亡诱导配体受体2嵌合抗体表达载体的构建、表达及其抗肿瘤活性分析

目的:构建抗人肿瘤坏死因子相关凋亡诱导配体(TRAIL)受体2(死亡受体5,DR5)的人-鼠嵌合抗体表达载体,获得稳定表达该嵌合抗体的细胞株,并分析嵌合抗体的抗肿瘤活性。方法:采用DNA重组技术,扩增抗人DR5的鼠源单克隆抗体(mAb)AD5-10的重链(HC)、轻链(LC)可变区基因片段,并将其分别插入含有人IgG重链、轻链恒定区基因的真核表达载体RpCI-neo,以重、轻链表达质粒共转染中国仓鼠卵巢细胞(CHO),筛选稳定表达抗人DR5嵌合抗体(hmAD5-10)的重组细胞。采用Western blot和间接ELISA检测嵌合抗体的表达量及其与抗原DR5的结合活性。采用MTS比色法检测嵌合抗体的生物学活性。并对重组细胞株进行无血清培养驯化。结果:获得了2株稳定表达嵌合抗体的重组细胞株CHO-A5和CHO-B11,抗体的表达水平分别为(0.36±0.11)mg/L和(0.16±0.01)mg/L,嵌合抗体与DR5有较好的结合活性,对体外培养的人T淋巴细胞白血病细胞SVT35有显著的杀伤作用。结论:在真核细胞中表达了具有生物学活性的抗DR5的人-鼠嵌合抗体,为其应用于肿瘤治疗研究奠定了基础。     

Construction and molecular characterization of mouse single-chain variable fragment antibodies against Burkholderia mallei and Burkholderia pseudomallei

We have selected two lipopolysaccharide (LPS) specific Burkholderia mallei mouse monoclonal antibodies (mAbs) and four anti-capsular B. pseudomallei-specific mAbs to generate mouse single-chain variable fragment (scFv) antibodies. This selection was made through extensive in vitro and in vivo assay from our library of mAbs against B. mallei and B. pseudomallei. We initially generated the mouse immunoglobulin variable heavy chain (VH) and light chain (VL) regions from each of these six selected mAbs using a phage display scFv technology. We determined the coding sequences of the VH and VL regions and successfully constructed two B. mallei-specific scFv phage antibodies consisting of two different VH (VH1 and VH2) and one Vλ1 families. Four scFvs constructed against B. pseudomallei had two VH (VH1 and VH6) and two VL (Vκ4/5 and Vκ21) families. All of six scFv antibodies constructed demonstrated good binding activity without any rounds of biopanning against B. mallei (M5D and M18F were 0.425 and 0.480 at OD405nm) and B. pseudomallei (P1E7, P2I67, P7C6, and P7F4 were 0.523, 0.859, 0.775, and 0.449 at OD405nm) by ELISA, respectively. A comparison of the immunoglobulin gene segments revealed that the gene sequences in complementarity-determining regions (CDRs) of three out of four B. pseudomallei-specific scFvs are highly conserved. We determined that the two B. mallei-specific scFvs have different CDRs in the VH, but the amino acid sequences of CDRs in the VL are conserved. This high sequence homology found in CDRs of VH or VL of these mAbs contributes to our better understanding and determination of binding to the specific antigenic epitope(s). The scFv phage display technology may be a valuable tool to develop and engineer mAbs with improved antigen-binding affinity.     

扩增抗体重链可变区的一个高效方法

从杂交瘤细胞或脾细胞中扩增抗体可变区，是构建单链抗体（Single-chain Fv fragment，ScFv）、克隆单克隆抗体和研究抗原与抗体相互作用的关键一步。而抗体可变区（Variable regions，Fv）高度变异，扩增相对困难，至今仍是一个难点，有待解决。我们在构建ScFv过程中，发现2株杂交瘤细胞用普通方法难于扩增出抗体重链可变区（Heavy—chain variable region，VH）抗体。于是提出了一种多序列比对和简并引物设计算法，并加以实现，设计出通用的鼠源性抗体VH引物；应用上述引物，采用反向多聚酶链反应（Reversepopymerase chain reaction，reversePCR）方法——3’RACE和5’RACE法，准确地扩增出2株杂交瘤细胞的VH基因。该算法很好地解决了引物特异性和最小化问题，具有简单、实现容易的特点，可用于基因家族中未知基因克隆和文库构建中的引物设计。与反向PCR方法结合，提高了难扩增的未知基因的成功率。     

Nucleotide sequence analysis of CDR3 elements of a panel of anti-peptide monoclonal antibodies recognizing parathyroid hormone-related protein.

Nucleotide sequences of heavy (VH) and light (VL) chain variable region complementarity determining regions have been determined from in vitro amplified mRNA isolated from a panel of monoclonal antibodies (mAb) raised to a synthetic 34mer peptide representing the N-terminal portion of human parathyroid hormone-related protein (PTHrP or parathyrin) reported to contain an immunodominant epitope. These mAb vary in affinity for the synthetic peptide and native PTHrP (Ka between 5.9 x 10(8) and 1.9 x 10(11)l/M). All 10 mAb studied were found were found to utilized restricted VH2, V kappa 2, JH4 and J kappa 1 family genes. Significant differences in the length and sequence of D elements were found; however 9/10 mAb utilize members of the DSP2 family. Significantly, two broad ranges of affinity could be determined based on the presence of Asp or Ala at residue 101 in JH.     

Baculovirus expression cassette vectors for rapid production of complete human IgG from phage display selected antibody fragments

For the expression of human intact IgG antibodies, we have constructed a set of baculovirus expression vectors designed to facilitate rapid insertion of heavy and light chain genes of Fab or scFv antibodies derived from phage display antibody libraries. By linking them to human constant or Fc regions, expression of complete human immunoglobulin molecules was achieved in insect cells by infection with recombinant baculovirus. The IgG expression cassette vectors are based on the backbone vector which contains two back to back polyhedron and p10 promoters. The IgG expression cassette elements, including the authentic IgG lambda or kappa and heavy chain signal sequences, as well as light chain (lambda or kappa) and heavy chain constant region genes are combined in a single vector and are controlled by the p10 and polyhedron promoter respectively. Either of VL or Fab-L and VH or Fab-Fd genes from common phage display systems can be directly inserted into one of the cassette vectors through in-frame cloning sites. This design of a single cassette vector combining heavy and light chain expression elements allowed rapid production and secretion of correctly processed and assembled intact immunoglobulins from recombinant baculovirus infected insect cells. The recombinant antibodies showed the expected molecular size of the H2L2 heterodimer in non reducing SDS-PAGE. No apparent differences were found between the expression level of heavy and light chains, and antigen binding function was preserved. For various antibodies, yields between 6 and 18 mg/l IgG were obtained.     

Improvement of anti-Burkholderia mouse monoclonal antibody from various phage-displayed single-chain antibody libraries

To improve anti-Burkholderia monoclonal antibody (MAb) binding affinity, six single chain variable fragments (scFvs) constructed previously were used as scaffolds to construct large highly-diversified phage-displayed mouse scFv random and domain libraries. First, we employed random mutagenesis to introduce random point mutations into entire variable regions, generating six random libraries. Additionally, the oligonucleotide-directed mutagenesis was targeted on complementarity-determining region 3 (CDR3) from each variable region of heavy (VH) and light chains (VL) derived from six scFvs, and generated eighteen domain libraries including six VH CDR3, six VL CDR3, and six combined VH/VL CDR3 mutated domains, respectively. We collected high scFvs binders through panning experiment over the large (size ~1 × 10?) random and domain libraries. The quality of the libraries was validated by successful selection of high-affinity clones. Random mutagenesis generated many mutant scFv clones having more than one amino acid changes around framework regions, but not many in CDRs. Surprisingly, the resulting eight higher scFv binders were selected from CDR3 mutations, but not from random mutations. Six of them resulted from CDR3 mutations of light chain, except for two scFvs from heavy chain, showing both Burkholderia pseudomallei and Burkholderia mallei had preferentially influenced the VL CDR3. Furthermore, all eight higher scFvs converted to full format human IgG1 antibodies were expressed transiently in 293T cell line. Five chimeric MAbs showed improved higher binding activity, as much as 0.2-0.3 at O.D. 405 nm, than positive control MAbs. These libraries could be valuable sources for selection of anti-Burkholderia antibodies and discovery of the relevant epitope(s) for developing effective vaccines or therapeutics.     

Human monoclonal striational autoantibodies isolated from thymic B lymphocytes of patients with myasthenia gravis use VH and VL gene segments associated with the autoimmune repertoire.

The production of autoantibodies reactive with components of skeletal muscle is characteristic of myasthenia gravis (MG) and is strongly associated with the presence of thymic epithelial tumors in patients with MG. The nucleotide sequences of the heavy and light chain variable regions (VH and VL) of three human monoclonal striated muscle antibodies (StrAb) isolated from thymic B lymphocyte lines from two patients with MG and thymoma were analyzed. The VH and VL gene segments used by these anti-striated muscle antibodies appear to be derived from the same repertoire of gene segments as have been found in other autoantibodies isolated from patients with a number of different autoimmune diseases. While the IgM StrAb SA-1A is a direct copy of germ-line VH and VL gene segments, analysis of the IgG StrAb SA-4A and SA-4B, which may be more representative of antibodies found in the serum of MG patients, suggests that the processes of antigenic selection and somatic mutation may contribute to the generation of autoantibodies in MG.     

米尔比霉素肟化物ScFv噬菌体展示抗体库的构建

目的构建具16元大环内酯共性结构小分子物质的特异性抗体库。方法免疫原MILO-BSA免疫小鼠后从脾细胞中提取总RNA,RT-PCR扩增得到全套抗体重链可变区基因(VH)及轻链可变区基因(VL),SOE-PCR将VH、VL片段拼接扩增得到单链抗体可变区基因片段(ScFv)。将ScFv基因克隆到噬菌粒载体pCANTAB5E中,电转化感受态大肠杆菌,辅助噬菌体超感染得到上清液即为噬菌体抗体库。结果RT-PCR扩增出长360bp左右的抗体重链可变区基因(VH)及340bp左右的轻链可变区基因(VL),SOE-PCR扩增得到750bp左右的ScFv基因片段,成功构建了库容量为2.4×106,滴度为2.0×1012pfu/mL的噬菌体单链抗体库。结论构建的抗体库目的片段连接率较高,多样性较好,为下一步16元大环内酯类小分子物质高特异性抗体的筛选奠了基础。     

抗人CD16单克隆抗体可变区基因的克隆和表达

目的：克隆抗人CD16单克隆抗体重，轻链可变区（VH，VL）基因并合成单链抗体（ScFv)基因。方法：从分泌抗人CD16单克隆抗体的杂交瘤细胞B88－9中提取总RNA，应用RT－PCR技术获得抗CD16单克隆抗体的VH，VL基因，用连接肽（Linker)肽VH和VL连接成具有VH－Linker-VL结构的ScFv基因，将其克隆到表达载体pcDNA3.1( )，并转杂COS－7细胞。结果：VH基因长度为354bp，属于鼠抗体可变区重链基因家族I（B）亚群，VL基因长度为333bp，属于鼠抗体可变区kappa轻链基因家族Ⅲ亚群，采用夹心ELISA方法检测到ScFv的表达。结论：抗人CD16单克隆抗体VH与VL基因的克隆和ScFv基因的构建为基于CD16的导向免疫治疗奠定了基础。     

HBV Pre S2 3B9单抗轻、重链可变区基因的分离,序列分析及单链可变区抗体基因的构建

3B9(杂交瘤细胞)是一株分泌HBV Pre S2抗原的单抗细胞,为从分子水平分析、了解3B9株进而为制备新一代基因工程抗体奠定基础,用分子生物学技术,提取3B9杂交瘤细胞总RNA,经反转录、PCR分离获得了3B9单抗的轻、重链可变区基因,并利用PCR及双脱氧核苷酸链末端终止法对3B9单抗的可变区基因的核苷酸序列进行了分析。结果表明,3B9单抗轻链隶属小鼠Ig Kappa轻链第Ⅱ亚组,JK_1基因重排;而3B9单抗重链隶属小鼠Ⅰg重链第Ⅱc亚组。在此基础上,作者用一编码疏水性多肽接头的DNA片断将3B9单抗轻、重链可变区基因连接,成功地构建了3B9单抗单链可变区抗体基因。为下一步克隆、表达有抗原结合活性的单链抗体打下了良好的物质基础。     

Rabbit antisera to the variable region domains of an anti-alpha(1----6) dextran using E. coli-produced VL and VH fusion proteins as immunogens.

Bacteria were engineered for the expression of mouse immunoglobulin light chain variable region (VL) and heavy chain variable region (VH) fusion proteins. cDNAs encoding the VL and VH of anti-alpha(1----6)dextran hybridoma protein 19.22.1 were inserted into the pATH 10 prokaryotic expression vector downstream of trp operon sequences. V domains joined to approximately 330 amino acids of the trp E gene product encoded by the expression plasmids accumulated at high levels in E. coli. In addition, the VL domain was expressed with a 15 amino acid extension at low levels in lon mutant bacteria. The trp E-VL and trp E-VH proteins were used to raise antisera in rabbits and the V specificity of the sera demonstrated.     

Molecular analysis of the murine lupus-associated anti-self response: involvement of a large number of heavy and light chain variable region genes

The mRNA encoding heavy and light chains of a hybridoma-derived monoclonal IgM, kappa anti-immunoglobulin (rheumatoid factor) and an IgG3, kappa anti-histone autoantibody from systemic lupus erythematosus and arthritis-prone MRL/Mp-lpr/lpr mice have been molecularly cloned, and the nucleotide sequences corresponding to their variable regions have been determined. To investigate whether autoantibodies with specificities frequently observed in lupus disease might share common structural components, the sequences obtained in this study have been compared with those of a monoclonal MRL/Mp-lpr/lpr IgM, kappa anti-DNA autoantibody previously analyzed in our laboratory (J. Exp. Med. 1985. 161: 805). The 3 immunoglobulins employed different heavy chain variable region (VH) genes belonging to the large J588 VH gene family, kappa light chain variable region (V kappa) genes from 3 different V kappa groups, and different diversity and joining segments. Our findings suggest that murine lupus-associated autoantibodies of different specificities do not have genetic components in common to signal their self-reactive nature and are encoded by a large number of immunoglobulin gene elements.     

Structural characterization of human monoclonal cold agglutinins: evidence for a distinct primary sequence-defined VH4 idiotype

Cold agglutinins that bind the developmentally regulated I red cell determinant occur naturally among human monoclonal IgM proteins. These autoantibodies are known to use light chains that derive mainly from the minor kappa III (kappa III) variable region subgroup. The kappa III subgroup is also highly expressed in monoclonal rheumatoid factors. However, while most monoclonal rheumatoid factors use structurally homologous heavy chains that derive from the VH1 family, information regarding the structure of the cold agglutinin heavy chains remains fragmentary. We demonstrate here that the kappa III cold agglutinin autoantibodies exclusively use heavy chains that derive from the VH4 family. Furthermore, these autoantibody heavy chains all express the same primary sequence-defined idiotype, corresponding to the second hypervariable region. These data indicate that cold agglutinins use a remarkably homogeneous subset of heavy chain variable regions. Moreover, unique patterns of preferential VH and VL pairing clearly distinguish the anti-I cold agglutinins from all other known monoreactive autoantibodies.     

Three monoclonal immunoglobulins, an IgG2(kappa), an IgM(kappa) and an IgM/A hybrid, in one patient. II. Sharing of common variable regions

Amino acid sequencing and haemagglutination inhibition studies were performed on three monoclonal immunoglobulins (an IgG2, an IgM and an IgM/A hybrid) isolated from a patient afflicted with a multiple gammopathy. The results demonstrated that all three proteins have shared idiotypic determinant(s). Furthermore, the light chains of all three paraproteins have identical NH2-terminal amino-acid sequences at all positions determined thus far. Similarly, the NH2-terminal amino-acid sequence of the gamma 2 chains is identical to that of the mu chain. The evidence strongly suggests a common ancestral clonal origin for cells which produce these paraproteins and that identical variable region (VH and VL) genes were used by the ESM lymphocyte subclones in the biosynthesis of the respective IgM, IgG, and IgM/A hybrid molecules. The occurrence of a mu/alpha hybrid chain, which shares identical V regions with a mu and a gamma chain, is consistent with the concept that IgM-producing cells can develop directly into cells producing other classes of immunoglobulins via separate pathways during B-cell maturation.