Mass spectrometry analyses of κ and λ fractions result in increased number of complementarity-determining region identifications

Sera from lung cancer patients contain antibodies against tumor-associated antigens. Specific amino acid sequences of the complementarity-determining regions (CDRs) in the antigen-binding fragment (Fab) of these antibodies have potential as lung cancer biomarkers. Detection and identification of CDRs by mass spectrometry can significantly be improved by reduction of the complexity of the immunoglobulin molecule. Our aim was to molecular dissect IgG into κ and λ fragments to reduce the complexity and thereby identify substantially more CDRs than by just total Fab isolation. We purified Fab, Fab-κ, Fab-λ, κ and λ light chains from serum from 10 stage I lung adenocarcinoma patients and 10 matched controls from the current and former smokers. After purification, the immunoglobulin fragments were enzymatically digested and measured by high-resolution mass spectrometry. Finally, we compared the number of CDRs identified in these immunoglobulin fragments with that in the Fab fragments. Twice as many CDRs were identified when Fab-κ, Fab-λ, κ and λ (3330) were combined than in the Fab fraction (1663) alone. The number of CDRs and κ:λ ratio was statistically similar in both cases and controls. Molecular dissection of IgG identifies significantly more CDRs, which increases the likelihood of finding lung cancer-related CDR sequences.     

Vasoactive intestinal peptide hydrolysis by antibody light chains

This paper describes evidence for hydrolysis of a neuropeptide, vasoactive intestinal peptide (VIP), by light chains purified from the IgG of a human subject positive for VIP binding antibodies. Purified IgG was digested with papain, resultant fragment antigen binding (Fab) fragments were reduced with 2-mercaptoethanol and alkylated with iodoacetamide, and light chains were purified by chromatography on immobilized antibodies to light chains and immobilized antibodies to heavy chains. Non-immunoglobulin components were undetectable in the light chain preparation, judged by sodium dodecyl sulfate-electrophoresis and Western blotting with anti-heavy and anti-light chain antibodies. The light chains hydrolyzed VIP with specific activity 32-fold greater than that of Fab, the pH optimum for light chain-mediated VIP hydrolysis was 7.0-7.5, and the hydrolytic activity was saturable (Vmax, 0.19 pmol/min/microgram light chains; substrate concentration at Vmax/2,380 nM).     

鼠源性抗雄性特异性抗原噬菌体Fab抗体的制备及分析

利用噬菌体抗体库筛选技术获得抗雄性特异性抗原的噬菌体Fab抗体,首次采用雄鼠脾细胞对鼠源性抗雄性特异性抗原噬菌体Fab抗体库进行3轮亲和富集和2轮雌鼠脾细胞吸附,对筛选后特异性噬菌体Fab抗体进行ELISA分析,重组率鉴定及基因测序分析。结果显示,5次筛选后的15个菌落中有9个能产生抗雄性特异性抗原特异性噬菌体抗体,噬菌体Fab抗体的基因重组率为60%,E5克隆的重链、轻链可变区序列分别属于VH1和VκⅣ基因家族,这为挑选出高亲和力的抗雄性特异性抗原噬菌体Fab抗体奠定了实验基础,将推进雄性特异性抗原及其抗体的研究进程,并为性别控制研究开创新途径。     

Biochemical studies of the excitable membrane of paramecium tetraurelia. IX. Antibodies against ciliary membrane proteins

The excitable ciliary membrane of Paramecium regulates the direction of the ciliary beat, and thereby the swimming behavior of this organism. One approach to the problem of identifying the molecular components of the excitable membrane is to use antibodies as probes of function. We produced rabbit antisera against isolated ciliary membranes and against partially purified immobilization antigens derived from three serotypes (A, B, and H), and used these antisera as reagents to explore the role of specific membrane proteins in the immobilization reaction and in behavior. The immobilization characteristics and serotype cross- reactivities of the antisera were examined. We identified the antigens recognized by these sera using immunodiffusion and immunoprecipitation with 35S-labeled ciliary membranes. The major antigen recognized in homologous combinations of antigen-antiserum is the immobilization antigen (i-antigen), approximately 250,000 mol wt. Several secondary antigens, including a family of polypeptides of 42,000-45,000 mol wt, are common to the membranes of serotypes A, B, and H, and antibodies against these secondary antigens can apparently immobilize cells. This characterization of antiserum specificity has provided the basis for our studies on the effects of the antibodies on electrophysiological properties of cells and electron microscopic localization studies, which are reported in the accompanying paper. We have also used these antibodies to study the mechanism of cell immobilization by antibodies against the i-antigen. Monovalent fragments (Fab) against purified i- antigens bound to, but did not immobilize, living cells. Subsequent addition of goat anti-Fab antibodies caused immediate immobilization, presumably by cross-linking Fab fragments already bound to the surface. We conclude that antigen-antibody interaction per se is not sufficient for immobilization, and that antibody bivalency, which allows antigen cross-linking, is essential.     

Linear epitope mapping by native mass spectrometry

The identification of antigenic epitopes is important for the optimization of monoclonal antibodies (mAbs) intended as therapeutic agents. MS has proven to be a powerful tool for the study of noncovalent molecular interactions such as those involved in antibody-antigen (Ab-Ag) binding. In this work, we described a novel methodology for mapping a linear epitope based on direct mass spectrometric measurement of Ab-Ag complexes. To demonstrate the utility of our methodology, we employed two approaches, epitope excision and epitope extraction, to study a model system consisting of a Fab antibody fragment with specificity toward the peptide aβ(1-40). In epitope excision, the Fab and aβ(1-40) complex was treated with proteolytic enzymes and the digested complexes were directly monitored by MS under native conditions. Mass differences between the Fab-aβ complex and the Fab control revealed the size of epitope peptides that were bound to the Fab. Using the epitope extraction approach, aβ(1-40) was first digested by Lys-C, and the fragment containing the epitope was selected by Fab binding. Data analysis allowed mapping of the epitope to aβ(16-27) which is in good agreement with previously unpublished data. The utility of the methodology was demonstrated by elucidating the binding epitopes for two full-length anti-aβ(1-40) mAbs.     

Enzymatic splitting of antibodies bound to immobilized antigen as a means of Fab fragments preparation]

A method of Fab fragments preparation by enzymatic splitting of antibodies bound to specific antigen immobilized on an insoluble support is described. The complex of rat muscle glyceraldehyde-3-phosphate dehydrogenase (GAPD), immobilized on Sepharose 4B, with anti-rat GAPD rabbit antibodies was digested with papain. The antigen was inaccessible to proteolysis under conditions employed. After 4 hrs of incubation with papain the antibody was completely split into non-precipitating fragments. The products of proteolysis not bound to Sepharose, were eluted with 0.1 M givcine buffer pH 2.5, and shown to correspond to Fab fragments.     

A new 85 kDa, breast tumour associated antigen — A potential diagnostic and prognostic agent

In an attempt to develop measures for early diagnosis and prognosis of the disease and to explore association of murine mammary tumour virus (MuMTV) or related virus in breast cancer, we purified a breast tumour associated antigen (BTAA) from the breast tumour tissues of untreated female cancer patients. The BTAA purified by DEAE discontinuous column chromatography followed by SE-HPLC was an 85 kDa glycoprotein. A high level of circulating antibodies against this antigen was observed, using ELISA, in all the untreated female breast cancer patients. The BTAA was not immunologically related to MuMTV antigens but strongly resembled an 83 kDa glycoprotein tumour associated antigen, purified from MuMTV induced mouse mammary tumour. In patients after surgical removal of the breast tumour, the circulating antibodies to the BTAA decreased gradually, but reappeared in the patients with secondary metastasis. In healthy age matched women or in female patients with carcinoma of tissues other than breast, no significant titre of the BTAA antibodies was observed.     

Guided selection of an anti-gamma-seminoprotein human Fab for antibody directed enzyme prodrug therapy of prostate cancer

Background The HAMA response is a major challenge when murine antibodies are repeatedly administered for antibody directed enzyme prodrug therapy in vivo. In this study we have achieved humanization of the anti-γ-seminoprotein E₄B₇ murine mAb by guided selection. Methods Using optimal Ig Fab primers, human Fd and CL gene repertoires were amplified by RT-PCR from PBMCs of prostate cancer patients. The human Lc gene repertoire was first paired with the murine Fd gene of E₄B₇ mAb to construct a pComb3X hybrid Fab display library. This hybrid library was screened with purified γ-seminoprotein antigen. The human Fd gene repertoire was then paired with the selected human Lc to construct a fully human Fab library. After four more rounds of panning, completely human Fab antibodies specific for γ-seminoprotein were selected and further identified. Results First, using the E₄B₇ Fd gene as a template, light chain shuffling was achieved by panning the hybrid library. Then, using the selected Lc as a template, a human Fab antibody against γ-seminoprotein was produced through heavy chain Fd shuffling. Western blotting, ELISA, and flow cytometry results demonstrated that the resulting human Fab antibody resembled the parental E₄B₇ mAb in that they both recognized the same epitope with similar affinities. Fluorescent cell staining and immunohistochemistry analysis further confirmed that this newly constructed human anti-γ-seminoprotein Fab antibody indeed specifically bound prostate cancer cells and tissue. Conclusions Through guided-selection, we successfully produced a human anti-γ-seminoprotein Fab antibody. This work lays the foundation for optimal antibody-directed enzyme prodrug therapy of prostate cancer using a fully human Fab antibody. Zhang Qing and Zhang Si-He are co-first authors on the publication.     

Construction and characterization of phage display library: recognition of mouse serologically detected male (SDM) antigen

Improvement of animal embryo sexing depends upon high-titer serologically detected male (SDM) antibody fragments. SDM sera collected from isogenic C57BL/7 female mice after inoculation with male spleen cells were characterized and used for construction of a recombinant Fab antibody library against SDM antigen, and used for analysis of the binding capacity and specificity to SDM antigen. The heavy-chain Fd and full-length light-chain kappa were amplified by RT-PCR from a mouse (#6) that'ed high-titer antiserum. The amplified product was inserted into the pComb3 vector followed by co-infections with the help phage VCSM 13 for construction of the phage library, which gave 1.5x10(7) colonies with the titer of 3.2x10(11) pfu/ml by a recombination rate of 80%. Sequence analysis of the PCR products of plasmid DNA of E5 clones showed that V(H) and V(kappa) had common characteristics shared by other known variable region of antibodies. The Fab antibody libraries against SDM antigen were enriched by three cycles of affinity enrichment with male spleen cells, and two cycles of non-specific absorption with female spleen cells. The ELISA results showed that 9 of 15 clones had binding capacity to the SDM antigen. This is the first report on a phage display library of SDM antigen. The mouse Fab antibody library could be used for identifying SDM antigen, and for the development of sex determination of early embryos in mammals.     

人源抗狂犬病毒单克隆抗体Fab段基因的获得和表达

运用噬菌体表面呈现（phage display）技术获得了人源抗狂犬病毒糖蛋白基因工程单克隆抗体Fab段基因及其表达。从狂犬病毒PM株Vero细胞疫苗免疫的人抗凝血中分离获得外周淋巴细胞,提取细胞总RNA,通过RTPCR方法,用一组人IgG Fab基因4特异性引物,从合成的cDNA中扩增了一组轻链和重链Fab段基因,将轻链和重链Fab段基因,将轻链和重链先后克隆入噬菌体载体pComb3,成功地建立了抗狂犬病毒抗原的方法,对此抗体库进行富积筛选表达,成功地获得了抗狂犬病毒的人源单抗Fab段基因及其在大肠杆菌中的有效表达,对其中一株单抗G10进行了较为系统的分析,发现它与一株鼠源中和性狂犬病毒糖蛋白特异性单抗存在竞争,证实该单抗能识别狂犬病毒糖蛋白,其序列资料分析表明,该单抗为一株新的抗狂犬病毒人源基因工程抗体。     

Antigen binding of human IgG Fabs mediate ERK-associated proliferation of human breast cancer cells

Serum-circulating antibody can be linked to poor outcomes in some cancer patients. To investigate the role of human antibodies in regulating tumor cell growth, we constructed a recombinant cDNA expression library of human IgG Fab from a patient with breast cancer. Clones were screened from the library with breast tumor cell lysate. Sequence analysis of the clones showed somatic hypermutations when compared to their closest VH/VL germ-line genes. Initial characterizations focused on five clones. All tested clones displayed stronger binding to antigen derived from primary breast cancers and established breast cancer cell lines than to normal breast tissues. In vitro functional studies showed that four out of five tested clones could stimulate the growth of MDA-MB-231 breast cancer cell lines, and one out of five was able to promote MCF-7 cell growth as well. Involvement of ERK2 pathway was observed. By 1H-NMR spectra and Western blot analysis, it was evident that two tested antibody Fabs are capable of interacting with sialic acid. Our study suggests a possible role for human antibody in promoting tumor cell growth by direct binding of IgG Fab to breast tumor antigen. Such studies prompt speculation regarding the role of serum antibodies in mediating tumor growth as well as their contribution to disease progression.     

Expression of furin-linked Fab fragments against anthrax toxin in a single mammalian expression vector

Human anti-recombinant protective antigen (rPA) Fab genes were previously cloned from single B cells of a donor immunized with anthrax vaccine using fluorescence activated cell sorting with fluorescein labeled rPA and single-cell PCR. The light and heavy chains were sub-cloned individually into mammalian expression vectors pSecTag2B or pEXPR44, respectively, and expressed in the same CHOK1 cells. Alternatively, the same heavy and light chains were linked together, using PCR, with an in-frame sequence coding for a furin cleavage site. This construct was cloned into pSecTag2B and expressed in CHOK1 cells. Once expressed, the individual chains combined in vivo to form a Fab fragment which was purified as a single protein when either method was utilized. The human Fab antibodies produced by this technique were functional when tested in Western blots using the recombinant PA antigen as the target.     

Fingerprinting the circulating repertoire of antibodies from cancer patients

Recognition of molecular diversity in disease is required for the development of targeted therapies. We have developed a screening method based on phage display to select peptides recognized by the repertoire of circulating tumor-associated antibodies. Here we isolated peptides recognized by antibodies purified from the serum of prostate cancer patients. We identified a consensus motif, NX(S/T)DK(S/T), that bound selectively to circulating antibodies from cancer patients over control antibodies from blood donors. We validated this motif by showing that positive serum reactivity to the peptide was specifically linked to disease progression and to shorter survival in a large patient population. Moreover, we identified the corresponding protein eliciting the immune response. Finally, we showed a strong and specific positive correlation between serum reactivity to the tumor antigen, development of metastatic androgen-independent disease, and shorter overall survival. Exploiting the differential humoral response to cancer through such an approach may identify molecular markers and targets for diagnostic and therapeutic intervention.     

Purification of antibody Fab and F(ab')2 fragments using Gradiflow technology

The Gradiflow, a preparative electrophoresis instrument designed to separate molecules on the basis of their size and charge, was used to purify antibody Fab and F(ab')2 fragments. The method described is charge based, utilizing the difference in the pI between the antibody Fab/F(ab')2 fragments and antibody Fc fragments that occur after enzyme digestion of whole antibody molecules. This method of purification was successful across a range of monoclonal and polyclonal antibodies. In particular, F(ab')2 fragments were purified from a number of mouse monoclonal antibodies (both IgG1 and IgG2a isotypes) and Fab fragments were purified from egg yolk IgY polyclonal antibodies. This is a rapid purification method which has advantages over alternative methods that usually comprise ion exchange and gel filtration chromatography. This method may be applicable to most antibody digest preparations.     

Primary antibody-Fab fragment complexes: a flexible alternative to traditional direct and indirect immunolabeling techniques.

Immunolabeling with immune complexes of primary and secondary antibodies offers an attractive method for detecting and quantifying specific antigen. Primary antibodies maintain their affinity for specific antigen after labeling with Fab fragments in vitro. Incubation of these immune complexes with excess normal serum from the same species as the primary antibody prevents free Fab fragments from recognizing immunoglobulin. Effectively a hybrid between traditional direct and indirect immunolabeling techniques, this simple technique allows primary antibodies to be non-covalently labeled with a variety of reporter molecules as and when required. Using complexes containing Fab fragments that recognize both the Fc and F(ab')2 regions of IgG, we show that this approach prevents nonspecific labeling of endogenous immunoglobulin, can be used to simultaneously detect multiple antigens with primary antibodies derived from the same species, and allows the same polyclonal antibody to be used for both antigen capture and detection in ELISA.     

抗金黄色葡萄球菌IgY的酶解稳定性及活性研究

目的:考察抗金黄色葡萄球菌特异性IgY的酶解稳定性和酶解产物的活性。方法:以灭活的金黄色葡萄球菌免疫产蛋母鸡,抗体经水稀释及盐析分离纯化。抗体在不同条件下经胃蛋白酶或胰蛋白酶消化。抗体及酶解产物的特异性和活性分别用ELISA法和凝集法检测,酶解产物用SDS-PAGE和Western-blotting鉴定。结果:免疫产生的抗体具有特异性,能与金黄色葡萄球菌凝集而抑制其生长。分离纯化后抗体纯度与标准IgY相近。IgY在pH<4时完全酶解;pH等于4时部分酶解生成Fab片断,具有特异性和凝集活性;pH>4时,抗体稳定,不发生酶解。结论:抗金黄色葡萄球菌特异性IgY及其酶解生成的Fab片断,具有特异性和抑菌活性,有望代替抗生素用于细菌感染性疾病的治疗。     

Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: Papain digestion of mAb and transient expression in mammalian cells

Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of antibody–antigen interactions. One route utilizes papain digestion of an intact monoclonal antibody for Fab fragment production. After digestion, separation of the Fab fragment from the Fc (fragment that crystallizes) and residual intact antibody was achieved using protein A affinity chromatography. In another route, His-tagged Fab fragments were obtained by transient expression of an appropriate construct in mammalian cells, and typical yields are 1–20 mg of Fab fragment per liter of cell culture. The His-tagged Fab fragments were first captured using immobilized metal affinity chromatography (IMAC). To provide high quality protein sample for crystallization, Fabs from either proteolytic digestion or from direct expression were further purified using size-exclusion chromatography (SEC) and/or ion-exchange chromatography (IEC). The purified Fab fragments were characterized by mass spectrometry, SDS–PAGE, dynamic light scattering, and circular dichroism. Crystallization experiments demonstrated that the Fab fragments are of high quality to produce diffraction quality crystals suitable for X-ray crystallographic analysis.     

Chimeric fab fragments of antibodies to recombinant Ebola virus glycoprotein

Previously, we have determined the nucleotide and amino acid sequences of the variable domains of three mouse monoclonal antibodies specific to the individual epitopes of the Ebola virus glycoprotein: GPE118 (IgG), GPE325 (IgM) and GPE534 (IgG) [1]. In the present paper, chimeric Fab fragments of Fab118, Fab325, and Fab534 antibodies were obtained based on the variable domains of murine antibodies by attaching CH1 and CL constant regions of human kappa-IgG1 to them. The recombinant chimeric Fab fragments were synthesized in the heterologous expression system Escherichia coli, isolated and purified using metal chelate affinity chromatography. The immunochemical properties of the obtained Fab fragments were studied by immunoblotting techniques as well as indirect and competitive ELISA using recombinant Ebola virus proteins: EBOV rGPdTM (recombinant glycoprotein of Ebola hemorrhagic fever virus without the transmembrane domain), NP (nucleoprotein) and VP40 (structural protein). The identity of recombinant chimeric Fab fragments, as well as their specificity to the recombinant glycoprotein of Ebola hemorrhagic fever virus (EBOV GP) was proved. The results of indirect ELISA evidence the absence of immunological cross-reactivity to NP and VP40 proteins of Ebola virus. The dissociation constants of the antigen-antibody complex K _d equal to 5.0, 1.0 and 1.0 nM for Fab118, Fab325 and Fab534, respectively, were determined; they indicate high affinity of the obtained experimental samples to EBOV GP. The epitope specificity of Fab fragments was studied using a panel of commercial neutralizing antibodies. It was found that all studied antibodies to EBOV GP are targeted to different epitopes, while the epitopes of the recombinant chimeric Fab fragments and original murine monoclonal antibodies (mAbs) coincide. All the obtained and studied mAbs to EBOV GP are specific to epitopes that coincide or overlap the epitopes of three commercial neutralizing mAbs to Ebola virus: epitopes Fab118 and Fab325 overlap the epitope of the known commercial mAb h13F6; Fab325 epitope also overlaps mAb c6D8 epitope; Fab534 epitope is located near mAb KZ52 conformational epitope, in the formation of which amino acid residues of GP1 and GP2 domains of EBOV GP are involved.     

RNA recognition by a human antibody against brain cytoplasmic 200 RNA

Diverse functional RNAs participate in a wide range of cellular processes. The RNA structure is critical for function, either on its own or as a complex form with proteins and other ligands. Therefore, analysis of the RNA conformation in cells is essential for understanding their functional mechanisms. However, no appropriate methods have been established as yet. Here, we developed an efficient strategy for panning and affinity maturation of anti-RNA human monoclonal antibodies from a naïve antigen binding fragment (Fab) combinatorial phage library. Brain cytoplasmic 200 (BC200) RNA, which is also highly expressed in some tumors, was used as an RNA antigen. We identified MabBC200-A3 as the optimal binding antibody. Mutagenesis and SELEX experiments showed that the antibody recognized a domain of BC200 in a structure- and sequence-dependent manner. Various breast cancer cell lines were further examined for BC200 RNA expression using conventional hybridization and immunoanalysis with MabBC200-A3 to see whether the antibody specifically recognizes BC200 RNA among the total purified RNAs. The amounts of antibody-recognizable BC200 RNA were consistent with hybridization signals among the cell lines. Furthermore, the antibody was able to discriminate BC200 RNA from other RNAs, supporting the utility of this antibody as a specific RNA structure-recognizing probe. Intriguingly, however, when permeabilized cells were subjected to immunoanalysis instead of purified total RNA, the amount of antibody-recognizable RNA was not correlated with the cellular level of BC200 RNA, indicating that BC200 RNA exists as two distinct forms (antibody-recognizable and nonrecognizable) in breast cancer cells and that their distribution depends on the cell type. Our results clearly demonstrate that anti-RNA antibodies provide an effective novel tool for detecting and analyzing RNA conformation.     

Production of recombinant humanized anti-HBsAg Fab fragment from Pichia pastoris by fermentation

In this report, we describe the high-yield secretory expression of the recombinant human anti-HBsAg Fab fragment from Pichia pastoris that was achieved by co-integration of the genes encoding the heavy and light chains (both under the control of alcohol oxidase promoter) into the genome of the yeast cells. The fed-batch fermentations were carried out in a 5 L scale. Both chains of the Fab were successfully expressed upon methanol induction. The absorbance (OD600) of the broth can reach 350 approximately 500 at the end of fed-batch phase. After the induction, the expression level of the recombinant Fab (soluble) reached 420 approximately 458 mg/L. The recombinant Fab fragment was purified from the crude culture supernatant by ion exchange chromatography and the purity of the recombinant Fab fragment was over 95%. The affinity activities of the crude fermentation supernatant and the purified Fab were analyzed by indirect ELISA, which showed that the purified recombinant Fab fragment had high affinity activity with hepatitis B surface antigen.