鉴定单克隆抗体识别蛋白质抗原表位方法的建立

为了建立鉴定治疗性单克隆抗体识别蛋白质抗原表位的方法,选择程序死亡受体-1(PD-1)作为目的蛋白。基于丙氨酸扫描策略,建立了定点突变技术和哺乳动物细胞表达系统相结合的抗原突变体快速表达方法,确定了真核表达元件扩增和细胞转染表达的条件。共表达了150个PD-1蛋白突变体,鉴定了这些突变体与抗PD-1抗体帕博利珠单抗的结合能力。根据蛋白突变体与抗体的结合力并结合蛋白结构分析确定了帕博利珠单抗的抗原表位,与已报道的基于晶体结构的抗原表位高度一致,表明本方法操作简单、准确性高,可用于治疗性单克隆抗体的抗原表位作图。     

Cell-mediated lysis of tumor targets directed by murine monoclonal antibodies of IgM and all IgG isotypes

Seven murine monoclonal antibodies to antigens expressed on T lymphoma targets were tested for directing antibody-dependent cellular cytotoxicity (ADCC). Peptone-induced peritoneal exudate macrophages, the LPS-stimulated RAW264.10 cell line, and human blood nonadherent mononuclear leukocytes were used as effector cells. All six IgG monoclonals tested, representing the four murine IgG isotypes and directed against four antigens (Thy-1.2, H-2k, Ly-2.1, Ly-9.2), were all active in ADCC. In contrast, an IgM anti-Thy-1.2 showed no activity despite very high C-cytotoxic titers. Thus, there does not seem to be any restriction among IgG classes for directing ADCC to tumor targets mediated by murine macrophages or human K cells.     

Differential endocytosis of IgM and IgD in murine B cell lines

The internalization of surface immunoglobulin (Ig) by B lymphocytes is the first step in the antigen-presenting function performed by these cells. Mature B cells coexpress on their surface IgM and IgD. At this time, there is controversy over whether these two isotypes serve different functions in the antigen-presenting process. The results presented here show that the intracellular pattern of distribution of IgM and IgD after internalization is strikingly different in the B cell lines studied. These findings support the hypothesis that the role of the two Ig classes in the antigen-presenting function may be different.     

Heterogeneity of microtubules recognized by monoclonal antibodies to alpha-tubulin

A set of four monoclonal antibodies against tubulin (TU-01, TU-02, TU-03, and TU-04) were produced using pig brain microtubule protein as antigen. Their characterization shows that all recognize antigenic determinants located on the tubulin alpha-subunit. However, peptide mapping of isolated alpha-tubulin, followed by immunoblotting with the monoclonal antibodies, shows that the antigenic determinants are located on different peptide fragments in at least three cases. The immunoreactivity with tubulins from different cells and tissues, ranging from eukaryotic microorganisms to man, was studied by immunoblotting and immunofluorescence. The antigenic determinants recognized by the antibodies are not uniformly distributed but, in some instances, are absent from tubulins of lower eukaryotic cells. These antibodies also make it possible to distinguish between different sets of microtubules within individual cells. Antigenically different microtubules are particularly evident in mouse spermatozoa and in some protozoa (T. vaginalis, H. muscarum, L. tropica, N. gruberi) possessing different sets of microtubules with different functions. These monoclonal antibodies can clearly identify the heterogeneity of tubulin or microtubules both from different organisms and within the same cell.     

Circulating microparticles carry oxidation-specific epitopes and are recognized by natural IgM antibodies

Oxidation-specific epitopes (OSEs) present on apoptotic cells and oxidized low density lipoprotein (OxLDL) represent danger-associated molecular patterns that are recognized by different arcs of innate immunity, including natural IgM antibodies. Here, we investigated whether circulating microparticles (MPs), which are small membrane vesicles released by apoptotic or activated cells, are physiological carriers of OSEs. OSEs on circulating MPs isolated from healthy donors and patients with ST-segment elevation myocardial infarction (STE-MI) were characterized by flow cytometry using a panel of OSE-specific monoclonal antibodies. We found that a subset of MPs carry OSEs on their surface, predominantly malondialdehyde (MDA) epitopes. Consistent with this, a majority of IgM antibodies bound on the surface of circulating MPs were found to have specificity for MDA-modified LDL. Moreover, we show that MPs can stimulate THP-1 (human acute monocytic leukemia cell line) and human primary monocytes to produce interleukin 8, which can be inhibited by a monoclonal IgM with specificity for MDA epitopes. Finally, we show that MDA⁺ MPs are elevated at the culprit lesion site of patients with STE-MI. Our results identify a subset of OSE⁺ MPs that are bound by OxLDL-specific IgM. These findings demonstrate a novel mechanism by which anti-OxLDL IgM antibodies could mediate protective functions in CVD.     

Carbohydrate sequences detected by murine monoclonal antibodies

The carbohydrate sequences of cell surface glycolipids change during differentiation and oncogenic transformation. To detect these structural changes, murine monoclonal antibodies have been produced in many different laboratories. Some of these antibodies are used to distinguish various cell types such as normal and transformed cells, while others are used to analyze developmentally regulated antigens. Recently, the structures of many of these carbohydrate antigens have been determined. The availability of these well-defined monoclonal antibodies will be useful for the study of the regulation and function of glycoconjugates.     

Serologic dissection of HLA-D specificities by the use of monoclonal antibodies

To study the gene products of the HLA complex, we produced two monoclonal antibodies, termed HU-18 and HU-23. They were active in complement-dependent cytotoxicity and detected B-cell alloantigens encoded by a locus (or loci) linked to HLA. When three types of HLA-DR4 homozygous B-cell lines with different HLA-D specificities were tested for reactivity with HU-18 and HU-23, they displayed distinct reaction patterns depending on the HLA-D specificities they possessed: EBV-Wa (HLA-DYT homozygous), negative for both HU-18 and HU-23; KT2 and KOB (HLA-DKT2 homozygous), positive only for HU-18; and ER (HLA-Dw4 homozygous), positive for both. These differential reaction patterns were further confirmed by testing against a panel of 17 HLA-DR4-positive peripheral blood lymphocytes with known HLA-D specificities. Thus, these monoclonal antibodies allow us to identify HLA-DYT, HLA-DKT2, and HLA-Dw4 solely by serologic methods. This is the first clearcut serologic identification of these three HLA-DR4-associated HLA-D specificities, which have been indistinguishable by conventional serology and identified only by cellular techniques. It is hoped that immunochemical investigations using HU-18 and HU-23 will advance our understanding of the HLA-D region on a molecular level.     

Serologic dissection of HLA-D specificities by the use of monoclonal antibodies

Three cytotoxic monoclonal antibodies, HU-11, HU-32, and HU-33, specific for human Ia-like antigens were used to analyze the two HLA-DR2-associated HLA-D specificities, HLA-Dw2 and HLA-Dw12. In the HLA-Dw2, DR2, MB1 homozygous B-cell line EB-CMG, the binding of radiolabeled HU-32 and HU-33 was strongly inhibited by the addition of nonlabeled HU-11, whereas no inhibition occurred in the HLA-Dw12, DR2, MB1 homozygous B-cell line EB-KT. To confirm this differential inhibition pattern further, F(ab')2 fragments were prepared from HU-11, and their ability to inhibit complement-dependent lysis mediated by HU-32 and HU-33 was assessed against a total of five homozygous typing cell lines homozygous for HLA-Dw2, DR2, MB1 or HLA-Dw12, DR2, MB1, including EB-CMG and EB-KT. Here again, the same differential inhibition pattern as that observed in the radiobinding inhibition assays was obtained. Thus, the data suggest that the two kinds of HLA-DR2-positive homozygous typing cell lines with distinct HLA-D specificity can be distinguished from each other by using solely serologic methods. This is the first clear-cut serologic distinction made between homozygous typing cells defining HLA-Dw2 and those defining HLA-Dw12, since no serologic means that enables one to distinguish one from the other has been available.     

Regional specificities of monoclonal anti-human apolipoprotein B antibodies

The usefulness of monoclonal antibodies as probes of protein structure is directly related to knowledge of the structures and locations of the epitopes with which they interact. In this report we provide a detailed map of 13 epitopes on apoB-100 defined by our anti-apoB monoclonal antibodies based on current information on the amino acid sequence of apoB-100. To localize antibody specificities to smaller regions along the linear sequence of the apoB-100 molecule we used a) thrombin- and kallikrein-generated fragments of apoB-100; b) beta-galactosidase- apoB fusion proteins; c) heparin; and d) antibody versus antibody competition experiments. Most of the monoclonal antibodies elicited by immunization with LDL were directed towards epitopes within the first 1279 amino terminal (T4/K2 fragments) or last 1292 carboxyl terminal amino acid residues (T2/K4 fragments) of apoB-100. One epitope localized to the mid-portion of apoB-100 was elicited by immunization with VLDL (D7.2). Saturating amounts of heparin bound to LDL did not inhibit the binding of any of the monoclonal antibodies to their respective epitopes on apoB-100, indicating that none of the antibody determinants is situated close to any of the reported heparin binding sites on LDL apoB. We examined the expression of apoB epitopes on VLDL subfractions and LDL isolated from a normolipidemic donor. The apparent affinities with which the antibodies interacted with their respective epitopes on the VLDL subfractions and LDL uniformly increased as follows: LDL greater than VLDL3 greater than VLDL2 greater than VLDL1, suggesting that each of the major regions of apoB-100 is progressively more exposed as normal VLDL particles become smaller in size and epitopes are most exposed in LDL. Previous experiments utilizing hypertriglyceridemic VLDL subfractions yielded similar results, but the rank order of VLDL subfractions and LDL was not the same for all antibodies tested. Thus, differences in apoB epitope expression on VLDL particles of differing sizes is a general phenomenon, but the expression of apoB epitopes in hypertriglyceridemic VLDL appears to be more heterogeneous than is the case for VLDL from normolipidemic donors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparative studies of the antigens recognized by sperm-immobilizing monoclonal antibodies.

Characteristic properties of the antigens recognized by sperm-immobilizing monoclonal antibodies (SI-mAbs) from different sources were compared by ELISA competitive inhibition assay, Western blot analysis, chromatographic analysis, and enzymatic digestion studies. Among 9 SI-mAbs, human mAb H6-3C4 and three mouse mAbs--2C6, 2B6, and 2E5--also possessed strong sperm-agglutinating activity. Binding of human mAb H6-3C4 to sperm was strongly inhibited by the three mouse mAbs (2C6, 2B6, and 2E5), but not by the rat or the other four mouse mAbs. SDS-PAGE revealed that mAb H6-3C4 and three mouse mAbs recognized the same antigen molecules of 15-25 kDa present in both sperm extracts and seminal plasma. Chemical treatments with trifluoromethanesulfonic acid and sodium metaperiodate destroyed the antigen determinants recognized by the above four mAbs, as detected by both ELISA and antibody absorption tests. Western blot analysis revealed that the antigens were susceptible to treatments with papain, proteinase K, and N-glycanase, but resistant to trypsin, V8 protease, and thermolysin. These results indicate that one of the major antigens recognized by mAbs with sperm-immobilizing action may be a sperm membrane-associated glycoprotein of 15-25 kDa and the epitope may involve N-linked oligosaccharides.     

Characterization of epitopes recognized by anti-Streptococcus mutans P1 monoclonal antibodies

Sequences contributing to epitopes recognized by a panel of monoclonal antibodies (mAbs) against the Streptococcus mutans surface protein P1 were delineated by Western blot and enzyme-linked immunosorbent assay using a battery of deletion constructs and recombinant polypeptides. mAbs that recognize complex discontinuous epitopes reconstituted by combining the alanine-rich and proline-rich repeat domains and varying degrees of flanking sequence were identified as well as mAbs that bound epitopes contained within contiguous segments of P1. Cross-reactivity with SspA and SspB from Streptococcus gordonii is also reported. This information enables insight into the structure and function of a streptococcal adhesin and its correlates of protection and furthers our understanding of the immunomodulatory and bacterial-adherence inhibition activities of anti-P1 mAbs.     

Study of antigenic epitopes recognized by monoclonal antibodies to recombinant interferon-gamma

Seven hybridomas (BG 1-7) which secreted monoclonal antibodies against recombinant interferon-gamma were produced. The ascites fluids containing four of the seven monoclonal antibodies (BG 1-4) neutralized the antiviral activity of both natural and recombinant interferon-gamma. Competition between labeled and unlabeled monoclonal antibodies for interferon-gamma in a solid phase immunoassay showed that BG 1 was competed by both BG 3 and BG 4 but not by BG 2; BG 2 was competed by BG 3 but not by BG 1 nor by BG 4. These results suggest that human interferon-gamma has at least two antigenic epitopes; one of the epitopes reacted with BG 1 & BG 4 while the other reacted with BG 2; BG 3 either binds to a region overlapping with the other two epitopes or reacts with both epitopes. The antigenic epitopes recognized by these four neutralizing monoclonal antibodies are likely at or closely related to the active sites of interferon-gamma.     

Trypanosoma lewisi: stage-specific surface antigens and exoantigens recognized by monoclonal antibodies

The stage-specific expression of surface antigens by Trypanosoma lewisi was investigated using monoclonal antibodies directed against this parasite. Hybridomas secreting monoclonal antibodies were produced by the fusion of SP2/0-Ag 14 mouse plasmacytomas with spleen cells from rats infected previously with the Taliaferro strain of T. lewisi. Additivity enzyme-linked immunosorbent assays and indirect immunofluorescent antibody tests indicated the determinant recognized by monoclonal antibody TL40.3 (IgM) was different from those recognized by monoclonal antibodies TL40.1 (IgA), TL40.2 (IgM), and TL40.6 (IgG2 alpha). Monoclonal antibody TL40.3 agglutinated trypanosomes collected 3 days after parasite inoculation while monoclonal antibodies TL40.1, TL40.2, and TL40.6 agglutinated trypanosomes collected 6 days after inoculation. Since agglutinin titers against trypanosomes from irradiated (700 rad from a 60Co source) and nonirradiated rats were similar, expression of the antigens recognized by the monoclonal antibodies appeared to be independent of the immunological state of the host and the morphology of the parasite. The reproduction of T. lewisi in in vitro trypanostatic assays was inhibited only when the monoclonal antibodies were present in concentrations greater than or equal to those needed to agglutinate the trypanosomes. Monoclonal antibodies TL40.1 and TL40.3, but not TL40.2 and TL40.6, agglutinated erythrocytes collected later in the infection from irradiated, infected rats. None of the monoclonal antibodies agglutinated erythrocytes from nonirradiated, infected rats, from irradiated, noninfected rats or from nonirradiated, noninfected rats. This suggests that immunocompetent rats may make blocking antibodies against the exoantigens recognized by monoclonal antibodies TL40.1 and TL40.3.     

Prolamin variation and evolution in Triticeae as recognized by monoclonal antibodies.

The isopropanol-soluble seed storage proteins, prolamins, were studied in 18 different genera of the tribe Triticeae by gel electrophoresis, Coomassie staining, and immunoblot assays. The monoclonal antibodies were originally raised against cultivated barley (Hordeum vulgare L.) hordein, and their reactions had been tested earlier on wild Hordeum species. The study showed that all the investigated Triticeae species produce prolamins and that structural similarities can be found throughout the tribe. The presence of the same antigenic sites in all the species indicates that the polypeptides contain well-conserved regions. They also indicate that the prolamins of the Triticeae species have a common evolutionary origin. In all the investigated species an antigenic site that is common to the B- and C-hordeins of barley was detected. Some of the reacting polypeptides also contained a site that is only present in the B-hordeins. The B-hordein specific site was found in all genera except Agropyron, Hordelymus, and Secale. This shows that although there are similarities between individual polypeptides, the composition of the various prolamin groups may vary between different genera. In the polyploid Elymus species different banding patterns were observed depending on what basic genomes were represented. The results suggest a direct correlation between the presence of a fast migrating polypeptide containing the B-hordein specific site and the presence of the H genome.     

Canine lymphoma-associated antigens defined by murine monoclonal antibodies

Summary Lymphoma in dogs resembles human non-Hodgkin's lymphoma in pathological presentation, immunophenotype, and response to therapy, thus representing a good model for comparative studies with human disease. Monoclonal antibodies (MAbs) were derived from mice immunized with a dog lymphoma cell line. Three MAbs were selected for further application in immunophenotyping and immunotherapy. The binding specificities, antigen characterization, and isotypes for these MAbs are described.Supported by NCI grant CA-10815     

Cell receptors for baboon endogenous virus recognized by monoclonal antibodies.

Hybridomas from mice immunized with baboon endogenous virus (BaEV) from A204(M7) cells produced several antiviral monoclonal antibodies and, in addition, antibodies D-12 and E-4, which appeared to be virus specific because they reacted with BaEV but not with Mason-Pfizer virus or RD-114 virus. However, they also bound to human virus-free cells, and they did not recognize BaEV from bat or canine host cells. Cell membrane targets for these antibodies comigrated with an 18,000-dalton protein, which may contain specific determinants of BaEV receptors since antibody masking of these cell sites prevented BaEV but not Mason-Pfizer virus or RD-114 virus adsorption. However, RD-114 virus interfered with BaEV adsorption. Thus, the two viral receptors must be adjacent, but the antibody D-12 and E-4 targets are not within the active site of RD-114 virus receptor. Conversely, cell coating with BaEV from bat or canine hosts inhibited antibody D-12 binding. Noncultivated human lymphocytes and cells from fetal organs bound much less antibody D-12 than did cells from established cell lines, with a correlation between amounts of antibody D-12 acceptor sites and BaEV receptors. Thus, in vivo, BaEV infection of human cells may be inefficient. In vitro, antibody D-12 treatment of chronically infected A204(M7) cells caused intracellular accumulation of viral proteins and decreased virus release, with no such effect on RD-114 virus-producing cells. Canine cells bound antibody D-12 only if coated with BaEV from A204(M7) cells, indicating that the human determinant coadsorbed with the virions to animal cells. Possibly, determinants of cell receptors participate in BaEV maturation and become associated with the virions.     

Topographic antigenic determinants recognized by monoclonal antibodies to sperm whale myoglobin

Monoclonal antibodies of high affinity (approximately 10(9) M-1) for sperm whale myoglobin were studied to pinpoint the antigenic determinants with which they interact. None of 6 different monoclonal antibodies tested reacted with any of the 3 CNBr cleavage fragments which encompass the whole sequence of myoglobin, an indication that they react with determinants present only on the native structure. To identify these sites, we compared the affinities of each antibody for a series of 14 mammalian myoglobins of known sequence and similar tertiary structure. Correlation of sequence differences with relative affinities allowed us, thus far, to identify critical antigenic residues recognized by 3 of the antibodies. Two of these antibodies recognize groups of residues which are far apart in primary structure but close together in the 3-dimensional structure of the native myoglobin molecule, i.e. topographic determinants. The third antibody distinguishes 140 Lys leads to Asn plus, probably, surface residues nearby. These determinants differ from previously reported antigenic sites on sperm whale myoglobin both in that they are topographic, rather than sequential, and in that almost all the critical residues recognized by these antibodies are outside the previously reported sites. Monoclonal antibodies are sensitive to subtle changes, e.g. Glu leads to Asp, in the antigenic site.     

Topographic antigenic determinants recognized by monoclonal antibodies on human choriogonadotropin beta-subunit

We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptides spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex.