Blocking of cytotoxic T cell function by monoclonal antibodies against the CD45 antigen (T200/leukocyte-common antigen)

Cytotoxic T cells are important effectors in graft rejection and antiviral immunity. A full identification of the functional surface molecules used by these cells may help in determining the best strategies for the therapeutic control of rejection responses. Many T cell surface molecules have been implicated in cytotoxic function through the ability of specific monoclonal antibodies to inhibit T cell activity in vitro. Such measures of "inhibition" must be affected by the avidity of interaction of antibody with the particular surface molecule, as well as the avidity of that surface molecule for any physiological ligand. We show that the introduction of an antiglobulin step substantially amplifies the inhibitory effects of certain CD3 and CD8 monoclonal antibodies, presumably by conferring multivalency. In addition the "antiglobulin" approach has permitted, for the first time, the demonstration that monoclonal antibodies to the "common" determinants of the leukocyte-common antigen family (CD45/LCA/T200) prevent cytotoxic T cell function.     

Inhibition of alloreactivity in vitro by monoclonal antibodies directed against restricted isoforms of the leukocyte-common antigen (CD45).

The leukocyte common antigen (LCA) is a protein tyrosine phosphatase and is identified by the CD45 cluster of monoclonal antibodies. CD45 is expressed in high-density on cells of hematopoietic lineage and generally is immunoprecipitated as 4 bands (220, 205, 190, and 180 KDa). Genetic studies have shown that a single gene produces additional forms of the molecule by alternate splicing including CD45RA (220, 205), CD45RO (180), and CD45RB (220, 205, 190). We have prepared a CD45RB Mab termed "MT3" that binds to a sialic acid dependent epitope. Since the LCA is one of the major targets of antilymphocyte globulin, we assessed a panel of CD45 and CD45R Mab for their ability to inhibit alloreactivity in vitro. MT3 (CD45RB) inhibits the allogeneic MLR and inhibits CD4+ T cells from expressing interleukin 2 receptors, and prevents CD4+ CD45RA- T cells from entering the proliferative phase of the cell cycle. Mem 93 (CD45RA) inhibits the generation of cytotoxic T cells. These data suggest that the CD45RB and CD45RA isoforms of the LCA may be appropriate targets for in vivo immunotherapy.     

Diagnosis of gastric cancers with fluorescein-labeled monoclonal antibodies to carcinoembryonic antigen

Forty gastric adenocarcinomas in 30 resected stomachs were examined by an endoscopic immunofluorescent technique using fluorescein isothiocyanate (FITC)-labeled antibodies to carcinoembryonic antigen (CEA). Fluorescence images of the tumors were obtained with a newly developed endoscopic television system for detecting faint fluorescence. Computer-assisted processing was used to enhance the detection and localization of the tumors visualized by the immunofluorescent technique. Twenty seven (90%) of 30 tumors showed positive fluorescence after treatment with FITC-labeled antibodies for 60 minutes, whereas only two (40%) of five cancers could be detected by the immunofluorescent technique after treatment with antibodies for less than 60 minutes. There was no significant relationship between positive fluorescence and the gross and histological types or the stages of the tumors. In contrast, no positive fluorescence could be demonstrated in cases of benign gastric lesions or after pretreatment with FITC-labeled alpha-fetoprotein. This study therefore suggests that the immunofluorescent technique using FITC-labeled anti-CEA antibodies can be useful in detecting the early stages of gastric cancer.     

Carcinoembryonic antigen and nonspecific cross-reacting antigen in thyroid cancer. An immunocytochemical study using polyclonal and monoclonal antibodies

The expression of carcinoembryonic antigen (CEA) and related antigens in formalin-fixed paraffin-embedded tissues of 200 primary thyroid carcinomas and 22 nonmalignant thyroid lesions was studied immunocytochemically by using three monoclonal antibodies recognizing different epitopes on CEA and related antigens. The monoclonal antibody 431/31 defined epitope (present only on CEA) was found exclusively on 77% of the medullary thyroid carcinomas and on 100% of hyperplastic C-cell nodules adjacent to infiltrating C-cell carcinomas. The monoclonal antibodies 374/14 and 250/183, defining epitopes present on CEA and the nonspecific cross-reacting antigens 95 or 55, reacted with 88% and 82%, respectively, of the medullary carcinomas, but labelled also many nonmedullary thyroid carcinomas (follicular 34% and 18%, papillary 42% and 30%, anaplastic 18% and 13%), follicular adenomas, and nonneoplastic follicle-cell lesions. Similar results were obtained by using a polyclonal CEA antiserum preabsorbed with lyophilized spleen tissue. The findings suggest that nonspecific cross-reacting antigens can be expressed by all types of thyroid carcinoma, while CEA is present only in C-cell tumors of this organ.     

Synergy between cyclosporine and anti-IL-2 receptor monoclonal antibodies in rats. Functional studies of heart and kidney allografts

Although cyclosporine has improved results of organ transplantation, treatment regimens using multiple agents are being evaluated both experimentally and clinically in attempts to diminish its often profound nephrotoxicity; some therapies act synergistically by differential inhibition of distinct steps of the rejection cascade. The effects on graft function of a full dose or a subclinical dose of CsA, ART-18, a monoclonal antibody (mAb) directed against the IL-2 receptor expressed on activated host cells, and a combination of low-dose CsA and ART-18, have been tested in rat recipients of both heart and kidney allografts. Renal graft function was assessed by several classic techniques; heart function by isolated perfusion methods. Full-dose CsA and combination treatment were most effective in both organ graft systems, with at least one-third of grafts surviving indefinitely. At seven days after transplantation, glomerular filtration rates and renal plasma flow of all grafted recipients were decreased as compared with normal; at 14 days, function in the best treatment groups had improved toward that of isografts. Similarly, cardiac output and stroke work index of best treatment groups were comparable to that of isografts. These functional studies complement previously reported immunological and immunohistological findings stressing that synergy occurs between subclinical doses of CsA and anti-IL-2-R mAb in two rat organ graft systems.     

Anti-epithelial membrane antigen monoclonal antibodies and radioimmunolocalization of colorectal cancer

The monoclonal antibodies LICR-LON M8 and 77-1, which react with epithelial membrane antigen, showed a strong reaction with colorectal cancer on immunohistochemistry. In a radioimmunolocalization study in patients with colorectal cancer, 111In-labelled M8 detected 13 of 16 tumour sites present in 16 patients. 111In-labelled 77-1 detected 10 of 15 tumour sites present in 14 patients. The high radioactive background in the liver prevented the detection of hepatic metastases in eight patients (five in the M8 and three in the 77-1 group). The mean (s.d.) tumour to normal colon ratio was 2.5(1.2) for M8 and 1.6(0.5) for 77-1 at day 6 after antibody administration. The mean (s.d.) tumour to blood ratio was 4.9(3.7) for M8 and 3.6(1.5) for 77-1. There was no statistical difference between these results. All scan-positive cancers reacted with M8 and 77-1 on immunohistochemistry. The results suggest that these monoclonal antibodies may have a role in the immunolocalization of colorectal cancer.     

New crossmatch technique eliminates interference by humanized and chimeric monoclonal antibodies

Humanized and chimeric antilymphocyte antibodies (Ab) are used to prevent and treat rejection and for treatment of human disease. Rituximab (RIT, anti-CD20), daclizumab (DAC; anti-CD25), alemtuzumab (ALE; anti-CD52), or infliximab (IFX) may interfere with Ab detection methods such as complement-dependent cytotoxicity (CDC) and flow cytometric crossmatch (FCXM). These agents are recognized as anti-human Ab or fix complement and are not differentiated from anti-allo-Ab. A new enzyme-linked immunosorbent assay crossmatch (XM) utilizing class I and II HLA antigens from donor cells called Transplant Monitoring System (TMS; GTI, Waukesha, Wisc) potentially precludes interference by eliminating non-major histocompatability complex antigens. To test this, normal sera (nonsensitized volunteers) were supplemented with 0.1 or 10 microg/mL of RIT, DAC, IFX or ALE, and were tested using three methods: the TMS T-cell CDCXM with antihuman globulin (AHG); and B-cell CDCXM without AHG; and FCXM with mean channel shifts of 45 and 150 indicating positive T-cell and B-cell crossmatch, respectively. No reactivity occurred with normal sera using any crossmatch technique. At 0.1 and 10 microg/mL, RIT interfered with CDC B-cell, but not T-cell crossmatch. RIT at 10, but not 0.1 microg/mL interfered with B-cell FCXM. No interference occurred with RIT in T-cell FCXM or TMS. ALE interfered with B-cell and T-cell CDC and FCXM but neither class I nor II TMS. DAC did not interfere with CDC or FCXM at 0.1 microg/mL, but gave false positive B-cell FCXM and CDCXM with some samples. No interference by DAC occurred using TMS. TMS may be useful to differentiate de novo donor-specific Ab after treatment with humanized or chimeric Ab.     

Immunohistologic diagnosis of multiple carcinomas. The role of monoclonal antibodies against carcinoembryonic antigen

The occurrence of multiple malignant lesions in the same patient requires that management decisions be made from an accurate pathological identification of the tumours. The authors describe five patients who had colorectal adenocarcinoma preceded or followed by adenocarcinomas in other organs. Definitive identification could not be established from routine histopathologic findings. The use of immunohistochemistry helped to clarify the relationship between the various tumours; in particular, a monoclonal antibody (D14) directed against carcinoembryonic antigen (CEA) made a notable contribution. Because of the increasing number of monoclonal antibodies with well-defined tumour specificities, their use in the documentation of multiple malignant tumours has great clinical potential.     

Polyreactivity and antigen specificity of human xenoreactive monoclonal and serum natural antibodies

Naturally occurring antibodies that react with xenogeneic antigens are a clinically important subset of antibodies because they initiate hyperacute rejection of organs transplanted between disparate species. This currently precludes the use of nonprimate organs for human transplantation. Most antibodies that arise after immunization are monoreactive, i.e., bind only to the immunogen. Similarly, some "natural" antibodies, e.g., isohemagglutinins, bind in a monoreactive manner. In contrast, other natural antibodies, e.g., those that bind to actin, are polyreactive (i.e., bind to multiple ligands). Such polyreactive antibodies may be derived predominantly from CD5+ B cells. In this study, we demonstrate that the majority of xenoreactive natural antibodies in human serum are polyreactive, as indicated by the ability of ssDNA and thyroglobulin (ligands commonly used as targets of polyreactive antibodies) to block the binding of the antibodies to xenogeneic antigens, whereas these ligands could not block the binding of antitetanus antibodies to tetanus toxoid. Furthermore, we compared the ability of 8 polyreactive and 7 monoreactive human mAb to bind to porcine antigens. All of the polyreactive mAb reacted with porcine antigens at mAb concentrations less than 3 micrograms/ml, while none of the monoreactive mAb reacted at concentrations less than 3 micrograms/ml. Each polyreactive mAb reacted with partially overlapping, but distinct sets of porcine cell surface moieties. These results indicate that human polyreactive mAb can bind to multiple xenogeneic antigens in a selective manner and that xenoreactive natural antibodies in human serum are largely polyreactive.     

抗人前列腺干细胞抗原单克隆抗体的制备及鉴定

目的制备并鉴定抗人前列腺干细胞抗原(PSCA)的单克隆抗体.方法以纯化的PSCA为抗原免疫Balb/c小鼠,经细胞融合、克隆化培养建立抗人PSCA的杂交瘤细胞株,采用免疫组化、Western blot等方法鉴定其特异性.结果利用小鼠杂交瘤技术,建立了1株分泌抗PSCA抗体的杂交瘤细胞系D3,经鉴定抗体类别为IgG1,能特异性识别PSCA,与其他因子无交叉反应.PSCA在5例正常前列腺组织、10例良性前列腺增生组织、38例前列腺癌组织中有不同程度的阳性染色,95%(38/40例)的前列腺癌PSCA染色呈阳性、强阳性染色,50%～100%的肿瘤细胞着色,与良性前列腺增生及正常前列腺组织相比,其染色强度及范围的差异有统计学意义(P＜0.01).结论成功制备了组织特异性的抗人PSCA单克隆抗体,为前列腺癌的诊断和免疫治疗提供了新的手段.     

Investigation of potential carbohydrate antigen targets for human and baboon antibodies

Yeh P, Ezzelarab M, Bovin N, Hara H, Long C, Tomiyama K, Sun F, Ayares D, Awwad M, Cooper DKC. Investigation of potential carbohydrate antigen targets for human and baboon antibodies. Xenotransplantation 2010; 17: 197–206. © 2010 John Wiley & Sons A/S. Abstract: Background: The continued presence of a primate antibody‐mediated response to cells and organs from α1,3‐galactosyltransferase gene‐knockout (GTKO) pigs indicates that there may be antigens other than Galα1,3Gal (αGal) against which primates have xenoreactive antibodies. Human and baboon sera were tested for reactivity against a panel of saccharides that might be potential antigen targets for natural anti‐non‐αGal antibodies. Methods: Human sera (n = 16) and baboon sera (n = 15) of all ABO blood types were tested using an enzyme‐linked immunoadsorbent assay for binding of IgM and IgG to a panel of synthetic polyacrylamide‐linked saccharides (n = 15). Human sera were also tested after adsorption on αGal immunoaffinity beads. Sera from healthy wild‐type (WT, n = 6) and GTKO (n = 6) pigs and from baboons (n = 4) sensitized to GTKO pig organ or artery transplants (of blood type O) were also tested. Forssman antigen expression on baboon and pig tissues was investigated by immunohistochemistry. Results: Both human and baboon sera showed high IgM and IgG binding to αGal saccharides, α‐lactosamine, and Forssman disaccharide. Human sera also demonstrated modest binding to N‐glycolylneuraminic acid (Neu5Gc). When human sera were adsorbed on αGal oligosaccharides, there was a reduction in binding to αGal and α‐lactosamine, but not to Forssman. WT and GTKO pig sera showed high binding to Forssman, and GTKO pig sera showed high binding to αGal saccharides. Baboon sera sensitized to GTKO pigs showed no significant increased binding to any specific saccharide. Staining for Forssman was negative on baboon and pig tissues. Conclusions: We were unable to identify definitively any saccharides from the selected panel that may be targets for primate anti‐non‐αGal antibodies. The high level of anti‐Forssman antibodies in humans, baboons, and pigs, and the absence of Forssman expression on pig tissues, suggest that the Forssman antigen does not play a role in the primate immune response to pigs.     

Prostate-specific antigenic domain of human prostate specific antigen identified with monoclonal antibodies

With the use of five murine monoclonal antibodies (1A5, 2A4, 3F1, F5 and 3A12) and an antigen-affinity purified goat polyclonal IgG antibody, the presence of a prostate-specific antigenic domain in human prostate-specific antigen molecule was identified. The results were based upon a series of quantitative competitive inhibition assays of each 125I-labeled monoclonal antibody and polyclonal antibody binding to prostate-specific antigen by unlabeled monoclonal antibodies as inhibitors, and immunohistochemical examination of an extensive panel of human tissue specimens. A cluster of two epitopes that are spatially related or in close topographical proximity and represent a prostate-specific antigenic domain are defined by the monoclonal antibodies 1A5, 2A4, 3F1, and F5, 3A12, respectively.     

Polyclonal and monoclonal antibodies to prostate-specific antigen can cross-react with human kallikrein 2 and human kallikrein 1

OBJECTIVES: The human tissue kallikrein family contains three closely related proteases: human kallikrein 1 (hK1), human kallikrein 2 (hK2), and prostate-specific antigen (PSA). The structural homology between these three proteins suggests potential cross-reactivity interference when different immunologic techniques are used. This study evaluated PSA and hK2 monoclonal antibody (mAb) and polyclonal antibody (pAb) reactivities to hK1, hK2, and PSA. METHODS: mAbs and pAbs to hK2 and PSA were evaluated using Western blot analysis on hK1, hK2, PSA, and seminal plasma. RESULTS: pAbs to PSA and hK2 recognized all three human kallikreins, as well as fragments of hK2 and PSA. An mAb with minimal (less than 0.4%) cross-reactivity between PSA and hK2 and a cross-reactive mAb were found. mAbs specific to PSA or hK2 did not cross-react with the less homologous hK1 protein. A PSA mAb raised specifically to PSA fragments recognized both PSA and hK2 but did not cross-react with hK1. pAbs to hK1 cross-reacted slightly with PSA and not at all with hK2. CONCLUSIONS: Both pAbs and mAbs to hK2 and PSA may exhibit immunocross-reactivity. pAbs to PSA or hK2 react with all three human tissue kallikreins. The potential for cross-reactivity should be considered in any clinical or research procedures that use hK1, hK2, and PSA antibodies.     

The specificity of nephritogenic antibodies. IV. Binding of monoclonal antithymocyte antibodies to rat kidney

Polyclonal rabbit antirat thymocyte serum (ATS) has been shown to form in situ glomerular immune aggregates following perfusion into normal rat kidney ex vivo. This may be due to the presence of T-cell-like epitopes in the rat kidney, or it may be a result of contaminating anti-connective-tissue antibodies in ATS. To exclude the latter possibility we investigated binding to the rat kidney of three different (mouse) monoclonal antirat thymocyte antibodies (anti-T-cell MoAbs), directed to Thy 1.1 antigen, as well as (control) anti-B-cell MoAbs. The MoAbs were incubated in vitro with kidney sections or perfused into the blood-free rat kidney ex vivo. It was shown using immunofluorescence (IF) and immunoelectron microscopy (IEM) (peroxidase technique) that the anti-T-cell MoAbs used, in contrast to anti-B-cell MoAbs, are able to bind with glomerular capillary walls, and with mesangial structures after incubation in vitro or perfusion ex vivo. Although staining patterns are not completely identical, the reaction product is clearly demonstrated throughout the glomerular basement membrane (GBM) and along the plasma membranes of endothelial and epithelial cells, after contact with either of the three anti-T-cell MoAbs used. It is concluded that the presence of T-cell-like epitopes in the rat kidney may lead to immune complex formation following contact with anti-T-cell MoAbs. The nephritogenicity of rabbit ATS, as well as of some batches of clinically used ATS, may also be explained by this mechanism rather than by the usually assumed presence of contaminating antibodies in these polyclonal antisera.     

Analysis of prostate specific antigen and alpha1-antichymotrypsin interaction using antipeptide monoclonal antibodies

PURPOSE: The synthetic peptides E30D and D10P that correspond to prostate specific antigen (PSA) sequences 60-91 and 78-89, respectively, and contain the kallikrein loop were used to immunize mice to obtain anti-PSA monoclonal antibodies (mAbs). MATERIALS AND METHODS: Antipeptide mAb characteristics were studied using biosensor technology and enzyme-linked immunosorbent assay, and analyzing the mAb effects on PSA-alpha1-antichymotrypsin (ACT) complex formation and PSA enzymatic activity. Epitope mapping of these mAbs was performed using overlapping peptide synthesis on nitrocellulose membrane. RESULTS: Anti-E30D mAbs bound PSA coated on the solid phase only, whereas anti-D10P mAbs recognized PSA in detection as well as in capture. However, these mAbs appeared to be anti-total PSA mAbs. Anti-E30D and anti-D10P mAbs were directed against linear epitopes corresponding to residues H74-Y77 and N84-R88, respectively, of the PSA sequence. Anti-D10P mAb recognition of PSA and PSA-ACT complex was equimolar, although an existing molecular model suggested that the sequence corresponding to anti-D10P mAb epitope was involved in the interaction site of PSA with ACT. Furthermore, we were unable to inhibit the enzymatic activity of PSA as well as PSA-ACT complex formation. Finally, the epitope N84-R88 overlapped the cleavage site R85-F86 of PSA. CONCLUSIONS: The linear anti-D10P mAb epitope is located outside of the PSA-ACT binding site. However, these mAbs may be of value for evaluating the presence of different molecular PSA forms in sera.