Tumor-associated antigens recognized by human monoclonal antibodies

Background: Nonhuman monoclonal antibodies (MoAbs) of desired specificities have been studied in cancer treatment and tumor targeting with minimal success. Attempts of using humanized chimeric antibodies have not improved significantly their clinical applications. We have engaged in the development of human MoAbs by incorporating the in vitro immunization protocols to the nodal lymphocytes of cancer patients. Three human MoAbs thus generated were found to be strongly reactive with various human malignancies. The antigens recognized by the three antibodies were selected for immunochemical and biochemical characterizations. Methods: The antigens investigated were AgSK1, PA 1-2 and PA 3-1. The patterns of each antigen expression in various human cancer cell lines were studied by the immunocytochemical staining technique. The expression of AgSK1 in association with cellular proliferation was examined by the flow cytometry analysis. In studying the biochemical natures of these antigens, their sensitivies toward various chemical and physical treatments were determined. The antigens that were shown to be proteins were subjected to SDS-PAGE and Western blot for estimations of molecular weights. Results: The AgSK1 was detected in 10 human carcinoma cell lines but in none of the melanoma cell lines. This suggests that SK1 may be an epithelial or carcinoma marker. The phenotypic expressions of AgSK1 were shown to be associated with proliferation of carcinoma cells. Biochemically AgSK1 was a sialophycoprotein with an estimated molecular weight of 42–44 kilodaltons (kDa). HuMAb PA1-2 demonstrated a unique staining pattern at both the cytoplasmic and intercellular interface. The stained filamentlike structures extending from cell to cell indicated that Ag PA1-2 might play a role in cellular interactions. Biochemically, Ag PA1-2 appeared to be an asialocarbohydrate. The Ag PA3-1 was a cytoplasmic glycoprotein expressed by all 13 cell lines. The estimated molecular weights of PA3-1 were 164, 104, and 40 kDa. Conclusions: Tumor-associated antigens recognized by the human MoAbs may be more relevant clinically than those recognized by the mouse immune system. Carcinoma-specific human MoAbs are desirable for cancer treatment and tumor localization.     

Quantitative evaluation of porcine endothelial cell antigens recognized by human natural antibodies: An analysis by Western blotting

Abstract: Xenoreactive natural antibodies recognize a series of glycoproteins in porcine endothelial cell membranes, the epitopes being Galα1–3Gal substitutions on those proteins. We recently identified the glycoprotein antigens as members of the integrin family, von Willebrand factor, and DM-GRASP. The antibodies that react with these structures are adsorbed during perfusion of a porcine organ, indicating that these antigens may be the biologically relevant targets. To evaluate the relative importance of the glycoproteins as targets of xenoreactive natural antibodies, immunoreactive electrophoretic bands containing these glycoproteins were analyzed by photodensitometry. The relevance of these antigens in the recognition of endothelial cell antigens by xenoreactive natural antibodies was assessed by an analysis of the differences between antigens recognized by normal human serum and those recognized by human serum that had been depleted of xenoreactive natural antibodies. Depletion of xenoreactive natural antibodies was performed by adsorption of serum on cultured porcine aortic endothelial cell monolayers and by perfusion through a porcine kidney. The analysis revealed that greater than 91% of the antibodies binding to porcine cell surfaces are specific for antigens identified in porcine endothelial cell membrane extracts. Quantitative analysis of antigens recognized by affinity purified antibodies confirmed that integrins are indeed the primary xenogeneic targets recognized on Western blots of porcine aortic endothelial cell membranes.     

Characterization of porcine endothelial cell determinants recognized by human natural antibodies

Abstract: Organs transplanted from pigs to primates are subject to hyperacute rejection. This immunologic reaction is initiated by the recipient's natural antibodies that bind to endothelial cell antigens of the organ, resulting in the activation of the complement system and rapid destruction of the graft. Various lines of evidence, particularly blocking studies, using purified carbohydrates have suggested that the endothelial cell determinant recognized by human natural antibodies is a terminal galactose in an α configuration (α-Gal). Although these studies are compelling, they fall short of proof because xenoreactive natural antibodies, being polyreactive, might bind to structures other than those used for blocking. Moreover, in vivo evidence that anti-α-Gal antibodies participate in hyperacute rejection has not been reported. Here we report that an enzyme specific for α-Gal, α-galactosidase, removes α-galactose residues from both intact porcine aortic endothelial cells and immobilized porcine aortic endothelial cell membrane extracts and that as a result of this, xenoreactive natural antibody binding is lowered by 70 to 80%. This decrease in binding of human IgM causes a corresponding decrease in complement activation. Similar results were obtained using the sera of other Old World primates. Digestion of immobilized porcine aortic endothelial cell membrane extracts with α-galactosidase produced a similar reduction in human IgM binding to gp 115/135. These results indicate that a terminal α-galactose is an important component of the gp 115/135 porcine endothelial cell antigen. We also report that perfusion of porcine organs I with primate serum removes anti-α-Gal IgM from the serum.     

Identification of porcine membrane antigens involved in the cytotoxic response mediated by human xenoreactive antibodies

Abstract: The identification of xenoantigens on the surface of endothelial cells is important for understanding the mechanism of hyperacute rejection and development of abrogating methods. The objective of our study was to identify the porcine antigens that, when bound by xenoreactive antibodies in human serum, result in cytotoxicity of porcine cells. Human AB and O sera were adsorbed with porcine aortae, erythrocytes, platelets, and a broad spectrum of immobilized carbohydrate moieties (Synsorbs). Aortae and erythrocytes were able to adsorb the xenoreactive antibodies that were cytotoxic to porcine cells (LLC-PK1), determined using an MTT cytotoxicity assay. Only carbohydrates having the αGal(1–3)βGal(1–4) moieties (Synsorbs 90, 115) were able to significantly reduce cytotoxicity with both types of sera. Western blots of porcine aortic endothelial cells (PAEC) and LLC-PK1 cell membrane extracts probed with unadsorbed sera indicate the binding of xenoreactive IgM to approximately 17 and 11 antigen bands, respectively, having molecular weights ranging from 20–133 kDa. Anti-IgG development showed 8 and 11 antigen bands on PAEC and LLC-PK1 membrane preparations, respectively. When blots were performed using adsorbed AB sera, the binding to all antigens was still observed. When Synsorb 90 bound antibodies were used to probe the blots, the majority of antigens were still detected. This suggests that the binding of xenoantibodies to the most prominent antigens, as detected by Western blot procedures may not be the ones to which cytotoxic xenoreactive antibodies bind. Alternative approaches are required to identify such antigens.     

IgM-enriched human introvenous immunoglobulin strongly inhibits complement-dependent porcine cell cytotoxicity mediated by human xenoreactive antibodies

Abstract:  Treatment with intravenous immunoglobulin preparations consisting of human IgG (IVIgG) prevents hyperacute rejection of pig xenografts transplanted into primates by inhibition of the classical complement pathway. Recent studies indicate that IVIg preparations mainly consisting of human IgM (IVIgM) have a stronger capacity than IVIgG to inhibit the complement system. IVIg preparations also contain xenoreactive antibodies (XAb) binding to pig cells. In the present study, we compared IVIgG and IVIgM for their capacity to inhibit xenogeneic complement activation, with special reference to the roles of IgG and IgM XAb present in these preparations. Xenogeneic complement activation was studied by exposure of pig cells (PK15) to human serum. For some experiments, IVIgG and IVIgM were depleted from XAb by immune absorption. Exposure of PK15 cells to human serum induced surface deposition of C4 and C3 and cytotoxicity, which could be inhibited in a dose-dependent fashion by both IVIgM and IVIgG. The efficacy of IVIgM was more than 10 times higher than that of IVIgG. IgG XAb were detected in both IVIgG and IVIgM whereas IgM XAb were only present in IVIgM. Depletion of XAb from the IVIg preparations did not modify the protective properties of IVIgG against cytotoxicity induced by human serum, whereas the IVIgM-mediated protection against xenogeneic cytotoxicity was only slightly improved. IgM-enriched IVIg is a potent inhibitor of xenogeneic complement activation and complement-dependent cytotoxicity of human serum to pig cells, irrespective of the presence of cytotoxic xenoreactive IgM antibodies in this preparation. Therefore, IVIgM has a promising therapeutic significance for the treatment of (hyper)acute xenograft rejection.     

Endothelial cell antigens recognized by IgA autoantibodies in patients with IgA nephropathy: partial characterization.

Antibodies directed against endothelial cells (AECA) have been described in IgA nephropathy. We have previously measured AECA of the IgA subclass (AECA-IgA) in the sera of patients with glomerulonephritis by an ELISA method, and have shown that patients with IgA nephritis (IgA N) and lupus nephritis (LN) had significantly greater AECA-IgA activity compared with normal controls [1]. In the current study, on Western blotting of the EC membrane components, 6 of 18 (33%) IgA N sera positive for AECA-IgA bound to molecules of 135 and 116 kDa and 10 of 18 (56%) to a molecule of 205 kDa. These EC membrane components recognized by AECA-IgA may be of importance in the pathogenesis of IgA nephropathy.     

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.     

Immunoselection of human lymphocytes producing xenoreactive natural antibodies against Galocl,3Gal terminal residues

Abstract: In the pig-to-human xenograft combination, human xenoreactive natural antibodies (XNA) bind to carbohydrate moieties, especially those with Galα1,3Gal terminal residues, expressed on the surface of most cells. The aim of this study was to select the cells that produce XNA by functionally characterizing a subset of cells for future studies on immunosuppression of XNA formation. We especially addressed the question whether XNA could be produced by CD5+ B lymphocytes, a subset of cells producing antibodies of high connectivity and polyreactivity, possibly involved in autoimmune processes. For that purpose we used porcine thyroglobulin (PTg), which expresses several Galα,3Gal terminal residues, for the immunoselection of XNA-producing cells. On FACS analysis, biotinylated PTg appeared to react with 5% of the B lymphocytes but the proportion of CD5+ B lymphocytes was not enriched in the PTg-reactive population. Similarly, magnetic beads coated with PTg were used to sort lymphocytes with Ig receptors recognizing PTg. Two and one-half percent of cells reacted, mainly B lymphocytes (89%), but they were not enriched for CD5+ B lymphocytes. When PTg-reactive lymphocytes were cultivated in presence of irradiated T cells and stimulated with PWM, the synthesis of Galα1,3Gal reactive antibodies, mainly of the IgG class, was demonstrated. The results of this study suggest that a high percentage of B lymphocytes react with Galα1,3Gal terminal residues. Such XNA-producing cells are not particularly common in the CD5+ subset.     

Definition of human melanoma-associated cell surface antigens by hybridoma monoclonal antibodies

Many human melanoma cell lines express HLA-D antigens in addition to HLA-A, -B, and -C specificities. Hybridoma antibodies produced after sensitization with melanoma cells are directed not only against tumor-associated antigens but against HLA and Ia-like specificities. Paired human melanoma cell lines and autologous lymphoblast lines were used to identify those monoclonal hybridoma antibodies that define human melanoma-associated antigens. Autologous lymphoblast lines were used to exclude hybridoma antibodies directed against HLA and Ia-like specificities. Using this method, we have identified several cell surface antigens that are expressed on the majority of human melanoma cell lines tested.     

Human xenoreactive natural antibodies of the IgM isotype activate pig endothelial cells

Abstract: Preformed, xenoreactive natural antibodies (XNA) and complement (C) are involved in the initiation of vascular rejection of organs transplanted between discordant species, presumably by stimulating donor organ endothelial cells (EC). Although C is known to play a role in the activation of EC, it has not been clear whether the antibodies serve only to anchor the initial components of C, and thus permit the C cascade to proceed, or whether the antibodies themselves deliver a signal to the EC. We have tested affinity-purified human IgM containing XNA (IgM-XNA) for its ability to stimulate in vitro the up-regulation of genes in pig EC. Northern blot analysis shows that IgM, which contains XNA, stimulates mRNA accumulation for certain genes (including IL-8, PAI-1, and ECI-7, a new gene that we have found is associated with EC activation), but not others known to be up-regulated in response to TNF, IL-1 or LPS. Our results show that XNA provide a signal to EC, and thus may themselves participate in activation of EC and consequent vascular rejection.     

Hybridoma antibodies reactive with human bladder carcinoma cell surface antigens

Hybridoma antibodies to the human bladder cancer cell line RT4 were prepared by fusing spleen cells from RT4-immunized BALB/c mice with the murine plasmacytoma line NS-1. When antibodies were characterized by direct serological testing, antibody A2 exhibited highly restricted specificity for an antigen found only on RT4. However, further analysis with absorption, a standard serologic technique that has not been widely applied to hybridoma antibodies because of their monoclonal nature, revealed that the antigen could also be found on 1 other bladder cancer cell line, 5637, and on the human cervical carcinoma line ME-180. Quantitative absorption assays suggest that this phenomenon of absorption-positive, direct test-negative cells may be related to the amount of antigen present on the cell surface. Another antibody, A80, detected an antigen with broader distribution. Both antibodies described heat-labile, trypsin-resistant antigens present on a restricted range of cells. The role of these antibodies in identifying subsets of malignant urothelial cells is being investigated.     

Human IgM xenoreactive natural antibodies can induce resistance of porcine endothelial cells to complement-mediated injury

Abstract: It is thought that human IgM xenoreactive natural antibodies (nAbs) can induce activation of porcine endothelial cells independent of complement. Therefore we hypothesized that pretreatment of porcine endothelial cells with anti-pig nAbs may affect the ability of the endothelial cells, when subsequently incubated with a source of nAbs and complement, to bind antibodies and complement components and to undergo complement-mediated cytotoxicity. We preincubated porcine endothelial cells at 37°C for 1 hr or 40 hr with a source of nAbs. We then incubated these pretreated endothelial cells with a complement source that contained a normal complement level and a low level of IgM nAbs for 1 hr to measure bound IgM and IgG and complement components, and for 4 hr to measure cytotoxicity. We found that preincubation for as long as 40 hr did not impair the binding of IgM and IgG, implying no antibody-induced loss of membrane antigens from the endothelial cells, or the binding of C3bi, C4d, C6, and C9 upon complement activation. In contrast, preincubation for 40 hr with a nAb source induced in the endothelial cells marked resistance to complement-mediated killing. Resistance could be induced with purified human IgM but not with purified IgG or IgM-depleted human serum. The ability of purified IgM to induce resistance was abrogated by removal of anti-pig xenoreactive nAbs by absorption with pig endothelial cells, and induction of resistance required protein synthesis. We conclude that prolonged incubation of human anti-pig nAbs with pig endothelial cells does not cause loss of endothelial cell membrane antigens or impairment in binding of nAbs or complement components; instead, it induces marked resistance to complement-mediated cytotoxicity. These observations may be of value to develop strategies that enhance survival of a xenograft.     

Human class II antigens. Implication of carbohydrate in the determinants recognized by several antihuman class II monoclonal antibodies

Several monoclonal antibodies (McAbs) to human class II antigens are described; three of these recognize monomorphic epitopes, and two are polymorphic. The biochemical nature of the reactive epitopes was investigated by determining the binding ability of the McAbs to protease- or glycosidase-treated antigen preparations. Both of the polymorphic McAbs recognized protein-defined epitopes. However, for one of the antibodies F5C9, carbohydrate residues were also implicated. The extent of carbohydrate involvement varied with class II antigens of different DR specificity, suggesting that the proximity of the F5C9 epitope to carbohydrate side chains can vary. One of the three monomorphic McAbs recognized a protein-defined epitope, while the other two antibodies detected epitopes in which carbohydrates were involved. These results indicate that both protein and carbohydrate residues of the human class II antigens can influence the epitopes recognized by murine McAbs. This could explain some of the apparent complexities of the anti-human class II McAbs.     

Demonstration of cell surface antigens and their antibodies by the peroxidase-antiperoxidase method.

The unlabeled antibody enzyme method, using the peroxidase-antiperoxidase immunocomplex for labeling, was applied to freshly prepared viable lymphocytes to detect antibodies in individuals preimmunized by pregnancies or blood transfusions. The "sandwich" incubations and washing steps were carried out with cells attached to poly(L-lysine) glass slides, in order to facilitate the handling of the samples and to save antisera and time. In comparison to the lymphocytotoxicity test, the described method is more sensitive and able to detect additional antibodies. Our findings indicate that further investigation of the use of this test for the demonstration of cell surface antigens and their antibodies appears to be worthwhile.     

Removal of xenoreactive antibodies by protein-A immunoadsorption: experience in 22 patients

Abstract: The presence of naturally occurring anti‐Galα1–3Gal (antiαGal) Ab in human serum is believed to be a major factor in the pathogenesis of hyperacute rejection of discordant organ xenografts such as the pig‐to‐human combination. Galα1–3Gal epitopes are expressed on pig tissues and the binding of anti‐Galα1–3Gal leads to endothelial cell activation and complement‐mediated hyperacute graft rejection. Several strategies have been suggested in donor animals or in the xenograft recipient to overcome the anti‐αGal barrier. Protein‐A immunoadsorption (PAIA) was developed for the in vivo removal of circulating Ab and it has been shown to be effective in cases where pathogenic auto or alloAb are present. The aim of our study was to analyze the effect of PAIA on total and xenoreactive serum anti‐αGal immunoglobulin levels in a group of patients treated with this technique for different diseases. After three consecutive sessions of PAIA, total and xenoreactive IgG and IgM immunoglobulin levels were decreased by more than 50% of pre‐treatment levels. So we conclude that PAIA is an effective method to significantly reduce circulating Ab, including xenogeneic IgM and IgG Ab. This mode of therapy might be considered as a tool to overcome hyperacute xenograft rejection. PAIA combined with other therapeutic approaches may well protect the xenograft.