Monoclonal antibodies to the antigens of human HeLa tumor cells

A panel of 6 hybridomas "XEJIMA" producing monoclonal antibodies specific to HeLa cells is prepared. Monoclonal antibodies do not bind to antigens of human diploid fibroblasts, human continuous B- and T-lymphocytes and animal cell lines. The specificity of monoclonal antibodies to cellular antigens of 5 HeLa-like cell lines and 6 human tumour cells lines, not contaminated with HeLa cells, is determined. Antibody containing ascitic fluid and culture media of hybridomas XEJIMA-3, -12, -13, and -22 significantly decrease the attachment of HeLa cells to the surface of culture flasks. Monoclonal antibodies XEJIMA-11, -12 and -13 block the multiplication of HeLa cells. The effect depends on serum concentration in the nutrient medium.     

Monoclonal antibodies to parasite antigens found in the serum of Dirofilaria immitis-infected dogs

We recently reported that parasite antigens are detectable in the serum of Dirofilaria immitis-infected dogs by counterimmunoelectrophoresis (CIE). Hybridoma cell lines that produce monoclonal antibodies specific for these antigens were obtained by immunizing mice with a partially purified antigen preparation, fusing spleen cells with SP-2 myeloma cells, and screening cell culture supernatants for antibody by ELISA and CIE inhibition. Antibodies specific for two epitopes shared by the two major circulating parasite antigens were identified. Immunoperoxidase studies showed that the epitopes recognized by the monoclonals were widely distributed in D. immitis, but the female uterus and eggs were particularly strongly labeled. A monoclonal antibody-based ELISA was developed to measure parasite antigens in dog sera. Parasite antigens were detected in 45 of 46 sera from infected dogs but were absent in sera from uninfected dogs and sera from dogs infected with Dipetalonema reconditum. Serum antigen content was significantly correlated with the number of female worms recovered from infected dogs (r = 0.82, p less than 0.001). Antigenemia was first detected 6 mo after infection, and antigen levels remained fairly stable between 9 and 21 mo after infection. Parasite antigen detection with this monoclonal antibody-based ELISA appears to be superior to previously described diagnostic methods for canine dirofilariasis in terms of sensitivity, specificity, and relation to infection intensity.     

The in vitro antibody response to cell surface antigens. II. Monoclonal antibodies to human leukemia cells.

A method has been developed for the production of monoclonal mouse antibody responses in vitro against human cell surface antigens. Limiting numbers of immune spleen cells were transferred to syngeneic, irradiated recipients whose spleen fragments were then cultured in vitro and stimulated to produce antibody. The majority of the antibody from any one fragment culture was likely to be the product of a single donor B cell and thus monoclonal. Evidence for this included a linear relationship between donor cell transferred and spleen fragments producing antibody, extremely restricted isoelectric focusing patterns of the individual antibody products, and unique reactivity patterns of these antibodies against a panel of human lymphoid cells. Different human B leukemia cells were seen as immunogenically distinct by the mouse. By using the monoclonal mouse antibodies as probes, a fine analysis of cell surface antigens is jow possible.     

Monoclonal antibodies to HLA-A,B, and Ia-like antigens inhibit colony formation by human myeloid progenitor cells

Hybridomas derived from the fusion of murine myeloma cells with splenocytes from mice immunized with human cultured lymphoid cells secreted monoclonal antibodies to human cell surface antigens. Serologic and immunochemical assays showed that 4 monoclonal antibodies (Ab Q2/47, Q2/61, Q2/70, Q2/80) recognize framework determinants of Ia-like antigens and 1 monoclonal antibody (Ab Q1/28) reacts with determinants expressed on the heavy chain of HLA-A,B antigens. Both anti-HLA-A,B and anti-Ia-like antigen monoclonal antibodies caused complement-dependent inhibition of granulocyte-macrophage colony formation by human bone marrow grown in soft agar. Mixing experiments excluded the possibility of an indirect effect on progenitor cells by lysis of auxiliary cells. These results indicate that human myeloid progenitor cells express HLA-A,B and Ia-like antigens.     

Monoclonal antibodies against human myelomonocytic cells: detection of certain lineage-specific antigens on CFU-GM progenitor cells

A series of monoclonal antibodies (mAb) were raised against nonlymphoid leukemic cell lines. Three of them have been characterized in detail. mAb H8 (IgG2), mAB U2 (IgG1), and mAb ML143 (IgM) were established with HEL, an erythroleukemia cell line, U937, a monocytoid (histiocytic) line, and ML-1, a myeloid cell line as immunogen, respectively. A 65 to 75 KD polypeptide was precipitated from monocytes by mAb H8, a 160 KD protein from monocytes by mAb U2, and two broad bands in the regions of 150 and 195 KD from granulocytes by mAb ML143. All three mAb stained peripheral blood monocytes and granulocytes, but not lymphocytes, platelets, and erythrocytes. The mAb reacted with immature myeloid cells in bone marrow, ranging from myeloblasts to mature myelomonocytic cells. They also were reactive with various nonlymphoid cell lines and leukemia of myelomonocytic origin. They did not react with B cell lines and B cell CLL cells. By complement-mediated cytolysis and/or an immune rosette method, antigens H8 and U2 were found to be expressed on the vast majority of CFU-GM (14 days) progenitors but not on BFU-E. Antigen ML143 was not expressed by either progenitor. Furthermore, ML143 antigen was found on T leukemia cell lines, a subpopulation of mitogen-activated T cells, and certain non-T/non-B ALL cells. This reactivity was not found with mAb H8 and U2. The relationship between these mAb and those reported are discussed. The possibility of using these mAb to obtain a markedly enriched CFU-GM progenitor population is also raised.     

Monoclonal antibodies to the surface antigens of Pseudomonas aeruginosa

Abstract A panel of 48 monoclonal antibodies was prepared against 8 O-serotype strains of Pseudomonas aeruginosa , and 43 of the antibodies reacted specifically with whole cells of the vaccine strain in an enzyme-linked immunosorbent assay (ELISA). 4 antibodies showed varying degrees of reactivity for more than one of the serotype strains, and one antibody bound to all of the serotype strains as well as strains of Pseudomonas putida and Pseudomonas fluorescens . The epitopes recognised by these antibodies were characterised by immunoblotting and the serotype-specific antibodies reacted only with lipopolysaccharide (LPS) of the vaccine strain. The antibodies that bound to more than one serotype strain were specific for outer-membrane proteins common to the serotype strains. The antibody that cross-reacted with all strains of P. aeruginosa apparently recognised an antigen associated with the core or lipid A components of LPS.     

Monoclonal antibodies to antigens associated with transitional cell carcinoma of the human urinary bladder

Summary Spleen cells from BALB/c mice immunized with cells derived from transitional cell carcinomas (TCC) of the human urinary bladder were fused with mouse myeloma Sp 2/0 Ag14 cells. Monoclonal antibodies from six established hybridomas were investigated for specificity in a cell ELISA and in indirect immunofluorescence against a large panel of fixed intact cells. Three of the antibodies reacted with half or more of the eight bladder tumors and with a few unrelated tumors. They did not react at all with malignant or normal cells of hematopoietic origin. A fourth antibody reacted with seven of eight bladder tumors. It also reacted weakly with a prostatic carcinoma, with five of six malignant or transformed B cell lines, and with a subpopulation of normal lymphocytes, but not with any of the other cells on the test panel. These four antibodies did not react with cells derived from normal urothelium. The results suggest that these antibodies might recognize cell-type-restricted antigens associated with malignancy. Another antibody reacted with almost all urothelium-derived cells. It also reacted with three of three melanomas but not with any other cells on the panel. The sixth antibody reacted with 32 of the 37 cells tested. The spectrum of reactivities displayed by the antibody suggested that it recognizes HLA antigens. Abbreviations used in this paper: TCC, transitional cell carcinoma of the urinary bladder; TAA, tumor-associated antigens; ELISA, enzyme-linked immunosorbent assay; NBCS, newborn calf serum; PBS, phosphate-buffered saline, pH 7.2; ALP, alkaline phosphatase; GDA, glutardialdehyde; BSA, bovine serum albumin; IF, indirect immunofluorescence; LCL, lymphoblastoid cell lines: B-lymphocytes transformed in vitro with Epstein-Barr virus     

Monoclonal antibodies to antigens abnormally expressed in breast cancer

We report the production, screening, and characterization of ten murine monoclonal antibodies directed at antigens that are expressed abnormally in human breast tumors. Immunoperoxidase staining of frozen and fixed tissues shows the antigens to be present at low levels on the luminal membrane of normal breast cells and at high levels in the cytoplasm and surface membrane of breast tumor cells. The ten antibodies appear to recognize six different epitopes on the basis of their quantitative differences in reactivity against four antigen preparations, as measured by ELISA. Immunoblots show that eight of the ten antibodies recognize a 300,000 MW molecule from breast tumor preparations; six of these antibodies also react with a second molecule from the same tumor preparations of 280,000 MW. Seven antibodies react with an antigen from milk fat globule membrane of 330,000 MW. It therefore appears that the two molecules from tumor tissue and the one molecule from normal tissue share common epitopes. Selected antibodies were tested for reactivity against 25 primary breast tumors and 14 pairs of primary and metastatic breast tumors. Three antibodies have broad reactivity and stain more than 80% of primary tumors; the three other antibodies identify subsets of those tumors. Results of staining pairs of primary and metastatic lesions show that metastases continue to express antigens of the primary lesion in a high percentage of cells.     

Monoclonal antibody OKT-9 recognizes the receptor for transferrin on human acute lymphocytic leukemia cells

The monoclonal antibody OKT-9 recognizes a surface protein of human lymphocytes that consists of a disulfide bonded homodimer of m.w. 200,000 intact and 95,000 reduced. A similar protein is precipitated by transferrin-agarose, but not by agarose alone. Peptide mapping by limited proteolysis shows that the proteins precipitated by OKT-9 antibodies and transferrin-agarose are homologous. It is concluded that OKT-9 antibodies recognize the transferrin receptor. Expression of receptors for transferrin may be useful as a marker for activated or dividing cells.     

Monoclonal antibodies in the treatment of leukemia

MoAb-based therapies are evolving into the first broad-spectrum class of targeted anti-leukemic therapy. Developments in many areas, including computer modeling of receptors and ligands, and increasing sophistication in recombinant technologies may result in a rapid increase in the number and complexity of MoAb's available. We can anticipate an increase in the number of safer conjugates being delivered to leukemia cells. Further understanding of the in vitro mechanisms involved in tumor cell killing by MoAb will be important in maximizing the efficacy of this approach.     

Monoclonal antibodies to carbohydrate antigens in autologous bone marrow transplantation

Normal and malignant myeloid cells express a highly immunogenic oligosaccharide, lacto-n-fucopentaose-III (LNF-III), that has been identified by numerous monoclonal antibodies (MoAb). We have been interested in the use of a particular monoclonal antibody to LNF-III, PM-81, in the treatment of patients with acute myelogenous leukemia using the antibody to treat bone marrow in vitro. Following in vitro treatment of bone marrow with PM-81 and another MoAb, AML-2-23, the remaining cells are used as an autograft in a patient treated with high-dose chemotherapy and radiotherapy. In order to enhance the ability of the MoAb to lyse leukemic cells in the remission bone marrow, we have explored the effect of neuraminidase treatment on leukemia cells. In this paper we describe that myeloid leukemia cells expressing low levels of LNF-III by immunofluorescence can be shown to have high levels of LNF-III after neuraminidase treatment. In addition, we show that normal bone marrow progenitor cells do not have cryptic LNF-III antigen, thus allowing the application of this finding to the clinical setting. Moreover, we have shown that leukemia colony-forming cells from one patient with acute myelogenous leukemia express cryptic LNF-III and that after exposure to neuraminidase there was an increased ability of PM-81 in the presence of complement to eliminate these colony forming cells. These data indicate that the LNF-III moiety is almost universally expressed on myeloid leukemia cells and their progenitors but not expressed on normal progenitors. Thus, it may be possible to enhance leukemia cell kill in vitro by neuraminidase treatment of bone marrow.     

Monoclonal antibodies to bovine and human acrosin

Monoclonal antibodies to human acrosin were required for studies of immunological interference with fertilization. Since human acrosin was not available in adequate amounts, monoclonal antibodies have been raised in mice against purified bovine acrosin and screened for cross-reaction with human sperm cells. Two of these antibodies are described, B4F6 and C2E5. Data from enzyme-linked immunosorbent assays, immunoblots, immunoprecipitation, and indirect immunofluorescence on sperm cells indicate that B4F6 binds only to bovine acrosin, and that C2E5 binds both to bovine and to human acrosin at a conformationally determined epitope. The antibodies do not inhibit the hydrolysis of benzoylarginine ethyl ester by acrosin, but C2E5 did inhibit the dissolution of the hamster zona pellucida by purified human acrosin. The antibodies have also been used for affinity purification of acrosin and proacrosin.     

Cellular resistance against troxacitabine in human cell lines and pediatric patient acute myeloid leukemia blast cells

Troxacitabine is a cytotoxic deoxycytidine analogue with an unnatural L-configuration, which is activated by deoxycytidine kinase (dCK). The configuration is responsible for differences in the uptake and metabolism of troxacitabine compared to other deoxynucleoside analogues. To determine whether troxacitabine has an advantage over other nucleoside analogues several cell lines resistant to cladribine and gemcitabine were exposed to troxacitabine, while blast cells from pediatric leukemia patients were tested for cross-resistance with other deoxynucleoside analogues. The gemcitabine resistant AG6000 (IC50: >3000 nM), and the cladribine resistant CEM (IC50: 150 nM) and HL-60 (IC50: >3000 nM) cell lines, all with no or decreased dCK expression, were less sensitive to troxacitabine than their wild type counterparts (IC50; A2780: 410, CEM: 71 and HL-60: 158 nM). dCK protein expression in CEM was higher than in HL-60, which, in turn, was higher than in A2780. Catalytically inactive p53 seems to increase the sensitivity to troxacitabine. The patient samples showed a large range of sensitivity to troxacitabine, similar to other deoxynucleoside analogues. Cross-resistance with all other deoxynucleoside analogues was observed.     

Kinetics of hybridization to human DNA of heterogeneous nuclear RNA isolated from normal human lymphoblasts and acute leukemia blast cells

Heterogeneous nuclear RNA was extracted from normal PHA-stimulated human lymphocytes and acute myeloid leukemia blast cells. Experiments were performed to determine the hybridization kinetics of these RNAs to human DNA. The best least squares solutions indicate in the hybridization reaction of both normal and leukemic RNA two main components. For leukemic cell RNA the rate constants of both components were significantly different from that of normal cell RNA. In particular, the difference between the rate constants of the second slower component suggests that the slowly hybridizing sequences in leukemic cell RNA have a degree of repetition higher than that of the corresponding sequences of normal cell RNA.     

Monoclonal antibodies to antigens of cholera vibrios of the Ogava serovar

A set of hybrids is obtained synthesizing monoclonal antibodies to the surface antigenic determinants of the choleric vibrio of the Ogava serovar. The antigenic structure of vibrios of the typical and atypical strains isolated from a man and from environmental objects is studied using a collection of highly specific immunoglobulins. The high-specific diagnostic preparations for identification of the 01-group cholera agent serovar may be created on their base.     

Monoclonal antibodies to human cytokeratins: application to various epithelial and mesothelial cells

Immunohistological analysis of human tissue using monoclonal antibodies against cytokeratins, which are confined to cells of epithelial origin, is a valuable technique. Using human epidermal keratins as antigen, we prepared monoclonal antibodies against cytokeratins (ZK1, ZK7, ZK61 and ZK99) and against a desmosomal protein (ZK31). Immunohistochemical staining of human skin sections using these antibodies showed a specific reaction with the epidermis: ZK1 stained the entire epidermis, ZK7 only the basal layer, ZK61 and ZK99 the suprabasal layers, and ZK31 the cellular interfaces. In order to test for antibody specificity, immunoblots with human epidermal and amnion epithelial cytokeratin polypeptides, as well as immunofluorescence microscopy of simple epithelia (glandular and simple columnar epithelia) were performed. ZK1, ZK61 and ZK99 reacted preferentially with cytokeratin polypeptides of stratified squamous epithelia and ZK7 recognized cytokeratins of stratified and simple epithelia. When the ZK antibodies were tested on mesothelial cells in pleural effusions, only ZK7 reacted with these cells. Biochemical analysis of cytokeratin accumulation in cells of primary and long-term cultures indicated that the cytokeratin pattern of mesothelial cells was quite unstable, while that of amnion epithelial cells showed only minor quantitative changes. The use of these antibodies to determine the epithelial origin of cells present in pleural effusions is proposed.     

Monoclonal antibodies to the species-specific and group antigens of Rickettsia prowazekii]

A number of hybridomas to different R. prowazekii determinants were obtained by the hybridization of spleen cells of BALB/c mice immunized with R. prowazekii corpuscular and soluble antigens. Some of the monoclonal antibodies (McAb) reacted with R. prowazekii thermolabile species-specific protein and did not react with R. typhi antigens (McAb of batches B4/4 and A-D3). McAb C5/2 and A3/2 reacted with the group thermostable antigen, common for R. prowazekii and R. typhi. McAb to the species-specific thermolabile antigen belonged to IgG2a. The McAb thus obtained permit the identification of R. prowazekii and R. typhi and the solution of the problem of the intragroup differentiation of rickettsiae belonging to the typhus group.     

Glycolipid antigens of human polymorphonuclear neutrophils and the inducible HL-60 myeloid leukemia line

Glycolipid and cell surface carbohydrate antigens of human polymorphonuclear neutrophils (PMN) and of HL-60 myeloid leukemia cells were analyzed with a panel of defined, monoclonal anti-carbohydrate antibodies. Antigenicities of intact PMN, HL-60, and retinoic acid-induced HL-60 (r.a.-HL-60) were studied by flow cytofluorometry. These three cell populations displayed quantitative differences, some of which were induction dependent, in their expression of lactosyl, N-acetyllactosaminyl, Y-hapten (Fuc alpha 1----2Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----R), and sialosyl-X-hapten (SA alpha 2----3Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----R) specificities. Structures reactive with antibodies specific for long-chain mono-, and di- or tri- alpha 1----3 fucosylated lacto-series glycolipids were also detected. Glycosphingolipids purified from organic extracts of these cells were analyzed to seek information concerning the chemical basis for these surface antigenic differences, to assess the structural and antigenic diversity of PMN and HL-60 glycolipids, and to quantitate chemically and antigenically prominent glycolipids. Binding of monoclonal antibodies to thin-layer chromatograms demonstrated that each of the specificities on intact cells was carried by one or more distinct glycolipids. The abundance of immunoreactive glycolipids in the extracts paralleled the relative staining intensities of the intact cell populations. Several "cryptic" glycolipid antigens, including alpha 2----6 sialosylated structures enriched five- to 10-fold in PMN extracts, were not detected on intact cells. Lactosylceramide accounted for two-thirds of the approximately 1.5 X 10(9) glycolipid molecules contained in each PMN. The remaining glycolipid antigens appeared to include structurally diverse fucolipids, fucogangliosides, and neutral and sialosylated glycolipids with Gal beta 1----4GlcNAc beta 1----R terminal core structure. The abundance, diversity, and induction-dependent expression of these structures suggest that they may participate in PMN maturation and function.