Human monoclonal antibodies to cytokeratins associated with squamous cell carcinoma

Human lymphocytes from a lymph node draining the tumor-bearing area of a patient with a large primary squamous cell carcinoma of the oral mucosa were fused with the nonproducer mouse myeloma, NS-1, to produce interspecies hybridomas. Of 95 hybridoma culture supernatants tested, 23 contained from 0.5 to 50 micrograms/ml of human IgM or IgG. Six supernatant fluids containing greater than 15 micrograms/ml of Ig were tested by indirect immunoperoxidase and immunofluorescence against sections of the autologous carcinoma. Five IgM (lambda) monoclonal antibodies stained the cytoplasm of autologous and allogeneic squamous carcinoma cells. All five monoclonal antibodies stained all layers of normal epidermis but each antibody stained the superficial keratin layer most intensely. Two of the five hybridoma antibodies were further tested. Both antibodies stained all types of normal epithelium; a network of fibers characteristic of intermediate filaments in cultured squamous carcinoma cells and cultured fibroblasts; Z lines in skeletal muscle; and axons in peripheral nerve fibers. We conclude that all five IgM monoclonal antibodies recognize cytokeratins associated with the autologous squamous cell carcinoma. Two of the five hybridoma antibodies recognize an antigenic determinant common to all types of intermediate filament proteins. These data indicate that cytokeratins released by squamous carcinoma cells induced an antibody response in this patient.     

Human monoclonal antibodies produced in transgenic BABkappa,lambda mice recognising idiotypic immunoglobulins of human lymphoma cells

Clonal idiotypic immunoglobulins of follicular lymphomas can be isolated by somatic fusion procedures. Idiotypic IgMs (Id-IgM) were isolated from two patients and used to immunise a strain of mice, deficient in mouse antibody production and engineered with yeast artificial chromosomes (YAC) containing fragments of the human immunoglobulin (Ig) micro/delta heavy chain and kappa/lambda light chain loci. Sequence analysis showed that hybridomas prepared from spleen cells of immunised mice expressed exclusively one of the six VH genes (VH1-2) present in the YAC transgene with different D/J rearrangements, and secrete fully human monoclonal antibodies (mAb) that recognised the tumour-specific IgM proteins. Further studies of the reactivity of the monoclonal anti-human Id-IgM antibodies revealed that they are specific for the individual protein of each patient and probably react with idiotypic determinants. In one case studied, the antibody recognised specifically the lymphoma cell expressing the corresponding idiotypic IgM and lysed those cells in the presence of complement. This is the first example of a human monoclonal antibody with such characteristics and may be of further use in the therapy of patients with B cell malignancies.     

Cell biology of human IgM-producing hybridomas derived from a fusion of human spleen lymphocytes with mouse myeloma cells

Hybrids were derived from the fusion of mouse myeloma cells with human spleen cells from a patient with active idiopathic thrombocytopenia. Of 288 initially seeded cultures, 186 were found to produce human Ig. The growth and Ig production rates, cloning efficiencies using different feeder layers and the karyotype were determined for 9 clones that stably produced human monoclonal IgM (2-100 micrograms/ml) for at least 9 months. All cells of the Ig-producing hybridoma clones were positive for cytoplasmic-Ig, whereas only 20-65% of cells expressed surface Ig (mu and chains). Human monoclonal antibodies in mass cultures were derived in serum-free PRMI 1640 medium. Two clones produced human IgM (nearly 2 mg/ml) in the ascitic fluid of nude mice. Feeder cells of peritoneal macrophages from Balb/c mice enabled more efficient recloning of human x mouse hybrids than did thymocytes. Nearly all subclones derived from 2 clones were found to produce the same monoclonal antibodies as the parental lines. Information on the individual parameters of a hybridoma cell line may be helpful in the large-scale production of human monoclonal antibodies.     

Use of 'single shot' intrasplenic immunization for production of monoclonal antibodies specific for human IgM

We report here the use of 'single shot' intrasplenic injection of human IgM for immunization of mice to obtain splenocytes for use in the production of hybridomas secreting antibodies against human IgM. Fusion was performed 3 days after intrasplenic injection of 20 micrograms of myeloma IgM. IgM-specific antibodies were found in 12% of the fusion wells; only 1 well contained antibodies which cross-reacted with other immunoglobulin classes. Two monoclonal antibodies (McAbs) have been fully characterized as specific for different epitopes on Fc mu. These antibodies can be used to detect IgM on the surface of human B cells by immunofluorescence and in solution by solid-phase radiobinding assay or single radial immunodiffusion. Both McAbs can also detect IgM fragments by immunoblotting from non-reducing SDS-polyacrylamide gels.     

Antibodies to histones in infectious mononucleosis.

A polyspecific human monoclonal (auto)antibody, isolated from a patient in the acute phase of infectious mononucleosis, was found to react with all subfractions (H1, H2A, H2B, H3 and H4) of histones. This finding prompted us to study the occurrence of antibodies to histones in sera of patients with infectious mononucleosis. It was found that IgM binding to histones was detectable both in control and patient sera; however, sera from patients showed binding values of IgM antibodies to histones significantly higher than those of healthy controls; moreover, both in control and patient groups anti-histone IgM activity was found to correlate with serum IgM concentration. These findings suggest that anti-histone IgM antibodies belong to the class of antibodies defined as "natural antibodies" and that their increase during infectious mononucleosis is due to Epstein-Barr virus-induced polyclonal B cell activation.     

Human monoclonal anti-nuclear antibodies produced by human-mouse heterohybridomas

To obtain human monoclonal anticentromere antibodies, mouse myelomas were fused with unfractionated mononuclear cells from the peripheral blood of a patient diagnosed as having the CREST variant of scleroderma: with only anticentromere antibodies. After a single fusion an heterohybridoma secreting a human antibody specific for nuclear antigens, as detected by indirect immunofluorescence staining, was isolated. The monoclonal antibody secreted by the clone was of the human IgM class. Indirect immunofluorescence staining of the antibody on HEp-2 cells showed multiple nuclear dots or a discrete speckled pattern resembling that of an anticentromere antibody. Immunoblot analysis showed antibody binding to a 33 kD antigen derived from the nuclear protein fraction. Enzyme-immunoassay results clearly showed that the antibody reacted with the chromosomal protein fraction and not calf thymus DNA.     

Interactions between natural autoantibodies to tumor-associated human ovarian cancer antigen and murine ovarian cancer cells CaO-1

The interactions between tumor cells and autoantibodies to tumor-associated human ovarian cancer antigen isolated from the plasma of a non-cancer patient are demonstrated using an original model of pseudomucinous murine ovarian carcinoma CaO-1. The use of natural auto-antibodies for active immunotherapy of tumors may be more effective and safe than murine monoclonal antibodies to CA 125, because there will be no reaction to a foreign protein. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 2, pp. 227–229, February, 1999     

Nature's best weapons to fight cancer. Revival of human monoclonal IgM antibodies

The unique features of monoclonal antibodies (specificity, effectiveness, purity and unlimited reproducibility) make them ideal tools for the specific treatment of all kind of diseases. The third generation of monoclonal antibodies for the treatment of human diseases will be, after murine and "humanised" murine immunoglobulins, fully human antibodies. The best source of human monoclonal antibodies are the antibody pools of cancer patients themselves with the best technique for generating them being conventional human hybridoma technology. This technique, will generate human monoclonal antibodies which will not only define important new targets on cancerous tissue, but will also provide the necessary therapeutic human antibodies in the fight against cancer.     

Towards the development of peptide mimotopes of carbohydrate antigens as cancer vaccines

Tumor-associated carbohydrate antigens are considered important targets in efforts to develop cancer vaccines. To further enhance vaccine efforts, we are developing peptide mimotopes of tumor-associated carbohydrate antigens that can elicit functional immune responses. Mapping peptide epitopes with anticarbohydrate antibodies can lend to defining structural relationships that can go undetected by screening of carbohydrate antigens alone. Here we contrast reactivity patterns for peptides using monoclonal antibodies (MAbs) directed to the neolactoseries related Lewis Y (LeY) and sialyl-Lewis X (sLeX) antigen and the GD3/GD2 ganglioside antigen. We observe that representative MAbs cross-react with a WRY-containing peptide and that this motif type is isolated by the respective monoclonal in peptide phage display screening. Primary immunization with multiple antigen peptide preparations with QS-21 adjuvant efficiently elicited cytotoxic IgM antibodies for a murine Meth A fibrosarcoma line expressing sLeX. The cytotoxicity of IgG polyclonal response was found to be as effective as IgM in mediating complement-dependent cytotoxicity against the Meth A line. These experiments suggest that peptide mimotopes of the LeY and sLeX tumor-associated carbohydrate antigen and QS-21 adjuvant could be considered as an immunogenic therapeutic vaccine in carcinoma and melanoma patients in the minimal residual disease setting.     

A highly conserved determinant on human rheumatoid factor idiotypes defined by a mouse monoclonal antibody

Different human IgM rheumatoid factor (IgM RF) idiotypes have been described defined by polyclonal rabbit anti-idiotypic antibodies. These antisera do not allow clear genetic analysis of the idiotypic determinants, be they cross-reactive or private. Therefore, we tried to obtain a set of monoclonal anti-idiotypic antibodies directed against RF idiotypes. Purified IgM RF serum from a patient with classical rheumatoid arthritis was used to immunize BALB/c mice. The spleen cells were fused with Sp 2/0 Ag 14, a nonsecreting mouse myeloma cell line, and a hybrid producing monoclonal anti-idiotypic antibody was selected. The mouse antibody, an IgG1 kappa, reacts with an identical or similar determinant located on (or close to) the binding site of all tested monoclonal or polyclonal IgM RF from totally unrelated patients with Waldenstr?ms's macroglobulinemias or rheumatoid arthritis. The monoclonal antibody also reacts with 2 rheumatoid arthritis patients' IgG RF and with a low proportion of normal polyclonal IgM without detectable RF activity. An hypothesis is proposed to explain the existence of a such highly conserved determinant on RF idiotypes.     

Human polyclonal and monoclonal IgG and IgM complement 3 nephritic factors: evidence for idiotypic commonality

Complement 3 nephritic factors (C3NeF) were isolated from the sera of patients with membranoproliferative glomerulonephritis (MPGN) and the supernatants of pokeweed mitogen-stimulated mononuclear cells from patients with MPGN. Three human monoclonal C3NeF antibodies (two IgGs, CK and PH, and one IgM, K3C4) were established. Using an exhaustive series of affinity columns, we isolated anti-C3NeF idiotypic antibodies (anti-IdNeF) (three from normal and two from patient sera). Anti-IdNeF preparations bound to F(ab')2-NeF and prevented its ability to stabilize C3bBb convertase. We have used the above reagents to address questions on the genesis and the diversity of C3NeF antibodies. The following results were obtained: All anti-IdNeF preparations bound to C3NeF isolated from patient sera, cell culture supernatants, and IgG and IgM monoclonal C3NeF. None of the monoclonal C3NeF bound to an extensive battery of common antigens, including Fc portion of IgG, TNP, beta-galactosidase, DNA, and bacterial products. These data indicate that C3NeF express one common idiotype and that these antibodies are not raised in response to an obvious antigen.     

Mouse monoclonal antibodies at the red cell surface--I. Generation of EAC4 and interaction with C1

C1 and C1 activity measurements were performed with EA and EAC4 prepared with rabbit anti-Forssman IgG or IgM and were compared to measurements with EA and EAC4 prepared with mouse monoclonal IgG2b and IgM anti-DNP antibodies on cells coupled with TNP: the amount of TNP per cell was optimal for antibody activity. No differences were found in the ability of EAC4 made with poly- or monoclonal IgM to measure C1 activity; in contrast, monoclonal IgM was capable of activating only about 30% of C1 when compared to activation by polyclonal IgM. Monoclonal vs polyclonal IgGs behaved in a similar manner but they were detecting only 50% of C1 or C1 activity when compared to IgM of the appropriate class. It was concluded that monoclonal antibodies were capable of generating EAC4 intermediate, and that the ability of monoclonal antibodies in the EAC4 complex to bind C1 and to detect C1 activity is not significantly different from that of polyclonal antibodies but that monoclonal antibodies are less efficient in activating C1 than polyclonal antibodies.     

Idiotypic Network in Myasthenia Gravis Demonstrated by Human Monoclonal B-Cell Lines

Human monoclonal anti-receptor and anti-idiotypic antibodies were obtained from Epstein-Barr virus-transformed lymphoblastoid cells of patients with myasthenia gravis. The majority of antibodies (85/117) were IgM, and all IgM antibodies had lambda light chains. A marked restriction to one recurrent idiotype was found, despite the additional presence of other idiotypes in the serum from all patients. The presence of idiotypes and anti-idiotypes in the same patient was verified by the demonstration of specific complex formation between an anti-receptor antibody and an anti-idiotypic antibody produced by two different clones. Studies of human monoclonal antibodies produced by lymphoblastoid cell lines provide important information about the B-cell repertoire in myasthenia gravis and demonstrate a basis for a functional antibody network in this disease.     

Monoclonal antibodies to human malignant mesothelioma

Murine monoclonal antibodies were used to identify tumor-cell membrane antigens on a new human mesothelioma cell line. Hybridomas were constructed by fusing SP2/0 mouse myeloma cells with spleen cells from Balb/C mice immunized by the human mesothelioma cell line MT-1. Hybridoma antibody was detected in 55/672 microculture wells that reacted to these MT-1 tumor cells by an indirect¹²⁵I-protein A binding assay. Six cultures produced antibody binding selectively to the MT-1 tumor cells but not to a human lymphoblastoid cell line. These six hybridomas were cloned: three were IgG and three were IgM antibodies. One monoclonal, MAb 45, reacted with 4 of 7 human mesothelioma cell lines but with only 1 of 11 carcinomas, 1 of 3 sarcomas, 4 of 11 melanomas, and 0 of 5 lymphoid lines. The other five monoclonals had a much broader cross-reactivity. Using an immunoperoxidase technique, MAb 45 bound to mixed-type malignant mesotheliomas but not to normal lung and pleura. The specificity of MAb 45 for diffuse mesotheliomas and the low cross-reactivity with carcinomas and normal adjacent tissues suggest that this monoclonal may be clinically useful.     

Normally occurring human anti-GM1 immunoglobulin M antibodies and the immune response to bacteria

Anti-GM(1) antibodies of the immunoglobulin M (IgM) isotype are normal components of the antibody repertoire of adult human serum. Using a sensitive high-performance thin-layer chromatography (HPTLC) immunostaining assay, we found that these antibodies were absent in the umbilical vein and children <1 month of age but could be detected after 1 month of age. Although most of the children older than 6 months of age were positive, there were still a few negative children. The appearance of anti-GM(1) IgM antibodies showed a perfect concordance with two well-characterized antibacterial antibodies, anti-Forssman and anti-blood group A, which indicates a similar origin. We also studied IgM reactivity with lipopolysaccharides (LPSs) from gram-negative bacteria isolated from stool samples from healthy babies and from Escherichia coli HB101 in serum from individuals of different ages. We found a positive reaction with both LPSs in all the children more than 1 month of age analyzed, even in those that were negative for anti-GM(1) antibodies. Anti-GM(1) IgM antibodies were purified from adult serum by affinity chromatography and tested for the ability to bind LPSs from different bacteria. This highly specific preparation showed reactivity only with LPS from a strain of Campylobacter jejuni isolated from a patient with diarrhea. We conclude that normally occurring IgM antibodies are generated after birth, probably during the immune defense against specific bacterial strains.     

Monoclonal antibodies defining a minor and a private idiotope on human rheumatoid factors

Two mouse monoclonal antibodies (mAb A216-5 and L 49-3) with antiidiotypic activity against two human monoclonal IgM rheumatoid factors (IgM RFs) were defined. Each of these monoclonal antibodies (two mouse IgG 1 K) reacted with an idiotope located on the heavy chain of the immunizing monoclonal IgM RF and was able to inhibit RF fixation to the antigen. These monoclonal antibodies did not react with other monoclonal IgM RFs from patients with macroglobulinemias or cryoglobulinemias and, therefore, did not recognize the known cross-reactive idiotopes of human monoclonal RFs. The presence of both 216-5 and 49-3 idiotopes on polyclonal IgM RFs from unrelated patients was undetectable by the inhibition assays. However, using a four-stage solid-phase radioimmunoassay, the 216-5 idiotope (minor), but not the 49-3 idiotope (private), was frequently present at a low concentration on polyclonal IgM RFs from patients suffering from rheumatoid arthritis, primary Sjögren syndromes, various infectious diseases, systemic vasculitis, and sarcoidosis and during aging. Interestingly, the 216-5 idiotope was undetectable among polyclonal IgM RFs of 12 normal adults. The main conclusions of these data are the following. (1) The definition of minor and private idiotopes of human RFs requires the use of assays able to detect low amounts of antibodies among polyclonal Ig. (2) The anti-IgG B cells which are sometimes clonally expanded during Waldenström diseases and cryoglobulinemias can also be activated during nonneoplastic diseases, among the other RF-secreting B cells. (3) Thein vivo expression of the IgM RF repertoire is different among normal adults and during various known IgM RF-inducing conditions.     

Delineation of four carcinoembryonic antigen (CEA) related antigens in normal plasma by transblot studies using monoclonal anti-CEA antibodies with different epitope specificities

Antigens related to the carcinoembryonic antigen (CEA) were isolated from normal human plasma by perchloric acid extraction, gel permeation chromatography and immunoaffinity chromatography using a monoclonal antibody with broad specificity and high affinity. The antigens were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. The binding of five monoclonal anti-CEA antibodies with different epitope specificities to the immobilized antigens was analyzed. Two antigens with mol. wts of greater than 200,000 and 177,000 bound all five antibodies, and two antigens with mol. wts of 114,000 and 85,000 bound three of the five antibodies. The findings reported indicate that even monoclonal antibodies with high specificity for colonic cancer CEA detect CEA-related antigens in normal human plasma.     

Monoclonal Antibodies against Pancreatic Islet-Cell-Surface Antigens Selected by Flow Cytofluorometry

BALB/c mice were immunized with human islets of Langerhans, and spleen cells from two mice, found to develop cell-surface antibodies against insulin-producing rat islet tumour RIN-5F cells, were fused with mouse myeloma cells. Antibody-producing hybrids were cloned on the basis of their production of surface antibodies reactive with paraformaldehyde-fixed RIN-5F cells by indirect immunofluorescence analysis in the fluorescence-activated cell sorter. Among 236 primary clones, eight stable cell lines producing islet-cell-surface antibodies were eventually cloned. Antibody 2G3 (IgM) reacted with viable normal rat islet cells and high insulin-producing rat islet tumour RIN5-A2 cells, while 3G3 (IgM) only reacted with RIN5-A2 cells. Antibody beta B1 (IgG1) reacted with all islet cells tested and detected an Mr21k component in immunoblotting experiments with RIN-5AH cell plasma membrane proteins electrophoretically transferred to nitrocellulose filters. Antibody 7F6 (IgM) reacted with all islet and non-islet cells tested and detected bands of Mr 66k and 27k by immunoblotting. Antibodies gamma B3, gamma B6, gamma C2, and 6B1 (all IgM) showed varying degrees of binding to different islet cells, but reacted only weakly with non-islet human cells. It is concluded that monoclonal antibodies against pancreatic islet cells may define specific endocrine islet-cell-surface determinants.     

Anti-tumor immunoglobulin M increases lung metastasis in an experimental model of malignant melanoma

Cancer metastasis involves distinct steps that depend on complicated tumor–host interactions. The hematogenous dissemination of tumor cells may be facilitated by factors that promote the arrest and adherence of cancer cells in capillaries. We examined whether anti-tumor monoclonal immunoglobulin M (IgM) antibodies promoted the hematogenous dissemination of B16 melanoma cells in syngeneic mice. IgM monoclonal antibodies were generated that selectively bind to B16 melanoma cells as compared to syngeneic fibroblasts, lymphocytes or Lewis lung carcinoma cells. Incubation of B16-BL6 or B16-F0 melanoma cells with these IgM anti-tumor antibodies significantly increased the number of lung colonies as compared with control antibodies. Moreover, intraperitoneal injection of specific antibody also significantly increased lung colonization. All anti-tumor antibodies promoted the aggregation of B16 melanoma cells. A chemically generated immunoglobulin G (IgG)-like fragment of an anti-tumor IgM antibody displayed greatly reduced tumor aggregation and, in contrast to intact IgM, did not significantly increase lung colonization of B16 melanoma cells. Neither intact IgM nor the IgG-like fragment enhanced the in vitro invasiveness of B16 melanoma cells across Matrigel-coated membranes. Our results, therefore, suggest that besides their beneficial anti-tumor effects, anti-tumor IgM antibodies may also promote the hematogenous dissemination of cancer cells. This revised version was published online in July 2006 with corrections to the Cover Date.