Heterohybridoma Cell Research Articles

HUMAN LYMPHOCYTOTOXIC MONOCLONAL AUTOANTIBODIES FROM A HIGHLY SENSITIZED RENAL DIALYSIS PATIENT

A panel of 5 human monoclonal autolymphocytotoxic antibodies (IRM-3, IRM-4, IRM-7, IRM-8, and IRM-10) of the IgM class was established from a highly sensitized renal dialysis patient (IRM), by the generation of mouse-human heterohybridomas. This panel was screened for reactivity against foreign and autoantigens by ELISA, and for reactivity against different tissue sections and HEp-2 slide preparations by indirect immunofluorescence. Cytotoxicity screening of heterohybridoma supernatants gave broad panel reactivity profiles, being cytotoxic against B cells from patient IRM and also against most B cells tested and less reactive with chronic lymphocytic leukemia B cells; T cells were the least sensitive target. Immunoblotting showed that monoclonal IRM displayed some heterogeneity in their binding profiles, although all of them recognized a cellular structure of 26 kDa. None of the heterohybridoma cell lines exhibited cytoplasmic nor surface staining with an anti-CD5 mAb. Results obtained showed that all the autolymphocytotoxic mAbs generated were also able to react against certain nuclear and cytoplasmic self-structures as well as foreign compounds. Monoclonal antibody IRM-7 and, to a lesser degree, IRM-10 exhibited multispecific properties similar to those observed for polyreactive or natural antibodies.

Influence of cell‐ and media‐derived factors on the integrity of a human monoclonal antibody after secretion into serum‐free cell culture supernatants

To investigate the effects of factors secreted by different cell lines on human monoclonal antibody (MAb) integrity, 600 mg of a human MAb, which specifically binds to human erythrocytes, were produced in a perfusion process. After purification by protein A affinity chromatography, the MAb was used for integrity testing in supernatants of several cell lines to investigate their potential to degrade the antibody in the extracellular environment. One insect cell line (IPLB-SF-21 AE) and four mammalian cell lines [CHO K1, BHK-21 (C13), C1271, P3-X63-Ag8.653], all of them commonly used for the production of recombinant proteins, and the human-human-mouse heterohybridoma cell line itself (H-CB-hahE), were adapted to serum-free culture media. For integrity testing all cell lines were cultivated in spinner flasks using serum-free media supplemented with 30 mug mL(-1) of purified MAb. MAb integrity was assayed by SDS polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing, both followed by Western blotting, and an antigen binding assay. None of the mammalian cells showed any detectable effects on antibody stability and integrity during exponential growth, whereas isoelectric focusing of monoclonal antibody taken from IPLB-SF-21 AE culture supernatants revealed a new band indicating a partial modification of the MAb by secreted factors of these cells. This observation did not correlate with the total proteolytic activity, which was measured in all supernatants and found to be lowest in the insest cell cultures. For mammalian cell cultures, it could be concluded from these findings that shifts of the antibody microheterogeneity pattern, which can be found normally as a result of variations in different production parameters, are not caused by extracellular factors once the product has been secreted into the supernatant. In addition to their well-known advantages in posttranslational modifications (e.g., formation of complex type N-glycans), mammalian cells appear to be more suitable as expression systems for human monoclonal antibodies to be used in vivo when compared with baculovirus-infected insect cells. (c) 1995 John Wiley & Sons, Inc.

The effects of oncogene transfection on growth and antibody production of human-mouse heterohybridomas

Human-mouse heterohybridomas producing human monoclonal antibodies show slower growth rates and lower peak cell densities than murine hybridomas. In order to improve the growth properties we transfected a heterohybridoma cell line with expression plasmids containing the oncogenes v-src, c-Ha-ras and SV40largeT. The plasmids were transferred by electroporation. Growth promoting activities of the plasmids were proven in NIH3T3 cells whereby a doubling of the maximum cell densities of this cell type was observed. The oncogene products were analyzed by means of northern blotting and immunofluorescence. After transfection of c-src and c-ras, a heterohybridoma cell line was derived which showed improved growth characteristics compared to the original cell line. Although specific antibody production was lower, antibody concentrations which accumulated in batch culture were higher due to increased maximum cell densities.

Cloning of human anti‐GM1 antibodies from motor neuropathy patients

Patients with multifocal motor neuropathy frequently have elevated titers of serum antibodies reactive with GM1 ganglioside. Although these antibodies may cause the syndrome, this has yet to be proven directly. As part of our studies on the nature and pathogenic potential of anti-GM1 antibodies, we have cloned B cells from the peripheral blood of 3 patients with multifocal motor neuropathy and generated four stable heterohybridoma cell lines secreting human monoclonal IgM anti-GM1 antibodies. In this report we describe the basic properties of these monoclonal antibodies in comparison with the patient's sera from which they were derived. The antibodies all differ in their pattern of reactivity with GM1 and other Gal(beta 1-3)GalNAc-containing glycoconjugates. They have widely varying thermal ranges and their reactivities are strongly influenced by the presence of accessory lipids. Affinity purification of the patient's sera with GM1 led to the identification of previously unrecognized paraproteins that were resolvable above the background of polyclonal anti-GM1 IgM. Our data demonstrate considerable heterogeneity in the immune response to GM1 both within individual sera and between different patients, which is likely to be of importance to their role in disease pathogenesis.

Flow cytometric analysis of the stability of antibody production by human × human × mouse heterohybridoma subclones

Flow cytometry has been utilized to evaluate the stability of antibody production by unstable subclones of a human × human × mouse heterohybridoma. Heterogeneity of cell-associated immunoglobulin heavy chain expression was demonstrated in different subclones and an increased frequency of cells containing low levels of heavy chain was found to correlate with low antibody productivity. However, the majority of cells were not completely devoid of heavy chains suggesting that the genetic information for the γ chain was not lost. In contrast, the gene encoding the κ light chain was shown to be absent from the subclones expressing low levels of heavy chain and these subclones also contained substantially reduced levels of heavy chain mRNA, suggesting that the production of this protein was controlled at the level of transcription or mRNA stability. In conclusion, the correlation of staining intensity as observed by flow cytometry and antibody productivity makes flow cytometry a suitable technique for the rapid evaluation of heterohybridoma cell lines used for antibody production.

Human Monoclonal λ Light Chain Protein Exhibits Specific Binding to the Variable Region of Monoclonal Anti-IgE Antibody

A monoclonal heterohybridoma cell line (B11-12) was developed by fusing EBV-transformed lymphocytes from a patient with hyper-IgE syndrome and SHM-D33 heteromyeloma cells. The resultant heterohybridoma secreted only human λ L chains exhibiting specific binding to mouse monoclonal Ab to human IgE (mAb-aIgE). B11-12 bound to four separate mAb-aIgE, but not to mAb of the same isotypes directed to other antigens. The mAb-aIgE differed in isotype (IgG1; IgG2b) demonstrating that B11-12 was recognizing an idiotypic, but not an isotypic determinant on the mAb-aIgE. Human IgE did not inhibit B11-12 binding to mAb-aIgE, suggesting the interaction between B11-12 and mAb-aIgE involved an idiotope outside the combining site of the mAb-aIgE. Human sera, including serum from the donor of the lymphocytes used to produce heterohybridoma B11-12, demonstrated the capacity to inhibit B11-12 binding to mAb-aIgE. This, along with the inability of these same sera to directly bind B11-12, suggests that the serum contained reactivity similar to B11-12. We propose that B11-12 demonstrates specificity resembling an anti-Id to anti-human IgE Ab.

Synteny mapping in the horse using horse‐mouse heterohybridomas

In a study of 35 horse-mouse heterohybridoma cell lines, synteny in the horse was found between LDHB, PEPB and IGF1 and between NP, MPI and IDH2. A synteny between ADA and PEPC was also indicated. The loci for horse immunoglobulin light chain (IgL) genes and for LDHA were independent.

The effects of hybridoma growth factor in conditioned media upon the growth, cloning, and antibody production of heterohybridoma cell lines

Interleukin 6 (IL-6)/hybridoma growth factor (HGF) has been shown to be the requirement for growth of murine hybridomas in vivo or in vitro. In this paper, two kinds of conditioned media (CM) from the culture supernatants of a human fibroblast cell line CRL1506 and a cloned Epstein-Barr virus (EBV) transformed human lymphoblastoid cell line (LCL) N23 were found to have IL-6 activity by strongly promoting the growth, antibody secretion (increase of one- to three-fold), and cloning efficiencies of heterohybridomas secreting human monoclonal anti-hemorrhagic fever with renal syndrome virus antibodies and LCL. Since these CM contained no detectable IL-2, and IL-4 had no effects on the growth of the cell lines, IL-6 was considered to be the main active component of the CM responsible for promoting hybridoma growth. This effect was further confirmed by IL-6-dependent cell line 7TD1 bioassay (IL-6 activity in the CM ranging from 1,000 to 10,000 units/ml). Moreover, we successfully established four EBV-transformed lymphoblastoid cell lines at single-cell level by adding an equal volume of CRL1506-CM to 10% FCS-RPMI1640 in limiting dilution. Finally, it is worth noting that the sensitivity of the heterohybridomas to the two kinds of CM was not the same and was not consistent with that of their parental myeloma cell lines. Thus, it suggests that the CM might contain more than one factor, and the choice of proper conditioned media should be very useful for human monoclonal antibody production.

Probing the human antibody repertoire to exogenous antigens. Characterization of the H and L chain V region gene segments from anti-hepatitis B virus antibodies.

Structural studies of human antibody V regions have been largely limited to those involving the fetal repertoire, autoantibodies, and malignant cell rearrangements, leaving the "normal" repertoire relatively unexplored. In this study we describe the nucleotide sequences of the H and L chain V regions of four antibodies specific for the surface Ag of the hepatitis B virus. Monoclonal cell lines were derived from healthy individuals who received standard immunizations with the serum-derived or recombinant hepatitis B virus vaccines by fusion of PBL to a heterohybridoma cell line, SPAZ-4. We utilized the polymerase chain reaction to amplify the H and L chain V regions for cloning and sequencing. The four antibodies express the following V region combinations: VHIII/V lambda V, VHIII/V kappa II, VHIV/V kappa I, VHV/V lambda III. When compared to germline genes with the closest sequence homology, all of the V regions appear to have undergone somatic mutation, ranging from 3.4 to 11.3% for the H chain, and 5.1 to 9.2% for the L chain. Analysis of the mutations shows them to be typical for an Ag-driven immune response.

Lupus-derived human monoclonal IgM anti-DNA antibody displays monospecificity, high affinity and private idiotype specificity.

A human monoclonal IgM k anti-DNA antibody, designated 2F7, was prepared by somatic hybridization of peripheral blood lymphocytes from a lupus patient with a human-mouse heterohybridoma cell line, K6H6/B5. 2F7 was tested for its antigen binding and idiotypic specificity by direct binding and inhibition enzyme-linked immunosorbent assays. 2F7 had a high binding activity to single-stranded DNA (ssDNA) but not to double-stranded DNA. It cross-reacted with single-stranded homopolymers with pyrimidine bases and double-stranded polynucleotides containing those homopolymers, suggesting that 2F7 recognizes a conformational determinant made up of both deoxyribose-phosphate backbone and specific nucleotide base. 2F7 did not cross-react with eight structurally unrelated self-antigens. Dissociation constant (Kd) of 2F7 for sonicated ssDNA was approximately 4.5 x 10(-9) M, indicating its relatively high affinity. Idiotypic characterization with rabbit anti-idiotype raised against 2F7 suggested that 2F7 expressed an idiotype at or near its antigen-binding sites that was not detected in sera from 20 unrelated lupus patients, 10 lupus family members and 10 normal individuals. These results suggest that certain IgM class anti-DNA antibodies in human systemic lupus erythematosus may arise by antigen stimulation and not simply by polyclonal B-cell activation.

Production of Heterohybridomas Secreting Autoreactive and Polyreactive Human Monoclonal Antibodies

Peripheral blood lymphocytes from two polytransfused renal dialysis patients were transformed by Epstein-Barr virus, fused to a heteromyeloma and cloned. Eight human monoclonal antibodies from the resulting clones were tested for their binding to a variety of antigens by ELISA, indirect immunofluorescence and immunoblotting. Antigens tested included B-cell lines, T and B lymphocytes, red blood cells, chronic lymphocytic leukaemic B cells, IgG, ssDNA, dsDNA, histones, nucleoprotamine, sperm nuclei, thymus and spleen extracts, MOLT4 cell lysates, affinity purified autoantigens, tetanus toxoid, bacterial lipopolysaccharide, insulin, and a tissue section screen. These human monoclonal antibodies reacted with more than one antigen to varying degrees and were autoreactive and polyreactive. One of these heterohybridoma cell lines exhibited cytoplasmic staining with an anti-CD5 monoclonal. Our findings support the concept that in adult individuals a subset of B cells produce heterogeneous IgM antibodies which can bind to a variety of different autoantigens and also to foreign antigens. These monoclonals were different from the autoantibodies usually seen in renal dialysis patients in the sense that they were not lymphocytotoxic.

Biological activity of a human monoclonal antibody to Bordetella pertussis lipooligosaccharide

The heterohybridoma cell line HBp2 secreting human monoclonal antibody (hMAb) directed against Bordetella pertussis was generated by fusing SP2/HPT heteromyeloma cells with human spleen lymphocytes, after in vitro stimulation for 6 days. The hybridoma was maintained in culture for more than 1 year with continuous antibody secretion. The hMAb HBp2, an IgM, reacted with untreated and proteinase K-treated B. pertussis outer membrane antigens, whereas the reactivity was lost when the antigen was treated with sodium periodate. Human MAb HBp2 was shown to be specific to B. pertussis LOS by immunoblotting of whole cell extracts after SDS-PAGE. In a dot enzyme immunoassay, HBp2 reacted with all B. pertussis strains and clinical isolates tested except for four atypical variant strains of the LOS B phenotype. Human MAb HBp2 also reacted with a clinical isolate of B. bronchiseptica. No reaction was observed against B. parapertussis and other gram-negative species. Together these studies suggested that HBp2 is reactive with carbohydrate epitopes present on the LOS A. Binding assays with live bacteria demonstrated that hMAb HBp2 reacted with cell surface exposed epitopes on B. pertussis but the antibody did not bind significantly to the surface on intact B. bronchiseptica cells. When examined for bactericidal activity in the presence of complement, hMAb HBp2 showed high lytic capability against B. pertussis while no killing was obtained against B. bronchiseptica. These experiments established that LOS A is a target for human bactericidal antibodies. This antigen merits further investigation as a potentially important component in human immunity to B. pertussis infection.

Cloning of human heterohybridoma cell lines using chronic lymphocytic leukemia B cells as a feeder layer

B cells from the peripheral blood of chronic lymphocytic leukemia patients were isolated by gradient density centrifugation and used without irradiation as a feeder layer in the cloning of human heterohybridoma cell lines by limiting dilution. Cloning efficiencies were high with all the cell lines tested. These feeder leukemia B cells could also be successfully used after having been stored in liquid nitrogen.

Cloning and sequencing of human λ immunoglobulin genes by the polymerase chain reaction

Universal oligonucleotide primers, designed for amplifying and sequencing genes encoding the rearranged human lambda immunoglobulin variable region, were validated by amplification of the lambda light chain genes from four human heterohybridoma cell lines and in the generation of a cDNA library of human V lambda sequences from Epstein-Barr virus-transformed human peripheral blood lymphocytes. This technique allows rapid cloning and sequencing of human immunoglobulin genes, and has potential applications in the rescue of unstable human antibody-producing cell lines and in the production of human monoclonal antibodies.

N‐Acetyl‐L‐galactosaminuronic acid as an epitope common to the O‐polysaccharides of Pseudomonas aeruginosa serotype A and H (Homma) recognized by a protective human monoclonal antibody

We have established a human--mouse heterohybridoma cell line producing a human monoclonal antibody TS-3G2 (IgG gamma 1, K). This monoclonal antibody specifically bound to O-polysaccharides belonging to plural Pseudomonas aeruginosa Homma serotypes, A and H, in contrast to serotype-specific monoclonal antibody which exclusively bound to strains belonging to a single specific serotype. The binding affinity for serotype A strains was higher than that for serotype H strains. Competitive enzyme immunoassay experiments with O-polysaccharide preparations derived from IID 1001, NCTC 8505 (serotype A) and IID 1009 (serotype H) and their derivatives demonstrated that the N-acetyl-L-galactosaminuronic acid residue in O-polysaccharide was essentially involved in the epitope for TS-3G2. Furthermore, a 6-deoxy-hexosamine residue neighboring the reducing terminal of N-acetyl-L-galactosaminuronic acid residues was also concerned with the epitope to some extent. In the experimental infection model of normal mice, the monoclonal antibody TS-3G2 showed a protective activity against both strains of serotype A and H.

Isolation and characterization of human fibroblast tenascin. An extracellular matrix glycoprotein of interest for developmental studies.

We have developed a biochemical method for purifying human tenascin from cultured fibroblasts or the culture medium. The method is a series of biochemical procedures including gel filtration, gelatin gel affinity chromatography and ion-exchange high performance liquid chromatography. The final preparation was identified as tenascin from its immunological cross-reactivity to antibody against chicken tenascin, strong hemagglutination activity which has been reported to be one of the biological functions of chicken tenascin, and from the electron microscopic study demonstrating a six-armed structure. Gel chromatography showed that intact human tenascin has an apparent molecular weight of over one million. Analysis of the purified tenascin with SDS-PAGE under reducing conditions demonstrated that tenascin consists of two kinds of subunits (250K and 190K). We established rat x mouse heterohybridoma cell lines which produce tenascin-specific antibodies. One monoclonal antibody (RCB1) was selected for immunohistochemical study and partially characterized. RCB1 bound native tenascin but not reduced and alkylated tenascin. Immunohistochemistry of normal and neoplastic tissues demonstrated that RCB1 bound the connective tissues surrounding the cancer nests and various normal tissues including interstitium of renal distal tubule, periosteum, endosteum, smooth muscles of digestive tract and media of arteries and arterioles.

Growth of rat-mouse hybridoma cells in immunosuppressed hamsters: An easy and effective method to prepare monoclonal antibodies from heterohybridoma cell lines

Rat-mouse hybridoma cells producing anti-mouse IgE antibodies were intraperitoneally or subcutaneously inoculated into newborn or suckling hamsters receiving rabbit anti-hamster thymocyte globulin from the day of birth twice a week for at least 3 weeks. The hybridoma cells were found to grow in the abdominal cavity of the hamsters as ascites tumor or in subcutaneous tissue as solid tumor without loss of antibody-secreting activities. For the production of ascites, 2-week-old hamsters were preferable to newborn hamsters. In 3-week-old hamsters, the hybridoma cells could scarcely survive. The antibody titers of the ascites were determined to be 10 5−10 6 in the ELISA and in the ability to neutralize the skin-sensitizing capacity of mouse IgE antibodies. The rat monoclonal antibodies were easily separated from ascites, serum or cell culture supernatant with affinity chromatography using Affigel protein A-Sepharose and anti-hamster IgG-Sepharose columns. The described method could be efficiently applicable for the proliferation of other hybridomas, such as human-human, human-mouse or hamster-mouse, etc.

Expression of Human Anti-Tetanus Toxoid Antibody in Transfected Murine Myeloma Cells

Mouse-human heterohybridoma cell line, GF4/1.1, secretes a human anti-tetanus toxoid (anti-TT) antibody of the γ3 isotype. This line was used as a source of mRNA for the construction of cDNA libraries and the subsequent isolation of the sequences encoding the human heavy and light chains. Expression vectors containing the cDNAs were constructed and introduced into a non-Ig producing, murine hybridoma cell line, together with a dihy-drofolate reductase (dhfr) selectable marker gene. Transfected cells producing human anti-TT antibody were grown in medium with increasing concentrations of the selecting drug (methotrexate). The level of antibody secretion was tested in cells that had adapted to the higher drug concentration and was found to increase in parallel with increases in the steady-state level of Ig mRNA. This increased productivity was not associated with detectable levels of gene amplification. Stable clones were obtained that secrete levels of human antibody in either open suspension cultures or within microcapsules that compare favorably with those of murine hybridomas.

Generation of a sheep × mouse heterohybridoma cell line (1C6.3a6T.1D7) and evaluation of its use in the production of ovine monoclonal antibodies

A stable aminopterin-sensitive sheep × mouse heterohybridoma cell line (1C6.3a6T.1D7) for use in the generation of sheep monoclonal antibodies is described. The line was first constructed by fusing the mouse myeloma line, NSO, to normal sheep lymphocytes obtained from the efferent lymphatic vessel of a cannulated popliteal lymph node. The line was rendered sensitive to aminopterin through a combination of irradiation and treatment with the anti-metabolite drug 6-thioguanine. Characterisation of the cloned cell line showed that it did not secrete sheep immunoglobulin (Ig) molecules, express Ig on the surface membrane, or express normal sheep B or T cell surface markers. The 1C6.3a6T.1D7 line has remained stable in tissue culture for over 2 years, showing no signs of reversion to aminopterin resistance. The 1C6.3a6T.1D7 cells have been used as fusion partners with lymphocytes from antigen primed sheep to generate sheep monoclonal antibodies to human chorionic gonadotropin (hCG) or a synthetic peptide analogue of the VP1 capsid protein of foot and mouth disease virus (FMDV). To optimise the efficiency of heterohybridoma generation, comparisons were made of peripheral blood, efferent lymph or excised lymph nodes as sources of antigen-stimulated lymphocytes for fusion. The results showed that lymphocytes prepared from either efferent lymph or lymph node on the fourth day following antigenic stimulation gave similar high fusion efficiencies, and both were vastly superior to peripheral blood lymphocytes. Results were also obtained which showed that the blast cells present in lymphoid tissues due to antigenic stimulation were the major cell types involved in the generation of viable antibody-secreting sheep × sheep × mouse heterohybridomas.

Production and characterization of rat monoclonal antibodies with an apparent specificity for certain lung cancers and their potential clinical application : Jitsukawa T, Iwahashi T, Sugawara I, Inagaki H, Mori S. Department of Basic Research, R & D Labs. Hoechst Japan Limited, Saitama 350. Jpn J Exp Med 1987;57:321-2

This paper describes two rat monoclonal antibodies (MAbs) (1A6 and 2H7, both IgG2a) directed against a human adenocarcinoma of the lung (A549). Enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry (Avidin-Biotin-complex method) as the screening of the hybridoma cells revealed that 1A6 and 2H7 MAb mainly reacted with adenocarcinoma of the lung, breast and pancreas, and 2H7 MAb also recognized large cell carcinoma, epithelia of the lung, trachea, tongue or small cell carcinoma of the lung. The antigen molecules recognized by 1A6 MAb was not detected by immunoprecipitation but the molecular size of the antigen by 2H7 was ca. 40Kd. The antibody-producing heterohybridoma cells could be transplanted into peritoneal cavities of nude mice to form ascites. We found these monoclonals useful for immunohistodiagnosis of surgical specimens from patients with lung cancer. Furthermore, we utilized 125I-labeled 2H7 MAb, aiming at localizing A549 tumor mass transplanted into nude mice subcutaneously. This antibody clearly localized the tumor mass. It is suggested that they may be useful as both diagnostic and therapeutic agents.