Significance of antigen, drug, and tumor cell targets in the preclinical evaluation of doxorubicin, daunorubicin, methotrexate, and mitomycin-C monoclonal antibody immunoconjugates

We tested drug monoclonal antibody immunoconjugates in vitro in 72 h 3H-thymidine assays and in vivo in athymic mice bearing human tumor xenografts of the same target cells. Experimental arms included control, monoclonal antibody, drug, drug + antibody, the test immunoconjugate, and a negative control immunoconjugate with an equivalent molar amount of drug for in vitro experiments, and the amount of drug conjugated to 500 micrograms of antibody in the animal experiments. Monoclonal antibodies included T101, an IgG2a that reacts with a rapidly modulating antigen, 9.2.27, an IgG2a that reacts with a slowly modulating antigen, and ME7, an IgG1 that reacts with a slowly modulating antigen. Cells used in testing included MOLT-4 (T lymphoma), 8392 (B lymphoma), and M21 (melanoma). Drugs tested were doxorubicin, daunorubicin, methotrexate, and mitomycin-C. M21 cells were resistant to daunorubicin in vitro but were inhibited by the 9.2.27 daunorubicin immunoconjugate. T101, 9.2.27, and ME7 cis-aconitate anthracycline immunoconjugates and mitomycin-C-glutarate immunoconjugates were specifically cytotoxic only for antigen positive cells in vitro and were superior to free drug in vivo. These results confirm that antigen specific-cytotoxic drug immunoconjugates can be produced that are superior to the same dose of free drug. However, each monoclonal antibody drug target system is unique and must be well-characterized for appropriate interpretation of data.     

Improved staining method for the simultaneous flow cytofluorometric analysis of DNA content, S-phase fraction, and surface phenotype using single laser instrumentation.

We have developed an improved technique for triple staining that permits the simultaneous flow cytofluorometric analysis of cell surface antigens, bromodeoxyuridine incorporation into DNA, and DNA quantification using 7-amino-actinomycin D. PHA-activated human peripheral blood lymphocytes were incubated with bromodeoxyuridine and stained for cell surface phenotype with phycoerythrin-labeled monoclonal antibodies. Stained cells were fixed serially with 1% paraformaldehyde and 45% ethanol. Fixed cells were sequentially stained with an anti-BrdUrd monoclonal antibody followed by a FITC-conjugated goat anti-mouse antibody and incubated with 7-amino-actinomycin D. Hypotonic buffer was employed for all procedures after fixation. Stained-fixed cells were analyzed by flow cytofluorometry for simultaneous green (525 nm), orange (570 nm), and red (greater than 650 nm) fluorescence. Utilizing this staining technique, we were able to analyze simultaneously cell phenotype, DNA synthesis, and total cellular DNA content with single laser excitation.     

A microinjected monoclonal antibody against human DNA polymerase-alpha inhibits DNA replication in human, hamster, and mouse cell lines

We have examined the effect that microinjection of a monoclonal antibody directed against human DNA polymerase-alpha (SJK-287) has on DNA synthesis in exponentially growing human, mouse, and hamster cell lines. We show that the SJK-287 antibody, when microinjected directly into the nuclei of cells is capable of inhibiting DNA synthesis in all three cell lines tested. Moreover, the effectiveness with which this antibody can inhibit ongoing DNA synthesis by the microinjection assay is closely correlated with the ability of the antibody to neutralize DNA polymerase-alpha activity fractionated from each cell line in vitro. Two other monoclonal antibodies of the same class, one directed against the cellular p53 protein (PAb122), and one directed against the c-myc protein (PM-8) were also tested for their ability to inhibit ongoing DNA synthesis by direct microinjection and in lysolecithin permeabilized cells. Both monoclonal antibodies failed to inhibit ongoing DNA synthesis in exponentially growing cells by these assays.     

Correlations between the 44D7 antigenic complex and the plasma membrane Na+-Ca2+ exchanger

The exchange of Na+ for Ca2+ across the plasma membrane is mediated by a carrier transport system known as the Na+-Ca2+ exchanger. We have recently reported the specific inhibition of Na+-Ca2+ exchanger activity in cardiac and skeletal muscle sarcolemmal vesicles by monoclonal antibody 44D7. In this review, we summarize the properties of the 44D7 monoclonal antibody and the antigenic complex reacting with this antibody. The 44D7 antibody was produced against human acute lymphocytic cells and recognizes a molecular complex composed of two subunits of the apparent molecular weights 95 000 and 38 000, linked by disulfide bonds. Two other monoclonal antibodies react with the same complex:4F2 which binds to the same epitope as 44D7 and specifically inhibits the Na+-Ca2+ exchanger activity, and 44H7 which reacts with a distinct epitope and does not inhibit exchanger activity. The 44D7 antibody reacts with nerve fibers in brain and proximal convoluted tubules of kidney, both known to possess Na+-Ca2+ exchanger activity. Reactivity of 44D7 antibody with tonsil and thymus sections is restricted to certain subpopulations of cells. The reactivity of the antibody is very weak with resting lymphocytes in suspension; however, activated T lymphocytes and leukemic cells show increased binding to 44D7 antibody. Several malignant cell lines express high levels of the 44D7 antigen. The reactivity of a human hepatoma with 44D7 antibody is much greater than that observed with normal hepatocytes. The inhibition by monoclonal antibody 44D7 of the Na+-Ca2+ exchanger activity and the similarity in tissue distribution of the 44D7 antigenic complex and the exchanger system suggests that these two molecules might be related.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of a mouse-human chimeric monoclonal antibody to CD20 with potent Fc-dependent biologic activity

Mouse monoclonal antibody 2H7 recognizes the CD20 cell surface phosphoprotein that is expressed in normal as well as malignant B cells. CD20 may be a useful target for therapy of B cell lymphomas, since damaged normal B cells can be replaced by their antigen-negative precursors. Monoclonal antibody 2H7 is an IgG2b (kappa) immunoglobulin which cannot mediate antibody-dependent cellular cytotoxicity with human lymphocytes or complement-dependent cytotoxicity with human serum. We have now generated a chimeric 2H7 antibody by substituting the mouse constant domains of 2H7 with the human gamma 1 and kappa domains. This new antibody has the same binding specificities as 2H7 but is highly effective in mediating antibody-dependent cellular cytotoxicity with human effector cells and complement-dependent cytotoxicity with human complement.     

Characteristics and distribution of endogenous RFamide-related peptide-1

We have recently identified RFamide-related peptide (RFRP) gene that would encode three peptides (i.e., RFRP-1, -2, and -3) in human and bovine, and demonstrated that synthetic RFRP-1 and -3 act as specific agonists for a G protein-coupled receptor OT7T022. However, molecular characteristics and tissue distribution of endogenous RFRPs have not been determined yet. In this study, we prepared a monoclonal antibody for the C-terminal portion of rat RFRP-1. As this antibody could recognize a consensus sequence among the C-terminal portions of rat, human, and bovine RFRP-1, we purified endogenous RFRP-1 from bovine hypothalamus on the basis of immunoreactivity to the antibody. The purified bovine endogenous RFRP-1 was found to have 35-amino-acid length that corresponds to 37-amino-acid length in human and rat. We subsequently constructed a sandwich enzyme immunoassay using the monoclonal antibody and a polyclonal antibody for the N-terminal portion of rat RFRP-1, and analyzed the tissue distribution of endogenous RFRP-1 in rats. Significant levels of RFRP-1 were detected only in the central nervous system, and the highest concentration of RFRP-1 was detected in the hypothalamus. RFRP-1-positive nerve cells were detected in the rat hypothalamus by immunohistochemical analyses using the monoclonal antibody. In culture, RFRP-1 lowered cAMP production in Chinese hamster ovary cells expressing OT7T022 and it was abolished by pre-treatment with pertussis toxin, suggesting that OT7T022 couples G(i)/G(o) in the signal transduction pathway.     

Stimulation of HLA-A,B,C by IFN-alpha. The derivation of Molt 4 variants and the differential expression of HLA-A,B,C subsets.

Spontaneous mutants with altered HLA-A,B,C response to interferon-alpha (IFN-alpha) were isolated from the human thymus leukemia cell line Molt 4. Using fluorescein isothiocyanate (FITC)-conjugated W6/32 (a monoclonal antibody to HLA-A,B,C) and the fluorescence-activated cell sorter, the cells with highest and lowest fluorescence after 24-48 h of IFN-alpha treatment were selected and expanded. After several cycles of selection, mutant clones with low (greater than 10% of wild-type) and high (three times better) response were obtained. A similar protocol was employed to derive high responder mutants with the monoclonal antibody YT76, which recognises a subset of HLA strongly induced by IFN-alpha. Stable clones were derived for which YT-HLA induction was 7-fold that of Molt 4 cells and for which HLA induction occurred at 100-fold lower concentrations of IFN-alpha. The high response phenotype of the mutants was not accompanied by a significant increase in the constitutive level of expression of HLA-A,B,C (in the absence of IFN). The increase in the level of HLA-A,B,C expression after IFN-alpha treatment is mostly accounted for by the increase in the expression of a subset of HLA molecules, detected by the monoclonal antibody YT76 including HLA-B molecules.     

Comparative activity of Sant7 and anti-IL-6, IL-6R monoclonal antibodies in a murine model of B-cell lymphoma

Interleukin-6 (IL-6) plays a central role in the pathogenesis of several autoimmune and inflammatory diseases as well as B-cell lymphoproliferative disorders. This work describes the effects of the recombinant or adenovirally-delivered IL-6 superantagonist Sant7, anti-IL-6 and IL-6 receptor monoclonal antibodies in a severe murine model of human B-cell lymphoma induced in SCID mice by transplantation of an LCL-41 cell line variant (isotype-switched IgM>IgG). Survival of 60% of the animals treated with anti-gp130 was observed up to day 33, while about 20% of the animals survived with anti-gp80 and Sant7 treatment. No survival was observed with the anti-IL-6 monoclonal antibody treatment. No significant change in serum and peritoneal levels of human IL-6 (hIL-6) and soluble human IL-6 receptor (shIL-6R) was observed in the recombinant Sant7-treated group towards the control group. The anti-gp80 monoclonal antibody induced significant increase of both hIL-6R and hIL-6 in serum and peritoneum. The anti-gp130 monoclonal antibody treatment determined a reduction of the seric shIL-6R and a significant increase of the seric hIL-6. Anti-IL-6 monoclonal antibody administration resulted in a reduction of serum and in an increase of peritoneal hIL-6. Treatment with adenoviral Sant7 was associated with a reduction of circulating shIL-6R, hIgG and mSAP. However, only marginal anti-tumor efficacy of the adenoviral Sant7 was observed. Overall, the present data suggest a potential for anti-hIL-6 therapy in B-cell lymphomas. Less severe animal models might be useful to better evaluate Sant7 efficacy alone or in combination with other anti-IL-6 therapeutics.     

Expression of markers shared between human natural killer cells and neuroblastoma lines

Summary Neuroblastoma is a tumor of neuroectodermal origin arising most commonly from the adrenal medulla. We have examined the ability of several monoclonal antibodies which recognize markers predominantly expressed on human natural killer (NK) cells to react with neuroblastoma cell lines in vivo derived sections of tumor. HNK-1 (Leu 7) is a monoclonal IgM antibody which recognizes a carbohydrate epitope on NK cells and a wide range of tumor cell types. We have shown that HNK-1 recognizes the human neuroblastoma lines SMS-KCNR, SMS-KAN, NMB/N7, and IMR/5. Expression of this antigen on cell lines can be slightly increased by retinoic acid-induced differentiation of the cells. N901 (NKH1), a monoclonal antibody raised against interleukin 2-dependent human NK cell lines also recognizes all human neuroblastoma cell lines examined. This expression is independent of differentiation induction and levels remain unaltered following retinoic acid treatment of the cell lines. Lastly, with monoclonal antibody 49H.8, it has been found that reactivity of the lines is weak until induction of differentiation, after which highly significant increases of reactivity are seen. 49H.8 recognizes several cryptic carbohydrate antigens with varying affinities, shown to identify mouse and rat NK cells. In contrast to other NK markers, human neuroblastoma cell lines did not express significant reactivity with B73.1, Leu 11b, or Leu 18. Immunohistochemical staining of sections of human neuroblastoma tumors correlated with the in vitro findings; however, staining with N901 and 49H.8 was only seen on frozen sections, not paraffin-embedded. The significance of shared NK cell-neuroblastoma/neuron antigens is currently under investigation.     

Leu-7(HNK-1)在人胃肠内分泌细胞中的表达

Leu-7(HNK-1)是一种分子量为110KD的糖蛋白,它是人类NK和K细胞特异性的表面抗原。Leu-7单克隆抗体可特异性识别NK和K细胞表面抗原。本文应用ABC免疫组织化学染色技术研究了Leu-7在人胃肠道的定位和分布。结果发现,Leu-7免疫反应(Leu-7-IR)细胞主要分布于胃腺和肠腺的底部。偶见于肠绒毛上皮中,呈典型内分泌细胞的形态特征。相邻切片法免疫双标记证明多数胆囊收缩素(CCK-8),生长抑素(SS),胃泌素(G34)或5-羟色胺(5-HT)免疫反应阳性细胞呈Leu-7阳性。我们的发现支持了在神经,内分泌和免疫系统间存在相同或相似抗原决定簇的假说。     

Aglycosylation of human IgG1 and IgG3 monoclonal antibodies can eliminate recognition by human cells expressing Fc gamma RI and/or Fc gamma RII receptors.

Aglycosylated human IgG1 and IgG3 monoclonal anti-D (Rh) and human IgG1 and IgG3 chimaeric anti-5-iodo-4-hydroxy-3-nitrophenacetyl (anti-NIP) monoclonal antibodies produced in the presence of tunicamycin have been compared with the native glycosylated proteins with respect to recognition by human Fc gamma RI and/or Fc gamma RII receptors on U937, Daudi or K562 cells. Human red cells sensitized with glycosylated IgG3 form rosettes via Fc gamma RI with 60% of U937 cells. Inhibition of rosette formation required greater than 35-fold concentrated more aglycosylated than glycosylated human monoclonal anti-D (Rh) antibody. Unlabelled polyclonal human IgG and glycosylated monoclonal IgG1 and anti-D (Rh) antibody inhibited the binding of 125I-labelled monomeric human IgG binding by U937 Fc gamma RI at concentrations greater than 50-fold lower than the aglycosylated monoclonal IgG1 anti-D (Rh) (K50 approximately 3 x 10(-9) M and approximately 6 x 10(-7) M respectively). Similar results were obtained using glycosylated and aglycosylated monoclonal human IgG1 or IgG3 chimaeric anti-NIP antibody-sensitized red cells rosetting with Fc gamma RI-/Fc gamma RII+ Daudi and K562 cells. Rosette formation could be inhibited by the glycosylated form (at greater than 10(-6) M) but not by the aglycosylated form. Haemagglutination analysis using a panel of murine monoclonal antibodies specific for epitopes located on C gamma 2, C gamma 3 or C gamma 2/C gamma 3 interface regions did not demonstrate differences in Fc conformation between the glycosylated or aglycosylated human monoclonal antibodies. These data suggest that the Fc gamma RI and Fc gamma RII sites on human IgG are highly conformation-dependent and that the carbohydrate moiety serves to stabilize the Fc structure rather than interacting directly with Fc receptors.     

Monoclonal antibody to the human glucose transporter that differentiates between the glucose and nucleoside transporters

A monoclonal antibody to the glucose transporter has been prepared with band 4.5 (Mr 45,000-65,000) from human erythrocyte ghosts as antigen. This antibody, designated 7F7.5, is of the IgG2b type. The antibody bound exclusively to proteins in the band 4.5 region of immunoblots of human erythrocyte ghosts separated on sodium dodecyl sulfate-polyacrylamide gels. Immobilized 7F7.5 antibody removed glucose transport activity from solubilized alkaline-treated ghosts. The material that was eluted from the immobilized antibody matrix migrated primarily in the band 4.5 region of electrophoretic gels and bound the antibody in immunoblots. To test the specificity of the antibody, glucose and nucleoside transporters in alkaline-treated human erythrocyte ghosts were affinity labeled with [3H]cytochalasin B and [3H]-S-(nitrobenzyl)thioinosine (NBMPR), respectively. Both of these transporters are band 4.5 proteins and "copurify" by DEAE-cellulose chromatography. A filter paper assay was developed to assess the presence of the labeled transporters. Immobilized 7F7.5 antibody bound 99% of the labeled glucose transporter. In contrast, only 3% of the specifically labeled nucleoside transporter bound to the immobilized antibody. Furthermore, the antibody did not remove nucleoside transport or NBMPR binding activities from detergent solution. The antibody recognized two tryptic fragments, Mr 23,000 and 18,000, which contain the cytochalasin B binding site of the glucose transporter. By immunoblot, the monoclonal antibody recognized the glucose transporter in cultured human IM9 lymphocytes, synovial cells, and HBL 100 mammary cells but not cells of murine or rat origin. These results indicate that the glucose and nucleoside transporters are distinct proteins which can be distinguished by monoclonal antibody 7F7.5. The method developed to quantitate covalently labeled glucose and nucleoside transporters should have broad applicability as a rapid and easy method for determining the recovery of affinity-labeled membrane proteins in detergent solution during purification. Because of the location of the epitope, the antibody itself should prove to be a valuable tool in establishing the molecular basis for the function and regulation of the glucose transporter.     

Simple preparation and characterization of cationic liposomes associated with a monoclonal antibody against glioma-associated antigen (immunoliposomes)

Abstract

In this study we prepared and characterized monoclonal antibody associated cationic liposomes (immunoliposomes) to be used as a vehicle for human gene therapy of malignant glioma. This association method is especially amenable to mass production. The immunoliposomes consist of N-(a-trimethylammonio-acetyl)-didodecyl-D-glutamate chloride (TMAG), dilauroyl phosphatidylcholine (DLPC), and dioleoyl phosphatidyl- ethanolamine (DOPE) in a molar ratio of 1:2:2 as TMAG:DLPC:DOPE. Their preparation required only the addition of a solution containing plasmid DNA and a monoclonal antibody against glioma-associated antigen to a lipid film of the above three lipids. The association of antibody on the surface of immunoliposomes was confirmed by an immunochemical procedure. Liposome-mediated LacZ gene transfection of human glioma cells resulted in p-galactosidase activity about 2- to 3-fold higher when immunoliposomes were used as compared to control liposomes that were not associated to antibody. Also, the production of human (3-interferon (HuIFN-P) into the medium was 2- to 7-fold higher when HuIFN-P gene was transfected. Based on the present results, the immunoliposomes associating a monoclonal antibody against glioma-associated antigen may become effective carriers for gene transfer to human glioma cells.     

High Yield of Human Monoclonal Antibody Produced by Stably Transfected Drosophila Schneider 2 Cells in Perfusion Culture Using Wave Bioreactor

Since it was first introduced in late 1990s Wave bioreactor has been used for protein production by mammalian and insect cell lines. However, using Wave bioreactor to produce human monoclonal antibody by stable Drosophila Schneider 2 (S2) cell transfectants has not been reported before. In this study, S2 cells were co-transfected with an inducible vector expressing human monoclonal antibody heavy and light chains, respectively, specific for hemagglutinin (HA) of H5N1 influenza virus. Stable S2 transfectant clone was selected by limiting dilution assay. Stable S2 transfectant clone that produce the highest amount of human monoclonal antibody was inoculated into two 2-l disposable cellbags, where cell growth and antibody production were compared between batch and perfusion cultures using Wave bioreactor. Here, we report that maximum viable cell density reached 1.06?×?10(7) cells/ml in batch culture; whereas 1.04?×?10(8)?cells/ml was achieved in perfusion culture. The maximum volumetric antibody productivity in batch culture was 52?mg/l/day; while perfusion culture yielded 1,437?mg/l/day. As a result, the total antibody production was 201?mg in batch culture and 8,212?mg in perfusion culture. The antibody produced by both cultures displays full neutralizing activity. Thus, our results provide strong support for using Wave bioreactor in perfusion culture for a large-scale production of human monoclonal antibody by stable S2 cell transfectants.     

Ki-M7 monoclonal antibody specific for myelomonocytic cell lineage and macrophages in human

We describe a new monoclonal antibody, termed Ki-M7, which is specific to human myelomonocytic cell lineage and macrophages, as tested by immunohistochemical methods. Ki-M7 recognizes an intracytoplasmic antigen of molecular weight 29,000. Ultrastructurally, the antigen is localized in the lysosome and phagosome compartments and seems to be involved in generation of oxygen radicals during the respiratory burst. Dendritic cells, such as dendritic reticulum cells of lymphoid follicles and interdigitating reticulum cells of lymphoid T-zones, considered as accessory cells of the B- and T-cell immune response, respectively, do not show any reactivity with monoclonal antibody Ki-M7. Ki-M7 seems to be an appropriate reagent to clearly differentiate between the phagocytosing and the immune accessory population of the human monocyte/macrophage system.     

Two monoclonal antibodies distinguish between human recombinant interferon-alpha 5s produced by Escherichia coli and by mouse cells

Two monoclonal antibodies against human IFN-alpha--one against natural leukocyte IFN-alpha and the other against recombinant human IFN-alpha 2 produced in E. coli--were prepared, and designated as HT-1, and 104-5-f, respectively. These monoclonal antibodies were used to examine the antigenicities of recombinant human IFN-alpha 5s produced by E. coli and by mouse cells. The HT-1 antibody could bind and neutralize recombinant human IFN-alpha 5 synthesized in mouse cells, but not recombinant human IFN-alpha 5 synthesized in E. coli. On the other hand, the 104-5-f antibody could bind and neutralize recombinant human IFN-alpha 5 synthesized in E. coli but not recombinant human IFN-alpha 5 synthesized in mouse cells. Then these monoclonal antibodies or sheep polyclonal antibody against human IFN-alpha were used to immunoprecipitate the radioactively labeled recombinant human IFN-alpha 5 synthesized either in E. coli or mouse cells, and analysed on polyacrylamide gel electrophoresis in the presence of NaDodSO4. The labeled recombinant human IFN-alpha 5 produced by mouse cells could be immunoprecipitated with the HT-1 monoclonal antibody or sheep anti-(human IFN-alpha) polyclonal antibody but not with the 104-5-f monoclonal antibody and showed a band of Mr. 17,500 on polyacrylamide gel electrophoresis in the presence of NaDodSO4. On the other hand, the labeled recombinant human IFN-alpha 5 produced by E. coli could be immunoprecipitated with the 104-5-f monoclonal antibody but not with the HT-1 monoclonal antibody and showed a band of similar Mr. on polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of the biologic response to insulin-like growth factor I in MCF-7 breast cancer cells by a new monoclonal antibody to the insulin-like growth factor-I receptor. The importance of receptor down-regulation.

We developed a mouse monoclonal antibody (4G11) against insulin-like growth factor I receptor by immunizing mice with mouse embryo fibroblasts overexpressing the human insulin-like growth factor-I receptor. Not only did the 4G11 antibody inhibit the binding of [ (125)I]insulin-like growth factor-I to the fibroblast receptor, but 4G11 antibody also potently down-regulated the insulin-like growth factor-I receptor. 4G11 Fab fragment inhibited ligand binding, but did not down-regulate the receptor, suggesting that receptor aggregation is required for down-regulation. 4G11 antibody also down-regulated the receptor in MCF-7 breast cancer cells, a panel of colon cancer cells and MG-63 osteosarcoma cells. Receptor recovery in MCF-7 cells after down-regulation by 4G11 antibody was slow, requiring 32 - 48 h for full recovery. Receptor down-regulation in MCF-7 cells by 4G11 antibody was confirmed by FACS analysis of intact and permeabilized cells. In contrast to 4G11 antibody, insulin-like growth factor-I did not down-regulate the receptor in MCF-7 cells. Down-regulation of the receptor by 4G11 antibody in MCF-7 cells resulted in inhibition of Akt and MAPK activation by insulin-like growth factor-I. We conclude that the ability of a monoclonal antibody to down-regulate the receptor may be an important antibody property in targeting the insulin-like growth factor-I receptor for the treatment of certain cancers.     

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.     

Antigenic, receptor-binding and mitogenic activity of proteolytic fragments of human growth hormone.

Analysis of the subtilisin-digested, two-chain form of human growth hormone (hGH) and its constituent polypeptide fragments has been aided by the use of monoclonal antibodies which bind specifically to four distinct epitopes on the native hormone. Using the SDS-polyacrylamide immunoblotting technique, it was shown that one epitope (shared with human chorionic somatomammotropin) detected by EB1 (or EB3) antibody was expressed to a similar extent by both the N-terminal (15 K) and C-terminal (7 K) polypeptides. This epitope is unique in that it represents a repeating determinant within the single chain structure of the hormone. Another three epitopes detected by monoclonal antibodies QA68/NA27, NA71 or NA39/EB2 were absent from the 7-K fragment but were expressed on the 15-K fragment to a similar extent to that on unmodified growth hormone. Binding of NA71 antibody was demonstrated only by radioimmunoassay since this, presumably conformational epitope could not be detected by immunoblotting. The functional importance of the 15-K peptide was demonstrated by its ability to bind specifically to hormone receptors on IM9 human lymphoblastoid cells and by its retention of mitogenicity for the NB2 rat lymphoma cell line. However, all tested monoclonal antibodies inhibited the binding of [125I]15-K to IM9 cell receptors by either steric hindrance or by an allosteric mechanism and therefore could not be further related topographically to the receptor-binding moiety of hGH.     

A hyman hybrid hybridoma producing a bispecific monoclonal antibody that can target tumor cells for attack by Pseudomonas aeruginosa exotoxin A

By fusing a human hybridoma producing an IgG2 antibody against human A431 epidermoid carcinoma cells with an Epstein-Barr virus-transformed human B lymphocyte producing an IgG2 antibody against Pseudomonas aeruginosa exotoxin A, we established a hybrid hybridoma producing a bispecific monoclonal antibody reacting with both A431 cells and the exotoxin. Human IgG was purified from the culture supernatant of the hybrid hybridoma, and the bispecific monoclonal antibody in the IgG preparation was further separated from the two parental antibodies by hydroxyapatite high-performance liquid chromatography. The human bispecific monoclonal antibody thus obtained efficiently targeted the antibody-reative cells, A431, for attack by the exotoxin in vitro.Abbreviations bs mAb Bispecific Monoclonal Antibody - HRP Horseradish Peroxidase - MHA Mixed Hemadsorption Assay - MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide - PEA Pseudomonas aeruginosa Exotoxin A - PEG Polyethylene Glycol