Cell surface antigens on human marrow cells: dissection of hematopoietic development using monoclonal antibodies and multiparameter flow cytometry

A single sample of bone marrow includes the entire range of the developmental process, since cells of all stages and lineages are present. By selecting appropriate monoclonal antibodies, marrow cells of different lineages can be identified, even in their immature forms. Maturationally different bone marrow cells can be distinguished on the basis of their cell surface antigen expression and physical characteristics on a flow cytometer. Antigenic markers can be used within a lineage to trace the development from colony-forming cells to functional blood cells. Changes in cellular markers are observed as smooth, quantitative increases or decreases in cellular antigen expression. After defining the composition of normal marrow, it is possible to identify perturbations from the steady state and to monitor the return to homeostasis. Cells at various stages can be enumerated, then isolated for further study. In particular, these studies provide for the identification, purification and manipulation of the earliest hematopoietic progenitor cells.     

Collection and analysis of hematopoietic progenitor cells from cynomolgus macaques (Macaca fascicularis): assessment of cross-reacting monoclonal antibodies

Previous studies have shown that hematopoietic progenitor cells can be isolated from human or nonhuman primate bone marrow (BM) cells. In the present study, we studied the cross-reactivity of 13 anti-human CD34, two anti-human c-Kit, and one anti-human CD133 monoclonal antibodies (mAbs) with cynomolgus macaque (Macaca fascicularis) BM cells, using flow cytometric analysis, cell enrichment, and clonogenic assay. Among the 13 anti-human CD34 mAbs assessed, six cross-reacted as previously reported by other groups. However, only three of these six mAbs (clones 561, 563, and 12.8) recognized cynomolgus CD34+ cells that formed progenitor colonies when grown in methylcellulose culture. Similarly, of the two anti-human c-Kit mAbs (clones NU-c-kit and 95C3) that were previously reported to cross-react with cynomolgus BM cells, only one (clone NU-c-kit) resulted in a similar outcome. The anti-human CD133 mAb (clone AC133) also cross-reacted with cynomolgus BM cells, although these cells did not give rise to colonies when grown in culture. These results suggest that antibodies that cross-react with nonhuman primate cells may not identify the hematopoietic cells of interest. In addition, while the CD34 mAb (clone 561) results in the selection of hematopoietic progenitor cells of all lineages when assessed in methylcellulose culture, the c-Kit(high) fraction (NU-c-kit) exclusively identifies erythroid-specific progenitor cells after growth in culture. It is important to consider these findings when selecting cross-reacting mAbs to identify cells of hematopoietic lineages in macaque species.     

Antigens on human plasma cells identified by monoclonal antibodies

Two monoclonal antibodies that define distinct plasma cell-associated antigens, termed PCA-1 and PCA-2, were developed against human plasma cell leukemia cells. These antigens are strongly expressed on human myelomas, plasma cell leukemia, and plasmacytoma tumor cells, but are not detected on other lymphoid malignancies of B, T, null, or myeloid origin. PCA-1 and PCA-2 are not expressed on either normal T or B lymphocytes, but are weakly expressed on granulocytes and monocytes. When pokeweed mitogen is used to induce human B lymphocyte differentiation, PCA-1 is expressed when other B cell determinants are lost and plasmacytoid morphology, intracytoplasmic immunoglobulins, and surface T10 staining characteristic of plasma cells appear. In contrast, PCA-2 cannot be induced and may therefore appear later in the B cell differentiation scheme. These antigens may be of utility for the study and regulation of normal and abnormal plasma cell growth, traffic, and tissue distribution and may aid in understanding heterogeneity within plasma cell dyscrasias.     

Recombinant human monoclonal antibodies

To produce human monoclonal antibodies in bacteria, a gene repertoire of IgM variable regions was isolated from human peripheral B lymphocytes by the polymerase chain reaction. Alternatively, synthetic antibody genes with random hypervariable regions are being generated that may provide libraries of even higher complexity. For the selection of specific monoclonal antibodies from these libraries, we have developed twoE. coli vector systems that facilitate the surface display of an antibody physically linked to its own gene. The phagemid pSEX encodes a fusion protein of an antigen binding domain (Fv-antibody) with the docking protein (pIII) of filamentous phages. Specific antibody genes can therefore be enriched by antigen affinity chromatography. The plasmid pAP1 encodes a fusion protein of an Fv-antibody with a bacterial cell-wall protein. Bacteria carrying this plasmid express functional Fv-antibodies tightly bound to their surface. This should enable the selection of single cells with a fluorescence-assisted cell sorter (FACS) using labeled antigen or by adsorption to immobilized antigen. These vectors permit three major principles of the antibody response to be mimicked inE. coli:

1. Generation of a highly complex antibody repertoire;
2. Clonal selection procedures for library screening; and
3. The possibility of increasing a given affinity by repeated rounds of mutation and selection.

     

Species crossreactivity of human progesterone receptor monoclonal antibodies: Western blot analysis

The crossreactivity of monoclonal antibodies (hPRa 1, 2, 3 and 6) generated against human progesterone receptor was examined in six mammalian and an avian species using the techniques of sodium-dodecylsulfate polyacrylamide gel electrophoresis and Western blot analysis. Immunoreactive bands were detected on protein blots of receptor-containing preparations from human endometrial carcinoma grown in nude mice, human T47D breast cancer cells, rabbit, cow and mouse uteri, and chick oviduct. No receptor-associated, immunoreactive bands were detected in rat, guinea pig or hamster uteri. The number and molecular weights of the receptor subunits detected varied between species, and only human progesterone receptor displayed electrophoretic microheterogeneity in its high molecular weight subunit. These data demonstrate that the human progesterone receptor antibodies recognize epitopes not common to all species.     

Cytotoxicity of monoclonal antibodies against individually immunophenotyped human leukemic cells

Summary Peripheral blood and bone marrow mononuclear cells from 12 patients with acute myeloid leukemia (AML), 2 patients with acute lymphatic leukemia, and 1 patient with chronic myeloid leukemia in blastic crisis were taken at diagnosis or in relapse. Cells were immunophenotyped with a panel of monoclonal antibodies (Moab) (OKIa, Leu M1, Leu M2, Leu M3, Leu M4, B1, Okt 11, J5) and the same antibodies were used in an in vitro cytotoxicity test. Of the 14 patients, 10 had antibody-binding cells, and the percentage of lysed cells was almost equal to that of blasts. The other 4 patients had few binding cells and little lysis. Acute leukemia with and without preceding myelodysplastic features did not differ in immunophenotype. Mean spontaneous release of ⁵¹Cr was 12.7% and complement alone caused an additional average release of 11.8%. Four single antibodies together with complement showed a mean ⁵¹Cr release of 0.7–32.4% above that found with complement alone. Combinations of Moabs resulted in ⁵¹Cr release at least 10% above the single most efficient Moab in 8 out of 12 patients. Not all blast cells showed antibody binding, nor were all antibody-binding cells susceptible to cytotoxicity.Normal bone marrow growth in vitro seemed to be stimulated by factors in complement and in the Moab. When this stimulation was compensated for by adding fetal calf serum, cytotoxicity tests prior to CFUc assays resulted in a mean decrease of 46% of colonies and 25% of clusters in normal bone marrow. CFUc are thus sensitive to the cytotoxicity, although CFU may also be resistant.Supported by Sw. Cancer Res. Found. Grant no. 699-B83-19XC Preliminary report given to Int. Soc. Hematol., Eur. Afr. Div. 5, 1983 Visiting scientist from Hacettepe University Ankara, TurkeyVisiting scientist from Hacettepe University Ankara, Turkey     

A novel platform to produce human monoclonal antibodies: The next generation of therapeutic human monoclonal antibodies discovery

A new technology has been developed by immunologix that allows human antibodies to be quickly generated against virtually any antigen. Using a novel process, naïve human B cells are isolated from tonsil tissue and transformed with efficiency up to 85%, thus utilizing a large portion of the human VDJ/VJ repertoire. Through ex vivo stimulation, the B cells class switch and may undergo somatic hypermutation, thus producing a human “library” of different IgG antibodies that can then be screened against any antigen. Since diversity is generated ex vivo, sampling immunized or previously exposed individuals is not necessary. Cells producing the antibody of interest can be isolated through limiting dilution cloning and the human antibody from the cells can be tested for biological activity. No humanization is necessary because the antibodies are produced from human B cells. By eliminating immunization and humanization steps and screening a broadly diverse library, this platform should reduce both the cost and time involved in producing therapeutic monoclonal antibody candidates.Key words: human, antibody, monoclonal, novel platform, naïve, B cell, therapeutic     

Development of human monoclonal antibodies: A review

This article describes the current status in the development of human monoclonal antibodies. Over the last ten years a lot of information about the human immune system has emerged. Combining these with the many new (bio-)technologies it is plausible that the long awaited breakthrough of this technology is close. This paper focuses on the classical cell-biological methods of achieving stable, antibody-producing human cell lines via cell fusion methods or virus derived transformations of human B-lymphocytes, as well as genetic engineering methods e.g. DNA libraries or phage display technology. The available in vitro immunization methods are critically reviewed and their impact on this topic is discussed. Therapeutic applications for cancer treatment or passive immunization against infectious diseases with antibodies derived by both ways are also reviewed.     

Binding of human lymphocyte-specific monoclonal antibodies to common marmoset lymphoid cells

Commercially available monoclonal antibodies which bind to human lymphocyte subsets were screened for their ability to bind to lymphoid cells from the common marmoset Callithrix jacchus. Anti-Leu-5 and T11 were the only pan T-cell antibodies which reacted strongly. None of the antibodies which bind human lymphocytes of the helper/inducer subpopulation reacted with C. jacchus cells and only one antibody, T8, specific for the cytotoxic/suppressor subset, bound to the marmoset cells. The two antibodies tested which bind human B cells, B1 and anti-HLA-DR, were also reactive with marmoset cells. The cellular specificity of the T11, T8, and B1 antibodies was determined by dual binding studies on the fluorescence-activated cell sorter. The B1 antibody bound only Ig+ cells and all Ig+ cells were B1+. The T11 and T8 antibodies bound only to Ig- marmoset lymphoid cells and, as in the human, all T8+ marmoset cells were also T11+. Thus, using these monoclonal antibodies in the common marmoset one can identify three populations of lymphoid cells: (1) T11+, T8+ cells; (2) T11+, T8- cells; (3) B1+ cells.     

Membrane antigens of human bronchial epithelial cells identified by monoclonal antibodies

Summary Subpopulations of normal bronchial epithelial cells were identified using a series of murine monoclonal antibodies. These antibodies were used to stain intact bronchial epithelial cells in culture by indirect immunofluorescence. LAM 2 reacted with 80%, LAM 6 with 75%, LAM 7 with 60%, and LAM 8 with 5% of these cells. Sections of human bronchial epithelium were also stained with these antibodies by immunoperoxidase methods. LAM 2 was found to bind with 80%, LAM 6 with 65%, LAM 7 with 50%, and LAM 8 with less than 1% of bronchial epithelial cells. LAM 2 stained both columnar epithelial cells and basal cells; LAM 6 stained mainly basal cells and only a small proportion of columnar cells; LAM 7 showed specificity for basal cells; LAM 8 distinctly stained single cells in the basal cell layer. These antibodies were previously shown to react with the surface membrane of human lung carcinomas, ranging from the broad reactivity of LAM 2 with small cell and non-small cell lung cancers to the highly restricted reactivity of LAM 8 with small cell carcinomas of the lung. Thus, membrane antigens have been identified in bronchial epithelial cells by monoclonal antibodies which exhibit a similar range of cellular reactivity in vitro as in vivo. Inasmuch as these antibodies recognize subsets of cells which could not be easily distinguished by morphologic characteristics, these reagents may be useful in classifying bronchial epithelial cells.     

Antigenic subsets of human breast epithelial cells distinguished by monoclonal antibodies

Three monoclonal antibodies raised to the human milk fat globule membrane bind, within the normal breast, to the surface of the luminal epithelial cells but not to the surrounding myoepithelial, connective tissue, or blood vessel cells. These antibodies distinguish three subsets of the epithelial cells that are not distinguishable by conventional histology. To show the arrangement of the cells in two dimensions over the sheet of epithelium, ducts were dissected out of normal breast tissue, opened up and laid flat as sheets of epithelium. The apical faces of the cells were strained, unfixed, using two-color immunofluorescence to contrast the subsets of cells stained by the different antibodies. The epithelium was then seen to be a mosaic of cells that express different surface antigens. The grouping and appearance of the cells stained by the different antibodies was characteristic. This may be just a random heterogeneity of antigen expression but alternatively the different cells may be in different physiological states. Regardless of its biological significance, the observation has practical consequences for the use of such antibodies in identifying cells and the study of antigenic heterogeneity in tumors.     

Immunohistochemical analysis of human peripheral blood and lymphoid tissues using monoclonal antibodies immunoreactive with non-lymphoid cells

Summary Immunoperoxidase methods were used to study human peripheral blood and lymphoid tissues using a panel of monoclonal antibodies to non-lymphoid cells. The majority of peripheral blood monocytes were immunoreactive for LeuM1, LeuM2, LeuM3 and LeuM5. Rare peripheral blood monocytes were immunoreactive for R4/23. The different antibodies showed characteristic patterns of immunoreactivity in peripheral lymphoid tissues. LeuM1 was immunoreactive with scattered cells in the paracortex of lymph node and tonsil and with many cells in the marginal zone of the spleen. LeuM2 was immunoreactive with endothelial cells in lymph node and tonsil. A few cells in the red pulp of the spleen were immunoreactive for LeuM2. LeuM3 and R4/23 showed distinctive immunoreactivity in germinal centers of secondary follicles, giving a lacy pattern. LeuM3 was also immunoreactive with endothelium in lymph node and tonsil and with sinus lining cells in lymph node. LeuM5 was immunoreactive with macropages in the germinal center, fibroblastic reticulum cells in the mantle zone and interdigitating reticulum cells in the paracortex of lymph node and tonsil.     

Immunohistochemical analysis of human peripheral blood and lymphoid tissues using monoclonal antibodies immunoreactive with non-lymphoid cells

Immunoperoxidase methods were used to study human peripheral blood and lymphoid tissues using a panel of monoclonal antibodies to non-lymphoid cells. The majority of peripheral blood monocytes were immunoreactive for LeuM1, LeuM2, LeuM3 and LeuM5. Rare peripheral blood monocytes were immunoreactive for R4/23. The different antibodies showed characteristic patterns of immunoreactivity in peripheral lymphoid tissues. LeuM1 was immunoreactive with scattered cells in the paracortex of lymph node and tonsil and with many cells in the marginal zone of the spleen. LeuM2 was immunoreactive with endothelial cells in lymph node and tonsil. A few cells in the red pulp of the spleen were immunoreactive for LeuM2. LeuM3 and R4/23 showed distinctive immunoreactivity in germinal centers of secondary follicles, giving a "lacy" pattern. LeuM3 was also immunoreactive with endothelium in lymph node and tonsil and with sinus lining cells in lymph node. LeuM5 was immunoreactive with macrophages in the germinal center, fibroblastic reticulum cells in the mantle zone and interdigitating reticulum cells in the paracortex of lymph node and tonsil.     

Murine monoclonal antibodies cytotoxic to human glioma cells in vitro.

Six monoclonal antibodies (mABs) against human glioma cells (T2) were produced. T2 cells grown as solid tumors in nude mice, were dissociated and used to immunize Balb/c mice. After fusion of splenocytes with myeloma cells, eight hybrids secreting mABs were selected according to their ability to react immunohistochemically with T2 cells, but not with normal adult human brain. Cytotoxicity of mABs was tested using (3H)-thymidine incorporation assays in vitro. Four mABs showed complement-mediated cytotoxicity for T2 cells, other human glioma cells (T1), and a human melanoma cell line. Incubation with one antibody, mAb2A1, lowered (3H)-thymidine incorporation in the T2 and T1 cells to ca. 10%, and in melanoma cells to ca. 35% of control levels. Another antibody, mAb3B2, displayed a similar cytotoxicity for T2 and T1 cells, but did not show measurable cytotoxicity for melanoma cells and rat primary astrocyte cultures. Moreover, this antibody did not crossreact with haematopoietic cells from patients bearing CNS tumors or normal subjects. MAb3B2, therefore, appears to recognize and epitope associated to human gliomas, will be a useful glioma tumor marker and may have some potential therapeutical value.     

ICO-13 monoclonal antibodies to the differentiation antigen of human hemopoietic cells

Monoclonal antibodies (Mab) ICO-13 of IgM isotype reacted in indirect surface immunofluorescence test with 88.5 +/- 3.4% of thymocytes and 7.1 +/- 2.4% of T cells, but failed to react with other peripheral blood cells from healthy donors. The antigen disappeared in cells stored at 4 degrees C overnight or at -196 degrees C in liquid nitrogen. The antigen detected by Mab ICO-13 was expressed on blast cells of acute lymphoblast leukemias and lymphomas. The antigen was absent in chronic lymphoblast leukemia and blast crisis of chronic myeloid leukemia.     

In situ detection of acetylaminofluorene-DNA adducts in human cells using monoclonal antibodies

The present study was performed to generate monoclonal antibodies capable of detecting N-acetoxy-2-acetylaminofluorene (NA-AAF)-derived DNA adducts in human cells in situ. As an immunogen, we employed NA-AAF-modified single-stranded DNA coupled electrostatically to methylated protein and we produced five different monoclonal antibodies. All of them showed strong binding to NA-AAF-modified DNA, but had undetectable or minimal binding to undamaged DNA. Competitive inhibition experiments revealed that the epitope recognized by these antibodies is N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) in DNA, although deacetylated N-(deoxyguanosin-8-yl)-2-aminofluorene in DNA is also recognized with slightly less efficiency. In contrast, these antibodies did not bind to 3-(deoxyguanosin-N(2)-yl)-2-acetylaminofluorene in DNA or to UV-induced lesions in DNA. Interestingly, they showed only minimal binding to small AAF-nucleoside adducts (dG-C8-AAF), indicating that DNA regions flanking a DNA-bound adduct, in addition to the adduct itself, are essential for the stable binding of the antibodies. Using an enzyme-linked immunosorbent assay with the most promising antibody (AAF-1), we detected the concentration-dependent induction of NA-AAF-modified adducts in DNA from repair deficient xeroderma pigmentosum (XP) cells treated with physiological concentrations of NA-AAF. Moreover, the assay enabled to confirm that normal human cells efficiently repaired NA-AAF-induced DNA adducts but not XP-A cells. Most importantly, the formation of NA-AAF-induced DNA adducts in individual nuclei of XP cells could be clearly visualized using indirect immunofluorescence. Thus, we succeeded in establishing novel monoclonal antibodies capable of the in situ detection of NA-AAF-induced DNA adducts in human cells.     

Hybridoma monoclonal antibodies in the analysis fo human cell surface antigens

Summary In the present paper, we will summarize studies we have performed on two distinct human lymphocyte cell surface antigens defined by monoclonal antibodies: Leu-1 and HLA-DR. Presented in the symposium on The Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association, Washington, D.C., June 7–11, 1981. This work was supported by USPHS-NIH Grants CA-21223, AI-11313, and CA-09302. This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England Nuclear Corporation, and Ortho Pharmaceutical Corporation.     

Ontogeny of the mouse hematopoietic system

In vertebrates the extraembryonic mesoderm of the yolk sac (YS) is the first site during embryogenesis where morphologically discernible hematopoiesis may be found. Later hematopoiesis shifts into the embryo proper, first to the liver, the major fetal hematopoietic site, then to definitive hematopoietic territories, the spleen and bone marrow. It is widely accepted that in the mouse this picture reflects the migration of pluripotent hematopoietic stem cells (HSC) from the YS accompanied by subsequent colonization of the hematopoietic tissues during embryogenesis. However, there is no conclusive evidence showing unequivocally the initiating role of the YS in murine adult hematopoiesis. Recently, we have demonstrated the important role of embryo body tissues in the development of CFU-S before the establishment of definitive hematopoiesis in the fetal liver. This finding suggests that the early development of the hematopoietic system in the mouse is more complex than has been previously proposed and we consider here the early hematopoietic events in the developing mouse embryo.     

Immunohistochemical analysis of human lung cancers with anti-ras p21 monoclonal antibodies

The expression of ras oncogene product p21 in human malignant pleurisy and primary lung cancer was studied immunocyto-histochemically with monoclonal antibodies (MoAbs) rp-28 and rp-35 against ras p21. In pleural effusion cells, cancer cells revealed more intensively positive reaction with MoAb rp-35 than with MoAb rp-28, especially in the plasma membrane, and no positive reaction was obtained in any kind of inflammation cells with the exception of faintly positive reaction in the cytoplasm of macrophages. In primary lung cancers, well or moderately differentiated adenocarcinoma tissues showed higher reactivity with MoAb rp-28 than those of poorly differentiated adenocarcinoma or any other histological subtype of lung cancer. With MoAb rp-35, intensively positive reaction was obtained in most of cases with all different histological subtypes of lung cancer. The staining in cancer cells was usually localized intensively to the plasma membrane and weakly to the cytoplasm with both MoAbs. Normal bronchial epithelial and glandular tissues showed only cytoplasmic staining. These two MoAbs, especially MoAb rp-35, may be useful in clinicopathological applications for the diagnosis of malignant pleurisy and primary lung cancer.