Immunohistochemical heterogeneity of alpha-tubulin in human epithelia revealed with monoclonal antibodies

Four monoclonal antibodies raised to alpha subunit of pig brain tubulin (TU-01, TU-02, TU-03, TU-04) were used to study immunohistochemical heterogeneity of alpha tubulin in human epithelia. Selective reactivity was detected in the skin and trachea/bronchi, whereas all other epithelia investigated reacted uniformly with all four monoclonal antibodies. In the skin TU-01 reacted very strongly with all layers except the basal layer; TU-02 reacted strongly with granular layer and was unreactive or only weakly reactive with others; TU-03 reacted very strongly with basal layer and weakly to moderately with superficial layers; TU-04 reacted strongly with the granular layer of epidermis and was unreactive with other layers. In the trachea and major bronchi TU-01 reacted with the entire epithelial layer; TU-02 reacted only with superficial layer; TU-03 reacted with superficial and basal layer; TU-04 reacted only with superficial layer. Different staining patterns obtained with these four monoclonal antibodies indicate that there is immunohistochemical heterogeneity of alpha tubulin in some but not all normal human epithelia.     

Immunohistochemical heterogeneity of alpha-tubulin in human epithelia revealed with monoclonal antibodies

Summary Four monoclonal antibodies raised to alpha subunit of pig brain tubulin (TU-01, TU-02, TU-03, TU-04) were used to study immunohistochemical heterogeneity of alpha tubulin in human epithelia. Selective reactivity was detected in the skin and trachea/bronchi, whereas all other epithelia investigated reacted uniformly with all four monoclonal antibodies. In the skin TU-01 reacted very strongly with all layers except the basal layer; TU-02 reacted strongly with granular layer and was unreactive or only weakly reactive with others; TU-03 reacted very strongly with basal layer and weakly to moderately with superficial layers; TU-04 reacted strongly with the granular layer of epidermis and was unreactive with other layers. In the trachea and major bronchi TU-01 reacted with the entire epithelial layer; TU-02 reacted only with superficial layer; TU-03 reacted with superficial and basal layer; TU-04 reacted only with superficial layer. Different staining patterns obtained with these four monoclonal antibodies indicate that there is immunohistochemical heterogeneity of alpha tubulin in some but not all normal human epithelia.     

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.     

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 phenotyping of human solid tumors with monoclonal antibodies in devising biotherapeutic strategies

Summary A panel of 14 monoclonal antibodies (MoAbs) (4 raised against breast cancer, 6 against colon cancer and 4 against melanoma) were used to phenotype frozen sections of tumor biopsies obtained from 110 patients, by avidin-biotin-peroxidase complex techniques. We observed heterogeneity of antigen expression among the multiple metastatic lesions of single patients, as well as among tumor lesions from different patients with similar tumor histotypes. A wide range of cross-reactivity of anti-(breast-carcinoma) and anti-(colon-carcinoma) MoAbs with other carcinoma histotypes and limited reactivity with melanoma and sarcoma was detected. Some of our anti-melanoma MoAbs were also found to cross-react with selected carcinomas. Nine of the 14 MoAbs most reactive with carcinomas of diverse histotypes have been identified. A mixture or cocktail of different MoAbs could be selected for each individual patient in order to achieve binding of MoAbs with most, if not 100% of tumor cells. This study illustrates the approach that we have taken to individualize the cocktail of MoAbs for the development of patient-specific therapeutic immunoconjugates.     

Structural characterization of human melanoma-associated antigen p97 with monoclonal antibodies

Monoclonal antibodies were used to study p97, a human melanoma-associated antigen (MAA). Four hybridomas, designated 4.1, 96.5, 118.1, and 8.2, were obtained by fusing mouse myeloma cells with spleen cells from mice immunized with human melanoma cells. Antibodies 4.1 and 8.2 were IgG1; antibodies 96.5 and 118.1 were IgG2a. Sequential immunoprecipitation (IP) and sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that all 4 antibodies recognized the same 97 kilodalton (kD) protein. Binding studies with 125I-labeled antibody showed that antibodies 4.1 and 96.5 bound the same epitope, p97a. Antibodies 118.1 and 8.2 defined epitopes p97b and p97c, respectively. Six monoclonal antibodies (M17, L1, L10, R10, I12, and K5) specific for gp95, a kD melanoma cell surface glycoprotein were also tested. Sequential IP showed that these antibodies bound p97; p97 and gp95 are thus identical. Binding studies showed that antibody m17 bound epitope p971, and antibodies L1, L10, and R19 bound epitope p97c. Antibodies I12 and K5 defined 2 other epitopes, p97d and p97e, respectively. SDS-PAGE under nonreducing conditions indicated that p97 is monomeric, probably with intrachain disulfide bonds. Cell-surface labeling of sialic acid residues and neuraminidase digestion showed that p97 is a sialoglycoprotein. Digestion of p97 with papain or trypsin produced a stable 40 kD fragment, which expressed epitopes p971, p97b, and p97c, but not p97d or p97e.     

Immunohistochemical study of angiotensin-converting enzyme in human tissues using monoclonal antibodies

Summary The localization of angiotensin-converting enzyme (ACE) in human tissues has been studied by the PAP-method with the use of monoclonal antibody 9B9 against human lung ACE. The enzyme was detected on the surface of endothelial cells in lung, myocardium, liver, intestine and testis as well as in the epithelial cells of the kidney proximal tubules and intestine. The monoclonal antibody 9B9 did not react with ACE in the epithelial cells of the testis seminiferous tubules. These data suggest that the antibody 9B9 recognizes epitope which is shared by the ACE molecule of endothelial cells and renal and intestinal epithelial cells but is not present in testicular ACE, or is not accessible there to the antibody.     

Immunohistochemical study of angiotensin-converting enzyme in human tissues using monoclonal antibodies

The localization of angiotensin-converting enzyme (ACE) in human tissues has been studied by the PAP-method with the use of monoclonal antibody 9 B9 against human lung ACE. The enzyme was detected on the surface of endothelial cells in lung, myocardium, liver, intestine and testis as well as in the epithelial cells of the kidney proximal tubules and intestine. The monoclonal antibody 9 B9 did not react with ACE in the epithelial cells of the testis seminiferous tubules. These data suggest that the antibody 9 B9 recognizes epitope which is shared by the ACE molecule of endothelial cells and renal and intestinal epithelial cells but is not present in testicular ACE, or is not accessible there to the antibody.     

Immunohistochemical localisation of arginase in human liver using monoclonal antibodies against human liver arginase

Summary Monoclonal antibodies against human liver arginase were raised in order to determine the exact distribution of arginase in human liver using a modified indirect unlabelled immunoperoxidase method. In normal human liver specific immunohistochemical staining was found in the cytoplasm of hepatocytes. Portal components (bile ducts and veins) and fibrous tissue were non-reactive, while erythrocytes were slightly positive. The specificity of the immunological reaction was confirmed by control tests. Spectrophotometry was used to quantitate the immunohistochemical reaction product, and the results indicated that arginase is homogeneously distributed in the liver lobule.Present address: Biologisches Institut der Universität Stuttgart, Ulmerstrasse 227, D-7000 Stuttgart 60, Federal Republic of Germany     

Immunohistochemical localisation of arginase in human liver using monoclonal antibodies against human liver arginase

Monoclonal antibodies against human liver arginase were raised in order to determine the exact distribution of arginase in human liver using a modified indirect unlabelled immunoperoxidase method. In normal human liver specific immunohistochemical staining was found in the cytoplasm of hepatocytes. Portal components (bile ducts and veins) and fibrous tissue were non-reactive, while erythrocytes were slightly positive. The specificity of the immunological reaction was confirmed by control tests. Spectrophotometry was used to quantitate the immunohistochemical reaction product, and the results indicated that arginase is homogeneously distributed in the liver lobule.     

Quantitative immunofluorescence, anti-ras p21 antibody specificity, and cellular oncoprotein levels

A general approach to investigating specificity and saturation of antibodies by quantitative immunofluorescence is applied to monoclonal antibodies generated against p21 or ras oligopeptides to quantify ras p21 oncoprotein in cultured cells. Ras 10, a panreactive mouse monoclonal antibody, appears to be a superior probe for detection of p21 in cell extracts or fixed cells because it binds a 21 kD protein on SDS-PAGE/western blots and labels the cytoplasmic membrane in a saturable and competitive manner. RAP-5, a widely used mouse monoclonal antibody generated against an oligopeptide of ras p21, does not recognize p21 in denaturing immunoblots or in immunofluorescence of cultured cells.     

Immunohistochemical analysis of pulmonary and pleural neoplasms with monoclonal antibodies B72.3 and CSLEX-1

Sequential paraffin sections of 222 epithelial lung tumors comprising all common histologic types, and 31 pleural mesotheliomas of all variants were immunostained with monoclonal antibodies (Mabs) B72.3 and CSLEX-1. Reactivity with Mabs B72.3 and CSLEX-1 respectively was noted in 7/57 and 4/57 squamous carcinomas, in 44/70 and 60/70 adenocarcinomas, 9/16 and 11/16 bronchioloalveolar carcinomas, 8/25 and 14/25 large cell undifferentiated carcinomas, 3/3 and 3/3 adenosquamous carcinomas, 0/11 and 0/11 carcinoids, 0/10 and 2/10 well differentiated neuroendocrine (NE) carcinomas, 4/13 and 5/13 intermediate cell NE carcinomas, 0/17 and 0/17 small cell NE carcinomas, and 0/31 and 1/31 mesotheliomas. In most instances, both Mabs stained the same tumors; however, reactivity with CSLEX-1 was more intense and extensive, and involved more cases. Therefore, regardless of conventional histologic type, staining with Mabs B72.3 and CSLEX-1 defines 4 subsets of lung tumors: one expressing both antigens, two expressing one but not the other, and one expressing neither. The possible biological and/or clinical significance of these subsets remains undetermined. When correlated with conventional histologic tumor types, our findings indicate: 1). both of these Mabs recognize most but not all adenocarcinomas and bronchioloalveolar carcinomas, and since CSLEX-1 stained more cases than B72.3, it may be argued that the former is a broader exocrine phenotype marker than the latter; 2). both of these Mabs select exocrine subsets of large cell undifferentiated carcinomas; 3). both of these Mabs stain exocrine cell subpopulations in well differentiated and intermediate cell NE carcinomas but not in carcinoids or small cell NE carcinomas, and 4). except for rare cases, neither B72.3 nor CSLEX-1 reacts with mesotheliomas regardless of variant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Generation and characterization of human anti-human IL-21 neutralizing monoclonal antibodies

Interleukin 21 (IL-21) is a type I four-helical bundle cytokine that exerts a variety of significant effects on many hematopoietic cells, including T and B lymphocytes and natural killer cells. IL-21 is produced predominantly by CD4⁺ T cells and natural killer T cells and, when aberrantly overexpressed, appears to play important roles in a wide variety of autoimmune disorders. To generate potential therapeutic reagents capable of inhibiting IL-21 for clinical use, we immunized human immunoglobulin transgenic mice with IL-21 and then identified and cloned a panel of human anti-human IL-21 binding monoclonal antibodies. IL-21 neutralizing and IL-21-binding, non-neutralizing antibodies were assigned to distinct epitope “bins” based on surface plasmon resonance competition studies. The most potent neutralizing antibodies had extremely high (sub pM) affinity for IL-21 and were able to block IL-21 activity in various biological assays using either an IL-21R-transfected pre-B-cell line or primary human B cells, and their neutralizing activity was, in some cases, superior to that of a soluble form of the high affinity heterodimeric IL-21 receptor. Characterization of this panel of IL-21 antibodies provided the basis for the selection of a therapeutic candidate antibody capable of inhibiting IL-21 activity for the treatment of autoimmune and inflammatory diseases.Key words: interleukin 21, IL-21, mAb, human Ig transgenic mice, autoimmunity     

Generation and characterization of human anti-human IL-21 neutralizing monoclonal antibodies

Interleukin-21 (IL-21) is a type I four-helical bundle cytokine that exerts a variety of significant effects on many hematopoietic cells, including T and B lymphocytes and natural killer cells. IL-21 is produced predominantly by CD4+ T cells and natural killer T cells and, when aberrantly overexpressed, appears to play important roles in a wide variety of autoimmune disorders. To generate potential therapeutic reagents capable of inhibiting IL-21 for clinical use, we immunized human immunoglobulin transgenic mice with IL-21 and then identified and cloned a panel of human anti-human IL-21 binding monoclonal antibodies. IL-21 neutralizing and IL-21-binding, non-neutralizing antibodies were assigned to distinct epitope “bins” based on surface plasmon resonance competition studies. The most potent neutralizing antibodies had extremely high (sub pM) affinity for IL-21 and were able to block IL-21 activity in various biological assays using either an IL-21R-transfected pre-B-cell line or primary human B cells, and their neutralizing activity was, in some cases, superior to that of a soluble form of the high affinity heterodimeric IL-21 receptor. Characterization of this panel of IL-21 antibodies provided the basis for the selection of a therapeutic candidate antibody capable of inhibiting IL-21 activity for the treatment of autoimmune and inflammatory diseases.     

Methods for production of monoclonal antibodies with specificity for human lung cancer cells

We have developed a screening strategy and technology to produce monoclonal antibodies with specificity for human lung cancer cells. Mice and rats were immunized with well-characterized tissue culture lines of human small cell lung cancer (SCLC), mouse myeloma x spleen hybrids formed by the technique of Kohler and Milstein, and the resulting culture fluids were screened for antibody binding phenotype using a radioimmunoassay. To facilitate testing large numbers of culture fluids, a 96-well, microtiter based, reusable, replicating device was designed. Using this, many hybridoma culture fluids were replica plated for antibody binding tests on a series of human target cell plates. Hybrids producing antibodies that reacted with the immunizing SCLC line and another independent SCLC line, but not with autologous B-lymphoblastoid cells derived from one of the patients, were identified, selected, and then repeatedly recloned using the same screening strategy. With this technology, hybridomas representing less than 0.5% of all hybrids generated could be isolated and stable antibody producing cultures derived. Such antibodies reacted with a panel of well-characterized SCLC lines and SCLC samples taken directly from patients but not with a variety of normal tissues. Using these antibodies we can demonstrate: tumor cell contamination of bone marrow specimens, marked heterogeneity of antigen expression on cells within individual SCLC lines and individual patients, and inhibition of clonal growth of SCLC lines in soft agarose assays. All of these findings have potential clinical and cell biologic application.     

Methods for production of monoclonal antibodies with specificity for human lung cancer cells

Summary We have developed a screening strategy and technology to produce monoclonal antibodies with specificity for human lung cancer cells. Mice and rats were immunized with well-characterized tissue culture lines of human small cell lung cancer (SCLC), mouse myeloma x spleen hybrids formed by the technique of Kohler and Milstein, and the resulting culture fluids were screened for antibody binding phenotype using a radioimmunoassay. To facilitate testing large numbers of culture fluids, a 96-well, microtiter based, resuable, replicating device was designed. Using this, many hybridoma culture fluids were replica plated for antibody binding tests on a series of human target cell plates. Hybrids producing antibodies that reacted with the immunizing SCLC line and another independent SCLC line, but not with autologous B-lymphoblastoid cells derived from one of the patients, were identified, selected, and then repeatedly recloned using the same screening strategy. With this technology, hybridomas representing less than 0.5% of all hybrids generated could be isolated and stable antibody producing cultures derived. Such antibodies reacted with a panel of well-characterized SCLC lines and SCLC samples taken directly from patients but not with a variety of normal tissues. Using these antibodies we can demonstrate: tumor cell contamination of bone marrow specimens, marked heterogeneity of antigen expression on cells within individual SCLC lines and individual patients, and inhibition of clonal growth of SCLC lines in soft agarose assays. All of these findings have potential clinical and cell biologic application. 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 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.     

Immunohistochemical analysis of normal and mutated ras oncogene p21 expression in human pulmonary and pleural neoplasms

In this study we examined 214 cases of primary human pulmonary neoplasms for the expression of a mutated form of the ras oncogene p21 product, recognized by the monoclonal antibody (MCA) DWP. Adjacent serial sections from these same cases had previously been used to demonstrate the frequency of ras p21 expression using the broadly reactive anti-ras p21 MCA RAP-5. Confirmation of the increased expression of p21 was accomplished using MCA Y13-259. The use of adjacent tissue sections from these cases allows the direct comparison of the expression of the mutated and non-mutated forms of ras p21. If reactivity with DWP would prove to be significantly more restrictive than that of the "pan" ras MCAs, RAP-5 and Y13-259, it would lend support to the possibility that DWP (and similar MCAs which detect other specific mutations) could be used to define subsets of these neoplasms based on their specific ras p21 phenotype. Since one would anticipate that the valine/cysteine substitution at position 12 of the ras p21 would occur at only low frequencies in human tumors, our results with DWP are consistent with this hypothesis. As previously reported, RAP-5 reacted with a high proportion of lung tumors (100/214 or 47%). In this report, we demonstrate the selective expression of the mutation recognized by the MCA DWP in only 5% of these same tumors (13/214), and that the expression of this mutated form is not restricted to any of the conventional histological subclasses of pulmonary neoplasms.