In vitro testing of the leukaemia monoclonal antibody WM-53 labeled with alpha and beta emitting radioisotopes.

We report the preparation and testing of a new alpha emitting radio-immunoconjugate (RIC) against acute myeloid leukaemia (AML) using CD33 positive monoclonal antibody WM-53 (specific for HL-60 cell line). Using cyclic anhydride of diethylenetriaminepentacetic acid (cDTPAa) as chelator, antibody was labeled with 213Bi (alpha), 149Tb (alpha), 153Sm (beta) and 152Tb (positron). In vitro testing showed high labeling efficiency (90-95%) and stability (11-19% leaching) with immunoreactivity virtually the same before and after labeling. DNA synthesis data and MTS cell survival were compared for all RICs. Only the alpha emitter was found to be capable of inhibiting DNA synthesis and had selective cell kill with activity as low as 2-3 microCi. The high stability and outstanding cytotoxicity of the 213Bi conjugate provides the basis for targeted alpha therapy for the control of metastatic and disseminated cancer such as AML.

Discrimination of von Willebrands disease (VWD) subtypes: direct comparison of von Willebrand factor:collagen binding assay (VWF:CBA) with monoclonal antibody (MAB) based VWF-capture systems.

Discrimination of von Willebrand's Disease (VWD) subtypes is important since it influences management. Qualitative [ie Type 2A, 2B, 2M] defects exhibit von Willebrand factor (VWF) discordance and give high VWF:Ag to VWF:'activity' ratios. Classically, VWF:'activity' is assessed using the VWF:RCof assay. The VWF:CBA is an ELISA-based VWF-functional adhesive assay which has consistently proved to be superior to VWF:RCof. A commercially available monoclonal antibody (MAB) based ELISA assay system claimed to mimic a VWF:RCof-like activity has also been recently described ('SE'), as has the production and characterisation of a large number [n = 10] of locally generated anti-VWF MAB. In the current study, we have adapted these MAB to in-house ELISA assays to assess their utility for VWD diagnosis and subtype discrimination, and to compare them with other assay systems. Thus, the VWF:CBA, VWF:RCof by agglutination, the SE assay, and in-house MAB based assays have been directly compared for their ability to discriminate Type 1 [n = 9] from Type 2 VWD samples [phenotypes 2A and 2B; n = 11]. In summary, MAB-based systems can be used to measure VWF and confirm a diagnosis of VWD, as well as exhibiting some VWD-subtype-discriminatory capabilities. However, better evidence of VWF-discordance was usually achieved using the VWF:RCof (agglutination) assay, while the greatest degree of VWF-discordance was consistently observed using the VWF:CBA assay. In conclusion, the VWF:CBA assay proved to offer the best diagnostic predictive tool for a Type 2 VWD defect, while MAB-based systems appear to be less effective in this regard.

Novel molecular mechanisms of dendritic cell-induced T cell activation.

In this study we have re-examined the molecular mechanisms involved in activation of T cells by dendritic cells (DC). Human peripheral blood DC (PBDC) were derived by 2 h adhesion followed by 7 day culture in a combination of granulocyte macrophage colony stimulating factor and IL-4, and depletion of residual T and B cells. These PBDC were used to induce autologous T cell proliferation in a CD3-dependent response, and antibodies against CD11a/18 and CD86 were used as control inhibitors of accessory function. Antibodies against five of the cell surface molecules that we have recently identified on the surface of DC, CD13, CD87, CD98, CD147 and CD148, and an antibody which recognizes a molecule that has not as yet been identified, all inhibited the CD3-induced T cell proliferation. These findings were observed not only when antibodies were present throughout the culture, but also when they were prepulsed on to the surface of the DC, suggesting the inhibition was mediated via the antigen-presenting cells rather than the T cell. The same set of antibodies also inhibited an allospecific mixed lymphocyte reaction, confirming that the inhibitory effect was not dependent on the use of a CD3 antibody as the stimulating agent. All the antibodies of known specificity inhibited both CD4 and CD8 T cells equally. Unlike CD87, CD98 and CD147 antibodies, which inhibited activation of both CD45RA (naive) T cells and CD45RO (memory) T cells, CD13 and CD148 appeared to be involved in activation of naive cells only. The molecules identified in this study have not previously been demonstrated to play a role as accessory molecules on DC, the cells that are pivotal for immune induction. Therefore they may provide new potential targets for modulation of the immune response at the APC level.

Use of a novel platelet function analyzer (PFA-100) with high sensitivity to disturbances in von Willebrand factor to screen for von Willebrand's disease and other disorders.

The PFA-100 is a new platelet function analyzer which uses whole blood and high shear stress blood flow to simulate primary hemostasis and assess platelet function. A small volume of blood is introduced into a disposable cartridge, and forced through a capillary tube. Platelet adhesion and aggregation is then initiated following exposure to either collagen/ADP [C/ADP] or collagen/epinephrine [C/Epi] coated membranes. Movement of blood through the capillary, and its subsequent occlusion is monitored and yields the measured endpoint (closure time [CT] in seconds). Using two approaches, we assessed the sensitivity of this system to disturbances in the function of von Willebrand Factor (VWF). Firstly, we assessed the ability of the PFA-100 to detect the presence of von Willebrands Disease (VWD). Using normal individuals (N = 18), CTs (in seconds; mean [range = mean +/- 2SD]) were (i) C/ADP, 95 [66-124], (ii) C/Epi, 128 [98-158]. A panel of 47 patients undergoing evaluation for clinical hemostatic defects inclusive of VWD were also evaluated. All samples from patients confirmed to have VWD following specific VWF studies [N = 9; 3 x Type 1, 1 x Type 3, 1 x Type 2A, 4 x Type 2B] gave prolonged CTs (>/= 200 s) for both C/ADP and C/Epi membranes; in contrast, all patients yielding normal CT values were found to yield normal VWF results (i.e., were found not to suffer from VWD). Patients with hemophilia (1 x hemophilia A, 1 x hemophilia C) gave normal PFA-100 CT, while those with clinical thrombocytopaenia (N = 3) gave prolonged PFA-100 CT. A number of other patient samples also gave abnormal CT values which in some cases could be linked to recent aspirin consumption. In the second evaluation process, and using normal blood, we have assessed the ability of various antibodies to influence the CT. Of the monoclonal antibody panel tested [N = 20], only a proportion of those against VWF [6/10] or gp1b/IX [CD42; 2/5] were found to be inhibitory (i.e., prolonged the CT). Data using polyclonal antibodies (against platelets, VWF, fibrinogen and fibronectin) is more complex but largely confirms the sensitivity of the system to VWF. On the basis of these results, we conclude that the PFA-100 is highly sensitive to disturbances in VWF and to the presence of VWD and may thus provide a valuable screening test for VWD in certain specific circumstances (i.e., acute need conditions or remote testing sites; normal CT result generally effective as negative predictor, i.e. not severe VWD). However, since abnormal CT values were obtained in clinical situations other than evident VWD, the PFA-100 cannot be used as a specific diagnostic tool to establish the presence of VWD. Thus, any abnormal PFA-100 CT result should be thoroughly evaluated by follow-up specific testing to establish the true clinical disorder affecting the individual under investigation, inclusive of appropriate VWF assays if VWD is clinically suspected.

MUC18, a member of the immunoglobulin superfamily, is expressed on bone marrow fibroblasts and a subset of hematological malignancies.

Despite the importance of bone marrow stromal cells in hemopoiesis, the profile of surface molecule expression is relatively poorly understood. Mice were immunized with cultured human bone marrow stromal cells in order to raise monoclonal antibodies to novel cell surface molecules, which might be involved in interactions with hemopoietic cells. Three antibodies, WM85, CC9 and EB4 were produced, and were found to identify a 100-110 kDa antigen on bone marrow fibroblasts. Molecular cloning revealed the molecule to be MUC18 (CD146), a member of the immunoglobulin superfamily, previously described as a marker of metastatic melanoma. In addition to the expected expression on melanoma cell lines and endothelial cells, a number of human leukemic cell lines were found to express MUC18, including all six T leukemia lines tested, one of five B lineage lines and one of four myeloid lines. Analysis of bone marrow samples from patients revealed positivity in 20% of B lineage ALL (n = 20), one of three T-ALL, 15% of AML (n = 13) and 43% of various B lymphoproliferative disorders (n = 7). No apparent reactivity was observed with mononuclear cells from normal peripheral blood or bone marrow, including candidate hemopoietic stem cells characterized by their expression of the CD34 antigen. However, positive selection of bone marrow mononuclear cells labeled with MUC18 antibody revealed a rare subpopulation (<1%) containing more than 90% of the stromal precursors identified in fibroblast colony-forming assays. The structure and tissue distribution of MUC18 suggest a functional role in regulation of hemopoiesis.

Assessment of the validity of the Adams and Fujita approximation for the higher oligomers of human spectrin.

Analysing the self-association behaviour of human erythrocyte spectrin is complicated by a large degree of nonideality. Adams and Fujita [1] proposed that, as a first order approximation, the logarithm of the activity coefficient of the protomer of a self-associating system can be considered to be linearly dependent on the total concentration of the protein, and that the same second virial coefficient could be considered to apply to all species. As a consequence of the Adams and Fujita approximation, the apparent equilibrium constant is equal to the thermodynamic equilibrium constant. The equilibrium concentrations at 30 degrees C of each oligomer spectrin species up to the 14-mer were determined after electrophoresis at low temperature. An apparent equilibrium constant for forming tetramer (K2,4) of (1.2 +/- 0.1) x 10(6) 1/mol was obtained, a value of (9.4 +/- 0.7) x 10(4) 1/mol was obtained for K4.6 and for all reactions forming oligomers higher than the hexamer an average approximate value of (2.7 +/- 0.4) x 10(5) 1/mol was obtained. The apparent equilibrium constants for the formation of all oligomer species of spectrin up to the tetrakaidecamer (14-mer) remain relatively independent of total spectrin concentration, and indicate that within the precision of the measurements a single virial coefficient is sufficient to account for the nonideality of spectrin self-association over the range 2-14 g/l, thus further justifying the use of the Adams and Fujita approximation for this protein over this concentration range.

Reinvestigation of the thermodynamics of spectrin self-association.

The thermodynamics of the self-association reactions of human spectrin have been reinvestigated by means of sedimentation equilibrium over the temperature range 18-40 degrees C. The experimental data were analysed in terms of a cooperative isodesmic model of association. The van't Hoff plot showed that the standard change in enthalpy for the heterodimer-tetramer step was temperature-dependent, leading to an estimate of -8.5 kJ mol-1 K-1 for the change in molar heat capacity, delta Cp. Curvature in the van't Hoff plots, not detected in previous studies, was revealed through the increased precision of the data and the wider temperature range examined. On the assumption that delta Cp reflects hydrophobic interactions in the tetramer that cannot be formed in the heterodimer, it can be estimated that approximately 50 CH2 groups per heterodimer participate in hydrophobic interactions in the tetramer that cannot be formed in the heterodimer.

A pilot clinical trial of two murine monoclonal antibodies fixing human complement in patients with chronic lymphatic leukaemia.

The use of monoclonal antibodies (MABs) for the therapy of malignant diseases offers the potential advantage of greater target cell specificity, and therefore less toxicity. A major limitation of this therapeutic approach has been the inability of most MABs to kill the cell once bound to the target antigen. We have previously reported the development of two murine IgM MABs, WM63 (CD48) and WM66 (unclustered), that react with panleucocyte antigens widely expressed on cells from lymphoproliferative disorders, and are lytic with human complement. These antibodies have subsequently been administered intravenously to patients with chronic lymphocytic leukaemia (CLL) in a Phase One trial. Seven patients with progressive CLL received increasing daily doses of WM66 (Patients 1-3) or WM63 (Patients 4-7), with one patient also receiving a continuous infusion of WM63 over 20 hours. All patients demonstrated a significant but transient reduction in the number of circulating leucocytes, and no overall effect on disease progression was observed. Antibody coating of circulating lymphocytes was seen in patients receiving WM-63. Patients receiving large doses of WM63 (cases 5-7) demonstrated a decline in complement levels during treatment. There were no major adverse reactions to WM66, but two patients developed dose limiting side effects to WM63. No human anti-mouse antibody (HAMA) responses were documented. These findings indicate that in vitro cytotoxicity mediated by Mabs fixing human complement correlates poorly with clinical responses, and support earlier observations which indicate that cell-mediated cytotoxicity is necessary for effective antibody therapy.

Characterization of a novel leucocyte surface membrane antigen recognized by the monoclonal antibody WM65.

WM65 is a murine mAb which recognizes a novel surface membrane antigen present on leukaemic and normal leucocytes. The present study further investigates the nature of this antigen, especially those features which relate to the possible therapeutic applications of the WM65 antibody. There are 1-3 x 10(4) molecules of this antigen present on normal leucocytes, and the same or greater numbers of antigen molecules are present on a variety of leukaemic cells. In vitro data showed that the WM65 antibody is internalized following interaction with its antigen on normal leucocytes. The affinity of this antibody was calculated using an ELISA method which required neither labelling of the antibody nor purification of the antigen and the affinity constant was found to be 3 x 10(7) +/- 2 x 10(7) (mol/L)-1. Further data are presented which suggest that this antigen is a differentiation antigen and an integral membrane protein. Despite the relatively low affinity of the WM65 antibody, a number of characteristics of the antigen suggest the antibody may possibly have therapeutic applications. These characteristics include its cellular distribution, the number of antigen molecules expressed on the cell surface and its ability to internalize in vitro.

Purification of two murine monoclonal antibodies of the IgM class by hydroxylapatite chromatography and gel filtration.

A two-step method using hydroxylapatite chromatography and gel filtration is described for the purification of two murine monoclonal antibodies of the IgM class. Ascites fluid from each hybridoma was diluted in sodium phosphate buffer (0.01 M, pH 6.8), loaded onto a hydroxylapatite column and eluted with a stepwise sodium phosphate gradient. The immunoreactive protein peaks were concentrated and subjected to gel filtration using either Sephadex G-200 or Sephacryl S-200HR. The biological activity of the end-products was confirmed by complement lysis assay and by indirect immunofluorescence and flow cytometry. The purity of the end-products as assessed by SDS polyacrylamide gel electrophoresis and densitometry was at least 90%. The methods described produced immunoreactive material with a high level of purity. The procedure for each antibody was reproducible and provides a reliable method for purification of monoclonal antibodies of the IgM class.

Reaction coupling of chelation and antigen binding in the calcium ion-dependent antibody binding of cyclic AMP.

Polyclonal antibodies, raised against cyclic AMP (cAMP) by the immunization of animals with a 2'-O-succinyl cAMP/bovine albumin conjugate, have been reported to be dependent upon the presence of calcium ion (Ca2+) for antigen binding. They also exhibit a major "bridge" effect whereby 2'-O-succinyl and 2'-O-acetyl derivatives are bound more avidly than the parent nucleotide. Since cAMP and these derivatives bind Ca2+ very weakly, they do not present substantially in the chelated form over the range of Ca2+ concentrations used. Thus direct antigen modification is excluded as an explanation for the observed ion dependence of the reaction. Instead, we propose a mechanism based on reaction coupling. The actual antigens are the Ca2+ chelates of these nucleotides, whose formation in the absence of antibody is rapid but not favored (as indicated by their weak association constants). When antibody is added, the chelates act as transient intermediates whose concentration remains low but which is replenished as they are consumed by antibody. The coupled reaction is driven by the antibody-antigen step which occurs more slowly but with a substantial gain in free energy. The reaction is limited by the availability of Ca2+. It also appears that the rabbit antibody-forming cell responds preferentially to the Ca(2+)-bound form of the 2'-O-succinyl cAMP/bovine albumin conjugate which may appear to be more "foreign" than the unbound form of the hapten containing the ubiquitous nucleotide cAMP.

A novel human leucocyte surface membrane antigen defined by murine monoclonal antibody.

A murine monoclonal antibody has been produced which identifies a novel human leucocyte differentiation antigen. The antibody, designated WM-66, of IgM subclass, was cytolytic with human complement. WM-66 was shown to react with virtually all normal T and B lymphocytes from peripheral blood and lymphoid tissues, as well as blood monocytes and approximately 40% of bone marrow mononuclear cells. The antibody also bound to the majority of cases of chronic B-cell malignancies, including chronic lymphatic leukaemia and non-Hodgkin's lymphoma, but not to cases of acute leukaemia or to the majority of leukaemic and lymphoblastoid cell lines. WM-66 also reacted with epithelium of bronchus and salivary gland ducts. A single band of relative molecular mass 65,000 Daltons was immunoprecipitated from membrane extracts of normal lymphocytes and the B-cell line Daudi. Treatment of a number of WM-66-negative B-cell lines with neuraminidase resulted in WM-66 binding, indicating that the antigen exists in a covert form masked by sialic acid residues on a wider spectrum of cell types than was initially apparent. The reactivity pattern of WM-66 indicates that it recognises a previously undescribed surface membrane molecule with broad non-lineage-specific distribution on leucocytes. This has recently been confirmed at the Fourth International Workshop on Human Leucocyte Differentiation Antigens. Although the biological function of the molecule recognised by WM-66 is unknown, the lytic properties of the antibody suggest a possible in vivo therapeutic role as an immunosuppressant or for treatment of lymphoid malignancy.

A novel non-lineage antigen on human leucocytes: characterization with two CD-48 monoclonal antibodies.

Two murine monoclonal antibodies have been produced which identify a novel surface antigen expressed on human leucocytes in a non-lineage-restricted distribution. Antibodies WM-63 and WM-68 were derived after immunization of mice with human T-CLL cells and the leukaemic cell line HSB-2. Both antibodies were shown to react with over 90 per cent of normal T and B lymphocytes from peripheral blood and tonsil, and also with monocytes from peripheral blood. A subset of bone marrow leucocytes, including granulocyte-macrophage progenitors, were also reactive. No activity with non-haemopoietic cells or tissues could be identified, however WM-63 and WM-68 showed binding to virtually all cases of chronic B cell malignancy, including chronic lymphatic leukaemia and non-Hodgkin's lymphoma, as well as a proportion of cases of acute leukaemia. Although the antigen recognized by these antibodies could not be immunoprecipitated from membrane extracts, it was removed from the surface of intact cells using the proteolytic enzymes protease and papain. Re-expression on cultured cells was inhibited by incubation with puromycin, cycloheximide, and tunicamycin, indicating that the epitopes detected by WM-63 and WM-68 are likely to be carbohydrate moieties on a protein backbone. Removal of the antigen from the cell surface by treatment with the enzyme phosphatidyl-inositol phospholipase C indicates that it is linked by a phosphatidyl-inositol bond. WM-63 and WM-68 were both recently clustered at the Fourth International Workshop on Human Leucocyte Differentiation Antigens into CD-48, together with four other monoclonal antibodies. Although no biological function has been ascribed to the molecule detected by these antibodies, its restriction to the haemopoietic lineage suggests a role in regulation of leucocyte function.

Expression of a novel human pan leucocyte differentiation antigen on leukaemic cells.

A murine monoclonal antibody has been produced which identifies a novel surface membrane antigen present on virtually all normal human leucocytes and leukaemic cells. The antibody, designated WM-65, reacted with over 95% of peripheral blood, tonsil and thymic lymphocytes, and with a similar proportion of monocytes and granulocytes. A majority of nucleated normal bone marrow cells were also reactive with WM-65; however, these included only a small proportion of myeloid progenitor cells. WM-65 reacted with a wide range of acute and chronic leukaemias of both myeloid and lymphoid types, and with corresponding cell lines, but did not react with non-haemopoietic cells. By immunoprecipitation and SDS-PAGE, WM-65 identifies a heavily glycosylated surface protein of molecular weight between 40 and 50 kD. This property, and the broad non-lineage-specific distribution of the antigen on haemopoietically-derived cells, indicates that WM-65 is different from other monoclonal antibodies with "leucocyte common" reactivity patterns. The extensive reactivity of WM-65 with leukaemic cells raises the possibility of therapeutic applications of the antibody in haematological malignancies.

Further studies on the heterogeneity of antigens recognised by CD-1 monoclonal antibodies: distribution of epitopes and analysis of serological binding patterns.

The binding patterns of 28 monoclonal antibodies (MAB) recognizing antigens belonging to Cluster of Differentiation One (CD-1) were analyzed in order to investigate heterogeneity within this cluster. Competitive binding assays using radiolabelled MAB provided detailed information on CD-1 antigenic heterogeneity, and demonstrated that at lease six epitopic regions are recognised as CD-1 MAB. Further studies, based on single cell suspension immunofluorescence assays (using thymocytes and subclones on the cell line Molt-4), suggested that the majority of MAB studied could be serologically separated into three groups. In view of the most recent information that CD-1 MAB recognize at least three different early T-cell differentiation molecules, our results indicate that there are three or more distinct epitopes on the 'gp49'(HTA-1/CD-1a) molecule, two on the 'gp45'(HTA-3/CD-1b) molecule and one on 'gp43'(HTA-2/CD-1c).

The effect of o,p'-DDD on adrenal steroid replacement therapy requirements.

Two patients with adrenal carcinoma treated with 2,2-bis (2-chlorophenyl-4-chlorophenyl)-1,1-dichloroethane (o,p'-DDD) as adjuvant therapy were studied. Both patients developed hypoadrenalism while on o,p'-DDD and apparently adequate dexamethasone replacement therapy. The hypoadrenalism was overcome by increasing steroid replacement therapy. Dexamethasone levels were measured in the serum by radioimmunoassay and shown to be lowered by o,p'-DDD therapy. A study of the absorption and disappearance of dexamethasone from the circulation in response to a (1 mg oral dose indicated that the steroid was absorbed normally but was cleared more rapidly from the circulation of these two patients than from normal controls. This may be due to a change in the type of metabolites excreted. It is suggested that many of the reported side-effects of o,p'-DDD may be due to hypoadrenalism and may be controlled by greatly increasing the steroid replacement dose. The adequacy of corticosteroid replacement therapy may best be assessed by monitoring the levels of ACTH.