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Six monoclonal IgG1-k antibodies (LK2, LK3r, LK4-55, LK5, LK6-55, LK7r) were raised against platelet membrane GPIIIa in order to study the structure-function relationship of this molecule. Antibodies were selected on their ability to react with GPIIIa by ELISA on adherent platelets, by immunoblot on platelet lysates and by fluorescence flow cytometry on intact platelets. Fluorescence reactivity varied from 3- to 202-fold greater than isotype control fluorescence. Two MoAbs reacted on immunoblot under reduced conditions (LK7r and LK3r). Two reacted with a 55 kD chymotrypsin/subtilisin digest of GPIIIa which is likely to exclude amino acids 121-348 (LK4-55 and LK6-55). Four of the MoAbs (LK5, LK3r, LK2 and LK4-55) inhibited tyrosine phosphorylation of one to four distinct bands on immunoblot. LK4-55 reacted with an N-terminal 66 amino acid fusion protein of GPIIIa near the PLA epitope (Leu 33). LK7r reacted with a 212-222 peptide reported to be an RGD fibrinogen binding site. LK2 reacted near a disintegrin-RGD binding site. Except for LK5, all inhibited ADP, collagen and thrombin-induced platelet aggregation in a heterogeneous fashion. Percentage inhibition of 125I-fibrinogen binding to platelets varied from 18% to 98%. No correlation was noted between inhibition of fibrinogen binding, location of MoAb binding on GPIIIa, reactivity of MoAb binding with GPIIIa, inhibition of thrombin-induced tyrosine phosphorylation or inhibition of platelet aggregation induced by ADP, collagen or thrombin. Thus MoAbs, binding to platelet GPIIIa at different sites, inhibit platelet aggregation in a heterogeneous manner.

Monoclonal antibody (MoAb) B724 to von Willebrand factor (vWF) completely inhibits its interaction with heparin, sulphatides and botrocetin and consequently botrocetin-induced binding of vWF to platelets. MoAb B724 has no effect on the binding of vWF to collagen or to ristocetin-treated platelets nor on vWF-dependent platelet aggregation induced with ristocetin and asialo-vWF-mediated platelet aggregation. MoAb B724 preferentially recognizes a conformation of native vWF, in solution, or immobilized through a coated antibody. It exhibits a markedly lower affinity for vWF immobilized onto collagen or plastic surfaces. Using proteolytic fragments of vWF, B724 epitope was localized within the 512-673 sequence of the A1 disulphide loop of vWF, MoAb B724 was used as second antibody in a two-site ELISA to test a series of patients with type 1, 2A, 2B and 2N vWD or haemophilia A and recombinant wild type or mutated vWFs. Results were compared with those obtained by control ELISAs performed using polyclonal antibodies. Using MoAb B724, strikingly lower levels of vWFAg were observed in plasma from most patients with type 2B vWD, and in seven out of the eight rvWF mutated close to or within the A1 disulphide loop. Therefore MoAb B724, which interferes with this loop involved in the function of vWF, appears to be a useful tool for rapid screening of conformational changes in this region.

Three synthetic peptides encompassing the entire cytoplasmic polypeptide sequence (amino acid residues 82-128) of glycophorin C (GPC) and glycophorin D (GPD) were used to immunize mice for the production of monoclonal antibodies (MoAbs). Only the synthetic peptide (GPC-peptide-1) corresponding to C-terminal residues 112-128 elicited a MoAb (named BGRL-100) which could react with native and denatured GPC and GPD. We characterized BGRL-100 by inhibition using GPC-peptide 1 and red cell sialoglycoproteins. The ability of BGRL-100 to interact with native GPC and GPD was assessed by immunoprecipitation with normal red cells (RBCs), and with denatured GPC and GPD by Western blotting of both normal RBCs and RBCs carrying GPC variants. Immunohistochemical staining of human tissue sections was performed using both BGRL-100 and a rat MoAb (named BRAC-1), which is specific for an extracellular domain of GPC and GPD. Both antibodies showed strong staining of erythroid lineage haemopoietic cells in fetal liver, sinusoids of adult liver and RBCs in the blood vessels of all tissues tested. Neither antibody reacted with epithelia from a range of human tissues. However, both MoAbs stained neural tissue in a distinctive fibrillar pattern. This suggests the presence of an analogue of erythroid GPC in neural tissues.

Two different approaches were used in order to define the epitope of three monoclonal antibodies (MoAbs) against the NH2-terminal part of the mature subunit of von Willebrand factor (vWF) which contains its factor VIII (FVIII) binding site. First, a vWF cDNA fragment library using the bacteriophage lambda gt11 expression vector was screened with radiolabelled MoAbs. The epitope of each MoAb was defined, following sequence analysis, by the overlapping DNA sequence of immunoreactive clones. MoAb 32B12, a potent inhibitor of FVIII/vWF interaction, binds within the Glu35-Ile81 sequence of vWF subunit. MoAb 14A12, a non-inhibitory antibody, recognizes a sequence within Thr141-Val220. MoAb 31H3, a partial inhibitory antibody, gives no positive clone. In the second method, a panel of 24 synthetic pentadecapeptides corresponding to the first NH2-terminal 105 amino acid residues was used to block the binding of inhibitor MoAbs to immobilized vWF in an ELISA system. The localization of MoAb 32B12 epitope was confirmed and restricted to the Met51-Ala60 sequence. The MoAb 31H3 binding to vWF is inhibited by two synthetic peptides with the overlapping sequence Cys66-Gly76. All these data confirm that the FVIII binding site of vWF is not limited to the binding area (Thr78-Thr96) of the previously described MoAbs inhibiting FVIII/vWF interaction but is composed of several key sequences.

The monoclonal antibody Ki-67 reacts with a nuclear antigen that is present only in proliferating cells. The proportion of Ki-67 positive cells may therefore serve as a reliable measurement for the growth fraction in normal and neoplasmic cell populations. We have tested the significance of the MoAb Ki-67 in the classification of monoclonal gammopathy and compared the results with the plasma cell labelling index. In benign monoclonal gammopathy the percentage of Ki-67 positive plasma cells (median 1.6%) was significantly lower than in untreated multiple myeloma (median 9.6). Among the patients with more than 10% Ki-67 positive plasma cells there were some very short survivors. The largest growth fractions (median 41.8%) were found in patients with relapsing multiple myeloma indicating here a different growth pattern more resembling the high-grade lymphomas. A linear correlation between the proportion of Ki-67 positive plasma cells and the labelling index was not found. Determination of the plasma cell growth fraction with the monoclonal antibody Ki-67 in monoclonal gammopathy may help to discriminate benign monoclonal gammopathy from multiple myeloma and will probably identify a subgroup of multiple myeloma patients with a poor prognosis, including those with relapsing multiple myeloma.

Immunophenotyping of frozen lymph node sections from seven patients with morphologically defined angioimmunoblastic lymphadenopathy (AILD) was performed with a panel of 20 monoclonal antibodies (MoAb). MoAb for identification of dendritic reticulum cells showed remnants of follicular structures in all cases. In four cases these 'burnt out' follicular structures were associated with clusters of polyclonal B-cells. Vascular proliferation and small amounts of intercellular collagenous fibrils were reliably revealed by MoAb CIV22 against basement membrane collagen. Cellular infiltrates of T4+ cells prevailed in four cases, of T8+ cells in one case. In one case conventionally identified blasts were of T- and B-cell origin. Expression of cell proliferation associated nuclear antigen identified by MoAb Ki-67 was present in more than 25% of lymph node cells in three patients with fatal outcome, but in less than 10% of cells in two patients with a better clinical course. Therefore MoAb Ki-67 may represent a valuable prognostic marker in AILD.

In this report a simple procedure for the production of murine monoclonal antibodies (MoAb) against the idiotype of malignant B cells is described. Mice were immunized with lymphoid cells from patients with B-cell chronic lymphocytic leukaemia (B-CLL). After fusion of the spleen cells, hybridoma supernatants were screened for anti-idiotypic MoAb in ELISA with immunoglobulins obtained from tumour-cell lysates, xenohybridomas and patients' sera. The anti-idiotypic MoAb were used to study tumour cells and serum immunoglobulins (Ig) from four different patients with B-CLL. It was found that the serum IgM and IgD in one patient shared the same idiotype. Evidence is presented that IgG-secreting cell populations are not restricted to lambda-Ig-light chain-expressing B-CLL cells. With the help of anti-idiotype MoAb accurate measurements of total and idiotype-positive serum immunoglobulin levels during chemotherapy were possible.

A patient having a B cell chronic lymphocytic leukaemia was treated with a monoclonal anti-idiotypic antibody (MoAb anti-id). Up to 24.5 g of MoAb anti-id has been administered to the patient over a period of 1 year without serious side effects. Despite a substantial amount of serum idiotype (id = 100 micrograms/ml) and a low expression of id on the tumour cells (+/- 6000 molecules per cell) clearance of serum id and a marked tumour reduction was obtained. Therapy resistance developed and coincided with a decreased clearance rate of circulating id-anti-id immune complexes and an increased modulation of cellular id expression, in vivo. This suggests that a decreased clearance rate of anti-id coated tumour cells provided more time for id modulation in vivo, resulting in therapy resistance. Therefore, the overall capacity of the natural effector system may have an important influence on the ultimate therapeutic effect of immunotherapy with MoAb anti-id. Although the partial remission obtained was not long-lasting, this study shows that MoAb anti-id therapy can be effective even when id expression on the tumour cells is low and a substantial amount of serum id is present.

Immunotoxins (ITs) appear to vary considerably in their killing efficiency towards antigen positive cells. In order to unravel the mechanisms underlying these differences, the parameters responsible for these variations were studied. The efficacy of the monoclonal antibodies (MoAbs) WT32 (CD3), OKT4 (CD4), T101 (CD5), WT1 (CD7) and WT82 (CD8) conjugated to ricin A-chain was expressed by the extent of protein synthesis inhibition of four leukaemic T cell lines (CEM, GH1, Jurkat and HPB-ALL). Large differences in cytotoxicity were observed. Efficient inhibition of protein synthesis was seen with anti-CD3 IT, anti-CD5 IT and anti-CD7 IT. In these cases the cytotoxicity was related to the antigen density on the target cell membrane. Anti-CD4 IT inhibited poorly and anti-CD8 IT was ineffective, even on cell lines with a high expression of the corresponding antigen. When antigen density and cytotoxicity were plotted for all CD antigens, no correlation could be found. Subsequently, internalization was studied with 125Iodine-labelled antibodies. Anti-CD7 showed the fastest internalization rate, followed by anti-CD3 and anti-CD5. Anti-CD4 and anti-CD8 antibodies were respectively moderately and hardly internalized. When the absolute amount of internalized MoAb was calculated, a highly significant correlation with cytotoxicity was found. We conclude that the degree of antigen expression is not so important as the absolute amount of antibody internalized in predicting the efficacy of ITs.