Inhibition of platelet adhesion to collagen by monoclonal anti-CD36 antibodies

Monoclonal anti CD36 antibodies capable of inhibiting platelet adhesion to collagen have not previously been identified. We have now prepared two groups of monoclonal antibodies. One group was prepared using, as immunogen, highly purified (99+%) CD36 prepared by a denaturing procedure. These antibodies (Mo series) reacted strongly with CD36 on protein blots but did not immunoprecipitate native CD36 from platelet lysates nor inhibit platelet adhesion to collagen. The second group of monoclonal antibodies (131 series) was prepared using CD36 purified to >95% by a non-denaturing procedure. These antibodies reacted with control platelets, but not Nak^a-negative platelets which lack CD36, as measured by flow cytometry and by immunoprecipitation. Three monoclonal antibodies of this latter group (131.4, 131.5 and 131.7) inhibited platelet adhesion to collagen in static systems under Mg²⁺-independent conditions but had little effect in the presence of Mg²⁺. 131.4 and 131.7 also inhibited adhesion to collagen using citrated whole blood in a parallel plate flow chamber at physiological shear rates (800 s^?1), whereas 131.5 was without effect. These are the first anti-CD36 monoclonal antibodies shown to be capable of inhibiting platelet adhesion to collagen and provide further evidence that CD36 plays a role in platelet–collagen interaction.     

Anti-CD36 antibodies in patients with lupus anticoagulant and thrombotic complications

Summary . Six patients with lupus anticoagulant with thrombotic complications, but not exhibiting systemic lupus erythematosus, demonstrated the presence in their plasma of antibodies directed against platelet antigens which were not detectable in two patients presenting with lupus anticoagulant but without thrombotic complications. Protein blotting of separated normal platelet proteins against patient plasma gave up to 18 bands of varying intensity indicative of multiple antiplatelet antibodies; one of these antibodies recognized a component with a mobility identical with CD36 (GPIV; m.w. 88,000) in 4/6 cases. Antibodies to CD36 and one or two other components were identified in 5/6 cases by immunoprecipitation from ¹²⁵I-labelled control platelets and 6/6 by dot blots against purified CD36. These results suggest that antiplatelet antibodies and, specifically, anti CD36 antibodies, occur frequently in the plasma of patients presenting with lupus anticoagulant and thrombotic complications.     

Targeting of saporin to Hodgkin's lymphoma cells by anti-CD30 and anti-CD25 bispecific antibodies

CD25 and CD30 represent suitable target molecules for bispecific antibody (bimAb)-driven toxin delivery to lymphoid tumour cells. We describe two new anti-CD30/anti-saporin bimAbs (termed CD30 × sap1 and CD30 × sap2), produced by hybrid hybridomas, which react against non-cross-reactive epitopes of the saporin molecule, and compared their effect with a bimAb reacting with saporin and with CD25 (CD25 × sap1). In a protein synthesis inhibition assay these bimAbs were able to enhance saporin toxicity (IC₅₀ 8.5 × 10^?9M in the absence of mAbs) with a similar activity: in the presence of 10^?9 M CD30 × sap1 bimAb the IC₅₀ was 2.75 × 10^?11 M , whereas with CD30 × sap2 bimAb the IC₅₀ was 6.5 × 10^?11 M and CD25 × sap1 bimAb displayed an IC₅₀ of 3 × 10^?11 M (as saporin). The combined use of the two anti-CD30 bimAbs further increased cytotoxicity by 100-fold, resulting in an IC₅₀ of 1.9 × 10^?13 M . A slightly less efficient improvement was obtained by combining the CD25 × sap1 bimAb with the CD30 × sap2 bimAb directed against a different toxin epitope (saporin IC₅₀ to 7 × 10^?13 M ). In contrast, no synergistic effect was observed using the combination of the anti-CD25 bimAb with the anti-CD30 bimAb reacting with the same epitope of saporin (IC₅₀ = 4.5 × 10^?11 M ). Analysis of FITC–saporin binding to L540 cells by flow cytometry demonstrated that the appropriate combinations of the two anti-CD30/anti-saporin bimAbs or of the anti-CD30/anti-saporin and anti-CD25/anti-saporin bimAbs had a cooperative effect on the binding of the ribosome-inactivating protein (RIP) to the cells, when compared with single bimAbs.     

Increased platelet CD36 constitutes a common marker in myeloproliferative disorders

Summary. The distribution of the major platelet membrane glycoproteins (GP), Ib, IX, IIb-IIIa and IV (or CD36), which play important roles as receptors for adhesive molecules in haemostasis and thrombosis, was studied in 34 patients with myeloproliferative disorders (MPD): 13 had essential thrombocythaemia (ET), 12 had polycythaemia vera (PV) and nine had chronic myelogenous leukaemia (CML). Only occasionally were modifications of the numbers of GPIb or GPIIb-IIIa measured using the binding of specific radiolabeled antibodies to platelets. In contrast, 2-3-fold increases of the total CD36 content and the surface CD36 expression were measured in almost all patients studied, using a radioimmunoassay and the direct binding of the radiolabelled antibody, FA6-152, to the platelet surface, respectively. These results indicate that the abnormality affected both the external and internal CD36 pools. Therefore platelet CD36 may be a useful tool for the diagnosis and the follow-up of MPD patients.
Surface CD36 has been proposed as a platelet receptor for thrombospondin, an adhesive glycoprotein that is released from platelets upon activation and promotes aggregate formation. Despite a 2-fold increase of CD36 molecules, resting and thrombin-activated platelets from ET patients expressed the same amount of thrombospondin as normal platelets, suggesting that there is not a direct correlation between the CD36 expression and thrombospondin binding either spontaneously or after activation.     

Characterization of platelet glycoproteins and platelet/endothelial cell antibodies in patients with thrombotic thrombocytopenic purpura

Platelets and sera from 12 patients with thrombotic thrombocytopenic purpura (TTP) and 12 healthy normal control subjects were examined. As determined by quantitative flow cytometry, prior to plasma exchange therapy platelet surface glycoprotein (GP) Ib levels were similar in TTP patients and normal controls (mean 20 188 and 20 226 molecules/platelet, respectively). Platelets from patients with TTP did, however, have significantly reduced levels of GPIIb/IIIa prior to plasmapheresis (mean 36 348 v 52 505 molecules/platelet in controls; P = 0.0004) and of GPIV (mean 13 321 v 26 212 molecules/platelet in controls; P = 0.0002). An increase in activated platelets, as determined by CD62 expression, was observed in 82% of patients. Increased platelet-associated immunoglobulins and/or complement was also seen in approximately 60% of the patients. In general, with return of platelet counts to normal levels following seven plasmaphereses, the above abnormalities were reversed, although often not to normal levels. Western blot analysis indicated the presence of antibodies reactive to platelet GPIV (88 kD) in 70% of pretreatment sera from patients with TTP; a similar band was observed in 80% of patient sera against microvascular endothelial cells. Immunofluorescence microscopic examination indicated the presence of antibody in pretreatment sera from patients with TTP to microvascular (73%) and large vessel (36%) endothelial cells. As measured by an indirect flow cytometric assay, pretreatment sera from 55% of patients with TTP were reactive with large vessel endothelial cells and 100% reacted with microvascular endothelial cells; reactivity was significantly greater against the microvascular endothelial cells (P = 0.0048) and was reduced following plasma exchange therapy. These results indicate abnormalities in platelet glycoprotein expression in TTP and suggest that anti-platelet and anti-endothelial cell antibodies play a role in the thrombocytopenia and vasculitis characteristic of this disorder.     

Immunotoxins containing saporin linked to different CD2 monoclonal antibodies: in vitro evaluation

In this study we describe immunotoxins prepared with different CD2 monoclonal antibodies (mAbs) and a ribosome-inactivating protein, saporin. The CD2 immunotoxins were tested on different models. Anti-CD2–saporin conjugates inhibited protein synthesis by a neoplastic CD2+ cell line (SKW-3) and by an interleukin 2 dependent polyclonal CD2+ lymphoid cell culture (T lymphoblasts), with IC₅₀s ranging from 10^-13 m to 10^-11 m (as saporin). Similar results were obtained with proliferation inhibition tests (³H-thymidine incorporation) on phytohaemagglutinin (PHA) driven lymphoid cultures and on mixed lymphocyte culture activated lymphocytes. Moreover a CD2–ricin A chain conjugate was less effective than an analogous immunotoxin containing the same CD2 mAb and saporin in inhibiting lymphocyte proliferation induced by PHA (IC₅₀ approximately 10^-9 m as ricin A chain versus 10^-12 m as saporin). The conjugates were not toxic on bone marrow stem cells. These results suggest that CD2–saporin immunotoxins could represent an effective tool for CD2+ lymphomas or leukaemias, and for T-dependent immune disorders, such as transplanted organ rejection and graft-versus-host disease.     

Anti-CD20 monoclonal antibodies: historical and future perspectives

Antibodies to CD20 have confirmed the hypothesis that monoclonal reagents can be given in vivo to alleviate human diseases. The targeting of CD20 on normal, malignant and auto-immune B-lymphocytes by rituximab has demonstrated substantial benefits for patients with a variety of B-cell lymphomas, as well as some with autoimmune disorders. There has been a notable increase in the survival rates from B-cell lymphoma in the decade since anti-CD20 therapy was introduced.     

Haemolytic uraemic syndrome is an immune-mediated disease: role of anti-CD36 antibodies

Haemolytic uraemic syndrome (HUS) is a disorder in which platelet microthrombi are formed that have a particular propensity to deposit in the kidney microvasculature, resulting in impaired renal function and thrombocytopenia. The mechanism of formation of these microthrombi is not known. In this study, we showed that plasma from five adult and six paediatric cases of HUS caused aggregation and release of adenosine triphosphate from normal platelets. The plasma reacted against platelet lysate in a protein blot and all samples showed reactivity against a band at 88 kDa, corresponding to the membrane antigen CD36. This was confirmed by probing with Mo91, a monoclonal antibody to CD36. CD36 was also identified in the immune complex formed by incubation of patient plasmas with normal platelet lysate. In other studies, bands of 32 and 7.7 kDa were obtained when purified verotoxin was protein blotted and probed with either patient plasma or with anti-CD36 antibody Mo91 suggesting structural homologies between CD36 and verotoxin. While a direct cause-effect relationship is not yet established, the data support the concept of an immunological pathogenesis for HUS and suggest that molecular mimicry involving one or both of the homologous domains in membrane-bound CD36 and verotoxin lead to the development of antibodies capable of inducing the pathophysiological events characteristic of HUS.     

Platelet surface antigens: Analysis by monoclonal antibodies

Summary Monoclonal antibodies were produced against human platelets. Four antibodies (PA1, PA2, PA3 and PA4) reacted specifically with platelets and megakaryocytes, but not with peripheral blood lymphocytes, granulocytes, erythrocytes or monocytes. The antibodies belonged to the mouse IgG subclass 2a (PA1, PA2, PA3), or 1 (PA4) respectively. PA1 and PA4 did not precipitate, their antigens have not yet fully been characterized. PA3 was directed against the glycoprotein (Gp) complex IIb/IIIa; PA2 precipitated Gp IIb/IIIa, and, in addition, Gp Ia. PA4 revealed specificity against the human platelet alloantigen Zw(a).     

Differential sensitivity of CD30+ neoplastic cells to gelonin delivered by anti-CD30/anti-gelonin bispecific antibodies

Summary. Lymphocyte activation antigens, such as CD30, represent suitable target molecules for antibody-driven drug delivery in haemopoietic malignancies. A ribosome-inactivating protein (RIP) type 1 of potential interest for mAb targeting is gelonin, which displays a lower toxicity, as compared to other RIPs. In this study, two anti-CD 3 0/anti-gelonin bispecific monoclonal antibodies (bimAbs), secreted by hybrid hybridomas, were used to deliver this RIP to CD30⁺ tumour cells. The two bimAbs, termed D4 and A18, were produced using the same anti-CD30 mAb and two anti-gelonin mAbs, directed to unrelated epitopes of the gelonin molecule. These bimAbs enhanced gelonin toxicity (IC₅₀ 5 × 10^?8 M, in the absence of mAbs) against the CD30⁺ L540 Hodgkin's lymphoma cell line in a protein synthesis inhibition assay. Thus, in the presence of 10^?9 M D4 bimAb, protein synthesis was inhibited with an ICs₀ of 5 × 1CT¹⁰M as gelonin, whereas with A18 bimAb the IC_S0 was 8 × 1CT¹¹ M. More interestingly, the combined use of the two bimAbs had a synergistic effect, since the IC₅₀ of gelonin reached 6 × 10^_12M. Among CD30 tumour cell lines, the Hodgkin's lymphoma L428 was also sensitive to gelonin delivered by bimAbs (IC₅₀ 6 × 1CT^U M), whereas the COLE Hodgkin's cell line and the T-ALL Jurkat were completely resistant to the toxic effect of gelonin and bimAbs. COLE and Jurkat cells were also resistant to a gelonin/anti-CD30 conventional immunotoxin, whereas they were sensitive to a saporin/anti-CD30 immunotoxin. This suggests that the resistance to gelonin is not related to a lack of internalization through the CD30 molecule but is associated with some property of the RIP.     

Selective internalization of monoclonal antibodies by B-cell chronic lymphocytic leukaemia cells

B-cell chronic lymphocytic leukaemia (B-CLL) cannot be cured by conventional chemotherapy, therefore, toxin-linked therapeutic monoclonal antibodies (mAbs) are increasingly examined for their potential to improve clinical outcome. The current study aimed to identify mAbs that were internalized by the B-CLL cells of 14 patients, using both flow cytometry and confocal laser scanning microscopy. Anti-CD5, CD22 and CD40 mAbs were effectively taken up by B-CLL cells, whereas mAbs against CD19, CD20, CD23 and CD45 were not. This study may form a basis for further research to identify antibodies that may serve as carriers for toxins to treat B-CLL.     

Circumsporozoite protein of Plasmodium gallinaceum characterized by monoclonal antibodies

Monoclonal antibodies (MoAb) were produced against both salivary gland sporozoites (SGS) and oocyst sporozoites (OS) of Plasmodium gallinaceum, an avian malaria parasite. By indirect immunofluorescence, all of the MoAbs reacted with both SGS and OS of P. gallinaceum and two of the MoAbs cross-reacted weakly with P. berghei sporozoites. None of the MoAbs reacted with sporozoites of six additional species of mammalian plasmodia. In Western blot analysis of extracts of either SGS or OS of P. gallinaceum, these MoAbs identified two polypeptides with molecular weights of approximately 76,000 and 64,000 D. The results of a MoAb inhibition of binding assay and a two-site one-antibody immunoradiometric assay indicate that the circumsporozoite protein of P. gallinaceum, like those of mammalian malaria parasites, contains a repetitive immunodominant epitope. Two of the anti-P. gallinaceum MoAbs were tested in a sporozoite neutralization assay and decreased, but did not abolish, the infectivity of sporozoites for chickens, indicating that the polypeptide of P. gallinaceum identified by immunoblot is probably the protective antigen.     

Enhancement of human platelet activation by the combination of low concentrations of collagen and rabbit anticardiolipin antibodies

Low concentrations of collagen and anticardiolipin antibodies (ACLA), which were raised in rabbits by immunization with cardiolipin (CL), co-operatively activated human gel-filtrated platelets (GFP). GFP activated by adding ACLA 5 min prior to collagen (ACLA + Col) showed strong responses in cytosolic Ca2+ mobilization and cell aggregation; the responses decreased after 1 min, however, when collagen was added prior to ACLA (Col + ACLA). Col + ACLA was 30% less effective than the ACLA + Col in: (1) the phosphorylation of pleckstrin and myosin light chain; and (2) the secretion of alpha- and dense granules. Indomethacin inhibited Ca2+ mobilization, pleckstrin phosphorylation and cell aggregation in platelets stimulated by ACLA + Col. The thromboxane B2 level in platelets induced by ACLA + Col was similar to that stimulated by low concentrations of collagen alone. ACLA + Col increased the activities of phospholipase C (PLC) as determined by formation of phosphatidic acid (PA), whereas indomethacin and adenosine 2',5'-diphosphate, an antagonist of the ADP P2Y1 receptor, inhibited PA formation. These results suggest that ACLA, thromboxane A2 derived from the collagen pathway and secreted ADP co-operatively augment PLC activity and lead to platelet aggregation.     

The in vivo mechanism of action of CD20 monoclonal antibodies depends on local tumor burden

Background

CD20 monoclonal antibodies are widely used in clinical practice. Antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity and direct cell death have been suggested to be important effector functions for CD20 antibodies. However, their specific contributions to the in vivo mechanism of action of CD20 immunotherapy have not been well defined.

Design and Methods

Here we studied the in vivo mechanism of action of type I (rituximab and ofatumumab) and type II (HuMab-11B8) CD20 antibodies in a peritoneal, syngeneic, mouse model with EL4-CD20 cells using low and high tumor burden.

Results

Interestingly, we observed striking differences in the in vivo mechanism of action of CD20 antibodies dependent on tumor load. In conditions of low tumor burden, complement was sufficient for tumor killing both for type I and type II CD20 antibodies. In contrast, in conditions of high tumor burden, activating FcγR (specifically FcγRIII), active complement and complement receptor 3 were all essential for tumor killing. Our data suggest that complement-enhanced antibody-dependent cellular cytotoxicity may critically affect tumor killing by CD20 antibodies in vivo. The type II CD20 antibody 11B8, which is a poor inducer of complement activation, was ineffective against high tumor burden.

Conclusions

Tumor burden affects the in vivo mechanism of action of CD20 antibodies. Low tumor load can be eliminated by complement alone, whereas elimination of high tumor load requires multiple effector mechanisms.     

Heterogeneity of platelet responsiveness to anti-CD36 in plasma associated with adverse transfusion reactions

BACKGROUND AND OBJECTIVES: Antibodies to CD36 (anti-CD36) are clinically important. As some platelet immunoglobulins produced by transfusion or pregnancy have been shown to induce platelet activation and to play roles in non-haemolytic transfusion reactions (NHTRs), we investigated the in vitro response of platelets to plasma containing anti-CD36. MATERIALS AND METHODS: Plasma containing anti-CD36, implicated in the development of NHTRs and subsequent thrombocytopenia, was incubated with CD36-positive platelets. Plasma-induced platelet activation was examined by evaluating platelet aggregation and RANTES (regulated on activation, normal, T-cell expressed, and presumably secreted) release. RESULTS: Platelet activation was induced by plasma alone in four out of 20 CD36-positive subjects. In seven subjects, platelet activation was synergistically induced by the combination of epinephrine priming and the plasma. The platelets of the nine remaining subjects failed to respond to the plasma. Platelet activation induced by either the plasma alone or by synergy with epinephrine required the involvement of Fc gamma RIIa. The different responsiveness of the platelets was partially associated with the surface levels of CD36 and Fc gamma RIIa, but not with Fc gamma RIIa polymorphisms. CONCLUSIONS: Plasma containing anti-CD36, implicated in the development of NHTRs, exhibited a platelet-activating capability. Additionally, platelets from healthy human subjects exhibited a considerable degree of heterogeneity in their responsiveness to this plasma. The heterogeneity of these responses may determine the occurrence of anti-CD36-related NHTRs.     

Refractory platelet transfusion in a patient with CD36 deficiency due to pseudothrombocytopenia

Type I CD36 deficiency is defined by the absence of CD36 on both platelets and monocytes. Pseudothrombocytopenia (PTCP) is characterized by a false reduction in the number of platelets in ethylenediaminetetraacetic acid (EDTA)-anticoagulated blood. Here we report a rare case of concomitant CD36 deficiency and PTCP. The patient was a 7-year-old boy who suffered comminuted fractures of the left humeral condyle. In the pre-operative examination, he was found to have thrombopenia and assumed to have idiopathic thrombocytopenic purpura. After immunotherapy and platelet transfusion, the platelet count remained low, suggesting that the patient was refractory to platelet transfusion. Serum was collected for the detection of platelet antibodies, and antibodies against CD36 were found. Flow cytometry verified the absence of CD36 on both the platelets and monocytes of this patient. However, the platelet count was normal when capillary blood smears were analysed; in addition, platelet coagulation was noted under the microscope when EDTA-anticoagulated peripheral blood was used. The patient underwent surgery without platelet transfusion and recovered uneventfully.     

Immunomodulation of transplant rejection using monoclonal antibodies and soluble receptors

The main objective of our studies has been to optimize the effects of monoclonal antibodies (MAbs) and other immunosuppressive reagents to enhance organ graft survival. One such agent is OKT3, a MAb that is directed against the CD3 component of the human T-cell receptor (TCR) complex. Treatment of a rejection episode with OKT3 results in a rapid and efficient clearing of circulating T cells and reversal of most rejection episodes. Its wider use in transplantation and in the treatment of immune-mediated disease is limited by adverse reactions that follow the initial dose, the production of neutralizing Abs, and the transient nature of the immunosuppression. We have engineered CDR-grafted humanized anti-CD3 MAbs that lack Fc-receptor binding activity through mutagenesis of amino acids in the Fc portion of the MAb. This results in an immunosuppressive anti-CD3 MAb that is less antigenic and one that does not induce the first-dose side effects. In addition, we have pursued a goal of developing a therapy that will induce donor-specific tolerance while maintaining overall recipient immune competency. Because antigen-specific T-cell activation depends not only on TCR-ligand interaction, but also on additional costimulatory signals mediated by accessory molecules such as CD28, blocking the binding of CD28 on T cells to its ligand B7, during TCR engagement, might modulate transplantation responses. Using a soluble fusion protein of human CTLA4, CTLA4-Ig, that binds B7 with high affinity, inhibition of human pancreatic islet rejection that occurs, at least in part, by affecting T-cell recognition of human B7⁺ antigen-presenting cells has been demonstrated. In addition, CTLA4-Ig induces long-term, donor-specific unresponsiveness.     

Effects of fibroblast extracellular matrix calcification on platelet adhesion in vitro

Calcified atherosclerotic lesions are more prone to rupture during angioplasty than non-calcified lesions and are associated with an increased risk of thrombotic complications following angioplasty. This study investigates the possible role of extracellular matrix (ECM) calcification for platelet adhesion. Human cultured fibroblasts (CRL-1635) were subjected to β-glycerophosphate (10 mM) for 10 to 16 days. Calcification was visualized by von Kossa staining and quantified by the O-cresolphthalein complexone method. Adhesion of calcein-labelled platelets was measured by fluorescence microscopy at static conditions and in a parallel-flow chamber at a shear rate of 1000 s^?1. β-glycerophosphate treatment resulted in a marked calcification of the ECM. In parallel, a small, albeit significant increase in platelet adhesion under static conditions was observed. In contrast, at flow conditions, the area covered by thrombi was significantly lower when calcified ECM was used. The number of thrombi was not significantly different which is compatible with a smaller thrombus size. Taken together, it appears unlikely that calcification of atherosclerotic lesions contributes to thrombotic complications by an increased platelet adhesion.     

The wonderful story of monoclonal antibodies

Monoclonal antibodies have become daily partners of both biologists and clinicians, as reagents and therapeutic agents. Behind their odd names and incredible diversity lies an amazing story of inventiveness and daring. This review tries to retrace the major steps of this saga, initiated by the search for anti‐rabbit red blood cell antibodies and currently culminating in amazing molecular constructions saving lives. After some historical and basic reminders, the fields of reagents and drugs will be addressed. This invaluable contribution of immunology to the understanding of both physiology and treatment clearly deserves to be fully recognized.