Intracellular routing rather than cross-linking or rate of internalization determines the potency of immunotoxins directed against different epitopes of sIgD on murine B cells.

Several variables influence the potency of an immunotoxin (IT) prepared with a monoclonal antibody (mAb) and ricin A chain (IT-A). These include the affinity of the mAb, the nature and density of the target antigen (Ag), the epitope on the target Ag bound by the mAb, the type of cell target, and the rate of endocytosis and route of internalization of the bound IT-A. In a previous report, we demonstrated that anti-delta mAbs directed against epitopes which are putatively more proximal to the plasma membrane make more effective IT-As than those directed against epitopes that are putatively more distal from the plasma membrane. It is known that the latter mAbs cross-link sIgD less effectively than the former. Therefore, in the present study, we determined whether the differential cytotoxicity of IT-As directed against these epitopes is related to their ability to cross-link their specific surface antigen (sIgD). We further determined whether they were internalized at different rates by normal B cells. Our results show that neither cross-linking nor rate of internalization account for the different potencies of anti-Fc vs anti-Fd IT-As. However, when these IT-As were used in the presence of the lysosomotropic agent chloroquine, the less potent IT-A became 100-fold more potent and was as cytotoxic as the effective anti-Fc IT-A. Taken together with the results of other studies, these findings further support the hypothesis that the epitope specificity of a given mAb may be an important factor in determining the intracellular routing of an IT-A after internalization.

CD4 peptide-protein conjugates, but not recombinant human CD4, bind to recombinant gp120 from the human immunodeficiency virus in the presence of serum from AIDS patients.

Sera from human immunodeficiency virus-positive (HIV+; Walter Reed stage 6) individuals inhibit the interaction between recombinant human CD4 and recombinant gp120 from HIV (rCD4 and rgp120, respectively), thereby interfering with the ability of soluble rCD4 to block infection with HIV or rCD4-toxin conjugates to kill HIV-infected cells. In this report we demonstrate that the inhibitory activity of such sera is caused primarily by anti-gp120 antibodies that do not recognize the CD4 interaction site on gp120. To circumvent the problem of inhibition, we have generated a construct containing a peptide of CD4 (residues 41-84) conjugated to ovalbumin (three to five peptides per molecule). This multivalent conjugate binds to rgp120 and binding is not inhibited by antibodies in HIV+ sera.

Evaluation of ricin A chain-containing immunotoxins directed against different epitopes on the delta-chain of cell surface-associated IgD on murine B cells.

Over the past decade, immunotoxins (IT) composed of mAb covalently coupled to toxins or their subunits have been developed for the treatment of malignancies and autoimmune diseases. Despite specific binding to target cells, not every mAb makes a therapeutically potent ricin A chain-containing IT (IT-A). A number of variables influence the potency of a mAb as an IT-A, including the affinity of the mAb, the nature and density of the cell surface Ag, and the type of target cell used. The present report investigates the influence of the epitope specificity of a mAb on the effectiveness of that mAb as an IT-A. Seven mAb directed against different regions of the mouse delta H chain of surface IgD, were conjugated to deglycosylated ricin A chain, and tested for their ability to kill murine B cells. The panel of IT-A had similar A chain activities and similar binding avidities. However, the mAb directed against epitopes in the Fc portion of surface IgD made more effective IT-A than those directed against epitopes in the Fd region. Overall, the anti-Fc-A were approximately 60- to 150-fold more toxic than the anti-Fd-A. Taken together with previous studies, these findings suggest that the epitope on a target Ag recognized by a given mAb is an important variable in determining the potency of a mAb as an IT-A.

Effect of nitroglycerin and aortic occlusion on myocardial blood flow.

To better characterize the cardiac and peripheral effects of nitroglycerin during aortic occlusion, we measured myocardial blood flow in 43 normal and pentobarbital (PB)-depressed dogs (groups I to VII). PB was continuously infused in groups IV to VII to maintain reduced cardiac output and contractility. In groups VI and VII nitroglycerin was administered at 2 micrograms/kg/min. Sequential injections of radioactive microspheres (10 micrometers) and hemodynamic measurements were performed during 2-hour occlusions of the infrarenal aorta. The hearts were divided into endocardium, midmyocardium, and epicardium; total and regional blood flows and the ratio of endocardial to epicardial blood flow (endo/epi) were calculated. The results (mean +/- SEM) were subjected to analysis of variance. Normal dogs that underwent aortic occlusion had predictable increases in peripheral vascular resistance. Coronary vascular resistance fell (P less than 0.05) and endo/epi ratios were maintained above 1. Following PB administration, the myocardial blood flow uniformly fell (1.08 +/- 0.34 to 0.55 +/-0.09 ml/min/gm, P less than 0.001), and the animals not treated with nitroglycerin demonstrated decreased endo/epi ratios (1.11 +/- 0-.07 to 0.83 +/- 0.08, P less than 0.001). Although nitroglycerin did not prevent decreases in total myocardial blood flow, endo/epi ratios were maintained above 1 in treated animals (group VI, 1.04 +/- 0.08; group VII, 1.18 +/- 0.17). Furthermore, the increases in left ventricular end diastolic pressure in the untreated animals were significantly greater than those in animals receiving nitroglycerin (P less than 0.01). Despite severe cardiac depression, nitroglycerin maintained normal transmural distribution favoring the endocardium. Since coronary and peripheral vascular resistances were not altered, this benefit most probably reflects decreased ventricular wall tension secondary to preload reduction.