No abstract

Monoclonal antibodies (mAbs) that exert antitumor activity can do so by virtue of their effector function and͞or their ability to signal growth arrest or cell death. In this study, we demonstrate that mAbs which have little or no signaling activity-i.e., anti-CD19, CD20, CD21, CD22 and Her-2-can become potent antitumor agents when they are converted into IgG-IgG homodimers. The homodimers exert antigrowth activity by signaling G 0 ͞G 1 arrest or apoptosis, depending upon which cell surface molecule they bind. This activity is specific and, in the case of the anti-CD19 mAb, did not require an Fc portion. These results offer the possibility that homodimers of other tumor-reactive mAbs which have little antitumor activity as monomers might be potent, antitumor agents.

Purpose: The purpose of this study was to prepare chimeric antihuman CD22 tetravalent monoclonal antibodies (MAbs) with high functional affinity, long persistence in the circulation, increased antitumor activity, and conserved effector function in vitro.Experimental Design: We investigated the association/ dissociation rates of these tetravalent antibodies using CD22؉ Daudi lymphoma cells. We then tested their ability to interact with Fc receptors on a human cell line (U937), to mediate antibody-dependent cellular cytotoxicity with human natural killer cells, to bind human C1q, to inhibit the in vitro growth of CD22 Daudi cells, and to persist in the circulation.Results: The rate of dissociation of the tetravalent MAbs versus the divalent antibody was considerably slower. These tetravalent MAbs inhibited the in vitro proliferation of CD22 Daudi cells at a concentration that was at least 100-fold lower than that of the divalent murine antibody. The tetravalent MAbs containing both the CH2 and CH3 domains and a chimeric recombinant divalent antibody bound similarly to Fc receptor, C1q, and mediate antibodydependent cellular cytotoxicity equally well with human natural killer cells. The persistence in the circulation of chimeric tetravalent MAbs was considerably longer than that of chemical homodimers. Conclusions:The tetravalent anti-CD22 MAbs with intact Fc regions should make effective therapeutic agents for B-cell tumors.

We have previously demonstrated that an anti-CD19 monoclonal antibody (MAb; HD37) inhibits the function of the Pglycoprotein (P-gp) pump in a multidrugresistant (MDR) B-lymphoma cell line, Namalwa/MDR1, and that this effect is not due to the recognition of a cross-reactive epitope on P-gp. In this study, we have used the same cell line to define the mechanisms responsible for the effect of HD37 on the P-gp pump. Using fluorescence resonance energy transfer (FRET), we show that CD19 and P-gp are constitutively associated in cells. In the absence of treatment with anti-CD19, 40% of P-gp molecules expressed by Namalwa/MDR1 cells reside in the low-density lipid (ie, cholesterol-rich) microdomains (lipid rafts). Following treatment of the cells with HD37 and disruption of the interactions between P-gp and CD19, P-gp translocated out of lipid rafts and CD19 translocated into lipid rafts. The effect of chemosensitization on Namalwa/MDR1 cells was specific for CD19; an anti-CD22 MAb had no such effect, although the cells express CD22. These results suggest that anti-CD19 might chemosensitize P-gp ؉ cells by interfering with interactions between CD19 and P-gp, rapidly resulting in the translocation of P-gp into a compartment on the plasma membrane where it is no longer active. IntroductionFollowing chemotherapy, multidrug-resistant (MDR) tumors often emerge; patients with such tumors have a poor prognosis. 1 A variety of agents can prevent MDR but all have side effects. These agents include analogs of drug transporter substrates (verapamil) and inhibitors of adenosine triphosphate (ATP) binding or utilization. 2,3 More recently, monoclonal antibodies (MAbs) against P-glycoprotein (P-gp; MRK16 and UIC2) have been used to inhibit the P-gp transporter. [4][5][6] However, P-gp is expressed on cells in many normal tissues (eg, kidney, brain, gut, and liver), and by targeting MDR tumors, those organs are targeted as well, resulting in potentially undesirable side effects. 7 Therefore, studies designed to find alternative nontoxic strategies to chemosensitize MDR tumors are needed.Our previous studies have shown that an anti-CD19 MAb, HD37, induces cell cycle arrest in human B-lymphoma cell lines in vitro and has significant antitumor activity in severe combined immunodeficient (SCID) mice with human lymphoma xenografts. 8 More recently, we demonstrated that HD37 can chemosensitize a P-gp ϩ lymphoma cell line in vitro and that this effect is due to its ability to inhibit the functional activity of P-gp. 9 P-gp is an ATP-driven pump expressed on many MDR tumors, 2,3 and several studies have suggested that the phosphorylation of P-gp can regulate drug efflux. 10,11 HD37 does not alter ATP levels and does not cross-react with P-gp. 9 P-gp is highly sensitive to its lipid environment and actively mediates cholesterol redistribution in the cell membrane. 12 Forty percent of P-gp resides in the low-density lipid (ie, cholesterolrich) microdomains of the plasma membrane (lipid rafts), 13,14 and this proportion increases when the ce...