Maria Ana Ghetie

Genetic engineering of an immunotoxin to eliminate pulmonary vascular leak in mice

Vascular leak syndrome is a major and often dose-limiting side effect of immunotoxins and cytokines. We postulated that this syndrome is initiated by damage to vascular endothelial cells. Our earlier studies identified a three–amino acid motif that is shared by toxins, ribosome-inactivating proteins, and interleukin-2, all of which cause this problem. We have now generated a panel of recombinant ricin A chains with mutations in this sequence or in amino acids flanking it in the three-dimensional structure. These have been evaluated alone and as immunotoxins for activity, ability to induce pulmonary vascular leak in mice, pharmacokinetics, and activity in tumor-xenografted mice. One mutant was comparable to the ricin A chain used before in all respects except that it did not cause vascular leak at the same dose and, when used as an immunotoxin, was more effective in xenografted SCID mice.

The GLP large scale preparation of immunotoxins containing deglycosylated ricin A chain and a hindered disulfide bond

The large scale preparation of two second generation immunotoxins containing murine monoclonal antibodies and deglycosylated ricin A chain is described. The procedure for the preparation of immunotoxins consists of the derivatization of antibody with SMPT and reduction of dgA with DTT followed by their reaction to establish a hindered interchain disulfide bond. The purification of the immunotoxin includes affinity chromatography on Blue-Sepharose to remove the free antibody and gel filtration on Sephacryl S-200HR to remove any high molecular weight material and free dgA. The two immunotoxins were prepared by GLP procedures and tested for yield, composition, purity, sterility and biological activity.

Recent developments in immunotoxin therapy

Immunotoxin (IT) research has been ongoing for 15 years. During the past 2 years, work has focused on several areas: on improvements and developments in first- and second-generation ITs; the preparation of new immunotoxin constructs with anti-tumor activity; novel animal models for preclinical evaluation of immunotoxins; and clinical trials, which are now entering Phase II or III in humans.

The epitope specificity and tissue reactivity of four murine monoclonal anti-CD22 antibodies☆

The CD22 antigen is expressed on the surface of normal human B cells and some neoplastic B cell lines and tumors. Previous cross-blocking studies using a panel of monoclonal anti-CD22 antibodies have defined four epitope groups, termed A-D. In the present studies, we have further dissected the epitopes recognized by four monoclonal anti-CD22 antibodies using immunoprecipitation and cross-blocking techniques, immunofluorescence analyses with a variety of cell lines, and immunoperoxidase analyses of 36 normal human tissues. Two of the antibodies, HD6 and RFB4, have been described previously, and two, UV22-1 and UV22-2, are described in this report. Our studies indicate that the four monoclonal antibodies show unexpected complexities in their reactivity with CD22+ and CD22- cells and their reactivity with solubilized CD22 molecules. The four antibodies, which recognize epitopes defined previously as CD22-A and CD22-B, further subdivide these epitope clusters into four determinants, A1, A2, B1, and B2. Furthermore, only two of the antibodies, RFB4 and UV22-2, are B cell-specific. In summary, our data indicate that RFB4 and UV22-2 would be the antibodies of choice for constructing immunotoxins to treat B cell tumors.

The Evaluation of Recombinant, Chimeric, Tetravalent Antihuman CD22 Antibodies

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 antibody-dependent 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.

Combination immunotoxin treatment and chemotherapy in SCID mice with advanced, disseminated Daudi lymphoma

We describe the use of an immunotoxin (IT) cocktail (anti‐CD22‐ and anti‐CD19‐ricin A chain) and any 1 of 3 chemotherapeutic drugs (doxorubicin, cytoxan or camptothecin) to treat advanced disseminated Daudi lymphoma in SCID mice (SCID/Daudi). In a previous report, we demonstrated that this regimen was curative when given the day following tumor cell inoculation. Here, we show that combination therapy in mice with advanced tumor significantly increased their survival, although it was not curative. Importantly, the outcome of therapy was dependent upon the temporal order in which IT and chemotherapy were administered. Thus, the best anti‐tumor effect was achieved when an IT cocktail was given before or at the same time as chemotherapy. When the IT was given after chemotherapy, there was no additional therapeutic benefit. Our results confirm the rationale of using combination therapy in the treatment of advanced B‐cell neoplasia and suggest that ITs should be administered prior to or during chemotherapy.

Rituximab but not other anti-CD20 antibodies reverses multidrug resistance in 2 B lymphoma cell lines, blocks the activity of P-glycoprotein (P-gp), and induces P-gp to translocate out of lipid rafts

The objective of this study was to investigate the ability of the anti-CD20 antibody, Rituximab (RTX), to inhibit the activity of P-glycoprotein (P-gp), and reverse multidrug resistance (MDR) in 2 P-gp+/CD20+ lymphoma cell lines. We determined whether RTX would chemosensitize the 2 P-gp+ cell lines in vitro, and inhibit the ability of the cells to efflux Rhodamine 123. One cell line was infected with an MDR1 vector and the other was generated by drug selection. We also determined whether RTX induced P-gp to translocate out of lipid rafts. RTX chemosensitized 2 different MDR cell lines, inhibited the activity of P-gp in both, and induced P-gp to translocate out of lipid rafts in the 1 cell line that was studied in greater detail. In contrast, 3 other anti-CD20 antibodies did not chemosensitize, inhibit the activity of P-gp, or induce it to translocate out of rafts, despite the fact that 1 antibody recognized the same epitope on CD20. Our results suggest that RTX can chemosensitize 2 CD20+/P-gp+ cell lines in vitro by inhibiting the activity of the P-gp pump. The inhibition of P-gp activity correlated with the ability of RTX to induce P-gp to translocate out of lipid rafts. Although the mechanisms by which RTX effects P-gp translocation and activity are not yet known, they are not associated with acid-sphingomyelinase activation in raft microdomains, as described for the antiproliferative activity of RTX.