John R. Murphy

Prolongation of cardiac allograft survival in murine recipients treated with a diphtheria toxin-related interleukin-2 fusion protein

A diphtheria toxin-related IL-2 fusion gene has been constructed that encodes a 68KD recombinant toxin in which the diphtheria toxin receptor-binding domain has been replaced with amino acids 2-133 of IL-2. This chimeric IL-2 toxin is cytotoxic for cells expressing the high-affinity IL-2 receptor but not for cells lacking this receptor. The ability of this IL-2 toxin to prolong allograft survival was examined in a murine vascularized, heterotopic heart transplant model in the strain combination B10.BR into C57B1/10. When given at a dose of 1.0 micrograms/day for 10 days, the IL-2 toxin significantly prolonged allograft survival in all recipients. CRM-45, a fragment of diphtheria toxin missing the binding domain, was ineffective, confirming the specificity of the therapy. The results demonstrate that this IL-2 toxin, which targets activated T cells expressing the IL-2 receptor, will prolong allograft survival, offering a new option for immunosuppressive therapy.

Metal stoichiometry and functional studies of the diphtheria toxin repressor

Diphtheria toxin repressor (DtxR) is a transition metal ion-activated repressor in Corynebacterium diphtheriae. DtxR is an iron sensor; metal-bound DtxR represses transcription of genes downstream of the tox operator. Wild-type DtxR [DtxR(wt)] and several mutant forms were overexpressed and purified from Escherichia coli. DtxR was isolated without bound metal. Metal reconstitution gave a binding stoichiometry of 2 per monomer for DtxR(wt) and 1 per monomer for DtxR(H79A) and DtxR(M10A). DNA binding of DtxR(H79A) and DtxR(M10A) indicates that metal site 2 is essential for activity. Metal binding lowers the dimerization Kd of DtxR from low micromolar to 33 nM. Gel electrophoretic mobility-shift assays show that Fe2+ and not Fe3+ activates DtxR for DNA binding. This finding suggests that gene regulation by DtxR may be sensitive not only to iron levels but also to redox state of the iron. Mutations in the tox operator sequence indicate that DtxR dimers binding to DNA may be highly cooperative.

Interleukin-2-diphtheria toxin fusion protein prolongs murine islet cell engraftment.

Allograft rejection is dependent upon complex cell-mediated processes, the primary effectors of which are activated T cells. As the expression of the cell surface protein interleukin 2 receptor is primarily limited to the subset of stimulated T cells, therapeutic agents that target this molecule may provide highly selective immunosuppression. A newly constructed chimeric IL-2 diphtheria toxin fusion protein specifically binds to and poisons activated T cells bearing the high-affinity IL-2R. We describe the in vivo effects of IL-2 toxin in preventing rejection of a crude pancreatic islet preparation transplanted across major and minor histoincompatibility barriers. IL-2 toxin administered once daily as the sole immunosuppressive agent prolongs islet graft survival and decreases the severity of the early mononuclear cell infiltrate into the graft site. Long-term survival of transplanted islets (greater than 100 days) was achieved following a short course (10 days) of more-intensive IL-2 toxin treatment. Thus IL-2 toxin, a highly selective immunosuppressive agent, leads to prolonged islet cell engraftment while sparing the resting or memory subset of the entire T cell repertoire.

Fusion protein toxins based on diphtheria toxin: Selective targeting of growth factor receptors of eukaryotic cells

Publisher Summary The construction of diphtheria toxin-based fusion protein toxins provides a means for the selective targeting and elimination of eukaryotic cells that express on their surface the targeted receptor. When bound to their respective cell surface receptors, the diphtheria toxin-based fusion protein toxins are internalized into the cell by receptor-mediated endocytosis. Productive delivery of the catalytic domain to the target cell cytosol requires passage through an acidic early endosomal compartment. As the lumen of the early endosome becomes acidified through the action of a vATPase, the transmembrane domain of the fusion protein spontaneously denatures and forms a pore, or channel, in the endosomal membrane and facilitates the translocation of the catalytic domain to the cytosol of the target cell. Because passage through an acid compartment is an essential step in the intoxication process, prior knowledge that a given surrogate ligand is internalized in an early endosomal compartment that becomes acidified is important.

A kinetic analysis of the effects of interleukin-2 diphtheria toxin fusion protein upon activated T cells

The interleukin-2 diphtheria toxin-related fusion protein (IL-2 toxin) inhibits protein and DNA synthesis IN rIL-2 (10(-10) M) stimulated T lymphoblasts in a dose-dependent fashion. However, prior to target cell death very low concentrations of rIL-2 and IL-2 toxin synergistically stimulate [3H] thymidine incorporation despite inhibition of [14C] leucine uptake. A sequential analysis of [3H] thymidine incorporation shows that high IL-2 toxin concentration (10(-9)-10(-7) M) stimulates DNA synthesis at 18 hr of culture and inhibits [3H] thymidine uptake after 24 hr, while low concentrations of IL-2 toxin (10(-12)-10(-10) M) exhibits stimulatory effects only after 24 hr of culture. Anti-Tac a monoclonal antibody directed against the p55 chain of the high affinity IL-2 receptor (IL-2R) blocks the stimulatory effects of high-dose IL-2 toxin, thereby proving that these effects are mediated through the IL-2 domain of the fusion protein. At 7 hr following interaction with IL-2R receptor (IL-2R)+ T cells, IL-2 toxin-treated cells evidence augmented transcription of the heat shock protein gene, an effect indistinguishable from those mediated by rIL-2. We conclude that interaction of IL-2 toxin with IL-2R+ T cells initially mimicks the stimulatory effects of IL-2 upon gene transcription and DNA synthesis yet concomitant inhibition of protein synthesis is evident.

DAB389 Interleukin-2 Receptor Binding Domain Mutations CYTOTOXIC PROBES FOR STUDIES OF LIGAND-RECEPTOR INTERACTIONS

Site-directed mutagenesis was used to generate point mutations in the diphtheria toxin-related fusion protein, DAB389 interleukin-2 (IL-2). Thr-439, in the IL-2 receptor binding domain of the fusion toxin, was changed to a Pro residue. The resultant fusion toxin, DAB389 IL-2(T439P), was 300-fold less cytotoxic than wild type DAB389 IL-2, partially as the result of a 100-fold decrease in binding affinity for the high affinity form of the IL-2 receptor. However, DAB389 IL-2(T439P) stimulated DNA synthesis to a greater extent than expected. Studies of intoxication kinetics indicated that the increased stimulation might result from an increased contact time between the mutated IL-2 receptor binding domain and the receptor, perhaps due to a decreased internalization rate. Another mutant, DAB389 IL-2(Q514D), in which a Gln residue at position 514 was changed to an Asp, was 2000-fold less cytotoxic than wild type DAB389 IL-2. This mutant had a 50-fold decrease in binding affinity, did not stimulate DNA synthesis and also had a reduced rate of intoxication. Gln-514 appears to play a role in receptor binding and activation, whereas Thr-439 appears to be involved with receptor binding and signaling internalization of the fusion toxin-receptor complex.

Interleukin 2 Toxin: A Step Toward Selective Immunomodulation

We have used protein engineering and recombinant DNA methodologies to genetically replace the eukaryotic cell receptor binding domain of diphtheria toxin with interleukin 2 (IL-2). The toxin-related T cell growth factor fusion gene has been cloned in Escherichia coli K12. Recombinant strains of E coli produce a 68,086 K hybrid toxin, IL-2 toxin that retains immunologic properties intrinsic to both its diphtheria toxin and IL-2 components. IL-2 toxin has been found to selectively inhibit protein synthesis in both human and murine T cell lines that bear high affinity IL-2 receptors, whereas the hybrid toxin is not active against cells that do not bear this receptor. The cytotoxic action of IL-2 toxin is specifically blocked by free IL-2 and monoclonal antibodies that bind to the p55 (Tac antigen) subunit of the high affinity IL-2 receptor. In addition, IL-2 toxin, like diphtheria toxin itself, must pass through an acidic compartment in order to deliver its adenosine diphosphate ribosyl transferase activity to the cytosol of target T cells. In a murine delayed type hypersensitivity (DTH) model system, we have shown that IL-2 toxin treatment induces a marked immunosuppression.

Interleukin-2 Receptor Directed Immunosuppressive Therapy

An ideal antirejection therapy should, of course, be effective in controlling rejection as well as selectively target only those T-cells that are committed to participate in rejection of the donor graft. Conventional immunosuppressive drugs exact unwanted side effects upon non-lymphoid tissues. The introduction of monoclonal antibodies as pharmacologic tools have been long awaited as therapeutic use of T-cell specific monoclonal antibodies can obviate many side effects on non-lymphoid tissues by providing new opportunities for a more targeted form of immunosuppressive therapy. Nonetheless, the pan-T cell antibodies, used with considerable success in transplantation, react with all T cells, while an ideal therapy would target only those lymphocytes committed to the unwanted immune reaction.

Abstract 1202: Target identification for a new type of ADC

Purpose: To explore the mechanism of action of a potent new type of antibody drug conjugate (ADC) called Extracellular Drug Conjugates or EDCs. Experimental Design: EDCs are a new type of ADC that have similar activity and components, yet EDCs are different in the following ways; 1) EDCs do require internalization, 2) EDCs are not pro-drugs but instead require the antibody and payload to remain permanently linked and 3) EDCs target two proteins in unison, requiring long non-cleavable linkers to span distances between bound antibody to the payload binding site. To define the MOA of the EDCs, high definition phase contrast imaging and protein marker experiments were conducted. In order to verify the targets of a set of EDCs consisting of Na,K-ATPase specific payloads, siRNA knockdown experiments were designed and tested. Results: First, we observed that after 24 hour exposure to each of the EDCs (or the free payload), cells rounded up, partially detached from the substrate, swelled, and lost plasma membrane integrity - morphology resembling necrosis. These observations were consistent with no induction apoptosis or autophagy. Second, our siRNA studies determined that alpha 1 of the Na,K-ATPase was a lethal target. Consistent with this observation on all tested cells lines tested, the EC50 values of each EDC decreased with decrease in alpha 1 expression. Additionally, when the corresponding antibody target expression decreased, overall activity of each EDC went down. Alpha 1 expression was also found to change the effects of the free payload, indicating that the EDCs and the free payload share the same target. Yet unlike the EDCs, payload alone was not affected by the expression of any antibody target used in the study. We also observed that siRNA knockdown of the Na,K-ATPase beta 1 or beta 3 subunits alone did not affect EDC or payload activity, yet a significant decrease in activity could be observed when both subunits were knocked down simultaneously. These results are also consistent with microarray data analysis using siRNAs against all 8 alpha and beta subunits which show that only alpha-1, beta-1, and beta-3 are expressed in the cell types tested. Conclusions: These results show that when payloads targeting the Na,K-ATPase are attached to certain antibodies via long flexible linkers, the resulting EDC activity specifically target the alpha 1 subunit of the Na,K-ATPase, of which we determined to be a lethal target. These results also show that EDC9s activity is dependent on the expression of the corresponding antibody target. In addition, the mechanisms of EDCs are similar to that of the free payload and appear to be necrosis like. These results support the continuing efforts to identify the detailed mechanism of the new ADCs which may lead to identifying patients most likely to respond to EDC based therapies. Citation Format: James R. Prudent, Chad Hall, David J. Marshall, Scott Harried, John Murphy. Target identification for a new type of ADC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1202.

Abstract 2964: An anti-CD20 extracellular antibody-drug conjugate for the treatment of B-cell malignancies

Purpose: CD20 is known to be a therapeutic antibody drug target as it is expressed on the surface of most B-cell neoplasms. Here, we assessed the anti-tumor activity of EDC9, a novel extracellular drug conjugate composed of Rituximab (a well know and FDA approved anti-CD20 monoclonal antibody) conjugated to a steroidal glycoside via a long, flexible and stable linker. Experimental Design: The anti-cancer activity and safety of EDC9 were examined and compared to Rituximab using in vitro and in vivo techniques. Results: We found that EDC9 showed picomolar cytotoxic activity that was independent of effector functions and exhibited cytotoxic activity through a mechanism that was dependent on CD20 expression, as cells not expressing CD20 were resistant and Rituximab alone could compete with EDC9, rendering it inactive. After 72 hours of EDC9 treatment at levels between 100 and 200 picomolar, no cells were determined to be viable by CellTiter-Glo or high definition phase contrast imaging. Importantly, when compared on the basis of toxicity to PBMC, EDC9 was found not to be more toxic than Rituximab and the activity of EDC9 was dependent on specific steroid chemistry. In a tumor xenograft model, EDC9 provided complete long-term remission of diffuse large B-cell human lymphoma line SU-DHL-8 tumors, while Rituximab alone did not. Conclusion: These results support efforts to further evaluate EDC9 (a novel CD20 specific antibody drug conjugate) and may lead to phase I clinical trials in patients with certain B-cell related malignancies. Citation Format: James R. Prudent, Chad A. Hall, David J. Marshall, John Murphy, Scott Harried. An anti-CD20 extracellular antibody-drug conjugate for the treatment of B-cell malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2964.