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| TARGA THERAPEUTIC PAYLOADS
Targa Therapeutic Payloads have very important advantages over conventional toxins for improving the therapeutic index of naked antibodies or other targeting moieties. These advantages include:
Targa Therapeutic Payloads: 4 Platform Technologies: Dr. Rosenblum has developed four Platforms of Targa Therapeutic Payloads over the past 17 years. The toxins in each Platform (except for Platforms 1 and 4, which have the same mechanism) have different mechanisms of action and offer advantages unique to specific types of targeting molecules and diseases.
Dr. Rosenblum has developed four molecularly distinct platform technologies of Targa Therapeutic Payloads with three different mechanisms of action. These Payloads offer advantages unique to specific classes of targeting molecules and diseases. Multiple mechanisms of cytotoxic action provide additional protection against drug resistance. Targa is developing its own proprietary drugs using these Therapeutic Payloads. In addition, Targa is interested in discussing the out-licensing of these Targa Therapeutic Payloads and providing key related services to other commercial groups interested in enhancing the potency and efficacy of their proprietary targeting molecules.
Gelonin is an N-glycosidase that cleaves adenine from mammalian ribosomal RNA thereby ablating protein synthesis and resulting in cell death. A recombinant, de-glycosylated version of gelonin (rGel) has been expressed in bacteria and is biologically equi-potent to the natural substance. As with many toxins of this class, a single molecule of rGel delivered to the cytoplasmic compartment is sufficient to irreversibly intoxicate a target cell. By itself, rGel does not bind to or become internalized into cells and thus has very little toxicity unless it is conjugated or fused to a targeting moiety. rGel can be chemically conjugated or genetically fused to many targeting moieties, such as antibodies or growth factors, that become internalized into the cells to which they bind. The original sequence of the gelonin molecule has been modified to include a free cysteine in an exposed external loop so that it can be chemically conjugated to targeting moieties through a site-specific disulfide linkage. This insures that no inactivation of the toxin molecule occurs during conjugation, and that the rGel remains tightly attached to the targeting moiety while it is in the bloodstream. rGel can also be genetically fused at either its N- or C-terminal end to targeting moieties. Like Targa’s other Targa Therapeutic Payloads, gelonin retains full biologic activity when fused in this manner. This gives them a unique flexibility and utility versus other payload technologies. rGel fusion constructs and chemical conjugates are cytotoxic to targeted cells at nanomolar concentrations. Conjugation or fusion of rGel to a targeting moiety increases its cytotoxicity by a factor of 100-1,000. rGel-containing conjugates and fusion constructs have plasma clearance and tissue distribution properties similar to those of the targeting moiety itself, in in vivo animal models. Numerous pre-clinical studies have been conducted with gelonin-based chemical conjugates and fusion constructs, and one product is currently in the clinic. Two human clinical trials of an rGel product that targets human leukemia cells are under way at M. D. Anderson Cancer Center. Human Clinical Trials Results with HuM195/rGel The first Therapeutic Payload-based targeted pharmaceutical is HuM195/rGel. In HuM195/rGel, rGel is chemically conjugated to a recombinant, humanized monoclonal antibody (HuM195) directed to the CD33 antigen that is expressed on the malignant cells in most patients with myelogenous leukemias. The HuM195 antibody was developed by Protein Design Labs (PDLI) and is also called Zamyl™. The naked Hum195 antibody was extensively clinically tested in Phase I, II and III
HuM195/rGel is currently being clinically developed for two indications:
A Phase I clinical trial testing HuM195/rGel for the intravenous treatment of patients with myelogenous leukemias is well-advanced. The trial is being conducted at the M. D. Anderson Cancer Center in Houston. Twenty patients have been treated with increasing doses of HuM195/rGel given four times over two weeks. A maximum tolerated dose has not yet been reached. No adverse events related to gelonin have been observed. Immunogenicity of the complex appears to be low as only 2 of 21 evaluable patients developed detectable Human Anti-Gelonin Antibodies (HAGA). Evidence of biological activity has been noted by the clinical investigator in 11 cases, to date. A Phase I trial of ex vivo purging of peripheral blood stem cells has recently opened at the M. D. Anderson Cancer Center. The goal of this trial is to determine the effectiveness of HuM195/rGel in removing leukemic cells from populations of hematopoietic stem cells to be used for autologous transplantation as part of the treatment for myelogenous leukemia. Residual leukemic cells are known to contaminate many preparations of hematopoietic stem cells and are thought to contribute to relapse of the disease after stem cell transplantation. If this approach works, it could open the door to much more widespread use of autologous stem cell transplantation. Future Directions with HuM195/rGel and/or Alternative Anti-CD33 Compounds The results of the ongoing clinical trials of HuM195/rGel may suggest further development of that compound, if Targa obtains all of the necessary intellectual property rights. Alternatively, these trials may serve as a proof of principle to justify the further develop of lower-cost, second-generation anti-CD33 payloaded compounds that Targa currently has under development.
Targa Designer RIP Payloads (TDRPs)™ Dr. Rosenblum has created and filed patents on a set of "Targa Designer RIP Payloads" that have the same mechanism of action as other Ribosomal Inhibitory Peptides (RIPs) such as gelonin, ricin and others, but have been genetically engineered to improve certain characteristics. For example, wherever possible, potentially antigenic sites not essential to cytotoxic function have been removed. Other plant-derived portions of the molecules, such as nucleic acid binding sites, have been replaced with human homologs. These changes have resulted in smaller, more compact molecules (e.g. ~19 kDa) that are thought to minimize potential antigenicity. Although gelonin has, to date, exhibited only minimal antigenicity in human clinical trials (2/22 patients as of 3/28/2003), Targa is planning to replace it in many future fusions and chemical conjugates with these second-generation Targa Designer RIP Payloads.
Tumor necrosis factor-alpha (TNF-a) is a 17 kDa human protein which exists in solution as a compact trimer. TNF-a is one of the primary cytotoxic proteins of the human immune system and its potent cell-killing effects are mediated through interaction with two cell-surface receptors. Studies in Dr. Rosenblum’s lab and elsewhere have demonstrated convincingly that TNF-a conjugated to a targeting moiety can generate impressive cytotoxic effects both in vitro and in vivo even when the tumor cells are resistant to unconjugated TNF-a itself. In vivo studies in tumor xenograft models also demonstrate specific antibody-mediated cytotoxic effects. As a recombinant version of a naturally-occurring human protein, TNF-a is likely to exhibit low immunogenicity. In contrast to both rGel and virtually all other known toxins used for pharmaceutical targeting, TNF-a does not have to be delivered to the interior of the tumor cell; it just needs to be concentrated by the targeting moiety at the surface of the cell where it can then bind to its cell-surface receptors and trigger its cytotoxic action. TNF-a, like rGel, can either be chemically conjugated or genetically fused to targeting moieties.
To destroy target cells, cytotoxic cells in the human immune system first release perforins to permeabilize the cell membrane of the cell being attacked. The serine protease granzyme B (GrB) is then released and enters the target cell through pores created by perforin. Granzyme B then generates an intense pro-apoptotic signal through direct cleavage of the effector caspases 3, 7 and 9. In addition, granzyme B can work directly on mitochondria causing release of Cytochrome C. Thirdly, Granzyme B cleaves nuclear matrix material. The end result of these three different mechanisms is an intense, irreversible, pro-apoptotic effect that commits the cell to death. Targa uses Granzyme B in a perforin-independent manner to kill cancer cells using these three distinct cytotoxic mechanisms. Dr. Rosenblum has developed a series of unique fusion constructs in which mature human granzyme B has been fused to various targeting moieties. The targeting moieties and their target receptors mediate endocytosis without the need for perforins. Fusion constructs containing GrB are biologically active without release of the molecule from the targeting moiety. Therefore, as in the case of the other Targa Therapeutic Payloads, there is no need for a cleavable linker. Cell-targeted GrB can be identified in the cytoplasm of tumor cells in vitro within 1 hr of the start of exposure. Targeting GrB to tumor cells results in an intense cytotoxic effect within 48 hours after drug exposure. Efficient killing of tumor cells in culture by GrB-containing recombinant constructs is produced by nanomolar concentrations.
Human
Granzyme B PDB id: 1FQ3
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Gelonin
is a small protein of 28 kDa that is extremely toxic when it gets inside
cells, but cannot get into cells in significant concentrations unless
it is coupled to a targeting moiety. This gives gelonin very low systemic
toxicity alone, or when used in conjunction with many targeting moieties.
