Targeted delivery of cytotoxic proteins to prostate cancer via conjugation to small molecule urea-based PSMA inhibitors.

Prostate cancer cells are characterized by a remarkably low proliferative rate and the production of high levels of prostate-specific proteases. Protein-based toxins are attractive candidates for prostate cancer therapy because they kill cells via proliferation-independent mechanisms. However, the non-specific cytotoxicity of these potent cytotoxins must be redirected to avoid toxicity to normal tissues. Prostate-Specific Membrane Antigen (PSMA) is membrane-bound carboxypeptidase that is highly expressed by prostate cancer cells. Potent dipeptide PSMA inhibitors have been developed that can selectively deliver and concentrate imaging agents within prostate cancer cells based on continuous PSMA internalization and endosomal cycling. On this basis, we conjugated a PSMA inhibitor to the apoptosis-inducing human protease Granzyme B and the potent Pseudomonas exotoxin protein toxin fragment, PE35. We assessed selective PSMA binding and entrance into tumor cell to induce cell death. We demonstrated these agents selectively bound to PSMA and became internalized. PSMA-targeted PE35 toxin was selectively toxic to PSMA producing cells in vitro. Intratumoral and intravenous administration of this toxin produced marked tumor killing of PSMA-producing xenografts with minimal host toxicity. These studies demonstrate that urea-based PSMA inhibitors represent a simpler, less expensive alternative to antibodies as a means to deliver cytotoxic proteins to prostate cancer cells.

Membrane traffic in animal cells: cellular glycoproteins return to the site of Golgi mannosidase I.

The recycling of cellular glycoproteins to the site of Golgi mannosidase I, an enzyme of asparagine-linked oligosaccharide synthesis, was studied in K562 human erythroleukemia cells. Cells were metabolically labeled in the presence of deoxymannojirimycin, a reversible inhibitor of Golgi mannosidase I. This generates glycoproteins with immature oligosaccharides in their normal locations. Transport to the mannosidase I compartment was then assessed by testing for the conversion of oligosaccharides into mature forms during reculture without deoxymannojirimycin. Transferrin receptor (TfR) was acted on by mannosidase I during reculture, suggesting that it returned to the region of the Golgi complex where this enzyme resides. The slow rate of this transport (t1/2 greater than 6 h) implies that it is probably different than TfR movement during transferrin internalization (t1/2 = 10-20 min) and TfR transport to the sialyltransferase compartment in the Golgi complex (t1/2 = 2-3 h) (Snider, M. D., and O. C. Rogers, 1985, J. Cell Biol., 100:826-834). The total cell glycoprotein pool was also transported to the mannosidase I compartment with a half-time of 4 h. Because this transport is 5-10 times faster than the rate of de novo glycoprotein synthesis in these cells, it is likely that most of the glycoprotein traffic through the Golgi complex is composed of recycling molecules.

Intracellular movement of cell surface receptors after endocytosis: resialylation of asialo-transferrin receptor in human erythroleukemia cells.

The intracellular movement of cell surface transferrin receptor (TfR) after internalization was studied in K562 cultured human erythroleukemia cells. The sialic acid residues of the TfR glycoprotein were used to monitor transport to the Golgi complex, the site of sialyltransferases. Surface-labeled cells were treated with neuraminidase, and readdition of sialic acid residues, monitored by isoelectric focusing of immunoprecipitated TfR, was used to assess the movement of receptor to sialyltransferase-containing compartments. Asialo-TfR was resialylated by the cells with a half-time of 2-3 h. Resialylation occurred in an intracellular organelle, since it was inhibited by treatments that allow internalization of surface components but block transfer out of the endosomal compartment. Moreover, roughly half of the resialylated molecules were cleaved when cells were retreated with neuraminidase after culturing, indicating that this fraction of the molecules had returned to the cell surface. These results suggest that TfR is transported from the cell surface to the Golgi complex, the intracellular site of sialyltransferases, and then returns to the cell surface. This pathway, which has not been previously described for a cell surface receptor, may be different from the route followed by TfR in iron uptake, since reported rates of transferrin uptake and release are significantly more rapid than the resialylation of asialo-TfR.

Transmembrane movement of oligosaccharide-lipids during glycoprotein synthesis.

The transport of sugar residues into the endoplasmic reticulum (ER) during glycoprotein synthesis was studied by examining the transmembrane orientations of the oligosaccharide-lipid precursors of asparagine-linked oligosaccharides. Using the lectin concanavalin A, the lipid-linked oligosaccharides Man3-5GlcNAc2 were found on the cytoplasmic side of ER-derived vesicles in vitro while lipid-linked Man6-9GlcNAc2 and Glc1-3Man9GlcNAc2 were found facing the lumen. These results suggest that Man5GlcNAc2-lipid is synthesized on the cytoplasmic side of the ER membrane and then translocated to the luminal side. Glc3Man9GlcNAc2-lipid is then completed on the luminal side where it serves as the donor in peptide glycosylation. Translocation of Man5GlcNAc2-lipid offers a mechanism for the export of sugar residues from the cytoplasm during glycoprotein synthesis. This translocation may be the reason for the participation of lipid-linked mono- and oligosaccharides in glycoprotein synthesis.

Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2.

The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N-terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.

Roles of erythropoietin, insulin-like growth factor 1, and unidentified serum factors in promoting maturation of purified murine erythroid colony-forming units.

We have used 75% to 90% pure murine erythroid colony-forming units (CFU-E) to delineate the processes and factors underlying their maturation. These CFU-E form 32 cell colonies and are drawn from what we term generation I of a six-generation long maturation sequence (Landschulz et al, Blood 79:2749, 1992). Applying assays of 59Fe-heme biosynthesis and colony numbers as measures of maturation and analyses of DNA degradation as an index of programmed cell death, we find that (1) erythropoietin (Epo) enhances maturation throughout most of its course; (2) Epo first seems able to forestall DNA degradation when CFU-E reach generation II; (3) the processes that Epo elicits thereafter start to persist when Epo is withdrawn; (4) insulin-like growth factor I (IGF-I) also forestalls DNA breakdown, but later loses effectiveness; (5) IGF-I adds little to maturation when Epo levels are high, but when Epo levels are low, enhances it substantially; and (6) for maturation to be entirely optimal, an unidentified serum factor(s) is probably required when Epo levels are high and is certainly needed when Epo levels are like those in normal animals. Quantitatively, about 40% of optimal in vitro erythropoiesis at normal Epo levels depends on Epo alone, another 30% or less on the addition of IGF-I, and the remaining 30% or more on the addition of unidentified serum factor(s). Applied together, these three or more factors lead to two-thirds of the maximum maturation realized with saturating Epo levels. Because we also find that heme accumulated in CFU-E culture can closely approach levels in red blood cells, we suppose that our conclusions apply as well to CFU-E maturation in vivo.

Onset of erythropoietin response in murine erythroid colony-forming units: assignment to early S-phase in a specific cell generation.

Murine erythroid colony-forming units (CFU-E) representing successive cell generations in a six-generation long in vitro maturation sequence were tested for their response to erythropoietin (Epo) by measurement of Epo-exposure times necessary to stimulate heme biosynthesis. Generation I CFU-E, which produce mainly 32-cell erythroid colonies, were isolated in 82% average purity from spleens of thiamphenicol-treated anemic animals via differential centrifugation. Generation II CFU-E, which produce mainly 16-cell colonies, were similarly isolated in 51% average purity. Although both types of CFU-E had equivalent dose sensitivity to and affinity for Epo, generation II CFU-E responded to shorter pulses of Epo than did generation I. Correlations between DNA cell-cycle profiles and 59Fe-heme biosynthesis resulting from pulsed exposures established that appreciable Epo response only begins when CFU-E attain early S-phase of generation II. Because CFU-E did not require Epo or other serum factors to pass from generation I to II and because the onset of Epo responsiveness coincided with the beginning of DNA replication in generation II, we suppose that differentiation has reprogrammed one or more of the events associated with generation II S-phase in CFU-E and that these alterations allow Epo to act. Further comparisons between CFU-E from generation I and II may allow us to identify the alterations in question and the nature of their interaction with Epo.

Construction of a human platelet alloantigen-1a epitope(s) within murine glycoprotein IIIa: identification of residues critical to the conformation of the antibody binding site(s).

The human platelet alloantigen 1 system (HPA-1) is determined by a polymorphism at position 33 in the N-terminus of human glycoprotein IIIa (GPIIIa). This naturally occurring substitution creates a conformation in the HPA-1a allelic form that can be antigenic when presented to an individual expressing the HPA-1b form. Anti-HPA-1a antibodies generated by this immune response can lead to the destruction of platelets, as seen in the clinical disorders, neonatal alloimmune thrombocytopenia (NAIT) and posttransfusion purpura (PTP). To understand better the structural requirements for recognition by these pathogenic antibodies, we investigated the N-terminal 66 amino acids from the HPA-1a form of human GPIIIa and the analogous amino acids from the nonimmunogenic murine homolog. Our objectives were to define further the boundaries of the HPA-1a epitope(s) in the N-terminus of human GPIIIa, to isolate the murine 5' nucleotide sequence and compare the deduced murine N-terminal sequence to that of human, and to mutate the murine sequence systematically to include an HPA-1a epitope(s). Murine amino acids that differed from human were changed by site-directed mutagenesis to the analogous residues in the HPA-1a form of human GPIIIa, starting and radiating from murine position 33 (site of human polymorphism). This systematic approach allowed us to pinpoint amino acids critical to a conformation recognized by anti-HPA-1a antibodies. Our results show that an HPA-1a epitope can be created within the N-terminus of murine GPIIIa and raise the possibility that murine models of HPA-1a sensitization can be developed.