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Background: While protein therapeutics are invaluable in managing numerous diseases, many require frequent injections to maintain therapeutically effective concentrations, due to their short half-life in circulation. PolyXen™, a platform and patented technology employing biodegradable, non-immunogenic and hydrophilic Polysialic Acids (PSA) for drug delivery, is being utilized to overcome such limitations, thereby potentially enabling the clinical utility of a broad range of protein therapeutics. Here, we report the recent progress on two development candidates, polysialylated deoxyribonuclease I (PSA-DNase) and polysialylated erythropoietin (PSA-EPO). Methods and Results: Chemical polysialylation of DNase I (DNase) using PSA with different chain length at various conjugation sites led to improved stability against proteases and thermal stress, and slightly reduced enzymatic activity. Polysialylation of EPO resulted in retention of protein structure and PSA-EPO remained biologically active. PSA-EPO had a significantly prolonged circulating half-life (e.g. t 1/2 of PSA-EPO = ~400 h in patients after subcutaneous administration, aimed for once monthly administration, vs. t 1/2 of EPO = ~22 h, administered twice or thrice weekly), and retained in vivo efficacy. Conclusion: This approach has been clinically validated in phase I (in healthy volunteers) and II studies of PSA-EPO [for managing anemia in patients with chronic kidney disease (CKD)].

Cells of the human lymphoblast line WI-L2 and its derivative TK-6 were synchronized by centrifugal elutriation and cell-cycle dependent mutation to 6TGR (HPRT) and OUAR (Na+, K+ ATPase) measured. Bromodeoxyuridine induced 6TGR and OUAR mutations within S phase while butylmethyl-sulfonate induced mutation displayed no cell-cycle dependence. The data indicate that centrifugal elutriation is a facile means to obtain a useful degree of synchrony for these cell lines.

Partially purified protein preparations containing megakaryocyte growth factor activity were prepared from human embryonic kidney (HEK) cell conditioned medium using ammonium sulfate precipitation, Cibicron blue affinity chromatography, and wheatgerm lectin affinity chromatography. Treatment of these preparations with neutralizing antibodies directed against erythropoietin (EPO) and interleukin 6 (IL6) resulted in a dramatic reduction in their capacity to stimulate megakaryocyte maturation in vitro. The presence of EPO in these preparations was confirmed by both immunoblotting and use of a mouse spleen erythroid progenitor cell proliferation assay routinely used to quantitate EPO activity in vitro. Northern blot analysis of HEK cell-derived mRNA with IL6 DNA probes revealed the presence of an IL6 transcript with a molecular size of 1.3 kb. Analysis of the HEK cell-derived preparation by ELISA confirmed the presence of immunologically reactive IL6. In addition, it was shown that purified recombinant human EPO and IL6 stimulated megakaryocyte maturation in the in vitro assay used in this study. These data indicate that the activity in HEK cell conditioned medium that stimulates megakaryocyte maturation in vitro is predominantly due to the presence of IL6 and EPO. Immunoneutralization studies of another HEK cell-derived preparation, which was inhibitory in the megakaryocyte maturation assay, demonstrated that it contained transforming growth factor beta (TGFβ), a potent inhibitor of megakaryocyte maturation. Taken together, these studies indicate that HEK cell conditioned medium, which has previously been reported to contain megakaryocyte growth factor activity, is comprised of a complex mixture of growth and differentiation factors, some of which promote and others that inhibit the process of megakaryopoiesis. © 1992 Wiley-Liss, Inc.

Mutants of a diploid human lymphoblast line resistant to 6-thioguanine (6TG) appear 6–16 generations after treatment with any of a diverse group of mutagens: methylnitrosourea (MNU), methylnitrosoguanidine (MNNG), ICR-191 5-bromodeoxyuridine (BUdR). A hypothesis is advanced that expression of the 6-thioguanine-resistant state may require the removal of essentially all pre-existing hypoxanthine—guanine phosphoribosyl transferase (HGPRT) molecules via division, dilution, and protein turnover. Design of protocols for quantitative mutation assays requires attention to this phenomenon.

The lag in phenotype expression of methylnitrosourea(MNU)-induced mutation to 6-thioguanine (6TG) resistance has been studied in a diploid human lymphoblastoid cell line. We find that a considerable period (8-12 days) elapses before new mutants appear in treated cultures; after 2 weeks, however, a stable maximum fraction is attained, as would be expected for a genetic mutation. We present preliminary data linking this phenotypic lag to the slow degradation rate of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and to an apparent requirement for very low (less than 0.2% normal) cellular HGPRT content in order for cells to be resistant to 10 mug 6TG/ml. A series of reconstruction experiments are presented, the results of which support the conclusion that selective pressures in the assay procedure do not bias the quantitative estimates of induced mutant fraction.

Terminal deoxynucleotidyl transferase (TdT, EC 2.7.7.31) is a unique DNA polymerase which catalyzes the polymerization of deoxyribonucleotides on the 3’-hydroxyl ends of preformed oligo- or polydeoxynucleotide initiators, in a template-independent manner (1). Its expression is restricted, in normal animals, to subsets of primitive lymphocytes, and, in disease states, to the blast cells of certain forms of acute leukemia and diffuse lymphoma (2). For immunobiologists TdT has emerged as a useful marker for characterizing subsets of pre-B and pre-T lymphocytes (3–7). For physicians caring for patients with leukemia and lymphoma, neoplastic cell TdT status has turned out to be a useful criterion for patient assignment to therapeutically meaningful categories (8).

The toxic and mutagenic effects of the alkylating agents N-methyl-N-nitrosourethane (MNUT) and ..beta..-propioactone (BPL) were quantitatively measured in human lymphoblasts and Salmonella typhimurium. Forward mutation to 6-thioguanine resistance was measured in the human lymphoblasts, and forward mutation to 8-azaguanine resistance was measured in the bacterial cells after equigenerational (1.5 doubling times) exposures. In both systems, the induced mutant fraction rose linearly as a function of concentration for BPL and was biphasic for MNUT. The responses of the two assay systems to eight alkylating agents were compared. The exposure of the cells to each alkylating agent was calculated as exposure concentration multiplied by the time of exposure, and allowance was made for the decomposition of the alkylating agents during exposure (integral exposure). Human cells were 2.5 to 13 times more sensitive than was S. typhimurium to the alkylating agents methyl methanesulfonate, ethyl methanesulfonate, propyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, methylnitrosourea, and MNUT. S. typhimurium cells were three times more sensitive to butyl methanesulfonate and 25 times more sensitive to BPL than were human cells.

Our primary reasons for using diploid human lymphoblast lines in studies of mutagenesis are that they can be grown in free suspension; they appear to be genetically stable for chromosome number, as well as immortal; and they are readily derived from humans of different genetic backgrounds and possibly different sensitivities to mutagenic agents. Of these properties, the most important in assessing the mutagenicity of a large number of suspect chemicals is the lymphoblasts' ability to grow in suspension culture. This characteristic offers the potential for preprogrammed, automatic handling, which probably could not be achieved with anchorage-dependent cells. In this chapter, we will introduce the basics of lymphoblast husbandry and some protocols (tricks) that we have used to facilitate their use in studying genetic change.