Philip C. Familletti

Interleukin-12 Antagonist Activity of Mouse Interleukin-12 p40 Homodimer in Vitro and in Vivo

Mo(p40)2 is a potent IL-12 antagonist that interacts strongly with the beta 1 subunit of the IL-12R to block binding of moIL-12 to the high-affinity mouse IL-12R. Mo(p40)2, alone or in synergy with the 2B5 mAb specific for the moIL-12 heterodimer, blocked IL-12-induced responses in vitro, Mo(p40)2 was thus used alone or with 2B5 mAb to examine the role of IL-12 in vivo, Mo(p40)2 caused a dose-dependent inhibition of both the rise in serum IFN-gamma levels in mice injected with endotoxin and the Th1-like response to immunization with KLH. Treatment with mo(p40)2 plus 2B5 anti-moIL-12 mAb also suppressed DTH responses to methylated bovine serum albumin but not specific allogeneic CTL responses in vivo. In each of these models, responses seen in mice treated with mo(p40)2 +/- 2B5 anti-moIL-12 mAb were similar to those observed in IL-12 knockout mice. Thus, mo(p40)2 can act as a potent IL-12 antagonist in vivo, as well as in vitro, and is currently being used to investigate the role of IL-12 in the pathogenesis of some Th1-associated autoimmune disorders in mice.

Characterization of a factor(s) which synergizes with recombinant interleukin 2 in promoting allogeneic human cytolytic T-lymphocyte responses in vitro

Supernatants from PHA-activated human peripheral blood mononuclear cells, depleted of virtually all IL-2 activity by an anti-rIL-2 immunoadsorbent column, contain a factor(s) which synergizes with rIL-2 in facilitating the generation of allogeneic human CTL responses in vitro. This factor, provisionally termed CTL maturation factor (TcMF), did not appear to promote CTL responses in the absence of rIL-2. Furthermore, it acted later than IL-2 in facilitating CTL responses and could not be replaced by recombinant IFN-gamma. In this report we show that rIFN-alpha, rIL-1 alpha, and rIL-1 beta likewise lack TcMF activity. The TcMF activity in lymphokine-containing culture supernatants could be eliminated by trypsin or pronase but not by neuraminidase or RNase. Gel filtration revealed two peaks of TcMF activity, one at 12,000 to 25,000 Da and the other at 45,000 to 65,000 Da. Isoelectrofocusing demonstrated substantial charge heterogeneity. The majority of TcMF activity was recovered between pI 4.0 and pI 5.5 with a minor component at pI 6.5, corresponding to the areas in which IL-1 activity was also found. However, TcMF activity could be separated from IL-1 by reverse-phase HPLC. Moreover, TcMF recovered following reverse-phase HPLC was also found to be depleted of IL-4 activity. These studies suggest that TcMF activity is mediated by a protein(s) distinct from IL-1, IL-2, IL-4, and interferon-alpha or-gamma.

Medium-scale ligand-affinity purification of two soluble forms of human interleukin-2 receptor.

Recombinant technology has facilitated the production of two soluble forms of human interleukin-2 receptor (IL-2R) in Chinese hamster ovary cells. We have developed a ligand-affinity method for the medium-scale purification of these two IL-2Rs, based on the biochemical interactions between the matrix-bound ligand (interleukin-2) and its soluble receptor. The affinity-purified IL-2R is further purified by anion-exchange chromatography followed by gel filtration. This method has provided enough highly pure IL-2R for structure and function studies and for use in practical applications such as high-flux drug screening assays and the receptor-affinity purification of human recombinant interleukin-2.

Purification of recombinant human secretory phospholipase A2 (Group II) produced in long-term immobilized cell culture

Recombinant human secretory phospholipase A2 (Group II) was expressed in long-term culture of immobilized Chinese hamster ovary cells utilizing a continuous-perfusion airlift bioreactor. The bioreactor was continuously perfused with cell-culture medium supplemented with 5% fetal calf serum at an average flow rate of 5 liters/day for 30 days. Recombinant phospholipase A2, at concentrations ranging from 100 to 500 micrograms/liter, was purified to apparent homogeneity by an efficient two-step procedure involving a silica-based cation-exchange resin and hydrophobic interaction chromatography (greater than 65% recovery of phospholipase A2). The purified recombinant protein has an apparent molecular weight of 16 kDa, identical to that of purified human placental or synovial fluid phospholipase A2, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Application of the purified protein onto several different gel filtration columns resulted in elution of the protein at molecular weights corresponding to 3.1-4.7 kDa, suggesting an interaction of the protein with the column resins. However, analytical ultracentrifugation experiments revealed that the protein behaves as a monomer (13.8-14.2 kDa) over a protein concentration range of approximately 10 micrograms/ml to 5 mg/ml. With autoclaved Escherichia coli membranes as substrate, the recombinant protein has catalytic properties (pH optimum, effects of bovine serum albumin, sodium chloride concentration, and requirement for calcium) similar to those of the protein purified from human placenta.

Isolation of proteins from crude mixtures with silica and silica-based adsorbents.

Silica and silica-based adsorbents have been used to isolate specific proteins from crude mixtures. Acid-activated Nugel silica and Nugel SP-Silica were used to extract human immune interferon from the conditioned medium of mixed lymphocyte cultures. Human interleukin-2 was extracted from the conditioned medium of Jurkat cells with LiChroprep RP-8. In each case, batch absorption was accomplished by gentle stirring in a microcarrier vessel. The adsorbate was collected by sedimentation, and either batch-eluted or packed into a column for gradient elution. A high degree of concentration and purification was achieved with a high recovery of biological activity.