IL-2R Subunits Research Articles

Biodistribution of anti-interleukin 2 receptor monoclonal antibodies correlates with their therapeutic efficacy following transplantation

IL-2R-targeted therapy prevents graft rejection in various experimental models and in man. However, the principles of optimal mAb selection remain elusive, as their efficacy in vivo does not always correlate with their characteristics in vitro. ART-18 and OX-39, mouse IgG1 mAbs, define distinct epitopes on the p55 subunit of the rat IL-2R. Treatment of LEW hosts with ART-18 prolongs survival of LBN cardiac allografts up to a month; in contrast, OX-39 never affects acute (8-day) rejection. In this study, we evaluated the biodistribution of 125I-labeled ART-18 and OX-39 administered iv to untreated and heart-grafted rats. ART-18 was cleared from the circulation (half-life time ca. 29 hr) and accumulated in host lymph nodes and spleen to a greater extent than OX-39 ( P < 0.001). In contrast, OX-39 was retained in blood (half-life time ca. 66 hr) and was eventually sequestered in liver, lungs, and kidneys, a pattern comparable to an irrelevant IgG1 (MOPC-21). ART-18 but not OX-39 entered specifically acutely rejecting allografts (allograft:native heart activity ratio = 4.0 and 2.3, respectively, P < 0.01). The distribution of ART-18 was IL-2R epitope but not mAb isotype specific as tissue accumulation of “hot” ART-18 was comparable in recipients conditioned with “cold” ART-18 of IgG1 or IgG2b isotype, but not in those treated with OX-39. Thus: (i) the biodistribution of anti-IL-2R mAbs is not random; the mAb “effective” in combating rejection (ART-18) penetrates preferentially host lymphoid tissues and the graft itself, whereas the biologically “ineffectual” mAb (OX-39) is retained in the circulation for prolonged periods; (ii) the epitope of IL-2R defined by the mAb is critical; a mAb may be “captured” by unrelated cells expressing a common epitope in vivo before reaching the related targets, and/or some epitopes may be more accessible than others for iv administered mAbs.

IL-2 regulation of tyrosine kinase activity is mediated through the p70-75 beta-subunit of the IL-2 receptor.

The stimulation of activated human T lymphocytes with IL-2 results in increased tyrosine kinase activity. IL-2 treatment of Tac+ T cells stimulates the rapid phosphorylation of multiple protein substrates at M of 116, 100, 92, 70 to 75, 60, 56, 55, 33, and 32 kDa. Phosphorylation on tyrosine residues was detected by immunoaffinity purification of protein substrates with Sepharose linked antiphosphotyrosine mAb, 1G2. Although phorbol ester stimulated serine phosphorylation of the IL-2R alpha (p55) subunit recognized by alpha TAC mAb, IL-2 did not stimulate any detectable phosphorylation of IL-2R alpha or associated coimmune precipitated proteins. In fact, the tyrosine phosphorylated proteins did not coprecipitate with alpha Tac antibody and similar phosphoproteins were stimulated by IL-2 in IL-2R alpha- human large granular lymphocytes which express only the 70 to 75 kDa IL-2R beta subunit of the high affinity IL-2R. Anti-Tac mAb could inhibit IL-2-stimulated tyrosine phosphorylation in activated T cells, which express both IL-2R subunits that together form the high affinity receptor complex, but not in large granular lymphocytes expressing only the IL-2R beta subunit. The data suggest that IL-2 stimulation of tyrosine kinase activities requires only the IL-2R beta subunit.

Four epitopes on the rat 55‐kDa subunit of the interleukin 2 receptor as defined by newly developed mouse anti‐rat interleukin 2 receptor monoclonal antibodies

Four new mouse monoclonal antibodies (mAb), ART-38, ART-35, ART-75 and ART-94, directed against the rat interleukin 2 receptor (IL 2R) have been developed. As shown by immunoprecipitation studies they all recognize specifically the 55-kDa subunit of the rat IL 2R. These mAb were compared to three previously characterized mouse mAb directed against the 55-kDa molecule of the rat IL 2R, namely the ART-18, ART-65 and OX-39 mAb. Out of all seven mAb, only ART-18 and OX-39 were found to inhibit the IL 2 binding to activated T cells, while IL 2R inhibited the binding of ART-18 alone. ART-18 was the only mAb found to inhibit the IL 2-dependent proliferation of cells carrying the IL 2R. Scatchard plot analyses showed gross differences in the numbers of mAb-binding sites ranging between 30,000 (ART-65) and 165,000 (ART-75) as well as in their affinities which ranged between 2.5 X 10(-9) M (ART-38) and 8.3 X 10(-10) M (OX-39). Cross inhibition studies revealed that the mAb recognize four different epitopes on the 55-kDa rat IL 2R subunit.