An IgG3-IL2 fusion protein activates complement, binds Fc gamma RI, generates LAK activity and shows enhanced binding to the high affinity IL-2R.
The therapeutic value of Interleukin 2 (IL-2) is limited by its short half life and systemic toxicity. One approach to overcoming these problems is to fuse this protein to an antibody, a protein with a long half life and the ability to target a unique antigen within the body. To examine the biochemical properties of such a molecule a fusion protein was constructed linking the N-terminus of human IL-2 to the C-terminus of IgG3. A similar fusion between IgG1 and IL-2 has previously been shown to bind antigen, generate antibody-dependent cellular cytotoxicity (ADCC) and stimulate T cell proliferation and cytotoxicity. We now extend these studies and show that the fusion protein, termed IgG3-IL2, is appropriately N-glycosylated within the IgG3 CH2 domain, binds the human high affinity Fc receptor (Fc gamma RI) with an affinity slightly lower than that of IgG3, and is able to activate complement via the classical pathway to lyse antigen coated sheep red blood cells (SRBC). When used to stimulate the proliferation of the IL-2 dependent cell line CTLL-2, IgG3-IL2 has a specific activity slightly lower than that of human recombinant IL-2 (hrIL-2). In marked contrast, when comparable unit concentrations, as defined by the standard CTLL-2 proliferation assay, are used to stimulate human peripheral blood lymphocytes (PBL), IgG3-IL2 generates significantly greater lymphokine activated killer (LAK) cell cytotoxicity than does hrIL-2. Competition studies show that IgG3-IL2 binds the intermediate affinity form of the IL-2 receptor (IL-2R), consisting of the beta and gamma subunits, with an affinity slightly less than that of hrIL-2. In contrast, IgG3-IL2 shows a greater affinity than hrIL-2 for the high affinity IL-2R, consisting of alpha, beta and gamma subunits. Our studies show that the IgG3-IL2 fusion protein possesses a combination of the biological properties of IgG3 and IL-2 including antigen binding, complement activation, Fc gamma RI binding, IL-2R binding and stimulation of both proliferation and LAK activity. This combination of activities may allow IgG3-IL2 to target humoral and cell-mediated immune activation to the site of an antigen of interest or target an antigen to IL-2R bearing cells or organs.
Harvill
E T
ET
Department of Microbiology and Molecular Genetics, University of California, Los Angeles 90024-1489, USA.
Morrison
S L
SL
D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003167:Complement Activation / Q000187:drug effects*; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000494:pharmacology*; D007376:Interleukin-2 / Q000235:genetics / Q000494:pharmacology*; D015979:Killer Cells, Lymphokine-Activated / Q000187:drug effects* / Q000276:immunology; D008213:Lymphocyte Activation / Q000187:drug effects; D017452:Receptors, IgG / Q000378:metabolism*; D015375:Receptors, Interleukin-2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*
D007074:Immunoglobulin G; D007376:Interleukin-2; D017452:Receptors, IgG; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins