title: IL-12: monoclonal antibodies specific for the 40-kDa subunit block receptor binding and biologic activity on activated human lymphoblasts.
abstract: IL-12, formerly known as cytotoxic lymphocyte maturation factor, is a cytokine that stimulates proliferation of PHA-activated human peripheral blood lymphoblasts and synergizes with low concentrations of IL-2 in the induction of lymphokine-activated killer cells. IL-12 is a 75-kDa heterodimer composed of disulfide-bonded 40-kDa and 35-kDa subunits. mAb prepared against a partially purified preparation of natural IL-12 have been characterized by 1) immunoprecipitation of 125I-labeled IL-12, 2) immunodepletion of IL-12 bioactivity, 3) Western blotting of IL-12, 4) inhibition of [125I]IL-12 binding to its cellular receptor, and 5) neutralization of IL-12 bioactivity. Twenty antibodies immunoprecipitate 125I-labeled IL-12 and immunodeplete IL-12 bioactivity as assessed in the T cell proliferation and lymphokine-activated killer cell induction assays. Western blot analysis demonstrated that each antibody binds to the 75-kDa heterodimer and to the 40-kDa subunit. An IL-12R-binding assay identified 12 individual antibodies that inhibited the binding of [125I]IL-12 to its cellular receptor. Two inhibitory antibodies, 4A1 and 7B2, were tested in the neutralization assay and found to block IL-12 bioactivity whereas one noninhibitory antibody, 8E3, was shown not to neutralize IL-12 bioactivity. Antibodies 4A1 and 8E3 can simultaneously bind to the 75-kDa heterodimer demonstrating that inhibitory and noninhibitory epitopes are spatially distinct on the 40-kDa protein. The ability of antibodies specific for the 40-kDa subunit of IL-12 to block receptor binding of [125I]IL-12 and to neutralize IL-12 bioactivity suggests that localized determinants on the 40-kDa subunit may be necessary for binding to the IL-12 cellular receptor.
authors.affiliation: Department of Molecular Genetics, Roche Research Center, Hoffmann-La Roche, Inc., Nutley, NJ 07110.
authors.forename: R
authors.initials: R
authors.lastname: Chizzonite
authors.forename: T
authors.initials: T
authors.lastname: Truitt
authors.forename: F J
authors.initials: FJ
authors.lastname: Podlaski
authors.forename: A G
authors.initials: AG
authors.lastname: Wolitzky
authors.forename: P M
authors.initials: PM
authors.lastname: Quinn
authors.forename: P
authors.initials: P
authors.lastname: Nunes
authors.forename: A S
authors.initials: AS
authors.lastname: Stern
authors.forename: M K
authors.initials: MK
authors.lastname: Gately
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D000939:Epitopes / Q000032:analysis; D006133:Growth Substances / Q000276:immunology*; D006801:Humans; D018664:Interleukin-12; D007378:Interleukins / Q000032:analysis / Q000276:immunology* / Q000378:metabolism; D007457:Iodine Radioisotopes; D008213:Lymphocyte Activation; D011233:Precipitin Tests; D051381:Rats; D011917:Rats, Inbred Lew; D011971:Receptors, Immunologic / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D006133:Growth Substances; D007378:Interleukins; D007457:Iodine Radioisotopes; D011971:Receptors, Immunologic; D018664:Interleukin-12