A humanized antibody that binds to the interleukin 2 receptor.

The anti-Tac monoclonal antibody is known to bind to the p55 chain of the human interleukin 2 receptor and to inhibit proliferation of T cells by blocking interleukin 2 binding. However, use of anti-Tac as an immunosuppressant drug would be impaired by the human immune response against this murine antibody. We have therefore constructed a "humanized" antibody by combining the complementarity-determining regions (CDRs) of the anti-Tac antibody with human framework and constant regions. The human framework regions were chosen to maximize homology with the anti-Tac antibody sequence. In addition, a computer model of murine anti-Tac was used to identify several amino acids which, while outside the CDRs, are likely to interact with the CDRs or antigen. These mouse amino acids were also retained in the humanized antibody. The humanized anti-Tac antibody has an affinity for p55 of 3 x 10(9) M-1, about 1/3 that of murine anti-Tac.     

Contribution of a p75 interleukin 2 binding peptide to a high-affinity interleukin 2 receptor complex.

There are at least two forms of cellular receptors for interleukin 2 (IL-2); one with a very high affinity and the other with a lower affinity. We identified a non-Tac IL-2 binding peptide with a relative molecular weight of 75,000 (p75). Cell lines bearing either the p55 Tac or the p75 peptide alone manifested low-affinity IL-2 binding, whereas a cell line bearing both peptides manifested both high- and low-affinity receptors. After the internalization of labeled IL-2 through high-affinity receptors, the p75 peptide could not be detected by cross-linking studies. Furthermore, fusion of cell membranes from low-affinity IL-2 binding cell lines bearing the Tac peptide alone with membranes from a cell line bearing the p75 peptide alone generated hybrid membranes bearing high-affinity receptors. These results suggest a multichain model for the high-affinity IL-2 receptor in which high-affinity receptors would be expressed when both Tac and p75 IL-2 binding peptides are present and associated in a receptor complex.     

Association of intercellular adhesion molecule 1 with the multichain high-affinity interleukin 2 receptor.

Previously, using flow cytometric resonance energy transfer and lateral diffusion measurements, we demonstrated that a 95-kDa protein identified by two monoclonal antibodies (OKT27 and OKT27b) interacts physically with the 55-kDa alpha protein of the high-affinity interleukin 2 (IL-2) receptor. In the present study, this 95-kDa protein (p95) was purified and amino acid sequence data were obtained that showed strong homology to the human intercellular adhesion molecule 1 (ICAM-1). The identity of the p95 protein with ICAM-1 was confirmed by sequential immunoprecipitations using OKT27 and an antibody, WEHI-CAM-1, that is directed toward ICAM-1. We confirmed the physical proximity of p95/ICAM-1 to the IL-2 receptor alpha subunit by demonstrating that radiolabeled IL-2 could be cross-linked to this protein expressed on activated T cells. In functional studies, the antibodies OKT27 and OKT27b inhibited T-cell proliferative responses to OKT3, to soluble antigen, and to heterologous cells (mixed lymphocyte reaction). However, these antibodies did not inhibit IL-2-induced proliferation of an IL-2-dependent T-cell line. Taken together with our previous observations, the present studies suggest that ICAM-1 is in proximity and interacts physically with the high-affinity IL-2 receptor. The association of ICAM-1 with the IL-2 receptor may facilitate the paracrine IL-2-mediated stimulation of T cells expressing IL-2 receptors by augmenting homotypic T-T-cell interaction, by receptor-directed focusing of IL-2 release by helper T cells, and by focusing IL-2 receptors of the physically linked cells to the site of lymphocyte function-associated antigen 1-ICAM-1-IL-2 receptor interaction.     

The multichain interleukin-2 receptor: a target for immunotherapy.

Activation of resting T-lymphocytes induces synthesis of interleukin-2 (IL-2) and expression of cell surface receptors for this lymphokine. In contrast to resting normal T-cells that do not express high-affinity IL-2 receptors (IL-2R), abnormal T-cells of patients with leukemia-lymphoma, certain autoimmune disorders, and individuals rejecting allografts express this receptor. Exploiting this difference in receptor expression, antibodies to the IL-2 receptor have been used effectively to treat patients with leukemia and lymphoma. One approach is to use monoclonal antibodies produced in mice; the disadvantage is that they are highly immunogenic. In an effort to reduce the immunogenicity of the mouse monoclonal antibodies, monoclonal-antibody-mediated therapy has been revolutionized by generating humanized antibodies produced by genetic engineering in which the molecule is human except for the antigen-combining regions, which are retained from the mouse. Further, to increase its cytotoxic effectiveness, the monoclonal antibody has been armed with toxins or radionuclides. Alternatively, IL-2 itself has been linked to a toxin to kill IL-2 receptor-bearing cells. Thus, IL-2 receptor-directed therapy provides a new method for treating certain neoplastic diseases and autoimmune disorders and for preventing allograft rejection.     

Interleukin 2 receptor beta chain expressed in an oligodendroglioma line binds interleukin 2 and delivers growth signal.

Interleukin 2 (IL-2) is a potent growth factor for T lymphocytes, playing a crucial role in the immune response. In view of the considerable evidence that the immunoregulatory cytokines (or lymphokines) also play a role in the growth and differentiation of cells in the central nervous system (CNS), we examined the operation of the IL-2 system in a cell line of CNS origin by expressing a cDNA encoding the beta chain of the human IL-2 receptor (IL-2R beta, a 75-kDa protein). When the cDNA was expressed in a human oligodendroglioma cell line, ONS-21, the IL-2R beta bound IL-2 with an affinity similar to that in lymphoid cells (Kd, approximately 2 nM). Furthermore, cell proliferation ([3H]thymidine incorporation) was stimulated by IL-2. These results demonstrate that the same cytokine receptor is functional in cells of the immune system and CNS and point to a molecular mechanism that is similar for growth-signal transduction between lymphoid and neural cells but that may be different in other cells, such as fibroblasts.     

Grb-IR: a SH2-domain-containing protein that binds to the insulin receptor and inhibits its function.

To identify potential signaling molecules involved in mediating insulin-induced biological responses, a yeast two-hybrid screen was performed with the cytoplasmic domain of the human insulin receptor (IR) as bait to trap high-affinity interacting proteins encoded by human liver or HeLa cDNA libraries. A SH2-domain-containing protein was identified that binds with high affinity in vitro to the autophosphorylated IR. The mRNA for this protein was found by Northern blot analyses to be highest in skeletal muscle and was also detected in fat by PCR. To study the role of this protein in insulin signaling, a full-length cDNA encoding this protein (called Grb-IR) was isolated and stably expressed in Chinese hamster ovary cells overexpressing the human IR. Insulin treatment of these cells resulted in the in situ formation of a complex of the IR and the 60-kDa Grb-IR. Although almost 75% of the Grb-IR protein was bound to the IR, it was only weakly tyrosine-phosphorylated. The formation of this complex appeared to inhibit the insulin-induced increase in tyrosine phosphorylation of two endogenous substrates, a 60-kDa GTPase-activating-protein-associated protein and, to a lesser extent, IR substrate 1. The subsequent association of this latter protein with phosphatidylinositol 3-kinase also appeared to be inhibited. These findings raise the possibility that Grb-IR is a SH2-domain-containing protein that directly complexes with the IR and serves to inhibit signaling or redirect the IR signaling pathway.     

Cloning and characterization of a binding subunit of the interleukin 13 receptor that is also a component of the interleukin 4 receptor.

Interleukins 4 (IL-4) and 13 (IL-13) have been found previously to share receptor components on some cells, as revealed by receptor cross-competition studies. In the present study, the cloning is described of murine NR4, a previously unrecognized receptor identified on the basis of sequence similarity with members of the hemopoietin receptor family. mRNA encoding NR4 was found in a wide range of murine cells and tissues. By using transient expression in COS-7 cells, NR4 was found to encode the IL-13 receptor alpha chain, a low-affinity receptor capable of binding IL-13 but not IL-4 or interleukins 2, -7, -9, or -15. Stable expression of the IL-13 receptor alpha chain (NR4) in CTLL-2 cells resulted in the generation of high-affinity IL-13 receptors capable of transducing a proliferative signal in response to IL-13 and, moreover, led to competitive cross-reactivity in the binding of IL-4 and IL-13. These results suggest that the IL-13 receptor alpha chain (NR4) is the primary binding subunit of the IL-13 receptor and may also be a component of IL-4 receptors.     

Building a multichain receptor: synthesis, degradation, and assembly of the T-cell antigen receptor.

The murine T-cell antigen receptor consists of at least seven chains and six different proteins. The two clonotypic chains alpha and beta are glycoproteins of 40-45 kDa present as a disulfide-linked heterodimer. Four clonally invariant chains include delta (a 26-kDa glycoprotein), gamma (a 21-kDa glycoprotein), epsilon (a 25-kDa protein), and zeta (a 16-kDa protein). zeta is found in the complex as a disulfide-linked homodimer. The clonotypic chains and the invariant chains form a noncovalent complex on the cell surface. We have developed antibodies against each of the chains and used them to examine the assembly of the mature complex in the murine antigen-specific T-cell hybridoma 2B4. Pulse-chase studies of metabolically labeled cells demonstrate that many of the chains are synthesized in great excess over the amount assembled into the mature complex. These excess chains, either as free components or as partially assembled complexes, are rapidly degraded. This degradation most likely takes place in the lysosomes. The complete complex is quite stable with a long half-life. A specific hierarchy of partially assembled complexes can be discerned.     

Characterization of a growth factor that binds exclusively to the erbB-2 receptor and induces cellular responses.

The erbB-2 oncogene encodes a 185-kDa transmembrane protein that has been suggested to be a growth factor receptor. We have previously identified and purified a 30-kDa growth factor (gp30) that is a ligand for the p185erbB-2 protein that at high concentrations induces growth inhibition of cells with erbB-2 amplification. We now report the purification and characterization of a protein from SKBr-3 human breast cancer cells with a molecular mass of 75 kDa (p75) that is a p185erbB-2 ligand. An affinity column coupled to the extracellular domain of p185erbB-2 was used for the purification. We found that p75 induced tyrosine phosphorylation of the erbB-2 oncoprotein, as determined by in vivo and in vitro phosphorylation and phosphoamino acid analysis. p75, as well as gp30, stimulated cell proliferation and colony formation of cells overexpressing erbB-2. The specificity of this effect was confirmed by showing that the antiproliferative effects of soluble erbB-2 extracellular domain were reversed by either p75 or gp30. p75 did not show binding to the epidermal growth factor receptor and had no growth effects on cells overexpressing epidermal growth factor receptor. These data show that SKBR-3 cells, which exhibit erbB-2 amplification and overexpression, secrete a growth factor that binds and activates p185erbB-2 specifically.     

A potent dimeric peptide antagonist of interleukin-5 that binds two interleukin-5 receptor alpha chains

Two series of peptides that specifically bind to the extracellular domain of the alpha chain of the human interleukin-5 receptor (IL-5Ralpha), but share no primary sequence homology to IL-5, were identified from libraries of random recombinant peptides. Affinity maturation procedures generated a 19-aa peptide that binds to the IL-5 receptor alpha/beta heterodimer complex with an affinity equal to that of IL-5 and is a potent and specific antagonist of IL-5 activity in a human eosinophil adhesion assay. The active form of the peptide is a disulfide-crosslinked dimer that forms spontaneously in solution. Gel filtration analysis, receptor-binding studies, and analytical ultracentrifugation reveal that the dimeric peptide binds simultaneously to two receptor alpha chains in solution. Furthermore, the dimer peptide, but not IL-5, can activate a chimeric receptor consisting of the IL-5Ralpha extracellular domain fused to the intracellular domain of the epidermal growth factor receptor, thus demonstrating that the peptide also promotes receptor dimerization in a cellular context. The functional antagonism produced by the bivalent interaction of the dimeric peptide with two IL-5R alpha chains represents a distinctive mechanism for the antagonism of cytokines that use heteromeric receptors.     

T lymphocyte activation in euthyroid Graves' ophthalmopathy: soluble interleukin 2 receptor release, cellular interleukin 2 receptor expression and interleukin 2 production

The present study was undertaken to examine the cellular control arm of the immune response with regard to T lymphocyte proliferation in euthyroid Graves' ophthalmopathy. Twenty patients with euthyroid Graves' ophthalmopathy (7 on antithyroid drugs and 13 on no treatment) and 18 healthy controls were studied in an infection-free period. Mitogen-stimulated cellular interleukin 2 (IL2) receptor expression, soluble interleukin 2 receptor release, and interleukin 2 production, were studied in peripheral blood mononuclear cells cultured for 24 h. The cellular IL2 receptor expression and soluble IL2 receptor release did not differ between the patients and healthy controls. In contrast, IL2 production in response to pokeweed mitogen stimulation was increased in lymphocytes from patients with Graves' ophthalmopathy. The IL2 release did not correlate with the quantities of cellular and soluble IL2 receptor. The mitogen-stimulated cellular IL2 receptor expression, IL2 receptor release, and IL2 production did not differ between patients with or without carbimazole therapy. Despite a suggested role of autoreactive T cells in mediating the development and propagation of autoimmune thyroid disease, this study fails to demonstrate a defective T lymphocyte activation state in patients with Graves' ophthalmopathy during an euthyroid state.     

Activation of erythropoietin receptor in the absence of hormone by a peptide that binds to a domain different from the hormone binding site

Applying a homology search method previously described, we identified a sequence in the extracellular dimerization site of the erythropoietin receptor, distant from the hormone binding site. A peptide identical to that sequence was synthesized. Remarkably, it activated receptor signaling in the absence of erythropoietin. Neither the peptide nor the hormone altered the affinity of the other for the receptor; thus, the peptide does not bind to the hormone binding site. The combined activation of signal transduction by hormone and peptide was strongly synergistic. In mice, the peptide acted like the hormone, protecting against the decrease in hematocrit caused by carboplatin.     

Identification of a direct interaction between interleukin 2 and the p64 interleukin 2 receptor gamma chain.

The interleukin 2 receptor (IL-2R) consists of at least two subunits, alpha and beta, both of which can bind interleukin 2 (IL-2). Recent studies have demonstrated the existence of a third subunit, a 64-kDa molecule termed IL-2R gamma chain, and have suggested that gamma chain functions to regulate the rate of IL-2 dissociation from the receptor. In the present report we have addressed whether the gamma chain modulates IL-2R affinity by contributing contact sites for IL-2 binding. Using reagents that allow the IL-2R complex to be immunoprecipitated through the IL-2 molecule itself, we demonstrate the existence of a stable IL-2-IL-2R gamma-chain complex. These studies thus establish that the IL-2R gamma chain directly contributes to the IL-2-binding site, consistent with the hypothesis that gamma chain influences IL-2R affinity through its direct interaction with IL-2.     

Identification of specific residues of human interleukin 2 that affect binding to the 70-kDa subunit (p70) of the interleukin 2 receptor.

Analogs of interleukin 2 containing defined amino acid substitutions and deletions were assayed for bioactivity and for competitive binding to the high-affinity human interleukin 2 receptor complex and its two component subunits, a 55-kDa subunit (p55 or TAC) and a 70-kDa subunit (p70). Substitution of Asp20 or deletion of Phe124 resulted in inactive analog proteins that were unable to interact with the high-affinity p55/p70 complex or the intermediate-affinity p70 subunit of the interleukin 2 receptor. These analogs, however, retained the capacity to compete for binding to the low-affinity p55 subunit. The presence of the carboxylic acid in the side chain of Asp20 was necessary for effective binding to the p70 protein. In contrast, substitution of Trp121 and Leu17 created analogs that were inactive in the bioassay and all three binding assays. The effects of these mutations on protein conformation were assessed by circular dichroism. These results demonstrate that specific residues in the NH2 and COOH termini of interleukin 2 are crucial for its structure and activity.