A T-cell-selective interleukin 2 mutein exhibits potent antitumor activity and is well tolerated in vivo.

Human interleukin 2 (IL-2; Proleukin) is an approved therapeutic for advanced-stage metastatic cancer; however, its use is restricted because of severe systemic toxicity. Its function as a central mediator of T-cell activation may contribute to its efficacy for cancer therapy. However, activation of natural killer (NK) cells by therapeutically administered IL-2 may mediate toxicity. Here we have used targeted mutagenesis of human IL-2 to generate a mutein with approximately 3,000-fold in vitro selectivity for T cells over NK cells relative to wild-type IL-2. We compared the variant, termed BAY 50-4798, with human IL-2 (Proleukin) in a therapeutic dosing regimen in chimpanzees, and found that although the T-cell mobilization and activation properties of BAY 50-4798 were comparable to human IL-2, BAY 50-4798 was better tolerated in the chimpanzee. BAY 50-4798 was also shown to inhibit metastasis in a mouse tumor model. These results indicate that BAY 50-4798 may exhibit a greater therapeutic index than IL-2 in humans in the treatment of cancer and AIDS.

Receptor-based design of cytokine therapeutics.

Cytokines hold huge potential for the treatment of disease due to their often fundamental roles in development and homeostasis. However, it is this same primary biological function that can both cause disease through dysregulation as well as prevent their therapeutic use due to systemic consequences arising from this inherent pleiotropy. Molecularly, this can be explained through an understanding of the receptor system specific to each cytokine and the cells on which they are expressed. This knowledge has been exploited to yield muteins (mutated proteins) that exhibit selective, and sometimes novel, biological properties dependent upon receptor subunit usage. In some cases, these muteins have been evaluated in clinical trials and have been approved for clinical use; in most instances, however, these muteins are not suitable for therapeutic application due to intrinsic characteristics of the muteins themselves or the cellular and receptor system to which they are directed. Ultimately, molecular insight to the biological processes governing disease pathology underlies the successful application of mutein-based therapy. The clinical success enjoyed by a subset of these proteins signals the advent of a new mode of therapeutic protein development.

Identification of critical regions in mouse granulocyte-macrophage colony-stimulating factor by scanning-deletion analysis.

Structure-function relationships for mouse granulocyte-macrophage colony-stimulating factor were examined by generating a series of small deletions scanning the entire length of the molecule. Deletions of three amino acids were introduced at intervals of five amino acids by site-directed mutagenesis of the mature mouse granulocyte-macrophage colony-stimulating factor gene. The mutant proteins were expressed in Escherichia coli and assayed for biological activity. This procedure identified four regions critical to activity. These critical regions were further delineated by additional three-amino acid deletion mutants. Larger deletions at each terminus were also made, as well as changes of specific amino acid residues. The four critical regions span amino acid residues 18-22, 34-41, 52-61, and 94-115. The disulfide bridge between Cys-51 and Cys-93 was also shown to be essential for activity, whereas that between Cys-85 and Cys-118 could be removed without loss of activity. The possible structural and/or functional roles of the critical regions are discussed.

High affinity ligand binding is not essential for granulocyte-macrophage colony-stimulating factor receptor activation.

The high affinity receptor of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is a heterodimer composed of two members of the cytokine receptor superfamily. GM-CSF binds to the alpha-subunit (GM-R alpha) with low affinity and to the receptor alpha beta complex (GM-R alpha beta) with high affinity. The GM-CSF.GM-R alpha beta complex is responsible for biological activity. Interactions of the N-terminal helix of mouse GM-CSF with mGM-R alpha beta were examined by introducing single alanine substitutions of hydrophilic residues in this region of mGM-CSF. The consequences of these substitutions were evaluated by receptor binding and biological assays. Although all mutant proteins exhibited near wild-type biological activity, most were defective in high affinity receptor binding. In particular, substitution of Glu-21 with alanine abrogated high affinity binding leaving low affinity binding unaffected. Despite near wild-type biological activity, no detectable binding interaction of this mutant with mGM-R beta in the context of mGM-R alpha beta was observed. Cross-linking studies showed an apparent interaction of this mutant protein with mGM-R alpha beta. The deficient receptor binding characteristics and near wild-type biological activity of this mutant protein demonstrate that mGM-CSF receptor activation can occur independently of high affinity binding, suggesting that conformational changes in the receptor induced by mGM-CSF binding generate an active ligand-receptor complex.

Identification of critical amino acid residues in human and mouse granulocyte-macrophage colony-stimulating factor and their involvement in species specificity.

Segments critical to the activity of human granulocyte-macrophage colony-stimulating factor (GM-CSF) were identified by scanning deletion analysis and compared with the critical regions previously identified in the homologous mouse GM-CSF protein. Three of the four critical regions thus identified are in equivalent positions in their respective polypeptides, while a fourth critical region of each is uniquely located. To investigate whether unique critical regions are responsible for the observed species specificity of human and mouse GM-CSF, all critical regions were substituted into their opposite homologue. This identified one specific, but different, critical region in each homologue that could not be replaced. Further characterization of the nature of the species specificity of these two proteins was accomplished by the generation of a series of human/mouse GM-CSF hybrids. Each hybrid protein was assayed for specific activity on human- and mouse GM-CSF-dependent cell lines. Significant differences in the specific activity of these hybrids was observed, suggesting that different segments of each molecule interact with their respective receptors. Based on these two approaches, individual amino acids were identified that could provide, at least in part, the interactions between these protein ligands and their respective receptors. These residues are Thr-78 and Met-80 in human GM-CSF and Asp-92, Thr-98, and Asp-102 in mouse GM-CSF.

The amino-terminal helix of GM-CSF and IL-5 governs high affinity binding to their receptors.

Transduction of the biological effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-5 (IL-5) requires the interaction of each cytokine with at least two cell surface receptor components, one of which is shared between these two cytokines. A strategy is presented that allowed us to identify receptor binding determinants in GM-CSF and IL-5. Mixed species (human and mouse) receptors were used to locate unique receptor binding domains on a series of human-mouse hybrid GM-CSF and IL-5 cytokines. Results show that the interaction of these two cytokines with the shared subunit of their high affinity receptor complexes is governed by a very small part of their peptide chains. The presence of a few key residues in the amino-terminal alpha-helix of each ligand is sufficient to confer specificity to the interaction. Comparison with other cytokines suggests that the amino-terminal helix of many of these proteins may contain the recognition element for the formation of high affinity binding sites with the alpha subunit of their multi-component receptors.

Cloning and overexpression of the colicin E1 immunity gene.

A DNA fragment containing only the putative immunity gene-coding sequence was cloned under the control of the trp and lambda PL promoters, generating pRKA11 and pIPL, respectively. Escherichia coli hosts containing either construction were immune to colicin E1. Cells harboring both pIPL and pNT204, which encodes a temperature-sensitive cI repressor, were sensitive to colicin E1 at 30 degrees C, but became immune after 0.5 h of incubation at 42 degrees C. In addition, pRKA11 directed the synthesis of a 14.5-kDA protein in maxicells, identical to that found with the wild-type immunity gene. This evidence identifies unequivocally the coding sequence of the immunity gene as that extending from bases 1214 to 1552 [OKA, A., et al., Mol. Gen. Genet. 172, 151-159 (1979)]. The entire immunity gene operon was also cloned under the control of the tac promoter, generating pTCU2, which, upon induction with isopropyl beta-D-thiogalactopyranoside, produced the imm gene product in amounts sufficient to be visualized by autoradiography.

Requirement of hydrophilic amino-terminal residues for granulocyte-macrophage colony-stimulating factor bioactivity and receptor binding.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein required for the proliferation and differentiation of granulocyte and macrophage precursors. Previous investigations have identified regions in human and murine GM-CSF that are required for bioactivity. In the present study, alanine substitution mutagenesis was undertaken to define more precisely specific amino-terminal residues in murine GM-CSF that are involved in bioactivity and receptor binding. Five double alanine mutants were identified that showed at least 10-fold reductions in bioactivity (K14AK20A, K14AE21A, H15AK20A, H15AE21A, K20AE21A). Each of these mutants maintained a normal N-linked glycosylation pattern when expressed in COS-1 cells, suggesting that native polypeptide backbone conformation was preserved. The purified prokaryotic expression products of two mutants (K14AE21A and H15AE21A) had a 100-fold decrease in bioactivity and a decrease in receptor binding, indicating that the side chains of K14, H15, and E21 are required for optimal receptor binding and maximal bioactivity.

Interleukin-4 variant (BAY 36-1677) selectively induces apoptosis in acute lymphoblastic leukemia cells.

Interleukin 4 (IL-4) suppresses the growth of acute lymphoblastic leukemia (ALL) cells, but its clinical usefulness is limited by proinflammatory activity due mainly to the interaction of cytokine with endothelial cells and fibroblasts. Stroma-supported cultures of leukemic lymphoblasts were used to test the antileukemic activity of an IL-4 variant, BAY 36-1677, in which the mutations Arg 121 to Glu and Thr 13 to Asp ensure high affinity for IL-4Ralpha/IL-2Rgamma receptors expressed by lymphoid cells, without activation of the IL-4Ralpha/IL-13Ralpha receptors mainly expressed by other cells. BAY 36-1677 (25 ng/mL) was cytotoxic in 14 of 16 cases of B-lineage ALL; the median reduction in cell recovery after 7 days of culture was 85% (range, 17%-95%) compared to results of parallel cultures not exposed to the cytokine. Twelve of the 14 sensitive cases had t(9;22) or 11q23 abnormalities; 3 were obtained at relapse. BAY 36-1677 induced apoptosis in leukemic lymphoblasts but did not substantially affect the growth of normal CD34+ cells, thus conferring a growth advantage to normal hematopoietic cells over leukemic lymphoblasts in vitro. BAY 36-1677 had antileukemic activity equal or superior to that produced by native IL-4, but it lacked any effects on the growth of endothelial cells and fibroblasts. The molecular manipulation of IL-4 to abrogate its proinflammatory activity has generated a novel and therapeutically promising cytokine for the treatment of high-risk ALL.

An immune cell-selective interleukin 4 agonist.

Interleukin 4 (IL-4) is a pleiotropic cytokine. Of the cell types responsive to IL-4, T cells express one IL-4 receptor (IL-4R) type, IL-4Ralpha/IL-2Rgamma (class I IL-4R), whereas endothelial cells express another type, IL-4Ralpha/IL-13Ralpha (class II IL-4R). It was hypothesized that IL-4 variants could be generated that would be selective for cell types expressing the different IL-4Rs. A series of IL-4 muteins were generated that were substituted in the region of IL-4 implicated in interactions with IL-2Rgamma. These muteins were evaluated in T cell and endothelial cell assays. One of these muteins, containing the mutation Arg-121 to Glu (IL-4/R121E), exhibited complete biological selectivity for T cells, B cells, and monocytes, but showed no activity on endothelial cells. Receptor binding studies indicated that IL-4/R121E retained physical interaction with IL-2Rgamma but not IL-13Ralpha; consistent with this observation, IL-4/R121E was an antagonist of IL-4-induced activity on endothelial cells. IL-4/R121E exhibits a spectrum of activities in vitro that suggest utility in the treatment of certain autoimmune diseases.

Antibodies to murine CD40 stimulate normal B lymphocytes but inhibit proliferation of B lymphoma cells.

A rat anti-mouse CD40 antiserum has been prepared by hyperimmunisation of Lewis rats with a highly purified preparation of the recombinant extracellular domain of murine CD40. This antiserum specifically binds CD40-expressing L cell transfectants, but not untransfected L cells, and induces vigorous proliferation of highly purified small dense B cells obtained from the spleens of unstimulated mice. Anti-CD40-induced B cell proliferation can be augmented by the addition of IL-4 and is inhibited by purified recombinant soluble mouse CD40. Interestingly the same anti-CD40 antiserum specifically inhibits the in vitro growth of A.20 murine B lymphoma cells. The specificity of this inhibition can be demonstrated by reversing the effect with purified recombinant soluble mouse CD40. These data implicate CD40 as a possible target for therapeutic intervention in the treatment of B lymphomas.