Recent structural information for complexes of cytokine receptor ectodomains bound to their ligands has significantly expanded our understanding of the macromolecular topology and ligand recognition ...mechanisms used by our three principal shared cytokine signaling receptors-gp130, gamma(c), and beta(c). The gp130 family receptors intricately coordinate three structurally unique cytokine-binding sites on their four-helix bundle cytokine ligands to assemble multimeric signaling complexes. These organizing principles serve as topological blueprints for the entire gp130 family of cytokines. Novel structures of gamma(c) and beta(c) complexes show us new twists, such as the use of a nonstandard sushi-type alpha receptors for IL-2 and IL-15 in assembling quaternary gamma(c) signaling complexes and an antiparallel interlocked dimer in the GM-CSF signaling complex with beta(c). Unlike gp130, which appears to recognize vastly different cytokine surfaces in chemically unique fashions for each ligand, the gamma(c)-dependent cytokines appear to seek out some semblance of a knobs-in-holes shape recognition code in order to engage gamma(c) in related fashions. We discuss the structural similarities and differences between these three shared cytokine receptors, as well as the implications for transmembrane signaling.
Interleukin-4 and Interleukin-13 are cytokines critical to the development of T cell-mediated humoral immune responses, which are associated with allergy and asthma, and exert their actions through ...three different combinations of shared receptors. Here we present the crystal structures of the complete set of type I (IL-4Rα/γ
c/IL-4) and type II (IL-4Rα/IL-13Rα1/IL-4, IL-4Rα/IL-13Rα1/IL-13) ternary signaling complexes. The type I complex reveals a structural basis for γ
c's ability to recognize six different γ
c-cytokines. The two type II complexes utilize an unusual top-mounted Ig-like domain on IL-13Rα1 for a novel mode of cytokine engagement that contributes to a reversal in the IL-4 versus IL-13 ternary complex assembly sequences, which are mediated through substantially different recognition chemistries. We also show that the type II receptor heterodimer signals with different potencies in response to IL-4 versus IL-13 and suggest that the extracellular cytokine-receptor interactions are modulating intracellular membrane-proximal signaling events.
T cell-engaging bispecific antibodies (TCB) are highly potent therapeutics that can recruit and activate cytotoxic T cells to stimulate an antitumor immune response. However, the development of TCBs ...against solid tumors has been limited by significant on-target toxicity to normal tissues. Probody therapeutics have been developed as a novel class of recombinant, protease-activated antibody prodrugs that are "masked" to reduce antigen binding in healthy tissues but can become conditionally unmasked by proteases that are preferentially active in the tumor microenvironment (TME). Here, we describe the preclinical efficacy and safety of CI107, a Probody TCB targeting EGFR and CD3. In vitro, the protease-activated, unmasked CI107 effectively bound EGFR and CD3 expressed on the surface of cells and induced T-cell activation, cytokine release, and cytotoxicity toward tumor cells. In contrast, dually masked CI107 displayed a >500-fold reduction in antigen binding and >15,000-fold reduction in cytotoxic activity. In vivo, CI107 potently induced dose-dependent tumor regression of established colon cancer xenografts in mice engrafted with human peripheral blood mononuclear cells. Furthermore, the MTD of CI107 in cynomolgus monkeys was more than 60-fold higher than that of the unmasked TCB, and much lower levels of toxicity were observed in animals receiving CI107. Therefore, by localizing activity to the TME and thus limiting toxicity to normal tissues, this Probody TCB demonstrates the potential to expand clinical opportunities for TCBs as effective anticancer therapies for solid tumor indications.
A conditionally active EGFR-CD3 T cell-engaging Probody therapeutic expands the safety window of bispecific antibodies while maintaining efficacy in preclinical solid tumor settings.
A 108 amino acid protein was designed and constructed from a reduced alphabet of seven amino acids. The 2.9 A resolution X-ray crystal structure confirms that the protein is a four helix bundle, as ...it was designed to be. Hydrogen/deuterium exchange experiments reveal buried amide protons with protection factors in excess of 1 x 10(6) in the range characteristic of well protected protons in functional folded proteins (10(3)-10(8)) rather than protons in rapid exchange (0-10(2)). The protein is monomeric at 1 mM, the concentration at which the exchange experiments were undertaken, indicating that the exchange factors are due to a unique stable tertiary structure fold, and not due to any higher order quaternary structure. Thermodynamic analysis provides an estimate of the free energy of folding of -9.3 kcal mole-1 at 25 degrees C, consistent with the free energy of folding derived from the protection factors of the most protected protons, indicating that global unfolding is required for exchange of the most protected protons.
An IL-4 antagonist was designed based on structural and biochemical analysis of unbound IL-4 and IL-4 in complex with its high-affinity receptor (IL-4Ralpha). Our design strategy sought to capture a ...protein-protein interaction targeting the high affinity that IL-4 has for IL-4Ralpha. This strategy has impact due to the potential relevance of IL-4R as a drug target in the treatment of asthma. To mimic the IL-4 binding surface, critical side chains for receptor binding were identified, and these side chains were transplanted onto a previously characterized, de novo-designed four-helix protein called designed helical protein 1 (DHP-1). This first-generation design resolved the ambiguity previously described for the connectivity between helices in DHP-1 and resulted in a protein capable of binding to IL-4Ralpha. The second-generation antagonist was based upon further molecular modeling, and it succeeded in binding IL-4Ralpha better than the first-generation. This protein, termed DHP-14-AB, yielded a protein with a cooperative unfolding transition (formula omitted) and an IC50 of 27 microM when in competition with IL-4 whereas DHP-1 had no affinity for IL-4Ralpha. The crystal structure of DHP-14-AB was determined to 1.9-A degrees resolution and was compared with IL-4. This comparison revealed how design strategies targeting protein-protein interactions require high-resolution 3D data and the incorporation of orientation-specific information at the level of side-chains and secondary structure element interactions. PUBLICATION ABSTRACT
An IL-4 antagonist was designed based on structural and biochemical analysis of unbound IL-4 and IL-4 in complex with its high-affinity receptor (IL- 4Ralpha). Our design strategy sought to capture a ...protein-protein interaction targeting the high affinity that IL-4 has for IL-4Ralpha. This strategy has impact due to the potential relevance of IL-4Ralpha as a drug target in the treatment of asthma. To mimic the IL-4 binding surface, critical side chains for receptor binding were identified, and these side chains were transplanted onto a previously characterized, de novo-designed four-helix protein called designed helical protein 1 (DHP-1). This first-generation design resolved the ambiguity previously described for the connectivity between helices in DHP-1 and resulted in a protein capable of binding to IL-4Ralpha. The second-generation antagonist was based upon further molecular modeling, and it succeeded in binding IL- 4Ralpha better than the first-generation. This protein, termed DHP-14-AB, yielded a protein with a cooperative unfolding transition (Formula: see text) and an IC sub(50) of 27 mu M when in competition with IL-4 whereas DHP-1 had no affinity for IL-4Ralpha. The crystal structure of DHP-14-AB was determined to 1.9-Aa resolution and was compared with IL-4. This comparison revealed how design strategies targeting protein-protein interactions require high-resolution 3D data and the incorporation of orientation-specific information at the level of side-chains and secondary structure element interactions.
Three molecules were investigated for their ability to distinguish variations in the microviscosity of the surrounding medium. Julolidinemalononitrile (JMN), p-(N-dimethylaminobenzylidene) ...malononitrile (BMN), and p-(N-dimethylaminocinnamylidene) malononitrile (CMN) were dissolved in media of various micro- and bulk viscosities. The fluorescence intensity of each dissolved probe and the bulk viscosity of each medium were measured. In solutions of low molecular weight substances, where the micro- and bulk viscosities are expected to correspond, the fluorescence behavior of each probe was a function of bulk viscosity and was independent of solution composition. In contrast, in aqueous solutions of methylcellulose, the fluorescence behavior of the probes corresponds to microviscosities significantly lower than the measured bulk viscosities. Thus, the probes are useful in resolving the microviscosity from bulk viscosity of neat liquid and solution systems. The sensitivity of the probes to viscosity is in the order JMN > BMN > CMN. Due to its limited water solubility, JMN is not particularly useful for pharmaceutical systems. CMN is the preferred probe for these applications due to its high fluorescence intensity over a large viscosity range.
The microenviromnent near the surface of dissolving polyethylene glycol 1450 (PEG 1450) samples was probed using microviscosity sensitive fluorescent probes. The microviscosity of 0.5 mm thick layers ...of solution was monitored by exciting the fluorescence of the soluble probe using a laser directed horizontally across the film at various distances above the solid. As the solid PEG dissolved slowly in an unstirred environment, fluorescence data revealed the development of a microviscosity gradient over the dissolving PEG. The microviscosity at each distance above the solid reached a plateau with respect to time which decreased with distance from the surface. Slow stirring homogenized the solution further from the surface, although a microviscous layer did develop close to the surface. These events are described in terms of a finite difference approximation to Fick's second law which takes into account the dependence of diffusivity on PEG concentration.
X-ray crystal structures of binary complexes of dUMP or dCMP with the Lactobacillus casei TS mutant N229D, a dCMP methylase, revealed that there is a steric clash between the 4-NH2 of dCMP and His ...199, a residue which normally H-bonds to the 4-O of dUMP but is not essential for activity. As a result, the cytosine moiety of dCMP is displaced from the active site and the catalytic thiol is moved from the C6 of the substrate about 0.5 Å further than in the wild-type TS−dUMP complex. We reasoned that combining the N229D mutation with mutations at residue 199 which did not impinge on the 4-NH2 of dCMP should correct the displacements and further favor methylation of dCMP. We therefore prepared several TS N229D mutants and characterized their steady state kinetic parameters. TS H199A/N229D showed a 1011 change in specificity for methylation of dCMP versus dUMP. The structures of TS H199A/N229D in complex with dCMP and dUMP confirmed that the position and orientation of bound dCMP closely approaches that of dUMP in wild-type TS, whereas dUMP was displaced from the optimal catalytic binding site.
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