Crystal Structure of Human TWEAK in Complex with the Fab Fragment of a Neutralizing Antibody Reveals Insights into Receptor Binding.

The tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine playing a key role in tissue regeneration and remodeling. Dysregulation of TWEAK signaling is involved in various pathological processes like autoimmune diseases and cancer. The unique interaction with its cognate receptor Fn14 makes both ligand and receptor promising targets for novel therapeutics. To gain insights into this important signaling pathway, we determined the structure of soluble human TWEAK in complex with the Fab fragment of an antibody selected for inhibition of receptor binding. In the crystallized complex TWEAK is bound by three Fab fragments of the neutralizing antibody. Homology modeling shows that Fab binding overlaps with the putative Fn14 binding site of TWEAK. Docking of the Fn14 cysteine rich domain (CRD) to that site generates a highly complementary interface with perfectly opposing charged and hydrophobic residues. Taken together the presented structure provides new insights into the biology of TWEAK and the TWEAK/Fn14 pathway, which will help to optimize the therapeutic strategy for treatment of related cancer types and autoimmune diseases

Interaction of the antibody with TWEAK.

<p>A) Ribbon representation of one Fab fragment binding to one TWEAK protomer (orange:TWEAK, blue:light chain, green:heavy chain). B) Stereo representation of the epitope recognition with interacting residues as labeled stick model and important hydrogen bond interaction highlighted as dashed lines. The binding is mainly mediated by CDR loop 1 and 2 of the heavy chain interacting with residues of the loops connecting strands D/E and B’/B and residues of strand G. In addition Y93 of CDR3 of the light chain interacts with a main chain N and stacks with the guanidinium group of R130 of TWEAK. C) Interestingly not only canonical CDR loops are involved in TWEAK binding, but an additional hydrogen bond is formed between light chain R68 of a non CDR loop with D75 of a second subunit of the trimeric TWEAK complex (gray).</p

Biochemical analysis of selected anti TWEAK antibodies.

<p>Biochemical analysis of selected anti TWEAK antibodies.</p

Crystallographic data collection and model refinement statistics.

<p>Crystallographic data collection and model refinement statistics.</p

Model of the TWEAK – Fn14 receptor interaction.

<p>A) Side view of the TWEAK trimer showing the solvent accessible electrostatic surface potential (red −4 kT to blue +4 kT). The positively charged patch indicating the possible receptor binding site (dashed ellipse) is covered by the antibody selected for inhibiting TWEAK-Fn14 interaction (cartoon model of Hv in green and Lv in blue). B) Same view as in A with the antibody and TWEAK surface set transparence. After superposition of cytokine-receptor structures APRIL-BCMA (blue; PDB ID 1XU2), APRIL-TACI (brown; PDB ID 1XU1), TALL-BCMA (red; PDB ID 1OQD) and TALL-BAFFR (green; PDB ID 1OQE) the CRD of the receptors co-localize and mark the putative binding site of Fn14 on TWEAK (only the CRD of the receptors is shown as colored cartoon model). C) The NMR model of the Fn14 CRD (blue; PDB ID 2RPJ) is placed at the putative receptor binding site of TWEAK according to the complex structures shown in B. The basic patch is indicated with the dashed ellipse. Only one of the three receptors is shown. D) Stereo view of the modeled TWEAK-Fn14 CRD interface. Upon rigid body and positional refinement of the putative TWEAK-Fn14 CRD complex a dense hydrogen bond network is formed at the interface. The perfect complementarities of charged and hydrophobic patches, as well as the involvement of Fn14 side chains already shown to play an important role in TWEAK binding support this model.</p

Structure of human TWEAK.

<p>A) Ribbon representations of the TWEAK trimer with one protomer colored orange and the symmetry related ones in gray (crystallographic 3-fold axis indicated as black triangle). On the left top view oriented as in 1A with N- and C-Terminus on the top. In the middle side view oriented as in 1B with labeled N- and C-Terminus. In the situation of the uncleaved precursor the membrane is located on top of the molecule. The disulfide bond is highlighted as stick model and beta strands are labeled according to TNF superfamily nomenclature. The dashed lines indicate flexible loops E-F and A-A’’ not visible in the electron density. On the right, bottom view of the TWEAK trimer. B) Solvent accessible electrostatic surface potential (red −4 kT to blue +4 kT) of the TWEAK trimer with the same orientations as in A. Resembling the high pI of TWEAK with 9.62 the complete upper surface is highly positively charged. A second basic patch is located at the side of the TWEAK trimer (dashed ellipse middle picture). This positively charged region is also found in other members of the TNF family (i.e. APRIL, BAFF) and coincides with their receptor binding site. C) Overview of the TWEAK-TWEAK interface as found in the homotrimer in the same orientation and labeled as in A (middle picture). Notable hydrogen bonds involved in the trimerization are indicated as dashed lines with the respective interacting amino acids as sticks. The hydrogen bonds with interacting atoms and distances are listed in the table.</p

Cross blocking assay.

1<p>The Molar Ratio (MR %) was calculated as the quotient of the secondary antibody binding signal to the primary antibody binding signal, both binding to the surface-presented TWEAK ligand.</p

Phase I clinical study of RG7356, an anti-CD44 humanized antibody, in patients with acute myeloid leukemia

RG7356, a recombinant anti-CD44 immunoglobulin G1 humanized monoclonal antibody, inhibits cell adhesion and has been associated with macrophage activation in preclinical models. We report results of a phase I dose-escalation study of RG7356 in relapsed/refractory acute myeloid leukemia (AML). Eligible patients with refractory AML, relapsed AML after induction chemotherapy, or previously untreated AML not eligible for intensive chemotherapy were enrolled and received intravenous RG7356 at dosages ≤ 2400 mg every other week or ≤ 1200 mg weekly or twice weekly; dose escalation started at 300 mg. Forty-four patients (median age, 69 years) were enrolled. One dose-limiting toxicity occurred (grade 3 hemolysis exacerbation) after one 1200 mg dose (twice-weekly cohort). The majority of adverse events were mild/moderate. Infusion-related reactions occurred in 64% of patients mainly during cycle 1. Two patients experienced grade 3 drug-induced aseptic meningitis. Pharmacokinetics increased supraproportionally, suggesting a target-mediated drug disposition (TMDD) at ≥ 1200 mg. Two patients achieved complete response with incomplete platelet recovery or partial response, respectively. One patient had stable disease with hematologic improvement. RG7356 was generally safe and well tolerated. Maximum tolerated dose was not reached, but saturation of TMDD was achieved. The recommended dose for future AML evaluations is 2400 mg every other week

Differential impact of prognostic parameters in hormone receptor-positive lobular breast cancer

Background Invasive lobular breast cancer (BC) is the second most common BC subtype. Prognostic parameters (tumor classification, lymph node status, histologic grade, Oncotype DX recurrence score [RS], progesterone receptor status, and Ki67 index) were retrospectively studied in a large, prospective clinical trial encompassing 2585 patients who had hormone receptor-positive early BC (the West German Study Group PlanB trial). Methods BCs were centrally reviewed and classified as lobular (n = 353; 14%) or nonlobular (n = 2232; 86%). The median follow-up was 60 months. Five-year disease-free survival (DFS) estimates were obtained using the Kaplan-Meier method. Prognostic parameters were evaluated using Cox proportional hazard models. Results Lobular BC was associated with higher tumor classification, higher lymph node status, lower histologic grade, lower Ki67 index, and low or intermediate RS. The prevalence of high RS (RS range, 26-100) was 3-fold lower in patients who had lobular BC compared with those who had nonlobular BC (8% vs 24%;P < .001). However, 5-year DFS estimates for lobular and nonlobular BC were similar (92.1% and 92.3%, respectively;P = .673). In multivariate analyses, prognostic parameters for DFS in lobular BC included grade 3 (hazard ratio, 5.06; 95% CI, 1.91-13.39) and a pathologic lymph node status (pN) of pN3 (hazard ratio, 12.16; 95% CI, 3.87-38.24), but not RS. By contrast, prognostic parameters in nonlobular BC included grade 3 (hazard ratio, 1.65; 95% CI, 1.11-2.44), pN3 (hazard ratio, 3.68; 95% CI, 1.60-8.46), and high RS (hazard ratio, 2.49; 95% CI, 1.69-3.68). Conclusions Lobular BC is associated with low and intermediate RS, although 5-year DFS is similar to that of nonlobular BC. The effect of the RS in lobular BC appears to be distinct from that in nonlobular BC. For risk assessment, the RS needs to be complemented by clinicopathologic parameters for therapy decision making