A cytokine-neutralizing antibody as a structural mimetic of 2 receptor interactions

Abstract

TGF-␤ isoforms are key modulators of a broad range of biological pathways and increasingly are exploited as therapeutic targets. Here, we describe the crystal structures of a pan-TGF-␤ neutralizing antibody, GC-1008, alone and in complex with TGF-␤3. The antibody is currently in clinical evaluation for idiopathic pulmonary fibrosis, melanoma, and renal cell cancer. GC-1008 recognizes an asymmetric binding interface across the TGF-␤ homodimer with high affinity. Whereas both cognate receptors, TGF-␤-receptor types I and II, are required to recognize all 3 TGF-␤ isoforms, GC-1008 has been engineered to bind with high affinity to TGF-␤1, 2, and 3 via a single interaction surface. Comparison with existing structures and models of TGF-␤ interaction with its receptors suggests that the antibody binds to a similar epitope to the 2 receptors together and is therefore a structurally different but functionally identical mimic of the binding mode of both receptors.cancer ͉ fibrotic diseases ͉ TGF-␤/antibody complex ͉ TGF-␤ signaling ͉ X-ray structure T he transforming growth factor ␤ (TGF-␤) superfamily of cytokines comprises Ͼ30 structurally related proteins that are involved in the regulation of a wide variety of biological processes such as cell proliferation, differentiation, and expression of extracellular matrix proteins (1, 2). Members of this family are Ϸ25-kDa homodimeric molecules with a similar structural framework in which the 2 monomers are covalently linked via a disulfide bridge (3-8). Three different isoforms of TGF-␤ are known in mammals that share a sequence identity of 70-82% (2). All 3 isoforms of TGF-␤ are expressed as latent or inactive propeptides that can be activated by a diverse number of physiologically and pathophysiologically associated mechanisms such as thrombospondin-1, integrin ␣v␤6, reactive oxygen species, and low pH (9, 10). Expression of TGF-␤ isoforms and the activation of latent TGF-␤ to mature active protein are tightly regulated in normal physiology, and a dysregulation of this process has been described in many pathological conditions leading to an enhanced activity of TGF-␤ in diseases such as fibrotic disease and some malignancies. Gene-deletion studies in vivo indicate that the 3 mammalian isoforms of TGF-␤ have nonoverlapping activities essential for vascular development and the regulation of immune cell function (11-13). However, in vitro, the biological activities of the 3 isoforms of TGF-␤ are almost identical. TGF-␤1 and TGF-␤3 trigger the cellular signaling cascade upon binding to the extracellular domains of 2 transmembrane receptors, known as TGF-␤ receptor types I and II, forming a ternary complex. This complex assembly occurs first through high affinity binding of the cytokines to their TGF-␤ receptor type II, followed by the recruitment of the TGF-␤ receptor type I (14, 15). The binding potency of TGF-␤ to its type II receptor is isoform-dependent, with the highest binding affinities for TGF-␤1 and 3 and a 10-to 20-fold-smaller binding affinity for 17). The formation ...

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