Structures of a pan‐specific antagonist antibody complexed to different isoforms of TGFβ reveal structural plasticity of antibody–antigen interactions

Various important biological pathways are modulated by TGFβ isoforms; as such they are potential targets for therapeutic intervention. Fresolimumab, also known as GC1008, is a pan‐TGFβ neutralizing antibody that has been tested clinically for several indications including an ongoing trial for focal segmental glomerulosclerosis. The structure of the antigen‐binding fragment of fresolimumab (GC1008 Fab) in complex with TGFβ3 has been reported previously, but the structural capacity of fresolimumab to accommodate tight interactions with TGFβ1 and TGFβ2 was insufficiently understood. We report the crystal structure of the single‐chain variable fragment of fresolimumab (GC1008 scFv) in complex with target TGFβ1 to a resolution of 3.00 Å and the crystal structure of GC1008 Fab in complex with TGFβ2 to 2.83 Å. The structures provide further insight into the details of TGFβ recognition by fresolimumab, give a clear indication of the determinants of fresolimumab pan‐specificity and provide potential starting points for the development of isoform‐specific antibodies using a fresolimumab scaffold.

[1]  D. Heitjan,et al.  Immunological effects of the TGFβ-blocking antibody GC1008 in malignant pleural mesothelioma patients , 2013, Oncoimmunology.

[2]  A. Hata,et al.  Targeting the TGFβ signalling pathway in disease , 2012, Nature Reviews Drug Discovery.

[3]  A. K. Reinhardt,et al.  TGF-β Isoform Specific Regulation of Airway Inflammation and Remodelling in a Murine Model of Asthma , 2010, PloS one.

[4]  A. Hinck,et al.  Ternary Complex of Transforming Growth Factor-β1 Reveals Isoform-specific Ligand Recognition and Receptor Recruitment in the Superfamily* , 2010, The Journal of Biological Chemistry.

[5]  M. Grütter,et al.  A cytokine-neutralizing antibody as a structural mimetic of 2 receptor interactions , 2008, Proceedings of the National Academy of Sciences.

[6]  A. Hinck,et al.  Cooperative assembly of TGF-beta superfamily signaling complexes is mediated by two disparate mechanisms and distinct modes of receptor binding. , 2008, Molecular cell.

[7]  Randy J. Read,et al.  Phaser crystallographic software , 2007, Journal of applied crystallography.

[8]  R. Akhurst,et al.  TGF beta inhibition for cancer therapy. , 2006, Current cancer drug targets.

[9]  K. Chung,et al.  Cytokines as targets in chronic obstructive pulmonary disease. , 2006, Current drug targets.

[10]  W. Seeger,et al.  [Pulmonary fibrosis--a therapeutic dilemma?]. , 2006, Medizinische Klinik.

[11]  Marc K. Halushka,et al.  Losartan, an AT1 Antagonist, Prevents Aortic Aneurysm in a Mouse Model of Marfan Syndrome , 2006, Science.

[12]  W. Seeger,et al.  Lungenfibrosen—ein therapeutisches Dilemma? , 2006, Medizinische Klinik.

[13]  C. Arteaga.  Inhibition of TGFβ signaling in cancer therapy , 2006 .

[14]  Joachim Nickel,et al.  A single residue of GDF-5 defines binding specificity to BMP receptor IB. , 2005, Journal of molecular biology.

[15]  J. Berzofsky,et al.  Transforming Growth Factor-β Production and Myeloid Cells Are an Effector Mechanism through Which CD1d-restricted T Cells Block Cytotoxic T Lymphocyte–mediated Tumor Immunosurveillance , 2003, The Journal of Experimental Medicine.

[16]  Ling Yu,et al.  TGF-β isoforms in renal fibrogenesis , 2003 .

[17]  J. Massagué,et al.  Mechanisms of TGF-β Signaling from Cell Membrane to the Nucleus , 2003, Cell.

[18]  T. Doetschman,et al.  Double-Outlet Right Ventricle and Overriding Tricuspid Valve Reflect Disturbances of Looping, Myocardialization, Endocardial Cushion Differentiation, and Apoptosis in TGF-β2–Knockout Mice , 2001 .

[19]  V. Petrov,et al.  Induction of cardiac fibrosis by transforming growth factor-beta(1). , 2000, Molecular genetics and metabolism.

[20]  K. Miyazono,et al.  Signaling inputs converge on nuclear effectors in TGF-β signaling , 2000 .

[21]  J. Pflugrath,et al.  The finer things in X-ray diffraction data collection. , 1999, Acta crystallographica. Section D, Biological crystallography.

[22]  W. Sebald,et al.  Crystal structure of human bone morphogenetic protein-2 at 2.7 A resolution. , 1999, Journal of molecular biology.

[23]  G. Boivin,et al.  TGFbeta2 knockout mice have multiple developmental defects that are non-overlapping with other TGFbeta knockout phenotypes. , 1997, Development.

[24]  D. Rifkin,et al.  Latent transforming growth factor-beta: structural features and mechanisms of activation. , 1997, Kidney international.

[25]  M. Sporn,et al.  Transforming growth factor beta 1: three-dimensional structure in solution and comparison with the X-ray structure of transforming growth factor beta 2. , 1996, Biochemistry.

[26]  M. Grütter,et al.  The crystal structure of TGF‐β3 and comparison to TGF‐β2: Implications for receptor binding , 1996, Protein science : a publication of the Protein Society.

[27]  M. Ferguson,et al.  Transforming growth factor–β3 is required for secondary palate fusion , 1995, Nature Genetics.

[28]  A. Kulkarni,et al.  Defective haematopoiesis and vasculogenesis in transforming growth factor-beta 1 knock out mice. , 1995, Development.

[29]  Jeffrey L. Wrana,et al.  Mechanism of activation of the TGF-β receptor , 1994, Nature.

[30]  D. Davies,et al.  Crystal structure of TGF‐β2 refined at 1.8 Å resolution , 1993 .

[31]  A. Kulkarni,et al.  Transforming growth factor-beta 1 knockout mice. A mutation in one cytokine gene causes a dramatic inflammatory disease. , 1993, The American journal of pathology.

[32]  M. Grütter,et al.  Refined Crystal Structure of Human Transforming Growth Factor β2 at 1·5 Å Resolution , 1993 .

[33]  Jeffrey L. Wrana,et al.  TGFβ signals through a heteromeric protein kinase receptor complex , 1992, Cell.

[34]  M. Grütter,et al.  An unusual feature revealed by the crystal structure at 2.2 Å resolution of human transforming growth fact or-β2 , 1992, Nature.

[35]  M. Sporn,et al.  Differential expression of the TGF‐β isoforms in embryogenesis suggests specific roles in developing and adult tissues , 1992, Molecular reproduction and development.

[36]  A. Brunger.  Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. , 1992 .

[37]  M. Sporn,et al.  Peptide Growth Factors and Their Receptors I , 1990, Handbook of Experimental Pharmacology.

[38]  Anita B. Roberts,et al.  Human transforming growth factor-β complementary DNA sequence and expression in normal and transformed cells , 1985, Nature.

[39]  C. Arteaga.  Inhibition of TGFbeta signaling in cancer therapy. , 2006, Current opinion in genetics & development.

[40]  P. Afonine,et al.  research papers Acta Crystallographica Section D Biological , 2003 .

[41]  Ling Yu,et al.  TGF-beta isoforms in renal fibrogenesis. , 2003, Kidney international.

[42]  J. Massagué,et al.  Mechanisms of TGF-beta signaling from cell membrane to the nucleus. , 2003, Cell.

[43]  T. Doetschman,et al.  Double-outlet right ventricle and overriding tricuspid valve reflect disturbances of looping, myocardialization, endocardial cushion differentiation, and apoptosis in TGF-beta(2)-knockout mice. , 2001, Circulation.

[44]  K. Miyazono,et al.  Signaling inputs converge on nuclear effectors in TGF-beta signaling. , 2000, Trends in biochemical sciences.

[45]  M. Ferguson,et al.  Transforming growth factor-beta 3 is required for secondary palate fusion. , 1995, Nature genetics.

[46]  R Wieser,et al.  Mechanism of activation of the TGF-beta receptor. , 1994, Nature.

[47]  D. Davies,et al.  Crystal structure of TGF-beta 2 refined at 1.8 A resolution. , 1993, Proteins.

[48]  M. Grütter,et al.  Refined crystal structure of human transforming growth factor beta 2 at 1.95 A resolution. , 1993, Journal of molecular biology.

[49]  A. Brünger.  Free R value: a novel statistical quantity for assessing the accuracy of crystal structures , 1992, Nature.

[50]  J. Massagué,et al.  TGF beta signals through a heteromeric protein kinase receptor complex. , 1992, Cell.

[51]  P. Kondaiah,et al.  Embryonic gene expression patterns of TGF beta 1, beta 2 and beta 3 suggest different developmental functions in vivo. , 1991, Development.

[52]  J. Massagué,et al.  The transforming growth factor-beta family. , 1990, Annual review of cell biology.