skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: A high-affinity human PD-1/PD-L2 complex informs avenues for small-molecule immune checkpoint drug discovery

Abstract

Immune checkpoint blockade of programmed death-1 (PD-1) by monoclonal antibody drugs has delivered breakthroughs in the treatment of cancer. Nonetheless, small-molecule PD-1 inhibitors could lead to increases in treatment efficacy, safety, and global access. While the ligand-binding surface of apo-PD-1 is relatively flat, it harbors a striking pocket in the murine PD-1/PD-L2 structure. An analogous pocket in human PD-1 may serve as a small-molecule drug target, but the structure of the human complex is unknown. Because the CC′ and FG loops in murine PD-1 adopt new conformations upon binding PD-L2, we hypothesized that mutations in these two loops could be coupled to pocket formation and alter PD-1’s affinity for PD-L2. Here, we conducted deep mutational scanning in these loops and used yeast surface display to select for enhanced PD-L2 binding. A PD-1 variant with three substitutions binds PD-L2 with an affinity two orders of magnitude higher than that of the wild-type protein, permitting crystallization of the complex. We determined the X-ray crystal structures of the human triple-mutant PD-1/PD-L2 complex and the apo triple-mutant PD-1 variant at 2.0 Å and 1.2 Å resolution, respectively. Binding of PD-L2 is accompanied by formation of a prominent pocket in human PD-1, as well asmore » substantial conformational changes in the CC′ and FG loops. The structure of the apo triple-mutant PD-1 shows that the CC′ loop adopts the ligand-bound conformation, providing support for allostery between the loop and pocket. This human PD-1/PD-L2 structure provide critical insights for the design and discovery of small-molecule PD-1 inhibitors.« less

Authors:
ORCiD logo [1]; ORCiD logo [2]
  1. Stanford Univ., CA (United States); Stanford Univ. School of Medicine, CA (United States)
  2. Stanford Univ., CA (United States); Stanford Univ. School of Medicine, CA (United States); Chan Zuckerberg Biohub, San Francisco, CA (United States)
Publication Date:
Research Org.:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1594989
Grant/Contract Number:  
AC02-76SF00515; DP1 DA043893; P41GM103393
Resource Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 116; Journal Issue: 49; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; PD-1; PD-L2; immune checkpoint; drug discovery

Citation Formats

Tang, Shaogeng, and Kim, Peter S. A high-affinity human PD-1/PD-L2 complex informs avenues for small-molecule immune checkpoint drug discovery. United States: N. p., 2019. Web. doi:10.1073/pnas.1916916116.
Tang, Shaogeng, & Kim, Peter S. A high-affinity human PD-1/PD-L2 complex informs avenues for small-molecule immune checkpoint drug discovery. United States. doi:10.1073/pnas.1916916116.
Tang, Shaogeng, and Kim, Peter S. Thu . "A high-affinity human PD-1/PD-L2 complex informs avenues for small-molecule immune checkpoint drug discovery". United States. doi:10.1073/pnas.1916916116. https://www.osti.gov/servlets/purl/1594989.
@article{osti_1594989,
title = {A high-affinity human PD-1/PD-L2 complex informs avenues for small-molecule immune checkpoint drug discovery},
author = {Tang, Shaogeng and Kim, Peter S.},
abstractNote = {Immune checkpoint blockade of programmed death-1 (PD-1) by monoclonal antibody drugs has delivered breakthroughs in the treatment of cancer. Nonetheless, small-molecule PD-1 inhibitors could lead to increases in treatment efficacy, safety, and global access. While the ligand-binding surface of apo-PD-1 is relatively flat, it harbors a striking pocket in the murine PD-1/PD-L2 structure. An analogous pocket in human PD-1 may serve as a small-molecule drug target, but the structure of the human complex is unknown. Because the CC′ and FG loops in murine PD-1 adopt new conformations upon binding PD-L2, we hypothesized that mutations in these two loops could be coupled to pocket formation and alter PD-1’s affinity for PD-L2. Here, we conducted deep mutational scanning in these loops and used yeast surface display to select for enhanced PD-L2 binding. A PD-1 variant with three substitutions binds PD-L2 with an affinity two orders of magnitude higher than that of the wild-type protein, permitting crystallization of the complex. We determined the X-ray crystal structures of the human triple-mutant PD-1/PD-L2 complex and the apo triple-mutant PD-1 variant at 2.0 Å and 1.2 Å resolution, respectively. Binding of PD-L2 is accompanied by formation of a prominent pocket in human PD-1, as well as substantial conformational changes in the CC′ and FG loops. The structure of the apo triple-mutant PD-1 shows that the CC′ loop adopts the ligand-bound conformation, providing support for allostery between the loop and pocket. This human PD-1/PD-L2 structure provide critical insights for the design and discovery of small-molecule PD-1 inhibitors.},
doi = {10.1073/pnas.1916916116},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 49,
volume = 116,
place = {United States},
year = {2019},
month = {11}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Save / Share:

Works referenced in this record:

Crystal structure of the complex between programmed death-1 (PD-1) and its ligand PD-L2
journal, July 2008

  • Lazar-Molnar, E.; Yan, Q.; Cao, E.
  • Proceedings of the National Academy of Sciences, Vol. 105, Issue 30
  • DOI: 10.1073/pnas.0804453105

Kinetic Rate Constant Prediction Supports the Conformational Selection Mechanism of Protein Binding
journal, January 2012


Nivolumab versus Docetaxel in Advanced Nonsquamous Non–Small-Cell Lung Cancer
journal, October 2015

  • Borghaei, Hossein; Paz-Ares, Luis; Horn, Leora
  • New England Journal of Medicine, Vol. 373, Issue 17
  • DOI: 10.1056/NEJMoa1507643

Structural Biology of the Immune Checkpoint Receptor PD-1 and Its Ligands PD-L1/PD-L2
journal, August 2017


Small-Molecule Inhibitors of Protein-Protein Interactions: Progressing toward the Reality
journal, September 2014


Drug penetration in solid tumours
journal, August 2006

  • Minchinton, Andrew I.; Tannock, Ian F.
  • Nature Reviews Cancer, Vol. 6, Issue 8
  • DOI: 10.1038/nrc1893

Structure-Based Druggability Assessment of the Mammalian Structural Proteome with Inclusion of Light Protein Flexibility
journal, July 2014


Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging
journal, November 2015

  • Maute, Roy L.; Gordon, Sydney R.; Mayer, Aaron T.
  • Proceedings of the National Academy of Sciences, Vol. 112, Issue 47
  • DOI: 10.1073/pnas.1519623112

Twenty years on: the impact of fragments on drug discovery
journal, July 2016

  • Erlanson, Daniel A.; Fesik, Stephen W.; Hubbard, Roderick E.
  • Nature Reviews Drug Discovery, Vol. 15, Issue 9
  • DOI: 10.1038/nrd.2016.109

MDM2 Small-Molecule Antagonist RG7112 Activates p53 Signaling and Regresses Human Tumors in Preclinical Cancer Models
journal, February 2013


B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion
journal, December 1999

  • Dong, Haidong; Zhu, Gefeng; Tamada, Koji
  • Nature Medicine, Vol. 5, Issue 12
  • DOI: 10.1038/70932

Deep mutational scanning: a new style of protein science
journal, July 2014

  • Fowler, Douglas M.; Fields, Stanley
  • Nature Methods, Vol. 11, Issue 8
  • DOI: 10.1038/nmeth.3027

Cytokine release syndrome
journal, June 2018

  • Shimabukuro-Vornhagen, Alexander; Gödel, Philipp; Subklewe, Marion
  • Journal for ImmunoTherapy of Cancer, Vol. 6, Issue 1
  • DOI: 10.1186/s40425-018-0343-9

Isolating and engineering human antibodies using yeast surface display
journal, July 2006

  • Chao, Ginger; Lau, Wai L.; Hackel, Benjamin J.
  • Nature Protocols, Vol. 1, Issue 2
  • DOI: 10.1038/nprot.2006.94

Chemical and physical instabilities in manufacturing and storage of therapeutic proteins
journal, December 2019


Impact of high-throughput screening in biomedical research
journal, March 2011

  • Macarron, Ricardo; Banks, Martyn N.; Bojanic, Dejan
  • Nature Reviews Drug Discovery, Vol. 10, Issue 3
  • DOI: 10.1038/nrd3368

Conformational kinetics reveals affinities of protein conformational states
journal, July 2015

  • Daniels, Kyle G.; Suo, Yang; Oas, Terrence G.
  • Proceedings of the National Academy of Sciences, Vol. 112, Issue 30
  • DOI: 10.1073/pnas.1502084112

Structure and Interactions of the Human Programmed Cell Death 1 Receptor
journal, February 2013

  • Cheng, Xiaoxiao; Veverka, Vaclav; Radhakrishnan, Anand
  • Journal of Biological Chemistry, Vol. 288, Issue 17
  • DOI: 10.1074/jbc.M112.448126

An Experimentally Determined Evolutionary Model Dramatically Improves Phylogenetic Fit
journal, May 2014


Treatment-Related Adverse Events of PD-1 and PD-L1 Inhibitors in Clinical Trials: A Systematic Review and Meta-analysis
journal, July 2019


Ultra-large library docking for discovering new chemotypes
journal, February 2019


Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy
journal, March 2017


High-affinity PD-1 molecules deliver improved interaction with PD-L1 and PD-L2
journal, August 2018

  • Li, Yanyan; Liang, Zhaoduan; Tian, Ye
  • Cancer Science, Vol. 109, Issue 8
  • DOI: 10.1111/cas.13666

Structural basis for blocking PD-1-mediated immune suppression by therapeutic antibody pembrolizumab
journal, June 2016

  • Na, Zhenkun; Yeo, Siok Ping; Bharath, Sakshibeedu R.
  • Cell Research, Vol. 27, Issue 1
  • DOI: 10.1038/cr.2016.77

Discovery of small molecule cancer drugs: Successes, challenges and opportunities
journal, March 2012


Clinical Pharmacokinetics of Therapeutic Monoclonal Antibodies
journal, January 2010


Structure and Dynamics of PD-L1 and an Ultra-High-Affinity PD-1 Receptor Mutant
journal, October 2016


FDA Approval Summary: Pembrolizumab for the Treatment of Microsatellite Instability-High Solid Tumors
journal, February 2019


Engaging neuroscience to advance translational research in brain barrier biology
journal, February 2011

  • Neuwelt, Edward A.; Bauer, Björn; Fahlke, Christoph
  • Nature Reviews Neuroscience, Vol. 12, Issue 3
  • DOI: 10.1038/nrn2995

Structural basis of checkpoint blockade by monoclonal antibodies in cancer immunotherapy
journal, October 2016

  • Lee, Ju Yeon; Lee, Hyun Tae; Shin, Woori
  • Nature Communications, Vol. 7, Issue 1
  • DOI: 10.1038/ncomms13354

High-resolution crystal structure of the therapeutic antibody pembrolizumab bound to the human PD-1
journal, October 2016

  • Horita, Shoichiro; Nomura, Yayoi; Sato, Yumi
  • Scientific Reports, Vol. 6, Issue 1
  • DOI: 10.1038/srep35297

Cancer immunotherapy — immune checkpoint blockade and associated endocrinopathies
journal, January 2017

  • Byun, David J.; Wolchok, Jedd D.; Rosenberg, Lynne M.
  • Nature Reviews Endocrinology, Vol. 13, Issue 4
  • DOI: 10.1038/nrendo.2016.205

Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade
journal, June 2017


Small molecules, big targets: drug discovery faces the protein–protein interaction challenge
journal, April 2016

  • Scott, Duncan E.; Bayly, Andrew R.; Abell, Chris
  • Nature Reviews Drug Discovery, Vol. 15, Issue 8
  • DOI: 10.1038/nrd.2016.29

Roll: a new algorithm for the detection of protein pockets and cavities with a rolling probe sphere
journal, October 2009


The PD-1/PD-L1 complex resembles the antigen-binding Fv domains of antibodies and T cell receptors
journal, February 2008

  • Lin, D. Y. -w.; Tanaka, Y.; Iwasaki, M.
  • Proceedings of the National Academy of Sciences, Vol. 105, Issue 8
  • DOI: 10.1073/pnas.0712278105

Pathways for Small Molecule Delivery to the Central Nervous System across the Blood-Brain Barrier
journal, January 2014

  • Mikitsh, John L.; Chacko, Ann-Marie
  • Perspectives in Medicinal Chemistry, Vol. 6
  • DOI: 10.4137/PMC.S13384

ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets
journal, January 2013

  • Souers, Andrew J.; Leverson, Joel D.; Boghaert, Erwin R.
  • Nature Medicine, Vol. 19, Issue 2
  • DOI: 10.1038/nm.3048

Pembrolizumab versus Ipilimumab in Advanced Melanoma
journal, June 2015

  • Robert, Caroline; Schachter, Jacob; Long, Georgina V.
  • New England Journal of Medicine, Vol. 372, Issue 26
  • DOI: 10.1056/NEJMoa1503093

Small Molecules as PD-1/PD-L1 Pathway Modulators for Cancer Immunotherapy
journal, March 2019


Structural and Functional Analysis of the Costimulatory Receptor Programmed Death-1
journal, March 2004


PD-L2 is a second ligand for PD-1 and inhibits T cell activation
journal, March 2001

  • Latchman, Yvette; Wood, Clive R.; Chernova, Tatyana
  • Nature Immunology, Vol. 2, Issue 3
  • DOI: 10.1038/85330

Structure of the Complex of Human Programmed Death 1, PD-1, and Its Ligand PD-L1
journal, December 2015