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

Lázár‐Molnár, Eszter; Scandiuzzi, Lisa; Basu, Indranil; Quinn, Thomas J.; Sylvestre, Eliezer; Palmieri, Edith; Ramagopal, U.A.; Nathenson, Stanley G.; Guha, Chandan; Almo, Steven C.

doi:10.1016/j.ebiom.2017.02.004

Cited by 51 publications

(53 citation statements)

References 75 publications

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“…Substantial efforts by us and others (31) to crystallize the human PD-1/PD-L2 complex were previously unsuccessful. Earlier studies (18,19,21) indicated that the PD-1 ligand-binding interface consists of a hydrophobic core, the CC′ loop, and the FG loop ( Figure 2A), and that formation of a complex with ligands results in loop movement and pocket formation in the hydrophobic core.…”

Section: Engineering Human Pd-1 Loop Variants With Enhanced Pd-l2 Affmentioning

confidence: 99%

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

Tang

Kim

2019

Preprint

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Immune checkpoint blockade of programmed death-1 (PD-1) by monoclonal antibody drugs has delivered breakthroughs in the treatment of cancer. Nonetheless, smallmolecule 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. Significance StatementImmune checkpoint blockade of programmed death-1 (PD-1) by monoclonal antibody drugs has transformed the treatment of cancer. Small-molecule PD-1 drugs have the potential to offer increased efficacy, safety, and global access. Despite substantial efforts such small-molecule drugs have been out of reach. We identify a prominent pocket on the ligand-binding surface of human PD-1 that appears to be an attractive small-molecule drug target. The pocket forms when PD-1 is bound to one of its ligands, PD-L2. Our high-resolution crystal structure of the human PD-1/PD-L2 complex facilitates virtual drug-screening efforts and opens additional avenues for the design and discovery of small-molecule PD-1 inhibitors. Our work provides a strategy that may enable discovery of small-molecule inhibitors of other "undruggable" protein-protein interactions.binding ( Figure 2C). As a result, we obtained a PD-1 triple mutant (Figures S1F-G) that contains all three substitutions identified from the first library: N74G, T76P, and A132V. PD-1 loop variants showed increased binding affinity and association kinetics for PD-L2 and PD-L1To validate the detected enhancement in affinity, we recombinantly expressed and purified human PD-1 and the loop variants, as well as the human PD-L2 and PD-L1 ectodomain proteins. Using bio-layer interferometry, we compared the binding of PD-L2 to wild-type PD-1 and t...

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Section: Engineering Human Pd-1 Loop Variants With Enhanced Pd-l2 Affmentioning

confidence: 99%

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

Tang

Kim

2019

Preprint

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“…In previous studies, high-affinity sPD-1 molecules have been reported to blockade the PD-1 axis. 30,31 However, we generated PD-1-based high-affinity PD-L1 binders with a unique mutation that played a dominant role in enhancing the binding strength. Our investigation demonstrated that the newly generated high-affinity PD-1 molecular could interact with PD-L1 on tumor cell surfaces and compete with antibodies specific to PD-L1 and PD-L2.…”

mentioning

confidence: 99%

High‐affinity PD‐1 molecules deliver improved interaction with PD‐L1 and PD‐L2

Liang

Tian

et al. 2018

Cancer Science

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The inhibitory checkpoint molecule programmed death (PD)‐1 plays a vital role in maintaining immune homeostasis upon binding to its ligands, PD‐L1 and PD‐L2. Several recent studies have demonstrated that soluble PD‐1 (sPD‐1) can block the interaction between membrane PD‐1 and PD‐L1 to enhance the antitumor capability of T cells. However, the affinity of natural sPD‐1 binding to PD‐L1 is too low to permit therapeutic applications. Here, a PD‐1 variant with approximately 3000‐fold and 70‐fold affinity increase to bind PD‐L1 and PD‐L2, respectively, was generated through directed molecular evolution and phage display technology. Structural analysis showed that mutations at amino acid positions 124 and 132 of PD‐1 played major roles in enhancing the affinity of PD‐1 binding to its ligands. The high‐affinity PD‐1 mutant could compete with the binding of antibodies specific to PD‐L1 or PD‐L2 on cancer cells or dendritic cells, and it could enhance the proliferation and IFN‐γ release of activated lymphocytes. These features potentially qualify the high‐affinity PD‐1 variant as a unique candidate for the development of a new class of PD‐1 immune‐checkpoint blockade therapeutics.

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“…This leads to an increase in van der Waals interactions. [34] Furthermore,ac rystal structure of aP D-L1 nanobody (single domain antibody) was published (PDB ID:5 JDS). Thenanobody KN035 competes with PD-1 for binding to PD-L1 mainly through asingle surface loop of 21 amino acids.…”

Section: Cocrystal Structures With Monoclonal Antibodiesmentioning

confidence: 99%

Immune Checkpoint PD‐1/PD‐L1: Is There Life Beyond Antibodies?

et al. 2018

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The PD-1/PD-L1 interaction has emerged as a significant target in cancer immunotherapy. Current medications include monoclonal antibodies, which have shown impressive clinical results in the treatment of several types of tumors. The cocrystal structure of human PD-1 and PD-L1 is expected to be a valuable starting point for the design of novel inhibitors, along with the recent crystal structures with monoclonal antibodies, small molecules, and macrocycles.

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

Cited by 51 publications

References 75 publications

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

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

High‐affinity PD‐1 molecules deliver improved interaction with PD‐L1 and PD‐L2

Immune Checkpoint PD‐1/PD‐L1: Is There Life Beyond Antibodies?

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