Structure-function analysis of immune checkpoint receptors to guide

Subscriber access provided by UNIV OF DURHAM

Perspective

Structure-function analysis of immune checkpoint receptors to guide emerging anticancer immunotherapy Rita Acúrcio, Anna Scomparin, Joao Conniot, Jorge A R Salvador, Ronit Satchi-Fainaro, Helena F Florindo, and Rita Cardoso Guedes J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00541 • Publication Date (Web): 18 Jul 2018 Downloaded from http://pubs.acs.org on July 19, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

Structure-function analysis of immune checkpoint receptors to guide emerging anticancer immunotherapy

Rita C. Acúrcioa, Anna Scomparinb, João Conniota, Jorge A. R. Salvadorc, Ronit Satchi-Fainarob, Helena F. Florindoa*, Rita C. Guedesa*

a

Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade

de Lisboa, 1649-003, Lisbon, Portugal; b

Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv

University, 6997801, Tel Aviv, Israel; c

Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Coimbra,

3000-548, Coimbra, Portugal; Centre for Neuroscience and Cell Biology, Coimbra, Portugal.

*Corresponding authors:

Helena F Florindo, [email protected] Rita C. Guedes, [email protected]

1 ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 60

ABSTRACT The modulation of immune checkpoint receptors has been one of the most successful, exciting and explored approaches for cancer immunotherapy. Currently several immune checkpoint modulators, mainly monoclonal antibodies, are showing remarkable results. However, the failure to show a response in most patients and the induction of severe immune-related adverse effects are the major drawbacks. Novel approaches concerning the development of immune modulatory small molecules have emerged as an alternative. Nevertheless, the lack of structural information about immune checkpoint receptors has hindered the rational design of those smallmolecule modulators, by preventing the use of methodologies such as computer-aided drug design. Herein, we provide an overview and critical analysis of the structural and dynamic details of immune checkpoint receptors (cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1) and glucocorticoidinduced TNFR-related protein (GITR)) and their interaction with known modulators. This knowledge is essential to advance the understanding of their binding mode and guide the design of novel effective targeted anticancer medicines.

Keywords: immune checkpoint; structure-function; small-molecule inhibitors;

computational-aided drug design; multitargeting; cancer immunotherapy


Page 3 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.

Introduction

Immunotherapy is currently a powerful strategy for the treatment of cancer. The idea of exploiting the host’s immune system to treat cancer is not new. It was first proposed by Rudolf Virchow 150 years ago1 and experimentally confirmed by William Coley2. In 1891, Coley injected streptococcal organisms into cancer patients, and the patient’s tumor disappeared, presumably due to the attack by the immune system2. In the last decades, immunotherapy has emerged as a therapeutic option supported by the great advances achieved in the clarification of the molecular mechanisms involved in cancer progression3. The immune system enrolls several subsets of cell types. Among them, T cells have received major attention to therapeutically activate antitumor immunity due to their ability to recognize antigen-major histocompatibility complexes (MHCs) and subsequently drive a broad range of immune responses (adaptive and innate effector mechanisms). Several approaches to activate antitumor immunity have been recently developed, and promising outcomes were reported in controlling mostly the disseminated form of this disease. In particular, the modulation of immune checkpoint receptors4 (Figure 1A) has shown very exciting outcomes. Immune checkpoints are immune regulators, either costimulatory or coinhibitory, that modulate the proliferation and activity of T cells, as well as those of other immune cells, enrolled in these pathways. Under normal conditions, immune checkpoints are essential in maintaining self-tolerance and ensuring adequate duration and amplitude of the physiological immune responses in peripheral tissues, avoiding collateral tissue damage5. Unfortunately, tumors also use these immune regulators as one of the major mechanisms to evade immune system recognition and destruction by suppressing their activation and effector functions. To date, several immune checkpoints have been identified and explored as potential therapeutic targets in oncology. Two inhibitory immune checkpoint receptors were intensively studied in the context of clinical cancer immunotherapy: cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1). Remarkable results have led to the regulatory approval of several monoclonal antibodies targeting CTLA-4 or PD-1 receptors 6–8. They include ipilimumab (Yervoy®; Bristol-Myers Squibb), pembrolizumab (Keytruda®; Merck), nivolumab (Opdivo®; Bristol-Myers Squibb) and atezolizumab 3 ACS Paragon Plus Environment


Page 4 of 60

(Tecentriq®; Genentech-Roche), as well as many others currently in clinical trials (Figure 1B and C). Additionally, other immune checkpoint receptors represent promising therapeutic targets based on newly discovered molecules that are now being evaluated in preclinical tumor models and/or clinical trials9,10 (Figure 1B). It is important to emphasize that all the approved immune checkpoint modulators in the clinic are monoclonal antibodies. These have revolutionized cancer therapy because of their remarkable clinical activity. However, they have drawbacks, including failure to show a response in most patients, administration by intravenous injection and severe immune-related adverse effects11–13. Recent efforts have dramatically evolved to devise novel approaches toward the development of immune modulatory small molecules that are more affordable and accessible potential alternatives than monoclonal antibodies. These new medicines exploit their benefits over recombinant protein approaches, namely: (i) high manufacturing and scale-up feasibility; (ii) possible oral bioavailability; (iii) greater diffusion rate within the tumor microenvironment; (iv) easier access to intracellular targets (targets not tractable by protein therapeutic agents); and (v) improved pharmacokinetics and/or pharmacodynamics14. These immune modulators may also offer the possibility of avoiding macrophage-mediated resistance observed in anti-PD1 therapy15.


Page 5 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 1 Costimulatory and inhibitory receptors regulating T-cell responses and translation into the clinic. (A) Representation of several ligand-receptor interactions between T cells and antigen-presenting cells (APCs). Several ligands bind to multiple receptors, some of which deliver costimulatory signals, while others lead to coinhibitory signals. One important family of membrane-bound ligands that binds both costimulatory and inhibitory receptors is the B7 family. B7 family members and their known ligands belong to the immunoglobulin superfamily. Ligands of other B7 receptors have not yet been identified. Tumor necrosis factor (TNF) family members that bind to cognate TNF receptor family molecules represent a second family. These receptors predominantly deliver costimulatory signals. (B) Agents currently in clinical trials for PD-1, PD-L1, GITR, LAG-3 and TIM-3. CTLA-4 has no new agents reported under clinical development. (C) Despite the extensive work devoted to the development of agents acting on immune checkpoint regulators, the number of approved drugs is extremely low. To date, approved drugs target only CTLA-4 and PD-1/PD-L1.

The development of new small-molecule products is a puzzling multidimensional problem that lasts, on average, 10 to 15 years. Drug discovery has traditionally had a basis in trial-and-error. Fortunately, during the last decades, this process has been revolutionized and has become more rational using approaches focused on the identification of agents. From the selection of potential hits to hit-to-lead campaigns, a long and challenging pathway occurs. In most pharmaceutical companies and in academia, beyond classical approaches, drug designers are taking advantage of using computational-aided drug design (CADD) to identify and select the best molecule candidates for synthesis that simultaneously are likely to display the desired ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties16,17. The use 5 ACS Paragon Plus Environment


Page 6 of 60

of computational methodologies, ligand and/or structure based, have been already proved as a synergic tool to boost this drug discovery process. The lack of structure and dynamics knowledge of the targets strongly limits the use of CADD methodologies18. Structural properties, binding regions, flexibility, key binding pocket interactions, and dynamics of the ligand-receptor interactions are critical information toward the development of new drugs. Usually, this information is mainly provided by crystallographic structures. Structural studies of immune checkpoint receptors have advanced significantly in recent years. New crystallographic structures were resolved, and new attempts to develop potential immune checkpoint modulators have occurred. However, this information is still scarce and mostly dispersed. This review intends to provide an overview and analysis of the most important immune checkpoint receptors currently explored for the development of specifically targeted cancer therapies (CTLA-4, PD-1 and GITR). It aims to perform an in-depth analysis of structural features to provide a better understanding of druggability and guide the rational design of novel effective and safe anticancer molecular targeted drugs. In addition, a critical evaluation will be provided regarding the impact of those structure-function analyses on the design of combinatorial cancer therapeutics. The information enclosed in this review is a step forward to the use of CADD in the rational development of novel immune checkpoint modulators but are also a great addition to the whole knowledge in this field.

2.

Immunologic checkpoint receptors: Principles learned for the structure, binding pockets and interaction

CTLA-4 CTLA-4, also known as CD152, is an inhibitory T-cell surface-expressed receptor that primarily regulates the amplitude of early-stage T-cell activation 19–21. This receptor is exclusively present in T cells, and it was the first immune checkpoint receptor to be targeted by monoclonal antibodies. Ipilimumab was the first immune checkpoint inhibitor used in cancer immunotherapy (Figure 1C). The United States Food and Drug Administration (FDA) approved this immune modulator in 2011 for the treatment of unresectable or advanced melanoma. 6 ACS Paragon Plus Environment

Page 7 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


CTLA-4 is a type 1 transmembrane glycoprotein of the immunoglobulin superfamily, 223 amino acids (aa) in length, comprising extracellular (125 aa), transmembrane (20 aa) and cytoplasmic (40 aa) domains. It is a covalent homodimer of 41-43 kDa19,22–24. Homodimerization of CTLA-4 is mediated by cysteine-dependent bonding at position 122 in the stalk region. In addition, N-glycosylation at positions 78 and 110 contribute to homodimer stabilization. Extracellular monomers of CTLA-4 display a two-layer βsandwich (Figure 2A) that exhibits the chain topology (C’’DEBA:GFCC’) found in the single immunoglobulin variable domain and three consecutive prolines in the FG loop, Met-Tyr-Pro-Pro-Pro-Tyr-Tyr, which have important implications in ligand binding. CTLA-4 binds to CD80 (B7-1) and CD86 (B7-2) (Figure 1A), which are expressed on antigen-presenting cells (APCs), leading to the down-regulation of T-cell activity. Both are also the ligands for the costimulatory receptor CD28, a homolog receptor of CTLA-4 (sharing 31% identity), but with lower overall affinity25–29. The role of CTLA-4 in controlling T-cell activation is dramatically demonstrated by the lethal systemic immune hyperactivation phenotype of CTLA-4-knockout mice30,31. In fact, CTLA-4 blockade results in the broad enhancement of immune responses that are dependent on T helper cells while conversely, CTLA-4 engagement on T regulatory cells (Tregs) increases their suppressive function. This central role on the modulation of T-cell activity elicited the promise of CTLA-4 as a target for cancer immunotherapy. Thus, several studies have been performed, leading to extensive structural analysis of this immune checkpoint receptor. In early 1997, before the crystallographic structures of CTLA-4 were released, an in

silico CTLA-4 3D structure was modeled. The structure obtained by homology modeling assessed the β-strands and loop regions, demonstrating that CTLA-4 adopted a single immunoglobulin variable domain fold32. In late 1997, the 3D structure of CTLA-4 was resolved using nuclear magnetic resonance (NMR) studies and proved to be in agreement with the first crystallographic structure33. Fortunately, to date, diverse crystal structures of CTLA-4 have been revealed. In 2000, the crystal structure of the extracellular domain of murine CTLA-4 was resolved by Ostrov et al. (PDBID: 1DQT, 2.0 Å)34. The CTLA-4 monomer was described as exhibiting 7 ACS Paragon Plus Environment


Page 8 of 60

a chain topology found in the single immunoglobulin variable domain. In this work, the CTLA-4/B7 complex was envisioned using manual docking, suggesting the formation of a periodic structure in which each CTLA-4 homodimer bridges two B7 molecules (Figure 2C). One year later, the crystal structures of CTLA-4/B7-1 (PDBID:1I8L) and CTLA-4/B7-2 (PDBID:1I85) complexes were also resolved at 3.0 and 3.2 Å resolution by Stamper et

al.35 and Schwartz et al.36, respectively. These studies allowed detailed characterization of the CTLA-4 homodimer interface stabilization (Figure 2B). They also concluded that no conformational changes or reorganization of this dimer interface occurs on binding to B7 proteins. Concerning the complex formed by CTLA-4 and B7 proteins, it was demonstrated that B7 binding sites are located distal to the dimer interface, leading to the formation of a periodic assembly of dimeric molecules (Figure 2C). This confirms the model previously described by Ostrov et al.34 encompassing CTLA-4 bridging to two B-7 molecules. The direct observation of this periodic network immediately provides a model describing the assembly of these molecules at the T-cell/APC interface. Once formed, the complex is stabilized by several interactions, such as ionic, 5 hydrogen bonds, and 28 van der Waals contacts involving several residues, including the Met99-Tyr-Pro-Pro-ProTyr-Tyr105 sequence, which adopts an unusual cis-trans-cis conformation essential for the interaction with B7 molecules. The other residues involved in the complex stabilization are Glu33 by the hydrogen bond and Arg35, Thr53 and Glu97 by ionic interaction35,36. Stamper and coworkers35 evidenced as well the potent inhibitory signaling of B7-1 for CTLA-4 (Kd = 0.2–0.4 µM)34, and the oligomeric high-avidity binding of the T-cell and APC interaction.


Page 9 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 2 Human CTLA-4 overall structure and interactions (PDBID: 3OSK). (A) Ribbon representation of the secondary structure of the CTLA-4 extracellular monomer, exhibiting the two-layer AGFCC’ and ABED IgSF domain topology. (B) Ribbon representation of the CTLA-4 extracellular homodimer. Each monomer exhibits three distinct CDR regions, CDR1 (BC loop), CDR2 (C'C" loop), and CDR3 (FC loop), which form the top surface of the molecule. Stick representation of homodimer interface focusing structure stabilization involving several residues (Pro119, Glu120, Cys122 and Pro123) and a disulfide linkage at Cys112. (C) Ribbon representation of the CTLA-4/B7-1 complex (PDBID: 1I8L). Two B7-1 (cyan) and two CTLA-4 (dark blue) protein units in the asymmetric unit. (D) Molecular association of CTLA-4 and B7-1 in the crystal lattice. ‘Skewed zipper’ arrays in the CTLA-4/B7-1 complex. In the perpendicular direction, across membranes, ligated receptors would span 140 Å.

In 2009, Schönfeld and coworkers disclosed the structure of an engineered lipocalin for CTLA-4 (PDBID: 3BX7, 2.1 Å)37. This study disclosed new information related to the CTLA-4 structure, namely, the site of recognition of lipocalin. The lack of a highresolution structure of the unbound form of human CTLA-4 was finally overcome by Yu et al.38 in 2013, with the resolution of a structure of the unbound CTLA-4 homodimer (PDBID: 3OSK; 1.8 Å). This new structure emphasized the rigid-body ligand recognition based on the absence of conformational changes compared with the previous resolved receptor-ligand structures34–37. A detailed structural analysis showed remarkable similarities between ligand-bound and unbound-CTLA-4 homodimers, undoubtedly revealing that CTLA-4 binds in a largely rigid body-like manner. However, comparing the B7 protein binding, 9 ACS Paragon Plus Environment


Page 10 of 60

unexpectedly and in contrast to the rigid-body binding evidenced by B7-1, the B7-2 binding to CTLA-4 exhibits elements of induced fit. These differences in the geometric fit of the respective interfaces possibly result from additional polar interactions established by Glu33 present in B7-1 and are replaced by Val in B7-2 that cannot establish the same interaction. To this point, the studies described in the literature were focused on the characterization of CTLA-4. However, the interest on CTLA-4 for immunotherapy resulted in the development of CTLA-4 immune checkpoint inhibitors. Thus far, therapeutic agents against CTLA-4 are focused on monoclonal antibodies; therefore, they lead to structural studies focused on the receptor-drug complex formed. More recently, in 2016, Lee and coworkers39 attained the crystal structure of the CTLA4/tremelimumab Fab (fully human IgG antibody; PDBID: 5GGV, 2.0 Å) complex, reporting the binding mode of the monoclonal antibody and conformational changes induced upon drug binding. This study demonstrated that the CTLA-4 homodimer interface is not impacted, and the antibody epitope partially occupies the B7-1/2 binding site of CTLA-4, showing a larger binding interface than with the receptor-ligand interface (Figure 3). Several CTLA-4 residues (Lys1, Ala2, Met3, Glu33, Arg35, Gln41, Ser44, Gln45, Val46, Glu48, Leu91, Ile93, Lys95, Met99, Pro102, Pro103, Tyr104, Tyr105, Leu106, Ile108 and Asn110) are involved in hydrogen bonds, salt bridges and van der Waals contacts with tremelimumab. Comparing the residues involved in the CTLA-4/B7 interaction previously described with those identified in the CTLA-4/tremelimumab complex formation, it is possible to conclude that tremelimumab and B7 proteins occupy the same binding sites. Consequently, the binding of tremelimumab to CTLA-4 efficiently competes with B7-1/2 binding, leading to blockade of CTLA-4 function in cancer39. In the crystal structures of CTLA-4/B7, the crystal packing generates an alternating periodic arrangement in which bivalent CTLA-4 homodimers connect bivalent B7-1/2 homodimers, providing a model that describes the assembly of CTLA-4 and B7-1/2 at the interface between T and cancer cells. The CTLA-4/tremelimumab complex distance (Figure 3) is incompatible with the oligomeric array of the CTLA-4/B7 complex. Considering the simple antagonism of the interaction between CTLA-4 and B7-1/2, the 10 ACS Paragon Plus Environment

Page 11 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


authors could predict that tremelimumab binding could prevent or disrupt this uniquely organized structure.

®

Fig. 3 CTLA-4/tremelimumab complex (PDBID: 5GGV). Hypothetical model for the prevention of an alternating arrangement of CTLA-4 (dark blue) and B7-1/2 (cyan) by tremelimumab (light blue) binding. The CTLA-4 dimer binds to two tremelimumab Fab fragments in the crystal. Tremelimumab binds in the vicinity spanning 150–190 Å in the perpendicular direction from the T-cell membrane.

The last crystal structures reported were the complex CTLA-4/ipilimumab (PDBID: 5TRU and 5XJ3, 3.0 and 3.2 Å) by Ramagopal and coworkers40, and Gao and coworkers41. These studies focused on the binding mode of ipilimumab to CTLA-4, as well as on the selectivity toward CTLA-4 over homologous CD28. Ipilimumab contacts the front β-sheet of CTLA-4 and intersects with the CTLA-4/Β7 recognition surface, indicating that direct steric overlap between ipilimumab and the B7 ligands is a major mechanistic contributor to ipilimumab function. The crystal structure also highlights the factors for the selectivity exhibited by ipilimumab toward CTLA-4. The authors stress that the detailed structural differences in the G strands of CTLA-4 and CD28 are the major reasons for the higher selectivity of ipilimumab. In addition, differences in the C′-strand β bulge probably contribute to the differences in the CC′ loop conformation. However, this difference is distal to the recognition interface and is not predicted to significantly impact the binding or selectivity40. Based on the CTLA-4-drug structures, both monoclonal antibodies bind to CTLA-4 with similar overall affinity and in the same binding site. However, these monoclonal antibodies exhibit a different behavior that, in part, can be due to depletion of targeted cells—e.g., immunosuppressive Tregs via Fc-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). 11 ACS Paragon Plus Environment


Page 12 of 60

ipilimumab is an IgG1, while tremelimumab is an IgG2, with different ADCC and CDC activities42,43. Table 1. CTLA-4 hot spots, residues and key interactions to target

The regulatory approvals of monoclonal antibodies led to a new era in cancer immunotherapy with clinical effects. Nevertheless, small-molecule drugs are still missing from this class of targeted therapies. As stated previously, modulating the immune system using small molecules may offer several advantages, but very limited studies targeting CTLA-4 modulation have been reported thus far. Uvebrant et al.44 and Classon et al.45 in 2004 and 2007, respectively, described pyrazoloquinoline derivatives as inhibitors of CTLA-4. Further investigations using these compounds were not reported. Consequently, the development of small molecules targeting this immune checkpoint receptor remain a challenge. Nevertheless, the extensive structural characterization of this immune regulator is evident, and all crystal structures disclosed provide crucial information. Structural


Page 13 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


analyses led to a deep knowledge on essential characteristics such as flexibility and binding domains, as well as on the residues and key interactions established between native ligands and inhibitors (Table 1). These multiple available crystal structures of CTLA-4 and detailed analyses warrant structure-based in silico studies. CADD techniques (docking or molecular dynamics) will be of upmost value to guide the design of targeted cancer therapeutic alternatives. Similar binding sites reported in the CTLA-4-drug complex structures indicate a hot-spot for CTLA-4 therapeutics and may serve as a target for future small-molecule modulator development. There is definitely room for the engineering of improved alternatives, particularly those leading to smallmolecule approaches, and several developments will certainly emerge in this field in the near future. PD-1/PD-L1

PD-1, also known as CD279, is a cell surface receptor that is expressed on different Tcell types such as T cells, B cells, natural killer (NK) cells or monocytes46,47. The major role of PD-1 is to limit the activity of T cells in peripheral tissues following an inflammatory response. Thus, it limits autoimmunity; however, when engaged by one of its ligands, triggers an immune evasion by compromising effector cytotoxic T-cell function. The mechanisms by which this happens include the apoptosis of tumorspecific T cells, inhibition of T-cell proliferation and secretion of cytokines. Persistent antigen exposure from tumor cells that have gained the ability to evade the host immune response leads to persistent upregulation of PD-1, resulting in T-cell exhaustion,

a

major

immune

resistance

mechanism

within

the

tumor

microenvironment48–51. PD-1 is a type 1 transmembrane glycoprotein of 50-55 kDa comprising an immunoglobulin variable-type extracellular domain, a transmembrane domain and a cytoplasmic tail, and is 288 aa in length (Figure 4A). It belongs to the CTLA-4 family, sharing 20% of sequence identity with it. PD-1 presents a monomeric structure, both in solution and at the cell surface, in contrast to CTLA-4 and other family members that are all disulfide-linked homodimers52.



Page 14 of 60

Fig. 4 PD-1 overall structure. (A) Schematic representation of PD-1 protein. Full-length PD-1 is separated into the ectodomain, transmembrane domain and intracellular domain. The ectodomain is composed of the signal peptide, N-loop, IgV domain and stalk region. The N-glycosylation sites are indicated with a solid green arrowhead. The numbers indicate amino acid positions. Ribbon representation of the PD-1 structure (blue). It consists of a two-layer β sandwich with the topology of IgSF domains and two β sheets (GFCC’ and ABED) stabilized by a disulfide bond (Cys34– Cys103). (B) PD-1/PD-L1 structure complex (PDBID: 4ZQK). PD-1 (dark blue) and PD-L1 (teal) are both shown using cartoon representation. (E) Close-up view of the PD-1/PD-L1 interface using cartoon representation. Residues involved in PD-1 and PD-L1 interaction are represented in dark blue and teal sticks, respectively.

PD-1 binds to PD ligand 1 (PD-L1) and PD ligand 2 (PD-L2), also known as B7-H1 and B7DC, respectively (Figure 1A). PD-1 ligands are type 1 transmembrane glycoproteins composed of immunoglobulin variable- and immunoglobulin constant-type domains, which share 37% sequence similarity and arise via gene duplication48,49,53–55. However, their regulation is highly divergent. PD-L1 is induced on activated hematopoietic and epithelial cells by the inflammatory cytokine interferon-ɤ, which is produced by some activated T and NK cells56. PD-L2 has a much higher selective expression on activated dendritic cells (DC) and some macrophages57. The use of the PD-1 pathway in cancer immunotherapy was furthered by the discovery of upregulated PD-1 ligands in many human cancers. Therefore, their blockade leads to T-cell activation, resulting in strengthened tumor recognition. Regulatory approval of several inhibitors of this pathway, such as nivolumab, pembrolizumab, atezolizumab, 14 ACS Paragon Plus Environment

Page 15 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


avelumab and duravalumab, and the extensive ongoing clinical development of many others, demonstrate the undoubted impact of PD-1/PD-L1 modulators for cancer immunotherapy (Figure 1B and 1C). Based on these outcomes, several studies concerning the characterization of the PD-1 signaling pathway have been conducted. Multiple crystal structures of PD-1 and its ligands have been reported. Because PD-1 is a member of the CTLA-4 coreceptor family, earlier work was focused on the characterization of PD-1 and understanding the similarities and differences within this family of coreceptors. In 2004, the first crystal structure of the extracellular domain of murine PD-1 was assessed by Zhang et al. (PDBID: 1NPU, 2.0 Å)58. It showed a monomer with a two-layer β sandwich topology and a ligand-binding surface significantly different from the other members of the CTLA-4 family. The CDR3 loop in CTLA-4 is characterized by a Met-Tyr-Pro-Pro-Pro-Tyr-Tyr sequence that plays a central role in ligand binding activity36. In PD-1, the same region presents high B factors and weak electron density compared with the rest of the molecule, indicating that this loop is highly flexible, in contrast to the rigid CDR3 loop of CTLA-4. Furthermore, mutagenesis studies showed that residues in the CDR3 loop of murine PD-1 (Leu-HisPro-Lys-Ala) were not critical for the PD-1/B7-H1 interaction. Murine and human PD-1CDR3 loop residues are not conserved, a finding that is consistent with their modest roles in ligand binding. Ligand binding assays have suggested that strands from the front faces make a major contribution to the receptor/ligand interface. The CDR3 loops reside at the periphery of the interface and do not make critical atomic contacts. This interpretation has suggested a strand-to-strand binding mode for the PD-1/PD-L complexes that is distinct from the loop-to-strand binding mode observed in the CTLA4/B7 complexes 58,59. The subsequent studies of Lin et al., Lázár-Molnár et al. and Freeman et al. were centered on the PD-1/PD ligand structure and the ligand-binding poses and pockets. The structures of murine PD-1/human PD-L1 and murine PD-1/murine PD-L2 complexes showed a 1:1 receptor/ligand stoichiometry, in contrast to the 1:2 of CTLA460–62. In 2008, Lin et al.60 disclosed the structure of the murine PD-1/human PD-L1 complex (PDBID: 3BIK, 2.7 Å). This study identified the residues involved in the PD-L1 ligand-binding interface as Met64, Asn66, Asn68, Ser73, Asn74, Gln75, Thr76, Lys78, Val90, Leu122, Gly124, Ile126, Leu128, Pro130, Lys131, Ala132, Ile134 and Glu136. 15 ACS Paragon Plus Environment


Page 16 of 60

Compared with human and murine PD-1 sequences, some differences are clear, suggesting that human PD-1 could bind with slight differences to PD-L1. In detail, Met64 corresponds to Val, Asn68 to Tyr, and Val90 to Gly. The critical residues of human PD-L1 for ligand binding are Phe19, Thr20 Tyr56, Met115, Ala121, Asp122, Tyr123, Lys124 and Arg125 (Figure 4C) and are highly conserved among different organisms. Additionally, their mutations lead to the abolishment of binding to PD-1. Lin et al.60 and Lázár-Molnár et al.61 have also suggested PD-1/PD-L organization and interaction at the cell-cell interface. Simple diffusive processes and mutual affinity lead to a compact complex of PD-1 to PD ligands in interacting cells. Although murine and human PD-1 share high sequence identity (64%), there is a strong need for structural studies involving human PD-1. Consequently, the structure of human PD-1 was assessed by Cheng and coworkers63 through NMR-based approaches, showing differences from its murine homolog. However, murine PD-1 has a “conventional” immunoglobulin variable-set domain, the human receptor lacks a C’’ strand, and, instead of the C’ and D strands, it presents a relatively long and flexible loop. Together, these variations are responsible for the different binding affinities of human and murine PD-1 to their ligands. Models of human PD-1/human PD-L1 and human PD-1/murine PD-L2 complexes were built by superimposing human PD-1 onto the solved complex structures assuming that human PD-1 would share a similar binding mode with the murine structures60,61. In the modeled complexes, the Tyr68 residue at the center of the binding region of human PD-1 (Asn in murine PD-1) interacts with a tyrosine (Tyr123 in human PD-L1, Tyr112 in murine PD-L2) conserved in the ligands of both species. The substitution of Tyr68 for Asn might otherwise be responsible for the weaker binding of the mouse proteins63. Although structural differences between human and murine PD-1 were reported by Cheng and coworkers63, their ligand-binding domains seem identical to those reported to date (Phe53, Asp55, Thr59, Ser60, Glu61, Asn66, Tyr68, Arg69, Ser73, Gln75, Thr76. Ala80, Gln88, Arg112, Thr120, Leu122, Cys123, Gly124, Ala125, Leu128, Lys131, Ala132, Gln133 and Ser137). The described residues are common to both PD-1 ligands, but some exclusively bind to PD-L2, such as Met70, Ser71, Asp77, Leu79, Phe72, Ser83, Phe106, and Ala149.


Page 17 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In addition to the crystallographic and NMR studies reported thus far, the absence of the crystal structure of human PD-1 and differences between the affinity of the ligands led to an intensive molecular modeling study reported in 2015 by Viricel et al.64. To establish an accurate model of human PD-1, as well as to predict the most likely binding conformation to its ligands, the Viricel et al. methodology involved homology modeling to generate a human PD-1 structure, followed by molecular dynamics simulations to study the stability and conformations of the models generated, proteinprotein docking and binding energy analysis. Once the human PD-1 homology model was generated, the docking and binding energy calculations agreed with the empirical results described by Cheng and coworkers63, identifying the missing C’’ strand and a long flexible loop in the human PD-164. Additionally, the binding energies resulted in a higher affinity of PD-L2 to PD-1, rather than PD-L1, due to the extension of the PD-L2 binding surface compared with that of PD-L1. The crystallographic structures described failed to assess human PD-1 or the human PD-1/PD-L complex. Later, in 2015, Zak et al.65 disclosed the crystal structure of the human PD-1/PD-L1 complex (PDBID: 4ZQK, 2.45 Å) (Figure 3D), leading to a clear definition of murine and human PD-1 differences in the PD-1/PD-L1 binding interface, as well as the definition of hotspot pockets (Tables 2 and 3). This study demonstrated that the human complex formation involves significant structural flexibility and reorganization, in contrast to the corresponding murine PD165. The residues demonstrating the highest flexibility were Tyr68, Thr76, Leu122, Cys123, Gly124 and Ile134. The detailed analysis of receptor-ligand interactions led to the identification of hydrophobic and polar interactions, as well as hydrogen bonds covering Val64, Asn66, Tyr68, Gln75, Thr76 Ile126, Leu128, Ala132, Ile134, and Glu136 of PD-1. This study also led to the identification of hotspots for PD-1 and PD-L1 (Tables 2 and 3).

Table 2. PD-1 hot spots, residues and key interactions to target



Page 18 of 60

The crystal structure reported by Zak et al.65 and the approval of drugs targeting the PD-1 receptor were the turning point in the studies regarding the PD-1/PD-L complex. Recent studies are now focused on the receptor/drug complex and established interactions. Several crystal structures of monoclonal antibodies approved, in clinical trials or in development, (nivolumab, pembrolizumab, avelumab, KN035 and BMS936559) that bind to PD-1 and PD-L1 were resolved. Na et al.66 disclosed the PD1/pembrolizumab complex (PDBID: 5JXE). Another structure of this complex was reported by Horita et al.67, with a higher resolution (PDBID: 5B8C, 2.2 Å). Lee and coworkers39 resolved the structure of PD-1/nivolumab (PDBID: 5GGR, 3.3 Å) and PD1/pembrolizumab (PDBID: 5GGS 2.0, Å). In 2017, Tan et al.68 disclosed the structure of the PD-1/nivolumab complex (PDBID: 5WT9, 2.4 Å). Those three crystal structures currently available for the PD-1/pembrolizumab complex provide detailed and significant information on the binding site of pembrolizumab (Figure 5A)39,66,67. This receptor/drug interface interaction pocket


Page 19 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


includes fifteen direct hydrogen bonds between residues, fifteen water-mediated hydrogen bonds, two salt bridges, and hydrophobic interactions. In total, 26 residues of PD-1 are involved in the interaction39,66,67. A structural comparison between the PD1/pembrolizumab and PD-1/PD-L1 complexes revealed that the epitope recognized by pembrolizumab largely overlaps the residues responsible for the PD-1/PD-L1 interaction (Figure 5B). The PD-1/nivolumab complex was also assessed, and two crystal structures are accessible. These structures suggest numerous hydrogen bonds involving Pro28, Asp29, Arg30, Ser60, Leu128, Ala129, Pro130 Lys131, and Ala132 residues, and van der Waals contacts with Ser27, Pro28, Pro31, Glu61, Ala129, Pro130, Lys131, Ala132 and Gln133 of PD-139,68. Similar to the PD-1/pembrolizumab complex, the PD-1/nivolumab complex indicates that the epitope recognized by nivolumab largely overlaps the residues involved in the PD-1/PD-L1 interaction (Figure 5A).

Fig. 5 PD-1 blockade using antibodies. The CDR loops and β-strands of nivolumab and pembrolizumab are involved in PD-1 blockade. (A) Overall structure surface of the PD-1/nivolumab Fab complex (PDBID: 5GGR). Interface region (orange) of nivolumab Fab (light blue) and PD-1 (blue). The residues of the PD1/nivolumab interface involved in the PD-1/PD-L1 interaction are highlighted. (B) Overall structure surface of the PD-1/pembrolizumab Fab complex (PDBID: 5GGS). Interface region (orange) of pembrolizumab Fab (light blue) and PD-1 (blue). The residues of the PD-1/Pembrolizumab interface involved in the PD-1/PD-L1 interaction are highlighted.

Inhibition of the corresponding ligand PD-L1 was also envisioned, and initial crystallographic studies focused on the characterization PD-L1 itself, first in 2008, by Lin et al.60 (PDBID: 3BIS, 2.6 Å), and later in 2010 by Cheng and coworkers69 (PDBID: 3FN3, 2.7 Å).



Page 20 of 60

Successful inhibition of PD-L1 by monoclonal antibodies led to a detailed structural elucidation of the drug/receptor complex formed using approved or currently in development antibodies (Figure 6). The groups of Liu70, Lee39,71 and Zhang72 resolved the crystallographic structure of PD-L1/avelumab, PD-L1/BMS-936559, PD-L1/KN035 and PD-L1/durvalumab complexes with 3.2, 2.8, 1.7 and 2.7 Å resolution (PDBID: 5GRJ, 5GGT, 5JDS and 5X8M), respectively. Lee and coworkers39 demonstrated that the epitope of BMS-936559 occupies a large part of the PD-1 binding site (Figure 6A). In addition, high avidity, resulting from IgG bivalency, is expected due to the high expression level of PD-L1 in many cancer types, leading to efficient blockade of the PD1/PD-L1 interaction39. The structure of the PD-L1/avelumab complex reported by both Liu et al.70 and Lee et al.71 revealed that avelumab binds to PD-L1 using both heavy and light chains and occupy the epitope region corresponding to PD-1 binding (Figure 6B). The structure reported by Zhang et al.72 in 2017 was the PD-L1/KN035 complex. This inhibitor is a nanobody that dissociates the PD-1/PD-L1 interaction by occupying the epitope region corresponding to PD-1 binding, as reported previously. The authors also focused on the nanomolar binding affinity of KN035 to PD-L1. This is due to harnessing both hydrophobic and ionic interactions on the PD-L1 binding surface and flexibility of the CDR loops, which can interact with interface residues (Figure 6C)72. The last crystal structure reported was the complex of PD-L1, and the last approved monoclonal antibody was durvalumab by Lee et al.71 Durvalumab binds perpendicularly to PD-L1 using 16 residues interacting through several hydrogen bonds, salt bridges and hydrophobic interactions. Most of the key interactions are established in the epitope region corresponding to PD-1 binding (Figure 6D). Based on the exciting outcomes from the blockade of PD-1 and/or its ligands, this is an attractive approach to enhance antitumor immunity. Although first-generation compounds (monoclonal antibodies) have already reached the market, the development of new immune checkpoint inhibitors remains an ongoing effort. Thus, the use of small-molecule modulators targeting PD-1 and PD-L1 has been recently described as an alternative to monoclonal antibodies.


Page 21 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 6 PD-L1 blockade by antibodies (A) Overall structure surface of the PD-L1/BMS-936559 Fab complex (PDBID: 5GGT. Interface region of BMS-936559 Fab (light blue) and PD-L1 (teal). The residues of the PD-L1/BMS-936559 interface involved in the PD-1/PD-L1 interaction are highlighted using stick representation. (B) Overall structure surface of the PD-L1/avelumab Fab complex (PDBID: 5GRJ). Interface region (yellow) of avelumab Fab (light blue) and PD-L1 (teal). The residues of the PDL1/avelumab® interface involved in the PD-1/PD-L1 interaction are highlighted using stick representation. Hydrophobic core of PD-L1 (Ile54, Tyr56, Met115 and Tyr 123), critical for the interaction with PD-1 hydrophobic residues, is now occupied by similar hydrophobic residues of avelumab. (C) Overall structure surface of the PD-L1/KN-035 complex (PDBID: 5JDS). Interface region of KN-035 Fab (light blue) and PD-L1 (teal). The residues of PD-L1/KN-035 interface (yellow) are highlighted using stick representation. (F) Overall structure surface of the PD-L1/durvalumab Fab complex (PDBID: 5X8M). Interface region (yellow) of durvalumab Fab (light blue) and PD-L1 (teal). The residues of the PDL1/durvalumab interface involved in the PD-1/PD-L1 interaction are highlighted using stick representation.

A library of nonpeptidic small molecules based on a (2-methyl-3-biphenylyl)methanol scaffold was patented by Bristol-Myers Squibb as useful immunomodulators targeting the PD-1/PD-L1 pathway73,74 (Figure 7). However, no detailed information was provided. To characterize the Bristol-Myers Squibb compounds, several crystal structures of PD-L1 with (2-methyl-3-biphenylyl)methanol low-molecular-mass inhibitors have been resolved in 2016 and 2017 by Holak and coworkers75–77—PDBID:



Page 22 of 60

5J89, 5J8O, 5N2F, 5N2D, 5NIX and 5NIU—with 2.2, 2.3, 1.7, 2.4, 2.2 and 2.0 Å resolution, respectively. The crystal structures of (2-methyl-3-biphenylyl)methanol compounds 1 and 2 (Figure 7A) and PD-L1 complexes were the first to be resolved in 201675. Structure characterization by X-ray and NMR studies revealed the disruption of the PD-1/PD-L1 interaction, as well as the binding of 1 and 2 to PD-L1 and not to PD-1. Although the compounds described display poor drug-like properties, they were instrumental in providing insight into protein-inhibitor interactions, thus constituting a proof of concept that targeting the PD-1 pathway is achievable not only with antibodies. This study provided a detailed description of the binding site. It showed the interaction of the compounds with PD-L1 through the hydrophobic surface, which was involved in the PD-1/PD-L1 interaction. This was in accordance with the hypothesized hotspots previously reported by the same authors in 201565. The 2017 studies reported by Holak and coworkers76,77 enforced the mechanism of inhibition of

(2-methyl-3-biphenylyl)methanol

derivatives

by

inducing PD-L1

dimerization upon ligand binding (Figure 7B). The last crystal structures resolved involving the small molecules 3 and 4 revealed the significant flexibility of the ligand binding pocket of PD-L1 and the possibility to generate larger structures. Although the residues involved are equal to those already described, Tyr56 movement creates an unexpected hydrophobic tunnel (Figure 7D) using [3-(2,3-dihydro-1,4-benzodioxin-6yl)-2-methylphenyl]methanol (3). Together with the characterization of the PD-L1 binding site and small-molecule inhibitors described, Holak et al.77 reported a generation of improved small-molecule inhibitors based on the (2-methyl-3-biphenylyl)methanol scaffold. They could generate derivative 4 and evaluate its activity in vitro. Structural and biochemical results confirmed their activity, being the first optimized molecules described thus far.


Page 23 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 5 Active small molecule acting on PD-L1. (A) The scaffold (2-methyl-3-biphenylyl)methanol was designed by Bristol-Myers Squibb. Small-molecule inhibitors 1 (IC50: 18 nM), 2 (IC50: 146 nM) and 3 (IC50: 80 nM) are from the original patented library. Inhibitor 4 is one of the optimized compounds described by Holak et al.71,72 (B) PD-L1 inhibition. Surface representation of two molecules of PD-L1 (teal) bridged by the small-molecule inhibitor (green) (PDBID:5N2F). Inhibition occurs by small-molecule-induced PDL1 dimerization. (C) Surface representation of the PD-L1 binding pocket (teal). Close-up view of the PDL1 binding pocket, 3 (green) binds at the hydrophobic cavity (Tyr56, Met115, Ile116, Ala121 and Tyr123). Additional interactions of 3 are depicted in yellow dashes (PDBID:5N2F). (D) PD-L1 binding site flexibility. Surface representation of the PD-L1 structure (teal) with inhibitor 1 (yellow) and 3 (green). The movement of Tyr56 makes the pocket of PD-L1 accessible in both sites, regarding inhibitor 3 (green). In the structure of inhibitor 1 (yellow), the pocket is a deep hydrophobic tunnel.



Page 24 of 60

Other small molecules have been described as immune checkpoint inhibitors (Figure 8). Among these are the 1,2,4-oxadiazole (5) and 1,2,4-thiadiazole moieties, currently in phase 1 of clinical trials78.

Fig. 8 Other active scaffolds acting on PD-L1. Small-molecule inhibitor 5 is derived from 1,2,4oxadiazole- inhibitors, and compound 6 is a peptidomimetic inhibitor; both are from Aurigene Discovery Technologies Limited. Potent macrocyclic peptidic inhibitor 7 (IC50: 5.2 nM) is from Bristol-Myers Squibb.

However, besides being reported as inhibitors of the PD-1/PD-L1 interaction, there are no data supporting their action. The hypotheses raised thus far suggest that these compounds act on the PD-1/PD-L1 pathway, and no direct binding was detected. Many other small-molecule inhibitors based on peptidomimetics79–81 or macrocyclic 24 ACS Paragon Plus Environment

Page 25 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


peptides82–84 were also reported, proving that the overall inhibition of the PD-1/PD-L1 interaction (Figure 9) is a possible and promising therapeutic approach for cancer immunotherapy85. Holak et al.84 reported structural insights on bioactive macrocyclic peptide inhibitors of the PD-1/PD-L1 pathway. X-ray crystallography was used to resolve two crystal structures with 1.0 and 2.3 Å resolution (PDBID: 5O45 and 5O4Y). The crystal structures revealed a different binding mode to PD-L1 than (2-methyl-3biphenylyl)methanol derivatives, raising an alternative to design new small-molecule inhibitors. Structural characterization shows that macrocyclic peptides can bind at the interface of PD-L1 coinciding with the PD-1 binding site (Figure 9). The upper part of the binding surface consists of hydrophobic interactions, while the lower part is dominated by polar interactions. The structure also revealed no major conformational changes in PD-L1 upon inhibitor binding.

Fig. 9 Macrocyclic peptidic inhibitor (7)/PD-L1 complex (PDBID: 5O45). (A) Detailed view of the PD-L1 (teal)/peptide (7) (green) interaction. The hydrophobic side chains interact with the cleft characteristic for the “face-on” binding mode. The polar zone of the interaction includes two hydrogen bonds with Glu58 and Asp61 by the backbone amines of the peptide.

Structural elucidation of the PD-1/PD-L1 pathway has been extensive in the last few years. The latest crystal structures disclosed are essential to move forward in the development of new inhibitors, particularly small-molecule inhibitors. Although many crystal structures demand a structure-based approach, it is also clear that PD-1 and PD-L1 are proteins with a highly flexible binding pocket. In addition, (2-methyl-3biphenylyl)methanol derivatives, being the only small molecules acting on the ligand 25 ACS Paragon Plus Environment


Page 26 of 60

PD-L1, are prone to be optimized. Their optimization has been challenging, and a deeper understanding of the mechanism of inhibition is fundamental to designing new scaffolds that will result in the discovery of new molecules targeting this pathway. Table 3. PD-L1 hot spots, residues and key interactions to target

GITR

Glucocorticoid-induced TNFR-related protein (GITR), also known as TNFRSF18, belongs to the TNFR family and is expressed after TCR engagement in CD4+ and CD8+ T cells86,87. GITR is expressed at low levels on resting T cells, and it is upregulated 24-72 h after TCR engagement, remaining on the lymphocyte surface for several days. By contrast, Treg cells constitutively and intensely express GITR56,88. This receptor is responsible for mediating costimulation, leading to the enhancement of T-cell proliferation and effector functions. GITR is a type 1 transmembrane glycoprotein of 241 aa in length, comprising an extracellular domain (residues 26-162), a 26 ACS Paragon Plus Environment

Page 27 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


transmembrane domain (residues 163-183) and a cytoplasmic domain (residues 184241). The receptor shares 14-28% sequence identity with other members of the TNFR family, but its crystal structure or other structural studies have not yet been reported. GITR is activated by the GITR ligand (GITRL), which is highly expressed on activated APC and endothelial cells (Figure 1A)89,90. GITRL is a type 2 transmembrane glycoprotein of 199 aa in length, comprising a cytoplasmic domain (residues 23-50), a transmembrane domain (residues 51-71) and an extracellular domain (residues 72-199). GITR ligation protects T cells from the activation of induced cell death, leading to an increase in memory T cells. Studies have demonstrated that GITR ligation can overcome tolerance to self and tumor antigens, making it an attractive target for the development of new therapeutics for cancer immunotherapy91. The antitumor effect of GITR stimulation has been demonstrated in different mouse tumor models (CT26, Meth A, B16F10, RENCA-TGL)92–99. The monoclonal antibody TRX518 (GITR Inc), fusion protein MEDI1873 (MedImmune LLC) and others are GITR agonists currently in phase 1 clinical trials (Figure 1B).

Fig. 10 Structures of TNF-related proteins GITRL and 4-1BBL (PDBID: 3B93 and 2X29). (A) Ribbon representation of the GITRL monomer showing the classic two-layer jelly roll fold of the THD. The βstrands are labeled with inner and outer sheets comprising the AA’HCF and B’BGDE strands, respectively. (B) Ribbon representation of the GITRL trimer. The shape of the GITRL trimer resembles a blooming flower. (C) GITRL residues whose mutation affected receptor binding are mapped onto the GITRL monomer surface and are colored yellow.

To date, limited studies concerning the GITR-GITRL pathway were reported. There is no crystal structure for GITR or any 3D model that was generated. By contrast, the crystal structures of GIRTL have already been disclosed. Chattopadhyay et al.100 reported in 2007 the only available crystal structure of human GIRTL with 2.3 Å resolution (PDBID: 3B93). Later, in 2008, Zhou et al.101 and Chattopadhyay et al.102 reported murine GITRL 27 ACS Paragon Plus Environment


Page 28 of 60

structures with 1.75, 2.49 and 2.09 Å resolution (PDBID: 2Q8O, 3B9I and 2QDN), respectively. Chattopadhyay et al.103 in 2009 also disclosed another murine GITRL structure with an improved resolution (1.76 Å, PDBID: 3FC0). Table 4. GITRL hot spots, residues and key interactions to target

The crystal structure of human GIRTL revealed the typical mechanism recognizing TNF ligands, in which a trimeric assembly engages three monomeric receptors resulting in a threefold-symmetric complex with 3:3 ligand:receptor stoichiometry104. GITRL demonstrates a classic β-sandwich “jelly roll” topology, which self-assembles into a noncovalently associated homotrimer (Figure 12A-C). The solvent-exposed loops near the inter-subunit clefts form the receptor-recognition surface. This involves 10 residues, including several containing hydrophobic and aromatic side chains, namely, Lys61, Leu94, Leu96, Tyr98, Asn135 and Tyr164 of one monomer and Thr139, Glu141, Tyr164 and Leu170 of the other monomer. Additionally, the GITRL trimer interfaces are remarkably small and lack the tightly packed aromatic/hydrophobic residues commonly observed in traditional TNF trimer 28 ACS Paragon Plus Environment

Page 29 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


interfaces (Figure 13B). Although murine GITRL shares 51% sequence identity with human GITRL, it does not recognize the human receptor, and human GITRL does not bind to the murine receptor, due to altered recognition interfaces105,106. Murine crystal structures demonstrated unique structural features based on the different dimeric assembly of GITR/GITRL, supporting the idea of a distinct mechanism for receptor binding, as well as a distinct mechanism for signaling in mice100–103. Although immune checkpoint activators are already in clinical trials, characterization of the GITR-GITRL pathway is still very limited, and considerable developments are needed to achieve new therapeutic options targeting this pathway (Table 4). The development of new approaches can be prompt by improving the know-how on structural features of this pathway. 3.

Future perspectives: can lessons learned from structure-functionality studies lead to the development of novel combination cancer therapies with immune checkpoint receptors?

The significant knowledge provided by recent crucial advances on molecular oncology and tumor immunology has revealed important pathways. Immune checkpoints mediate cancer-immune cell crosstalk within the tumor site toward a tolerant microenvironment. Despite the limited information available on the structural analysis of these promising immune regulators, the clinical evidence obtained thus far may anticipate an unprecedented control of cancer disease. The adequate targeting and modulation of these cancer-related immune evasion receptors within the tumor microenvironment are highly desirable. To date, more than 700 clinical trials have focused on the evaluation of different combinations of immune checkpoint modulators, mainly involving anti-PD-1/PD-L1. Currently, the rationale behind combination therapy in preclinical evaluations is not well established. The deep knowledge on the structure-function relationship of immune checkpoint receptors can improve the rationale for suitable combination therapy approaches.



Page 30 of 60

The combination of anti-CTLA-4 and anti-PD-1 inhibitors is more effective than treatment with single agents, as reported in clinical trials for melanoma (e.g., NCT01844505- NCT01783938- NCT01927419)107,108. These promising results led to the approval of the combination in clinical use. In addition, this combination is currently being tested for several other cancer types (e.g., renal carcinoma NCT03274258NCT03297593, non-small cell lung carcinoma NCT03083691-NCT03262779, thyroid cancer NCT03246958, and Kaposi sarcoma NCT03219671). The combination of anti-PD-1 and anti-LAG-3 antibodies showed interesting evidence of synergism in preclinical evaluations109,110, and it is under clinical development for different types of cancer (NCT02658981, NCT02966548). LAG3 is among the next generation of coinhibitory receptors that is found on the cell surface of CD4+ and CD8+ T cells, Treg cells and a subset of natural killer (NK) cells. LAG3 has a higher binding affinity to MHC class II complex, compared with CD4

111,112

. LSECtin is a molecule

member of the DC-SIGN family found in many tumors and is shown to be another ligand of LAG3, supporting its role as a negative regulator of CD8+ and NK cell expansion within tumors112,113. Another emerging immune checkpoint receptor is TIM-3, expressed on IFN-Υproducing CD4+Th1, CTL cells, Treg, as well as on monocytes, NK cells and DC. Galectin9, phosphatidyl serine, high-mobility group protein B1 (HMGB1) and Caecam-1 are TIM-3 ligands, but their impact on the function this coinhibitory receptor is not fully known114. However, similar to other coinhibitory receptor proteins, recent studies demonstrated the potential synergistic effect of anti-TIM-3 antibodies when combined with other receptor blockade strategies. The combination of anti-PD-1 and anti-TIM-3 antibodies is in the early clinical development (phase I) for advanced or metastatic solid tumors (NCT02817633, NCT03099109, and NCT02608268). The anti-OX40 monoclonal antibody shows synergistic activity with anti-PD1 in ovarian cancer115. Interestingly, a recent study revealed the importance of the dosing schedule and order of the administration of the two antibodies, highlighting the advantages of a first treatment with the agonist antibody against OX40, followed by immune checkpoint inhibition by anti-PD-1116. This combination is currently in early stage


Page 31 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


clinical trials for different types of solid tumors, including breast cancer, melanoma or colorectal cancer (NCT02737475, NCT02528357). Anti-OX40 antibody is also used in combination with anti-PD-L1 (NCT02410512). Some investigations are being conducted using the combination of the 3 immune checkpoint modulators117 anti-OX40, tremelimumab, and durvalumab (NCT02705482). Even if a clear clinical efficacy has been achieved specifically using monoclonal antibodies against anti-PD-L1/PD-1 in advanced melanoma patients, only a small percentage of patients has responded, and the development of resistance has emerged as a drawback. In fact, not much is known regarding the mechanisms underlying the failure of these targeted checkpoint therapies. Most immune checkpoint modulator candidates under development and that have shown clear advantages in pre- and clinical investigations are monoclonal antibodies. However, small-molecule immune-oncology-based treatments are emerging as a new type of immunotherapy to modulate these immune checkpoint receptors. These new potential immunotherapies present economic advantages, constituting a cost-effective alternative. In addition, the possibility to be administered orally and easily combined with other approaches, such as indoximod® (NewLink Genetics), an IDO1 inhibitor in phase 2 clinical trials. Furthermore, small molecules can easily be modulated to improve their pharmacokinetic/pharmacodynamic properties, leading to more effective treatments. These low-molecular-weight inhibitors can indeed be chemically conjugated or physically entrapped in lipidic or polymeric systems. Additionally, the use of supramolecular deliver systems can improve the physicochemical properties of the drugs (i.e., solubility and stability) but also alter pharmacokinetics and biodistribution, leading to improved pharmacological activity118. This technology has already been successfully applied for antifungal, antibiotics and anticancer drugs 119. In the future, following the data released regarding the efficacy and safety of these small molecules currently in clinical trials, it will be undoubtedly valuable to compare the application of these small molecules and antibodies as immunotherapeutic options to fight cancer. 31 ACS Paragon Plus Environment


Page 32 of 60

Moreover, cancer is a multifactorial disease and being clear that the control of those malignant cells encompass combinatorial treatment targeting complementary pathways within tumor microenvironment. Development of small molecules can also open new avenues for new combination regimens. In fact, the design of a small molecule to target receptors at different cells, such as cancer and T cells, may constitute promising tools in the near future. This allows for the administration of lower doses, in addition to higher efficacy due to the close proximity of immune and targeted cancer cells. 4.

Conclusions

There is no doubt regarding the massive success of immune checkpoint modulators in cancer immunotherapy. This review explores the structural bases of the most promising targets among several receptors already identified (CTLA-4, PD-1 and GITR), opening new opportunities for the unique role that computational tools can have on the design of new and effective but safe targeted therapies. PD-1/PD-L1 is currently accepted as the most promising immune regulator, but other checkpoints cannot be underestimated and must be explored. In this review, exhaustive work was devoted to assemble meticulously and critically the structural characterization of immune checkpoint receptors and their modulation. Specific structural features have been considered: conformations, ligands, key interactions and residues, flexibility, gating or binding pockets/regions. The impact that these in silico studies could have on screening is particularly promising, further guiding the design and development of novel compounds with optimal binding affinities. Clearly, these structural insights of immune checkpoint receptors obtained through crystallographic or modeling studies will be of utmost importance in the development of novel agents targeting the receptors herein highlighted. It will guide not only in silico studies to identify novel modulators but will also be instrumental to advance cancer biology and tumor immunology fields. AUTHOR INFORMATION Corresponding authors: e-mail: Helena F Florindo, [email protected] and Rita C. Guedes, [email protected] Biographies 32 ACS Paragon Plus Environment

Page 33 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Rita C. Acúrcio received her B.S. (2012) in Chemistry at Faculty of Sciences in Porto University, and her M.S. (2014) in Organic Chemistry and Natural Products at Aveiro University. In 2015, she was a Research Fellow in the Molecular Microbiology and Biotechnology group at Research Institute for Medicines (iMed.ULisboa). She was awarded a Ph.D. fellowship in 2015 from the National Portuguese Science Foundation to pursue her Ph.D. research project at the Faculty of Pharmacy University of Lisbon, focused on the discovery and development of small-molecule modulators of key players within immune system, as novel therapeutic approaches against cancer. Anna Scomparin is a Research Associate in the laboratory of Prof. Ronit Satchi-Fainaro, Sackler Faculty of Medicine, Tel Aiv University. She has graduated her M.Sc. degree in Pharmaceutical Chemistry and Technology at the Department of Pharmaceutical Sciences, University of Padua in 2006. In 2010, she obtained her Ph.D. in Molecular Sciences at the University of Padua. In her studies, she acquired expertise in polymer chemistry, pharmaceutical sciences and tumor biology research. She was involved in the development of several nanomedicines for cancer therapy, ranging from polysaccharides-based polymer therapeutics, novel nanocarriers for oligonucleotides targeted specifically to tumor tissues, and polymer-based nanoparticles for melanoma targeted vaccination. Anna published 25 manuscripts, presented her research in 30 meetings worldwide and is an inventor on 3 filed patents. João Conniot is a postdoctoral researcher developing immunotherapeutic strategies for immune modulation, which have been assessed in several. In parallel, he started working on the scale-up of these novel nano-vaccines, as they showed to be promising immune modulation tools and potential off-the-shelf products. Conniot’s ongoing and future research is focused on the design, development and characterization of alternative therapeutic schemes for melanoma treatment, combining nano-vaccine platforms with immune checkpoint modulators and other therapies available in the clinic. The results of his research provide deep insight on the synergistic effect of combined targeting in cancer vaccines and immunotherapy, enabling improved outcomes for melanoma treatment strategies. Jorge A. R. Salvador has a degree in Pharmaceutical Sciences, a Master degree in Organic & Technological Chemistry, and a PhD in Pharmaceutical Chemistry. He is a Full Professor at Faculty of Pharmacy, University of Coimbra in Portugal. He is a Non33 ACS Paragon Plus Environment


Page 34 of 60

Executive Member of the Board of CHEM4FARMA, a start-up Pharmaceutical Company located in Portugal. His research activity has been focused on the development of new drugs, mainly for cancer treatment. He has published more than 90 papers, 12 book chapters and 10 patents (2 US Patents). He is the author/coauthor of 200 presentations in national and international/workshops. Over the last years, he has conducted several research projects in the field of pharmaceutical/medicinal chemistry which has contribute to his significant expertise in drug discovery. Ronit Satchi-Fainaro is a Full Professor and Chair of the Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Israel. Her research group has been developing novel strategies to advance the field of cancer angiogenesis and theranostic nanomedicines. She has published more than 100 papers and is named inventor on more than 45 patents. She was awarded numerous prestigious grants and prizes, among them JULUDAN Prize for the Advancement of Technology in Medicine, the Teva Pharmaceutical Industries Founders Award for the Discovery of new molecular mechanisms and targets for new therapeutic approaches, the European Research Council Consolidator Award and the Saban Family Foundation-Melanoma Research Alliance Team Science Award. Helena F. Florindo graduated in Pharmaceutical Sciences in 2003 (University of Lisbon) and obtained her Ph.D. degree in Pharmaceutical Tecnhology in 2008 (University of Lisbon), in collaboration with the University of London. Currently, she is an Assistant Professor at the Faculty of Pharmacy, University of Lisbon. She is the Group Leader of the BioNanoSciences – Drug Delivery & Immunotherapy research group, at the Research Institute for Medicines. Both nanotechnology and immune-oncology fields have motivated her multidisciplinary research focused on the rational development of functionalized nanobiomaterials as novel immunotherapies for cancer treatment. It includes the characterization of the anti-tumor effects induced by the combination of nano-vaccines with nano-therapeutics designed to modulate the functions of key cells within tumor microenvironment, such as T cells, myeloid-derived cells and tumor cells. Rita C. Guedes did her studies in chemistry at University of Lisbon, where she also completed her PhD degree in Chemistry (Computational Chemistry) in 2003. Currently, she is an Assistant Professor at the department of medicinal chemistry at the Faculty of Pharmacy, University of Lisbon. She participates on several funded Doctoral and 34 ACS Paragon Plus Environment

Page 35 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Master Programs focused on medicinal chemistry (infectious, oncologic, and neurodegenerative diseases) on a national and international level. She has published more than 70 papers. Her research lab at iMed.ULisboa (Universidade de Lisboa) identifies and optimizes small molecules to target proteins involved in inflammation, cancer and infectious diseases using computational methods like molecular docking, virtual screening, homology modeling, pharmacophore modeling, de novo design, molecular dynamics simulations and quantum mechanics calculations for drug discovery campaigns.

ACKNOWLEDGEMENTS RA and JC are supported by the Fundação para a Ciência e a Tecnologia, Ministério da

Ciência,

Tecnologia

e

Ensino

Superior

(FCT-MCTES)

(PhD

grants

PD/BD/128238/2016 and SFRH/BD/87150/2012). The authors thank the funding received from the European Structural & Investment Funds through the COMPETE Programme and from National Funds through FCT under the Programme grant SAICTPAC/0019/2015 and UID/DTP/04138/2013 (HF, RG). The MultiNano@MBM

project was supported by The Israeli Ministry of Health, and FCT-MCTES, under the frame of EuroNanoMed-II (ENMed/0051/2016; HF, RS-F).

ABBREVIATIONS USED CTLA-4, cytotoxic T-lymphocyte-associated antigen 4; PD-1, programmed cell death protein 1; PD-L1, programmed cell death protein ligand 1; TNF, tumor necrosis factor; GITR, glucocorticoid-induced TNFR-related protein; GITRL, glucocorticoid-induced TNFR-related ligand; LAG3, Lymphocyte-activation gene 3; TIM3, T-cell immunoglobulin and mucin-domain containing-3; ADMET, absorption, distribution, metabolism, excretion, and toxicity; CADD, computer-assisted drug design; Treg, T regulatory cells; NMR, nuclear magnetic resonance; APC, antigen-presenting cells; IgG, immunoglobulin G; NK, natural killer; DC, dendritic cells; ADDC, antibody-dependent cell-mediated cytotoxicity; ADC, complement-dependent cytotoxicity; NK, natural killer; HMGB1, high-mobility group protein B1.

REFERENCES (1)

Balkwill, F.; Mantovani, A. Inflammation and Cancer: Back to Virchow? Lancet 2001, 357, 539–545. 35 ACS Paragon Plus Environment


Page 36 of 60

(2)

Coley, W. B. II. Contribution to the Knowledge of Sarcoma. Ann. Surg. 1891, 14, 199–220.

(3)

Makkouk, A.; Weiner, G. J. Cancer Immunotherapy and Breaking Immune Tolerance: New Approaches to an Old Challenge. Cancer Res. 2015, 75, 5–10.

(4)

Zou, W.; Chen, L. Inhibitory B7-Family Molecules Microenvironment. Nat. Rev. Immunol. 2008, 8, 467–477.

(5)

Chen, L.; Flies, D. B. Molecular Mechanisms of T Cell Co-Stimulation and CoInhibition. Nat. Rev. Immunol. 2013, 13, 227–242.

(6)

Pardoll, D. M. The Blockade of Immune Checkpoints in Cancer Immunotherapy. Nat. Rev. Cancer 2012, 12, 252–264.

(7)

Sharma, P.; Allison, J. P. Immune Checkpoint Targeting in Cancer Therapy: Toward Combination Strategies with Curative Potential. Cell 2015, 161, 205– 214.

(8)

Topalian, S. L.; Drake, C. G.; Pardoll, D. M. Immune Checkpoint Blockade: A Common Denominator Approach to Cancer Therapy. Cancer Cell 2015, 27, 451– 461.

(9)

Ira Mellman, George Coukos, G. D. Cancer Immunotherapy Comes of Age. J. Clin. Oncol. 2011, 29, 4828–4836.

(10)

Page, D. B.; Postow, M. A.; Callahan, M. K.; Allison, J. P.; Wolchok, J. D. Immune Modulation in Cancer with Antibodies. Annu. Rev. Med. 2014, 65, 185–202.

(11)

Baldo, B. A. Adverse Events to Monoclonal Antibodies Used for Cancer Therapy: Focus on Hypersensitivity Responses. Oncoimmunology 2013, 2, e26333.

(12)

Michot, J. M.; Bigenwald, C.; Champiat, S.; Collins, M.; Carbonnel, F.; PostelVinay, S.; Berdelou, A.; Varga, A.; Bahleda, R.; Hollebecque, A.; Massard, C.; Fuerea, A.; Ribrag, V.; Gazzah, A.; Armand, J. P.; Amellal, N.; Angevin, E.; Noel, N.; Boutros, C.; Mateus, C.; Robert, C.; Soria, J.C.; Marabelle, A.; Lambotte, O.; Immune-Related Adverse Events with Immune Checkpoint Blockade: A Comprehensive Review. Eur. J. Cancer 2016, 54, 139–148.

(13)

Kroschinsky, F.; Stölzel, F.; von Bonin, S.; Beutel, G.; Kochanek, M.; Kiehl, M.; Schellongowski, P.; Intensive Care in Hematological and Oncological Patients (iCHOP) Collaborative Group. New Drugs, New Toxicities: Severe Side Effects of Modern Targeted and Immunotherapy of Cancer and Their Management. Crit. Care 2017, 21, 89.

(14)

Adams, J. L.; Smothers, J.; Srinivasan, R.; Hoos, A. Big Opportunities for Small Molecules in Immuno-Oncology. Nat. Rev. Drug Discov. 2015, 14, 603–622.

(15)

Arlauckas, S. P.; Garris, C. S.; Kohler, R. H.; Kitaoka, M.; Cuccarese, M. F.; Yang, K. S.; Miller, M. A.; Carlson, J. C.; Freeman, G. J.; Anthony, R. M.; Weissleder, R.; Pittet, M. J. In Vivo Imaging Reveals a Tumor-Associated Macrophage – Mediated Resistance Pathway in Anti – PD-1 Therapy. Sci. Trans. Med. 2017, 9, eaal3604.

in

the

Tumour


Page 37 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(16)

Liao, C.; Sitzmann, M.; Pugliese, A.; Nicklaus, M. C. Software and Resources for Computational Medicinal Chemistry. Future Med. Chem. 2011, 3, 1057–1085.

(17)

Devi, R. V.; Sathya, S. S.; Coumar, M. S. Evolutionary Algorithms for de Novo Drug Design - A Survey. Appl. Soft Comput. J. 2015, 27, 543–552.

(18)

Triggle, D. Comprehensive Medicinal Chemistry II: Computer-Assisted Drug Design; Elsevier Science, 2006.

(19)

Schwartz, R. H. Costimulation of T Lymphocytes: The Role of CD28, CTLA-4, and B7/BB1 in Interleukin-2 Production and Immunotherapy. Cell 1992, 71, 1065– 1068.

(20)

Lenschow, D. J.; Walunas, T. L.; Jeffrey, A. CD28 / B7 System of T Cell Costimulation. Annu. Rev. Immunol. 1996, 14, 233–258.

(21)

Rudd, C. E.; Taylor, A.; Schneider, H. CD28 and CTLA-4 Coreceptor Expression and Signal Transduction. Immunol. Rev. 2009, 229, 12–26.

(22)

Brunet, J. F.; Denizot, F.; Luciani, M. F.; Roux-Dosseto, M.; Suzan, M.; Mattei, M. G.; Golstein, P. A New Member of the Immunoglobulin Superfamily-CTLA-4. Nature 1987, 328, 267–270.

(23)

Dariavach, P.; Mattéi, M. G.; Golstein, P.; Lefranc, M. P. Human Ig Superfamily CTLA-4 Gene: Chromosomal Localization and Identity of Protein Sequence between Murine and Human CTLA-4 Cytoplasmic Domains. Eur. J. Immunol. 1988, 18, 1901–1905.

(24)

Lindsten, T.; Lee, K.; Harris, E.; Petryniak, B.; Craighead, N. Characterization of CTLA-4 Structure and Expression on Human T Cells. J. Immunol. Immunol. 1993, 151, 3489–3499.

(25)

Hathcock, K. S.; Laszlo, G.; Dickler, H. B.; Bradshaw, J.; Linsley, P.; Hodes, R. J. Identification of an Alternative CTLA-4 Ligand Costimulatory for T Cell Activation. Science 1993, 262, 905–907.

(26)

Linsley, P. S.; Clark, E. A.; Ledbetter, J. A. T-Cell Antigen CD28 Mediates Adhesion with B Cells by Interacting with Activation Antigen B7/BB-1. Proc. Natl. Acad. Sci. U. S. A. 1990, 87, 5031–5035.

(27)

Azuma, M.; Ito, D.; Yagita, H.; Okumura, K.; Phillips, J. H.; Lanier, L. L.; Somoza, C. B70 Antigen Is a Second Ligand for CTLA-4 and CD28. Nature 1993, 366, 76–79.

(28)

Freeman, G. J.; Gribben, J. G.; Boussiotis, V.; Daley, J.; Gray, G.; Nadler, L. M. Activated human B lymphocytes express three CTLA-4 counterreceptors that costimulate T-cell activation. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 11059– 11063.

(29)

Linsley, P. S.; Brady, W.; Urnes, M.; Grosmaire, L. S.; Damle, N. K.; Ledbetter, J. A. CTLA-4 Is a Second Receptor for the B Cell Activation Antigen B7. J Exp Med 1991, 174, 561–569.

(30)

Tivol, E. A.; Borriello, F.; Schweitzer, A. N.; Lynch, W. P.; Bluestone, J. A.; Sharpe, A. H. Loss of CTLA-4 Leads to Massive Lymphoproliferation and Fatal Multiorgan 37 ACS Paragon Plus Environment


Page 38 of 60

Tissue Destruction, Revealing a Critical Negative Regulatory Role of CTLA-4. Immunity 1995, 3, 541–547. (31)

Waterhouse, P.; Penninger, J.; Timms, E.; Wakeham, A.; Shahinian, A.; Lee, K.; Thompson, C.; Griesser, H.; Mak, T. Lymphoproliferative Disorders with Early Lethality in Mice Deficient in Ctla-4. Science 1995, 270, 985–987.

(32)

Bajorath, J.; Linsley, P. S. Molecular Modeling of Immunoglobulin Superfamily Proteins : Predicting the Three Dimensional Structure of the Extracellular Domain of CTLA-4 ( CD152 ). Pharm. Res. 1997, 4, 117–123.

(33)

Metzler, W. J.; Bajorath, J.; Fenderson, W.; Shaw, S.-Y.; Constantine, K. L.; Naemura, J.; Leytze, G.; Peach, R. J.; Lavoie, T. B.; Mueller, L.; Linsley, P. S. Solution Structure of Human CTLA-4 and Delineation of a CD80/CD86 Binding Site Conserved in CD28. Nat. Struct. Biol. 1997, 4, 527.

(34)

Ostrov, D.; Shi, W.; Schwartz, J.-C.; Almo, S.; Nathenson, S. Structure of Murine CTLA-4 and Its Role in Modulating T Cell Respnsiveness. Science 2000, 290, 816– 819.

(35)

Stamper, C. C.; Zhang, Y.; Tobin, J. F.; Erbe, D. V; Ikemizu, S.; Davis, S. J.; Stahl, M. L.; Seehra, J.; Somers, W. S.; Mosyak, L. Crystal Structure of the B7-1/CTLA-4 Complex That Inhibits Human Immune Responses. Nature 2001, 410, 608–611.

(36)

Schwartz, J. C.; Zhang, X.; Fedorov, a a; Nathenson, S. G.; Almo, S. C. Structural Basis for Co-Stimulation by the Human CTLA-4/B7-2 Complex. Nature 2001, 410, 604–608.

(37)

Schönfeld, D.; Matschiner, G.; Chatwell, L.; Trentmann, S.; Gille, H.; Hülsmeyer, M.; Brown, N.; Kaye, P. M.; Schlehuber, S.; Hohlbaum, a M.; Skerra, a. An Engineered Lipocalin Specific for CTLA-4 Reveals a Combining Site with Structural and Conformational Features Similar to Antibodies. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 8198–8203.

(38)

Yu, C.; Sonnen, A. F. P.; George, R.; Dessailly, B. H.; Stagg, L. J.; Evans, E. J.; Orengo, C. A.; Stuart, D. I.; Ladbury, J. E.; Ikemizu, S.; Gilbert, R. J. C.; Davis, S. J. Rigid-Body Ligand Recognition Drives Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) Receptor Triggering. J. Biol. Chem. 2011, 286, 6685–6696.

(39)

Lee, J. Y.; Lee, H. T.; Shin, W.; Chae, J.; Choi, J.; Kim, S. H.; Lim, H.; Won Heo, T.; Park, K. Y.; Lee, Y. J.; Ryu, S. E.; Son, J. Y.; Lee, J. U.; Heo, Y.-S. Structural Basis of Checkpoint Blockade by Monoclonal Antibodies in Cancer Immunotherapy. Nat. Commun. 2016, 7, 13354.

(40)

Ramagopal, U. A.; Liu, W.; Garrett-Thomson, S. C.; Bonanno, J. B.; Yan, Q.; Srinivasan, M.; Wong, S. C.; Bell, A.; Mankikar, S.; Rangan, V. S.; Deshpande, S.; Korman, A. J.; Almo, S. C. Structural Basis for Cancer Immunotherapy by the First-in-Class Checkpoint Inhibitor Ipilimumab. Proc. Natl. Acad. Sci. 2017, 114, E4223-E4232.

(41)

He, M.; Chai, Y.; Qi, J.; Zhang, C. W. H.; Tong, Z. Remarkably Similar CTLA-4 Binding Properties of Therapeutic Ipilimumab and Tremelimumab Antibodies. 38 ACS Paragon Plus Environment

Page 39 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Oncotarget 2017, 8, 67129–67139. (42)

Romano, E.; Kusio-kobialka, M.; Foukas, P. G.; Baumgaertner, P.; Meyer, C. Ipilimumab-dependent cell-mediated cytotoxicity of regulatory T cells ex vivo by nonclassical monocytes in melanoma patients. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 6140-6145.

(43)

Furness, A. J. S.; Vargas, F. A.; Peggs, K. S.; Quezada, S. A. Impact of Tumour Microenvironment and Fc Receptors on the Activity of Immunomodulatory Antibodies. Trends Immunol. 2014, 35, 290–298.

(44)

Uvebrant, K.; Da Graca Thrige, D.; Rosen, A.; Akesson, M.; Berg, H.; Walse, B.; Bjork, P. Discovery of Selective Small-Molecule CD80 Inhibitors. J. Biomol. Screen. 2007, 12, 464–472.

(45)

Huxley, P.; Sutton, D. H.; Debnam, P.; Matthews, I. R.; Brewer, J. E.; Rose, J.; Trickett, M.; Williams, D. D.; Andersen, T. B.; Classon, B. J. High-Affinity Small Molecule Inhibitors of T Cell Costimulation: Compounds for Immunotherapy. Chem. Biol. 2004, 11, 1651–1658.

(46)

Terme, M.; Ullrich, E.; Aymeric, L.; Meinhardt, K.; Desbois, M.; Delahaye, N.; Viaud, S.; Ryffel, B.; Yagita, H.; Kaplanski, G.; Prévost-Blondel, A.; Kato, M.; Schultze, J. L.; Tartour, E.; Kroemer, G.; Chaput, N.; Zitvogel, L. IL-18 Induces PD1-Dependent Immunosuppression in Cancer. Cancer Res. 2011, 71, 5393–5399.

(47)

Fanoni, D.; Tavecchio, S.; Recalcati, S.; Balice, Y.; Venegoni, L.; Fiorani, R.; Crosti, C.; Berti, E. New Monoclonal Antibodies against B-Cell Antigens: Possible New Strategies for Diagnosis of Primary Cutaneous B-Cell Lymphomas. Immunol. Lett. 2011, 134, 157–160.

(48)

Freeman, G. J.; Long, a J.; Iwai, Y.; Bourque, K.; Chernova, T.; Nishimura, H.; Fitz, L. J.; Malenkovich, N.; Okazaki, T.; Byrne, M. C.; Horton, H. F.; Fouser, L.; Carter, L.; Ling, V.; Bowman, M. R.; Carreno, B. M.; Collins, M.; Wood, C. R.; Honjo, T. Engagement of the PD-1 Immunoinhibitory Receptor by a Novel B7 Family Member Leads to Negative Regulation of Lymphocyte Activation. J. Exp. Med. 2000, 192, 1027–1034.

(49)

Keir, M. E.; Liang, S. C.; Guleria, I.; Latchman, Y. E.; Qipo, A.; Albacker, L. A.; Koulmanda, M.; Freeman, G. J.; Sayegh, M. H.; Sharpe, A. H. Tissue Expression of PD-L1 Mediates Peripheral T Cell Tolerance. J Exp Med 2006, 203, 883–895.

(50)

Dong, H.; Strome, S. E.; Salomao, D. R.; Tamura, H.; Hirano, F.; Flies, D. B.; Roche, P. C.; Lu, J.; Zhu, G.; Tamada, K.; Lennon, V. a; Celis, E.; Chen, L. TumorAssociated B7-H1 Promotes T-Cell Apoptosis: A Potential Mechanism of Immune Evasion. Nat. Med. 2002, 8, 793–800.

(51)

Blank, C.; Brown, I.; Peterson, A. C.; Transgenic, T. C. R.; Cells, C. D. T.; Spiotto, M.; Iwai, Y.; Honjo, T.; Gajewski, T. F. PD-L1 / B7H-1 Inhibits the Effector Phase of Tumor Rejection by T Cell Receptor ( TCR ) Transgenic CD8 + T Cells PD-L1 / B7H-1 Inhibits the Effector Phase of Tumor Rejection by T Cell Receptor. Cancer Res. 2004, 64, 1140–1145.



Page 40 of 60

(52)

Greenwald, R. J.; Freeman, G. J.; Sharpe, A. H. The B7 Family Revisited. Annu Rev Immunol. 2005, 23, 515–548.

(53)

Francisco, L. M.; Salinas, V. H.; Brown, K. E.; Vanguri, V. K.; Freeman, G. J.; Kuchroo, V. K.; Sharpe, A. H. PD-L1 Regulates the Development, Maintenance, and Function of Induced Regulatory T Cells. J. Exp. Med. 2009, 206, 3015–3029.

(54)

Dong, H.; Zhu, G.; Tamada, K.; Chen, L. B7-H1, a Third Member of the B7 Family, Co-Stimulates T-Cell Proliferation and Interleukin-10 Secretion. Nat. Med. 1999, 5, 1365–1369.

(55)

Tseng, S. Y.; Otsuji, M.; Gorski, K.; Huang, X.; Slansky, J. E.; Pai, S. I.; Shalabi, A.; Shin, T.; Pardoll, D. M.; Tsuchiya, H. B7-DC, a New Dendritic Cell Molecule with Potent Costimulatory Properties for T Cells. J. Exp. Med. 2001, 193, 839–846.

(56)

Shimizu, J.; Yamazaki, S.; Takahashi, T.; Ishida, Y.; Sakaguchi, S. Stimulation of CD25(+)CD4(+) Regulatory T Cells through GITR Breaks Immunological SelfTolerance. Nat. Immunol. 2002, 3, 135–142.

(57)

Zhong, X.; Tumang, J. R.; Gao, W.; Bai, C.; Rothstein, T. L.; Rothstein, T. L. PD-L2 Expression Extends beyond Dendritic Cells/macrophages to B1 Cells Enriched for VH11/VH12 and Phosphatidylcholine Binding. Eur. J. Immunol. 2007, 37, 2405– 2410.

(58)

Zhang, X.; Schwartz, J. C.; Guo, X.; Bhatia, S.; Cao, E.; Lorenz, M.; Cammer, M.; Chen, L.; Zhang, Y.; Edidin, M.; Nathenson, S. G.; Almo, S. C. Structural and Functional Analysis of the of the costimulatory receptor programmed death-1. Immunity 2004, 20, 337–347.

(59)

Wang, S.; Bajorath, J.; Flies, D. B.; Dong, H.; Honjo, T.; Chen, L. Molecular Modeling and Functional Mapping of B7-H1 and B7-DC Uncouple Costimulatory Function from PD-1 Interaction. J. Exp. Med. 2003, 197, 1083–1091.

(60)

Lin, D. Y. W.; Tanaka, Y.; Iwasaki, M.; Gittis, A. G.; Su, H. P.; Mikami, B.; Okazaki, T.; Honjo, T.; Minato, N.; Garboczi, D. N. The PD-1/PD-L1 Complex Resembles the Antigen-Binding Fv Domains of Antibodies and T Cell Receptors. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 3011–3016.

(61)

Lázár-Molnár, E.; Yan, Q.; Cao, E.; Ramagopal, U.; Nathenson, S. G.; Almo, S. C. Crystal Structure of the Complex between Programmed Death-1 (PD-1) and Its Ligand PD-L2. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 10483–10488.

(62)

Freeman, G. J. Structures of PD-1 with Its Ligands: Sideways and Dancing Cheek to Cheek. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 10275–10276.

(63)

Cheng, X.; Veverka, V.; Radhakrishnan, A.; Waters, L. C.; Muskett, F. W.; Morgan, S. H.; Huo, J.; Yu, C.; Evans, E. J.; Leslie, A. J.; Griffiths, M.; Stubberfield, C.; Griffin, R.; Henry, A. J.; Jansson, A.; Ladbury, J. E.; Ikemizu, S.; Carr, M. D.; Davis, S. J. Structure and Interactions of the Human Programmed Cell Death 1 Receptor. J. Biol. Chem. 2013, 288, 11771–11785.

(64)

Viricel, C.; Ahmed, M.; Barakat, K. Human PD-1 Binds Differently to Its Human Ligands: A Comprehensive Modeling Study. J. Mol. Graph. Model. 2015, 57, 131– 40 ACS Paragon Plus Environment

Page 41 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


142. (65)

Zak, K. M.; Kitel, R.; Przetocka, S.; Golik, P.; Guzik, K.; Musielak, B.; Dömling, A.; Dubin, G.; Holak, T. A. Structure of the Complex of Human Programmed Death 1, PD-1, and Its Ligand PD-L1. Structure 2015, 23, 2341–2348.

(66)

Na, Z.; Yeo, S. P.; Bharath, S. R.; Bowler, M. W.; Balıkçı, E.; Wang, C.-I.; Song, H. Structural Basis for Blocking PD-1-Mediated Immune Suppression by Therapeutic Antibody Pembrolizumab. Cell Res. 2016, 27, 147–150.

(67)

Horita, S.; Nomura, Y.; Sato, Y.; Shimamura, T.; Iwata, S.; Nomura, N. HighResolution Crystal Structure of the Therapeutic Antibody Pembrolizumab Bound to the Human PD-1. Sci. Rep. 2016, 6, 35297.

(68)

Tan, S.; Zhang, H.; Chai, Y.; Song, H.; Tong, Z.; Wang, Q.; Qi, J.; Wong, G.; Zhu, X.; Liu, W. J.; Gao, S.; Wang, Z.; Shi, Y.; Yang, F.; Gao, G. F.; Yan, J. An Unexpected NTerminal Loop in PD-1 Dominates Binding by Nivolumab. Nat. Commun. 2017, 8, 14369.

(69)

Chen, Y.; Liu, P.; Gao, F.; Cheng, H.; Qi, J.; Gao, G. F. A Dimeric Structure of PDL1: Functional Units or Evolutionary Relics? Protein Cell 2010, 1, 153–160.

(70)

Liu, K.; Tan, S.; Chai, Y.; Chen, D.; Song, H.; Zhang, C. W.-H.; Shi, Y.; Liu, J.; Tan, W.; Lyu, J.; Gao, S.; Yan, J.; Qi, J.; Gao, G. F. Structural Basis of Anti-PD-L1 Monoclonal Antibody Avelumab for Tumor Therapy. Cell Res. 2016, 27, 151– 153.

(71)

Lee, H. T.; Lee, J. Y.; Lim, H.; Lee, S. H.; Moon, Y. J.; Pyo, H. J.; Ryu, S. E.; Shin, W.; Heo, Y. S. Molecular Mechanism of PD-1/PD-L1 Blockade via Anti-PD-L1 Antibodies Atezolizumab and Durvalumab. Sci. Rep. 2017, 7, 5532.

(72)

Zhang, F.; Wei, H.; Wang, X.; Bai, Y.; Wang, P.; Wu, J.; Jiang, X.; Wang, Y.; Cai, H.; Xu, T.; Zhou, A. Structural Basis of a Novel PD-L1 Nanobody for Immune Checkpoint Blockade. Cell Discov. 2017, 3, 17004.

(73)

Chupak, L. S.; Zheng, X. Compounds Useful as Immunomodulators. WO2015034820A1, 2015.

(74)

Abdel-Magid, A. F. Inhibitors of the PD-1/PD-L1 Pathway Can Mobilize the Immune System: An Innovative Potential Therapy for Cancer and Chronic Infections. ACS Med. Chem. Lett. 2015, 6, 489–490.

(75)

Zak, K. M.; Grudnik, P.; Guzik, K.; Zieba, B. J.; Musielak, B.; Dömling, A.; Dubin, G.; Holak, T. A. Structural Basis for Small Molecule Targeting of the Programmed Death Ligand 1 ( PD-L1 ). Oncotarget 2016, 7, 30323-30335.

(76)

Guzik, K.; Zak, K. M.; Grudnik, P.; Magiera, K.; Musielak, B.; Törner, R.; Skalniak, L.; Dömling, A.; Dubin, G.; Holak, T. A. Small-Molecule Inhibitors of the Programmed Cell Death-1/Programmed Death-Ligand 1 (PD-1/PD-L1) Interaction via Transiently Induced Protein States and Dimerization of PD-L1. J. Med. Chem. 2017, 60, 5857-5867.

(77)

Skalniak, L.; Zak, K. M.; Guzik, K.; Magiera, K.; Musielak, B.; Pachota, M.; 41 ACS Paragon Plus Environment


Page 42 of 60

Szelazek, B.; Kocik, J.; Grudnik, P.; Tomala, M.; Krzanik, S.; Pyrc, K.; Dömling, A.; Dubin, G.; Holak, T. A. Small-Molecule Inhibitors of PD-1/PD-L1 Immune Checkpoint Alleviate the PD-L1-Induced Exhaustion of T-Cells. Oncotarget 2017, 8, 72167–72181. (78)

Sasikumar, P. G. N.; Ramachandra, M.; Naremaddepalli, S. S. S. 1,2,4-Oxadiazole Derivatives as Immunomodulators. US20150073024, 2015.

(79)

Sasikumar, P. G. N.; Ramachandra, M.; Vadlamani, S. K.; Vemula, K. R.; Satyam, L. K.; Subbarao, K.; Shrimali, R. K.; Kandepu, S. Immunosuppression Modulating Compounds. EP2585099A2, 2013.

(80)

Sasikumar, P. G. N.; Ramachandra, M.; Naremaddepalli, S. S. S. Peptidomimetic Compounds as Immunomodulators. WO2013132317A8, 2013.

(81)

Sasikumar, P. G. N.; Ramachandra, M.; Vadlamani, S. K.; Shrimali, K. R.; Subbarao, K. Therapeutic Compounds for Immunomodulation. WO2012168944A1, 2012.

(82)

Miller, M. M.; Mapelli, C.; Allen, M. P.; Bowsher, M. S.; Boy, K. M.; Gillis, E. P.; Langley, D. R.; Mull, E.; Poirier, M. A.; Sanghvi, N. Macrocyclic Inhibitors of the Pd-1/pd-l1 and cd80(b7-1)/pd-l1 Protein/protein Interactions. WO2014151634A1, 2014.

(83)

Sasikumar, P. G. N.; Ramachandra, M.; Naremaddepalli, S. S. S. Cyclic Peptidomimetic Compounds as Immunomodulators. WO2015033303A1 2015.

(84)

Magiera-Mularz, K.; Skalniak, L.; Zak, K. M.; Musielak, B.; Rudzinska-Szostak, E.; Berlicki, Ł.; Kocik, J.; Grudnik, P.; Sala, D.; Zarganes-Tzitzikas, T.; Shaabani, S.; Dömling, A.; Dubin, G.; Holak, T. A. Bioactive Macrocyclic Inhibitors of the PD1/PD-L1 Immune Checkpoint. Angew. Chemie - Int. Ed. 2017, 56, 13732–13735.

(85)

Weinmann, H. Cancer Immunotherapy: Selected Targets and Small-Molecule Modulators. ChemMedChem 2016, 11, 450–466.

(86)

Nocentini, G.; Giunchi, L.; Ronchetti, S.; Krausz, L. T.; Bartoli, a; Moraca, R.; Migliorati, G.; Riccardi, C. A New Member of the Tumor Necrosis Factor/nerve Growth Factor Receptor Family Inhibits T Cell Receptor-Induced Apoptosis. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 6216–6221.

(87)

Nocentini, G.; Ronchetti, S.; Cuzzocrea, S.; Riccardi, C. GITR/GITRL: More than an Effector T Cell Co-Stimulatory System. Eur. J. Immunol. 2007, 37, 1165–1169.

(88)

Shevach, E. M.; Stephens, G. L. The GITR-GITRL Interaction: Co-Stimulation or Contrasuppression of Regulatory Activity? Nat. Rev. Immunol. 2006, 6, 613–618.

(89)

Snell, L. M.; Lin, G. H. Y.; McPherson, A. J.; Moraes, T. J.; Watts, T. H. T-Cell Intrinsic Effects of GITR and 4-1BB during Viral Infection and Cancer Immunotherapy. Immunol. Rev. 2011, 244, 197–217.

(90)

Tone, M.; Tone, Y.; Adams, E.; Yates, S. F.; Frewin, M. R.; Cobbold, S. P.; Waldmann, H. Mouse Glucocorticoid-Induced Tumor Necrosis Factor Receptor Ligand Is Costimulatory for T Cells. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 42 ACS Paragon Plus Environment

Page 43 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


15059–15064. (91)

Schaer, D. A.; Murphy, J. T.; Wolchok, J. D. Modulation of GITR for Cancer Immunotherapy. Curr. Opin. Immunol. 2012, 24, 217–224.

(92)

Hu, P.; Arias, R. S.; Sadun, R. E.; Nien, Y. C.; Zhang, N.; Sabzevari, H.; Lutsiak, M. E. C.; Khawli, L. A.; Epstein, A. L. Construction and Preclinical Characterization of Fc-mGITRL for the Immunotherapy of Cancer. Clin. Cancer Res. 2008, 14, 579– 588.

(93)

Cote, A. L.; Zhang, P.; O’Sullivan, J. A.; Jacobs, V. L.; Clemis, C. R.; Sakaguchi, S.; Guevara-Patino, J. A.; Turk, M. J. Stimulation of the Glucocorticoid-Induced TNF Receptor Family-Related Receptor on CD8 T Cells Induces Protective and HighAvidity T Cell Responses to Tumor-Specific Antigens. J. Immunol. 2011, 186, 275–283.

(94)

Ramirez-Montagut, T.; Chow, A.; Hirschhorn-Cymerman, D.; Terwey, T. H.; Kochman, A. A.; Lu, S.; Miles, R. C.; Sakaguchi, S.; Houghton, A. N.; van den Brink, M. R. M. Glucocorticoid-Induced TNF Receptor Family Related Gene Activation Overcomes Tolerance/Ignorance to Melanoma Differentiation Antigens and Enhances Antitumor Immunity. J. Immunol. 2006, 176, 6434–6442.

(95)

Zhou, P.; L’italien, L.; Hodges, D.; Schebye, X. M. Pivotal Roles of CD4+ Effector T Cells in Mediating Agonistic Anti-GITR mAb-Induced-Immune Activation and Tumor Immunity in CT26 Tumors. J. Immunol. 2007, 179, 7365–7375.

(96)

Placke, T.; Salih, H. R.; Kopp, H.-G. GITR Ligand Provided by Thrombopoietic Cells Inhibits NK Cell Antitumor Activity. J. Immunol. 2012, 189, 154–160.

(97)

Tu, Y. K.; Hong, Y. Y.; Chen, Y. C. Finite Element Modeling of Kirschner Pin and Bone Thermal Contact during Drilling. Life Sci. J. 2009, 6, 23–27.

(98)

Ko, K.; Yamazaki, S.; Nakamura, K.; Nishioka, T.; Hirota, K.; Yamaguchi, T.; Shimizu, J.; Nomura, T.; Chiba, T.; Sakaguchi, S. Treatment of Advanced Tumors with Agonistic Anti-GITR mAb and Its Effects on Tumor-Infiltrating Foxp3+CD25+CD4+ Regulatory T Cells. J. Exp. Med. 2005, 202, 885–891.

(99)

Xiao, X.; Shi, X.; Fan, Y.; Zhang, X.; Wu, M.; Lan, P.; Minze, L.; Fu, Y.-X.; Ghobrial, R. M.; Liu, W.; Li, X. C. GITR Subverts Foxp3+ Tregs to Boost Th9 Immunity through Regulation of Histone Acetylation. Nat. Commun. 2015, 6, 8266.

(100) Chattopadhyay, K.; Ramagopal, U. a; Mukhopadhaya, A.; Malashkevich, V. N.; Dilorenzo, T. P.; Brenowitz, M.; Nathenson, S. G.; Almo, S. C. Assembly and Structural Properties of Glucocorticoid-Induced TNF Receptor Ligand: Implications for Function. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 19452–19457. (101) Zhou, Z.; Tone, Y.; Song, X.; Furuuchi, K.; Lear, J. D.; Waldmann, H.; Tone, M.; Greene, M. I.; Murali, R. Structural Basis for Ligand-Mediated Mouse GITR Activation. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 641–645. (102) Chattopadhyay, K.; Ramagopal, U. a; Brenowitz, M.; Nathenson, S. G.; Almo, S. C. Evolution of GITRL Immune Function: Murine GITRL Exhibits Unique Structural and Biochemical Properties within the TNF Superfamily. Proc. Natl. Acad. Sci. U. 43 ACS Paragon Plus Environment


Page 44 of 60

S. A. 2008, 105, 635–640. (103) Chattopadhyay, K.; Ramagopal, U. A.; Nathenson, S. G.; Almo, S. C. 1.8 Å Structure of Murine GITR Ligand Dimer Expressed in Drosophila Melanogaster S2 Cells. Acta Crystallogr. Sect. D Biol. Crystallogr. 2009, 65, 434–439. (104) Bodmer, J. L.; Schneider, P.; Tschopp, J. The Molecular Architecture of the TNF Superfamily. Trends Biochem. Sci. 2002, 27, 19–26. (105) Gurney, A. L.; Marsters, S. A.; Huang, R. M.; Pitti, R. M.; Mark, D. T.; Baldwin, D. T.; Gray, A. M.; Dowd, a D.; Brush, a D.; Heldens, a D.; Schow, a D.; Goddard, A. D.; Wood, W. I.; Baker, K. P.; Godowski, P. J.; Ashkenazi, A. Identification of a New Member of the Tumor Necrosis Factor Family and Its Receptor, a Human Ortholog of Mouse GITR. Curr. Biol. 1999, 9, 215–218. (106) Yu, K.; Kwon, B.; Ni, J.; Zhai, Y.; Ebner, R.; Kwon, B. S. A Newly Identified Member of Tumor Necrosis Factor Receptor Superfamily (TR6) Suppresses LIGHTmediated Apoptosis. Biochemistry 1999, 274, 13733–13736. (107) Larkin, J.; Chiarion-Sileni, V.; González, R.; Grob, J.; Cowey, C.; Lao, C. Combined Nivolumab and Ipilimumab or Monotherapy in Previously Untreated Melanoma. N Engl J Med. 2015, 373, 23–34. (108) Wolchok, J. D.; Chiarion-Sileni, V.; Gonzalez, R.; Rutkowski, P.; Grob, J.-J.; Cowey, C. L.; Lao, C. D.; Wagstaff, J.; Schadendorf, D.; Ferrucci, P. F.; Smylie, M.; Dummer, R.; Hill, A.; Hogg, D.; Haanen, J.; Carlino, M. S.; Bechter, O.; Maio, M.; Marquez-Rodas, I.; Guidoboni, M.; McArthur, G.; Lebbé, C.; Ascierto, P. A.; Long, G. V.; Cebon; J.; Sosman, J.; Postow, M. A.; Callahan, M. K.; Walker, D.; Rollin, L.; Bhore, R.; Hodi, F. S.; Larkin, J. Overall Survival with Combined Nivolumab and Ipilimumab in Advanced Melanoma. N. Engl. J. Med. 2017, NEJMoa1709684. (109) Huang, R.-Y.; Eppolito, C.; Lele, S.; Shrikant, P.; Matsuzaki, J.; Odunsi, K. LAG3 and PD1 Co-Inhibitory Molecules Collaborate to Limit CD8 + T Cell Signaling and Dampen Antitumor Immunity in a Murine Ovarian Cancer Model. Oncotarget 2015, 6, 27359–27377. (110) Woo, S.; Turnis, M. E.; Goldberg, M. V; Bankoti, J.; Nirschl, C. J.; Bettini, M. L.; Gravano, D.; Vogel, P.; Liu, L.; Tangsombatvisit, S.; Grosso, J.F.; Netto, G.; Smeltzer, M.P.; Chaux, A.; Utz, P.J.; Workman, C.J.; Pardoll, D.M.; Korman, A.J. Drake, C.G.; Vignali, D.A. Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape. Cancer Res. 2012, 72, 917–927. (111) Li, N.; Workman, C. J.; Martin, S. M.; Vignali, D. A. A. Biochemical Analysis of the Regulatory T Cell Protein Lymphocyte Activation Gene-3 (LAG-3; CD223). J. Immunol. 2004, 173, 6806–6812 (112) Andrews, L. P.; Marciscano, A. E.; Drake, C. G.; Vignali, D. A. A. LAG3 (CD223) as a Cancer Immunotherapy Target. Immunol. Rev. 2017, 276, 80–96. (113) Liu, W.; Tang, L.; Zhang, G.; Wei, H.; Cui, Y.; Guo, L.; Gou, Z.; Chen, X.; Jiang, D.; Zhu, Y.; Kang, G.; He, F.; Characterization of a Novel C-Type Lectin-like Gene, 44 ACS Paragon Plus Environment

Page 45 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


LSECtin: Demonstration of Carbohydrate Binding and Expression in Sinusoidal Endothelial Cells of Liver and Lymph Node. J. Biol. Chem. 2004, 279, 18748– 18758. (114) Hastings, W. D.; Anderson, D. E.; Kassam, N.; Koguchi, K.; Edward, A.; Kent, S. C.; Zheng, X. X.; Strom, T. B.; Hafler, D. A.; Vijay, K. TIM-3 Is Expressed on Activated Human CD4+ T Cells and Regulates Th1 and Th17 Cytokines. Eur J Immunol. 2009, 39, 2492–2501. (115) Guo, Z.; Wang, X.; Cheng, D.; Xia, Z.; Luan, M.; Zhang, S. PD-1 Blockade and OX40 Triggering Synergistically Protects against Tumor Growth in a Murine Model of Ovarian Cancer. PLoS One 2014, 9, e89350. (116) Messenheimer, D. J.; Jensen, S. M.; Afentoulis, M. E.; Wegmann, K. W.; Feng, Z.; Friedman, D. J.; Gough, M. J.; Urba, W. J.; Fox, B. A. Timing of PD-1 Blockade Is Critical to Effective Combination Immunotherapy with Anti-OX40. Clin. Cancer Res. 2017, 23, 6165-6177. (117) Linch, S. N.; McNamara, M. J.; Redmond, W. L. OX40 Agonists and Combination Immunotherapy: Putting the Pedal to the Metal. Front. Oncol. 2015, 5, 1–14. (118) Markovsky, E.; Baabur-Cohen, H.; Eldar-Boock, A.; Omer, L.; Tiram, G.; Ferber, S.; Ofek, P.; Polyak, D.; Scomparin, A.; Satchi-Fainaro, R. Administration, Distribution, Metabolism and Elimination of Polymer Therapeutics. J. Control. Release 2012, 161, 446–460. (119) Tiwari, G.; Tiwari, R.; Sriwastawa, B.; Bhati, L.; Pandey, S.; Pandey, P.; Bannerjee, S. K. Drug Delivery Systems: An Updated Review. Int. J. Pharm. Investig. 2012, 2, 2–11. TABLE OF CONTENTS GRAPHIC



Fig. 1 Costimulatory and inhibitory receptors regulating T-cell responses and translation into the clinic. (A) Representation of several ligand-receptor interactions between T cells and antigen-presenting cells (APCs). Several ligands bind to multiple receptors, some of which deliver costimulatory signals, while others lead to coinhibitory signals. One important family of membrane-bound ligands that binds both costimulatory and inhibitory receptors is the B7 family. B7 family members and their known ligands belong to the immunoglobulin superfamily. Ligands of other B7 receptors have not yet been identified. Tumor necrosis factor (TNF) family members that bind to cognate TNF receptor family molecules represent a second family. These receptors predominantly deliver costimulatory signals. (B) Agents currently in clinical trials for PD-1, PD-L1, GITR, LAG-3 and TIM-3. CTLA-4 has no new agents reported under clinical development. (C) Despite the extensive work devoted to the development of agents acting on immune checkpoint regulators, the number of approved drugs is extremely low. To date, approved drugs target only CTLA-4 and PD-1/PD-L1. 761x523mm (96 x 96 DPI)

ACS Paragon Plus Environment

Page 46 of 60

Page 47 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 2 Human CTLA-4 overall structure and interactions (PDBID: 3OSK). (A) Ribbon representation of the secondary structure of the CTLA-4 extracellular monomer, exhibiting the two-layer AGFCC’ and ABED IgSF domain topology. (B) Ribbon representation of the CTLA-4 extracellular homodimer. Each monomer exhibits three distinct CDR regions, CDR1 (BC loop), CDR2 (C'C" loop), and CDR3 (FC loop), which form the top surface of the molecule. Stick representation of homodimer interface focusing structure stabilization involving several residues (Pro119, Glu120, Cys122 and Pro123) and a disulfide linkage at Cys112. (C) Ribbon representation of the CTLA-4/B7-1 complex (PDBID: 1I8L). Two B7-1 (cyan) and two CTLA-4 (dark blue) protein units in the asymmetric unit. (D) Molecular association of CTLA-4 and B7-1 in the crystal lattice. ‘Skewed zipper’ arrays in the CTLA-4/B7-1 complex. In the perpendicular direction, across membranes, ligated receptors would span 140 Å. 650x487mm (96 x 96 DPI)



Fig. 3 CTLA-4/tremelimumab® complex (PDBID: 5GGV). Hypothetical model for the prevention of an alternating arrangement of CTLA-4 (dark blue) and B7-1/2 (cyan) by tremelimumab (light blue) binding. The CTLA-4 dimer binds to two tremelimumab Fab fragments in the crystal. Tremelimumab binds in the vicinity spanning 150–190 Å in the perpendicular direction from the T-cell membrane. 407x276mm (96 x 96 DPI)


Page 48 of 60

Page 49 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 4 PD-1 overall structure. (A) Schematic representation of PD-1 protein. Full-length PD-1 is separated into the ectodomain, transmembrane domain and intracellular domain. The ectodomain is composed of the signal peptide, N-loop, IgV domain and stalk region. The N-glycosylation sites are indicated with a solid green arrowhead. The numbers indicate amino acid positions. Ribbon representation of the PD-1 structure (blue). It consists of a two-layer β sandwich with the topology of IgSF domains and two β sheets (GFCC’ and ABED) stabilized by a disulfide bond (Cys34– Cys103). (B) PD-1/PD-L1 structure complex (PDBID: 4ZQK). PD-1 (dark blue) and PD-L1 (teal) are both shown using cartoon representation. (E) Close-up view of the PD-1/PD-L1 interface using cartoon representation. Residues involved in PD-1 and PD-L1 interaction are represented in dark blue and teal sticks, respectively. 655x428mm (96 x 96 DPI)



Fig. 5 PD-1 blockade using antibodies. The CDR loops and β-strands of nivolumab and pembrolizumab are involved in PD-1 blockade. (A) Overall structure surface of the PD-1/nivolumab Fab complex (PDBID: 5GGR). Interface region (orange) of nivolumab Fab (light blue) and PD-1 (blue). The residues of the PD1/nivolumab interface involved in the PD-1/PD-L1 interaction are highlighted. (B) Overall structure surface of the PD-1/pembrolizumab Fab complex (PDBID: 5GGS). Interface region (orange) of pembrolizumab Fab (light blue) and PD-1 (blue). The residues of the PD-1/Pembrolizumab interface involved in the PD-1/PD-L1 interaction are highlighted. 614x299mm (96 x 96 DPI)


Page 50 of 60

Page 51 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 6 PD-L1 blockade by antibodies (A) Overall structure surface of the PD-L1/BMS-936559 Fab complex (PDBID: 5GGT. Interface region of BMS-936559 Fab (light blue) and PD-L1 (teal). The residues of the PDL1/BMS-936559 interface involved in the PD-1/PD-L1 interaction are highlighted using stick representation. (B) Overall structure surface of the PD-L1/avelumab Fab complex (PDBID: 5GRJ). Interface region (yellow) of avelumab Fab (light blue) and PD-L1 (teal). The residues of the PD-L1/avelumab® interface involved in the PD-1/PD-L1 interaction are highlighted using stick representation. Hydrophobic core of PD-L1 (Ile54, Tyr56, Met115 and Tyr 123), critical for the interaction with PD-1 hydrophobic residues, is now occupied by similar hydrophobic residues of avelumab. (C) Overall structure surface of the PD-L1/KN-035 complex (PDBID: 5JDS). Interface region of KN-035 Fab (light blue) and PD-L1 (teal). The residues of PD-L1/KN-035 interface (yellow) are highlighted using stick representation. (F) Overall structure surface of the PDL1/durvalumab Fab complex (PDBID: 5X8M). Interface region (yellow) of durvalumab Fab (light blue) and PD-L1 (teal). The residues of the PD-L1/durvalumab interface involved in the PD-1/PD-L1 interaction are highlighted using stick representation. 827x681mm (96 x 96 DPI)



Fig. 7 Active small molecule acting on PD-L1. (A) The scaffold (2-methyl-3-biphenylyl)methanol was designed by Bristol-Myers Squibb. Small-molecule inhibitors 1 (IC50: 18 nM), 2 (IC50: 146 nM) and 3 (IC50: 80 nM) are from the original patented library. Inhibitor 4 is one of the optimized compounds described by Holak et al.71,72 (B) PD-L1 inhibition. Surface representation of two molecules of PD-L1 (teal) bridged by the small-molecule inhibitor (green) (PDBID:5N2F). Inhibition occurs by small-molecule-induced PD-L1 dimerization. (C) Surface representation of the PD-L1 binding pocket (teal). Close-up view of the PDL1 binding pocket, 3 (green) binds at the hydrophobic cavity (Tyr56, Met115, Ile116, Ala121 and Tyr123). Additional interactions of 3 are depicted in yellow dashes (PDBID:5N2F). (D) PD-L1 binding site flexibility. Surface representation of the PD-L1 structure (teal) with inhibitor 1 (yellow) and 3 (green). The movement of Tyr56 makes the pocket of PD-L1 accessible in both sites, regarding inhibitor 3 (green). In the structure of inhibitor 1 (yellow), the pocket is a deep hydrophobic tunnel. 512x647mm (96 x 96 DPI)


Page 52 of 60

Page 53 of 60


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



Fig. 8 Other active scaffolds acting on PD-L1. Small-molecule inhibitor 5 is derived from 1,2,4-oxadiazoleinhibitors, and compound 6 is a peptidomimetic inhibitor; both are from Aurigene Discovery Technologies Limited. Potent macrocyclic peptidic inhibitor 7 (IC50: 5.2 nM) is from Bristol-Myers Squibb. 272x416mm (96 x 96 DPI)


Page 54 of 60

Page 55 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 9 Macrocyclic peptidic inhibitor (7)/PD-L1 complex (PDBID: 5O45). (A) Detailed view of the PD-L1 (teal)/peptide (7) (green) interaction. The hydrophobic side chains interact with the cleft characteristic for the “face-on” binding mode. The polar zone of the interaction includes two hydrogen bonds with Glu58 and Asp61 by the backbone amines of the peptide. 520x266mm (96 x 96 DPI)



Fig. 10 Structures of TNF-related proteins GITRL and 4-1BBL (PDBID: 3B93 and 2X29). (A) Ribbon representation of the GITRL monomer showing the classic two-layer jelly roll fold of the THD. The β-strands are labeled with inner and outer sheets comprising the AA’HCF and B’BGDE strands, respectively. (B) Ribbon representation of the GITRL trimer. The shape of the GITRL trimer resembles a blooming flower. (C) GITRL residues whose mutation affected receptor binding are mapped onto the GITRL monomer surface and are colored yellow. 556x222mm (96 x 96 DPI)


Page 56 of 60

Page 57 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. CTLA-4 hot spots, residues and key interactions to target 502x422mm (96 x 96 DPI)



Table 2. PD-1 hot spots, residues and key interactions to target 499x418mm (96 x 96 DPI)


Page 58 of 60

Page 59 of 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 3. PD-L1 hot spots, residues and key interactions to target 498x415mm (96 x 96 DPI)



Table 4. GITRL hot spots, residues and key interactions to target 497x420mm (96 x 96 DPI)


Page 60 of 60

Page 61 of 60


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Structure-function analysis of immune checkpoint receptors to guide

Recommend Documents