Received: 2 May 2018 | Revised: 18 July 2018
DOI: 10.1002/med.21530
REVIEW ARTICLE
Immunomodulators targeting the PD‐1/PD‐L1
protein‐protein interaction: From antibodies to
small molecules
Jeffrey Yang1 | Longqin Hu1,2
1
Department of Medicinal Chemistry, Ernest
Mario School of Pharmacy, Rutgers, The State
University of New Jersey, Piscataway,
New Jersey
2 recent decade, especially in the area of immune checkpoint
Cancer Pharmacology Program, Rutgers
Cancer Institute of New Jersey,
New Brunswick, New Jersey
Correspondence
Longqin Hu, PhD, Department of Medicinal
Chemistry, Ernest Mario School of Pharmacy,
Rutgers, The State University of New Jersey,
160 Frelinghuysen Road, Piscataway,
NJ 08854.
Email: LongHu@pharmacy.rutgers.edu
Med Res Rev. 2018;1-37.
| Accepted: 23 July 2018
Abstract
Cancer immunotherapy has made great strides in the
blockade. The outstanding efficacy, prolonged durability of
effect, and rapid assimilation of anti‐PD‐1 and anti‐PD‐L1
monoclonal antibodies in clinical practice have been
nothing short of a medical breakthrough in the treatment
of numerous malignancies. The major advantages of these
therapeutic antibodies over their small molecule counter-
parts have been their high binding affinity and target
specificity. However, antibodies do have their flaws
including immune‐related toxicities, inadequate pharmaco-
kinetics and tumor penetration, and high cost burden to
manufacturers and consumers. These limitations hinder
broader clinical applications of the antibodies and have
heightened interests in developing the alternative small
molecule platform that includes peptidomimetics and
peptides to target the PD‐1/PD‐L1 immune checkpoint
system. The progress on these small molecule alternatives
has been relatively slow compared to that of the
antibodies. Fortunately, recent structural studies of the
interactions among PD‐1, PD‐L1, and their respective
antibodies have revealed key hotspots on PD‐1 and PD‐
L1 that may facilitate drug discovery efforts for small
molecule immunotherapeutics. This review is intended to
discuss key concepts in immuno‐oncology, describe the
successes and shortcomings of PD‐1/PD‐L1 antibody‐based
therapies, and to highlight the recent development of small
wileyonlinelibrary.com/journal/med © 2018 Wiley Periodicals, Inc. | 1
2 | YANG AND HU

molecule inhibitors of the PD‐1/PD‐L1 protein‐protein

interaction.

KEYWORDS
immune checkpoint, immunomodulator, PD‐1/PD‐L1 protein‐pro-
tein interaction, programmed cell death‐1, small molecule inhibitor

1 | INTRODUCTION

For more than a century, the field of cancer immunotherapy has experienced several periods of acceptance and
skepticism. The notion of harnessing one’s immune system to seek and destroy cancer cells was officially
introduced by Paul Ehrlich in 1909 as the cancer immune surveillance hypothesis.1 Before Ehrlich in the late 19th
century, the idea was first tested by William Coley, now known as the father of cancer immunotherapy. He
developed a cancer vaccine based on bacterial toxins, which was shown to induce spontaneous and long‐lasting
remission in several patients with inoperable sarcomas.1 Unbeknownst to Coley, his proof‐of‐concept study
highlighted the potential role of the immune system in killing tumor cells. Because of the potential hazards,
inconsistent efficacy, and unknown mechanism of action, Coley’s primitive form of immunotherapy was not readily
accepted by medical oncologists and overshadowed by traditional chemotherapy, radiation, and surgery. Since
then, the scientific community’s knowledge of cancer biology and immunology, combined with advances in immune
monitoring tools (eg, enzyme‐linked immunosorbent assay [ELISA], polymerase chain reaction [PCR], and
serological analysis of recombinant tumor cDNA expression libraries [SEREX]) and immune‐based technologies
(eg, hybridoma technology), has rapidly evolved and has led to major discoveries that have ushered in a renaissance
phase for cancer immunotherapy.
Indeed, the discovery of immune checkpoint molecules, the regulators of immune cell activity, has brought forth
a class of groundbreaking cancer immunotherapies in the early decades of the 21st century (Figure 1). As a specific
example to illustrate the success of this treatment modality, the therapeutic landscape of melanoma has changed
dramatically from the introduction of these immune checkpoint inhibitors to the clinic.2 Before immune checkpoint
blockers, the mainstay of therapy was either high‐dose interleukin‐2 or nontargeted cytotoxic chemotherapy (eg,
alkylating agents like dacarbazine, temozolomide; antimicrotubule agents alone or in combination with platinating
agents such as paclitaxel/carboplatin). Their unfavorable toxicity profile, along with poor survival outcomes,
instigated the development of genomically targeted therapies such as the BRAF (eg, vemurafenib) and MEK (eg,
trametinib) inhibitors. In a pivotal phase 3 trial BRIM‐3, more patients responded to vemurafenib treatment (48%;
106 of 219 patients; two complete responses [CR] and 104 partial responses [PR]) as compared to those in the
dacarbazine treatment group (5%; 12 of 220 patients; all PRs).3 In addition, vemurafenib significantly improved
survival when compared to dacarbazine (progression‐free survival [PFS] of 5.3 months vs 1.6 months) in patients
with advanced melanoma possessing the BRAF V600E mutation.3 Although these targeted therapies extended
patients’ lives for a few more months, patients eventually succumbed to the disease as the response to therapy was
short‐lived (median duration of response: 6.7 months).4 In 2011, ipilimumab, the first cytotoxic T‐lymphocyte‐
associated protein‐4 (CTLA‐4)‐targeting monoclonal antibody, was approved for patients with advanced melanoma.
Following closely behind, nivolumab and pembrolizumab, both programmed cell death‐1 (PD‐1)‐targeting
monoclonal antibodies, were also approved for advanced melanoma in 2014. When compared to traditional
chemotherapy and targeted therapies, these immune checkpoint inhibitors not only further increased overall
survival (OS) and PFS (ie, an earlier rightward shift in the survival curve) but also promoted durable responses (ie, a
“plateauing” effect in the survival curve rather than a drop to zero). In 2013, the immune checkpoint modulators
were heralded as the “Breakthrough of the Year” by Science.5 Immune checkpoint modulators have refueled
immense excitement in cancer immunotherapy research, resulting in a surge in pipeline activity across several
YANG AND HU | 3

F I G U R E 1 Major events in the discovery and development of immune checkpoint modulators, 1893‐2018.
BC, bladder cancer; cHL, classical Hodgkin lymphoma; CRC, colorectal cancer; CTLA‐4, cytotoxic T‐lymphocyte‐
associated protein‐4; dMMR, mismatch repair‐deficient; HCC, hepatocellular carcinoma; HNSCC, head and neck
squamous cell carcinoma; MSI‐H, high‐level microsatellite instability; mAb, monoclonal antibody; NSCLC, non‐small
cell lung cancer; PD‐1, programmed cell death‐1; PD‐L, programmed cell death ligand; PMBCL, primary mediastinal
B‐cell lymphoma; RCC, renal cell carcinoma

pharmaceutical and biopharmaceutical companies. The US Food and Drug Administration (FDA) has expanded the
indications of nivolumab and pembrolizumab to other advanced solid and hematologic malignancies. Additional
immune checkpoint inhibitors targeting the ligand of PD‐1 (ie, PD‐L1) have also been introduced: atezolizumab
(2016), avelumab (2017), and durvalumab (2017). These fast‐paced developments have positioned the immune
checkpoint modulators to become a multibillion dollar market in the near future.
In spite of all this, there have been modest efforts to design small molecule modulators of immune
checkpoints with comparable potency and efficacy and possibly fewer safety concerns. The goal of this review
is to stimulate interest in and to encourage the pursuit of the discovery and development of small molecule
immunomodulators of the PD‐1/PD‐L1 immune checkpoint pathway. First, we provide a basic understanding of
how the immune system responds to cancer cells and how the tumor manages to evade the immune system by
taking advantage of regulatory immune checkpoints. Next, we describe the biological significance of the PD‐1
and PD‐L1 immune checkpoint molecules and summarize the efficacy and safety of recent FDA‐approved,
antibody‐based PD‐1/PD‐L1 inhibitors, as well as their limitations. Finally, we compare and contrast these
therapeutic monoclonal antibodies to their small molecule counterparts, assess the progress made in the
development of small molecule inhibitors of this pathway, and conclude by providing a persuasive rationale for
pursuing this exciting and challenging endeavor.
4 | YANG AND HU

2 | E S T AB L I S HM E N T O F A N E F F E C TI V E A N T I T U M O R
IMMUNE RESPONSE

Cancer is characterized as a heterogeneous disease composed of diverse cell types, resulting from an accumulation
of somatic gene mutations and epigenetically dysregulated genes.6 The high mutation rates observed in certain
tumors gives rise to a multitude of tumor‐associated antigens or neoantigens, making some cancers more
susceptible to the immune system than others.7,8 The host immune system must recognize all of these antigens as
foreign or “nonself” and subsequently generate an effective antitumor immune response that is specific, durable,
and adaptable (Figure 2).9,10 The antigens produced by tumors are introduced to and captured by antigen‐
presenting cells (APC)—for example, dendritic cells, macrophages, or activated B lymphocytes—in peripheral
tissues. Upon antigen binding, the naive APCs engulf and phagocytose the antigen and migrate into the lymphatic
system. The APCs mature while in transit by upregulating the expression of major histocompatibility complex
(MHC) and costimulatory molecules on their cell surfaces. The processed antigen is also bound onto a MHC
molecule. Once in the lymph nodes and lymph organs (eg, thymus, spleen), these mature, activated APCs encounter
and engage with naive T lymphocytes. The APC presents its MHC‐antigen complex to a T lymphocyte possessing a
specific T cell receptor (TCR) which recognizes the unique complex. This interaction confers specificity to the
immune response, ensuring that only those cells expressing the specific tumor‐associated antigen will be targeted
and eliminated. In addition, it constitutes the first of two signals required for T cell priming and activation. The
second activation signal requires the binding of the costimulatory molecules CD80 (B7‐1) or CD86 (B7‐2) on the
APC to its cognate receptor CD28 on the T lymphocyte.11,12

F I G U R E 2 The establishment and propagation of an antitumor immune response. The antitumor immune response
can be visualized as a self‐propagating process as described previously.9 The steps of the “cancer‐immunity cycle”
involve: (1) antigen release by cancer cells; (2) antigen recognition and processing by antigen‐presenting cells;
(3) priming or activation of T lymphocytes upon exposure to antigen; (4) migration of activated T lymphocytes to
the tumor site; (5) tumor penetration by T lymphocytes; and (6) recognition and elimination of cancer cells by
T lymphocytes and other activated immune cells (eg, antibody‐producing plasma cells). The final step of this cycle results
in the release of additional tumor antigens and the enhancement and expansion of the immune response against
the tumor. APC, antigen‐presenting cell; CD, cluster of differentiation; CTLA‐4, cytotoxic T‐lymphocyte‐associated
protein‐4; Fas, first apoptosis signal; MHC, major histocompatibility complex; PD‐1, programmed cell death‐1;
PD‐L, programmed cell death ligand; TCR, T‐cell receptor
YANG AND HU | 5

Upon activation, the antigen‐specific T lymphocytes undergo rapid proliferation and differentiation into active,
tumor‐directed effector T cells. These effector T cells must now find their way to the tumor by trafficking through
the vasculature and infiltrating into the tumor bed. They then can directly eradicate the tumor cells by releasing
pore‐forming perforins and serine protease granzymes (ie, the perforin‐granzyme pathway) or by triggering death‐
inducing pathways (eg, Fas/Fas ligand pathway).13–15 Effector T cells may also indirectly kill tumor cells by
activating other immune cells such as antibody‐producing plasma B cells. Memory T cells can also result from the
initial activation event and serve to establish immunologic memory, ready to stimulate a more rapid immune
response upon re‐exposure to tumor cells expressing the same antigen, thereby providing durable protection
against cancer.16 As cancer mutates over time, an overwhelming number of antigens are created. Additional
antigens are also released from tumor cells killed by the initial immune response. It is crucial for the activated
immune system to rapidly adapt and distinguish between the many different antigens. To respond to these various
antigens, APCs detect the new antigens and stimulate the development of entirely different populations of antigen‐
specific T and B lymphocytes, generating new antitumor immune responses in a process termed antigen
spreading.17,18 Interference by tumor cells and certain components of the tumor microenvironment in any of these
key steps of the cancer‐immunity cycle—broadly grouped into T‐cell generation, T‐cell infiltration, and tumor‐cell
killing—inhibits T‐lymphocyte‐mediated tumor immunity and promotes uncontrollable tumorigenesis. Immu-
notherapies like the immune checkpoint inhibitors have been developed to reverse these disruptions and produce a
dynamic anticancer response against malignant cells.

3 | IMMUNE TOLE RANCE AND TUMOR IMMUNE EVASION

Immune tolerance, the natural or induced unresponsiveness of the immune system to self and “nonself” antigens, is
essential in terminating an ongoing immune response, preventing autoimmunity, and establishing immune
homeostasis.19–21 In brief, immune tolerance can occur early in lymphocyte development in the thymus and bone
marrow (termed “central” tolerance), as well as after lymphocyte maturation and release to the periphery (termed
“peripheral” tolerance). In the thymus and bone marrow, naive lymphocytes are exposed to various tissue‐specific
self‐antigens. Those lymphocytes possessing TCRs with high affinity for self‐antigen‐MHC complexes are deleted
centrally (or negatively selected). This central mechanism of tolerance is far from perfect, and some autoreactive
immune cells do fall through the cracks and reach peripheral tissues. As a result, there are several peripheral
tolerance mechanisms, which can be divided into “T‐cell intrinsic” mechanisms—for instance, ignorance, anergy,
phenotypic skewing, and deletion or apoptosis—and “T‐cell extrinsic” mechanisms, for example, tolerogenic
dendritic cells and T regulatory (Treg) cells.19
Regulating tumor growth and development has been hypothesized to be a complex, dynamic process that
requires balancing host‐protective and tumor‐promoting actions, as described by the “cancer immunoediting”
hypothesis.22 On one end, the immune system efficiently surveys the periphery and blocks the growth,
development, and survival of cancer cells (termed the “elimination” phase), resulting in tumor remission. While on
the other end, the tumor cells evade detection and destruction by coopting mechanisms of immunological tolerance
and, consequently, dampening the antitumor response (termed the “escape” phase). The end result is a progressive
disease that leads to metastasis and eventual death. Between these two extremes, there is an “equilibrium” phase in
which the tumor survives elimination and persists but does not have a chance to grow or spread because of the
protective actions of the immune system. Effectively, the cancer is maintained in a dormant state. At some point,
the cancer leaves this “equilibrium” state and progresses to the “escape” phase as a consequence of immune
dysfunction and tumor immune resistance.
Several tolerance mechanisms—in particular, anergy or exhaustion, ignorance, deletion, and cell‐based
suppression—have been adopted by the tumor cell to escape from immune surveillance.23,24 Of relevance to this
review is tumor‐induced anergy, or functional inactivation or unresponsiveness, and exhaustion of lymphocyte
6 | YANG AND HU

activity through immune checkpoints. Immune checkpoint molecules are receptors and their corresponding ligands,
which upon binding either strengthen (costimulatory) or inhibit (coinhibitory) the initial signal for T lymphocyte
activation. These receptor‐ligand interactions regulate the responsiveness of peripheral T lymphocytes to tumor
antigens presented on MHC molecules. In essence, T lymphocyte activation can be understood as a balance
between the costimulatory signals and the coinhibitory signals, with whichever side being stronger dominating the
overall responsiveness of T lymphocytes toward cancer cells.

3.1 | Costimulatory immune checkpoints: unleashing the immune system

Again, for optimal activation of effector T lymphocytes, two signals are required: (1) a primary activation signal
from the interaction between TCR and a MHC‐antigen complex and (2) a costimulatory signal that bolsters the first.
The most prominent example of costimulation is the engagement of the CD28 receptor on T lymphocytes with its
ligand CD80 on APCs. CD28 costimulation of T lymphocyte activation is associated with an increased expression of
the T cell growth factor interleukin‐2 (IL‐2), which promotes the development and survival of immunocompetent T
lymphocytes.25,26 In the absence of CD28 signaling, naive T lymphocytes are not exposed to IL‐2 and are unable to
proliferate and differentiate into effector T lymphocytes, resulting in the formation of anergic T lymphocytes.12,27
The biological consequences of blocking CD28 costimulation have been extensively studied. In several in vivo
studies of the immune function in animals lacking CD28 or treated with inhibitors of CD28 signaling (eg, CTLA4‐Ig),
with and without other costimulation blockade, a diminished immune response toward infectious microbes28,29 and
organ transplants30–32 and a reduction in the severity of several autoimmune disorders—including multiple
sclerosis,33 myocarditis,34 thyroiditis35—have been demonstrated. In December 2005, the FDA approved abatacept
as the first‐in‐class antagonist of CD28 costimulation for the treatment of rheumatoid arthritis after it showed both
statistically and clinically significant improvements in patients refractory to standard‐of‐care disease‐modifying
antirheumatic drugs (DMARD) such as methotrexate and the tumor necrosis factor (TNF)‐α inhibitors, yet again
emphasizing the importance of CD28 signaling in promoting immune function.36
Several costimulatory immune checkpoints can be expressed upon activation of naive T lymphocytes, resulting
in an enhancement in T cell proliferation, survival, and activity. A partial list of these costimulatory molecules
include the inducible costimulator (ICOS)/ICOS ligand (ICOSL), 4‐1BB (CD137) receptor/4‐1BB ligand (4‐1BBL),
and OX40 receptor/OX40 ligand (OX40L).37–40 Although much focus has been placed on reversing the inhibitory
signals on T lymphocytes, pharmaceutical companies have started to target immune stimulatory pathways by
developing agonistic antibodies to strengthen the activity of tumor‐specific T lymphocytes and further improve
responses to anti‐PD‐1/PD‐L1 and anti‐CTLA‐4 immunotherapy. ICOS/ICOSL is a member of the CD28/B7
immunoglobulin superfamily whose expression is upregulated on the surface of CD4+ and CD8+ T cells following
their activation.41,42 Of interest, a study has demonstrated that the therapeutic effects of the anti‐CTLA‐4
monoclonal antibody ipilimumab can be enhanced by the simultaneous engagement of the ICOS/ICOSL pathway.42
Currently, JTX‐2011, an ICOS agonist monoclonal antibody, is being studied in the phase 1/2 trial ICONIC as
monotherapy and in combination with nivolumab, ipilimumab, or pembrolizumab in adult patients with advanced
solid tumors. 4‐1BB/4‐1BBL and OX40/OX40L are costimulatory members of the TNF receptor superfamily.38
Urelumab and utomilumab are two agonistic anti‐4‐1BB monoclonal antibodies undergoing clinical development.
Initially, the clinical trials of urelumab were halted due to liver toxicity observed at doses ≥ 1 mg/kg in a phase 1/2
trial.43 It was later brought back and tested at a lower fixed dose of 8 mg with a variety of different chemotherapies
such as rituximab, cetuximab, elotuzumab, and nivolumab.44 Although limited clinical activity was observed at this
lower dose in these various combinations, urelumab seemed able to broaden the patient population responding to
nivolumab, demonstrating similar response rates between patients with PD‐L1 positive and PD‐L1 negative
tumors.44 In comparison, utomilumab has displayed a superior safety profile relative to urelumab in several clinical
trials examining its use in solid and hematologic malignancies.44 In a phase 1b trial, utomilumab was combined
with pembrolizumab to treat patients with advanced solid cancers and demonstrated two confirmed CRs (one in
YANG AND HU | 7

small‐cell lung cancer, one in renal cell carcinoma [RCC]) and four confirmed PRs (one each in anaplastic thyroid
carcinoma, non‐small cell lung cancer [NSCLC], head and neck squamous cell carcinoma, and RCC).45 The two
patients achieving CRs sustained the response for more than 6 months.45 Currently, utomilumab is being
investigated in combination with avelumab and an anti‐OX40 monoclonal antibody. With regard to OX40, it has
been shown to be expressed on activated effector T lymphocytes46,47 and immunosuppressive Treg cells.48 While
OX40 regulates the activation of CD4+ and CD8+ T lymphocytes, it abrogates the immune‐suppressing activity of
Treg cells and depletes intratumoral Treg cells.49 These two complementary mechanisms of OX40 have provided
convincing rationale to develop several OX40 agonists, six fully human monoclonal antibodies—MEDI6469,
MEDI0562, PF‐04518600, MOXR0916, GSK3174998, and INCAGN01949—and one OX40L‐Fc fusion protein,
MEDI6383. Several phase 1/2 studies are underway for these OX40 agonists in combination with other cancer
treatment modalities.

3.2 | Coinhibitory immune checkpoints: overcoming obstacles

Tumor cells can limit the cytotoxic actions of effector T lymphocytes by blocking T cell‐surface costimulatory
receptors and ligands or hijacking the coinhibitory mechanisms normally used to regulate immune responses.
Structurally related to the CD28/B7 family of receptors and ligands, cytotoxic T‐lymphocyte‐associated
protein‐4 (CTLA‐4 or CD152) is a major negative regulator of T lymphocyte activation.50,51 In other words, it
serves as the “immunological brake” for an overactive immune system. This cell surface molecule is inducibly
and exclusively expressed on T lymphocytes upon activation.51 CTLA‐4 binds with greater affinity than CD28
to the ligands CD80 and CD86 on the surface of APCs and outcompetes CD28 for these costimulatory
ligands.52 In addition, CTLA‐4 is able to capture and remove these ligands by the process of trans‐endocytosis,
effectively attenuating tumor‐specific T‐cell proliferation by enforcing an activation threshold.53 Definitive
evidence for the immune inhibitory role of CTLA‐4 comes from CLTA‐4 deficient mouse models. Massive
expansion of peripheral T cells is observed, and these mice succumb to lethal multiorgan lymphocytic
infiltration and the resulting tissue destruction,54 presumably because of nonselective expansion of both
autoreactive and tumor‐specific T lymphocytes. Neoplastic lymphoid and myeloid cells and cells from
malignant solid tumors have been shown to constitutively express CTLA‐4.55,56 Translating this discovery into
a potential breakthrough cancer therapy was achieved with the approval of the first‐in‐class anti‐CTLA‐4
monoclonal antibody ipilimumab for the treatment of metastatic melanoma after a clear survival advantage
was demonstrated in patients refractory to all other systemic therapies.57 The clinical efficacy of ipilimumab in
select patients confirms that tumors are able to modulate the antitumor immune response by coopting
immunoregulatory pathways such as CTLA‐4. CTLA‐4 blockade reverses the tumor‐induced inhibitory effects
of CTLA‐4 and reactivates suppressed T lymphocytes to eliminate the tumor. Due to the high incidence of
severe immune‐related adverse events (irAE) associated with the use of ipilimumab, the PD‐1/PD‐L1 immune
checkpoint inhibitors have become the mainstay therapy for various malignancies.
Aside from CTLA‐4 and PD‐1/PD‐L1, of which an extensive discussion follows in the next section of this
review, additional inhibitory checkpoint molecules include, but not limited to, the lymphocyte activation gene 3
(LAG‐3, CD223), T‐cell immunoglobulin and mucin‐domain containing 3 (TIM‐3), and V‐domain Ig suppressor of
T‐cell activation (VISTA, B7‐H5).39,40 These additional inhibitory pathways have been proposed as a
mechanism of immune escape to CLTA‐4‐ and PD‐1/PD‐L1‐targeted immunotherapies.24 In several preclinical
studies, some of these immune inhibitory pathways have been upregulated as a compensatory mechanism
against PD‐1/PD‐L1 and/or CTLA‐4 blockade.58–60 A few studies have also shown that tumor‐infiltrating T
lymphocytes coexpress various coinhibitory immune checkpoint molecules, with a majority of effector T cells
simultaneously expressing at least four of these inhibitory receptors, resulting in T lymphocytes that display a
more severely exhausted phenotype.61–63 Intuitively, combinations of anti‐PD‐1/PD‐L1 or anti‐CTLA‐4
monoclonal antibodies with antibodies blocking these alternative coinhibitory receptors and ligands may
8 | YANG AND HU

unlock cancer immunity in more patients. With respect to LAG‐3, there are two antagonistic monoclonal
antibodies (relatlimab and LAG525) and one soluble fusion protein (IMP321, Immuntep) in phase 1/2 clinical
development. IMP321 has been tested in combination with paclitaxel for the treatment of metastatic breast
cancer and was demonstrated to produce an objective response rate of 50%, with 90% of patients receiving
clinical benefit without progression of the disease.64 Relatlimab is currently being investigated in combination
with nivolumab in patients with advanced melanoma. LAG525 is presently being studied in a few ongoing phase
1 and 2 clinical trials in combination with an experimental anti‐PD‐1 antibody spartalizumab for various
advanced solid and hematologic malignancies. Only one anti‐TIM‐3 monoclonal antibody, MBG453, is known
and is being investigated in phase 1/2 clinical trials in combination with spartalizumab for the treatment of
advanced malignancies. Originally, there had been an anti‐VISTA monoclonal antibody, JNJ‐61610588, in a
phase 1 trial, but it has been terminated.

4 | THE PROGRAMME D CELL D EATH ‐1 IMMU NE CH ECKP OINT

PA THW AY

The PD‐1/PD‐L1 signaling pathway has been identified and validated as a major inhibitory immune checkpoint
signaling pathway that results in T lymphocyte anergy and exhaustion.65 Unlike CTLA‐4 which primarily affects T
cells at an early stage in lymphatic tissues (ie, interference in the generation of active T lymphocytes), PD‐1
interacts with its ligands primarily within the periphery and tumor microenvironment and reduces the tumor‐cell
killing abilities of T lymphocytes.66 Under normal physiological conditions, this pathway serves to induce and
maintain peripheral tolerance, ultimately protecting against excessive inflammatory tissue damage and limiting
autoimmunity. Deficiencies in this regulatory pathway have caused autoimmune conditions, such as lupus‐like
glomerulonephritis,67 arthritis,67 autoimmune dilated cardiomyopathy,68 diabetes,69 colitis,70 and multiple
sclerosis71 in animal models. Intriguingly, a role for PD‐1 and its ligands in pathogen immune evasion has also
been extensively studied. Smith et al72 have shown that the trematode parasite Schistosoma mansoni can induce
anergy in T lymphocytes by selectively upregulating PD‐L1 on macrophages. Subsequent blockade of PD‐L1
restores T cell activation. In patients experiencing an active tuberculosis (TB) infection, the expression of PD‐1 and
its ligands have been found to be upregulated on T cells, resulting in T‐cell unresponsiveness to the infectious
pathogen.73 Upon antagonism of the PD‐1/PD‐L1 pathway, the proliferation of Mycobacterium tuberculosis‐specific
T lymphocytes is enhanced, allowing control of the disease.74 These select examples illustrate the importance of the
PD‐1/PD‐L1 pathway as a negative regulator of immune function.

4.1 | Structural characteristics and expression of the PD‐1 receptor

The programmed cell death‐1 receptor (PD‐1, CD279, PDCD1) is a member of the CD28/B7/CTLA‐4 family of
T cell regulatory receptors and functions as an immune inhibitory receptor.37 The receptor is encoded by the PD‐1
gene located on chromosome 2 at band q37.75,76 It is characterized as a 288 amino acid type I membrane protein,
composed of a signal sequence, an N‐terminal extension, an extracellular immunoglobulin (Ig) variable (V) domain, a
stalk region, a single hydrophobic transmembrane domain, and an intracellular domain.77,78 The intracellular
domain contains two structural motifs, each containing a tyrosine residue: (1) immunoreceptor tyrosine‐based
inhibitory motif (ITIM) and (2) immunoreceptor tyrosine‐based switch motif (ITSM).75,77 Phosphorylation of the
tyrosine residue in the ITSM recruits inhibitory phosphatases, such as Src homology region 2 domain‐containing
phosphatase‐1 and ‐2 (SHP‐1/2), which in turn dephosphorylate certain downstream signaling molecules of the
TCR/CD28 signal (eg, PI3K‐Akt‐mTOR, Lck‐ZAP‐70, Ras‐MEK‐ERK, p38, BATF) and attenuate the activation signal
(Figure 3).77,79,80 The extracellular domain binds to two ligands of the PD‐1 receptor, PD‐L1 and PD‐L2.
Interestingly, the PD‐1 receptor and its ligands possess binding domains that structurally resemble the
YANG AND HU | 9

F I G U R E 3 Induction of T lymphocyte exhaustion via the PD‐1:PD‐L1/PD‐L2 immune checkpoint pathway and
its reversal by therapeutic monoclonal antibodies. Normally, binding of the MHC‐antigen complex on cancer cells
to the T cell receptor triggers a cascade of intracellular events that leads to increased T cell proliferation and
cytokine production, among many other physiological events. When PD‐L1 on cancer cells binds to its receptor
PD‐1, phosphorylation of the intracellular ITIM and ITSM domains of PD‐1 occurs, resulting in recruitment of
phosphatases, SHP‐1 and SHP‐2. These phosphatases dephosphorylate TCR proximal signaling molecules (eg,
PI3K/Akt and other kinases), ultimately resulting in an inactivated or “exhausted” T lymphocyte. This inhibition can
be reversed by blocking the PD‐1:PD‐L1 protein‐protein interaction through the administration of monoclonal
antibodies that target either protein. IgC, constant immunoglobulin domain; IgV, variable immunoglobulin domain;
ITIM, immunoreceptor tyrosine‐based inhibitory motif; ITSM, immunoreceptor tyrosine‐based switch motif; MHC,
major histocompatibility complex; PD‐1, programmed cell death‐1; PD‐L, programmed cell death ligand; SHP, Scr
homology region 2 domain‐containing phosphatase; TCR, T cell receptor; Treg, regulatory T lymphocytes

antigen‐binding Fv domains of antibodies and T cell receptors.81 The IgV domain of human PD‐1 (hPD‐1), same for
human PD‐L1 (hPD‐L1) and human PD‐L2 (hPD‐L2), is organized into nine parallel β‐strands (ABCC′C′′DEFG) with
loops connecting the strands, with an additional disulfide bond connecting the B and F strands to form a two‐
layered sandwich structure with two faces (ABED and GFCC′; Figure 4A).82 The hPD‐1:hPD‐L1 pair utilizes the
front GFCC′ faces of their V‐domain to interact with one another at an acute angle, burying a total surface area of
approximately 1900 Å2.82 hPD‐1 also interacts with the GFCC′ β sheet of hPD‐L1 with its CC′ loop, C′C′′ loop, and
FG loop.82 The interaction is facilitated by both hydrophobic interactions and polar interactions around a central
hydrophobic core, made up of five nonpolar residues from hPD‐1 (Val64, Ile126, Leu128, Ala132, and Ile134) and
five from hPD‐L1 (Ile54, Tyr56, Met115, Ala121, and Tyr123; Figure 4B).83 Using per‐residue energy
decomposition and virtual alanine scanning mutagenesis, a recent study determined that six residues on the
surface of PD‐L1—Tyr56, Gln66, Met115, Asp122, Tyr123, and Arg125— are hotspots that contribute greater than
2 kcal/mol of energy to the interaction between PD‐L1 and PD‐1.84 These same residues are also involved in the
interaction between PD‐L1 and various monoclonal antibodies and small molecules reported to date, indicating
their importance as potential targets for small molecule or peptide inhibitor design. Compared to CD28 and CTLA‐
4, which can form disulfide‐bonded homodimers, PD‐1 exists as a monomer on the cell surface because it lacks the
analogous C‐terminal cysteine residue to form a disulfide bridge.81 PD‐1 is inducibly expressed on activated T cells,
Treg cells, natural killer T cells, B cells, and myeloid cells—for example dendritic cells and macrophages—to maintain
immune homeostasis as a negative feedback mechanism.85
10 | YANG AND HU

F I G U R E 4 Characterization of the interaction between human PD‐1 (hPD‐1) and human PD‐L1 (hPD‐L1; modified
from PDB ID: 4ZQK with MOE). A, Canonical designations of the strands and loops within hPD‐1 and hPD‐L1. The
hPD‐1 and hPD‐L1 proteins interact with the front faces of their IgV‐domain GFCC′ β sheet at an acute angle
(indicated by the red letters). Note that the C′C′′ loop, C′′ strand, and C′′D loop are not shown for PD‐1. B, The key
amino acids involved in the interaction between hPD‐1 (light blue ribbon model; navy blue amino acid residues) and
hPD‐L1 (green ribbon model; light green amino acid residues) are illustrated. Black dashed lines indicate hydrogen
bonding and water‐mediated interactions. Red spheres indicate water molecules. Amino acids that constitute the
central hydrophobic core of the hPD‐1/hPD‐L1 interface are indicated in yellow. Strands on both PD‐1 and PD‐L1 are
indicated by red letters. Adapted from Zak et al83

4.2 | Structural characteristics, expression, and binding properties

of the PD‐1 ligands, PD‐L1 and PD‐L2
PD‐1 has two ligands, PD‐L1 (CD274, B7‐H1) and PD‐L2 (CD273, B7‐DC), which are both members of the same
CD28/B7/CTLA‐4 family as CD80 and CD86 and are encoded by the PD‐L genes situated on chromosome 9 at
band p24.37,86 Structurally, both ligands are type I membrane proteins, consisting of a single hydrophobic
transmembrane domain, a short intracellular domain, and two Ig‐like domains within the extracellular region—
a N‐terminal IgV domain and a C‐terminal Ig constant (C) domain joined by a short linker.81,83,87 The
extracellular IgV domain of PD‐L1 and PD‐L2 interacts with the extracellular IgV domain of PD‐1 to
downregulate the immune response by recruiting phosphatases such as SHP‐1/‐2 as described earlier.81,87 A
crystal structure of two complexed hPD‐L1 proteins has been published and suggests that PD‐L1 may form
homodimers (Figure 5A). It illustrated that the β strands forming the BED and AGFCC′C′′ sheets of the IgV
domain (referred to as domain 1 [D1]) on both PD‐L1 proteins form three hydrogen bonds (Figure 5B).88 In
addition, the ABED and CFG β sheets on the IgC domain of both PD‐L1 proteins (referred to as domain 2 [D2])
made five additional hydrogen bond interactions (Figure 5C).88 These eight hydrogen bond interactions
between the D1 and D2 interfaces of the PD‐L1 proteins appear to be responsible for the dimerization, but in
vivo confirmation of this phenomenon is required. In addition to PD‐1, PD‐L1 is a ligand of B7‐1 or CD80.89
Similar to the PD‐1:PD‐L1 interaction, B7‐1:PD‐L1 interaction also prevents the activation of effector T
lymphocytes.89–91 Therefore, it may be more advantageous to target PD‐L1 to hinder the two inhibitory signals
of T cell activation, PD‐1:PD‐L1 and B7‐1:PD‐L1.
The expression profile of the two PD‐1 ligands is distinct. The predominant ligand for PD‐1 is PD‐L1, which is
both constitutively and inducibly expressed by T and B cells, dendritic cells, macrophages, mesenchymal stem cells,
and bone marrow–derived mast cells and on nonhematopoietic cells, such as in the heart, skeletal muscle, placenta,
lung, thymus, spleen, kidney, and liver.92,93 Its expression is normally upregulated in response to inflammation, in
particular to proinflammatory cytokines such as interferon (IFN)‐γ and TNF‐α.94,95 Unlike PD‐L1, the expression of
PD‐L2 is more restricted and is induced only on monocytes and dendritic cells upon interleukin‐4 stimulation.96,97
YANG AND HU | 11

F I G U R E 5 Characterization of the PD‐L1:PD‐L1 dimerization interface (modified from PDB ID: 3FN3 with MOE).
A, Full view of the PD‐L1/PD‐L1 (shown as purple and pink ribbon models) homodimer structure. B, Representation of the
three hydrogen bond interactions (indicated by black dashed lines) between the IgV domains of the two PD‐L1 proteins.
C, Representation of the five hydrogen bond interactions (indicated by black dashed lines) between the IgC domains of the
two PD‐L1 proteins. Adapted from Chen et al.88 Amino acids on the purple‐colored PD‐L1 are colored light pink, while
those on the pink‐colored PD‐L1 are colored yellow

PD‐L2 is also expressed on activated T lymphocytes.98 The difference in expression of PD‐L1 and PD‐L2 suggests
that these two ligands may serve different roles in inducing and maintaining peripheral tolerance. PD‐L1 may play a
more general role in protecting peripheral tissues from excessive inflammation. Conversely, the restricted
expression of PD‐L2 on APCs may indicate a more specialized role for PD‐L2 in regulating the activation and
differentiation process of T lymphocytes. More research is needed to elucidate the biological functions of PD‐L1
and PD‐L2 in regulating the immune system.
The binding affinities of PD‐L1 and PD‐L2 to PD‐1 have been reported in the literature but vary with large
discrepancies, ranging from 10.4 nM to 8.2 μM78,89,99–105 and 11.3 nM to 4.7 μM,89,99–104 respectively. PD‐L1 binds
to B7‐1 with an affinity ranging between 1.4 and 35.4 μM.89,100,102,103 The methods used to quantify these
biomolecular interactions include surface plasmon resonance (SPR), isothermal titration calorimetry, microscale
thermophoresis, and the magneto‐nanosensor platform. These inconsistencies may be attributed to the various
protein constructs used in the studies. As one example, the monomeric PD‐1/PD‐1 ligand interactions are generally
weaker than the bivalent interactions. With regard to the SPR and magneto‐nanosensor platforms, immobilization
of a binding partner to the sensor chip surface may result in binding affinity measured to be different from those
found in solution or on the cell surface. In addition, it has been proposed that PD‐1 and PD‐L1 are not purposefully
optimized for maximal binding affinity in vivo and that the modest binding affinity of PD‐1 to its ligands could be a
result of natural selection for transient immune regulation.106 However, this claim needs to be examined carefully.
In all studies, the binding affinity of PD‐L2 to PD‐1 is consistently higher than that of PD‐L1 to PD‐1. Generally, the
B7‐1:PD‐L1 interaction is weaker than the PD‐1:PD‐L1 and PD‐1:PD‐L2 interactions. The difference in binding
affinity of PD‐L1 and PD‐L2 to PD‐1 is discussed by Ghiotto et al101 to be a result of their different binding
12 | YANG AND HU

mechanism. Based on kinetic analyses using SPR, the data obtained for the PD‐1:PD‐L2 interaction fits well with
the 1:1 Langmuir binding model.101 In contrast, the PD‐1:PD‐L1 binding was characterized as a two state reaction
model in which a conformation change is required for efficient binding of PD‐L1 to its receptor.101 Recently, the
first complete in situ kinetic analysis of the PD‐1/PD‐1 ligand/B7‐1 system was performed at the cell surface by Li
et al.107 The measured in situ parameters exhibited a similar trend as the solution measurements, with PD‐1:PD‐L2
being stronger than PD‐1:PD‐L1 and PD‐1:PD‐L1/PD‐L2 being stronger than B7‐1:PD‐L1. The authors also stated
that solution‐based affinity measurements in the micromolar range were weaker than the in situ parameters
measured in their study, suggesting that the in vivo interactions could be in the nanomolar to submicromolar range.
Further kinetic studies are required to have a better grasp of the affinity of the interactions in the PD‐1/PD‐1
ligand/B7‐1 system.

4.3 | The biological significance of the PD‐1/PD‐L1 signaling pathway

in tumor immunity
PD‐1 is highly expressed on tumor‐infiltrating lymphocytes,108 whereas PD‐L1 and PD‐L2 are elevated on a
variety of cancers, with PD‐L2 expression across tumor types being much less prevalent than PD‐L1.93,109–119
The overexpression of PD‐L1 in several cancers has been correlated with poor prognosis.120 Stemming from
these observations, it was hypothesized that tumors use PD‐L1 to evade T cell recognition by functioning as a
stop signal that limits the tumor‐killing actions of already‐activated T lymphocytes. Indeed, several animal
studies have proven that PD‐L1 expression on tumor cells prevents T cell‐mediated tumor immunity. In an in
vivo study conducted by Dong et al,121 they transferred activated T lymphocytes into two groups of
immunodeficient mice: (1) those injected with mock‐transfected P815 tumor cells (mock/P815) as the control
group and (2) those injected with B7‐H1‐transfected P815 tumor cells (B7‐H1/P815). While the number of T
lymphocytes greatly increased in the mock/P815 group, the T lymphocytes in the B7‐H1/P815 group
underwent significant apoptosis; furthermore, upon examination of the peritoneal cavities, a larger number of
B7‐H1/P815 tumor cells was measured. These observations meant that tumor‐associated PD‐L1 increased
apoptosis of activated tumor‐specific T lymphocytes and, as a result, promoted tumor growth. Most notably,
when they infused an anti‐B7‐H1 monoclonal antibody into mice harboring B7‐H1/P815 cells, the growth of
the B7‐H1/P815 cells were inhibited in vivo. In a subsequent study by the same group, using the same B7‐H1/
P815 mouse model, they demonstrated that these PD‐L1‐expressing P815 tumor cells were less susceptible to
cytotoxic T cell‐mediated lysis in vitro and had markedly enhanced tumorigenicity and invasive potential in vivo
as compared to those tumor cells lacking PD‐L1.122 Again, administration of an anti‐PD‐L1 antibody reversed
these observations.
More recently, Juneja et al123 provided additional evidence of the role of PD‐1 and PD‐L1 as “molecular
shields” for MC38 colorectal adenocarcinoma cells against an antitumor immune response. The absence of PD‐
1 receptors on the immune cells of PD‐1−/− mice resulted in complete clearing of PD‐L1‐expressing MC38
tumors. While in the wild‐type mice, growth of PD‐L1‐expressing MC38 tumor cells was quite robust.
Moreover, in the absence of PD‐L1 on the host cells in the tumor microenvironment, tumor‐derived PD‐L1
molecules were shown to be sufficient in limiting antitumor immunity by suppressing activated CD8+ tumor‐
infiltrating lymphocytes in PD‐L1−/− mice with MC38 tumors. Altogether, these findings suggest that the PD‐1/
PD‐L1 immune checkpoint pathway confers a selective advantage for tumors cells to grow and survive through
the inhibition of T lymphocyte activity, illustrating the potential of PD‐1 and PD‐L1 as targets for the
development of cancer immunotherapy. Indisputably, the discovery of this biological target has been translated
into life‐changing medicines for the treatment of metastatic diseases in patients who had little to no success
with traditional therapies and had a very low chance for survival.
YANG AND HU | 13

5 | ANTIBODY ‐BASED PD ‐1 /P D ‐L1 IMMUN E CH ECK POINT INHIBITORS

5.1 | A breakthrough product of translational medicine: the clinical success and efficacy
of antibodies targeting the PD‐1/PD‐L1 immune checkpoint pathway
The FDA has approved five antibody‐based PD‐1/PD‐L1 immune checkpoint inhibitors (anti‐PD‐1: nivolumab and
pembrolizumab; anti‐PD‐L1: atezolizumab, avelumab, and durvalumab) for the treatment of a wide variety of
cancers (Supporting Information Table S1). Many ongoing clinical trials are evaluating their use as single agents and
in combination with other therapeutic approaches. Most recently, the FDA has granted breakthrough therapy
designation status to the investigational drug cemiplimab (REGN2810) for the treatment of adults with metastatic
cutaneous squamous cell carcinoma (CSCC) and adults with locally advanced and unresectable CSCC, showing that
there is still a substantial amount of interest in developing PD‐1/PD‐L1 inhibitors. This section aims to briefly
summarize the clinical efficacy and exciting developments associated with these anti‐PD‐1/PD‐L1 monoclonal
antibodies.
It is undeniable that the advent of monoclonal antibodies that target and block the PD‐1 receptor and its
ligands PD‐L1 and PD‐L2 have resulted in unprecedented durable responses in select patients with advanced
cancers. The success story of the PD‐1/PD‐L1 inhibitors originates from the unexpected results obtained in a phase
1 dose‐escalation clinical trial that assessed the clinical activity of nivolumab.124,125 In this trial, 39 patients with
various advanced cancers (melanoma, NSCLC, RCC, castration‐resistant prostate cancer, and colorectal cancer)
were initially enrolled. As results were analyzed, it soon became apparent that nivolumab was able to induce
significant tumor regression in several patients. One patient with metastatic colorectal cancer received five doses
of nivolumab and experienced an impressive antitumor response for at least 21 months. In another patient with
multiorgan metastatic RCC, the first dose of nivolumab resulted in tumor regression at all sites except one, which
eventually converted to an overall PR after two additional doses that lasted for at least 16 months without further
therapy. Cumulative response rates were reported to be 18% for NSCLC, 28% for melanoma, and 27% for RCC; no
objective responses were found in patients with prostate or colorectal cancer. Amazingly, ~65% of patients
receiving nivolumab experienced durable responses lasting for at least a year. What stood out in this trial was the
fact that patients with NSCLC, which was previously believed to be nonimmunogenic, responded to nivolumab. This
finding has resulted in clinical trials testing for the activity of these anti‐PD‐1/PD‐L1 inhibitors in other
nonimmunogenic cancers, such as pancreatic and breast cancer.
Turning to more recent studies, these checkpoint inhibitors are beginning to be studied and used in earlier
stages of cancer as adjuvant therapy—for instance, nivolumab in melanoma126 and durvalumab in stage III
NSCLC.127 In a phase 3 randomized clinical trial comparing the efficacy of nivolumab versus ipilimumab for
adjuvant therapy in patients with resected stage IIIB, IIIC, and IV melanoma,126 nivolumab exhibited a statistically
significant 35% decline in disease recurrence or death as shown by the higher 12‐month rate of recurrence‐free
survival (70.8% vs 60.8%; hazard ratio [HR], 0.65; p < 0.001). Patients also tolerated nivolumab much better than
ipilimumab as evidenced by the number of treatment‐related grade 3 or 4 adverse events (14.4% vs 45.9%) and
number of discontinuation due to adverse events (9.7% vs 42.6%) occurring in the two treatment groups. These
results have allowed nivolumab to replace high‐dose ipilimumab as the preferred adjuvant immunotherapy regimen
in the National Comprehensive Cancer Network guidelines for melanoma. Similarly, durvalumab was shown to
significantly prolong progression‐free survival as compared to placebo as a consolidation therapy in patients with
stage III, locally advanced, unresectable NSCLC.127 In those patients that responded to durvalumab, the median
PFS from randomization was 16.7 months vs 5.6 months (P < 0.001) in favor of durvalumab. The 12‐month and 18‐
month PFS were 55.9% vs 35.3% (P < 0.001) and 44.2% vs 27.0% (P < 0.001), respectively. In addition, 72.8% of
patients on durvalumab vs 46.8% on placebo had ongoing response at 18 months. Finally, the median time to death
or distant metastasis was longer with durvalumab than with placebo (23.2 vs 14.6 mo, P < 0.001). These findings are
very promising and exemplify the durability of responses attained with the use of immunotherapy.
14 | YANG AND HU

Finally, of historic importance is the recent approval of pembrolizumab for the treatment of all advanced solid
tumor types classified as having high microsatellite instability (MSI‐H) or deficient DNA mismatch repair (dMMR),
the first cancer treatment to be approved based on a common biomarker rather than on tumor origin in the body.
Cancers classified as such have higher mutation rates, making them highly immunogenic and susceptible to immune
attack. In five uncontrolled, open‐label, multicohort, multicenter, single‐arm trials,128 totaling 149 patients,
pembrolizumab was shown to produce CRs and PRs in ~40% of the treated patients. Response to pembrolizumab in
some patients lasted for at least 22 months, with 78% of patients with response duration of at least 6 months.
Overall, the outstanding success and efficacy of the anti‐PD‐1/PD‐L1 inhibitors have been unbelievable.

5.2 | Structural basis of PD‐1/PD‐L1 immune checkpoint blockade by the anti‐PD‐1 and
anti‐PD‐L1 therapeutic antibodies
Several crystal structures of the anti‐PD‐1 and anti‐PD‐L1 monoclonal antibodies have been published78,129–132
and illustrate the structural basis of PD‐1 and PD‐L1 blockade by the antibodies. A thorough understanding of the
epitopes of these therapeutic antibodies will be instructive in the identification of key amino acid residues to target
in the design of small molecule inhibitors of this immune checkpoint. This section is meant to briefly describe
general features of the antibody:PD‐1 and antibody:PD‐L1 binding interactions.
The binding affinities of nivolumab and pembrolizumab have been measured to be 1.45 nM and 27 pM,
respectively.78,129 Furthermore, their half‐maximal inhibitory concentrations (IC50) have been found to be 2.52 nM
and ~0.1 to 0.3 nM, respectively.133,134 To explain this potent inhibition by both antibodies, the structures of the
Fab fragments of both antibodies in complex with PD‐1 were examined.78,129,135,136 Nivolumab makes contact with
PD‐1 predominantly at its N‐terminal loop, as well as the BC and FG loops (Figure 6A and 6B). Ten hydrogen bond
interactions are made between the N‐loop of PD‐1 and the heavy chain of the Fab (VH) of nivolumab.78 The FG loop
of PD‐1 makes five hydrogen bond interactions with the light chain (VL) and VH of nivolumab.78 Finally, an
additional hydrogen bond interaction occurs between the VH of nivolumab and the BC loop of PD‐1.78 The
interaction between nivolumab and PD‐1 is unique because the N‐terminal loop is located outside of the IgV
domain of PD‐1. Nonetheless, nivolumab is still able to block PD‐L1 from binding to PD‐1 by steric clash with its VL
at the FG loop, where both binding sites overlap. On the other hand, the Fab fragment of pembrolizumab binds
primarily to the C′D loop of PD‐1, with additional contacts with the C, C′, and F strands (Figure 6A and 6B).137 The
picomolar affinity of pembrolizumab for PD‐1 is mainly attributed to its extensive hydrogen bond, water‐mediated
hydrogen bond, and salt bridge interactions with the C′D loop and C′ strand of PD‐1.136 The binding site of
pembrolizumab overlaps greatly with that for PD‐L1 at the C, C′, and F strands, allowing it to hinder PD‐L1 from
binding to PD‐1 with high potency.129 Overall, nivolumab and pembrolizumab bind to PD‐1 at distinct sites with
almost no overlap. In contrast, the PD‐1 binding site of the antibody Fab fragments coincide with that of PD‐L1,
providing a structural rationale for the antibody‐mediated abolishment of the PD‐1/PD‐L1 interaction (Figure 6B).
A potentially attractive strategy for inhibiting the PD‐1/PD‐L1 PPI could involve designing small molecules that
target the loops of PD‐1 and induce and stabilize a conformation of the PD‐1 protein, which does not allow binding
to the PD‐1 ligands.
All clinically used anti‐PD‐L1 antibodies—atezolizumab, avelumab, and durvalumab—exhibit picomolar affinities
for PD‐L1 (400, 42.1, and 667 pM, respectively).130,138,139 Aligning very well with the binding affinities, the IC50
values for the three anti‐PD‐L1 antibodies have been reported in the FDA Pharmacology Review documents to be
82.8, 70, and 100 pM, respectively, as measured by competitive binding assays. Again, structures of the interaction
between the Fab fragments of these antibodies and the surface of PD‐L1 help to elucidate these highly potent
activities.130,139,140 Atezolizumab mainly uses its VH to form extensive hydrogen bond, salt bridge, and hydrophobic
interactions with the GFCC′ β sheet of PD‐L1. In addition, atezolizumab also makes contact with the BC, CC′, C′C′′,
and FG loops of PD‐L1. The epitope‐binding region of avelumab on PD‐L1 includes the GFCC′ β sheet, the C′′
strand, and the CC′ loop. Similar to the other anti‐PD‐L1 antibodies, durvalumab also binds to the GFCC′ β sheet of
YANG AND HU | 15

F I G U R E 6 Structural basis of PD‐1/PD‐L1 checkpoint blockade by anti‐PD‐1 therapeutic antibodies, nivolumab

and pembrolizumab (modified from PDB ID: 4ZQK, 5WT9, and 5JXE with PyMOL). A, Overall structure of the hPD‐
1 (blue ribbon model surrounded by light gray surface) and hPD‐L1 (green ribbon model) complex, the hPD‐1 and
nivolumab‐Fab complex, and the hPD‐1 and pembrolizumab‐Fab complex. The heavy chain (VH) and light chain (VL)
of the antibody‐Fab fragment are represented with purple and light pink ribbon models, respectively. B, Illustration
of the binding site of PD‐L1 (blue), nivolumab (red), and pembrolizumab (yellow) on PD‐1 (gray surface
representation). The images on the last row are turned 90° to the left with respect to the images on the second
row. The images on the second and last rows are oriented in the same relative position with respect to one another
in the same row

PD‐L1, as well as the CC′ loop. However, durvalumab also makes major hydrogen bond interactions with the
N‐terminal A strand of PD‐L1. In general, all anti‐PD‐L1 antibodies mainly interact with the GFCC′ face of the IgV
domain of PD‐L1 using their VH and VL. The major differences in their binding interaction with PD‐L1 are the
orientation of the VH and VL on the surface of the PD‐L1 and the varying contribution of the loops of PD‐L1 in
stabilizing the antibody/protein interactions (Figure 7A and 7B). In general, all the anti‐PD‐L1 antibodies compete
with PD‐1 for the same surface area on PD‐L1 to block the PD‐1/PD‐L1 interaction. Interestingly, the binding of the
three anti‐PD‐L1 antibodies and PD‐1 all involve the Tyr56, Glu58, Arg113, Met115, and Tyr123 residues on
16 | YANG AND HU

F I G U R E 7 Structural basis of PD‐1/PD‐L1 checkpoint blockade by anti‐PD‐L1 therapeutic antibodies—

atezolizumab, avelumab, and durvalumab (modified from PDB ID: 4ZQK, 5X8L, 5GRJ, and 5X8M with PyMOL).
A, Overall structure of the hPD‐1 (blue ribbon model) and hPD‐L1 (green ribbon model surrounded by light gray
surface) complex, the hPD‐L1 and atezolizumab‐Fab complex, the hPD‐L1 and avelumab‐Fab complex, and the
hPD‐L1 and durvalumab‐Fab complex. The heavy chain (VH) and light chain (VL) of the antibody‐Fab fragment are
represented with purple and light pink ribbon models, respectively. B, Illustration of the binding site of PD‐1 (blue),
atezolizumab (red), avelumab (yellow), and durvalumab (green) on PD‐L1 (gray surface representation). All images
on the second row are oriented in the same relative position with respect to one another

PD‐L1.131 These amino acids may be pivotal in the design of small molecule inhibitors of the PD‐1/PD‐L1 protein‐
protein interaction (PPI). Because these residues lie on the relatively flat GFCC′ β sheet of PD‐L1, it may be difficult
to design small molecule inhibitors targeting only this region. To develop small molecules with high binding affinity
and potency, it may be necessary to incorporate elements into small molecules that allow them to make
interactions with the loops of these proteins, as well as the GFCC′ β sheet. Aside from these FDA‐approved
antibodies, an experimental anti‐PD‐L1 antibody BMS‐936559 has been reported with the structure deposited in
the Protein Data Bank (PDB) under the PDB ID 5GGT, detailing its interaction with PD‐L1.135
A relatively novel drug platform has been pursued in recent years to develop a single domain antibody, termed
a nanobody, KN035.106 When KN035 was fused to a Fc fragment, it bound to PD‐L1 specifically with an affinity of
3 nM and inhibited the PD‐1/PD‐L1 interaction with an IC50 value of 5.25 nM.106 This nanobody platform evolved
from the desire to develop therapeutic entities smaller than the monoclonal antibodies and with more favorable
physiochemical properties to enable more potent antitumor activity. It was found to dose‐ and time‐dependently
YANG AND HU | 17

F I G U R E 8 Binding interaction between the nanobody KN035 (pink ribbon model) and the human PD‐L1 (hPD‐
L1; purple ribbon model; modified from PDB ID: 5JDS with MOE). A, KN035 interacts with the C′CFG strands of
the IgV domain of the hPD‐L1 using its CDR1 (blue ribbon model) and CDR3 (red ribbon model) domains. B, Key
residues involved in the interaction between KN035 and hPD‐L1 are illustrated with stick models. The left panel is
a zoomed‐in image of the interaction between KN035 and hPD‐L1. The right panel is the same image as the left
panel but rotated 180° to show a back side view. Amino acid residues colored yellow belong to KN035. Amino acid
residues colored pink are part of hPD‐L1, with those colored cyan indicating the five key hotspot residues (Ile54,
Tyr56, Glu58, Gln66, and Arg113). Red spheres indicate water molecules. Black dashed lines indicate various types
of interactions between residues, such as hydrogen bonding interactions, water‐mediated interactions, and π‐π
stacking interactions (eg, KN035:Phe101 and PD‐L1:Tyr56)

induce T‐lymphocyte cytokine production and demonstrated strong antitumor activity comparable to that of
durvalumab. It exhibited a relatively long half‐life of 72 hours in vivo.106 Currently, KN035 is being tested in several
phase 1 clinical trials in patients with advanced solid tumors. The nanomolar binding affinity to PD‐L1 is believed to
be achieved through the various hydrophobic and polar interactions that KN035 makes with the CC′FG strands of
the IgV domain of PD‐L1, using its complementarity‐determining region (CDR) 3 and CDR1 domains, that is, the FG
loop and BC loop, respectively (Figure 8A). From structural and mutagenesis studies, five key hotspot residues on
PD‐L1 (Ile54, Tyr56, Glu58, Gln66, and Arg113) were determined to be crucial for the interaction between KN035
and PD‐L1 (Figure 8B).106 These residues, along with those already discussed, map out potential surfaces on the
PD‐L1 protein to which small molecules can be designed to bind and inhibit the PD‐1/PD‐L1 PPI.
In general, monoclonal antibodies can potentially initiate antibody‐dependent cell‐mediated cytotoxicity
(ADCC) and complement‐mediated cytotoxicity (CMC). ADCC is a well‐established mechanism in which monoclonal
antibodies (bound to a specific target on the tumor) interact with the Fc receptor (FcR) on FcγR + immune effector
18 | YANG AND HU

cells using their Fc domain to trigger cell‐mediated destruction of tumor cells.141 CMC is the result of complement
binding to the monoclonal antibodies and activating a membrane attack complex, which leads to cell death by
formation of pores in the cell membrane.142 ADCC/CMC would be appropriate if the target to be eliminated were
the tumor cells like in the case of trastuzumab against HER2‐overexpressing breast cancer cells or rituximab
against CD20‐expressing lymphoma cells. However, it would be undesirable and counterproductive for the anti‐PD‐
1 and anti‐PD‐L1 monoclonal antibodies to eliminate PD‐1/PD‐L1‐expressing immune cells as this would result in
minimal or no enhancement in immune activity against tumor cells. IgG1 antibodies are known to induce ADCC and
CMC, while IgG4 antibodies have reduced tendency to cause ADCC and do not bind complement, thereby
preventing CMC.143 Engineered IgG1 antibodies that contain specific mutations to the Fc domain have also been
studied and shown to reduce or eliminate ADCC activity.144 Of the currently approved PD‐1/PD‐L1‐blocking
monoclonal antibodies, they are either of the IgG4 isotype (nivolumab and pembrolizumab) or of the IgG1 isotype
with modifications made to the Fc domain to prevent ADCC (atezolizumab, avelumab, and durvalumab). Therefore,
there should be little concern for ADCC and CMC to occur and interfere with the immune‐boosting effects of these
anti‐PD‐1 and anti‐PD‐L1 monoclonal antibodies.

5.3 | A double‐edged sword: limitations of therapeutic monoclonal antibodies

Despite the promising tumor shrinkage, durable responses and prolonged survival observed in clinical trials, the
anti‐PD‐1/PD‐L1 monoclonal antibodies are not without flaws. A major limitation of these therapies is the failure to
elicit a response in a majority of cancer patients. The response to these PD‐1/PD‐L1‐targeted immunotherapies
varies across different tumor types, ranging from ~13% in patients with bladder cancer to ~70% in classical
Hodgkin lymphoma, with an overall response rate of 20% to 40% in most solid tumors.128,145–148 Several
mechanisms of immune escape to immune checkpoint blockade have been described to explain this wide variability
in response to immunotherapies or the lack thereof.23,24,149 Some of these escape mechanisms include poor tumor
immunogenicity or low tumor mutational burden, reduced maturation of APCs such as dendritic cells, suboptimal T‐
cell activation, impaired T‐cell trafficking and infiltration, stroma‐dependent exclusion of T lymphocytes from the
tumor parenchyma, reduced tumor recognition by immune cells through the downregulation of MHCs on cancer
cells, and the presence of alternative immunosuppressive factors (eg, LAG‐3, TIM‐3, VISTA) and cells (eg, Treg cells,
tumor‐associated macrophages, myeloid‐derived suppressor cells) in the tumor microenvironment. To overcome
these mechanisms of immune escape and to elevate response rates, there is a need to rationally combine these
agents with other treatments that can activate T lymphocytes and have them traffic to the tumor site, infiltrate
successfully, and ultimately eliminate the tumor cells.
Several clinical trials are currently examining such combinations. One combination regimen is that of nivolumab
and ipilimumab. In the phase 3 trial CheckMate‐067, previously untreated patients with unresectable stage III or IV
melanoma displayed a greater survival advantage when treated with the combination of ipilimumab and nivolumab
(median PFS: 11.5 months; median OS not reached at 36‐month follow‐up; 3‐year OS rate: 58%) as compared to
ipilimumab alone (median PFS: 2.9 months; median OS: 19.9 months; 3‐year OS rate: 34%) or nivolumab alone
(median PFS: 6.9 months; median OS: 37.6 months; 3‐year OS rate: 52%).150,151 More recently, in the phase 2 trial
CheckMate‐142, nivolumab and ipilimumab again exhibited high response rates (55%; 65 of 119 patients) and
encouraging PFS (71%) and OS (85%) at 12 months in patients with dMMR/MSI‐H metastatic colorectal cancer.152
Responses were shown to be durable, with 94% of responders having ongoing response at data cutoff, and 83%
having responses lasting at least 6 months.152 These results indicate that the combination of two or more agents
with distinct but complementary immune‐bolstering mechanisms can increase the percentage of patients
responding to therapy and the durability of such responses. However, the combination of nivolumab and
ipilimumab was associated with increased toxicity, with a greater number of grade 3 and 4 treatment‐related
adverse events and treatment discontinuations.150,152 Therefore, caution must be taken in testing different
combination regimens, ensuring that patient safety is not sacrificed for greater efficacy.
YANG AND HU | 19

Safety is another major drawback with these agents and is inherent in their mechanism of action. These agents
boost the body’s natural defense system against cancer by reactivating immune cells such as T lymphocytes.
Careless activation of T lymphocytes, especially autoreactive T lymphocytes, may wreck havoc on normal tissues,
resulting in autoimmune reactions, commonly termed irAEs. When compared to ipilimumab, nivolumab has a better
safety profile as evidenced by the lower treatment‐related grade 3 or 4 adverse events (16.3% vs 27.3%150 and
25.4% vs 55.2%126 in two separate studies). Again, the higher toxicity of ipilimumab is most likely a result of its
nonselective activation and expansion of T lymphocytes as previously explained. In contrast, the PD‐1/PD‐L1
inhibitors are much better tolerated and all have similar manageable toxicity profiles. The most common drug‐
related adverse events of any grade were fatigue, pruritis, and rash, in addition to musculoskeletal pain, decreased
appetite, nausea, and diarrhea.128,145–148 irAEs are unique in that they are not self‐limiting. In addition, they have a
delayed onset—occurring even after treatment interruption or termination—and are potentially life‐threatening,
resulting in temporary or permanent discontinuation of treatment and a reduction in the quality of life of cancer
patients.153 It is therefore important to quickly and promptly diagnose and treat these irAEs and to monitor patient
labs periodically while on these agents. Because the immune system is present in all parts of the body, nearly all
organs can be affected by immune‐related toxicities. These toxicities commonly affect the skin (maculopapular rash,
pruritis, vitiligo), the gastrointestinal tract (diarrhea, colitis), the endocrine glands (hyperthyroidism, hypothyroid-
ism, hypophysitis, type I diabetes mellitus), the lungs (pneumonitis), the liver (hepatitis), the kidneys (interstitial
nephritis, lupus‐like glomerulonephritis), and the nervous system (peripheral neuropathy, Guillain‐Barré syndrome,
myasthenia gravis).153–155 With respect to the median time to onset, irAEs can be classified as early (<2 months) or
late (>2 months): skin, 5 weeks; gastrointestinal, 7.3 weeks; hepatic, 7.7 weeks; pulmonary, 8.9 weeks; endocrine,
10.4 weeks; and renal, 15.1 weeks (although these toxicities can develop at any time due to the wide confidence
intervals reported in clinical studies).153 This unique spectrum of toxicities and their variable kinetics pose a
tremendous challenge for even the most experienced physicians. The only drug therapies that are recommended by
the manufacturers to alleviate these reactions are systemic immunosuppressants such as corticosteroids, most
commonly prednisone. However, the use of these immunosuppressants may diminish the therapeutic efficacy of the
anti‐PD‐1/PD‐L1 inhibitors, causing a major treatment dilemma. As with any therapeutic protein, it is possible to
develop neutralizing antibodies against these agents, potentially limiting their therapeutic benefit and increasing
the incidence of anaphylaxis and autoimmunity. In fact, treatment‐emergent neutralizing antibodies have been
detected in patients administered these PD‐1/PD‐L1 inhibitors. Fortunately, the development of these
antitherapeutic antibodies do not appear to have significantly impacted the efficacy, safety, or pharmacokinetics
(PK).128,145–148
With regard to the PK, unsurprisingly, these monoclonal antibodies exhibit relatively low volumes of
distribution at steady state (range, 4.72 to 7.92 L) and prolonged elimination half‐lives (range, 6.1 days to 27
days).128,145–148 The low volume of distribution indicates a low tendency to penetrate cellular membranes and
barriers. Because of their large size (~150 000 Da), it is difficult for these antibodies to reach the exhausted
intratumoral T lymphocytes and bind to either PD‐1 or PD‐L1. It also makes it challenging to treat tumors located
at immune‐privileged sites, such as the brain, eyes, uterus, and testicles. Although a long half‐life and prolonged
target occupancy have advantages (eg, lower doses, less frequent administration, and prolonged duration of action),
it becomes problematic when toxicities occur as a consequence of the drug therapy.
Other shortcomings of these therapeutic monoclonal antibodies include the high costs associated with their
administration and manufacturing. All of the anti‐PD‐1/PD‐L1 agents are administered by intravenous infusion
every 2 or 3 weeks, depending on the cancer being treated. Because of the route of administration, patients can
only receive doses of these drugs in a hospital setting, requiring additional expenditure by patients with respect to
travel and healthcare services on top of their copay or coinsurance. The high commercial prices of these drugs have
also placed a great strain on hospital budgets. The manufacturing of monoclonal antibodies is very unpredictable,
possibly giving rise to a heterogeneous population of antibodies, owing to the fact that living cell cultures are used
to create them. This unpredictability may affect the efficacy, safety, and quality of these medications. Furthermore,
20 | YANG AND HU

monoclonal antibodies are highly sensitive to external conditions and require refrigeration and special storage to
prevent structural degradation. These limitations of the current antibody‐based PD‐1/PD‐L1 inhibitors provide a
strong argument in favor of the development of nonantibody–based inhibitors of this immune checkpoint pathway.

6 | SMA LL MOLE CU LE P D ‐1/ PD ‐L 1 IMMU NE CH ECKP OINT IN HIBITORS

In this section of the review, we present several promising small molecules targeting the PD‐1/PD‐L1 immune
checkpoint pathway and review certain design strategies for creating small molecules to tackle this challenging PPI.
We conducted a literature search in the PubMed and SciFinder patent databases to identify small molecule
inhibitors published between 2011 and 2018 that were shown to possess relatively high inhibitory activity against
the PD‐1/PD‐L1 PPI. It should be noted that the small molecules are selected to illustrate representative design
strategies and are not meant to be inclusive. Several small molecule inhibitors have been listed in Supporting
Information Table S2, showing the emerging interest in this area of research.

6.1 | Nonpeptide, small molecule inhibitors

Some of the drawbacks of antibody‐based therapeutics can be overcome using small molecule immune checkpoint
inhibitors that include peptidomimetics and peptides. Small molecules would be more readily absorbed and have
higher oral bioavailability, making it easier to formulate and administer to patients. They can be modified
structurally to have larger volumes of distribution, allowing them to tackle extravascular targets, accumulate in
high concentration in the tumor microenvironment, and treat cancers in difficult‐to‐reach anatomical locations.
They can be designed to have shortened half‐lives to control for irAEs. In addition, the synthesis of small molecules
is much more straightforward, and uniformity and stability can be properly maintained across batches. Most
importantly, they would spare patients, healthcare institutions, and society as a whole from high drug costs. For
these reasons, there has been a lot of interest in searching for small molecule immune checkpoint inhibitors.
The structures of two series of small molecule inhibitors of PD‐1/PD‐L1 containing a biphenyl core structure
have been disclosed: one consisting of the (2‐methyl‐3‐biphenylyl)methanol scaffold (eg, BMS‐202, 1) and another
based on the (3‐(2,3‐dihydro‐1,4‐benzodioxin‐6‐yl)‐2‐methylphenyl)methanol scaffold (eg, BMS‐200, 2;
Figure 9).156,157 The IC50 values of these small molecule inhibitors, as evaluated by an europium‐allophycocyanin

F I G U R E 9 Structures of a select group of small molecule PD‐1/PD‐L1 inhibitors with the (2‐methyl‐3‐
biphenylyl)methanol and (3‐(2,3‐dihydro‐1,4‐benzodioxin‐6‐yl)‐2‐methylphenyl)methanol core structures156,157
YANG AND HU | 21

homogenous time‐resolved fluorescence (HTRF) binding assay, range from 920 pM to 14.25 μM.156,157 These
compounds were recently found to form complexes with PD‐L1 using two‐dimensional 1H‐15N heteronuclear
multiple quantum coherence (HMQC) NMR structural studies, where the compounds bind specifically to PD‐L1 and
occupy a deep hydrophobic channel‐like pocket between a pair of PD‐L1 proteins.158 Derivatives with a similar
structure as BMS‐202 created a deep, hydrophobic cleft, closed off at one end by a tyrosine residue (ie, Tyr56).159
In contrast, those with a similar structure as BMS‐200 opened up the cleft by pushing away the Tyr56 residue and
formed a tunnel through the PD‐L1 dimer.158 The change in conformation of the PD‐L1 dimer associated with the
binding of different ligands illustrate the plasticity of the ligand binding site. Based on NMR and size‐exclusion
chromatography studies, it was concluded that these small molecules induced PD‐L1 dimerization, which may be
responsible for occluding the interaction between the ligand and its cognate receptor.158–160 The compounds
dimerize the two PD‐L1 proteins using their GFCC′ faces. At the present time, more studies are needed to validate
this phenomenon and to determine if it is physiologically significant. Moreover, the BMS compounds were
partitioned into smaller fragments, which then were screened for binding to PD‐L1 using 1H‐15N HMQC NMR.
Intriguingly, it was found that the smallest fragments with the ability to bind and dimerize PD‐L1 were the (2‐
methyl‐3‐biphenylyl)methanol and (3‐(2,3‐dihydro‐1,4‐benzodioxin‐6‐yl)‐2‐methylphenyl)methanol fragments from
the respective series.158,160 These fragments largely contribute to the activity of these compounds and may serve
as potential starting points in fragment‐based drug discovery efforts.
With regard to their in vitro activity, the small molecules, specifically BMS‐1001 (3) and BMS‐1166 (4; Figure 9),
antagonized the inhibitory effects of cell surface PD‐L1 and soluble PD‐L1 (sPD‐L1) and reactivated Jurkat T‐
lymphocytes in a dose‐dependent manner.160 Although their activity was significantly less pronounced when
compared to therapeutic antibodies, these compounds proved that it is possible to target the PD‐1/PD‐L1 axis with
small molecules. Further optimization of these lead compounds may lead to more potent inhibitors capable of
matching the potency and efficacy of therapeutic antibodies.
Three major “hotspots” or “pockets” were identified on PD‐L1 based on the binding of the BMS compounds to
the hPD‐L1 protein (Figure 10): (1) a hydrophobic pocket consisting of the side chains of Tyr56, Glu58, Arg113,
Met115, and Tyr123; (2) a second nearby hydrophobic cleft composed of Met115, Ala121, and Tyr123; and (3) an
extended groove made up of the main chain and side chains of Asp122, Tyr123, Lys124, and Arg125.83 Indeed,
BMS‐202 makes contact with the bordering amino acids of hotspots 1 and 3 and inserts its methylphenyl moiety
into the relatively hydrophobic hotspot 2 to obstruct the binding of PD‐1 to PD‐L1 (Figure 11A‐C). Most notably,

F I G U R E 1 0 Illustration of the three major “hotspots” on the PD‐L1 protein surface (black ribbon model;
modified from PDB ID: 5J89 with MOE). Key amino acid residues are indicated in cyan. The molecular surfaces
represented in the zoomed‐in images are color‐coded, such that red = hydrophilic regions, white = neutral regions,
and blue = lipophilic regions
22 | YANG AND HU

F I G U R E 1 1 Structural basis of PD‐1/PD‐L1 checkpoint blockade by a small molecule, BMS‐202 (modified from
PDB ID: 5J89 with MOE). A, Crystal structure of BMS‐202 (light green stick model) on the surface of hPD‐L1 (gray
ribbon structure with hotspot residues indicated in yellow). B, Receptor representation of the docked BMS‐202
molecule (green stick model) on PD‐L1 (gray) with the location of hotspot residues indicated in yellow.
Red = hydrophilic regions, white = neutral regions, and blue = lipophilic regions. C, Detailed characterization of the
binding mode of BMS‐202 in between the GFCC′ β strands of the two PD‐L1 proteins. The image on the right is the
same image on the left but rotated 180° along the vertical axis. Red spheres represent water molecules. Dashed
light blue lines indicate hydrophobic, hydrogen bond, and water‐mediated interactions. Subscripts before amino
acid abbreviation indicate A = bottom PD‐L1 structure (blue ribbon model) and B = top PD‐L1 structure (magenta
ribbon model) as is used in PDB ID: 5J89

the hotspot residues proposed for these small molecules are the same residues (ie, Tyr56, Glu58, Arg113, Met115,
Tyr123) used in the binding interaction between the anti‐PD‐L1 antibodies and PD‐L1, demonstrating that these
amino acids are essential for the interaction between PD‐1 and PD‐L1 and that small molecules can be designed to
fit into the grooves created by these hotspot residues to inhibit the interaction.
CA‐170 is a small molecule that inhibits multiple immune checkpoints, specifically PD‐L1/L2 and VISTA, with
low nanomolar potencies.161 CA‐170 was derived from a focused small molecule library rationally designed based
on the structure of the interaction hotspots between PD‐1 and PD‐L1. Functional screening was then performed to
identify compounds capable of selectively disrupting PD‐L1/2 and VISTA without cross‐reactivity with other
immune checkpoints. In preclinical ex vivo data, CA‐170 demonstrated dose‐dependent enhancement in the
proliferation of PD‐L1, PD‐L2, and VISTA‐inhibited T lymphocytes (half‐maximal effective concentration
[EC50] = 17, 13, and 37 nM, respectively) and in INF‐γ production.162,163 Several subsequent in vivo studies have
YANG AND HU | 23

shown that CA‐170 can activate both peripheral and intratumoral T lymphocytes at doses of 10 and 100 mg/kg.164
CA‐170 administered orally on a once‐daily schedule could significantly reduce the number of B16/F10 melanoma
lung metastases and the growth rate of implanted mouse MC38 colon carcinoma tumors.162,163 In addition, CA‐170
significantly inhibited the growth of CT26 colon tumors when used in combination with cyclophosphamide.163 All
these studies showed that CA‐170 had comparable efficacy to a blocking anti‐PD‐1 comparator antibody. CA‐170
also displayed a clean preclinical safety profile with a >100‐fold therapeutic window.161 In 2016, this drug
candidate became the first small molecule to be progressed into phase 1 clinical trials for the treatment of
advanced solid tumors and lymphoma.165,166 Emerging clinical data from a small number of patients show early
signs of antitumor activity, including tumor shrinkages and prolonged stable disease, with an acceptable safety
profile and approximately dose proportional PK profile.164 The most promising feature of CA‐170 is that it exhibits
sustained immune pharmacodynamics (PD) in vitro and in vivo, suggesting that drug efficacy may extend beyond
drug clearance.161,163

6.2 | Peptide‐based and peptidomimetic inhibitors

Peptide‐based therapeutics have certain advantages over their small molecule and antibody counterparts. Due to
their larger size, they can bind more easily to the relatively flat surface of the protein‐protein interface and cover a
much wider portion of its surface, which may translate into more potent and selective inhibition of the interaction.
Peptides can also offer a lower degree of toxicity and minimal nonspecific and drug‐drug interactions.167 Moreover,
because they are not as large as antibodies, they may retain a small molecule’s oral bioavailability and ability to
penetrate into tumor cells. The major disadvantage, among others, of this drug platform is the oral and plasma
stability of peptides.167 One method to improve stability involves replacing L‐amino acids with D‐amino acids to
decrease substrate recognition and binding affinity of proteolytic enzymes. Another method for overcoming this
limitation involves the modification of side chains or the backbone of peptides to produce peptidomimetics that can
withstand enzymatic degradation. Cyclizing the N‐termini and C‐termini of a linear peptide offers a third way to
impart enhanced stability against gastrointestinal proteases and peptidases.
A series of hydrolysis‐resistant D‐peptide antagonists was reported to target the PD‐1/PD‐L1 PPI.168 Initially,
an L‐peptide inhibitor was discovered using phage display technology in which a peptide library displayed on M13
phage was screened for binding to the L‐version of the IgV domain of PD‐L1 expressed by E. coli. However, this L‐
peptide inhibitor showed no in vivo activity when injected into tumor‐bearing mice, mainly due to rapid hydrolysis
in the blood. To circumvent this problem, the researchers decided to design D‐peptide inhibitors, which are
generally more resistant to degradation than their L‐enantiomeric counterparts. Using mirror‐image phage display,
they discovered the D‐peptide DPPA‐1 (sequence NYSKPTDRQYHF). DPPA‐1 binds to the L‐enantiomeric form of
PD‐L1 with an affinity of 0.51 μM as measured by SPR spectroscopy.168 In addition, D
PPA‐1 could effectively
disrupt the PD‐1/PD‐L1 interaction up to concentrations of 1 mg/mL without much toxicity, suggesting a high
therapeutic index of the D‐peptides.168 This peptide inhibitor also inhibited tumor growth and prolonged animal
survival.168 When DPPA‐1 was conjugated to fluorescent cyanine 5.5, in vivo near‐infrared fluorescence imaging
showed that this inhibitor accumulated at the site of the tumor tissue. It was found to be nontoxic to tumor cells,
indicating that the activity of DPPA‐1 might involve the activation of the antitumor immune system instead of direct
cytotoxicity. Although these results are encouraging, a peptide inhibitor composed of unnatural D‐amino acids has
the potential to pose an immunogenic and safety risk.169–171
Another peptide inhibitor targeting PD‐L1 was identified using bacterial surface display methods, the targeting
PD‐L1 peptide (TPP‐1; sequence: SGQYASYHCWCWRDPGRSGGSK) exhibited approximately 3‐fold greater
selectivity for hPD‐L1 over mouse PD‐L1 (mPD‐L1) and hPD‐L2 and an affinity of ~95 nM for hPD‐L1.172 It blocked
the interaction of PD‐1 and PD‐L1, which resulted in the reversal of PD‐L1‐mediated inhibition of T‐cell activation
as measured by production levels of IFN‐γ and proliferation of T lymphocytes at 20 μM concentration. Relative to
the positive control durvalumab, TPP‐1 did not produce as great of an extent of T‐cell activation. A xenograft
24 | YANG AND HU

mouse model demonstrated that TPP‐1 (4 mg/kg) and durvalumab (0.1 mg/kg) significantly inhibited tumor growth
by 56% and 71%, respectively, relative to the control mice treated with a scrambled TPP‐1 peptide.172 It is
important to note that inhibition of tumor growth by TPP‐1 was not observed in the absence of T cells, indicating
that the inhibitory activity of TPP‐1 relies on the reactivation of the immune system.172 The expression of IFN‐γ
and granzyme B was examined by immunohistochemistry and was found to be significantly increased in tumor
tissues.
Another peptide inhibitor Ar5Y_4 was reported to target PD‐1, unlike the two peptides described above.
Ar5Y_4 exhibits a binding affinity of ~1.38 μM for PD‐1.173 It restores 67% of the Jurkat T cells’ production of IL‐2
but does so at a relatively high concentration of 250 μM, indicating that further optimization of this peptide
structure is necessary to obtain more potent cellular activity.173 The goal was to design a peptide ligand for PD‐1
that incorporated five key hotspot residues derived from PD‐L1 (eg, Tyr56, Arg113, Ala121, Asp122, Tyr123 as
determined by three in silico hotspot prediction tools) linked by different helices and scaffold fragments derived
from a library. Ar5Y_4 has the following amino acid sequence: GNWDYNSQRAQLYNQ, with underlined residues
representing the hotspot residues. Note that Ala121 was replaced with a tryptophan to introduce novel
interactions with PD‐1. To examine if these hotspot residues were responsible for the binding affinity of Ar5Y_4,
alanine scanning was performed. Replacement of any of the residues with alanine resulted in a ~8 to 21‐fold
reduction in the affinity of Ar5Y_4, suggesting that these residues are indeed necessary for binding to PD‐1.173
Other peptides with varying combinations of hotspot residues on PD‐L1 (eg, Ala121, Asp122, and Tyr123; Arg113,
Ala121, Asp122, Tyr123; Arg113, Met115, Ala121, Asp122, and Tyr123) were found to have lower binding affinity
to PD‐1 than Ar5Y_4, with the KD ranging from ~3 to ~370 μM.173
AUNP‐12 with the putative structure 5 shown in Figure 12 is a branched 29‐amino acid peptide engineered to
contain certain sequences from the extracellular PD‐L1/L2 binding domain of the human PD‐1 protein.174 This
inhibitor is expected to bind PD‐L1/L2 and inhibit the three key receptor‐ligand interactions in the PD‐1/PD‐1
ligand/B7‐1 system (ie, PD‐1:PD‐L1, PD‐1:PD‐L2, and B7‐1:PD‐L1) with sub‐nanomolar potency.174 It is purported
to inhibit both primary tumor growth and metastasis and to sustain antitumor immune activity for at least 24 hours
with minimal toxicities. Conducting a brief structure‐activity relationship (SAR) study (Figure 12), the BC loop (6,
sequence SNTSESF), FG loop (sequence LAPKA), D strand (sequence FRVTQ), and G strand (sequence QIKE) of the
PD‐1 ectodomain appear to be essential for maintaining potent activity as measured by the CFSE mouse splenocyte
proliferation assay.174 If any of these segments are deleted (7 to 9) or replaced (10 to 12), the activity of the
peptide inhibitor drops dramatically. In addition, acylation can either enhance (13 and 14) or dampen (15 and 16)
the activity of Aur‐08. Palmitoylation of the lysine in the FG loop (14) or G strand (13) fragment gives about similar
activity as compared with Aur‐08. However, varying the side‐chain length of the acyl group (15) and/or the location
of the acylation to other amino acids (16) will cause a decrease in the activity relative to Aur‐08. As a quick aside,
the PD‐1/PD‐L1 immune checkpoint pathway has been noted as a cause of immunosuppression in septic
individuals.175 Recently, Aur‐08 was shown to confer a 2‐fold survival advantage against two‐hit Candida albicans
sepsis for mice treated with this anti‐PD‐L1 peptide relative to mice treated with an inactive scrambled peptide
(59.4% vs 30.3%, P = 0.015).176 Stemming from the work done with AUNP‐12, several series of small peptides and
peptidomimetics (Figure 13) have been disclosed in patents177–181 and listed in Supporting Information Table S2 for
further reference. Peptides built with the natural loops and strands of either PD‐1 or PD‐L1 may be another
effective strategy to develop potent inhibitors of the PD‐1 and PD‐L1 immune checkpoints.
Using peptide discovery platform system (PDPS) technology, three series of macrocyclic peptide inhibitors with
varying number of amino acids in the ring system (Figure 14) have been identified to target both the PD‐1/PD‐L1
and CD80/PD‐L1 interactions.182 Of these three series, the representative structures for the 15‐mer (21 with
IC50 = 9 nM) and 14‐mer (22 with IC50 = 7 nM) series were more potent than that of the 13‐mer series (23 with
IC50 = 153 nM) in terms of their inhibitory activity on the PD‐1/PD‐L1 interaction. Based on a Jurket T lymphocyte‐
based model of the PD‐1/PD‐L1 immune checkpoint blockade, 21 and 22 dose‐dependently restored activity of T
lymphocytes with EC50 values of 566 and 293 nM, respectively.183 In comparison, the positive controls durvalumab
YANG AND HU | 25

F I G U R E 1 2 Structures of select peptide inhibitors targeting the PD‐1/PD‐1 ligand/B7‐1 system.174 The
structure‐activity relationship of the peptide‐based inhibitors of PD‐1/PD‐L1 is depicted relative to Aur‐08. These
inhibitors incorporate four key sequences from the human PD‐1 ectodomain: the BC loop (sequence SNTSESF,
denoted in red), the FG loop (sequence LAPKA, denoted in green), the D strand (sequence FRVTQ, denoted in blue),
and the G strand (sequence QIKE, denoted in purple). The most potent inhibitors are indicated in the dashed or
solid boxes. The compound identification numbers provided correspond to those used in the referenced patent.
The percentages listed beneath each peptide indicate the percentage of splenocytes rescued by compounds
screened at 100 nM concentration in a CFSE (carboxyfluorescein diacetate succinimidyl ester)‐based mouse
splenocyte proliferation assay

(EC50 = 0.199 nM) and nivolumab (EC50 = 1.27 nM) were more effective at inhibiting the PD‐1/PD‐L1 interac-
tion.183 Remarkably, the peptides restored comparable levels of activity to the tested cells at their maximal activity
as measured by the RLUmax relative to the antibodies, ranging between 2.70 to 2.96 and 2.62 to 3.25 for the
peptides and antibodies, respectively.183 Based on structural studies, both 21 (Figure 15A) and 22 (Figure 15B) bind
partially at the site where PD‐1 interacts with PD‐L1, providing a rationale for the effective inhibition of the PD‐1/
PD‐L1 interaction.183 Both peptides interact with the surface of PD‐L1 using hydrophobic and polar interactions.
When compared to the anti‐PD‐L1 antibodies, avelumab and BMS‐936559, these peptides mimicked only ~37% of
the PD‐L1/antibody interactions.183 Therefore, it is essential to study the interactions between the antibodies and
their corresponding protein target, either PD‐1 or PD‐L1, to design more potent inhibitors of this immune
checkpoint pathway.
An engineered PD‐1 ectodomain has been reported as a high‐affinity competitive antagonist of PD‐L1 and acts
as a “decoy” receptor for the PD‐1 ligand.184 The motivation for creating high‐affinity PD‐1 variant (mimic)
polypeptides was to circumvent the issues associated with the large size of monoclonal antibodies. Due to the poor
binding affinity of the wild‐type ectodomain, they set out to enhance the affinity of PD‐1 for PD‐L1 using directed
evolution with yeast‐surface display.184 They obtained variants with 15 000 to 40 000‐fold enhanced affinity after
five rounds of selection and found a strong trend toward convergence onto a consensus sequence of 10 amino acid
26 | YANG AND HU

F I G U R E 1 3 Structures of peptide‐based (top row) and peptidomimetic (bottom row) inhibitors of PD‐L1.177–181 The
activities of these inhibitors were evaluated with a CFSE‐based proliferation assay and reported as the rescue percentage
of mouse splenocyte proliferation from PD‐L1‐mediated exhaustion, which have been listed under each inhibitor shown.
The compounds were all tested at a concentration of 100 nM

F I G U R E 1 4 Structures of representative macrocyclic peptide inhibitors of the PD‐1:PD‐L1 and CD80:PD‐L1

interactions.182 The IC50 values reported under each macrocyclic peptide indicates the concentration at which 50%
of the interaction between hPD‐1 and hPD‐L1 is inhibited. The IC50 values are derived from a homogeneous time
resolved fluorescence (HTRF)‐based assay

substitutions.184 Two high‐affinity consensus (HAC) PD‐1 polypeptides were generated that differed by an
isoleucine or valine at position 41, referred to as HAC‐I (KD ~107 pM) and HAC‐V (~110 pM), respectively.184 The
high affinity of HAC‐V has been explained to be the result of novel binding interactions (ie, additional hydrogen
bonds and a salt bridge) introduced by some of the single point mutations made to the wild‐type PD‐1 (Figure
16).185 The sequence of these PD‐1 mimic polypeptides are the same as the wild‐type sequence at residues 26–147
with the amino acid substitutions at positions 39 to 41, 43, 45, 49, 53, 97, 100, and 107.186 From competitive
YANG AND HU | 27

F I G U R E 1 5 Structural basis of checkpoint blockade by macrocyclic peptide inhibitors 21 (A, white ball‐and‐stick
model) and 22 (B, white ball‐and‐stick model; modified from PDB ID: 5O4Y and 5O45 with MOE, respectively). The
surface of the PD‐L1 protein is colored as follows: red = hydrophilic regions, gray = neutral regions, blue = lipophilic
regions). The first panel in each row depicts the top view of the binding interaction of each peptide to the surface of
hPD‐L1. The second panel in each row is the same image but rotated 90° up to present the front side view of the
binding interaction. The binding of the peptides and hPD‐1 to hPD‐L1 are overlaid in the final panel of each row
(green ribbon model = hPD‐L1; pink ribbon model = hPD‐1; cyan stick model = peptide; red ribbon model = area of
overlap between PD‐1 and peptide)

binding studies in human (SK‐MEL‐28) and murine (B16‐F10) melanoma cell lines, HAC‐V blocked binding of wild‐
type PD‐1 to PD‐L1 with IC50 values of 210 pM (vs 8.2 μM for wild‐type) and 69 nM (vs 2.6 μM for wild‐type),
respectively.184 These HAC‐PD‐1 selectively inhibited PD‐L1 and not PD‐L2. To determine if these polypeptides
could reach tumor cells inaccessible to antibody binding, a fluorescently labeled HAC‐PD‐1 and anti‐PD‐L1
antibody were injected into an engineered hPD‐L1‐expressing murine CT26 colon carcinoma model. The results
showed widespread staining of the tumor with the HAC‐PD‐1 as compared to the staining of peripheral and
perivascular regions of the tumor with the antibody. HAC‐PD‐1 was also shown to have no detectable effects on
circulating T lymphocyte levels as compared to the tested antibody, validating that these polypeptides did not
induce ADCC or CMC. In syngeneic murine CT26 colon tumor models, a HAC microbody (HACmb; ie, HAC‐PD‐1
conjugated to the dimeric CH3 domain of human IgG1 to augment the potency of mPD‐L1 blockade) and a
comparative anti‐mouse PD‐L1 antibody significantly slowed tumor growth and were only effective in
immunocompetent mice, indicating that HACmb activated the adaptive immune system to trigger its antitumor
effects. A similar experiment was carried out in mice with much larger tumor volume, representing a more
challenging situation to treat. Astonishingly, HACmb maintained its ability to significantly reduce tumor growth in
larger tumors over the duration of their study compared to the antibody. Finally, the HAC‐PD‐1 polypeptide was
applied to immuno‐PET as a noninvasive means to measure tumor expression of PD‐L1. The PET tracer was
constructed from their polypeptide by conjugation to a thiol‐reactive bifunctional chelate DOTA‐maleimide,
followed by radiolabeling with 64Cu (64Cu‐DOTA‐HAC).184 The tracer was rapidly and specifically taken up by hPD‐
L1‐positive tumors and provided favorable tumor‐to‐background ratios, which persisted for at least 24 hours.
Overall, this approach of generating high‐affinity PD‐1 ectodomains to target PD‐L1 produced promising results,
28 | YANG AND HU

F I G U R E 1 6 Structural basis of checkpoint blockade by high‐affinity polypeptide mimic HAC‐V (modified from
PDB ID: 5IUS with MOE). The amino acid substitutions made in HAC‐V (blue ribbon model with amino acid
residues in the dark blue) have been highlighted in yellow. Red sphere represents a water molecule. Black dashed
lines indicate key π‐alkyl interactions, hydrogen bonding interactions, and water‐mediated interactions between
residues on hPD‐L1 (green ribbon model with amino acid residues in light green) and those derived from single
point substitutions, explaining the enhancement in affinity of the HAC‐PD‐1 mimic polypeptides. The position
numbers of the amino acid residues for HAC‐V is based on the original position numbers indicated in Maute et al184

highlighting its superior efficacy against challenging large tumor models relative to antibodies and potential
feasibility as a clinical imaging agent.

7 | CONC LU DIN G REMARKS

Cancer immunotherapy has become the newest addition to the war chest—including surgery, radiation therapy,
traditional nontargeted cytotoxic chemotherapy, and oncogene‐targeted therapy—in the fight against cancer. This
milestone in cancer treatment was achieved as a result of the remarkable responses to monoclonal antibodies
targeting immune checkpoint pathways. Yet, the impressive clinical development of the anti‐PD‐1/PD‐L1
monoclonal antibodies is in stark contrast to that of small molecule immunomodulators of this same pathway.
Even though quite a few small molecule PD‐1/PD‐L1 inhibitors have been reported (Supporting Information Table
S2), many of them have relatively modest inhibitory activity. One major reason for the lackluster progress in the
development of small molecule inhibitors is the inherent challenges of targeting PPIs. Compared to traditional drug
targets such as G‐protein coupled receptors and enzymes, where there is a clear, deep pocket for small molecules
to bind, the surfaces of proteins are relatively flat, large (~1500 to 3000 Å2), and devoid of suitable pockets for
small molecules to effectively bind and disrupt PPIs. However, over the last decade, this perspective has been
dispelled by several successful examples of small molecules inhibiting established PPIs.187–189 It has become
evident that protein surfaces are studded with pockets, crevices, and indentations, amenable to the binding of small
molecules.190 In addition, it has been shown that a molecule does not necessarily need to cover the entire surface
of a PPI, but rather a select group of amino acids, termed “hotspots,” to modulate the interaction. These hotspots
are analogous to the active site of an enzyme; they contribute most to the binding energy and overall stability of
protein‐protein complexes.191 Moreover, the surface area of a hotspot is generally approximately 600 Å2, which
makes it feasible for a small molecule to bind and alter PPIs as such molecules can cover an average of
YANG AND HU | 29

approximately 300 to 1000 Å2.188,189 Evidently, identifying these hotspot residues is critical to facilitating the
medicinal chemist’s pursuit of small molecule modulators of PPIs. Lastly, often times, small molecules are designed
to align with the Lipinski rule of five to ensure adequate oral bioavailability. However, this empirical rule may
impose unnecessary difficulties for medicinal chemists in the identification of potent and selective agents against
PPIs. Small molecules targeting PPIs will likely have molecular masses greater than 500 Da if they are to make
optimal contact with hotpots on the target with high affinity and selectivity.
The advent of monoclonal antibodies directed toward PD‐1 or PD‐L1 has established a foundation on which
small molecules can be successfully designed. There is now a wealth of structural information from recently
published crystal structures of the PD‐1:PD‐L1 interaction, PD‐1:antibody or PD‐L1:antibody interactions, and PD‐
L1:small molecule interactions to guide drug discovery efforts. These structures hint at potential hotspots located
on the surfaces of PD‐1 and PD‐L1, specifically the GFCC′ face, that can be exploited by small molecules. Small
molecules may be designed with enhanced potency relative to those already reported in the literature by mimicking
the diverse interactions of the monoclonal antibodies. Furthermore, it has been shown in structural studies that the
binding sites of PD‐1 and PD‐L1 exhibit more plasticity than previously thought. This unexpected adaptability of
the contact surfaces may expose small pockets or grooves on the protein, which small molecules can access and
outcompete the natural protein partner for binding.189
There are still many aspects of the PD‐1/PD‐1 ligand/B7‐1 immune checkpoint system that require clarification
and additional research to facilitate the development of small molecule immunomodulators. First, the binding
affinity of the PD‐1/PD‐L1 PPI is crucial for evaluating the likely efficacy of potential PPI modulators that will have
to compete with the natural protein partners.188 The strength of the PPI dictates the ease with which it may be
modulated. Therefore, the discrepancies in the binding affinity measurements for the PD‐1/PD‐L1 PPI, ranging
from single‐digit micromolar to nanomolar affinity, will need to be reconciled. Second, there are currently no known
structures published for the human PD‐1/PD‐L2 and B7‐1/PD‐L1 interactions. The mouse PD‐1/PD‐L2 interaction
has been studied,87 but there is likely to be differences in the details of the binding modes. For instance, despite
mPD‐1 being able to bind to hPD‐L1, the sequence identity between hPD‐1 and mPD‐1 is only 64%.83 Therefore,
understanding how PD‐L1 and PD‐L2 bind to the PD‐1 and B7‐1/CD80 receptors in humans will elucidate the
complex communication network of this immunoregulatory system, which can aid in the design of selective small
molecule inhibitors. Third, there is much uncertainty regarding the natural state of the PD‐L1 protein on the cell
surface. Finally, several patents have disclosed small molecule compounds, which have been proposed to target the
PD‐1/PD‐L1 immune checkpoint. However, with the exception of the BMS compounds, there has been no
validation of whether or not these small molecules bind directly to PD‐1 or PD‐L1 to inhibit the PPI, hindering
interests in further optimizing these reported immunomodulators. Structural information concerning their mode of
interaction will also be helpful in guiding the rational development of compounds with novel scaffolds that vary
from the biphenyl moiety present in the BMS compounds. In spite of these current challenges and unknowns in the
discovery of small molecule immunomodulators, the PD‐1/PD‐L1 PPI is a promising target for small molecules.
Today, much effort and resources have been allocated toward the pursuit of potentially curative therapeutic
combinations against cancer in the hopes of “raising the tail of the survival curve” (ie, improving the percentage of
patients responding to therapy and surviving up to a specified number of years) while maintaining a relatively clean
safety profile.192 Moving forward, small molecules will have a promising role to play in this endeavor. First, they
may be more feasible to incorporate into various combinations as compared to the monoclonal antibodies. The
manufacturing, formulation, storage, transport, handling, administration, and stability of a single protein‐based drug
is already cumbersome and costly. The idea of combining two or more protein therapeutics into a single dosage
form will pose daunting new challenges for manufacturers, distributors, healthcare providers, and patients.193
Second, as previously discussed, the PK/PD of monoclonal antibodies are often exaggerated due to their prolonged
residence time in the body. Any resulting adverse reactions from combination therapy would be difficult to
terminate with treatment discontinuation alone, requiring management with immunosuppressants, which have the
potential to negatively affect therapeutic efficacy. On the other hand, small molecules have shorter half‐lives,
30 | YANG AND HU

enabling reversal of adverse reactions with prompt treatment discontinuation. Overall, small molecule
immunomodulators offer greater flexibility than the monoclonal antibodies and have the potential to augment
the therapeutic benefits of existing cytotoxic, targeted, and immunotherapeutic agents. Numerous small molecules
with immunomodulatory effects are currently being pursued, including indoleamine‐2,3‐dioxygenase (IDO)
inhibitors, toll‐like receptor agonists, and RAR‐related orphan receptor gamma T (RORγt) inhibitors.194 Most
notably, preclinical studies of IDO inhibitors have shown that these small molecule immunomodulators can
empower the efficacy of cytotoxic chemotherapy, radiotherapy, and immune checkpoint therapy without additional
toxicity.195 Introducing small molecule modulators of both costimulatory and coinhibitory immune checkpoint
molecules would nicely complement these other small molecules. Analogous to the impact that small molecules
have had in highly active antiretroviral therapy, or HAART, for the treatment of HIV/AIDS, small molecule
combinations (eg, the combination of small molecule immunomodulators with current genomically targeted
therapies) will transform cancer from a death sentence into a stable, manageable chronic disease in the future.

OR CID

Jeffrey Yang http://orcid.org/0000-0002-5647-1464

Longqin Hu http://orcid.org/0000-0002-1799-5652

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AUTHOR ’S B IO G R APH I ES
Jeffrey Yang received his PharmD degree in 2017 from the Ernest Mario School of Pharmacy at Rutgers, the State
University of New Jersey. He is currently pursuing his PhD degree in Medicinal Chemistry as part of the joint
PharmD/PhD program at the Ernest Mario School of Pharmacy, Rutgers University under the supervision of
Professor Longqin Hu. His doctoral dissertation work involves the design, synthesis, and evaluation of small
molecule immunomodulators of the PD‐1/PD‐L1 immune checkpoint pathway as novel cancer immunotherapies.
He is interested in a career in drug discovery and development in the fields of oncology, immunology, and infectious
diseases.

Longqin Hu received his Bachelor of Pharmacy degree from the Second Military Medical University in Shanghai,
China in 1984 and his PhD in Medicinal Chemistry from the University of Kansas in 1993. He did his postdoctoral
research in Biochemistry as an NIH NRSA postdoctoral fellow at the University of Delaware between 1993 and
1996. He started his academic career first at the University of Oklahoma and moved to Rutgers University in 1999.
He is currently a Professor of Medicinal Chemistry and Chair in the Department of Medicinal Chemistry, Ernest
Mario School of Pharmacy, Rutgers, the State University of New Jersey. His research interests include peptide
chemistry, drug design, targeted drug delivery, targeted anticancer prodrugs, inhibitors of crystallization for kidney
stones, and small molecule inhibitors of protein‐protein interactions and protein kinases.

S U P P O R T I N G I N F O RMA T I O N
Additional supporting information may be found online in the Supporting Information section at the end of the
article.

How to cite this article: Yang J, Hu L. Immunomodulators targeting the PD‐1/PD‐L1 protein‐protein
interaction: From antibodies to small molecules. Med Res Rev. 2018;1‐37.
https://doi.org/10.1002/med.21530