REVIEW

The Next Decade of Immune

Checkpoint Therapy
Padmanee Sharma1,2,3, Bilal A. Siddiqui1, Swetha Anandhan1, Shalini S. Yadav3, Sumit K. Subudhi1,
Jianjun Gao1, Sangeeta Goswami1,2, and James P. Allison2,3

ABSTRACT Immune checkpoint therapy (ICT) can provide durable clinical responses and
improve overall survival. However, only subsets of patients with specific tumor
types respond to ICT. Thus, significant challenges remain, including understanding pathways of resist-
ance, optimizing patient selection, improving management of immune-related adverse events, and
identifying rational therapeutic combinations. These challenges will need a focused approach encom-
passing both clinical and basic research, with the integration of reverse translational studies. This
integrated approach will lead to identification of potential targets for subsequent clinical trials, which
will guide decisions as we develop novel combination strategies to maximize efficacy and minimize
toxicities for patients.

Significance: ICTs induce durable antitumor responses for subsets of patients with cancer. Recent
evidence suggests that rational combinatorial strategies can improve response by overcoming primary
and adaptive resistance mechanisms, although these may carry an increased risk of immune-mediated
toxicities. This review surveys the current understanding of mechanisms of response and resistance to
ICTs and active areas of investigation, and proposes a path forward to improving efficacy and minimiz-
ing toxicities through better patient selection and rational combinations.

INTRODUCTION Despite the unprecedented durable clinical responses

observed in subsets of patients, most patients do not respond,
Metastatic cancers remain incurable in most patients as
and some patients develop resistance to therapy after initial
conventional therapies that target tumor cells usually can-
response. Furthermore, ICTs can result in life-threatening
not provide a durable response. The discovery of immune
toxicities, known as immune-related adverse events (irAE).
checkpoints that regulate immune responses transformed
Therefore, research studies are ongoing to understand path-
cancer care, offering long-term clinical responses and the
ways of resistance to ICT and mechanisms of irAEs. In this
possibility of cure in many more patients with metastatic
review, we outline the fundamentals of ICT and the current
cancer (1). In 2011, the FDA approved the first immune
state of ICT in the clinic. We propose that development of
checkpoint targeting agent, ipilimumab—a mAb targeting
effective ICT combinations will require a reverse translational
CTLA4—which opened the field of immune checkpoint ther-
approach, in which hypotheses are generated from in-depth
apy (ICT). Subsequently, mAbs blocking other checkpoints
immune monitoring studies of patients’ samples and then
such as PD-1 or PD-L1 have received FDA approvals to treat
tested in appropriate preclinical models, thereby yielding
a number of tumor types alone and in combination with
important insights to guide rational therapeutic strategies
other agents.
in subsequent clinical trials. The last ten years provided an
understanding of how T cells can be targeted with anti-
CTLA4 and anti–PD-1/PD-L1 to provide clinical benefit.
1
Department of Genitourinary Medical Oncology, The University of Texas Bolstered by the latest technologies, in the next decade we will
MD Anderson Cancer Center, Houston, Texas. 2Department of Immunology, better understand how to combine ICT with agents targeting
The University of Texas MD Anderson Cancer Center, Houston, Texas. 3The
Immunotherapy Platform, The University of Texas MD Anderson Cancer,
multiple other cell subsets and pathways in the innate and
Center, Houston, Texas. adaptive immune system. The future of ICT offers ongoing
Corresponding Author: Padmanee Sharma, Genitourinary Medical Oncol- promise to improve outcomes in patients with cancer.
ogy and Immunology, The University of Texas MD Anderson Cancer Center,
1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-552-3980;
E-mail: padsharma@mdanderson.org BACKGROUND
Cancer Discov 2021;11:838–57 T cells are the soldiers of the immune response. Activation
doi: 10.1158/2159-8290.CD-20-1680 of T cells requires a coordinated and stepwise process involv-
©2021 American Association for Cancer Research. ing two primary signals (Fig. 1; ref. 2). T cells express the T-cell

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The Next Decade of Immune Checkpoint Therapy REVIEW

A T-cell activation B T-cell inhibition

PD-L1/L2
PD-1

MHCII-Ag TCR MHCII-Ag TCR

CTLA4
CD28
CD28

B7 B7

Cytokine release

C T-cell reactivation using immune

checkpoint antibodies

PD-L1/L2
PD-1

Other coinhibitory
molecules
MHCII-Ag TCR
CD28
CTLA4

B7

Cytokine release

Figure 1.  Regulation of T-cell activation. A, T-cell activation requires both signal 1, TCR engagement with the MHC–peptide antigen complex (MHC-
Ag) on an APC or a target cell, and signal 2, interaction of the costimulatory receptor CD28 on the T cell with costimulatory B7 molecules (CD80/CD86).
B, In response to T-cell activation, the immune checkpoints CTLA4 and PD-1 are upregulated on the T cell and bind to B7 and PD-L1/L2, respectively, to
inhibit T-cell activation. C, Immune checkpoint antibodies targeting CTLA4 or PD-1/PD-L1 block these inhibitory interactions, reactivating T cells.

receptor (TCR), which signals via the CD3 complex upon the
interaction of the TCR with MHC plus its cognate peptide anti-
gen on antigen-presenting cells (APC). T cells also constitutively
express a costimulatory receptor, CD28, which binds to the
B7 family of costimulatory molecules that are predominantly
expressed on professional APCs. To activate or turn T cells “on,”
both the TCR signal via CD3 (signal 1) and the CD28 costimu-
latory signal via B7 interactions (signal 2) are required (Fig. 1A).
Interaction of APCs with T cells that bear the specific TCR for a
given antigen leads to activation of T cells and T cell–mediated
responses. T cells then differentiate and clonally expand, with
subsequent formation of memory T cells (3).
CD4 and CD8 T cells constitute the majority of T cells in
the immune system. CD4 T cells differentiate into a variety
of subtypes: Th1, Th2, Th17, follicular Th (Tfh), T regulatory
(Treg), and Th9 cells. These subsets have distinct transcrip-
tion factors that regulate their function and cytokine profiles,
which affect immune responses that range from effector cell
responses for tumor rejection to regulatory cell responses to
suppress effector cells. These transcription factors include
T-bet for Th1 cells, BCL6 for Tfh, RORγ for Th17 cells, and
FOXP3 for Tregs. Although CD4 T cells are known as helper
cells, accumulating evidence suggests a cytotoxic function
demonstrated by certain CD4 T cells (4). CD8 T cells are typi-
cally regarded as a uniform population with cytotoxic func-
tion; however, recent studies have highlighted the diversity of
the CD8 T-cell population (5–9).
In general, Th1 CD4 cells and CD8 T cells are the effector
cells responsible for driving antitumor immune responses.
However, these immune responses are controlled by multiple
mechanisms. Upon T-cell activation via the TCR and CD28,
T cells proliferate and produce cytokines, but also upregulate
inhibitory molecules that attenuate T-cell activation (Fig. 1B).
Therefore, T cells have limited time to function before they are
reined in to prevent damage to normal cells. Multiple inhibi-
tory mechanisms exist, both cell-intrinsic and cell-extrinsic,
to control T-cell responses. The signals that control antitu-
mor immunity and immune suppression are comprised of a
tightly regulated process that forms the yin and yang of the
immune response.

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CTLA4-Mediated Negative Costimulation
CTLA4 is a canonical inhibitory molecule that is expressed
immediately following engagement of TCR, with peak
expression around 48 to 72 hours following T-cell activa-
tion. It bears structural homology to CD28 and binds to
B7-1 (CD80) and B7-2 (CD86) molecules on APCs with
higher affinity than CD28, leading to competitive inhibition
of costimulatory CD28 signaling, thereby dampening T-cell
signaling (10, 11). In early studies, an anti-CTLA4 antibody
prevented CTLA4 from engaging B7, leading to prolonged
T-cell responses and eradication of cancer cells (1). Simi-
larly, the in vivo administration of anti-CTLA4 antibodies
enhanced the antitumor effect of tumor-infiltrating T cells,
leading to regression of established tumors and long-lived
immunity in tumor models (1).
Antibody blockade of CTLA4 was proposed as a way to
enhance T-cell responses in the setting of cancer. By removing
the brakes from T cells, it was hypothesized that T cells would
have a longer time to function, which would enable tumor
rejection. Anti-CTLA4 opened an entire new field termed
“ICT.” The success of anti-CTLA4 led to the identification of
other pathways that regulate T-cell responses, including both
costimulatory and coinhibitory pathways (Fig. 1C; refs. 1, 12).
PD-1–Mediated Negative Costimulation
The primary biological function of PD-1 is to maintain
peripheral tolerance. PD-1 engages with its ligands PD-L1 and
PD-L2, widely expressed on nonlymphoid tissues, primarily
dampening T-cell activation in the periphery. PD-1 interacts
with an SHP2 tyrosine phosphatase domain, leading to dephos-
phorylation of signaling molecules downstream of the TCR
and inhibiting TCR-mediated activation of IL2 production and
T-cell proliferation (13). Therefore, whereas CTLA4 inhibits sig-
nal 2 (CD28-B7), PD-1/PD-L1 inhibits signal 1 (TCR).
PD-L1 expression has been described on a variety of tumor
types, and is directly correlated with poor prognosis in several
cancers (14, 15). Preclinical models demonstrated that PD-1
blockade results in tumor rejection through reinvigoration
of exhausted CD8 T cells that express PD-1, LAG3, and TIM3
and expansion of CD8 T cells, a hallmark of response to anti–
PD-1 therapy (16).
Distinct Mechanisms of Action of CTLA4 and PD-1
Although both CTLA4 and PD-1 are inhibitory molecules
that attenuate T-cell activation, they have different mecha-
nisms of action, and inhibition of CTLA4 and PD-1 leads to
distinct immune responses. Anti-CTLA4 primarily functions
in T-cell priming and expands clonal diversity. Anti-CTLA4
predominantly affects CD4 T cells, with multiple studies
identifying increased frequency of a Th1 subset of CD4 T
cells that express inducible costimulator (ICOS) as a result of
anti-CTLA4 therapy (17–19). Increased frequency of ICOS+
CD4 T cells in peripheral blood acts as a pharmacodynamic
biomarker of anti-CTLA4 therapy (20), whereas increased
frequency of ICOS+ CD4 T cells in tumor tissues, possibly in
the context of tertiary lymphoid structures, tends to correlate
with improved clinical outcomes with ICT (21). Anti-CTLA4
can also promote T-cell trafficking into immunologically
“cold” tumors. Investigation into the mechanisms of anti­
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Sharma et al.
tumor activity of CTLA4 blockade demonstrated the contri-
bution of both the effector T-cell and Treg compartments in
preclinical models (22, 23). Of note, although CTLA4 block-
ade was shown to cause selective depletion of intratumoral
Tregs through antibody-dependent cell-mediated cytotoxic-
ity (ADCC) in murine models and ex vivo experiments on
human tumor samples (24–26), subsequent studies compar-
ing pretreatment and posttreatment tumor samples from
patients who received anti-CTLA4 therapy, either as treme-
limumab (IgG2 antibody) or as ipilimumab (IgG1 anti-
body), did not observe a decrease in intratumoral Tregs after
therapy (27). It is possible that the expression of specific Fc
receptors on APCs or other cell types may be limited in some
human tumors, thereby affecting Treg depletion by anti-
CTLA4 therapy in patients with cancer. In future studies,
it may be important to ascertain the expression of specific
Fc receptors within the tumor microenvironment (TME) to
determine whether ICT antibodies will mediate ADCC to
affect Treg frequency. In general, anti-CTLA4 expands CD4
effector T cells (Teff) with subsequent increase in the Teff/
Treg ratio in responding tumors (22).
Anti–PD-1/PD-L1 antibodies predominantly affect exhausted
CD8 T cells. Anti–PD-1/PD-L1 also does not appear to expand
clonal diversity nor promote T-cell trafficking into tumors. Pre-
clinical studies have shown that concurrent targeting of CTLA4
and PD-1/PD-L1 can improve therapeutic efficacy when com-
pared with each alone (28–30). mAbs targeting CTLA4, PD-1,
and PD-L1 have formed the foundation of effective ICT in
patients and are now in widespread clinical use across a number
of tumor types (31–34).
CURRENT ICTs IN THE CLINIC
There are now more than 50 FDA approvals for ICTs in
human cancers, which are briefly discussed in this section
and summarized in Table 1. The clinically approved ICTs
have been comprehensively reviewed in other sources (35, 36).
Anti-CTLA4 Monotherapy
Ipilimumab (anti-CTLA4) was the first ICT to gain FDA
approval in 2011 for the treatment of metastatic melanoma,
on the basis of phase III trials demonstrating improvements
in overall survival compared with vaccine or chemotherapy
(37, 38). Initial studies were challenging because of the para-
doxical phenomenon of pseudoprogression, in which patients
initially experienced tumor growth before subsequent regres-
sion. The benefit of ICT would not have been adequately
captured by traditional metrics of tumor measurement
(39–41). Survival therefore proved to be the most impor-
tant indicator of ICT benefit. Indeed, approximately 20% of
patients who received ipilimumab achieved long-term sur-
vival greater than three years, with survival of 10 years noted
for some patients (38, 42). Although ipilimumab remains
the only anti-CTLA4 therapy to have approval in many coun-
tries, including FDA approval, other mAbs are being stud-
ied, including tremelimumab and quavonlimab (43). New
forms of anti-CTLA4 therapies are also under development,
including glycoengineered antibodies that seek to enhance
ADCC and prodrug forms with reduced systemic activity to
improve safety (44).
The Next Decade of Immune Checkpoint Therapy REVIEW

Table 1. FDA-approved ICTs

Year of
Agent/target Indication approval
1. Ipilimumab/anti-CTLA4  1. Unresectable or metastatic melanoma 2011
 2. High-risk stage III melanoma after complete resection (adjuvant) 2015
 3. Pediatric patients 12 years and older with unresectable or metastatic melanoma 2017
2. Nivolumab/anti–PD-1  1. Unresectable or metastatic melanoma 2014
 2. Metastatic squamous NSCLC with progression on or after platinum-based chemotherapy 2015
 3. Metastatic nonsquamous NSCLC with progression on or after platinum-based 2015
chemotherapy
 4. Patients with metastatic RCC who have received prior therapy 2015
 5. cHL that has relapsed or progressed after autologous hematopoietic stem cell transplan- 2016
tation and posttransplantation brentuximab vedotin or after ≥3 lines of therapy
 6. Recurrent or metastatic HNSCC with disease progression on or after platinum-based 2016
therapy
 7. Locally advanced or metastatic UC with disease progression during or following platinum- 2017
based chemotherapy
 8. Adult and pediatric patients with MSI-H or dMMR metastatic colorectal cancer that has 2017
progressed following chemotherapy
 9. HCC previously treated with sorafenib 2017
10. Melanoma with lymph node involvement (adjuvant) or metastatic disease following 2017
complete resection
11. Unresectable advanced, recurrent, or metastatic esophageal squamous cell carcinoma 2020
after prior fluoropyrimidine- and platinum-based chemotherapy
12. Advanced RCC, first line in combination with cabozantinib 2021
3. Pembrolizumab/anti–PD-1  1. Advanced or unresectable melanoma that no longer responds to other drugs 2014
 2. Metastatic NSCLC with PD-L1–positive tumors and disease progression after other 2015
treatments
 3. First-line treatment of unresectable or metastatic melanoma 2015
 4. Recurrent or metastatic HNSCC with disease progression on or after platinum-based 2016
chemotherapy
 5. First-line treatment of metastatic NSCLC, with high PD-L1 expression and no EGFR or 2016
ALK genomic tumor alterations
 6. Adult and pediatric refractory cHL or disease relapse after three or more prior lines of 2017
therapy (extended in 2020 to adult patients with relapsed or refractory cHL and pediatric
patients with refractory cHL or cHL relapsed after two or more lines of therapy)
 7. First-line treatment of metastatic nonsquamous NSCLC, irrespective of PD-L1 expres- 2017
sion (in combination with carboplatin/pemetrexed)
 8. Locally advanced or metastatic UC with disease progression on or after platinum-based 2017
chemotherapy or within 12 months of neoadjuvant or adjuvant platinum chemotherapy
or patients who are not eligible for cisplatin-containing chemotherapy and whose tumors
express PD-L1 (CPS ≥10), or in patients who are not eligible for any platinum-containing
chemotherapy regardless of PD-L1 status
 9. Adult and pediatric unresectable or metastatic solid tumors that are MSI-H or dMMR 2017
10. Recurrent locally advanced or metastatic gastric or gastroesophageal junction adeno- 2017
carcinoma, with PD-L1–positive tumors and disease progression on or after two or more
prior lines of therapy
11. Recurrent or metastatic cervical cancer, with tumor PD-L1 CPS ≥ 1 and disease progres- 2018
sion on or after prior chemotherapy
12. Primary mediastinal B-cell lymphoma, refractory disease, or relapse after two or more 2018
lines of therapy
13. Metastatic, nonsquamous NSCLC (in combination with pemetrexed and cisplatin/ 2018
carboplatin)
14. Metastatic, squamous NSCLC (in combination with carboplatin and paclitaxel or 2018
nab-paclitaxel)

(continued)

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Table 1. FDA-approved ICTs (Continued)

Year of
Agent/target Indication approval
15. HCC, following treatment with sorafenib 2018
16. Merkel cell carcinoma, recurrent locally advanced or metastatic 2018
17. Melanoma with lymph node involvement following complete resection 2019
18. Locally advanced or metastatic NSCLC with PD-L1 TPS ≥ 1% 2019
19. Advanced RCC, first line (in combination with axitinib) 2019
20. HNSCC, first-line treatment for metastatic or unresectable recurrent disease with tumor 2019
PD-L1 CPS ≥ 1
21. HNSCC, first-line treatment for metastatic or unresectable recurrent disease (in combi- 2019
nation with platinum and fluorouracil)
22. SCLC, metastatic, with disease progression on or after platinum-based chemotherapy 2019
and at least 1 other prior line of therapy
23. Esophageal cancer, recurrent locally advanced or metastatic squamous cell, with tumor 2019
PD-L1 CPS ≥ 10 and disease progression after one or more prior lines of systemic therapy
24. Endometrial carcinoma, advanced, non–MSI-H/dMMR with disease progression after 2019
prior systemic therapy and not candidate for curative surgery or radiation (in combination
with lenvatinib)
25. UC, high-risk, BCG-unresponsive, non–muscle invasive with carcinoma in situ with or 2020
without papillary tumors, ineligible for or declined cystectomy
26. Recurrent or metastatic cutaneous squamous cell carcinoma not curable by surgery or 2020
radiation
27. Unresectable or metastatic TMB-high (≥10 mut/Mb) solid tumors that have progressed 2020
on prior treatment with no satisfactory alternative treatment options.
28. Unresectable or metastatic MSI-H or dMMR colorectal cancer, first line 2020
29. Locally recurrent unresectable or metastatic triple-negative breast cancer with tumor 2020
PD-L1 CPS ≥ 10 (in combination with chemotherapy)
4. Ipilimumab + nivolumab/  1. BRAFV600 wild-type unresectable or metastatic melanoma 2015
anti-CTLA4 + anti–PD-1  2. BRAFV600 wild-type and BRAFV600 mutation–positive unresectable or metastatic 2016
melanoma
 3. Poor/intermediate risk previously untreated advanced RCC 2018
 4. Previously treated MSI-H/dMMR colorectal cancer 2018
 5. HCC following treatment with sorafenib 2020
 6. Metastatic NSCLC with PD-L1 ≥1% or in combination with two cycles of platinum-doublet 2020
chemotherapy regardless of PD-L1 status
 7. Unresectable malignant pleural mesothelioma 2020
5. Durvalumab/anti–PD-L1  1. Unresectable stage III NSCLC with nonprogressive disease following concurrent 2018
platinum-based chemotherapy and radiotherapy
 2. SCLC, extensive stage, first line (in combination with etoposide and carboplatin/cisplatin) 2020
6. Atezolizumab/anti–PD-L1  1. Locally advanced or metastatic UC, with disease progression during or following 2016
platinum-based chemotherapy, either before or after surgery
 2. Metastatic NSCLC with disease progression during or following platinum-based chemo- 2016
therapy, and progression on an FDA-approved targeted therapy if the tumor has EGFR or
ALK gene abnormalities
 3. Locally advanced or metastatic UC not eligible for cisplatin chemotherapy whose tumors 2017
express PD-L1
 4. First-line, metastatic, nonsquamous SCLC without EGFR or ALK genomic alterations 2018
(in combination with bevacizumab, paclitaxel, and carboplatin)
 5. SCLC, extensive stage, first line (in combination with etoposide and carboplatin) 2019
 6. Unresectable locally advanced or metastatic triple-negative breast cancer with PD-L1 2019
≥1% (in combination with nab-paclitaxel)
 7. Metastatic NSCLC without EGFR or ALK genomic alterations (in combination with 2019
carboplatin/nab-paclitaxel)

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Table 1. FDA-approved ICTs (Continued)

Agent/target
7. Avelumab/anti–PD-L1
8. Cemiplimab/anti–PD-1
Year of
Indication approval
 8. Metastatic NSCLC, first line, PD-L1 high (tumor cells ≥50% or immune cells ≥10%) 2020
without EGFR or ALK genomic alterations
 9. Metastatic or unresectable HCC, first line (in combination with bevacizumab) 2020
10. BRAFV600 mutation–positive unresectable or metastatic melanoma in combination with 2020
cobimetinib and vemurafenib
 1. Adult and pediatric patients with metastatic Merkel cell carcinoma, including those who 2017
have not received prior chemotherapy
 2. Locally advanced or metastatic UC with disease progression during or following platinum- 2017
based chemotherapy
 3. Advanced RCC, first line (in combination with axitinib) 2019
 4. Maintenance treatment for locally advanced for metastatic UC that has not progressed 2020
with first-line platinum-containing chemotherapy
 1. Metastatic or locally advanced cutaneous squamous cell carcinoma 2018

Abbreviations: BCG, Bacillus Calmette–Guérin; cHL, classic Hodgkin lymphoma; CPS, combined positive score; dMMR, mismatch-repair deficiency;
HCC, hepatocellular carcinoma; HNSCC, head and neck squamous cell carcinoma; MSI-H, microsatellite instability-high; NSCLC, non–small cell lung
cancer; RCC, renal cell carcinoma; SCLC, small cell lung cancer; TPS, tumor proportion score; TMB, tumor mutational burden; UC, urothelial carcinoma.
Updated as of February 1, 2021.

Anti–PD-1/PD-L1 Monotherapy either monotherapy, in different cancer types including mel-

In 2014, the first PD-1 inhibitors were approved for the
treatment of unresectable or metastatic melanoma (45, 46).
The PD-1/PD-L1 inhibitors are now approved across mul-
tiple tumor types, including skin, genitourinary, lung, head
and neck, breast, lymphoma, gynecologic, and gastrointes-
tinal cancers. These approvals are summarized in Table 1.
The PD-1 mAbs include nivolumab (fully human IgG4),
pembrolizumab (humanized IgG4), and cemiplimab (fully
human IgG4). The PD-L1 mAbs include durvalumab (fully
human IgG1κ), atezolizumab (humanized IgG1), and ave-
lumab (fully human IgG1). Anti–PD-1 therapies have also
received approval for treatment of patients with cancer who
are diagnosed with tumors that have microsatellite instability–
high (MSI-H)/mismatch-repair deficiency (dMMR; refs. 47,
48) or high tumor mutational burden (TMB-high) regardless
of tumor type (49). In addition, anti–PD-1/PD-L1 therapies
were also approved by the FDA as maintenance therapy, which
is administered after initial treatment of locally advanced or
metastatic disease. Clinical benefit with maintenance ICT
in specific settings was demonstrated in patients with non–
small cell lung cancer (NSCLC; ref. 50), extensive-stage small
cell lung cancer (51, 52), and bladder cancer (53), leading to
FDA approvals in these tumor types.
Combination of Anti-CTLA4 and Anti–PD-1/PD-L1
Despite the promising responses with ICT monotherapies,
these responses are observed in only 20% to 30% of treated
patients. Therefore, combination therapy was pursued in
the clinic based on preclinical data demonstrating that anti-
CTLA4 and anti–PD-1/PD-L1 function through distinct
pathways, and combined inhibition enhanced antitumor
responses in murine models (28).
Multiple clinical trials with combined blockade of CTLA4
and PD-1/PD-L1 demonstrated higher response rates than
anoma (54, 55), hepatocellular carcinoma (56), renal cell
carcinoma (RCC; refs. 57, 58), NSCLC (59), malignant pleural
mesothelioma (60), and colorectal cancer (MSI-H/dMMR;
ref. 61). Long-term follow-up has demonstrated durable sur-
vival benefit for a subset of patients with melanoma and RCC,
and combination therapy with ICT is under active investiga-
tion for a number of tumor types (55, 58).
Combination of ICT with Other Therapeutic Agents
The quest to improve response rates has also prompted
combinations with other established cancer drugs, for exam-
ple, chemotherapy, which is hypothesized to kill immuno-
suppressive cells in the TME while increasing exposure to
tumor antigens via the induction of tumor cell death, thereby
enhancing recognition and elimination of tumor cells by the
host immune system (62). Toward this end, combinations of
ICTs with concurrent and/or sequential chemotherapy are
under extensive investigation, with phase III clinical data dem-
onstrating overall survival and/or progression-free survival
benefit in NSCLC (63–66), small cell lung cancer (51, 52), tri-
ple-negative breast cancer (67), bladder cancer (53), and head
and neck squamous cell carcinoma (68). However, the impact
of different chemotherapy agents on the immune response
within the TME has not been fully elucidated. Similarly,
improved clinical responses have been demonstrated with
the combination of ICT and targeted therapy. Combinations
of VEGF-targeted agents with anti–PD-1 have demonstrated
clinical benefits in endometrial carcinoma (pembrolizumab
plus lenvatinib; refs. 69, 70), RCC (pembrolizumab plus axi-
tinib; nivolumab plus cabozantinib; avelumab plus axitinib),
and hepatocellular carcinoma (atezolizumab plus bevaci-
zumab; refs. 71–75). Recently, the addition of atezolizumab
to targeted therapy was also shown to improve progression-
free survival in BRAF-mutated melanoma (76).

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Although these combination therapies offer the possi-
bility of improving cancer-specific outcomes, they remain
limited to specific tumor types and disease settings. For
the field of cancer immunotherapy to move forward suc-
cessfully, with improved clinical benefit for many more
patients, we will require rational combination therapies,
which should be based on a reasonable understanding of
the mechanism of action of each agent and its impact on
immune responses.
Immune-Related Adverse Events
As combination strategies are pursued, there is an increased
risk of development of irAEs. Mitigation strategies, therefore,
will require a deeper mechanistic understanding of the toxici-
ties. These irAEs are distinct from the predictable side effects
associated with conventional cancer therapies. The irAEs
can affect any organ, most commonly skin, gastrointestinal
tract, and endocrine system (including the thyroid, adrenal,
and pituitary glands). The severity of irAEs can range from
mild to even fatal. Clinical guidelines for the management
of irAEs have been reviewed elsewhere (77). Although guide-
lines recommend high-dose corticosteroids for severe irAEs,
often with prolonged tapers, this approach can be associated
with serious iatrogenic side effects, highlighting the need for
mechanistically informed, steroid-sparing immunosuppres-
sive approaches. Early recognition and treatment of irAEs is
essential to prevent progression to severe events, and major
efforts are under way to develop biomarkers for earlier pre-
dictions of irAEs, including blood-based biomarkers such as
CD8 T-cell clonal expansion, serum autoantibodies (reflective
of B-cell response), and technology-based approaches with
patient symptom reporting (78–81).
Published data indicate that nonfatal irAEs may be asso-
ciated with improved outcomes in patients with cancer
(82–96). Therefore, the development of strategies that can
enable patients to continue on ICT, including the use of
selective immunosuppressive agents, will be important as the
field of ICT moves forward. For example, the recent develop-
ment of a murine model of myocarditis, one of the most
feared complications of ICT, provided a rationale for the use
of CTLA4-Ig (abatacept), which blocks T-cell costimulation,
although an open question remains as to whether this will
interfere with antitumor immunity (97, 98). Similarly, in
a study of patients with melanoma who developed colitis
after ICT, single-cell RNA sequencing (scRNA-seq) and TCR
sequencing identified tissue-resident colitis-associated CD8
T cells, consistent with the clinical observation that colitis
symptoms tend to occur early after initiation of ICT, and
also provided support for the use of the α4β7 antibody ved-
olizumab to decrease inflammatory immune responses in
ICT-associated colitis (99).
Overall, as multiple mechanisms related to irAEs become
clear, including both T cell– and B cell–mediated pathways, a
major question will be to address whether the mechanisms of
irAEs can successfully be decoupled from those of antitumor
efficacy. Future ICT clinical trials may include components
of selective, prophylactic immunosuppression for high-risk,
biomarker-selected patients to prevent severe irAEs, or pos-
sibly include administration of targeted immunosuppression
to enable continuation of ICT in patients.
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IMPROVING CLINICAL OUTCOMES WITH
RATIONAL ICT COMBINATIONS
To obtain a greater understanding of mechanisms of
response and resistance to ICT, we propose that reverse trans-
lational studies on individual agents be undertaken, whereby
each agent can be studied in small cohorts of patients, with
longitudinal samples collected and analyzed thoroughly to
generate hypotheses regarding immunologic mechanisms
that can be tested in appropriate preclinical models, and with
translation of the preclinical data to new clinical trials. These
data sets may provide relevant information that can help with
the design of rational clinical trials as well as avoid the pit-
falls of failed clinical trials. We have adopted this integrated
strategy to understand the pathways regulating immune
responses and to guide rational combinatorial strategies, as
outlined below.
Addressing Primary and Adaptive
Resistance Mechanisms
Prostate cancer is considered a “cold” tumor due to the fact
that it has few mutations, as compared with “hot” tumors
such as melanoma and NSCLC. As a result of a low number
of mutations, and therefore few antigens for T-cell recogni-
tion, as noted by scarce tumor-infiltrating T cells, prostate
cancer has been predominantly resistant to treatment with
ICT (100–102). The mechanisms by which prostate tumors
exclude T cells or fail to elicit robust infiltration by T cells
represent primary resistance mechanisms to ICT. Without
tumor-infiltrating T cells, the blockade of a T cell–inhibitory
pathway such as PD-1/PD-L1 is unlikely to provide clinical
benefit. For example, a recent phase III clinical trial of the
androgen receptor antagonist enzalutamide plus anti–PD-L1
failed to meet its primary endpoint of improved overall
survival (103). Previous studies on human prostate cancers
demonstrated few tumor-infiltrating T cells, with minimal
to no expression of PD-1 or PD-L1, and lack of significant
clinical benefit with anti–PD-1 monotherapy in patients with
metastatic prostate cancer (104, 105). Therefore, the phase III
combination study would have benefited from a smaller clini-
cal trial to truly determine whether enzalutamide was capa-
ble of increasing tumor-infiltrating T cells with subsequent
increase in PD-1 and PD-L1 expression within the TME (106).
Similarly, a phase III combination study of anti-CTLA4 plus
radiotherapy in patients with metastatic prostate cancer also
failed to meet its primary endpoint (100, 107). The combina-
tion was conducted without understanding the immunologic
impact of anti-CTLA4 or radiotherapy on the prostate TME,
including sites of metastatic disease, which are predominantly
bone metastases in patients with metastatic prostate cancer.
To address the immunologic impact of anti-CTLA4 on the
prostate TME, a small clinical trial evaluated pretreatment
and posttreatment tumor samples, which demonstrated that
anti-CTLA4 was capable of increasing tumor-infiltrating T
cells (105). However, anti-CTLA4 also led to an increase in
compensatory inhibitory mechanisms, also known as adaptive
resistance mechanisms, including increased frequency of cells
expressing PD-1/PD-L1 and VISTA within the TME (105).
VISTA is an inhibitory checkpoint that is highly expressed on
myeloid cells [including macrophages, dendritic cells (DC),
The Next Decade of Immune Checkpoint Therapy
monocytes, neutrophils], and moderately expressed on T cells
(108). This yin and yang of the immune response, where ther-
apy may drive antitumor immune responses but also increase
immunosuppressive mechanisms, is an important concept to
take into consideration as new immunotherapeutic strategies
are developed.
The data from this small clinical trial generated hypotheses
regarding combination therapies with anti-CTLA4 plus anti–
PD-1 and/or anti-VISTA. These hypotheses were tested in
preclinical studies, and the data led to a new study with anti-
CTLA4 plus anti–PD-1 (109). The new combination clinical
trial elicited antitumor responses in some patients, but these
responses were predominantly in patients with soft-tissue
metastases as opposed to bone metastases (109). Immune
monitoring data of posttreatment tumor samples collected
from patients indicated that soft-tissue metastases were infil-
trated by Th1 cells, whereas bone metastases were infiltrated
by Th17 cells (110). The increased Th1 responses provided an
explanation for why soft-tissue metastases responded better
than bone metastases to ICT.
But why did ICT drive Th1 responses in soft-tissue metas-
tases and Th17 responses in bone metastases? A preclinical
model was designed to answer this question. Data from these
experiments demonstrated high levels of IL6 and TGFβ in
bone metastases as opposed to soft-tissue metastases, which
are important cytokines for skewing T-cell response toward
Th17 instead of Th1 (110). These preclinical data are now
being used to develop a new combination therapy clinical
trial with ICT plus an agent to target TGFβ.
On the basis of these data, the concept of organ-specific
immune microenvironments will need to be addressed as the
field of ICT moves forward in the next decade. Treatment of
patients with metastatic disease will need to take into consid-
eration whether certain organs, such as bone and liver, have
distinct immune microenvironments that will require differ-
ent immunotherapeutic strategies (111).
Immunosuppressive Myeloid Cells
The immune microenvironment is unique to each tumor
type and plays a critical role in response and resistance to
ICT. Therefore, it is important to understand the composi-
tion and phenotypic states of intratumoral immune cells
of different tumor types to develop a tumor-specific ICT
strategy. Analysis of the immune microenvironment of five
different tumor types, including those that respond relatively
well to ICT and those that do not, revealed that ICT-sensitive
and ICT-resistant tumor types have distinct immune micro-
environments. Interaction of tumor cells with the immune
microenvironment guides the constitution and phenotypes
of immune subpopulations.
Immunologically cold tumors, such as prostate cancer and
glioblastoma multiforme (GBM), have few tumor-infiltrating
T cells but higher frequency of immune-suppressive myeloid
cells. Comparison of tumor-infiltrating lymphocytes (TIL)
from GBM and multiple other tumor types led to the iden-
tification of a GBM-specific CD73hi myeloid cell population,
which persists even after treatment with anti–PD-1 (112).
CD73 is an extracellular ectonucleotidase that plays a role in
the adenosine immunosuppressive pathway, inhibiting T-cell
activation and proliferation. Preclinical studies with CD73
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knockout mice showed improved survival with ICT compared
with control mice, demonstrating a direct role for CD73
(112). These series of studies helped in the identification of
CD73 as a specific immunotherapeutic target to improve
antitumor immune responses to ICT in GBM, generating a
combination strategy for a future clinical trial in GBM.
Other myeloid-specific targets, including chemokine recep-
tors (CXCR4, CCR2, and CSF1R), intracellular mediators
(IDO1 and PI3Kϒ), and proangiogenic agents (VEGF receptors,
semaphorin 4D and angiopoietin-2), are also being explored
in an attempt to limit the function of immunosuppressive
(M2) macrophages while promoting the function of M1 mac-
rophages, which facilitate antitumor immune responses (113).
The differentiation of macrophages into M1 or M2 subsets
is a complex biological process that is being investigated.
Many different signals, including cytokine and epigenetic
signals, appear to play a role in driving macrophage differ-
entiation. Interestingly, the process of phagocytosis, which
is required for clearing tumor cells, also seems to play a
role in driving M2 differentiation in an attempt to suppress
immune responses and avoid further cell death (114), indicat-
ing the complexity of how immune responses are regulated.
Detailed understanding of the signals and biological pro-
cesses involved in the differentiation of macrophages will be
needed to effectively target these cells.
Epigenetics and Immune Response
As outlined previously, the functional and phenotypic
states of immune subpopulations play an important role
in determining response to ICT. Cellular plasticity is often
driven by epigenetic mechanisms. Although the role of the
epigenetic machinery in regulating cancer cell plasticity has
been characterized (115), we lack mechanistic insight into
the epigenetic regulation of immune subsets, especially in the
context of ICT.
It was demonstrated that ipilimumab induces the expres-
sion of EZH2, a key epigenetic enzyme that plays an impor-
tant role in cellular plasticity (115–118). CD28 signaling leads
to increased EZH2 expression on T cells (119). Anti-CTLA4
increases CD28 signaling, thereby leading to increased EZH2
expression on T cells. On the basis of these observations, a
series of preclinical studies were designed that identified a
potential role for EZH2 in driving adaptive resistance to anti-
CTLA4 therapy. The combination of EZH2 inhibition with
anti-CTLA4 therapy led to improved survival in the preclinical
studies (116). These studies formed the basis for a clinical trial
of the rational combination of an EZH1/2 inhibitor (DS3201)
with ipilimumab in patients with genitourinary malignancies
with primary resistance to ICT (NCT04388852). This repre-
sents the tip of the iceberg in terms of epigenetic regulation of
immune cells, providing the foundation for the combination
of epigenetic modulators with ICT (120).
It is clear that we currently have the tools at our disposal to
interrogate human immune responses in great depth and we
will need to take advantage of these tools, and combine the
human data with appropriate preclinical models, to identify
rational targets for combination strategies that will improve
clinical outcomes for patients. Patient selection will also play
an important part in developing effective treatments, and
we will need to consider multiple factors, including both
REVIEW Sharma et al.

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Figure 2.  Biomarkers of response and resistance to ICT. Tumor cell–specific and immune cell–specific biomarkers associated with response and
resistance to ICT, and combinatorial biomarkers that may predict response to ICT. DC, dendritic cell; MSI, microsatellite instability; Teff, effector T cell;
TLS, tertiary lymphoid structure; TMB, tumor mutational burden; Treg, regulatory T cell.

tumor-related factors and immune-related factors, to identify
appropriate populations.
PREDICTIVE BIOMARKERS FOR OPTIMAL
PATIENT SELECTION
One of the major challenges in the field of ICT is the lack of
a robust predictive biomarker for optimal patient selection.
Traditionally, biomarkers have focused primarily on tumor-
intrinsic factors or single immune-specific markers. As our
understanding of determinants of antitumor immunity has
improved, we appreciate that single biomarkers in isolation
are insufficient. Combinatorial biomarker strategies that cap-
ture attributes of the host and tumor–immune ecosystem will
be essential (Fig. 2; refs. 121–124).
TMB and Neoantigen Load as
Predictive Biomarkers
High TMB is associated with response to ICT in numerous
cancer types, most notably melanoma and NSCLC (49). High

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The Next Decade of Immune Checkpoint Therapy
TMB may contribute to the formation of neoantigens that
are recognized by the immune system (125, 126). On a tissue-
agnostic basis, anti–PD-1 (pembrolizumab) received FDA
approval for the treatment of advanced pediatric and adult
solid tumors that have high TMB (≥10 mutations/megabase).
This is a promising step on which to build, and future stud-
ies on TMB as a predictive biomarker will address threshold
validation as well as provide a deeper understanding of the
immunogenicity of different mutational patterns (127).
Although high TMB led to FDA approval, it should be
noted that certain tumor types with moderate TMB carry rel-
atively high response rates to ICT, such as clear cell RCC (128,
129). In addition, low TMB does not preclude generation of
effective antigen responses, nor does high TMB guarantee
response to ICT (130–132). For example, it was shown that
anti-CTLA4 therapy can induce effective neoantigen-specific
T-cell responses in patients with prostate cancer, even in the
background of low TMB (131). Therefore, one approach to
enhance antigen-specific T-cell responses may be the combi-
nation of ICT with personalized neoantigen vaccines, which
have been shown to modulate T cells in the TME and enhance
responses to ICT, including in both immunologically “cold”
tumors such as glioblastoma and “hot” tumors such as mela-
noma (133–135). As vaccine therapies evolve to encompass
appropriate formulations, adjuvants, and delivery methods
that can elicit effective antitumor immune responses, it may
be possible to take advantage of tumor mutations to generate
neoantigen vaccines in combination with ICT for the devel-
opment of successful therapeutic strategies.
DNA Damage Repair Pathways: MSI-H/dMMR and
Homologous Repair Defects
There are fundamental links between DNA damage and
immunogenicity. Tumors with DNA damage repair (DDR)
defects, exemplified by MSI-H/dMMR status, were predicted
to have high rates of neoantigen formation due to their
high mutational load (approximately 10-fold compared with
that of MMR-proficient tumors) and therefore higher rates
of response to ICT (136). An initial study in patients with
colorectal cancer demonstrated significantly higher rates of
response to pembrolizumab in dMMR patients compared
with MMR-proficient patients (47), a finding that was later
extended to multiple tumor types (48). These data led to
the tumor-agnostic approval of pembrolizumab for MSI-H/
dMMR solid tumors (48).
Additionally, homologous repair defects, such as patho-
genic mutations in BRCA1/2, can lead to enhanced immune
response by multiple pathways including the release of cyto-
solic DNA, promotion of type I IFN signaling, and upreg-
ulation of PD-L1 (137–139). In a study of patients with
urothelial carcinoma, higher response rates to anti–PD-1/
PD-L1 antibodies were observed in patients with known
or likely deleterious DDR alterations (140). Emerging evi-
dence indicates that specific DDR mutations have differential
effects on the tumor–immune microenvironment, and there-
fore mutations will need to be selected carefully as candidate
predictive biomarkers (141). In all, an integrated approach
incorporating events in specific driver genes and mutational
signatures will be critical to develop additional genetic bio-
markers of response (142).
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PD-L1 Expression as a Predictive Biomarker
IHC evaluation of PD-L1 expression on tumor and/or
immune cells is frequently performed to select patients for
treatment with anti–PD-1/PD-L1 antibodies. A correla-
tion between PD-L1 expression on tumor cells and clinical
response is seen in certain circumstances (e.g., in NSCLC);
however, in other cases, substantial responses can be observed
in some patients with PD-L1–negative tumors, which high-
lights the difficulty of using PD-L1 as a single biomarker
(34, 143, 144). PD-L1 expression is dynamic due to multiple
factors, including T-cell responses and IFNγ signaling. There-
fore, variations in the timing of biopsy of the tumor as well
as the site of biopsy may influence whether PD-L1 expres-
sion is detected or not. Although a range of assays have been
approved, there remains significant variation in antibodies,
tissue handling techniques, and thresholds, which limits
the uniform benefit of PD-L1 expression as an isolated bio-
marker, and efforts are under way to integrate PD-L1 expres-
sion with other biomarkers (described below; ref. 127).
TILs and Exclusion of TILs as Biomarkers of
Response and Resistance
TILs have a long history in cancer immunotherapy, with
demonstration in the 1980s that adoptive transfer of TILs
derived from patients’ melanoma tumors can lead to cancer
regression (145). It was later shown that TILs, including CD8
T cells, correlated with clinical outcomes in multiple tumor
types, including ovarian cancer and bladder cancer (146,
147). Subsequent research on the correlates of response of
anti-CTLA4 therapy demonstrated that the extent of ICT-
induced tumor necrosis was directly related to the ratio of
intratumoral CD8 T cells to FOXP3+ Tregs (148). TILs are
therefore under active investigation as a biomarker to guide
selection of patients for treatment with ICT, although signifi-
cant variability exists in definitions and thresholds (149, 150).
In a prospective study, the AMADEUS trial (NCT03651271,
ongoing) was designed to evaluate the benefit of classifying
tumors into immunologically “hot” and “cold” tumors based
on CD8+ T-cell density (≥15% and <15%, respectively), with
anti–PD-1 (nivolumab) monotherapy administered as treat-
ment for “hot” tumors, whereas anti-CTLA4 (ipilimumab)
plus anti–PD-1 (nivolumab) combination is administered
as treatment for “cold” tumors in an attempt to take advan-
tage of the ability of anti-CTLA4 to drive T cells into “cold”
tumors. In addition, the Immunoscore is an approach that
aims to standardize and quantify CD3 and CD8 T cells infil-
trating and surrounding a tumor (149, 151). However, due
to variability in immune cell infiltration in different tumor
types, its applicability across cancers is challenging. Multiple
studies indicate that the location of T cells in the tumor
(whether at the invasive margins or in tumor parenchyma)
and the phenotype of the T cells (whether cells are memory-
like, exhausted, and/or activated) will need to be taken into
consideration (152–154).
In the same vein, T-cell exclusion is a mechanism of resist-
ance to ICT, exemplified by “cold” tumors such as prostate
cancer and pancreatic cancer, which have few T cells in the
TME. Certain oncogenic pathways may enable tumors to
exploit this mechanism of immune evasion. For example,
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tumor cell–intrinsic WNT/β-catenin activation has been
shown to lead to exclusion of T cells within the TME (123,
155, 156). In addition, PTEN loss is also associated with
increased signaling through the PI3K–AKT pathway and
correlated with exclusion of CD8 T cells and poor clinical
responses to ICT (124). Thus, patient selection for treatment
and strategies to overcome T-cell exclusion in the TME will
need to be considered in future ICT trials.
IFNg Gene Signature as a Predictive Biomarker
IFNγ is a key cytokine produced by activated T cells, as
well as natural killer (NK) and NK T cells, and is critical
for effective antitumor immunity. Preclinical work using
IFNγ-blocking antibody and murine models lacking essential
IFNγ signaling molecules demonstrated a key role for IFNγ
in immune surveillance and tumor rejection (157, 158). In
addition, knockdown of IFNγ-receptor 1 led to impaired
antitumor responses with anti-CTLA4 in a murine model
of melanoma (159). Consistent with the preclinical data,
patients with melanoma harboring genomic defects in IFNγ
pathway genes did not respond to anti-CTLA4 (160). Also,
patients with melanoma tumors harboring loss-of-function
mutations in JAK1 and JAK2, downstream IFNγ signaling
molecules, were resistant to anti–PD-1 therapy, highlighting
the utility of IFNγ as a potential biomarker.
IFNγ induces a host of target genes that have been used
to develop gene-expression signatures, which correlate with
responses to ICT. The gene-expression signatures can be ret-
rospectively labeled as “high” or “low” based on median cutoff
values. Multiple studies have reported that tumors identified
as having “high” expression of IFNγ-related gene signatures
have better clinical responses to ICT (131, 161, 162). However,
the gene panels used to design the IFN-gene signatures were
not consistent across these studies. In addition, consistent with
the yin and yang concept of immune responses, IFNγ drives
expression of proteins such as PD-L1 and IDO1, which may act
to suppress immune responses in the TME. Additional studies
are ongoing to develop reproducible assays for detection of
IFNγ-related genes and signatures that may be used as a pro-
spective biomarker for selection of patients for ICT.
Tertiary Lymphoid Structures as a
Predictive Biomarker
Tertiary lymphoid structures (TLS) have recently emerged
as biomarkers of response to ICT across several tumor types,
including lung cancer, melanoma, RCC, sarcoma, and urothe-
lial cancer (163–167). TLS or TLS-like structures comprised
of T cells, B cells, DCs, and other APCs may play a significant
role in antitumor T-cell responses, likely due to further prim-
ing and activation of T cells within the TME, which may
represent another important event in the cancer immunity
cycle. In contrast to other biomarkers of response which give
a snapshot of a single time point and single cell type, TLS are
dynamic structures comprised of multiple cell types. Multiple
chemokines and cytokines are involved in attracting these cell
types and forming the TLS, including CXCL13 (B cells) and
IL2 (T cells; ref. 168).
TLS represent sites of interactions between multi-
ple immune cell types and capture an ongoing antitumor
response (168). Future studies using newer technologies will
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be essential to understand which cells are interacting within
the TLS, and how they differ from the typical structures
within lymph nodes. Finally, because TLS appear to be asso-
ciated with responses to ICT across multiple tumor types, it
will be important to define the specific mechanisms driving
TLS formation to determine whether these mechanisms can
be exploited to induce the formation of TLS in otherwise
nonresponding tumors.
Combinatorial Biomarkers
Although many of the biomarkers described above, includ-
ing tumor-intrinsic and host-specific variables, correlate with
response to ICTs, many of the studies have been retrospective
and need further validation through prospective clinical tri-
als. Furthermore, there is a critical need to bridge preclini-
cal mechanistic studies with clinical investigations and vice
versa to develop reproducible predictive biomarkers. There
is growing recognition that single biomarker strategies do
not incorporate the dynamic and complex interaction of
the tumor and host immune system and that combinatorial
biomarkers will be essential to optimize patient selection.
With inherent inter- and intratumor heterogeneity, a complex
interplay of diverse cell types, and the continuously evolving
tumor immune microenvironment, it is clear that the utility
of isolated biomarkers is limited. A combination approach
integrating multiple data sets across different platforms from
diverse studies will be essential for developing more sensitive
and robust biomarkers of response.
Recent studies demonstrated that the combination of
existing biomarkers has better predictive capacity than single
biomarkers. For example, TMB plus a T cell–inflamed gene-
expression profile or TMB plus PD-L1 expression showed an
improved prediction performance compared with single bio-
markers (130). A recent study of more than 1,000 ICT-treated
patients across seven tumor types showed that a multivari-
able predictor of response outperformed TMB alone (169).
Such combinatorial biomarkers, tested in prospective clinical
trials, will be key for optimal patient selection.
It was also recently demonstrated that two novel biomark-
ers correlate with response to anti–PD-1/PD-L1 therapies.
A retrospective analysis identified mutations in ARID1A in
tumor tissues and expression of the chemokine CXCL13 as
predictive biomarkers for ICT (170). Preclinical studies dem-
onstrated association of ARID1A and CXCL13 with antitumor
immunity in mice (170). Importantly, the combination of
ARID1A mutation plus CXCL13 showed improved predictive
capacity compared with either of the single biomarkers alone
(170). This retrospective study highlighted the importance of
integrating tumor mutational status with the immune micro-
environment to predict responses to ICT, which led to a pro-
spective clinical trial to test ARID1A mutation plus CXCL13
as a combinatorial biomarker strategy for patient selection.
NEXT STEPS FOR ICT
The field of ICT has expanded across many different tumor
types, with improved benefit for patients with cancer. In addi-
tion, as the field continues to highlight other pathways that
can be targeted, there are now a vast number of combinatorial
approaches than can be tested in patients. Therefore, it will
The Next Decade of Immune Checkpoint Therapy
Further understanding of mechanisms of
primary and adaptive resistance to ICT
Development of robust predictive biomarkers for
optimal patient selection
Integration of newer technologies to obtain deeper
biological insights
Mechanistic and clinical understanding of irAEs
Incorporation of reverse translational strategies to
develop rational combinations
Figure 3.  Future directions in ICT. Figure highlighting the focus areas in the coming decade in the field of ICT that will play a critical role in improving
patient outcome.
be important to efficiently design future studies with atten-
tion to the mechanisms of action of each agent as well as an
understanding of resistance mechanisms that evolve over
time to control immune responses. The field of ICT is still
growing, but as we gain knowledge in multiple areas, as out-
lined below, the field will mature to provide clinical benefit
for even more patients with cancer (Fig. 3).
Integration of ICT with Other Therapies
Over the past decade, ICT has become established as the
fourth pillar of cancer therapy, along with chemotherapy/
targeted therapy, radiotherapy, and surgery. We have seen
some success in combining ICT with chemotherapy and
targeted therapy. The next decade will provide scientific
insights into integrating ICT with these pillars of cancer care
to benefit more patients. Exciting preclinical data on radio-
therapy plus ICT should provide insight into translating it to
clinical practice. Although surgical options in the metastatic
setting are extremely limited, we are exploring combinations
of ICT with surgery in the metastatic setting. These studies
will broaden the field of ICT across all aspects of oncology
as a unifying foundation for rational combination strate-
gies with chemotherapy, targeted therapy, radiotherapy, and
surgery. In addition, a number of other potential candi-
dates such as other immune checkpoints and costimulatory
receptors, epigenetic modulators, and metabolic targets hold
promise for improving the durable clinical responses seen
with ICT.
Moving from Metastatic to Earlier Disease Stages
Treatment of patients with earlier stages of disease, with
therapy given in either the neoadjuvant or adjuvant set-
ting, has the potential to prevent disease relapse and lead
to improved survival. Neoadjuvant ICT offers the possibil-
ity of surgical downstaging and provides ample tissue for
examination of an intact TME, potentially yielding insight
into posttreatment changes that can inform subsequent
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The
next
decade
of ICT
clinical trials. The first neoadjuvant ICT trial was conducted
in 2006 and provided safety data indicating that ICT can
be given prior to surgery (17). The study was conducted in
patients with localized bladder cancer who received anti-
CTLA4 before proceeding to cystectomy (17). This study
was also the first ICT trial in patients with bladder can-
cer, which provided critical data demonstrating antitumor
responses in patients with bladder cancer, thereby leading
to large clinical trials with ICT in patients with metastatic
bladder cancer, with subsequent FDA approval. In addi-
tion, the immune monitoring studies performed on the
trial highlighted the reverse translational approach and
identified ICOS+ CD4 T cells as a novel subset of T cells
associated with anti-CTLA4 therapy (17, 18, 171, 172). The
trial also led to the first combination ICT neoadjuvant trial
with anti-CTLA4 plus anti–PD-L1 in patients with bladder
cancer, which demonstrated clinical responses and correla-
tion of TLS with responses (21). Clinical trials with ICT in
the neoadjuvant setting have now demonstrated clinical
benefit in multiple tumor types, including melanoma (173,
174), Merkel cell carcinoma (175), NSCLC (176), MMR-
deficient colorectal cancer (177), urothelial carcinoma (21,
178), and breast cancer (179–181). Additional studies are
being planned, especially as investigators consider whether
to implement survival or other clinical endpoints, includ-
ing pathologic complete responses, which will enable faster
readout and review for possible approval (182–188).
Administration of ICT in the neoadjuvant setting enables
T and B cells to have access to the antigens that are present
on tumor tissues, which is in contrast to adjuvant studies
where ICT is given after tumor tissues have been removed,
thereby limiting the amount of tumor antigens that T and
B cells encounter. Nonetheless, adjuvant ICT has been suc-
cessful in high-risk melanoma, which led to FDA approval
(189–193). A number of additional studies are currently
ongoing and will provide additional insight into the use of
ICT in the adjuvant setting.
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Targeting Innate Immune Responses
Although much attention has traditionally focused on
adaptive immunity, there is growing interest in the role of
innate immune responses to boost overall antitumor immu-
nity. The innate immune system is composed of NK cells,
dendritic cells, macrophages, monocytes, neutrophils, eosin-
ophils, basophils, and mast cells. Therapeutic approaches
include stimulating innate responses with microbial peptides
or oncolytic viruses, which have previously been shown to
enhance local and systemic antitumor immunity in combina-
tion with ICT and may be able to improve payload to enhance
agonist signals (194–196). The presence of tumor-infiltrating
DCs expressing the transcription factor BATF as well as the
CD103 marker has been shown to be essential for presenta-
tion of neoantigens and activation of cytotoxic T cells in the
TME (197, 198). Therefore, approaches that enhance and/or
activate these DCs may improve local antitumor responses.
Systemic approaches to stimulating innate immunity, such
as via agonism of stimulator of interferon response cGAMP
interactor 1 (STING) or Toll-like receptors (TLR), have been
challenging (199–201). Therefore, intratumoral delivery rather
than systemic delivery of agents to target innate immune cells
may provide an avenue to ensure presentation of tumor-
specific antigens, enable effective T-cell responses, and also
reduce systemic toxicities. Intratumoral agents under inves-
tigation in clinical trials include mRNAs encoding CD70,
CD40 ligand, and TLR4 (202) and oncolytic viruses such as
talimogene laherparepvec, or T-VEC (203, 204), which is FDA-
approved for melanoma and is being explored in a number
of clinical trials. However, successful DC activation in a sup-
pressive TME continues to remain a challenge and needs to
be further investigated.
Targeting Microbiome to Drive Innate and
Adaptive Immune Responses
Furthermore, as our understanding of host effects on the
immune system has grown, it is clear that the gut micro-
biome exerts influence over the effectiveness of ICTs (127).
Responses to ICTs have been correlated with commensal gut
flora, and there appear to be substantial differences in the
diversity and composition in patients who respond to ICT
versus nonresponders (205, 206). Challenges in investigating
the gut microbiome include differences in sampling, analysis
platforms, and population heterogeneity, though these can
be addressed with the development of standard sequencing
and analytic tools. Manipulation of the gut microbiome,
such as through fecal microbiota transplantation, antibiotic
therapy, or diet, may serve as an adjunct strategy to enhance
responses to ICT. These strategies are still in early stages
of being explored but will provide important data that will
affect treatment with ICT.
Driving Agonist Signals for Combination
Therapy with ICT
On the basis of our understanding of T-cell activation,
there is growing interest in driving agonist signals via costim-
ulatory receptors. However, clinical experience with a CD28
superagonist that resulted in severe toxicities has led the field
to proceed with caution as additional agonist antibodies are
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Sharma et al.
evaluated (207). Both OX40 and 4-1BB/CD137 agonists have
been tested in clinical trials. Preclinical work with OX40
and 4-1BB showed efficacy; however, a phase Ib study of an
OX40 agonist combined with atezolizumab demonstrated
safety but limited efficacy in patients (208–210). In addition,
phase I studies with CD137-directed mAb urelumab caused
significant elevation of liver enzymes (211), and a second
anti-CD137 antibody, utomilumab, was well tolerated but
had limited efficacy (212). These studies highlight significant
knowledge gaps regarding the use of agonist antibodies in
terms of understanding expression of the target proteins,
sequencing of the agonist antibody with ICT and choice of
ICT agent (anti-CTLA4 vs. anti–PD-1/PD-L1). Currently, a
clinical trial with anti-CTLA4 plus an agonist anti-ICOS
antibody is ongoing (NCT03989362) based on preclinical
data demonstrating enhanced antitumor immune responses
in murine models of melanoma and prostate cancer (172). In
addition, the ICOS agonist GSK3359609 is also under study
in a phase III clinical trial (NCT04128696; ref. 213).
The development of immune agonists faces a number
of challenges, including target affinity, epitope selection,
receptor occupancy, Fc gamma receptor interactions, anti-
body isotype, and appropriate in vitro assays to clearly distin-
guish between an antagonist antibody with activity against
a costimulatory molecule expressed on immunosuppressive
cells versus an agonist antibody with activity against the same
costimulatory molecule expressed on effector cells (214). As a
result, the development of an agonist antibody to target T-cell
responses may require additional experimental steps and
careful immune monitoring studies of patients to help design
appropriate combination trials with current ICT agents.
As the field builds on current data with agonist antibodies,
novel approaches are being developed to deliver agonist sig-
nals in the TME. For example, bispecific antibodies are being
explored, including a bispecific antibody to target CD28 plus
a tumor-associated antigen (NCT03972657; ref. 215). This
approach of targeting the costimulatory pathway within the
TME, as opposed to systemic targeting, may provide a safer
way to provide agonist signals to T cells. As these processes
are optimized, costimulatory receptor targeting is becoming
a feasible strategy to enhance the efficacy of ICT.
Incorporation of Newer Technologies
In the future, spatiotranscriptomic tools such as Digital
Spatial Profiling and CO-Detection by indEXing (CODEX)
may allow us to decipher the cell types and their inter-
actions within the tumor. In addition, the incorporation
of single-cell high-throughput technologies into clinical
and preclinical studies will enable better characterization
of cell subsets regulating antitumor responses. Assessing
single-cell transcriptomics, proteomics, and chromatin state
through scRNA-seq, Cellular Indexing of Transcriptomes and
Epitopes by Sequencing (CITE-seq), and single-cell Assay for
Transposase-Accessible Chromatin sequencing (scATAC-seq)
will enable identification of cellular identity at a single-cell
level. Furthermore, integration of various platforms assisted
by novel bioinformatic tools will allow us to understand the
regulatory pathways governing response and resistance to
ICT. In addition, integration of longitudinal blood-based
assays such as circulating-tumor DNA (ctDNA; ref. 216),
The Next Decade of Immune Checkpoint Therapy
which are easier to implement and less invasive as opposed
to longitudinal tumor biopsies, is under investigation as a
method to monitor responses to ICT.
Additionally, the development of better bioinformatics
algorithms that help with neoantigen prediction will enable
identification of more relevant therapeutic targets. Progress
in the prediction of shared MHC class I antigens in clinical
samples has been exciting. A recent study developed a pipe-
line to determine tumor epitope immunogenicity that might
enable prediction of effective antitumor responses with much
higher accuracy than conventional methods (217). However,
although developing prediction algorithms for class I anti-
gens is necessary, it has recently been shown that MHC class II
neoantigens are also involved in effective tumor responses
and will need to be considered (218).
The successful implementation of these technologies will
require close collaboration between computational biologists
and immunologists to avoid simplification of the complex
biological and functional processes of immune responses,
thereby maintaining fidelity to the dynamic nature of cell
subsets within the immune system. For example, PD-1
expression on a T cell may represent an “activated” T cell
or an “exhausted” T cell. The simple identification of PD-1
through a bioinformatics approach may not provide suf-
ficient understanding regarding the complexity of the T-cell
response. Therefore, appropriate functional studies or other
immunologic assays may be necessary to identify additional
biomarkers that should be included in the bioinformatic
analyses for a more accurate definition of a given immune cell
state. Careful attention to these types of details will be neces-
sary for advancing the field of ICT.
CONCLUSIONS
Over the next decade, the field of cancer immunotherapy
will need to grow vertically, to gain a deeper understanding
of different immune cell subsets and pathways that regulate
their responses, as well as horizontally, to integrate with other
fields, including cancer biology, epigenetics, computational
biology, and many others. ICT will also need to expand across
all of oncology for the development of combination strategies,
including combinations with surgery, radiotherapy, chemo-
therapy, targeted therapy, and other immunotherapy agents
such as novel immune checkpoints and chimeric antigen
receptor T cells. As the field continues to grow, it will need to
remain grounded in the strength of basic immunology while
it fosters close collaborations with other scientific disciplines.
The future of ICT is promising, with ample opportunity to
further improve outcomes for patients with cancer.
Authors’ Disclosures
P. Sharma reports personal fees from Jounce Therapeutics, Onc-
olytics, Earli, Glympse Bio, Lava Therapeutics, Lytix, Phenomics,
Codiak, Constellation, Achelois, Dragonfly, BioAtla, Infinity Pharma,
Hummingbird, and Polaris outside the submitted work; in addition,
P. Sharma has a patent for ARID1A plus CXCL13 as combinatorial
biomarker pending and a patent for combination of anti-CTLA4
plus agonist targeting of ICOS for improved antitumor immune
responses licensed to Jounce Therapeutics. S.K. Subudhi reports per-
sonal fees from Dendreon, Kahr Medical, Ltd., Cancer Expert Now,
Inc., Bayer Healthcare Pharmaceuticals, Apricity Heatlh, LLC, Dava
April 2021 CANCER DISCOVERY 10TH ANNIVERSARY ISSUE | 851
Downloaded from cancerdiscovery.aacrjournals.org on April 9, 2021. © 2021 American Association for Cancer
Research.
REVIEW
Oncology, Bristol-Meyers Squibb, Amgen, Inc., and Exelixis, Inc.
outside the submitted work. J.P. Allison reports personal fees from
Jounce Therapeutics, Codiak Biosciences, Achelois, Lava Therapeu-
tics, Lytix, Earli, Phenomics, Dragonfly, Hummingbird, ImaginAb,
Forty-Seven, and Polaris outside the submitted work. No disclosures
were reported by the other authors.
Received November 23, 2020; revised February 1, 2021; accepted
February 5, 2021; published first April 2, 2021.
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The Next Decade of Immune Checkpoint Therapy
Padmanee Sharma, Bilal A. Siddiqui, Swetha Anandhan, et al.

Cancer Discov 2021;11:838-857.

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