Targeting PD-1 Or PD-L1 in Metastatic Kidney Cancer – Combination Therapy in the First Line Setting

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Author Manuscript Published OnlineFirst on January 16, 2020; DOI: 10.1158/1078-0432.CCR-19-3323 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Targeting PD-1 or PD-L1 in Metastatic Kidney Cancer – Combination Therapy in the First Line Setting

David H. Aggen, Charles G. Drake, and Brian I. Rini

Running Title: Combination PD-1 and PD-L1 Immunotherapy for Kidney Cancer

Corresponding Author: David H. Aggen, MD, PhD [email protected] Memorial Sloan Kettering Cancer Center Sidney Kimmel Center for Prostate and Urologic Cancers 353 E. 68th St – Room 421 New York, NY 10065

Charles G. Drake, MD, PhD [email protected] Herbert Irving Cancer Center New York-Presbyterian/Columbia University Medical Center 177 Fort Washington Avenue, 6GN-435 New York, NY 10032

Brian I. Rini, MD [email protected] Vanderbilt University Medical Center Division of Hematology/Oncology 2220 Pierce Ave. 777 Preston Research Building Nashville, TN 37232

Keywords Kidney Cancer PD-1 PD-L1 Tyrosine Kinase Inhibitors Immunotherapy

Declaration of Interest/Disclosure D. Aggen: Consulting fees from Boehringer Ingelheim C. Drake: Consulting fees from BMS, Merck, Genentech/Roche, and Pfizer B. Rini: Research funding and consulting fees from Merck, Pfizer, Ax, Peloton Therapeutics, BMS, and Genentech/Roche.

Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 16, 2020; DOI: 10.1158/1078-0432.CCR-19-3323 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract: Recent FDA approvals of regimens targeting programmed death 1 (PD-1) in

combination with anti-CTLA-4 or with vascular endothelial growth factor (VEGF) tyrosine kinase

inhibitors (TKIs) are reshaping front-line therapy for metastatic kidney cancer. In parallel,

therapeutics specific for programmed death ligand 1 (PD-L1), one of the two major ligands for

PD-1, are under continued investigation. Surprisingly, not all PD-1 and PD-L1 agents lead to

similar clinical outcomes, potentially due to biological differences in the cellular expression and

regulation of these targets. Here, we review current clinical data on combination immune

checkpoint inhibitor therapy in metastatic kidney cancer and discuss the relevant biology of PD-

1 and PD-L1. The design of future rational combination therapy trials in metastatic renal cell

carcinoma will rely upon an understanding of this biology, along with an evolving understanding

of immune cell populations and their functional states in the tumor microenvironment.

Introduction

The discovery of PD-1 and PD-L1 as a mechanism of peripheral T cell tolerance spurred the development of multiple therapeutics blocking their critical interaction.(1) In the context of kidney cancer, the PD-1 specific therapies nivolumab(2,3) and pembrolizumab(4), as well as the

PD-L1 antibody avelumab(5) are FDA-approved in combination with other therapies for

metastatic renal cell carcinoma (RCC). In the past 14 months, 4 phase III trials have tested the

hypothesis that immunotherapy (I/O)-based combinations are efficacious as first-line therapy in

metastatic RCC (Table 1). Combination therapies based on anti-PD-1 antibodies,

pembrolizumab plus axitinib and nivolumab plus ipilimumab, have improved overall (OS)

relative to sunitinib.(3,4) In contrast, combination therapies with anti-PD-L1 antibodies

including avelumab plus axitinib (Javelin-101)(5) or atezolizumab plus bevacizumab (ImMotion

151)(6), have not yet demonstrated an overall survival benefit. Overall survival data in these

anti-PD-L1 combination trials is still immature, and ultimately a survival benefit might be

observed with further follow-up. Despite this finding, it is notable that occasional complete

responses were reported with both anti-PD-1 and anti-PD-L1 combination regimens. Caution

should be exercised in cross-trial comparison due to potential differences in baseline patient

characteristics. However, the OS benefits documented in anti-PD-1 combination therapy trials,

but not in anti-PD-L1 immunotherapy studies highlights potential advantages to targeting PD-1 as compared to PD-L1 in specific clinical contexts.

Observations in other tumor types support the concept of non-equivalence between PD-

1 and PD-L1 targeted therapeutics. In bladder cancer, the anti-PD-1 agent pembrolizumab

showed an OS benefit compared to 2nd line treatment in Keynote-045(7), whereas the anti-PD-

L1 antibody atezolizumab did not show an OS benefit when compared to 2nd line chemotherapy in a similar patient population in the IMVigor211 trial(8). In NSCLC, the anti-PD-1 antibody pembrolizumab improved OS relative to 2nd line chemotherapy in Keynote-010(9), whereas the anti-PD-L1 antibody avelumab failed to improve OS in a similar cohort of patients(10).

Notwithstanding the limitations of cross-trial comparisons, the discrepancies in clinical outcome

between PD-1 and PD-L1 antibodies beg a lingering question: are these therapeutics

equivalent?

Fundamental differences in the biologic mechanisms of anti-PD-1 and anti-PD-L1 may

underlay these potentially disparate clinical outcomes; thus, understanding these nuances is

critical to the design of next generation combinatorial strategies. Herein, we describe some key

distinctions in PD-1 and PD-L1 biology in terms of cell type specific expression, differential regulation, and the physiologic effects of blockade. The relative contribution of antibody directed cell cytotoxicity (ADCC) for PD-L1 therapeutics is also discussed. Finally, we summarize ongoing clinical activity using these therapeutics in combination regimens.

The PD-1 / PD-L1 Interaction Attenuates a T Cell Response

T cell activation is initiated by the engagement of a T cell receptor (TCR) with its cognate

peptide-MHC complex - along with an appropriate costimulatory signal (Signal 2). In this

setting, the primary biologic function of PD-1 is to maintain a desirable range of T cell activation

so as to prevent rampant autoimmunity.(11) Upon T cell activation, PD-1 is upregulated within

12-36 hours and its interaction with PD-L1 and/or PD-L2 down-modulates T cell proliferation

and effector function (Figure 1).(12) Biochemically, PD-1 binding to either PD-L1 or PD-L2(13)

activates the tyrosine phosphatase SHP-2 in the PD-1 expressing cell; this directly dampens T

cell activation by dephosphorylating the TCR and costimulatory molecules like CD28.(14) In the setting of chronic antigen stimulation, PD-1 preserves T cell clones that might otherwise undergo activation-induced cell death. As a consequence of its biology, PD-1 expression is both

a marker of initial T cell activation as well as a marker of several states of functional exhaustion.

Those states are defined in part by the co-expression of additional immune checkpoint

molecules like LAG-3 and TIM-3.(15) Consequently, not all PD-1 expressing T cells behave as

functionally exhausted T cells, and additional cell surface markers and epigenetic signatures are

required to more completely define immune cell subsets with diminished effector capacity

(recently reviewed in detail).(16)

PD-1 and PD-L1 Protein Expression: Shared but Distinct Cellular Compartments

In general, PD-1 is expressed on activated / exhausted CD8 and CD4 T cells, although

expression has been reported on a number of other populations including B cells and

macrophages.(17) PD-L1 may be expressed on both tumor cells or other cells in the TME,

including dendritic cells, macrophages and other myeloid populations.(18) Controversy exists

regarding the most important PD-L1 expressing population – with some studies suggesting that

tumor expression is most critical(19,20) and other studies highlighting expression on myeloid

populations.(21,22) The second major PD-1 ligand, PD-L2, has a more restricted expression pattern with predominant expression on endothelial cells, monocytes, and dendritic cells. In a study evaluating PD-L2 expression in 7 distinct tumor types, RCC had amongst the lowest level

of tumor PD-L2 expression with relatively high stromal and endothelial cell expression of this

ligand.(23) The expression of PD-L2 in the TME is in general under-appreciated, especially as

PD-1 has a higher binding affinity for PD-L2 than for PD-L1.(24) Indeed, the potential of PD-L2

to promote T cell tolerance provides one potential explanation for the lack of overall survival

benefit with PD-L1 combination therapies in kidney cancer.

PD-1 and PD-L1 Are Dynamically Regulated by Cell Extrinsic and Intrinsic Factors

As described above, PD-1 expression is initiated by T cell activation. Expression is

further modulated by a number of signals in the TME including TGF-(25), and IFN-, which

promote upregulation of PD-1 on both T cells and macrophages.(26) PD-1 biology is somewhat

complex, with at least 10 transcriptional factor complexes that function in modulating PD-1

activity dependent on the state of T cell activation (reviewed in detail elsewhere).(27) In acute

infection, antigen clearance leads to eventual down-regulation of PD-1, while in the context of

cancer and chronic viral infection persistent antigen exposure drives continued PD-1 expression

on antigen-specific T cells.(28)

PD-L1 expression on immune and tumor cell subsets is largely induced by TH1 cytokines

like interferon-gamma (IFN-. Following IFN- exposure, tumor and immune cells up-regulate

PD-L1 through a transcriptional program involving the JAK1/STAT signaling pathway.(29)

Clinically, this is important, since mutations in the JAK/STAT pathway and antigen presentation machinery have been implicated in primary and acquired resistance to PD-1 therapy in melanoma.(30) At the genomic level, copy number alterations (CNA) in the PD-L1 gene in

tumor cells may also lead to increased levels of PD-L1 expression. CNA in PD-L1 at chromosome

9p24 are associated with increased tumor mutation burden(31) and are enriched in a rare but

unique RCC subset with sarcomatoid pathologic features(32). The latter is of keen interest as

gene signatures associated with sarcomatoid RCC pathology were enriched in patients

responding to atezolizumab and bevacizumab in IMMotion 151.(33) Subgroup analysis of

patients with sarcomatoid pathologic features from Checkmate 214(34) (nivolumab +

ipilimumab) and Keynote 426 (pembrolizumab + axitinib)(35) also demonstrated improved ORR

and OS relative to sunitinib.

Additional tumor intrinsic factors may also drive PD-L1 expression to promote immune tolerance and tumor immune evasion. In clear cell RCC, HIF2 activation secondary to Von

Hippel Landau (VHL) deficiency promotes PD-L1 expression in vitro.(36,37) However, clinical data supporting this association are not yet available. VHL inactivation is estimated to occur in

>90% of RCC patients either through direct mutation or promoter hyper-methylation, and one would anticipate the number of PD-L1 expressing tumor samples in RCC would be dramatically higher if this association were absolute.(38) For example, in Checkmate 214, only 20-30% of

RCC patients had PD-L1 positive tumor cells.(3) Similarly, in the COMPARZ trial evaluating pazopanib versus sunitinib, 36% of patients had PD-L1 positive specimens.(39) As a consequence, the association between PD-L1 expression and VHL deficiency certainly requires additional investigation.

Metabolic and Epigenetic Programs Modulated by the PD-1 / PD-L1 Axis

PD-1 ligation with PD-L1 or PD-L2 induces T cell functional exhaustion by causing distinct metabolic changes within the T cell. PD-1 binding switches the T cell energy source to fatty acid oxidation with concomitant attenuation of glycolysis.(40) This metabolic switch assists in determination of T cell effector versus memory cell fates and promotes the maintenance of functional CD8 exhaustion. Similarly, attenuation of glycolysis in CD4 T cells, which may or may not be independent of PD-1 signaling, promotes regulatory T cell commitment.(41) Thus PD-1 and additional costimulatory molecules, such as 4-1BB, are implicated in driving immune cell metabolic programs that lead to T cell dysfunction.

Ongoing PD-1 engagement with its cognate ligands also results in epigenetic

reprogramming of T cells, which may prevent effective rescue by immune checkpoint blockade.

These observations were initially based on murine studies utilizing the LCMV virus that mimics

chronic antigen stimulation as is observed in cancerous states.(42) In LCMV murine models,

functionally exhausted T cell remained in a PD-1HI exhausted state even after clearance of

antigen, demonstrating that epigenetic mechanisms likely underlie long-lived functional

exhaustion and PD-1 expression.(43)

More recent data suggest that distinct epigenetic profiles define states of functional T

cell exhaustion.(16) Elegant work identified the nuclear transcription factor TOX as a central

regulator of epigenetic and transcriptional programs driving T cell exhaustion.(44,45) TOX

expression increases following chronic antigen stimulation, leading to a decrease in markers of

self-renewal in T cells - including the key transcription factor TCF1.(46) Conversely, deletion of

TOX restored CD8 T cell function and differentiation to effector and memory phenotypes. Taken

together, these studies show that that TOX is a critical driver of early T cell exhaustion.

Advancements in single-cell analysis(47) and epigenetic profiling will be critical in further

defining the functional and phenotypic heterogeneity within these exhausted states, and

clinical interventions aimed at altering the epigenetic phenotype of T cells remains an area of

active interest.(48-50)

Functional Consequences of PD-1 / PD-L1 Blockade in the Clinic

Anti-PD-1 agents can restore the functionality of exhausted T cells through direct

ligation of PD-1 on CD4 and CD8 T lymphocytes, and based on that principle may rescue an immune response relatively independent of tumor PD-L1 expression (Figure 2A). Direct T cell

binding by an anti-PD-1 therapeutic may afford significant advantages relative to an anti-PD-L1

treatment, potentially via more rapid T cell expansion. In melanoma patients treated with anti-

PD-1, peripheral blood profiling showed that expansion of a PD-1+ effector T cell pool after

immune checkpoint blockade correlated with clinical response.(51) Relevant neoadjuvant

studies illustrate peripheral occupancy of PD-1, with peripheral blood responses detected

within 3 weeks on therapy.(52) Peripheral blood profiling also shows that anti-PD-1

therapeutics rapidly stimulate T cells in the periphery, enabling tumor cell lysis, relatively

independent of tumor volume/burden. The kinetics of T cell expansion mediated by direct

engagement of PD-1 on effector T cells may not be achievable with anti-PD-L1 agents targeting

tumor and immune cells. With adoption of immunotherapy into the neoadjuvant setting in

clinical trials of kidney cancer including PROSPER-RCC(53), we will gain further insights into the

mechanistic and kinetic differences in PD-1 and PD-L1 occupancy and immune blockade.

Similarly, adjuvant trials for high-risk RCC evaluating atezolizumab (NCT03024996) and pembrolizumab (NCT03142334) have completed accrual, and peripheral blood studies from these trials will enhance our understanding of the relative benefit of peri-operative PD-L1 and

PD-1 blockade.

PD-L1 targeted therapies, in contrast, can induce immune tumor rejection through

multiple mechanisms (Summarized in Figure 2B-2D). First, anti-PD-L1 therapies prevent ligation

with PD-1 on immune cells like anti-PD-1 therapeutics. PD-L1 blockade also prevents ligation

with the costimulatory molecule B7.1 (CD80) either in cis or in trans, which may provide a

secondary mechanism for T cell reinvigoration.(54) Secondly, anti-PD-L1 therapeutics may also

drive direct tumor cell killing through antibody-dependent cellular cytotoxicity (ADCC), in this

case via PD-L1 expressed on tumor cells (Figure 2C). In murine models, anti-PD-L1 that bind Fc

receptors that mediate ADCC let to tumor regression, whereas a similar effect was not

observed with anti-PD-1 therapies in those models.(55)

Despite these theoretical advantages, there are now 2 randomized phase III trials in

metastatic RCC using anti-PD-L1 therapeutics that have not yet shown an overall survival

benefit (Table 2). Overall survival data in these trials is still immature and longer follow-up is awaited. While the mechanisms underlying this difference may be challenging to dissect, one possibility is that interaction between PD-1 and PD-L2 is unaffected by PD-L1 blockade, such

that interactions between PD-L2 in the TME and PD-1 on T cells provides some level of ongoing

suppression. A 2nd theoretical concern involves binding to non-tumor cell expressing isoforms

of PD-L1, sequestering antibody that might be important in blocking the PD-1 / PD-L1

interaction. Accordingly, relevant data suggest that PD-L1 expression on exosomes(56) and

secreted variants of PD-L1(57) may suppress anti-PD-L1 responses. A final potential mechanism

of interest is that expression of PD-L1 on immune cells might deplete immune effector cells

through ADCC in certain circumstances (Figure 2D, reviewed below).

Antibody Isotype Effects on Clinical Activity

An underappreciated aspect of immune checkpoint blockade is the relative contribution

of T cell mediated tumor killing versus the potential for antibody-dependent cellular

cytotoxicity (ADCC) or complement dependent cytotoxicity. In ADCC, FC gamma receptors

(primarily FcRIII) on the surface of macrophages and NK cells bind to the Fc portion of

antibodies resulting in depletion of tumor or subsets of immune cells (Figure 2C and 2D).

Specific IgG subtypes are more likely to promote ADCC, with IgG1 and IgG3 antibody subtypes with a higher binding affinity for Fc receptors (Supplemental Table 1).(58) Thus, anti-PD-L1 antibodies of the IgG1 isotype may lead to depletion of both tumor and immune cells. Indeed, avelumab, an IgG1 isotype antibody, can mediate ADCC and lead to effective direct tumor cell killing. In theory ADCC can also occur on PD-L1 positive CD8 effector cells leading to elimination of immune effectors. However, no definitive evidence of the latter phenomena has been appreciated clinically. Importantly, recently presented subgroup analysis from the Javelin-101 showed no difference in activity of combination avelumab plus axitinib treatment in patients with Fc-gamma receptor RIII polymorphisms, demonstrating that ADCC may be only a minor mechanism in anti-PD-L1 immunotherapy.(59)

Ongoing efforts are focused on improving the efficiency of antibody-induced cellular cytotoxicity with immune checkpoint blockade antibodies. Through modification of glycosylation and fucosylation sites, antibodies can be engineered to have differential effects on ADCC and cellular depletion.(60) To promote ADCC, an Fc-modified (non-fucosylated version) of anti-CTLA-4 is in early phase clinical trials with the goal of regulatory T cell depletion

(NCT#03110107). Future immunotherapy combination approaches may leverage the ability to selectively deplete immunosuppressive cell subsets and potentiate antitumor responses.

PD-1 vs. PD-L1 in combination with VEGF TKIs

In RCC, the addition of a VEGF TKI to an anti-PD-1 or PD-L1 antibody exploits a number of potentially synergistic mechanisms. VEGF in the tumor ecosystem promotes immunosuppression by decreasing T cell trafficking to tumors, increasing immunosuppressive

cytokines and initially increasing regulatory T cells. Treatment with anti-angiogenic therapies

mitigates a number of the immunosuppressive effects of VEGF in preclinical models.(61,62) For

example, the use of sunitinib in a preclinical RCC model decreases immunosuppressive myeloid-

derived suppressor cells (MDSCs), a potential mechanism of adaptive immune resistance to PD-

1 immunotherapy.(63) More recent preclinical data with axitinib showed anti-tumor efficacy

not only through vascular remodeling but also through depletion of tumor-promoting mast cells

and tumor-associated macrophages.(64) In human RCC specimens, treatment with antiangiogenic therapy increased infiltration of CD4 and CD8 effector T cells supporting the hypothesis that VEGF inhibition might potentiate the response to immune checkpoint blockade by promoting T cell infiltration.(65) Clearly, the immune effects of VEGF TKIs support non-

redundant mechanisms of immune activation distinct from the PD1/PD-L1 axis, with TKI

immune remodeling affecting the myeloid and T cell compartment.

Although the MDSC-specific effects of TKIs provide a good rationale for combining anti-

PD-L1 with VEGF therapy, there are some data suggesting that VEGF TKIs may dampen a T cell response to cancer. Chronic inhibition of VEGF with TKIs can induce a hypoxic state within the

TME with a concomitant accumulation of HIF-1.(66) Multiple studies show that HIF-1

accumulation induces a compensatory immunosuppressive state through recruitment of

MDSCs(67), tumor-associated macrophages(68,69), and Tregs(70). Additionally, accumulation

of HIF-1 alters PD-L1 expression on immune cell subsets.(71) That observation is supported by

data from human RCC samples showing that TKIs may decrease PD-L1 expression, rendering

anti-PD-L1 blockade more challenging. Of note, on-treatment biopsies from patients treated

with pazopanib or sunitinib showed transient decreases in PD-L1 expression by

immunohistochemistry.(72)

In some model systems, VEGF TKI’s also decrease immune cell PD-1 expression.(73)

The decrease in PD-1 expression, however, is not absolute, and blockade of remaining PD-1 on

T cells with anti-PD-1 therapeutics may explain the improved OS noted with combination anti-

PD-1 with VEGF TKIs. Taken together, these collected observations lend support to a hypothesis that limited or intermittent VEGF TKI therapy in combination regimens might allow an even greater immune response, but at present all TKI combinations in the phase III setting have been taken continuously. Further, HIF1 inhibition may be a therapeutic approach to enhance the clinical benefit of VEGF TKI-based combinations.

Consensus First-Line Therapy Approaches in Metastatic RCC

Both nivolumab plus ipilimumab and pembrolizumab plus axitinib are now consensus first line treatments for metastatic RCC. At present the choice of first line therapy for a given patient is not driven by a randomized, comparative trial, but rather by treatment side effect profile, prognostic risk group, perceived benefits of complete and overall response rate and

MD/patient preference. Avelumab plus axitinib is also FDA approved for first-line RCC, but so far, an overall survival benefit relative to sunitinib has not been documented. Finally, the FDA

application for drug approval of atezolizumab plus bevacizumab was withdrawn by the manufacturer, although there may in fact be subgroups of patients with specific gene signatures that benefit from this combination.(74) Taken together, these data support the use

of a PD-1 based immunotherapy combination, either with pembrolizumab plus axitinib or

nivolumab plus ipilimumab for first-line therapy of metastatic RCC.

Future Combination Therapy Approaches for Metastatic RCC

The impressive response rates and overall survival for patients treated with combination

anti-PD-1 plus anti-CTLA-4 or anti-PD-1 plus VEGF TKI therapy with a favorable side effect profile and tolerability begs the question of utilizing a triplet therapy in the first line setting.(75)

Combination nivolumab, ipilimumab, and cabozantinib has been administered safely across GU

malignancies, and the activity of this triplet will be tested in a phase II expansion cohort and a

randomized, phase 3 trial.(76) Triplet therapy, however, likely over treats some patients, such

that biomarker-based strategies to select patients for the appropriate mechanism and intensity

of therapy is an unmet need. One additional combination for first-line treatment,

pembrolizumab + lenvatinib, is currently being tested in large phase III trials. Table 3 provides a

complete listing of trials currently accruing for RCC.

A potential approach to mitigate the toxicities of I/O-I/O combinations is to incorporate

other anti-inflammatory medications into the first-line treatment regimens. To this end, clinical

trials are on-going exploring cytokine targets including anti-IL1NCT04028245), anti-IL-6, and

anti-IL8(77) (NCT03400332) to augment the immune response and potentially improve regimen

tolerability. Another potential approach might be to block TNF- in the combination therapy

setting. A recent publication in animal models highlighted this approach, demonstrating

increased activity of combination immunotherapy when TNF- blockade was added to anti-PD-

1 plus anti-CTLA-4.(78) The wealth of treatment options available for RCC also raises the

questions of optimal therapeutic sequencing which will be addressed in an upcoming trial.(79)

In contemporary cohorts of metastatic RCC patients, ~50% of patients will not receive a second

line treatment due to either disease progression or declining performance status.(80) In real-

world data sets, it is estimated that >80% of patients do not receive any second line

treatment.(80,81) As a consequence, maximizing the efficacy of first-line therapy is of utmost

importance.

Conclusions

There are now 3 FDA approved combination immunotherapies for the treatment of

first-line kidney cancer, but only anti-PD-1-based combinations to date have illustrated an

overall survival benefit. Blockade of PD-1 permits direct reprogramming of T cells, whereas

anti-PD-L1 exerts those effect in an indirect fashion and permits binding between PD-1 and PD-

L2. Although in contemporary models the activity of anti-PD-1 + VEGF TKI appears to be

additive, the remarkable gains in ORR, PFS and OS will likely necessitate that anti-PD-1 therapeutics remain the backbone of first-line treatment for renal cell carcinoma. For the foreseeable future, the selection of first-line treatment will be guided by side-effect profile, risk group and patient preference, while the next generation of first-line therapies may require clinically validated biomarkers to select the appropriate treatment regimen.

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Figure Legends

Figure 1. PD-1 / PD-L1 Targeted Therapeutics in Renal Cell Carcinoma. Overview of current immunotherapy targets in renal cell carcinoma. The PD-1 antibodies pembrolizumab, nivolumab, and spartalizumab prevent interaction with PD-L1 and PD-L2. In contrast, the PD-L1 antibodies avelumab, atezolizumab, and durvalumab prevent PD1 ligation, but leave PD-1 and

PD-L2 ligation unopposed.

Figure 2. Mechanisms of PD-1 and PD-L1 Targeting Immunotherapy. A) PD-1 blockade exerts direct effects on immune cells upon ligation by driving distinct metabolic and epigenetic programs that reverse T cell dysfunction. B) PD-L1 blockade on immune cells masks PD-1 ligation. PD-1 can bind PD-L2. C) Antibody directed cell cytotoxicity (ADCC) from PD-L1 ligation on tumor cells permits tumor cell killing. D) PD-L1 expression on T cells permits ADCC of immune cells overexpressing PD-L1.

Tables

Table 1. Summary of Completed Phase III Trials in Metastatic RCC Evaluating Combination

Immune Therapies

Nivolumab + Pembrolizumab + Avelumab + Axitinib Atezolizumab + Ipilimumab Axitinib Bevacizumab

Trial CheckMate 214 Keynote 426 Javelin 101 ImMotion 151

N 1096 861 1096 915

PD-L1+ (%)* 23.0% 59.3% 61.0% 40.0%

PD-L1 Assay/Cutoff Dako 28-8 (≥1% TC) Dako 22C-3 (CPS≥1%) Ventana SP263 (≥ 1% IC) Ventana SP-142 (>1% IC)

Favorable 23.0% 31.9% 21.3% 20.0%

Risk Intermediate 61.0% 55.1% 61.3% 69.0% Category Poor 17.0% 13.0% 16.3% 12.0%

Liver Metastases (%) 24.5% 15.0% NR 17.0%

Median Follow-Up (m) 32.4 12.8 12.0 24.0

ORR** 41.0% 59.3% 51.4% 37.0%

CR** 11.0% 5.8% 3.4% 5.0%

PFS (m) Combination Arm 9.7 15.1 13.8 11.2

Sunitinib Arm 9.7 11.1 8.4 8.4

HR (CI) 0.85 0.69 0.69 0.83 (95.0% CI 0.73 – 0.98) (95% CI 0.57 – 0.84) (95.0% CI 0.56 – 0.84) (95.0% CI 0.70 – 0.97)

OS (m) Combination Arm NR NR NR 33.6

Sunitinib Arm 37.9 NR NR 34.9

HR (CI) 0.71 0.53 0.78 0.93 (95.0% CI 0.59 – 0.86) (95% CI 0.38 – 0.74) (95% CI 0.55 – 1.08) (95% CI 0.76 – 1.14)

*Percent PD-L1 positive in combination I/O arm. PD-L1 cutoff and compartment evaluated differs in each trial. **ORR and CR rate in combination I/O arm TC= Tumor cells, IC= Immune Cells, CPS= Combined positive score calculated as the number of (total PD-L1 + TC and IC)/divided by total number of TC x 100 NR= Not reported

HR with statistically significant confidence intervals are in Bold.

Table 2. Prospects and Limitations of Anti-PD-1 and Anti-PD-L1 Immunotherapies for

Combination Therapy in RCC

Advantages Disadvantages

- Targets T cells directly - Distinct metabolic and epigenetic changes upon PD1 binding reverse T cell exhaustion - No direct tumor effects from antibody Anti-PD-1 - Does not require tumor PD-L1 expression - In vitro VEGF TKIs increase PD-1 expression on immune for activity cells providing a potential resistance mechanism - PD-1 occupancy on T cells observed within 3 weeks on treatment

- Does not block PD-1 binding to PD-L2 - PD-L1 exists on exosomes and in soluble forms which may act as a “decoy” receptor for antibody therapy - VEGF TKI treatment permits PD-L1 upregulation on tumor cells providing a potential resistance mechanism - Targets tumor cells directly - Limited engagement of anti-PD-L1 with PD-L1+ tumor T Anti-PD-L1 - May permit ADCC of tumor cells cells allows for continued T cell dysfunction - May also target immunosuppressive TAMs - Potential for ADCC and elimination of PD-L1+ positive that express PD-L1 in the TME immune cells subsets (NK cells, DCs, anti-tumor TAM) - Kinetics of PD-L1 occupancy are not yet defined and complete saturation to block PD-1 ligation may not be possible

Table 3. On-going Immunotherapy Trials in Renal Cell Carcinoma

Therapy Number Phase Trial ID Estimated Completion Date

First Line Metastatic RCC Trials Pembrolizumab + Lenvatinib or Everolimus + Lenvatinib vs. Sunitinib (CLEAR) 1050 III NCT02811861 February 2021

Nivolumab + Ipilimumab Followed by Nivolumab +/- Cabozantinib (PDIGREE) 1046 III NCT03793166 September 2021

Nivolumab + Ipilimumab +/- Cabozantinib (COSMIC-313) 676 III NCT03937219 November 2021

Nivolumab + Cabozantinb vs. Sunitinib 638 III NCT03141177 May 2024

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Perioperative (Neoadjuvant RCC Trials) Nivolumab – PROSPER RCC 805 III NCT03055013 November 2023

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Royal Marsden 19 Pilot NCT02446860 Late 2019

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Adjuvant RCC Immunotherapy Trials Durvalumab vs. Durvalumab/Tremelimumab vs. Observation 1750 III NCT03288532 December 2037

Pembrolizumab vs. Observation 950 III NCT03142334 December 2025

Nivolumab + Ipilimumab vs. Observation 800 III NCT03138512 July 2023

Atezolizumab vs. Observation 778 III NCT03024996 April 2024

Figure 1:

CD8 T cell

PD-1 Anti-PD-1 TAM PD-L2 Pembrolizumab DC Nivolumab Spartalizumab TCR PD-1

VEGF PepMHC PD-L1 B7.1/2 B7.1/2 Pep Anti-PD-L1 MHC Avelumab CD28 CTLA4 Atezolizumab TCR Durvalumab

Anti-CTLA4 Tumor lpilimumab CD4 cell VEGF Tremelimumab VEGF TKIs Axitinib Cabozantinib Sunitinib Bevacizumab Pazopanib Tavozanib Sitravitanib

Figure 2: A Prevents PD-L1 interaction B Prevents PD-1 interaction C ADCC on tumor cell D ADCC on immune cell PD-1 Blockade PD-L1 blockade

Direct T-cell engagement Direct tumor engagement Direct tumor engagement Direct T-cell engagement

T cell = Anti-PD-1 T cell = Anti-PD-L1 = Anti-PD-L1 = Anti-PD-L1

TAM TAM PD-1 B7.1 Fc (CD80) PD-1 PD-1 receptor Fc TCR PD-1 PD-L2 PD-L2 TCR receptor pep pep pep MHC TCR PD-L1 DC MHC PD-L1 DC Lytic enzymes MHC Peforin Lytic enzymes PD-L1 Granzymes Peforin TNF Granzymes T cell TNF Tumor Tumor Tumor cell cell cell

• Allows metabolic and • Blocks interaction with PD-1 on • Anti-PD-L1 binds to tumor • Anti-PD-L1 binds to immune epigenetic reprogramming immunocytes and B7.1 (CD80) cells expressing PD-L1 cells expressing PD-L1 of T cells • Does not interact directly with T • Tumor cell killing via Fc-receptor • Potential for immune cell • Blocks interaction between cells mediated antibody-dependent killing and depletion of either PD-1 and PD-L1/PD-L2 cellular cytotoxicity effector T cells or immunosuppressive cells (M2- like TAMs, MDSCs)

Targeting PD-1 or PD-L1 in Metastatic Kidney Cancer - Combination Therapy in the First Line Setting

David H Aggen, Charles G. Drake and Brian I. Rini

Clin Cancer Res Published OnlineFirst January 16, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-19-3323

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2020/01/16/1078-0432.CCR-19-3323.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

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