Chinese Journal of Cancer
`
`Immune checkpoints in cancer clinical trials
`
`Elad Sharon‘, Howard Streicher‘, Priscila Goncalves2 and Helen X. Chen1
`
`Abstract
`
`Immunology-based therapy is rapidly developing into an effective treatment option for a surprising
`
`range of cancers. We have learned over the last decade that powerful immunologic effector cells may
`
`be blocked by inhibitory regulatory pathways controlled by specific molecules often called "immune
`
`checkpoints." These checkpoints serve to control or turn off the immune response when it is no longer
`
`needed to prevent tissue injury and autoimmunity. Cancer cells have learned or evolved to use these
`
`mechanisms to evade immune control and elimination. The development of a new therapeutic class
`
`of drugs that inhibit these inhibitory pathways has recently emerged as a potent strategy in oncology.
`
`Three sets of agents have emerged in clinical trials exploiting this strategy. These agents are antibody-
`
`based therapies targeting cytotoxic T-lymphocyte antigen 4 (CTLA4), programmed cell death 1 (PD-1),
`
`and programmed cell death ligand 1 (PD-L1). These inhibitors of immune inhibition have demonstrated
`
`extensive activity as single agents and in combinations. Clinical responses have been seen in melanoma,
`
`renal cell carcinoma, non—small cell lung cancer, and several other tumor types. Despite the autoimmune
`
`or inflammatory immune-mediated adverse effects which have been seen, the responses and overall
`
`survival benefits exhibited thus far warrant further clinical development.
`
`Key words Immune checkpoints, PD-1, PD—L1, CTLA4, immunotherapy, immune modulators
`
`Immune checkpoints refer to regulatory pathways in the
`immunome that inhibit a portion of an active immune response
`against a specific target or set of targetsm. Immune checkpoints
`are therefore seen as a normal part of the immune system’s
`regulatory cascade and necessary to modulate and maintain immune
`homeostasis. These regulatory pathways are redundant, multi-
`faceted, and complex (Figure 1). Their presence and evolutionary
`development are thought to reflect the fact that a normal immune
`response can also be potentially deadly to a host if not properly
`regulated, as evidenced by autoimmune disease.
`As immunotherapy has gained a foothold in the anticancer
`armamentarium, there is a commonly held belief among
`oncologists that cancers thwart these regulatory mechanisms to
`evade immune detection and continue their growth and spread.
`However, the whole picture is more complex.
`In many chronic
`infections, immune checkpoints are exploited by parasitic and viral
`
`Authors' Affiliations: ‘Cancer Therapy Evaluation Program, Division of
`Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda,
`MD 20892, USA; 2Medical Oncology Service, Center for Cancer
`Research, National Cancer Institute, Bethesda, MD 20892, USA.
`Corresponding Author: Elad Sharon, 9609 Medical Center Drive,
`5W502, Bethesda, MD 20892, USA. Tel: +1-240-276-6565; Fax: +1-
`240-276-7894; Email: sharone@mail.nih.gov.
`dol: 10.5732/cjc.014.10122
`
`pathogens as evidenced by T-cell exhaustion in these conditions“.
`Cancer, however, arises from within the host. While some immune
`checkpoints are engaged to avoid immune-mediated eradication,
`others are merely a part of the normal immune response to stimuli.
`We are only beginning to understand these pathways.
`As a therapeutic class, drugs that inhibit these co-inhibitory
`signaling pathways, also known as immune checkpoint inhibitors,
`have emerged as a mainstay in melanoma therapy, with potential
`roles in the treatment of renal cell carcinoma (RCC), non—small cell
`lung cancer (NSCLC), urothelial cancer, head and neck cancer,
`ovarian cancer, and various lymphomasm”. In fact, ClinicalTrials.
`gov now lists dozens of trials with immune checkpoint inhibitors in a
`broad range of indications. As the targets of therapy are components
`of the immune system, there is currently no theoretical reason
`to exclude any potential histologies or tumor types from clinical
`evaluation. However, from a drug development perspective, this can
`be a challenge, as the resources are constantly constrained. It will
`be important to optimize the development of strategies to efficiently
`explore the therapeutic potentials.
`While a variety of agents could be deemed to interact with
`immune checkpoint inhibitors, the focus of this review are limited
`to the most advanced agents in clinical trials, those targeting the
`cytotoxic T-lymphocyte antigen 4 (CTLA4), programmed cell death 1
`
`434 www.cjcsysu.com
`
`CA CA Chinese Anti-Cancer Association
`Genome Ex. 1016
`
`Page 1 ofll
`
`Genome Ex. 1016
`Page 1 of 11
`
`

`

`Elad Sharon et al.
`
`Immune checkpoints in cancer clinical trials
`
`(PD-1), and programmed cell death ligand 1 (PD-L1) pathway. The
`early trials involving CTLA4- and PD-1lPD—L1—targeting agents have
`shown how the addition of just one blocking antibody can reveal
`a hidden immune response, with potentially massive therapeutic
`benefit for patients. Immune modulators targeting other mechanisms
`(Table 1). such as indoleamine 2,3-dioxigenase (IDO)“2'13],
`
`lymphocyte-activation gene 3 (LAG3)“‘“, T-cell membrane protein 3
`(TIM3)M, signal transducer and activator of transcription 3 (STAT3)“6],
`glucocorticoid-induced tumor necrosis factor receptor (TNFR) family-
`related protein (GITR)“7], and agents against the human inhibitory
`killer IgG-like receptor (anti-KIR) targeting natural killer (NK) cells
`have also entered the clinical trials arenanml.
`
`Lu mph node.-
`
`Peripheral ti55 Lie
`
`
`
`TL'R Mlli'
`II-
`
`PL]
`
`1
`
`I‘L'I-LI
`
`.! I1| i -F‘[}-'|
`
`L arl'l'J’IJ-L'I
`
`Jammie
`I
`
`NK cell.
`
`.Inli (Tin-1
`
`Figure 1. Regulatory pathways In Immune-oncology. The immune system has multiple levels of co-stimulatory (shown
`in green) and co-inhibitory (shown in red) pathways, helping to maintain immune homeostasis in the midst of responding
`to antigenic stimulation. Antibodies blocking cytotoxic T-lymphocyte antigen 4 (CTLA4), programmed cell death 1 (PD-1),
`and programmed cell death ligand 1 (PD-L1) have shown remarkable clinical activity and further development is underway.
`Agonist antibodies, which directly interact with co-stimulatory pathways such as 41 BB and CD40, are also in clinical
`development. Other means of affecting the immune responses are being explored, including direct actionon regulatory T (Treg)
`cells and natural killer (NK) cells. MHC, major histocompatibility complex; TCR, T-cell receptor.
`
`Table 1. Other immunotherapeutic agents in development
`
`Target
`
`Name
`
`lndication(s)
`
`Company
`
`Phase
`
`Clinical Trials.gov identifier (selected trials)
`
`Heme, hematologic tumors; ATL, acute T-cell leukemia; CTCL, cutaneous T-cell lymphoma; AML, acute myeloid leukemia.
`
`87.1
`B?H3
`LAGS
`
`CD137
`
`KIR
`
`CCR4
`CDZ7
`0x40
`
`CD40
`
`Galiximab
`MGA271
`IMP321
`EMS-986016
`BMS-663513
`PF—05082566
`IPH2101
`
`KW-0761
`CDX—1127
`MEDl-6469
`
`CP-870,893
`
`Lymphoma
`Solid tumors
`Solid tumors
`Solid tumors
`Solid tumors
`Lymphoma
`Myeloma, AML
`
`Biogen Idec
`Macrogenics
`lmmuntep
`Bristol-Myers Squibb
`Bristol-Myers Squibb
`Pfizer
`Innate Pharma (BMS)
`
`Kyowa Kirin
`ATL, CTCL
`Solid tumors & Heme CellDex Therapeutics
`Solid tumors
`Medlmmune/AZ
`
`Pancreatic
`
`Genentech
`
`Phase II
`Phase I
`Phase II"
`Phase I
`Phase II”
`Phase I
`Phase II
`
`Phase l/ll
`Phase I
`Phase I
`
`Phase I
`
`NCT00516217
`NCT01391143
`NCT00349934
`NCT01968109
`NCT00309023
`NCT01307267
`NCT01248455
`NCT01256073
`
`NCT00920790
`NCT01460134
`NCT02205333
`
`NCT01456585
`
`www.cjcsysu.com
`
`Chin J Cancer; 2014; Vol. 33 Issue 9 435
`Genome Ex. 1016
`
`Page 2 of 11
`
`Genome Ex. 1016
`Page 2 of 11
`
`

`

`Elad Sharon et al.
`
`Immune checkpoints in cancer clinical trials
`
`CTLA4 Inhibition and Human Cancers
`
`CTLA4 background
`
`CTLA4 is one of several co-inhibitory molecules that aid in
`modifying the T-cell response to antigen activation. The primary role
`of CTLA4 is to modify T-lymphocyte response to stimuli. CTLA4
`regulates the clonal burst size during priming and secondary
`expansion, which is thought to be proportional to the activation
`strength of the complex formed by the T-cell receptor—major
`histocompatibility complex (TCR—MHC complex), both in terms of
`binding affinity and co-stimulation by accessory signals. CTLA4,
`also known as cluster of differentiation (CD) 152, is an inhibitory
`molecule found on T cells, and its counterpart is CD28. CD28 is a co-
`stimulatory signal, but it is important to note that CTLA4 has higher
`avidity for its ligands than CD28, which suggests a dominance of
`inhibitory signals in immune activation. The ligands for both CD28
`and CTLA4 are known as B7 proteins, which are found on antigen-
`presenting cells (APCs). There are two types of B7 proteins: B7-1 (also
`known as CD80) and B7-2 (also known as CD86)[21'22].
`In normal circumstances, T lymphocytes are thought to express
`CTLA4 on their surface immediately upon response to antigenic
`stimulation of TCR. Because a blocking antibody would attenuate
`the inhibitory signal, an anti-CTLA4 antibody would appear to have a
`clear role in enhancing antitumor immunity, as the interaction of B7
`with CD28 would thereby be enhanced. Indeed, in preclinical models,
`this is exactly what was seenlz’]. However, B7 is rarely present in the
`tumor microenvironment, leading to a second hypothesis for how anti-
`CTLA4 antibodies may enhance immune-mediated tumor rejectionm'.
`CTLA4 is differentially expressed among separate T-Iymphocyte
`subsets. Indeed, higher levels of surface expression of CTLA4 are
`seen in regulatory T cells (Treg cells) as compared with effector
`T cells (Teff cells). Researchers have established that higher Teffl
`Treg ratios in both CD4—positive and CD8-positive tumor-infiltrating
`lymphocytes are associated with enhanced tumor eradication when
`CTLA4 blockade is employed in tumor modelslz‘m.
`Other mechanisms of action involving Treg cells have been
`hypothesized. For example, Treg cells are known to reduce B7
`expression on both human and animal APCSW. While B7 is thought
`to directly signal CTLA4, leading to its co-inhibitory effect, the reverse,
`or CTLA4 signaling to APCs via B7, also occurs. CTLA4 reverse
`signaling via B7 can lead to increased expression of IDO. IDO leads
`to a reduced level of local tryptophan stores in the microenvironment,
`which inhibits T-lymphocyte activation and proliferationm. Lastly,
`recently discovered alternatively spliced mRNA molecules encode
`soluble CTLA4 molecules lacking transmembrane domains. These
`soluble inhibitors could also mediate immune suppression, either by
`down-regulating B7 expression as noted above or by blocking the
`potential interaction of B7 and the co-stimulatory molecule CD28”.
`
`lpilimumab
`
`The first anti-CTLA4 agent in clinical development, ipilimumab,
`was approved by the United States Food and Drug Administration
`
`(FDA) and the European Medicines Agency for the treatment
`of metastatic melanoma following research showing improved
`survivalm]. lpilimumab is a fully humanized immunoglobulin (lg) G1
`kappa monoclonal antibody that antagonizes CTLA4 and prevents
`ligand binding”.
`A phase III combination study of ipilimumab at 3 mglkg with a
`glycoprotein 100 (gp100) peptide vaccine was conducted with 676
`patients with stage III or IV unresectable melanoma, randomized in
`a 3:1 :1 ratio. The results showed the median overall survival (OS) in
`the ipilimumab plus gp100 group was 10.0 months [95% confidence
`interval (CI), 8.5 to 11.5 months], as compared with 6.4 months
`(95% CI, 5.5 to 8.7 months) in the gp100 alone group [hazard ratio
`(HR) for death, 0.68; P < 0.001]. The median OS in the ipilimumab
`alone group was 10.1 months (95% CI, 8.0 to 13.8 months; HR for
`death with ipilimumab alone as compared with gp100 alone, 0.66;
`P = 0.003). Grade 3 or 4 immune-related adverse events occurred
`in 10% to 15% of patients treated with ipilimumab and in 3% of
`patients treated with gp100 alone. The investigators concluded that
`ipilimumab, with or without a gp100 peptide vaccine, as compared
`with gp100 alone, improved OS in patients with previously treated
`metastatic melanoma. Adverse events can be severe, long-lasting, or
`both, but most are reversible with appropriate treatment, particularly
`corticosteroids”.
`
`Another supportive study in metastatic melanoma involved 502
`patients in a 1:1 randomized trial of ipilimumab at a dose of 10 mglkg
`plus dacarbazine at a dose of 850 mg/m2 of body surface area versus
`dacarbazine and placebo. The OS was longer in the group receiving
`ipilimumab-dacarbazine therapy than in the dacarbazine-placebo
`arm (11.2 months vs. 9.1 months). Survival rates were also higher
`for the ipilimumab-dacarbazine arm at 1 year (47.3% vs. 36.3%),
`2 years (28.5% vs. 17.9%), and 3 years (20.8% vs. 12.2%), with
`an HR for death at 0.72 (P < 0.001). Grade 3 or 4 adverse events
`were recorded in 56.3% of patients in the experimental arm and only
`27.5% in the control arm“. These findings further support the use of
`ipilimumab in metastatic melanoma.
`The preliminary results of the first adjuvant trial were reported
`at the 2014 Annual Meeting of the American Society of Clinical
`Oncology (ASCO). The European Organization for Research and
`Treatment of Cancer (EORTC) conducted a trial with 951 patients
`with stage III melanoma following complete resection (EORTC 18071,
`NCT00636168). Patients were randomized to receive ipilimumab at
`a dose of 10 mglkg versus placebo every 3 weeks for 4 doses. The
`median relapse-free survival was 26.1 months for the ipilimumab arm
`and 17.1 months for the placebo arm, with an HR of 0.75 (95% CI,
`0.64 to 0.90; P = 0.001). The adverse event profile was thought to
`be generally consistent with that seen in advanced melanoma, but a
`higher incidence of endocrinopathies were reported“.
`While the results were largely seen as positive, there are still
`unresolved issues that may limit the ability of regulatory agencies
`to approve ipilimumab in the adjuvant setting. In particular, patients
`with stage III melanoma have the option of high-dose interferon,
`which is the standard of care in the United States“. In addition, as
`noted above, the dose chosen in EORTC 18071 trial was 10 mglkg
`of ipilimumab, higher than the dose used in the metastatic setting.
`
`436 Chin J Cancer; 2014; Vol. 33 Issue 9
`
`Chinese Journal of Cancer
`
`Genome Ex. 1016
`
`Page 3 of 11
`
`Genome Ex. 1016
`Page 3 of 11
`
`

`

`Elad Sharon et al.
`
`Immune checkpoints in cancer clinical trials
`
`Another large adjuvant trial, E1609 (NCT01274338), addresses the
`questions of the optimal dose of ipilumumab and its comparative
`effectiveness to high-dose interferon.
`In this trial, conducted by the
`Eastern Cooperative Oncology Group (ECOG) under the sponsorship
`of the United States National Cancer Institute (NCI), 1,500 patients
`with stage III or IV melanoma that has been fully resected are
`randomized to three arms: high-dose interferon, ipilimumab at 3 mg/
`kg, and ipilimumab at 10 mglkgm]. The trial is close to completing
`accrual and the outcome data are eagerly awaited.
`Other indications with ipilimumab are still under clinical
`development. On ClinicalTrials.Gov, a search with the term
`“ipilimumab" returns 191 trials, over 100 of which remain open. Much
`of the attention in ipilimumab clinical development has moved to
`combination therapy, particularly with the anti-PD-1 agent nivolumab.
`The development of ipilumumab brought up interesting ques-
`tions about the best endpoints of efficacy assessment for this class
`of agents. In the phase III melanoma trials with ipilimumab described
`above, improvements in OS seen in the metastatic setting were not
`accompanied by significant radiographic responses to therapy. This
`has complicated efficient drug development, as response rate is
`less likely to serve as a readout of efficacy, at least when ipilimumab
`is used as a monotherapy. Monotherapy is still being explored in
`various settings, however.
`In one example, in a single institution
`case series, 5 patients with recurrent glioblastoma multiforme (GBM)
`who were treated with ipilimumab were followed. Patients had time
`to progression ranging from 1 to 6 months, and 1 patient remains
`recurrence-free 19 months after therapy, suggesting penetration of
`this drug across the blood-brain barrierm].
`Currently, there are no biomarkers to predict which patients or
`tumor types are more likely to respond to anti-CTLA4 therapies.
`Some analysis suggested that a high mutational load may be
`associated with a clinical benefit from ipilimumab.
`In particular,
`one group of researchers has focused on identifying a “neoantigen
`signature” that correlates with benefit. Findings are preliminary,
`but this suggests that tumor genetics might explain the divergent
`outcomes among patients treated with ipilimumablas'.
`
`Tremelimumab
`
`Tremelimumab (formerly CP-675,206) is a human lgG2
`monoclonal antibody specific for CTLA4. In a large, single-arm phase
`II trial, 241 response-evaluable patients with advanced refractory or
`relapsed melanoma were treated at a dose of 15 mglkg intravenously
`every 3 months. Objective responses were observed in 6.6% of those
`patients, which led to a phase III trial in a similar patient population“.
`In the phase III trial in advanced melanoma, 655 patients were
`enrolled and randomly assigned to treatment with tremelimumab or
`chemotherapy. Median OS by intent to treat was 12.6 months (95%
`CI, 10.8 to 14.3 months) for tremelimumab and 10.7 months (95%
`Cl, 9.36 to 11.96 months) for chemotherapy (HR, 0.88; P = 0.127).
`Of note, while objective response rates were similar in the two arms
`(10.7% in the tremelimumab arm and 9.8% in the chemotherapy
`arm), duration of response was significantly longer among patients
`treated with tremelimumab (35.8 months vs. 13.7 months, P =
`
`www.cjcsysu.com
`
`0.0011)“ Regardless, the study failed to demonstrate a statistically
`significant survival advantage of treatment with the investigational
`drug over standard-of-care chemotherapy.
`While the results were disappointing, there was a possibility that
`the dosing schedule of tremelimumab (once every 3 months) was
`insufficient to achieve results similar to that of ipilimumab. As a result,
`ongoing efforts with tremelimumab as a monotherapy have used more
`frequent dosing. The preferred dose and schedule for tremelimumab
`for ongoing trials has generally been 10 mglkg intravenously, every
`4 weeks for 6 months. A phase II, single-ann study of tremelimumab
`as a second-line treatment was conducted with 29 patients with
`malignant mesothelioma. Results showed a partial response in 4
`patients (13.8%) and stable disease in 11 patients (37.9%), with OS
`of 11.5 months“. As a result, a phase III, double-blinded, randomized
`trial is currently undenlvay using tremelimumab in mesotheliomam].
`Tremelimumab is also being tested in various combinations in a
`variety of other tumor types.
`
`PD-1lPD-L1 Inhibition and Human
`Cancers
`
`PD-l/PD-Ll background
`
`Compared to the limited development with CTLA4, there is
`an immense level of interest and investment by pharmaceutical
`companies in the PD-1/PD-L1 pathway (Table 2). Seven major
`companies have entered the fray, with multiple phase III registration
`trials already underway in a variety of malignancies, most notably
`melanoma, RCC, and NSCLC (Table 3). This enthusiasm is justified,
`as clinical trial results thus far confirm that targeting the PD-1/PD-
`L1 pathway represents the most exciting opportunity for advancing
`cancer immunotherapy to date.
`Whereas CTLA4 is involved in central tolerance and control, the
`PD-1 receptor is critical in peripheral tolerance. PD-1 is expressed on
`T lymphocytes during thymic development. Similar to CTLA4, PD-1
`becomes expressed on CD4— and CD8—positive T lymphocytes during
`antigenic stimulation, serving as a co-inhibitory signal. In addition,
`PD-1 is expressed on numerous other immune cells, including
`natural killer T cells, B cells, monocytes, and certain dendritic cell
`subsets“°"‘”. PD-1 has two main ligands, namely PD-L1 (also known
`as B7-H1) and PD-L2 (also known as B7-DC). While PD-L2 has a
`much higher affinity for PD-1,
`it is expressed chiefly on activated
`dendritic cells, macrophages, certain B-cell subsets, and other
`immune cells. PD-L1 is more widely expressed on hematologic and
`non-hematologic tissuesm.
`As a co-inhibitory signal, PD-1 engagement results in reduced
`cytokine production, cytolytic activity, and lymphocyte proliferationl‘”.
`PD-1 is up—regulated in T lymphocytes following viral infections and
`down-regulated following viral clearance. In chronic viral infections,
`however, CD8-positive T lymphocytes express PD-1 constitutively,
`likely through gene demethylation, contributing to what has been
`termed a T-cell exhaustion phenotype. In human immunodeficiency
`virus (HIV) infection, for example, high levels of PD-1 expression
`allow for persistent interaction between PD-L1 expressed by APCs
`
`Chin J Cancer; 2014; Vol. 33 Issue 9 437
`Genome Ex. 1016
`
`Page 4 of 11
`
`Genome Ex. 1016
`Page 4 of 11
`
`

`

`Elad Sharon et al.
`
`Immune checkpoints in cancer clinical trials
`
`Table 2. Agents targeting PD-1/PD-L1 in clinical development
`
`Company
`
`Agent targeting PD-1
`
`Agent targeting PD-L1
`
`CT-01 1
`
`Pidilizumab (humanized lgG1 mAb)
`
`Medlmmune/AZ
`
`AMP-514
`
`MEDI4736 (fully human mAb)
`
`EMD Serono
`
`N/A
`
`MSBDO107180
`
`PD-1, programmed death 1 receptor; PD-L1, programmed cell death ligand 1;
`available.
`
`lgG4, immunoglobulin G4; mAb, monoclonal antibody; N/A, not
`
`NCT02008227
`
`Table 3. Selected ongoing PD-1/PD-L1 trials in melanoma, renal cell cancer (RCC), and non—small cell lung cancer
`(NSCLC)
`
`Sponsor
`
`Setting
`
`Comparison
`
`Primary
`endpoint
`
`Clinical trials.
`Sample Primary
`size
`completion date gov identifier
`
`Bristol-Myers Squibb NSCLC
`
`3rd-line squamous cell
`
`||
`
`Nivolumab vs. docetaxel
`
`OS
`
`264
`
`Jan 2016
`
`NCT01642004
`
`Merck
`
`Melanoma
`
`ist-/2nd-Iine (ipi-nai've)
`
`III
`
`Bristol-Myers Squibb Melanoma 2nd-Iine post-ipi
`
`Bristol-Myers Squibb Melanoma 1st-/2nd-line
`
`Genentech
`
`NSCLC
`
`2nd-line
`
`Genentech
`
`NSCLC
`
`2nd-Iine
`
`III
`
`|||
`
`II
`
`III
`
`Pembrolizumab vs.
`ipilimumab
`
`Nivolumab vs.
`chemotherapy
`
`Nivolumab vs.
`chemotherapy
`
`OS & PFS 645
`
`Feb 2015
`
`NCT01866319
`
`OS
`
`405
`
`May 2015
`
`NCT01721746
`
`OS
`
`410
`
`Sept 2015
`
`NCT01721772
`
`MPDL3280A vs. docetaxel OS
`
`287
`
`Mar 2016
`
`NCT01903993
`
`MPDL3280A vs. docetaxel 03
`
`850
`
`Jun 2017
`
`ORR, overall response rate; OS, overall survival; PFS, progression-free survival; ipi, ipilumumab. Other footnotes as in Table 2.
`
`and subsequent T-cell dysfunction. T-lymphocyte activation is
`essentially blocked in this setting. In an experiment using B cells from
`HIV-infected individuals, researchers were able to show increased
`responses to HIV antigens in the presence of PD-1 blocking
`antibodies in vitro‘m. This suggests that an active immune response
`can be induced if effective blockade of the PD-1IPD-L1 pathway
`can be implemented. A clinical trial sponsored by the US National
`Institutes of Health is currently planned to investigate that possibility
`
`using the anti-PD-L1 antibody, BMS—936559 (NCT02028403).
`Similarly, tumors use the same pathway, meant to induce
`peripheral immune tolerance, to evade T-lymphocyte-mediated
`immune eradication. Tumors evading an active immune response
`are thought to express PD-L1 following T lymphocyte infiltration
`and expression of interferon-gamma. Expression of PD-L1 has
`been associated with a poor prognosis in a wide variety of human
`tumorsm'm”. Interestingly, in one analysis of glioma cases, a better
`
`438 Chin J Cancer; 2014; Vol. 33 Issue 9
`
`Chinese Journal of Cancer
`
`Genome Ex. 1016
`
`Page 5 ofll
`
`Genome Ex. 1016
`Page 5 of 11
`
`

`

`Elad Sharon et al.
`
`Immune checkpoints in cancer clinical trials
`
`prognosis was seen in patients whose tumor-adjacent brain tissue
`expressed PD-L1 and whose tumors did not express PD-L1. This
`suggests a general role for PD-L1 in protecting normal tissue from
`immune attack, though it would be important to see these findings
`replicated elsewhere and in other tumor typeslsz].
`In general, the results of PD-1/PD-L1 blockade have been
`encouraging. Higher response rates and durable responses were
`seen with PD-1/PD-L1 blocking antibodies than with CTLA4 blockade
`in melanoma, RCC, and unexpectedly, NSCLC. In addition, although
`similar immune-mediated adverse events were seen with both CTLA4
`
`and PD-1/PD-L1 agents, the severity of adverse events has generally
`been lower, with the caveat that fatal pneumonitis was seen in 1% of
`the patients on the nivolumab phase I trial described below“.
`
`PD-l—targeting agents
`
`Nivolumab
`
`Nivolumab (BMS-936558) is a fully human IgG4 monoclonal
`antibody targeting PD-1. A phase II” study was reported in 2012
`in the New England Journal of Medicine in parallel with a plenary
`session at the 2012 ASCO Annual Meeting“. Patients on the trial had
`advanced melanoma, NSCLC, castration-resistant prostate cancer,
`RCC, or colorectal cancer (CRC). Patients received nivolumab at
`doses of 0.1 to 10.0 mglkg of body weight every 2 weeks for up to 12
`cycles until disease progression or a complete response occurred.
`Of the 296 patients enrolled, only 14% experienced grade 3 or 4
`drug-related adverse events, but there were 3 deaths from pulmonary
`toxicity. No maximum tolerated dose (MTD) was defined. Cumulative
`response rates (all doses) were 18.4% (14/76) among patients with
`NSCLC. 27.6% (26/94) among patients with melanoma, and 27.3%
`(9/33) among patients with RCCW. A subsequent analysis of the
`104 patients in that trial with metastatic melanoma was performed.
`The investigators reported that the median OS in nivolumab-treated
`metastatic melanoma patients was 16.8 months, and 1- and 2-year
`survival rates were 62% and 43%, respectively. Among the 33
`patients (30.8%) with objective tumor regressions, the Kaplan-Meier
`estimated median response duration was 2 years. Of note, responses
`were not observed in prostate cancer and colon cancer patients on
`the trialls‘".
`
`While preliminary results from the phase II” trial suggested
`that PD-L1 could be an appropriate biomarker for patient selection,
`subsequent analyses have shown numerous PD-L1—negative
`patients had responded to treatment with nivolumab, although lower
`rates are seen. Given the variability and limitation of the available
`PD-L1 assays, a true absence of PD-L1 expression in those patients
`cannot be confirmed. At this time it remains to be determined whether
`
`PD-L1 is a predictive marker of response for PD-1 pathway inhibitors.
`At the 2014 ASCO Annual Meeting, preliminary results of other
`nivolumab clinical trials were reported. In a small dose-escalation trial
`in Japan, 3 of 13 patients with platinum-resistant ovarian cancer had
`objective responses to nivolumab. The initial 10 patients were treated
`at a dose of 1 mglkg, and 2 responders were from that cohort. The
`subsequent cohort that received 3 mglkg had only 3 patients, but 1
`was a responder. These results, though preliminary, were promisingm.
`
`www.cjcsysu.com
`
`In addition, investigators reported early results using nivolumab at
`a dose of 3 mglkg every 2 weeks in patients with treatment-naive
`advanced NSCLC. The initial results on 20 patients revealed an
`objective response rate of 30%. Two patients had a greater than
`80% target lesion reduction at 18 weeks. Of the 15 evaluable tumor
`samples, 9 were PD-L1—positive (defined as greater than 5% PD-
`L1 expression using a Dako kit), and the response rate was 67% in
`PD-L1—positive patients; whereas no responses were observed in
`the 6 PD-L1—negative patientsl55‘. Combinations of nivolumab with
`conventional chemotherapy or epidermal growth factor receptor
`(EGFR) inhibitors were also reported. but results are difficult to
`interpret in the absence of randomized comparisonslfi'sn.
`
`Pembrolizumab
`
`Pembrolizumab (MK-3475, formerly lembrolizumab) is a
`humanized IgG4 monoclonal antibody targeting PD-1. Pembro-
`lizumab has been very successful in treating melanoma and NSCLC,
`similar to nivolumab. Significant differences cannot be assessed
`in the absence of a randomized trial comparing the two agents.
`However, binding affinities of the agents are different. Nivolumab is a
`fully human IgG4, and pembrolizumab is humanized. In phase I trials,
`neither agent has been found to have a maximally tolerated dose.
`That said, more time and energy has been spent on searching for an
`appropriate dose for pembrolizumab.
`In the first major publication involving pembrolizumab, Hamid et
`al.” reported that patients with advanced melanoma were analyzed
`after being treated with three separate dosing strategies: 10 mg/
`kg of body weight every 2 or 3 weeks or 2 mglkg every 3 weeks.
`Ultimately, 135 patients with advanced melanoma were treated.
`Adverse events were similar to those found in patients treated with
`nivolumab, including fatigue, rash, pruritus, and diarrhea. Response
`rates across all dose levels were 38% (95% Cl, 25% to 44%).
`Investigators found no difference among those with and without
`prior ipilimumab therapy. Responses were durable, and the median
`progression-free survival (PFS) among the 135 patients was longer
`than 7 months“.
`
`A subsequent prospective, randomized analysis was performed
`using both the 2 mglkg and the 10 mglkg doses, given every 3 weeks
`to patients with ipilimumab-refractory advanced melanoma. There
`were 89 patients in the 2 mglkg cohort and 84 patients in the 10
`mglkg cohort. The response rate was 26% at both doses. Safety
`profiles were similar and there were no deaths reported”. Other
`attempts to analyze patients with melanoma have been reported,
`including an analysis of 411 melanoma patients treated across
`multiple dose levels and multiple trials, which was reported at the
`2014 ASCO Annual Meeting. Median OS data was not available, but
`1-year OS rate over all dose cohorts was 71%. Response rates were
`encouraging as well, ranging from 26% to 57%, varying based on
`ipilimumab prior treatment, dose, and schedule”.
`Investigators also reported preliminary results of a phase I trial
`of previously treated patients with locally advanced or metastatic
`NSCLC. Enrolled patients with PD-L1 detected in their tumors
`by a preliminary immunohistochemical assay were randomized
`to pembrolizumab at a dose of 10 mglkg every 2 weeks or every
`
`Chin J Cancer; 2014; Vol. 33 Issue 9 439
`Genome Ex. 1016
`
`Page 6 ofll
`
`Genome Ex. 1016
`Page 6 of 11
`
`

`

`Elad Sharon et al.
`
`Immune checkpoints in cancer clinical trials
`
`3 weeks. In addition, some patients with tumors without PD-L1
`expression who had received at least two prior lines of therapy were
`treated at a dose of 10 mglkg every 2 weeks. Ultimately, 102 patients
`comprised the every-2-week cohort (including 43 whose tumors did
`not express PD-L1), and 119 patients comprised the every-3-week
`cohort. Investigators reported that 48% of patients experienced drug-
`related adverse events, with 6% experiencing grade 3/4 adverse
`events. As in the nivolumab trial reported above, pneumonitis was
`a concern, with 3 cases of drug-related grade 3/4 pneumonitis. The
`Response Evaluation Criteria in Solid Tumors (RECIST) response
`rate in all patients was 21%. The response rate was slightly higher
`(24%) for patients with PD-L1—positive tumors (more specifically.
`31% on the every-2-week cohort and 22% on the every-3-week
`cohort). RECIST response rates were 8% for patients without PD-L1
`expression“.
`
`Pidilizumab
`
`Pidilizumab is a humanized lgG1 antibody targeting PD-1. The
`agent was initially evaluated in a phase I trial targeting hematologic
`malignancies. Presently, there are a number of clinical trials
`undenivay in both hematologic and solid tumors”.
`The results of two pidilizumab clinical trials were recently
`published in peer-reviewed journals. In a single-center, single-arm,
`phase II trial, 32 patients with relapsed follicular lymphoma received
`pidilizumab at a dose of 3 mglkg every 4 weeks for 4 infusions with
`up to 8 additional infusions administered. In addition, rituximab was
`given at a dose of 375 mg/m2 of body surface area every week for
`4 weeks. Investigators reported that 19 of 29 evaluable patients
`achieved an objective response, with complete responses in 15
`patients (51.7%)“.
`An additional phase II trial involved patients with diffuse large
`B-cell lymphoma (DLBCL) following autologous hematologic stem cell
`transplantation (AHSCT). Sixty-six patients were treated with 3 doses
`of pidilizumab in the first 1 to 3 months after AHSCT. The PFS rate
`was 72% at 6 months after AHSCT (90% CI, 60% to 82%), meeting
`the primary endpoint. Thirty-five patients had measurable disease
`following AHSCT, and the response rate in those patients was 51 %m.
`Investigators also presented results on the use of pidilizumab
`in metastatic melanoma at the 2014 ASCO Annual Meeting. In this
`trial, 103 patients were randomized in a 1:1 ratio to receive either
`1.5 mglkg or 6 mglkg every 2 weeks for 27 doses. Response rates
`were lower than those observed