V O L U M E
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`2 8
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`䡠 N U M B E R 1 9
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`J U L Y 1
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`2 0 1 0
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`JOURNAL OF CLINICAL ONCOLOGY
`
`O R I G I N A L R E P O R T
`
`From the Johns Hopkins University
`School of Medicine and the Sidney
`Kimmel Comprehensive Cancer Center,
`Baltimore, MD; Henry Ford Health
`Systems, Detroit, MI; Carolina BioOn-
`cology Institute, Huntersville, NC;
`Washington University School of Medi-
`cine Siteman Cancer Center, St Louis,
`MO; and Medarex, Bloomsbury, NJ,
`and Milpitas, CA.
`
`Submitted November 21, 2009;
`accepted March 12, 2010; published
`online ahead of print at www.jco.org on
`June 1, 2010.
`
`Supported in part by a grant from the
`Melanoma Research Alliance Founda-
`tion (L.C., D.M.P., S.L.T.).
`
`I.L. and S.L.T. contributed equally to
`this work.
`
`Presented in part at the 44th Annual
`Meeting of the American Society of
`Clinical Oncology (ASCO) May 30-June
`3, 2008, Chicago, IL, and the 45th
`Annual Meeting of ASCO, May 29-June
`2, 2009, Orlando, FL.
`
`Terms in blue are defined in the glos-
`sary, found at the end of this article
`and online at www.jco.org.
`
`Authors’ disclosures of potential con-
`flicts of interest and author contribu-
`tions are found at the end of this
`article.
`
`Clinical Trials repository link available on
`JCO.org.
`
`Corresponding author: Suzanne L.
`Topalian, MD, Department of Surgery,
`Johns Hopkins University School of
`Medicine, 1550 Orleans St, CRB 2,
`Room 508, Baltimore, MD 21231;
`e-mail: stopali1@jhmi.edu.
`
`© 2010 by American Society of Clinical
`Oncology
`
`0732-183X/10/2819-3167/$20.00
`
`DOI: 10.1200/JCO.2009.26.7609
`
`Phase I Study of Single-Agent Anti–Programmed Death-1
`(MDX-1106) in Refractory Solid Tumors: Safety, Clinical
`Activity, Pharmacodynamics, and Immunologic Correlates
`Julie R. Brahmer, Charles G. Drake, Ira Wollner, John D. Powderly, Joel Picus, William H. Sharfman,
`Elizabeth Stankevich, Alice Pons, Theresa M. Salay, Tracee L. McMiller, Marta M. Gilson, Changyu Wang,
`Mark Selby, Janis M. Taube, Robert Anders, Lieping Chen, Alan J. Korman, Drew M. Pardoll, Israel Lowy,
`and Suzanne L. Topalian
`
`A
`
`B
`
`S
`
`T
`
`R
`
`A
`
`C
`
`T
`
`Purpose
`Programmed death-1 (PD-1), an inhibitory receptor expressed on activated T cells, may suppress
`antitumor immunity. This phase I study sought to determine the safety and tolerability of anti–PD-1
`blockade in patients with treatment-refractory solid tumors and to preliminarily assess antitumor
`activity, pharmacodynamics, and immunologic correlates.
`Patients and Methods
`Thirty-nine patients with advanced metastatic melanoma, colorectal cancer (CRC), castrate-
`resistant prostate cancer, non–small-cell
`lung cancer (NSCLC), or renal cell carcinoma (RCC)
`received a single intravenous infusion of anti–PD-1 (MDX-1106) in dose-escalating six-patient
`cohorts at 0.3, 1, 3, or 10 mg/kg, followed by a 15-patient expansion cohort at 10 mg/kg. Patients
`with evidence of clinical benefit at 3 months were eligible for repeated therapy.
`
`Results
`Anti–PD-1 was well tolerated: one serious adverse event, inflammatory colitis, was observed in a
`patient with melanoma who received five doses at 1 mg/kg. One durable complete response
`(CRC) and two partial responses (PRs; melanoma, RCC) were seen. Two additional patients
`(melanoma, NSCLC) had significant lesional tumor regressions not meeting PR criteria. The serum
`half-life of anti–PD-1 was 12 to 20 days. However, pharmacodynamics indicated a sustained mean
`occupancy of ⬎ 70% of PD-1 molecules on circulating T cells ⱖ 2 months following infusion,
`regardless of dose. In nine patients examined, tumor cell surface B7-H1 expression appeared to
`correlate with the likelihood of response to treatment.
`
`Conclusion
`Blocking the PD-1 immune checkpoint with intermittent antibody dosing is well tolerated and associated
`with evidence of antitumor activity. Exploration of alternative dosing regimens and combinatorial
`therapies with vaccines, targeted therapies, and/or other checkpoint inhibitors is warranted.
`
`J Clin Oncol 28:3167-3175. © 2010 by American Society of Clinical Oncology
`
`INTRODUCTION
`
`Genetic and epigenetic aberrations occur com-
`monly in human tumors and produce altered anti-
`genic profiles that can be selectively recognized by
`the adaptive immune response.1 A dynamic inter-
`play exists between host and tumor, and the ability
`of the tumor to evade immune recognition often
`determines the clinical course of the disease.2 The
`successes of passive immunotherapies, such as
`monoclonal antibodies (mAbs) directed against
`tumor or vascular cell surface molecules or adop-
`tive transfer of tumor-specific T cells, validate the
`potential of immunotherapy to eradicate established
`
`metastatic cancers. However, active immunothera-
`peutic strategies designed to enhance endogenous
`antitumor responses, such as cancer vaccines, have
`been far less successful.
`Augmenting specific antitumor CD4⫹ and
`CD8⫹ T cell responses is a major goal of cancer
`immunotherapy. Important insights explaining the
`limitations of T cell– based cancer immunothera-
`pies have come from the discovery of inhibitory
`coreceptors and pathways termed immune check-
`points, which restrain T cell functions in normal
`physiologic settings and may be exploited by tu-
`mors.3 Preclinical cancer models demonstrate that
`inhibitory signals mediated by coreceptors on
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`tumor-specific T cells impede antitumor immunity and suggest that
`blockade of such interactions can release the brakes on immune re-
`sponsiveness leading to tumor elimination. The most extensively
`studied inhibitory T cell coreceptor, CTLA-4 (CD152), has been
`evaluated in patients with advanced cancers. As originally pre-
`dicted by murine models, anti–CTLA-4 therapy in humans resulted
`in objective tumor regressions including durable complete re-
`sponses (CRs) in some patients. However, as anticipated from the
`uncontrolled lymphoproliferation observed in CTLA-4 null mice,4
`anti–CTLA-4 therapy was associated with a significant frequency of
`serious immunologic adverse events (AEs).5 Thus, investigators have
`searched for new checkpoint blocking agents with more favorable
`therapeutic profiles.
`Programmed death-1 (PD-1, CD279) is an inhibitory coreceptor
`expressed on antigen-activated and exhausted T and B cells.6 It bears
`homology to CTLA-4 but provides distinct immune-inhibitory sig-
`nals. In contrast to early lethality in CTLA-4 knockout mice, PD-1
`knockouts demonstrate modest late-onset strain- and organ-specific
`autoimmunity.7,8 There are two known ligands for PD1: B7-H1/PD-L1
`(hereafter B7-H1), the predominant mediator of PD-1–dependent
`immunosuppression, and B7-DC/PD-L2. In murine tumor models,
`B7-H1 expression confers immune resistance, and interrupting PD-1:
`B7-H1 interactions has antitumor effects.9-11 B7-H1 is highly upregu-
`lated in many murine and human tumors (either in tumor cells or
`nontransformed cells in the tumor microenvironment such as
`antigen-presenting cells),12 and its expression is associated with poor
`outcome for patients with certain epithelial cancers.13,14 These find-
`ings have focused attention on PD-1:B7-H1 blockade as a strategy for
`cancer immunotherapy. On the basis of these considerations, we ini-
`tiated a phase I clinical trial of PD-1 blockade with the fully human
`mAb MDX-1106 in 39 patients with advanced treatment-refractory
`solid tumors. We report here the safety, antitumor activity, pharma-
`codynamics, and correlative in vitro results from this trial.
`
`PATIENTS AND METHODS
`
`MDX-1106
`MDX-1106 (BMS-936558/ONO-4538) is a genetically engineered, fully
`human immunoglobulin G4 (IgG4) mAb specific for human PD-1 (Appendix
`Fig A1A, online only). Mice transgenic for human Ig loci were immunized with
`Chinese hamster ovary cell PD-1 transfectants and a PD-1/human IgG1 Fc
`fusion protein. MDX-1106 contains an engineered hinge region mutation
`(S228P) designed to prevent exchange of IgG4 molecules; the IgG4 isotype
`minimizes cellular and complement-mediated cytolytic functions. MDX-1106
`⫽ 2.6 nmol/L by Scatchard analysis to
`binds PD-1 with high affinity (KD
`polyclonally activated human T cells), blocks its interactions with both B7-H1
`and B7-DC (Appendix Fig A1B), and enhances tumor antigen-specific T cell
`proliferation and secretion of cytokines in vitro.15
`
`Patients
`Eligible patients had treatment-refractory metastatic melanoma,
`castrate-resistant prostate cancer, renal cell carcinoma (RCC), non–small-cell
`lung cancer (NSCLC), or colorectal cancer (CRC), and had no cancer therapy
`for at least 4 weeks before enrollment. Patients were ⱖ 18 years old with a life
`expectancy of ⱖ 12 weeks, an Eastern Cooperative Oncology Group (ECOG)
`performance status of 0 or 1, adequate organ function, and no ongoing sys-
`temic infections or history of autoimmune disease. Concurrent antineoplastic
`therapies, systemic steroids, and prior treatment with anti–CTLA-4 were not
`permitted. Patients with treated stable brain metastases were eligible.
`
`Study Design and Procedures
`This multi-institutional, first in-human, open-label, phase I, dose-
`escalation study was approved by local institutional review boards. All partic-
`ipating patients signed informed consent. The primary objectives were to
`characterize the safety and tolerability of a single dose of MDX-1106 in patients
`with selected malignancies and to determine the maximum-tolerated dose
`(MTD) and pharmacokinetics. Secondary objectives included assessing anti-
`tumor activity, pharmacodynamics, and immunologic end points. Sequential
`cohorts of six patients received a 60-minute intravenous infusion of MDX-
`1106 at 0.3, 1, 3, or 10 mg/kg and were evaluated for toxicities on a weekly basis
`for 8 weeks. Dose-limiting toxicity (DLT) was defined as a treatment-related
`grade ⱖ 3 AE or laboratory abnormality occurring ⱕ 28 days postdose. The
`MTD was the highest dose at which no more than one of six patients experi-
`enced a DLT. Fifteen additional patients were planned to be enrolled at the
`MTD or the highest planned dose (10 mg/kg) to confirm safety.
`Patients were restaged radiographically (Response Evaluation Criteria in
`Solid Tumors [RECIST] 1.0) at 8 and 12 weeks. Patients with progressive
`disease were taken off study. Those with stable disease or evidence of lesional
`tumor regression, no AE grade ⱖ 3, and no evidence of human antihuman Ab
`at a 1:10 serum dilution received additional doses of MDX-1106 at weeks 12
`and 16 and were then observed for 3 months and restaged. Those with contin-
`ued clinical benefit could receive two more doses, spaced by 4 weeks. Each
`re-treatment phase was 16 weeks. Patients with objective partial responses
`(PRs) or CRs were observed, with optional re-treatment on progression.
`Responses are reported as of January 2010.
`
`Pharmacokinetics
`Serum samples were collected serially before and up to 85 days after the
`first dose of MDX-1106. MDX-1106 serum concentrations were determined
`with a quantitative enzyme-linked immunosorbent assay (ELISA) capable of
`detecting ⱖ 1.2 ␮g/mL, using 96-well plates coated with chimeric PD-1/
`human IgG1 Fc protein (R&D Systems, Minneapolis, MN).
`
`Tumor Biopsies
`Sections of formalin-fixed, paraffin-embedded tumor specimens ar-
`chived before protocol entry or core-needle or excisional biopsies obtained
`immediately pre- and post-therapy were subjected to hematoxylin and
`eosin staining and immunohistochemistry (IHC) to detect lymphoid infil-
`trates (anti-CD3, anti-CD4, and anti-CD8) and B7-H1 expression (murine
`antihuB7-H1, clone 5H1; previously described13). Pigmented melanoma sam-
`ples were bleached before staining and visualized with a red chromogen.
`Tumors were considered B7-H1–positive if ⱖ 5% of tumor cells showed
`membranous staining with 5H1.
`
`Immunologic Assessments
`Delayed-type hypersensitivity reactions to Candida albicans and tetanus
`toxoid were assessed along with viral antigen recall reactions (details are in-
`cluded in the Appendix, online only). Peripheral blood lymphocyte (PBL)
`phenotypes were also assessed. Serially collected blood was analyzed for the
`presence and activation status of various lymphocyte subsets, as detailed in
`the Appendix.
`
`PD-1 Receptor Occupancy (pharmacodynamics)
`MDX-1106 binding to PD-1 molecules on circulating CD3⫹ PBLs was
`investigated with flow cytometric analysis of serially collected blood samples
`(see PBL phenotyping schedule in the Appendix). Peripheral blood mononu-
`clear cells were preincubated (30 minutes at 4°C) with a saturating concentra-
`tion (20 ␮g/mL) of either unlabeled huIgG4 (isotype control) or MDX-1106,
`washed extensively, and then costained with anti-CD3 fluorescein isothiocya-
`nate and murine antihuIgG4 biotin (Invitrogen, Carlsbad, CA) plus
`streptavidin-phycoerythrin. PD-1 occupancy by infused MDX-1106 was esti-
`mated as the ratio of the percent of CD3⫹ cells stained with antihuIgG4 after in
`vitro saturation with isotype control Ab (indicating in vivo binding) to that
`observed after MDX-1106 saturation (indicating total available binding sites).
`
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`Single-Agent Anti–PD-1 Therapy for Refractory Solid Tumors
`
`Table 1. Patient Characteristics
`
`Table 2. Treatment Characteristics and Clinical Response to Therapy
`
`Characteristic
`
`No. of Patients
`
`Sex
`Male
`Female
`Age, years
`Median
`Range
`Tumor histology
`Colorectal cancer
`Melanoma
`Prostate cancer
`NSCLC
`Renal cell carcinoma
`ECOG PS
`0
`1
`Prior therapies
`Median
`Range
`Chemotherapyⴱ
`Radiation therapy
`Surgery
`Immunotherapy
`Biologics†
`Hormonal therapy
`
`62
`42-84
`
`4
`1-13
`
`22
`17
`
`14
`10
`8
`6
`1
`
`13
`26
`
`36
`13
`39
`14
`26
`8
`
`%
`
`56.4
`43.6
`
`35.9
`25.6
`20.5
`15.4
`2.6
`
`33.3
`66.7
`
`92.3
`33.3
`100
`35.9
`66.7
`20.5
`
`Abbreviations: NSCLC, non–small-cell lung cancer; ECOG, Eastern Coopera-
`tive Oncology Group; PS, performance status.
`ⴱIncludes molecularly targeted therapies.
`†Includes monoclonal antibody therapies.
`
`RESULTS
`
`Patients and Treatments
`Thirty-nine patients with advanced metastatic NSCLC, mela-
`noma, castrate-resistant prostate cancer, RCC, or CRC received
`MDX-1106 in four escalating dose cohorts of 0.3 to 10 mg/kg and an
`expansion cohort at 10 mg/kg, from October 2006 through June 2009
`(Tables 1 and 2). Their median age was 62 years. All had progressive
`treatment-refractory disease, and they had undergone a median of
`four prior therapies.
`
`Treatment-Related Toxicities
`MDX-1106 was well-tolerated: no DLTs were observed after one
`dose, and an MTD was not defined in this study. Grade ⱖ 2 adverse
`clinical and laboratory events are summarized in Appendix Table A1
`(online only). Most frequent were decreased CD4⫹ lymphocyte
`counts (14 patients, 35.9%), lymphopenia (10 patients, 25.6%), fa-
`tigue and musculoskeletal events (six patients each, 15.4%). No pa-
`tient developed human antihuman Ab, even after multiple doses.
`Immune-related AEs (irAEs) were of special interest because of
`the presumed mechanism of action of anti–PD-1 and prior experience
`with anti–CTLA-4.5 No grade ⱖ 3 irAE occurred in the 28-day period
`following the first dose of anti–PD-1. One patient with metastatic
`ocular melanoma developed grade 3 inflammatory colitis following
`five doses (1 mg/kg) administered over 8 months (Appendix Fig A2,
`online only), which responded to steroids and infliximab. One patient
`
`Dose
`(mg/kg)
`
`No. of
`Patients
`
`0.3
`1
`3
`10
`
`Total
`
`6
`6
`6
`21
`
`39
`
`Total No. of Doses
`
`1
`
`6
`3
`3
`15
`
`27
`
`2
`
`0
`1
`0
`1
`
`2
`
`3
`
`0
`1
`2
`4
`
`7
`
`5
`
`0
`1
`1
`0
`
`2
`
`11
`
`0
`0
`0
`1
`
`1
`
`Best Response
`(duration in months)ⴱ
`
`N/A
`1 MXR (1)
`1 CR (21⫹)†
`2 PR (3⫹, 16⫹)‡§
`1 MXR (1)
`1 CR, 2 PR, 2 MXR
`
`Abbreviations: N/A, not applicable; MXR, mixed response defined as regres-
`sion in some lesions but concomitant progression in others; CR, complete
`response; PR, partial response.
`ⴱCR and PR by Response Evaluation Criteria in Solid Tumors 1.0 criteria.
`†This patient with stage IV colorectal cancer had previously shown progres-
`sive disease after receiving chemotherapy regimens including bevacizumab
`and cetuximab.
`‡PR duration of 3⫹ months was preceded by an MXR in this patient with
`melanoma lasting 20 months. Previous therapies that were ineffective in-
`cluded high-dose interleukin-2 and temozolomide.
`§PR duration of 16⫹ months was preceded by an MXR in this patient with renal
`cell carcinoma lasting 4 months. Previous therapies that were ineffective included
`sunitinib, sorafenib, and an experimental histone deacetylase inhibitor.
`
`(10 mg/kg) experienced grade 2 hypothyroidism requiring hormone
`replacement. Two patients (at 3 and 10 mg/kg) developed grade 2
`polyarticular arthropathies requiring oral steroids and were not fur-
`ther treated; in retrospect, both had potentially contributory predis-
`posing factors, one with a history of Lyme arthritis and polymyalgia
`rheumatica, the other with a preexisting antinuclear antibody titer
`⬎ 1:1000.
`
`Antitumor Activity
`One patient with CRC (3 mg/kg) achieved a CR, and two patients
`with RCC (10 mg/kg) and melanoma (10 mg/kg) experienced PRs to
`therapy. A 67-year-old male with CRC metastatic to intra-abdominal
`lymph nodes received five doses of MDX-1106 and experienced a CR
`persisting 21⫹ months. A 72-year-old male with multiorgan meta-
`static RCC had a mixed response after one dose of MDX-1106, with
`progression in a pancreatic metastasis but regression in other sites; this
`evolved to an overall PR after two additional doses, lasting 16⫹
`months without further therapy (Fig 1A). A 51-year-old female with
`melanoma metastatic to multiple lymph nodes and liver sites initially
`experienced a mixed response, with regression at all sites except an
`enlarging subpectoral lymph node, and achieved a PR after receiving
`11 doses of MDX-1106 over 24 months (Fig 1B). Two additional
`patients had significant lesional or mixed tumor regressions (defined
`as regression in individual lesions with concomitant progression at
`other sites), including one with NSCLC (1 mg/kg) and another with
`melanoma (10 mg/kg). In total, 12 patients with stable disease or
`lesional tumor regressions at the first disease assessment received
`multiple doses of MDX-1106 (Table 2).
`
`Tumor Biopsies
`B7-H1 expression on tumors may affect the ability to respond to
`PD-1 blockade. To explore this, tumor biopsies from nine patients
`undergoing treatment with MDX-1106 were analyzed for B7-H1 ex-
`pression with IHC (Appendix Table A2, online only). In two cases,
`both pre- and post-treatment samples were available; in the others,
`only pretreatment (six cases) or post-treatment (one case) samples
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`A
`
`B
`
`Brahmer et al
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`01/15/08
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`04/22/08
`
`07/22/08
`
`03/25/08
`
`04/14/09
`
`Pre-Rx
`
`12 wk post Dose 1
`
`8 wk post Dose 3
`
`Pre-Rx
`
`4 wk post Dose 1
`
`4 wk post Dose 3
`
`Fig 1. Objective tumor responses in patients with metastatic renal cell carcinoma (RCC) and melanoma after repeated dosing with anti–programmed death-1
`monoclonal antibody (MDX-1106) at 10 mg/kg. (A) Patient 4033 with RCC experienced a partial response (PR) after receiving three doses of MDX-1106. Regression
`of metastases in mediastinal lymph nodes and bone (scapula) demonstrated on contrast-enhanced computed tomography scans are representative of lesions at other
`sites including lung, muscle, pancreas, and pericolic lymph node. Date of first treatment was January 29, 2008. (B) Patient 3019 experienced a PR after receiving 11
`doses of MDX-1106. Serial core-needle biopsies of a regressing axillary lymph node metastasis were stained with anti-CD8, revealing a moderate post-treatment
`infiltrate. Infiltration of CD4⫹ cells was not observed (not shown). 20⫻ objective. Rx, treatment; wk, week.
`
`were accessible. Tumor cell staining for B7-H1 expression fell into
`three patterns: negative, intracytoplasmic, or membranous (cell sur-
`face). Among nine patients studied, four exhibited membranous
`B7-H1 staining (Fig 2): three of these patients experienced tumor
`regressions following MDX-1106 therapy; the fourth was treated in
`the 0.3-mg/kg cohort where no responses were observed. Con-
`versely, among five patients whose tumors failed to express B7-H1
`at the cell surface, there was no evidence of clinical response.
`B7-H1 staining patterns were consistent in five patients from
`
`whom multiple biopsies were available (AppendixTable A2). In
`this small sample size, the correlation between membranous
`B7-H1 expression on tumor cells and the likelihood of tumor
`regression following PD-1 blockade suggested potential signifi-
`cance (two-sided P ⫽ .0476; Fisher’s exact test).
`Melanoma patient 3019, who experienced a PR to anti–PD-1
`therapy, underwent pre- and post-treatment biopsies of an axillary
`lymph node metastasis for characterization of intratumoral lymphoid
`infiltrates by IHC. Whereas the pretreatment biopsy contained only
`
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`Single-Agent Anti–PD-1 Therapy for Refractory Solid Tumors
`
`Isotype Control
`
`Anti-B7-H1
`
`A
`
`B
`
`Fig 2. Membranous pattern of B7-H1 ex-
`pression demonstrated on (A) renal cell car-
`cinoma cells in a tumor thrombus from
`patient 4033, and (B) melanoma cells in an
`axillary lymph node metastasis from patient
`3019. Both patients experienced partial re-
`sponses after anti–programmed death-1
`monoclonal antibody (MDX-1106) therapy.
`40⫻ objective.
`
`sparse lymphoid cells, subsequent tumor regression was accompanied
`by a moderate infiltration of CD8⫹, but not CD4⫹, T cells (Fig 1B).
`
`Reactivity to Recall Antigens and PBL Phenotypes
`No significant effects of MDX-1106 therapy on delayed-type
`hypersensitivity responses against C albicans or tetanus toxoid or
`antiviral recall responses by interferon gamma ELISpot analysis were
`observed in 15 and six patients examined, respectively. The effects of a
`single 10-mg/kg dose of MDX-1106 on PBL numbers, subset profiles,
`and activation status were analyzed in 17 patients (Fig 3). Twenty-four
`hours postdose, total lymphocyte as well as CD3, CD4, and CD8
`numbers declined and then rebounded from days 2 through 29 and
`declined again from days 29 through 85. These trends were not ob-
`served for CD19 (B lymphocyte) or CD56 (natural killer) cells (not
`shown), suggesting a selective effect on T cells. Percentages of CD3,
`CD4, and CD8 cells declined steadily over the 85-day observation
`period, with a reciprocal increase in CD19 and CD56 cell percentages
`(Appendix Fig A3A, online only). No significant changes were ob-
`served in expression of the T cell activation markers CD25, CD45RO,
`or HLA-DR by CD4⫹ or CD8⫹ PBLs (Appendix Fig A3B).
`
`PD-1 Receptor Occupancy (pharmacodynamics)
`PD-1 has predominant cell surface expression, unlike CTLA-4,
`which is displayed only transiently at the T cell surface. Thus, it was
`possible to develop flow cytometric methods to evaluate the pharma-
`
`codynamics of infused MDX-1106, estimating PD-1 occupancy on
`circulating T cells over time. Standard pharmacokinetic measure-
`ments of MDX-1106 serum concentrations yielded an approximate
`serum half-life (t1/2) of 12 days (0.3-, 1-, or 3-mg/kg dose) to 20 days
`(10 mg/kg), with maximum concentration (Cmax) and AUC directly
`related to dose. However, pharmacokinetics and pharmacodynamics
`were unexpectedly discordant in 15 patients studied (Fig 4). PD-1
`occupancy appeared to be dose-independent, with a mean peak occu-
`pancy of 85% (range, 70% to 97%) and a mean plateau occupancy of
`72% (range, 59% to 81%) observed at 4 to 24 hours and ⱖ 57 days,
`respectively, after one infusion (Fig 4A). These data are consistent with
`the high affinity of MDX-1106 for PD-1—in vitro, 0.04 ␮g/mL MDX-
`1106 is sufficient to occupy ⬎ 70% PD-1 molecules on T cells (not
`shown)—suggesting that even when serum levels are undetectable
`(⬍ 1.2 ␮g/mL), sufficient concentrations persist to maintain plateau
`PD-1 occupancy. Occupancy eventually decayed after 85 days (Fig 4B,
`top and middle panels). In patients receiving repeated infusions of
`MDX-1106 at 10 mg/kg, troughs and peaks of PD-1 occupancy
`around each dose were observed (Fig 4B, middle and bottom panels),
`although 100% occupancy was not achieved. In vitro experiments
`indicate that PD-1 occupancy analyses of cryopreserved PBLs may
`underestimate occupancy on fresh PBLs (not shown). It is unknown
`whether these findings in circulating lymphocytes reflect PD-1 occu-
`pancy on lymphocytes in the tumor, secondary lymphoid organs,
`and/or tissues.
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`treatment will be necessary to make direct comparisons with anti–
`CTLA-4.
`While efficacy was not a primary end point of this trial, we found
`evidence of antitumor activity, with one CR (ongoing at 21 months)
`and two PRs (ongoing at 3 and 16 months) in patients with CRC,
`melanoma, and RCC; significant lesional regressions were seen in two
`additional patients with melanoma and NSCLC. The observed clinical
`activity of MDX-1106 is most likely exerted through immunologic
`mechanisms rather than direct tumoricidal effects, since nonhe-
`matologic tumors do not express PD-1. Importantly, tumor re-
`gressions were seen in patients with colon and lung cancer,
`suggesting that the capacity of anti–PD-1 to enhance antitumor
`immunity extends beyond the classically immunogenic tumor types
`of melanoma and RCC.
`Two possible scenarios for T cell inhibition via PD-1:B7-H1
`ligation have been proposed in the context of cancer.16,17 First, lym-
`phocytes may encounter B7-H1 expressed on activated antigen-
`presenting cells during priming or reactivation in lymphoid organs. In
`addition, tumor-specific effector lymphocytes may be inhibited on
`contact with B7-H1–positive tumor cells. In the latter case, direct
`tumor assessment for B7-H1 expression may provide a powerful pre-
`dictive biomarker for responsiveness to anti–PD-1. Analysis of serial
`tumor biopsies may provide additional insights into specific antitu-
`mor mechanisms resulting from PD-1 blockade. A regressing lesion in
`one melanoma patient on this study revealed a significantly increased
`and selective CD8⫹ T cell infiltrate post-therapy. While this kind of
`analysis needs to be performed on much larger numbers of patients, in
`light of the acute decline in CD3⫹ T cell numbers observed post-
`treatment, it is interesting to speculate that anti–PD-1 may cause
`redistribution of lymphocyte subsets from the blood into tumor and
`tissue sites.
`There is a single previous report18 of cancer therapy with a differ-
`ent anti–PD-1 antibody, CT-011, in 17 patients with hematologic
`malignancies. One CR was reported in a non-Hodgkin’s lymphoma
`patient. Because the CT-011 trial evaluated patients with a completely
`distinct and nonoverlapping set of cancers relative to this study, it is
`difficult to compare the two antibodies at this time.
`The promising safety profile and evidence of durable clinical
`activity in this phase I trial is encouraging, and early results from a
`follow-up trial (NCT00730639) appear to confirm the tolerability and
`activity of anti–PD-1 in patients for whom multiple prior therapies
`failed. However, it is important to consider factors that might
`improve the response rate. Despite the consistently high level of
`PD-1 occupancy observed on PBLs and the fact that T cells continu-
`ously redistribute between blood, lymph, and tissue, repeated dosing
`of MDX-1106 at shorter intervals might increase occupancy and tissue
`penetration and should be explored. Patient selection is an important
`issue, since anti–PD-1 may have suboptimal effects in patients with
`compromised immune systems. This notion is supported by preclin-
`ical models demonstrating that anti–PD-1 is most effective against
`immunogenic tumors in intact mice. Another important factor in
`determining outcomes may be intratumoral expression of B7-H1,
`either by tumor cells or by nontransformed cells in the tumor micro-
`environment. As suggested by our preliminary biopsy analysis, tumor
`cell surface expression of B7-H1 may be a predictor of responsiveness
`to PD-1 blockade. Finally, on the basis of murine models showing that
`anti–PD-1 is highly synergistic in combination with tumor vaccines,19
`we anticipate that more effective uses of this agent for cancer therapy
`
`1,500
`
`(17)(15)
`
`(14)
`
`1,000
`
`(16)
`
`(12)
`
`(13)
`
`Lymphocytes
`CD3
`CD4
`CD8
`
`(10)
`
`500
`
`0
`
`1
`
`2
`
`20
`40
`Time (days)
`
`60
`
`80
`
`100
`
`1,500
`
`2,000
`
`1,500
`
`1,000
`
`500
`
`0
`
`1
`
`2
`
`Time (days)
`
`A
`
`Lymphocytes (per mm3)
`
`B
`
`Lymphocytes (per mm3)
`
`Fig 3. Effects of a single dose of anti–programmed death-1 monoclonal
`antibody (MDX-1106; 10 mg/kg) on circulating lymphocyte numbers. (A) Twenty-
`four hours postdose, a decline in total lymphocyte as well as CD3, CD4, and CD8
`numbers was observed (two-sided P ⫽ .004, .002, ⬍ .001, and .01 respectively;
`Wilcoxon signed rank test). These parameters followed similar trends, rebound-
`ing from days 2 through 29 (two-sided P ⬍ .001; two-sided P ⫽ .01 for CD8), and
`declining again from days 29 through 85 (two-sided P ⬍ .001; mixed model test
`for trend with knots [changes in slopes] and repeated measures). Means ⫾
`standard error of mean are shown; numbers of patients studied at each time
`point are indicated in parentheses.
`(B) Paired analysis of total
`lymphocyte
`numbers in 16 patients, comparing immediate pretreatment samples (day 1) with
`24-hour post-treatment samples (day 2). A significant decline at day 2 was
`observed (two-sided P ⫽ .004; Wilcoxon signed rank test). Dotted line indicates
`the lower limit of normal lymphocyte counts.
`
`DISCUSSION
`
`We report results from a dose-escalation trial of a fully human anti–
`PD-1 mAb, MDX-1106, in 39 patients with refractory metastatic can-
`cers. MDX-1106 was well tolerated to the maximum planned dose of
`10 mg/kg. While both anti–PD-1 and anti–CTLA-4 therapies are
`associated with irAEs, as predicted by preclinical models and consis-
`tent with the physiologic roles of these molecules, these toxicities
`appear to be less frequent and milder in patients receiving anti–PD-1.
`Among 39 patients treated with MDX-1106, a single attributable seri-
`ous AE, inflammatory colitis, occurred. Low-grade irAEs occurred in
`two patients with polyarticular arthropathies requiring oral steroids
`and one patient with hypothyroidism requiring hormone replace-
`ment. However, expanded experience with multidose anti–PD-1
`
`3172
`
`© 2010 by American Society of Clinical Oncology
`
`JOURNAL OF CLINICAL ONCOLOGY
`
`Genome Ex. 1030
`Page 6 of 9
`
`

`

`Single-Agent Anti–PD-1 Therapy for Refractory Solid Tumors
`
`100
`
`B
`
`100
`
`0.3 mg/kg
`
`[MDX-1106] (ug/mL)
`PD-1 occupancy (%)
`
`300
`
`200
`
`100
`
`[MDX-1106] (ug/mL)
`
`A
`
`Pt. 4025
`
`100
`
`200
`300
`Time (days)
`
`400
`
`500
`
`600
`
`Pt. 4033
`
`400
`
`500
`
`600
`
`80
`
`60
`
`40
`
`20
`
`0
`
`100
`
`80
`
`60
`
`40
`
`20
`
`PD-1 Occupancy (%)
`
`PD-1 Occupancy (%)
`
`PD-1 Occupancy (%)
`
`80
`
`60
`
`40
`
`20
`
`0
`
`100
`
`PD-1 Occupancy (%)
`
`80
`
`60
`
`40
`
`20
`
`0
`
`0
`
`20
`
`40
`60
`Time (days)
`
`80
`
`1 mg/kg
`
`[MDX-1106] (ug/mL)
`PD-1 occupancy (%)
`
`300
`
`200
`
`100
`
`0
`
`[MDX-1106] (ug/mL)
`
`20
`
`40
`60
`Time (days)
`
`80
`
`0
`
`100
`
`200
`300
`Time (days)
`
`Pt. 3019
`
`100
`
`200
`300
`Time (days)
`
`400
`
`500
`
`600
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`PD-1 Occupancy (%)
`
`100
`
`PD-1 Occupancy (%)
`
`80
`
`60
`
`40
`
`20
`
`0
`
`100
`
`PD-1 Occupancy (%)
`
`80
`
`60
`
`40
`
`20
`
`0
`
`3 mg/kg
`
`[MDX-1106] (ug/mL)
`PD-1 occupancy (%)
`
`300
`
`200
`
`100
`
`[MDX-1106] (ug/mL)
`
`0
`
`20
`
`40
`60
`Time (days)
`
`80
`
`10 mg/kg
`(n = 10)
`
`[MDX-1106] (ug/mL)
`PD-1 occupancy (%)
`
`300
`
`200
`
`100
`
`[MDX-1106] (ug/mL)
`
`0
`
`20
`
`40
`60
`Time (days)
`
`80
`
`Fig 4. Pharmacodynamics of anti–programmed death-1 (PD-1) monoclonal antibody (MDX-1106). (A) PD-1 occupancy on circulating CD3⫹ T cells after one infusion
`of MDX-1106 is shown for single patients (Pts.) each receiving 0.3, 1, or 3 mg/kg, and for 10 patients receiving 10 mg/kg (mean ⫾ standard error of mean; solid squares).
`Serum concentrations of MDX-1106 at the same time points are indicated (open diamonds). (B) Long-term PD-1 occupancy analysis in patients receiving one (top panel)
`or multiple doses (middle and bottom panels) of MDX-1106 at 10 mg/kg. All patients received infusions at day 1; additional infusions are indicated by arrows. Results
`in (B) middle and bottom panels are representative of five patients receiving multiple doses.
`
`www.jco.org
`
`© 2010 by American Society of Clinical Oncology
`
`3173
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`

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`Brahmer et al
`
`will involve combinatorial therapies with other agents that boost en-
`dogenous antitumor immunity. Treatment regimens combining
`MDX-1106 with vaccines, molecularly targeted therapies, or other
`immunomodulators are already under evaluation in the laboratory
`for near-term clinical development.
`
`AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS
`OF INTEREST
`
`Although all authors completed the disclosure declaration, the following
`author(s) indicated a financial or other interest that is relevant to the subject
`m