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  • Cancer Therapy: Clinical

    CTLA4 Blockade Induces Frequent Tumor Infiltration by ActivatedLymphocytes Regardless of Clinical Responses in Humans

    Rong Rong Huang1, Jason Jalil2, James S. Economou3,4, Bartosz Chmielowski2, Richard C. Koya3,Stephen Mok3, Hooman Sazegar2, Elizabeth Seja2, Arturo Villanueva2, Jesus Gomez-Navarro5,John A. Glaspy2,4, Alistair J. Cochran1, and Antoni Ribas2,3,4

    AbstractBackground: CTLA4 blocking monoclonal antibodies provide durable clinical benefit in a subset of

    patients with advanced melanoma mediated by intratumoral lymphocytic infiltrates. A key question is

    defining whether the intratumoral infiltration (ITI) is a differentiating factor between patients with and

    without tumor responses.

    Methods: Paired baseline and postdosing tumor biopsy specimens were prospectively collected from 19

    patients with metastatic melanoma, including 3 patients with an objective tumor response, receiving the

    anti-CTLA4 antibody tremelimumab within a clinical trial with primary endpoint of quantitating CD8

    cytotoxic T-lymphocyte (CTL) infiltration in tumors. Samples were analyzed for cell density by automated

    imaging capture and further characterized for functional lymphocyte properties by assessing the cell

    activation markers HLA-DR and CD45RO, the cell proliferation marker Ki67, and the regulatory T-cell

    marker FOXP3.

    Results: There was a highly significant increase in ITI by CD8 cells in biopsy samples taken aftertremelimumab treatment. This included increases between 1-fold and 100-fold changes in 14 of 18

    evaluable cases regardless of clinical tumor response or progression. There was no difference between the

    absolute number, location, or cell density of infiltrating cells between clinical responders and patients with

    nonresponding lesions that showed acquired intratumoral infiltrates. There were similar levels of expres-

    sion of T-cell activation markers (CD45RO, HLA-DR) in both groups and no difference in markers for cell

    replication (Ki67) or the suppressor cell marker FOXP3.

    Conclusion: CTLA4 blockade induces frequent increases in ITI by T cells despite which only a minority

    of patients have objective tumor responses. Clin Cancer Res; 17(12); 41019. 2011 AACR.

    Introduction

    Costimulatory and co-inhibitory molecules are keyplayers in the activation step of the adaptive immunesystem and regulate the expansion and effector functionsof antigen-specific T cells (1). CTLA4 has a pivotal role inthis interaction, dampening immune responses to self-antigens (2). Ipilimumab, a fully human IgG1 anti-CTLA4

    antibody (formerly MDX-010; Bristol-Myers Squibb), hasshown improvement in overall survival relative to a peptidevaccine in a phase 3 randomized clinical trial in patientswith metastatic melanoma previously treated with stan-dard-of-care therapies (3), presenting the therapeutic activ-ity of this class of antibodies. Despite this success, theclinical experience shows that the objective response rateof patients with metastatic melanoma treated with ipili-mumab, or the IgG2 anti-CTLA4 antibody tremelimumab(formerly CP-675,206; Pfizer), is low, in the range of 5% to15%, and they both have similar rates of inflammatory andautoimmune toxicities (grade 3) in approximately 20%of patients in pivotal phase 2 trials in second-line therapyfor melanoma (4, 5). However, most patients with objec-tive tumor regression have durable responses, the longestongoing since 2001 (6). The proof of concept of antitumoractivity and patient benefit with CTLA4 blockade have beenachieved, but there is a clear need to determine whatdifferentiates patients who respond from those who showtumor progression.

    Multiple groups have studied how anti-CTLA4 antibo-dies impact the human immune system and the mechan-isms that determine tumor response or progression.

    Authors' Affiliations: Departments of 1Pathology and Laboratory Med-icine, 2Medicine, Division of Hematology/Oncology, and 3Surgery, Divisionof Surgical Oncology, and 4Jonsson Comprehensive Cancer Center,University of California, Los Angeles (UCLA), Los Angeles, California;and 5Pfizer Global Research and Development, New London, Connecticut

    Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

    Current address for J. Gomez-Navarro: Millennium-Takeda, Boston, Mas-sachusetts.

    Corresponding Author: Antoni Ribas, Division of Hematology-Oncology,11-934 Factor Building, UCLA Medical Center, 10833 Le Conte Avenue,Los Angeles, CA 90095. Phone: 310-206-3928; Fax: 310-825-2493.E-mail: aribas@mednet.ucla.edu

    doi: 10.1158/1078-0432.CCR-11-0407

    2011 American Association for Cancer Research.

    ClinicalCancer

    Research

    www.aacrjournals.org 4101

    Research. on July 15, 2018. 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

    Published OnlineFirst May 10, 2011; DOI: 10.1158/1078-0432.CCR-11-0407

    http://clincancerres.aacrjournals.org/

  • Analysis of the effects of anti-CTLA4 antibodies in patientshas been mainly based on the study of peripheral bloodsamples (718). Studying the effects of CTLA4 blockingantibodies in tumor samples allows analysis of the inter-action between an activated immune system and its cancercell targets. Preclinical models suggest a key role for CTLA4both in the infiltration of T lymphocytes into peripheraltissues including tumors and in the modulation of theduration of the interaction between T cells and cells pre-senting with cognate antigens (19, 20). These data predictthat the use of CTLA4 blocking antibodies should increaseintratumoral infiltration (ITI) by lymphocytes and retaintumor antigenspecific T cells within tumors. Clinical datato date show lymphocytic ITI in tumor biopsies of patientresponding after the administration of anti-CTLA4 anti-bodies (16, 17, 21, 22).

    In a prior study, we analyzed 15 tumor biopsy samplestaken at different time points from 7 patients treated withtremelimumab, with lesions biopsied when there wasclinical evidence of either response or progression (22).Clinically responding lesions had diffuse intratumoralinfiltrates by CD8 T cells that were markedly increasedin cases for whom comparison with a baseline biopsy wasavailable. These T-cell infiltrates were massive at the peakof the response at around 1 to 2 months after the firstantibody infusion, occupying much of the biopsiedregressing lesions. Interestingly, expression of FOXP3and indoleamine-2,3-dioxygenase, 2 proteins associatedwith immunosuppressive cells in the tumor microenvir-onment [regulatory T cells (Treg) and plasmacytoiddendritic cells, respectively], were actually increased inthe regressing lesions, particularly at the sites of immunecellmelanoma cell interaction (22). The retrospectivenature of that analysis (22) may have induced bias;patients with responding tumors were prone to be biop-sied at one stage of the response whereas those withdisease progression were primarily biopsied when the

    therapy effects may be overwhelmed by melanomaprogression.

    Therefore, a key question remains whether the presenceor degreeof ITI of T cells differentiates betweenpatientswithand without objective tumor responses in prospectivelyconducted tumor biopsy samples taken at a defined timepoint. Therefore, we conducted a clinical trial with pairedbaseline and postdosing tumor biopsy specimens collectedwithin 1 and 2 months from the first dose of the CTLA4blocking antibody.Ourmain finding is a remarkable induc-tion of immune cell infiltrates by CD4 andmostly CD8 Tcells after the administration of tremelimumab. This waspresent both in lesions that went on to objective tumorresponse and in half of the lesions that progressed.

    Materials and Methods

    Clinical trial designThirty-two patients with measurable advanced mela-

    noma (stages IIIcIV) with metastatic lesions amenableto outpatient biopsies were enrolled in this phase II clinicaltrial (UCLA IRB# 06-06-093 IND# 100453, clinical trialregistration NCT00471887). Patients received single-agenttremelimumab at 15 mg/kg every 3 months with baselineand approximately day 30 to 60 postdosing biopsies.Samples were coded with the study denomination of GAand a patient-specific number. Adverse events attributed totremelimumab were graded according to the NCI commontoxicity criteria version 2.0 (23). Patients who experiencedthe following adverse events at any time during the pre-vious cycle were considered to have a dose limiting toxicity(DLT) and treatment with tremelimumab was discontin-ued: grade 4 treatment-related adverse event; grade 3 orhigher hypersensitivity reaction; grade 2 or higher colitis;and/or autoimmune reaction in a critical organ (brain, eye,liver, thyroid, hypophysis). Objective clinical responseswere recorded following a modified Response EvaluationCriteria in Solid Tumors (RECIST; ref. 24), wherein skinand subcutaneous lesions evaluable only by physical exam-ination were considered measurable if adequately recordedusing a photographic camera with a measuring tape orruler; there was no minimum size restriction for theselesions.

    Sample procurement and immunohistochemicalquantitation of CD4 and CD8 cells

    Biopsies samples were formalin-fixed and paraffin-embedded and stained for immunohistochemistry (IHC)as previously described (22) with anti-CD4 (clone 4B12;NeoMarkers) and anti-CD8 (clone C8/144B; Dako). TheSimple-PCI Imaging System (version 5.2.1.1609; CompixInc. Imaging System) was utilized to quantitatively evaluateT-cell infiltration. The frequency of intratumoral and peri-tumoral lymphocytes was assessed by analyzing 10 tumorareas from each sample at 200magnification. The densitywas compared between pretreatment and posttreatmentbiopsies. All samples we