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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 CD8þ

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 CD8þ cells in biopsy samples taken after

tremelimumab 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); 4101–9. �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: [email protected]

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

�2011 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 4101

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

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Analysis of the effects of anti-CTLA4 antibodies in patientshas been mainly based on the study of peripheral bloodsamples (7–18). 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 antigen–specific 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 CD8þ 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 immunecell–melanoma 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 CD4þ andmostly CD8þ 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 IIIc–IV) 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 CD4þ and CD8þ 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 200�magnification. The densitywas compared between pretreatment and posttreatmentbiopsies. All samples were analyzed without the knowledgeof the patients’ clinical outcomes.

Translational Significance

After 10 years of clinical development, it is stillunclear how anti-CTLA4 blocking monoclonal antibo-dies mediate antitumor responses and why only a fewpatients have durable responses. The anticipatedmechanism of action is the stimulation of a cytotoxicT-lymphocyte (CTL) response against the cancer, whichrequires studying such cells within tumors. We docu-mented, using paired tumor biopsy samples frompatients treated with the CTLA4 blocking antibodytremelimumab, a remarkable intratumoral infiltrationby activated CTLs in most patients regardless of having atumor response. This evidence suggests that the keydeterminant of tumor response or resistance residesin the interaction between CTLs and their tumor targets,as opposed to the more frequently studied effects ofCTLA4 blocking antibodies on cells or proteins detect-able in the blood.

Huang et al.

Clin Cancer Res; 17(12) June 15, 2011 Clinical Cancer Research4102




Immunohistochemical staining for T-cell activation,proliferation, and regulatory markersPostdosing biopsy specimens with significant increase in

T-cell infiltrates were stained by immunohistochemicaldouble staining with anti-HLA-DR (clone TAL, 1B5; Dako)and anti-CD45RO (clone UCHL1; Dako), and single stain-ing with Ki67 (clone MIB-1; Dako) or anti-FOXP3 (clone236A/E7; Abcam).

Statistical analysisThe statistical design of this clinical trial was based on the

assumption of a 20% or higher probability of increasedCD8þ infiltration in posttreatment biopsies detected byIHC. This assumption was based on the lower boundary ofchange in CD8þ infiltration from our prior studies (22).Two scores using semiquantitative analysis of immunohis-tochemical data (0 to 2þ, 1þ to 3þ) were assessed. Abinomial test was used at 5% level of significance. If thetrue probability of infiltration increased by at least 2 scorelevels in at least 50% of the tremelimumab-treated patients,then 20 to 21 evaluable patients would provide 90% powerto reject the null hypothesis. The Mann–Whitney rank-sumtest was used to compare values obtained from assessmentof the pre- and posttreatment samples. Analyses were doneusing the SigmaPlot software package, and all tests were 2-sided with the significance level set at P ¼ 0.05.

Results

Patient characteristics, clinical response, andtoxicitiesThirty-two patients were enrolled (Table 1). Themajority

of patients had M1c metastatic melanoma (visceral metas-tasis and/or high lactate dehydrogenase level) and morethan half of the patients had received prior systemic ther-apy, most frequently a chemotherapy-containing regimen.There were 9 patients with clinically relevant toxicitiesprospectively defined as DLTs that precluded continueddosing with tremelimumab. One patient had immunethrombocytopenia purpura (ITP), which developed within1 week after the first dose; grade 3 colitis in 5 patients, 2during the first cycle and the other 3 while on chronicmaintenance dosing; 1 patient with a grade 3 skin rash inthe first cycle; and 2 with grade 2 hypophysitis, both duringthe second or later cycles. Three patients had an objectiveand durable tumor response, all with a complete responseof in-transit metastasis (patients GA18, GA29, and GA33).One additional patient had an objective response in supra-clavicular and laterocervical lymph nodes meeting partialresponse criteria, followed by slow disease progression ofnodal metastasies 7 months after initiating dosing withtremelimumab (patient GA5). This patient died from anunrelated cause (infectious osteomyelitis after an acciden-tal wound) 20 months after starting tremelimumab, withactive nodal metastases of melanoma localized in thesupraclavicular area but without widespread systemicmetastasis. There are 7 patients alive beyond 2 years, the3 patients with a complete response (35þ, 30þ and 28þ

months from study start), and 4 patients who are alive withmetastatic melanoma (follow-up between 29þ to 41þmonths) despite not having an objective response to tre-melimumab (patients GA7, GA19, GA26, and GA32).

Biopsy sample procurementPaired tumor biopsy samples before and after the first

infusion with tremelimumab were obtained in 21 of the 32patients enrolled in this clinical trial. Reasons for obtainingonly a baseline biopsy were absence of melanoma in thebiopsy specimen in 1 case, toxicity within the first cycleresulting in inability to return for the postdosing biopsy in

Table 1. Patient characteristics (all patients)

Characteristic Number of patients

SexFemale 9Male 23

AgeMean 52Range 27–86

EthnicityCaucasian 28Hispanic 3Asian 1

Prior therapiesNo prior therapy 13Biological only 4Chemotherapy-based 13

StageIIIc 4M1a 3M1b 3M1c 22

DLTs (grade)ITP 1 (4)Colitis 5 (3)Rash 1 (3)Hypophysitis 2 (2)

ResponseNot evaluable 1Progressive disease 27Partial response 1Complete response 3

Alive for >18 moUnrelated death 1 (20)Alive with disease 4 (33þ, 27þ, 23þ, 21þ)Maintained response 3 (27þ, 22þ, 20þ)

BiopsiesPre and post 21Pre onlyScreen fail 1Progression 7Toxicity 2Sample lost 1

Intratumoral Infiltrates Following CTLA4 Blockade

www.aacrjournals.org Clin Cancer Res; 17(12) June 15, 2011 4103




Tab

le2.

Details

ofpatientswho

provided

pre-an

dpos

tdos

ingbiopsy

spec

imen

s

Patient

stud

y#

Age

Prior

trea

tmen

tsStage

Biopsy

site

DLT

sTim

ingof

postdosing

biopsy

Res

pons

eSites

of

progress

ion

Progress

ion-

free

survival

(mo)

OS(m

o)

Commen

tsFo

ldch

ange

inCD8þ

cell

infiltrate

GA5

65Non

eM1c

LN70

PR

LN7

20Diedfrom

unrelated

caus

es

1.80

GA7

62DC

IIIc

Skin

41PD

Skin

241

þAWD

0.08

GA8

48IL-2,TIL

M1c

Skin

33PD

13

1.04

GA9

52Non

eM1c

LN35

PD

LN,bon

e3

142.37

GA10

63GM-C

SF,

TMZ

M1c

Skin

23PD

LN,lung

22

0.00

GA11

47Non

eM1c

LN54

PD

LN,ad

rena

l,brain

27

1.40

GA12

76Non

eM1c

Skin

ColitisG3

43Offdue

toAE/PD

220

4.93

GA13

37TM

ZM1a

s.c.

Hyp

ophy

sitis

G2

33PD

LN,sk

in3

131.46

GA14

38Non

eM1c

s.c.

35PD

s.c.,mus

cle

315

6.98

GA17

78Non

eM1c

Skin

51PD

LN,brain

22

4.44

GA18

49GM-C

SF

M1a

Skin

40CR

–35

þ35

þOng

oing

CR

52.71

GA19

55TM

ZM1c

Skin

ColitisG3

34PD

LN3

3621

.95

GA21

71TM

Zþ

thalidom

ide

M1c

Skin

49PD

LN,lung

,liver,

spleen

,ad

rena

l3

80.00

GA24

81Non

eM1c

Skin

28PD

LN,lung

,brain

23

2.75

GA26

68Non

eM1b

LNColitisG3

34PD

Lung

223

þAWD

0.88

GA27

52Non

eM1c

Skin

43PD

LN6

11>1

00GA29

79Non

eIIIc

Skin

ColitisG3

33CR

–30

þ30

þOng

oing

CR

12.26

GA30

32DC,DTIC,

bioch

emothe

rapy,

IL-2,TC

RACT,

TIL

M1c

Skin

51PD

Skin,

s.c.,LN

24

15.68

GA31

49Non

eIIIc

Skin

Hyp

ophy

sitis

G2

34CR

–28

þ28

þOng

oing

CR

Abbreviations

:LN,lym

phno

des

;s.c.,su

bcu

tane

ous;

G,tox

icity

grad

e;DC,d

endritic

cells;IL-2,

interle

ukin

2;TM

Z,tem

ozolom

ide;

DTIC,d

acarbaz

ine;

TCRACT,

T-ce

llrece

ptor

tran

sgen

icad

optiv

ece

lltran

sfer

therap

y;TIL,

tumor-infiltratin

glympho

cyte

adop

tivece

lltran

sfer

therap

y;GM-C

SF,

gran

uloc

ytemac

ropha

geco

lony

-stim

ulatingfactor;CR,

complete

resp

onse

;PR,partia

lres

pon

se;PD,progres

sive

disea

se;AE,ad

verseev

ents;AWD,alivewith

disea

se.

Huang et al.





2 patients (ITP and colitis), and early disease progression in7 patients who withdrew consent before the proposedpostdosing biopsy. The postdosing sample from 1 of the21 patients with paired biopsies could not be retrieved foranalysis (patient GA32). The postdosing biopsy specimenfrom patient GA25 did not contain melanoma. The pre-senting characteristics and outcome of the remaining 19patients with pre- and postdosing biopsies available foranalysis are presented in Table 2.

Tumor infiltration by T lymphocytesUp to 10 randomly selected fields per sample were

analyzed for ITI (when T cells were mixed within themelanoma cells) and peritumoral infiltration (PTI, whenT-cell infiltrates are located peripheral to the tumor massand in collagen bundles that dissected the tumor mass).Overall, there was a marked and highly statistically sig-nificant increase in ITI by CD8þ cells in biopsy samplestaken after tremelimumab treatment (Figs. 1 and 2). Themean pretreatment CD8þ cell count was 289 cells/mm2

(SEM ¼ 61), and the postdosing density of these cellsincreased to 955 (SEM ¼ 191, P ¼ 0.005; Fig. 2). Thedifference in ITI for CD4þ cells was also significantlyincreased but at a lower magnitude (mean predosing104 � 32 vs. mean postdosing 428 � 156, P ¼ 0.018;Fig. 2). Analysis of PTI by lymphocytes was not feasible in 5cases with metastatic melanoma to the lymph nodes

because the great majority of peritumoral cells were nodallymphocytes. Among the remaining cases, there were nosignificant changes in PTI by CD8þ or CD4þ cells (Sup-plementary Fig. S1), as there was no evidence of tumoradjacent infiltration by T cells in skin biopsies beyond themetastases (data not shown).

Analysis of tumor-infiltrating lymphocytes dependingon clinical response

Among the 4 patients with objective clinical responses, 2cases (patients GA5 andGA29) had amarked increase in ITIof CD8þ cells (Figs. 1 and 2; Supplementary Fig. S2).Melanoma cells showed degenerative changes, with disas-sociated connection and abundant lymphocytes betweenand around the melanoma cells. Pretreatment biopsiesshowed intactmelanomacells,with little orno intratumoralpenetration by T cells. The posttreatment biopsy in patientGA18, who had a durable complete response, showedcomplete regression of the melanoma, with no residualmelanoma cells and abundant infiltrating lymphocytesat the regressed melanoma site (Fig. 1). The postdosingbiopsy from the fourth patient with a clinical response(patient GA31) also showed no residual melanoma cells.This completely regressed melanoma included abundantmelanin pigment, both free and in macrophages, andextensive scar tissue without lymphocytic infiltration andwas therefore interpreted as showing late-stage regression

Figure 1. Immunohistochemicalanalysis of CD8þ cell infiltrationbefore and after tremelimumab in4 representative patients.Specimens of pre- (Pre-Tx) andposttreatment (Post-Tx)tremelimumab tumor biopsiesfrom 2 patients who progressed toa durable complete response aftertremelimumab treatment (GA18and GA29) are compared with 2representative samples of patientswho had disease progression aftertherapy (GA12 and GA14).Although there were no intactmelanoma cells evident in thepostdosing sample GA18, thehistologic changes supported thatthe biopsy derived from aregressing lesion with lymphocyticinfiltrates and the values for thiscase are considered as ITI.

GA18

10x

Pre-Tx biopsies Post-Tx biopsies

40X10x40X

GA12

GA14

10x

10x 40X

40X10x

10x40X

40X

10X 40X10X40X

GA29

Patients with clinical progression

Patients with clinical objective response






(Supplementary Fig. S2). Many postdosing samples frompatients with clinically progressive disease also showedsignificant lymphocytic ITI (Figs. 1 and 2; SupplementaryFig. S2). In 8 of 16 patients with disease progression, theincrease in CD8þ cell density was greater than the averageincrease for the whole series. In addition, 6 cases hadincreases in CD4þ cell density greater than the averageoverall series increase over the baseline biopsy. There wasno correlation between the density of ITI after tremelimu-mab treatment in responder and nonresponder patients orbetween patients alive 2 years or more after study initiationand patients who died from melanoma less than 2 yearsafter treatment initiation (Fig. 2).

Characterization of intratumoral infiltrates inpatients with postdosing increases

Because T-cell infiltrates increased in patients with andwithout a clinical response, we were interested in studyingwhether the functional characteristics of infiltrating T cellsdifferenced between these samples. HLA-DR is a surfacemarker of T-cell activation after exposure to CTLA4 block-ing antibodies (8, 9, 25), whereas CD45RO is a marker ofprior cognate antigen-exposed T cells. Together they markcells with a surface phenotype of T effector or T effectormemory cells (26). The combined analysis of HLA-DR/CD45RO staining showed a marked increase in double-positive cells in postdosing biopsies in all cases, irrespectiveof whether they had an objective response or not or were

alive beyond 2 years from study initiation (Fig. 3 andTable 3). Given the increase in the number of T cells inpostdosing biopsies, we stained the samples for the cellproliferation marker Ki67 to determine the extent of cellreplication within tumors. There was no change in thefrequency of Ki67-positive nuclei among lymphocytic infil-trates when postdosing biopsies were compared with base-line biopsies (Fig. 3 and Table 3). In our prior analysis oflesions regressing after tremelimumab treatment (22), wenoted an increase in FOXP3þ cells. The current studyconfirmed this finding. Three postdosing samples frompatients who had a durable complete response had amarked increase in FOXP3þ cells (Fig. 3). In the 8 non-responding tumors, there was an increase in FOXP3þ cellsin 5 tumors, 2 had no apparent change, and 1 had adecrease in FOXP3þ cells. When comparing the infiltratesbetween durably responding and nonresponding patients,the trend was in favor of higher infiltrates of FOXP3þ cellsin responding lesions (Table 3).

Discussion

An intratumoral infiltrate with activated T cells hasprognostic significance in patients with cancer, in whichprimary tumors with larger and more diffuse T-cell infil-trates displaying an effector functional phenotype haveimproved survival (26, 27). The major goal of tumorimmunotherapy is to induce such intratumoral infiltrates

Intratumoral CD8 infiltration

Pre Post

CD

8 ce

ll de

nsity

0

500

1,000

1,500

2,000

2,500

3,000

Paired t test P = 0.005

Intratumoral CD4 infiltration

Pre Post

CD

4 ce

ll de

nsity

0

500

1,000

1,500

2,000

2,500

3,000


Peritumoral CD8 infiltration

Pre Post

CD

8 ce

ll de

nsity

0

1,000

2,000

3,000

4,000


Clinical responseNo clinical response

Peritumoral CD4 infiltration

Pre Post

CD

4 ce

ll de

nsity

0

500

1,000

1,500

2,000

2,500

3,000


Clinical responseNo clinical response

Figure 2. Quantitativeimmunohistochemical analysis ofITI and PTI by CD4þ and CD8þ

cells. Results are expressed as theabsolute number of positivelystaining cells/mm2 averaged over10 fields. Open circles, patientswith an objective clinicalresponse. Closed triangles,patients with stable disease orprogression.

Huang et al.





by using a therapeutic intervention. It should obviouslyrequire demonstration that T cells do effectively infiltratetumor lesions to exert their cytotoxic activity. However,given the practical limitations of obtaining serial biopsy

specimens in patients with metastatic cancers, there isa paucity of data for studying immune infiltrates intumors of patients receiving immunotherapy. This pointis particularly relevant for CTLA4 blocking antibodies, as

GA18

Pre-Tx Post-Tx

GA12

Pre-Tx Post-Tx

HLA

-Dr+

/CD

45R

O+

activ

ated

T c

ells

Ki 6

7+ P

rolif

erat

ion

cells

FO

XP

3+T

reg

Figure 3. Immunohistochemical analysis of HLA-DR and CD45RO double staining, and Ki67 and FOXP3 single staining, in 2 representative samples withpostdosing intratumoral lymphocytic infiltrates. Specimens of pre- (Pre-Tx) and posttreatment (Post-Tx) tremelimumab tumor biopsies from a patient whoprogressed to a durable complete response after tremelimumab treatment (GA18) are compared with samples from a patient who had disease progressionafter therapy (GA12). Top row, HLA-DR and CD45RO double staining. Middle row, Ki67 staining. Bottom row, FOXP3 staining.

Table 3. Analysis of functional phenotypes of TILs

Cell density (number/mm2)in all subjects

Cell density (number/mm2)according to clinical responses

Pretreatment Posttreatment Change P 3 patientswith CR

8 patientswith PD

P

HLADR/CD45RO

83.68 � 81.42 440.09 � 288.12 þ356.40 � 257.33 0.0010 407.34 � 329.38 452.37 � 294.89 0.4238

Ki67 1,075.38 � 355.95 835.12 � 515.23 �240.26 � 690.48 0.28 266.23 � 137.72 130.27 � 162.73 0.1167FOXP3 35.20 � 30.06 167.35 � 162.37 þ132.15 � 160.35 0.0029 993.76 � 734.18 775.63 � 458.70 0.3340

NOTE: The analysis was done in samples from 3 patients with an objective response (casesGA5, GA18 andGA29) and from 8 patientswith disease progression and marked postdosing increases in TILs (cases GA9, GA12, GA14, GA17, GA19, GA24, GA27, and GA30).This analysis did not include the fourth patient with an objective tumor response (GA31), as the postdosing biopsy showed completelyresolvedmetastatic lesion without residual lymphocytes. (i) Summary of results of immunohistochemical staining for markers of T-cellactivation (HLA-DR, CD45RO), proliferation (Ki67) and suppressor cell (FOXP3) in 11 patients with increased TILs after treatment. (ii)Comparison of the postdosing intratumoral infiltrates in 3 patients with a complete response versus 8 patients with progressivedisease. Results are expressed as the number of positively staining cells/mm2 averaging 10 fields.






preclinical data suggest that the mechanism of tumorregression should be mediated by the intratumoral accu-mulation of T cells, with little evidence of changes in thesystemic circulation. In the current studies, we analyzedtumor biopsy specimens from patients receiving anti-CTLA4 antibodies for the treatment of advanced mela-noma. The main goal was to compare baseline andpostdosing samples for the presence and functional char-acteristics of lymphocytic infiltrates. Contrary to conclu-sions based on prior experience with biopsies conductedlate in the treatment with tremelimumab (22), the currentstudies with biopsies at 1 to 2 months after the first doseof tremelimumab show sharp increases in tumor-infil-trating lymphocyte (TIL) in half of the patients whoshowed disease progression. Quantitative analysis ofthe T-cell infiltrates did not differentiate clinical respon-ders and nonresponders. Additional analyses to deter-mine whether there was a difference in the functionalityof these cells by using phenotypic markers also indicatethat the cellular infiltrate induced by tremelimumab doesnot differ significantly between clinical responders andnonclinical responders.

Postdosing intratumoral lymphocyte infiltrates could beas a result of cell mobilization and increased ITI induced byCTLA4 blockade, a possibility supported by some precli-nical models (19, 20, 28). Alternatively, the increase maybe due to active tumor antigen–specific lymphocyte pro-liferation with release of the so-called CTLA4 cell-cyclecheckpoint with G1 arrest (29–32). The dominant effectof CTLA4-inhibiting lymphocyte replication is evidencedby studies in CTLA4 knockout mice, which die within daysof postnatal antigen exposure due to massive lymphocyteproliferation and peripheral tissue infiltration (33, 34). Tostudy whether active lymphocyte replication within tumorscaused the postdosing increase in TILs, we compared pre-and postdosing samples for the nuclear expression of thecell replication marker Ki67. The data showed no suchchange, suggesting that tumors are not the site of lympho-cyte replication after CTLA4 blockade. This information iscomplemented by our recent experience in using wholebody imaging with positron emitting tomography (PET) tostudy tumor and lymphoid organs for a differential uptakeof radiolabeled PET traces in patients treated with treme-limumab (35). After treatment with tremelimumab, therewas increased 30-deoxy-3’-[18F]fluorothymidine ([18F]FLT)uptake in the spleen in most patients, which is reflective ofcell replication in this large lymphoid organ (35). The PETimaging data, together with the morphologic data from theanalysis of tumors presented herein, suggest that tremeli-mumab induces lymphocyte replication in lymphoidorgans that, in turn, leads to increased infiltration oftumors inmost patients, whether or not they have a clinicaltumor response. Because the changes in intratumoral T-cell

infiltrates are well beyond what can be detected in blood orin normal tissues, lymphocyte trafficking changes are themost likely explanation for the observed results in thisbiopsy series.

Studies analyzing immune parameters after CTLA4blockade have not yet provided a reproducible explanationof why clinical tumor responses are infrequent despiteevidence of immune activation in most patients. Multiplestudies reported lymphocyte activation in blood (7–10,12–16, 18, 36), and our current data confirm immuneactivation within tumors (17). It is difficult to reconcile thefrequent immune responses to CTLA4 blockade with infre-quent clinical evidence of tumor regression. Reportedmechanisms of tumor escape to tumor immunotherapyinclude downregulation of MHC and tumor antigen–pro-cessing and antigen-presenting machinery (37), or theeffects of oncogenes on sensitivity or resistance to apoptosisinduced by immune effector cells (38–40).

In conclusion, postdosing melanoma tumor biopsy spe-cimens from more than half of patients treated with theCTLA4 antagonistic antibody tremelimumab showincreased T-lymphocyte infiltrates. This increase is pro-nounced in patients who show an objective tumorresponse but is indistinguishable quantitatively and phe-notypically from infiltrates in half of the patients whoshowed disease progression. These data indicate that, inmost patients, therapeutic CTLA4 blockade induces thedesired immune stimulation resulting in T-cell infiltrationof tumors. Because only a minority have clinical responses,then differences on how tumors respond to the T cellinfiltrates is likely to be a major cause of resistance toanti-CTLA4 antibodies.

Disclosure of Potential Conflicts of Interest

J. Gomez-Navarro was an employee of Pfizer Inc. at the time of this work.A. Ribas received honoraria from Pfizer for the participation in advisoryboards during the conduct of this study.

Acknowledgments

We thank the manuscript review by Dr. Margaret Marshall from PfizerInc., New London, CT.

Grant Support

This work was funded in part by Pfizer Inc., the Melanoma ResearchFoundation, the NIH grant 2U54 CA119347, The Fred L. Hartley FamilyFoundation, the Jonsson Cancer Center Foundation and the Caltech-UCLAJoint Center for Translational Medicine (all to A. Ribas).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 25, 2011; revised April 19, 2011; accepted April 21,2011; published OnlineFirst May 10, 2011.

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2011;17:4101-4109. Published OnlineFirst May 10, 2011.Clin Cancer Res Rong Rong Huang, Jason Jalil, James S. Economou, et al. HumansActivated Lymphocytes Regardless of Clinical Responses in CTLA4 Blockade Induces Frequent Tumor Infiltration by

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