SPECIAL ISSUE
`
`Pence Immunology and
`Immunotherapy
`
`UNIV. CHICAGO EX. 2035
`
`| DO NOT REMOVE FROM
`i) CURRENT PERIODICALS
`ROOM
`
`Genome & Co. v. Univ. of Chicago
`PGR2019-00002
`
`

`

`ubsidiaries unless otherwise specified. CO365
`
`STaoemBeSR
`
`OEMcaf
`Cebeecin]
`
`

`

`OAVVAY osRe
`\S
`
`» 418128
`
`How cells dampen
`gene noise
`
`
`3 APRIL 2015 * VOLUME 348 + ISSUE 6230
`
`NENS
`
`IN BRIEF
`
`20 AS EBOLA WANES, TRIALS JOCKEY FOR
`PATIENTS
`Researchers debate ending sometrials
`to allow others to go forward
`By K. Kupferschmidt
`
`42 INFANTS EXPLORE THE UNEXPECTED
`Infants are morelikely to explore objects
`that behave in unexpected ways, such as
`passing through walls By L. Schulz
`» RESEARCH ARTICLE P.91
`
`12 Roundupof the week’s news
`
`FEATURES
`
`22 DEEPWATER HORIZON:AFTER THE OIL
`Five years on, the world’s largest
`accidental marinespill has left subtle
`scars on the Gulf of Mexico
`By W. Cornwall
`: 27 Critics question plans to spray
`: dispersant in future deepspills
`: By W. Cornwall
`>» EDITORIAL P. 11; BOOKS ET AL. P. 49; PODCAST
`
`
`
`LETTERS
`
`32 NEXTGEN VOICES
`
`PERSPECTIVES
`
`36 APRUDENT PATH FORWARD FOR
`GENOMIC ENGINEERING AND GERMLINE
`GENE MODIFICATION
`A framework for open discourse on
`the use of CRISPR-Cas9 technology
`to manipulate the human genomeis
`urgently needed By D. Baltimore etal.
`
`38 DEFINING THE EPOCH WE LIVE IN
`Is a formally designated “Anthropocene”
`a good idea? By W. F Ruddimanet al.
`
`40 TRACKING ANTIWEAR FILM FORMATION
`Atomic force microscopyvisualizes the
`formation of a lubricating film
`By U. D, Schwarz
`» REPORT P.102
`
`IN DEPTH
`
`14 EGGS’ POWER PLANTS ENERGIZE
`NEW IVF DEBATE
`Firm adding energy-generating
`mitochondria to egg cells has already
`produced human pregnancies
`By J. Couzin-Frankel
`
`15 ACHILD-KILLING TOXIN EMERGES
`FROM SHADOWS
`Scientists link mystery deaths in India
`to consumptionof lychees By P Pulla
`
`17 ‘THE BLOB’ INVADESPACIFIC,
`FLUMMOXING CLIMATE EXPERTS
`Persistent mass of warm wateris
`reshuffling ocean currents, marine
`ecosystems, and inland weather
`By E. Kintisch
`
`18 HOAX-DETECTING SOFTWARE SPOTS
`FAKE PAPERS
`Springer jumps into sham submissions
`
`arms race By J. Bohannon
`
`44 HOW YOUNG STARS GROW AND
`BECOME FOCUSED
`Observations 18 years apart capture
`early changes of a massive star
`By M. G. Hoare
`» REPORT P. 114
`
`45 MULTIPLYING CANCER IMMUNITY
`A soluble ligand of an innate
`immunoreceptor arms natural
`killers for tumor attack
`By A, Steinle and A. Cerwenka
`>» REPORT P. 136; CANCER IMMUNOLOGY
`AND IMMUNOTHERAPYSECTION P. 54
`
`46 EBOLAAND BEYOND
`Recent experiences in confronting
`the Ebola epidemic suggest principles
`for vaccine efficacy trials in challenging
`environments By M. Lipsitch et al.
`
`BOOKS ETAL.
`
`49 p53
`By S. Armstrong, reviewed by
`A. Mandinova and S. W. Lee
`» CANCER IMMUNOLOGY AND
`IMMUNOTHERAPYSECTIONP. 54
`
`49 ASEAIN FLAMES
`By C. Safina
`» EDITORIALP. 11; NEWS STORYP. 22
`
`51 CORNELIA PARKER
`M. Griffiths, curator, reviewed by D. Dixon
`» VIDEO
`
`
`
`DEPARTMENTS
`
`11 EDITORIAL
`A community for disaster science
`By Marcia McNutt
`» NEWS STORY P. 22; BOOKS ETAL. P. 49;
`PODCAST
`
`150 WORKING LIFE
`A careeris like a love affair
`By Madeleine Jacobs
`
`
`
`
`
`PHOTO(BOTTOM):©GERALDHERBERT/AFYCORBIS:
`
`4) MicroRNAs SILENCE THE
`NOISY GENOME
`Evolution may have selected for a
`dampening service for genes whose
`SICIENGOSEANwes saxaxsuas sdpicvarvetten res ea otvaieerezceethes
`noise may have otherwise been too high
`
`New: Products .iccicucveyssveteacdsunaraipre 141
`By Y. Hoffman and Y. Pilpel
`
`» REPORT P.128
`Science Careers i.jieisscischavessienn co etteees 142
`
`SCIENCE sciencemag.org
`
`3 APRIL 2015 + VOL 348 ISSUE 6230
`
`5
`
`

`

`ap 4 ma ¢
`‘oe\\
`PRINTNAY
`
`UUNIENYo
`
`
`
`40 &102
`
`A look at what keeps engines
`running smoothly
`
`109 THERMOELECTRICS
`Dense dislocation arrays embedded
`in grain boundaries for high-
`performance bulk thermoelectrics
`S.J. Kim et al.
`
`SPECIAL SECTION
`
`Cancer Immunology
`and Immunotherapy
`
`INTRODUCTION
`
`ON THE COVER
`
`54 Realizing the promise
`REVIEWS
`
`56 The future of immune checkpoint
`therapy P Sharma and J. P. Allison
`
`62 Adoptivecell transfer as personal-
`ized immunotherapy for human
`cancer S.A. Rosenberg and N. P. Restifo
`
`
`
`Cancer immunotherapy
`harnesses the power
`of the immunesystem
`to kill tumors. These
`therapies aim to activate
`and expandT cells, such
`as those shown in blue,
`to specifically kill tumors
`(black). Current approaches
`69 Neoantigens in cancer
`include antibodies targeting inhibitory
`120 PLANT BIOLOGY
`immunotherapy T: N. Schumacher
`proteins onTcells, adoptive T cell therapy, and
`and R. D. Schreiber
`Suppression of endogenous
`tumorvaccines, among others. See page 54.
`gene silencing by bidirectional
`Illustration: Valerie Altounian/Science
`cytoplasmic RNA decay in
`Arabidopsis X. Zhang et al.
`
`114 STELLAR PHYSICS
`Observing the onset of outflow
`collimation in a massive protostar
`C. Carrasco-Gonzdlez et al.
`» PERSPECTIVE P. 44
`
`117 VIROLOGY
`Mutation rate and genotype
`variation of Ebola virus from Mali
`case sequences 7: Hoenen et al.
`
`124 CANCER IMMUNOLOGY
`Mutational landscape
`determinessensitivity to PD-1
`blockade in non-small cell lung
`cancer N. A. Rizvi et al.
`» CANCER IMMUNOLOGY AND
`IMMUNOTHERAPY SECTION P. 54
`
`128 GENE EXPRESSION
`MicroRNAcontrol of protein
`expression noise J. M. Schmiedelet al.
`» PERSPECTIVE P. 41
`
`132 EPIGENETICS
`Restricted epigenetic
`inheritance of H3K9 methylation
`P.N.C.B. Audergon et al.
`» RESEARCH ARTICLE P.90
`
`74 T cell exclusion, immune
`privilege, and the tumor microenvi-
`ronment J. A. Joyce and D. T. Fearon
`
`80 Cancer and the microbiota
`WS. Garrett
`
`RESEARCH
`
`IN BRIEF
`
`87 From Science and other journals
`
`RESEARCH ARTICLES
`
`90 EPIGENETICS
`Epigenetic inheritance uncoupled
`from sequence-specific recruitment
`K. Ragunathan et al.
`RESEARCH ARTICLE SUMMARY;FOR FULL TEXT:
`dx.doi.org/10.1126/science.1258699
`» REPORT P.132
`
`SEE ALSO » PERSPECTIVEP45 » BOOKS ET
`AL. P.49 » REPORTS PP. 124 &136 » REPORT BY
`B. M. CARRENO ETAL. 10.1126/science.aaa3823
`» SCIENCE CAREERS STORY BY R. BERNSTEIN
`
`
`
`95 RIBOSOME
`Thestructure of the human
`mitochondrial ribosome A. Amunts et al.
`» RESEARCH ARTICLE BYB. J. GREBER ET AL.
`10.1126/science.aaa3872
`
`REPORTS
`
`99 MOLECULAR PHYSICS
`Production oftrilobite Rydberg molecule
`dimers with kilo-Debye permanent
`electric dipole moments D. Booth et al.
`
`102 TRIBOLOGY
`Mechanismsof antiweartribofilm growth
`revealed in situ by single-asperity sliding
`contacts N. N. Gosvamietal.
`» PERSPECTIVE P. 40
`
`
`
`136 ANTITUMOR IMMUNITY
`A shed NKG2Dligand that
`91 COGNITIVE DEVELOPMENT
`promotes natural killer cell
`activation and tumorrejection
`106 FRUSTRATED MAGNETISM
`Observing the unexpected enhances
`W. Deng etal.
`Large thermal Hall conductivity of
`infants’ learning and exploration
`» PERSPECTIVE P. 45; CANCER IMMUNOLOGY
`A. E. Stahl and L. Feigenson
`neutral spin excitations in a frustrated
`AND IMMUNOTHERAPYSECTION P. 54
`» PERSPECTIVE P. 42
`quantum magnet M. Hirschberger etal.
`
`SCIENCE(ISSN 0036-8075)is published weekly on Friday, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue, NW, Washington, DC 20005. Periodicals
`mail postage (publication No. 484460)paid at Washington, DC, and additional mailing offices. Copyright © 2015 by the American Association for the Advancementof Science,Thetitle SCIENCEis a registered trademark of the AAAS.
`Domestic individual membership and subscription(51 issues): $153 ($74 allocated to subscription). Domesticinstitutional subscription(51 issues): $1282; Foreign postage extra: Mexico, Caribbean (surface mail) $55;other countries (air
`assist delivery) $25,First class,airmail, student, and emeritus rates on request, Canadianrates with GST available upon request, GST #1254 88122. Publications Mail Agreement Number 1069624. Printed In the U.S.A.
`Change of address: Allow 4 weeks,giving old and new addresses and 8-digit account number. Postmaster: Send change of address to AAAS, P.O, Box 96178, Washington, DC 20090-6178. Single-copy sales: $10.00 currentissue.
`$15.00 backissue prepaid includes surface postage: bulk rates on request. Authorization to photocopy material for internal or personal use undercircumstancesnotfalling within the fair use provisions of the Copyright Actis granted by
`AAASto libraries and other users registered with the Copyright Clearance Center (CCC) Transactional Reporting Service, provided that $30.00per article is paid directly to CCC, 222 Rosewood Drive, Danvers, MA.01923.The identification
`code for Science is 0036-8075, Scienceis indexed in the Reader's Guide to PeriodicalLiterature and in several specialized indexes.
`
`SCIENCE sciencemag.org
`
`3 APRIL 2015 + VOL 348 ISSUE 6230
`
`7
`
`

`

`CANCER IMMUNOLOGY AND IMMUNOTHERAPY
`IAG RVRKouuCem
`
`
`This material may be protected by Copyrightlaw(Title 17 U.S. Code)
`
`
`
`
`REVIEWS
`
`that inhibits BRAF (J, 2). These targeted ther-
`apies have led to promisingclinical responses,
`albeit generally of short duration, in patients
`whose tumors express the appropriate target
`biomarker.
`
`The future of immune
`checkpoint therapy
`
`Padmanee Sharma’’”* and James P. Allison’
`
`Immune checkpoint therapy, which targets regulatory pathwaysin T cells to enhance
`antitumor immune responses, has led to important clinical advances and provided a new
`weapon against cancer. This therapy has elicited durable clinical responses and, in a
`fraction of patients, long-term remissions where patients exhibit no clinical signs of cancer
`for many years. The way forward for this class of novel agentslies in our ability to
`understand human immuneresponsesin the tumor microenvironment. This will provide
`valuable information regarding the dynamic nature of the immune responseand regulation
`of additional pathways thatwill need to be targeted through combination therapies to
`provide survival benefit for greater numbersof patients.
`
`Tumor or
`
`
`
`
`
`
`APC's
`(dendritic cells,
`macrophages)
`
`
` T cell
`
`
`Fig. 1. Activation of T cells requires two signals. T cell activation occurs
`only after interaction between T cell receptor (TCR) and antigenin the context of
`MHC(signal 1) plus CD28 costimulation (signal 2).
`
`In the past two decades, remarkable advances
`in basic science have led to new strategies for
`the treatment of cancer, which are justifiably
`generating optimism that it may soon be pos-
`sible to cure a subset of patients with some types
`of cancer. We now havedetailed knowledge
`of the molecular basis of cancer to allow a more
`“personalized” treatment based on genomic se-
`quencing of an individual's cancercells to identify
`specific mutations in genes. These mutations
`can then be targeted with compoundsto block
`the downstream pathways that drive cancer
`development and progression. Therefore, each
`specific mutation serves as the predictive bio-
`markerfor selecting patients for treatment with
`a given agent. For example, patients with mela-
`noma whose tumors harbor the BRAFV600E
`mutation, which enables constitutive activa-
`tion of the BRAFsignaling pathway, would be
`selected to receive treatment with an agent
`
`
`
`‘Department of Immunology, M.D. Anderson Cancer Center,
`Houston, TX, USA. *Genitourinary Medical Oncology, M.D.
`Anderson Cancer Center, Houston, TX, USA.
`“Corresponding author. E-mail: padsharma@mdanderson.org
`(P.S.); jallison@mdanderson.org (J.P A.)
`
`56
`
`3 APRIL 2015 « VOL 348 ISSUE 6230
`
`sciencemag.org SCIENCE
`
`Theclinical success of genomically targeted
`agents laid the foundation for other cancer ther-
`apies, including the prerequisite to identify pre-
`dictive biomarkersfor selection of patients for
`treatment. Eventually, as the field of cancer im-
`munotherapy foundclinical success with agents
`based on a greater understanding of how to
`unleash T cell responses by targeting immune
`checkpoints, it became clear that the frame-
`work used for identification of predictive bio-
`markers for genomically targeted agents would
`present a challenge. As opposed to mutated genes
`in tumors that permanently mark a tumor, the
`immuneresponse is dynamic and changes rap-
`idly. Therefore, the issue facing the field of can-
`he field of immune check-
`cer immunotherapy may not be the
`point therapy has joined the
`identification of a single biomarker
`NoTcell
`ranks of surgery, radiation,
`to select a subset of patients for treat-
`chemotherapy, and targeted
`proliferation
`ment. Instead, we must assess the
`therapy as a pillar of cancer
`effectiveness of an evolving immune
`therapy. Three new immune check-
`response, define the immune re-
`sponse that contributes toclinical
`point agents have now been ap-
`proved by the U.S. Food and Drug
`benefit, and then, hopefully, drive
`Administration (FDA) for the treat-
`every patient’s immune response
`ment of melanoma, and there is a
`in that direction through combi-
`high expectation that these agents,
`nation therapies.
`and others in this class, will also
`be approved over the next several
`years for treatmentof patients with
`lung cancer, kidney cancer, bladder
`cancer, prostate cancer, lymphoma,
`and many other tumortypes. The
`antibodyagainst CTLA-4 ipilimu-
`mab was approved in 2011, and
`two antibodies against PD-1 (pem-
`brolizumab and nivolumab) were
`approved in 2014. These drugs rep-
`resenta radical and disruptive change
`in cancer therapy in two ways. First,
`they do not target the tumorcell,
`but target molecules involved in
`regulation of T cells, the soldiers
`of the immunesystem. And, perhaps in a more
`radical shift, the goal of the therapy is not to
`activate the immune system to attack particular
`targets on tumorcells, but rather to remove in-
`hibitory pathways that block effective antitumor
`T cell responses. Immune checkpoint therapy,
`with anti-CTLA-4 having longer follow-up than
`other agents, leads to durable clinical responses
`that can last a decade and more, but only in a
`fraction of patients. There are ongoing studies
`to identify predictive biomarkers with which to
`select patients for treatment with a particular
`agent, but the complexity of the immuneresponse
`has madethis difficult.
`
`proliferation
`
`,
`
`Tumor microenvironment:
`Cancercells and host
`immune responses
`Tumors are composed of manycell
`types, including the cell of origin
`with genetic alterations and a myr-
`iad of othercells, such as fibroblasts,
`endothelial cells, and eventually, per-
`haps, a variety of immunecells. Ini-
`tially the immuneinfiltrates may be
`scarce, but eventually may contain
`natural killer (NK) cells and mac-
`rophages with lytic capacity and,
`perhaps most importantly, T cells.
`T cells attack tumorcells that ex-
`press tumor-specific antigens in the form of com-
`plexes of tumor-derived peptides bound to major
`histocompatibility complex (MHC) molecules on
`the cell. The tumor antigens can be derived from
`oncogenic viruses, differentiation antigens, epige-
`netically regulated molecules such as cancer testes
`antigens, or neoantigens derived from mutations
`associated with the process of carcinogenesis (3).
`T cells survey the microenvironment and become
`activated when tumor antigens are recognized.
`They then proliferate and differentiate, ultimate-
`ly leading to the T cell’s ability to attack and de-
`stroy cells that express relevant antigens. However,
`regulation of T cell responses is an extremely
`complex process consisting of both stimulatory
`and inhibitory cell intrinsic signaling pathways,
`which limit T cell responses against cancer and
`prevent eradication of tumors.
`Recognition of antigen-MHC complexes by
`the T cell antigen receptoris not sufficient for
`
`

`

`
`
`—
`
`activation of naive T cells—additional costim-
`ulatory signals (4, 5) are required that are
`provided by the engagement of CD28 on the T
`cell surface with B7 molecules (CD80 and CD86)
`on the antigen-presenting cell (APC) (Fig. 1).
`Expression of B7 molecules is limited to subsets
`of hematopoietic cells, especially dendritic cells,
`which have specialized processes for efficient
`antigen presentation. With the exception of cer-
`tain lymphomas, cancercells do not express B7
`molecules, and henceare largely invisible to the
`immune system. This can be overcome by an in-
`flammatory response, such as the killing of tu-
`mor cells, which permits APCs, such as dendritic
`cells, to take up antigen and present antigen
`bound to MHC along with B7 molecules for ef-
`fective activation of T cells.
`After encountering tumorantigen in the con-
`text of B7 costimulation, initially in tumor-draining -
`lymph nodes, tumor-specific T cells may acquire
`effector function andtraffic to the tumorsite to
`mount an attack on the tumor. Infiltration of
`Tcells into the tumor microenvironmentis a
`critical hurdle that must be overcomefor an ef-
`fective antitumor immune response to occur.
`However, once T cells are in the tumor micro-
`environment, the success of the assault is deter-
`mined by their ability to overcome additional
`barriers and counter-defenses they encounter
`from the tumorcells, stroma, regulatory T cells,
`myeloid-derived suppressorcells, inhibitory cyto-
`kines, and other cells in the complex tumor mi-
`croenvironment that act to mitigate antitumor
`immuneresponses.
`In the 1980s, tumor antigens from human
`melanomas were foundto elicit T cell responses
`(6), which drove efforts to use vaccination strat-
`egies to mobilize the immune system to attack
`cancer, The vaccines generally consisted of some
`form of the antigen (for example, peptide or DNA
`vaccines), as well as additional components
`to enhance responses (for example, cytokines).
`
`While there were anecdotal successes, in hun-
`dreds of trials there was scant evidence of re-
`producible clinical responses (7). This failure
`to induce effective immune responses by attempt-
`ing to turn T cell response “on” with antigenic
`vaccines led many to become skeptical of the
`potential of immunotherapy as a strategy for
`cancer treatment.
`
`Regulation of T cell responses
`Furtherinsights into the fundamental mecha-
`nisms that regulate early aspects of T cell ac-
`tivation may provide one of many possible
`explanations for the limited effectiveness of
`these early vaccine trials. By the mid-1990s,it
`was becoming clear that T cell activation was
`even more complex, and in addition to initiat-
`ing proliferation and functional differentia-
`tion, T cell activation also induced an inhibitory
`pathway that could eventually attenuate and
`terminate T cell responses. Expression of ctla-4,
`a gene with very high homology to CD28,is ini-
`tiated by T cell activation, and, like CD28, CTLA-+
`binds B7 molecules, albeit with much higher
`affinity. Although CTLA-4 was first thought to
`be another costimulatory molecule (8), two lab-
`oratories independently showed that it opposed
`CD28 costimulation and down-regulated T cell
`responses (9, 10). Thus, activation of T cells re-
`sults in induction of expression of CTLA-4, which
`accumulates in the T cell at the T cell-APC inter-
`face, reaching a level where it eventually blocks
`costimulation and abrogates an activated T cell
`response (Fig. 2).
`Based on knowledge ofthe function of CTLA4,
`we proposed that blocking its interaction with
`the B7 molecules might allow T cell responses
`to persist sufficiently to achieve tumor eradica-
`tion. We hypothesized that this could be achieved
`by releasing the endogenous immune responses,
`perhaps even without specific knowledge of
`
`the antigenic targets of those responses or even
`
`the type of cancer. We also proposed that com-
`bination treatment with an antibody against
`CTLA~4: and agents that directly killed tumorcells
`to release antigens for presentation by APCs to
`T cells would improve antitumorresponses. Our
`hypotheses were tested in many different ex-
`periments in mice (JJ-15), with data generated
`to support the concept, leading to the develop-
`ment of ipilimumab, an antibody against hu-
`man CTLA~: forclinical testing. Ipilimumabled
`to considerable improvementin overall survival
`for patients with metastatic melanoma (16, 17),
`which led to FDA approval in 2011.
`The preclinical successes of anti-CTLA-4 in
`achieving tumorrejection in animal models and
`the ultimate clinical success opened a newfield
`of immune checkpoint therapy (78, 19). It is now
`known that there are many additional immune
`checkpoints. Programmedcell death-1 (PD-1)
`was shown in 2000 to be another immune check-
`
`point that limits the responses of activated T
`cells (20). PD-1, like CTLA-4, has twoligands,
`PD-L1 and PD-L2, which are expressed on many
`cell types. The function of PD-1 is completely
`distinct from CTLA-+ in that PD-1 does notinter-
`fere with costimulation, but interferes with sig-
`naling mediated by the T cell antigen receptor
`(4). Also, one of its ligands, PD-L1 (B7-H1), can be
`expressed on many cell types (Fig. 2), including T
`cells, epithelial cells, endothelial cells, and tumor
`cells after exposure to the cytokine interferon-y
`(IFN-y), produced by activated T cells (27). This
`has led to the notion that rather than function-
`ing early in T cell activation, the PD-1/PD-L1 path-
`way acts to protect cells from T cell attack.
`
`Immune checkpoint therapyin the clinic
`Ipilimumab, a fully human antibody to human
`CTLA~, entered clinical trials in the late 1990s and
`early 2000s. As predicted, tumor regression was
`. observed in patients with a variety of tumortypes.
`Phase I/II trials showed clinical responses in
`
`Activated T cells
`up-regulate immune
`checkpoint molecules
`such as CTLA-4 and PD-1,
`which act to abrogate
`|,
`
`/)
`
`T cell responses cs\ re
`
`Antibody blockade of
`immune checkpoints
`enhances T cell responses
`
`
` Immune
`
`Immune
`checkpoint
`_
`2 therapy
`
`Activated T cells make
`
`IFN-ywhichincreases ® @
`PD-Llexpression @ ©
`a
`8
`
`@
`© . a
`e
`
`e
`
`
`
`at this time
`would show
`
`PD-L1-positive
`cells
`
`PD-1
`
`CTLA-4
`
`checkpoint
`: therapy
`
`anti-
`CTLA-4
`
`Biopsy
`at this time
`would show
`
`:
`Utell
`
`PD-Ll-negative 9—————-__
`cells
`EnhancedT cell infiltration
`into tumor tissue
`
`Fig. 2. Blockade of immune checkpoints to enhance T cell responses.After T cell activation, T cells express immune checkpoints such as CTLA-4. and PD-L. A
`biopsy of tumors taken from patients before treatrnent with immune checkpoint therapy (so priorto infiltration of activated Tcells into tumortissues) mayindicate
`lack of PD-L1 expression. However, uponTcell activation,T cells can traffic to tumors, up-regulate expression of immune checkpoints such as CTLA-4 and PD-1,
`and produce cytokines such as IFN-y, which leads to expression of PD-L1 on tumorcells and othercells, including T cells, within the turnor tissues.
`
`SCIENCE sciencemag.org
`
`3 APRIL 2015 » VOL 348 ISSUE 6230
`
`57
`
`

`

`SPECIAL SECTION CANCER IMMUNOLOGY AND IMMUNOTHERAPY
`
`
`
`
` CTLA-4: blockade (25, 26). The most pronounced
`
`difference was an increase in T cells that ex-
`press inducible costimulator (ICOS), a T cell
`surface molecule that is a closely related mem-
`ber of the extended CD28/CTLA-4 family. We
`confirmed our gene expression studies by flow
`cytometry. ICOS* T cells were increased in tumor
`tissues from patients treated with ipilimumab
`(36). The increase in the frequency of ICOS* T
`cells in tumorinfiltrates was accompanied by
`similar increases in the blood. These data, cou-
`pled with otherstudies, showed that an increase
`in the frequency of ICOS* CD4- T cells served as
`a pharmacodynamic biomarker of anti-CTLA-+
`treatment(37).
`To test our hypothesis that ICOS* CD4 T cells
`might play a role in the therapeutic effect of
`CTLA-4: blockade, we conducted studies in mice.
`In wild-type C57BL/6 mice, anti-CTLA-4: treat-
`ment resulted in tumorrejection in 80 to 90%
`of mice, but in gene-targeted mice that were
`deficient for either ICOSorits ligand, the ef-
`ficacy was less than 50% (38). The loss of ef-
`ficacy of CTLA-4 blockade in the absence of an
`intact ICOS pathway indicates the critical im-
`portance of ICOS to the therapeutic effects of
`treatment with anti-CTLA-4 antibodies. The im-
`portant role played by ICOS in the effectiveness
`of CLTA-4 blockade suggested that providing
`an agonistic stimulus for the ICOS pathway
`during anti-CTLA-4 therapy might increaseits
`effectiveness. To test this notion, we conducted
`studies in mice to provide an agonistic signal
`through ICOS in combination with CTLA-4 block-
`ade. We found that combination therapy resulted
`in an increase in efficacy that was about four
`to five times as large as that of control treatments
`(39). Thus, ICOSis a stimulatory checkpoint that
`provides a novel target for combination immu-
`notherapy strategies. Antibodies for ICOS are
`being developed for clinical testing, which are
`expected to start within the next year,
`Whereas some presurgical and tissue-based
`trials are focused on evaluating human immune
`responses in the tumor microenvironment, other
`studies have focused on evaluating components
`of the cancer cells that may contribute to clin-
`ical benefit with anti-CTLA-4. Genetic analyses
`of melanoma tumors revealed that higher num-
`bers of mutations, termed “mutational load,”
`and creation of new antigens that can be recog-
`nized by T cells as a result of these mutations,
`termed “neoantigens,” correlated with clinical
`responses to anti-CTLA-+ therapy (3, 40). These
`studies provide a strong rationale to integrate
`genetic analyses of the tumor with immunepro-
`filing of the tumor microenvironment for a more
`comprehensive evaluation of mechanisms that
`contribute to clinical responses with anti-CTLA-+
`therapy.
`
`Tissue-based immune monitoring:
`Anti-PD-1/PD-L1 therapy
`Given that immune checkpoint therapy only
`benefits a fraction of patients, there are ongoing
`efforts to identify predictive biomarkers that
`could be used to select patients for treatment.
`
`sciencemag.org SCIENCE
`
`patients with melanoma(22), renal cell carcinoma
`(23), prostate cancer (24), urothelial carcinoma
`(25), and ovarian cancer (26). Two phaseIII clin-
`ical trials with anti-CTLA-4 (ipilimumab) were
`conducted in patients with advanced melanoma
`and demonstrated improved overall survival for
`patients treated with ipilimumab (76, 17). Impor-
`tantly, durable responses were observed in about
`20% of patients living for more than 4 years, in-
`cluding a recent analysis indicating survival of
`10 years or more for a subset of patients (27).
`Antibodies targeting the PD-1/PD-L1 axis have
`also shown clinical responses in multiple tu-
`mortypes. Anti-PD-L1 antibodies led to tumor
`regression in patients with melanoma, renal
`cell carcinoma, non-small cell lung cancer (28),
`and bladder cancer (29). Phase I clinical trials
`with anti-PD-1 (nivolumab) demonstrated sim-
`ilar clinical responses (30). Recently, a large
`phase I clinical trial with the anti-PD-1 antibody
`MK-3475 was shown to lead to responserates of
`~37 to 38% in patients with advanced melanoma
`(31), with a subsequent study reporting an over-
`all response rate of 26% in patients who had
`progressive disease after prior ipilimumab treat-
`ment(32), which led to FDA approval of MK-
`3475 (pembroluzimab) in September 2014. A
`phaseIII trial of a different anti-PD-1 antibody
`(nivolumab) also showed clinical benefit in pa-
`tients with metastatic melanoma. In this trial,
`the objective response rate was 4.0% and over-
`all survival rate was 72.9% for patients treated
`with nivolumab as compared to an objective
`response rate of 13.9% and overall survival rate
`of 42.1% for patients treated with dacrabazine
`chemotherapy (33). Nivolumab received FDA
`approval in December 2014 as a treatment for
`patients with metastatic melanoma. In addi-
`tion, nivolumab was FDA-approved in March
`2015 for patients with previously treated ad-
`vanced or metastatic non-small cell lung cancer
`based on a phaseIIIclinical trial, which reported
`an improvement in overall survival for patients
`treated with nivolumab as comparedto patients
`treated with docetaxel chemotherapy.
`That CTLA-4 and PD-1 regulate distinct in-
`hibitory pathways and have nonoverlapping
`mechanismsof action suggested that concurrent
`combination therapy with both might be more
`efficacious than either alone. This was indeed
`shown to be the case in preclinical studies in
`murine models (34). In 2013, a phaseI clinical
`trial with anti-CTLA4: (ipilimumab) in combi-
`nation with anti-PD-1 (nivolumab) demonstrated
`tumor regression in ~50% of treated patients
`with advanced melanoma, most with tumorre-
`gression of 80% or more (35). There are ongoing
`clinical trials with anti-CTLA-4 plus anti-PD-1,
`or anti-PD-L1, in other tumor types, with pre-
`liminary data indicating promising results, which
`highlight this novel combination as an effective
`immunotherapy strategy for cancer patients.
`
`Tissue-based immune monitoring:
`Anti-CTLA-4 therapy
`Properly designed presurgical or tissue-based
`trials, where treatmentis administered before
`
`58
`
`3 APRIL 2015 + VOL 348 ISSUE 6230
`
`surgical resection of tumors, can provide val-
`uable insight into the cellular and molecular
`mechanisms of immune checkpoint therapy
`by providing sufficient tissues to conduct a bat-
`tery of analyses. Data gathered from analysis of
`tumortissue can then guide rational searches
`for relevant markers in the blood. We designed
`the first presurgical clinical trial with anti-CTLA-4.
`(ipilimumab), which was administered to 12
`patients with localized bladder cancerprior to
`radical cystectomy (36). The endpointsof this
`study were safety and access to samples for im-
`mune monitoring. We did not view this trial
`as a neoadjuvant study, which administers ther-
`apy prior to surgery for clinical benefit, but as a
`presurgical study to provide mechanistic insights
`regarding the impact of anti-CTLA-4 therapy
`on the tumor microenvironment. Unexpectedly,
`
`“Because of the very nature
`ofimmune
`oint ther-
`apy, the development of
`pharmacodynamic, predic-
`tive, or prognostic biomark-
`ersfaces unique challenges.”
`
`the trial enabled us to detect a clinical signal
`for anti-CTLA-4 as a therapeutic agent for pa-
`tients with bladder cancer since three patients
`had no residual tumors identified within the
`cystectomy samples. This trial was also success-
`ful in establishing the safety of anti-CTLA-4 in
`the presurgical setting, which would be impor-
`tant for future trials, and obtaining patients’
`matched tumor and blood samples for immune
`monitoring. This work laid the foundation for
`using presurgical trials as an important tool to
`evaluate human immuneresponses in the tumor
`microenvironment, which should be included
`in the current paradigm of phase I, II, and III
`clinicaltrials.
`The collection of fresh tumor samples at the
`time of surgery can provide sufficient tissue for
`genetic, phenotypic, and functional studies, as
`well as material for immunchistochemical (THC)
`analyses, which can provide extensive insight
`into the biologic impact of the immunotherapy
`agent on the tumor microenvironment. For ex-
`ample, high-quality mRNA can be obtained for
`gene expression studies comparing posttreatment
`tumortissues to pretreatment tumortissues or
`untreated samples obtained from a stage-matched
`control group ofpatients. These types of studies
`allow unbiased analyses of the samples to iden-
`tify novel genes and pathways that are affected
`by therapy. In ouripilimumabtrial, gene array
`data revealed that most of the differences be-
`tween treated and untreated samples could be
`attributed to pathways involved in T cell signal-
`ing, which is not surprising given the large in-
`creases in T cell infiltrates in tumortissues after
`
`
`
`
`

`

`
`
`Because the PD-1 ligand PD-L1 (and sometimes
`PD-L2) can be expressed on tumorcells and im-
`mune cells in the tumor microenvironment,
`there have been efforts to use expression of PD-
`L1 as a criterion for selecting patients for treat-
`ments with antibodies targeting the PD-1/PD-L1
`pathway.
`Theinitial phase I trial with anti-PD-1 therapy
`(nivolumab) reported that PD-L1 expression
`on tumorcells, measured on pretreatmentar-
`chival samples by immunohistochemical (IHC)
`methods, may potentially serve as a predictive
`marker to indicate which patients would bene-
`fit from treatment (30). Patients with PD-Li-
`positive tumors (25% staining for PD-L1 on tumor
`cells) had an objective response rate of 36% (9
`of 25 patients) whereas patients with PD-L1-
`
`
`
`
`Immunogenic tumor
`microenvironment
`
`Immune checkpoint therapy
`and durable clinical benefit
`
`negative tumors did not show any objective
`clinical responses (0 of 17 patients). However,
`in subsequenttrials, some patients whose tu-
`mors were deemed to be PD-Li-negative had
`clinical responses to anti-PD-1 and anti-PD-L1
`treatments with either tumor regression or sta-
`bilization of disease. For example, on a phase I
`trial with anti-PD-1 (nivolumab), patients with
`PD-L1-positive tumors had an objective response
`rate of 44% (7 of 16) and patients with PD-LI-
`negative tumors had an objective response rate
`of 17% (8 of 18) (41). Although PD-L1 expression
`in tumortissues does correlate with higher re-
`sponserates, it is not predictive for clinical ben-
`efit. Furthermore, current data indicate that the
`differences in response rates do not translate to
`differences in surviva