SPECIAL ISSUE
`
`_ DO NOT REMOVE FROM
`
`I CURRENT PERIODICALS
`
`Cancer Immunology and
`Immunotherapy
`
`UNIV. CHICAGO EX. 2035
`
`ROOM
`
`Genome & Co. v. Univ. of Chicago
`PGR2019—00002
`
`

`

`
`
`ThermcIFisheri‘ enuhci
`
`.N1nghisrestrvedfifltrademnrk 1r: thepr.
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`
`3 APRIL 2015 - VOLUME 34-8 0 ISSUE 6230
`
`EELS
`
`IN BRIEF
`
`20 AS EBOLA WANES. TRIALS JOCIIEY FOR
`PATIENTS
`Researchers debate ending some trials
`to allow others to gO forward
`By K. Kapferschmidt
`
`42 INFANTS EXPLORE THE IINEXPECTED
`Infants are more likely to explore objects
`that behave in unexpected ways, such as
`passing through walls By L. Schulz
`r RESEARCH ARTICLE P. 91
`
`12 Roundup of the week‘s news
`
`FEATURES
`
`IN DEPTH
`
`14 ECDS’ POI'IER PLANTS ENERCIZE
`NE’I'I WF DEBATE
`Firm adding energy-generating
`mitochondria to egg cells has already
`produced human pregnancies
`By J. Cousin-Wankel
`
`15 A CHILD-KILLING TOXIN EIIIERGES
`FROIII SHADOWS
`Scientists link mystery deaths in India
`to consumption of lychees By P. Patio
`
`1? 'THE BLOII' INVADES PACIFIC.
`FLUAINOXING CLIMATE EXPERTS
`Persistent mass of warm water is
`reshuffling ocean currents, marine
`ecosystems, and inland weather
`By E. Ktnrisch
`
`13 HOAX-DETECTING SOFTWARE SPOTS
`FAIIE PAPERS
`Springer jumps into sham submissions
`arms race ByJE Bahama
`
`
`
`SCIENCE sciencemag.org
`
`22 DEEPWATER HORIZON: AFTER TIIE OIL
`Five years on, the world's largest
`accidental marine spill has left subtle
`scars on the Gulf of Mexico
`By W: Cornwall
`
`27 Crltlcs questlen plans to spray
`dispersant In future deep spllls
`By W. Cornwall
`r EDITORIAL P. 11: BOOKS ETAL. P. 49: PODCAST
`
`
`
`LETTERS
`
`32 NEXTGEN 1I'IIIICES
`
`PERSPECTIVES
`
`36 A PRlIDENT PATH FORWARD FDR
`GENOAIIC ENGINEERING AND GERAILINE
`GENE MODIFICATION
`A framework for open discourse on
`the use Of CRISPR—CasQ technology
`to manipulate the human genome is
`urgently needed By D. Baltimore et a1.
`
`38 DEFINING TIIE EPOCH WE LIVE IN
`Is a formally designated "iAnthrOpocene”
`a good idea? By W. E Ruddtman et a1.
`
`40 TRACKING ANTII'IEAR FILIII FORMATION
`Atomic force microscopy visualizes the
`formation of a lubricating film
`By U. D. Software
`r REPORT P. 102
`
`41 IIIIcroRNAs SILENCE THE
`NOISY CENOIAE
`Evolution may have selected for a
`dampening service for genes whose
`noise may have otherwise been too high
`By Y. Hofi‘man and K Pilpel
`r REPORT P.128
`
`44 HOW YOUNG STARS GRO’N AND
`BECOME FOCUSED
`'
`Observations 18 years apart capture
`early changes of a massive star
`By M. G. Home
`» REPORT P. 114
`
`45 HOLTIPL‘I'INC CANCER IAIIIIIINITY
`A soluble ligand of an innate
`immunoreceptor arms natural
`killers for tumor attack
`By A. Statute and A. Cerwenko
`r REPORT P. 136: CANCER IMMUNOLOGY
`AND IMMUNOTHERAPY sECTION P. 54
`
`46 EDOLA AND BEYOND
`Recent experiences in confronting
`the Ebola epidemic suggest principles
`for vaccine efficacy trials in challenging
`environments ByM. Lipsitch. et a].
`
`BOOKS ETAL.
`
`49 p53
`By S. Amt-mag, reviewed by
`A. Mandfnom and S. W Lee
`p CANCER IMMUNOLOGY AND
`IMMUNOTHERAPY SECTION P154
`
`49 A SEA IN PLANES
`ByC. Sarina
`s EDITORIAL P. 11: NEws STORY P. 22
`
`51 CORNELIA PARKER
`M. (la-mime, curator; reviewed by D. Dir-0n
`, VIDEO
`
`
`DEPARTMENTS
`
`11 EDITORIAL
`A community for disaster science
`By Marcia McNuIt
`5 NEWS STORY P. 22: BOOKS ETAL P. 49:
`PODCAST
`
`150 ’NORIIING LIFE
`A career is like a love affair
`ByMadelei-ne Jacobs
`
`ScienceStaffS
`New Products.......
`..... 141
`
`Science Careers ......................................... 142
`
`3 APRIL 2015 . VOL 343 ISSUE 6230
`
`5
`
`
`
`
`
`more(BOTTOM):QGERALDHERBERT/nP/CORBI‘S
`
`

`

`/
`
`a
`
`,
`
`I
`
`7
`Z
`
`V /
`
`\\\“’...‘\\\ m
`HI, “\\\\\\“
`r;%.
`
`
`40 8: 102
`A look at what keeps engines
`running smoothly
`
`
`
`INTRODUCTION
`
`SPECIAL SECTION
`
`Cancer Immunology
`and Immunotherapy
`ON THE COVER
`
`54 Realizing the promise
`REvIEws
`
`56 The future of immune checkpoint
`therapy P. Shanna and J. P. Allison
`
`62 Adoptive cell transfer as personal-
`ized immunotherapy for human
`cancer S. A. Rosenberg and N. P. Restifo
`
`69 Neoantigens in cancer
`immunotherapy T. N. Schumacker
`and R. D. Schmiber
`
`74 T cell exclusion, immune
`privilege, and the tumor microenvi»
`ronmcnt .1 A. Joyce and D. T. Foam
`
`80 Cancer and the microbiota
`W 3. Garrett
`
`
`
`Cancer immunotherapy
`harnesses the power
`of the immune system
`to kill tumors. These
`therapies aim to activate
`and expand T cells, such
`as those shown in blueI
`to specifically kill tumors
`(black). Current approaches
`include antibodies targeting inhibitory
`proteins on T cells, adoptive T cell therapy, and
`tumor vaccines, among others. See page 5%.
`Illustration: Vaten‘e Absentee/Science
`
`SEE ALSO b PERSPECI IVE; P. 45 b BOOKS ET
`AL P. 4'3 r REPORTS P9124 8:136 II REPORT BY
`8. M. CARRILNO ETAL. 10 1126.1’5CIcncc-naaES28
`5 SCIENCE CAREERS STORY BY R. BERNSTEIN
`
`RESEARCH
`
`IN BRIEF
`
`8? From Science and other journals
`
`RESEARCH ARTICLES
`
`90 EPIGENETIGS
`Epigenetic inheritance uncoupled
`from sequence-specific recruitment
`K. Regunothan et a1.
`RESEARCH ARTICLE SUMMARY; FOR FULL TEXT:
`dx.doi.orgf10.llZB/sclenceJZSSGQQ
`» REPORT P. 132
`
`95 RIBOSOME
`The structure of the human
`mitochondrial ribosome A. Amunts et al.
`, RESEARCH ARTICLE BY 3. J. GRERER ETAL.
`lOJlZS/scienceaaaBSTZ
`
`REPORTS
`
`99 MOLECULAR PHYSICS
`Production of trilobite Rydberg molecule
`dimers with kilo-Debye permanent
`electric dipole moments D. Booth et a1.
`
`
`
`102 TRIBOLOGY
`Mechanisms of antivvear tribofilm growth
`revealed in situ by single-asperity sliding
`contacts N N. Gostumt et al.
`) PERSPECTIVE P. 40
`
`109 THERMOELEOTRIOS
`Dense dislocation arrays embedded
`in grain boundaries for high-
`performance bulk thermoelectrics
`S. I. Kim et al.
`
`114 STELLAR PHYSICS
`Observing the onset of outflow
`collimation in a massive protostar
`C. Corrosco-Gonzétez et a1.
`, PERsPECTIvE P. 44
`
`117 VIROlOGY
`Mutation rate and genotype
`variation of Ebola virus from Maii
`case sequences I: Hoenen et al.
`
`120 PLANT BIOLOGY
`Suppression of endogenous
`gene silencing by bidirectional
`cytoplasmic RNA decay in
`Ambtdopszls X. Zhang et al.
`
`124 RANGER IMMUNOLOGY
`Mutational landscape
`determines sensitivity to PD—I
`blockade in non—small cell lung
`cancer N. A. Rr‘zm‘ et a1.
`5 CANCER IMMUNOLOGY AND
`IMMUNCTHERAPv SECTION P. 54
`
`123 GENE EKPRESOION
`MicroRNA control of protein
`expression noise J. M. Schmiedet et al.
`D PERSPECTIVE P. 41
`
`132 EPIGENETIGS
`Restricted epigenefic
`inheritance of H3K9 methylation
`R N. C. B. Audergtm et al.
`r RESEARCH ARTICLE P, so
`
`
`
`136 ANTITUROR IMMUNITY
`A shed NKG2D ligand that
`91 OOGNITI'IIE DEVELOPMENT
`promotes natural killer cell
`activation and tumor rejection
`106 FRUSTRATEIJ MAGNETISM
`Observing the unexpected enhances
`W. Deng et al.
`infants’ learning and exploration
`Large thermal Hall conductivity of
`» PERSPECTIVE P. 45: CANCER IMMUNOLOGY
`neutral spin excitations in a frustrated
`A. E. Starr: and L. Fetgenson
`AND IMMUNOTHERAPY SECTION P. 54
`» PERSPECTIVE P. 42
`quantum magnet M. Hirschberger et al.
`
`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 ‘I’brk Avenue. NW. Washington. DC 20005. Period-.cals
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`3 APRIL 2015 - VOL 348 ISSUE 6230
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`7
`
`

`

`CANCER rMMUNOLOGYAND IMMUNO THERAPY
`SPECIAL sac-now
`
`
`This materia' may be Protected by Copyright 'aW (Title 17 ”-5- Code)
`
`
`
`
`that inhibits BRAF (I, 2). These targeted ther-
`apies have led to promising clinical responses,
`albeit generally of short duration, in patients
`whose tumors express the appropriate target
`biomarker.
`
`REVIEWS
`
`The future of immune
`
`checkpoint therapy
`
`Padrnanee Shannan“ and James P. Allison”
`
`Immune checkpoint therapy, which targets regulatory pathways in Tcells 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 agents lies in our ability to
`understand human immune responses in the tumor microenvironment. This will provide
`valuable information regarding the dynamic nature of the immune response and regulation
`of additional pathways that will need to be targeted through combination therapies to
`provide survival benefit for greater numbers of patients.
`
`Tumor or
`
`
`No Tcell
`
`proliferation
`
`
`The clinical success of genomically targeted
`agents laid the foundation for other cancer ther-
`apies, including the prerequisite to identify pre-
`dictive biomarkers for selection of patients for
`treatment Eventually, as the field of cancer im-
`munotherapy found clinical 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 bier
`markers for genomically targeted agents would
`present a challenge. As opposed to mutated genes
`in tumors that permanently mark a tumor, the
`immune response is dynamic and changes rap-
`idly. Therefore, the issue facing the field of can—
`cer immunotherapy may not be the
`identification of a single biomarker
`to select a subset of patients for treat-
`ment. Instead, we must assess the
`effectiveness of an evolving immune
`response, define the immune re-
`sponse that contributes to clinical
`benefit, and then, hopefully, drive
`every patient’s immune response
`in that direction through combi-
`nation therapies.
`
`
`
`
`
`he field of immune check-
`point therapy has joined the
`ranks of surgery, radiation,
`chemotherapy, and targeted
`therapy as a pillar of cancer
`therapy. Three new immune check-
`point agents have now been ap-
`proved by the [1.8. Food and Drug
`Administration (FDA) for the treat-
`ment of melanoma, and there is a
`high expectation that these agents,
`and others in this class, will also
`be approved over the next several
`years for treatment of patients with
`lung cancer, kidney cancer, bladder
`cancer, prostate cancer, lymphoma,
`and many other tumor types. The
`antibody against C'I‘LA-4 ipilimu-
`mab was approved in 2011, and
`two antibodies against PD—l (pem-
`brolizumab and nivolumab) were
`approved in 2014-. These drugs rep-
`resent a radital and disruptive cliange
`in cancer therapy in two ways. First,
`they do not target the tumor cell,
`but target molecules involved in
`regulation of T cells, the soldiers
`of the immune system. And, perhaps in a more
`radical shift, the goal of the therapy is not to
`activate the immune system to attack particular
`targets on tumor cells, but rather to remove in-
`hibitory pathways that block effective antitumor
`T cell responses. Immune checkpoint therapy,
`with anti-Clue 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
`agenlz, but the complexity of the immune response
`has made this difficult.
`
`APC's
`(dendritic cells.
`macrophages}
`
`
`
`
`Tcell
`proliferation
`
`Fig. 1. Activation of T cells requires two signals. T cell activation occurs
`only after interaction between Tcell receptor (TCR) and antigen in 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 have detailed knowledge
`of the molecular basis of cancer to allow a more
`“personalized" treatment based on genomic se-
`quencing of an individual's cancer cells to identify
`specific mutations in genes. These mutations
`can then be targeted with compounds to block
`the downstream pathways that drive cancer
`development and progression. Therefore, each
`specific mutation serves as the predictive bio-
`marker for selecting patients for treatment with
`a given agent For example, patients with mela—
`noma whose tumors harbor the BRAFVGOOE
`mutation, which enables constitutive activa-
`tion of the BRAF signaling pathway, would be
`selected to receive treatment with an agent
`
`
`
`1Department oi Immunology. M.[). Anderson Cancer Center.
`Houston. TX. USA. aGenitourinary Medical Oncology. MD.
`Anderson Cancer Center. Houston. TX. USA.
`'Corrasponding author. E—mail: padsharma®mdandersonorg
`(P3): jallison®mdandersoaorg (M7 A.)
`
`56
`
`3 APRIL 2015 . v01, 3+3 ISSUE 6230
`
`Ttnnor microenvironment:
`Cancer cells and host
`immune responses
`
`I'r
`
`sciencemagcrg SCIENCE
`
`Tumors are composed of many cell
`types, including the cell of origin
`with genetic alterations and a myr-
`iad of other cells, such as fibroblasts,
`endothelial cells, and eventually, per-
`haps, a variety of immune cells. Ini-
`tially the immune infiltrates 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 tumor cells that ex—
`press tumor-specific antigens in the form of com-
`pletes of tumorderived peptides bound to major
`histocompatibility complex (MHC) molecules on
`the cell. The tumor antigens can be derived from
`oncogenic viruses, difierentiation antigens, epige-
`netimlly regulated molecules such as cancer testw
`antigens, or neoantigens derived from mutations
`associated with the process of carcinogenesis (3).
`T cells survey the microcnvironment 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 stimulaton
`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 receptor is not sufficient for
`
`

`

`
`
`__.._--""
`
`activation of naive T cells—additional costirn-
`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 (AFC) (Fig. 1).
`Expression of B7 molecules is limited to subsets
`of hematopoietic cells, especially dendritic cells,
`wtiich have specialized processes for efficient
`antigen presentation. With the exception of cer—
`min lymphomas, cancer cells do not express B7
`molecules, and hence are 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.
`Afier encountering tumor antigen in the con-
`text of B7 mstimulation, initially in nnnordraining .
`lymph nodes, tumor—specific T cells may acquire
`effector function and traffic to the tumor site to
`mount an attack on the tumor. Infiltration of
`’1‘ cells into the tumor microenvironment is a
`critical hurdle that must be overcome for 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 tumor cells, stroma, regulatory T cells,
`myeloid-derived suppressor cells, inhibitory cyto-
`kines, and other cells in the complex tumor mi-
`croenvironment that act to mitigate antitumor
`immune responses.
`In the 19805, tumor antigens from human
`melanomas were found to 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).
`
`Activated T cells
`up-regulate immune
`checkpoint molecules
`such as CTLA-4 and PD-l.
`.I'.
`which act to abrogate
`-
`
`Tcell responses We'
`.
`«:'
`t
`
`
`Immune
`checkpoint
`
`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 response-3 by attempt-
`ing to turn T cell response "on" with antigenic
`vaccines led many to become skeptical of the
`potential of inununotherapy as a strategy for
`cancer treatment
`
`Regulation of Tcell responses
`Further insights 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-19905, 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 odd-‘14,
`a gene with very high homology to CD28, is ini-
`tiated by T cell activation, and, like CD28, CI‘LA4
`binds B7 molecules, albeit with much higher
`affinity. Although CILA—d 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 ETTA-4, which
`accumulates in the T cell at the T cell—AFC inter-
`face, reaching a level where it eventually blocks
`costimulation and abrogares an activated T cell
`response (Pig. 2).
`Based on knowledge of the function of (ETTA-43
`we proposed that blocking its interaction with
`the B7 molecules might allow T cell responses
`to persist sufl‘iciently 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
`CIlA—e and agents that directly killed tumor cells
`to release antigens for presentation by APC's to
`T cells would improve antitumor responses. Our
`hypotheses were tested in many different ex-
`periments in mice (11—15), with data generated
`to support the concept, leading to the develop-
`ment of ipilimnmab, an antibody against hu-
`man CI'LA-Kt for clinical testing. Ipilimumab led
`to considerable improvement in overall survival
`for patients with metastatic melanoma (16, 1?},
`which led to FDA approval in 2011.
`The preclinical successes of anti-CI‘LA-e in
`achieving tumor rejection in animal models and
`the ultimate clinical success opened a new field
`of immune checkpoint therapy (18, 1.9). It is now
`known that there are many additional immune
`checkpoints. Programmed cell death—1 (PD-1)
`was shown in 2000 to be another immune check—
`
`point that limits the responses of activated T
`cells (20}. PD-I, like C'I‘lA-‘t, has two ligands,
`PD—Ll and PD-L2, which are expressed on many
`cell types. The function of PD—l is completely
`distinct from CI'LA-‘L in that PD-l does not inter—
`fere with costimuiation, but interferes with sig-
`naling mediated by the T cell antigen receptor
`(4.). Also, one of its ligands, PD-Ll (B7-HI), can be
`expressed onmanycelltypesfig 2),including'l‘
`cells, epithelial cells, endothelial cells, and tumor
`cells after exposure to the cytokine interferon-y
`(IFN-y), produced by activated T cells (21). This
`has led to the notion that rather than function-
`ing early in T cell activation, the PIZHJPDle path—
`wayarastopmtectcellsfmmTcellattadc
`
`Immune checkpoint therapy in the clinic
`Ipilimumah, a fuliy human antibody to human
`Cflnsgenteied clinical nialsinthelate199£lsand
`early 2000s As predicted, tumor regression was
`.observedinpafientswithavarietyofmmortypes
`Phase IfII trials showed clinical responses in
`
`Antibody blockade of
`immune checkpoints
`enhances T cell responses
`
`Activated T cells make
`
`lFN-ywhichincreases. o
`PD—Liexpresslon o 0
`
`o
`
`o
`
`
`
`Tumor CE“
`
`Biopsy
`at this time
`at this time
`would show
`would show
`PD-Ll—negative ~—-—-——* PD-Ll—positive
`cells
`Enhancechell infiltration
`cans
`into tumor tissue
`
`
`
`Fig, 2. Blockade of immune checkpoints to enhance Tcell responses. After Tcell activation, Tcells express immune checkpoints such as CTLA—4 and PD—l. A
`biopsy of tumors taken from patients before treatment with immune checkpoint therapy (so prior to infiltration of activated Tcells into tumor tissues) may indicate
`lack of PD-Ll expression. However: upon Tcell activation. Tcells can traffic to tumors, Lip-regulate expression of immune checkpoints such as CTLA-4 and PD-l.
`and produce cytokines such as IFN-y. which leads to expression of PD—L] on tumor cells and other cells. lnciudlng T cells. within the tumor tissues.
`
`sCIEBNJE scienccmagcrg
`
`3 APRIL 2015 - vOL sac ISSUE sass
`
`57
`
`

`

`SPECIA 1. SECTION CANCER IMMUNOLOGYAND IMMUNE) THEMPY
`
`
`
`
`patients with melanoma (22), renal cell carcinoma
`(23), prostate cancer (24), urothelial carcinoma
`(25}, and ovarian cancer (26). Two phase lll clin-
`ical trials with anti-CTLA-‘t (ipilimumab) were
`conducted in patients with advanced melanoma
`and demonstrated improved overall survival for
`patients treated with ipilimumab (16, 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 (2?).
`Antibodies targeting the PD—lfPD-Ll axis have
`also shown clinical responses in multiple tu—
`mor types. Anti—PD—Ll 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-l (nivolumab) demonstrated sim-
`ilar clinical responses (30). Recently, a large
`phase I clinical trial with the anti-PD-l antibody
`MEL-3475 was shown to lead to response rates of
`~37to 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
`phase III trial of a different anti-PD—l antibody
`(nivolumab) also showed clinical benefit in pa-
`tients with metastatic melanoma. In this trial,
`the objective response rate was 40% 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 20M 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 phase III clinical trial, which reported
`an improvement in overall survival for patients
`treated with nivclumab as compared to patients
`treated with docetaxel chemotherapy.
`That CTLA-4 and PD-l regulate distinct in
`hibitory pathways and have nonoverlapping
`mechanisms of 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 phase I clinical
`trial with anti-CTLA—4 (ipilimumab) in combi-
`nation with anti—PD-l (nivolumab) demonstrated
`tumor regression in ~50% of treated patients
`with advanced melanoma, most with tumor re-
`gression of 30% or‘ more (.35). There are ongoing
`clinical trials with anti-CILA—It plus anti—PD—l,
`or anti-PD-Ll, 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 treatment is administered before
`
`
`
`
`53
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`3 APRIL 2015 - v01. ass Issue 6230
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`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
`tumor tissue can then guide rational searches
`for relevant markers in the blood. We designed
`the first pmurgical clinical trial with antiCI'LA-a
`(ipilimumab), which was administered to 12
`patients with localized bladder cancer prior to
`radical cystectomy (36). The endpoints of 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-C'ILA—rt therapy
`on the tumor microcnvironment. Unexpectedly,
`
`“Because of the very nature
`of immune dwdqmint ther-
`apy; the development of .
`phannaoodyrwmic, predic-
`tive, or prognostic biomark-
`ers faces unique challenges.”
`
`the trial enabled us to detect a clinical signal
`for anti-CTLA-c 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-C'I‘LA-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 immune responses in the tumor
`microenvironment, which should be included
`in the current paradigm of phase I, II, and III
`clinical trials.
`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 irrununohistochernical (THC)
`analyses, which can provide extensive insight
`into the biologic impact of the immunotherapy
`agent on the tumor microenvironment. For ex—
`ample, high-quaiity mRNA can be obtained for
`gene expression swdios comparing posttreatment
`tumor tissues to pretreatment tumor tissues or
`untreated samples obtained from a stage-matched
`control group of patients. These types of studies
`allow unbiased anabses of the samples to iden-
`tify novel genes and pathways that are affected
`by therapy. In our ipilimumab trial, 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 tumor tissues after
`
`
`
`CTLA—tt 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 CD28fCI‘LA—4- family. We
`confirmed our gene expression studies by flow
`cytometry. ICOS+ ’1‘ cells were increased in tumor
`tissues from patients treated with ipilimumab
`(36). The increase in the frequency of ICOS+ T
`cells in tumor infiltrates was accompanied by
`similar increases in the blood. These data, cou—
`pled with other studies, showed that an increase
`in the frequency of ICOS+ CD4- T cells served as
`a pharmacodynamic biomarker of anti-CTLA—t
`treatment (37).
`To test our hypothesis that ICOS" CD4 T cells
`might play a role in the therapeutic effect of
`CTLA—4 blockade, wc conducted studies in mice.
`In wild-type C57BU6 mice, anti-CI'LA-d treat-
`ment resulted in tumor rejection in 80 to 90%
`of mice, but in gene-targeted mice that were
`deficient for either ICOS or its ligand, the ef-
`ficacy was less than 50% (38). The loss of ef—
`ficacy of CTLA—‘l‘ blockade in the absence of an
`intact ICOS pathway indicates the critical im-
`portance of ICOS to the therapeutic effects of
`treatment with anti-C'I'LA-d 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 increase its
`effectiveness. To test this notion, we conducted
`studies in mice to provide an agonistic signal
`through ICOS in combination with CI'LA—t 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, ICOS is 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 tissuebased
`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-CTIA—d. Genetic analyses
`of melanoma. tumors revealed that higher num~
`bers of mutations, termed “mutational loa ,"
`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-CI'LAA- therapy (3, 40). These
`studies provide a strong rationale to integrate
`genetic analyses of the tumor with immune pro-
`filing of the tumor microenvironment for a more
`comprehensive evaluation of mechanisms that
`contribute to clinical responses with anti-CILA4
`therapy.
`
`Tissue~based immune monitoring:
`Anti-PD-l/ 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.
`
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`
`Because the PD—l ligand PD-Ll (and sometimes
`PD-L‘Z) can be expressed on tumor cells and im-
`mune cells in the tumor microenvimnment,
`there have been efforts to use expression of PD-
`L1 as a criterion for selecting patients for treat-
`ments with antibodies targeting the PD-liPD-Ll
`pathway.
`The initial phase 1 trial with anti-PD-l therapy
`(nivolumab) reported that PD-Ll expression
`on tumor cells, measured on pretreatment ar-
`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-Ll—
`positive tumors (25% staining for PD—Ll on tumor
`cells) had an objective response rate of 36% (9
`of 25 patients) whereas patients with PD-Ll—
`
`negative tumors did not show any objective
`clinical responses (0 of 1'? patients). However,
`in subsequent trials, some patients whose tu-
`mors were deemed to be PD-Ll-negative had
`clinical responses to anti-PD-l and anti-PD-L'l
`treatments with either tumor regression or sta—
`bilization of disease. For example, on a phase I
`trial with anti-PD-l (nivolumab), pau‘ents with
`Pull—positive tumors had an objective response
`rate of 44% (7 of 16) and patients with PD-Ll—
`negative tumors had an objective response rate
`of 17% (3 of 18) (41). Although PD-Ll expression
`in tumor tissues does correlate with higher re—
`sponse rates, 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