Extended Abstracts forthe 45th International Symposium of
the Princess Takamatsu Cancer Research FundRECENT ADVANCES IN
CANCER IMMUNOTHERAPY
November 18-20, 2014
Tokyo, Japan
Edited by

Shimon Sakaguchi
Jedd D. Wolchok
Yuji Heike
Yuzuru Kanakura
Yutaka Kawakami
Haruo Sugiyama

Princess Takamatsu Cancer Research Fund

CopyrightⒸ2015 by the Princess Takamatsu Cancer Research Fund
No part of this Extended Abstracts may be reproduced, stored in retrieval
systems or transmitted, in any form or by any means, without permission
from the Princess Takamatsu Cancer Research Fund.

Princess Takamatsu Cancer Research Fund

1-14-15-102 Takanawa, Minato-ku, Tokyo 108-0074, Japan
Telephone: +81-3-3441-0111
Facsimile: +81-3-3441-0112
E-mail:info@ptcrf.or.jp
Printed in Japan
ISSN 2186-8603

B

CONTENTS
KEYNOTE ADDRESS

Immune Regulation for Cancer Immunotherapy
Shimon Sakaguchi ..........................................................................................1

TUMOR IMMUNOENVIRONMENT

Translating Cancer Immunoediting Principles to Cancer Immunotherapy
Robert D. Schreiber.........................................................................................6

Cancer as a Disease of the Metaorganism
Giorgio Trinchieri..........................................................................................11
Combination Immunotherapy Based on the Cancer Cell’s Characteristics
and Patients’ Immune Status
Yutaka Kawakami.........................................................................................18

The Immune Contexture of Human Cancers: Shaping of Clinically
Efficient Landscapes
Wolf H. Fridman ...........................................................................................23

Mediators of Tumor Induced Immune Suppression in Malignant
Melanoma
Rolf Kiessling.................................................................................................30i

IMMUNE REGULATION

Therapeutic Strategies Based on Overcoming Immune ResistanceMechanisms within the Tumor Microenvironment
Thomas F. Gajewski......................................................................................35

Reprogramming the Tumor Microenvironment for Efficient Tumor
Rejection
Günter J. Hämmerling .................................................................................43

Harnessing Regulatory T Cells in Cancer Immunotherapy
Hiroyoshi Nishikawa ...................................................................................48

CHEMO IMMUNOTHERAPY

Immunogenic Cell Death in Cancer Therapy
Guido Kroemer .............................................................................................52

Role of Immune-suppressive Cells in the Survival of Gastrointestinal
Cancer Patients Treated with Standard Chemotherapy
Yuji Heike.......................................................................................................59

ii

IMMUNE CHECKPOINTS

Immune Checkpoint Blockade in Cancer Therapy:
New Insights and Opportunities
James P. Allison .............................................................................................61

Therapeutic Modulation of the Immune Microenvironment of Cancer
Drew M. Pardoll............................................................................................63

Immunotherapy that Targets the Tumor and the Host
Ronald Levy...................................................................................................65

Combination Checkpoint Blockade and the Role of‘Passenger’ Mutations in Clinical Response to Ipilimumab
Jedd D. Wolchok ...........................................................................................70
Therapy-induced Antibody Responses
Glenn Dranoff................................................................................................76

ANTIBODY THERAPY

Clinical Application of Anti-CCR4 Monoclonal Antibody
Ryuzo Ueda ...................................................................................................82
Drugging the Undruggable with Monoclonal Antibodies
David A. Scheinberg.....................................................................................88iii

CANCER VACCINE

WT1 (Wilm's Tumor Gene) Peptide Cancer Vaccine
Haruo Sugiyama ...........................................................................................93

Synergy of Therapeutic Vaccination against HPV16 Oncogenic Proteinsand Standard Chemotherapeutics
Cornelis J. M. Melief .....................................................................................97

NAKAHARA MEMORIAL LECTURE

Cancer Immunotherapy: the Narrow Road between Inefficacy and Toxicity
Thierry Boon................................................................................................101

CANCER VACCINE

Optimizing Therapeutic Cancer Vaccines
Pedro J. Romero ..........................................................................................105

Cancer Immunotherapy Using Novel Tumor-associated Antigenic
Peptides and Human iPS Cell-derived Dendritic Cells
Yasuharu Nishimura...................................................................................111
Dendritic Cell Vaccination against Cancer: from Preclinical Research to
Phase III Trial
Gerold Schuler.............................................................................................118iv

T CELL THERAPY

Immunotherapy in Allogeneic Hematopoietic Cell Transplantation
Robert S. Negrin..........................................................................................125

Cish Attenuates Proximal TCR Signalling and Its Deletion Enhances
Tumor Destruction
Nicholas P. Restifo ......................................................................................129

Engineering T Cell Responses to Effectively Target Tumors: Insights from
Preclinical Models and Clinical Results
Philip D. Greenberg....................................................................................134

Adoptive Immunotherapy with TCR Gene-transduced Lymphocytes
Hiroshi Shiku...............................................................................................139
Designing CAR T Cells for Cancer Therapy
Carl H. June .................................................................................................145

CONCLUDING REMARKS

Jedd D. Wolchok .........................................................................................151
List of Participants ..............................................................................................154
List of Previous International Symposia Series............................................175v

1

IMMUNE REGULATION FOR CANCER IMMUNOTHERAPY

Shimon Sakaguchi

WPI Immunology Frontier Research Center, Osaka University
3-1 Yamadaoka, Suita, Osaka 565-0871, Japan(shimon@ifrec.osaka-u.ac.jp)

Cancer immunotherapy is now in a new era as illustrated by recent clinical results

showing effectiveness of immune-checkpoint blockade and cell therapy with tumor-killing

T cells. Therapeutic vaccination with tumor antigens has been widely explored in the clinic

as an antigen-specific cancer immunotherapy since the molecular characterization of tumor

antigens that are recognized by tumor-reactive antibodies and cytotoxic T-lymphocytes(CTLs) in cancer patients. One of the key issues for improving the efficacy of the current

cancer immunotherapy is to control various immunosuppressive elements present incancer patients. In particular, given that many tumor-associated antigens recognized byautologous tumor-reactive CTLs are antigenically normal or slightly modified self-

constituents, tumor immunity is, in part, an autoimmunity. This means that themechanisms for maintaining immunological self-tolerance may hinder effective tumor
immunity in cancer patients.

Central and peripheral mechanisms of self-tolerance are indeed operating on T cells

recognizing tumor-associated self or quasi-self antigens1). As a typical illustration of this,CD8 + T cells recognizing Melan-A/MART-1, a self-antigen expressed by normalmelanocytes and some melanoma cells, are subjected to negative selection in the thymus;

and those having escaped thymic deletion are subjected to active suppression in the

periphery by regulatory T (Treg) cells, rendered anergic, or stay dormant2-6). In addition,

cancer cells or antigen-presenting cells (APCs) may attenuate effector activity of tumorreactive T cells via co-inhibitory molecules (such as PD-1 and CTLA-4) expressed byeffector T cells and via immuno-suppressive molecules (e.g., cytokines such as TGF-b, IL10, and tryptophan metabolites) produced by cancer cells and stromal cells including

2

S. Sakaguchi

APCs. A current key issue is therefore to determine which mechanism(s) of peripheral self-

tolerance or which aspect(s) of immune suppression can be practically manipulated to

evoke and enhance effective tumor immunity.

Among key mechanisms of peripheral self-tolerance, recent studies have shown that

Treg cells play an essential role in sustaining self-tolerance and immune homeostasis by

suppressing a wide variety of physiological and pathological immune responses against

self and nonself antigens, as well as quasi-self tumor antigens7). The most physiologically

relevant Treg cell population is CD25+CD4+ Treg cells, which are naturally present in the
immune system as ~10% of CD4+ T cells, specifically expressing the transcription factor
Foxp3 (forkhead box P3 transcription factor), and largely produced by the thymus as afunctionally distinct population with a self-skewed TCR repertoire7,8). Evidence is now

accumulating in humans and rodents that Foxp3 + CD25 + CD4 + Treg cells dominantly

infiltrate into tumor tissues, forming a sizable fraction of tumor-infiltrating lymphocytes,

and apparently impeding immune responses to tumor cells9). Depletion of Treg cells is

indeed able to evoke effective tumor immunity in otherwise non-responding animals
without serious autoimmunity if the duration and the degree of Treg depletion iscontrolled10). As a key mechanism of Treg cell-mediated suppression, Treg cells appear tocontrol APC functions, in particular, down-regulate their expression of the co-stimulatory

molecules CD80 and CD86 to various extents, thereby driving responder T cells into

distinct cell fates, depending on TCR affinity of the latter for tumor-associated or self-

antigens6,11). For example, with low expression of CD80 and CD86 by APCs, high affinity T
cells for a self- or tumor-associated antigen become deleted by apoptosis, intermediate ones

rendered anergic, and low affinity ones kept dormant (ignorant) 6) . Since anergic or

dormant CD8+ T cells can be distinguished functionally and phenotypically (e..g., theformer being phenotypically naïve but highly expressing the co-inhibitory molecule CTLA-4), both populations specific for a particular self/tumor antigen (e.g., Melan-A/MART-1)

have been shown to be present in the peripheral blood of healthy individuals and also

tumor tissues where Treg cells are abundant (6, and our unpublished data). In tumor
immunology setting, it is dormant naïve type T cells specific for tumor antigens that areable to become activated and expand upon tumor antigen vaccination, hence capable oftumor killing, because anergic type T cells are profoundly hypo-responsive and prone todie by apoptosis upon stimulation6).

Thus, a variety of T-cell populations are present in tumor tissues and presumably inregional lymphoid tissues; e.g., Treg cells, naïve T cells, anergic T cells, exhausted T cells,

and activated T cells. Each can be phenotypically distinguished in tumor tissues by theexpression of specific cell surface molecules (Figure 1). For example, tumor-infiltrating

Treg cells, in particular terminally differentiated and most suppressive effector Treg cells,Keynote Address3

are generally high in the expression of CTLA-4, PD-1, and the chemokine receptor CCR4.

Anergic CD8+ T cells are much higher in CTLA-4 expression than Treg cells or activated T

cells6). PD-1 is expressed by exhausted T cells, some anergic CD8+ T cells, and early
activated CD8+ T cells in addition to tumor-infiltrating Treg cells12). It is therefore likely thatagonistic, antagonistic, blocking, or cell-depleting antibodies specific for these molecules

may exert different effects on anti-tumor immune responses as a whole. For example, cell-

depleting anti-CCR4 depletes effector Treg cells; an anti-CTLA-4 mAb has a Treg-depleting

effect in tumor tissues, but not in the circulation, in mice13,14). Anti-PD-1 appears to be
blocking and able to re-juvenile exhausted CD8+ T cells12). Whether anti-PD-1 has a Tregdepleting effect in tumor tissues remains to be determined. It is certainly envisaged thatproper combinations of these antibodies specific for different T-cell surface molecules andwith distinct T-cell function-modulatory effects will synergistically enhance anti-tumor
immunity.

In addition to T-cell components discussed above, tumor microenvironments arecomposed of various cellular elements including cancer cells, cancer stromal cells, andvascular cells, which may suppress tumor immunity by various ways. It is hoped thatcoordinate targeting of these T and non-T cellular elements and their products, together

with antigenic stimulation such as tumor antigen vaccination, will make cancer

immunotherapy a major and key modality of cancer treatment in near future.

Figure 1

T cells in tumor tissues

4

S. Sakaguchi

References
1. Sakaguchi S, Powrie F, Ransohoff RM. Re-establishing immunological self-tolerance inautoimmune disease. Nat Med. 18:54-58, 2012.
2. Coulie PG, Brichard V, Van Pel A, Wölfel T, Schneider J, Traversari C, Mattei S, De
Plaen E, Lurquin C, Szikora JP, Renauld JC, Boon T. A new gene coding for a differentiation antigen recognized by autologous cytolytic T lymphocytes on HLA-A2
melanomas. J Exp Med. 180:35-42, 1994.
3. Kawakami Y, Eliyahu S, Delgado CH, Robbins PF, Sakaguchi K, Appella E, Yannelli JR,
Adema GJ, Miki T, Rosenberg SA. Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection. Proc
Natl Acad Sci USA. 91:6458-6462, 1994.
4. Zippelius A, Pittet MJ, Batard P, Rufer N, de Smedt M, Guillaume P, Ellefsen K,
Valmori D, Liénard D, Plum J, MacDonald HR, Speiser DE, Cerottini JC, Romero P.
Thymic selection generates a large T cell pool recognizing a self-peptide in humans. J Exp
Med. 195:485-494, 2002.
5. Pinto S, Sommermeyer D, Michel C, Wilde S, Schendel D, Uckert W, Blankenstein T,
Kyewski B. Misinitiation of intrathymic MART-1 transcription and biased TCR usage
explain the high frequency of MART-1-specific T cells. Eur J Immunol. 44:2811-2821,
2014
6. Maeda Y, Nishikawa H, Sugiyama D, Ha D, Hamaguchi M, Saito T, Nishioka M, Wing JB,
Adeegbe D, Katayama I, Sakaguchi S. Detection of self-reactive CD8+ T cells with ananergic phenotype in healthy individuals. Science. 346:1536-40, 2014.
7. Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T cells and immune tolerance.
Cell. 133:775-787, 2008.
8. Miyara M, Yoshioka Y, Kitoh A, Shima T, Wing K, Niwa A, Parizot C, Taflin C, Heike T,
Valeyre D, Mathian A, Nakahata T, Yamaguchi T, Nomura T, Ono M, Amoura Z,
Gorochov G, Sakaguchi S. Functional delineation and differentiation dynamics ofhuman CD4+ T cells expressing the FoxP3 transcription factor. Immunity. 30:899-911,
2009.
9. Nishikawa H, Sakaguchi S. Regulatory T cells in cancer immunotherapy. Curr Opin
Immunol. 27:1-7, 2014.
10. Ko K, Yamazaki S, Nakamura K, Nishioka T, Hirota K, Yamaguchi T, Shimizu J,
Nomura T, Chiba T, Sakaguchi S. Treatment of advanced tumors with agonistic anti-GITR
mAb and its effects on tumor-infiltrating Foxp3+CD25+CD4+ regulatory T cells. J Exp
Med. 202:885-891, 2005

Keynote Address

5

11. Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T, Miyara M, Fehervari Z, Nomura T,
Sakaguchi S. CTLA-4 control over Foxp3+ regulatory T cell function. Science. 322:271275, 2008.
12. Kamphorst AO, Ahmed R. Manipulating the PD-1 pathway to improve immunity. Curr
Opin Immunol. 25:381-388, 2013.
13. Sugiyama D, Nishikawa H, Maeda Y, Nishioka M, Tanemura A, Katayama I, Ezoe S,
Kanakura Y, Sato E, Fukumori Y, Karbach J, Jäger E, Sakaguchi S. Anti-CCR4 mAb
selectively depletes effector-type FoxP3+CD4+ regulatory T cells, evoking antitumor
immune responses in humans. Proc Natl Acad Sci USA. 110:17945-17950, 2013.
14. Simpson TR, Li F, Montalvo-Ortiz W, Sepulveda MA, Bergerhoff K, Arce F, Roddie C,
Henry JY, Yagita H, Wolchok JD, Peggs KS, Ravetch JV, Allison JP, Quezada SA. Fcdependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy ofanti-CTLA-4 therapy against melanoma. J Exp Med. 210:1695-1710, 2013.

Shimon Sakaguchi, M.D., Ph.D.

1989-1991
1992-1994

1994-1997

1998-20112007-2011

2011-

2013-

Assistant Professor, The Scripps Research Institute
PRESTO Investigator, Japan Science and Technology
AgencyHead of Immunopathology, Tokyo Metropolitan
Institute of Gerontology
Professor, Institute for Frontier Medical Sciences,
Kyoto University
Director, Institute for Frontier Medical Sciences, Kyoto
University
Professor, WPI Immunology Frontier Research Center,
Osaka University
Distinguished Professor, Osaka University

Specialty and Present Interest:
Immune Regulation, Autoimmune Disease, Tumor Immunity

6

TRANSLATING CANCER IMMUNOEDITING PRINCIPLES TOCANCER IMMUNOTHERAPY

Matthew M. Gubin, Jeffrey P. Ward, Takuro Noguchi andRobert D. Schreiber
Department of Pathology and Immunology
Washington University School of Medicine
660 South Euclid Ave, St. Louis, MO 63110, USA(schreiber@immunology.wustl.edu)

Cancer Immunoediting is the process by which the immune system controls and shapes

cancer. We originally envisaged and subsequently showed that, in its most complex form,

cancer immunoediting occurs in three phases: Elimination (also known as cancer
immunosurveillance, the host protective phase of the process), Equilibrium (the phase inwhich tumor cells that survive immune elimination remain under immunologic growth

control resulting in a state of functional tumor dormancy) and Escape (the phase whereclinically apparent tumors emerge because immune sculpting of the tumor cells has

produced variants that display either reduced immunogenicity or enhanced

immunosuppressive activity)1-3) (Figure 1). Strong experimental data have been obtainedusing mouse cancer models to demonstrate the existence of each phase of the cancer

immunoediting process and compelling clinical data suggest that a similar process also

occurs during the evolution of certain types of human cancer4). Our efforts now focus on
elucidating the molecular and cellular mechanisms that underlie each phase of cancer

immunoediting, identifying the critical checkpoints that regulate progression from one

phase of the process to the next and defining the targets and mechanisms underlying thecancer immunoediting process.

In 2012, we reported one of the first experimental uses of cancer exome sequencing andepitope prediction algorithms to show that (a) mutant proteins in highly immunogenic

tumor cells derived from methylcholanthrene treated immunodeficient mice represent

immunodominant, tumor-specific mutant antigens for CD8 + T cells; (b) these highly

immunogenic tumor-specific mutant antigens represent major targets of cancer

immunoediting and (c) that T cell dependent immunoselection for tumor cells that lackTumor Immunoenvironment

Figure 1

7

Cancer Immunoediting

expression of strong tumor specific mutant antigens is a major mechanism by which

immunoediting occurs5).

More recently, we asked whether our genomics/bioinformatics based approach could

identify the antigens in progressively growing T3 sarcomas that render them susceptible tocheckpoint blockade immunotherapy6). T cell lines generated from anti-PD-1 treated micethat had rejected T3 progressor sarcoma cells displayed restriction to H-2Kb but not to H2Db, suggesting that anti-PD-1 promotes T cell responses to only a limited number ofantigens. We then identified expressed non-synonymous mutations in T3 tumor cells using
cDNA Capture Sequencing and generated a prioritized list of potential H-2Kb binding

epitopes based on predicted binding affinity. Filters were then applied to this list to take

into account the likelihood that the mutant protein would undergo proteasomal processing

and whether the predicted mutant neo-epitope displayed an equal or enhanced ability tobind to MHC class I compared to the corresponding wild type sequence while also

deprioritizing predicted epitopes from hypothetical proteins. Based on these criteria, twounequivocal “best candidates” were predicted —mutant forms of Laminin a subunit 4(mLama4) and a glucosyltransferase (mAlg8) (Figure 2). To test the validity of these8

R. D. Schreiber et al.

A.

B.

C.

D.

E.

Figure 2

Mutations in Lama4 and Alg8 form top predicted d42m1-T3 epitopes
A. Potential H-2Kb binding epitopes predicted by in silico analysis of all missense mutations in the T3 sarcoma. B. Median affinity values for the top 62 predicted H-2Kb epitopes. C.
Median affinity values of H-2Kb epitopes after filtering. D. Screening for specificities of
CD8+ TILs from aPD-1 treated, T3 sarcoma bearing mice using H-2Kb tetramers loaded with
top 62 H-2Kb epitopes. E. IFN-g and TNF-a induction in CD8+ TILs from aPD-1 treated, T3
sarcoma bearing mice following culture with irradiated splenocytes pulsed with the top 62 H2Kb peptides. Data are presented as per cent CD8+ TILs expressing IFN-g, TNF-a or for
both. Data are representative of two independent experiments.

predictions, we synthesized 8 amino acid peptides representing the top 62 predicted H-2Kb

epitopes and assessed whether they were recognized by CD8+ TILs isolated from T3 tumors

when presented by H-2Kb. Only the top two predicted epitopes—mLama4 and mAlg8—scored positive when either used in labelled H-2Kb tetramers to stain CD8+ TILs from T3

tumors (as assessed by flow cytometry) or when the CD8+ TILS from T3 tumors wereincubated with irradiated splenocytes that had been pulsed with each of the predicted H2Kb epitopes and T cells subsequently analyzed by flow cytometry for intracellular IFNg

and TNFa production. These findings were further validated by showing that: (a) T3

tumor-specific CD8+ T cell lines generated from mice that had rejected T3 tumors following
treatment with anti-PD-1 recognized the same two epitopes; (b) the mLama4 and mAlg8

Tumor Immunoenvironment

9

epitopes were detected by mass spectrometry within the pool of peptides bound to H-2Kb
expressed on T3 tumor cells; (c) CD8+ T cells expressing TCRs for either mLama4 or mAlg8

accumulated over time in T3 tumors in anti-PD-1-treated, tumor-bearing mice, reaching

maximal numbers just prior to tumor rejection; (d) vaccination of naïve WT mice withmutant but not WT forms of Lama4 or Alg8 induced strong CD8+ T cell responses; and (e)naïve mice vaccinated prophylactically against mLama4 plus mAlg8 in the presence of poly

I:C controlled outgrowth of T3 tumors. Most importantly, the mLama4 plus mAlg8 vaccine

also caused the elimination of established T3 tumors, in a manner that was very similar to T3

tumor bearing mice treated with either anti-PD-1 and/or anti-CTLA-4 (Figure 3). Thesefindings reveal that the combination of genomics, bioinformatics and immunologic
approaches can indeed identify key tumor-specific mutant proteins that can function

therapeutically as tumor-specific mutant rejection antigens and thus provide a basis fortheir use in the development of personalized therapeutic cancer vaccines.

TumorͲSpecificAntigenVaccine

CheckpointBlockadeTherapy
DPDͲ1+D
DCTLAͲ4

p=0.0002
PolyI:C

HPVcontrol
DayspostͲtransplant
SLPVaccineond+3,+9,+15,+21

Figure 3

DPDͲ1Percentmousesurvival

Percentmousesurvival

mLama4+mAlg8

DCTLAͲ4

p=0.001

ControlDayspostͲtransplant
CheckpointmAbs ond+3,+6,+9

Combined mLama4 plus mAlg8 vaccine is nearly as effective as checkpoint blockade
immunotherapy.
Groups of mice were treated with 1 x 106 T3 sarcoma cells and on day +3, when tumors had
become established, groups of mice were treated either with: Left Panel: a synthetic long peptide (SLP) vaccine consisting of mLama4 plus mAlg8 plus 100mg poly I:C on days +3, +9, +15
and +21 (controls included treatment with an irrelevant SLP plus poly I:C vaccine or poly I:C
alone) or Right Panel: 200mg each anti-PD-1, or anti-CTLA-4 or the combination of anti-PD1 plus anti-CTLA-4 administered on days +3, +6 and +9. Tumor growth was measured
and plotted as the mean of two perpendicular diameters. Mice were euthanized whentumors reached 20mm in diameter, which was the point used to generate the Kaplan-Meiersurvival curves.

10

R. D. Schreiber et al.

References
1. Shankaran V, Ikeda H, Bruce AT, White JM, Swanson PE, Old LJ, Schreiber, RD. IFNg andlymphocytes prevent primary tumour development and shape tumour immunogenicity.
Nature. 2001;410:1107-1111.
2. Dunn GP, Bruce AT, Ikeda H, Old LJ, Schreiber RD. Cancer immunoediting: from
immunosurveillance to tumor escape. Nature Immunology. 2002;3:991-998.
3. Koebel CM, Vermi W, Swann JB, Zerafa N, Rodig SJ, Old LJ, Smyth MJ, Schreiber, RD.
Adaptive immunity maintains occult cancer in an equilibrium state. Nature.
2007;450:905-908.
4. Schreiber RD, Old LJ, Smyth MJ. Cancer immunoediting: integrating immunity’s roles incancer suppression and promotion. Science. 2011;331:1565-1570.
5. Matsushita H, Vesely, MD, Koboldt DC, Rickert, CG, Uppaluri R, Magrini VJ, Arthur, CD,
White JM, Chen YS, Shea LK, Hundal J, Wendl MC, Demeter R, Wylie T, Allison JP,
Smyth MJ, Old LJ, Mardis ER, Schreiber RD. Cancer exome analysis reveals a T celldependent mechanism of cancer immunoediting. Nature. 2012;482:400-404.
6. Gubin MM, Zhang X, Schuster H, Caron E, Ward JP, Noguchi T, Ivanova Y, Hundal J,
Arthur CD, Krebber W-J, Mulder GE, Toebes M, Vesely MD, Lam, SK, Korman AJ,
Allison JP, Freeman GJ, Sharpe AH, Pearce EL, Schumacher TN, Aebersold R,
Rammensee H-G, Melief CJM, Mardis ER, Gillanders WE, Artyomov MN, Schreiber
RD. Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens. Nature. 2014;515:577-581.

Robert D. Schreiber, Ph.D.

1976-1985

1985-1990
1990-

1998-

2008
20132013-

Assistant - Associate Member, Dept. of Molecular
Immunology, Research Institute of Scripps Clinic
Professor, Dept. of Pathology, Washington University
School of Medicine
Alumni Endowed Professor of Pathology &Immunology, Washington University School of
Medicine
Program Leader, Tumor Immunology, The Alvin J.
Siteman Cancer CenterCo-founder, Igenica Biotherapeutics, Inc.
Director, Center for Human Immunology and
Immunotherapy Programs, Washington University,
School of Medicine
Co-founder, Jounce Therapeutics, Inc.

Specialty and Present Interest:
Cancer Immunoediting, Personalized Cancer Immunology, Interferon-Gamma Receptor
Signaling

11

CANCER AS A DISEASE OF THE METAORGANISM

Giorgio Trinchieri

Cancer and Inflammation Program, Center for Cancer Research
National Cancer Institute, National Institutes of Health
Building 37, Room 41-46A, 9000 Rockville Pike, Bethesda, MD 20892, USA(trinchig@mail.nih.gov)

Commensal microorganisms colonize barrier surfaces of all multicellular organisms,

including those of humans. For more than 500 million years commensal microorganisms

and their hosts have coevolved and adapted to each other1). As a result, the commensal

microbiota affects many immune and non-immune functions of their hosts, and de facto thetwo together comprise one metaorganism1).

The commensal microbiota communicates with the host via biologically active

molecules. The interplay between the host immune system and the microbiota prevents

tissue-damaging inflammatory responses to the commensals and controls the growth ofindigenous pathobionts while it sets the stage for immune responses against pathogenic

infections2-4). This homeostatic immune regulation may be disrupted by changes in the

microbial community that alter the symbiotic relationship with the microbiota and theresultant microbial imbalance is commonly referred to as dysbiosis5). Many regulatory

mechanisms involved in these local interactions have been elucidated6). In addition to local

immunity, the commensal microbiota regulates systemic inflammation, innate resistanceand adaptive immunity affecting both resistance to infection and autoimmunity 7-12) .

Recently, it has been reported that microbial imbalance may play a critical role in thedevelopment of multiple diseases, such as cancer, autoimmune conditions and increased

susceptibility to infection2).

The commensal microbiota not only may affect the development, progression andimmune evasion of cancer but it has also important effects on the response to cancer

immune- and chemo-therapy 3-5) . Myeloid cells are a major component of the tumor
microenvironment where they play a dual role inducing anti-tumor immune responses but

12

G. Trinchieri

mostly promoting immune evasion, tumor progression and metastases formation6). Thus,

strategies aiming at reprogramming the tumor microenvironment represent a promising

immunotherapy approach. Myeloid cells respond to environmental factors including

signals derived from commensal microbes that modulate their function and reactivity thus

impacting the response to cancer therapy5).

The gut microbiota influences the response to cancer immunotherapy andchemotherapy by affecting the differentiation and functions of myeloid cells in the tumor

microenvironment (Figure 1). Intratumoral injection of CpG-oligodeoxynucleotide (CpGODN) combined with antibody neutralization of IL10 signaling is a very effective treatmentof large transplanted subcutaneous tumors in conventional mice but it is largely ineffective

in GF or antibiotics-treated mice5). Within hours following CpG-ODN and anti-IL10R
treatment tumors undergo an extensive hemorrhagic necrosis that is dependent on TNF

and nitric oxide (NO) production by tumor-infiltrating myeloid cells7). DCs are thenactivated, and they migrate to the draining lymph nodes where they induce a CD8 T cellmediated tumor-specific response required for tumor eradication. In antibiotics-treated or

Figure 1

The gut microbiota influences the response to cancer immunotherapy and chemotherapy byaffecting the differentiation and functions of myeloid cells in the tumor microenvironment
with different mechanisms involved in the different types of immune- or chemo-therapy. For
details, please see text.

Tumor Immunoenvironment

13

GF mice, tumor-infiltrating myeloid-derived cells fail to produce inflammatory cytokines,

including TNF and IL12, in response to CpG-ODN5). Oral gavage of antibiotics-treatedmice with LPS partially rescued the deficient response to CpG-ODN5). Tumors fromantibiotics-treated mice contained a lower number of monocyte-derived Ly6C+ MHC-class

II + macrophage-like cells while the number of Ly6C high MHC class II - inflammatory

monocytes was equivalent to that of control mice without antibiotics treatment5). Tumorassociated myeloid-cell subsets have been shown to be mostly derived from circulating

inflammatory monocytes that differentiate in situ8-10). Although inflammatory monocytes

appear to infiltrate the tumors in equivalent number, their differentiation after reaching thetumor microenvironment is altered in the absence of gut microbiota and this may affecttheir response to CpG-ODN5). The fecal microbiota composition in mice showing high andlow TNF responses to CpG-ODN appeared distinct. In particular, the abundance of several

individual Gram + and Gram - bacterial spp positively correlated with the CpG-ODN
response, whereas the presence of commensal Lactobacillus spp decreased the response5). In

vivo association experiments confirmed that the Gram- Alistipes shaii enhances the CpG-ODN response while L. fermentum attenuates it5).

The effect of the microbiota on chemotherapy was analyzed utilizing platinumcompounds (e.g. oxaliplatin, cisplatin) 5) . These compounds mediate genotoxicity byforming platinum DNA-adducts followed by formation of intrastrand cross-links that

inhibit protein synthesis and proliferation, and induce apoptosis in part downstream of theataxia telangiectasia and rad3-related (ATR) kinase recruitment to DNA damage and p53

activation 1 1 ) . The therapeutic effect of oxaliplatin and cisplatin on mouse sterile

subcutaneous transplanted tumors was dramatically reduced in antibiotics-treated or GF
mice5). In antibiotics-treated mice, platinum adducts to tumor-cell DNA were formed at alevel comparable to that observed in control mice, however, already at 48 hour post-

treatment there was a significant decrease in DNA damage and cytotoxicity. Antibiotics
treatment of mice largely suppressed all the gene expression modification induced in thetumor by oxaliplatin. In antibiotics-treated mice tumor-infiltrating myeloid cells failed to

produce reactive oxygen species (ROS) via the NADPH oxidase NOX2. ROS are needed forthe oxaliplatin antitumor effect. Thus, the microbiota affects oxaliplatin early tumor

genotoxicity by systemically priming tumor-associated myeloid cells for ROS production.

The effects of microbiota deprivation in the response to CpG-ODN and platinumcompounds were evident at very early times following treatment, suggesting that tumorassociated myeloid cells were primed for responsiveness to therapy by the preexisting

microbiota composition. However, chemotherapy and radiation therapy in addition to theirantitumor effect also induce damage of the intestinal mucosa affecting mucosal

permeability and inducing dysbiosis and bacterial transmucosal translocation. Adoptive

14

G. Trinchieritransfer of tumor-specific cytotoxic CD8+ T cells is an efficient therapy for cancer both inmice and in the clinic12). For best survival of the T cells and effectiveness of the transfer,

lympho- and myelo-ablation are necessary13). Both in human patients and in mice, total

body irradiation (TBI) improves therapy efficacy by increasing DC activation andhomeostatic cytokine levels13). Due to the mucosal damage effect of TBI, commensal gut

bacteria were found to infiltrate the mesenteric lymph nodes of irradiated animals andelevated endotoxin levels were observed in their sera13). The ability of TBI to improvetumor regression induced by adoptive T-cell transfer was reduced in animals treated withbroad spectrum antibiotics, by neutralization of serum LPS using polymyxin B, or in mice

genetically deficient in CD14 or TLR4 that are unable to respond to LPS. Administration of

LPS or LPS-containing serum from irradiated animals to non-irradiated lymphopenic mice

was able to enhance the number and function of transferred CD8+ T cells, leading to longterm cure of mice with large transplanted tumors13).

Cyclophosphamide (CTX) treatment of conventionally raised animals induces dysbiosis

and mucositis. Due to the mucosal damage, Gram+ bacteria translocate into the mesentericdraining lymph nodes, prime pathogenic effector Th17 cells and memory Th1 cells, all ofwhich were not observed in microbiota-depleted mice3). Thus, the activation of antigen-presenting cells and the subsequent induction of antitumor immune responses bychemotherapy-induced immunogenic cell death not only depend on the release ofendogenous mediators of inflammation as previously shown14), but also on the priming
and/or activating effects mediated by commensal bacteria and/or by their products.

In the past few years there has been very promising progress in the therapy ofmelanoma, kidney and lung cancers in terms of boosting the patient’s immune response
against the tumor using immune checkpoint inhibitors such as antibodies blocking theCTLA4 or PD-1 checkpoint receptors 1 5 ) . The role of the commensal microbiota inmodulating the response to cancer immunotherapy, immunogenic chemotherapy andadoptive T-cell transfer suggest the possibility that the microbiota may also modulate theclinical effectiveness of this new class of anti-cancer drugs.

In conclusion, there is a considerable body of evidence, both in humans and inexperimental animals, that the commensal microbiota – bacteria, fungi, and viruses – exerts

important effects on carcinogenesis, tumor progression, and the response to therapy. The

effect of the microbiota on cancer can be local, situated at the level of the organism barriers

in which cancer originates, or can be systemic, through the physiological communication of

the organism and the microbiota through intact membrane or following alteration of

barrier permeability in pathology. While many mechanisms of the local effects have beencharacterized in recent years, our understanding of the systemic effects is currently much
more rudimental. A detailed understanding of these mechanisms both in experimental

Tumor Immunoenvironment

15

animals and in humans will teach us how to target them therapeutically and could bringmuch progress in cancer prevention and treatment.

References
1. Costello, E. K., K. Stagaman, L. Dethlefsen, B. J. Bohannan, and D. A. Relman. 2012. The
application of ecological theory toward an understanding of the human microbiome.
Science 336: 1255-1262.
2. Belkaid, Y., and T. W. Hand. 2014. Role of the microbiota in immunity and inflammation.
Cell 157: 121-141.
3. Viaud, S., F. Saccheri, G. Mignot, T. Yamazaki, R. Daillere, D. Hannani, D. P. Enot, C.
Pfirschke, C. Engblom, M. J. Pittet, A. Schlitzer, F. Ginhoux, L. Apetoh, E. Chachaty, P. L.
Woerther, G. Eberl, M. Berard, C. Ecobichon, D. Clermont, C. Bizet, V. GaboriauRouthiau, N. Cerf-Bensussan, P. Opolon, N. Yessaad, E. Vivier, B. Ryffel, C. O. Elson, J.
Dore, G. Kroemer, P. Lepage, I. G. Boneca, F. Ghiringhelli, and L. Zitvogel. 2013. The
intestinal microbiota modulates the anticancer immune effects of cyclophosphamide.
Science 342: 971-976.
4. Trinchieri, G. 2012. Cancer and inflammation: an old intuition with rapidly evolving
new concepts. Annu. Rev. Immunol. 30: 677-706.
5. Iida, N., A. Dzutsev, C. A. Stewart, L. Smith, N. Bouladoux, R. A. Weingarten, D. A.
Molina, R. Salcedo, T. Back, S. Cramer, R. M. Dai, H. Kiu, M. Cardone, S. Naik, A. K. Patri,
E. Wang, F. M. Marincola, K. M. Frank, Y. Belkaid, G. Trinchieri, and R. S. Goldszmid.
2013. Commensal bacteria control cancer response to therapy by modulating the tumor
microenvironment. Science 342: 967-970.
6. Goldszmid, R. S., A. Dzutsev, and G. Trinchieri. 2014. Host immune response to infection
and cancer: unexpected commonalities. Cell Host Microbe 15: 295-305.
7. Guiducci, C., A. P. Vicari, S. Sangaletti, G. Trinchieri, and M. P. Colombo. 2005.
Redirecting in vivo elicited tumor infiltrating macrophages and dendritic cells towards
tumor rejection. Cancer Res. 65: 3437-3446.
8. Franklin, R. A., W. Liao, A. Sarkar, M. V. Kim, M. R. Bivona, K. Liu, E. G. Pamer, and M.
O. Li. 2014. The cellular and molecular origin of tumor-associated macrophages. Science
344: 921-925.
9. Movahedi, K., D. Laoui, C. Gysemans, M. Baeten, G. Stange, J. Van den Bossche, M.
Mack, D. Pipeleers, P. In't Veld, P. De Baetselier, and J. A. Van Ginderachter. 2010.
Different tumor microenvironments contain functionally distinct subsets ofmacrophages derived from Ly6Chigh monocytes. Cancer Res. 70: 5728-5739.

16

G. Trinchieri

10. Shand, F. H., S. Ueha, M. Otsuji, S. S. Koid, S. Shichino, T. Tsukui, M. Kosugi-Kanaya, J.
Abe, M. Tomura, J. Ziogas, and K. Matsushima. 2014. Tracking of intertissue migration
reveals the origins of tumor-infiltrating monocytes. Proc. Natl. Acad. Sci. U. S. A. 111:
7771-7776.
11. Siddik, Z. H. 2003. Cisplatin: mode of cytotoxic action and molecular basis of resistance.
Oncogene 22: 7265-7279.
12. Restifo, N. P., M. E. Dudley, and S. A. Rosenberg. 2012. Adoptive immunotherapy for cancer: harnessing the T cell response. Nat. Rev. Immunol. 12: 269-281.
13. Paulos, C. M., C. Wrzesinski, A. Kaiser, C. S. Hinrichs, M. Chieppa, L. Cassard, D. C.
Palmer, A. Boni, P. Muranski, Z. Yu, L. Gattinoni, P. A. Antony, S. A. Rosenberg, and N.
P. Restifo. 2007. Microbial translocation augments the function of adoptively transferred self/tumor-specific CD8+ T cells via TLR4 signaling. J. Clin. Invest. 117: 2197-2204.
14. Kroemer, G., L. Galluzzi, O. Kepp, and L. Zitvogel. 2013. Immunogenic cell death incancer therapy. Annu. Rev. Immunol. 31: 51-72.
15. Brahmer, J. R., and D. M. Pardoll. 2013. Immune checkpoint inhibitors: making
immunotherapy a reality for the treatment of lung cancer. Cancer Immunol Res 1: 85-91.

Tumor Immunoenvironment

Giorgio Trinchieri, M.D.

1971-1975
1973-1976

1975-1977
1976-1999

1977-1979

1979-1999
1986-1999

1999-2006
1999-2004

2004-2006
2006-

2008-

2009-

2012-

17

Member, Basel Institute of Immunology
Assistant Professor of Medical Genetics, School of
Medicine, Univ. of Torino
Associate Scientist, The Wistar Institute
Faculty, Graduate Group of Immunology, University ofPennsylvania
Investigator, Swiss Institute for Experimental Cancer
Research
The Wistar Institute: 1979-1987 Associate Professor,
1987-1999 Professor, 1991-1999 Chairman,
Immunology Program, 1999 Hilary Koprowski Chair
Wistar Professor of Medicine (Associate 1986-90),
Hematology/Oncology Section, Department ofMedicine, University of Pennsylvania
Adjunct Professor, The Wistar Institute
Director, Laboratory for Immunological Research,
Schering Plough
Fogarty Scholar, Laboratory of Parasitic Diseases,
NIAID, NIH
Director, Cancer and Inflammation Program and Chief,
Laboratory of Experimental Immunology, CCR, NCI,
NIH
Associate Director, Basic Science, NIH Center for
Human Immunology
Adjunct Investigator, Laboratory of Parasitic Diseases,
NIAID
Adjunct Investigator, University of Maryland

Specialty and Present Interest:
Microbiome, Inflammation and Cancer Therapies

18

COMBINATION IMMUNOTHERAPY BASED ON
THE CANCER CELL’S CHARACTERISTICS AND
PATIENTS’ IMMUNE STATUS

Yutaka Kawakami

Division of Cellular Signaling, Institute for Advanced Medical Research
Keio University School of Medicine
35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan(yutakawa@z5.keio.jp)

Pretreatment immune-status in tumor microenvironments varies among cancer patients.

High infiltrations of CD8+ T-cells, CD3+ T-cells, FOXP3+ T-cells and B-cells in cancer tissuesof stage I-II colon cancer patients were found to be significantly correlated with favorable

prognosis after surgery. FOXP3+ T-cells in colon cancers may contain helper T-cells which
may be partly activated by intestinal bacteria. High FOXP3+ T-cells are generally correlatedwith poor prognosis in other types of cancers (e.g. lung cancer, oral squamous cancer, andcervical cancer which we have evaluated). Pretreatment immune-status in blood also varies

among patients. We measured more than 50 cytokines and chemokines in blood of patientswith various cancers by using multiplex analysis. High pretreatment levels of somecytokines were found to correlate with poor prognosis after various cancer

immunotherapies including peptide vaccines and antigen pulsed dendritic cell vaccine. In

the peptide vaccine protocol, delayed clinical effect (delayed separation of Kaplan-Meiercurve) was not observed in the patients with high blood cytokine levels. Multivariate

analyses showed that some cytokines were significant independent factors along withinduction of tumor antigen specific T-cells and IgG Abs for post-immunotherapy

prognosis. Therefore, the evaluation of pretreatment immune-status in tumor and blood

may be useful for cancer therapies to predict prognosis and treatment response, thereby

selecting appropriate patients and applying personalized therapy.

The immune-status may be defined by balance between positive and negative pathways

in the anti-tumor T-cell responses which are regulated by cancer cell characteristics (e.g.

immunogenic mutations, oncogene activation), patients’ immune-reactivity (e.g. HLA type,

polymorphism of immune-regulating molecules) and environmental factors (e.g. smoking,Tumor Immunoenvironment

19

diet, intestinal microbiota, previous history of infection). The positive pathway, inductionof tumor antigen specific T-cells, may be regulated by quantity and quality of immunogenic

mutated antigens derived from DNA mutations as well as immunogenic shared antigens.

We found that tumor infiltrating T-cells (TIL) frequently recognize mutated unique peptide

antigens derived from passenger mutations in melanoma1,2). We also found that immune
responses to frameshift mutations occurs in patients with microsatellite instability positive(MSI + ) colon cancers having abundant DNA mutations due to dysfunction of DNA

mismatch repair enzymes, and also high CD8+ T-cell infiltrations in tumors3). In cervicalcancer, TIL frequently recognize HPV oncoviral proteins, E6 and E7. Enhancement of theanti-tumor T-cell induction may be achieved by release of immunogenic antigens by

induction of immunogenic cancer cell death (e.g. chemotherapy, molecular targeted

therapy, radiation), stimulation of DC with strong adjuvants (e.g. TLR3, STING

stimulators), and immune-checkpoint blockade (e.g. anti-CTLA-4 Ab, PD-1/PD-L1
blocking Ab)

In the negative pathway, suppression of anti-tumor immune responses, constitutive

activation of various signaling pathways caused by genetic alternations such as activatedoncogenes in cancer cells triggers multiple immunosuppressive cascades via production of

immunosuppressive cytokines such as TGF-b, IL10, IL6 and VEGF, and via generation of
immunosuppressive cells such as Tregs, DCregs and MDSCs. In human melanoma,

BRAF/MAPK, STAT3, or Wnt/b-catenin is constitutively activated in some patients, andtheir activation generates immunosuppressive condition such as impaired function of DC

and T-cells partly due to production of immunosuppressive cytokines and chemokines
such as IL6, IL10, VEGF and CCL2 4 , 5 ) .

These cytokines generate various

immunosuppressive cells including MDSCs, tDCs, and Tregs through activation of STAT3

in the immune cells 6 ) . Increase of TGF-b in tumor microenvironments decreases

accumulation of anti-tumor T-cells in tumors through impairment of DCs, increase of

MDSCs and Tregs in tumors and sentinel lymph nodes7). TGF-b induced epithelial tomesenchymal transition (EMT) of melanoma may result in enhanced metastasis throughsnail induced production of immunosuppressive cytokines and chemokines such as TSP-1,
TGF-b and CCL2 along with the increased cancer cell motility8). In heal and neck squamouscancers, expression of CCL22 and CCR4 in cancer cells not only enhances their lymph nodemetastasis, but also induce M2-like macrophages producing CCL22 which further recruit
CCR4+ Tregs in tumor and sentinel lymph nodes9). In human ovarian cancers, NF-kB is

constitutively activated and generates immunosuppressive microenvironments such as

impaired DC function and accumulation of immunosuppressive MDSCs partly due to highproduction of IL6, and IL810). Constitutive activation of NF-kB in cancer cells may also

inhibit IFN-a production by pDC via expression of ILT7 ligand11), which may result in20

Y. Kawakami

decrease of anti-tumor T-cell induction.

Administration of various inhibitors against these oncogene products such as MAPK,

b-catenin and NF-kB are useful for reversal of the immunosuppressive conditions in tumor

microenvironments through reversal of impaired DC function and inhibition of

immunosuppressive MDSCs and Tregs by acting on both cancer cells and immune cells inmouse models implanted with human cancer cells4,5,10). In syngeneic murine tumor models,either NK-kB dependent IL6 producing tumor or non-IL6 producing tumor, administrationof NF-kB inhibitors enhanced induction of anti-tumor T-cells accompanied by restoration ofT-cell stimulatory activity of DCs. However, administration of inhibitor alone was not

sufficient to inhibit tumor growth because of local negative feedback (adaptive resistance)

by PD-1/PD-L1 interaction in tumor microenvironments. Combined administration of anti-

PD-L1 Ab with the NF-kB inhibitors resulted in synergistic inhibition of tumor growth.

Therefore, we are attempting to develop combination immunotherapy using appropriate
signal inhibitors, immune checkpoint blockers, and immunostimulating adjuvants. Someof the combinations have demonstrated superior anti-tumor activities. Personalized
combination immunotherapy based on the immune-status of patients is a promisingstrategy12).

References
1. Robbins PF, El-Gamil M, Li YF, Kawakami Y, Loftus D, Appella E, Rosenberg SA. A
mutated b2-catenin gene encodes a melanoma - specific antigen recognized by tumor infiltrating lymphocytes. J Exp Med. 1996;183:1185-1192.
2. Kawakami Y, Wang X, Shofuda T, Sumimoto H, Tupesis JP, Fitzgerald E andRosenberg SA. Isolation of a new melanoma antigen, MART-2, containing a mutatedepitope recognized by autologous tumor infiltrating T lymphocytes. J Immunol.
2001;166: 2871-2877.
3. Ishikawa T, Fujita T, Suzuki Y, Okabe S, Yuasa Y, Iwai T, Kawakami Y. Tumor-specificrmmunological recognition of frameshift-mutated peptides in colon cancer withmicrosatellite instability. Cancer Res. 2003;63:5564-5572.
4. Sumimoto H, Imabayashi F, Iwata T, Kawakami Y. The BRAF-MAPK signaling pathway
is essential for cancer immune evasion in human melanoma cells. J Exp Med.
2006;203:1651-1656.
5. Yaguchi T, Goto Y, Kido K, Mochimaru H, Sakurai T, Tsukamoto N, Kudo-Saito C,
Fujita T, Sumimoto H, Kawakami Y. Immune suppression and resistance mediated byconstitutive activation of Wnt/b-catenin signaling in human melanoma cells. J
Immunol. 2012;189:2110-2117.

Tumor Immunoenvironment21

6. Iwata-Kajihara T, Sumimoto H, Kawamura N, Ueda R, Takahashi T, Mizuguchi H,
Miyagishi M, Takeda K, Kawakami Y. Enhanced cancer immunotherapy using STAT3depleted dendritic cells with high Th1-inducing ability and resistance to cancer cellderived inhibitory factors. J Immunol. 2011;187:27-36.
7. Nakamura S, Yaguchi T, Kawamura N, Kobayashi A, Sakurai T, Higuchi H, Takaishi H,
Hibi T, Kawakami Y. TGF-b1 in tumor microenvironments induces immunosuppression in the tumors and sentinel lymph nodes and promotes tumor progression. J
Immunother. 2014;37:63-72.
8. Kudo-Saito C, Shirako H, Takeuchi T, Kawakami Y. Cancer metastasis is accelerated
through immunosuppression during EMT of cancer cell. Cancer Cell. 2009;16:195-206.
9. Tsujikawa T, Yaguchi T, Ohmura G, Ohta S, Kobayashi A, Kawamura N, Fujita T,
Nakano H, Shimada T, Takahashi T, Nakao R, Yanagisawa A, Hisa Y, Kawakami Y.
Autocrine and paracrine loops between cancer cells and macrophages promote lymph
node metastasis via CCR4/CCL22 in head and neck squamous cell carcinoma. Int J
Cancer. 2013;132:2755-2766.
10. Nishio H,Yaguchi T, Sugiyama J, Sumimoto H, Umezawa K, Iwata T, Susumu N, Fujii T,
Kawamura N, Kobayashi A, Park JH, Aoki D, Kawakami Y, Immunosuppression
through constitutively activated NF-kB signaling in human ovarian cancer and its
reversal by a NF-kB inhibitor, Br J Cancer. 2014;110:2965-74.
11. Tsukamoto N, Okada S, Onami Y, Sasaki Y, Umezawa K, Kawakami Y, Impairment ofpDC for IFN production by the ligand for immunoglobulin-like transcript 7 (ILT7)
expressed on human cancer cells. Clin Cancer Res. 2009;15:5733-5743.
12. Kawakami Y, Yaguchi T, Sumimoto H, Kudo-Saito C, Iwata-Kajihara T, Nakamura S,
Miyzaki J, Kawamura N, Hoon Park J, Popivanova B, Tsujikawa T. Improvement ofcancer immunotherapy by combining molecular targeted therapy. Frontiers in Oncol.
2013;36:1-7,

22

Y. Kawakami

Yutaka Kawakami, M.D., Ph.D.

1984-1985
1985-1987

1989

1989-1997
19972005-

Instructor, Div. Hematology, Keio University
Research Associate, Dept. Immunology, University ofSouth Florida
Visiting Researcher, Dept. Biology,
California Institute of Technology
Visit Scientist, Surgery Branch, NCI, NIH
Professor, Div. Cellular Signaling, Inst. for Advanced
Medical Res, Keio Univ.
Director, Inst. for Adv. Med. Res., Keio Univ.

Specialty and Present Interest:
Tumor Immunology, Tumor Microenvironment, Cancer Immunotherapy

23

THE IMMUNE CONTEXTURE OF HUMAN CANCERS:SHAPING OF CLINICALLY EFFICIENT LANDSCAPES

Etienne Becht, Nicolas Giraldo, Jeremy Goc, Claire Germain,Marie Caroline Dieu-Nosjean, Catherine Sautès-Fridman andWolf H. Fridman

Cordeliers Research Centre, INSERM UMRS 1138 and University Paris Descartes
15 rue de l’Ecole de Médecine, 75006, Paris, France(herve.fridman@crc.jussieu.fr)
Tumors grow in a complex tissue where vascular and lymphatic vessels, inflammatory

and immune cells, present at different levels, influence cancer spreading and patient’s

clinical outcome. The density, location and functional orientation of tumor-infiltrating
lymphocytes from the immune contexture which composition is strongly correlated withpatient’s survival 1 ) . Thus, in the vast majority of cancers, a high infiltration of
Th1/cytotoxic T cells is associated with good disease free and/or overall survival1), both inprimary and metastatic sites, as exemplified in colorectal cancer2,3). There are however,exceptions to this rule as in Hodgkin lymphoma4) and renal cell cancer5) where a strong

infiltration of CD8+ T cells is associated with a poor prognosis. Moreover, increasing

evidence support that the potential beneficial effect of Th1/cytotoxic T cells may behampered by other components of the tumor microenvironment, such as regulatory T cells,or myeloïd derived suppressor cells as well as by tumor cell expression and production of

immunossuppressive molecules (reviewed in 6).

The requirements for shaping a clinically efficient immune landscape were analyzed.

We found that the site of education of adaptive immune cells was essential.

Thus, intumors with high density of Tertiary Lymphoïd Structures (TLS), CD8 T cells and B cells+

were recruted, activated and educated without passing through the tumoral
immunosuppressive milieu and become effector and antibody producing cells, with apositive impact on patient’s survival7,8). CD8+ T cells, educated outside TLS were associatedwith a bad prognosis. It was particularly exemplified in Non Small Cell Lung Cancer(NSCLC) and Colorectal cancer (CRC). In NSCLC, high density of CD8+ T cells correlatewith longer disease free survival (DFS) and overall survival (OS) both in early10) and24

W. H. Fridman et al.

advanced stage (up to stage III)7) tumors. Mature Dendritic Cells (DC), expressing the DC-

Lamp marker, were almost exclusively found in TLS10,11). The combined densities of CD8+ T

cells and DC-Lamp+ cells identified cohorts with the highest (Lo/lo) and the lowest (Hi/Hi)

risk of relapse7,10) suggesting that the enumeration of TLS-associated mature DC improves

the prognostic impact of CD8+ T lymphocytes. Indeed, the analysis of the cohort of patientswith high densities of intratumoral CD8+ T cells, stratified according to the density of DC-

Lamp+ cells identified a cohort of patients with high CD8+ T cells but low numbers of DC-

Lamp+ cells and few TLS, with short DFS and OS7). These data suggest that when CD8+ T
cells are not likely educated in TLS, they may not be active in controlling tumor spread10).

In CRC, in which the paradigm of the pronostic impact of the infiltration by CD8+ memory

T cells and a Th1 orientation has been established1,12,13) leading to the definition of the

Immunoscore14,15), tumors in which the gene encoding CXCL13, a chemokine involved in theformation of tumor-associated TLS11), is deleted identifies a group of patients with poor
prognosis16).

In Renal Cell Cancer (RCC), as in Lymphomas 16,17) a high density of CD8 + T cells

correlates with shorter DFS and OS in primary5) and metastatic3) patients. We revisited thissurprising findings by analyzing cohorts of primary and metastatic clear cell RCC (ccRCC)

and not only confirmed it on larger cohorts of primary ccRCC, but also identified a groupof tumors where there were high densities of DC Lamp+ cells, lacking characterisitics ofmature DC and few TLS. In these tumors, CD8+ T cells are associated with poor prognosis18).

Since the respective positive and negative prognostic impacts of CD8+ T cells in CRC and

RCC are found in metastatic locations in the lung3,18,19), it suggests that the tumor cells,

rather than the organ, shape the immune contexture.

If the tumor cells shape their immune microenvironment molecular subgroups oftumors from the same origin should identify different immune infiltrates. We establishedimmune metagenes, based on the selective expression of genes in purified immunologicalpopulations, and applied them to larger cohorts of ccRCC and CRC6). A cohort of primarytumors of metastatic ccRCC has been classified according to tumor-associated moleculartyping20). Four groups were identified, group 4 being the one with the worst prognosis interms of survival 21) . Group 4 appeared to be the one with the highest expression of

immune, inflammatory angiogenic and myeloïd cell attracting genes and where there was ahigh density of CD8++ and PD1+ lymphocytes as well as PD-L1+ tumors20). In CRC, several

molecular subgroups have been defined21-28). The application of the immune metagenes tomolecular subgroups identified a cohort with microsatellite instability and the best

prognosis. Another subgroup had a strong Th1/CD8+ signature together with angiogenic
and myeloid signatures and was associated with a poorer prognosis (E. Becht et al., in

preparation). The analysis of large collections of colorectal and renal cell cancers, classified

Tumor Immunoenvironment

25

according to tumor cell specific signatures, revealed an association of tumoral subtypes
with immune signatures correlating with good or bad prognosis. The analysis ofrepresentative tumor cell lines revealed that the malignant cells express and produce themolecules found in subtypes of patient’s tumors that modulate the immune contexture.

The identification of these immunomodulatory tumoral subtypes is essential to guide theexploding field of immunomodulatory therapies.

References
1. Fridman W, Pagès F, Sautès-Fridman C & Galon J. The immune contexture in humantumours: impact on clinical outcome. Nature Reviews Cancer (2012) 12: pp. 298-306.
2. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pagès C, Tosolini M,
Camus M, Berger A, Wind P, Zinzindohoué F, Bruneval P, Cugnenc P, Trajanoski Z,
Fridman W & Pagès F. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science (2006) 313: pp. 1960-1964.
3. Remark R, Alifano M, Cremer I, Lupo A, Dieu-Nosjean M, Riquet M, Crozet L,
Ouakrim H, Goc J, Cazes A, Fléjou J, Gibault L, Verkarre V, Régnard J, Pagès O,
Oudard S, Mlecnik B, Sautès-Fridman C, Fridman W & Damotte D. Characteristics andclinical impacts of the immune environments in colorectal and renal cell carcinomalung metastases: influence of tumor origin. Clin. Cancer Res. (2013) 19: pp. 4079-4091.
4. Scott DW, Chan FC, Hong F, Rogic S, Tan KL, Meissner B, Ben-Neriah S, Boyle M,
Kridel R, Telenius A, Woolcock BW, Farinha P, Fisher RI, Rimsza LM, Bartlett NL,
Cheson BD, Shepherd LE, Advani RH, Connors JM, Kahl BS, Gordon LI, Horning SJ,
Steidl C & Gascoyne RD. Gene expression-based model using formalin-fixed paraffinembedded biopsies predicts overall survival in advanced-stage classical hodgkin lymphoma. J. Clin. Oncol. (2013) 31: pp. 692-700.
5. Nakano O, Sato M, Naito Y, Suzuki K, Orikasa S, Aizawa M, Suzuki Y, Shintaku I,
Nagura H & Ohtani H. Proliferative activity of intratumoral CD8+ T-lymphocytes as aprognostic factor in human renal cell carcinoma: clinicopathologic demonstration of
antitumor immunity. Cancer Res. (2001) 61: pp. 5132-5136.
6. Giraldo N, Becht E, Remark R, Damotte D, Sautès-Fridman C & Fridman W. The
immune contexture of primary and metastatic human tumours. Current Opinion in
Immunology (2014) 27: pp. 8-15

26

W. H. Fridman et al.

7. Goc J, Germain C, Vo-Bourgais TKD, Lupo A, Klein C, Knockaert S, de Chaisemartin L,
Ouakrim H, Becht E, Alifano M, Validire P, Remark R, Hammond SA, Cremer I,
Damotte D, Fridman W, Sautès-Fridman C & Dieu-Nosjean M. Dendritic cells in tumorassociated tertiary lymphoid structures signal a th1 cytotoxic immune contexture andlicense the positive prognostic value of infiltrating cd8+ t cells. Cancer Res. (2014) 74: pp.
705-715.
8. Germain C, Gnjatic S, Tamzalit F, Knockaert S, Remark R, Goc J, Lepelley A, Becht E,
Katsahian S, Bizouard G, Validire P, Damotte D, Alifano M, Magdeleinat P, Cremer I,
Teillaud J, Fridman W, Sautès-Fridman C & Dieu-Nosjean M. Presence of b cells in tertiary lymphoid structures is associated with a protective immunity in patients with lungcancer. Am. J. Respir. Crit. Care Med. (2014) 189: pp. 832-844.
9. Dieu-Nosjean M, Goc J, Giraldo NA, Sautès-Fridman C & Fridman WH. Tertiary lymphoid structures in cancer and beyond. Trends in Immunology (2014) 35: pp. 571-580.
10. Dieu-Nosjean M, Antoine M, Danel C, Heudes D, Wislez M, Poulot V, Rabbe N,
Laurans L, Tartour E, de Chaisemartin L, Lebecque S, Fridman W & Cadranel J. Longterm survival for patients with non-small-cell lung cancer with intratumoral lymphoidstructures. J. Clin. Oncol. (2008) 26: pp. 4410-4417.
11. de Chaisemartin L, Goc J, Damotte D, Validire P, Magdeleinat P, Alifano M, Cremer I,
Fridman W, Sautès-Fridman C & Dieu-Nosjean M. Characterization of chemokines andadhesion molecules associated with t cell presence in tertiary lymphoid structures inhuman lung cancer. Cancer Res. (2011) 71: pp. 6391-6399.
12. Galon J, Fridman W & Pagès F. The adaptive immunologic microenvironment in colorectal cancer: a novel perspective. Cancer Res. (2007) 67: pp. 1883-1886.
13. Mlecnik B, Tosolini M, Kirilovsky A, Berger A, Bindea G, Meatchi T, Bruneval P,
Trajanoski Z, Fridman W, Pagès F & Galon J. Histopathologic-based prognostic factors ofcolorectal cancers are associated with the state of the local immune reaction. J. Clin.
Oncol. (2011) 29: pp. 610-618.
14. Galon J, Pagès F, Marincola FM, Angell HK, Thurin M, Lugli A, Zlobec I, Berger A,
Bifulco C, Botti G, Tatangelo F, Britten CM, Kreiter S, Chouchane L, Delrio P, Arndt H,
Asslaber M, Maio M, Masucci GV, Mihm M, Vidal-Vanaclocha F, Allison JP, Gnjatic S,
Hakansson L, Huber C, Singh-Jasuja H, Ottensmeier C, Zwierzina H, Laghi L, Grizzi F,
Ohashi PS, Shaw PA, Clarke BA, Wouters BG, Kawakami Y, Hazama S, Okuno K,
Wang E, O'Donnell-Tormey J, Lagorce C, Pawelec G, Nishimura MI, Hawkins R,
Lapointe R, Lundqvist A, Khleif SN, Ogino S, Gibbs P, Waring P, Sato N, Torigoe T,
Itoh K, Patel PS, Shukla SN, Palmqvist R, Nagtegaal ID, Wang Y, D'Arrigo C, Kopetz S,
Sinicrope FA, Trinchieri G, Gajewski TF, Ascierto PA & Fox BA. Cancer classification
using the immunoscore: a worldwide task force. J Transl Med (2012) 10: p. 205.

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15. Bindea G, Mlecnik B, Tosolini M, Kirilovsky A, Waldner M, Obenauf AC, Angell H,
Fredriksen T, Lafontaine L, Berger A, Bruneval P, Fridman WH, Becker C, Pagès F,
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immune cells reveal the immune landscape in human cancer. Immunity (2013) 39: pp. 782795.
16. Asano N, Oshiro A, Matsuo K, Kagami Y, Ishida F, Suzuki R, Kinoshita T, Shimoyama Y,
Tamaru J, Yoshino T, Kitamura K, Fukutani H, Morishima Y & Nakamura S. Prognostic
significance of t-cell or cytotoxic molecules phenotype in classical hodgkin's lymphoma: aclinicopathologic study. J. Clin. Oncol. (2006) 24: pp. 4626-4633.
17. Rajnai H, Heyning FH, Koens L, Sebestyén A, Andrikovics H, Hogendoorn PCW,
Matolcsy A & Szepesi Á. The density of cd8+ t-cell infiltration and expression of bcl2 predicts outcome of primary diffuse large b-cell lymphoma of bone. Virchows Arch. (2014)
464: pp. 229-239.
18. Giraldo N, Becht E, Pagès F, Skliris G, Verkarre V, Vano Y, Mejean A, Saint-Aubert N,
Lacroix L, Natario I, Lupo A, Alifano M, Damotte D, Cazes A, Triebel F, Freeman G, DieuNosjean M, Oudard S, Fridman W & Sautès-Fridman C. Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic
renal cell cancer. Clin Cancer Res. (2015).
19. Halama N, Michel S, Kloor M, Zoernig I, Pommerencke T, von Knebel Doeberitz M,
Schirmacher P, Weitz J, Grabe N & Jäger D. The localization and density of immune
cells in primary tumors of human metastatic colorectal cancer shows an association
with response to chemotherapy. Cancer Immun. (2009) 9: p. 1.
20. Beuselinck B, Job S, Becht E, Karadimou A, Verkarre V, Couchy G, Giraldo N, RiouxLeclercq N, Molinié V, Sibony M, Elaidi R, Teghom C, Patard J, Fridman W, SautèsFridman C, de Reyniès A, Oudard S & Zucman-Rossi J. Molecular subtypes of clear cell
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Schiappa R, Guenot D, Ayadi M, Kirzin S, Chazal M, Fléjou J, Benchimol D, Berger A,
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22. The Cancer Genome Atlas. Comprehensive molecular characterization of human colonand rectal cancer. Nature (2012) 487: pp. 330-337.
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Hodgson JG, Weinrich S, Bosman F, Roth A & Delorenzi M. Gene expression patterns
unveil a new level of molecular heterogeneity in colorectal cancer. J. Pathol. (2013) 231: pp.
63-76.

28

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24. De Sousa E Melo F, Wang X, Jansen M, Fessler E, Trinh A, de Rooij LPMH, de Jong JH, deBoer OJ, van Leersum R, Bijlsma MF, Rodermond H, van der Heijden M, van Noesel CJM,
Tuynman JB, Dekker E, Markowetz F, Medema JP & Vermeulen L. Poor-prognosis
colon cancer is defined by a molecularly distinct subtype and develops from serrated precursor lesions. Nat. Med. (2013) 19: pp. 614-618.
25. Roepman P, Schlicker A, Tabernero J, Majewski I, Tian S, Moreno V, Snel MH, Chresta
CM, Rosenberg R, Nitsche U, Macarulla T, Capella G, Salazar R, Orphanides G, Wessels
LFA, Bernards R & Simon IM. Colorectal cancer intrinsic subtypes predict chemotherapy
benefit, deficient mismatch repair and epithelial-to-mesenchymal transition. Int. J.
Cancer (2014) 134: pp. 552-562.
26. Sadanandam A, Lyssiotis CA, Homicsko K, Collisson EA, Gibb WJ, Wullschleger S,
Ostos LCG, Lannon WA, Grotzinger C, Del Rio M, Lhermitte B, Olshen AB,
Wiedenmann B, Cantley LC, Gray JW & Hanahan D. A colorectal cancer classification system that associates cellular phenotype and responses to therapy. Nat. Med. (2013) 19:
pp. 619-625.
27. Schlicker A, Beran G, Chresta CM, McWalter G, Pritchard A, Weston S, Runswick S,
Davenport S, Heathcote K, Castro DA, Orphanides G, French T & Wessels LFA.
Subtypes of primary colorectal tumors correlate with response to targeted treatment incolorectal cell lines. BMC Med Genomics (2012) 5: p. 66.
28. Dienstmann R, Guinney J, Delorenzi M, de Reynies A, Roepman P, Sadanandam A,
Vermeulen L, Schlicker A, Missiaglia E, Soneson C, Marisa L, Homicsko K, Wang X,
Simon I, Laurent-Puig P, Wessels L, Medema J, Kopetz S, Friend S & Tejpar S.
Colorectal cancer subtyping consortium (crcsc) identification of a consensus of molecularsubtypes. J. Clin. Oncol. (2014) 32: p. 5s.

Tumor Immunoenvironment

Wolf H. Fridman, M.D., Ph.D.

1972-1976

1976-1983

1983-2005

2005-2013

29

Scientist at the Tumor Immunology Laboratory Hopital
St Louis, Paris
Head, Cellular Immunology Laboratory Scientific
Research Institute on Cancer, Villejuif
Director, Inserm U255, Chairman of the Department ofClinical Biology, Institut Curie, Paris
Director, Cordeliers Research Centre
Chairman, Department of Immunology, European
Hospital Georges Pompidou, Paris

Specialty and Present Interest:
Tumor Immunology, Colorectal Cancer, Renal Cell Cancer, Tumor Microenvironment

30

MEDIATORS OF TUMOR INDUCED IMMUNE SUPPRESSION
IN MALIGNANT MELANOMA

Rolf Kiessling

Department of Oncology/Pathology, Karolinska Institutet
171 76 Stockholm, Sweden(rolf.kiessling@ki.se)

Tumour-induced immune dysfunction is a serious challenge to immunotherapy forcancer. Immune suppressive mechanisms do not only affect the tumor microenvironment,

but also result in decreased systemic immunity. An intact adaptive and innate cellularimmunity are keys to the success of immunotherapy of cancer, why methods to restore

immunity in patients with cancer are attractive to combine with immunotherapy. This isparticularly important in adoptive T cell therapy with Tumor Infiltrating Lymphocytes(TIL), to avoid that ex-vivo expanded TIL cells are inactivated when re-injected into an

immune suppressive environment. At Cancercenter Karolinska we treat patients withadvanced malignant melanoma in the closed clinical trial MAT011) and the ongoing trial

MAT02 (ClinicalTrials.gov: NCT01946373) with a combination of TIL cells and autologous

Dendritic Cells (DCs) derived from the same tumor as the TIL cells were isolated from.

Methods are being developed to prolong the life span of injected TIL cells and to makethese resistant to the immune suppressive mechanisms in the patient. We have focused on

methods to counteract the central role that myelomonocytic cells and their products have in
immune suppression. It is well established that tumor-associated macrophages,

eosinophils, neutrophils and myeloid-derived suppressor cells (MDSC's) are of major

importance in tumor induced immune suppression. In particular MDSC's secrete a broadrepertoire of inflammatory mediators providing them with powerful tools to inhibit tumor

reactive T cells and natural killer cells. These immune suppressive mediators include freeoxygen radicals including reactive oxygen species (ROS) and nitric oxide (NO), arginase,indoleamine 2,3-dioxygenase, prostaglandins, the pro-inflammatory heterodimer S100A8/9

and cytokines, such as granulocyte–macrophage colony-stimulating factor (GM-CSF) andTumor Immunoenvironment

31transforming growth factor-beta (TGF-b ) which all have proven potent in suppressing anti-

tumour cellular immunity (for review see 2). Our studies have focused on the monocytic

MDSCs (CD14 + HLA-DR lo/- ) in peripheral blood of patients with advanced malignant

melanoma3-6). The characteristics and suppressive properties of these mo MDSC's are

summarized in table 1. These mo MDSC suppress T cell and NK functions ex vivo andcorrelate with lower numbers of antigen-specific T cells, but the exact mechanisms

underlying this effect is unclear. We have recently analyzed in vitro the effects of tumor-

derived factors in inducing phenotypic and functional alternations on healthy humanmonocytes or CD34+ hematopoietic stem cells (HSCs) (3, 4 and unpublished data). Thiswas done by co-culturing monocytes or CD34+ hematopoietic stem cells (HSCs) with early-

passage tumor cells isolated from metastatic lymph-nodes or addition of tumor-

conditioned medium. Our results demonstrated that tumor-derived factors promoted

monocytes to acquire an MDSC-like phenotype and suppressive functions of T cells andNK cells where the COX-2/PGE2 pathway played a dominant role. Mouse models arebeing explored to test the in vivo relevance of targeting MDSC and factors responsible for

their expansion and effector functions. This was exemplified by a murine model, where

4T1 mammary carcinoma tumor cells were disabled from cyclooxygenase-2 (COX-2)
production and subsequently their in vivo NK cytotoxicity was evaluated by live imaging4).

MDSCs and regulatory T cells (T reg) constitute an important target for cancer immune

therapy, and new methods to deplete cancer patients from these inhibitory cells will lead tobetter clinical responses. We have recently studied the effect that treatment with the check-point inhibitor ipilimumab, which targets the CTLA-4 molecule, has on MDSC and T reg

cells in patients with advanced malignant melanoma7). Our results, as summarized in table
2, showed that T reg's are depleted in ipilimumab treated patient.

As a new finding weobserved an almost total decrease in the frequency of granulocytic MDSCs, while

monocytic MDSC were not affected by this treatment. In ongoing experiments we are now

analysing the mechanisms behind this drastic effect of CTLA-4 blockade on granulocytic

MDSCs, and also ask to what extent analysis of MDSCs and their products (iNOS,
Table 1

Characteristics of monocytic MDSCs as studied at Cancercenter Karolinska(From references 3-6 and unpublished results)

32

R. Kiessling

Arginase) can be used in clinical practise to predict responsiveness to checkpoint inhibitors.

Reactive oxygen species (ROS) is another product of MDSCs which severely can impair

cancer immune therapy. Oxidative stress produced by MDSCs and other cells in the tumor

stroma can severely impair the function of both T cells and NK cells, leading to decrease in

their signal transducing capacity and at high concentrations to apoptosis8, 9). In ongoingexperiments we are “arming” T cells by gene transfer of the anti-oxidant enzymes catalase,which catalyzes H2O2 to be enzymatically reduced to water and oxygen, thereby

protecting the T cells from functional defects or apoptosis induced by ROS. We haveshown that T cells transduced with bicistronic construct expressing both catalase and achimeric antigen receptors (CAR) are able to maintain functional anti-tumor activities evenwhen confronted with high concentrations of H 2 O 2 as well as H 2 O 2 producing cells,

granulocytes and MDSC (Ligtenberg et al, submitted for publication). Also, the protection

offered to the CAR-CAT bicistronic construct transduced T cells was found to be extended

to bystander cells, allowing for NK cells to functionally eliminate their K562 model cells

under H2O2 induced oxidative stress.
Table 2

Effect of ipilimumab treatment on Treg and MDSC in patients with advanced malignant
melanoma (From reference 7 and unpublished results)

References
1. Poschke I, Lovgren T, Adamson L, Nystrom M, Andersson E, Hansson J, Tell R,
Masucci GV, Kiessling R. 2014. A phase I clinical trial combining dendritic cell vaccination
with adoptive T cell transfer in patients with stage IV melanoma. Cancer Immunol
Immunother 63: 1061-71
2. Mao Y, Poschke I, Kiessling R. 2014. Tumour-induced immune suppression: role ofinflammatory mediators released by myelomonocytic cells. J Intern Med 276: 154-70

Tumor Immunoenvironment

33

3. Mao Y, Poschke I, Wennerberg E, Pico de Coana Y, Egyhazi Brage S, Schultz I, Hansson J,
Masucci G, Lundqvist A, Kiessling R. 2013. Melanoma-educated CD14+ cells acquire amyeloid-derived suppressor cell phenotype through COX-2-dependent mechanisms.
Cancer Res 73: 3877-87
4. Mao Y, Sarhan D, Steven A, Seliger B, Kiessling R, Lundqvist A. 2014. Inhibition oftumor-derived prostaglandin-e2 blocks the induction of myeloid-derived suppressor
cells and recovers natural killer cell activity. Clin Cancer Res 20: 4096-106
5. Poschke I, Mao Y, Adamson L, Salazar-Onfray F, Masucci G, Kiessling R. 2012.
Myeloid-derived suppressor cells impair the quality of dendritic cell vaccines. Cancer
Immunol Immunother 61: 827-38
6. Poschke I, Mougiakakos D, Hansson J, Masucci GV, Kiessling R. 2010. Immature
immunosuppressive CD14+HLA-DR-/low cells in melanoma patients are Stat3hi and over-express CD80, CD83, and DC-sign. Cancer Res 70: 4335-45
7. Pico de Coana Y, Poschke I, Gentilcore G, Mao Y, Nystrom M, Hansson J, Masucci GV,
Kiessling R. 2013. Ipilimumab treatment results in an early decrease in the frequency of
circulating granulocytic myeloid-derived suppressor cells as well as their Arginase1
production. Cancer Immunol Res 1: 158-62
8. Ando T, Mimura K, Johansson CC, Hanson MG, Mougiakakos D, Larsson C, Martins da
Palma T, Sakurai D, Norell H, Li M, Nishimura MI, Kiessling R. 2008. Transduction
with the antioxidant enzyme catalase protects human T cells against oxidative stress. J
Immunol 181: 8382-90
9. Harlin H, Hanson M, Johansson CC, Sakurai D, Poschke I, Norell H, Malmberg KJ,
Kiessling R. 2007. The CD16- CD56bright NK cell subset is resistant to reactive oxygen
species produced by activated granulocytes and has higher antioxidative capacity than the
CD16+ CD56dim subset. J Immunol 179: 4513-9

34

R. Kiessling

Rolf Kiessling, M.D., Ph.D.

1994-

Professor of Experimental Oncology, Karolinska
Institutet, Stockholm. Consultant in oncology andclinical immunology at the Oncology clinic, Karolinska
University HospitalSpecialty and Present Interest:
Senior Consultant in oncology and clinical immunology at the Oncology clinic,
Karolinska University Hospital. R.K. is performing studies both in basic and clinicaltumor immunology and has participated as principal investigator or co-investigator in several
investigator initiated clinical trials, initiated from his laboratory

35

THERAPEUTIC STRATEGIES BASED ON OVERCOMINGIMMUNE RESISTANCE MECHANISMS WITHINTHE TUMOR MICROENVIRONMENT

Thomas F. Gajewski

University of Chicago
5841 S. Maryland Avenue, Chicago, IL 60637 USA(tgajewsk@medicine.bsd.uchicago.edu)

1. Characterization of the T cell-inflamed and non-T cell-inflamed tumor
Based on the lack of correlation between the induction of anti-tumor T cell responses as

microenvironment.

measured in the blood and clinical responses in melanoma, we began over 10 years ago toprobe the tumor microenvironment for predictive biomarkers for clinical response. Two
major categories of melanoma metastases have been observed based on gene expression

profiling and confirmatory assays. One subgroup of patients has an inflamed phenotype

that includes expression of chemokines, T cell markers, and other immunoregulatory

factors including a type I IFN signature. In contrast, the other major subset lacks thisphenotype and appears to display immune “exclusion” 1-3). The T cell-inflamed tumor
microenvironment subset shows the highest expression of negative regulatory factors,

including PD-L1, IDO, and FoxP3+ Tregs4), and evidence for T cell-intrinsic anergy has also

emerged aided by a recently defined functional role of EGR25,6). In addition, the mechanismof induction of these inhibitory mechanisms has been elucidated—PD-L1 and IDO are

induced by IFN-g, and Tregs are largely recruited by the chemokine CCL22, both being

produced by activated CD8+ effector T cells7). Among the T cells within the tumor site are

CD8+ T cells specific for defined tumor antigens8-10). Moreover, these T cells represent thestarting point for tumor-infiltrating lymphocyte (TIL)-based adoptive therapy formelanoma11). Thus, the T cell-inflamed tumor microenvironment appears to represent asituation of chronic T cell activation trapped in the negative feedback phase of an adaptive

immune response. In contrast, the non-T cell-inflamed tumors appear to have minimalcommunication with the host immune response and demonstrate what appears to be
immune exclusion.

36

T. F. Gajewski

The optimal immunotherapeutic interventions may be distinct in the T cell-inflamed

2. Immunotherapeutic interventions targeting the T cell-inflamed tumor subset.

versus non-T cell-inflamed tumor phenotypes12). As a predictive biomarker, early data

analyzing the clinical responders in melanoma to vaccines, high-dose IL-2, the anti-CTLA-4

mAb ipilimumab, and anti-PD-1 mAbs largely fall within this group13-18). These data argue
that the tumor microenvironment must support chemokine-mediated T cell trafficking into

the tumor microenvironment for current immunotherapies to succeed.

The presence of defined immune-inhibitory pathways in these inflamed tumors argues

that blockade of these pathways might restore effective T cell function and lead to

improved tumor control. Strategies to block PD-1/PD-L1 interactions, inhibit IDO

function, deplete Tregs, and reverse T cell anergy have all shown success in preclinicalmodels12,19-21). Anti-PD-1 and PD-L1 mAbs have shown remarkable clinical responses inmetastatic cancer patients, and one of these (pembrolizumab) was FDA approved in

September, 2014 for melanoma22,23). Early clinical biomarker data have suggested again thatmajority of responding patients have a baseline T cell infiltrate, supporting our working

model 1 8 ) . The presence of multiple inhibitory mechanisms in the same tumor
microenvironment argues that combination therapies may be advantageous. Preclinicaldata have indicated synergy between anti-CTLA-4 +/- anti-PD-L1 +/- IDO inhibition,

among others21). The mechanism of synergy is striking, as it correlates with a marked

improvement of IL-2 production and proliferation of tumor-infiltrating CD8+ T cells21).

Clinical translation of these combination immunotherapies is promising and ongoing, withresponse rates of approximately 50% in early studies of anti-CTLA-4 mAb combined with

anti-PD-1 or with an IDO inhibitor24,25). Taken together, these observations have generated
tremendous enthusiasm that manipulation of immune regulatory pathways may ultimatelytranslate into clinical activity in the majority of patients having the T cell-inflamed tumor

microenvironment phenotype.

3. Understanding of innate immune sensing that gives rise to the T cell-inflamed
We believe that a next major hurdle in the cancer immunotherapy field is to understandphenotype—importance of the STING pathway.

why a major subset of patient tumors excludes the host immune response and fails togenerate the T cell-inflamed signature. One approach has been to work towards
identification of the innate immune sensing pathways that mediated spontaneous T cellpriming against sterile tumors. Based on the presence of a type I IFN signature in the

inflamed tumors, we utilized mouse models to determine whether type I IFN signaling was

required upstream from endogenous T cell priming against tumor antigen. Indeed this was

the case, and the effect was mediated though activity on the CD8a+ subset of dendritic cells

Immune Regulation

37(DCs) that carry out antigen cross-presentation26-28). Moving upstream to identify therequisite pathways for induction of IFN-b production by DCs, we uncovered a critical role

for the STING pathway29). STING-deficient mice failed to control immunogenic tumors,
were defective in spontaneous T cell priming, and showed absent IFN-b production bytumor-infiltrating DCs. The STING pathway is involved with cytosolic DNA sensing, andindeed we identified tumor-derived DNA within the cytosol of DCs in vivo. A major

implication of these observations is that the non-T cell inflamed tumors likely have failed toengage the host innate immune pathways, in particular STING. A corollary is that

deliberate activation of the STING pathway may be an attractive novel therapeutic

consideration.

An additional approach for determining mechanisms of immune evasion in the non-T

4. Identifying molecular correlates involved in T cell exclusion.

cell-inflamed tumors has been to analyze molecular features of patients correlating with thepresence or absence of this phenotype, combined with mouse models for mechanistic
studies. Three hypothetical sources of inter-patient heterogeneity are being evaluated:

somatic differences at the level of the tumor cells, germline polymorphisms in immune

regulatory genes, and environmental differences in the composition of the intestinal

microbiome. Preliminary data are supporting functional relevance of all three of thesevariables. Within the tumor cells, we have identified the first oncogene pathway which

Figure 1

Identification of the STING pathway as a major mechanism of innate immune sensing in cancer.
Current evidence indicates that tumor-derived DNA can become taken up and localized tothe cyctosol of tumor-infiltrating DCs, where cGAS becomes activated. This generates
intracellular cGAMP, which promotes STING aggregation and downstream phosphorylationof TBK1 and IRF3. This leads to transcription of the IFN-b gene and also results in a broad
DC activation profile. These activated DCs then are capable of cross-priming CD8+ T cells
which ultimately home back to the tumor and support tumor growth control.

38

T. F. Gajewski

appears to mediate exclusion of the host immune response, which is tumor-intrinsic

activation of the b-catenin pathway. Germline polymorphisms of high statisticalsignificance have been identified that correlate with high presence of T cell infiltration.

Finally, mouse preclinical experiments have indicated that altering only the intestinal

microbiota can profoundly influence immune-mediated tumor control in vivo. A thoroughanalysis of these factors in patients with a range of advanced cancers should be performed

to uncover similar mechanisms beyond melanoma. Each of these mechanisms can beenvisioned to give rise to new therapeutic interventions.

5. Future perspectives: potential strategies to promote immunity in non-T cellIn contrast to the T cell-inflamed melanomas, new paradigms may be needed toinflamed tumors.

promote de novo inflammation in cases of the non-T cell-infiltrated tumor

microenvironment. Based on recent data implicating the STING pathway and endogenous

type I IFN production in spontaneous immune priming, STING agonists are being

evaluated to promote activation of this pathway. Tremendous anti-tumor activity has been

observed in preclinical experiments, supporting plans for clinical translation. Increasing IFN-babundance in the tumor microenvironment also is an effective strategy. Coupling IFN-b totumor-targeting mAbs has shown interesting activity in mouse models that works through an

immunologic mechanism30). Direct administration of high doses of IFN-b within the tumor

microenvironment also is therapeutic, although the majority of this activity is through an anti-

angiogenic effect31). Recent work has suggested that targeted radiation to the tumor also
promotes STING pathway activation and type I IFN production in vivo32).

Additional therapeutic strategies can be envisioned based on data emerging from thegenomic analysis of patient specimens. Our current data already have suggested thatpharmacologic strategies to inhibit the b-catenin pathway may provide one means ofreversing an immune-inhibitory oncogene pathway. It also might be feasible to pursue

pharmacologic approaches to functionally mimic the effect of favorable germline

polymorphisms. Finally, manipulation of the intestinal microbiome to facilitate anti-tumor
immunity and potentiate the T cell-inflamed tumor microenvironment phenotype also is
attractive to consider, for example through development of appropriate probiotics. Thus,

as these molecular mechanisms continue to be unraveled, novel therapeutic strategies tosupport natural T cell responses against tumors can be envisioned. Combining suchinterventions with blockade of immune inhibitory pathways ultimately may be necessary
for maximal therapeutic efficacy at the population level.

Immune Regulation

Figure 2

39

Hypothetical mechanisms of inter-patient heterogeneity that could contribute to the presenceor absence of a spontaneously generated T cell-inflamed tumor microenvironment.
Genomics approaches are being pursued to analyze germline polymorphisms, tumor
somatic changes, and intestinal microbiome species that may associate with the presence orabsence of a T cell-inflamed tumor microenvironment in individual patients. Integration ofthese genomics platforms should provide a compresensive assessment of molecular correlates
to host anti-tumor immune responses, and also identifiy new therapeutic approaches forintervention. Inasmuch as the T cell-inflamed tumor microenvironment is associated withclinical response to immunotherapies, it is envisioned that a multivariate predictive biomarker algorithm will ultimately be created from these data.

References
1. Gajewski TF. 2007. Failure at the effector phase: immune barriers at the level of themelanoma tumor microenvironment. Clin Cancer Res 13: 5256-61
2. Gajewski TF, Schreiber H, Fu YX. 2013. Innate and adaptive immune cells in the tumor
microenvironment. Nat Immunol 14: 1014-22
3. Harlin H, Meng Y, Peterson AC, Zha Y, Tretiakova M, Slingluff C, McKee M, Gajewski
TF. 2009. Chemokine expression in melanoma metastases associated with CD8+ T-cell
recruitment. Cancer Res 69: 3077-85
4. Spranger S, Spaapen RM, Zha Y, Williams J, Meng Y, Ha TT, Gajewski TF. 2013. UpRegulation of PD-L1, IDO, and Tregs in the Melanoma Tumor Microenvironment Is
Driven by CD8+ T Cells. Sci Transl Med 5: 200ra116
5. Zheng Y, Zha Y, Spaapen RM, Mathew R, Barr K, Bendelac A, Gajewski TF. 2013. Egr2dependent gene expression profiling and ChIP-Seq reveal novel biologic targets in T
cell anergy. Mol Immunol 55: 283-91

40

T. F. Gajewski

6. Zheng Y, Zha Y, Driessens G, Locke F, Gajewski TF. 2012. Transcriptional regulator
early growth response gene 2 (Egr2) is required for T cell anergy in vitro and in vivo. J Exp
Med 209: 2157-63
7. Spranger S, Spaapen R, Zha Y, Williams J, Meng Y, Ha TT, Gajewski TF. 2013.
Upregulation of PD-L1, IDO and Tregs in the melanoma tumor microenvironment isdriven by CD8+ T cells. Science Translational Medicine: In Press
8. Harlin H, Kuna TV, Peterson AC, Meng Y, Gajewski TF. 2006. Tumor progression
despite massive influx of activated CD8+ T cells in a patient with malignant melanoma
ascites. Cancer Immunol Immunother 55: 1185-97
9. Zippelius A, Batard P, Rubio-Godoy V, Bioley G, Lienard D, Lejeune F, Rimoldi D,
Guillaume P, Meidenbauer N, Mackensen A, Rufer N, Lubenow N, Speiser D, Cerottini
JC, Romero P, Pittet MJ. 2004. Effector function of human tumor-specific CD8 T cells inmelanoma lesions: a state of local functional tolerance. Cancer Res 64: 2865-73
10. Mortarini R, Piris A, Maurichi A, Molla A, Bersani I, Bono A, Bartoli C, Santinami M,
Lombardo C, Ravagnani F, Cascinelli N, Parmiani G, Anichini A. 2003. Lack of terminally differentiated tumor-specific CD8+ T cells at tumor site in spite of antitumor immunity

to self-antigens in human metastatic melanoma. Cancer Res 63: 2535-45
11. Rosenberg SA, Dudley ME. 2004. Cancer regression in patients with metastaticmelanoma after the transfer of autologous antitumor lymphocytes. Proc Natl Acad Sci USA
101 Suppl 2: 14639-45
12. Gajewski TF, Woo SR, Zha Y, Spaapen R, Zheng Y, Corrales L, Spranger S. 2013. Cancer
immunotherapy strategies based on overcoming barriers within the tumor microenvironment. Curr Opin Immunol 25: 268-76
13. Ji RR, Chasalow SD, Wang L, Hamid O, Schmidt H, Cogswell J, Alaparthy S, Berman D,
Jure-Kunkel M, Siemers NO, Jackson JR, Shahabi V. 2011. An immune-active tumor
microenvironment favors clinical response to ipilimumab. Cancer Immunol Immunother
14. Vansteenkiste JF, Zielinski M, Dahabreh IJ, Linder A, Lehmann F, Gruselle O, Therasse P,
Louahed J, Brichard VG. 2008. Association of gene expression signature and clinicalefficacy of MAGE-A3 antigen-specific cancer immunotherapeutic (ASCI) as adjuvant
therapy in resected stage IB/II non-small cell lung cancer (NSCLC). J Clin Oncol 26:
Abstract 7501
15. Gajewski TF, Louahed J, Brichard VG. 2010. Gene signature in melanoma associatedwith clinical activity: a potential clue to unlock cancer immunotherapy. Cancer J 16: 399403
16. Sullivan RJ, Hoshida Y, Brunet J, Tahan S, Aldridge J, Kwabi C, Gardiner E, McDermot D,
Golub T, Atkins MA. 2009. A single center experience with high-dose IL-2 treatment forpatients with advanced melanoma and pilot investigation of a novel gene expression
signature as a predictor of response. J. Clin. Oncol. 27:15S: Abstract 9003

Immune Regulation

41

17. Gajewski TF, Zha Y, Thurner B, Schuler G. 2009. Association of gene expression profile inmelanoma and survival to a dendritic cell-based vaccine. J. Clin. Oncol. 27: 9002
18. Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJ, Robert L, Chmielowski B,
Spasic M, Henry G, Ciobanu V, West AN, Carmona M, Kivork C, Seja E, Cherry G,
Gutierrez AJ, Grogan TR, Mateus C, Tomasic G, Glaspy JA, Emerson RO, Robins H,
Pierce RH, Elashoff DA, Robert C, Ribas A. 2014. PD-1 blockade induces responses by
inhibiting adaptive immune resistance. Nature 515: 568-71
19. Kline J, Brown IE, Zha YY, Blank C, Strickler J, Wouters H, Zhang L, Gajewski TF. 2008.
Homeostatic proliferation plus regulatory T-cell depletion promotes potent rejection of
B16 melanoma. Clin Cancer Res 14: 3156-67
20. Blank C, Brown I, Peterson AC, Spiotto M, Iwai Y, Honjo T, Gajewski TF. 2004. PDL1/B7H-1 inhibits the effector phase of tumor rejection by T cell receptor (TCR) transgenic
CD8+ T cells. Cancer Res 64: 1140-5

21. Spranger S, Koblish HK, Horton B, Scherle PA, Newton R, Gajewski TF. 2014.
Mechanism of tumor rejection with doublets of CTLA-4, PD-1/PD-L1, or IDO blockade
involves restored IL-2 production and proliferation of CD8+ T cells directly within thetumor microenvironment. J Immunother Cancer 2: 3
22. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, Powderly JD,
Carvajal RD, Sosman JA, Atkins MB, Leming PD, Spigel DR, Antonia SJ, Horn L, Drake
CG, Pardoll DM, Chen L, Sharfman WH, Anders RA, Taube JM, McMiller TL, Xu H,
Korman AJ, Jure-Kunkel M, Agrawal S, McDonald D, Kollia GD, Gupta A, Wigginton JM,
Sznol M. 2012. Safety, Activity, and Immune Correlates of Anti-PD-1 Antibody inCancer. N Engl J Med
23. Hamid O, Robert C, Daud A, Hodi FS, Hwu WJ, Kefford R, Wolchok JD, Hersey P,
Joseph RW, Weber JS, Dronca R, Gangadhar TC, Patnaik A, Zarour H, Joshua AM,
Gergich K, Elassaiss-Schaap J, Algazi A, Mateus C, Boasberg P, Tumeh PC,
Chmielowski B, Ebbinghaus SW, Li XN, Kang SP, Ribas A. 2013. Safety and tumor
responses with lambrolizumab (anti-PD-1) in melanoma. N Engl J Med 369: 134-44
24. Wolchok JD, Kluger H, Callahan MK, Postow MA, Rizvi NA, Lesokhin AM, Segal NH,
Ariyan CE, Gordon RA, Reed K, Burke MM, Caldwell A, Kronenberg SA, Agunwamba
BU, Zhang X, Lowy I, Inzunza HD, Feely W, Horak CE, Hong Q, Korman AJ,
Wigginton JM, Gupta A, Sznol M. 2013. Nivolumab plus ipilimumab in advanced
melanoma. N Engl J Med 369: 122-33
25. Gibney G, Hamid O, Gangadhar T, Lutzky J, Olszanski A, Gajewski T, Chmielowski B,
Boasberg P, Zhao Y, Newton R, Scherle P, Bowman J, Maleski J, Leopold L, Weber J. 2014.
Preliminary results from a phase 1/2 study of INCB024360 combined with ipilimumab(ipi) in patients (pts) with melanoma. J Clin Oncol 32:5s (suppl; abstr 3010)

42

T. F. Gajewski

26. Fuertes MB, Woo SR, Burnett B, Fu YX, Gajewski TF. 2013. Type I interferon response andinnate immune sensing of cancer. Trends Immunol 34: 67-73
27. Fuertes MB, Kacha AK, Kline J, Woo SR, Kranz DM, Murphy KM, Gajewski TF. 2011.
Host type I IFN signals are required for antitumor CD8+ T cell responses throughCD8{alpha}+ dendritic cells. J Exp Med
28. Diamond MS, Kinder M, Matsushita H, Mashayekhi M, Dunn GP, Archambault JM,
Lee H, Arthur CD, White JM, Kalinke U, Murphy KM, Schreiber RD. 2011. Type I interferon is selectively required by dendritic cells for immune rejection of tumors. J Exp
Med 208: 1989-2003
29. Woo S-R, Fuertes Mercedes B, Corrales L, Spranger S, Furdyna Michael J, Leung
Michael YK, Duggan R, Wang Y, Barber Glen N, Fitzgerald Katherine A, Alegre M-L,
Gajewski Thomas F. 2014. STING-Dependent Cytosolic DNA Sensing Mediates Innate
Immune Recognition of Immunogenic Tumors. Immunity 41: 830-42
30. Yang X, Zhang X, Fu ML, Weichselbaum RR, Gajewski TF, Guo Y, Fu YX. 2014.
Targeting the tumor microenvironment with interferon-beta bridges innate and adaptive
immune responses. Cancer Cell 25: 37-48
31. Spaapen RM, Leung MY, Fuertes MB, Kline JP, Zhang L, Zheng Y, Fu YX, Luo X, Cohen
KS, Gajewski TF. 2014. Therapeutic Activity of High-Dose Intratumoral IFN-beta
Requires Direct Effect on the Tumor Vasculature. J Immunol 193: 4254-60
32. Deng L, Liang H, Xu M, Yang X, Burnette B, Arina A, Li X-D, Mauceri H, Beckett M,
Darga T, Huang X, Gajewski Thomas F, Chen Zhijian J, Fu Y-X, Weichselbaum Ralph R.
2014. STING-Dependent Cytosolic DNA Sensing Promotes Radiation-Induced Type I
Interferon-Dependent Antitumor Immunity in Immunogenic Tumors. Immunity 41:843-52

Thomas F. Gajewski, M.D., Ph.D.

1997200220022004
2009
2010-2012

Assistant Professor; Director, Melanoma Oncology,
University of Chicago
Leader, University of Chicago Medicine
Comprehensive Cancer Center (UCCCC) Immunology
and Cancer Program
Director, Human Immunologic Monitoring Facility
Associate Professor with Tenure, University of Chicago
Professor with Tenure, University of Chicago
President, Society for Immunotherapy of Cancer (SITC)43

REPROGRAMMING THE TUMOR MICROENVIRONMENTFOR EFFICIENT TUMOR REJECTION

Rafael Carretero1, Ibrahim M. Sektioglu1, Natalio Garbi1,3,
Philipp Beckhove2 and Günter J. Hämmerling1

Division of Molecular Immunology
Division of Translational Immunology
DKFZ - German Cancer Research Center Heidelberg, Germany
3
Present address: Institutes of Molecular Medicine and Experimental Immunology
University of Bonn, Germany(hammerling@dkfz.de)
1

2

To date, immunotherapy of cancer has met limited clinical success. Responsive cases,

however, demonstrate that immunotherapy can be a powerful approach. They also showthat for improvement of immunotherapy the mechanisms of tumor rejection need to be

explored in more detail. Clinical and experimental studies have identified several
mechanisms that prevent tumor rejection. Tolerance mechanisms including regulatory T

cells (Tregs) play an important role as they result in impaired activation of tumor-specific T

lymphocytes. Of crucial importance are also insufficient infiltration of T cells into tumors

and an inhibitory tumor microenvironment.

We have reported that the usually aberrant phenotype of the tumor vasculature

Danger signals enhance T cell infiltration.

significantly contributes to the lack of infiltration. An aberrant vasculature is alsocharacteristic for Rip.Tag mice that develop autochthonous insulinomas in a multi-stepprocess closely resembling the clinical situation of human pancreatic neuroendocrine

tumors. Vaccination or transfer of tumor-specific T lymphocytes failed to eliminate thetumors, because the aberrant tumor endothelium formed a barrier against cell infiltration.
We have observed that the endothelial barrier could be overcome by induction of an

inflammatory microenvironment in the tumor by danger signals, e.g. by irradiation or

immunostimulatory TLR ligands. Both of these auxiliary treatments resulted innormalization of the tumor vasculature, expression of adhesion molecules such as VCAM1
on vessels, efficient T cell infiltration, and tumor eradication1,2), see Figure 1.

44

G. J. Hämmerling et al.

These findings raised two major questions, namely 1. the mechanisms by which

irradiation and TLR ligands exert their dramatic effects, and 2. whether or not vessel

normalization is important for T-cell infiltration.

In order to study a potential role of vessel normalization, we performed gene expression

Vessel normalization.

profiling and found regulator of G protein signalling 5 (RGS5) to be strongly expressed on

the aberrant tumor vasculature, but largely absent on normal vessels. RGS5 k.o. mice were

produced and crossed with RipTag5 mice. The RGS5-/- x RipTag mice developed

insulinoma with about the same kinetics as RGS5-positive RipTag mice. Detailed analysis

revealed that RGS5-negative tumors exhibited a normalized vasculature. In addition,

tumor hypoxia was decreased. Importantly, immunization or adaptive T cell transfer

resulted in T cell infiltration and survival of the animal3). These data are schematicallypresented in Figure 2.

These results demonstrate that normalization of tumor vessels is sufficient to permit T

cell infiltration without the requirement for danger signals. Probably the decreased

hypoxia is of importance as it is known to influence T cell functions.

Next, we investigated the mechanisms by which danger signals might control T cell

infiltration and tumor rejection.

TLR ligands.

We have observed that labelled CpG binds to tumor associated macrophages2). In order

to see if this binding and subsequent activation of the macrophages was important, wedepleted macrophages with clodronate liposomes and observed that now tumor rejection

was impeded. Mechanistically, polarization of immunosuppressive M2-like tumor

macrophages towards immunostimulatory M1-like iNOS+ macrophages was found to berequired for tumor rejection.

With regard to radiation , local radiation with a low dose of 2 Gy combined with T cell

Local low dose radiation.

transfer was sufficient to support vessel normalization, infiltration of T cells and tumor

rejection. Interestingly, macrophages were again crucial for this process, in particular theiNOS positive M1 polarized macrophages, because blocking of iNOS in vivo with thespecific inhibitor 1400w abrogated tumor rejection4).

In another set of studies we adressed the question as to how efficient Treg depletion

From Tregs to eosinophils.

would affect spontaneous tumor immunity. 90%depletion of Tregs (but not 70% depletion)

Immune Regulation

45

Figure 1

T cell infiltration and tumor rejection after induction of inflammation by irradiation or TLR
stimulation.
RipTag mice carrying established tumors were irradiated or treated with CpG-ODN and thentransferred with activated tumor-specific T cells. Irradiation or CpG-ODN treatment lead tovessel normalization and T cell infiltration (left part), resulting in survival of the animals(right hand part).

Figure 2

RGS5 (regulator of G protein signalling) knockout results in normalization of the tumor
vasculature, T cell infiltration and increased survival.
The left hand part shows the typically aberrant vasculature of wild-type (RGS5+/+)
RipTag insulinoma which prevents infiltration of tumor-specific T cells. In RGS5-/- mice thevasculature is normalized and permits strong T cell infiltration, thereby enhancing survival(right hand part).

46

G. J. Hämmerling et al.

in tumor bearing Foxp3.LuciDTR mice resulted in a cytokine storm and at the same time inactivation of endogeneous tumor specific T cells, normalization of the tumor vasculature,

CD8 T cell infiltration and tumor eradication5). Interestingly, not only T cells were found to
infiltrate the tumor after Treg depletion but also eosinophils. Tumor eosinophilia is often

observed in the clinic, but its function is a long standing question and so far only a matterof speculations6,7). Surprisingly, depletion of eosinophils by specific Siglec F antibody

impaired tumor eradication. Cotransfer studies with activated eosinophils and T- cells

revealed that eosinophils migrated into the tumor where they produced chemokines that

attracted T cells into the tumor. Concommitantly the tumor macrophages were skewed
towards M1 and the vasculature was normalized (unpublished data).

The results presented hereindicate that different approaches of T cell mediated therapy

Conclusions.

of cancer function via the same mechanisms, namely reprogramming of the tumor

microenvironment, with macrophage polarization towards M1 and vessel normalization, as

depicted below in Figure 3. We suggest that for successful clinical cancer immunotherapy

vaccination or adoptive T cell transfer should be combined with modulators of the tumor

microenvironment, such as TLR ligands or local low dose tumor irradiation which

Figure 3

Immune Regulation

47

polarizes tumor macrophages, thereby enhancing T cell infiltration. Indeed, in preliminary

patient studies, local irradiation of pancreatic tumors resulted in accumulation of iNOS+ M1

macrophages and increased T cell infiltration4). Together, these observations open the way

for novel therapeutic interventions.

References
1. Ganss, R., R. Ryschich, E. Klar, B. Arnold, G.J. Hämmerling, Combination of T cell therapy and trigger of inflammation induces remodeling of the vasculature and tumor eradication. Cancer Research, 62, 1462-1470, 2002
2. Garbi, N., Arnold, B., Gordon, S., Hämmerling, G.J., Ganss, R., CpG motifs as proinflammatory factors render autochthonous tumors permissive for infiltration anddestruction. J. Immunol. 172(10), 5861-5869, 2004
3. Hamzah J, Jugold M, Kiessling F, Rigby P, Manzur M, Marti HH, Rabie T, Kaden S,
Gröne HJ, Hämmerling GJ, Arnold B, Ganss R. Vascular normalization in RGS5-deficient tumors promotes immune destruction. Nature 453(7193):410-414, 2008
4. Klug F, Prakash H, Huber PE, Seibel T, Bender N, Halama N, Pfirschke C, Voss RH,
Timke C, Umansky L, Klapproth K, Schäkel K, Garbi N, Jäger D, Weitz J, SchmitzWinnenthal H, Hämmerling GJ, Beckhove P.
Low-Dose Irradiation Programs+
Macrophage Differentiation to an iNOS /M1 Phenotype that Orchestrates Effective T

Cell Immunotherapy. Cancer Cell. 24(5):589-602, 2013
5. Li X, Kostareli E, Suffner J, Garbi N, Hämmerling GJ. Efficient Treg depletion induces T
cell infiltration and rejection of large tumors. Eur.J.Immunol. 40(12):3325-35, 2010
6. Lotfi, R., Lee, J.J. & Lotze, M.T. Eosinophilic granulocytes and damage-associatedmolecular pattern molecules (DAMPs): role in the inflammatory response withintumors. J Immunother 30, 16-28 (2007).
7. Rosenberg, H.F., Dyer, K.D. & Foster, P.S. Eosinophils: changing perspectives in healthand disease. Nature Reviews. Immunology 13, 9-22 (2013).

Günter J. Hämmerling, Prof. Dr. rer. nat.

1979-

Professor and Chairman, Division of Molecular
Immunology, Tumor Immunology Program, German
Cancer Research Center, Heidelberg, Germany

Specialty and Present Interest:
Tumor Immunity, Peripheral Tolerance, Antigen Presentation

48

HARNESSING REGULATORY T CELLS IN
CANCER IMMUNOTHERAPY

Hiroyoshi Nishikawa

Experimental Immunology, Immunology Frontier Research Center, Osaka University
3-1 Yamadaoka, Suita, Osaka 565-0871, Japan(nisihiro@ifrec.osaka-u.ac.jp)

Regulatory T cells (Tregs) hinder cancer immuno-surveillance and also prevent theinduction of effective anti-tumor immunity. It is therefore becoming an important priority

in the cancer immunotherapy field to find strategies for controlling Tregs, in addition tostimulating effector T cells by immunostimulatory signals including immune checkpoint

blockade and antigen-specific cancer vaccines1,2). Indeed, depletion of Tregs enhances
spontaneous and vaccine-induced anti-tumor immune responses, and the stimulation ofTregs by immunization with self-antigens induces enhanced chemically-induced primarytumor development and increased numbers of pulmonary metastasis following injection oftransplantable tumor cells in animal models3-7). In humans, the presence of high numbers ofTregs or a low ratio of CD8 + T cells to Tregs in tumor tissues is correlated with anunfavorable prognosis in various types of cancers1,8,9). Thus, an understanding of thedetailed mechanisms of Treg suppression against tumor-antigen-specific T cells is crucial.

Yet, how Tregs stably suppress self-(tumor)-antigen-specific CD8+ T cells remains obscure.

We investigated proliferation, cytokine production, and the cell fate of self-(tumor)-antigen-specific CD8 + T cells in peripheral blood mononuclear cells (PBMCs) whenstimulated in vitro with self-antigen Melan-A/MART-1 (Melan-A) peptide in the presenceor absence of natural FoxP3+CD25+CD4+ Tregs. Melan-A-specific CD8+ T cells activated withphysiologically-stimulated Tregs underwent one cell division and stopped further

proliferation. These Melan-A-specific CD8+ T cells had an anergic phenotype (hypo-proliferative and cytokine hypo-producing upon TCR re-stimulation). Such anergic T cells

possessed lower affinity T-cell receptors (TCRs) for the stimulating self-antigen thanantigen-activated proliferating T cells. Importantly, Melan-A-specific CD8+ T cells obtainedImmune Regulation

49

from Treg-absent or -present cell cultures had substantial differences in gene expression

profiles. Particularly, Melan-A-specific CD8+ T cells suppressed by Tregs were distinct fromother T cells in being phenotypically naïve, e.g., CC chemokine receptor 7 (CCR7) positive,

but expressing high levels of several co-inhibitory molecules, such as cytotoxic T-

lymphocyte-associated antigen-4 (CTLA-4). This unique phenotype enabled in vivo
detection of hitherto elusive anergic T cells as a distinct T-cell subpopulation in healthy

individuals, and revealed concurrent generation of anergic T cells and activated T cells in

an immune response. Mechanistically, in accordance with the ability of Treg cells to down-

modulate the expression of the co-stimulatory molecules CD80 and CD86 on antigen-presenting cells (APCs)10,11), blockade of these molecules on APCs was able to drive lowaffinity self-reactive T cells into anergy with similar phenotype and function to the anergic

T cells observed following Treg suppression. Melan-A-specific CD8+ T cells with thisanergic phenotype were detected ex vivo in PBMCs from healthy individuals in contrast

with the effector or memory phenotype of the majority of Melan-A-specific CD8+ T cells in

vitiligo, and therefore contributed to the tolerance of self-(tumor)-antigens12). This Treg-

dependent induction of anergy is likely operating in tumor immunity, indicating theimportance of controlling Tregs to allow the efficient activation of self-(tumor)-antigenspecific CD8+ T cells.

To control Tregs, we attempted to find specific markers of tumor (malignant

melanoma)-infiltrating Tregs. FoxP3 + CD4 + T cells can be dissected into threesubpopulations by the expression levels of FoxP3 and the cell surface molecules CD45RA

and CD25: FoxP3loCD45RA+CD25lo cells, designated naive or resting Tregs, FoxP3hiCD45RACD25hi cells, designated effector Tregs, which are terminally differentiated and highly

suppressive; and FoxP3loCD45RA-CD25lo non-Tregs, which do not possess suppressive

activity but can secrete pro-inflammatory cytokines2,10). In melanoma tissues, Effector Tregs

were predominant among tumor-infiltrating FoxP3+ T cells and much higher in frequency
compared with those in peripheral blood. These melanoma infiltrating effector Tregs

specifically expressed CC chemokine receptor 4 (CCR4). With peripheral blood
lymphocytes from melanoma patients, ex vivo depletion of CCR4+ T cells (effector Tregs)

and subsequent in vitro stimulation of the depleted cell population with the cancer/testis
antigen NY-ESO-1 efficiently induced NY-ESO-1-specific CD4+ T cells. Non-CCR4-depleted

cells failed to induce these cells. The magnitude of the responses was comparable to thoseseen following total removal of FoxP3+ Tregs by CD25+ T-cell depletion. CCR4+ T-cell

depletion also augmented in vitro generation of NY-ESO-1-specific CD8 + T cells inmelanoma patients. Furthermore, in vivo administration of anti-CCR4 mAb markedly

reduced the effector Treg fraction and augmented NY-ESO-1-specific CD8+ T cell responses

in an adult T-cell leukemia-lymphoma patient whose leukemic cells expressed NY-ESO-1.

50

H. Nishikawa

Collectively, these findings indicate that anti-CCR4 mAb treatment is instrumental for

evoking and enhancing anti-tumor immunity in cancer patients by selectively depleting

effector Tregs13). We propose that selective reduction of effector Tregs, rather than whole

Treg population can augment anti-tumor immune responses without significant

autoimmunity, and CCR4 can be a possible target for selective Treg control.
References

1. Nishikawa H, Sakaguchi S. Regulatory T cells in tumor immunity. Int J Cancer
2010;127:759-67.
2. Nishikawa H, Sakaguchi S. Regulatory T cells in cancer immunotherapy. Curr Opin
Immunol 2014;27:1-7.
3. Onizuka S, Tawara I, Shimizu J, et al. Tumor rejection by in vivo administration of antiCD25 (interleukin-2 receptor a) monoclonal antibody. Cancer Res 1999;59:3128-33.
4. Shimizu J, Yamazaki S, Sakaguchi S. Induction of tumor immunity by removing
CD25+CD4+ T cells: a common basis between tumor immunity and autoimmunity. J

Immunol 1999;163:5211-18.
5. Nishikawa H, Kato T, Tanida K, et al. CD4+CD25+ T cells responding to serologicallydefined autoantigens suppress anti-tumor immune responses. Proc. Natl Acad Sci USA
2003;100:10902-6.
6. Nishikawa H, Kato T, Tawara I, et al. Definition of target antigens for naturally occurring
CD4+CD25+ regulatory T cells. J Exp Med 2005;201:681-86.
7. Nishikawa H, Kato T, Tawara I, et al. Accelerated chemically-induced tumor development mediated by CD4+CD25+ regulatory T cells in wild-type hosts. Proc Natl Acad Sci
USA. 2005;102:9253-57.
8. Curiel TJ, Coukos G, Zou L, et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 2004;10:942-49.
9. Sato E, Olson SH, Ahn J, et al. Intraepithelial CD8+ tumor-infiltrating lymphocytes and ahigh CD8+/regulatory T cell ratio are associated with favorable prognosis in ovariancancer. Proc Natl Acad Sci USA 2005;102:18538-43.
10. Sakaguchi S, Miyara M, Costantino CM, et al. FOXP3+ regulatory T cells in the humanimmune system. Nat Rev Immunol 2010;10:490-500.
11. Qureshi OS, Zheng Y, Nakamura K, et al. Trans-endocytosis of CD80 and CD86: amolecular basis for the cell-extrinsic function of CTLA-4. Science 2011;332:600-3.
12. Maeda Y, Nishikawa H, Sugiyama D, et al. Detection of self-reactive CD8+ T cells with ananergic phenotype in healthy individuals. Science 2014;346:1536-40.

Immune Regulation

51

13. Sugiyama D, Nishikawa H, Maeda Y, et al. Anti-CCR4 mAb selectively depletes effectortype FoxP3+CD4+ regulatory T cells, evoking antitumor immune responses in humans.
Proc Natl Acad Sci USA 2013;110:17945-50.

Hiroyoshi Nishikawa, M.D., Ph.D.

2003-2006

2006-2010
2010-

2012-

Research fellow, LICR at Memorial Sloan Kettering
Cancer Center
Assistant Professor, Department of Cancer Vaccine,Mie University Graduate School of Medicine
Associate Professor, Experimental Immunology, IFReC,
Osaka University
Adjunct Associate Professor, Department of Oncology,
Roswell Park Cancer InstituteSpecialty and Present Interest:
Cancer Immunology, Immunotherapy, Immunosuppression, Regulatory T Cells

52

IMMUNOGENIC CELL DEATH IN CANCER THERAPY

Guido Kroemer

University of Paris Descartes, INSERM U1138, Centre de Recherche des Cordeliers
Hopital Européen George Pompidou, Institut Gustave Roussy, Villejuif/Paris, France(kroemer@orange.fr)

The supreme goal of anticancer therapy is the physical elimination of tumor cell by

causing their death. In physiological settings, cell death occurs as a continuous byproductof cellular turnover. This type of cell death is non-immunogenic or even tolerogenic, thus

avoiding the induction of autoimmune disease. However, the iatrogenic cancer cell death

elicited by radiotherapy and a specific class of chemotherapeutic agents (such as
anthracyclines and oxaliplatin) can be immunogenic. Immunogenic cell death (ICD)

involves subtle changes in the external surface of the plasma membrane, as well as therelease of soluble immunogenic signals. These changes in the cell surface and the
pericellular microenvironment must occur in a defined temporal sequence that constitutesa ‘code’ or ‘key’. This ‘code’ is decoded by a series of receptors expressed by cells from theinnate immune system, in particular dendritic cells (DC, the ‘lock’). If all the changes
accompanying immunogenic cell death are present and – presumably – occur in the rightorder, as well as at the correct intensity, they allow for the DC-mediated cross-presentationof tumor-specific antigens to T lymphocytes and for triggering of an efficient anticancer

immune response1-6). Thus far, we have been able to identify four hallmarks of ICD.

Immunogenic cell death is characterized by the pre-apoptotic cell surface exposure of

Calreticulin exposure.

calreticulin (CRT), which determines the uptake of tumor-associated antigens by DC. The
precocious release of CRT is the result of an endoplasmic reticulum stress response (alsocalled unfolded stress response) causing the translocation of CRT from the endoplasmic

reticulum lumen to the cell surface. Tumors that lack CRT expression or that lack elements

Chemo Immunotherapy

53of the CRT exposure pathway are relatively resistant to anticancer chemotherapy (ascompared to normal control tumors) unless recombinant CRT protein is injected into thetumor7-9).

During the apoptotic blebbing phase, dying cells can release adenosine triphosphate

ATP release.

(ATP) through a pathway that is particularly efficient if the cells have undergone

premortem induction of autophagy, likely due to a the autophagy-mediated increase of
intralysosomal ATP stores. The release of ATP from apoptotic cells causes the P2Y2

purinergic receptor-mediated attraction of DC precursors into the tumor bed. ATP released

from dying cancer cells also acts on another type or purinergic receptors, namely ionotropic

P2RX7 receptors. P2RX7 receptor-dependent activation of the NLRP3 inflammasome in DC

allows these cells to release interleukin-1b and hence to polarize tumor antigen-specific
CD8 T lymphocytes towards a Tc1 cytokine pattern. Tumors that are autophagy deficient

fail to elicit an anticancer immune response following treatment with anthracyclines or

oxaliplatin and hence are resistant to anticancer chemotherapy with these agents.

Moreover, mice that lack P2RX7 expression or mice in which P2Y2 has been blocked
pharmaceutically fail to mount anticancer immune responses post-chemotherapy10-17).

At a post-apoptotic stage, as the plasma membrane undergoes permeabilization during

HMGB1 exodus.

secondary necrosis, the protein high mobility group box 1 (HMGB1) is released.

Extracellular HMGB1 acts on toll-like receptor 4 (TLR4), the activation of which is required

for the optimal presentation of antigens from dying tumor cells. This effect is likely

obtained by virtue of the capacity of TLR4 to inhibit the lysosomal degradation of ingested

tumor antigens. TLR4 ligation also provides a signal for the transcriptional upregulation of

the gene coding for interleukin-b. Tumor cells that lack HMGB1 expression fail to elicit ananticancer immune response, and HMGB1-negative tumors become refractory to

chemotherapy with anthracyclines or oxaliplatin. Moreover, HMGB1-expressing tumorstransplanted to mice lacking TLR4 or its adaptor MYD88 fail to respond to chemotherapy

in vivo18,19).

Recently, we identified yet another signal that contributes to the immunogenicity of cell

Type 1 intefereron production.

death. We observed that cancer cells expressing the double-stranded RNA sensor TLR3 canproduce type-1 interferons (IFN-1) after treatment with anthracyclines or oxaliplatin in vitro
or in vivo. Apparently, IFN-1 then must act on its receptor (IFNAR) in an autocrine or

54

G. Kroemer

paracrine fashion to favor the anticancer immune response. Knockout of IFNAR in the
immune system has no major inhibitory effect on the anticancer immune response elicitedby dying tumor cells. However, knockout of IFNAR in cancer cells curtails their capacity toelicit such a response. Downstream of IFNAR, tumor cells produce the chemokine CXCR10,

which then acts to attract T lymphocytes (and perhaps other immune cells) into the tumor

bed20).

Altogether, there results indicate the existence of four hallmarks of ICD, namely (i) CRT

exposure, (ii) ATP release, (iii) HMGB1 exodus and (iv) IFN-1 secretion. Each of thesehallmarks is essential for the immunogenicity of cell death in a non-redundant fashion(Figure 1). At present it appears plausible, yet remains to be explored, that thesecharacteristics of ICD must be produced by several cancer cells located in close proximity

to each other (rather than on single, isolated cells). This ‘design’ of the system might

constitute a fail-safe mechanism that reduces the likelihood of immune reaction when
apoptosis is a sporadic event.

Figure 1

Central hypothesis of the present research project. Chemotherapy must induce (at least)
four molecular species to be exposed on the surface of cancer cells or to appear in soluble
form in their vicinity. These ligands then act on a series of specific receptors that ultimately
stimulate the capacity of DCs to prime/activate cytotoxic T cells specific for tumor-associated cell antigens.

Our current working hypothesis suggests that the immune system dictates the long-term success of anti-cancer therapies, and that this immune response is determined byimmunogenic tumor cell death (20,21). Thus, therapeutic failure can result from theincapacity of cancer cells to undergo ICD (rather than cell death as such). Only
chemotherapeutics that elicit ICD may mediate a long-term therapeutic success. In

addition, tumors that are unable to undergo ICD are intrinsically incurable. Similarly,

genetically or acquired defects that reduce the capacity of the immune system to sense ICDassociated signals reduce the efficacy of anticancer chemotherapy with anthracyclines orChemo Immunotherapy

55

oxaliplatin20,21). We are currently developing therapeutic strategies that restore deficient
ICD signals with the objective of ameliorating present and future chemotherapies.

In addition, we are currently screening large drug libraries for the identification ofdrugs that are able of inducing ICD using the established hallmarks of this cell deathmodality23-25). So far, we have found that approximately 5% of all currently used anticancer

agents (6 out of 114) are endowed with the capacity of stimulating all hallmarks of ICD. In

contrast, we found that among the mechanistic set of anticancer agents that is being

developed by the National Cancer Institute (NCI) of the United States of America, only

0.1% of the drugs (1 out of 879) act as bona fide ICD inducers. This suggests that clinicaldevelopment of successful anticancer agents has led to a selection in favor of componentsthat are able to induce ICD, contrasting with the merely pre-clinical (and nonimmunological) development that has yielded the collection of cytotoxicals assembled by

the NCI. We surmise that prescreening of prospective anticancer agents for ICD induction

might constitute a strategy for ameliorated selection of successful drugs.
References

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Hamai A, Obeid M, Coutant F, Metivier D, Pichard E, Aucouturier P, Pierron G,
Garrido C, Zitvogel L, Kroemer G. Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death. J Exp Med. 2005 Dec 19;202(12):1691-701.
2. Zitvogel L, Kepp O, Kroemer G. Decoding cell death signals in inflammation andimmunity. Cell. 2010 Mar 19;140(6):798-804.
3. Kroemer G, Galluzzi L, Kepp O, Zitvogel L. Immunogenic cell death in cancer therapy.
Annu Rev Immunol 2013 Mar 21;31:51-72.
4. Ma Y, Aymeric L, Locher C, Mattarollo SR, Delahaye NF, Pereira P, Boucontet L,
Apetoh L, Ghiringhelli F, Casares N, Lasarte JJ, Matsuzaki G, Ikuta K, Ryffel B,
Benlagha K, Tesnière A, Ibrahim N, Déchanet-Merville J, Chaput N, Smyth MJ,
Kroemer G, Zitvogel L. Pivotal contribution of IL-17 producing g/d T cells to the efficacy of chemotherapy. J Exp Med. 2011 Mar 14;208(3):491-503.
5. Ma Y, Mattarollo SR, Adjemian S, Yang H, Aymeric L, Hannani D, Portela Catani JP,
Duret H, Teng MW, Kepp O, Wang Y, Sistigu A, Schultze JL, Stoll G, Galluzzi L,
Zitvogel L, Smyth MJ, Kroemer G. CCL2/CCR2-dependent recruitment of functional
antigen-presenting cells into tumors upon chemotherapy. Cancer Res. 2014 Jan
15;74(2):436-45.

56

G. Kroemer

6. Hannani D, Locher C, Yamazaki T, Colin-Minard V, Vetizou M, Aymeric L, Viaud S,
Sanchez D, Smyth MJ, Bruhns P, Kroemer G, Zitvogel L. Contribution of humoral
immune responses to the antitumor effects mediated by anthracyclines. Cell Death
Differ. 2014 Jan;21(1):50-8.
7. Obeid M, Tesniere A, Ghiringhelli F, Fimia GM, Apetoh L, Perfettini JL, Castedo M,
Mignot G, Metivier D, Larochette D, van Edert P, Ciccosanti F, Piacentini F, Zitvogel L,
Kroemer G. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat
Med. 2007 Jan;13(1):54-61.
8. Panaretakis T, Kepp O, Brockmeyer U, Tesniere A, Durchschlag M, Joza, N, Pierron G,
van Endert P, Yuan J, Zitvogel L, Madeo F, Williams DB, Kroemer G. Mechanisms of preapoptotic calreticulin exposure in immunogenic cell death. EMBO J. 2009 Mar
4;28(5):578-90.
9. Senovilla L, Vitale I, Martins I, Tailler M, Pailleret C, Michaud M, Galluzzi L, Adjemian S,
Kepp O, Niso-Santano M, Shen S, Mariño G, Criollo A, Boilève A, Job B, Ladoire S,
Ghiringhelli F, Sistigu A, Yamazaki T, Rello-Varona S, Locher C, Poirier-Colame V,
Talbot M, Valent A, Berardinelli F, Antoccia A, Ciccosanti F, Fimia GM, Piacentini M,
Fueyo A, Messina NL, Li M, Chan CJ, Sigl V, Pourcher G, Ruckenstuhl C, CarmonaGutierrez D, Lazar V, Penninger JM, Madeo F, López-Otín C, Smyth MJ, Zitvogel L,
Castedo M, Kroemer G. An immunosurveillance mechanism controls cancer cell ploidy.
Science. 2012 Sep 28;337(6102):1678-84.
10. Ghiringhelli F, Apetoh L, Tesniere A, Aymeric L, Ma Y, Ortiz C, Vermaelen K,
Panaretakis T, Mignot G, Ullrich E, Perfettini JL, Schlemmer F, Tasdemir E, Uhl M,
Génin P, Civas A, Ryffel B, Kanellopoulos J, Tschopp J, André F, Lidereau R,
McLaughlin NM, Haynes NM, Smyth MJ, Kroemer G, Zitvogel L (equal senior and cocorresponding author). Activation of the NLRP3 inflammasome in dendritic cells
induces IL-1b-dependent adaptive immunity against tumors.
Nat Med 2009
Oct;15(10):1170-8.
11. Zitvogel L, Kepp O, Galluzzi L, Kroemer G. Inflammasomes in carcinogenesis and anticancer immune responses. Nat Immunol. 2012 Mar 18;13(4):343-51.
12. Michaud M, Martin I, Sukkuwala A, Adjemian S, Ma Y, Pellegati P, Shen S, Kepp O,
Scoazec M, Mignot G, Rello-Varona S, Tailler M, Menger L, Vacchelli E, Galluzzi L,
Ghiringhelli F, Galluzzi L, di Virgilio F, Zitvogel L, Kroemer G. Autophagy-dependent
anticancer immune responses induced by chemotherapeutic agents in mice. Science.
2011 Dec16;334:1573-1577.
13. Ma Y, Adjemian S, Mattarollo SR, Yamazaki T, Aymeric L, Yang H, Portela Catani JP,
Hannani D, Duret H, Steegh K, Martins I, Schlemmer F, Michaud M, Kepp O,
Sukkurwala AQ, Menger L, Vacchelli E, Droin N, Galluzzi L, Krzysiek R, Gordon S,
Taylor PR, Van Endert P, Solary E, Smyth MJ,Zitvogel L, Kroemer G. Anticancer
chemotherapy-induced intratumoral recruitment and differentiation of antigen-presenting cells. Immunity. 2013 Apr 18;38(4):729-41

Chemo Immunotherapy

57

14. Ma Y, Galluzzi L, Zitvogel L, Kroemer G. Autophagy and the cellular immune
response. Immunity. 2013 Aug 22;39(2):211-27.
15. Rao S, Tortola L, Perlot T, Wirnsberger G, Novatchkova M, Nitsch R, Sykacek P, Frank L,
Schramek D, Komnenovic V, Sigl V, Aumayr K, Schmauss G, Fellner N, Handschuh S,
Glösmann M, Pasierbek P, Schlederer M, Resch GP, Ma Y, Yang H, Popper H, Kenner L,
Kroemer G, Penninger JM.. A dual role for autophagy in a murine model of lung cancer.
Nat Commun. 2014 Jan 20;5:3056.
16. Ko A, Kanehisa A, Martins I, Senovilla L, Chargari C, Dugue D, Mariño G, Kepp O,
Michaud M, Perfettini JL, Kroemer G, Deutsch E. Autophagy inhibition radiosensitizes invitro, yet reduces radioresponses in vivo due to deficient immunogenic signalling. Cell
Death Differ. 2014 Jan;21(1):92-9.
17. Martins I, Wang Y, Michaud M, Ma Y, Sukkurwala AQ, Shen S, Kepp O, Métivier D,
Galluzzi L, Perfettini JL, Zitvogel L, Kroemer G. Molecular mechanisms of ATP secretion
during immunogenic cell death. Cell Death Differ. 2014 Jan;21(1):79-91.
18. Apetoh L, Ghiringhelli F, Tesniere A, Obeid M, Ortiz C, Criollo A, Mignot G, Maiuri MC,
Ullrich E, Saulnier P, Yang H, Amigorena S, Ryffel B, Barrat FJ, Saftig P, Levi F,
Lidereau R, Nogues C, Mira JP, Chompret A, Joulin V, Clavel-Chapelon F, Bourhis J,
André F, Delaloge S, Tursz T, Kroemer G, Zitvogel L. Toll-like receptor 4-dependent
contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat
Med. 2007 Oct;13(9):1050-1059.
19. Yamazaki T, Hannani D, Poirier-Colame V, Ladoire S, Locher C, Sistigu A, Prada N,
Adjemian S, Catani JP, Freudenberg M, Galanos C, André F, Kroemer G, Zitvogel L.
Defective immunogenic cell death of HMGB1-deficient tumors: compensatory therapy
with TLR4 agonists. Cell Death Differ. 2014 Jan;21(1):69-78.
20. Sistigu A, Yamazaki T, Vacchelli E, Chaba K, Enot DP, Adam J, Vitale I, Goubar A,
Baracco EE, Remédios C, Fend L, Hannani D, Aymeric L, Ma Y, Niso-Santano M, Kepp O,
Schultze JL, Tüting T, Belardelli F, Bracci L, La Sorsa V, Ziccheddu G, Sestili P, Urbani F,
Delorenzi M, Lacroix-Triki M, Quidville V, Conforti R, Spano JP, Pusztai L, PoirierColame V, Delaloge S, Penault-Llorca F, Ladoire S, Arnould L, Cyrta J, Dessoliers MC,
Eggermont A, Bianchi ME, Pittet M, Engblom C, Pfirschke C, Préville X, Uzè G,
Schreiber RD, Chow MT, Smyth MJ, Proietti E, André F, Kroemer G, Zitvogel L. Cancercell-autonomous contribution of type I interferon signaling to the efficacy ofchemotherapy. Nat Med. 2014 Nov;20(11):1301-1309.
21. Zitvogel L, Apetoh L, Ghiringhelli F, André F, Tesniere A, Kroemer G. The anticancer
immune response: indispensable for therapeutic success? J Clin Invest. 2008
Jun;118(6):1991-2001.
22. Zitvogel L, Galluzzi L, Smyth MJ, Kroemer G. Mechanism of action of conventional andtargeted anticancer therapies: reinstating immunosurveillance. Immunity. 2013 Jul
25;39(1):74-88.

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G. Kroemer

23. Menger L, Vacchelli E, Adjemian S, Martins I, Ma Y, Shen S, Yamazaki T, Sukkurwala AQ,
Michaud M, Mignot G, Schlemmer F, Sulpice E, Locher C, Gidrol X, Ghiringhelli F,
Modjtahedi N, Galluzzi L, André F, Zitvogel L, Kepp O, Kroemer G. Cardiac glycosides
exert anticancer effects by inducing immunogenic cell death. Sci Transl Med. 2012 Jul
18;4(143):143ra99.
24. Sukkurwala AQ, Martins I, Wang Y, Schlemmer F, Ruckenstuhl C, Durchschlag M,
Michaud M, Senovilla L, Sistigu A, Ma Y, Vacchelli E, Sulpice E, Gidrol X, Zitvogel L,
Madeo F, Galluzzi L, Kepp O, Kroemer G. Immunogenic calreticulin exposure occurs
through a phylogenetically conserved stress pathway involving the chemokine CXCL8.
Cell Death Differ. 2014 Jan;21(1):59-68.
25. Kepp O, Senovilla L, Kroemer G. Immunogenic cell death inducers as anticancer
agents. Oncotarget. 2014 Jul 30;5(14):5190-1.

Guido Kroemer, M.D., Ph.D.

2000-2010

2010-

2011-

2011201220132013-2014-

Research Director at INSERM (French Medical
Research Council)
Professor at the Medical Faculty of University Paris
Descartes
President of the European Academy of Tumor
Immunology
Director of the LabEx Immuno-Oncology
President of the European Cell Death Organization
Director of the Paris Alliance of Cancer ResearchInstitutes
Director of the Cell Biology Platform at Gustave
Roussy, Villejuif
Deputy Director of the Cordeliers Research Center,
Paris

Specialty and Present Interest:
Apoptosis, Autophagy, Chemotherapy, Endoplasmic Reticulum Stress, Necrosis

59

ROLE OF IMMUNE-SUPPRESSIVE CELLSIN THE SURVIVAL OF GASTROINTESTINAL CANCERPATIENTS TREATED WITH STANDARD CHEMOTHERAPY

Yuji Heike

Immunotherapy and Cell Therapy Service
St. Luke’s International Hospital
9-1 Akashi-Cho, Chuo-ku, Tokyo 104-8560(heiyuji@luke.ac.jp)
Immunotherapy Research Field
Exploratory Oncology Research & Clinical Trial Center
National Cancer Research Center

Recent progress in cancer immunotherapy has been astonishing, and several immune

checkpoint inhibitory antibodies have been able to induce remarkable therapeutic benefits

against several cancers. Cancer immunotherapy is now securing a position as a standard

therapy.

Thinking back to the history of cancer treatment, chemotherapy including moleculartarget agents has been a standard therapy against advanced cancers. Many oncologists
have imagined that because chemotherapy damages the immune status of the patient, it isinconsistent with immunotherapy. However, in recent years, some anti-cancer drugs havebeen found to induce positive immune reactions against cancers, emerging a new conceptof ‘immunogenic chemotherapy’.

Several reports have suggested the responsiveness to first-line chemotherapy as apredictive marker of the responsiveness of second and subsequent chemotherapies, eventhough the mechanisms of each anti-cancer drug differ. Various factors, including type ofgene mutation, epigenetic change, and genetic polymorphisms, have been reported to be
associated with response to each drug; however, the reasons why the same patients

respond to agents with different mechanisms remain unclear. So, we examined what

immunological factors affect the response and survival of cancer patients treated withstandard chemotherapeutic regimens.

In our study, we prospectively enrolled patients with colorectal, gastric, and esophageal

cancers who were due to undergo standard chemotherapies at the National Cancer Center

Hospital from June 2013 to present. After obtaining written informed consent, we collected

peripheral blood before and under treatment, and at 6 months after initiation of

60

Y. Heikechemotherapy. We evaluated myeloid-derived suppressor cells, T cell subsets including
regulatory T cells and natural killer cells using flow cytometry.

Our study showed that the high frequency of immune-suppressive cells such as MDSCs

were strongly associated with poor survival even in patients with colorectal and gastriccancer, who received the standard chemotherapeutic regimens. Tumor-infiltrating immune

cells represent a local immunological status. Our study also showed that tumor-infiltrating

myeloid cells were also involved in prognosis of patients with cancers.

Furthermore, the depletion of MDSC significantly suppressed the tumor growth andprolonged survivals in murine cancer models treated with chemotherapeutic reagents.

Therefore, the immune-suppressive cells may be a novel promising target ingastrointestinal cancer patients treated with standard chemotherapies.

Yuji Heike, M.D., Ph.D.

1992-1998

1998-2002

2002-2006

2006-20122012-2013

2013-

Research Staff and Chief Investigator, Pharmacology
Division, National Cancer Center Research InstituteStaff Doctor, Department of Internal Medicine,National Shikoku Cancer Center
Chief Investigator, Pharmacology Division,
National Cancer Center Research Institute
Medical Staff, Stem Cell Transplantation Service,
National Cancer Center Hospital
Unit Head of Immunotherapy Research Field,
Translational Research Group, and Translational
Medicine Department, Phase I Group, Exploratory
Oncology Research & Clinical Trial Center,
National Cancer Center
Chief, Immunotherapy and Cell Therapy Service,
St. Luke’s International Hospital

61

IMMUNE CHECKPOINT BLOCKADE IN CANCER THERAPY:NEW INSIGHTS AND OPPORTUNITIESJames P. Allison and Padmanee Sharma

The University of Texas MD Anderson Cancer Center
4014 Portsmouth, Houston, TX 77027, USA(jallison@mdanderson.org)

The existence of multiple non-redundant l inhibitory pathways that limit T cell

responses offers novel strategies for mobilizing the immune system to attack cancer cells.

The best characterized of these immune checkpoints is CTLA-4, which inhibits CD28

mediated costimulation. Antibodies to CTLA-4 have proven effective against multiple

tumor types in both pre-clinical and clinical studies. Ipilimumab, an antibody to human

CTLA-4, showed long term (>4.5 years) survival benefit in about 23% of patients in arandomized, placebo-controlled trial in late stage melanoma. In 2011 it was approved by

the FDA for treatment of late stage melanoma and is now a standard of care for thatdisease. A recent retrospective study of almost 5,000 patients showed an inflection point at

about 2.5 years with essentially no deaths of about 20% of patients for 10 years following

treatment.

The mechanism(s) of action of anti-CTLA-4 are still being elucidated. We and others

have shown that CLTA-4 limits T cell proliferation by a cell intrinsic mechanism. However,there is also evidence that anti-CTLA-4 has to engage the target on both effector (Teff) andregulatory (Treg) T cells. We have recently uncovered a mechanism whereby anti-CTLA-4

antibodies expand Treg in lymph nodes but cause their depletion in the tumor
microenvironment. Thus anti-CTLA-4 exerts its anti-tumor effects by multiple

mechanisms. We have also shown that CTLA-4 blockade results in a 2-5 fold increase in
the frequency of CD4 T cells expression ICOS (inducible costimulator) in both tumor

tissues and blood. This population contains that vast majority of tumor antigen specificcells that produce IFNg and TNFa. The appearance of the ICOS+ CD4 cells serves as apharmacodynamic marker of a biological effect of anti-CTLA-4 activity. Using mouse

62

J. P. Allison et al.

models, we have shown that the ICOS/ICOSL pathway is critical for optimal anti-tumor
activity of anti-CTLA-4. Furthermore, we have shown that agonist stimulation of ICOS

coupled with CTLA-4 blockade results in enhanced anti-tumor efficacy in mouse models,suggest that ICOS is a compelling molecule to develop as a target for agonistic targeting ofcostimulatory checkpoints.

PD-1, another checkpoint, recruits a phosphatase and seems to interfere with T cell

antigen receptor mediated signaling. It has two ligands, PD-L1 and PD-L2, which are both

expressed on dendritic cells. However, many tumor cells also express PD-L1. Antibodies

to PD-1 and PD-L1 have both shown objective responses against several tumor types inclinical trials with response rates of about 25% . A recent phase II trial of a combination ofanti-PD-1 and anti-CTLA-4 in melanoma showed objective responses in about 50% of latestage melanoma patients. Our studies indicate that the mechanisms of anti-PD-1 mediatedtumor immunity are distinct from those of anti-CTLA-4, at least as for the role of ICOS+

CD4 T cells.

These studies and their implications for cancer therapy are discussed.

James P. Allison, Ph.D.

1978-2004
1984-2004

1997-2012
2004-2012
2012-

Assistant Biochemist
Professor, Division of Immunology
Director, Cancer Research Laboratory
Univ. of California, Berkeley
Investigator, Howard Hughes Medical Institute
Chair, Immunology Program
Director, Ludwig Center for Cancer Immunotherapy
Memorial Sloan-Kettering Cancer Center
Professor and Chair, Dept. of Immunology
Executive Director, Immunotherapy Platform
Univ. of Texas MD Anderson Cancer Center

63

THERAPEUTIC MODULATION OF THE IMMUNEMICROENVIRONMENT OF CANCER

Drew M. Pardoll

Department of Oncology
Johns Hopkins University School of Medicine(dpardol1@jhmi.edu)

Major advances have been made in the immune-based therapy of cancer by antibody

blockade of immune inhibitory pathways such as CTLA-4 and PD-1. Anti-PD-1 antibodies

have produced objective responses in one third to one half of patients with advanced,

chemotherapy refractory melanoma and renal cancer and one quarter of patients with non-small cell lung cancer. Recently, significant response rates to anti-PD-1 antibodies havebeen shown in bladder cancer, head and neck cancer, gastroesophageal cancer, ovariancancer and lymphoma. These responses are highly durable, the majority lasting
significantly greater than one year and beyond cessation of therapy. Further, expression bytumor cells of ligands for PD-1 is associated with higher response to anti-PD-1 therapy. In

exploring the basis for up-regulation of the major PD-1 ligand, PD-L1, on tumor cells, wefound that its expression is not constitutive, but rather, is highly associated withlymphocytic infiltration. We identified IFN-g as an immune signal sensed by the tumor cellthat induces PD-L1 expression. In addition to IFN-g, genes associated with Th1 responses,

CTL responses and other inhibitory molecules, such as LAG-3, are up-regulated inlymphocytic infiltrates associated with PD-L1+ tumor cells. These findings indicate thatmultiple counterbalancing immune effector and inhibitory pathways are operative in the
immune microenvironment. They led us to hypothesize a new mechanism of PD-L1

expression in tumors, termed adaptive resistance, distinct from a constitutive mechanism of
PD-L1 expression in tumors. The adaptive resistance mechanism implies that othertherapies such as vaccines may induce antitumor responses that in turn induce up-

regulation of PD-L1. In such a circumstance, vaccination and PD-L1 blockade might
produce synergistic anti-tumor activity. Using novel vaccine formulations, we indeed

64

D. M. Pardoll

demonstrate IFN-g dependent induction of PD-L1 on tumors and synergistic activity suchthat the vaccine/anti-PD-1 combination.

Drew M. Pardoll, M.D., Ph.D.

Present

Abeloff Professor of Oncology, Medicine,
Pathology and Molecular Biology and Genetics at theJohns Hopkins University, School of Medicine
Director, Cancer Immunology in the Sidney Kimmel
Comprehensive Cancer Center

65

IMMUNOTHERAPY THAT TARGETS
THE TUMOR AND THE HOST

Ronald Levy

Stanford Medical School
Center for Clinical Science Research 269 Campus Drive
Room 1105, Stanford, CA 94305, USA(levy@stanford.edu)

Cancer therapies can target the tumor or the host microenvironment, and especially thehost immune system. An exciting challenge is to combine these therapies in ways that aresynergistic.

The therapeutic power of antibodies can be enhanced by stimulating antibody

Improving the power of monoclonal antibodies

dependent cellular cytotoxicity (ADCC), a primary mechanism of anti-tumor effect. One

way to accomplish this is to target CD137, an activation molecule that appears on host NK

cells after they encounter tumor cells coated by an anti-tumor antibody1-3). CD137 is also
expressed on activated T cells and therefore the antibody against CD137 might alsoenhance an underlying host anti tumor T cell immune response. Clinical trials are now

under way to test the effect of an antibody against CD137 as a single agent and incombination with Rituximab in lymphoma, with Traztuzumab in breast cancer and with
Cetuximab in colon cancer.

Abscopal tumor responses can be induced in patients with lymphoma by local in situ

In situ therapeutic vaccination

vaccination. CpG oligonucleotide, a ligand for a Toll-like receptor, is injected into one siteof tumor and low dose radiation is directed to the same site4-6).

In animal models this treatment generates an anti-tumor T cell immune response. These

T cell responses can be enhanced by intra-tumoral injections of antibodies against CTLA4

and OX407). These antibodies work by depleting the negative T regulatory cells (Tregs) in66

R. Levy

the local tumor microenvironment, unleasing T effector cells to attack tumor at distant sites.

Very low doses of checkpoint antibodies can be used to avoid autoimmunity. Suchcombinations of low dose radiation with immune enhancing agents may change the way

we employ radiotherapy in the future- to help trigger immune responses rather than tosterilize the local tumor site.

Small molecule inhibitors of survival pathways in lymphoma cells, such as B cell

Combination of immunotherapies with small molecule-targeted therapies

receptor signaling, also have effects on the immune system. A case is point is Ibrutinib, aImmune Checkpoints

67

covalent inhibitor of Bruton’s tyrosine kinase (Btk), recently approved for the treatment of
CLL and MCL and with significant clinical activity in other lymphoid malignancies.

Ibrutinib inhibits other members of the Tec kinase family, such as Itk, an importantsignaling molecule in subsets of T cells. By doing so, it can sculpt the T cell immune
response and direct it against the tumor. Recent preclinical experiments indicate that

Ibrutinib can enhance the T cell immune response induced by in situ vaccination as well asother immunotherapeutic maneuvers.

68

R. Levy

References
1. Kohrt HE, Houot R, Goldstein MJ, Weiskopf K, Alizadeh AA, Brody J, Müller AM,
Pachynski R, Czerwinski D, Coutre S, Chao MP, Chen L, Tedder TF, Levy R., CD137 stimulation enhances the anti-lymphoma activity of anti-CD20 antibodies. Blood. Blood.
2011 Feb 24;117(8):2423-32 PMCID: PMC3062409
2. Kohrt HE, Houot R, Weiskopf K, Goldstein MJ, Scheeren F, Czerwinski D, Colevas AD,
Weng WK, Clarke MF, Carlson RW, Stockdale FE, Mollick JA, Chen L, Levy R.
Stimulation of natural killer cells with a CD137-specific antibody enhances trastuzumab
efficacy in xenotransplant models of breast cancer. J Clin Invest. 2012 Mar 1;122(3):106675. Epub 2012 Feb 13. PMID: 22326955; PMCID: PMC3287235.
3. Kohrt HE, Colevas AD, Houot R, Weiskopf K, Goldstein MJ, Lund P, Mueller A, SagivBarfi I, Marabelle A, Lira R, Troutner E, Richards L, Rajapaska A, Hebb J, Chester C,
Waller E, Ostashko A, Weng WK, Chen L, Czerwinski D, Fu YX, Sunwoo J, Levy R.
Targeting CD137 enhances the efficacy of cetuximab. J Clin Invest. 2014 Jun
2;124(6):2668-82. PubMed PMID: 24837434; PubMed Central PMCID: PMC4089447.
4. Li J, Song W, Czerwinski DK, Varghese B, Uematsu S, Akira S, Krieg AM, Levy R.,
Lymphoma Immunotherapy with CpG Oligodeoxynecleotides Requires Expression of the
Toll Like Receptor 9, either in the host or on the tumor itself. J. Immunol 2007 179:24932500. PMID: 17675511Joshua D. Brody, Weiyun Z. Ai,
5. Roch H., Levy R. T cell modulation combined with intratumoral CpG cures lymphoma ina mouse model without the need for chemotherapy. Blood, 9 April 2009, Vol. 113, No. 15,
pp. 3546-3552. PMID: 18941113
6. Joshua D. Brody, Weiyun Z. Ai,j, Debra K. Czerwinski, James A. Torchia, Mia Levy,
Ranjana H. Advani, Youn H. Kim, Richard T. Hoppe, Susan J. Knox, Lewis K. Shin,
Irene Wapnir, Robert J. Tibshirani, Ronald Levy, In Situ Vaccination With a TLR9
Agonist Induces Systemic Lymphoma Regression: A Phase I/II Study, J. Clin Onc. June 162010 PMCID: PMC2954133
7. Marabelle A, Kohrt H, Sagiv-Barfi I, Ajami B, Axtell RC, Zhou G, Rajapaksa R, Green MR,
Torchia J, Brody J, Luong R, Rosenblum MD, Steinman L, Levitsky HI, Tse V, Levy R.
Depleting tumor-specific Tregs at a single site eradicates disseminated tumors. J Clin
Invest. 2013 Jun 3;123(6):2447-63. PMID: 2372817

Immune Checkpoints

Ronald Levy, M.D.

1968-1969

1969-1970

1970-1972

1972-1973

1973-1975

1975-1981

1981-1987
1987-

1987-

1993-

69

Internship Internal Medicine, Massachusetts General
Hospital
Residency Internal Medicine, Massachusetts General
Hospital
Clin. Assoc., Immunology Branch, National Cancer
Institute
Fellow, Department of Medicine, Division of Oncology,
Stanford University
Fellow of the Helen Hay Whitney Foundation,
Department of Chemical Immunology, The Weizmann
Institute of Science
Assistant Professor of Medicine, Division of Oncology,
Stanford University
Associate Professor of Medicine, Division of Oncology,
Stanford University
Professor of Medicine, Division of Oncology, Stanford
University School of Medicine
Robert K. Summy and Helen K. Summy Professor,
Stanford University
Chief of the Division of Oncology, Stanford University
School of Medicine

70

COMBINATION CHECKPOINT BLOCKADE AND
THE ROLE OF ‘PASSENGER’ MUTATIONS IN
CLINICAL RESPONSE TO IPILIMUMAB

Jedd D. Wolchok, Margaret K. Callahan, Michael A. Postow,
Alexandra Snyder Charen, Taha Merghoub and Timothy A. Chan
Ludwig Center and Human Oncology Pathogenesis Program
Departments of Medicine and Radiation Oncology
Memorial Sloan Kettering Cancer Center
1275 York Avenue, New York, NY 10065, USA(Wolchokj@mskcc.org)

The past decade has been a period of significant progress in the area of cancer

immunotherapy, enabled by significant advances in basic science allowing for a better
understanding of molecular mechanisms of immune regulation and immune cell dynamics.

These successes reflect a close collaboration between basic, translational and clinicalscientists.

Blockade of cytotoxic T lymphocyte antigen 4 (CTLA-4) was suggested to be an

impactful means to augment tumor immunity based on the pioneering studies of Jim
Allison, who hypothesized that impairment of this critical molecular brake would
overcome tumor-induced immune suppression and allow for more robust immune

response to tumors.1) In 2011, the US FDA approved ipilimumab for the treatment ofmetastatic melanoma with two randomized phase 3 trials demonstrating prolongation of

overall survival, the first intervention ever to demonstrate a survival benefit in patientswith metastatic melanoma.2,3) Investigation of blockade of the programmed death-1 (PD-1)

pathway followed very shortly thereafter. PD-1 is a marker on T cells which, while being
up-regulated after activation, serves to mark T cells as being exhausted. When engaging

one of its ligands (PD-L1 or PD-L2), PD-1 can lower the threshold for apoptosis in T cells.4,5)

A growing number of antibodies that block either PD-1 or PD-L1 have entered clinical trials
with some recently approved (nivolumab, pembrolizumab) for metastatic melanoma andothers currently in later stage investigation for additional cancers. It was not completely
surprising that PD-1 blockade showed clinical efficacy in melanoma and renal cell

carcinoma. However, Julie Brahmer and Suzanne Topalian at Johns Hopkins observed thatpatients with non-small cell lung cancer could respond to PD-1 blockade with nivolumab,

Immune Checkpoints

71

forever changing that notion that melanoma and renal cell carcinoma were the only solid

tumors amenable to immune modulation.6,7) The list of malignancies where PD-1 pathway

blockade has been demonstrated significant activity also now includes head and necksquamous cell carcinoma, urothelial bladder cancer, ovarian cancer, gastric cancer and both

Hodgkin’s and non-Hodgkin’s lymphomas.

Given the activity noted with both CTLA-4 and PD-1 blockade and the complementarymechanisms of action as well as supportive pre-clinical data, clinical trials are now

investigating combination checkpoint blockade. The most mature data has come from a

Phase 1 study combining ipilimumab and nivolumab in patients with melanoma whichshowed an overall response rate of 40% across dose cohorts with over 50% of patients insome cohorts showing rapid and deep (>80% reduction) tumor regressions (Figure 1).8)

These responses were seen in the context of manageable mechanism-based toxicity, which

had previously been observed in trials of CTLA-4 and PD-1 pathway blockade as
monotherapies. Patients treated with combined checkpoint blockade tend to have rapidand deep responses, materially distinct from those observed in monotherapy trials. Such

responses are generally durable, even in some cases when treatment was stopped early due

to toxicity. Unlike in studies of PD-1 blockade as monotherapy, there was no significant

difference in clinical activity based on tumor expression of PD-L1, suggesting the
inflammation mediated by ipilimumab could enhance PD-L1 or other relevant marker

Figure 1 Rapid and Durable Changes in Target Lesions

72

J. D. Wolchok et al.

expression that sensitizes to PD-1 blockade (Figure 2). However, this remains ahypothetical explanation that requires mechanistic confirmation.

Early studies of the immune dynamic correlates in patients treated with the combination

therapy show a high spike in proliferating CD4+ and CD8+ T cells occurring within days

after the first dose. This was not evident in patients treated with nivolumab after receiving

prior ipilimumab. Pharmacokinetic analysis of patients treated sequentially with

ipilimumab followed by nivolumab suggest that the plasma concentration of ipilimumabremaining when nivolumab is initiated could be important as a higher response rate wasnoted for patients with higher than the mean concentration. Phase 2 and 3 trials of thiscombination are ongoing in melanoma with phase 1 programs in numerous other tumor

types. Similar strategies are being investigated using a combination of other CTLA-4

blocking antibodies such as tremelimumab and other inhibitors of the PD-1 pathway suchas the anti-PD-L1 antibody, MEDI-4736. The above approaches are also being considered

as combinatorial partners both with other immunotherapies as well as with treatments that

directly affect the cancer cell.

Recently, attention is being paid to the mechanisms underlying the clinical efficacy ofcheckpoint blockade in certain malignancies. One hypothesis has been that cancers thathave a high mutational load may be more amenable to immune modulation by virtue of thelarger number of potential neo-epitopes present, fostering baseline immune recognition

Figure 2 Response Rate by PD-L1 Status (5% cutoff)Immune Checkpoints

73

that can then be potentiated by checkpoint blockade. This is supported by referring to thespectrum of mutational loads across cancer, in which the majority of the malignancies thathave been shown to respond to these immunotherapeutic strategies have a higher numberof mutations.9) We have recently found that metastatic melanoma patients having longterm clinical activity after ipilimumab treatment have a significantly greater median

number of non-synonymous passenger mutations, compared with patients who do notrespond or those who have only short-term (< 6 months) regression (Figure 3).10)

The use of whole exome sequencing has allowed us to explore the additional hypothesisthat the higher likelihood of durable response and long-term survival observed may notsimply be explained by a higher quantity of mutations but rather that the individual
immunologic quality of mutations is also important. This could serve to explain thepresence of outliers in the prior analysis, where some patients with low mutation burden

had long-term survival while others with quite higher numbers of mutations (1000-1500)

did not respond to ipilimumab treatment. Using a novel bioinformatics platform, we havefound that there are substrings of class I epitopes (tetrapeptides) which are shared by theclinical responders and not found in the non-responders. Intriguingly, the vast majority ofthese favorable substrings are also found in known epitopes derived from proteins foundin bacteria and viruses. This suggests the biologically interesting hypothesis that a highFigure 3 Mutational Load Correlates with Clinical Outcome

74

J. D. Wolchok et al.

mutational load can lead to a greater probability of a tumor-derived peptides appearing
structurally similar to truly foreign antigens.

The confluence of advances in multiple areas of cancer research have allowed for thedevelopment of new and effective strategies to durably control cancer. Our task now isfurther understanding the mechanistic basis of such activity and to leverage the recent

knowledge gained in the area of mutational neo-epitopes to devise strategies to initiate

tumor immunity in patients who are not currently responding and to predict responses
before treatment is initiated.

References
1. Leach DR, Krummel MF, Allison JP. Enhancement of antitumor immunity by CTLA-4
blockade. Science. 1996;271:1734-6.
2. Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, Gonzalez R,
Robert C, Schadendorf D, Hassel JC, Akerley W, van den Eertwegh AJ, Lutzky J,
Lorigan P, Vaubel JM, Linette GP, Hogg D, Ottensmeier CH, Lebbe C, Peschel C, Quirt I,
Clark JI, Wolchok JD, Weber JS, Tian J, Yellin MJ, Nichol GM, Hoos A, Urba WJ.
Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J
Med. 2010;363:711-23.
3. Robert C, Thomas L, Bondarenko I, O'Day S, Weber J, Garbe C, Lebbe C, Baurain JF,
Testori A, Grob JJ, Davidson N, Richards J, Maio M, Hauschild A, Miller WH, Jr.,
Gascon P, Lotem M, Harmankaya K, Ibrahim R, Francis S, Chen TT, Humphrey R,
Hoos A, Wolchok JD. Ipilimumab plus dacarbazine for previously untreated metastaticmelanoma. N Engl J Med. 2011;364:2517-26.
4. Hirano F, Kaneko K, Tamura H, Dong H, Wang S, Ichikawa M, Rietz C, Flies DB, Lau JS,
Zhu G, Tamada K, Chen L. Blockade of B7-H1 and PD-1 by monoclonal antibodies
potentiates cancer therapeutic immunity. Cancer Res. 2005;65:1089-96.
5. Iwai Y, Ishida M, Tanaka Y, Okazaki T, Honjo T, Minato N. Involvement of PD-L1 on
tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1
blockade. Proc Natl Acad Sci USA. 2002;99:12293-7.
6. Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, Drake CG, Camacho
LH, Kauh J, Odunsi K, Pitot HC, Hamid O, Bhatia S, Martins R, Eaton K, Chen S, Salay
TM, Alaparthy S, Grosso JF, Korman AJ, Parker SM, Agrawal S, Goldberg SM, Pardoll
DM, Gupta A, Wigginton JM. Safety and activity of anti-PD-L1 antibody in patientswith advanced cancer. N Engl J Med. 2012;366:2455-65.

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75

7. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, Powderly JD,
Carvajal RD, Sosman JA, Atkins MB, Leming PD, Spigel DR, Antonia SJ, Horn L, Drake
CG, Pardoll DM, Chen L, Sharfman WH, Anders RA, Taube JM, McMiller TL, Xu H,
Korman AJ, Jure-Kunkel M, Agrawal S, McDonald D, Kollia GD, Gupta A, Wigginton JM,
Sznol M. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N
Engl J Med. 2012;366:2443-54.
8. Wolchok JD, Kluger H, Callahan MK, Postow MA, Rizvi NA, Lesokhin AM, Segal NH,
Ariyan CE, Gordon RA, Reed K, Burke MM, Caldwell A, Kronenberg SA, Agunwamba
BU, Zhang X, Lowy I, Inzunza HD, Feely W, Horak CE, Hong Q, Korman AJ,
Wigginton JM, Gupta A, Sznol M. Nivolumab plus ipilimumab in advanced melanoma. N
Engl J Med. 2013;369:122-33.
9. Lawrence MS, Stojanov P, Polak P, Kryukov GV, Cibulskis K, Sivachenko A, Carter SL,
Stewart C, Mermel CH, Roberts SA, Kiezun A, Hammerman PS, McKenna A, Drier Y,
Zou L, Ramos AH, Pugh TJ, Stransky N, Helman E, Kim J, Sougnez C, Ambrogio L,
Nickerson E, Shefler E, Cortes ML, Auclair D, Saksena G, Voet D, Noble M, DiCara D, Lin
P, Lichtenstein L, Heiman DI, Fennell T, Imielinski M, Hernandez B, Hodis E, Baca S,
Dulak AM, Lohr J, Landau DA, Wu CJ, Melendez-Zajgla J, Hidalgo-Miranda A, Koren A,
McCarroll SA, Mora J, Lee RS, Crompton B, Onofrio R, Parkin M, Winckler W, Ardlie K,
Gabriel SB, Roberts CW, Biegel JA, Stegmaier K, Bass AJ, Garraway LA, Meyerson M,
Golub TR, Gordenin DA, Sunyaev S, Lander ES, Getz G. Mutational heterogeneity in cancer and the search for new cancer-associated genes. Nature. 2013;499:214-8.
10. Snyder A, Makarov V, Merghoub T, Yuan J, Zaretsky JM, Desrichard A, Walsh LA,
Postow MA, Wong P, Ho TS, Hollman TJ, Bruggeman C. Genetic Basis Underlying
Clinical Response to CTLA-4 Blockade in Melanoma. N Engl J Med. 2014;In press.

Jedd D. Wolchok, M.D., Ph.D.
2006-20092009-

2013-

2014-

Associate Director, Ludwig Center for Cancer
Immunotherapy
Associate Member, Ludwig Cancer Research
Associate Professor of Medicine, Weill Medical Collegeof Cornell University
Lloyd J. Old/Virginia and Daniel K. Ludwig Chair in
Clinical Investigation
Chief, Melanoma & Immunotherapeutics Service

76

THERAPY-INDUCED ANTIBODY RESPONSES

Glenn Dranoff

Cancer Vaccine Center and Department of Medical Oncology
Dana-Farber Cancer Institute and Department of Medicine
Brigham and Women’s Hospital and Harvard Medical School
Boston, MA 02215, USA
Correspondence at: Dana-Farber Cancer InstituteDana 520C, 450 Brookline Avenue, Boston, MA 02215, USA(glenn_dranoff@dfci.harvard.edu)

Detailed histopathologic analysis of resected tumors in large cohorts of clinicallyannotated patients has established the prognostic importance of intra-tumoral immune
infiltrates 1-3) . A coordinated humoral and cellular response with associated tertiary

lymphoid structures is tightly correlated with prolonged survival after standard oncologic

therapy4). A systems biology approach has linked CD8+ cytotoxic T lymphocytes, a Th1

cytokine signature, CD4+ T follicular helper cells, and B cells to favorable outcomes5). Whilethese endogenous reactions develop in only a small subset of patients, their association

with durable disease control suggests that a major goal for active immunotherapy should
be to enhance the proportion of patients who mount this type of immune response.

Vaccination with irradiated, autologous tumor cells engineered to secrete granulocyte-

macrophage colony stimulating factor (GM-CSF) engenders a coordinated cellular andhumoral anti-tumor responses in some patients with solid or hematologic malignancies6).

Examination of metastases resected after vaccination, but not before therapy, reveals theinduction of brisk CD4+ and CD8+ T cell and CD20+ B cell infiltrates associated with tumor

destruction7). A tumor-specific vasculopathy with lymphoid and granulocytes disrupting

small tumor-blood vessels in association with zonal areas of ischemic necrosis is also
observed7). The subsequent administration of fully human monoclonal antibodies thatblock the negative T cell costimulatory molecule cytotoxic T lymphocyte associatedantigen-4 (CTLA-4) intensifies these therapeutic effects, resulting in long-term survival in
the absence of serious inflammatory pathology 8) . Pathologic analysis of metastases

obtained after the sequential vaccination/immune checkpoint blockade revealed a linear

relationship between the extent of tumor necrosis and the natural logarithm of the ratio of

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77

infiltrating CD8 + cytotoxic T cells and FoxP3 + Tregs. Moreover, the tumor-specificvasculopathy was again observed. Together, these results suggest that identification ofstrategies to enhance the proportion of patients that generate high CD8+ cytotoxic T cell to

FoxP3+ Treg ratios and responses that target the tumor vasculature should be a highpriority.

The generation and analysis of GM-CSF deficient mice uncovered an unexpected dualrole for the cytokine in tumor immunity9). The development of chronic inflammatory

disease in the lung and susceptibility to colitis revealed a key role for GM-CSF in immune

tolerance 10,11) . This pathway involves the ability of GM-CSF to promote the efficientphagocytosis of apoptotic cells by dendritic cells and macrophages through theupregulation of phosphatidylserine binding proteins such as milk fat globule epidermal

growth factor-8 (MFG-E8); binding of MFG-E8 to cognate avb3/5 integrins on phagocytes inturn triggers upregulation of Twist, TGF-b, and CCL22, which are critical for the
homeostasis of FoxP3 + Tregs 9) . GM-CSF deficiency thus results in attenuated Treg

responses that likely contribute to the inflammatory pathology. Notwithstanding thistolerance mechanism, GM-CSF also contributes to the induction of protective T and B
effector responses when a second activating signal is provided, such as concurrent

engagement of toll-like receptors. In this setting, stimulation through diverse TLRs
downregulates the tolerance pathway and instead switches GM-CSF function towards
dendritic cell maturation and tumor protection.

A recent clinical trial combining systemic GM-CSF protein and anti-CTLA-4 antibodies(ipilimumab) in advanced melanoma patients supports the concept of a dual role for GM-

CSF in tumor immunity12). In the study, 245 patients with unresectable stage III or IV
metastatic melanoma were randomized to ipilimumab alone or combined with GM-CSF.

At a median follow-up of 13.3 months, patients that received GM-CSF and ipilimumab
showed a reduced incidence of serious gastrointestinal and pulmonary pathology,

consistent with the murine studies that delineated a key role for the cytokine inmaintaining immune homeostasis at mucosal interfaces. GM-CSF also improved the one-year survival, increasing from the 52.9% with ipilimumab alone to 68.9% for combined GM-

CSF and ipilimumab. Although the precise mechanism for the therapeutic advantage

remains to be clarified, one interesting possibility might involve the ability of GM-CSF toenhance anti-CTLA-4 mAb mediated antibody dependent cellular cytotoxicity (ADCC)
through tumor infiltrating myeloid cells. Studies in murine models established a key

requirement for Fc receptors and ADCC in the therapeutic effects of anti-CTLA-4 mAbs,

likely because tumor-infiltrating Tregs express the highest levels of CTLA-4 of any cell in atumor-bearing host13). The selective reduction of Tregs in the tumor microenvironment

might allow GM-CSF function to be skewed more toward protective tumor immunity.

78

G. Dranoff

The delineation of a dual role for GM-CSF in tumor immunity suggests novel strategies

to enhance the overall therapeutic effects of the cytokine. In one approach, a dominantnegative inhibitory form of MFG-E8 that retains the ability to bind phosphatidylerine but isimpaired in signaling through avb3/5 integrins (MFG-E8-RGE) could be used to block thetolerance pathway14). Pre-clinical studies in several murine models demonstrated thatvaccination with irradiated tumor cells engineered to co-express GM-CSF and MFG-E8-

RGE stimulates higher levels of tumor immunity compared to GM-CSF or MFG-E8-RGE

alone9). The improved tumor protection involves the generation of a higher CD8+ T effector
to FoxP3+ Treg ratio and a higher titer of anti-tumor antibodies (Figure 1). To translate thistherapeutic concept to “first-into-human” testing in patients, we have generated human
K562 cell lines that stably express human GM-CSF and the human MFG-E8-RGE mutant.
Studies that use peripheral blood mononuclear cells from healthy donors have revealed

that the human MFG-E8-RGE mutant attenuates the tolerance functions of human GM-CSF.

Based on these results, we plan to submit an Investigator Sponsored New Drug

Application to the FDA to advance this vaccination strategy to patient testing.

In a second approach, material science engineering has been applied to develop a morepotent cellular vaccine strategy based on the dual functions of GM-CSF. Porous, poly-(lactide-coglycolide) scaffolds have been generated that provide precise spatial andtemporal control over the release of encapsulated agents15-17). The polymers have been

Figure 1

Schema of combined GM-CSF/MFG-E8-RGE secreting tumor cell vaccines. The dominantnegative inhibitor MFG-E8-RGE binds phosphatidylserine present in the vaccine microenvironment, but is unable to signal through avb3/5 integrins to promote Treg responses. A
higher ratio of CD8+ Teffectors to FoxP3+ Tregs is generating, enhancing anti-tumor immunity. Reprinted from (6).

Immune Checkpoints

79

extensively used in the context of resorbable sutures and manifest a very favorable safety

profile. The inclusion of recombinant GM-CSF protein, TLR ligands (including agonists of

TLR3, 4, or 9), and necrotic tumor cell lysate in the scaffolds results in a potent andsustained local immune response following implantation in subcutaneous pockets inimmunocompetent mice (Figure 2). This combination of agents results in a broad dendritic

cell response that includes CD8 + and CD11b + classical dendritic cell subsets andplasmacytoid dendritic cells. The cross-talk among these subsets leads to the production ofhigh levels of Th1 cytokines but the attenuation of immunoregulatory cytokines,

engendering a high ratio of CD8+ T effectors to FoxP3+ Tregs that accomplish the rejectionof established tumors. A “first-into-human” trial of this material scaffold vaccine has been

initiated in advanced melanoma patients.

In addition to enhancing T cell immunity, GM-CSF based vaccines elicit a broad

humoral reaction directed towards intracellular, surface, and secreted tumor proteins18).

Through screening tumor derived cDNA expression libraries and protein arrays with serafrom patients who achieved long-term clinical responses to autologous GM-CSF secreting

tumor cell vaccines and CTLA-4 blockade, we identified multiple targets of high titerantibodies. A subset of these likely plays critical roles in tumor pathogenesis. For example,

antibodies directed against major histocompatibility complex chain-related protein A(MICA) antagonize the immunosuppressive effects of soluble MICA, thereby promoting

innate and adaptive anti-tumor cytotoxicity 19) . Moreover, antibodies to angiogeneic

cytokines including vascular endothelial growth factor-A (VEGF-A), angiopoietin-1/2, andmacrophage migration inhibitory factor likely contribute to the tumor-selective

vasculopathy through blocking the angiogeneic activities of these factors20). Methods toFigure 2

Material engineered scaffold vaccines elicit potent vaccine responses. Porous, PLGA scaffolds
incorporating recombinant GM-CSF protein, CpG oligonucleotides, and necrotic B16 celllysates were implanted into a subcutaneous pocket of a C57Bl/6 mouse. A brisk infiltrate ofdendritic cells, macrophages, granulocytes, T and B cells, and stromal elements is evident.

80

G. Dranoff

isolate the rare memory B cells that produce these functionally relevant antibodies havebeen developed21). These isolated monoclonal antibodies provide important insights into

the mechanisms through which humoral immunity contributes to tumor destruction andare attractive candidates for clinical development as novel immunotherapies.
References

1. Clark W, Elder D, Guerry D, Braitman L, Trock B, Schultz D, et al. Model predicting survival in stage I melanoma based on tumor progression. J Natl Cancer Inst. 1989;81:1893904.
2. Mihm M, Clemente C, Cascinelli N. Tumor infiltrating lymphocytes in lymph nodemelanoma metastases-a histopathologic prognostic indicator and an expression of local
immune response. Lab Invest. 1996;74:43-7.
3. Azimi F, Scolyer RA, Rumcheva P, Moncrieff M, Murali R, McCarthy SW, et al. Tumorinfiltrating lymphocyte grade is an independent predictor of sentinel lymph node status
and survival in patients with cutaneous melanoma. J Clin Oncol. 2012;30:2678-83.
4. Dieu-Nosjean MC, Antoine M, Danel C, Heudes D, Wislez M, Poulot V, et al. Longterm survival for patients with non-small-cell lung cancer with intratumoral lymphoidstructures. J Clin Oncol. 2008;26:4410-7.
5. Bindea G, Mlecnik B, Tosolini M, Kirilovsky A, Waldner M, Obenauf AC, et al.
Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape inhuman cancer. Immunity. 2013;39:782-95.
6. Jinushi M, Hodi FS, Dranoff G. Enhancing the clinical activity of granulocytemacrophage colony-stimulating factor-secreting tumor cell vaccines. Immunol Rev.
2008;222:287-98.
7. Soiffer R, Lynch T, Mihm M, Jung K, Rhuda C, Schmollinger J, et al. Vaccination with irradiated, autologous melanoma cells engineered to secrete human granulocytemacrophage colony stimulating factor generates potent anti-tumor immunity inpatients with metastatic melanoma. Proc Natl Acad Sci USA. 1998;95:13141-6.
8. Hodi FS, Butler M, Oble DA, Seiden MV, Haluska FG, Kruse A, et al. Immunologic andclinical effects of antibody blockade of cytotoxic T lymphocyte-associated antigen 4 in previously vaccinated cancer patients. Proc Natl Acad Sci USA. 2008;105:3005-10.
9. Jinushi M, Nakazaki Y, Dougan M, Carrasco DR, Mihm M, Dranoff G. MFG-E8 mediateduptake of apoptotic cells by APCs links the pro- and anti-inflammatory activities of
GM-CSF. J Clin Invest. 2007;117:1902-13.
10. Dranoff G, Crawford AD, Sadelain M, Ream B, Rashid A, Bronson RT, et al.
Involvement of granulocyte-macrophage colony-stimulating factor in pulmonary
homeostasis. Science. 1994;264:713-6.

Immune Checkpoints

81

11. Dranoff G. Granulocyte-macrophage colony stimulating factor and inflammatory bowel
disease: establishing a connection. Gastroenterology. 2011;141:28-31.
12. Hodi FS, Lee SJ, McDermott DF, Rao UNM, Butterfield LH, Tarhini AA, et al.
Multicenter, randomized phase II trial of GM-CSF (GM) plus ipilimumab (Ipi) versus Ipi
alone in metastatic melanoma: E1608. J Clin Oncol. 2013;31:suppl; abstr CRA9007.
13. Bulliard Y, Jolicoeur R, Windman M, Rue SM, Ettenberg S, Knee DA, et al. Activating Fc
gamma receptors contribute to the antitumor activities of immunoregulatory receptor-targeting antibodies. J Exp Med. 2013;210:1685-93.
14. Asano K, Miwa M, Miwa K, Hanayama R, Nagase H, Nagata S, et al. Masking of phosphatidylserine inhibits apoptotic cell engulfment and induces autoantibody production in
mice. J Exp Med. 2004;200:459-67.
15. Ali OA, Emerich D, Dranoff G, Mooney DJ. In situ regulation of DC subsets and T cells
mediates tumor regression in mice. Sci Transl Med. 2009;1:8ra19.
16. Ali OA, Huebsch N, Cao L, Dranoff G, Mooney DJ. Infection-mimicking materials toprogram dendritic cells in situ. Nat Mater. 2009;8:151-8.
17. Ali OA, Mooney DJ. Immunologically active biomaterials for cancer therapy. Curr Top
Microbiol Immunol. 2011;344:279-97.
18. Dranoff G. Targets of protective tumor immunity. Ann N Y Acad Sci. 2009;1174:74-80.
19. Jinushi M, Hodi FS, Dranoff G. Therapy-induced antibodies to MHC class I chain-related
protein A antagonize immune suppression and stimulate antitumor cytotoxicity. Proc
Natl Acad Sci USA. 2006;103:9190-5.
20. Schoenfeld J, Jinushi M, Nakazaki Y, Wiener D, Park J, Soiffer R, et al. Active
immunotherapy induces antibody responses that target tumor angiogenesis. Cancer
Res. 2010;70:10150-60.
21. Franz B, May KF, Jr., Dranoff G, Wucherpfennig K. Ex vivo characterization and isolationof rare memory B cells with antigen tetramers. Blood. 2011;118:348-57.

Glenn Dranoff, M.D.

1985-1988

1989-1994

1989-1994
1994-

Resident in Internal Medicine, Massachusetts General
Hospital
Clinical Fellow in Medical Oncology, Dana-Farber
Cancer InstitutePost-doctoral Fellow, Whitehead Institute
Faculty, Dana-Farber Cancer Institute and Harvard
Medical School

82

CLINICAL APPLICATION OF ANTI-CCR4
MONOCLONAL ANTIBODY

Ryuzo Ueda, 1Susumu Suzuki, 2Kazuhiro Yoshikawa and3
Takashi Ishida1

Dept. Tumor Immunology, 2Advanced Medical Research Center
Aichi Medical University School of Medicine
1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan(uedaryu@aichi-med-u.ac.jp)
3
Dept. Hematology/Oncology, Nagoya City University
1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi 467-8501, Japan1

Immunotherapy offers an attractive approach to treat cancer by specifically targeting

tumor cells and by modulating the host immune reactions. A number of antibodies haverecently been developed and used for the treatment of malignant diseases. Here, we

present a review of our research strategies for developing novel therapeutics using ahumanized monoclonal antibody for treatment of hematological malignancies and cancers.

Research to develop the anti-CCR4 monoclonal antibody (Mogamulizumab) as an

Clinical application for Lymphoid Neoplasms

anticancer antibody drug for adult T-cell leukemia-lymphoma (ATL) started in 1999, with acompany that successfully produced a mouse monoclonal antibody (KM2160) against achemokine receptor, CCR4. The target molecule CCR4 is a 7 transmembrane G protein

coupled receptor (GPCR), which recognizes two functional ligands, TARC (thymus andactivation-regulated chemokine; CCL17) and MDC (macrophage-derived chemokine;
CCL22). It is expressed mainly by Th2 cells and FOXP3+ T regulatory (Treg) cells amongnormal lymphocytes. CCR4 is expressed on most ATL cells1), and a subgroup of peripheral

T-cell lymphomas (PTCL)2).

ATL is an aggressive peripheral T-cell neoplasm caused by human T-cell lymphotropic

virus type 1. The disease is resistant to conventional chemotherapy, and there currently

exist very limited treatment options; thus, it has a poor prognosis, with a medianprogression-free survival (PFS) and overall survival (OS) of 7.0 and 12.0 months,

respectively, when using the best combination chemotherapy in previously untreated ATL
patients. Therefore, the development of alternative treatment strategies for patients with

Antibody Therapy

83

ATL is an urgent issue.

Mogamulizumab is a first-in-class defucosylated humanized anti-CCR4 monoclonal

antibody, which is engineered to exert higher antibody-dependent cell-mediatedcytotoxicity (ADCC) activity using a defucosylation technology we named Potelligent

technology, developed by the Kyowa Hakko Kirin Co. Ltd. The ADCC activity ofMogamulizumab has a 2–3 log stronger activity than conventional antibodies. Furthermore,

Mogamulizumab has no complement dependent cytotoxicity (CDC) activity, no

neutralizing activity and does not directly induce apoptosis3). Since this agent showed
potent antitumor activities in patients with relapsed ATL (NCT00355472, NCT00920790)4,5)

or PTCL and cutaneous T-cell lymphoma (CTCL) (NCT01192984)6), it was approved for thetreatment of relapsed/refractory ATL in 2012 as an orphan drug, and for PTCL in 2014, inJapan. (Figure 1). Excellent treatment results have been reported in line with previous

studies3-6). For example, Mogamulizumab monotherapy demonstrated significant responses

in patients with relapsed ATL with an acceptable toxicity profile. An overall response rateof 50% and median PFS and OS values of 5.2 and 13.7 of months, respectively, wereobserved. The most common adverse events were infusion reactions (89%) and skin rashes(63%), which were manageable and reversible in all cases. It was also reported that

objective responses were noted for 13 of 37 patients (35%; 95% confidence interval [CI], 20%

to 53%), including five patients (14%) with complete responses in a multicenter phase II

study for patients with relapsed PTCL and CTCL.

Figure 1 History of Translational Research of anti-CCR4 mAb(Mogamulizumab)

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R. Ueda et al.

CCR4 is expressed on CD45RA-FOXP3high CD4+ Treg cells [effector Treg (eTreg) cells], but

Feasibility of anti-CCR4 monoclonal antibody towards cancer immunotherapy

is not expressed on CD45RA+FOXP3high CD4+ Treg cells [naïve Treg (nTreg) cells]7), therefore,anti-CCR4 monoclonal antibodies deplete only eTreg cells but not nTreg cells8). Tumor sites

were shown to be intensively infiltrated with FOXP3+CCR4+ eTreg cells8,9), which can be

depleted by anti-CCR4 monoclonal antibody treatment8). In addition, the depletion ofeTreg cells by anti-CCR4 monoclonal antibodies effectively augmented anti-tumor

immunity compared with depletion by anti-CD25 monoclonal antibodies in an in vitro

study8). Furthermore, in some ATL patients, in vivo administration of Mogamulizumab

markedly reduced the eTreg-cell fraction and augmented anti-tumor immunity resulting inlong lasting objective responses5,10).

These results and observations indicate that CCR4 is an attractive target for cancer

therapy, because eTreg cells play an important role in tumor escape from host immunity inthe tumor microenvironment. Based on this concept, we are now conducting an

investigator initiated phase la/lb trial of Mogamulizumab for CCR4-negative advanced orrecurrent solid tumors, specifically aiming to deplete eTreg cells and boosting antitumor

immunity (NCT01929486) (Figures 2, 3).

It has been observed in phase Ia trial that KW-0761 is tolerated at dose range between

0.1 mg/kg to 1.0 mg/kg, demonstrating effective elimination of eTreg population in
peripheral blood. Phase Ib trials currently underway.

Figure 2 Monotherapy of Mogamulizumab for solid tumor by investigator initiated phase Ia/Ib trials
Chief Investigator: R Ueda
Co-investigators: E Nakayama, M Oka, H Udono, Y Doki, H Wada, H Nishikawa, S Iida,
T Ishida, H Inagaki, T Doki, K Kakimi, K Funakoshi, E Sato

Antibody Therapy

85

Figure 3 Collaboration members of Treg depletion project

On the other hand, alterations of Treg-cell distribution or frequencies may contribute tovarious types of autoimmune diseases. In ATL patients who were administered

Mogamulizumab, skin rash was frequently observed11). It is therefore a matter of someurgency to establish the safest and most effective treatment strategies for using

Mogamulizumab in cancer patients12). In the near future, a new cancer immunotherapy

combined with check point inhibitors and depletion of Treg cells with Mogamulizumab
will be offered to appropriate cancer patients (Figure 4).

Figure 4 CCR4 immunotherapy in combination with immune checkpoint blockade

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R. Ueda et al.

Reference
1. Ishida, T., Utsunomiya, A., Iida, S., Inagaki, H., Takatsuka, Y., Kusumoto, S., Takeuchi, G.,
Shimizu, S., Ito, M., Komatsu, H., Wakita, A., Eimoto, T., Matsushima, K. and Ueda R.
Clinical significance of CCR4 expression in adult T-cell leukemia/lymphoma:its close
association with skin involvement and unfavorable outcome. Clinical Cancer Res. 2003;
9:3625-3634.
2. Ishida, T., Inagaki, H., Utsunomiya, A., Takatsuka, Y., Komatsu, H., Iida, S., Takeuchi, G.,
Eimoto, T., Nakamura, S. and Ueda R. CXC chemokine receptor 3 and CC chemokine
receptor 4 expression in T-cell and NK-cell lymphomas with special reference to clinicopathological significance for peripheral T-cell lymphoma, unspecified. Clin Cancer Res.
2004;10:5494-500.
3. Niwa, R., Hosaka, S.E., Sakurada, M., Shinkawa, T., Uchida, K., Nakamura, K.,
Matsushima, K., Ueda R., Hanai, N. and Shitara, K. Defucosylated chimeric anti-CC
chemokine receptor 4 IgG1 with enhanced antibody-dependent celluar cytotoxicity
shows potent therapeutic activity to T-cell leukemia and lymphoma. Cancer Res.,
2004;64:2127-2133.
4. Yamamoto K, Utsunomiya A, Tobinai K, Tsukasaki K, Uike N, Uozumi K, Yamaguchi K,
Yamada Y, Hanada S, Tamura K, Nakamura S, Inagaki H, Ohshima K, Kiyoi H, Ishida T,
Matsushima K, Akinaga S, Ogura M, Tomonaga M, Ueda R. Phase I study of KW-0761, adefucosylated humanized anti-CCR4 antibody, in relapsed patients with adult T-cellleukemia-lymphoma and peripheral T-cell lymphoma. J Clin Oncol. 2010;20; 28(9):1591-8.
5. Ishida T, Joh T, Uike N, Yamamoto K, Utsunomiya A, Yoshida S, Saburi Y, Miyamoto T,
Takamoto S, Suzushima H, Tsukasaki K, Nosaka K, Fujiwara H, Ishitsuka K, Inagaki H,
Ogura M, Akinaga S, Tomonaga M, Tobinai K, Ueda R. Defucosylated anti-CCR4 monoclonal antibody (KW-0761) for relapsed adult T-cell leukemia-lymphoma: a multicenter
phase II study. J Clin Oncol, 2011; 30(8):837-42.
6. Ogura M, Ishida T, Hatake K, Taniwaki M, And K, Tobinai K, Fujimoto K, Yamamoto K,
Miyamoto T, Uike N, Tanimoto M, Tsukasaki K, Ishizawa K, Suzumiya J, Inagaki H,
Tamura K, Akinaga S, Tomonaga M, Ueda R. Multicenter phase II study of mogamulizumab (KW-0761), a defucosylated anti-CCR4 antibody, in patients with relapsed
peripheral T-cell lymphoma and cutaneous T-cell lymphoma. J Clin. Oncol.
2014;32:1157-63.
7. Miyara M, Yoshioka Y, Kitoh A, Shima T, Wing K, Niwa A, Parizot C, Taflin C, Heike T,
Valeyre D, Mathian A, Nakahata T, Yamaguchi T, Nomura T, Ono M, Amoura Z,
Gorochov G, Sakaguchi S. Functional delineation and differentiation dynamics ofhuman CD4+ T cells expressing the FoxP3 transcription factor. Immunity. 2009; 30:899-911.

Antibody Therapy

87

8. Sugiyama D, Nishikawa H, Maeda Y, Nishioka M, Tanemura A, Katayama I, Ezoe S,
Kanakura Y, Sato E, Fukumori Y, Karbach J, Jager E, Sakaguchi S. Anti-CCR4 mAb
selectively depletes effector-type FoxP3+CD4+ regulatory T cells, evoking antitumor
immune responses in humans. Proc Natl Acad Sci USA. 2013;110:17945-50.
9. Kurose K, Ohue Y, Sato E, Yamaguchi A, Eikawa S, Isobe M, Nishio Y, Uenaka A, Oka
M,Nakayama E. Increase in activated Treg in TIL in lung cancer and in vitro depletion ofTreg by ADCC using an anti-human CCR4 mAb (KM2760). J Thorac Oncol. 2014; Oct 16.
[Epub ahead of print]
10. Suzuki S, Masaki A, Ishida T, Ito A, Mori F, Sato F, Narita T, Ri M, Kusumoto S,
Komatsu H, Fukumori Y, Nishikawa H, Tanaka Y, Niimi A, Inagaki H, Iida S, Ueda R.
Tax is a potential molecular target for immunotherapy of adult T-cell leukemia/lymphoma. Cancer Sci. 2012;103:1764-73.
11. Ishida T, Ito A, Sato F, Kusumoto S, Iida S, Inagaki H, Morita A, Akinaga S, Ueda R.
Stevens-Johnson Syndrome associated with mogamulizumab treatment of adult T-cellleukemia/lymphoma. Cancer Sec, 2013; 104:647-50.
12. Ishida T and Ueda R. Immunopathogenesis of lymphoma: focus on CCR4. Cancer Sci.,
2011;102:44-50.

Ryuzo Ueda, M.D., Ph.D.

1969-1976
1976-1980

1980-1995

1995-20102008-2012
2012-

Clinical Fellow, Nagoya University School of Medicine
Research Fellow, Memorial Sloan-Kettering Cancer
CenterSenior, Head, Chief, Laboratory of Chemotherapy,
Aichi Cancer Center Research Institute
Professor, Internal Medicine (Medical Oncology and
Immunology), Nagoya City University Medical School
Specially appointed Professor, Tumor Immunology,
Graduate School of Medical Sciences, Nagoya City
University
Professor, Tumor Immunology, Aichi Medical Schoolof Medicine

Specialty and Present Interest:
Hematology/Oncology and Tumor Immunology
Molecular Target for Cancer, Monoclonal Antibody Therapy

88

DRUGGING THE UNDRUGGABLE WITH MONOCLONALANTIBODIESDavid A. Scheinberg

Memorial Sloan Kettering Cancer Center
1275 York Avenue, New York, NY 10065, USA(d-scheinberg@ski.mskcc.org)

Most small molecule drugs currently available are directed to less than 5% of theproteins in the cell, typically those that are enzymes and therefore “druggable.”

Monoclonal antibodies (mAb) may be useful for a small number of proteins, all outside of

the cell, which are not druggable by small molecules. However, the vast majoarity ofproteins, particularly the important oncogenic proteins and mutated proteins, are inside of

the cell and not accessible to mAb. Here we describe an approach to using mAb to target

an important intracellular protein in cancer. The Wilms’ tumor oncogene protein (WT1) is

one such intracellular, oncogenic transcription factor that is over-expressed in a wide rangeof human cancers. Wt1 is a validated target for T cell-based immunotherapy, used in

multiple clinical trials of vaccines and cellular therapies. RMFPNAPYL, “RMF”, is a WT1-

derived CD8 T cell epitope presented by HLA-A0201 on the surface of cancer cells.

We hypothesized that a “TCR-like” antibody or “TCR mimic” antibody (TCRm)

specific for the WT1 peptide/HLA-A0201 complex might be an effective therapeutic agent.

(Figure 1) We generated a high avidity (KD= 0.1 nM), fully human monoclonal antibody

IgG1 (mAb), ESK1, specific for the WT1 RMF peptide/HLA-A0201 complex, using phagedisplay technology1-3). ESK1 binds cancer and leukemia cell lines as well as primary cancer

leukemia cells, which are both WT1+ and HLA-A0201+, as shown by flow cytometry andradioimmunoassays. ESK1 was not active alone, or with complement, but it mediatedantibody dependent cell cytotoxicity (ADCC) against several different types tumor cells in

vitro. Surprisingly, ESK1 antibody substantially reduced established disseminated, humanleukemias in NSG mouse models, at doses as low as 50 to 100 ug. Therapeutic effects weremore pronounced and more durable when combined with human CD34-, CD3-, human NK

Antibody Therapy

89

and monocyte effectors in the NSG mice. We concluded that ESK1 is a potential

therapeutic agent for a wide range of human leukemias and cancers over-expressing theWT1 oncoprotein.

In order to make the ESK1 more potent, we engineered a second-generation mAb,

ESKM, which displayed enhanced antibody dependent cell-mediated cytotoxicity (ADCC)

function due to altered fucosylation on the Fc. In ADCC assays in vitro ESKM was morepotent than native ESK1 against a variety of cancers including mesothelioma and leukemia.

While ESK antibodies mediated ADCC against several hematopoietic cancer cell lines andfresh cancer cells and solid tumor cells at concentrations below 1µg/ml, but ESKM wasabout 5-10 fold more potent in vitro against these multiple cancer cell lines. ESKM also wasmore potent in vivo against JMN mesothelioma. ESK was effective against SET2 AML andfresh ALL xenografts. HLA-A*02:01+ transgenic mice were used to assess ESKM toxicity.

No changes in cell numbers or cell types in the blood spleen or BM were observed,

including murine stem cells. ESKM had a shorter half-life (4.9 vs 6.5 days). The
biodistribution pattern in C57BL6/J mice was the same as the native IgG1. There was nodifference in half-life or biodistribution in HLA-A*02:01+ transgenic mice compared to theparent strain, at therapeutic doses of ESKM. Therapeutic doses of ESKM in these micecaused no pathologic tissue damage. These data support the concept that an Fc-enhanced

mAb can improve efficacy against a low-density, tumor-specific, peptide/MHC target. We

plan to develop such an mAb against this important intracellular oncogenic protein.

Additional experiment were done to try other formats of the ESK mAb that might beeven more effective. One new effective immunotherapy for cancer that directs T cell

cytotoxicity against cells expressing a cell surface protein is recognized by an antibody scFv

is Bi-specific T cell engager antibody (BiTE) therapy. Therefore, we generated the first BiTE

Figure 1

Schematic of TCrm mAb function

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D. A. Scheinberg

construct derived from our TCR-mimic ESK mAb. ESK-BiTE was able to selectively

activate and induce proliferation of cytolytic human T cells. ESK-BiTE was also able to kill

multiple leukemias and cancers in vitro and in mice. These activities occurred despite theultra-low density at the cell surface of the peptide/HLA-A2 complex.

In an effort to add multi-functionality and multi-valency to our antbody platform wealso explored use of nanomaterial carriers. We developed a nanoscale isotope generator

that elaborates 4 alpha particle emitting elemnts at the tumor cell site4,5). This targeted
nanogenerator is the most potent cancer cytotoxic agent known, requiring only 1 deliveredalpha to kill a cell.

Single-walled carbon nanotubes (SWNTs) provided an even more versatile platform.

SWNT can deliver imaging agents or drugs to tumors and offer several advantages overapproaches based other nanomaterials or antibodies. The nanotubes can carry 100 times

more cargo than an mAb, but are still rapidly eliminated from circulation by renal

filtration, like a small molecule, due to their narrow dimension of 1 -2 nanometers and their

Figure 2

SWNT can be appended with multiple ligands (left). One hundred times more Ac-225 can be
attached to a SWNT than an mAb (upper right). Use of morpholinos allows rapid, highaffinity addressable labels to assemble the parts (lower right).

Antibody Therapy

91high aspect ratio of 300:1. Nanotubes can carry fluorophores or radioisotopes, and wereshown to selectively bind to cancer cells in vitro and in tumour-bearing xenografted mice.

SWNT conjugates can be labelled with both high specific activity alpha-particle andgamma-ray emitting isotopes for imaging. Using nanotubes modified with morpholino

oligonucleotide sequences to bind to cancer cells that have been pre-targeted with

antibodies modified with oligonucleotide strands complementary to those on the

nanotubes, we showed that SWNTs can target tumors in a two-component self-assembling

approach6-8). (Figure 2)

The binding of the SWNT was also found to lead to internalization of theantibody/nanotube complexes. Because the SWNT conjugates labelled with alpha-particle

generating 225 Ac were found to clear rapidly, unlike the slower clearing mAB,

radioisotope toxicity was mitigated. The two step self assembly process was shown to be

therapeutically effective in vivo. Thus using SWNT-oligonucleotide conjugates as selfassembling, tumor-targeted devices may be feasible.
References

1. Dao T, Yan S, Veomett N, Pankov D, Zhou L, Korontsvit T, Whitten J, Tan T, Liu H, Scott
A, Whitten J, Maslak P, Casey E, Tan T, Liu H, Zakhaleva V, Curcio M, Doubrovina E,
O’Reilly RJ, Liu C and David A. Scheinberg DA. Targeting the intracellular WT1 oncogene product with a therapeutic human antibody. Sci Transl Med 5, 176Ra33 (2013)
2. Dubrovsky L, Pankov D, Brea EJ, Dao T, Scott A, Yan S, O’ Reilly RJ, Liu C, andScheinberg DA. A TCR-mimic antibody to WT1 bypasses tyrosine kinase inhibitor
resistance in human BCR-ABL+ leukemias. Blood, (epub 2014) PMID 24723681
3. Veomett N, Dao T, Liu H, Xiang J, Dubrovsky L, Pankov D, Whitten JA, Park SM,
Korontsvit T, Zakhaleva V, Curcio M,. Kharas MG, O’Reilly RJ, C, and Scheinberg DA.
Therapeutic Efficacy of an Fc-Enhanced TCR-Like Antibody to the Intracellular WT1
Oncoprotein. Clin Cancer Res., (epub 2014) PMID 24850840
4. McDevitt MR, Ma DS, Lai LT, Simon J, Frank RK, Borchardt P, Wu K, Pellegrini V,
Curcio MJ, Miederer M, Bander NH, Scheinberg DA. Tumor therapy with targetedatomic nanogenerators. Science 294:1537-1540. 2001. PMID: 11711678,
5. Jurcic J, Larson SM, Sgouros G, McDevitt MR, Scheinberg DA. Antibody targetedalpha-particle immunotherapy of leukemia. Blood 100:1233-1239. 2002. PMID:
12149203, Journal in Progress.
6. Ruggiero A, Villa CH, Bander E, Rey DA, Bergkvist M, Batt CA, Manova-Todorov K,
Deen WM, Scheinberg DA, McDevitt MR. Paradoxical glomerular filtration of carbon
nanotubes. Proc. Nat Acad Sci USA 107(27):12369-74 . 2010. PMID: 20566862 [PubMed in process]92

D. A. Scheinberg

7. Villa C; McDevitt M; Escorcia F, Rey D; Bergkvist M, Batt C; Scheinberg D. Synthesis
and biodistribution of oligonucleotide-functionalized, tumor-targetable carbon nanotubes. Nano Letters 8(12):4221-4228 [Epub ahead of print] 2008. PMID: 19367842,
Journal in Progress.
8. Mulvey JJ, Villa CH, Mcdevitt MR, Escorcia FE, Casey E, Ruggiero A, Scheinberg DA.
Targeted Self-Assembly On Tumors Of Functionalized Carbon Nanotube andMonoclonal antibodies. Nat Nanotechnol. 2013 8:763-71. doi: 10.1038/nnano.2013.190.
Epub 2013 Sep 29. PMID 24077028

David A. Scheinberg, M.D., Ph.D.

1999-

20002001-

20022005-

2009-20142012-

2013-20132013-

Attending Physician; Department of Medicine,
Leukemia Service and Clinical, Memorial Hospital
Attending Physician; Hematology Laboratory
Service/Department of Clinical Laboratories
Member, MSKCC
Professor of Pharmacology, Weill Cornell College ofMedicine
Incumbent of the Vincent Astor Chair, MSKCC
Professor of Medicine, Weill Cornell College ofMedicine
Co-Chairman, Pharmacology Program, Weill Graduate
School of Medical Sciences
Chairman, Molecular Pharmacology and Chemistry,
SKI
Chairman, Experimental Therapeutics Center, MSKCC
Professor, Gerstner-Sloan-Kettering Graduate School,
SKI
Chairman, Nanotechnology Center, MSKCC
Tri-institutional Program in Chemical Biology; Faculty
member, SKI
Director (interim) Sloan-Kettering Institute
Director, Tri-Institutional Therapeutics Discovery
Institute

93

WT1 (WILM'S TUMOR GENE) PEPTIDE CANCER VACCINE

Haruo Sugiyama

Department of Functional Diagnostic Science
Osaka University Graduate School of Medicine
1-7 Yamada-Oka, Suita, Osaka 565-0871, Japan(sugiyama@sahs.med.osaka-u.ac.jp)

Wilms’ tumor gene 1 (WT1) is isolated as a gene responsible for childhood renal tumor,

Wilms’ tumor, encodes a transcription factor that regulate many genes, and plays animportant role in cell proliferation and differentiation, embryogenesis, leukemogenesis andtumorigenesis.

I discovered that WT1 was a good molecular marker for detection of minimal residual

leukemic cells in leukemia patients1). Quantitation of WT1 mRNA (WT1 mRNA assay) inperipheral blood mononuclear cells made it possible to detect one leukemic cell in 100,000

normal mononuclear cells. WT1 mRNA assay becomes an essential clinical test fortreatment of leukemia and myelodysplastic syndrome (MDS) and is commercially available

in Japan and EU, and soon in USA. WT1 mRNA assay is covered by national healthinsurance in 2007 for AML and in 2011 for MDS in Japan.

I also discovered that WT1 protein was expressed in not only leukemia but also almost

all types of solid tumors and thus was a pan-tumor associated antigen. I identified a

WT1235 (235-243 aa) and WT1126 (126-134 aa) peptides as an HLA-A*2402- or HLAA*0201- restricted WT1 peptide, respectively. I started for the first time (First – in – Man) aphase 1 clinical study of WT1 vaccination for patients with leukemia, MDS, lung cancer, or
breast cancer in 2001 2) . Patients were intradermally injected with an HLA-A*2402-

restricted, natural (CMTWNQMNL) or modified (CYTWNQMNL) 9-mer WT1 peptide

emulsified with Montanide ISA51 adjuvant at 0.3, 1.0, or 3.0 mg per body at 2-weekintervals, with toxicity and clinical and immunological responses. Toxicity consisted onlyof local erythema at the WT1 vaccine injection sites. Three of the four AML patients weresuccessively being injected WT1 vaccine and are in complete remission over 11 years. A

94

H. Sugiyama

phase II clinical study, in which WT1 vaccination was intensified and repeated every weekat a dose of 3.0 mg / body of modified WT1 peptide for three months, were started from

2004, and safety of this study was confirmed. Clinical effect of WT1 vaccination issatisfactory for glioblastoma multiforme with relapse and disease control rate is 47.7%3).

Adverse effect is only erythema at WT1 vaccine injection sites. Thirty-two patients withadvanced pancreatic cancer were biweekly WT1-vaccinated and received gemcitabine(1000mg/m2) on days 1, 8, and 15 of a 28-day cycle. This combination therapy was welltolerated. The frequencies of grades 3-4 adverse events for this combination therapy weresimilar to those for gemcitabine alone. Objective response rate and disease control rate was

20.0% (6/30 evaluable patients) and 75.0%, respectively. Clinical response was 7 PR

(21.9%), 17 SD (53.1%), 7 PD (21.9%) and 1 unevaluable. Median survival rate was 8.1

months and 1-year survival rate was 29%.

We have been performing randomized

phase Ⅱ clinical study of WT1 vaccine +gemcitabine vs. gemcitabine alone4).

Until now, over 750 patients with AML, CML, MDS, myeloma, glioblastoma multiform,

pancreatic, lung, breast, thyroid, colon, gynecological, renal, or other cancers were WT1

vaccinated. However, there is no WT1 vaccination-related death, indicating that WT1

vaccination is very safe.

The National Cancer Institute of USA evaluated 75 popular tumor associated antigens

for the clinical utility and ranked WT1 antigen as a top among 75 tumor associatedantigens5).

In cure-oriented cancer therapy, complete eradication of quiescent cancer stem cells isessential. Only immunity against cancer is considered to be able to eradicate non-dividing

quiescent cancer stem cells. Therefore, we proposed a novel strategy for cancer treatment

as follows. At present time, WT1 peptide vaccine is usually used as the last line treatment

under the impaired immune conditions of cancer-bearing patients and thus immune

response induced is limited to weak. In a novel strategy, WT1 peptide vaccination will bestarted as soon as possible when patients were diagnosed as having cancer and on the baseline of WT1 immunity induced, surgery, chemotherapy and /or radiotherapy should be

performed. In such situation, for example, in the combination of WT1 vaccination andchemotherapy, some chemotherapies sometimes dominantly suppress immune
suppressive cells, resulting in relative increase in WT1 immunity against cancers.

Nowadays, two WT1 killer peptide vaccines (one for HLA-A*24:02+ patients andanother for HLA-A*02:01+ patients) and one WT1 helper peptide vaccine(HLA class Ⅱ-

restricted, but universal peptide) are under phases Ⅰ and Ⅱ clinical studies, respectively, by
two pharmaceutical companies.

Cancer Vaccine

95

References
1. Sugiyama H. WT1 (Wilms' tumor gene 1): biology and cancer immunotherapy. Jpn J
Clin Oncol 2010; 40: 377-87.
2. Oka Y, Tsuboi A, Taguchi T, Osaki T, Kyo T, Nakajima, Elisseeva OA, Oji Y, Kawakami
M, Ikegame K, Hosen, N, Yoshihara S, Wu F, Fujiki F, Murakami M, Masuda T, Nishida S,
Shirakata T, Nakatsuka S, Sasaki A, Udaka K, Dohy H, Aozasa K, Noguchi S, Kawase I,
Sugiyama H. Induction of WT1 (Wilms’ tumor gene)-specific cytotoxic T lymphocytes by
WT1 peptide vaccine and the resultant cancer regression. Proceedings of National
Academy of Science USA 2004; 101:13885-13890.
3. Izumoto S, Tsuboi A, Oka Y, Suzuki T, Hashiba T, Kagawa N, Hashimoto N, Maruno M,
Elisseeva, OA, Shirakata T, Kawakami M, Oji Y, Nishida S, Ohno S, Kawase I,
Hatazawa J, Morita S, Sakamoto J, Sugiyama H, Yoshimine T. Phase II clinical trial ofWilms tumor 1 peptide vaccination for patients with recurrent glioblastoma multiforme. J Neurosurg 2008; 108: 963-971.
4. Nishida S, Koido S, Takeda Y,Honma S, Kimita H, Takahara A, Morita S, Ito T,
Morimoto S, Hara K, Tsuboi A, Oka Y, Yanagisawa S, Toyama Y, Ikegami M, Kitagawa T,
Eguchi H, Wada H, Nagano H, Nakata J, Nakae Y, Hosen N, Oji Y, Tanaka T, Kawase I,
Kumanogoh A, Sakamoto J, Doki Y, Mori M,Ohkusa T, Tajiri H, Sugiyama H. Wilms
Tumor Gene 1 (WT1) Peptide–based Cancer Vaccine Combined With Gemcitabine forPatients With Advanced Pancreatic Cancer. J Immunother 2014; 37: 105-14.
5. Cheever MA, Allison JP, Ferris AS, Finn OJ, Hastings BM, Hecht TT, Mellman I,
Prindiville SA, Viner JL, Weiner LM, Matrisian LM. The prioritization of cancer antigens: a national cancer institute pilot project for the acceleration of translational
research. Clin Cancer Res 2009; 15: 5323-37.

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H. Sugiyama

Haruo Sugiyama, M.D., Ph.D.

1975-1979
1979-1980
1980-1983
1983-1994

1994-1995
1995

2003

Present

Ph. D. course of Institute for Microbial Diseases
Research Fellow, Institute for Microbial Diseases
Medical Fellow, Osaka University Hospital
Assistant Professor, Department of Medicine III, Osaka
University Medical School
Lecturer, Department of Medicine III, Osaka University
Medical School
Professor, Department of Functional Diagnostic
Science, Osaka University Medical School
Professor, Department of Functional Diagnostic
Science, Osaka University Graduate School of Medicine
Professor and Chairman, Departments of Functional
Diagnostic Science, of Cancer Vaccine, of Cancer Stem
Cell Biology and of Cancer Immunology, Osaka
University

Specialty and Present Interest:
Tumor Immunology, Hematology

97

SYNERGY OF THERAPEUTIC VACCINATIONAGAINST HPV16 ONCOGENIC PROTEINS AND
STANDARD CHEMOTHERAPEUTICS

Cornelis J.M. Melief 1,8, Marij J.P. Welters2 , Tetje C. van der Sluis1,
Helene van Meir3, Suzanne van Duikeren1, Suzanna Huppelschoten4,
Ekaterina S. Jordanova5, Arjen Sloots1, Judith R. Kroep2, Gemma G. Kenter5,
Vincent T.H.B.M. Smit6, Ferry Ossendorp1, Bob van de Water4,
Mariette I.E. van Poelgeest7, Jacobus Burggraaf3, Ramon Arens1 andSjoerd H. van der Burg2
Department of Immunohematology and Blood Transfusion, Leiden University Medical
Center, E3-Q, P.O. Box 9600, 2300 RC, Leiden, The Netherlands
2
Clinical Oncology, Leiden University Medical Center
3
Center for Human Drug Research
4
Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University
5
Center for Gynaecological Oncology Amsterdam, Free University Amsterdam
6
Dept. of pathology, Leiden University Medical Center
7
Dept. of gynaecology, Leiden University Medical Center
8
ISA Pharmaceuticals, J.H. Oortweg 19-21, 2333 CH, Leiden, The Netherlands(melief@isa-pharma.com)

1

We previously developed a vaccine, comprising human papillomavirus (HPV) type 16

E6 and E7 overlapping synthetic long peptides (HPV16-SLP), that was clinically active inpatients with HPV16 + high-grade vulvar intraepithelial neoplasia 1) . Complete lesion
regression correlated with a strong vaccine-prompted HPV16-specific effector T-cell

response1,2). However, in patients with HPV16-induced metastatic cervical cancer vaccineinduced T-cell responses were weaker and did not result in improved clinical outcome3,4),

although some patients benefited clinically from subsequent treatment withchemotherapeutic agents3), suggesting that the combination of both modalities can make adifference. In a preclinical mouse model of HPV16-induced cervical cancer we studied

whether SLP vaccination could be combined with chemotherapy to eradicate tumors. First

we tested the effect of SLP vaccination in combination with individual chemotherapeutic

agents. In most mice that received either peptide vaccination or chemotherapy treatment,

only a temporary regression in tumor size was observed. Importantly, combined chemo-

98

C. J.M. Melief et al.

immunotherapy induced complete tumor eradication in nearly all mice 5 ) . The

chemotherapeutic agents cisplatin and carboplatin displayed the strongest synergy with

SLP vaccination. This synergy was not due to increased sensitivity of cisplatin treatedtumor cells to CTL-mediated killing, or to a stronger vaccine-induced circulating tumorspecific T cell response. Analysis of the intra-tumoral immune response revealed thatcombined treatment with cisplatin and SLP vaccination resulted in a strong increase in thedensity of intra-tumoral, Tumor Necrosis Factor alpha (TNFa) and Interferon gamma (IFN-g) producing CD8+ CTLs. Tumor cells incubated with TNFa and IFN-g, together withcisplatin strongly enhance their chemokine expression, compatible with the abundantleukocyte infiltration in the tumor upon combined chemo-immunotherapy. Accordingly,

when combined with systemic cisplatin treatment, SLP vaccine-induced CTLs appeared tomigrate earlier into the tumor beds. Moreover, analysis of the tumor cells in vivo showedthat combined treatment not only caused a decrease in the proliferative capacity of tumor

cells, but also a TNFa- induced enhancement of cisplatin- mediated tumor cell death. Cell

death was accompanied by an increased expression of pro-apoptotic molecules.

Importantly, when SLP vaccination and cisplatin were combined, the synergistic effect of

this combination on tumor eradication in vivo was abolished by treatment of the mice withi.p. injections of monoclonal antibody against TNFa5). We then tested the effect of SLP
vaccination together with the combination carboplatin and paclitaxel, which is a standardof care clinical chemotherapy doublet. It was observed that abnormally high mononuclear

myeloid cell populations found in the blood and in tumors of mice were normalized tomuch lower levels by this chemotherapy doublet, again permitting a marked increase in the

response to SLP vaccination and showing synergy in tumor eradication. A new clinicalpilot study on the constitution of immune cells in the blood of late stage cervical cancer
patients also revealed increased numbers of mononuclear myeloid cells, associated withlow T-cell reactivity against common microbial recall antigens and a lower stimulatory

capacity of antigen presenting cells, indicating an immunosuppressed status. When thesepatients were subsequently treated with the standard carboplatin-paclitaxel chemotherapy

a normalized systemic immune profile similar to that found in healthy subjects wasobserved at a specific time point during this chemotherapy. Together these data indicatethat standard chemotherapy has a variety of immunostimulatory effects, including better
attraction of T cells into tumors and greater sensitivity of tumors to apoptosis by TNFa

produced by T cells as well as better expansion of T cells through depletion of myeloid

derived suppressor cells without suppression of T cell function or numbers. These

combined effects lie at the basis of synergy between SLP vaccination and chemotherapy inmouse tumor eradication. Therefore a pilot clinical trial was started in which 12 late stage
cervical cancer patients were treated with standard chemotherapy in combination with aCancer Vaccine

99

single dose of HPV16-SLP vaccination. Comprehensive immune monitoring confirmed thebeneficial effect of myeloid cell depletion associated with a robust induction of HPV16-

specific T-cell responses after the single dose of HPV16-SLP vaccination, that were

sustained throughout several cycles of chemotherapy. Currently, we have started amulticenter trial in which HPV16-SLP vaccination is combined with carboplatin-paclitaxel

and type I interferon to assess clinical and immunological outcome in a group of HPV16+

metastatic cervical cancer patients. The treatment scheme of this trial is shown in Table 1.

Each patient cohort consists of 6 patients with recurrent or metastatic cervical cancer. 4

dose levels of HPV16-SLP vaccine (termed ISA101) will be given and at each dose level

patients will receive pegylated interferon alpha 2b (Pegintron) or not. Interferon alpha 2b is

expected to promote the CD8 component in the response and to promote dendritic cell
activation for increase in both CD4 and CD8 responses.
Table 1

As of this writing (November 2014), 18 patients have been recruited into the study.
The results will be reported in due course.

References
1. Kenter GG, Welters MJ, Valentijn AR, Löwik MJ, Berends-van der Meer DM, Vloon AP,
Essahsah F, Fathers LM, Offringa R, Drijfhout JW, Wafelman AR, Oostendorp J, Fleuren
GJ, van der Burg SH, Melief CJ. Vaccination against HPV-16 oncoproteins for vulvar
intraepithelial neoplasia. N Engl J Med 2009;361:1838-1847.

100

C. J.M. Melief et al.

2. Welters MJ, Kenter GG, de Vos van Steenwijk PJ, Löwik MJ, Berends-van der Meer,
DM, Essahsah F, Stynenbosch, LF, Vloon AP, Ramwadhoebe, TH, Piersma SJ, van der
Hulst, JM, Valentijn RP, Fathers LM, Drijfhout JW, Franken KL, Oostendorp, J, Fleuren GJ,
Melief CJ, van der Burg, SH. Success or failure of vaccination for HPV16-positive vulvar
lesions correlates with kinetics and phenotype of induced T-cell responses. Proc Natl
Acad Sci USA 2010;107:11895-11899.
3. Kenter GG, Welters MJ, Valentijn AR, Lowik MJ, Berends-van der Meer DM, Vloon AP,
Drijfhout JW, Wafelman AR, Oostendorp J, Fleuren GJ, Offringa R, van der Burg SH,
Melief CJ. Phase I immunotherapeutic trial with long peptides spanning the E6 and E7
sequences of high-risk human papillomavirus 16 in end-stage cervical cancer patientsshows low toxicity and robust immunogenicity. Clin Cancer Res 2008;14:169-177.
4. van Poelgeest MI, Welters MJ, van Esch EM, Stynenbosch LF, Kerpershoek G, vanPersijn van Meerten EL, van den Hende, M, Löwik MJ, Berends–van der Meer, DM,
Fathers LM, Valentijn AR, Oostendorp J, Fleuren GJ, Melief CJ, Kenter GG, van der
Burg SH. HPV16 synthetic long peptide (HPV16-SLP) vaccination therapy of patients withadvanced or recurrent HPV16-induced gynecological carcinoma, a phase II trial. J
Transl Med 2013;11:8
5. van der Sluis TC, van Duikeren S, Huppelschoten S, Jordanova ES, Nejad EB, Sloots A,
Boon L, Smit VT, Welters MJ, Ossendorp F, van de Water B, Arens R, van der Burg SH,
Melief CJ. Vaccine-induced Tumor Necrosis Factor alpha producing T-cells synergize
with cisplatin to promote tumor cell death. Clin. Cancer Res. e-publication 2014

Cornelis J. M. Melief

1973-1974

1975

1985

1991

2011-

Postdoctoral Fellow, the New England Medical Centerand Dana Farber Cancer Center
Staff Member, Department of Cell-mediatedImmunology, the Netherlands Red Cross Blood
Transfusion Service
Head of the Dept. of Tumor Immunology at theNetherlands Cancer InstituteHead of the Dept.of Immunohematology and Blood
Transfusion at Leiden University Medical Center andTumor Immunology Group in the department.
Emeritus Professor, Departments of
Immunohematology and Blood Transfusion, Leiden
University Medical Center and Chief Scientific Officer(CSO) of ISA Pharmaceuticals

101

CANCER IMMUNOTHERAPY: THE NARROW ROAD
BETWEEN INEFFICACY AND TOXICITY

Thierry Boon, Christophe Lurquin and Christophe BourdeauxLudwig Institute for Cancer Research, Brussels Branch
Avenue Hippocrate 75, Bte B1.74.03, B. 1200 Brussels, Belgium(Thierry.Boon@bru.licr.org)

It is now established that most melanoma patients produce a spontaneous T cell

response against their tumor. In many patients this attempt at eliminating the tumor
evidently fails and a large number of anti-tumor T cells remain in an inactive state in thecirculation and within tumor deposits. Inside the tumors, this anergic state appears to be

reinforced by an immunosuppressive environment. For the vaccinated patients who show

tumor regression, we propose the following sequence of events: a very small number ofactive anti-vaccine T cells penetrates the tumor and attacks some tumor cells. As a result,these CTL are restimulated and produce cytokines which focally reverse the local

immunosuppression, creating instead an immunostimulatory environment. This
reawakens many of the inactive anti-tumor T cells that are already present in significant

numbers in situ. Naïve anti-tumor T cells may also be activated. The mobilization of allthese T cells, most of which are directed against tumor antigens other than the vaccine

antigen, provides the large numbers of effector cells required to reject the tumor.

Why is it that following therapeutic vaccination with tumor-specific antigens tumors

regress in a small minority of patients whereas no sign of regression is observed in mostpatients? A first possibility is that this may be due to differences in the quantity or qualityof T cells responding to the vaccine. However, the lack of improvement of clinical outcome
following highly immunogenic vaccination suggests that the strength of the response to thevaccine is not the limiting factor. A second possible difference between regressing andnon-regressing patients could be the degree of expression of the relevant antigens on thesurface of tumor cells. It is well established that tumors can lose the expression of some or

all of their HLA molecules. However, we derived melanoma cell lines from a large number

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T. Boon et al.

of non-responder patients, and almost all of them proved to be sensitive to anti-vaccine

CTL in vitro. We therefore suspect that loss of antigen expression accounts for only a small

fraction of the treatment failures. A third possible explanation for the individual

differences in clinical response is that patients vary in the degree of immunosuppression

present in their tumor. A highly immunosuppressive tumor environment could render theanti-vaccine T cells inactive soon after penetrating the tumor, preventing them frominitiating the cascade of events that leads to tumor rejection. The responder patients would

be those who have a lower degree of immunosuppression in their tumor.

The anergic state of tumor-infiltrating lymphocytes and the immunosuppressive tumor

environment are often considered to result from an active immunosuppressive mechanismof tumor cells acquired in the course of the selection for resistance to the immune response.

Agents such as IDO, TDO and TGF-b are produced in many tumors and unquestionably

contribute to inducing anergy of infiltrating lymphocytes. But it is also possible that when

the immune system makes a rather weak attempt to eliminate a tissue, as expected in theabsence of pathogen-associated molecular patterns binding to Toll-like, Nod and RIG

receptors, after a length of time it enters an inactive state. Examples of such "immune

fatigue" are observed in human transplantation: a small number of recipients can bewithdrawn from treatment with immunosuppressive agents without deleterious effects on

the grafted organ. This process, whereby an incomplete response evolves into an anergic

state could be common to all progressive metastatic tumors, whereas the presence and thenature of immunosuppressive factors secreted by tumor cells are likely to vary from one

tumor to another.

The common anergic state could involve a degree of

immunosuppression within the tumor resulting in potential paralysis of incoming T

lymphocytes.

If the degree of intratumor immunosuppression is the limiting factor for the success of

vaccination against tumors, the use of cytokines to alleviate this immunosuppression could

improve the outcome. We feel that local cytokine treatment of metastases should be tested

in the clinic as an adjunct to vaccination. This local treatment could enable the anti-vaccine
T cells to initiate the rejection process.

Moreover, the amount of agents used for local

treatment would be orders of magnitude below those used for systemic treatments, thereby

reducing the occurrence of adverse side-effects. We realize that this approach will lead totherapeutic progress only if the immune rejection response of the treated metastasis leads

to the production of anti-tumor T cells of such quantity and quality that they successfully
attack untreated metastases disseminated elsewhere in the body.

We carried out preclinical studies using the mouse HY system, which involves T cell

responses to antigens expressed in male and not in female tissues. This model is attractive

as it has several features shared with the immune response to human tumors. Whilst inNakahara Memorial Lecture103

inbred mouse strain C57BL/6 (H2b) females reject syngeneic male skin grafts, in CBA (H2k)

they do not, even though we have observed that a CTL response directed against HY

antigens does occur following grafting. Thus the process observed in CBA is similar to thatobserved in progressing human tumors: a failed attempt at rejection followed by thepersistence of ineffective memory T cells. We observed that local immunostimulatory

treatments involving certain combinations of cytokines and TLR ligands can reactivate

stalled T cell responses against male mouse skin grafts resulting in graft rejection. Local

injections of a mixture of low-dose IL-12 and interferon alpha proved effective but required

prolonged treatment. Similar results were obtained with a combination of Gardiquimod , a

TLR7 ligand , and IL-2.

The purpose of this report is to stimulate clinical cancer immunotherapy researchcombining vaccination with local and systemic immunostimulatory agents. We areconvinced that local immunostimulation should be supplemented with vaccination against

specific tumor antigens. The influx into the tumor of anti-tumor T cells, which have been

freshly produced by the vaccination and therefore have not yet been rendered anergic, may

prove to be a key condition for success. Considering the absence of toxicity observed with

anti-tumor vaccination and the extremely low dose of immunostimulatory agents applied

locally, we expect this combination treatment to produce very little or no toxicity. The
combinations of immunostimulatory agents described here could be used in a first step.

Additional agents could be put into play later according to the observations that will bemade. Agents that counteract immunosuppressive factors produced or elicited by cancer

cells may be particularly useful. A low systemic dosage of anti-PD1, anti-PDL1 or anti-

CTLA-4 might provide added efficacy without provoking autoimmune side effects.

Perhaps our cancer immunotherapy strategies should emulate the immune system byadministering a multiplicity of agents that complement each other.
Reference

1. Bourdeaux C, Lurquin C, Jacquemart I, Lethé B, Brasseur F, van Baren N, Baurain JF,
Dyson J, Van Snick J, Uyttenhove C, Boon T (2014) Local immunostimulation leading torejection of accepted male skin grafts by female mice as a model for cancer immunotherapy. Proc Natl Acad Sci USA 111(9) : 3502-3507.

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T. Boon et al.

Thierry Boon, Ph.D.

1970-1971

1971-1975
1975-1980
1980-2004

1978-20091980-

Research associate, Rockefeller University, New York,USA (Laboratory of Dr N. Zinder)
Research Fellow, Centre National de la Recherche
Scientifique, France (Laboratory of Dr F. Jacob, Institut
Pasteur, Paris)
Lecturer, Université catholique de Louvain, Belgium
Director, Institute of Cellular Pathology, Brussels,
Belgium
Director, Ludwig Institute for Cancer Research,
Brussels Branch, Belgium
Professor, Université catholique de Louvain, Belgium

Specialty and Present Interest:
Immunology, Immunotherapy, Melanoma, Cancer Vaccines

105

OPTIMIZING THERAPEUTIC CANCER VACCINES

Pedro J. Romero1, Rachel Perret1, Alena Donda1, Natalia Botelho1,
Stéphanie Corgnac1, Benjamin Tschumi1, Dietmar Zehn2and
Lianjun Zhang1

Ludwig Cancer Research Center, Faculty of Biology and Medicine
Swiss Vaccine Research Institute, Lausanne University Hospital, University of Lausanne
Chemin des Boveresses 155, 1066 Epalinges, Switzerland(pedro.romero@hospvd.ch)1

2

Therapeutic cancer vaccination aims at boosting naturally acquired as well as priming

de novo tumor antigen specific T cell responses that may control and/or eliminate tumors.

The molecular characterization of T cell-defined tumor antigens in the early 90s opened theway to design vaccines based on targeting one or more well defined tumor antigens.

Extensive preclinical and clinical testing of a large variety of vaccine formulations

addressed to many types of cancer has provided a wealth of information. Modern

vaccination consistently elicits tumor specific T cell responses in the majority of treatedcancer patients. Clinical benefit is achieved in only a proportion of those patients and thebest estimates indicate that one in four patients would experience disease stabilization,

mixed tumor responses or even objective tumor regression. Two or three cancer vaccines

have been approved for clinical use in three countries and there are at least 15 vaccines inpivotal clinical trials. However, some of them have recently failed their end points despite

the promising signals in the phase II clinical trials. There is therefore a need to improve
vaccines and optimize vaccination strategies so as to reach consistent clinical efficacy.

In our studies, we have identified several parameters for vaccine optimization. Peptide-based vaccines can be enhanced via the substitution of selected amino acid residues that

increase immunogenicity yet preserve antigenicity and tumor reactivity1,2). Long synthetic

peptides effectively target professional antigen presenting cells and may offer both class I

and class II restricted T cell epitopes. Subunit vaccines are strictly dependent on the co-administration of potent immunological adjuvants. TLR agonists are effective adjuvants

that signal dendritic cell activation and maturation. Both poly(I:C), a double stranded RNA

analog interacting with TLR-3, and CpG-oligodeoxynucleotides, ligands for TLR-9, are106

P. J. Romero et al.

potent in both pre-clinical models and in cancer patients3,4). Invariant NKT cells may also

provide strong immunological adjuvant activity5). Further, vaccine optimization efforts are

currently focused on identifying approaches to improve and sustain T cell function in thestrongly suppressive tumor microenvironment6), as well as in understanding the molecularmechanisms underlying the induction of long lived memory T cells that may provide

robust immunity over prolonged periods of time7).

Different adjuvants differ in their ability to induce a high CD8 T cell effector to
We used a double adoptive transfer of TCR transgenic T cells specific for class I andregulatory T cell ratio in response to peptide immunization:

class II restricted peptide antigens in which the latter was also knocked in for a eGFP-Foxp3

reporter gene. Tracking in this way tumor-specific CD8, CD4 and Treg responses to pepti invarios adjuvant formulations, we could show that vaccines containing TLR-9 CpG-ODN or

TLR-3 ligand poly(I:C) preferentially induce strong proliferation of antigen-specific effector

T cells, while minimizing expansion of antigen-specific Tregs. High Teff:Treg rations werelinked to strong Th1 cytokine production in the lymph nodes early after immunization andresulted in polyfunctional CTLs with enhanced tumor infiltration and protective function(Figure 1)3).

Therapeutic cancer vaccines based on synthetic peptides have provided encouraging

Targeting the cross-presenting dendritic cell subset improves polypeptide vaccination:clinical signals in both intravaginal neoplasia (HPV long synthetic peptide cocktail)8) andmetastatic renal cell carcinoma (synthetic naturally presented MHC-I restricted peptides)9).

However, the same cocktail of HPV antigenic long synthetic peptides vaccine failed tomodify tumor progression when used to vaccinate advanced cervical carcinoma patients.

Moreover, two phase III randomized placebo-controlled clinical trials of vaccination with a

Figure 1

OT-I Teff accumulation in TIL depends on the adjuvant used and correlates with tumor
protection.

Cancer Vaccine

107

MAGE-A3 recombinant fusion protein failed their primary efficacy endpoints in locallyadvanced local carcinoma and in metastatic melanoma. In these cases, no detectable tumor
protection was accompanied by modest induction of specific CD8 T cell responses. A
possible explanation for these outcomes is the limited cross-priming of T cell immunity bysoluble polypeptide antigen. It would be desirable to target these antigens to the uniquedendritic cell subset specialized in cross-presentation. This cell type is present in spleen

and in dermal dendritic cells at low frequencies, is dependent on the master transcription

factor Batf3 and is characterized by the expression of CD8aa homodimers, CD141,
CLEC9A and high levels of TLR3. The cross-presenting DCs are the majority of cells

outlined by this phenotype and can be identified by their expression of XCR1 chemokine

receptor (Figure 2). Its ligand, XCL1, is unique among the chemokine families and isabundantly produced by activated CD8 T cells. It has been suggested that targeting

antigens through this ligand-receptor pair may increase the cross-priming of specific CD8 T

cells to soluble protein immunogens10). We have used either the free XCL1 polypeptide or
Fc-XCL1 recombinant fusion proteins to assess the impact on the relative immunogenicity

of soluble recombinant protein or long synthetic peptide and TLR agonist as compared toimmunization with TLR agonist alone. The results indicate that immunization with XCL1

leads to increased frequencies of specific CD8 T cells, enhanced recall responses andFigure 2

Cross priming dendritic cells.
The human orthologues are BDCA3+ (CD141, thrombomodulin), CLEC9A+, XCR1+ and
TLR3+. Adapted from Merad et al, 2013 & Heath et al, 2013.

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P. J. Romero et al.

increased functionality. Importantly, preliminary results also suggest that therapeutic
vaccination including the XCL1 chemokine may lead to enhanced ability of the responding

CD8 T cells to control tumor growth in a model of OVA-expressing lymphoma (Figure 3).

It has been shown that feeding rapamycin to mice acutely infected with theUnderstanding molecular switches steering T cell differentiation:

lymphochoriomeningitis virus (LCMV) may increase the quantity and/or quality of LCMV

antigen-specific CD8 T cells11). This effect has been confirmed in additional models of

infection and of vaccination with recombinant viral vectors. Increasing the memory T cell

responses induced by vaccination with tumor antigens may contribute to long lived tumor

protection. Since rapamycin selectively inhibits mTORC1 in several cell types but also
mTORC2 in others, we have set out to dissect the role of mTORC1 and mTORC2 inregulating T cell differentiation using conditional deletion of Raptor and Rictor in T cells.

A mouse line in which Raptor, a regulatory component of the mTORC1 complex, wasdeleted in T cells had a profoundly reduced effector CD8 T cell response in response toinfection with LCMV. The few effector phenotype cells generated displayed severely

reduced cytokine secretion in response to antigenic peptide challenge and had reduced

content of perforin and granzymes. Moreover, the memory response was also nearly

absent in comparison to mTORC1 competent littermates (Figure 4). Thus, in contrast to theeffect of mTORC1 inhibition mediated by rapamycin, genetically mediated ablation of themTORC1 signaling led to near complete inhibition of the memory response as well as of theacute effector CD8 T cell response. Preliminary results with the mouse line with aconditional deletion of Rictor, a key regulatory component of the mTORC2 complex,

suggest a twofold reduction in the effector CD8 T cell response accompanied by anaccelerated induction of higher frequencies of memory CD8 T cells. The latter appeared
functionally competent and able to mediate enhanced recall responses to antigen ascompared to the Rictor wild type littermates. Experiments in progress explore themechanisms involved as well as the relationship between the effect of rapamycin on CD8 T
cell differentiation and that of genetic modulation of the two branches of mTORC

signaling. Moreover, we are also performing metabolic profiling of differentiating CD8 T
cells to understand the extent to which mTOR signaling mediated metabolic changes indifferentiating T cells determine their ultimate functional fates. We expect that insightsfrom these studies would inform the design of cancer vaccines able to induce optimized

CD8 T cell responses capable of mediating potent, long lived protective anti-tumor
immunity.

Cancer Vaccine

Figure 3

XCL1 enhances CD8 T cell induction by vaccination with long synthetic peptide.

Figure 4

Loss of specific CD8 T cell memory formation in the absence of Raptor.

109

References
1. Speiser, D. E. et al. Unmodified self antigen triggers human CD8 T cells with stronger
tumor reactivity than altered antigen. Proc Natl Acad Sci USA 105, 3849-3854 (2008)
2. Valmori, D. et al. Enhanced generation of specific tumor-reactive CTL in vitro by selected
Melan-A/MART-1 immunodominant peptide analogues. J Immunol 160, 1750-1758(1998)

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3. Perret, R. et al. Adjuvants that improve the ratio of antigen-specific effector to regulatory
T cells enhance tumor immunity. Cancer research 73, 6597-6608 (2013)
4. Speiser, D. E. et al. Rapid and strong human CD8+ T cell responses to vaccination withpeptide, IFA, and CpG oligodeoxynucleotide 7909. The Journal of clinical investigation
115, 739-746 (2005).
5. Corgnac, S. et al. CD1d-antibody fusion proteins target iNKT cells to the tumor and trigger
long-term therapeutic responses. Cancer Immunol Immunother 62, 747-760 (2013)
6. Sierro, S. R. et al. Combination of lentivector immunization and low-dose chemotherapy
or PD-1/PD-L1 blocking primes self-reactive T cells and induces anti-tumor immunity.
European journal of immunology 41, 2217-2228 (2011)
7. Speiser, D. E. & Romero, P. Molecularly defined vaccines for cancer immunotherapy, andprotective T cell immunity. Seminars in immunology 22, 144-154 (2010)
8. Kenter, G. G. et al. Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia. The New England journal of medicine 361, 1838-1847 (2009)
9. Walter, S. et al. Multipeptide immune response to cancer vaccine IMA901 after single-dose
cyclophosphamide associates with longer patient survival. Nature medicine 18, 12541261 (2012)
10. Kroczek, R. A. & Henn, V. The Role of XCR1 and its Ligand XCL1 in Antigen CrossPresentation by Murine and Human Dendritic Cells. Frontiers in immunology 3, 14 (2012)
11. Araki, K. et al. mTOR regulates memory CD8 T-cell differentiation. Nature 460, 108-112(2009)

Pedro J. Romero, M.D.

1985-1989

1989-2011
20032011-

2014-

Postdoctoral fellow, Dept of Medical and Molecular
Parasitology, New York University School of Medicine
Ludwig Institute for Cancer Research, Lausanne branchProfessor, Faculty of Biology and Medicine, Universityof Lausanne
Ludwig Research Cancer Center, University of
Lausanne
Director, Division of Fundamental Oncology,
Department of Oncology, University Hospital,
Lausanne

Specialty and Present Interest:
Immunology, Tumor Immunology and Immunotherapy of Cancer

111

CANCER IMMUNOTHERAPY USING NOVEL TUMORASSOCIATED ANTIGENIC PEPTIDES AND HUMANiPS CELL-DERIVED DENDRITIC CELLS

Yasuharu Nishimura

Department of Immunogenetics
Graduate School of Medical Sciences, Kumamoto University
1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan(mxnishim@kumamoto-u.ac.jp)

Y. Nakamura and his colleagues analyzed the gene expression profiles of various

cancerous and normal tissues using a genome-wide cDNA microarray covering more than

27,000 genes, and subsequently many novel oncofetal or cancer testis tumor-associatedantigens (TAAs) were identified1-6). These TAAs are overexpressed in human malignant

tumors including head and neck squamous cell cancer (HNSCC) , esophageal, lung,pancreatic, and urinary bladder cancers etc., but not in many adults normal tissues except

for testis, placenta or fetal organs. Furthermore, these TAAs are involved in promotion ofmalignant cell proliferation and the high expressions of TAAs were correlated with poor

prognosis of the patients.

Subsequently we have identified many these TAAs-derived short peptides (SPs)

recognized by cytotoxic T lymphocytes (CTLs) in the context of Japanese common HLA-A2

or -A24 by using HLA transgenic mice in vivo and human PBMCs in vitro (Figure 1)1~6).

Moreover we succeeded in propagation in vivo of TAAs-derived SPs-specific CTLs in the

majority of patients with HNSCC by immunization with these SPs without any serious

adverse effects in a phase II clinical trial with 37 HNSCC patients. Positive clinicalresponses including complete response were observed in a fraction of patients and thestrong correlation between magnitude of CTL responses induced in the vaccinated patientsand prolonged overall survival periods was also observed7). Now the phase II clinical trial,aiming at prevention of recurrence and metastasis of HNSCC has been started in patients
who have received curative operations.

It is well known that CD4+ T cells have important role in the immune system andpromote effective T-cell-mediated anti-tumor immunity. CD4+ helper T (Th) cells are

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Y. Nishimura

essential for sensitization of tumor-specific CD8+ T-cells to induce the expansion anddifferentiation of TAA-derived SPs-specific CTLs, and generation and maintenance of long-

lasting CTL responses8). Th cells also have a crucial role to pave the way for entry of CTLs

at the tumor microenvironments. Especially T-helper type 1 (Th1) cells have a crucial rolein elimination of tumor and IFN-g produced by Th1 cells exhibits anti-angiogenic effects.

Therefore, it is important to identify TAA-derived peptides that can activate not only CTLs

but also tumor-specific Th1 cells for induction of effective tumor immunity in tumorbearing hosts. Professional antigen presenting cells including dendritic cells (DCs) has acapacity to process extracellular antigens and present antigens-derived SPs in the context of

HLA class I molecules via so called cross-presentation pathway leading to the activation of

a TAA-derived SPs-specific CTL responses. Recent clinical study have shown thattargeting Th cells and CTLs using both HLA class I- and II-restricted epitopes enhanced

vaccine-specific immune responses and improved clinical responses9). As a result, a single

polypeptide encompassing epitopes for both Th1 cells and CTLs may be an ideal peptide

Figure 1

Identification of tumor-associated antigens (TAAs)-derived short peptides (SPs) carrying
cytotoxic T lymphocyte (CTL) epitopes using HLA-class I transgenic mice and their application to the clinical trials of cancer immunotherapy.
We have identified many TAAs-derived SPs recognized by cytotoxic T lymphocytes(CTLs) in the context of Japanese common HLA-A2 or -A24 by using HLA transgenic mice invivo and human PBMCs in vitro. We have succeeded in induction of SPs-specific CTL in manyhead and neck squamous cell cancer patients and a prolongation of survival period in a small
fraction of patients by vaccination with three CT antigens-derived SPs.

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vaccine for cancer immunotherapy.

Based on these information, we attempted to identify TAA-derived long peptides (LPs)that can induce both TAA-specific Th1 cells and CTLs in order to further improve TAA

peptides-based cancer immunotherapy. We focused on three cancer testis antigens, Cell
division cycle associated 1 (CDCA1), Kinesin family member 20A (KIF20A) andLymphocyte antigen 6 complex, locus K (LY6K). We have identified highly immunogenic

TAAs-derived LPs encompassing both Th1 cell- and CTL-epitopes. The 24-26 mer LPs

encompassed naturally processed Th cell epitopes which are presented by various frequent

HLA-class II molecules (Figure 2). Based on the findings, CDCA1-, KIF20A- and LY6K-

LPs are useful in at least 90% of the Japanese population10-12). These Th1-cell epitopes can
induce CTLs as well as Th1 cells in both human in vitro and HLA class I transgenic mice invivo by possible crosspresentation. Moreover we observed an augmentation of TAA-

specific Th1 cell responses in cancer patients immunized with TAA-derived CTL epitope

Figure 2

Development of effective cancer immunotherapy using TAA-derived long peptides carrying
both HLA-II restricted Th1 cell and CTL epitopes.
We attempted to identify TAA-derived long peptides (LPs) that can induce both TAAderived LPs-specific Th1 cells and TAA-derived SPs-specific CTLs in order to further
improve TAA peptides-based cancer immunotherapy. We have succeeded in identification ofhighly immunogenic TAAs-derived LPs encompassing both Th1 cell- and CTL-epitopes.

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Y. Nishimura

SPs vaccines.

We observed the synergistic effect of these 3 TAA-derived LPs on induction of antigen-specific and CD107a-positive CTLs. These findings indicate that the LPs may be able toaugment the induction of antigen-specific CTLs in combination with immunotherapy using

CTL-epitope SPs and improve clinical outcomes in cancer expressing TAAs. The
checkpoint blockade by specific antibodies to cytotoxic T lymphocyte antigen 4 (CTLA-4),

programmed cell death -1 (PD1), or it’s ligand PD-L1 etc. in combination with therapeutic

vaccines has been illustrated13). Thus, checkpoint blockade may be a good candidate for
combination therapy with TAAs-derived LPs vaccine.

We previously succeeded in generation of immune competent DCs from both mouseand human ES (ES-DC) and iPS cells (iPS-DC) 14~18). In mouse model, these ES-DC and iPSDC stimulated both TAA-specific CTLs and Th cells to inhibit tumor growth in vivo 14, 16).

Human iPS-DC also activated HLA-I-restricted CTLs reactive to TAA-derived SPs to killtumor cells in vitro. Moreover, we succeeded in generation of TAP-deficient human iPS-

DCs expressing an allogeneic HLA-A2 gene, and these iPS-DCs could induce HLA-A2-

Figure 3

Targeted disruption of TAP2 gene in human iPS cells to generate universally applicabledendritic cell source.
We have succeeded in generation of TAP-deficient human iPS-DCs expressing an allogeneic HLA-A2 gene, and these iPS-DCs could induce HLA-A2-restricted and the TAAderived SPs-specific CTLs in vitro. This technology may well provide universally applicabledendritic cells of which HLA class I molecules expressed on the cell surface are compatible to
the recipient's (patients') HLA-class I molecules and useful for iPS-DC-based cancer
immunotherapy.

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115

restricted and the TAA-derived SPs-specific CTLs in vitro19). We are planning to develop acancer immunotherapy by using these TAA-derived peptides and human iPS-DCs (Figure 3).

In conclusion, the CDCA1-, KIF20A- and LY6K-derived LPs provide a tool for

propagation of tumor-specific Th1 cells and CTLs. Our studies support a possible clinicalapplication of LPs-based cancer immunotherapy. We also propose a usefulness of HLAclass I compatible TAP-deficient human iPS cell-derived DC for the future cell-based cancer

immunotherapy.

References
1. Yoshitake, Y., Nakatsura, T., Monji, M., Senju, S., Matsuyoshi, H., Tsukamoto, H.,
Hosaka, S., Komori, H., Fukuma, D., Ikuta, Y., Katagiri, T., Furukawa, Y., Ito, H.,
Shinohara, M., Nakamura, Y., and 1. Nishimura, Y. Proliferation potential-related protein, an ideal esophageal cancer antigen for immnotherapy, identified using complementary DNA microarray analysis. Clin. Cancer Res. 2004;10: 6437-6448.
2. Nakatsura, T., Komori, H., Kubo, T., Yoshitake, Y., Senju, S., Katagiri, T., Furukawa, Y.,
Oagawa, M., Nakamura, Y., and Nishimura, Y. Mouse homologue of a novel humanoncofetal antigen, Glypican-3, evokes T cell-mediated tumor rejection without autoimmune reactions in mice. Clin. Cancer Res. 2004;10: 8630-8640.
3. Komori, H., Nakatsura, T., Senju, S., Ikuta, Y., Yokomine, K., Harao, M., Yoshitake, Y.,
Motomura, Y., Beppu, T., Matsui, M., Torigoe, T., Sato, N., Baba, H., and Nishimura, Y.
Identification of HLA-A2- or HLA-A24-restricted CTL epitopes possibly useful for
glypican-3-specific immunotherapy of hepatocellular carcinoma. Clin. Cancer Res.
2006;12: 2689-2697.
4. Imai, K., Hirata, S., Irie, A., Senju, S., Ikuta, Y., Yokomine, K., Harao, M., Inoue, M.,
Tsunoda, T., Nakatsuru, S., Nakagawa, H., Nakamura, Y., Baba, H., and Nishimura, Y.
Identification of a novel tumor-associated antigen, Cadherin 3/P-cadherin, as a possible
target for immunotherapy of pancreatic, gastric and colorectal cancers. Clin. Cancer
Res 2008;14: 6487-6495.
5. Harao, M., Hirata, S., Irie, A., Senju, S., Nakatsura, T., Komori, H., Ikuta, Y., Yokomine, K.,
Imai, K., Inoue, M., Harada, K., Mori, T., Tsunoda, T., Nakatsuru, S., Daigo, Y., Nomori,H., Nakamura, Y., Baba, H., and Nishimura, Y. HLA-A2-restricted CTL epitopes of anovel lung cancer-associated cancer testis antigen, cell division cycle associated 1, caninduce tumor-reactive CTL. Int. J. Cancer 2008;123: 2616-2625.
6. Imai, K., Hirata, S., Irie, A., Senju, S., Ikuta, Y., Yokomine, K., Harao, M., Inoue, M.,
Tomita, Y, Tsunoda, T., Nakagawa, H., Nakamura, Y., Baba, H., and Nishimura, Y.
Identification of HLA-A2-restricted CTL epitopes of a novel tumor-associated antigen,
KIF20A, overexpressed in pancreatic cancer. Brit. J. Cancer 2011;104: 300-307.

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7. Yoshitake , Y., Fukuma, D., Yuno, A. , Hirayama, M. , Nakayama, H. , Tanaka. T.,
Nagata, M. , Takamune, Y., Kawahara, K., Nakagawa, Y., Yoshida, R., Hirosue, A., Ogi,
H., Hiraki, A. , Jono, H., Hamada, A., Yoshida, K., Nishimura, Y., Nakamura, Y., andShinohara, M. Phase II clinical trial of multiple peptide vaccination for advanced headand neck cancer patients revealed induction of immune responses and improved OS. Clin.
Cancer Res. 2015;21: 312-321.
8. Dobrzanski MJ. Expanding roles for CD4 T cells and their subpopulations in tumor
immunity and therapy. Frontiers in oncology. 2013;3: 1-19.
9. Melief CJ, van der Burg SH. Immunotherapy of established (pre) malignant disease bysynthetic long peptide vaccines. Nat Rev Cancer 2008;8: 351–360.
10. Tomita, Y., Yuno, A., Tsukamoto, H., Senju, S., Kuroda, Y., Hirayama, M., Irie, A.,
Kawahara, K., Yatsuda, J., Hamada, A., Jono, H., Yoshida, K., Tsunoda, T., Kohrogi, H.,
Yoshitake, Y., Nakamura, Y., Shinohara, M. and Nishimura, Y. Identification of promiscuous KIF20A long peptides bearing both CD4+ and CD8+ T-cell epitopes: KIF20A-specificCD4+ T-cell immunity in patients with malignant tumor. Clin. Cancer Res. 2013;19:4508-4520.
11. Tomita,Y., Yuno, A., Tsukamoto, H., Senju, S., Yoshimura, S., Osawa,R., Kuroda,Y.,
Hirayama,M., Irie,A.,Hamada,A., Jono,H.,Yoshida,K., Tsunoda,T., Kohrogi,H.,
Yoshitake,Y., Nakamura,Y., Shinohara, M. and Nishimura, Y. Identification of CDCA1long peptides bearing both CD4+ and CD8+ T-cell epitopes: CDCA1-specific CD4+ T-cell
immunity in cancer patients. Int. J. Cancer 2014;134: 352–366.
12. Tomita, Y., Yuno, A., Tsukamoto, H., Senju, S., Kuroda, Y., Hirayama, M., Imamura, Y.,
Yatsuda, J., Sayem, M. A., Irie, A., Hamada A., Jono, H., Yoshida, K., Tsunoda, T,
Daigo, Y., Kohrogi, H., Yoshitake, Y., Nakamura,Y., Shinohara, M. and Nishimura, Y.
LY6K-specific CD4+ T-cell immunity in patients with malignant tumor: Identification of
LY6K long peptide encompassing both CD4+ and CD8+ T-cell epitopes.
OncoImmunology 2014;3: e28100-1-15.
13. Pardoll D. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev
Cancer 2012;12: 252–264.
14. Motomura, Y., Senju, S., Nakatsura, T., Matsuyoshi, H., Hirata, S., Monji, M., Komori, H.,
Fukuma, D., Baba, H., and Nishimura, Y. Embryonic stem cell-derived dendritic cells
expressing Glypican-3, a recently identified oncofetal antigen, induce protective immunity
against highly metastatic mouse melanoma, B16-F10. Cancer Res. 2006;66: 2414-2422.
15. Senju, S., Suemori, H., Zembutsu, H., Uemura, Y., Hirata, S., Fukuma, D., Matsuyoshi, H.,
Shimomura, M., Haruta M., Fukushima, S., Matsunaga, Y., Katagiri, T., Nakamura, Y.,
Furuya, M., Nakatsuji, N., and Nishimura, Y. Genetically manipulated human embryonic stem cell-derived dendritic cells with immune regulatory function. Stem cells
2007;25: 2720-2729.

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16. Matsunaga, Y., Fukuma, D., Hirata, H., Fukushima, S., Haruta, M., Ikeda, T., Negishi, I.,
23. Nishimura, Y. and Senju, S. Activation of antigen-specific cytotoxic T lymphocytes by
b2-microglobulin or TAP1 gene disruption and the introduction of recipient-matched
MHC class I gene in allogeneic embryonic stem cell-derived dendritic cells. J.Immunol.
2008;181: 6635-6643.
17. Senju, S., Haruta, M., Matsunaga, Y., Fukushima, S., Ikeda, T., Takahashi, K., Okita, K.,
Yamanaka, S., and Nishimura, Y. Characterization of dendritic cells and macrophages
generated by directed differentiation from mouse induced pluripotent stem cells. StemCells 2009;27: 1021-1031.
18. Senju, S., Haruta, M., Matsumura, K., Matsunaga, Y., Fukushima, S., Ikeda, T.,
Takamatsu, K., Irie, A., and Nishimura, Y. Generation of dendritic cells andmacrophages from human induced pluripotent stem cells aiming at cell therapy. Gene
Therapy 2011;18: 874-883.
19. Haruta, M., Tomita, Y., Yuno, A., Matsumura, K., Ikeda, T., Takamatsu, K., Haga, E.,
Koba, C., Nishimura, Y., and Senju, S. TAP-deficient human iPS cell-derived myeloid celllines as unlimited cell source for dendritic cell-like antigen presenting cells. Gene
Therapy 2013;20: 504-513.

Yasuharu Nishimura, M.D., Ph.D.

1970-1976
1978-1982

1982-1984

1984-1985

1985-1988

1986-1992

1992-2003

2003-2008
2008-

Kyushu University, Faculty of Medicine: B.M.
Kyushu Univ., Grad. Sch. Med. Sci. (Int. Med.): M.D.,
Ph.D.
Research Associate: Dept. of Human Genetics, Med.
Res. Inst., Tokyo Med. Dent. Univ., Tokyo
Research Associate: Dept. of Genetics, Med. Inst.
Bioregulation., Kyushu Univ.
Research Fellow supported by the NIH, Fogarty
International Fellowship: Div. of Pediat. Oncol., DanaFarber Cancer Inst. Boston MA, U.S.A.
Associate Professor: Dept. of Gentics, Med. Inst.
Bioregulation, Kyushu Univ.
Professor: Div. Immunogenetics, Dept. of Neuroscienceand Immunology, Kumamoto Univ. Grad. Sch. Med.
Sci., Kumamoto Univ., Kumamoto
Professor: Dept. Immunogenetics, Grad. Sch. Med. Sci.,
Kumamoto Univ.
Vice Dean, Grad. Sch. Med. Sci., Kumamoto Univ.

Specialty and Present Interests:
Tumor Immunology, Translational research of cancer immunotherapy using novel tumorassociated antigenic peptides and human iPS cell-derived dendritic cells and macrophages.

118

DENDRITIC CELL VACCINATION AGAINST CANCER:
FROM PRECLINICAL RESEARCH TO PHASE III TRIAL

Gerold Schuler

Department of Dermatology, University Hospital Erlangen
Internistisches Zentrum, Ulmenweg 18, 91054 Erlangen, Germany(gerold.schuler@uk-erlangen.de)Cancer-specific T cells are often present in patients suffering from so-called

immunogenic tumors with cutaneous melanoma being the best studied example. The
emergence of such T cells, likely induced by dendritic cells (DC) that have taken up dying

tumor cells and received maturation stimuli sufficient to let them switch from thetolerogenic to the immunogenic mode, cannot have a significant impact on the clinicalcourse at the metastatic stage simply because the T cells are made useless by various

immunosuppressive mechanisms in the tumor microenvironment. Checkpoint blockade
can unleash these T cells, and has yielded unequivocal clinical results even in initial trials

thus being primarily responsible for cancer immunotherapy being selected as the scientificbreakthrough of the year 2013 and for convincing even the most conservative
chemotherapy-oriented oncologists that T cell-mediated immunotherapy of cancer will

have a bright future. The question now is whether cancer vaccines will finally play a roleor not in melanoma or other tumors. Given the above scenario in retrospect it is not

surprising that vaccines even if appropriately immunogenic (i.e. inducing sufficientamounts not only of functionally competent CD4+ but also CD8+ tumor-specific T cells)
could produce clinical effects only in subsets of metastatic melanoma patients. It is now

also understandable, that vaccines that probably have not induced tumor-specific CD8+ T
cells or primarily resulted in CD4+ T cell responses were totally disappointing.

We like other academic groups continue to study cancer vaccines addressing thefollowing hypotheses: 1.) in tumors such as melanoma, which we are studying, it will be

rewarding to combine vaccination with checkpoint blockers (for enhanced efficacy and lessside effects, as already suggested by the results of a recent pilot trial performed by the K.

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Thieleman's group) or treat in an adjuvant or neoadjuvant (e.g. regressing tumors in

response to BRAF inhibitors) setting with minimal tumor load and lessimmunosuppression for less relapse and prolonged survival (as for example suggested byour own trial results and those of Carl Figdor's group); 2.) in non-immunogenic tumors

appropriate cancer vaccines might be critical in the metastatic and even more so in theadjuvant setting to induce tumor-specific immunity which (only) then can be pushed bycheckpoint blockade or other interventions; 3.) it will be critical to develop highly

immunogenic vaccine strategies, also capable of driving memory T cell differentiation, and4.) capable of inducing T cells specific for mutated passenger and driver mutations.

Seminal work by the T. Boon group has shown that although Cancer Testis (CT) antigens

can function as rejection antigen a second wave of endogenously induced ("endogenous

vaccination") T cells directed to tumor-specific mutations seems critical in regressing

individual metastases (1) and literature cited therein). This observation not only for the firsttime proved that regressions occur because of vaccination and not despite vaccination, butalso revealed the relevance of mutation-specific T cells at the effector arm. The importanceof such T cells has recently been supported by refined immunomonitoring data inmelanoma patients with regressions upon adoptive T cell transfer or ipilimumab treatment

by S. A. Rosenberg and T. Schumacher, respectively2,3) ; and 5.) the hypothesis that vaccineseven if clinically effective will reach clinical practice only if their production is feasible,

reproducible and cost-effective. An illustrative example for this logistical aspect is

Dendreon's Provenge TM vaccine, approved by the FDA in 2010 as first active cancer
immunotherapy ever. It is a DC-based vaccine which was simply too complicated to

manufacture requiring one apheresis for each vaccine shot so that it was also for this reason

commercially not viable finally now resulting in bankruptcy of the company.

We have been working for many years to optimize dendritic cell (DC) vaccination, i.e.

the adoptive transfer of antigen-laden DC as vaccine strategy. We use monocyte-derived

DC, whereby immature DC are generated by exposing blood monocytes to GM-CSF + IL-4

over 6 days followed by addition of an inflammatory cocktail (IL-1beta + IL-6 + TNF alpha+ PGE2) to reproducibly yield very homogenous populations of mature DC that can beloaded with antigen by adding exogenous peptides or by transfection of mRNA after rather

than before maturation using a patented electroporation approach 4) . Monocytes are
elutriated from aphereses directly into culture bags for DC generation, and this process is(already) semi-closed, highly reproducible and effective (yield 400-600 million mature DC),

cost-effective (altogether 4,000 Euro per vaccine shot production cost), and GMP

production has been approved already for phase III trials according to the rules of the EMA

every company would have to follow in a pivotal trial. In cooperation with Miltenyi Biotec

Inc. their Prodigy cell processing system is currently adapted to avoid also the last open

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G. Schuler

step, namely electroporation, to allow a semiautomated highly standardized DC vaccine
production. An automated closed system is also under development by Argos Therapeutics

Inc. The message is that production at least of monocyte-derived DC will no longer posean obstacle for large pivotal trials and commercialization, the only critical issue is to makethe DC vaccine effective.

Our monocyte-derived DC vaccines have been tested over the years in several trials,

and I have reported results of two so far unpublished ones, one using peptide-loaded first

generation and another one employing RNA-transfected second generation DC vaccines,
and encompassing CT as well as melanocyte differentiation antigens. These trials areillustrative in that advanced metastatic cutaneous melanoma patients were vaccinated andfollowed up for years before the modern checkpoint or BRAF inhibitor therapies became
available. The vaccines were uniformly immunogenic, inducing IFN gamma producing

CD4+ T cells (often strongly ex vivo detecable) and polyfuntional CD8+ T cells (rarely clearlyex vivo detectable indicating room for improvement) that lysed target cells in part with highaffinity. Of note was the fact that overall survival was definitely comparable to what has

been observed in non-resectable metastatic melanoma patients upon second line anti-CTL-

A4 (ipilimumab) therapy with 25% of stage IV (70% M1c) melanoma patients living 50

months after start of DC vaccination and formation of a “shoulder” of long-term survivors.

As expected based on what we know today there was no clear correlation between

detection of vaccine-induced T cells, even after we have recently concluded more extensive

immunomonitoring studies. Strikingly, there was, however, a statistically significant

correlation between emerging transient eosinophilia after the first series of vaccine shotsand later long-term survival (defined as beyond at least 2 years). The relevance andmechanism of the eosinophilia observed is unclear at present but it is noteworthy thateosinophilia is a well-known phenomenon after IL-2 application, is observed in the courseof GVHD, and appears to evolve also in ipilimumab-treated patients with prolonged

survival. The phenomenon possibly indicates successful in vivo effects of DC on thepatient's immune system (strong T cell activation, but perhaps also changes of the innate
immune system) with eosinophilia being just a simple biomarker readout. Within an EU

consortium we (Carl Figdor's and our DC vaccine group in cooperation with Duccio

Cavalieri as systems biologist) have observed that certain transcriptome changes occur in

PBMC after the first series of DC vaccines in patients who later show prolonged survival

supporting such an interpretation (manuscript in preparation). To enhance the stimulatory

activity of our DC we introduced a highly engineered CD40L mRNA (obtained from Argos

Therapeutics Inc.) into our cocktail-matured DC while Argos uses another maturation
method5). These CD40L-DC like the so-called Tri-Mix DC (generated by transfecting

immature DC with mRNA coding for constitutively active TLR-4, CD70 and non-Cancer Vaccine

121

engineered CD40L mRNA) displayed an enhanced T cell stimulatory capacity in vitro.

When we explored the DC transfected with RNA coding for defined antigens (with orwithout mRNA coding for CD40L or TriMix) in various cohorts we found that the

intravenous route, we had already tested in our first in man trial, appeared to produce
equivalent or better results than the intracutaneous route (as also found by Kris

Thieleman's group6)) so that this is now our preferred administration route. Of interest isalso that i.v. administration resulted in higher eosinophilia and constitutional symptoms(including elevated body temperature first observed about 8 hours after the second or third

but not the first DC vaccination), both preferably occurring in patients which later turn out

to survive longer.

We use such second generation DC transfected with PCR amplified total tumor mRNA

now in an ongoing phase III trial for adjuvant vaccination after resection or destruction of

so-called monosomy 3 uveal melanomas which in contrast to uveal melanomas displaying

both chromosomes 3 metastasize in the large majority of patients. The DC type we use has
already been proven capable of inducing in vivo de novo responses to mutated (but not wildtype) GNAQ using overlapping 9mer peptide libraries as readout.

In an effort to further improve our DC vaccine we have studied in more detail howhelper T cells help human DC to stimulate CD8+ T cells7). Helper T cells in seminal studies

have been shown to license DC for CD8+ T cell induction notably by expressing CD40L anddelivering a stimulus to CD40 expressing DC. Often this signal has been studied using

immature DC which were then induced to mature hereby switching DC into theirimmunogenic mode and “licensing” them to prime CD8+ T cells. We have now found thatmature DC albeit they prime well do not imprint a "memory type" differentiation programin CD8+ T cells unless helper T cells, CD8+ T cells and DC stay together forming a tricellular complex 7) . Only then antigen-specific CD8 + T cells are primed which upon

subsequent antigen-specific restimulation prove highly proliferate and functionallycompetent (enhanced lytic capability and cytokine production). If helper T cells are taken

away after the initial contact with and activation by the DC (so-called sequential model)

priming is similar but induced antigen-specific CD8+ T cells do not proliferate well upon

subsequent restimulation. The message here is that while maturation of DC is irreversible

the subsequent activation stimulus delivered via antigen-specifically stimulated helper T
cells to DC is transient. Importantly we found that our effort to imitate "T helped" DC bytransfecting highly engineered CD40L mRNA into cocktail-matured DC was just like TriMix DC not fully successful (see Figure 1) in that the activated state of the DC was transientlasting only a few hours. We, therefore, next explored expression of constitutively active
stabilized IKK8) to directly activate NF-B over a prolonged duration which resulted in athird generation of "Super Designer DC" capable of inducing highly proliferative CD8+ T

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G. Schuler

cells without need for antigen-specific help (see Figure 1). These DC are the ones we finallywill use in future trials. In preclinical studies we have shown that they can reliably prime

CD8+ T cells of high quality and proliferative potential including T cells recognizing

peptides derived from driver mutations such as NRAS. These DC which can soon be
produced using a semi-automated closed system thus represent a universal yet costeffective vaccine platform suitable for addressing the hypotheses and open questions

mentioned above.

Figure 1

Monocyte-derived DC were cytokine cocktail-matured and electroporated with MelanA
RNA (cmDC+ MelA) or were not loaded with antigen (no Ag). In addition, DC were electroporated with RNA encoding activators in addition to MelanA (activator+MelA). CD40L,
TriMix, and constitutively active IKK (caIKK) were used as activators. TriMix is a mix of constitutively active TLR4, CD40L, and CD70, which was electroporated into immature DC,
CD40L and caIKK were electroporated into cocktail-matured DC. Four hours (left handside) or 24 h (right hand side) after electroporation, these DC were used to stimulate autologous CD8+ T cells in a 1:10 ratio. One week after stimulation, part the T cells were re-stimulated with cryopreserved DC similar to the first stimulation. The percentage of MelanAspecific T cells was analyzed by MHC-tetramer staining after priming (1st stimulation) andtwo re-stimulations (2nd and 3rd stimulation). The average values of 4 (CD40L 4 h), 2(CD40L 24h), 7 (TriMix 4 h), 9 (Trimix 24 h), 5 (caIKK 4 h), and 3 (caIKK 24h) independent
experiments with SEM are shown. (part of the data was published in: Pfeiffer et al. Eur J
Immunol. 2014 Aug 6. doi: 10.1002/eji.201344417)

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References
1. Corbière V, Chapiro J, Stroobant V, Ma W, Lurquin C, Lethé B, van Baren N, Van den
Eynde BJ, Boon T, Coulie PG. Antigen spreading contributes to MAGE vaccinationinduced regression of melanoma metastases. Cancer Res. 2011; 71(4):1253-62.
2. Lu YC, Yao X, Li YF, El-Gamil M, Dudley ME, Yang JC, Almeida JR, Douek DC,
Samuels Y, Rosenberg SA, Robbins PF. Mutated PPP1R3B is recognized by T cells used totreat a melanoma patient who experienced a durable complete tumor regression. J
Immunol. 2013; 190(12):6034-42.
3. van Rooij N, van Buuren MM, Philips D, Velds A, Toebes M, Heemskerk B, van Dijk LJ,
Behjati S, Hilkmann H, El Atmioui D, Nieuwland M, Stratton MR, Kerkhoven RM,
Kesmir C, Haanen JB, Kvistborg P, Schumacher TN. Tumor exome analysis reveals
neoantigen-specific T-cell reactivity in an ipilimumab-responsive melanoma. J Clin
Oncol. 2013; 31(32): e439-42.
4. Schaft N, Dörrie J, Thumann P, Beck VE, Müller I, Schultz ES, Kämpgen E, Dieckmann D,
Schuler G. Generation of an optimized polyvalent monocyte-derived dendritic cell vaccineby transfecting defined RNAs after rather than before maturation. J Immunol. 2005;
174(5):3087-97.
5. Calderhead DM, DeBenedette MA, Ketteringham H, Gamble AH, Horvatinovich JM,
Tcherepanova IY, Nicolette CA, Healey DG. Cytokine maturation followed by CD40L
mRNA electroporation results in a clinically relevant dendritic cell product capable ofinducing a potent proinflammatory CTL response. J Immunother. 2008; 31(8):731-41.
6. Wilgenhof S, Van Nuffel AM, Benteyn D, Corthals J, Aerts C, Heirman C, Van Riet I,
Bonehill A, Thielemans K, Neyns B. A phase IB study on intravenous synthetic mRNAelectroporated dendritic cell immunotherapy in pretreated advanced melanoma
patients. Ann Oncol. 2013; 24(10):2686-93.
7. Hoyer S, Prommersberger S, Pfeiffer IA, Schuler-Thurner B, Schuler G, Dörrie J, Schaft N.
Concurrent interaction of DCs with CD4+ and CD8+ T cells improves secondary CTL
expansion: It takes three to tango. Eur J Immunol. 2014 Sep 11 [Epub ahead of print].
8. Pfeiffer IA, Hoyer S, Gerer KF, Voll RE, Knippertz I, Gückel E, Schuler G, Schaft N,
Dörrie J. Triggering of NF-kB in cytokine-matured human DCs generates superior DCs for
T-cell priming in cancer immunotherapy. Eur J Immunol. 2014; 44(11):3413-28.

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Gerold Schuler

1975-1977

1977-1983
1983-1985

1985-1995

1990-1991

1993-1995

1995-

Postdoctoral Fellow, Depts. of Histology andBiochemical Pharmacology, University of Innsbruck
Doctor-in-training at the Depts. of Dermatology,
Internal Medicine, and Plastic Surgery, University of
Innsbruck
Two year Fellowship (Max Kade Foundation) at theRockefeller University (c/o Prof. Dr. R. M. Steinman)
Senior Physician, Dept. of Dermatology and
Venereology, University of Innsbruck
6 Month sabbatical at the Rockefeller University (c/oProf. Dr. R. M. Steinman)
Vice-chairman and Associate Professor for
Experimental Dermatology, at the Dept. of
Dermatology, University of Innsbruck
Chairman, Dept. of Dermatology, University Hospital
ErlangenSpecialty and Present Interest:
Dendritic Cell Biology and Vaccination; Adoptive T Cell Therapy (Regulatory T Cells,
TCR-transfected T Cells)

125

IMMUNOTHERAPY IN ALLOGENEIC HEMATOPOIETICCELL TRANSPLANTATION

Robert S. Negrin

Division of Blood and Marrow Transplantation
Center for Clinical Sciences Research Building, Room 2205
269 W. Campus Drive, Stanford, CA 94305-5623, USA(negrs@stanford.edu)

Allogeneic hematopoietic cell transplantation (HCT) is a curative therapy for patientswith complex hematological malignancy and one of the first clinical settings demonstratingthat immune effects can eradicate cancer. In this setting donor derived T cells are criticalfor graft vs tumor (GVT) effects yet can also result in graft vs host disease (GVHD).

Developing clinical strategies to reduce GVHD while maintaining GVT are key toimproving outcomes. We have explored several different strategies including the timed

addition of CD4+CD25+FoxP3+ regulatory T cells (Treg) as well as ex vivo expanded cytokine
induced killer (CIK) cells initially in animal models with extension to the clinic.

We have utilized bioluminescence imaging (BLI) to evaluate the fate of adoptivelytransferred T cells and have developed luciferase (luc) expressing transgenic animals whichallow for the isolation of different cell populations that can be followed after theadministration of luciferin the substrate of luc. Utilizing these animal models we haveidentified that allogeneic T cells rapidly infiltrate nodal sites and begin to proliferate
resulting in the upregulation of key molecules that allow for entry into GVHD target

tissues such as the skin, gastrointestinal tract and liver. Once activated GVHD is difficult tocontrol especially once target tissue infiltration has occurred. A number of different

strategies have been explored including the use of Treg and CIK cells. Treg have potently

resulted in the control of GVHD mainly through reducing the proliferation of thealloreactive T cells1). Yet Treg do not appear to interfere with GVT responses in these animalmodels due to the activity of the alloreactive conventional CD4+ and CD8+ T (Tcon) cells thatcan recognize foreign cells, in this case, cancer cells. Limitation of cell proliferation which

controls GVHD does not appear to limit GVT effects, especially when adequate numbers of

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R. S. Negrin

Tcon are administered and the tumor burden is low2). Recently, a novel approach of using

the monoclonal antibody 4C12 which reacts with DR5, a TNF superfamily member has

resulted in dramatic expansion of donor Treg and reduction in GVHD risk when donors aretreated with a single injection of antibody. Further another population of donor derived

invariant natural killer T (iNKT) cells have potently suppressed GVHD in these murine
models3). Interestingly, the iNKT cells require donor Treg for optimal function.

The use of donor derived Treg have been translated to the clinic with excellent results

both by the groups from the University of Perugia and University of Minnesota. These
initial studies demonstrate that Treg can be isolated from donors and in the latter study

expanded ex vivo with apparent reductions in GVHD risk4,5). Interestingly, in an updatefrom the University of Perugia, low relapse rates have been observed following adoptive

transfer of Treg and Tcon6).

Another strategy to enhance the GVT effects of allogeneic transplantation involve theuse of cellular populations that have limited capacity for GVHD yet are capable orrecognizing and eliminating malignant cells. Cytokine induced killer (CIK) cells are an
expanded cell population prepared by the timed addition of interferon-g, CD3 stimulation

with monoclonal antibodies such as OKT3 and interleukin-2. Following culture for 14-21

days there is marked expansion primarily of T cells with co-expression of NK markers suchas CD56 and NKG2D. CIK cells have been studied in both murine and human models. In

murine models, CIK cells have been shown to have potent anti-tumor activity and in the

allogeneic setting, do not result in graft-versus-host disease (GVHD) 7 , 8 ) . Using

bioluminescent imaging CIK cells traffic to tumor sites primarily due to the inflammatory

nature of the tumor and chemokine gradient9,10). Once at the tumor site, the cells arecapable of recognizing tumor cells primarily through an NKG2D mediated mechanism,

although other cytotoxic mechanisms are operative. CIK cells have been utilized in avariety of different clinical settings both in solid tumors, as well as hematologic
malignancies. Our group has been particularly interested in the setting of allogeneic

hematopoietic cell transplantation and it is well recognized that graft-versus-tumor (GVT)

effects are critical. We have expanded CIK cells from both related and unrelated donors
and demonstrated that this is a feasible approach, responses occur and GVHD is notexacerbated11). We have developed a preparative regimen using total lymphoid irradiation

and anti-thymocyte globulin (TLI/ATG) which is safe and results in a low incidence of
GVHD, yet relapse remains a persistent problem12). We have explored the use of TLI/ATG

in a series of patients with myelodysplastic syndrome (MDS)13) using this as a model system

for assessing the overall benefit of allogeneic transplantation and the planned addition of
CIK cells. With TLI/ATG conditioning patients with MDS have an ~40% two year survivalwith ~50% risk of relapse with a low incidence of GVHD and transplant-related mortality

T Cell Therapy

127(TRM). Due to the high risk of relapse, we have added CIK cells in 25 patients at day+21

similarly selected. Up to 2x108 CIK cells/kg can be obtained and these cells do not result inexacerbation of GVHD or TRM. Relapse risk appears to be reduced, as well as

improvement in overall survival. Another approach actively under investigation is the useof CIK cells to deliver other anti-tumor biotherapies. We have focused on the use of CIK

cells and oncolytic viruses since oncolytic viruses have been shown to result in anti-tumor

effects, yet require injection directly into the tumor site. Upon infection with mutant strainsof vaccinia virus there is an eclipse period of 48-72 hours that is advantageous since this is

the period of time it takes for CIK cells to migrate to the tumor sites. Upon release of thevirus there is direct cytotoxicity to the tumor cells associated with further upregulation of

NKG2D ligand expression. This strategy has been effective in a variety of different murine

models and is being readied for clinical translation14). In sum, CIK cells could be useful todevelop alternative approaches to cell mediated anti-tumor responses.
References
1. Beilhack A, Schulz S, Baker J, et al: In vivo analyses of early events in acute graft-versushost disease reveal sequential infiltration of T-cell subsets. Blood 106:1113-22. Epub
2005 Apr 26., 2005
2. Edinger M, Hoffmann P, Ermann J, et al: CD4+ CD25+ regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation. Nat Med 9:1144-1150, 2003
3. Schneidawind D, Pierini A, Alvarez M, et al: CD4+ invariant natural killer T cells protectfrom murine GVHD lethality through expansion of donor CD4+CD25+FoxP3+ regulatory T
cells. Blood, 2014
4. Di Ianni M, Falzetti F, Carotti A, et al: Tregs prevent GVHD and promote immune
reconstitution in HLA-haploidentical transplantation. Blood 117:3921-8, 2011
5. Brunstein CG, Miller JS, Cao Q, et al: Infusion of ex vivo expanded T regulatory cells in
adults transplanted with umbilical cord blood: safety profile and detection kinetics.
Blood 117:1061-70, 2011
6. Martelli MF, Di Ianni M, Ruggeri L, et al: HLA-haploidentical transplantation with regulatory and conventional T-cell adoptive immunotherapy prevents acute leukemia
relapse. Blood 124:638-44, 2014
7. Schmidt-Wolf IG, Negrin RS, Kiem HP, et al: Use of a SCID mouse/human lymphoma
model to evaluate cytokine-induced killer cells with potent antitumor cell activity. J
Exp Med 174:139-49, 1991

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8. Baker J, Verneris MR, Ito M, et al: Expansion of cytolytic CD8+ natural killer T cells withlimited capacity for graft-versus-host disease induction due to interferon gamma production. Blood 97:2923-31., 2001
9. Edinger M, Cao YA, Verneris MR, et al: Revealing lymphoma growth and the efficacy of
immune cell therapies using in vivo bioluminescence imaging. Blood 101:640-8, 2003
10. Nishimura R, Baker J, Beilhack A, et al: In vivo trafficking and survival of cytokineinduced killer cells resulting in minimal GVHD with retention of antitumor activity.
Blood 112:2563-74, 2008
11. Laport GG, Sheehan K, Baker J, et al: Adoptive immunotherapy with cytokine-induced
killer cells for patients with relapsed hematologic malignancies after allogeneic
hematopoietic cell transplantation. Biol Blood Marrow Transplant 17:1679-87, 2011
12. Lowsky R, Takahashi T, Liu YP, et al: Protective conditioning for acute graft-versushost disease. N Engl J Med 353:1321-31, 2005
13. Benjamin J, Chhabra S, Kohrt HE, et al: Total lymphoid irradiation-antithymocyte globulin conditioning and allogeneic transplantation for patients with myelodysplastic syndromes and myeloproliferative neoplasms. Biol Blood Marrow Transplant 20:837-43,
2014
14. Thorne SH, Negrin RS, Contag CH: Synergistic antitumor effects of immune cell-viral biotherapy. Science 311:1780-4, 2006

Robert S. Negrin, M.D.

2000-2002
2006-2007

20092000-

President, International Society of Cellular Therapy
President, American Society of Blood and Marrow
Transplantation
Associate Editor, Blood
Chief, Division of Blood and Marrow Transplantation,
Stanford University

Specialty and Present Interest:
Clinical Hematopoietic Cell Transplantation, Cellular Immunology

129

CISH ATTENUATES PROXIMAL TCR SIGNALLING AND
ITS DELETION ENHANCES TUMOR DESTRUCTION

Douglas C. Palmer, Geoffrey C. Guittard, Lawrence E. Samelson andNicholas P. Restifo
National Cancer Institute
NCI/Surgery Branch, 9000 Rockville Pike Building, 10 Room, 3W-5762
Bethesda, MD 20892, USA(restifon@mail.nih.gov)

In his Nobel lecture in 1980, George Snell described how tumor transplantation

experiments led him to the discovery of MHC. Zinkernagel and Dougherty, who won theNobel Prize in 1996, continued to provide insight into the structures recognized by T cells.

These fundamental discoveries have laid the groundwork for the molecular definition oftumor rejection antigens, including those derived from mutated antigens, and this work
continues at the present time.

Although the target structures that are recognized by anti-tumor T cells are ofparamount importance, it has become clear that the mere recognition of antigen/MHC

structures is insufficient to destroy tumor cells. Molecular elements downstream of TCR

recognition can truncate T cell functions, although these elements remain incompletely

elucidated. One class of potentially critical negative regulators of immune cell function are

the Suppressors of Cytokine Signalling (SOCS), a family of eight proteins (SOCS 1-7, andthe founding member Cish) that share a central SH2 domain and a ‘SOCS box’ critical totheir functions as E3 ligases (see Figure 1).

We characterized a Cish-/- mouse and found it to be healthy and breed well. Cish-/-micewere more resistant to infectious challenge with vaccinia virus. Deletion of Cish resulted inbetter survival, viral clearance and increased numbers of CD8 + IFNg + T cells. Gene-

expression profiling revealed that Cish-deficient CD8+ T cells more robustly expressed

genes downstream of TCR-signalling indicating that Cish was likely acting very proximal

to the TCR signalling, to truncate the signalling event itself. Biochemical analysis of thesignalling machinery yielded the clear result that Cish was physically interacting withphospholipase Cg1 (PLCg1), resulting in its degradation. Activated PLCg1 converts

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phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and diacylglycerol,consequently potentiating calcium flux, Protein Kinase C activation and NFAT and NFkB

transcriptional activity, properties found to be enhanced in Cish-/- mice. We observed thatthe adoptive transfer of Cish-/- tumour-reactive CD8+ T cells resulted in increased T cell
expansion, pronounced and sustained tumour regression, and enhanced ex vivo target-

reactivity. Thus, Cish plays a significant role in CD8+ T cell immunity by attenuatingproximal TCR signalling and immunity to pathogens and cancer and that the deletion of

Cish unleashed PLCg1 activation and durability (Figure 2). This approach may be useful inthe augmentation of cancer regression.

Figure 1

CIS is the founding member of the SOCS family of evolutionarily conserved proteins.
Abbreviations – SOCS: Suppressors of Cytokine Signalling; KIR: Kinase inhibitory region;
ESS: Extended SH2 domain. Adapted from Palmer DC and Restifo NP reference 1.

Figure 2

SOCS molecules form E3 ligases, targeting proteins for degradation. Adapted from Palmer
DC and Restifo NP reference 1.

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References
1. Palmer DC, Restifo NP. Suppressors of Cytokine Signaling (SOCS) in T cell differentiation,
maturation and function. Trends Immunol. 2009; 30(12): 592-602.
2. Restifo NP, Dudley ME, Rosenberg SA. Adoptive immunotherapy for cancer: harnessing
the T cell response. Nat Rev Immunol. 2012 Mar 22;12(4):269-81.Overwijk WW, Theoret
MR, Finkelstein SE, Surman DR, de Jong LA, Vyth-Dreese F, Dellemijn TA, Antony PA,
Spiess PJ, Palmer DC, Heimann D, Klebanoff CA, Yu Z, Hwang L, Feigenbaum L,
Kruisbeek AM, Rosenberg SA, NP Restifo NP. Induction of tumor regression andautoimmunity by reversing the functionally tolerant state of self-reactive CD8+ T cells. J
Exp Med. 2003; 198 (4): 569-580.
3. Restifo NP.A "big data" view of the tumor "immunome". Immunity. 2013 Oct 17; 39(4):631-2.
4. Paulos CM, Wrzesinski C, Kaiser A, Hinrichs CS, Chieppa M, Cassard L, Palmer DC, BoniA, Muranski P, Yu Z, Gattinoni L, Antony PA, Rosenberg SA, Restifo NP. Microbial
translocation augments the function of adoptively transferred self/tumor-specific CD8+ T

cells via TLR4 signaling. J Clin Invest. 2007; 117(8):2197-204.
5. Klebanoff CA, Gattinoni L, Torabi-Parizi P, Kerstann K, Cardones AR, Finkelstein SE,
Palmer DC, Antony PA, Hwang ST, Rosenberg SA, Waldmann TA, Restifo NP. Central
memory self/tumor-reactive CD8+ T cells confer superior antitumor immunity compared with effector memory T cells. Proc Natl Acad Sci USA. 2005;102(27): 9571-6. Epub
2005 Jun 24.
6. Gattinoni L, Zhong XS, Palmer DC, Ji Y, Hinrichs CS, Yu Z, Wrzesinski C, Boni A,
Cassard L, Garvin LM, Paulos CM, Muranski P, Restifo NP. Wnt signaling arrests effector
T cell differentiation and generates CD8+ memory stem cells. Nat Med. 2009 Jul;15(7): 80813.
7. Restifo NP.Big bang theory of stem-like T cells confirmed. Blood. 2014 Jul 24;124(4):476-7.
8. Gattinoni L, Lugli E, Ji Y, Pos Z, Paulos CM, Quigley MF, Almeida JR, Gostick E, Yu Z,
Carpenito C, Wang E, Douek DC, Price DA, June CH, Marincola FM, Roederer M,
Restifo NP. A human memory T cell subset with stem cell-like properties. Nat Med.
2011 Sep 18;17(10):1290-7.
9. Restifo NP, Esquivel F, Kawakami Y, Yewdell JW, Mulé JJ, Rosenberg SA, Bennink JR.
Identification of human cancers deficient in antigen processing. J Exp Med. 1993 Feb 1;
177(2):265-72.
10. Restifo NP, Spiess PJ, Karp SE, Mulé JJ, Rosenberg SA. A nonimmunogenic sarcoma
transduced with the cDNA for interferon gamma elicits CD8+ T cells against the wild-type
tumor: correlation with antigen presentation capability. J Exp Med. 1992 Jun
1;175(6):1423-31.

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11. Restifo NP. Can antitumor immunity help to explain "oncogene addiction"? Cancer
Cell. 2010 Nov 16;18(5):403-5.
12. Restifo NP, Bacík I, Irvine KR, Yewdell JW, McCabe BJ, Anderson RW, Eisenlohr LC,
Rosenberg SA, Bennink JR. Antigen processing in vivo and the elicitation of primary
CTL responses. J Immunol. 1995; 154(9):4414-22.
13. Antony PA, Restifo NP. CD4+CD25+ T regulatory cells, immunotherapy of cancer, andinterleukin-2. J Immunother. 2005; 28(2):120-8.
14. Antony PA, Piccirillo CA, Akpinarli A, Finkelstein SE, Speiss PJ, Surman DR, Palmer DC,
Chan CC, Klebanoff CA, Overwijk WW, Rosenberg SA, Restifo NP. CD8+ T cell immunity against a tumor/self-antigen is augmented by CD4+ T helper cells and hindered by naturally occurring T regulatory cells. J Immunol. 2005; 174(5):2591-601.
15. Restifo NP, Marincola FM, Kawakami Y, Taubenberger J, Yannelli JR, Rosenberg SA.
Loss of functional beta 2-microglobulin in metastatic melanomas from five patients
receiving immunotherapy.J Natl Cancer Inst. 1996;88(2):100-8.
16. Restifo NP, Rosenberg SA.Use of standard criteria for assessment of cancer vaccines.
Lancet Oncol. 2005;6(1):3-4.
17. Rosenberg SA, Restifo NP, Yang JC, Morgan RA, Dudley ME. Adoptive cell transfer: aclinical path to effective cancer immunotherapy. Nat Rev Cancer. 2008;8(4):299-308.
18. Rosenberg SA, Yang JC, Restifo NP. Cancer immunotherapy: moving beyond current vaccines. Nat Med. 2004;10(9):909-15.
19. Hinrichs CS, Kaiser A, Paulos CM, Cassard L, Sanchez-Perez L, Heemskerk B,
Wrzesinski C, Borman ZA, Muranski P, Restifo NP. Type 17 CD8+ T cells display

enhanced antitumor immunity.Blood. 2009;114(3):596-9
20. Muranski P, Borman ZA, Kerkar SP, Klebanoff CA, Ji Y, Sanchez-Perez L, Sukumar M,
Reger RN, Yu Z, Kern SJ, Roychoudhuri R, Ferreyra GA, Shen W, Durum SK,
Feigenbaum L, Palmer DC, Antony PA, Chan CC, Laurence A, Danner RL, Gattinoni L,
Restifo NP. Th17 cells are long lived and retain a stem cell-like molecular signature.
Immunity. 2011;35(6):972-85.
21. Roychoudhuri R, Hirahara K, Mousavi K, Clever D, Klebanoff CA, Bonelli M, Sciumè G,
Zare H, Vahedi G, Dema B, Yu Z, Liu H, Takahashi H, Rao M, Muranski P, Crompton JG,
Punkosdy G, Bedognetti D, Wang E, Hoffmann V, Rivera J, Marincola FM, Nakamura A,
Sartorelli V, Kanno Y, Gattinoni L, Muto A, Igarashi K, O'Shea JJ, Restifo NP. BACH2
represses effector programs to stabilize T(reg)-mediated immune homeostasis. Nature.
2013;498(7455):506-10.
22. Khong HT, Restifo NP. Natural selection of tumor variants in the generation of "tumor
escape" phenotypes. Nat Immunol. 2002 Nov;3(11):999-1005

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Nicholas P. Restifo, M.D.

1987
1989

1992
1997-

New York University (MD degree)
Post-doctoral fellowship Memorial Sloan-Kettering
Cancer Center, NY
Senior Investigator, National Cancer Institute (NCI)
Principal Investigator, NCI

Specialty and Present Interest:
Immunotherapy; Immunoablation; Adoptive Cell Therapy; Novel Mouse Tumor Models;
T Cell Development; T Memory Stem Cells

134

ENGINEERING T CELL RESPONSES TO EFFECTIVELYTARGET TUMORS: INSIGHTS FROM PRECLINICAL MODELSAND CLINICAL RESULTS

Philip D. Greenberg, Sebastian Ochsenreither, Tom Schmitt,Andrea Schietinger, Ingunn Stromnes, Sunil Hingorani, Shannon Oda,
Rachel Perret, Kristin Anderson, Merav Bar and Aude Chapuis
University of Washington and Fred Hutchinson Cancer Research Center
1100 Fairview Ave North, Mail Stop D3-100, Seattle, WA 98109-1024, USA(pgreenberg@fhcrc.org)

Effective cellular therapy for human malignancies requires first identifying andvalidating an appropriate antigenic target, and then establishing in each patient a tumorreactive T cell response of high avidity and high magnitude that is safe and can infiltrate

and retain function in the tumor microenvironment. We have been exploring in preclinicalmodels and clinical trials methods to reproducibly provide such responses by transfer ofgenetically engineered T cells. We examined purified human leukemic stem cells for overexpression of genes in comparison to purified human hematopoietic stem cells as well as

normal somatic tissues. Our analysis revealed that WT1, a gene known to be associatedwith promoting leukemic transformation, and demonstrated to be an immunogenic target

in human cancers by the Sugiyama lab, is expressed in comparative abundance in humanleukemic stem cells1). Preclinical studies were then performed in a mouse model, andrevealed that CD8 T cells specific for this oncogene with even higher avidity than can bedetected in normal repertoires could be safely administered, with no evidence of toxicity to
the normal tissues known to express low but detectable levels of WT12). These studies led

to performing our initial clinical trial in patients with leukemia who relapsed after

hematopoietic cell transplant (HCT), a group of patients with a very poor prognosis. For
this trial, we transferred WT1-specific CD8 T cells clones isolated and expanded in vitro

from the HCT donor, and demonstrated that such T cells were safe, mediated in vivo anti-

leukemic activity, and were associated with maintenance of long-term remissions in somepatients3). However, generating sufficient numbers of WT1-specific CD8 T cells with highavidity for the target in each patient represented a substantive problem. To overcome thisobstacle and create a standardized reagent for treatment of patients whose tumor expresses

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the target antigen and shares an MHC restricting allele, we pursued methods to geneticallyengineer patient T cells to acquire high avidity for the tumor target by introducing andachieving high level expression of the genes encoding the V and V genes of a TCR
demonstrated to have high affinity for the target epitope (Figure 1). This required

developing strategies to overcome the potential problem of mispairing of the introduced

TCR chains with the endogenous TCR chains4,5), and incorporating changes in the geneconstruct such as codon optimization to enhance expression. We then screened a large
number of normal repertoires for the presence of high avidity WT1-specific CD8 T cells,

and selected the T cell clone expressing the highest affinity TCR. We have now have now

initiated a trial in which this high affinity, WT1-specific, A2-restricted TCR is being

introduced into patient CD8 T cells with a lentiviral vector and the transduced cells arebeing infused into the patient. The early results from this trial appear promising in termsof both evidence of antileukemic activity and the capacity for the transferred cells to persist

in the patients, and we plan to begin very shortly another trial in patients with non-small

cell lung cancer (NSCLC) utilizing this same TCR, as WT1 is also commonly overexpressed

in NSCLC.

However, isolating high affinity TCRs for many tumor antigens that are also normal

self-antigens may not be readily achieved from normal repertoires. Therefore, we havedeveloped strategies to enhance the affinity of isolated TCRs with retention of specificity,

including both saturation mutagenesis of CDR3 regions2) and an in vitro thymic selection

Figure 1

Impart Target Specificity by Introduction of High Affinity TCR Genes

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P. D. Greenberg et al.

system that allows for capture of a more diverse set of high affinity specific TCR genes

during TCR gene rearrangement. The strategies being pursued are designed to minimize

the risk of off-target toxicity from promiscuous epitope recognition, and we are further

using bioinformatics as well as modeling in the mouse to decrease the potential for suchoff-target toxicity.

Unfortunately, providing a high avidity T cell response does not necessarily result intumor eradication, as there are other substantive obstacles that can preclude even a T cell

expressing a high affinity TCR from being effective. These impediments include thedevelopment of T cell dysfunction by cell intrinsic and extrinsic pathways6), particularly
within the tumor microenvironment7), and we are using genetically engineered mousemodels to elucidate the cellular and molecular pathways8) that need to be modulated toachieve meaningful therapeutic benefit in a variety of solid tumor settings, including

pancreatic 7) and ovarian cancer. Our preclinical therapy studies, particularly in thepancreatic cancer model, already appear very promising, and we plan to use the insights

derived from these studies to initiate within the next 1-2 years clinical trials in humanpancreatic and ovarian cancers. The genetically-engineered mouse models we are using
are making it possible to assess T cell therapy targeting a tumor antigen that is newly

expressed in a solid tumor that develops “spontaneously” as a consequence of regulated

tissue-specific expression of an oncogene and for tumor antigens also detected at low levels

in normal tissues. These studies are revealing the importance of not only cell extrinsic

mechanisms of regulation and dysfunction that render T cells unresponsive, particularly

via inhibitory pathways commonly operative within the tumor microenvironment thatinterfere with an effector response such as the accumulation of regulatory CD4 T cells (Treg),
myeloid derived suppressor cells (MDSC), and tumor-associated macrophages (TAM), butalso the cell intrinsic mechanisms that ultimately can epigenetically dictate maintenance ofa tolerant or non-responsive state. These cumulative mechanisms highlight the difficulties

eliciting and/or sustaining responses to tumor antigens. Therefore, we are using themodels to evaluate strategies to sustain function and anti-tumor activity by modulating,selecting, or genetically modifying T cells to be resistant to obstacles that prevent tumor

eradication or re-establish a tolerant state. As different tumor types exhibit uniquecharacteristics and are capable of engaging distinct pathways, improved understanding of
the immunobiology of the particular tumor to be treated will likely prove essential for

designing effective therapies. However, the relatively straightforward means to use

synthetic biology to genetically engineer T cells to acquire novel capacities to overcome

inhibitory signals and function in the tumor microenvironment suggests that cancer

therapy with engineered T cells will likely find an increasing role in the treatment of
human cancers.

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137

References
1. Ochsenreither S, Majeti R, Schmitt T, Stirewalt D, Keilholz U, Loeb KR, Wood B, Choi YE,
Bleakley M, Warren EH, Hudecek M, Akatsuka Y, Weissman IL, Greenberg P. Cyclin-A1
represents a new immunogenic targetable antigen expressed in acute myeloid leukemia
stem cells with characteristics of a cancer-testis antigen. Blood. 2012; 119:5492-501
2. Schmitt TM, Aggen DH, Stromnes IM, Dossett,ML, Richman SA, Kranz DM, Greenberg
PD. Enhanced-affinity murine TCRs for tumor/self-antigens can be safe in gene therapy
despite surpassing the threshold for thymic selection. Blood. 2013; 122:348-56
3. Chapuis AG, Ragnarsson GB, Nguyen HN, Chaney CN, Pufnock JS, Schmitt TM,
Duerkopp N, Roberts IM, Pogosov GL, Ho WY, Ochenreither S, Wölfl M, Bar M, Radich
JP, Yee C, Greenberg PD. Transferred WT1-reactive CD8+ T cells can mediate
4.
5.

6.

7.
8.

antileukemic activity and persist in post-transplant patients. Sci Transl Med. 2013;
5:174ra27
Kuball J, Dossett ML, Wolfl M, Ho WY, Voss R-H, Fowler C, Greenberg PD.
Facilitating matched pairing expression of TCR-chains introduced into human T-cells.
Blood, 2007; 109:2331-8
Provasi E, Genovese P, Lombardo A, Magnani Z, Liu PQ, Reik A, Chu V, Paschon DE,
Zhang L, Kuball J, Camisa B, Bondanza A, Casorati G, Ponzoni M, Ciceri F, Bordignon C,
Greenberg PD, Holmes MC, Gregory PD, Naldini L, Bonini C. Editing T cell specificity
towards leukemia by zinc finger nucleases and lentiviral gene transfer. Nat Med. 2012;
18:807-15
Schietinger A, Greenberg PD. Tolerance and exhaustion: defining mechanisms of T cell
dysfunction. Trends Immunol. 2013; 35:51-60
Stromnes IM, Brockenbrough JS, Izeradjene K, Carlson MA, Cuevas C, Simmons RM,
Greenberg PD, Hingorani SR. Targeted depletion of an MDSC subset unmasks pancreatic ductal adenocarcinoma to adaptive immunity. Gut. 2014; 63:1769-81
Schietinger A, Delrow JJ, Basom RS, Blattman JN, Greenberg PD. Rescued tolerant CD8 T
cells are preprogrammed to reestablish the tolerant state. Science. 2012; 335:723-7

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P. D. Greenberg et al.

Philip D. Greenberg, M.D.

1978-1982

1982-1988
1988-

1991-

Assistant Professor of Medicine, Departments ofMedicine/Oncology and of Immunology at the
University of Washington (UW), and Assistant Member
at the Fred Hutchinson Cancer Research Center(FHCRC)
Associate Professor of Medicine/Oncology and of
Immunology at the UW, and Associate Member at theFHCRCProfessor of Medicine/Oncology and of Immunology at
the UW, and Member at the FHCRC
Head, Program in Immunology, at the FHCRC

Specialty and Present Interest:
Cellular and Molecular Immunology, Tumor Immunobiology, Adoptive T Cell Therapy,
Genetic Engineering of T Cells

139

ADOPTIVE IMMUNOTHERAPY WITHTCR GENE-TRANSDUCED LYMPHOCYTESHiroshi Shiku

Department of Immuno-GeneTherapy
Mie University Graduate School of Medicine
2-174 Edobashi, Tsu, Mie 514-8507, Japan(shiku@clin.medic.mie-u.ac.jp)

Engineering the antigen receptor gene in patients’ lymphocytes is one promisingstrategy to create antigen-specific lymphocytes without senescent phenotypes. The

strategy provides an opportunity to extend the application of adoptive T cell therapy forpatients with various types of cancer (Figure 1). However, this concept has not been welltested in patients with epithelial cancer or hematological malignancy.

Figure 1

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H. Shiku

We completed a phase I clinical trial of TCR gene therapy targeting MAGE-A4 to treat

esophageal cancer patients without lympho-depleting pre-conditioning (Figure 2). MAGE-

A4-specific TCR a and b chains were isolated from a human T cell clone that recognizes

MAGE-A4 143-151 peptide in an HLA-A*24:02 restricted manner1). This T cell clone did notshow any cross reactivity to the peptides with a homology to the MAGE-A4 epitope. A

retroviral vector that encodes these TCR chains without any artificial modification wasconstructed2,3). The trial was designed as a cell-dose escalation consisting of three cohorts,

2x108, 1x109 and 5x109 cells/patient. The treatment was tolerable with no adverse events
associated with transferred cells. In all patients, the transferred lymphocytes were detectedin their peripheral blood in a dose-dependent manner during the first 14 days. The infused

cells persisted more than 5 months after the transfer and sustained the reactivity to theantigen-expressing tumor cells. Three patients showed SD or long tumor free status. These

results suggest that this approach may extend the availability of adoptive T cell therapy for

epithelial cancer patients by providing tumor-reactive and long surviving lymphocytes
reducing the risk of intensive pre-treatments.

We have developed a unique retroviral vector that encodes tumor-specific TCR andsiRNAs that specifically down-regulate endogenous TCR in lymphocytes (Figure 3). This

vector could enhance the expression of the transduced TCR and minimize the risk to create
mis-paired TCR4,5).

Utilizing this novel “siTCR vector”, we have started a phase I trial of WT1-targeted TCR

gene therapy for patients with leukemia/MDS6), and also of MAGE-A4 target TCR genetherapy for patients preconditioned with cyclophosphamide and fludarabine.

Development of TCR-gene therapy with allogeneic T cells utilizing siTCR vector
An alternative use of this novel siTCR vector is its application for the use of allogeneic

silencing endogenous TCR expression

lymphocytes for infusion. Down-regulation of expression of endogenous TCR genes may

facilitate to avoid GVHD (graft versus host disease) when T cells transduced with tumor

specific TCR with this vector are infused to allogeneic recipients. We explored thepossibility by analyzing whether siTCR gene transduction lead to reduction of endogenous

TCR expression. In fact, thus treated lymphocytes showed apparent reduction of

expression of endogenous TCR with dominant reduction of proliferation when stimulatedby allogeneic lymphocytes in mixed lymphocytes culture test. When these receptor genemodified human lymphocytes were infused into immune deficient NOG mice, reduced or

essentially no GVHD against xenogeneic hosts of NOG mice was observed while infusionof control lymphocytes of the same donor without siTCR gene transduction resulted insevere GVHD. The use of siTCR transduced allogeneic donor derived lymphocytes inT Cell Therapy

Figure 2

Figure 3

141

142

H. Shiku

combination with hematopoietic stem cell transplantation is a reasonable next step.
CAR (Chimeric antigen receptor)gene-transduced T cells specific for WT-1 derived
Another major approach of antigen receptor gene modified lymphocytes is CAR gene

peptides with MHC

transduced lymphocytes. For this, strict tumor specificity of targeted antigen molecules isessential. Since there is no readily available cell surface antigens recognized by antibodies

with strict sense, we focused our effort for raising antibodies against peptide-MHC

complexes expressed on tumor cells. A number of intracellular tumor specific molecules

are known to be expressed on cell surface as peptide-MHC complexes (Figure 4). Screening

with phage libraries of human immunoglobulin resulted in obtaining antibodies reactivewith a complex of WT-1 derived peptide with HLA-A2402. The scFv of thus obtained

antibodies allowed us to construct retrovirus vectors expressing CAR in combination withsignal transduction element of T cells. CAR expressing lymphocytes recognize T2A24 cells

pulsed with cognate WT-1 derive peptides in the context of HLA-A2402. They also reactwith a number of cell lines co-expressing WT-1 derived peptides and HLA-A2402

indicating their recognition of the complexes derived from endogenous WT-1 molecules. A

manner of WT-1 derived peptide recognition by these CAR T cells was analyzed incomparison with readily available WT-1 TCR gene transduced lymphocytes recognizing

Figure 4

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143

the same peptides. CAR expressing T lymphocytes showed apparently different peptide

recognition pattern from TCR gene transduced T cells. However, both of them still

demonstrated clear WT-1 derived peptide specificity, showing no reactivity with any otherpeptides of sequence similarity in the range of our study. These results may indicate apossible use of CAR T cells for peptide MHC complexes may provide an alternative
approach for adoptive cell therapy.

References
1. Miyahara Y., Naota H., Wang L, et al. Determination of cellularly processed HLAA2402-restricted novel CTL epitopes derived from two cancer germ line genes, MAGE-A4
and SAGE. Clin. Cancer Res. 11:5581-9, 2005.
2. Hiasa A., Hirayama M., Nishikawa H., et al. Long-term phenotypic, functional andgenetic stability of cancer-specific T cell receptor (TCR) ab genes transduced to CD8+ T
3.

4.
5.
6.

cells. Gene Ther. 15:695-9, 2008.
Hiasa A., Nishikawa H., Hirayama M., et al. Rapid alphabeta TCR-mediated responses ingammadelta T cells transduced with cancer-specific TCR genes. Gene Ther. 16:620-8,
2009.
Okamoto S., Mineno J., Ikeda H., et al. Improved expression and reactivity of transduced tumor-specific TCRs in human lymphocytes by specific silencing of endogenous
TCR. Cancer Res. 69:9003-11, 2009.
Okamoto S, Amaishi Y, Goto Y, et al. A promising vector for TCR gene therapy:
Differential effect of siRNA, 2A peptide, and disulfide bond on the introduced TCR
expression. Mol. Ther. Nucleic Acids. 1:10, 2012.
Ochi T., Fujiwara H., Okamoto S., et al. Novel adoptive T-cell immunotherapy using a
WT1-specific TCR vector encoding silencers for endogenous TCRs shows marked
antileukemia reactivity and safety. Blood. 118:1495-503, 2011

144

H. Shiku

Hiroshi Shiku, M.D.

1972-1979

1984-1995

1994-2006

2006-

Research Fellow, Research Associate, Associate, SloanKettering Institute for Cancer Research, New York
Professor, Department of Immunology and Oncology,
Nagasaki University School of Medicine
Professor and Chairman, The Second Department ofInternal Medicine, Mie University School of Medicine
Professor, Department of Immuno-Gene Therapy, Mie
University Graduate School of Medicine

Specialty and Present Interest:
Cancer Immunotherapy: Adoptive cell therapy with gene-engineered T cells. Cancer vaccine with nanoparticle (CHP) and designed long peptides. Immuno suppression inhibitors.

145

DESIGNING CAR T CELLS FOR CANCER THERAPYCarl H. June

Department of Pathology and Laboratory Medicine andAbramson Cancer Center
Perelman School of Medicine
University of Pennsylvania
Smilow Center for Translational Research
3400 Civic Center Boulevard, Building 421
Philadelphia, PA 19104-5156, USA(cjune@exchange.upenn.edu)
It is now realized that T cell immune surveillance of cancer occurs in many cases. One

approach that has now been developed to deal with tolerance to tumors has beencheckpoint blockade, which can enable the natural immune response to respond to

antigens encountered in the tumor microenvironment. In this seminar I will outline recent

results in the area of synthetic biology, which uses engineered T cells to overcome immune

tolerance. There are three types of T cell therapy that are being actively tested in the clinic1).

In one case tumor infiltrating lymphocytes are obtained from surgical biopsies. This

therapy is most advanced in melanoma, and trials for patients with metastatic melanoma

are currently underway with phase 3 trials in Europe. Gene transfer technologies can also

be used to produce engineered T cell therapies. One approach is a chimeric antigen

receptor or CAR T cell approach. Another is the use of engineered T cells that express T cell

receptors of known specificity and affinity.

There are number of considerations for successful T cell therapy with adoptivelytransferred T cells. It is necessary to have an adequate number of T cells in order toeradicate large numbers of tumor cells. If a kilogram of tumor represents approximately
1x1012 T cell tumor cells, then it is likely that a similar number of T cells will be required foran equivalent effector to target ratio in vivo. The failure to achieve a critical mass of T cells
numerically may explain many previous trials with disappointing results following

therapeutic vaccines. There are two potential solutions to overcome inadequate effector T
cell function in cancer patients with adopted cell transfer therapy.

On the one hand it ispossible to infuse very large numbers of T cells, as has been done in tumor infiltrating

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C. H. June

lymphocyte therapy2). The other approach is to infuse smaller numbers of cells that areprogrammed to expand extensively in the patient3,4).

There are three essential factors to consider when developing engineered T celltherapies (Figure 1). One issue under consideration is the nature of the lymphocyte subset

to be infused. At present it is not known whether the optimal cell type is a memory cell ora naïve cell. Related to this is the question of the proportion of CD4 helper cells and CTLs.

Secondly it is important to consider the aspects of genetic engineering that are used toreprogram the cells 5) . Finally it is necessary to have an optimized approach for the
manufacturing of the T cells.

The design of CAR T cells has become increasingly sophisticated (Figure 2). The initial

CAR T cells were so-called first-generation CARs and only had signaling from the T cell
receptor zeta chain or related molecules. More recently second and third generation CAR T

Figure 1

Essential factors for augmenting adoptive cellular immunotherapy.

Figure 2

Design of CAR T Cells.

T Cell Therapy

147

cells have been designed, incorporating progressively more complicated signaling

domains. A number of strategies have been used to make CARs more potent or to enhancetheir proliferative capacity or their cytokine secretion. For example it has been shown that

so-called Signal 1 from the Lafferty model6) can be enhanced by encoding a ubiquitin-

resistant linker of activated T cells (LAT)7). A number of costimulatory domains have beenused to augment Signal 2. These include CD28, and the tumor necrosis family members 4-

1BB, Ox40, and CD27, for example. Recent studies have shown that the ICOS signaling

domain, a molecule related to CD28, provides CAR T cells with enhanced persistence inpreclinical animal models. These studies were initiated by Crystal Paulos and showed thatwhen T cells were activated through their natural ICOS receptor molecule with agonistic

antibodies, that this promoted the proliferation of TH 17 cells and T follicular helper cells

that had highly potent effector functions (Figure 3). In contrast, if the T cells were
costimulated with a CD28 dominant costimulatory signal, then the TH17 cells wererestrained in their antitumor activity and inflammatory properties8). More recently, Sonia

Guedan has demonstrated that when ICOS is expressed in CD4 cells by encoding thecytosolic domain in the CAR endodomain that this leads to enhanced persistence of theCD4 cells and that those cells promote the persistence of CD8 CAR T cells9). This latter

effect is independent of the costimulatory domain it is expressed in the CD8 CAR T cell.

Figure 3

Distinct Cosignaling by CD28 and ICOS: Implications for ACT Therapy

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Other studies in our laboratory have recently identified examples of CAR designs that have

either a continuous and ligand-independent growth phenotype, or are quiescent in theabsence of stimulation by surrogate antigen. Finally, studies by Yangbing Zhou haverecently shown that it is possible to increase the therapeutic index of CARs by affinitytuning of the single chain variable fragments.

Ongoing research is defining the characteristics of CAR design that lead to acontinuously active CAR. These T cells are capable of proliferating for months without

stimulation or addition of exogenous cytokines. Factors that dictate ligand independent
growth of the CAR T cells depend on the particular scFv used, and so far they have only

been observed with CD28 endodomains. The growth is independent of exogenous ligands

and there is evidence for constitutive signaling through CD28 involving AKT, NF-kappaB

and the MAP kinase pathway. The continuous CAR T cells have constitutive secretion of

Th1 and Th2 cytokines, and they tend to highly express T-bet, Eomes, GATA3, and Bcl-xL.
Transformation not observed, and surprisingly, CAR T cells with the continuous growthphenotype are less effective in pre-clinical tumor models. One implication of this researchis that it is important to screen newly designed CAR T cells for constitutive activity.

The initial CAR T cell trials were conducted in patients with HIV, using a first-

generation CAR10). At the University of Pennsylvania CAR T cell trials in cancer patientstargeting CD19 were initiated in 201011). There were three unique aspects of this the CAR,

termed CART19, that were used in this trial that have not been tested previously. First alentiviral vector was used to express the CAR. Secondly, a short duration T cell culture

process using antibody-coated beads was used for T cell manufacturing. Thirdly, the CAR

employed a TNF family member for co-stimulation, CD1 37, also referred to as 4-1BB. The

initial patients that were treated had refractory chronic lymphocytic leukemia12). All three of
the initial patients responded to the CART19 therapy. The patients had large bulky tumors

and it was possible to conclude that each CART19 cell or its progeny through cell division

killed more than 1000 tumor cells. As of December 2014 the patients continue to express the
CARs and the remissions have been durable in two of the three patients having noevidence of leukemia for more than 4 years. The level of CAR T cell proliferation in vivo isa predictive biomarker of clinically beneficial antitumor responses. In more recent studies

the CART19 T cell approach has been used to treat patients with acute lymphoblastic
leukemia 9ALL). We recently reported the first 30 patients on this phase 1 trial and astriking 90% rate of complete remission was observed in both pediatric and adult cases13).

Durable B cell aplasia is associated with a low risk of relapse of ALL following CART19 T
cell therapy.

There have been a number of toxicities associated with CAR T cell therapy. B cell aplasia

is observed in responding patients following therapy with CD19 or CD20 specific CARs.

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Tumor lysis syndrome has been observed in many patients, and this is managed as perstandard oncology practices and the unique aspect of this is that it may be delayed for 20 to

50 days after infusion of the CAR T cells. Cytokine release syndrome characterized initiallywith fever occurs in many patients, and this is related to the tumor burden in the patient.

Finally, a subset of patients develops macrophage activation syndrome which ischaracterized in the serum by very high levels of ferritin, C-reactive protein and evidenceof coagulopathy. The hallmark of this syndrome are very high levels of IL-6 and interferon
gamma in the serum. This syndrome is rapidly reversed with the IL-6 receptor antibody

tocilizumab, indicating that this inflammatory disorder is dependent on IL-6 (Figure 4).

In summary, engineered T cells are finally on the path towards widespread use. A

number of trials are now underway that should lead to commercial approval in the UnitedStates and in Japan.

Figure 4

Tocilizumab Anti-Cytokine Therapy for Cytokine Release Syndrome. A patient withleukemia was given tocilizumab on day 10 after CART19 therapy, with immediate resolutionof fever.

References
1. Maus MV, Fraietta JA, Levine BL, Kalos M, Zhao Y, June CH. Adoptive
Immunotherapy for Cancer or Viruses. Annu Rev Immunol. 2014;32:189-225.
2. Hinrichs CS, Rosenberg SA. Exploiting the curative potential of adoptive T-cell therapy
for cancer. Immunol Rev. 2014;257:56-71.
3. June CH. Principles of adoptive T cell cancer therapy. J Clin Invest. 2007;117:1204-12.
4. June CH. Adoptive T cell therapy for cancer in the clinic. J Clin Invest. 2007;117:1466-76.

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5. Kalos M, June CH. Adoptive T cell transfer for cancer immunotherapy in the era of synthetic biology. Immunity. 2013;39:49-60.
6. Lafferty KJ, Cunningham AJ. A new analysis of allogeneic interactions. AustJ Exp Biol
Med Sci. 1975;53:27-42.
7. Kunii N, Zhao Y, Jiang S, Liu X, Scholler J, Balagopalan L, et al. Enhanced function of redirected human T cells expressing linker for activation of T-cells that is resistant to ubiquitylation. Hum Gene Ther. 2013;21:27-37.
8. Paulos CM, Carpenito C, Plesa G, Suhoski MM, Varela-Rohena A, Golovina TN, et al. The
inducible costimulator ICOS is critical for the development of human TH17 cells.
Science Translational Medicine. 2010;2:55-78.
9. Guedan S, Chen X, Madar A, Carpenito C, McGettigan SE, Frigault MJ, et al. ICOSbased chimeric antigen receptors program bipolar TH17/TH1 cells. Blood.
2014;124:1070-80.
10. Walker RE, Bechtel CM, Natarajan V, Baseler M, Hege KM, Metcalf JA, et al. Long-term invivo survival of receptor-modified syngeneic T cells in patients with human immunodeficiency virus infection. Blood. 2000;96:467-74.
11. Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor-modified T
cells in chronic lymphoid leukemia New England Journal of Medicine. 2011;365:725-33.
12. Kalos M, Levine BL, Porter DL, Katz S, Grupp SA, Bagg A, et al. T cells expressing
chimeric receptors establish memory and potent antitumor effects in patients withadvanced leukemia. Science Translational Medicine. 2011;3:95ra73.
13. Maude S, Frey N, Shaw P, Aplenc R, Barrett D, Bunin N, et al. Sustained Remissions with
Chimeric Antigen Receptor T Cells For Leukemia. N Engl J Med. 2014;371:1507-17.

Carl H. June, M.D.

2001-

2004-

Professor of Pathology and Laboratory Medicine,
University of Pennsylvania School of Medicine
Professor of Medicine, University of Pennsylvania
School of Medicine

Specialty and Present Interest:
Lymphocyte biology, with a major translational focus on ex vivo T cell engineering forcancer and HIV cell based therapies.

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CONCLUDING REMARKS

Jedd D. Wolchok

Ludwig Center at Memorial Sloan Kettering
1275 York Avenue, New York, NY, 10065 USA(wolchokj@mskcc.org)

The 45th Annual Symposium of the Princess Takamatsu Cancer Research Fund was

focused on the very timely and important topic of Recent Advances in Cancer
Immunotherapy. The recognition of the research area is particularly significant since

Science declared cancer immunotherapy to be the “Breakthrough of the Year” in 2013.

Many of the most international important physicians and scientists in the field traveled tojoin distinguished colleagues in Japan to discuss the most up to date information in thefield. The Symposium indeed covered the most important areas of research progress thathave resulted, in recent years, in unprecedented advances for patients using highly

innovative approaches.

Much has been learned about the dynamic interaction that exists between the immune

system and cancer. It is now well known that T cells can infiltrate tumors early in theirdevelopment and that important prognostic information can be gained from quantifying

the extent and quality of the infiltrate. Further, the identification of the importance ofmutational neo-epitopes has validated the importance of the concept of cancer immune

surveillance and has given new energy to the quest to develop truly specific cancer

vaccines. This area will be complemented by advances in understanding of mechanisms ininnate immunity, including the STING pathway. The recognition that tumors are ‘seen’ by

the immune system has allowed for the development of approaches that enhance immune

reactivity. One such area is immunologic checkpoint blockade and two classes ofcheckpoint blocking antibodies have recently gained regulatory approval after

demonstration of significant clinical impact. CTLA-4 blockade with ipilimumab was thefirst intervention ever to improve overall survival in metastatic melanoma and PD-1

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blockade has led to impressive clinical responses in both sold tumors and hematologic

malignancies. Most recently, combinations of these agents have resulted in rapid anddurable regressions in a large subset of patients with melanoma and renal cell carcinoma.

Successes such as these have given rise to additional investigations involving agonist

agents targeting CD137 alone and in combination with antibodies to antigens on tumor

cells. While tumor targeting antibodies have been relatively limited in the past to surface

markers on B cell malignancies, Her-2 and EGFR, more current technologies are allowing

for the development of antibodies specifically directed to MHC-peptide complexes,

allowing for recognition of important intracellular molecules.

For tumors which may not have favorable antigens engendering a baseline immune

response, recent developments in the expansion and engineering of T cell products havegiven way to a variety of approaches which introduce highly activated and specific T cells

into the repertoire. The use of expanded tumor infiltrating lymphocytes and T cells bearing

transgenic T cell receptors and chimeric antigen receptors are all showing great promise in

the treatment of a variety of malignancies.

Further, the elegant identification and characterization of suppressive cell populations

such as Treg cells and myeloid derived suppressor cells allowed for an additional avenue

for cancer immunotherapy. Suppression or inhibition of these populations could certainly

lead to additional therapeutic benefit for patients with cancer.

The logical conclusion from this truly remarkable Symposium is that the future ofcancer treatment will most certainly be replete with treatment programs that include

immunotherapeutic approaches. The inherently complementary mechanistic nature of theapproaches yields clear opportunities for them to be rationally combined to achieve evengreater clinical impact. The collegial and collaborative environment of the Princess
Takamatsu Cancer Research Fund Symposium has provided an optimal venue for suchvaluable efforts to succeed.