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CANCER SCIENCE 9
Cancer immunotherapy
Taormina, March 9-13, 2013
C O L L O Q U E S
M É D E C I N E
E T
R E C H E R C H E
SCIENTIFIC REPORT BY APOORVA MANDAVILLI
Fondation IPSEN is placed under the auspices of Fondation de France
7 Foreword by Inder M. Verma
11 PART I: Harnessing the power of T cells
13 Steven A. Rosenberg
Autologous T cells as a drug for the treatment of metastatic cancer
19 Philip D. Greenberg
Targeting tumors with T cells: "You can't always get what you want, but..."
25 Carl H. June
CARs as an example of synthetic biology for cancer therapy
31 Ronald Levy
Targeting the immune system while targeting the cancer.
Therapy customized for the patient vs. Ôoff-the-shelfÕ
37 Malcolm K. Brenner
Will cellular immunotherapies ever become an 'industry standard'?
43 PART II: Exploiting checkpoint blockades
45 Rafi Ahmed
T-cell memory and exhaustion
49 James P. Allison
Immune checkpoints and cancer: New insights and opportunities
55 Antoni Ribas
ACT immunotherapy and BRAF targeted therapy for melanoma
61 PART III: Inducing tumor immunity with vaccines
63 Glenn Dranoff
Mechanisms of protective tumor immunity
69 Karin Jooss
CONTENTS
Development of combinatorial cancer vaccines
75 Elizabeth M. Jaffee
Tipping in the balance from a procarcinogenic to an anticancer response
in pancreatic cancer
81 PART IV: Tracking the immune response
83 Jérôme Galon
From the immune contexture to the immunoscore in the era of cancer immunotherapy
89 Robert D. Schreiber
Cancer immunoediting: From basic mechanisms to novel immunotherapies
95 Klaus Rajewsky
Modeling Epstein-Barr virus infection, immune surveillance and human B-cell
lymphomas in mice
101 George Coukos
Endothelial regulation of T-cell homing in tumors
107 PART V: Decoding immune response players
109 Ton Schumacher
(Neo)-antigens and tumor-reactive TCR repertoires in human tumors
115 Miriam Merad
Dissecting the tumor myeloid niche
121 Sebastian Amigorena
Regulatory T cells control immune responses to self and non-self antigens
127 Irving L. Weissman
Normal and neoplastic stem cells
133
136
141
145
Abbreviations and glossary
Participants
Cancer Science meeting series
Fondation IPSEN
7
FOREWORD by INDER M. VERMA
9
In 1891, William Coley, a New York surgeon, began intratumoral injections of live or inactivated
Streptococcus pyogenes and Serratia marcescens in an effort to reproduce the spontaneous remissions
of sarcomas observed in rare-cancer patients who had developed skin rashes and inflammation.
The so called ÔColey's toxinsÕ acted by stimulating antibacterial phagocytes that might kill bystander
tumor cells.
Some significant responses were recorded over the ensuing 40 years, but successes were sporadic,
difficult to reproduce and not obtained in a scientifically rigorous fashion. This early and risky
approach of administration of infectious agents was never embraced by oncologists who continued
to rely on surgery, radiation therapy and, ultimately, chemotherapy.
But this was the early approach to cancer immunotherapy Ñ a powerful therapeutic option for cancer
that uses T cells to seek and destroy tumor cells. It is possible to grow large numbers of T cells in
vitro and avoid the harmful side effects of harsh drugs or vaccines.
The 9th IPSEN Foundation Cancer Series meeting was devoted to discussing the potential Ñ and
limitations Ñ of immunotherapy for cancer in beautiful and sunny Taormina, a storied city in Sicily,
facing the mighty, smoldering Mount Etna .
The first speaker, Steven A. Rosenberg, a pioneer in the field, described studies that demonstrate
the potential of adoptive T-cell immunotherapy to mediate the regression of metastatic cancers.
In sequential clinical trials of 93 patients with metastatic melanoma using autologous tumorinfiltrating lymphocytes selected for antitumor activity, 20 achieved a complete tumor regression
and have ongoing complete responses.
In another clinical trial of metastatic melanoma, 14 patients were given MART-1 melanosomal
antigen adoptive cell transfer (ACT), together with MART-1 peptide-pulsed dendritic cell vaccination.
Of the 14, 9 showed evidence of tumor regression, but the responses were not durable. Antoni Ribas
described the testing of ACT and BRAF inhibitor therapy combinations in patients with BRAF-mutant
metastatic melanoma.
Carl H. June described novel immunotherapy approaches based on the engineering of antibody
fragments and cells Ñ such as chimeric-antigen receptor (CAR)-engineered or T-cell receptorengineered cells Ñ to provide long-lasting antitumor effects.
Effective T-cell therapy will require many factors, including an appropriate antigenic target, a T-cell
response of high avidity and high magnitude, and the ability of the T cells to infiltrate and retain
function in the tumor microenvironment. Philip D. Greenberg suggested that T cells can be made
to express high-affinity T-cell receptors (TCRs) and overcome the problems posed by the tumor
microenvironment.
The combination of second-generation sequencing and immunomonitoring may potentially be used to
identify T-cell reactivity against patient-specific neo-antigens formed by mutations. Ton Schumacher
argued that the ability to describe patient-specific tumor-reactive T-cell responses accurately should
form a first step towards the development of personalized cancer immunotherapy.
10
Many approaches to immunotherapy require customization for each patient, which limits their
application. In contrast, rituximab, a generic antibody against CD20, became a blockbuster drug for
B-cell lymphoma. Ronald Levy described the advantages of Ôoff-the-shelfÔ immunotherapy for cancer.
Overall, however, immunotherapies for cancer have failed to make the transition into licensed drugs
that are standard of care. Malcolm K. Brenner argued that at least part of the delay can be attributed
to the dissimilarity between the business models needed to bring standard small-molecule drugs to
success and those required for cellular immunotherapies.
Efficacious cancer immunotherapies are likely to require combinations of strategies that enhance
tumor-antigen presentation and antagonize negative immune regulatory circuits. Glenn Dranoff
suggested that therapy for tumors may not just be similar to antivirals, but may also involve tapping
into the anti-parasitic response.
Cancer immunoediting is the process by which the immune system controls and shapes cancer.
Robert D. Schreiber is using genomic sequencing approaches to define the antigens that induce
tumor rejection following checkpoint blockade and to develop individualized cancer vaccines.
Chronic antigen stimulation during cancer can lead to functional exhaustion of CD8+ T cells. A
characteristic feature of these exhausted CD8+ T cells is sustained expression of inhibitory receptors
such as PD1. Rafi Ahmed described a new role for PD1 in regulating T-cell differentiation and
discussed strategies for enhancing PD1-directed immunotherapy.
CTLA-4 is a critical down-regulator of T-cell responses, and CTLA-4 blockade allows prolonged T-cell
responses. PD1, another checkpoint, has a different mechanism of action. It recruits a phosphatase
and seems to interfere with T-cell antigen receptor-mediated signaling. James P. Allison suggested
that because CTLA-4 and PD-1 have different mechanisms of action, a combination of the two might
be better than either alone.
Karin Jooss presented results from a clinical trial of metastatic castration-resistant prostate cancer
suggesting that higher numbers of activated memory and effector T cells, low numbers of regulatory
T cells, increases in Th2 and Th17 rates and seroconversion to multiple tumor antigens are all
associated with a favorable clinical outcome and prolonged overall survival.
Pancreatic cancer has the poorest survival rates reported for solid tumors, and is highly resistant
to traditional forms of therapy. Elizabeth M. Jaffee's research is focused on elucidating the immune
mechanisms within the tumor microenvironment with the goal of tipping the balance in favor of an
anticancer immune response.
There is significant evidence indicating that only patients who show strong infiltration of tumors by
activated T cells have a positive outcome long-term. George Coukos described multiple approaches
to combat the endothelial cell control of the tumor immune response.
The anatomic extent of tumor classification, the TNM, has so far been the most widely used to
predict the prognosis of cancer patients. However, this classification provides limited prognostic
information in estimating the outcome in cancer and does not predict response to therapy. Jérôme
Galon presented the ÔimmunoscoreÕ, which has a prognostic value superior to that of the TNM
classifications.
Miriam Merad discussed the contribution of tissue-resident macrophages and bone marrowderived macrophages to tumor homeostasis. Sebastian Amigorena proposed that regulatory T cells
are important regulators of the homeostasis of CD8+ T-cell priming, and play a critical role in the
induction of high-avidity primary responses to non-self antigens and effective memory.
Genetic mouse models of human cancer can provide insights into functionally relevant pathogenic
pathways. Klaus Rajewsky presented two examples of this basic approach.
Tissue stem cells are highly regulated by a number of pathways, including programmed cell death
11
(PCD) and programmed cell removal (PCR), which is independent of PCD. Irving L. Weissman
proposed that the PCD/PCR pathways might play a role in a number of pathogenic states, from
persistent infections by microbes that hide in cells, to cells that respond to persistent damage or
immune signals.
Cancer immunotherapy has experienced its highs and lows, and we must all thank David Baltimore
for his vision and persistence in challenging me to overcome my skepticism and help organize the
meeting on this fascinating subject. The progress in the field is remarkable, the outcome for some
patients is nothing short of miraculous. The use of novel therapeutics that can overcome immune
checkpoint blockade is revolutionizing treatments of incurable cancers.
As always, Jacqueline Mervaillie, Yves Christen and their terrific staff, Astrid de Gérard and
Céline Colombier-Maffre, made the whole experience memorable. Once again Apoorva Mandavilli
has been able to capture complexities of the subject and render it into a highly readable monograph.
Inder M. Verma
13
PART I: H a r n e s s i n g t h e p o w e r o f T c e l l s
Steven A. Rosenberg
Autologous T cells as a drug for the treatment of metastatic cancer
Philip D. Greenberg
Targeting tumors with T cells:
"You can't always get what you want, but..."
Carl H. June
CARs as an example of synthetic biology for cancer therapy
Ronald Levy
Targeting the immune system while targeting the cancer.
Therapy customized for the patient vs. off-the-shelf
Malcolm K. Brenner
Will cellular immunotherapies ever become an 'industry standard'?
Harnessing the power of T cells
Autologous T cells as a drug for
the treatment of metastatic cancer
A report on a lecture by
Steven A. Rosenberg
National Cancer Institute – NIH, Bethesda, USA
The adoptive transfer of antitumor T cells can mediate regression of established metastatic cancers in patients.
A series of three sequential clinical trials treated patients with metastatic melanoma using autologous tumorinfiltrating lymphocytes (TIL) selected for antitumor activity. Of the 93 patients, 20 achieved a complete tumor
regression of widespread cancer and 19 of the 20 patients have ongoing complete responses at 63 to 108 months.
New techniques utilizing exome sequencing have been used to identify multiple mutated antigens recognized by
TIL. Because melanomas are the only histologic type of cancer that readily gives rise to TIL with demonstrable
antitumor activity, a series of clinical trials are using cytokine genes and antitumor T-cell receptors (TCR)
transduced into normal peripheral lymphocytes for use in adoptive cell transfer. The cellular and molecular
mechanisms of T-cell destruction of cancers are under active investigation. Ongoing studies are evaluating
chimeric antibody receptors targeting mesothelin, VEGFR2 and EGFRvIII. Steven A. Rosenberg described
studies that demonstrate the power and potential of adoptive T-cell immunotherapy to mediate the regression
of established metastatic cancers in humans.
Adoptive cell therapy is a way to use T cells as a drug, much like a chemotherapeutic agent, to attack the
cancer. One of the advantages of this approach, as opposed to a vaccine or an indirect way of simulating
T cells, is that it is possible to grow large numbers of these effector cells in vitro.
In fact, it is possible to grow about a fifth of the total number of lymphocytes in the human body. High avidity
cells can be identified with in vitro assays or created in vitro using genetic engineering.
Most importantly, the host can be manipulated to provide a favorable tumor microenvironment prior to
administering the cells in ways that are not possible with other approaches. For example, if you eliminate
regulatory T cells (Tregs) and myeloid suppressor cells, that's not the time to apply a vaccine, but it might
be the ideal time to introduce a T cell as an antitumor drug.
This particular iteration of the approach uses T cells
that infiltrate the tumor. We excise the tumor and
grow multiple cultures to identify the cells within
that tumor that have high reactivity against the
cancer. We then take the most active cells, based on
in vitro assays, and grow them to large numbers. If
we then lymphodeplete the host to eliminate Tregs
and make space for the T cells, we see dramatic
antitumor effects.
A chemotherapy regimen, such as cyclophosphamide
and fludarabine, given temporarily for about 8 days,
eliminates all circulating T cells before they naturally
recover. Animal models show that lymphodepletion
improves the antitumor effects. With total-body
irradiation (TBI), the hematopoietic cells recover in
about 10 days.
Figure 1
An ongoing complete
response in a patient
who received T-cell therapy.
15
16
There are results from three sequential trials. One trial of 43 patients, who got their own T cells with the
most antitumor activity, showed a 49% objective response rate. Of the 43, 5 patients, or 12%, showed a
complete regression of their metastatic cancer and have remained so for more than 7 years1.
With 200 cGy of TBI in 25 patients, the response rate was about the same, but 20% had complete regressions.
Finally, in the last trial of 25 patients, who got 1,200 cGy of TBI, the response rate went up to 72%. Overall,
40% of all of the patients had complete regressions of their metastatic disease, and all but one has an
ongoing response for up to 9 years.
There are a few things worth noting about the response. First, it happens fast. The T cells are primed to
attack the tumor directly. One patient's tumors, which were present throughout his liver and lungs, were
all gone by day 34. The patient has an ongoing complete response 10 years after the treatment. Another
patient had multiple subcutaneous deposits that went away over a 12-day period.
In another patient, there were multiple lung metastases and a malodorous local lesion that continued to
grow, despite multiple surgical excisions, radiation therapy and chemotherapy. By day 22 after treatment,
the tumor was blackening. Two months later, however, the tumor was starting to regrow. The tumor went
away completely after the second treatment. This was the only patient who was treated twice. In all the
other cases, one treatment is enough: The T cells survive and continue to act.
Finally, this treatment can work in all sites of the body, including the brain. One patient who had 4 brain
metastases underwent a complete regression over the course of 3 months. There's no relationship
between having a regression in the site of the disease and the tumor bulk, or between any prior treatment
and the likelihood of a complete regression.
Host factors:
There is a lot of variation in the host factors involved in regression, but at a highly statistically significant
level, patients who receive cells with long telomeres are more likely to have a complete regression. Those
who receive cells that are a little less differentiated because of the expression of CD27 as a marker are also
more likely to have a complete regression.
There is an inverse correlation between the likelihood of a complete response and the presence of
CD4+FOXP3+ Tregs. The lymphopenic environment eliminates Tregs and myeloid-derived suppressor
cells, but it also eliminates competition for homeostatic cytokines such as interleukin-7 (IL-7) and IL-15
that are vital for survival.
In the lymphopenic host, these antitumor T cells can proliferate thousands of fold in vivo, persist, infiltrate
organs and recognize cancer antigens. In a population of patients with widespread melanoma who were all
heavily pretreated, about 30% of those patients are surviving close to 10 years after treatment.
Figure 2
Multiple subcutaneous
deposits in this patient
disappeared over 12 days.
So far, there has been only
one recurrence among the
20 patients who have had a
complete response. It appears
that these patients have been
cured and somehow the tumorinfiltrating lymphocytes (TIL)
are capable of eliminating the
last cancer cell.
Among cancers, melanoma
appears to be uniquely immunogenic. Melanoma cells are
just as susceptible to lysis by a
Harnessing the power of T cells
T cell as a breast cancer cell or prostate cancer cell, but they give rise to immune reactions. And they're
susceptible to IL-2, anti-CTL4, anti-PD40, anti-PD1. They can also generate TIL that recognize cancerassociated antigens.
These observations raise two intertwining questions: Why is melanoma uniquely immunogenic? What do
TIL recognize that enables the in vivo destruction of every last cancer cell in the patient?
Melanoma's uniqueness does not appear to be related to the normal melanocyte antigens such as MART-1
and GP100 that were first described in 19942. We developed high-affinity TCRs against these antigens, and
treated 36 patients with their own cells, genetically engineered with the high-affinity receptors.
The treatment gave a 25% objective regression rate, but a large percent of the patients developed severe
eye and ear toxicity. In contrast, of the 93 patients treated with TIL, only one had eye and ear toxicity. The
TIL recognize something other than these melanoma melanocyte antigens.
That suggests that the TIL might be recognizing mutated antigens, and possibly driver mutations that
are important for maintaining the malignant phenotype. Exome sequencing of 33 consecutive melanomas
showed a wide variation in the number of non-synonymous mutations, with a median number of 234.
This is a large number of mutations compared with virtually any other tumor except smoking-induced
squamous cell cancer of the lung. In fact, this may explain why anti-PD1 produces responses in melanoma
and lung cancer patients, but not other histologies.
Target practice:
We've developed a new approach to rapidly identify exactly the cancer antigens that the TILs recognize.
Exome sequencing of either a fresh tumor or an early cultured line determines the exact exomic mutations
that are present. We then obtain the sequence of 9-10 amino acids on either side of that mutation, use an
algorithm to predict the best binders to that patient's HLA antigens and test those peptides.
In virtually every patient so far, we've
been able to identify the exact mutations
that the TIL recognize. For example, of the
595 non-synonymous mutations we found
in one patient, we were able to identify
that the TIL recognize PLEKHM2, which
plays a role in microtubule formation. In
another case, it was the GAS7 protein,
which is primarily expressed in growtharrested cells and HAUS3, which also
plays a role in microtubule formation.
Now that it's possible to identify the
mutations, we are trying to selectively
target them, either using tetramer
selection of the reactive cells or by in
vitro sensitization to generate reactive
lymphocytes against them. This approach
should be applicable to other tumors that
have exomic mutations.
There are also ways to preselect
populations of TIL with antitumor activity.
Among the infiltrating CD8+ T cells within
these melanomas, about 10% are PD1+
Figure 3
Exomic analysis identified
the mutation in this
patient, who had multiple
lung metastases.
17
18
and only about 2-3% of them are 4-1BB+. Surprisingly, it's the PD1 populations that have the greatest
anti tumor activity based on 3 different measurements: up-regulation of interferon-γ in a co-culture,
up-regulation of 4-1BB, and the ability to bind tetramers. LAG3+ and TIM3+ cells also have anti tumor
reactivity; 2% of all the CD8s that are 4-1BB+ appear to have all of the antitumor activity.
That was true for each of the other 5 patients studied. This enables us to study a tiny fraction of the
population, 2-10% of all the cells within the tumor, and expand them for therapy. As expected, the PD1+
cells have many fewer TCR clonotypes than the PD1- cells.
Interestingly, virtually all of the most common TCRs in the expanded population come from the PD1+
population. These common TCRs are the ones that recognize the mutations.
Essentially, sorting for the 2% of the cells that are 4-1BB+ enriches for reactivity against multiple tumor
antigens. Administering those cells without first screening for antitumor activity would shorten their time
in cell culture, retaining their proliferative potential.
Altering the genetic makeup of these lymphocytes using viruses to introduce cytokine genes would improve
the cells' activity. Similarly, other molecules would prevent apoptosis or improve their survival.
Of all of the cytokines that can be introduced, single chain IL-12, which is important for innate immunity,
has a dramatic effect on improving TIL ability to mediate tumor regression. The problem is that IL-12 is
toxic, and can be fatal3.
We developed an inducible vector that would only secrete IL-12 at the tumor site, only when the T cells
encounter the specific antigen. The clinical trial began with low doses but has progressed to higher
doses. The overwhelming majority of the patients have ongoing responses in the absence of any IL-2
administration.
To expand the recognition against a variety of tumors, we've tried to use TCRs that recognize cancer
antigens in autologous lymphocytes, and give these normal lymphocytes the ability to recognize tumor.
We've done that with both conventional TCRs or with a chimeric antigen receptor (CAR) in which the variable
regions of the heavy and light chains are combined. The huge power of this approach is that although it's
difficult to find TCRs that recognize colon or prostate cancer, you only have to find one cell.
Critical challenges:
The most critical challenge hindering the development of effective human cancer immunotherapy is the
identification of appropriate antigens to target. There are 5 possible kinds of antigens: those expressed on
cancers and on non-essential normal tissues (CD19 and thyroglobulin); shared antigens unique to cancer
(cancer-testes antigens); mutations unique to each cancer (EGFRvIII); critical components of the tumor
stroma (VEGFR2, FAP); and differentiation antigens overexpressed on cancers compared with normal
tissue (MART-1, gp100, CEA, HER-2).
About 22,000 people die each year of B-cell lymphomas in the U.S. alone. CD19 is expressed in more
than 90% of these lymphomas. And there are good antibodies that recognize CD19 on both malignant and
normal B cells.
T cells can be genetically engineered to express an anti-CD19 CAR. The first patient had undergone many
treatments. He received the anti-CD19 CAR-transduced T cells in 2009. He has an ongoing regression of
the disease4.
His bone marrow biopsies showed extensive leukemia before treatment and nearly absent B-lineage cells
after. In contrast, T cells and NK cells return. So far, 14 patients have been treated, 8 with diffuse large
B-cell lymphoma, and 6 with chronic lymphocytic leukemia. The response rate is about 80%, and many of
the responses are ongoing.
Harnessing the power of T cells
Treatment for common tumors can target the
cancer testes antigen. First described in 1990,
these are antigens expressed during fetal
development. They're not in any normal adult
tissue but the male testis, which doesn't express
class I and so is immunologically protected.
19
Figure 4
Treatment with T cells
eliminated brain, lung and
neck metastases in this
patient.
This antigen is up-regulated in large number of
cancers from multiple tissues. The NY-ESO-1
family of cancer testes antigens is expressed in
30-40% of common cancers including bladder
cancers, non-small cell lung cancer and ovarian
cancers.
In a clinical trial of synovial cell sarcoma, a TCR
that recognizes NY-ESO-1 produced regression
in 75% of the patients. In any of these cases,
there's no relationship between the size of the
tumor and the likelihood of seeing a response.
A mutation that's unique to the cancer and not
in any normal cell also exists in high-grade
glioblastoma. EGFRvIII is expressed in a third of
high-grade glioblastomas, which are 100% fatal
regardless of the treatment. It's not expressed in
any normal tissues and is probably responsible
for the malignant phenotype of the cell.
A CAR that recognizes EGFRvIII with high specificity is being tested as a treatment for glioblastoma.
Another clinical trial is testing VEGFR-2, the vascular protein that is over-expressed on tumor vasculature.
The ability to genetically modify human T cells opens possibilities to improve the effectiveness of cell
transfer immunotherapy and extend it to patients with common epithelial cancers.
References
1. D
udley M.E. et al. J. Clin.
Oncol. 26, 5233-5239
(2008)
2. Kawakami Y. et al. J. Exp.
Med. 180, 347-352 (1994)
erkar S.P. et al. Cancer
3. K
Res. 70, 6725-6734 (2010)
4. Kochenderfer J.N. et al.
Blood 119, 2709-2720
(2012)
Harnessing the power of T cells
Targeting tumors with T cells:
"You can't always get what you
want, but..."
A report on a lecture by
Philip D. Greenberg
Department of Immunology – University of Washington, Seattle, USA
Modulating T-cell immunity to treat human cancers is showing increasing promise, but serious obstacles
remain to generating a T-cell response that provides therapeutic benefit. Effective therapy will require many
factors, including an appropriate antigenic target, a T-cell response of high avidity and high magnitude, and the
ability of the T cells to infiltrate and retain function in the tumor microenvironment. A clinical trial targeting one
promising antigen associated with the leukemic phenotype, WT1, has shown the potential benefit of establishing
potent T-cell responses by adoptive T-cell transfer. However, cloning T cells for therapy from individual patients
is limited by the quality of the T-cell repertoire and responses that can be elicited in each patient. This could
be overcome by cloning the T-cell receptor (TCR) from characterized high avidity leukemia-reactive T cells,
and putting such TCRs into vectors for introducing them
into patient T cells. However, many pro-oncogenic
targets are detected at low levels in normal cells, and the thymic selection process appears to be overprotective,
eliminating T cells expressing high-affinity TCRs. Philip D. Greenberg suggested that T cells can be made to
express high-affinity TCRs and overcome the problems posed by the tumor microenvironment.
To make T-cell therapy effective, the first step is to find targets. We have focused mostly on self-proteins
over-expressed by the tumor and important for the malignant phenotype. This makes it difficult for the
tumor to lose such antigens as a means to escape an immune response, and would permit developing a
therapy that would be effective for a large number of patients rather than a single patient.
It's important that the antigen be found only at low levels in normal tissues below immune detection,
because toxicity from recognition of normal tissues could otherwise be unacceptable. Ideally, the antigen
would also be present in the cancer stem cells. Finally, the antigen must be able to elicit a T-cell response.
Based on gene expression profiling of leukemia, WT1 immediately stood out as a candidate. The vast
majority of acute myelogenous leukemia cells dramatically over-express WT1 compared with normal
tissues. Moreover, nearly all leukemia stem cells (LSC) also dramatically over-express WT1, whereas
hematopoietic stem cells (HSC) express only low levels.
A more complete analysis of normal tissues revealed that WT1 is normally expressed at low but detectable
levels in a few tissues, most notably, the uterus, kidney and HSC. Although there is no evidence these
tissues can be recognized by WT1-specific CD8+ T cells, it does raise questions about safety, and all clinical
trials, particularly with enhanced affinity TCRs, will need to proceed with caution.
To find alternative targets, in collaboration with Ravi Majeti and Irving Weissman at Stanford University, we
compared gene expression in LSC to HSC and a panel encompassing the other somatic cells. We ultimately
found 55 genes that seemed relatively leukemia-specific and tried to validate each one for uniqueness and
potential immunogenicity. This greatly narrowed the choices, and the only gene besides WT1 that appeared
to be both a reasonable target and is immunogenic is CCNA1, which encodes the cyclin A1 protein1.
Cyclins are highly conserved proteins that regulate cell cycle progression. There are two different cyclin
A genes. CCNA2 is ubiquitously expressed and regulates cell-cycle progression through mitosis, so it is
obviously not a viable target. By contrast, CCNA1, despite being similar to CCNA2 in the highly conserved
kinase domain, is disparate at other parts of the gene.
21
22
Figure 1
Profile of candidate
targets.
CCNA1 is expressed almost exclusively in male germ cells, with the exception of many cancers including
leukemia. Some immunohistochemistry experiments suggested it may be expressed at low levels in the
brain, but that has not been borne out by gene expression analysis. Knocking down CCNA1 gene expression
in leukemic cell lines reduces proliferation and increases apoptosis, consistent with it having a role in
maintaining the malignant phenotype.
Mice lacking CCNA1 are viable but predictably, because of its role in meiosis in males, the male mice are
sterile. Transgenic mice that over-express CCNA1 in the myeloid lineage show abnormal myelopoiesis
at birth, and many of the mice develop AML at 7 months of age. The data overall suggest that the gene is
important for the leukemic phenotype and could represent a potential target.
CCNA1 expression is detected not only in LSC, but throughout differentiation of leukemic cells, including in
95% of leukemic blasts. It has proven relatively easy to generate high avidity CD8+ T-cell responses to this
antigen, presumably because it is not detected in normal somatic cells and thus contains a large number
of potentially immunogenic epitopes.
Compared with WT1, CCNA1 mRNA is detected at much higher levels and is more selectively expressed
in malignant cells. Although WT1 expression is found in more than 90% of leukemias, CCNA1 expression
curiously appears to increase in leukemias that down-regulate WT1, suggesting that targeting both
antigens might further limit the potential for immune escape.
Both WT1 and CCNA1 can be detected in solid tumors as well as in leukemia. Based on the very promising
safety and immunogenicity profile, we are developing cyclin A1 as a T-cell therapy target, and hope to test
it after completing the second WT1 trial.
Trial and error:
The WT1 trial in leukemia patients has yielded several interesting and promising results. The 11 treated
participants all received multiple T-cell infusions, with the highest dose being more than 10 billion cells.
All of the infused cell products were CD8+ CTL clones derived from single cells. The clones were generated
and expanded in vitro in the presence of T-cell growth factors, interleukin-2 (IL-2), IL-7, and IL-15, and,
for the last 4 patients, the cells were also exposed to IL-21, which has been shown to limit terminal
differentiation of proliferating T cells.
The inclusion of IL-21 appeared to be important and useful: Cells cloned and expanded without exposure
to IL-21 exhibited a differentiated effector phenotype, whereas clones generated in the presence of IL-21
exhibited qualities of polyfunctional central memory CD8+ T cells.
Harnessing the power of T cells
These cells also exhibited enhanced persistence in vivo after cell transfer2, and expressed markers of
central memory cells, including expression of CD28, CD27 and CD127, as well as CD62L and CCR7, which
direct T cells to lymph nodes.
Of the 37 patients enrolled in this trial, only 11 were treated because, as a condition for treatment, patients
had to have detectable relapsed leukemia and be able to return to our center for T-cell therapy. Despite
receiving salvage chemotherapy for their relapsed leukemia before we could treat them with T cells, 7
patients still had overt progressive disease, with 2 of 7 showing a reduction of their leukemic burden
following T-cell therapy.
There were 4 patients who had either no detectable disease (3/4) after salvage chemotherapy or minimal
detectable disease (1/4), but all of these patients had a poor prognosis with relapse likely within months,
and predicted median survivals of less than 1 year. All of these patients received cells that had been
exposed to IL-21, and all exhibited prolonged T-cell persistence in vivo.
For this patient group, fewer than 3% are predicted to be alive after 2 years. Although 1 patient did expire
after 28 months, the other 3 are disease-free and alive after 4 years. As these patients did not have
detectable disease, it is difficult to prove that the treatment had anti-leukemic activity, but the survival
data are encouraging and worthy of further study.
To overcome barriers to efficacy, one crucial factor is making sure that all patients receive T cells
that are of high avidity for the leukemia. Although we selected the highest-avidity clone that we could
generate for each patient, the avidities were variable.
A more reproducible strategy for ensuring that highavidity cells will be available for each patient would be to
introduce Ôoff-the-shelfÕ characterized high-affinity TCR
genes into CD8+ T cells.
We screened about 100 normal donors, and identified
the HLA-A2 restricted CD8 clone, denoted C4, with the
highest avidity. We isolated the TCR genes from this
clone, and a clinical trial of CD8+ T cells transduced with
the TCR from this clone is enrolling leukemia patients.
Another trial with CD8+ T cells transduced with the TCR
from this clone in lung cancer patients Ñ another tumor
that expresses WT1 Ñ is pending.
Contact regions:
Is it possible to make a higher-affinity TCR, and would it be safe? When a TCR sits on a peptide/MHC complex,
the contact points between the TCR and the peptide/MHC are in the CDR regions. The CDR3 regions of the
TCR α and β chains predominantly contact the peptide, and the other CDR regions predominantly contact
the MHC and orient the TCR on the MHC molecule.
These structures suggest that modifying the CDR3 region would most likely increase affinity with retention
of specificity3. Saturation mutagenesis of CDR3 regions, as developed by our collaborator David Kranz, has
provided a high-throughput strategy for generating such TCRs with higher affinity.
As mouse expression of WT1 in normal tissues is virtually identical to that of humans, we have used mice
as models to understand the potential risks of using an enhanced affinity TCR. We isolated a high-avidity
WT1-specific CD8 T-cell clone from B6 mice, cloned the TCR genes and generated a library of mutants by
directed mutagenesis of the CDR3 region. From this library, we identified two high-affinity TCRs for further
study.
Figure 2
Mouse model to assess
safety of enhanced affinity
human TCRs targeting
WT1.
23
24
CD8 T cells were transduced with the wild type or one of the high-affinity TCRs, and transferred into a B6
mouse. After 18 days, we immunized the mice with a recombinant Listeria vector expressing WT1 to expand
persisting cells. We analyzed the mice 6 days later for immune responses, and at later time points for
survival and autoimmunity.
After immunization, about 4% of the cells in the peripheral blood expressed the TCR. However, these cells
rapidly returned to a resting state, with no evidence of engagement or activation by any normal tissue.
Additionally, there was no sign of any T-cell infiltration or toxicity in the kidney, the lung or the bone
marrow, or of depletion HSCs, even with a TCR having about 300-500 fold greater affinity than any TCR
found in the normal peripheral repertoire.
If these cells are indeed safe, why are we not finding cells with such high-affinity TCRs in the normal hosts?
To assess this, we introduced these TCRs into HSCs and examined development of T cells in the thymus.
Cells expressing the enhanced-affinity TCR were largely deleted in the thymus, and the cells that survived
down-regulated CD8 and the TCR, which greatly lowered the functional avidity of the T cells, allowing them
to avoid deletion.
By contrast, when these same enhanced TCRs were put into peripheral T cells, the T cells did not downregulate their TCR or CD8, and did not cause autoimmunity to any normal tissues. The events in the thymus
likely reflect the high level of thymic WT1 expression, which appears similar to levels in embryonic tissues.
These results suggest that thymic deletion is highly efficient but potentially overprotective, removing or
modifying cells that wouldn't necessarily mediate self-reactivity in the periphery of adults.
Understanding which antigens exhibit this kind of pattern is critical, because it suggests there is a window
for safely increasing TCR affinity, and thereby producing T cells with much better recognition of tumor
targets while still avoiding the potential for toxicity.
Figure 3
Antitumor activity of
transferred SV40-specific
TN, TEM and TCM against
established liver tumors.
Harnessing the power of T cells
25
Of mice and men:
Interestingly, the mouse WT1specific TCRs restricted to Db
recognize the same epitope
that's immunodominant in
HLA-A2 humans, although Db
and A2 have different anchor
residues. What's more, the
sequence of the human C4
TCR in the CDR3α region is
similar to the sequence of the
CDR3α region of the mouse
TCR we cloned and modified,
suggesting again this region is
a peptide contact point.
Figure 4
Transferred T cells
become progressively
exhausted in tumor
environment.
We have preliminarily introduced
mutations into the human C4
TCR that model the changes
we identified to enhance the
murine TCR. We have already identified a TCR with a single point mutation that binds tetramers with higher
affinity, and kills WT1 targets with much greater efficiency.
Now that we have generated TCRs that can be expressed in T cells, what kind of T cell do we want to give a
patient? In leukemia, central memory cells, at least in the mouse, seem to be best. But is this also true for
solid tumors? To assess therapeutic activity, we used a model of ÔspontaneousÕ hepatocellular carcinoma
in which the tumor arises from induced expression of the SV40 T antigen oncogene, and we have used the
SV40 T antigen as the CD8+ T cell target antigen4.
We transferred purified na•ve, effector memory, or central CD8+ T cells into tumor-bearing mice and
immunized them 24 hours later to induce in vivo effector cells. We then analyzed mice at day 10, the peak
of the response to the vaccine, and at 25 days.
Each of these cell subsets exhibited very different activities, with the transferred na•ve T cells exhibiting
the greatest antitumor activity and the central memory cells exhibiting the least. Additionally, the central
memory cells were the population most rapidly deleted after transfer.
This is consistent with central memory T cells having acquired a cell-intrinsic program to return to a
quiescent state, and thus may not tolerate exposure to persistent antigen and chronic stimulation. These
studies require further analysis, but suggest that the nature of the tumor setting may dictate the type of
T cell that should be used to most effectively target it therapeutically.
References
1. O
chsenreither S. et al.
Blood 119, 5492-5501
(2012)
2. C
hapuis A.G. et al. Sci.
Transl. Med. 5, 174ra27
(2013)
arcia K.C. et al. Nat.
3. G
Immunol. 10, 143-147
(2009)
4. Schietinger A. et al.
Science 335, 723-727
(2012)
Harnessing the power of T cells
CARs as an example of synthetic
biology for cancer therapy
A report on a lecture by
Carl H. June
Abramson Family Cancer Research Institute – University of Pennsylvania
Philadelphia, USA
Novel immune therapy approaches based on the engineering of antibody fragments and cells — such as
chimeric-antigen receptor (CAR)-engineered or T-cell receptor-engineered cells have the promise to overcome
tolerance to tumors and provide long-lasting antitumor effects.
The first set of patients treated with CAR T cells
had chronic HIV infection. These CAR T cells generated by γ-retroviral vector engineering are still persisting,
with no signs
of integrational mutagenesis after a decade or more of follow up. The patients continue to express
CAR T cells at high levels. CAR T cells directed against CD19 have a potent and durable clinical activity in B-cell
malignancies, with the drawback that the on-target cytokine release syndrome can be severe. The longevity of
engineered T cells may lead to lifelong tumor cell control, but may also mean that any side effects of the T-cell
transfer (such as B-cell aplasia) may be difficult to control or eliminate. Most importantly, the clinical effects
observed in the treated chronic lymphocytic leukemia and acute lymphoblastic leukemia patients justify larger
clinical trials with anti-CD19 CARs. Carl H. June presented data that fuel the hope that CAR therapy may change
clinical practice for hematological or even solid malignancies.
The idea of using synthetic biology to enhance the immune system is gaining increasing traction. Small
details in the manufacturing and construction can make a huge difference. In the area of cellular
engineering, synthetic biology has been used to enhance the function of cells above their baseline
properties. Engineered T cells and antibodies can both address the issue of tolerance.
The main issue in cancer is that the immune system recognizes tumors with a low affinity, if at all. Highaffinity interactions that can fully activate T cells are associated with the kind of inflammation that occurs
with a viral infection.
Chimeric antigen receptors (CARs) are personalized in that the T cells are generally from the patients
themselves, although allogenic cells can also be used. This approach is MHC-independent because it
targets surface molecules on target cells rather than the peptide MHC complex.
The first CAR trials were done in people with
HIV/AIDS, using a single ζ chain as a signaling
domain. Analyzing patients from that initial trial
has shown that those cells have half-lives in
patients of more than 17 years1. The initial trials
used retroviruses; ongoing trials now rely on
HIV-based lentiviruses, although some trials
still use retroviruses with promising results.
Researchers are also testing tandem cycling
domains with either CD28 or CD137 (4-1BB)
to enhance the function of the CAR. In T-cell
malignancies, the target has often been CD19 or
CD20, with a single ζ chain as a signaling domain
or tandem signaling domains. Our group has
worked on testing the 4-1BB signaling domain,
and others have been testing CD28.
Figure 1
Marrow biopsy of this ALL
patient shows expansion
of CD19 CAR T cells and
reciprocal loss of ALL
blasts.
27
28
Using a lentiviral vector, we expressed a CD19 single chain with permutations of either CD28 or 4-1BB
or TCR-ζ. These are all expressed brightly on the surface of T cells. If they are introduced into primary T
cells, there is an initial amplification with 4 to 5 population doublings, and then the cells die unless they're
re-stimulated.
However, one cell population keeps dividing, and that's the one encoding 4-1BB-ζ. They don't continue
to divide infinitely, but there is a large expansion that's due to constitutive activity of the 4-1BB signaling
domain, which is a member of the tumor necrosis factor (TNF) family. We've used this BB-ζ as a signaling
domain in our trials2.
The initial patients were adults with end-stage chronic lymphocytic leukemia (CLL). The patients had all
progressed on FDA-approved therapies, and only then got an infusion of the T cells. With the treatment
based on the HIV-based lentivirus and the BB-ζ CAR, there's a short T-cell manufacturing period of 10
days. The first vector lot, in July 2010, treated 3 patients; as of December 2012, 12 patients were treated.
Of the 12 patients, 10 had CLL, with a median of 5 prior regimens; 9 of the 12 had clinical responses, which
is high for a phase I trial, and 3 had p53 deletions or deletions in the 17p chromosomal region. Surprisingly,
all of them remain in remission, the longest of them for more than 2 years. The follow-up for the remaining
patients is short, however.
Scaling out:
The patients achieve remission after receiving modest cell doses between 15 and 150 million T cells. Thus,
the real issue now for the field is not how do we scale up but rather how do you scale this out. And the
answer to that is that we don't know.
At our center, this approach requires scientists with Ph.D.s to do the cell culture. Novartis licensed the
technique last August, and will attempt to do this using robotics. Our hypothesis is that T-cell engineering
can be automated like automobile production.
To assess response, bone marrow and blood is easiest to access; lymph nodes are more difficult because
they can contain residual masses even with complete responses. A waterfall plot of the CLL patients shows
that there's a high response rate, but 3 of the 12 did not respond, for reasons that are not yet clear.
The initial 3 patients had late-stage refractory and relapse CLL, and 2 of the 3 had p53 deletions, which
confers resistance to chemotherapy. These tumors were large, up to 7 pounds based on CAT-scan
assessments and measurements conducted on peripheral blood and bone marrow biopsies.
Figure 2
This patient, who had
relapsed three times after
chemotherapy, showed
complete response to CAR
T-cell therapy.
The remissions are now beyond 2 years. In
those patients, CAR cells expanded in vivo,
without the addition of interleukin-2 (IL-2). That
suggests that the patients have both targetdriven expansions through CD19, as well as
homeostatic expansions of 10,000-fold or more
from the infused cell mass3.
Looking at copies of the CAR per microgram of
genomic DNA, there's an exponential expansion
in the patients that peaks between 10 days to a
month after infusion of the cells. What has not
been seen on previous trials is that they persist
as memory CAR cells, with sustained expression
of the CD19 antibody, after a single infusion
of engineered cells. And these cells when reisolated from the patient are functional: They
can kill a CD19-expressing target4.
Harnessing the power of T cells
The best correlate of response so far is the degree of expansion of CAR T cells in the patients. These
patients have at least 1,000 CARs per microgram of genomic DNA within a month after the infusion. The
patients who don't respond have had little expansion. So this predicts a target-dependent expansion of the
CAR cells.
These patients don't have any residual normal B cells left, so they cannot be vaccinated. They also have few
T cells because of previous chemotherapy, and have not regained thymic function. The pediatric ALL cases
show a different pattern of immune reconstitution. They regain thymic function after 6-8 months and then
have an out-flux of new T cells.
The initial studies showed that the cells were not rejected even though we used a mouse antibody for the
CAR. Perhaps because of their profound B-cell aplasia, the patients don't make antibodies, and they also
do not mount T-cell responses against the CARs.
Target dependence:
In patients with CLL, the cells persist at higher levels in the bone marrow than in the blood. They also do
traffic to the central nervous system, but seem to have a niche in the bone marrow. That may because stem
cells in the bone marrow become common lymphoid progenitor cells and evolve into B cells.
There is probably a relationship between the tumor burden and CAR T-cell proliferation. If we treated
patients with earlier-stage (low burden) disease, the CAR expansion probably would not occur to that
magnitude.
That means that the toxicity is on-target, but it can be severe and may be related to tumor burden. In the
U.S., patients will have to be followed for 15 years according to present regulations to establish long-term
safety because it's a gene transfer using an integrating vector. However, there have been no issues so far.
CLL is an indolent disease. Would this CAR approach work in more rapidly growing malignancies?
We have tested the approach in the Ôpedi-CAR trialÕ, with 5 children who had pre-B acute lymphoblastic
leukemia (ALL). We could not begin this trial even though it's the same drug, CD19-CAR, until we had safety
data for adult patients with CLL, and therefore the clinical data are not as mature.
We treated the first child in April 2012; 4 of the 5 children have had a complete response. The activity in these
responses in relapsed pediatric ALL patients is striking, and perhaps even better than in CLL. We speculate
that perhaps this is because the T-cell proliferative capacity is larger in children than in older adults.
Figure 3
Efficient trafficking of
CTL019 T Cells to the
central nervous system
in ALL.
29
30
The first patient, reported in The New York Times, was sort of a "Lazarus case". She had relapsed 3 times
after chemotherapy and was in a DNR status at the time. She had a marrow that was packed with 60% ALL.
And then, 6 weeks later, after the last chemotherapy failed, she got a T-cell infusion.
Therefore, in this case, there was no chemotherapy that can be ascribed as having accomplished the
remission. The T cells were infused over 3 days at 10 million CAR cells per kilogram. She was aplastic at
that point based on white blood counts and lymphocytes and neutrophils, and then she went into remission
about 12 days later. The neutrophils came back, and the lymphocytes were all CAR T cells at 4,000 cells per
microliter. She had an exponential expansion at 12 days after the infusion of the CARs5.
In her bone marrow aspirate 6 days after infusion, 3% of the cells were CARs; at least 75% were CAR T
cells on day 23. She also had a reciprocal loss of CD19+ and CD20+ blasts in the marrow. She entered into
an MRD-level remission, and she remains in a complete response in ALL.
At baseline, 98% of the initial reads by sequencing were one dominant B-cell clone. Over a period of
6 months after treatment, however, we haven't been able to find that clone. It disappeared by clinical
assessment within 23 days, suggesting that the treatment rapidly triggers a deep molecular remission.
Side effects:
In the peripheral blood, the CARs have the morphology of activated T cells. Surprisingly, patients have also
had cells that trafficked through the cerebral spinal fluid (CSF), but they do not have any CNS side effects.
Tracking the blasts in both bone marrow and peripheral blood by flow cytometry shows that the ALL cells
are CD45-dim, and CD34+/CD19+. At a month after treatment, one patient lost this peripheral population of
cells, and had a complete response. But at 2 months, she relapsed, and had CD34+/CD19- cells, indicating
a loss of target.
The mechanism of this target loss is so far unknown, but it may indicate a biphenotypic leukemia or that a
subclone was present at baseline. The other patients have not had a target loss, but with any kind of cancer
therapy, multimodalities against different targets are likely to be necessary.
Figure 4
B-cell aplasia and
emergence of CD19 escape
in an ALL patient.
The side effects of the treatment in
these patients includes B-cell aplasia,
tumor lysis syndrome Ñ albeit delayed
Ñ and cytokine release syndrome.
Unexpectedly, the responding patients
have all had macrophage activation
syndrome, characterized by high IFN-γ
and IL-6 levels. There is also evidence
of hemophagocytosis in the bone marrow biopsies. This was not predicted in
our initial modeling in xenograft mice.
The cytokine release syndrome is
characterized clinically with high
non-infectious fevers, and high levels
of IFN-γ and IL-6, and it can lead to
multi-organ failure if not reversed. It
may occur in part as a result of the
high tumor burden. Strikingly, it's IL-6
dependent.
Harnessing the power of T cells
31
Treatment with tocilizumab, an FDA-approved drug for juvenile rheumatoid arthritis that blocks
IL-6, immediately reverses the syndrome in both children and adults. It's much more effective than
corticosteroids.
The first patient with pediatric ALL who was treated developed a fever of between 37 and 41 degrees.
Biochemical markers of tumor lysis syndrome peaked in the peripheral blood between day 6 and 12. She
was treated because of high fever and multi-organ failure with high dose methyl prednisolone, then Enbrel,
a TNF blocker, and then with tocilizumab. Only tocilizumab aborted the toxicity.
In the serum, as shown by Luminex analysis, IFN-γ and IL-6 went up 1,000-fold above baseline.
Hemophagocytosis and macrophage activation syndrome returned to normal when the tumor disappeared.
Cytokine patterns in the pediatric ALL are the same as reported in adults with CLL, so it's not targetdependent: The concomitant death of CLL blasts and ALL blasts can both lead to this pattern of cytokines.
We have completed this phase I study, and are analyzing the responders and non-responders. We've also
designed a dose-finding study, randomizing patients to low or high doses of the CAR cells. And with Renier
Brentjens at Memorial Sloan-Kettering Cancer Center in New York, we're doing a study funded by the
National Cancer Institute to compare two different CARs, one with a CD28 signaling domain, and the other
with the 4-1BB signaling domain. Novartis is starting larger phase II trials with CLL and ALL.
The larger question is, will CAR technology work for solid tumors? What it will take to make that work is
a big challenge. Target escape can happen, and combinations of CARs will be required for more complex
genetics in solid tumors. The other big issue now is scaling out Ñ as opposed to scaling up Ñ and learning
to do it robotically.
References
1. S
choller J. et al. Sci. Transl.
Med. 4, 132ra53 (2012
2. M
ilone M.C. et al. Mol. Ther.
17, 1453-1464 (2009)
orter D.L. et al. N. Engl. J.
3. P
Med. 365, 725-733 (2011)
4. Kalos M. et al. Sci. Transl.
Med. 3, 95ra73 (2011)
5. Grupp S.A. et al. N. Engl. J.
Med. 368, 1509-1518 (2013)
Harnessing the power of T cells
Targeting the immune system while
targeting the cancer. Therapy customized for the patient vs. Ôoff-the-shelfÕ
A report on a lecture by
Ronald Levy
Division of Oncology – Stanford University, Stanford, USA
Many approaches to immunotherapy require the production of a protein, gene or cell that is customized for each
patient. While scientifically exciting, these approaches strain the limits of available resources and are not ready
for widespread application.
Instead, Rituximab, a generic antibody against the CD20 molecule present on B
cells, became a blockbuster drug for B-cell lymphoma. Ritixumab and many other monoclonal antibodies work
by antibody-dependent cytotoxicity mediated by NK cells. Second-generation antibodies, engineered to interact
better with Fc receptors on NK cells, are being tested in the clinic. However, the killing activity of effector cells
seems to be the rate-limiting step, and new antibodies against targets that enhance the activity of the killer cells,
such as CD47 and CD137, are likely to boost the effectiveness of monoclonal antibody therapy. Two companies,
Pfizer and Bristol Myers-Squibb each have an antibody against CD137 in the clinic. An alternative approach
is to vaccinate or immunize against cancer by injecting immune stimulants and checkpoint antibodies locally
into one site of tumor and thereby induce an immune response that attacks tumors throughout the body. This
approach works even for sanctuary sites, such as the brain. Ronald Levy described the advantages of ‘off-theshelfÕ immunotherapy for cancer.
There are many small molecule drugs that target critical signaling pathways in cancer. Like BRAF inhibitors
for melanoma, BTK inhibitors will be blockbuster drugs, inducing high frequency of durable remissions in
patients. But resistance to BRAF inhibitors has quickly developed in melanoma. Resistance of lymphomas
to BTK inhibitors is just beginning to be described.
My goal eventually is to induce remissions using these small-molecule targeted therapies and then to
enhance the immune system to sustain these remissions. Hopefully we will be able eventually to eliminate
the use of cytotoxic chemotherapy.
When we started making monoclonal antibodies against lymphoma, we produced so-called anti-idiotype
antibodies, which are highly customized to each individual's B-cell clone. The famous first patient we
worked on was Philip Karr. After he had failed to respond to all the conventional treatments, we treated
him with an antibody made for his own tumor. The tumor responded dramatically, and he is now in his 90s,
more than 35 years later, and free of his lymphoma.
Over the following 10 years, we did this
for about 50 more patients and saw a
high frequency of clinically significant
tumor responses, and we launched a
company called IDEC. It turned out that
customization was not the problem; the
real driver of cost was manufacturing
and labeling it to the specifications of
the Food and Drug Administration,
along with the requirements for
demonstration of quality, purity and
safety the agency requires.
Figure 1
Monoclonal antibody
targets.
33
34
Instead, the company made a monoclonal antibody against CD20, a molecule present on B cells. This
antibody, called rituximab, is not at all customized. It temporarily wipes out all normal B cells. However,
surprisingly, this does not make patients immunodeficient. Rituximab is now the biggest selling cancer
drug in the world, with $5 billion of annual sales.
There are several other successful antibodies approved for cancer, including Herceptin for breast cancer
and Erbitux for colon cancer. However, these drugs cure very few people. How do these antibodies work?
There is evidence that they generate direct signals to kill the tumor cells. There is also evidence for
complement-mediated killing, for antibody-dependent cellular cytotoxicity (ADCC), antigen presentation
and cross-priming.
Rituximab loses its therapeutic effect when the Fc receptor (FcR) is not present on the killer cells,
suggesting that the FcR is required for antitumor cytotoxicity. There is also genetic evidence supporting
this. When a particular polymorphism in the FcR is homozygous for valine, which binds more strongly to
the Fc of the antibody, there is an 80% chance that the lymphoma responds to rituximab. That drops to 40%
with either the homozygous phenylalanine or the heterozygous form of the FcR.
We give the drug to everybody because 40% is not bad for a therapy without many side effects. So we don't
use this test in the clinic, but it gave us clues to how the antibody works, and gave the industry clues for
making a better drug.
Combination therapy:
A number of companies produce second-generation anti-CD20 monoclonal antibodies by engineering
the sequence of this antibody for better ADCC in vitro. But results in the clinic have not borne out. For
example, in a head-to-head comparison of GA101 versus rituximab for follicular lymphoma, the endpoint
of progression-free survival was equivalent.
This suggests that if the rate-limiting step is not the strength of binding to the FcR, but rather the killer
cell's ability to do the job.
If that is true, why not goose the killer cell rather than make a better antibody? We have a second antibody
that enhances the activity of the first antibody by activating the killer. A gene expression profile of this
killer cell shows that when it encounters an antibody-coated target cell, many things happen, including an
increase in the level of CD137.
Figure 2
FcγRs are required for
antitumor cytotoxicity of
therapeutic antibodies.
CD137, also known as 4-1BB, is a signaling motif
and a member of TNF superfamily of receptors. It
is expressed on T cells, monocytes and dendritic
cells, and, most importantly, on NK cells when
they become activated. An activating antibody
against CD137 enhances ADCC.
Human CD56+ NK cells normally do not express
CD137, but when they are exposed to a tumor
coated by rituximab, nearly all the cells express
the marker. This effect is not specific for rituximab
or lymphoma: The same thing happens with
trastuzumab and a HER2+ breast cancer cell, or
with cetuximab and an EGFR+ colon cancer cell.
The expression of CD137 in NK cells peaks at
about 24 hours, so the timing for bringing in the
second antibody is crucial.
Harnessing the power of T cells
Mouse experiments show that each antibody, for CD20 or CD137, has some therapeutic effect but together,
they have a synergistic effect that cures the animals of their tumor1. This effect can be tracked with optical
imaging. Similarly, in a nude mouse with a human breast cancer xenograft, a combination of Herceptin
(trastuzumab) and the agonizing antibody produces a synergistic therapeutic effect and long-term survival
of the animals2.
Targeting with the first antibody is critical. In an animal with two tumors, one with HER2, and the other
without, growth of the targetable tumor slows in response to trastuzumab alone. With the second antibody,
the animals are cured of the targetable tumor, but not the non-targetable tumor. This establishes the
principle that the first antibody has to see its target, and then the second antibody can make the effect
more powerful.
Emerging approaches:
Two companies, Pfizer and Bristol Myers-Squibb each have an antibody against CD137 in the clinic. Pfizer's
is a human IgG2 agonistic monoclonal antibody and blocks the ligand. We have a phase I trial in solid
tumors in combination with rituximab for lymphoma.
Bristol Myers-Squibb's drug is a human IgG4, agonistic monoclonal antibody, and does not block the ligand.
There is a similar phase I trial in solid tumors and lymphoma, and then in combination with rituximab.
These trials are ongoing.
My conclusion about antibodies is that we've just scratched the surface. They kill cancer by ADCC. They
increase ADCC not so much by reengineering the antibodies, but by going after the NK cells that do the
killing. The NK cells dial up CD137 when they encounter antibody-coated tumor cells Ñ and there are other
targets to go after as well.
An appropriately timed agonistic antibody against CD137 will enhance the activity of rituximab against
lymphoma, Herceptin against breast cancer, cetuximab against head-and-neck and colon cancer, and is a
general strategy for enhancing the efficacy of a monoclonal antibody.
In a completely different approach we have found that injecting the proper immune stimulants locally into
one tumor can induce an immune response that attacks tumors throughout the body.
When we kill some tumor cells, they
release their antigens. We can then
inject an immune stimulant and, in
addition, using antibodies against
targets on regulatory T cells (Tregs),
we can take the brakes off the immune
response, resulting in tumor regression
at sites throughout the body.
The first molecule we investigated (CpG)
is a ligand for a receptor that is present
on
lymphoma
antigen-presenting
cells. It is a small piece of DNA with a
certain sequence of unmethylated Cs
and Gs. The toll-like receptor 9 (TLR9)
recognizes these motifs of unmethylated
DNA in bacteria and fungi, and is set up
to trigger the innate immune system of
DCs and B cells.
Figure 3
Phase I clinical trial of
rituximab and anti-CD137
monoclonal antibody.
35
36
This works in animal models, and we set up a clinical trial with patients who have lymphoma, with
measurable tumor in many parts of the body. We used a low dose of radiation, just enough to kill some
tumor cells at the local site, and then injected the CpG.
For example, in one 38-year-old patient, we treated and injected a tumor containing lymph node behind
his neck and then monitored tumors at other parts of the body. Over time, the patient showed a complete
regression of lymph nodes throughout his body, bone marrow, as well as subcutaneous masses.
In a trial of 15 patients, several completely regressed their tumors, a couple of patients had about 50%
reduction, and some had more minor effects3.
Amplified responses:
To improve this response rate we can take the brakes off the killer T cells, by eliminating the negative
Tregs. Tregs that are present at tumor sites express the CTLA4 and OX40 molecules.
How do we know Tregs recognize the cognate antigen? If a transgenic animal whose T cells all recognize
ovalbumin has two tumors, identical except for the fact that only one of them expresses ovalbumin, there
are many more Tregs in the tumor that has the antigen than in the tumor that does not.
Depletion of intra-tumoral Tregs using anti-CTLA4 or anti-OX40 triggers systemic antitumor immune
response. It is possible to inject the antibodies directly into the tumor, which can keep the dosage low and
help avoid autoimmune toxicity.
At the site of injection of the antibodies, the FOXP3+ CD4+ Tregs disappear because they are killed by the
ADCC.
On the other side of the body, the tumor regresses as a result of this local immunization maneuver. You can
get this effect with one-thousandth of the usual systemic dose of the antibodies.
This approach works even in sanctuary sites such as the brain. In the animal model with tumors on both
sides of the body, if there is a third tumor in the brain, the local in situ vaccination induces a systemic T-cell
immune response, which eliminates tumors throughout the body, including the brain. The brain even has
memory of this response. If the same tumor is reinjected into the brains of surviving animals, the animals
reject the tumor.
Figure 4
Enhancing vaccination.
In terms of drug development, antiCTLA4 is available, and anti-OX40 is
being developed by several groups
and will soon be available in the
clinic.
Based on these preclinical results
we are conducting a clinical trial
in patients with melanoma, colon
cancer and lymphoma. We are
using the anti-CTLA4 antibody,
ipilumimab, at one-tenth of the
currently
approved
dose
for
melanoma. We are hoping to prove
that we can deplete Tregs from the
locally injected site, that we will
induce a systemic T-cell response
and we hope to see regression of
tumor at uninjected sites in the body.
Harnessing the power of T cells
37
In summary, in situ vaccination with intratumoral TLR9 ligand induces CD8 anti tumor T-cell immunity.
T cell antitumor immunity can be enhanced by antibodies against immunologic checkpoints. Combinations
of antibodies are synergistic. Distant uninjected tumors can be eradicated, even in the brain. Most
importantly, this therapy is Ôoff-the-shelf,Õ with customization only at the point of delivery4.
References
1. K
ohrt H.E. et al. Blood 117,
2423-2432 (2011)
2. K
ohrt H.E. et al. J. Clin.
Invest. 122, 1066-1075
(2012)
rody J.D. et al. J. Clin.
3. B
Oncol. 28, 4324-4332
(2010)
4. Brody J. and R. Levy Blood
114, 4477-4485 (2009)
Harnessing the power of T cells
Will cellular immunotherapies ever
become an 'industry standard'?
A report on a lecture by
Malcolm K. Brenner
Department of Molecular and Human Genetics – Baylor College of Medicine
Houston, USA
Despite more than two decades of clinical application, cellular immunotherapies for cancer have, almost without
exception, failed to make the transition into licensed drugs that are standard of care for patients with malignant
disease. At least part of the delay can be attributed to the dissimilarity between the business models needed to
bring standard small-molecule drugs to success and those required for cellular immunotherapies. Unlike small
molecules, cellular immunotherapies are usually individualized medicines, intended to be curative rather than
ameliorative. They have complex intellectual property, continuing high manufacturing costs, and they require
iterative cycles of preclinical and clinical development. One historic success story for cellular immunotherapy
is the treatment of EBV-associated lymphoid and epithelial malignancies by tumor-specific T cells. At least 4
factors contribute to this success: the infused T cells target strong and unique antigens, presented with ample
accessory signals and co-stimulation; the T cells contain polyclonal and multispecific memory population;
the tumor lacks potent immune escape mechanisms; and the post-transplant environment favors lymphoid
expansion. Malcolm K. Brenner elucidated the factors needed to make cellular immunotherapies the standard
of care.
The idea of using T cells to treat cancer has had a long history. Despite a few striking successes, however,
there are no licensed products based on this approach.
Among the few successes is the effective and safe treatment of Epstein Barr Virus(EBV)-associated
malignancy after stem cell transplantation. EBV infects more than 90% of the population, followed by
lifelong latency. A limited array of viral latency proteins, which are usually benign, occasionally trigger
malignant change in B cells, T cells and some epithelial cells.
The expression of latent antigens falls into three patterns, which correspond with the various stages of
the viral life cycle. Type 3 latency expresses many different antigens, is highly immunogenic, and can only
occur in patients who are severely immune-compromised Ñ for example, post-transplant lymphoma or
HIV-associated lymphoma.
Type 2 latency is much weaker, with more restricted expression of antigens, and can occur in people with
normal immune systems. It includes Hodgkin's lymphoma, non-Hodgkin's lymphoma and nasopharyngeal
carcinoma. Type 1, which includes Burkitt's lymphoma, expresses the fewest antigens.
More than 20 years ago, we began treating type 3 latency tumors in people who had received allogeneic
stem cell transplants. About 10 to 15% of them developed rapidly progressive and inevitably fatal B-cell
lymphomas. We made cytotoxic T lymphocytes (CTLs) against EBV-infected B cells and gave them to the
patients after genetically marking them so we could track their fate in vivo.
Small cell numbers, about 10 million cells per patient or sometimes even less, resulted in more than 3-log
proliferation in vivo, and long-term persistence of more than 10 years. In the more than 130 patients who
received this as prophylaxis, none of them developed the disease compared with 12% of controls. There
has also been complete and sustained resolution of the tumor in 11 of 13 people with resistant lymphoma.
39
40
One of the two patients who didn't respond was moribund at the time of infusion and died within two days.
The second case was instructive. This person received a mismatched cell line that only recognized one
portion of one EBV antigen and initially responded, but subsequently relapsed.
These studies give us the four rules for successful T-cell therapy: strong and unique antigens presented
with ample co-stimulation, T cells that contain polyclonal and multi-specific memory population so that
they can expand and persist long-term, a tumor that lacks potent immune escape mechanisms, and an
environment that favors some form of lymphoid expansion.
Critical conditions:
However, not all of these conditions need to be present to have some success. For example, even with
weak antigens and a tumor with some escape mechanisms, the results have been reasonably positive. Of
23 patients with EBV-associated Hodgkin or non-Hodgkin EBV lymphoma of Type 2 latency (i.e. only weak
tumor-associated antigens expressed) more than half had complete responses sustained long-term.
What's clear, though, is that if none of those criteria are present, the treatment will not be successful. That
was illustrated with the first few years of the chimeric antigen receptor (CAR) T-cell therapy (see June,
page 25).
What everyone's been doing since then is trying to compensate for these deficiencies one by one. The first
compensation is to try and include co-stimulation as the T cell encounters the antigens so that it doesn't
get just an antigen signal but gets all the accessory signals as well.
Figure 1
Incomplete activation of
first generation CARdirected T cells.
There are two ways of doing this: either modify
the artificial receptor so that it incorporates a
co-stimulatory endodomain, such as 41BB or
CD28, or use complementation from the native
receptor1.
The idea is that when a first generation CARdirected T cell encounters a tumor, it gets an
antigen-directed signal, but it doesn't get an
accessory signal Ñ for example, through CD28
or B7. If the CAR incorporates a co-stimulatory
endodomain, then it gets both an antigen signal
and a co-stimulatory signal at the same time,
improving activation and increasing killing.
To evaluate the influence of the CD28 co-stimulatory endodomain on performance, we made a CAR that
targets CD19, present on most B-cell malignancies. One receptor had just the signal transduction domain
and one also had the co-stimulatory domain. These were distinguishable on PCR.
The conventional CAR without the co-stimulation was present at low levels or not at all in peripheral blood,
whereas the cells that contained the co-stimulatory domain had increased in number. These cells could
persist for up to 6 months.
B-cell malignancies are clonal, and because they're malignant, express either k- or λ -Ig light chains. If
you eradicate the malignant clone and either all normal κ- or all λ-B cells, it will spare at least half the
immune repertoire and ensure that the patient is not B cell-deficient throughout life.
We made κ-directed CD28 CAR and have given it to 6 patients with κ B-cell malignancies. One patient with a
large resistant B-cell lymphoma showed a complete response within 6 weeks of giving a single dose of the
κ-CAR activated T cells. In a second patient with Waldenstrom's, an IgM κ-myeloma, pretreatment reduced
the size of large nodes to about half size by 6 weeks post-infusion.
Harnessing the power of T cells
Careful choreography:
It's not just B cells that are amenable to CAR killing. When a patient who had EBV-Hodgkin's disease with
residual disease in the jaw and the spine was treated with a CD30-directed CAR, there was an apparent
tumor flare but, by 4 months, the disease was gone. He has so far remained free of disease.
The problem, however, is that co-stimulation is extremely complex. There are many interactions that need
to occur in a carefully choreographed manner. It's difficult to replicate that with one or two constitutively
expressed co-stimulatory endodomains on a single CAR.
That may not matter for hematologic malignancies because they may be able to provide many of these costimulatory signals. But most solid tumors require more care and consideration.
We modified EBV-specific CTLs and got them to express a CAR directed to the solid tumor. These cells
encounter the viral antigens on the EBV-infected B cell, which is an excellent antigen-presenting cell and
provides the necessary and physiologically sequenced co-stimulation. The CAR-CTL is then licensed and
can then kill the tumor cell2.
We did the first study of this approach for
neuroblastoma, the most common extracranial
solid tumor in children. The disease is treatable,
but if it relapses, it's essentially incurable.
Neuroblastomas express a target antigen,
GD2 (disialogangloside), which is expressed at
high density on the tumor cells. A monoclonal
antibody to GD2 gets some clinical response,
but isn't curative on its own.
In the study, we transduced patients with
EBV-directed CTLs and GD2-recognizing CAR,
incorporating a distinct oligonucleotide in the
transducing vectors so we could distinguish
which cell type was expanding in vivo. The first
patient received activated T cells transduced
with a short oligo vector, and the EBV-CTLs
transduced with the longer oligo vector.
We reversed the order in the second patient so that we could attribute any differences in cell survival in vivo
to the differences in the biology of the cells, rather than to differential effects of the vectors.
As we had hoped, the CAR-CTLs persisted longer than the CAR-T cells. Overall, of 11 patients treated with
relapsed resistant disease, 3 entered complete remission and several others had some, albeit transient,
tumor response.
Multiple antigens:
Overall, however, solid tumors are extremely heterogeneous, not just among patients but within them. It's
unlikely that a single antigen will be present on all tumor cells in every patient, or present only on tumor
cells. The solution to that problem is to make these CARs multi-specific.
That can be done by expressing either two different CARs Ñ on the same cell or on different cells Ñ or
expressing distinct tumor-targeting receptors that are native and chimeric in their origin.
One candidate antigen for this approach is HER2. Some tumors that express high levels of HER2 can be
effectively targeted with the monoclonal antibody to HER2, but there are many others, such as glioblastoma
multiforme, that express HER2 at a low level.
Figure 2
CT showing hypodense
neuroblastoma lesions in
liver after GD2-directed
T cells.
41
42
These tumors can be completely resistant to
even the highest levels of the antibody. However,
they are sensitive to the antibody expressed as a
CAR on T cells, presumably because of the higher
overall avidity. HER2-specific T cells efficiently
kill tumor cells, both ex vivo human cells and in
vivo in orthotopic xenograft models3.
Figure 3
HER2-specific T cells
induce regression
of autologous GBM
xenografts.
Glioblastoma is also associated with expression
of at least some CMV proteins. It's unclear
whether these proteins are present in the tumor
itself, in the tumor vasculature or in tumorassociated tissue. It's also not clear whether
they're associated with the malignant process.
Regardless, they offer an opportunity for direct
targeting or, at the very least, for getting costimulation at the site of the tumor.
CMV-specific CTLs recognize CMV through their native receptor. And they can be transduced with a
chimeric receptor directed to HER24. These cells kill MHC-matched CMV-positive cells even if they're
HER2-negative, and kill HER2+ MHC-mismatched cells, even if they're CMV-, so they kill both through
their chimeric and their native receptor.
The plan was to give these cells to people with advanced glioblastoma who had recurrent disease after
frontline therapy. Unfortunately, these patients have a short life expectancy. For various reasons, we had
to delay the start of this protocol for a year and when we did begin it, were only allowed to give 10,000 cells
per patient.
Not surprisingly, those first patients did not respond. More recently, we have been able to give 10 million
cells, and are beginning to see a hint of a response. One patient has had stable disease for 6 months, which
is not at all typical of late-stage patients. A second patient's tumor shrank from about 43 mm to 24 mm and
then to 21 mm after a single dose of the cells.
Immune escape:
Another challenge in treating solid tumors is that they have a great many different immune escape
mechanisms (see Coukos, page 101). It may be that these tumors need so many escape mechanisms
because none of them alone is sufficient. Subverting just one or two of them might be able to tip the
balance into effective activity.
TGF-β is secreted by many tumor cells in the stroma, and inhibits CTL proliferation and functionality. A
known dominant negative variant of TGF-β stops signal transduction because of its truncated intracellular
domain. When this variant is expressed in CTLs, SMAD is not phosophorylated, and it doesn't trigger the
downstream signaling normally produced by TGF-β.
Among people with non-Hodgkin's lymphoma who failed to respond to EBV-directed CTLs, at least two
patients have shown sustained responses, suggesting that these CTLs have properties superior to those
of the original CTLs.
For therapies to be successful, there also needs to be an environment that favors homeostatic lymphoid
expansion. Blocking immune checkpoints is clearly the most exciting opportunity in this arena, but there
are other ways.
Harnessing the power of T cells
43
One of them is to administer recombinant growth factors, and another is to have the engineered T cells
express receptors or cytokines themselves. The crudest Ñ but nonetheless currently most effective Ñ way
is simply to lymphodelete the patient. We have begun to do that with GD2 CTLs directed to neuroblastomas.
One child with advanced neuroblastoma had a stem cell transplant followed by administration of GD2expressing virus-specific T cells. The cells showed a near three-log expansion in peripheral blood. By
about 4 weeks after infusion, more than 50% of the T cells in the child's marrow were expressing CARs.
This enormous expansion of T cells occurs even though this CAR lacks any endogenous co-stimulatory
endodomain. The immunological response was associated with a near-complete tumor response, and this
bedridden child was able to play basketball, albeit briefly.
This emphasizes how active the immune system can be if properly stimulated and treated. The keys are
to target the T-cell therapies to strong and unique combinations of antigens; to ensure co-stimulation,
either by incorporating endodomains or by using virus-specific T cells; to incorporate countermeasures to
immune escape mechanisms; and to ensure that the environment favors lymphoid expansion.
How do we enable these successes to be standard practice? Some of this is a matter of process development,
but there are also fundamental conceptual obstacles.
These complex biologic therapies are often individualized, and rarely linear in their development. Academic
involvement often continues well into early phase II trials. Pharmaceutical companies prefer drugs that
must be taken for a long time, not those that are curative with a single dose. Shortage of trained clinical
researchers and daunting amounts of paperwork are also both obstacles.
Fortunately, drug companies are
beginning to recognize the value of agents
that are targeted to relatively small
numbers of patients. We have developed
a research collaboration with Celgene
and BlueBird Bio in which the skill sets of
each component should complement one
another. Ultimately, what is needed is an
economic model, based on effectiveness
that will lead to resource reallocation,
not an unsustainable resource increase.
Figure 4
Generation of CMV-specific
CTL using Ad5f35 vectors.
References
1. S
avoldo B. et al. J. Clin.
Invest. 121, 1822-1826
(2011)
2. Savoldo B. et al. Blood
110, 2620-2630 (2007)
hmed N. et al. Clin.
3. A
Cancer Res. 16, 474-485
(2010)
4. Leen A.M. et al. Nat.
Med. 12, 1160-1166
(2006)
45
PART II: E x p l o i t i n g c h e c k p o i n t b l o c k a d e s
Rafi Ahmed
T-cell memory and exhaustion
James P. Allison
Immune checkpoints and cancer: New insights and opportunities
Antoni Ribas
ACT immunotherapy and BRAF targeted therapy for melanoma
Exploiting checkpoint blockades
T-cell memory and exhaustion
A report on a lecture by
Rafi Ahmed
Basic Immunology/Virology Division – Emory University, Altanta, USA
Acute viral infections result in the generation of a long-lived and self-renewing pool of highly functional memory
CD8+ T cells. These memory CD8+ T cells play an important role in faster control of infection upon re-exposure
to the same pathogen. In contrast, chronic antigen stimulation during persistent viral infections or during cancer
can lead to functional exhaustion of CD8+ T cells. A characteristic feature of these exhausted CD8+ T cells is
sustained expression of inhibitory receptors such as PD1. It is now well established that PD1 plays a major
role in T-cell exhaustion and that blockade of the PD1 inhibitory pathway can restore function in exhausted
T cells. The role of PD-1 in T-cell exhaustion was first described in mice during chronic LCMV infection and
these observations have been extended to other chronic infections in mice, non-human primates and humans.
Most recently, PD1-directed immunotherapy has shown promising results in a phase I clinical trial in cancer
patients. Rafi Ahmed described a new role for PD1 in regulating T-cell differentiation and discussed strategies
for enhancing PD1-directed immunotherapy.
A mouse model of lymphocytic choriomeningitis virus (LCMV) infection is useful for studying what happens
during CD8+ T-cell differentiation in either an acute or a chronic setting. Some strains of LCMV cause an
acute infection, whereas others cause chronic infections.
The other advantage to this model is that the T-cell epitopes in chronic and acute infection are identical, so
the same CD8+ T-cell epitopes can be tracked in either setting.
LCMV is a pantropic virus, infecting many different cells in the mouse including dendritic cells (DCs) and
many non-lymphoid tissues. In the early phase, a CD8+ T cell sees an antigen presented by a DC. The
system generates an optimal T-cell response Ñ the right DC presenting the antigen, the right inflammatory
signals, and the appropriate CD4+ T-cell help.
Under these conditions, the T cells undergo rapid proliferation. They go through a full cell cycle within 6 to
8 hours, or roughly 3 cycles in a 24-hour period, resulting in a 10-fold expansion. The division starts 2 days
after infection, with 3 doublings per day, so there are rapid increases up to 100,000-fold or even greater.
This rapid proliferation is coupled to
differentiation. The cells express genes
involved in killing target cells, including
high expression of perforin, granzyme and
inflammatory cytokines, and are critical in
controlling acute infection. After the infection
is resolved, 95% of the effector CD8+ T cells
die. The other 5% survive to generate a longlived pool of functional memory cells.
In a chronic infection, the virus outpaces
the T-cell response and then persists and
disseminates in many tissues. Instead of
resulting in a functional pool of T cells, the T
cells become functionally exhausted.
Figure 1
CD8+ T-cell differentiation
during acute vs. chronic
viral infection.
47
48
In the ideal scenario, a memory T cell is activated upon re-encounter or re-exposure to virus and produces
interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α)-and interleukin-2 (IL-2). This response is remarkably
rapid: The cells secrete IFN-γ within minutes and kill the virus-infected cells within a couple of hours.
Their limiting factor is location Ñ if the memory T cells are not in contact with the virus Ñ but once they
see antigen, the response is rapid. They become effectors first and also have the capacity to proliferate
extensively.
Under conditions of antigen persistence, there is a hierarchical loss of function: The higher the antigen
load, or the longer the duration of antigen's presence, the more of these functions are lost. The first
function lost is the ability to make IL-2 and to undergo proliferation. So, the cells may continue to make low
amounts of IFN-γ and TNF-α, but their proliferation capacity is diminished and they are not efficient killers.
There is also an inverse correlation with CD4+ help. The lower the help from CD4+ T cells, the faster the
CD8+ T cells become exhausted. These results have since been extended not only to other mouse models
of chronic infection, but also to infections in people. HIV infection or hepatitis infection also result in T-cell
exhaustion, as do many tumors.
These results raise a number of questions: What is the mechanism of T-cell exhaustion? More importantly,
is it possible to reverse exhaustion and get functional memory cells?
Gene signature:
Gene profiling has shown that several inhibitory receptors, such as CTLA4, LAG3 and PD1 are expressed
at high levels in exhausted cells but not in memory cells1. One receptor that really stands out not only in
terms of the expression profile but also the functional effect is PD1.
In a chronically infected mouse, the T cells express high levels of PD1. Antibodies against either the
inhibitory receptor PD1 or its ligand PD-L1 partially restores function. And that is accompanied by a
reduction in the viral load. This was one of the first demonstrations that it is possible to restore function in
exhausted T cells using immunotherapy2.
PD1 is an inhibitory receptor with two ligands, PD-L1 and PD-L2. PD-L1 is promiscuously expressed
on most cells, and highly expressed in many tumors. Expression of the PD-1 ligands is upregulated by
cytokines such as IFN-γ and TNF-α. PD-L2 has a much more restricted expression, and its role is not as
well understood.
PD1's interaction with PD-L1, either in an infected cell or tumor cell, inhibits or attenuates T-cell receptor
(TCR) signaling. So, this has the opposite action to that of a co-stimulatory molecule.
It turns out that HIV-specific CD8+ T cells are functionally exhausted, and express high levels of PD1 on
the cell surface. In vitro, treating cells with antibodies to either PD1 or PD-L1 restores some function back
to these cells.
In the SIV monkey model, PD1 blockade results in expansion of SIV-specific CD8+ T cells with improved
functional quality. This enhanced T-cell immunity is associated with reductions in viral load. Importantly,
the PD-1 blockade also prolongs the survival of SIV-infected monkeys.
Figure 2
Synergistic effect of
therapeutic vaccination
and PD-1/PD-L1 blockade
during chronic infection.
Interestingly, in the case of hepatitis C infection, HCVspecific CD8+ T cells in the blood are also functionally
exhausted, but their expression of PD1 is not very high.
However, in the liver, the target organ for this disease, the
cells show high levels of PD1 and more severe exhaustion.
We examined the therapeutic potential of PD1 blockade
in chronic HCV infection in chimpanzee models. Only 3
chimpanzees have been chronically infected with HCV
so far.
Exploiting checkpoint blockades
Before PD1 blockade, one chimp infected for 12 years with
HCV had no detectable HCV-specific T cells in the blood,
and only minimal levels in the liver. PD1 blockade in these
chimps had no effect. Two other chimps were both 2 years
post-infection, but had slightly different outcomes in
terms of their T-cell response. PD1 blockade again had no
effect on one of these chimps, but on the other chimp Ñ
which, during the acute phase, had made a broad attempt
to bring the viral load down Ñ it showed a striking effect.
49
Figure 3
Lessons from blockade experiments.
In this second chimp, PD1 blockade dropped the viral titer more than 100-fold, and was accompanied by
an increase in the CD8+ T-cell response. This tells us something about where this kind of therapy might
or might not work.
If the T cells are not there to begin with, or if the response is extremely narrow, PD1 therapy may have
its limitations in a viral model. However, if the T-cell response is reasonably broad, and the cells are
exhausted, the blockade may be very effective. That's because PD1 doesn't create new T cells, it merely
restores function in exhausted T cells that are already there.
Combination therapies:
There have been extremely promising results with PD1 therapy
for cancer3. There is much interest in particular in combining PD1
with therapeutic vaccination, existing drugs, or with the blockade
of other inhibitory receptors, such as CTLA-4, LAG-3 or TIM-3.
Figure 4
The progression from
naïve cells to exhaustion.
In mouse models, a combination of PD1 with therapeutic
vaccination is synergistic4. This was surprising because the amount
of antigen created by the therapeutic vaccine Ñ in this case a POX
virus vector Ñ is extremely low compared with the total amount of
antigen already in the mouse. The combination works because the
vaccine targets DCs, making the antigen-presenting cells (APCs)
much more functional.
However, at least in this viral model, there isn't much synergy with anti-PD1 and anti-CTLA4. In contrast, a
combination of anti-PD1 and anti-LAG3 has shown clear synergy. Blocking TIM-3 or LAG-3 by itself shows
some rescue of T cells, but does not reduce the viral load. Also, depleting regulatory T cells increases
T cells, and rescues exhaustion, but has minimal effect on viral load. The viral load only comes down when
PD1 blockade is added to the mix.
A combination of IL-2 and PD1 blockade has shown the most striking synergy in terms of increasing T-cell
numbers, rescuing T-cell exhaustion, and in reducing the viral load5.
References
Together, these results suggest that PD1 is important. There are two aspects to resolving chronic infection.
First, T-cell function needs to be restored. PD1 does this, but there are probably other ways to do it. Once
function is restored, the T cells still have to kill the tumor or virally infected cell. These T cells still express
high levels of PD1, however, and when they contact the target, they still receive a negative signal from the
PD1/PD-L1 interaction.
1. W
herry E.J. et al. Immunity 27, 670-684 (2007)
This interaction is a critical aspect of PD1's effects. Other immune therapies, such as IL-2 or anti-CTLA4
don't eliminate the PD1/PD-L1 interaction, which tells the T cells not to kill. This might be the reason only
PD1 blockade, by blocking that interaction, succeeds in reducing the viral load.
3. R
ibas A. N. Engl. J. Med.
366, 2517-2519 (2012))
2. Barber D.L. et al. Nature
439, 682-687 (2006)
a S.J. et al. J. Exp. Med.
4. H
205, 543-555 (2008)
5. West E.E. et al. J. Clin.
Invest. 123, 2604-2615
(2013)
Exploiting checkpoint blockades
Immune checkpoints and cancer:
New insights and opportunities
A report on a lecture by
James P. Allison
Dept. of Immunology – MD Anderson Cancer Center – The University of Texas
Houston, USA
Decades of efforts to produce therapeutic vaccines for the treatment of cancer have been disappointing. That’s
because although T-cell responses are initiated by engagement of the antigen receptor, they are shaped by
co-stimulatory as well as multiple inhibitory pathways that act in concert to shape the magnitude, quality
and location of the immune response. CTLA-4 is a critical down-regulator of T-cell responses, and CTLA-4
blockade allows prolonged T-cell responses. Anti-CTLA-4 antibodies exert their antitumor effects by multiple
mechanisms. The best studied such antibody is ipilimumab, developed by Bristol Meyers-Squibb. Ipilimumab
has been used in more that 17,000 patients. In early trials, objective responses were observed in metastatic
melanoma, castration-resistant prostate cancer, renal cell carcinoma, ovarian cancer and others. PD1, another
checkpoint, has a different mechanism of action. It recruits a phosphatase and seems to interfere with T cell
antigen receptor mediated signaling. Antibodies to PD1 and PD-L1 have both shown objective responses against
several tumor types in clinical trials. James P. Allison argued that because CTLA-4 and PD1 have different
mechanisms of action, a combination of the two might be better than either alone.
There are three reasons for doing active immunotherapy. The first is that T cells have specificity for
peptides, including those that are generated by mutations in a cancer cell during carcinogenesis. Second,
T cells persist for a long time and retain the memory that can keep cancer cells from recurring. Finally,
the immune system can adapt to changes in the tumor cells, so there is no resistance to immunotherapy
as there is to other drugs.
So far, however, cancer vaccines have been disappointing because researchers did not take into account
the fact that there are signals that serve to limit immune responses. It was only in the mid-1990s and later
that CTLA-4 was recognized as a critical inhibitor of immune responses. Without it, mice die when they're
about 3 weeks old.
Monoclonal antibodies that block CTLA-4 can take the brakes off the immune response, allowing T cells to
eliminate tumors. Because this approach targets the immune system and not the tumor, it may work for
any kind of tumor. It could also be used as a monotherapy to take advantage of the necrotic cell death that
might occur during tumor growth and
benefit from cross-priming. It can also
be used to increase the effectiveness
of other cancer therapies, including
radiation, some kind of chemotherapy,
and the new 'targeted' therapies.
In a colorectal carcinoma transplanted
into mice, blocking CD28 makes
the tumor grow faster. If α-CTLA-4
antibodies are injected, the tumor grows
for a time but is then rejected. The
mice are permanently immune. This
approach turned out to be effective in
many different kinds of tumors in many
different kinds of mice.
Figure 1
CTLA-4 blockade enhances
tumor-specific immune
responses.
51
52
In many cases, this requires combinations of drugs. For example, with B16 melanoma, a GVAX tumor cell
vaccine with GM-CSF doesn't work well therapeutically. Similarly, α-CTLA-4 antibodies by themselves
have no effect on the growth of the tumor. But combining those two approaches cures essentially 100% of
the mice.
Within tumors, there are regulatory T cells (Tregs) that make FOXP3, interleukin-10 (IL-10), TGF-β and
other factors that can interfere with an immune response. The tumor has a few other kinds of immune
cells.
With the addition of GVAX and an α-CTLA-4 antibody, the vasculature becomes activated and begins
expressing ICAM and VCAM, and then the T cells extravasate into the tumor. The tumor still has some
FOXP3+ cells, but the vast majority of the cells are now T effector cells (Teffs), that are CD4+, FOXP3- or
CD8+.
Overall, in virtually every animal model, in human bladder cancer and melanoma, this approach results in
an increase in the ratio of Teffs to Tregs1. That increase correlates ultimately with tumor rejection.
Multiple actions:
When FOXP3+ Tregs were first identified and shown to express more CTLA-4 than Teffs do, there was a
controversy in the field about CTLA-4's function. In 1996, we showed that TCR transgenic T cells that lack
CTLA-4 divide many more times in a mouse than controls do.
Similarly, in a colitis model, CD25+ cells, which later turned out to be Tregs, block colitis. Injecting
α-CTLA-4 removes this block, suggesting that the antibody stops the action of Tregs.
We developed a way of getting uni-compartmental blockade by humanizing mice so that they express antihuman α-CTLA-4. We can then sort cells to enrich for human Teffs, for example, and mix them with mouse
Tregs or vice versa.
Using this method, we showed that blocking CTLA-4 only on Tregs in RAG -/- reconstituted lymphocyte
chimaeras doesn't confer any therapeutic benefit. In contrast, blocking CTLA-4 on just the Teffs cures
about half the mice. Interestingly, blocking CTLA-4 in both the Teff and Treg compartments is required for
optimal effect.
Figure 2
Clinical response –
melanoma.
We used TRP1 model mice to study the effects of α-CTLA-4 on tumor-specific CD4+ T cells. The model
generates CD4+ T cells with a T-cell receptor reactive to a peptide derived from TRP1, a melanoma
antigen, and presented on MHC class II. When the cells are harvested and put into wild type mice, many of
them convert to FOXP3+ cells. So they are
congenically marked and can be used to
track tumor-specific CD4+ Teff (FOXP3-)
cells and Tregs (FOXP3+).
In the lymph node, α-CTLA-4 results in
the expansion of the Teffs, as expected,
but also of the Tregs. This suggests that in
both cell types, CTLA-4 limits proliferation.
Because the Tregs expand more, however,
the Teff/Treg ratio decreases.
The opposite happens in tumors, where
α-CTLA-4 results in Teff expansion, but
the Treg numbers stay flat. As a result,
there is a big increase in the Teff/Treg
ratio.
Exploiting checkpoint blockades
Experiments with mice showed that α-CTLA-4 rapidly eliminates Tregs in the tumor. It was clear that this
as not a result of conversion, or complement- or NK-mediated killing.
Immune depletion:
The other possibility is that the Tregs are eliminated by Fc receptors. There are 4 types of Fc receptors in
mice. These receptors bind to the Fc region of antibodies and can mediate the depletion of antibody-coated
targets or immune complexes. Macrophages, NK cells and others express Fc receptor.
In wild type mice, anti-CTLA-4 depletes TRP1+ Treg levels from 14% to 1%. But in mice lacking Fcγ receptor,
there is no decrease, indicating that Fcγ mediates the Treg depletion. The Fcγ receptor also depletes
endogenous polyclonal Tregs by more than 50%. In particular, it's FcγRIV that mediates the depletion of
these Tregs. FcγRIV binds α-CTLA-4 with high avidity, compared with other Fc receptors.
Within a tumor, the majority of cells that express FcγRIV are CD11b+ Ly6ChiLy6G-macrophages. These cells
are enriched in the tumor microenvironment, comprising about 57% of the tumor. In contrast, there are
very few of them in lymph nodes.
There are two other factors that are important. One is that on the cell surface, CTLA-4 levels are high on
both Teffs and Tregs in the tumor, but about 2 logs brighter on the Tregs than on the Teffs. The other is that
in the tumor microenvironment, FcγRIV expression is elevated in the macrophages.
All of these factors together create a perfect storm of Tregs that express higher levels of CTLA-4 than
Teffs do, binding enough antibody to be picked up by macrophages, and killed by antibody-dependent
cell-mediated cytotoxicity (ADCC). FcγRIV
expression is also markedly higher in the
tumor than in lymph node cells. This perfect
storm depletes T regs from the tumor, but not
from the lymph nodes, where their absence
would result in autoimmunity.
In bladder cancer, up to 90% of the T cells in
the tumors are FOXP3+. That number drops
after treatment with ipilimumab Ñ a fully
human anti-CTLA-4 antibody made by BristolMyers Squibb Ñ which binds to Fc receptors.
The major function of CTLA-4 blockade seems
to be to release the CTLA-4 inhibition on Teffs,
the cell-intrinsic effect. It also has a cellextrinsic effect in depleting Tregs, mediated
by Fc receptors on myeloid cells. There may
be other mechanisms as well.
Powerful drug:
Ipilimumab has been used to treat more than 17,000 patients. Objective responses have been seen in
virtually every tumor type tested, with the biggest numbers in melanoma and prostate cancer. The side
effects include colitis, hepatitis and hypophysitis, but these are usually manageable with systemic steroids.
In a randomized placebo-controlled trial of ipilimumab for late-stage melanoma, there was a medium
increase in the survival by a few months2. No other drug has shown this sort of survival benefit in a
randomized controlled trial. What's more, the benefit from this therapy appears to be long-term and
durable.
Figure 3
Improving survival with
combination therapy.
53
54
The Food and Drug Administration approved ipilimumab in 2011 for first-line and second-line therapy for
metastatic melanoma. It's now the standard of care, and a combination of the drug with chemotherapy has
been shown to be better than chemotherapy alone.
There are several critical issues remaining for further development of ipilimumab. First, how can we
increase the response rate? We need to know its mechanism of action, how to predict responders, and
which drugs to combine it with to raise the response rate. For example, combination with vaccines,
conventional therapies such as gemcitabine or cisplatinum or targeted therapies, may all increase the
efficacy of CTLA-4 blockade.
CTLA-4 was the first identified checkpoint, but there are others, including PD1, B7-H3, B7-H4, Vista, TIM3 and LAG-3. Most of these are negative checkpoints rather than positive ones because it's important to
downregulate immune responses.
PD1, which has 2 ligands, doesn't work like CTLA-4 Ñ it has nothing to do with CD28 or the blocking of
co-stimulatory signals. It recruits a phosphatase to the synapse, directly interfering with T-cell antigen
signaling.
In a phase II trial of 296 patients with metastatic cancer, an antibody to PD1 showed 28% response rate in
melanoma, 18% in non-small cell lung cancer and 27% in renal cancer. For some reason, however, it did
not show any response in colorectal cancer or with castration-resistant prostate cancer.
Because PD1 and CTLA-4 operate via different ligands and mechanisms, it seemed reasonable to combine
the antibodies. Each of the antibodies has about a 25% response rate on its own. The clinical trial to test
the combination is ongoing, but the response rate appears to be high3.
ICOS effects:
We made one interesting observation in a pre-surgical trial of anti-CTLA-4 in bladder cancer patients:
Tumor tissue in these patients showed an increase in the ICOS+ fraction of CD4+ T cells after anti-CTLA-4
therapy4.
ICOS has several functions. It's thought to be associated with follicular T helper cells, with a role in Th2
cytokine regulation, germinal center formation and T/B cell collaboration. ICOS is also implicated in
the function of IL-10-producing Tregs induced by plasmacytoid dendritic cells. It's necessary for IFN-γ
production and containment of viral infection, and it may promote survival of activated T cells, including
both Tregs and Teffs.
Figure 4
anti-CTLA-4/GVAX
therapy activates tumor
vasculature and increases
tumor infiltration by CD4
and CD8 effector cells.
The ICOShi cells from the peripheral blood
of these patients recognize NY-ESO-1.
Melanoma patients have two patterns
of ICOS expression: either a transient or
no increase during anti-CTLA-4 therapy,
or a persistent increase that lasts the
entire 3 months of therapy. Those who
have a persistent increase survive
longest, suggesting that ICOS elevation is
important either to the immune response
or to anti-CTLA-4's effects.
Experiments in mice suggest that ICOS
is important for the latter: Mice lacking
either ICOS or its ligand don't reject
tumors after anti-CTLA-4 therapy.
Exploiting checkpoint blockades
55
To take advantage of this dependency, we made a vaccine called IVAX for cells that express the ICOS ligand.
Irradiated mice are given wild type tumors and then treated with either the wild type vaccine or the ICOS
ligand expressing vaccine and anti-CTLA-4.
The results are very clear. The wild type vaccine plus anti-CTLA-4 has about a 23% response rate. With
the ICOS ligand, that rate skyrockets to almost 90%. The IVAX vaccine doesn't work in ICOS knockout mice,
indicating that it works by signaling through ICOS.
The IVAX and anti-CTLA-4 combination increases the density of CD8+ and CD4+ FOXP3- Teffs, but the density
of Tregs doesn't change. Interestingly, it more than doubles the production of CD107a and granzyme B by
CD8+ Teffs. It also increases the production of IFN-γ and TNF-α by CD4+ Teffs by about 30-fold. Overall,
there is about a 90-fold increase in the cells' ability to kill tumors.
Engaging ICOS may be another strategy to induce both CD4 and CD8 effector cells as well as for tumor
rejection. There's a number of ways to do this, including agonistic monoclonal antibodies, fusion proteins,
multimeric ligands and cellular vaccines.
Until now, with conventional therapies and targeted therapies, we have mainly looked for an increase
in median survival. But with ipilimumab and anti-PD1 antibodies, it's possible to get long-term, durable
responses. Our job now is to extend this to more types of cancers and test the right combinations in many
more trials.
References
1. Q
uezada S.A. et al. J. Clin.
Invest. 116, 1935-1945
(2006)
2. Yuan J. et al. Proc. Natl.
Acad. Sci. USA 108, 1672316728 (2011)
urran M.A. et al. Proc.
3. C
Natl. Acad. Sci. USA 107,
4275-4280 (2010)
4. Simpson T.R. et al. Curr.
Opin. Immunol. 22, 326332 (2010)
Exploiting checkpoint blockades
ACT immunotherapy and BRAF
targeted therapy for melanoma
A report on a lecture by
Antoni Ribas
Division of Hematology and Oncology – UCLA, Los Angeles, USA
Genetic engineering of peripheral blood lymphocytes to express cancer-specific T-cell receptors (TCR) allows
the generation of large quantities of tumor-specific T cells for adoptive cell
transfer (ACT). In a clinical trial of
metastatic melanoma, 14 patients were given MART-1 melanosomal antigen ACT together with MART-1 peptide
pulsed dendritic-cell vaccination; 9 of the 14 showed evidence of tumor regression, but the responses were not
durable. Analysis using newly developed microfluidics and nanotechnology-based immune monitoring assays
shows an acquired but transient expansion of T cells, and a shift in functionality towards an immune suppressive
phenotype. Targeted oncogene inhibition with small-molecule BRAF inhibitors has given unprecedently high, but
short-lived, response rates in patients with BRAF-mutant metastatic melanoma. Combining immunotherapy
with BRAF targeted therapy may result in improved treatments for advanced melanoma. A BRAF-driven murine
model of melanoma, SM1, exposed to the BRAF inhibitor vemurafenib as well as TCR-engineered ACT, results in
superior antitumor responses compared with either therapy alone. The BRAF inhibitor paradoxically increases
MAPK signaling, in vivo cytotoxic activity, and intratumoral cytokine secretion by adoptively transferred cells. This
phenomenon is the pathogenic basis for the development of secondary cutaneous squamous cell carcinomas in
patients. Antoni Ribas described findings supporting the testing of ACT and BRAF inhibitor therapy combinations
in patients with BRAF-mutant metastatic melanoma.
It's a great time to be working on melanoma because interesting research has led to improved patient
care with drugs blocking PD1 or CTLA4, as well as targeted therapy to block driver oncogenes. These
treatments are getting reproducible and clinically meaningful benefits to patients.
There are at least 6 anti-PD1 antibodies in clinical development, including nivolumab from Bristol-Myers
Squibb. The Merck antibody MK-3475 (lambrolizumab) also recently reported an objective response rate in
the first 83 patients with metastatic melanoma.
Early data from a phase I trial of advanced melanoma, MK-3475 gave a 40% response rate with those
who had previous received ipilumbimab, and up to 50% responses in patients na•ve to ipilimumab. The
responses tend to be durable: Of nearly 40 patients, only 4 have progressed after having an objective
response.
For example, one of the clinical
responses was in a patient previously
treated for lung, pleural and liver
metastases from melanoma. The
patient had progressed after receiving
chemotherapy, high dose interleukin-2
(IL-2) and ipilumimab, and was in a
wheelchair and on oxygen support at
the time of the first infusion. After 3
months of MK-3475, this patient is off
oxygen support, continues to respond,
and has returned to playing tennis.
Figure 1
Clinical activity in a patient
with lung, pleural and
liver metastases from
melanoma.
57
58
Another patient had a tumor called desmoplastic melanoma. This tumor is relentless, even with repeated
surgeries. This patient had progressed with lung metastases after ipilumbimab, but showed an impressive
response with MK-3475.
The drug's mechanism of action is infiltration of CD8+ T cells. Taking biopsies of skin metastases from a
patient shows that at the beginning, there are few cytotoxic T lymphocytes (CTLs) in the tissue. But as the
tumor responds, there are many more of them.
At baseline, these lymphocytes express the surface marker CD45Ro, suggesting that they have seen
antigens before, and also express the homing markers, CD62L, CCR7 and CD27, usually seen in T central
memory cells. After infusion, the phenotype changes from central memory markers to effector markers.
Most patients with metastatic melanoma have few detectable lymphocytes with a specific T-cell receptor
(TCR) recognizing a melanoma antigen called MART-1. This can be corrected by TCR engineering of T cells
obtained from patients and used in adoptive cell transfer (ACT) therapy. In a clinical grade preparation, we
take white blood cells from a patient through a leukapheresis and infect them with a retrovirus to introduce
tumor-specific TCRs.
Infusing a billion of these cells back into the patient essentially changes the patient's immune system to
have a high proportion of T lymphocytes specific for the MART1 antigen. These cells home to the tumor, and
are highly active in their antitumor activity, as seen by both PET and CAT scans.
Transient responses:
MART1 is expressed in all melanocytes, including in the pigmented cells in the eye and ear. As a result,
the therapy may cause some patients to develop middle ear toxicity and lose pigment from skin and hair
follicles.
One patient who responded developed pancytopenia and was given corticosteroids, which wipes out the
TCR transgenic T cells. The patient developed S100+/MART1+ bone marrow metastases from melanoma,
suggesting that tumors that progress maintain their antigen.
In many cases, the initial tumor responses have been followed by frequent relapses. This may be because
the immune system stops working, or because the tumor changes such that the immunotherapy no longer
works.
Figure 2
Clinical activity in a
patient with a metastatic
desmoplastic melanoma.
Most existing immune monitoring
assays don't provide information about
the polyfunctionality of a lymphocyte.
They are usually flow cytometrybased, and give only a small number
of readouts. The development of
new nanotechnology-based immune
monitoring assays allows us to
separate T-cell specificities based on
the TCR, and see what the cells do
when they encounter the antigen. The
newly developed microfluidic device
has several chambers, with each
chamber holding one cell, decorated
with barcoded primary antibodies that
allow us to have multiple readouts.
There are typically 20 antibody
pairs to different secreted proteins,
chemokines and cytokines.
Exploiting checkpoint blockades
So far, we have learned that the cells given to the patients first expand and then as they contract Ñ because
that's what a natural immune response would be doing Ñ they shift their function1. At the beginning, the
cells make multiple cytokines including perforin, granzymes and interferon-γ, but they later shift to a Th2
response, making IL-4 and TGF-β.
So one challenge is maintaining the lymphocytes' function, perhaps by pharmacological inhibition of
checkpoints such as CTLA4 and PD1. Another possibility is to create an endogenous population that would
generate a progeny of TCR-engineered lymphocytes. This could be done by genetically modifying stem
cells: It's possible to engineer the bone marrow of mice and let them continuously produce T cells that
have specificity for a TCR.
We have done this kind of experiment with humanized mice, which have a human thymus, or with deeply
immunodeficient mice that have human HLA-A2.1. One important benefit of doing it this way is that it takes
away the possibility of mis-pairing between the transgenic TCRs and the endogenous TCRs.
Evolutionary regression:
What about changes in the tumor? One of the patients with an objective response had about 30 metastases.
A responding lesion continues to be MART1+ but the pattern of expression of MART1 seems to be different,
more patchy than homogeneous. Another lesion that was progressing was actually MART1 Ñ and also
showed many infiltrating T cells despite the absence of the antigen.
Another lab also showed that after targeting tumors with gp100, another melanosomal antigen, some
parts of the tumor remain positive for the antigen, and others are negative.
T cells infused into a mouse first produce tumor necrosis factor-α. This then signals to the tumor to
dedifferentiate Ñ to go back in evolutional time and instead of being a melanoma that expresses MART1
and gp100, express nerve growth factor receptor (NGFR), which comes from the neural crest lineage.
Could that be happening in these patients? At baseline, the tumor is S100+/MART1+. Post dosing, it
continues to be S100+, but loses MART1. The MART1- cells express NGFR. These cells correlate with the
presence of tumor-infiltrating lymphocytes. This suggests that in some patients, these tumors can develop
adaptive resistance through dedifferentiation as in the mouse, and that may be one of the escape routes.
Two drugs approved for
metastatic mwelanoma, the
CTLA4-blocking antibody
ipilumimab, and the BRAF
inhibitor vemurafenib, are
very different. The first is an
immunotherapy with a low
frequency of responses, but
once a patient responds, it
tends to be long-term (see
Rosenberg, page 13).
That's different from targeted
therapy: If we select patients
with a BRAF V600 mutant
melanoma, the majority
of patients respond to a
treatment that specifically
blocks BRAF, but the response
tends to be transient.
Figure 3
PET and CT images of
objective tumor responses
with adoptive cell transfer
of TCR engineered cells.
59
60
The ideal therapy would be both highly active and durable. Combining immunotherapy and targeted therapy
with ipilimumab (anti-CTLA4) and vemurafenib (BRAF inhibitor) results in liver toxicity2, but the concept
has merit.
One possibility is that when BRAF is blocked, the dying tumor presents several antigens. Another is that the
BRAF inhibitor may act on the immune system independently of the tumor and directly activate T cells. Or,
because BRAF controls many immunosuppressive proteins downstream of the oncogene, blocking BRAF
may result in a more permissive tumor microenviroment for immune cells.
To set up a model for testing these possibilities, we established cell lines from mice that have the BRAF
oncogene driven by a melanocyte promoter. The SM1 cell line expresses the BRAFV600 mutation, as well as
cross-chromosomal aberrations similar to that of human tumors, suggesting that it can serve as a good
model.
The tumor is aggressive, so the mice have to be sacrificed within 2 to 3 weeks, making it difficult to do
antibody therapy. There is a big difference between the mice that receive both the ACT and the BRAF
inhibitor compared with either therapy alone3.
There is no change in the number of T cells specific to the tumor, suggesting that indirect antigen
presentation may not be the main driver. However, there's evidence of direct activation of T cells with
the BRAF inhibitor. The BRAF inhibitor doubles the tumor cell lysis, even with no tumor antigen present,
suggesting that its effect is directly on the T cells as opposed to the tumor.
Paradoxical activation:
Patients who receive BRAF inhibitors develop an interesting side effect, which is squamous cell carcinoma.
This is the result of a mechanism of paradoxical MAPK activation. BRAF controls the MAPK pathway that
includes RAF/MEK/ERK and signals the cell to grow and survive. In the case of some mutations, this
pathway is always on, and may lead to melanoma.
Blocking BRAF turns off the pathway and patients respond. In a wild type cell, the pathway is controlled
mostly by heterodimers of BRAF and CRAF that are regulated upstream by RAS. If wild type BRAF is
blocked in these cells, the pathway can still go down through CRAF. If there is an upstream mutation, for
example in HRAS, blocking BRAF transactivates this pathway.
This may also happen with lymphocytes. Blocking BRAF in SM1 decreases phospho-ERK levels and shuts
down the MAPK pathway. But in T cells, where the MAPK pathway is under the control of the TCR, P-ERK
levels actually go up. This paradoxical activation is specific for the MAPK pathway4. Immunoprecipitation of
CRAF results in increased MEK phosphorylation in a kinase assay.
One of the best ways to understand the effects of the MAPK pathway is by looking at the pathway's output.
Gene expression profiling of SM1 and the PMEL-1 T cells exposed to vemurafenib shows genes that are up
in the PMEL-1 T cells and down in SM1 are associated with the paradoxical MAPK activation, and those that
are up in both with negative feedback regulation.
How does BRAF targeted therapy increase the activity of tumor immunotherapy given that it has no indirect
effect on T cells? The gene expression profiling reveals a list of secreted proteins, including vascular
endothelial growth factor, CSF1 and CCL2.
If the SM1 cell line is treated with anti-CTLA4, anti-PD1, anti-TIM3, anti-CD137 or anti-41BB (CD137), only
the 4-1BB works. Adding the BRAF inhibitor to the 4-1BB antibody gives true regressions that don't occur
with any of the other antibodies in this model5.
The BRAF inhibitor significantly decreases CCL2 production, as predicted. This is part of the mechanism by
which BRAF inhibition works, in combination with immunotherapy. BRAF inhibition is not effective in mice
lacking CCR2, the receptor for CCL24.
Exploiting checkpoint blockades
In conclusion, PD1 antibodies
are an exciting new addition
to
tumor
immunotherapy.
This approach is effective for
melanoma, kidney cancer and
lung cancer, and may work in
several other tumors.
61
Figure 4
Transient response
and progression with
persistent MART-1
expression.
Resistance to TCR-engineered
ACT can be mediated both by
changes in function of T cells and
by adaptive de-differentiation of
melanoma cells. Combinatorial
effects of the BRAF inhibitor and
immunotherapy are mediated by
diverse mechanisms, including
paradoxical MAPK activation
in lymphocytes and changes in
chemokine gradients.
References
1. M
a C. et al. Nat. Med. 17,
738-743 (2011)
2. Ribas A. et al. N. Engl. J.
Med 368, 1365-1366 (2013)
oka R.C. et al. Cancer
3. K
Res. 72, 3928-3937 (2012)
4. Su F. et al. N. Engl. J. Med.
366, 207-215 (2012)
5. Knight D.A. et al. J. Clin.
Invest. 123, 1371-1381
(2013)
63
PART III: I n d u c i n g t u m o r i m m u n i t y
with vaccines
Glenn Dranoff
Mechanisms of protective tumor immunity
Karin Jooss
Development of combinatorial cancer vaccines
Elizabeth M. Jaffee
Tipping in the balance from a procarcinogenic
to an anticancer response in pancreatic cancer
Inducing tumor immunity with vaccines
Mechanisms of protective tumor
immunity
A report on a lecture by
Glenn Dranoff
Department of Immunology – Dana-Farber Cancer Institute, Boston, USA
Efficacious cancer immunotherapies are likely to require combinations of strategies that enhance tumor-antigen
presentation and antagonize negative immune regulatory circuits. Vaccination with irradiated, autologous
melanoma cells engineered to secrete GM-CSF followed by antibody blockade of CTLA-4 accomplishes
clinically significant tumor destruction with minimal toxicity. The extent of tumor necrosis in post-treatment
biopsies is linearly related to the natural logarithm of the ratio of infiltrating CD8+ effector T cells to FOXP3+
regulatory T cells. Detailed analysis of the patients affords a rich opportunity to identify antigens associated
with immune-mediated tumor destruction and to delineate mechanisms of therapeutic immunity. One intriguing
antigen identified is progranulin, a protein expressed in many tumors that promotes invasion and metastasis.
Progranulin also modulates the tumor microenvironment and functions, overall, as a negative immunoregulatory
cytokine. The host can mount a potent antibody response that specifically targets driver pathways, including that
of progranulin, in cancer. Progranulin is also important in the liver fluke, Opishorchis. Glenn Dranoff suggested
that therapy for tumors may not just be similar to antivirals, but may also involve tapping into the anti-parasitic
response.
The generation of endogenous antitumor immunity proceeds through several steps, beginning with the
acquisition of tumor antigens by professional antigen-presenting cells (APCs) such as dendritic cells (DCs).
They process this material to MHC class I and class II and CD1D presentation pathways and migrate to the
regional lymph nodes, where they stimulate antigen-specific T, NK-T and B cells.
These cells then migrate systemically in an attempt to mediate an antitumor reaction. Some of the pathways
by which tumors impede these reactions have been elucidated, and have given rise to a number of targeted
therapy strategies aimed at overcoming defects at the level of antigen presentation, T-cell activation or in
the tumor microenvironment.
Large randomized clinical trials have
shown that these manipulations
can produce clinically meaningful
benefits, with the Food and Drug
Administration approvals of
checkpoint blockades and cancer
vaccines and, almost certainly in
the near future, PD1 blockade.
Still, it's clear that most patients
don't benefit from these
treatments. How can we improve
on these results? One approach
is to study the long-term
responders and see what they
teach us about mechanisms of
protective tumor immunity in
humans.
Figure 1
GVAX induces tumor
infiltrates.
65
66
Figure 2
Activity of GVAX +
ipilumimab highlights the
intra-tumoral CD8/FOXP3
ratio.
Our approach involves two
therapeutic strategies. One is
to engineer autologous tumor
cells to produce the cytokine
GM-CSF, which improves DC
presentation of the antigens.
About two-thirds of the time,
that leads to a measurable
increase in tumor immunity.
This is most convincingly
demonstrated by an examination
of a metastasis distant from
where the vaccine is given.
Before therapy, there is no
significant host response, but
after vaccination, there is a
nascent immune response
characterized by a coordinated
and antibody T-cell response.
When a checkpoint blockade
such as an anti-CTLA4 antibody
(ipilumimab) is layered on top
of the vaccine, a substantial
proportion of patients who get the sequential therapy go on to achieve long-term survival1. So far, the longest
is up to 10 years. Importantly, these responses have been achieved with minimal toxicity, suggesting that
there is selectivity for tumor targeting in this cohort of patients.
One of the lessons from this group of patients is that cytotoxic T cells are an important component of the
response. This is illustrated by the pathologic study of a metastasis removed after the sequential therapy.
A biopsy of a tumor from a patient that failed to respond to the sequential treatment showed a paucity of
CD8+ T effector cells (Teffs) and an abundance of FOXP3+ regulatory T cells (Tregs).
In contrast, metastases from patients that benefited from treatment showed abundant CD8+ T cells and
few Tregs. Based on the analysis of several patients, there is an almost linear relationship between the
amount of treatment-induced tumor necrosis and the Teff/Treg ratio.
In order to understand the mechanisms underlying this ratio, it is important to learn more about the
biology of GM-CSF. Studies of GM-CSF-deficient mice reveal that the homeostatic role of the cytokine is in
immune tolerance. Without GM-CSF, the animals develop chronic inflammatory disease, in part because
the cytokine acts on myeloid cells to make them efficient at ingesting apoptotic cells in the periphery,
supporting Treg homeostasis2. When this mechanism is perturbed, Treg numbers are diminished.
If this immunoregulatory function of GM-CSF had been discovered first, we might not have thought to use
GM-CSF as an immune stimulant. We subsequently learned that GM-CSF can switch from tolerance mode
to one of protective immunity when there's a second signal provided concurrently. This can be, for example,
a TLR ligand, which leads to maturation of APCs and an induction of a stronger Teff response. Clearly, the
preexisting balance of Teff and Treg populations is likely to have a big impact on the therapeutic effect of
these vaccines.
Inducing tumor immunity with vaccines
Microsphere delivery:
To explore this in more detail, we have collaborated with material science engineers, and are developing
a new vaccine strategy for clinical testing. This approach involves microspheres composed of polymers of
lactic acid and glycolic acid, which are biodegradable3.
These polymers can be fabricated in double emulsion technique to contain interesting biologic agents
within the particle. Unlike free recombinant proteins, which have a short half-life, the proteins encased in
these microspheres would have prolonged delivery.
These devices were initially used in tissue engineering ex vivo. We became intrigued by the possibility of
using them for cellular immunotherapy following subcutaneous implantation of the devices. Recombinant
GM-CSF in the device recruits and activates DCs, and CpGs trigger TLR9 signaling to block Tregs and
immunosuppressive pathways. Necrotic tumor cell lysate provides the target antigens for vaccine response.
At the site of implantation of the device into syngeneic mice, this combination elicits a broad DC reaction.
This probably better reflects what happens in vivo during a potent immune response. The mixture of DC
subsets is associated with a favorable cytokine profile, including interleukin-12 and type I and type II
interferons.
Importantly, this system has allowed us to validate the model of GM-CSF's dual functions. GM-CSF and
the lysate generate high levels of homeostatic tolerance cytokines. But if TLR is thrown into the mix, these
cytokines are suppressed.
Concordantly, these sophisticated vaccines generate a much more favorable Teff/Treg ratio.
The previous generation of vaccines composed of GM-CSF alone had about a 1:1 Teff/Treg ratio, but the
triple combination can increase it significantly, in some animals up to almost 50. As a result, the approach
has achieved true regressions of virulent tumors, something we were never able to achieve before4.
Careful histological studies of the implantation sites over time have shown some striking phenomena.
One month after implantation, there is tremendous local reaction in the scaffold implantation site along
with regional lymphoid hyperplasia. At three months, the rim is even more impressive, with an emerging
presence of a large mast cell population. Several months after device implantation,the residual scaffold is
still visible, and there are classic multinucleated giant cells.
This response has continued to evolve. Even at latest time point available, almost a year and a half, there's
still microscopic evidence of ongoing immune activation, with residual necrotic tumor cell lysate. These
studies have taught us that the relatively short-lived persistence of cells can be dramatically changed
using sophisticated material engineering devices.
Drug delivery is essential, and it's critical to
have both spatial and temporal control over
the release of immune modulators. The
requirement for more systemic therapies in
the past may in part reflect the fact that those
vaccines were inefficient. This approach is
likely to have a more favorable therapeutic
index in terms of targeting tumors. It
requires tremendous collaboration between
engineers, cell processing labs and the
clinical investigative team.
Figure 3
Scaffold implantation site
day 30.
67
68
Complex interactions:
Tumor immunity in patients is incredibly complex. This was hinted at by the first patient who received
ipilumimab. He was first vaccinated and then got the antibody, but he succumbed to his disease. However,
postmortem results showed masses throughout his body, with a histological picture of hemorrhagic tumor
necrosis.
These vaccines induce a B- and T-cell response. Surprisingly, sera collected after treatments to screen
tumor-derived cDNA expression libraries contain antibodies against a number of angiogenic cytokines
including VEGF-A, angiopoietin-1,2 and macrophage migration inhibitory factor. Therapy-induced
antibodies are able to neutralize all of the known in vitro effects of the angiogenic cytokines5.
Using a similar approach, we also identified as a target of these high-titer antibodies a protein called
progranulin. Progranulin is one of the most highly conserved, ancient secreted proteins. This secreted
factor contributes to the transformed phenotype. Progranulin is important: It is over-expressed in many
tumors, it manifests angiogenic capacity, it's a bona fide growth factor and survival factor and it promotes
invasion and metastasis. Our studies have indicated that the therapy-induced antibodies block the
oncogenic activities of progranulin.
It turns out that healthy people only rarely develop antibodies to progranulin. Even among cancer patients,
most do not develop progranulin antibodies. But among those who develop vasculitis, about 30-40%
manifest anti-progranulin antibodies.
Progranulin has also been linked to one cause of frontal temporal lobar dementia, although this phenotype
doesn't manifest until well into life. This raises a cautionary note about targeting progranulin, but people
have had antibodies for more than 10 years without any central nervous system toxicity.
Figure 4
GVAX and ipilumimab
evoke hemorrhagic tumor
necrosis.
Overall, these findings suggest
that the host can mount a potent
antibody response that specifically
targets driver pathways in
cancer. In collaboration with Kai
Wucherpfennig's laboratory at
Dana-Farber, we have developed
methods to pull out the antigenspecific memory B cells from
these patients and have a panel
of interesting human monoclonal
antibodies
that
may
be
attractive candidates for clinical
development.
It seems really surprising that a
tumor vaccine or CTLA4 blockade
is able to provoke patients to
develop powerful neutralizing
antibodies against secreted critical
factors that might be important
for homeostasis. Why might this
happen? A recent paper described
Opishorchis, a liver fluke that
affects hundreds of millions of
people in Asia.
Inducing tumor immunity with vaccines
69
Opishorchis is known to cause cholangiocarcinoma, a classic chronic inflammation-driven tumor.
Interestingly, the genomic analysis of this worm reveals a granulin factor. This factor is secreted out of the
worm and affects biliary epithelium, the target for transformation, in the hamster model.
It's not easy for CTLs to attack a multicellular target at the single-cell level. The host's strategy is to
isolate worms in fibrous capsules that prevent the worms and their eggs from disseminating, and reduce
fecundity. Multiple innate effectors are important components of the antitumor response.
In the worm, they attempt to cause a slow death by attrition. An interesting possibility is that the host
develops antibodies to things such as the fluke progranulin as part of this strategy of containment, and the
antibodies in cancer patients may be the residue of the host learning to live with worm. These observations
suggest that therapy for tumors may not just be similar to antivirals, but may also involve tapping into the
anti-parasitic response.
References
1. H
odi F.S. et al. Proc. Natl.
Acad. Sci. USA 105, 30053010 (2008)
2. J
inushi M. et al. J. Clin. Invest.
117, 1902-1913 (2007)
3. Ali O.A. et al. Nat Mater. 8,
151-158 (2009)
4. Ali O.A. et al. Sci. Transl.
Med. 1, 8ra19 (2009)
5. Schoenfeld J. et al. Cancer
Res. 70, 10150-10160 (2010)
Inducing tumor immunity with vaccines
Development of combinatorial
cancer vaccines
A report on a lecture by
Karin Jooss
Cancer Vaccines & Immunopharmacology – Pfizer Inc., La Jolla, USA
The effects of a GM-CSF-secreting allogeneic prostate cancer vaccine (GVAX prostate) and the anti-CTLA4 antibody
ipilimumab were investigated in a phase I dose-escalation/expansion trial of patients with asymptomatic
metastatic castration-resistant prostate cancer. At the two highest doses of ipilimumab, patients lived
significantly longer than predicted by the Halabi nomogram. These patients demonstrated increases in the
frequency of activated CD4+ and CD8+ T cells. Increase in CD4+ memory T cells and a drop in regulatory T
cells during treatment were associated with improved overall survival. GVAX/ ipilimumab treatment induces a
Th2/Th17 cytokine profile. Profound up-regulation of interleukin 5-producing CD4+ cells was also associated
with improved overall survival. Antibodies to PSMA, PNPO and NRP2 significantly correlated with survival.
Interestingly, clinical response also coincides with hypophysitis, a side effect that coincides with the increase in
Th17 cells. In summary, the trial results suggest higher numbers of activated memory and effector T cells, low
numbers of regulatory T cells, increases in Th2 and Th17 rates and seroconversion to multiple tumor antigens
are all associated with a favorable clinical outcome and prolonged overall survival. Karin Jooss suggested that
together, these data may provide an immune profile to predict clinical outcome and point to a mechanism of
action for combined Prostate GVAX and anti-CTLA4 immunotherapy.
Prostate cancer is one of the most common malignancies in elderly men and is the second leading cause
of cancer deaths in Western countries, with 240,000 new cases every year and about 30,000 deaths in
the U.S. alone. Prostate cancers are usually treated with hormonal maneuver. Immunotherapies are
generally tested in the asymptomatic castration-resistant phase, in men who are resistant to the hormonal
maneuver.
The prostate GVAX vaccine is based on two allogenic cell lines, LNCaP and PC-3, that are modified to
secrete GM-CSF. The cells are sub-lethally irradiated and intradermally injected. The GM-CSF secretion
leads to the recruitment of dendritic cells, which pick up the antigens and prime the T cells.
In preclinical models, the slow and durable release of GM-CSF from the GVAX cells leads to durable
infiltration of DCs at the vaccine site. That's distinctly different from injecting recombinant GM-CSF,
which is quickly cleared from the vaccination site. This vaccine was combined clinically with anti-CTLA4
(ipilumimab), which blocks a T-cell inhibitory signal.
In the trial, 28 patients received the vaccine at a steady dose every 2 weeks, for a total of 13 doses over 6
months. The anti-CTLA4 was given monthly, for a total of 6 infusions, with 3 patients given escalating doses
of 0.3, 1.0, 3.0 and 5.0 mg/kg. There was activity at 3 mg/kg, so 16 patients were added in an expansion dose
cohort1.
One of the objectives of the study was to do immune monitoring, so the patients had monthly blood draws to
look for antibodies before and after treatment. We categorized partial response (5 patients) as having more
than a 50% decline in PSA levels, progressive disease (11 patients) as more than 25% increase in PSA, and
stable disease (12 patients) if they didn't fall in either of those two categories.
Patients who demonstrated a partial or stable disease PSA response had a significantly prolonged median
survival when compared with those who had progressive disease. This suggests that stabilization of disease
or partial response based on PSA readout translates to clinical benefit in prostate cancer.
71
72
The Halabi nomogram also allows us to predict median survival when patients present with castrationresistant prostate cancer. In this case, patients with PSA stabilization or PSA decline demonstrated
significantly prolonged survival than the median survival predicted by the Halabi nomogram, suggesting
that there is true clinical activity in these patients. The PSA declines were durable, ranging from 6 to
31 months. By bone scan, 11 patients had stable disease and 2 had regressing bone and lymph node
metastases.
Figure 1
Treatment schedule: GVAX
prostate + ipilumimab.
The patients in this trial had a unique side effect profile: 7 of the 28 patients overall, and 5 of the 5 partial
responders, had hypophysitis. The endocrine side effects, including secondary adrenal insufficiencies and
secondary hypothyroidism, go hand in hand with clinical activity.
The frequency of these side effects is 100% in those with clinical benefit, compared with less than 2% in
melanoma patients given anti-CTLA4. The results suggest that the treatment combination changes the
side-effect profile compared with the immune modulator alone.
Multiple analyses:
The objective of immune monitoring was to try and identify immune parameters that correlate with clinical
activity and that may be useful for clinical response prediction. We were also interested in understanding
whether this side effect profile is antigen-specific. The data were intended to generate hypotheses that
would need further validation with larger sample size.
We performed many different analyses, including serology for tumor-specific antibodies, analysis of
peripheral blood for regulatory T cell (Treg) and T effector cell (Teff) ratio, activation status and effector/
memory phenotype. We also explored tumor antigen-specific reactivity, cytokine profiles and peripheral
blood dendritic cells and myeloid derived suppressor cells (MDSC), looking for signatures that predict
clinical activity.
There is evidence that several antigens cloned by antibody approaches, such as NY-ESO-1, have shown
subsequent coordinate CTL responses and vice versa. We thought therefore that this serology analysis
might suggest antigens to go after.
One approach for this is low-throughput, simply going through the literature and expressing a panel of
prostate cancer-specific tumor antigens, and screening pre- and post-vaccination for antibodies. However,
because we used a whole-cell vaccine, we also did a high-throughput analysis, screening samples with a
ProtoArray from Invitrogen for 8,000 human proteins and SEREX analysis with phage-display libraries (see
Dranoff, page 63).
We selected the prostate-specific antigens PSA, PSMA PSCA and PAP, cancer testes antigens Ñ because
NY-ESO-1 responses had been found in previous prostate cancer patients Ñ and general cancer antigens.
Inducing tumor immunity with vaccines
There is evidence demonstrating that the induction of tumor-specific auto-antibodies can correlate with
favorable clinical outcome Ñ for example, with NY-EOSO-1 in melanoma, or with GVAX monotherapy in
prostate cancer.
There were 12 patients in the initial dose
escalation cohort. Only the patients who
received 3 or 5 mg/kg of anti-CTLA4 induced
PSMA-specific antibodies. However, with
GVAX prostate monotherapy, not even a
single patient developed these antibodies,
so the addition of the immune modulator
is important. The patients also had NYESO-1 antibodies and, interestingly,
antibodies to the androgen receptor,
suggesting that the induction of androgen
receptor-specific antibodies may achieve
the same as the hormonal maneuver with
androgen deprivation therapy.
The SEREX analysis identified FLJ14668,
NRP2 and PNPO, and the protein array
identified another set of antigens,
including TRAP1, NNAT, SELS and LIMCH.
This list shows that there are many shared
antigens between the pituitary gland and
the GVAX prostate cell lines LN-CaP and
PC-3 used in the vaccine.
Strong overlap:
Induction of PSMA, NRP2 and PNPO is associated with clinical benefit, and the patients seem to live longer
if they develop antibody responses to more than one of the antigens. However, many of the other antibodies
did not translate into therapeutic benefit. Interestingly, in the T-cell arm, there is a correlation between the
number of induced antibody responses and the frequency of T helper cells producing IL-5 in the circulation.
Comparing the antibody responses to LN-CaP and PC-3 in a patient who received 3 mg/kg dose of antiCTLA4 with RNA from 80 different pituitary glands shows that there is a significant overlap between the
two. Interestingly, many of the hits for LN-CaP and PC-3 were present before treatment. In the pituitary
gland, 80% of the shared antibody responses where induced post-GVAX.
Overall, GVAX and anti-CTLA4 therapy induces multiple prostate tumor-associated auto-antibodies
responses that are not seen with GVAX monotherapy. Striking increases of IL-5-secreting CD4 T cells
correlate with the number of induced auto-antibodies in the patients. And there is a strong overlap of
induced antibodies to the GVAX cell lines and the pituitary gland.
A powerful vaccine not only drives CD4 and CD8 T-cell responses but also induces antibody-dependent
cell-mediated cytotoxicity (ADCC). In the case of GVAX, antibodies may play an important role in the effector
function.
It is generally believed that Th1 responses are beneficial in cancer vaccines, and Th2 is perhaps not a desired
outcome for a vaccine. The evidence on Th17 is mixed. With this treatment, however, not much happened
with IL-2, tumor necrosis factor-α(TNF-α) or interferon-γ(IFN-γ), whereas IL-4 and IL-5-secreting CD4 T
cells increased post-treatment, suggesting that this treatment skews toward a Th2 response.
Figure 2
Clinical results: GVAX
prostate + ipilumimab.
73
74
Patients who showed an
increase in the IL-5-secreting
CD4 T cells lived significantly
longer than those who did not2.
These patients also have higher
numbers of induced antibody
responses. But there is little
enhancement in Th1 cells.
Figure 3
Treatment resulted in
regression of metastases
and stable disease.
As for Th17, there is some
evidence that high levels of
Th17 cells correlate with a
low Gleason score and better
prognosis3. But there is also
evidence that high levels of Th17
cells correlate with a faster time
to progression4.
In our patients, there is a slight but statistically significant increase of these cells during treatment.
However, patients who had a partial response or stable disease showed a more pronounced increase of
these cells compared with those who had progressive disease.
Thus, this increase correlates with clinical benefit and clinical benefit correlates with immune-related
adverse events. Higher frequency of Th17 cells are found in the patients who developed hypophysitis and
endocrine pathologies. The increase of Th17 cells coincides in time with the onset of adverse events,
suggesting that these cells may contribute to the autoimmune-related adverse events.
Biphasic curve:
The PSA levels initially drop in response to treatment, then rise, and then show a pronounced drop. It is
possible that the second drop in PSA is less immune-mediated and is more related to the hypophysitis.
In fact, in the old days, prostate cancer patients were treated by adrenal gland resection, which leads to a
strong PSA drop.
In terms of T-cell activation, there is a distinctly different expansion in the periphery of CD8 T cells
expressing HLA-DR in the clinical responders but not in those with progressive disease. There is also
a subsequent drop in these CD8 cells that corresponds with the increase in PSA on the biphasic PSA
response.
Here again, the T-cell expansion is not remarkable, and it it possible that the strong PSA drop results from
the pituitary events observed. The treatment increases T-cell activation across the board, but this has little
association with survival.
However, prostate cancer patients have high pretreatment levels of CD4 T cells expressing CTLA4 when
compared to healthy donors. This tumor-related elevation has a strong predictive value for survival after
treatment. In contrast, high levels of CD4+CD25highFoxP3+ Tregs pretreatment is associated with reduced
survival, as is an increase in the percent of the Tregs on treatment.
An unsupervised cluster analysis, based on just 28 patients, suggests that the dominant predictors of
survival are high pretreatment numbers of CD4+/CTLA4+ cells. High pretreatment levels of circulating
MDSCs in prostate cancer patients are associated with a poor outcome. In contrast, increases in granulocyte
MDSCs on treatment are associated with poor survival.
Inducing tumor immunity with vaccines
75
In summary, vaccines may need to be combined with immune modulators such as anti-CTLA4 or anti-41BB
that expand T cells. But MDSCs or Treg compartments should also be taken into account. For example,
Sutent has been shown in renal cell carcinoma patients to decrease the level of MDSCs. The responder
frequency of patients may go up significantly if, along with a vaccine, both of these immune checkpoints
are concurrently modulated.
Many questions remain: Is this treatment-specific? How about other immunotherapies in the same prostate
cancer setting? Is this disease specific and disease stage-specific? The answers from prostate cancer may
apply to many other cancers.
Figure 4
Baseline CD4+/CTLA4+ is
a dominant predictor of
survival.
References
1. v an den Eertwegh A.J.
et al. Lancet Oncol. 13,
509-517 (2012)
2. Santegoets S.J. et
al. Cancer Immunol.
Immunother. 62,
245-256 (2013)
fanos K.S. et al. Clin.
3. S
Cancer Res. 14, 32543261 (2008)
4. Derhovanessian E. et al.
Int. J. Cancer 125, 13721379 (2009)
Inducing tumor immunity with vaccines
Tipping in the balance from a
procarcinogenic to an anticancer
response in pancreatic cancer
A report on a lecture by
Elizabeth M. Jaffee
Department of Oncology – Johns Hopkins University, Baltimore, USA
Pancreatic Cancer has the poorest survival rates reported for solid tumors, and is highly resistant to traditional
forms of therapy. The approval of the first cancer vaccine (Provenge) for prostate cancer and the first inhibitor of an
immune checkpoint have opened the floodgates for a whole new class of anticancer agents. New understanding
of the immune pathways within the pancreatic tumor microenvironment has identified new targets for developing
cancer-specific vaccines and new targets for modulating the tumor microenvironment. These approaches may
allow more potent activation and improved access of vaccine-induced immune responses. Elizabeth Jaffee's
research is focused on elucidating the immune mechanisms within the tumor microenvironment with the goal of
tipping the balance in favor of an anticancer immune response. Recent advances in molecular technologies and
the development of relevant pancreatic cancer mouse models make it possible to dissect the inhibitory pathways
within the pancreatic tumor microenvironment. This will in turn lead to new therapeutic opportunities to treat
this deadly cancer.
Pancreatic cancer remains a major challenge for therapy, whether immune-based or any other kind of
treatment. The survival curve has not changed much over the past 30 or 40 years. The majority of patients
die within 1 to 2 years, and the 5-year survival for this disease is still under 5%.
Figure 1
The inflammatory response in pancreatic cancer
is a progressive, dynamic
process.
77
78
Pancreatic tumors are typically considered non-immunogenic. They are usually evaluated at a late stage
of progression, when they have already started to metastasize. Also, based on mouse models, the cancer
appears to develop as an inflammatory response to accumulated genetic changes.
The resected tumor may look immunologically inactive compared with other cancers. However, in
preclinical models that recapitulate progression from normal to premalignancy and malignancy, it appears
that inflammation is initially present, and progresses with the different genetic changes.
The tumor microenvironment is particularly important to understanding pancreatic cancer and progression.
The earliest changes in the disease are typically telomere shortening, but the first mutation that occurs is
usually in KRAS. This occurs early, at the time of normal to premalignant lesion. Pancreatic intraepithelial
neoplasias, or PanINs go through multiple stages from stage I to stage III before invasive cancer.
The inflammatory response in pancreatic cancer is a progressive, dynamic process, interrelated with
cancer genetics. Starting at the time of the earliest mutation, a number of inflammatory cells, including
macrophages, regulatory T cells (Tregs) and neutrophils, can be seen.
The cells progressively become more pro-carcinogenic, secreting cytokines and chemokines to facilitate
tumor development, until high-grade lesions form. By the invasive stage of the disease, new antigens,
including mesothelin, are expressed at high levels.
Can targeting an early genetic mutation that's important to the cancer slow down this tumor progression?
Can invasive human pancreatic cancer be converted from an immunologically inactive to an immunologically
active state? These two questions are active areas of research.
Early response:
For the first question, KPC mice, which mimic human pancreatic ductal adenocarcinoma (PDA), are
being used. These mice are genetically altered to have a dominant active KRAS mutant allele and a p53
inactivating mutation, driven by the pancreas-specific PDX1 promoter.
Figure 2
Regulatory T cells
infiltrate early in mouse
models of pancreatic
cancer.
The mice show a stepwise progression
to PDA via multiple stages of
premalignant lesions. PanINs begin at
4 to 6 weeks of age, and the mice with
both mutations have a median survival
of 4 to 5 months.
This model also shows the characteristic
stroma response. Unlike some other
cancers in which Tregs have been
reported to come in later in the disease
progression, in this model, there is an
early infiltration of CD4+FOXP3+ Tregs,
starting in panIN-1, and increasing with
progression.
There's some genetics to suggest why
this happens. For example SMAD4,
which regulates TGF-β signaling, is
often up-regulated in this cancer. There
are few effector T cells at this stage.
Inducing tumor immunity with vaccines
Targeting mutated KRAS along with depletion of Tregs can slow the progression of these PanINs. The
effects of a listeria monocytogenes vaccine, genetically modified to target mutated KRAS, can be compared
with or without depletion of Tregs using a combination of low-dose cyclophosphamide and a monoclonal
antibody to the interleukin-2 receptor1.
The listeria vaccine is an important vector for several reasons. From an immune activation point of view,
it targets dendritic cells and macrophages. It's an obligate intracellular organism, which means that in
targeting, it gets its antigen not only into the exogenous pathway but also Ñ by opening the class II antigenprocessing compartment Ñ into the cytosol.
So, this is a way to naturally induce both the CD4 and the CD8 T-cell responses. The vector also has innate
properties including activation of TLRs 2, 5 and 9. It's easy to engineer this vector to express multiple
antigens.
In phase I trials, it has proven to be safe, and has been modified so that it's unable to induce cell-to-cell
propagation, preventing virus shedding. The phase I trial identified an optimal dose, and the phase II trial
has just been completed with optimistic results, so it is likely to go into phase III trials soon.
The vector induces responses in both the wild type and the mutant mice, and there isn't a significant
difference between the two types.
Combined effects:
With the vaccine alone, there is some depletion of Tregs in the spleen, but when you get closer to the
malignancy, the depletion is inadequate. Even so, immunizing against mutated KRAS in the early panIN
stages depletes Tregs and prolongs survival.
Immunizing after 2 months fails to slow progression, however. Pathological analysis shows that giving the
vaccine with Treg depletion slows progression from early to late-stage panIN and PDA. In this case, there
is more infiltration of both CD4 and CD8 T cells with Treg depletion than without.
The combination immunotherapy induces Th17- and
Th1-type cytokines in the CD4 T cells found in the tumor
microenvironment. In contrast, there is much less of the Th2type response.
Overall, it's clear that immune suppression occurs early
in pancreatic cancer. Like therapeutic vaccines, however,
vaccines for primary prevention are likely to need immune
modulation.
To determine whether invasive pancreatic cancer in people
can be converted from an immunologically inactive to an
immunologically active state, we have been working with GVAX
(see Dranoff, page 63). The vaccine in this case comprises two
allogenic cell lines developed from primary tumors that are
genetically modified to express GM-CSF. The GM-CSF brings
dendritic cells to the tumor site.
The vaccine is given intradermally, which is the optimal way to
give it. It goes to the lymph node, where hopefully it activates
both CD4 and CD8 cells2.
Figure 3
Lymphoid aggregates
develop in pancreatic
tumors following
vaccination.
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80
Much of the clinical research in patients has been done in the adjuvant or metastatic setting. The studies
moved to neoadjuvant setting about 2 years ago, and are powered to look at immunity in these patients.
Patients who are surgical candidates are vaccinated two weeks before surgery and go on to a second
vaccine once they've recovered. They then get adjuvant chemo or radiation based on prior studies and once
a month, they receive a 3rd, 4th, 5th or 6th vaccine.
If they remain disease-free, they get boosters every 6 months. This approach is based on published studies
of T-cell responses against mesothelin.
Giving low-dose intravenous cyclophosphamide one day prior to vaccination depletes Tregs about 50% of
the way, and allows for a better T-cell response measured in the periphery. Some studies suggest that
metronomic cyclophosphamide, given 3 out of 4 weeks in each cycle, may be able to more completely
deplete Tregs.
Surprisingly, the majority of vaccinated patients develop lymphoid aggregates in their pancreatic tumors,
which is atypical in this disease.
From an immunohistochemistry perspective, the aggregates have organized T- and B-cell zones, and
germinal center-like structures. They are also Ki67-positive, indicating that they are actively proliferating.
Inverse relationship:
There seems to be an inverse relationship between the number of infiltrating FOXP3+ T cells and the
number of lymphoid aggregates. Those who have fewer infiltrating FOXP3+ T cells also survive longer, and
have many more CD8 T cells. The same is true in non-aggregate intra-tumoral areas.
Microdissection and gene array analysis of lymphoid aggregates shows that in those who survive longer
than 3 years, Treg signals are down, Th1 and Th17 are up and Th2 is mixed. GM-CSF often pushes patients
toward an allergic eosinophil-type response, and many of those who do well with this therapy long-term
develop some hives and other allergic reactions.
RT-PCR and flow cytometry and immunohistochemistry confirm the array data. The Th1 transcription
factor TBET is increased in lymphoid aggregates in patients whose overall survival is greater than 3
years. RORγC, the Th17 transcription factor, also appears to be increased. There appears to be some colocalization of RORγC and IL-17A.
Figure 4
PD-L1 expressing cells
and PD-1+ cells are colocalized in the germinal
centers of lymphoid
aggregates.
Inducing tumor immunity with vaccines
81
To examine whether there are antigen-specific T cells in the tumor microenvironment, we are looking at
mesothelin. This is not the only antigen, but it's the only one found to be important in vaccinated patients
who had done well, versus those who hadn't3.
Mesothelin was originally identified by differential expression using SAGE. Using overlapping peptides of
10 to 15 amino acids revealed that this one protein can provide 6 HLA2 epitopes and 8 HLA1 epitopes4.
Until this point, most people were looking at the number of cytokine-expressing cells and trying to correlate
with outcome, but that has not been effective. Rather, the induction and expansion of the T-cell repertoire
seems to give a better response.
However, it is difficult to predict outcome after the first vaccine. It's only after four boosts that those who
have this expansion seem to go on to longer disease-free survival.
By tetramer analysis, it looks like these mesothelin-specific T cells are getting into the tumors, although
the degree varies among patients. If it's in the periphery, there's a chance it gets into the lymphoid
aggregates, but if it's not in the periphery, it's not in the aggregates.
The first patient to complete neo-adjuvant and 4 adjuvant vaccines went on to long-term follow-up/boost
study. He received a boost every 6 months. After the 2nd boost, the patient felt great, but a routine CAT scan
showed a mass in the tail of his pancreas. Resection showed that this was in fact chronic inflammation and
not a tumor.
I'd like to propose that there is a balance in the tumor microenvironment between pro-carcingenic and
anti-inflammatory signals. Vaccines play a role in tipping this balance in favor of an anticancer response.
The right immune-modulating agents can also help effectively activate T cells.
For example, in a small study, patients who had failed two chemotherapies received either ipilimumab
alone or in combination with vaccine. Compared with ipilimumab alone, the combination prolonged survival
by 2 months. After 14 weeks of treatment, the patients show radiographic regressions.
Overall, we cannot say that vaccines don't work until we have optimally tested them. The optimal situation
is to test vaccines with immune-modulating agents. Without that, we won't make progress.
References
1. L
e D.T. et al. Clin. Cancer
Res. 18, 858-868 (2012)
2. Lutz E. et al. Ann. Surg.
253, 328-335 (2011)
3. L
aheru D. et al. Cancer
Ther: Clin. Cancer Res.
14, 1455-1463 (2008)
4. Thomas A.M. et al. J.
Exp. Med. 200, 297-306
(2004)
83
PART IV: T r a c k i n g t h e i m m u n e r e s p o n s e
Jérôme Galon
From the immune contexture to the immunoscore
in the era of cancer immunotherapy
Robert D. Schreiber
Cancer immunoediting: From basic mechanisms
to novel immunotherapies
Klaus Rajewsky
Modeling Epstein-Barr virus infection, immune surveillance and
human B-cell lymphomas in mice
George Coukos
Endothelial regulation of T-cell homing in tumors
Tracking the immune response
From the immune contexture to
the immunoscore in the era of
cancer immunotherapy
A report on a lecture by
Jérôme Galon
Integrative Cancer Immunology Lab. Ñ Inserm U872, Paris, France
The anatomic extent of tumor classification, the TNM, has so far been the most widely used to predict the
prognosis of cancer patients. However, this classification provides limited prognostic information in estimating
the outcome in cancer and does not predict response to therapy. Based on large-scale approaches and quantitative
measurements, tumors from human colorectal cancer with a high density of infiltrating memory and effector
memory T cells are less likely to disseminate to lymphovascular and perineural structures and to regional lymph
nodes. The combination of immune parameters associating the nature, the density, the functional orientation
and the location of immune cells within the tumor is essential for accurately defining the impact of the local host
immune reaction on patients prognosis. These immune criteria form the basis for the ‘immune contexture’ of a
tumor. Based on quantification of the intratumor immune reaction, Jérôme Galon presented the ‘immunoscore’,
which has a prognostic value superior to that of the TNM classifications. An ongoing worldwide task force aims
to propose an international standardized assay to routinely measure the immune status of a cancer patient.
In the past few years, there have been successes with some cancer therapies that boost the patients'
natural immune response. It's important to understand this response in order to differentiate between
those who respond to therapies and those who do not. Understanding the immune response may also help
identify the parameters associated with metastasis and with long-term survival, as well as the evolution of
the response along with the tumor.
Systems biology analyses of the tumor microenvironment have shown that in colon cancer, for example,
all of the subsets of immune cells are present within the tumor, including T helper cells, regulatory T cells
(Tregs) and T effector cells (Teffs), macrophages, dendritic cells and NK cells.
The early steps of metastatic invasion is dubbed VELIPI for venous emboli (VE) lymphatic invasion (LI)
perineural invasion (PI). Deep characterization of this stage by flow cytometry with 410 parameters shows
that there are markers associated with those sites of early metastasis. These are markers of subsets of T
cells, particularly the Th1 subset and memory subsets of markers.
Memory T cells and, in particular a subset of memory cells called effector memory T cells, correlate
with the absence of early-metastatic invasion, and with a good clinical outcome for people with colorectal
carcinoma.
Gene expression profiling of 75 patients shows that in an unsupervised correlation matrix, there are
clusters of genes that correlate strongly with each other, and with inflammation, adaptive immunity and
immune suppression-related genes such as interleukin-10 (IL-10), TGF-β, vascular endothelial growth
factor (VEGF) and FOXP31.
However, only the genes from the adaptive immunity cluster correlate with clinical outcome. The better
the coordination of these genes, the least the risk of tumor relapse. For example, those who have a high
expression of only granzyme B but not other T-cell genes are not protected against recurrence.
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86
Figure 1
Coordinated adaptive
immune response
correlated with relapse.
One important aspect of the tumor is its heterogeneity, especially in terms of the location of the immune
cells. We micro-dissected tumors in 415 patients with colorectal carcinoma, quantifying the immune
populations in the center and the invasive margins of the tumor.
Software analysis can determine the precise cell count and Ñ given the surface area of the tissue analyzed
Ñ the density of any immune subpopulation in a tumor in a particular region. Based on the analysis of 6,640
immunohistochemistry spots and a 15-year follow-up, those who have a high density of adaptive immune
cells in both the center and invasive margins of the tumor seem to have an excellent outcome with a low
risk of tumor recurrence.
However, that advantage is lost when there is a low density of the immune cells in either of the two tumor
regions. The worst outcome is in those patients who have a low density of T cells in both tumor regions.
Coordinated response:
In the classical tumor invasion parameters of the UICC-AGCC-TNM classification, patients with colon
cancer are classified as being in stage 1 through 4, from early-stage disease to metastasis. If the T cells
and memory T cells are quantified in both the center and the invasive margins of the tumor, however, those
who have a high density of those cells in both tumor regions have an excellent outcome over 15 years,
regardless of the stage of the disease.
For example, patients with lymph-node metastasis have a similar outcome to those with earlier-stage
disease if both sets of people have the same density of immune cells in the center and invasive margins
of the tumor. These results suggest that the coordinated adaptive immune reaction, more than tumor
invasion parameters, predicts the clinical outcome.
The most striking observation comes from the statistical analysis: In a Cox multivariate analysis, the
immune pattern remains significant, but most strikingly, the classical stages become insignificant and
statistically dependent on the immune reaction.
Tracking the immune response
Based on these analyses, the Ôimmune contextureÕ of the tumor has four parameters critical to survival:
the nature of the immune response, the functional orientation of the immune response, the density of the
cells and their location.
To understand why some people have a strong immune infiltrate, with more than 15-year survival, and
others have a weak infiltrate and survive less than 2 years, we created ARACNE, or Algorithm for the
Reconstruction of Accurate Cellular Networks, using gene ontology, Cytoscape and ClueGO software,
experimental data and gene expression profiling of 107 colorectal carcinoma tumors.
The biggest nodes in this analysis are the Th1 cytotoxic-related genes, which are the most associated
with survival of the patients and with a strong T-cell infiltration. The search tool for predicting functional
interaction relied on public databases such as the conserved genomic neighborhood databases, receptor
ligand protein-protein databases, functional genomics databases, Affymetrix data with DNA microarrays
and all of PubMed.
This analysis showed that the two families that are most predictive are the chemokines CX3CL1, CXCL9,
CXCL10 and CCL5, and adhesion molecules such as MADCAM1, VCAM and ICAM1. Patients with low
expression of CX3CL1, CXCL9 and CXCL10 have very few CD8 T cells, whereas those who have high
expression of the chemokines are fully infiltrated with T cells. In particular, CXCL9 and CXCL10 correlate
with a high density of macrophages and memory T cells, whereas CX3CL1 correlates with Th1-activated
CTLs.
Looking at chromosomal instability in cytokine regions reveals a clear correlation between a deletion of
a region on chromosome 4 and lower expression of the cytokines present in this region. In particular,
deletion of one of the
4 cytokines present
on that chromosome,
IL-15, correlates with
poor prognosis in the
patients. There is also
a correlation between
IL-15 expression and
proliferation of T and
B cells in the lymphoid
islets, the center and
the invasive margins
of the tumor. Finally,
the expression of IL15 itself correlates
strongly with patient
survival.
Network analysis:
To explore how the immune response evolves along with the tumor, we analyzed gene expression and
other factors in tumors from hundreds of patients at different stages. This analysis reveals that networks
of cells correlate together in the center and the invasive margins of the tumor.
For example, the more granulocytes there are in the invasive margins, the more there are in the center.
There is also a different level of density of the cells in different areas. For example, B cells surround the
tumor, but only rarely enter the tumor.
Evaluating the cell distribution by tumor stage shows that in the center of the tumor, adenocarcinoma
patients at T1 already have many CD3 and memory T cells and few macrophages. As the tumor progresses
Figure 2
Gene predictive network
based on experimental
+ in silico data.
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from T1 to T2, macrophage numbers go up, and from T2 to T3, the number of B cells increases. By T3,
compared with T1, the numbers of CTLs and activated Th1 cells have fallen. All of the subsets of the T cells,
except Th17, are associated with a good outcome.
Based on this, we have a defined the immune contexture, which is composed of the type of immune cells
associated with patient survival. The presence of CD8+ CD3+ and CD45RO+ cells, their density and their
location are all important factors. Also, the expression of genes including IFN-γ, CTL-related genes such
as granulozyme, chemokines, adhesion molecules, as well as IL-15, are the most striking and important
parameters associated with the natural long-term survival of the patients2.
Patients with optimal immune reactions are those who have high density of memory T cells, a good immune
contexture, high coordination, and with low expression of VEGF and few Th17+ cells. Even 10 years after
diagnosis, the risk of relapse for those patients is less than 10%.
Figure 3
Understanding evolution of
the immune response with
systems biology.
For those patients who
have high T-cell infiltration,
but with either increased
expression of VEGF or loss
of coordination in terms
of the spatial distribution,
the risk of relapse rises
to 50%. Among patients
who are heterogeneous
between the two tumor
regions, depending on
the level of Th17, the risk
of relapse goes up from
10% to 25%. In the worstcase scenario, for patients
who have a low density of
T cells and no immune
coordination, the risk of
relapse at 2 years is 80%.
Simple score:
To simplify these complex scenarios, we have developed an immune score, which provides a simple and
powerful way to quantify the immune response. This is a powerful new addition to diagnosis because the
immune response is rarely taken into account when classifying tumors.
The gold standard of cancer classification is the TNM classification, which assesses the severity of the
disease based on the extension into the primary organ (T), lymph node invasion (N), metastasis and distant
metastasis (M). That classification was proposed in 1932, and has been revised many, many times, but
always still based on the TNM features.
There are additional features taken into account, especially by pathologists, such as the histological grade
of the tumor or venous emboli. They may also test for some genetic markers. But in general, these do not
predict the outcome of the patient.
By contrast, the immune score quantifies two subsets of immune cells, the CTLs and memory T cells, in
both regions of the tumor Ñ the center and the invasive margins. Those who have an immune score of 4
have high levels of both markers in both regions. There's a direct relationship between the immune score
and survival of the patient.
Tracking the immune response
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Figure 4
Immunoscore using whole
slide FFPE.
In a Cox multivariate analysis, the immune status of the patient is powerful, whereas the classic parameters
of the TNM become insignificant. The immune score can be a useful predictor, especially for early-stage
cancer. About 25% of patients with early-stage colorectal cancer will have cancer recurrence, for example,
but there is no way to predict those high-risk patients. In a cohort of 411 patients validated with a second
cohort of 188 patients, more than 95% of those with an immunoscore of 4 remained disease-free for more
than 18 years.
The fact that CTLs or memory T cells are associated with a good prognosis has been shown in all solid
tumors (see Coukos, page 101). A meta-analysis of 124 papers looking at patient outcome and the
prognostic power of subsets of T cells clearly showed that, for all 20 tumor types analyzed, CTLs and Th1
cells are associated with a good outcome. The data are less clear for other types of cells, especially Tregs.
Th17 cells are associated with a bad outcome3.
To enable the use of immunoscore in routine clinical practice, we are trying to apply it using regular slide
sections. We have also developed software that automatically detects the invasive margin of the tumor, for
example, and counts the cell density in each region for each marker.
A worldwide consortium, supported by 17 national societies, is validating the score worldwide in more than
9,000 patients across 23 big centers4. Those results should soon be available.
In conclusion, the immune contexture could have huge implications for patients, to classify their tumors,
as well as to predict their prognosis. Second, the markers could also be predictive for response to
treatment, for immune-based and other therapies. The immunoscore may also help stratify patients as
having different types of immune defects, which may help develop new immunotherapies.
References
1. G
alon J. et al. Science 313,
1960-1964 (2006)
2. Tosolini M. et al. Cancer
Res. 71, 1263-1271 (2011)
ridman W.H. et al. Nat.
3. F
Rev. Cancer 12, 298-306
(2012)
4. Galon J. et al. J. Transl.
Med. 10, 205 (2012)
Tracking the immune response
Cancer immunoediting:
From basic mechanisms
to novel immunotherapies
A report on a lecture by
Robert D. Schreiber
Division of Immunology Ñ Washington University, St Louis, USA
Cancer immunoediting is the process by which the immune system controls and shapes cancer. 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 in which tumor cells that
survive immune elimination remain under immunologic control resulting in functional tumor dormancy); and
escape (the phase in which clinically apparent tumors emerge that display either reduced immunogenicity or
enhanced immunosuppressive activity). Strong experimental data using mouse models of cancer have shown
the existence of each phase of this process. Compelling clinical data suggest that a similar process may also
occur during the evolution of certain types of human cancer. Efforts to elucidate the molecular and cellular
mechanisms that underlie each of the phases have helped to identify the nature of antigens seen
by immunity in
nascent cancers. They have also shown that immunoselection is a major mechanism of immunoediting and that
edited tumors can still be controlled by the immune system if natural mechanisms that prevent autoimmunity
are suspended. Robert D. Schreiber is using genomic sequencing approaches to define the antigens that induce
tumor rejection following checkpoint blockade and to develop individualized cancer vaccines.
The term Ôcancer immunoeditingÕ, first proposed 12 years ago, explains part of the complex interactions
that occur between the immune system and the developing tumor1. It is an extrinsic tumor-suppressor
mechanism that occurs only after cellular transformation and intrinsic tumor suppressor mechanisms
such as p53 and Rb.
In its most complex form, cancer immunoediting exists in three phases. The first phase is elimination,
which is a modification of the older, perhaps more controversial, concept of cancer immunosurveillance.
The innate and the adaptive immune systems need to work together to recognize a tumor long before it
becomes clinically apparent and, in many cases, completely destroy the tumor2.
However, sometimes that process doesn't go to completion. In that case, residual tumor cells that survive
the elimination phase enter a second phase called equilibrium. Although the immune system is incapable
of killing those last few cancer cells, it nevertheless keeps them in relatively functional growth dormancy,
preventing their outgrowth. It is in equilibrium that immunoediting occurs, and it is the longest phase of
this three-step phase3.
If in fact the tumor becomes edited to an extent that its immunogenicity is reduced below a threshold level,
then the immune system can no longer keep these cells from growing out. The tumor cells begin to grow
progressively and become a clinically apparent disease. It's in context of this expanding tumor that the
immunosuppressive environment is set up, blocking the immune system from functioning.
Most of the work on cancer immunoediting has been focused on defining the process and on identifying the
cells and molecules involved. There are details that need to be worked out, including the targets of cancer
immunoediting, the mechanisms involved, and whether edited tumors are immunogenic and can serve as
targets for cancer immunotherapy.
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92
Figure 1
The three Es of cancer
immunoediting.
To try and define tumor antigens and what happens to them in the course of interaction with the immune
system, we used a specific tumor, d42m1, a sarcoma derived from immunodeficient RAG2 knockout mice.
These mice are highly immunodeficient, and don't have any T, B or NK cells. Because the tumors in these
mice have never been in contact with the immune system, they retain a lot of their mutations, and remain
highly immunogenic.
About 80% of na•ve, syngeneic wild type mice reject transplanted d42m1 tumor cells. In the remaining 20%
of cases, the tumor grows and then begins to regress, but then eventually grows out and kills the mouse.
When cell lines derived from these Ôescape tumorsÕ are transplanted into na•ve, syngeneic wild type mice,
they grow progressively. This shows that it is possible to recapitulate the immunoediting process by
passaging the cell lines through immunocompetent mice. It also suggests that the tumor is immunogenically
heterogeneous.
Molecular heterogeneity:
The majority of tumor clones retain high immunogenicity and are rejected when injected into wild type
na•ve mice; a couple of the clones grow progressively, in a manner that looks just like escape tumors. One
possibility is that the molecular basis of this heterogeneity is at the level of antigens.
We performed exome sequencing on 3 of the regressor tumors, 2 of the progressor clones and 3 escape
tumors using an effective, inexpensive and relatively quantitative method that we developed called cDNA
capture sequencing.
Tracking the immune response
MCA sarcomas have a profound number of non-synonymous expressed mutations, ranging from 2,000 to
3,000 mutations per tumor, similar to that of carcinogen-induced lung cancers in smokers. The various
d42m1 derivatives look remarkably similar because they are all phylogenetically related, but with subtle
differences.
When the sequences from these tumors are pipelined into immunoepitope prediction algorithms, the
computer predicts which of the mutations in a given sequence might bind the two class I molecules
expressed in the 129 background: H2-Kb and H2-Db.
There are mutations that are present in only the regressor clones, but not in the progressor clones or the
escape tumors. This suggests there might be epitopes that are shared among the regressor forms.
To investigate this, we made a CD8+ T-cell clone from a mouse that had rejected d42m1 and then incubated
these T cells in vitro with the different regressor and progressor clones and escape tumors. The CTL clone
recognizes and reacts with parental d42m1 and the three regressor clones, but not with the progressor
clones or any of the escape tumors. Because all of the T cells in this clone share the same T-cell receptor,
this result shows that the regressor clones must share a particular antigen.
Filtering the data to look only at the Db and Kb epitopes simplifies the diagrams considerably. We then made
the assumption that the best antigens in this tumor would be those in which the wild type sequence would
bind to the class I molecule poorly or not at all, but the mutated sequence would bind with high affinity.
With that additional filter, of the 2,700 mutations, 2 rose to the surface. One is a mutation in spectrin-β2
that produces an epitope that would bind to H2-Db. The other is a mutation in erythrocyte protein band 4
that would bind as a peptide to H2-Kb.
In vitro, it is pretty clear
that the C3 d42m1-specific
CTL clone recognizes
the regressor clones and
shows H2-Db restriction.
Based on the genome
sequencing approach and
predictive algorithms, the
R913L mutated form of
spectrin-β2 seems to be a
major rejection antigen of
the d42m1 tumor.
Rejection antigen:
You can test this by going back to the old ways of cloning tumor antigens, especially expression cloning
of the tumor antigen, using as a readout the activation of the tumor-specific T-cell clone. For the d42m1
tumor, this turns out to be the same R913L mutation in spectrin-β2. Interestingly, this latter approach took
several months whereas the predictive algorithms are as quick as a week.
The mutant form of spectrin-β2 is in the parental d42m1 but is not expressed in the three escape versions
of the tumor and is not in any other sarcoma from any other mouse. Perhaps most importantly, normal
cells from the very mouse that d42m1 was derived from do not carry this mutation.
To validate this as the correct antigen, we first took cohorts of wild type na•ve syngeneic mice and injected
into them either the d42m1 tumor cell line, or one of the escape versions of the tumor. We then looked at the
Figure 2
Mutant (R913L) Spectrin-β2
is the major rejection
antigen of the d42m1 MCA
sarcoma.
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infiltration of CD8+ T cells into the
tumor in the draining lymph node,
using a fluorescent tetramer of
H2-Db bound to a peptide derived
from mutant spectrin-β2.
Figure 3
Enforced expression of
mutant Spectrin-β2 in
d42m1 escape tumor cells
results in rejection.
In mice injected with parental
d42m1, there is a time-dependent
increase in mutant spectrin-β2
specific T cells in both tumor and
the draining lymph node. They
reach maximum levels at day 11,
which for this particular tumor is
the day just prior to the rejection
of the tumor. In contrast, there
is no infiltration of the tumor
antigen-specific T cells into the
tumor that lacks the mutant form
of spectrin-β2.
Finally, to fulfill Koch's postulates, we showed that if expression of the mutant spectrin-β2 is enforced in
the escape tumors to the level seen in the parental tumor, the tumor now becomes highly immunogenic,
and is rejected. What's more tetramer-positive CD8+ T cells infiltrate the tumor.
This suggests that mutant spectrin-β2 is a physiologically relevant major rejection antigen of the d42m1
sarcoma. It also indicates that immunoediting can produce tumor cells that lack immune-dominant tumorspecific rejection antigens4.
Although this work suggests what immune editing is, it doesn't reveal how it occurs. One possibility is
that it is an epigenetic mechanism, but our transplant system does not take long enough for epigenetic
changes to occur. However, as shown in our model, a highly effective T cell-dependent immunoselection
mechanism does indeed occur.
Every single clone of the d42m1 tumor that expresses mutant spectrin-β2 is rejected when transplanted
into wild type mice, and every tumor clone that lacks mutant spectrin-β2 grows progressively. This indeed
suggests that there is immunoselection at work here.
Darwinian selection:
In one last experiment, we made an artificial mixture of these homogenous clones, mixing 95% of the clones
that contain the mutant spectrin-β2 rejection antigen, and 5% of a clone that lacks the antigen. When this
mixture is injected into na•ve wild type mice, it completely recapitulates the behavior of the parental d42m1
tumor cell line. That is, 80% of the mice reject the tumor and 20% show these escape tumors.
From this work, we concluded that cancer immunoediting, at least of d42m1, is the result of a Darwinian
immunoselection process, acting on a heterogeneous tumor cell population that displays different levels
of strong antigens. This results in the evolution of tumor cell variants lacking major rejection antigens.
Although this example shows immunoediting of the antigens, a similar selection process could
result in variants with other mutations that compromise immunogenicity or that favor induction of
immunosuppression Ñ for example, mutations in the MHC proteins, any of the MHC class I pathway
proteins or in the IFN-γ receptor signaling pathway.
Is an edited tumor really non-immunogenic? Or does it express an immunogenicity that is just below the
immune radar Ñ that is, can cancer immunotherapy control edited tumors?
Tracking the immune response
95
If mice bearing edited MCA sarcoma tumors from wild type mice are treated when the tumors are small
with anti-CTLA4, about 70% Ñ 4 of the 13 tumors in the experiment Ñ grow progressively, unaffected
by the checkpoint blockade therapy. The other 9 are sensitive to CTLA4 inhibition and are rejected. This
suggests that there is residual immunogenicity in at least a high percentage of edited MCA sarcomas.
Similarly, when mice bearing
either a d42m1 clone that lacks
the major rejection antigen or
an escape tumor that also lacks
the antigen are treated with a
variety of checkpoint blockade
antibodies against CTL4, PD1
or PD-L1, the tumors are now
seen by the immune system and
are rejected. Proving that this is
an immunologic rejection, the
rejection can be prevented by first
depleting CD8+ or CD4+ T cells or
by blocking IFN-γ.
Figure 4
Mutant ALG8 and LAMA4
are recognized by CD8+ T
cells from α-PD-1 Tx mice
that reject d42m1-T3.
Genomics approach:
We next aimed to use genomics and immune epitope prediction methods to identify the antigens responsible
for anti PD1-induced rejection of the d42m1 tumors. When d42m1 tumors are treated with anti-PD1 on day
3, 6 and 9, the mice reject their tumors. We let the animals rest for a period for 2-3 weeks, and harvested
their spleens. We then generated CTL lines by stimulating them in vitro a couple of times with irradiated
tumor cells.
These T-cell lines, called CTL-62, -73 and -74 after the mice they are derived from, see the parental d42m1
tumor as well as the progressor and escape tumors. But they do not see irrelevant tumors. The patterns
are remarkably similar, suggesting once again that these T cells may see shared antigens present in the
tumor clones. In this case, the restriction element is H-2Kb.
Using predictive algorithms we found that two of the three top predicted epitopes turn out to be recognized
by the T cells, leading to IFN-γ induction. One is laminin alpha subunit 4, and the other is ALG8, a glucosidase
homolog. The CTL 74 cell line selectively recognizes the mutant peptides from LAMA4 and ALG8.
Several questions still remain. For example, can we detect mLAMA4 or mALG8-specific T cells entering
d42m1-T3 tumors and draining lymph nodes, following anti-PD1 treatment using H2-Kb tetramer staining?
Can we prevent d42m1-T3 tumor growth either prophylactically or therapeutically by vaccinating with a
mLAMA4 or mALG8 vaccine? If so, can this approach be extended to fully edited tumors from MCA-treated
wild type mice?
This technique may be useful for longitudinally analyzing the extent of immune editing that tumors undergo
naturally or as a consequence of therapy. It may also help identify patients who might best benefit from
cancer immunotherapy including checkpoint blockade. The ultimate goal is also to rapidly identify tumor
antigens, making it possible to develop individualized tumor vaccines or immunotherapy.
References
1. S
hankaran V. et al. Nature
410, 1107-1112 (2001)
2. Koebel C.M. et al. Nature
450, 903-907 (2007)
esely M.D. et al. Annu
3. V
Rev Immunol. 29, 235-71
(2011)
4. Matsushita H. et al. Nature
482, 400-404 (2012)
Tracking the immune response
Modeling Epstein-Barr virus
infection, immune surveillance and
human B-cell lymphomas in mice
A report on a lecture by
Klaus Rajewsky
Immune Regulation and Cancer Prog. – Max-DelbrŸck-Cter for Molecular Med.
Berlin, Germany
Burkitt's lymphoma derives from germinal center B cells, is driven by overexpression of the c-MYC oncogene,
and presumably relies on survival signals through the B-cell antigen receptor. This pathogenic scenario can be
reconstructed in mice, where combined overexpression of c-MYC and PI3K signaling in germinal center B cells
results in tumors resembling Burkitt's lymphoma in terms of location, cellular origin, cell-surface markers
and gene expression. Most strikingly, they accumulate tertiary somatic mutations also recurrent in human
Burkitt lymphoma. This preclinical model opens a way to test therapies and address the urgent problem of
tumor dissemination into the central nervous system. More than 90% of the population is latently infected with
Epstein-Barr-Virus (EBV), a B cell-transforming herpes virus. In a primary EBV infection, T and NK cells rapidly
clear transformed B cells, establishing life-long immunity. However, some latently infected B cells persist
and are under permanent immune surveillance; serious pathologies arise when that immune surveillance is
weakened. Mouse models with key EBV proteins expressed in the B-cell lineage can pinpoint basic mechanisms
of both EBV-driven lymphomagenesis and immune surveillance. Genetic mouse models of human cancer can
thus provide insights into functionally relevant pathogenic pathways. Klaus Rajewsky presented two examples
of this basic approach.
There is evidence that both antigen-dependent and ÔtonicÕ survival signals through the B-cell antigen
receptor (BCR) are important for driving B-cell lymphomas. The importance of tonic BCR-derived survival
signals has become apparent in a mouse model developed for one particular lymphoma, which is Burkitt's
lymphoma.
Most B-cell lymphomas originate from the germinal center Ñ either from the B cells participating in
the germinal center reaction, or from their progeny. The germinal center is an environment in which
programmed DNA breaks and cell proliferation occur concomitantly. As a result, there are many forms of
germinal center-derived B-cell lymphomas, driven by different chromosomal translocations.
One such malignancy is Burkitt's lymphoma, characterized in 100% of the cases by translocation of the
MYC gene into the immunoglobulin (Ig) loci. The tumors are typical germinal center tumors with ongoing
somatic hypermutations of the Ig-V region genes. They have a high mitotic index, and a high frequency of
apoptosis.
The tumors express many of the markers that are typical
for germinal centers B cells. The sporadic form of Burkitt's
lymphoma mostly localizes in the abdomen and originates
from the gut-associated, chronically activated lymphoid
tissue.
In an attempt to model Burkitt's lymphoma, we combined
deregulated MYC expression in germinal center B cells
with the induction of an anti-apoptotic signaling pathway in
the same cells, which would rescue the cells from MYC's
pro-apoptotic properties. Here the question was, which prosurvival signal keeps the MYC-transformed Burkitt tumor
cells alive?
Figure 1
P110- and MYC-expressing
animals develop
aggressive Burkitt–like
tumors.
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A characteristic feature of Burkitt's lymphoma is that the MYC translocations always involve the nonproductive Ig heavy or light chain loci, but never the productive loci. This suggests that the lymphoma cells
are selected for BCR expression. Relating to this finding, we showed years ago that the BCR on mature
B cells is a survival determinant. Ablation of the BCR or its signaling subunit from the surface of the cells
leads to cellular apoptosis.
More recently we have shown that it is PI3K signaling downstream of the BCR which keeps the cells alive. In
the case of Burkitt's, gene expression data indeed suggest that both MYC and PI3K activity may drive these
tumors. That is consistent with the finding that in Burkitt's, in contrast to many other B-cell lymphomas,
NF-κB signaling is nearly off.
Indeed, NF-κB signaling is toxic for cell lines derived from Burkitt's tumors. Based on these ideas, we
hypothesized that in Burkitt's lymphoma, the pro-apoptotic activity of MYC is balanced by the BCRdependent pro-survival activity of PI3K signaling1.
Combined effects:
We generated a straightforward genetic mouse model of Burkitt's, combining two conditional gain-offunction alleles Ñ one for c-MYC and another for PI3K signaling Ñ together with a germinal center-specific
Cre allele. After about 250 days or so, the compound mutant mice all die from lymphomas that are striking
phenocopies of Burkitt's lymphoma.
In most cases, the lymphomas originate from Peyer's patches, as seen in sporadic Burkitt's. They also
resemble Burkitt's lymphoma in many other respects. Thus, the tumors are monoclonal and display
ongoing somatic hypermutation. By global gene expression profiles, the tumor cells are not only similar to
human Burkitt's lymphoma, but also exhibit a germinal center B-cell signature.
Figure 2
PI3K pathway activation
in primary Burkitt’s
lymphoma.
Tracking the immune response
In addition, the tumors acquired tertiary mutations that are seen in human Burkitt's lymphoma. For
example, a mutation in the CyclinD3 gene hits the same codon in human and mouse. This mutation leads
to the elimination of a phosphorylation site which in the wild type promotes degradation of cyclinD3, so that
in the mutant tumor cells the protein is stabilized. Also, PI3K activation has been found to be a common
feature in human Burkitt's lymphoma and the mouse tumors1,2.
Overall, this is a useful preclinical model of human Burkitt's lymphoma. The model suggests treatments Ñ
for example, PI3K inhibition Ñ and also opens the way to an analysis of the problem of tumor dissemination.
In that latter respect, a main clinical problem in human Burkitt's lymphoma is that the tumors easily
disseminate from the primary site into the central nervous system (CNS). This often necessitates
intrathecal chemotherapy from the very beginning.
Primary mouse tumors transplanted into immunocompetent mice don't generate tumors. However, when
primary tumors from the Peyer's patches are transferred to an immunodeficient recipients, the recipients
die within a few weeks. Most of the animals die from paralysis and tumor infestation of the CNS. Thus, this
animal model should allow us to find out what controls tumor dissemination into the CNS.
Viral infection:
The second topic, tumor immune surveillance, may be related to the control of Burkitt's lymphoma
dissemination. It brings us into the field of lymphomagenesis caused by viral infection, specifically
infection by Epstein-Barr Virus (EBV). This cannot be modeled by infecting mice with EBV because the virus
doesn't propagate in these animals. Instead, we introduced selected EBV proteins into mice by genetic
manipulation.
More than 90% of people worldwide carry latent infections with EBV. Acute infection in young adults leads
to a disease called infectious mononucleosis, which is characterized by viral transformation of the B cells.
The virus selectively transforms and expands B cells, but then immune surveillance sets in.
The human immune system is specialized in recognizing EBV-infected cells, and eliminates the cells within
2-3 weeks. However, in about one in a million B cells the virus persists in a latent form. A fatal disease
called X-linked lymphoproliferative syndrome (XLP) is characterized by the inability of children to fight EBV
virus.
Another clinically important aspect of EBV infection is that under conditions of immusuppression Ñ for
example, post-transplantation or in people with AIDS Ñ the immune surveillance can be broken, the virus
can reactivate and spread from latently infected cells, resulting in explosive expansion of infected B cells
and their malignant transformation.
The malignancies that can develop include endemic Burkitt's lymphoma and Hodgkin's lymphoma. The
most dangerous are lymphomas that develop in XLP patients, in post-transplant lymphoproliferative
disorder or in AIDS patients.
We focused on two main EBV proteins that are expressed in many EBVdriven tumors and play key biological roles: Latent membrane protein 2A
(LMP2A) and LMP1. These molecules are interesting because they are
mimics of BCR signaling and B-cell co-stimulation.
LMP2A is constitutively active with an ITAM motif in the cytoplasmic tail
that mediates constitutive BCR signaling. LMP1 is an EBV-evolved mimic
of an active CD40 co-receptor. It is also constitutively active if introduced
into cells, and activates a variety of pathways Ñ NF-κB, ERK, JNK and STAT
Ñ that are important for B-cell expansion and survival. In fact, it is able to
generate B-cell lymphomas by itself in transgenic mice at a low frequency3.
Figure 3
T cell-deficient CD19-cre;
LMP1flSTOP mice develop
lymphomas in liver or
spleen.
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To express LMP proteins in B cells conditionally, we crossed CD19creERT2 with ROSA26LMP1 and/or
-2A stop-flox mice. The CreERT2 protein is expressed only in B cells because of the B cell-specific CD19
promoter.
This is an inducible system in which, upon tamoxifen application, the CreERT2 protein translocates into
the nucleus and eliminates the stop cassette in front of the LMP1/2A cDNA. LMP1 and/or LMP2A is then
expressed together with reporter genes. Recombination is seen in only 3-5% of B cells in this system,
mimicking infection by the virus in just a fraction of the cells.
Explosive expansion:
In a first simple experiment, we administered tamoxifen to adult mice carrying the CD19creERT2 allele
together with the conditional signal-on alleles for LMP1 and LMP2A or just LMP1. In the controls, there
was no change in splenic B and non-B cell numbers upon induction of LMP proteins.
By contrast, there was a rapid and dramatic expansion of LMP+ B-cell blasts in the spleen of mutant mice,
exactly as in human mononucleosis. By day 20, however, the LMP+ B cells had been completely cleared,
and the spleen again had normal levels of (LMP-) B cells.
The LMP+ B cells disappear because they are cleared by T cells which are activated and expand in
response to them, very much like in humans. The animal is then immune against further expression of
these proteins. If T cells are depleted before tamoxifen application the animals are unable to deal with the
situation, and die within a few weeks from an explosive expansion of the B-cell blasts. This is reminiscent
of the uncontrolled expansion of EBV infected B cells in XLP patients.
We also wanted to study LMP1-driven lymphomagenesis by breaking CD4 T cell-mediated surveillance. If
LMP1 expression is targeted to B cells on a T cell-deficient background, all of the animals die within 2-3
months from aggressive B-cell lymphomas in the liver or spleen4.
So this system can be used to study the generation and surveillance of LMP1-driven tumors in a mouse
model in vivo. Apparently, a single EBV-encoded protein, LMP1, plays a dominant role in both processes.
As the lymphomas arising in the T cell-deficient animals are monoclonal, the tumor cells must have
accumulated genetic alterations in addition to LMP1 expression. Next-generation DNA and RNA
sequencing will help to identify such additional mutations, with the prospect of functionally analyzing
their role in lymphoma pathogenesis and therapeutic potential in connection with EBV+ lymphomas in
immunosuppressed patients.
Figure 4
Human EBV+ PTLDs
express high levels of
NKG2D ligands.
With respect to immune surveillance, CD4+ and CD8+ T cells
recognize the LMP1+ B cells in an MHC-restricted manner.
In an in vitro killing assay, CD8+ cells, but not CD4+ T cells
kill the LMP1+ tumors. Surprisingly, however, in vivo, it's
the CD4+ T cells, and not the CD8+ T cells, that clear the
tumor. Experiments are underway to explore the basis of this
unexpected and clinically potentially important finding.
Another interesting feature of this system is that NK cells
may slow disease progression in T cell-deficient mice,
reminiscent of the role of NK cells in the control EBVinfected cells in people. The LMP1-driven lymphoma cells in
the mouse express stress antigens such as RAE1 that are
recognized by activated NK cells.
Tracking the immune response
101
Interestingly, these stress antigens are also expressed on cells in human post-transplant
lymphoproliferative disorder. That suggested a potential therapeutic approach, involving the production
of a fusion protein between the NK activating receptor NKG2D and an immunoglobulin Fc fragment. This
protein can control the growth of the LMP1-driven tumors in vivo, as shown in a collaboration with Glenn
Dranoff's laboratory at the Dana-Farber Cancer Institute in Boston4 (see Dranoff, page 63).
One central question is, why are the LMP1+ cells so immunogenic? Our evidence suggests that this is not
due to an evolutionary adaptation of the TCR repertoire to EBV epitopes (which has not taken place in mice),
but rather to a high immunogenicity of the EBV-infected B cells due to their specific mode of activation.
That involves proliferative expansion, combined with strong upregulation of a host of antigen-presenting,
adhesion and co-stimulatory molecules.
References
1. S
ander S. et al. Cancer
Cell 22, 167-179 (2012)
2. Schmitz R. et al. Nature
490, 116-120 (2012)
3. K
ulwichit W. et al. Proc.
Natl. Acad. Sci. USA 95,
11963-11968 (1998)
4. Z
hang B. et al. Cell 148,
739-751 (2012)
Tracking the immune response
Endothelial regulation of T-cell
homing in tumors
A report on a lecture by
George Coukos
Ludwig Institute for Cancer Research – UNIL, Epalinges, Switzerland
There is significant evidence indicating that only patients who show strong infiltration of tumors by activated T
cells have a positive outcome long-term. Gene expression analysis suggests an inverse relationship between
T-cell infiltration and angiogenesis. For example, there is an inverse correlation between activated T cells and
the expression of the endothelin B receptor, which is responsible for promoting angiogenesis. In combination
with a tumor vaccine, an inhibitor of the endothelin B receptor can surmount the endothelial barrier and allow
T cells to infiltrate the tumor. Adoptive transfer experiments also suggest that with a massive number of T
cells, the endothelial barrier is no longer important. Complement component 3, a mediator of inflammation,
and anaphylatoxin C5A are both required for the T-cell infiltration into the tumor. Complement is also required
for the fundamental adhesion of T cells on endothelial cells. There is also a second killing mechanism in which
the effector T cells that manage to adhere to endothelial cells are eliminated through expression of the death
factor FAS ligand. A combination of aspirin and bevacizumab decreases FAS ligand expression, resulting in a
massive infiltration of activated T cells into tumors. George Coukos described multiple approaches to combat
the endothelial cell control of the tumor immune response.
There are two main patterns of immune cell infiltration in ovarian cancer. In one, the T cells make it
into the epithelial component of the tumor (tumor islets), and in the other, they are present only in the
surrounding stroma. This dichotomous pattern has significant implications for disease outcomes, in that
patients who have intraepithelial (or intratumoral) T cells survive significantly longer. This conclusion is
based on analyzing the outcome in about 1,800 patients across more than 10 studies.
There are several questions about this immune infiltrate, including whether the cells are tumor-reactive,
and if they are, why they cannot reject the tumor. It's also important to determine what contributes to the
low or absent tumor immunogenicity, and what can be done to boost the tumor's immunogenicity.
In an orthotopic model that reliably reproduces ovarian cancer, the ovarian tumors do not show tumorinfiltrating lymphocytes (TIL), and so were classified as non-immunogenic. However, it turns out that
T cells are present in the very early stages of tumor development.
As tumors progress, they lose CD3+ and
CD4+ T cells, and primarily CD8+ T cells,
while there is progressive accumulation
of macrophages and myeloid-derived
suppressor cells and dendritic cells in
this microenvironment. Therefore, lack
of tumor immunogenicity is acquired, it's
not endogenous1.
One of the contact or paracrine factors
related to the strengthening of the tumor
is PD1. In fact, injecting PD1-blocking
antibodies early on completely rescues
the immune decline in the tumor. Later
on in tumor development, however, this
becomes more and more difficult.
Figure 1
Intratumoral T cells,
recurrence and survival in
epithelial ovarian cancer.
103
104
Recent gene expression profiling classified 251 ovarian tumors into various genotypes based on gene
expression analysis. One large group, which comprises approximately 40% of tumors, has no T cells by
immunohistochemistry and no T-cell signatures by gene expression profiling, but it has an accentuated
stroma, angiogenesis or pericyte gene signature.
On the other hand, two other subgroups, which hold about 35% of the tumors, have intraepithelial T cells
by immunohistochemistry. One of them has a prominent T-cell gene signature, while in the other there is
absence of stroma and angiogenesis gene signatures. This suggests an inverse association between T-cell
infiltration and the development of a robust angiogenesis signature.
This is consistent with the observation that tumors that have no intraepithelial T cells are more likely to
have increased expression of vascular endothelial growth factor (VEGF) in the tumor microenvironment. A
lot of work since then has led to a unifying model in which tumor-related paracrine mechanisms support
immunosuppression, stroma repair and angiogenesis at the same time.
In fact, many cellular players of the inflammatory infiltrate in tumors and the tumor cells can support
the same pro-angiogenic and immunosuppressive functions: Many immunosuppressive cells support
angiogenesis and many angiogenesis mechanisms are immunosuppressive.
Vascular control:
Is there a mechanism by which the tumor endothelium actively regulates trafficking in tumors? If the
endothelium has a pro-angiogenic programming, would that prevent the extravasation of T cells into the
tumor microenvironment?
To address that question, we performed laser-capture micro-dissection of endothelial cells from ovarian
cancer specimens and normal ovaries, harvested the cells and analyzed their gene expression profile.
Comparing the isolated tumor endothelial cells to normal endothelial cells showed that the tumor
endothelial cells have a distinct signature. What's more, among the tumor endothelial cells, there are
tumor endothelial signatures associated with the presence or absence of intraepithelial T cells.
Among the genes upregulated in the tumor endothelium associated with the absence of intraepithelial T
cells is endothelin B receptor (ETBR). Endothelin B is already implicated in promoting angiogenesis and has
been implicated in the regulation of the vasculature. There is an inverse correlation between intraepithelial
T cells and tumor ETBR expression: Tumors with a lot of TILs have very little ETBR if any, and vice versa2.
In vitro and in vivo work has shown that tumors produce the endothelin ligands, which, via a paracrine
mechanism, speak to the endothelin receptors expressed on vascular cells. ETBR is responsible for
upregulating nitric oxide within the endothelial cells. That dysregulates the expression of ICAM-1 on the
tumor endothelium, leading to the absence of the intraepithelial T cells, both in humans and in mouse
models.
Figure 2
The tumor-endothelial
barrier hypothesis.
A cyclic peptide called BQ-788 specifically inhibits
ETBR. In the ovarian cancer model, the peptide
alone has no efficacy, and neither does an ID8
tumor vaccine. But in combination, there is a
synergy at both the early and late stages of peptide
injection in the subcutaneous model, as well as
in the intraperitoneal model. This validates the
evidence that blocking ETBR on the vasculature
massively increases T-cell infiltration into the
tumor.
Overall, this indicates that there is an endothelial
barrier, which dynamically and actively regulates
Tracking the immune response
the homing of T cells. The obvious next question is whether the endothelial barrier is surmounted in some
way in the many patients who respond to vaccines or to the adoptive transfer of T cells.
Adoptive transfer experiments indicate that the magnitude of the T cells matters, both in humans and in
mice. With a low amount of T cells, the endothelial barrier is still present, and ETBR is required to enhance
homing of T cells. But if this system is flooded with a critical mass of T cells, then ETBR is no longer
required.
Complementary effects:
Based on the initial gene expression profiling, the most upregulated gene in the endothelium of tumors
that have intraepithelial T cells is complement component 3 (C3), a major mediator of inflammation.
Activated T cells, and both tumor necrosis factor-α and interferon-γ can upregulate C3 in the endothelium.
Exposure to Th1 cytokines also upregulates the expression of anaphylatoxin C5a, one of the main effector
molecules in the complement cascade. In fact, T-cell media not only increase the activation of complement
in the endothelial cells, but induces precipitation of complement complexes right on the endothelial cell
surface.
To address whether the activation of complement is a cause or consequence of T-cell activation, we created
chimeric mice by transferring wild-type bone marrow into either wild-type mice, or into C3 knockout mice.
In the wild-type mice, both the tumor-infiltrating leukocytes as well as the endothelial and stroma cells
express complement. In the C3 knockout mice, the bone marrow-derived cells express complement but
the endothelium does not.
In the wild-type mice, an effective dose of adoptively transferred T cells can induce massive infiltration
of T cells in tumors and regression of tumors. However, adoptive transfer of T cells has no effect in C3
knockout mice. This indicates that C3 is required for the effective infiltration of adoptively transferred
T cells, and this C3 is derived from the endothelium.
The reverse situation is also true Ñ that is,
hyperactivated complement can make T cells
effective. For example, a low dose of T cells,
which has no effect in wild-type mice, has a
significant effect on tumor regression in DAF
knockout mice, which have a constitutive
hyperactivation of complement. This again
indicates that complement tightly regulates the
efficacy of T-cell adoptive transfer.
C5a anaphylatoxin is also upregulated in tumor
endothelial cells. C5a is the main effector for
complement cascade activation, so adoptive
transfer is ineffective in C5a receptor knockout
mice. Loss of complement does not affect
the engraftment of T cells in the spleen, but
significantly impairs the engraftment of T cells
in the tumor, and the ability of T cells to affect
the tumor microenvironment.
Once again, we created chimeric mice by
transferring wild-type bone marrow into the C5a
receptor knockout mice so that the endothelium
does not express the C5a receptor, but the
Figure 3
ETBR is upregulated in
tumor endothelium in
tumors without intraepithelial TIL.
105
106
T cells do. In the absence of C5a receptor, the infiltration of T cells into the tumor microenvironment is
impaired, as is the T cells' ability to control tumor growth.
Complement is also required for the fundamental adhesion of T cells on endothelial cells. C5a receptor
antagonists impair T-cell adhesion on endothelial cells. Antibodies blocking ICAM1 or VCAM1 also have a
similar effect. By contrast, endothelial cells treated with a recombinant C5a peptide are able to support the
adhesion of T cells much more potently.
Together these results suggest that C5a anaphylatoxin activates ICAM1- and VCAM1-mediated adhesion.
Compstatin, a C3 inhibitor, completely abrogates the upregulation of VCAM1 in endothelial cells by IFN-γ.
Similarly, a C5a receptor antagonist also completely blocks the responsiveness of endothelial cells to
IFN-γ. This suggests that the induction of VCAM1 by Th1 cytokines is mediated by C5a3.
Geographic dichotomy:
Overall, these inflammatory adhesive interactions are critically important for the extravasation of T cells.
Reprogramming of the tumor endothelium can prevent this adhesiveness to T cells and is mediated in part
by ETBR. VEGF upregulates ETBR, contributing further to this.
However, these mechanisms do not explain the geographic dichotomy of T cells in the stroma, or why
regulatory T cells (Tregs) may still make it into the tumor while effector T cells (Teffs) do not. It turns out
that the tumor endothelium expresses death molecules that eliminate Teffs, and this is under the control
of paracrine factors known to promote angiogenesis and stroma repair.
We looked at a tissue microarray collection comprising 1,300 tumors from different solid tumors and found
Fas ligand (FasL) expression in vessels of all tumor types. For example, between 30 and 70% of endothelial
cells from ovarian cancer express FasL.
This expression is tightly regulated such that, in many tumors, FasL is expressed in the tumor endothelial
cells within tumor islets, but not in the adjacent stroma. When freshly isolated endothelial cells are
incubated with T cells, the majority of activated T cells undergo apoptosis. A FasL antibody or a Fas antibody
attenuates the apoptosis.
Figure 4
Two mechanisms explain
the geographic dichotomy
of T cells in the stroma.
Tracking the immune response
107
Paracrine mediators, overexpressed by ovarian cancer cells at steady state and even more under hypoxia,
upregulate FasL in endothelial cells. A critical triad of factors Ñ interleukin-10, VEGF and prostaglandin
E2 Ñ synergize to upregulate FasL.
A combination of aspirin and bevacizumab decreases FasL, resulting in a massive infiltration of T cells
into the tumor in the absence of any immune manipulation. This again demonstrates that these tumors
are inherently immunogenic, but have important barrier mechanisms that prevent the accumulation and
engraftment of T cells.
The regression of tumor is entirely dependent on CD8+ T cells: If the CD8+ cells are depleted from the
mice, the tumor continues to grow uninterrupted in spite of the addition of the two drugs.
In summary, there are two mechanisms Ñ one in which the tumor endothelium reprograms to become
non-sticky so that the Teffs can't attach, and a killing mechanism in which the Teffs that manage to adhere
are eliminated through expression of FasL.
Endothelial cells that express FasL kill Teffs, but activated Tregs resist FasL-induced cell death. The
expression of FasL on the vessels correlates inversely with the CD8/FOXP3 ratio.
Collectively, these data argue strongly that the endothelial compartment of tumors is an important
immunoregulatory system, which is under the control of paracrine mechanisms within the tumor
microenvironment. This control seems to be extraordinarily tightly regulated because it can be different a
few microns away in the epithelial compartment of the tumor.
These mechanisms are important, especially with low-affinity or low-frequency T cells. When this affinity
increases, as it does with adoptive transfer, they may be less relevant. In the case of low-affinity T cells, it
may make sense to block angiogenesis at the same time.
In a pilot study at the University of Pennsylvania, we are using a whole-tumor antigen loaded on dendritic
cells, along with bevacizumab and aspirin, and cyclophosphamide to eliminate Tregs4. We are also
developing DNA vaccines to target targets unique to tumor vasculature, such as tumor endothelial marker
1 (TEM1).
In conclusion, once you break down the tumor vascular barrier, you enable homing of preexisting T cells
against the tumor, and allow the system to reject the tumors.
References
1. Z
hang L. et al. N. Engl. J.
Med. 348, 203-213 (2003)
2. B
uckanovich R.J. et al.
Nat. Med. 14, 28-36 (2008)
unez-Cruz. S. et al.
3. N
Neoplasia 14, 994-1004
(2012)
4. Kandalaft L.E. et al.
Oncoimmunology 2,
e22664 (2013)
109
PART V: D e c o d i n g i m m u n e r e s p o n s e p l a y e r s
Ton Schumacher
(Neo)-antigens and tumor-reactive TCR repertoires
in human tumors
Miriam Merad
Dissecting the tumor myeloid niche
Sebastian Amigorena
Regulatory T cells control immune responses
to self and non-self antigens
Irving L. Weissman
Normal and neoplastic stem cells
Decoding immune response players
(Neo)-antigens and tumor-reactive
TCR repertoires in human tumors
A report on a lecture by
Ton Schumacher
Department of Immunology – The Netherlands Cancer Institute
Amsterdam, The Netherlands
There is strong evidence that human cancers, and in particular melanoma, can be recognized and destroyed by
T lymphocytes. An important gap in our understanding of these therapies, however, is which antigens on cancer
cells are the targets in T cell-mediated tumor destruction. Knowledge of such antigens would be of obvious
value, as it would allow one to move towards approaches in which tumor-specific T-cell activity is boosted in a
more directed manner. New technologies to analyze T-cell reactivity have recently become available. Analyses
of T-cell reactivity against the entire group of shared tumor-associated antigens in melanoma patients have
shown that the overall magnitude of the T-cell responses against these widely studied shared antigens is
extremely modest. These data indicate that T-cell reactivity against non-shared tumor-associated antigens may
be of greater clinical importance. The combination of second-generation sequencing and immunomonitoring
could subsequently be shown to allow the identification of T-cell reactivity against patient-specific neo-antigens,
formed by mutations. Ton Schumacher argued that the ability to describe patient-specific tumor-reactive T-cell
responses accurately should form a first step towards the development of personalized cancer immunotherapy.
When an animal is infected with a pathogen, the few T cells that recognize that pathogen expand to large
numbers, and this population then contracts to form T-cell memory. This process always occurs with the
same kinetics. Although T-cell populations show reproducible behavior, however, we do not know how that
reproducibility is achieved. This is because population behavior cannot be used to infer single-cell capacity.
In principle, there might be two ways to achieve reproducibility in the behavior of T-cell populations. One is
control at the single-cell level, in which different na•ve T cells, with the same affinity T-cell receptor (TCR),
produce the same type of progeny. In this case, the system is set up such that each na•ve T cell responds
in the same way.
The second system involves control by
averaging. In this system, there may
be divergent differentiation paths for
individual families, and reproducibility
manifests itself only at the population
level.
To follow this T-cell differentiation
process by lineage tracing, we
developed a technique called Ôviral
barcoding.Õ The first version of this had
a barcode library with 2,500 unique
genetic tags, and a GFP marker.
First, thymocytes are infected with this
retroviral library to avoid activating
na•ve T cells. The thymocytes are then
injected into recipient mice where they
develop into na•ve T cells, each with
their own genetic tag.
Figure 1
Division of labor? Families
for immediate action
and families for multiple
recalls?
111
112
In infected mice, those T cells differentiate and cell populations of interest can be isolated. For each
cell population, we do technical replicates to determine how much a barcode is present within a given
population. These technical replicates are essential because only if they are accurate can you draw any
conclusions about the clonal composition of a cell population1.
The results show that clonal expansion of individual T cells varies on orders of magnitude. There are
T cells that generate massive families and there are many, many T cells that generate minute families.
Because the number of total T cells in the mouse is known, this read data can be converted into absolute
cell numbers to calculate the size of each family.
Reproducibly, in each mouse, the most prominent T-cell family has some 100,000 progeny. By comparison,
the median family in the same mouse has 200 cells, meaning that there is a 400-fold difference in clonal
expansion.
This profound diversity in the clone size may be because, irrespective of TCR, some families are more fit
than others. Alternatively, there is a Ôdivision of laborÕ between na•ve T cells. Whereas some cells primarily
produce the rapid deployment force, others may primarily contribute the backup troops. In that case, there
should be differences in phenotype, and also in recall responses, among families.
Family ties:
First, we looked at phenotypic disparity between T-cell families. At the peak of infection, at day 7 or 11,
we sorted cells on the basis of 3 markers, CD62L, CD27 and KLRG1. CD62L is a marker that's lost early
during T-cell responses, CD27 is lost late during T-cell responses, and KLRG1 is acquired late during
T -cell responses.
The vast majority of families have few members that are CD62L+, but some families are largely comprised
of T cells that express CD62L. Many families are mostly CD27+, but at the same time, there are families
that are largely CD27-. For KLRG1, there are similarly many families that are highly positive, but there's a
long tail of families that can be fully KLRG1-.
It turns out that the moment a family becomes bigger, the proportion of members that express CD62L
becomes less and less. Loss of CD62L may therefore be co-regulated with clonal expansion.
However, this is not the case for all markers. The fraction of CD27+ cells per family correlates to a very
limited extent with family size, and the fraction of KLRG1+ cells does not at all correlate with family size.
This shows that there is disparity along multiple dimensions, and no single mechanism can therefore
regulate diversity.
Figure 2
UV-induced peptide exchange
allows generation of 1,000s of
pMHC multimers.
If there were a division of labor in the T-cell
population, there would be families with
immediate action and families for multiple recalls.
We tested this and indeed, there are many families
that are important in the response to a primary
infection, but then get lost in the secondary and
tertiary response, or vice versa. In line with this,
in a cluster analysis, the secondary and tertiary
responses cluster well together and are quite
distinct from the primary response.
These data show that reproducibility of T-cell responses does not occur at the single-cell level, but only at
the population level Ñ control by averaging2.
The second conclusion that we can draw is that sister cells behave alike. For example, within a large
family, the cells are completely CD27- or completely CD27+. There are two potential explanations for this:
Decoding immune response players
Either the sister cells in the same family receive the same environmental cues, or, in an extreme case,
there may be imprinting in a grandmother cell.
Finally, the first division of na•ve T cells may be asymmetric, with an unequal distribution of molecules,
including those associated with cell fate. However, asymmetric division can't explain physiological diversity,
because each family would have to look the same. Even if asymmetric division does occur, it would at the
very least need to be combined with subsequent effects.
So, how do T-cell families diverge? How do na•ve T cells generate different kinds of offspring? One potential
explanation is that there are multiple competing internal stochastic processes. These would have to be
early after T-cell activation, because if they occur late after T-cell activation, there would be already be so
many daughters that they would cancel out through statistics.
The alternative, and preferred, explanation is that during an immune response, there are many local niches
that each give different signals to the locally expanding family. It would be interesting to define systems in
which early signals received by T cells can be coupled to late fates.
Identifying antigens:
Immunotherapy can be a highly effective treatment for some cancers. In one approach, tumor-infiltrating
lymphocytes (TILs) are grown and expanded to large numbers, and then given to a melanoma patient
together with high-dose interleukin-2.
This approach has shown about a 50% response in trials in multiple centers. But there are important
questions about which cytotoxic T cells mediate the cancer regression. Once you know the essential
antigens that the T cells target on the tumor, you could try to specifically boost their numbers rather than
give an undefined cell product.
There are two large categories of antigens that the T cells might recognize on a tumor. One is self antigens,
to which tolerance has to be incomplete. The big advantage of these antigens is that they are shared by
patients, so it is easy to study them. The field has really focused on this class of antigens.
Figure 3
Partial response upon
anti-CTLA4 treatment.
113
114
The second class of antigens, neo-antigens, are epitopes that arise as a consequence of tumor-specific
mutations. These are, in large part, patient-specific. Most mutations in human cancer are passenger
mutations and have in fact been generally ignored.
To make progress in this field, we first needed to develop technology to measure T-cell responses in
cancer patients in a high-throughput fashion. We could then use this to measure reactivity against shared
antigens in patients, and try to measure T-cell reactivity against patient-specific neo-antigens.
We developed a technology inspired by tetramer technology. We produce massive amounts of MHC molecule
and load it with a peptide that is sensitive to ultraviolet light. To show how this technology works we
prepared crystals of these MHC molecules. When the crystal is exposed to UV light, the electron density of
the peptide disappears because it is cleaved into small parts, each having a lower affinity.
The crystal can be soaked in a solution with a new peptide, which can then bind the now-vacant binding
groove. This crystal structure is identical to an MHC complex generated conventionally. This method can
be used to make hundreds or thousands of different peptide-MHC multimers. We have developed this for
some 20 HLA class I alleles.
The second issue is pragmatic. In a clinical trial, the amount of material that can be obtained from a patient
is extremely limited. So we developed a multiplex detection system, in which each MHC molecule is labeled
with two colors, thereby creating combinatorial codes. Using this, we can measure more than 35 T-cell
responses in a single test tube3.
Antigen recognition:
To try to reveal T-cell responses against neo-antigens, we used a sequencing approach. Exome sequencing
can be used to identify tumor-specific mutations. RNASeq can then be used to determine which of the
mutated genes are expressed. Using this info, we can predict T-cell epitopes for each mutation, make MHC
multimers for all the predicted epitopes, and measure T-cell responses against those predicted epitopes.
Figure 4
Exploiting patient-specific
neo-antigens in TIL therapy?
For one patient who experienced a partial response upon anti-CTLA4 treatment, we obtained tumor
fragments, isolated the tumor cells and sequenced them by exome sequencing. In this one tumor, there
were more than 1,000 mutations that alter a protein-encoding part of the genome, meaning that there is
plenty of opportunity for the immune system to recognize something.
We then predicted potential epitopes around all those mutations and made MHC multimers for those
alleles for which we had this technology operational. In the first patient, we saw two T-cell responses, one
minor and one quite major. The T cells specifically recognize cells expressing the mutant epitope.
Based on peripheral blood samples from various points in time, we were also able to show that anti-CTLA4
treatment boosts the T-cell response against this mutant epitope by about 5-fold. In a second patient, we
saw T-cell responses against three mutated epitopes.
Decoding immune response players
115
This is the first demonstration that this kind of exome information in cancer patients can be used to follow
the course of cancer immunotherapy. Further experiments have shown that neo-antigen recognition is
biased to really high-affinity binders.
Can you exploit these patient-specific neo-antigens in TIL therapy? We have developed peptide-exchange
MHC streptamers for clinical use. The idea is to enrich the T-cell populations that recognize neo-antigens
of interest including, perhaps, driver mutations.
Overall, these data show that exome-based analysis of neo-antigen-specific T-cell responses in human
cancer is feasible. In the majority of melanoma patients, there will be some reactivity against neo-antigens.
To develop personalized immunotherapies to target these antigens, there are two possible approaches.
The first is to try to generate T-cell products that selectively target defined neo-antigens. As an alternative,
one could develop vaccines that contain neo-epitopes, perhaps combined with antibodies against T-cell
checkpoint molecules.
It's also important to try and find out how common neo-antigen-specific T-cell reactivity is in other human
malignancies. If it is linear with mutation load, this approach is going to be primarily of large value for
melanoma, non-small cell lung cancer and perhaps colorectal cancer, tumors with a high mutation load.
If it's not linear, this may be of value for even more tumor types.
Finally, an important question for therapy is whether there is a hidden repertoire of neo-antigens that do
not induce immune responses unless the system is pushed, perhaps because of immunodominance or
because presentation is less efficient4. If these epitopes are present, it may be possible to push the system
and try to induce immune responses against them.
References
1. S
chepers K. et al.
J. Exp. Med. 205,
2309-2318 (2008)
2. Gerlach G. et al. Science
340, 635-639 (2013)
adrup S.R. et al. Nat.
3. H
Methods 6, 520-526 (2009)
4. Heemskerk B. et al. EMBO
J. 32, 194-203 (2013)
5. Van Rooij N. et al. J. Clin.
Oncol. In press (2013)
Decoding immune response players
Dissecting the tumor myeloid niche
A report on a lecture by
Mirian Merad
Department of Oncology – The Mount Sinai School of Medicine, New York, USA
Despite accumulating evidence suggesting local self-maintenance of tissue macrophages in the steady state, the
dogma remains that tissue macrophages derive from circulating monocytes. Novel parabiosis, fate mapping and
adoptive transfer approaches have shown that monocytes contribute minimally to tissue-resident macrophages
in the steady state. After depletion of macrophages, the majority of repopulation occurs by local proliferation in a
stochastic manner and is not restricted to a specific macrophage population. These results establish that similar
to Langerhans cells and microglia, several tissue macrophages self-renew in situ and can repopulate locally
after tissue injuries. These results are consistent with results obtained in humans showing that macrophages
can form in the absence of circulating monocytes, suggesting that tissue-resident macrophages and circulating
monocytes should be classified as independent mononuclear phagocyte lineages. Because existing tools can
distinguish monocytes and infiltrating macrophages from resident macrophages, more stringent distinctions
should be made for these two populations. Miriam Merad argued that this will be important in understanding
the contribution of tissue-resident macrophages and bone marrow-derived macrophages to tumor homeostasis.
Tissue-resident phagocytes represent a heterogeneous group of hematopoietic cells, mostly of myeloid
origin. They include macrophages, identified in the late 1800s, and dendritic cells (DCs), discovered in the
1970s. These two compartments have common features: They populate all tissues, constantly sample
environmental antigens, have constitutive MHC II expression and large sensing machinery, and they
express large number of toll-like receptors (TLRs).
However, macrophages and DCs also have some
differences in function. For example, they express
different TLR and protease machinery, which might affect
their antigen repertoire expression. Another importance
difference is that DCs have a unique ability to migrate from
peripheral tissues to the draining lymph node. In contrast,
macrophages don't migrate, and this may affect their ability
to modulate peripheral or central tolerance.
DCs have been shown to induce and control innate and
adaptive immune responses. They appear to play an
important role in the priming of both CD4+ and CD8+
T-cell responses. They have also been shown to promote
the homing of T cells to tissues of injury, and they play a
role in humoral immunity, and in peripheral and central
tolerance1.
Yet controversy remains on the exact contribution of DCs
and macrophages to tissue immunity. Resolving the debate
is important because the knowledge of the cells that control
the induction of tissue immunity response affect our ability
to modulate immune response in people with defective
immune function.
Figure 1
There are few infected
dendritic cells in the lung.
117
118
Recent results have established that DCs derive from a lineage that is different from macrophages.
Macrophages are thought to derive from monocytes, whereas DCs derive from a DC-restricted lineage.
The commitment to DC lineage is associated with the expression of receptor tyrosine kinase 3 and the
production of the FLT3 ligand by the stroma, leading to FLT3 and STAT3 signaling.
These progenitors have been identified in mouse and human bone marrow, and give rise to spleen, lymph
node and thymic DC. In lymphoid tissue, DCs include two compartments: CD8+ DC and CD11b+ DC. The
CD8+ DCs have a superior ability to prime CD8+ T-cell response, whereas CD11b+ cells are more potent at
driving CD4+ T-cell response. The transcription factors IRF8, ID2 and BATF3 are required to generate CD8+
DCs, whereas IRF4 and NOTCH2 are thought to control the differentiation of the CD11b+ DCs.
Despite their critical localization at the interface with the environment, non-lymphoid tissues DC have been
much less studied than lymphoid tissue DC. Non-lymphoid DCs were thought to derive from monocytes,
adding to the confusion between the relative contribution of macrophages and DCs to tissue immunity.
Distinct compartments:
Non-lymphoid tissue DCs include two phenotypically distinct DC compartments that can be distinguished
by the expression of CD103. This integrin is also a ligand for E-cadherin, an adhesion molecule that is
expressed by all epithelial cells. The dermis contains another population that expresses CD11b and lacks
CD103.
CD103+ and CD11b+ cells, as well as macrophages, populate most tissues, including the dermis, lung,
liver and lamina propria. In addition, the epidermis has another phagocyte population called epidermal
langerhans cells.
Figure 2
Dendritic cell composition
in human lungs and the
lungs of humanized mice
are remarkably similar.
Similar to CD8+ DC, the CD103+
DC compartment requires FLT3,
IRF8, ID2 and BATF3 for their
development. FLT3 knockout has
no DCs at all whereas the BATF3
knockout still has CD11b+ DCs.
So that has become an interesting
tool to study the contribution of
particular compartments in tissue
immune response.
The Immunological Genome Project
is an NIH-funded consortium of
researchers, whose goal is to
make publicly available the gene
expression profile of all mouse
immune cells. The project has
revealed that all CD8+ DCs and
CD103+ DCs, regardless of their
origin, segregate together, away
from macrophages. In contrast,
the CD11b+ population spans the
principal component analysis.
The power of the consortium is
also to be able to do computational
analyses. Using a clustering
approach, we have profiled more
Decoding immune response players
than 244 populations of cells and 800 replicates, and used a clustering approach to identify a module of
strongly co-expressed genes. An algorithm called Ontogenet identifies activators and repressors of each of
these modules. For example, CD103+ and CD8+ DCs share 28 genes that are absent in CD8- DCs.
One interesting molecule among these is XCR1, a chemokine receptor that binds to CXL1. XCR1 has been
shown to be important for the induction of CD8 effector response. Even in humans, XCR1 is expressed only
in a subset of DCs, and not in any other hematopoietic cells.
Based on this computational analysis, we also identified some activators of the DC compartment. Some
of these Ñ for example BATF3 and ZIRF8 Ñ were previously known, but we identified new ones such as
MYCL1.
Infectious response:
By FACS analysis, lung tissue consists of two subsets of DCs, the CD103+ DCs and CD11b+ DCs. The
CD103+ DCs line the airway epithelia as well as the blood and lymphatic vessels. The CD11b+ DCs are
present much deeper in the parenchyma.
In the case of a flu infection, most of the infected cells in the lung are macrophages and epithelial cells,
rather than DCs. In contrast, in the draining lymph node, only the langerin+ CD103+ DCs are infected.
Following virus infection, both CD103+ and CD11b+ DCs migrate quickly to the lymph node, but the CD103+
DCs migrate much quicker than the CD11b+ DCs.
The lung migratory CD103+DCs are also the only one that drive the flu-specific response and present viral
antigens to CD8+ T cells during the first day of infection. Eliminating only the CD103+ DCs strongly reduces
flu-specific CD8 response, and inhibits clearance of the virus from the lung.
The CD103+ DCs don't prime better because they are most susceptible to infection. Rather, they induce the
CD8+ T-cell response because of their superior ability to cross-present virus-infected cells. The CD11b+
DCs are able to induce CD4+ T-cell response, perhaps because, like macrophages, they have a superior
ability to degrade the material they uptake.
In lymphoid tissue, the CD103+ DCs express several antiviral genes in the steady state. This may be useful
because it may prevent migratory DCs from transporting virus to lymphoid organs. But it also shows that
the cells have a superior ability to prime CD8+ response.
Figure 3
Microglia are present in
the brain rudiment of E10
embryos.
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120
Knowledge about human DCs has lagged behind mostly because the phenotype of human DCs is different
from that of mice. Human DCs express different antigens, so it has been difficult to find the equivalent of
CD103 or CD11b. Transcriptome analysis suggests that humans also have two different DC compartments.
These studies have revealed that CD1C+ and CD141+ DCs in humans are the equivalent of mouse CD11b+
and CD103+ DCs, respectively. The CD141+ DCs have a superior ability to drive CD8 immunity and expansion
of CD8+ T cells. A subset of the CD141+ cells also express BDCA1.
The CD141+ cells express a similar profile of genes to that of CD103+ DCs, including TLR3, XCR1 and
CLEC9a Ñ also called DNGR1 Ñ which is apparently important for the cross-presentation of necrotic cellassociated antigen. The cells also express langerin, albeit at lower levels than in mice.
The CD141+ population is enriched in the human lung and in the lung of the humanized mouse. Interestingly,
the CD103+ population in mice is also present in high numbers in the lung.
In humanized mice, there is no difference in CD8 response to flu virus: Both compartments are equally
potent in driving CD8+ T cells. However, only the CD1c+ positive compartment drives expression of CD103,
the same molecule that is expressed in mouse DCs. CD103 is an important molecule in intraepithelial
T cells, it loads the retention of T cells in the epithelial compartment2.
Origin of macrophages:
Macrophages comprise a different compartment. In the brain, macrophages are not populated by
circulating precursors. They remain in the tissues throughout the mouse life, completely independently of
circulating precursors. These microglia can be detected in E10 mouse embryos.
To investigate whether primitive macrophages contribute to this adult microglia pool, we used a RUNX1
fate-mapping system, which helps trace the progeny of primitive macrophages. This approach shows that,
at least in mice, adult microglia derive from the primitive pool of macrophages3.
Langerhans cells, or epidermal phagocytes, are also maintained locally, completely independent of the
circulating precursors. Similar to the brain, yolk sac macrophages seed the skin rudiment at an early
age after conception. However, in contrast to the brain, most yolk sac macrophages are almost entirely
replaced by fetal liver precursors around E13.5 3.
In fact, most macrophage populations, including bone marrow macrophages, peritoneal macrophages,
red blood macrophages and lung macrophages, are maintained completely independently of circulating
monocytes. These compartments all seem to derive from a second wave of fetal liver monocytes4.
Figure 4
F4/80+ cells localize in the
stroma and within dead
tumor cell zones.
Langerhans cells and microglia are absent
in mice that lack the CSF1 receptor. CSF1 is
also called macrophage colony stimulating
factor (M-CSF), a cytokine that is important
for maintaining the macrophage pool in tissue.
In contrast, two compartments are present in
mice that have a natural mutation for CSF1, the
op/op mice.
A newly discovered cytokine called IL-34 binds
the CSF1 receptor with higher affinity than
CSF1 does. It turns out that IL-34 drives the
differentiation of monocytes into macrophages.
Strikingly, IL-34 is expressed at high levels in
the skin and in the brain, and it's the only CSF1
receptor ligand that's present in the epidermis5.
Decoding immune response players
121
In the dermis, both IL-34 and CSF1 are present. If IL-34 is deleted the Langerhans compartment is
completely lost. But CSF1 op/op mice have a network of Langerhans cells. So IL-34 is the cytokine that
maintains Langerhans cell homeostasis in the adult skin.
IL-34 is expressed in neurons in specific areas of both the mouse and human brain. In its absence, there is
a reduction of microglia, specifically in the areas of the brain that produce IL-34.
Overall, there are different cytokines that contribute to the maintenance of macrophages in tissue. CSF1
may be the predominant population, but in the lung, for example, macrophages are maintained mostly
by CSF2. It is important to continue to dissect the factors that maintain homeostasis of tissue-resident
macrophages as these factors likely contribute to the maintenance of tissue integrity.
References
1. M
erad M. et al. Annu.
Rev. Immunol. 31, 563604 (2013)
2. Yu Cl. et al. Immunity 38,
818-830 (2013)
inhoux F. et al. Science
3. G
330, 841-845 (2010)
4. Hashimoto D. et al.
Immunity 38, 792-804
(2013)
5. Greter M. et al. Immunity
37, 1050-1060 (2012)
Decoding immune response players
Regulatory T cells control
immune responses to self and
non self antigens
A report on a lecture by
Sebastian Amigorena
Institut Curie – Inserm U932 ÔImmunitŽ et CancerÕ, Paris, France
In the absence of FOXP3+ regulatory T cells (Tregs), fatal multiple organ autoimmunepathologies arise, leading
to death in both humans and mice. Tregs are known to suppress self-reactive autoimmune responses. Previous
reports have suggested that Tregs not only inhibit immune responses to non-self antigens, but may also
contribute to clearance of viral or parasite infections. But how Tregs regulate the priming of T lymphocytes
to non-self antigens remains unclear. In the past few years, Tregs have been shown to control the stability of
contacts between dendritic cells and CD4+ T cells, thereby regulating the induction of effective T-cell activation,
expansion, effector functions and memory. In the absence of Tregs, even low-affinity peptides induce stable
dendritic cell-T cell contacts, but the blockade of CCL-3/4/5 chemokines in vivo or in vitro inhibits these stable
contacts. Treg depletion also induces the activation and expansion of a population of low-avidity CD8+ T cells.
Impaired avidity of the primary immune response results in reduced persistence and expansion of memory CD8+
T cells to Listeria monocytogenes, causing incomplete protection against challenge by the bacteria. Sebastian
Amigorena proposed that Tregs are important regulators of the homeostasis of CD8+ T-cell priming and play a
critical role in the induction of high-avidity primary responses to non-self antigens and effective memory.
When regulatory T cells (Tregs) were first discovered, there was a conceptual shift in the field of immunology
in general, and in cancer immunity in particular, because of their importance in the immune response to
cancer.
Tregs control antitumor immune responses.
Blocking Tregs, in contrast, doesn't affect
anti-infectious immune responses or non-self
challenges, indicating that Tregs can distinguish
between the two kinds of immune responses.
There are mainly two populations of Tregs:
natural Tregs differentiate in the thymus and
emerges as mature Treg cells and induced
Tregs mature in the periphery from na•ve cells,
under the effect of TGF-β and interleukin-2 (IL2). These two populations can both block CD4+
and CD8+ T cell-mediated immune responses.
From imaging experiments, it's clear that natural
Tregs represent a motile swarm of inhibitory
cells. For the immune response to occur despite
this depends on affinity. The affinity of self and
non-self immune responses is not the same
because of thymic selection.
The group of cells that emerge from the thymus
don't recognize self antigens with high affinity,
but there are some that recognize self with
Figure 1
Regulatory T cells promote
CD8+ T-cell memory.
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124
intermediate or low affinity. In contrast, there is no selection in the thymus for non-self antigens and the
mature T cells that are generated express a wide repertoire of T-cell receptors (TCRs), covering every
possible specificity and affinity.
A transgenic T cell with OT1 is specific for a peptide from ovalbumin. There is a series of peptides available
that have mutations in different positions. Researchers have tested and isolated peptides that bind to MHC
with a similar affinity, but bind to the TCR with lower affinity. If we now load antigen-presenting cells (APCs)
and mature dendritic cells (DCs) with these two peptides and then deplete Tregs, there are differences in
the efficiency with which Tregs inhibit these two types of interactions.
With the high-affinity peptide, there is a significant increase in the expansion only at low concentrations.
This falls when there is more antigen, so there is some inhibition of this high-affinity peptide. The lowaffinity peptide by itself induces almost no expansion in the presence of Tregs.
When Tregs are depleted from the beginning of the reaction, there is some expansion, albeit not as much
as with the high-affinity peptide. However, that increase doesn't fall with a rise in antigen concentration. If
in contrast, the Tregs are depleted on day 2 or 3, we lose this effect.
Dynamic interactions:
The priming of T cells by DCs occurs in three distinct phases of dynamic interactions1,2. During the first
phase, T cells and DCs establish short interactions that last 1 to 2 minutes and then they move apart.
Then at some point, one DC and one T cell form a stable interaction that lasts for several hours, and that
allows for long-lasting exchange of information. And then again, there is a phase of labile and unstable
interactions that lasts for another day or so.
Figure 2
Regulatory T cells
represent a motile swarm
of inhibitory cells.
The length of this last phase depends
on the processing of the antigen and the
maturation of the DC, so it can be short
under certain conditions where there
is a lot of antigen from the beginning.
If resting DCs are used in the second
phase, you don't induce maturation using
LPS or a strong adjuvant, and lose this
stable phase of interaction. The result
of this lack of stable interactions is
tolerance, no CD8+ T-cell memory and
no interferon-γ production. Mature DCs
that don't express ICAM1 also cannot
sustain the stable interactions3.
Two papers published in 2006 showed that Tregs can regulate the stability of the interaction between DCs
and CD4+ T cells or CD8+ T cells. Tregs make the interactions unstable, thereby affecting the efficiency of
priming. Later studies suggested that PD1 could be the key factor in this inhibition.
For example, the OT1 cells with the high-affinity peptide interact stably with DCs. The Tregs then make
contact with those DCs, but that doesn't affect the interaction of the OT1s with the DCs. However, in the
case of the low-affinity peptide, the same interactions occur, but at some point, the OT1s detach from the
DCs after the Tregs make contact with the DCs.
Analyzing the mean velocity of the T cells shows that with the high-affinity peptide, the velocity in the first
phase is already low, and remains low during the stable interactions. Depleting Tregs makes no difference
to the velocity. In contrast, with the low-affinity peptide, the T cells have a high velocity in the presence of
Tregs. In the absence of Tregs, the contacts are stable and can last for several hours.
Decoding immune response players
These observations suggest that Tregs somehow inhibit low-avidity T cell-DC interactions, but not highavidity interactions. When Tregs are depleted, a peptide that is not able to induce stable interactions by
itself can now mediate stable interactions and effective priming.
Affinity selection:
What the thymus does is select for TCRs that are high- or low- affinity, not for peptides that are high- or
low- affinity. Based on the above results, the hypothesis is that the same thing would occur with high- and
low-affinity TCRs: Tregs would inhibit the initial interactions between low-avidity T-cell clones and DCs.
The T-cell repertoire for non-self antigens, for which there is no thymic selection, includes a wide repertoire
of TCRs that have varying affinities for that peptide. The proportion of low- and high- affinity TCRs is not
random, but can be somehow controlled because during memory responses, the affinity is always higher
than during primary responses.
We predicted that, if Tregs also inhibit the low-affinity responses of T cells to non-self, depleting Tregs
during a non-self response should change the overall affinity of the response.
We tested this prediction using tetramers. MHC class I tetramers bind to T cells according to the affinity
of the TCRs. The more they bind, the higher the affinity. When priming happens in the absence of Tregs,
there is a strong expansion and proliferation of a low-affinity T-cell response. Tregs seem to be required
to obtain high-affinity primary CD8+ T-cell responses.
Another way to measure this is to make dilutions of the tetramers; the rate of decay of the binding of the
tetramers is proportional to the affinity of the whole population. In wild type mice, the high-affinity TCRs
retain labeling at dilutions at which the low-affinity TCRs are already negative. This suggests that the
response in wild type mice is variable:
Some respond with high affinity and
others with low affinity. But when Tregs
are depleted, all of the mice respond
with low affinity.
If Treg depletion is delayed to day 3,
there is no longer any difference in the
affinity of the CD8+ T-cell response.
This again suggests that something
happens at the beginning, just as in
the OT1 transgenic system. Even when
Tregs are depleted at day 3 or day 5,
there is still an increase in the number
of tetramer+ cells, suggesting that
Tregs also regulate the expansion of the
CD8+ T cells, but do so independently of
the affinity.
Relevance for protection:
Listeria monocytogenes is a potent inducer of CD8+ T-cell responses. However, the bacteremia in the
primary infection is mainly controlled by the innate immune response. In the spleen and in the liver, the
bacteremia goes down to zero before the CD8+ T-cell response comes up. We therefore analyzed the effect
of Treg depletion during primary infection on the efficiency of the memory response during re-infection
(challenge).
Figure 3
Tregs control the motility
of T cells during priming.
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126
We depleted Tregs from the mice before the infection, infected the mice and then looked at the number
of bacteria in the spleen on days 3, 5 and 7. In the presence or absence of Tregs, the bacteria are totally
cleaned up from the mice, spleen, liver or other organs, indicating that Tregs don't alter the innate immune
response to this infection.
However, there is a big difference in the high-affinity response, as seen with the dilution experiments,
meaning that Tregs promote the expansion of high-avidity CD8+ T cells. When Tregs are depleted, the
response is much more variable, and in most mice, there is a strong decrease in the affinity of the primary
response.
The results overall suggest that Tregs inhibit the interactions between low-avidity T-cell clones and DCs
even in the case of infectious antigens. They inhibit the priming of low-avidity clones and limit the expansion
of both high- and low-avidity clones.
Co-stimulatory molecules and other molecules in DCs have no effect on how Tregs regulate this response.
In lymph nodes, gene expression analysis with or without Treg depletion shows changes in a series of
chemokines, mainly CCL3, CCL4 and CCL5.
Depletion of Tregs increases the amounts of CCL3, CCL4 and CCL5 (after ex vivo stimulation), suggesting
that Tregs normally negatively regulate their production. These chemokines are ligands of CCR5, which
is known to regulate DC-T cell interaction. Low-affinity interactions may be more dependent on the
chemokines than the high-affinity ones. Inhibiting these chemokines with monoclonal antibodies abolishes
the effect on both the affinity and the stability of the interactions.
These experiments suggest that Tregs impose an Ôavidity thresholdÕ for the priming of T-cell responses, so
that only avidities above a certain limit result in T-cell expansion and responses4. Depleting Tregs lowers
that threshold, resulting in the expansion of low-affinity T cells.
Figure 4
Working model: Tregs
inhibit the initial
interactions between lowavidity T-cell clones and
dendritic cells.
Decoding immune response players
127
Adoptive transfer experiments show that the primary T-cell response generated in the absence of Tregs
is not different from the response observed in the presence of Tregs. The low-affinity CD8+ T cells protect
from infection as efficiently Ñ or even better Ñ than the high-affinity ones. That's because the low-affinity
population looks much more like an effector population.
These results suggest that the difference in affinity affects the memory response. Reinfection experiments
with Listeria show that in fact, mice primed in the absence of Tregs produce defective memory CD8+ T-cell
responses. What's more, the memory cells produce lower amounts of IFN-γ.
To be effective, the memory response needs to be of the right strength4. Tregs are required in order to get
the right strength during priming, allowing the induction of effective protection in the memory phase. What
we would like to propose is that regulation by Tregs depends on the affinity of TCR peptide recognition.
Therefore, Tregs suppress anti-self responses, but promote responses to non-self antigens.
References
1. M
ittelbrunn M. et al. Blood
113, 75-84 (2009)
2. Hugues S. et al. Curr. Opin.
Immunol. 18, 491-495
(2006)
choler A. et al. Immunity
3. S
28, 258-270 (2008)
4. Pace L. et al. Science 338,
532-536 (2012)
Decoding immune response players
Normal and neoplastic stem cells
A report on a lecture by
Irving L. Weissman
Department of Developmental Biology – Stanford University, Stanford, USA
Tissue stem cells are highly regulated by a number of pathways, including programmed cell death (PCD) and
programmed cell removal (PCR), which is independent of PCD. In mouse acute myelogenous leukemia, the
leukemic stem cells express high levels of CD47, an age marker on mouse red blood cells that prevents PCR.
CD47 is a ligand for macrophage SIRP-α, which blocks phagocytosis of adhered CD47+ red blood cells. The
protein is found in mouse and human primary and metastatic cancers. Antibodies that block CD47 cause in vitro
primary cancer cells to be phagocytosed by mouse or human macrophages, indicating that CD47 is a cancer
‘don’t eat me’ signal, countering the various ‘eat me’ signals involved in PCR. In all tumors tested, several
weeks of treatment with anti-CD47 antibodies shrinks primary tumors and eliminates metastases. Anti-CD47
antibodies synergize with the anti-CD20 antibody rituximab to eliminate primary non-Hodgkin’s lymphoma
transplants into immunodeficient mice. These studies reveal that evasion of PCR, like the evasion of PCD, are
requirements for malignant tumor progression. Irving Weissman proposed that the PCD/PCR pathways might
play a role in a number of pathogenic states, from persistent infections by microbes that hide in cells, to cells
that respond to persistent damage or immune signals.
The only self-renewing cells in the blood-forming system Ñ other than B and T memory cells Ñ are
hematopoietic stem cells (HSCs), which account for about 1 in 20,000 cells in the bone marrow of adult mice
or adult humans. Because they are the only self-renewing cells, HSCs are the only important functioning
cells in a bone marrow transplant.
Others had proposed based on experiments in acute myelogenous leukemia that cells that are CD34+/
CD38- are leukemia stem cells (LSC). We knew at the time that those two markers are not only on the selfrenewing stem cells, but also on multipotent progenitors, which normally don't self-renew.
The Atom Bomb Casualty Commission froze bone marrows at Hiroshima hospital from patients who got
leukemia after the bomb. In a subset of the samples with the AML-1/ETO+ translocation, leukemia is
seeded not by the self-renewing stem cell fraction but by the multipotent progenitors.
These cells comprise about 5-15% of the cells in the bone marrow. Interestingly, the HSCs that have
the same translocation, and are the clonal pre-leukemia precursors, don't show any abnormality, and
transfer normal hematopoiesis for all lineages. That suggests that although the AM1/ETO translocation is
necessary, it's not sufficient to turn a cell into a leukemia cell1.
Chronic myelogenous leukemia in the chronic phase, before it goes to blast crisis, is a clonal disorder.
The BCR/ABL translocation is not limited to the myeloid cells that are over-produced, but is present in the
granulocytes, monocytes, erthyrocytes and B cells. In fact, the translocation is in the stem cells.
Second, the numbers of the stem cells that have the BCR/ABL translocation increase at the expense of
normal stem cells in those patients. So the BCR/ABL-containing HSCs not only self-renew, but have a
competitive advantage.
Still, in that disease and in every leukemic disease, there is a powerful set of regulations to keep the
HSC numbers constant. The same translocation in myeloid blast crisis is five steps downstream, at the
granulocyte/monocyte progenitors (GMPs), which over-express activated nuclear β-catenin. In every
129
130
patient, the GMPs have nuclear unphosphorylated β-catenin, and in all of them, axin shuts down the
proliferation of the leukemic clone.
Looking at each step of the β-catenin pathway in 7 patients, of all the LSC transcripts, the only error was
in GSK3-β. This is the enzyme held in the APC-axin cytoplasmic complex that phosphorylates β-catenin for
proteasomal destruction. In 4 of the 7 patients, the transcript had spliced out the kinase domain.
In another example, we found that it is possible to isolate HSCs from JAK2 kinase-induced myeloproliferative
disease, polycythemia vera, in which the body over-produces red blood cells. HSCs that have JAK2 kinase
over-produce red cells, and an inhibitor of JAK2 kinase blocks that property. However, the same patients
also have normal HSCs.
Polycthemia vera can progress to AML that have the same JAK2 mutation, so in this case also pre-leukemic
progression is in the HSC pool, but the subsequent leukemia is at progenitor stage downstream.
Figure 1
Hematopoietic hierarchy.
Competitive advantage:
We proposed a long time ago that the genetic alterations that lead to leukemia are hardly ever the ones
that confer the ability to self-renew. Yet in every leukemia we've seen in humans or induced in mice, the
LSC at the highly active phase is not at the stem cell stage but at a downstream progenitor. We have never
seen a stem cell leukemia.
If BCR/ABL, AML1/ETO, Jak kinase or PML/RARA occur in a downstream progenitor, they're not enough
by themselves to produce the leukemia phenotype. We propose that the pre-leukemic progression occurs
in a clone derived from HSC.
Decoding immune response players
The first event would form a clone, and that
HSC clone would start outcompeting the
others until a second event Ñ for example,
BCL2 or FAS knockout, p53 knockdown to get
rid of programmed cell death or any other
event. So there is a whole set of events, genetic
or epigenetic, to get to a LSC clone.
To test this hypothesis critically, we isolated
each leukemia and did exome sequencing to
look for SNPs. We excluded the ones that are
also in T cells because they could be allelic
variations. For example, we found a patient
with one allele of TET2 with a stop codon, and
the second event in that particular patient was
a subclone that had the other allele of TET2, and then a transcription-related factor, and then FLT3. In fact,
FLT3 was not the initiating mutation in any of the 6 patients we analyzed.
Each of the patients fit the hypothesis Ñ they made all of the genomic alterations in the progression from
a normal HSC to an LSC. This is probably true in glioblastoma, lung cancer and breast cancer: A subclone
of the normal stem cell goes through pre-cancerous progression, eventually all the events are gathered
together, and it breaks out.
We only looked in this series at genetic changes, but if done right, it may also pick up epigenetic changes.
Comparing the gene expression profile of normal HSC, normal multipotent progenitors and the LSC
generated a list of genes that are expressed in the LSC but not in the HSC. For example, CD96 is overexpressed by about 40-fold in about half of acute myelogenous leukemia (AML) samples, CD44 expression
is increased by about 3-fold in all tested AML, and CD47 is up by more than 5-fold in mouse AML and all
tested human AML.
Don’t eat me:
CD47 is a Ôdon't eat meÕ signal that is expressed in young red blood cells. As the cells age, this marker
fades away and Ôeat meÕ signals come up and the cells are eaten by a macrophage. CD47 is a ligand for the
SIRPα receptor on macrophages. It has an ITIM motif that activates SHIP1 and SHIP2 phosphatases, which
dephosphorylate the actin-myosin skeleton. As a result of the signal from CD47, they're paralyzed and
can't eat the cell to which they're attached, no matter what other receptors they have.
CD47 is normally up-regulated when bone marrow stem cells are forced to be mobilized into the blood,
and its expression decreases again as the cells go into a bone marrow niche. Its levels are higher in human
mobilized peripheral blood, and even higher in cord blood stem cells.
This is a useful characteristic when transplanting human hematopoiesis into immune-deficient mice: If
CD47 is missing, the transplant doesn't take because any large number of stem cells would all get eaten,
whereas it only takes about 100 cells to save the mice if the cells express CD47.
Increased expression of CD47 on myeloid leukemia cells may contribute to pathogenesis by facilitating the
evasion of phagocytosis. If that's true, the prediction would be that increased expression of CD47 in human
AML is associated with a worse clinical outcome.
To test this hypothesis, we looked at a number of arrays of normal karyotype AML patients. Those with the
top 27% mRNA level of CD27 all died within 5 years compared with those in the lower 73%, despite being in
the same hospitals, with access to the same doctors and treatment paradigms.
In vitro, macrophages don't eat leukemia cells that express CD47. When these cells expressing CD47 are
Figure 2
Young cells overexpress
CD47 to evade phagocytosis.
131
132
introduced into the femur
of immune-deficient mice,
they spread to all bone
marrow sites and kill the
mice. That suggests that
CD47 is a metastasis gene
for leukemia.
Figure 3
Anti-CD47 antibodies can
eliminate established
metastasized tumors.
An antibody called B6H12
blocks the interaction of
human CD47 with human
SIRP-α. When LSC from
patients are treated with this antibody to block CD47, the macrophages now eat the cells. It's important that
the antibody blocks the functional interaction of CD47 with SIRP-α. Antibodies that block this interaction
from SIRP-α's side have the same effect.
If the same leukemias analyzed in vitro are transferred into immunodeficient mice, within 4 months the
mice are completely riddled with the human leukemia. However, 2-3 weeks of the B6H12 antibody clears
the leukemic cells, and the bone marrow from these mice now cannot transfer the leukemia to another
animal.
We didn't know what the Ôeat meÕ signal was, but one promising candidate was calretulin. Some, but not all,
pre-cancer cells express calreticulin, and emergent cancer clones overcome this with CD47. Calreticulin
is not the only 'eat me' signal, however.
Synergistic effects:
The Fc-FcR interaction is important for the function of rituximab as an antibody in vivo. Like NK cells,
macrophages also have the Fc Receptor. We investigated the combination of anti-CD47 antibody with
rituximab for synergy in eradicating non-Hodgkin's lymphoma.
If immunodeficient mice with non-Hodgkin's lymphoma are given two weeks of treatment with rituximab
alone, they go into remission, followed by a relapse. The same thing happens with anti-CD47 alone. But the
two antibodies synergize in vivo: The anti-CD47 blocks the Ôdon't eat meÕ signal and rituximab provides a
better, more solid Ôeat meÕ signal. This approach is effective in many solid tumors2.
ÔEat meÕ signals are on in every cancer. If BCL2 expression is enforced only in the myeloid lineage, the
circulating neutrophils that result don't die, and yet the number of neutrophils in the body doesn't increase.
That's because BCL2 blocks programmed cell death (PCD), but not programmed cell removal (PCR) of
neutrophils3.
It turns out that PCR is sufficient for the homeostasis of granulocytes in the body. This system enables
dying cells to pop open inside of macrophages instead of in tissue spaces. That's important because when
a cell dies in the tissue space, it can cause inflammation.
We propose that all cancers have to defeat both PCD and PCR4,5. All cancers defeat PCD and there are
many ways to do it. But so far, the only one that blocks PCR is CD47.
We tested whether this system is involved myelodysplastic syndrome (MDS)5. People with MDS don't have
neutrophils, red cells or platelets, and a significant proportion of these people go on to develop AML. When
MDS cells are transplanted into immunodeficient mice, the MDS clone outcompetes the normal HSC over
time.
The MDS clone does not make granulocytes, monocytes or progenitors. That's because these MDS
progenitors up-regulate calreticulin, and they are eaten by macrophages. If calreticulin peptides are
blocked, the progenitors are not eaten by macrophages.
Decoding immune response players
133
Figure 4
Analysis of single HSC
to identify pre-leukemic
clones.
In summary, in MDS, the initiating events turn on PCD and PCR. The successful clones overcome PCD, but
they are still susceptible at the oligo lineage Ñ that is, the megakaryocyte, erythrocyte, GMP lineages Ñ
and so they can outcompete at the stem cell level but they can't make blood.
This tells us that calreticulin should be a target in the pre-leukemic phase. Because leukemia comes out
of the multipotent progenitor stage, fixing the oligo lineage progenitors so they can make blood should
treat the disease. The final step in refractory anemia with excess blasts and in AML that comes up is
always CD47 up-regulation. It may not be the only event between MDS and AML, but it's a late event and
it's probably epigenetic.
Because SIRP-α is present on mouse and human macrophages and on mouse and human dendritic cells,
we wanted to know whether anti-CD47-mediated phagocytosis enhances antigen presentation. Anti-CD47
blocks the interaction with SIRP-α, enables the cells to be phagocytosed, and that causes CD8+ T cells
from the animal to proliferate in response. It doesn't have that effect on CD4+ cells6.
We're launching a clinical trial with humanized anti-CD47. This will be an all-comers trial because it works
on all cancers. We need relevant assays to find out if we're activating T cells during the experimental
clinical trial, so I'm challenging you to come up with realistic assays.
References
1. M
iyamoto T. et al. Proc.
Natl. Acad. Sci. USA 97,
7521-7526 (2000)
2. Willingham S.B. et al.
Proc. Natl. Acad. Sci. USA
109, 6662-6667 (2012)
3. L
agasse E. and I. Weissman J.
Exp. Med. 179, 1047-1052 (1994)
4. Chao M. P. et al. Nat. Rev.
Cancer 12, 58-67 (2011)
5. Pang W.W. et al. Proc. Natl. Acad.
Sci. USA 110, 3011-3016 (2013)
6. Tseng D. et al. Proc. Natl.
Acad. Sci. USA Epub ahead
of print May 20 (2013)
A B B R E V I AT I O N S A N D G LO S S A R Y
Abbreviations and glossary
Abbreviations
ADCC
antibody-dependent cell-mediated cytotoxicity
NSCLC non-small cell lung cancer
AML
acute myelogenous leukemia
PDAC pancreatic ductal adenocarcinoma
APC antigen-presenting cells
PSA prostate-specific antigen
CAR chimeric antigen receptor
TCGA the cancer genome atlas project
CTL
cytotoxic T lymphocyte
TCR T-cell receptor
DC dendritic cell
Teff T effector cell
EBV Epstein-Barr Virus
TGF-β transforming growth factor beta
GM-CSF granulocyte macrophage colony-stimulating factor
TIL tumor-infiltrating lymphocytes
IFN interferon
TNF tumor necrosis factor
IL interleukin
Treg regulatory T cells
NHL non-Hodgkin's lymphoma
VEGF vascular endothelial growth factor
Glossary
Terms in italics are defined elsewhere in the Glossary.
adaptive immune system
also called acquired immune system, it is composed of highly specialized,
systemic cells and processes that eliminate or prevent the growth of
pathogens.
adoptive cell transfer
the passive transfer of immune-derived or other cells into a recipient Ñ
for example, transfer of T lymphocytes to enhance immunity against cancer
antigen
a substance that evokes the production of one or more antibodies
antibody
an immune protein that can identify foreign objects through an antigen and
then neutralize them
B cell
a subgroup of white blood cells or lymphocytes, and a vital part of the
immune system
cancer stem cell
cells in a tumor that possess the ability to self-renew and give rise to all
other cell types
chimeric antigen receptor
engineered receptors that graft a molecule with specificity, such as a
monoclonal antibody, to immune cells such as T effector cells
135
136
cytokines
small signaling molecules, including immunomodulating agents such as
interleukins and interferons
dendritic cell
immune cells whose function is to present antigens to other cells of the
immune system
exome
protein-coding portion of the genome
immunotherapy
treatment of a disease by inducing or enhancing or suppressing an immune
response
leukemia
a type of cancer of the blood or bone marrow characterized by an abnormal
increase of immature white blood cells, or lymphocytes
lymphocytes
a kind of white blood cell in the vertebrate immune system, specifically a
landmark of the adaptive immune system
lymphoma
a type of blood cancer that occurs when B or T cells of the immune system
divide faster than normal cells or live longer than they are supposed to
macrophage
memory T cell
metastasis
monoclonal antibody
mutation
cellular organelles whose job is to engulf and digest cellular debris
a subset of infection- and potentially cancer-fighting T cells that have
previously encountered and responded to an antigen
the spread of cancer from one organ to another non-adjacent part
antibodies made by identical immune cells that can bind a specific protein
or antigen
a change in the nucleotide sequence of the genome
stem cells
cells characterized by the ability to renew themselves through mitotic cell
division and differentiate into a diverse range of specialized cell types
stroma
the connective, functionally supportive framework of a biological cell, tissue,
or organ
T regulatory cell
a subpopulation of T cells that modulate the immune system, maintain
tolerance to self-antigens, and abrogate autoimmune disease
wild type
gene of interest with no known mutations; animal carrying such a gene;
often designated as +/+ if both alleles are wild type, or +/- if one allele is wild
type, the other mutated (-/- indicates that both genes are mutated
PA R T I C I PA N T S
Participants
Group picture
139
140
Participant List
AHMED Rafi
Basic Immunology/Virology Div. – Emory University
Altanta, USA
[email protected]
COLOMBIER-MAFFRE Céline
Fondation IPSEN
Boulogne-Billancourt, France
[email protected]
ALLISON James P.
Dept. of Immunology – MD Anderson Cancer Center
The University of Texas
Houston, USA
[email protected]
COUKOS George
Ludwig Institute for Cancer Research – UNIL
Epalinges, Switzerland
[email protected]
AMIGORENA Sebastian
Institut Curie – Inserm U932 ÔImmunitŽ et CancerÕ
Paris, France
[email protected]
BALTIMORE David
Division of Biology – California Institute of Technology
Pasadena, USA
[email protected]
BARBACID Mariano
Centro Nacional de Investigaciones Oncologicas
Madrid, Spain
[email protected]
BISHOP J. Michael
The G.W. Hooper Foundation – University of California
San Francisco, USA
[email protected]
BONINI Chiara
Laboratory of Experimental Hematology
San Raffaele Foundation Scientific Institute
Milan, Italy
[email protected]
BRENNER Malcolm K.
Department of Molecular and Human Genetics
Baylor College of Medicine
Houston, USA
[email protected]
CANTLEY Lewis
Cancer Center – Weill Cornell Medical College
New York, USA
[email protected]
CHRISTEN Yves
Fondation IPSEN
Boulogne-Billancourt, France
[email protected]
DE THE Hugues
Dept. de Biologie Cellulaire – Inserm, CNRS, Univ. Paris 7
Paris, France
[email protected]
DE GERARD Astrid
Fondation IPSEN
Boulogne-Billancourt, France
[email protected]
DRANOFF Glenn
Dept. of Immunology – Dana-Farber Cancer Institute
Boston, USA
[email protected]
EVANS Ronald
Gene Expression Laboratory
Salk Institute for Biological Studies
La Jolla, USA
[email protected]
GALON Jérôme
Integrative Cancer Immunology Lab. – Inserm U872
Paris, France
[email protected]
GREENBERG Philip D.
Dept. of Immunology – University of Washington
Seattle, USA
[email protected]
HUNTER Tony
Molecular and Cell Biology Laboratory
Salk Institute for Biological Studies
La Jolla, USA
[email protected]
JAFFEE Elizabeth M.
Department of Oncology – Johns Hopkins University
Baltimore, USA
[email protected]
Participants
JOOSS Karin
Cancer Vaccines & Immunopharmacology – Pfizer Inc.
La Jolla, USA
[email protected]
JUNE Carl H.
Abramson Family Cancer Research Institute
University of Pennsylvania
Philadelphia, USA
[email protected]
RAJEWSKY Klaus
Immune Regulation and Cancer Program
Max-DelbrŸck-Cter for Molecular Medicine
Berlin, Germany
[email protected]
RIBAS Antoni
Division of Hematology and Oncology – UCLA
Los Angeles, USA
[email protected]
KLAUSNER Richard
The Column Group
San Francisco, USA
[email protected]
ROSENBERG Steven A.
National Cancer Institute – NIH
Bethesda, USA
[email protected]
LEVITZKI Alexander
Unit of Cellular Signaling
The Hebrew University of Jerusalem
Jerusalem, Israel
[email protected]
SCHMIDLIN Fabien
Department of Translational Biology
Ipsen Innovation
Les Ulis, France
[email protected]
LEVY Ronald
Division of Oncology – Stanford University
Stanford, USA
[email protected]
SCHREIBER Robert D.
Division of Immunology – Washington University
St Louis, USA
[email protected]
LOUVARD Daniel
Laboratoire Morphogense, Signalisation Cellulaire
Institut Curie
Paris, France
[email protected]
SCHUMACHER Ton
Department of Immunology
The Netherlands Cancer Institute
Amsterdam, The Netherlands
[email protected]
MAK Tak Wah
Ontario Cancer Inst. – University of Health Network
Toronto, Canada
[email protected]
THOMPSON Craig B.
Cancer Biology and Genetics Program – MSKCC
New York, USA
[email protected]
MANDAVILLI Apoorva
The Simons Foundation
New York, USA
[email protected]
THURIEAU Christophe
Ipsen Innovation
Les Ulis, France
[email protected]
MELIEF Cornelis J.M.
Dept. of Immunohematology & Blood Transfusion
Leiden University
Leiden, The Netherlands
[email protected]
VERMA Inder M.
Lab. of Genetics – Salk Institute for Biological Studies
La Jolla, USA
[email protected]
MERAD Miriam
Dept. of Oncology – The Mount Sinai School of Med.
New York, USA
[email protected]
MERVAILLIE Jacqueline
Fondation IPSEN
Boulogne-Billancourt, France
[email protected]
PARMIANI Giorgio
Lab. of Immuno-biotherapy of Melanoma and Solid
Tumors – San Raffaele Foundation Scientific Institute
Milan, Italy
[email protected]
WEINBERG Robert A.
Whitehead Institute for Biomedical Research – MIT
Cambridge, USA
[email protected]
WEISSMAN Irving L.
Dept. of Developmental Biology – Stanford University
Stanford, USA
[email protected]
ZINKEWICH-PEOTTI Karen
Ipsen Innovation
Les Ulis, France
[email protected]
141
CANCER SCIENCE MEETING SERIES
Cancer Science meeting series
CAN CANCER BE TREATED AS A CHRONIC DISEASE? • Agra, February 14-16, 2005
organizing committee
Inder M. Verma (Salk Institute for Biological Studies, La Jolla, USA), Yves Christen (Fondation IPSEN, Paris, France), Jacqueline Mervaillie
(Fondation IPSEN, Paris, France)
speakers and discussants
Jerry Adams (University of Melbourne, Parkville, Australia), Kari Alitalo (University of Helsinki, Helsinki, Finland), Mariano Barbacid
(Centro Nacional de Investigaciones Oncologicas, Madrid, Spain), Lewis Cantley (Harvard Medical School, Boston, USA), Kishore Chaudhry
(Indian Council for Medical Research, New Delhi, India), Suzanne Cory (University of Melbourne, Parkville, Australia), Titia De Lange
(Rockefeller University, New York, USA), Hugues de Thé (CNRS, Université de Paris, Paris, France), Vishva Dixit (Genentech, South San
Francisco, USA), Wolf Hervé Fridman (Inserm, Paris, France), Philip D. Greenberg (University of Washington, Seattle, USA), Daniel Haber
(M.G.H. Cancer Center and Harvard Medical School, Charlestown, USA), Jean-Luc Harousseau (Hôtel Dieu, Nantes, France), Gilbert Lenoir
(Institut Gustave Roussy, Villejuif, France), Daniel Louvard (Institut Curie, Paris, France), Pier Giuseppe Pelicci (European Institute of
Oncology, Milan, Italy), Annapoorni Rangarajan (Indian Institute of Science, Bangalore, India), Charles L. Sawyers (University of California,
Los Angeles, USA), Kumar Somasundaram (Indian Institute of Science, Bangalore, India), Michael Stratton (Wellcome Trust Sanger
Institute, Hinxton, USA), Inder M.Verma (Salk Institute for Biological Studies, La Jolla, USA).
ARE INFLAMMATION AND CANCER LINKED? ¥ Cape Town, February 12-15, 2006
organizing committee
Inder M. Verma (Salk Institute for Biological Studies, La Jolla, USA), Yves Christen (Fondation IPSEN, Paris, France), Jacqueline
Mervaillie (Fondation IPSEN, Paris, France)
speakers and discussants
Sebastian Amigorena (Institut Curie, Paris, France), Frances Balkwill (Barts and London Queen Mary's School of Medicine, London, UK),
David Baltimore (California Institue of Technology, Pasadena, USA), Mariano Barbacid (Centro Nacional de Investigaciones Oncologicas,
Madrid, Spain), Anton Berns (Netherlands Cancer Institute, Amsterdam, The Netherlands), Yinon Ben Neriah (Hebrew University,
Jerusalem, Israel), Hans Clevers (Hubrecht Laboratory, Utrecht, The Netherlands), Hugues de Thé (CNRS, Université de Paris, France),
Vishva Dixit (Genentech Inc., South San Francisco, USA), Ronald Evans (Salk Institute for Biological Studies, La Jolla, USA), Richard A.
Flavell (Yale University School of Medicine, New Haven, USA), Wolf Hervé Fridman (Inserm, Paris, France), Vandana Govan (University
of Capetown, Capetown, Republic of South Africa), Dhirendra Govender (University of Capetown, Capetown, Republic of South Africa),
Jean-Luc Harousseau (Hôtel-Dieu, Nantes, France), Tony Hunter (Salk Institute for Biological Studies, La Jolla, USA), Alain Israel (Institut
Pasteur, Paris, France), Rudolf Jaenisch (Massachusetts Institute of Technology, Cambridge, USA), Michael Karin (University of California
San Diego, La Jolla, USA), Grish Kotwal (University of Capetown, Capetown, Republic of South Africa), Gilbert Lenoir (Institut Gustave
Roussy, Villejuif, France), Daniel Louvard (Institut Curie, Paris, France), Albero Mantovani (Istitio Clinico Humanitas, Milan, Italy), Carlos
Martinez-A (National Center for Biotechnology, Madrid, Spain), Bongani Mayosi (University of Capetown, Capetown, Republic of South
Africa), Anthony W. Segal (University College London, London, UK), Tadatsugu Taniguchi (University of Tokyo, Tokyo, Japan), Tomas Tursz
(Institut Gustave Roussy, Villejuif, France), Inder M. Verma (Salk Institute for Biological Studies, La Jolla, USA), Timothy Cragin Wang
(Columbia University Medical Center, New York, USA), Robert A. Weinberg (Massachusetts Institute of Technology, Cambridge, USA).
METASTASIS AND INVASION ¥ Spineto, May 20-23, 2007
organizing committee
Inder M. Verma (Salk Institute for Biological Studies, La Jolla, USA), Yves Christen (Fondation IPSEN, Paris, France), Jacqueline Mervaillie
(Fondation IPSEN, Paris, France)
speakers and discussants
Kari Alitalo (University of Helsinki, Helsinki, Finland), David Baltimore (California Institute of Technology, Pasadena, USA), Mariano
Barbacid (Centro Nacional de Investigaciones Oncologicas, Madrid, Spain), J. Michael Bishop (University of California, San Francisco, USA),
Lewis Cantley (Harvard Institute of Medicine, Boston, USA), Ann Chambers (University of Western Ontario, London, Canada), Gerhard
Christofori (University of Basel, Basel, Switzerland), Hans Clevers (Hubrecht Laboratory, Utrecht, The Netherlands), Philippe Clezardin
(Inserm, Lyon, France), Mario Colombo (Istituto Nazionale Tumori, Milano, Italy), Paolo Comoglio (University of Torino, Torino, Italy), Hugues
de Thé (CNRS, Université de Paris, Paris, France), Wolf Hervé Fridman (Inserm, Paris, France), Christian Gespach (Inserm, Paris, France),
Douglas Hanahan (University of California, San Francisco, USA), Jean-Luc Harousseau (Hôtel-Dieu, Nantes, France), Richard Hynes
(Massachusetts Institute of Technology, Cambridge, USA), Alain Israel (Institut Pasteur, Paris, France), Gilbert Lenoir (Institut Gustave
Roussy, Villejuif, France), Arnold J. Levine (Institute of Advanced Study, Princeton, USA), Daniel Louvard (Institut Curie, Paris,France), Tak
Wah Mak (University of Toronto, Toronto, Canada), Joan Massagué (Memorial Sloan Kettering Cancer Center, New York, USA), Luigi Naldini
(University Medical School Milan, Milan, USA), Daniel Peeper (Netherlands Cancer Institute, Amsterdam, The Netherlands), Jeffrey Pollard
(Albert Einstein College of Medicine, New York, USA), Shahin Rafii (Cornell University Medical College, New York, USA), Inder M. Verma
(Salk Institute for Biological Studies, La Jolla, USA), Robert A. Weinberg (Massachusetts Institute of Technology, Cambridge, USA), Zena
Werb (University of California, San Francisco, USA), Owen N. Witte (Howard Hughes Medical Institute, Los Angeles, USA).
METABOLISM AND CANCER ¥ Villa Caletas, March 9-11, 2008
organizing committee
Inder M. Verma (Salk Institute for Biological Studies, La Jolla, USA), Yves Christen (Fondation IPSEN, Paris, France), Jacqueline
Mervaillie (Fondation IPSEN, Paris, France)
speakers and discussants
Kari Alitalo (University of Helsinki, Helsinki, Finland), Mariano Barbacid (Centro Nacional de Investigaciones Oncologicas, Madrid, Spain),
J. Michael Bishop (University of California, San Francisco, USA), Mina Bissell (University of California San Francisco, Berkeley, USA), Joan
Brugge (Harvard Medical School, Boston, USA), Lewis Cantley (Harvard Medical School, Boston, USA), Lynda Chin (Dana-Farber Cancer
Institute, Boston, USA), Hans Clevers (Hubrecht Laboratory, Utrecht, The Netherlands), Hugues de Thé (CNRS, Université de Paris, Paris,
France), Ronald Depinho (Dana-Farber Cancer Institute, Boston, USA), Ivan Dikic (Goethe University Medical School, Frankfurt, Germany),
Vishva Dixit (Genentech Inc., South San Francisco, USA), Gerard Evan (University of California, San Francisco, USA), Ronald Evans (Salk
Institute for Biological Studies, La Jolla, USA), Wolf Hervé Fridman (Inserm, Paris, France), Tony Hunter (Salk Institute for Biological
Studies, La Jolla, USA), William Kaelin (Harvard Medical School, Boston, USA), Gilbert Lenoir (Institut Gustave Roussy, Villejuif, France),
Arnold Levine (Simons Center for Systems Biology, Princeton, USA), Daniel Louvard (Institut Curie, Paris, France), Steven McKnight
(University of Texas Southwestern Medical Center, Dallas, USA), Carol Prives (Columbia University, New York, USA), Neal Rosen (Memorial
Sloan-Kettering Cancer Center, New York, USA), David Sabatini (Whitehead Institute for Biomedical Research, MIT, Cambridge, USA),
Gregg Semenza (Johns Hopkins University School of Medicine, Baltimore, USA), Reuben Shaw (Salk Institute for Biological Studies, La
Jolla, USA), George Thomas (University of Cincinnati, Reading, USA), Craig B. Thompson (University of Pennsylvania, Philadelphia, USA),
Inder M. Verma (Salk Institute for Biological Studies, La Jolla, USA), Karen Vousden (Beatson Institute for Cancer Research, Glasgow, UK),
Robert A. Weinberg (Massachusetts Institute of Technology, Cambridge, USA).
143
144
MOLECULAR TARGETS OF CANCER THERAPY ¥ Jaipur, February 14-18, 2009
organizing committee
Inder M. Verma (Salk Institute for Biological Studies, La Jolla, USA), Yves Christen (Fondation IPSEN, Paris, France), Jacqueline
Mervaillie (Fondation IPSEN, Paris, France)
speakers and discussants
Julian Adams (Infinity Pharmaceuticals Inc., Cambridge, USA), James P. Allison (Memorial Sloan-Kettering Cancer Center, New York,USA),
José Baselga (Vall d'Hebron University Hospital, Barcelona, Spain), Anton Berns (The Netherlands Cancer Institute, Amsterdam,The
Netherlands), J. Michael Bishop (University of California, San Francisco, USA), Arul Chinnaiyan (University of Michigan, Ann Arbor, USA),
Frederic de Sauvage (Genentech Inc., South San Francisco, USA), Hugues de Thé (CNRS, Université de Paris, Paris, France), Vishva Dixit
(Genentech Inc., South San Francisco, USA), Ronald Evans (Salk Institute for Biological Studies, La Jolla, USA), Wolf Hervé Fridman
(Inserm, Paris, France), Gary Gilliand (Harvard University, Boston, USA), Tony Hunter (Salk Institute for Biological Studies, La Jolla, USA),
Richard Klausner (The Column Group, San Francisco, USA), John Kuriyan (University of California, Berkeley, USA), Alexander Levitzki
(The Hebrew University of Jerusalem, Jerusalem, Israel), Ronald Levy (Stanford University, Stanford, USA), Tak Mak (University of Toronto,
Toronto, Canada), Victoria Richon (Merck Research Laboratoires, Boston, USA), Neal Rosen (Memorial Sloan-Kettering Cancer Center,
New York, USA), Rajiv Sarin (Actrec, Mumbai, India), William Sellers (Novartis, Cambridge, USA), Kovan Shokat (University of California,
San Francisco, USA), Vinay Tergaonkar (Institute for Molecular and Cell Bilogy, Singapore), Craig B. Thompson (University of Pennsylvania,
Philadelphia, USA), Harold Varmus (Memorial Sloan-Kettering Cancer Center, New York, USA), Gregory Verdine (Harvard University,
Cambridge,USA), Inder M. Verma (Salk Institute for Biological Studies, La Jolla, USA), K. Vijayraghavan (National Center for Biological
Sciences, Bangalore, India), Barbara Weber (GlaxoSmithKline, Collegeville, USA).
STEM CELLS AND CANCER ¥ Bariloche, March 6-10, 2010
organizing committee
Inder M. Verma (Salk Institute for Biological Studies, La Jolla, USA), Yves Christen (Fondation IPSEN, Paris, France), Jacqueline
Mervaillie (Fondation IPSEN, Paris, France)
speakers and discussants
David Baltimore (California Institue of Technology, Pasadena, USA), Mariano Barbacid (Centro Nacional de Investigaciones Oncologicas,
Madrid, Spain), Juan Carlos Belmonte (Salk Institute for Biological Studies, La Jolla, USA), Anton Berns (The Netherlands Cancer Institute,
Amsterdam, The Netherlands), J. Michael Bishop (University of California, San Francisco, USA), Lewis Cantley (Beth Israel Deaconess
Medical Center, Boston, USA), Hans Clevers (Hubrecht Institute, Utrecht, The Netherlands), John Dick (UHNRES, University of Toronto,
Toronto, Canada), Peter Dirks (University of Toronto, Toronto, Canada), Vishva Dixit (Genentech Inc., South San Francisco, USA), Ronald
Evans (Salk Institute for Biological Studies, La Jolla, USA), Fred H. Gage (Salk Institute for Biological Studies, La Jolla, USA), Gary Gilliland
(Harvard University, Boston, USA), Jean-Luc Harousseau (Hôtel Dieu, Nantes, France), Tony Hunter (Salk Institute for Biological Studies,
La Jolla, USA), Rudolf Jaenisch (Whitehead Institute for Biomedical Research, MIT, Cambridge, USA), Catriona Jamieson (University
of California, San Diego, USA), Gilbert Lenoir (Institut Gustave Roussy, Villejuif, France), Daniel Louvard (Institut Curie, Paris, France),
Tak Wah Mak (Princess Margaret Hospital UHN, Toronto, Canada), Sean Morrison (University of Michigan, Ann Arbor, USA), Catherine
O'Brien (UHNRES, Toronto, Canada), Jeremy Rich (Cleveland Clinic, Cleveland, USA), Charles L. Sawyers (Memorial Sloan-Kettering
Cancer Center, New York, USA), David T. Scadden (Massachusetts General Hospital, Cambridge, USA), Craig B. Thompson (University
of Pennsylvania, Philadelphia, USA), Inder M. Verma (Salk Institute for Biological Studies, La Jolla, USA), Jane Visvader (The Walter and
Elisa Hall Institute of Medical Research, Melbourne, Australia), Robert A. Weinberg (Whitehead Institute for Biomedical Research, MIT,
Cambridge, USA), Irving L. Weissman (Stanford School of Medicine, Stanford, USA), Max S.Wicha (University of Michigan Comprehensive
Cancer Center, Ann Arbor, USA), Hong Wu (University of California Los Angeles School of Medicine, Los Angeles, USA).
EPIGENETICS AND CANCER ¥ Swakopmund, March 19-23, 2011
organizing committee
Inder M. Verma (Salk Institute for Biological Studies, La Jolla, USA), Yves Christen (Fondation IPSEN, Paris, France), Jacqueline
Mervaillie (Fondation IPSEN, Paris, France)
speakers and discussants
Geneviève Almouzni (Institut Curie, Paris, France), David Baltimore (California Institue of Technology, Pasadena, USA), Stephen B. Baylin
(The Sidney Kimmel Comprehensive Cancer Center, Baltimore, USA), Adrian Bird (University of Edinburgh, Edinburgh, UK), J. Michael
Bishop (University of California San Francisco, USA), Xiaodong Cheng (Emory University School of Medicine, Atlanta, USA), Susan Clark
(The Garvan Institute of Medical Research, Darlinghurst, Australia), Hugues de Thé (University of Paris, Paris, France), Joseph Ecker (Salk
Institute for Biological Studies, La Jolla, USA), Robert Eisenman (Fred Hutchinson Cancer Research Center, University of Washington,
Seattle, USA), Ronald Evans (Salk Institute for Biological Studies, La Jolla, USA), Edward E. Harlow (Constellation Pharmaceuticals,
Cambridge, USA), Kristian Helin (University of Copenhagen, Copenhagen, Denmark), Tony Hunter (Salk Institute for Biological Studies, La
Jolla, USA), Jean-Pierre Issa (M.D. Anderson Cancer Center, University of Texas, Houston, USA), Rudolf Jaenisch (Whitehead Institute for
Biomedical Research, Massachusetts Institute of Technology, Cambridge, USA), Peter A. Jones (Epigenome Center, University of Southern
California, Los Angeles, USA), Peter W. Laird (Epigenome Center, University of Southern California, Los Angeles, USA), Gilbert Lenoir
(Institut Gustave Roussy, Villejuif, France), Daniel Louvard (Institut Curie, Paris, France), Charles L. Sawyers (Memorial Sloan-Kettering
Cancer Center, New York, USA), Maarten van Lohuizen (The Netherlands Cancer Institute, Amsterdam, The Netherlands), Ali Shilatifard
(Stowers Institute for Medical Research, Kansas City, USA), Craig B. Thompson (Memorial Sloan-Kettering Cancer Center, New York, USA),
Inder M. Verma (Salk Institute for Biological Studies, La Jolla, USA), Robert A. Weinberg (Whitehead Institute for Biomedical Research,
MIT, Cambridge, USA), Irving L. Weissman (Stanford University School of Medicine, Stanford, USA), Richard Young (Whitehead Institute for
Biomedical Research, MIT, Cambridge, USA).
MOUSE MODELS OF HUMAN CANCER: ARE THEY RELEVANT? ¥ Our Preto, March 10-14, 2012
organizing committee
Inder M. Verma (Salk Institute for Biological Studies, La Jolla, USA), Yves Christen (Fondation IPSEN, Paris, France), Jacqueline
Mervaillie (Fondation IPSEN, Paris, France)
speakers and discussants
David J. Adams (Wellcome Trust Sanger Institute, Cambridge, UK), Laura Attardi (Stanford University School of Medicine, Stanford, USA),
Mariano Barbacid (Centro Nacional de Investigaciones Oncologicas, Madrid, Spain), Daniela Basseres (Universidade de Sao Paulo, Sao
Paulo, Brazil), Anton Berns (The Netherlands Cancer Institute, Amsterdam, The Netherlands), J. Michael Bishop (University of California
San Francisco, San Francisco, USA), Lewis Cantley (Harvard University, Boston, USA), Mario Capecchi (University of Utah, Salt Lake City,
USA), Roger Chammas (Universidade de Sao Paulo, Sao Paulo, Brazil), Lynda Chin (UT MD Anderson Cancer Center, Houston, USA),
Hugues de Thé (Inserm, CNRS, Université Paris Diderot, Paris, France), Vishva Dixit (Genentech, Inc., South San Francisco, USA), Gerard
Evan (University of Cambridge, Cambridge, UK), Ronald Evans (Salk Institute for Biological Studies, La Jolla, USA), Douglas Hanahan
(Institute for Experimental Cancer Research – EPFL, Lausanne, Switzerland), Tony Hunter (Salk Institute for Biological Studies, La Jolla,
USA), Tyler Jacks (MIT, Cambridge, USA), Claudio Joazeiro (Scripps Research Institute, La Jolla, USA), Leisa Johnson (Genentech Inc.,
South San Francisco, USA), Jos Jonkers (The Netherlands Cancer Institute, Amsterdam, The Netherlands), Michael Karin (University of
California SD, La Jolla, USA), Daniel Louvard (Institut Curie, Paris, France), Scott Lowe W. (Memorial Sloan-Kettering Institute, New York, USA),
Tak Wah Mak (University of Alberta, Toronto, Canada), Joan Massagué (Memorial Sloan-Kettering Institute, New York, USA), Luis Parada
(UT Southwestern Medical Center, Dallas, USA), Neal Rosen (Memorial Sloan-Kettering Institute, New York, USA), Charles L. Sawyers
(Memorial Sloan-Kettering Institute, New York, USA), Reuben Shaw (Salk Institute for Biological Studies, La Jolla, USA), David Tuveson
(Cancer Research UK, Cambridge, UK), Inder M. Verma (Salk Institute for Biological Studies, La Jolla, USA), Robert A. Weinberg (MIT,
Cambridge, USA).
CANCER SCIENCE 9
CANCER IMMUNOTHERAPY • Taormina, March 9-13, 2013
organizing committee
Inder M. Verma (Salk Institute for Biological Studies, La Jolla, USA), Yves Christen (Fondation IPSEN, Paris, France), Jacqueline
Mervaillie (Fondation IPSEN, Paris, France)
Cancer Science 9 ― Cancer immunotherapy
2FI174
Fondation Ipsen
ndation-ipsen.org
ourt Cedex ― Tel.: 33(0)1 58 33 50 00 ― Fax: 33 (0)1 58 33 50 01
Cancer Science meeting series
speakers and discussants
Cancer immunotherapy
Taormina, March 9-13, 2013
C O L L O Q U E S
M É D E C I N E
E T
R E C H E R C H E
Rafi Ahmed (Emory University, Atlanta, USA), James P. Allison (Memorial Sloan-Kettering Cancer Center, New York, USA), Sebastian
Amigorena (Institut Curie-Inserm, Paris, France), David Baltimore (California Institute of Technology, Pasadena, USA), Mariano
Barbacid (Centro Nacional de Investigaciones Oncologicas, Madrid, Spain), J. Michael Bishop (University of California San Francisco,
San Francisco, USA), Chiara Bonini (Fondazione San Raffaele Del Monte Tabor, Milan, Italy), Malcolm K. Brenner (Baylor College of
Medicine, Houston, USA), Lewis Cantley (Cancer Center – Weill Cornell Medical College), George Coukos (University of Pennsylvania,
Philadelphia, USA), Hugues de Thé (Inserm, CNRS, Université Paris Diderot, Paris, France), Glenn Dranoff (Dana-Farber Cancer
Institute, Boston, USA), Ronald Evans (Salk Institute for Biological Studies, La Jolla, USA), Jérôme Galon (Inserm, Paris, France), Philip
D. Greenberg (University of Washington, Seattle, USA), Tony Hunter (Salk Institute for Biological Studies, La Jolla, USA), Elizabeth M.
Jaffee (Johns Hopkins University, Baltimore, USA), Karin Jooss (Pfizer Inc., La Jolla, USA), Carl H. June (University of Pennsylvania,
Philadelphia, USA), Richard Klausner (The Column Group, San Francisco, USA), Alexander Levitzki (Unit of Cellular Signaling –
The Hebrew University of Jerusalem, Jerusalem, Israel), Ronald Levy (Stanford University School of Medicine, Stanford, USA), Daniel
Louvard (Institut Curie, Paris, France), Tak Wah Mak (University of Health Network, Toronto, Canada), Cornelis J.M. Melief (Leiden
University Medical Center, Leiden, The Netherlands), Miriam Merad (The Mount Sinai School of Medecine, New York, USA), Giorgio
Parmiani (Fondazione San Raffaele Del Monte Tabor, Milan, Italy), Klaus Rajewsky (Max-Delbrück-Center for Molecular Medicine, Berlin,
Germany), Antoni Ribas (University of California Los Angeles, Los Angeles, USA), Steven A. Rosenberg (National Cancer Institute – NIH,
Bethesda, USA), Robert D. Schreiber (Washington University School of Medicine, St Louis, USA), Ton Schumacher (The Netherlands
Cancer Institute, Amsterdam, The Netherlands), Craig B. Thompson (Memorial Sloan-Kettering Cancer Center, New York, USA), Inder M.
Verma (Salk Institute for Biological Studies, La Jolla, USA), Robert A. Weinberg (Massachusetts Institute of Technology, Cambridge, USA),
Irving L. Weissman (Stanford University School of Medicine, Palo Alto, USA).
CANCER GENOMICS • Chantilly, April 12-15, 2014 (in preparation)
organizing committee
Inder M. Verma (Salk Institute for Biological Studies, La Jolla, USA), Yves Christen (Fondation IPSEN, Paris, France), Jacqueline
Mervaillie (Fondation IPSEN, Paris, France)
speakers and discussants
Geneviève Almouzni (Institut Curie – CNRS UMR 218, Paris, France), Alan Ashworth (The Institute of Cancer Research, London, UK)
David Baltimore (California Institute of Technology, Pasadena, USA), Mariano Barbacid (Centro Nacional de Investigaciones Oncologicas,
Madrid, Spain), José Baselga (Breast Cancer Medicine Service – MSKCC, New York, USA), Anton Berns (The Netherlands Cancer Institute,
Amsterdam, The Netherlands), J. Michael Bishop (University of California San Francisco, San Francisco, USA), Elizabeth H. Blackburn
(University of California San Francisco, San Francisco, USA), James E. Bradner (Dana-Farber Cancer Institute, Boston, USA) Brigitte
Bressac-De Paillerets (Inserm UMRS 946 ´Variabilité Génétique et Maladies Humaines´, Villejuif, France), Andrea Califano (Columbia
University, New York, USA) Lewis Cantley (Weill Cornell Medical College, New York, USA), Mario Capecchi (University of Utah, Salt Lake
City, USA), Lynda Chin (UT MD Anderson Cancer Center, Houston, USA), Arul Chinnaiyan (University of Michigan, Ann Arbor, USA) Hugues
De Thé (Inserm, CNRS, Université Paris 7, Paris, France), Vishva Dixit (Genentech, Inc., South San Francisco, USA), James R. Downing
(St Jude Children´s Research Hospital, Memphis, USA), Levi A. Garraway (Harvard University, Boston, USA), Gary Gilliland (Merck & Co.,
Inc., Whitehouse Station, USA), Todd R. Golub (Dana-Farber Cancer Institute, Cambridge, USA), Douglas Hanahan (Ecole Polytechnique
Fédérale de Lausanne, Lausanne, Switzerland), Jules A. Hoffmann (Institut de Biologie Moléculaire et Cellulaire, UPR 9022 CNRS,
Strasbourg, France), Tony Hunter (Salk Institute for Biological Studies, La Jolla, USA), Tyler Jacks (Massachusetts Institute of Technology,
Cambridge, USA), William G. Kaelin (Dana-Farber Cancer Institute, Boston, USA), Peter W. Laird (University of Southern California, Los
Angeles, USA), Eric S. Lander (Broad Institute – MIT – Harvard University, Cambridge, USA), Arnold J. Levine (Institute for Advanced Study,
Princeton, USA), Daniel Louvard (Institut Curie, Paris, France), Tak Wah Mak (Ontario C¬¬ancer Institute, Toronto, Canada), Elaine R.
Mardis (Washington University School of Medicine, Saint Louis, USA), Paul Nurse (The Royal Society, London, UK), Nickolas Papadopoulos
(The Johns Hopkins University, Baltimore, USA), Stefan Pfister (German Cancer Research Center (DKFZ), Heidelberg, Germany), Klaus
Rajewsky (Max-Delbrück-Center for Molecular Medicine, Berlin, Germany), Charles L. Sawyers (Human Oncology & Pathogenesis
Program – MSKCC, New York, USA), William R. Sellers (Novartis Institutes for Biomedical Research, Cambridge, USA), Philip A. Sharp
(Massachusetts Institute of Technology, Cambridge, USA), Louis M. Staudt (National Cancer Institute – NIH, Bethesda, USA), Michael
Stratton (Wellcome Trust Sanger Institute, Hinxton, UK), Craig B. Thompson (Cancer Biology and Genetics Program – MSKCC, New York,
USA), Harold E. Varmus (National Cancer Institute – NIH, Bethesda, USA), Inder M. Verma (Salk Institute for Biological Studies, La Jolla,
USA), Karen Vousden (Beatson Institute for Cancer Research, Glasgow, UK), Robert A. Weinberg (Whitehead Institute for Biomedical
Research – MIT, Cambridge, USA), Irving L. Weissman (Stanford University, Stanford, USA), Owen N. Witte (University of California Los
Angeles – HHMI, Los Angeles, USA), Jessica Zucman-Rossi (Inserm U674 ´Génomique Fonctionnelle des Tumeurs Solides´, Paris, France).
In preparation
145
FO N D AT I O N I P S E N
Fondation IPSEN
The Fondation IPSEN, created in 1983 under the
auspices of the Fondation de France, has two
objectives: the distribution of knowledge and
encouraging the exploration of emerging areas
of research.
Contributing to the development
and distribution of knowledge
One mission of the foundation is to promote
interaction between researchers and clinicians
by creating ÔcrossroadsÕ and forums for
fruitful exchanges. Today, with the extreme
specialization of knowledge and the increasing
mass of information that many find difficult to decipher, such exchanges are indispensable. For this to be
effective, the foundation has focused on some of the crucial biomedical themes of our time: the spectacular
developments in neuroscience and the scientific study of cognitive mechanisms, the challenges of
neurodegenerative pathologies, the omnipresence of genetics and molecular biology, the growing field
of endocrine interactions, and the problems of aging populations and theories of longevity. More recently,
activities have expanded into an area that is exciting for both its medical and fundamental challenges and
that is currently in a phase of rapid development: cancer science.
Another goal of the Fondation IPSEN is to initiate, in partnership with the specialists and institutions involved,
discussions and exchanges on the major scientific challenges of the future. Rather than trying to provide
definitive knowledge, or to replace the work of large research organizations, the aim of these discussions
is to emphasise multidisciplinary approaches at the boundaries of several disciplines, an approach that is
essential for understanding the complexity and originality of human beings and their pathologies.
To fulfil these commitments, the foundation organises several series of international Colloques MŽdecine
et Recherche, as well as several series of annual meetings in collaboration with scientific journals and
institutions. Also, the Fondation IPSEN is funding awards to encourage research and publishing reports on
its meetings. For each of these activities, the foundation brings together partners from the scientific and
clinical world, who can independently report on the current state of knowledge and discuss the main issues
in the areas on which the foundation has chosen to focus.
Over the past 30 years, the Fondation IPSEN has established its place in the scientific and medical landscape
and intends to continue to be at the forefront in forming links, initiating multidisciplinary exchanges and
contributing to the spread of knowledge, with time, intelligence, good will and above all, the collaboration
of leaders in current biomedical research.
The Colloques Médecine et Recherche series
The Colloques Médecine et Recherche were created in 1987, with the first series dedicated to AlzheimerÕs
disease. Its success stimulated the establishment of other several dedicated series: neurosciences,
longevity, endocrinology, the vascular tree and more recently cancer. Meetings in each series are held
annually, bringing leading international specialists together to present their most recent work, sometimes
even before publication. Through these meetings, the Fondation IPSEN has over the years developed a
large, international network of experts.
By focusing on emerging fields of knowledge, the meetings have supported the development of many
new topics and have impacted on scientific advances in areas such as gene therapy and stem cells in the
central nervous system, the role of cerebral amyloidosis in neurodegeneration, the contribution of genetic
factors in resistance to disease, the benefits of neuronal grafts, biological markers of AlzheimerÕs disease,
apolipoprotein E, brain-somatic cross-talk, relationships between brain and longevity, hormonal control of
cell cycle to name a selection.
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The series are organized around topics where active research is having or is likely to have a major impact
on our knowledge:
¥ Neurosciences – Started in 1990, this series of conferences has both enabled the identification of
the major themes to emerge in this area and has supported not only the remarkable expansion of the
neurosciences in the past fifteen years but also the effort to integrate its subdisciplines, from molecular
mechanisms to human cognition.
¥ Alzheimer and neurodegenerative diseases – Since 1987, this topic has been explored at annual meetings
that have followed or even anticipated the development of the new field of ÔalzheimerologyÕ, which has gone
beyond histology and neurochemistry to establish the underlying pathological mechanisms.
¥ Cancer Science – Annual experts meetings are organized in collaboration with Inder M. Verma (Salk
Institute for Biological Studies, La Jolla, USA) and the participation of remarkable leading opinion makers
in the field. Challenging topics (see pp.141-143) have generated outstanding discussions among the
participants.
¥ Endocrinology – Established in 2002, this series examines the involvement of the endocrine system in the
integration of all bodily functions. One example is the recent discovery of many hormones important in the
control of metabolism, such as leptin and ghrelin. As aspects of brain-somatic crosstalk, such topics have
impacts far beyond studies of hormones and the endocrine organs.
¥ Longevity – Launched in 1996, this series examines the challenges and paradoxes of medicine by focusing
on a positive aspect, cases of exceptional resistance to the effects of aging, rather than on disease. The
evolution of research dedicated to aging into research dedicated to longevity represents a remarkable
development in this field.
¥ Vascular Tree – This series, begun in 2004, aims to examine the various steps that lead to development
of the vascular system, its growth in harmony with that of other organs, its degeneration, death and the
possibilities for its regeneration. A new vision is emerging of blood vessels not as simple ÔpipesÕ but as
living, complex organs with interactions throughout the body.
Partnerships
Long ago, the Fondation IPSEN has developed partnerships with international institutions and organisations,
to encourage cooperation between experts in various disciplines. These partners include: the World Health
Organisation (WHO), the Fondation Nationale de Gérontologie (FNG) and Harvard University.
Additional series of meetings and partnerships have been implemented since 2007:
¥ Biological Complexity series (Salk Institute for Biological Studies, Nature Publishing Group, and Fondation
IPSEN): Transcription Diseases (La Jolla, 2007), Genes, Circuits and Behavior (La Jolla, 2008), Processes
of Aging (La Jolla, 2009), Sensory Systems (La Jolla, 2010), Future Concepts and Trends (La Jolla, 2010),
Immunity and Inflammation (La Jolla, 2012), Molecular biology of psychiatric disorders (La Jolla, 2013),
Genes and physiology (La Jolla, 2014, in preparation).
¥ Emergence and Convergence series (Nature Publishing Group and Fondation IPSEN): Small RNAs in
Development, Immunology and Cancer (New York, 2007), Genome Variation (Seattle, 2007), Epigenetics and
Behavior (Houston, 2008), Multiple Sclerosis: From Pathogenesis to Therapy (Paris, 2009), Mitochondrial
Dysfunction in Neurological Diseases (Durham, 2008), Epigenetic Dynamics in the Immune System (San
Antonio, 2010).
¥ Exciting Biologies series
– Cell Press, Massachusetts General Hospital, and Fondation IPSEN:
Biology in Motion (Evian, 2007), Biology of Cognition (Chantilly, 2008), Biology in Balance (Buenos Aires,
2009), Biology of Recognition (Singapore, 2010).
ell Press, DMMGF, and Fondation IPSEN:
–C
Cellular Development: Biology at the Interface (Kobe, 2011), in collaboration with The Riken Instititute,
Forces in Biology (Dublin, 2012), Biology of Boundaries (Savudrija, 2013, in preparation)
Fondation IPSEN
¥ Days of Molecular Medicine series (AAA Science, Karolinska Institute, Hong Kong University, DMMGF, and
Fondation IPSEN): Tissue Engineering and Stem Cells: Driving Regenerative Medicine Forward (Hong Kong,
2011), The translational science of rare diseases: From rare to care (Vienna, 2012), in collaboration with the
Institute of Molecular Biotechnology of the Austrian Academy of Sciences
¥ Bridging Biomedical Worlds series (AAA Science, Science Translational Medicine, and Fondation IPSEN):
Turning obstacles into opportunities for stem cell therapy (Beijing, 2014, in preparation)
Awards to Encourage Research
The Fondation IPSEN awards prizes to researchers who achieved outstanding and pioneering research.
Currently, four awards are given annually:
Posters advertising the
Fondation IPSEN prizes.
¥ The Neuronal Plasticity Award has been given each year since 1990 to three researchers working on the
same theme: Albert Aguayo, Anders Björklund and Fred H. Gage; Ursula Bellugi, Wolf Singer and Torsten
Wiesel; Philippe Ascher, Kjell Fuxe and Terje Lomo; Per Andersen, Masao Ito and Constantino Sotelo;
Mariano Barbacid, Yves Barde and Hans Thoenen; Jacques Melher, Brenda Milner and Mortimer Mishkin;
Friedrich Bonhoeffer, Cory Goodman and Marc Tessier-Lavigne; Antonio Damasio, Richard Frackowiak
and Michael Merzenich; Heinrich Betz, Gerald Fischbach and Uel McMahan; Masakazu Konishi, Peter
Marler and Fernando Nottebohm; Tomas Hökfelt, Lars Olson and Lars Terenius; Albert Galaburda, John
Morton and Elizabeth Spelke; Arturo Alvarez-Buylla, Ron McKay and Sam Weiss; François Clarac, Sten
Grillner and Serge Rossignol; James Gusella, Jean-Louis Mandel and Huda Zoghbi; Ann Graybiel, Trevor
Robbins and Wolfram Schultz; Mary Kennedy, Morgan Sheng and Eckart Gundelfinger; Nikos Logothetis,
Keiji Tanaka and Giacomo Rizzolatti; Jean-Pierre Changeux, Peter Kalivas and Eric Nestler; Alim-Louis
Benabid, Apostolos P. Georgopoulos, Miguel A. L. Nicolelis; Thomas Insel, Bruce McEwen and Donald
Pfaff; Helen Neville, Isabelle Peretz, Robert Zatorre; Catherine Dulac, Michael Meaney and J. David Sweatt;
Tim V.P. Bliss, Richard G.M. Morris and Yadin Dudai.
¥ The Endocrine Regulations Award, first given in 2002, has been received by Wylie Vale, Robert Lefkowitz,
Pierre Chambon, Tomas Hökfelt, Roger Cone, William Crowley, Ronald Evans, Gilbert Vassart, Shlomo
Melmed, Paolo Sassone-Corsi, Jeffrey M. Friedman and Bert O'Malley.
¥ The Jean-Louis Signoret Neuropsychology Award: since 1992, the recipients have been Eric Kandel,
Jacques Paillard, Rodolfo Llinas, Steven Kosslyn, Alfonso Caramazza, Jean-Pierre Changeux, Emilio
Bisiach, Joseph LeDoux, Joaquim Fuster, Stanislas Dehaene, Deepak Pandya, Utah Frith, Antonio and
Hanna Damasio, Marc Jeannerod, Faraneh Vargha-Khadem, Alvaro Pascual-Leone, Elizabeth Warrington,
Pierre Maquet, Giacomo Rizzolatti, Patricia Kuhl, Cathy Price and Jean Decety.
¥ The Longevity Award, created in 1996, has been bestowed on: Caleb Finch, Vainno Kannisto, Roy L.
Walford, John Morley, Paul and Margret Baltes, Justin Congdon, George Martin, James Vaupel, Linda
Partridge, Sir Michael Marmot, Cynthia Kenyon, David Barker, Gerald McClearn, Jacques Vallin, Judith
Campisi, Tom Kirkwood, Linda Fried and Gary Ruvkun.
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International Publications
Proceedings of the conferences organised by the Fondation IPSEN are published in English and distributed
by international publishers:
¥ Research and Perspectives in AlzheimerÕs Disease (Springer, 27 titles)
¥ Research and Perspectives in Neurosciences (Springer, 21 titles)
¥ Research and Perspectives in Longevity (Springer, 5 titles)
¥ Research and Perspectives in Endocrinology (Springer, 11 titles)
¥ WHO/Fondation IPSEN series (Springer, 7 titles)
¥ Brain and Mind Collection
Books and brochures
recently published by
the Fondation IPSEN.
In addition, since 1986 the Fondation IPSEN
has published over 230 issues of Alzheimer
Actualités, a newsletter dedicated to
AlzheimerÕs disease; in 1993, a bi-annual
journal, the Bulletin du Cercle de Neurologie
Comportementale was started; and in 2005,
the first report on the annual conferences
dedicated to Cancer Science appeared.
The foundation also has widely distributed
information in various forms to the medical
professions and families of patients, as
well as produced teaching films that have
received awards from specialized festivals.
Illustration: HervŽ Coffinires
Photos: Fondation IPSEN
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65, quai Georges Gorse ― 92650 Boulogne-Billancourt Cedex ― Tel.: 33(0)1 58 33 50 00 ― Fax: 33 (0)1 58 33 50 01