The present invention relates to a novel and improved method
of cancer immuno-therapy and to a kit for use in this method. Specifically, the
present invention relates to a method of providing lymphocytes obtained from sentinel
lymph nodes of a cancer patient and expanding them in vitro.
Cancer, a term frequently used to indicate any of the various
types of malignant neoplasms, is one of the leading causes of death in humans. At
present, cancer accounts for approximately 23 % of all deaths in the world, with
only cardiovascular diseases claiming more lives. During the lifetime of an individual
cancer may develop in any tissue of any organ and at any age with the transformed
cells invading surrounding tissues or metastasizing to several sites in the body.
At present there is growing evidence that apart from endogenous factors also certain
activities and environmental conditions, such as smoking, exposure to specific compounds
contained in food or released by industrial processes account for the development
of and enhance the risk for certain types of cancers and tumors.
Once cancer has been diagnosed, treatment decisions become
paramount. In the past, both local and regional therapies have been applied, specifically
surgery and/or radiation, which are usually combined with systemic therapy, i.e.
administration of anti-neoplastic drugs.
Surgery is the oldest form of cancer therapy, but suffers
from various shortcomings. It will be , successful only, if the tumor is detected
in an early developmental stage, at which cells of the primary tumor have not started
to disseminate yet, and the treatment success varies greatly between the cancer
sites. Radiation is utilized in addition to surgery or alone in cases, where the
primary tumor is difficult to access by surgical means, such as in nodular and diffuse
non-Hodgkin's lymphomas, squamous cell carcinoma of the head and neck, mediastinal
germ-cell tumors, prostate cancer, or early stage breast cancer.
Along with any of these above two treatment regimen anti-neoplastic
drugs are administered to a cancer patient, which are to prevent cell division or
spread of neoplastic cells (chemotherapeutic treatment). Even though these agents
are toxic for essentially all fast proliferating cells, in the adjuvant setting,
chemotherapy may bring about a limited improvement reducing mortality, e.g. in colon
cancer from 51% to 40% after more than five years of follow up.
Recently, two additional treatment regimes have been developed,
the photodynamic therapy and tumor immuno-therapy.
In photodynamic therapy, photosensitizing compounds and
lasers are utilized to produce tumor necrosis. Tumor localizing photosensitizing
compounds are systemically administered to a patient and subsequently activated
by a laser. Upon absorbing light of the appropriate wavelength the sensitizer is
converted to an excited state, with cytotoxicity being mediated by the interaction
between the sensitizer and molecular oxygen within the tissue treated to generate
cytotoxic singlet oxygen.
Tumor immuno-therapy on the other hand tries to take benefit
from the inherent task the immune system fulfils in the individual as an instrument
for preserving the physical integrity of the body.
In principle, the mammalian immune system has two general
mechanisms to protect the body, the non-specific or innate immune response and the
specific or acquired immune response. In contrast to the innate response, which
un-specifically combats any foreign invading material, the specific response is
tailored to a particular substance (antigen), which is effected by clonal selection.
Acquired immunity is mediated by specialized cells, the B-cells, that produce antibodies
as effector molecules (implementing the humoral immune response), and T-cells that
mediate their effectivity through the cell as such (cell mediated immunity).
The cells of the specific immune system generically combat
and destroy each entity that has been recognized as not belonging to the body, i.e.
as foreign or "non-self", while at the same time desisting from any attack to substances/antigens
that are known to determine "self'. Thus, any exogenous biological and non-biological
entity entering/invading the individual's body, is attacked, but also any endogeneous
matter, the immune system never has learnt about to represent "self' and recognizes
In an individual the immune system usually provides an
immunologic surveillance and prevents the development of cancer. Cancer immunity
is mediated mainly by T-cells and NK cells, wherein transformed tumor cells are
destroyed after being recognized as (tumor-associated) foreign material. i.e. antigens
that are not known as belonging to "self" (T cells) or from lack of MHC class I
expression (NK cells).
Medicine tried to exploit these mechanisms in cancer treatment
and so far, two general methods have been applied in tumor immuno-therapy. According
to a first method the innate immune response is stimulated by administering substances
to tumor patients, such as interleukin-2, tumor necrosis factor or interferon. However,
this approach did not prove to be quite successful, while showing strong side effects
due to the high toxicity the administered substances show at the effective concentrations.
Another focus has been on the specific immune system, taking
advantage of the immune system's endogenous capability to distinguish between "self"
and "non-self". Generally the immune system attacks transformed cells, since they
exhibit antigens recognized to be foreign or "non-self'. However, in some instances
tumor cells may escape the immune system in vivo, partly because the tumor associated
antigen is not capable to elicit an immune response, or is not capable to effect
a sufficient stimulation of T-cells, or for the reason that the tumor cells produce
factors that down-regulate the immune response in vivo.
In order to assist the immune system in performing its
endogeneous task mononuclear cells of a cancer patient have been isolated from peripheral
blood and transferred into a culture, where they have been stimulated and expanded.
After expansion they were infused back to the patient, however, giving success response
rates of only up to 35 %. One of the drawbacks resides in that peripheral blood
contains only small numbers of lymphocytes with functioning activity against tumor
In order to overcome this deficiency a variation of such
a procedure has been proposed, which includes isolating and expanding populations
of lymphocytes that have infiltrated tumors in vivo, so-called tumor-infiltrating
lymphocytes. Yet, this process suffers from the disadvantage of requiring a specific
type of lymphocytes, which are present only in a rather late stage of tumor development
and in low numbers. Furthermore, the tumor infiltrating lymphocytes are subjected
to immunosuppressive substances from the tumor and finally the isolation of such
lymphocytes is not always an easy task to achieve.
Therefore, a problem of the present invention resides in
overcoming the above disadvantages of the prior art and providing a novel and improved
method for immuno-therapy
This problem has been solved by a method for treating and/or
preventing the recurrence of cancer comprising the steps of providing lymphocytes
obtained from sentinel lymph nodes and activating and expanding the lymphocytes
thus obtained in vitro.
In the figures,
- Fig. 1 shows the results of a peroperative identification of sentinel node(s).
Identification of sentinel nodes was performed by subserosal injections of Patent
blue dye (A) at four sites around the tumor (B). Usually within five minutes, one
or more blue coloured lymph nodes appear in the mesentery (C).
- Fig. 2 shows the results obtained from a characterization of lymphocytes. Data
from a patient with Dukes B (A) and Dukes C (B). Specimens from the tumor (CC),
sentinel node (SN) and non-sentinel node (LN) were stained with hematoxylineosin
(left panels) (40x). Arrows indicate the presence of metastatic colon cancer cells
in a sentinel node (B, left panel, SN). Lymphocytes from the tumor (CC), sentinel
node (SN), non-draining lymph nodes (LN) were stained with antibodies against CD4
and the activation marker CD69 and analyzed using flow cytometry (middle panels),
the percentage of double positive activated CD4 T helper cells are indicated in
the upper right corner. Cells from the tumor, (CC), sentinel node (SN) and lymph
node (LN) were incubated with a 10- or 100-fold dilution of autologous colon cancer
extract in a day 5-7 time course study. Cells were pulsed 16h before harvesting
with 1 pCi 3H-Thymidine. Peak proliferation occurred at day 5 (right panels).
- Figs. 3 shows the results of an unspecific activation and intracellular FACS
analyses for metastasis. Proliferative responses against Concanavalin A stimulation
(A) and intracellular FACS analyses using cytokeratin-20 antibody (B). Cells from
peripheral blood (PEL), the tumor (CC), sentinel node (SN) and lymph node (LN) were
investigated in a time course proliferation assay stimulating with 10 pg/mL of Concanavalin
A (A). Cells from the tumor (CC), sentinel node (SN) and lymph node (LN) were permeabilized
with saponin followed by incubation with an anti cytokeratin-20 antibody and detection
with an anti mouse IgG FITC conjugated antibody (B). The dotted line represent control
samples incubated with secondary antibody only and in the overlay plot both the
primary and secondary antibodies were included in the incubations. The numbers indicate
the percentage of cytokeratin-20 positive tumor cells.
- Fig. 4 shows a hypothetical scheme of how antigenic material from the tumor
is transported by the lymph vessels to the draining lymph node, identified by tracer
dye, where presentation of antigenic peptides and activation of T cells occur. Hypothetical
scheme Dukes B (A). In the tumor there is a rapid turnover of cells, lack of oxygen
and nutritions causing a hostile environment attracting macrophages and dendritic
cells. Debris from tumor cells are phagocytosed by these professional antigen presenting
cells (MC) and transported by the lymph vessels to the draining sentinel node, which
can be detected peroperatively by peritumoral injection of Patent blue. In the sentinel
node the APCs load peptides, processed in the endosomal/lysosomal pathway and derived
from tumor antigens, mainly into the HLA class II pocket. The class II-tumor peptide
complex is transported to the surface of the APC and is recognized by CD4-t- T helper
cells which provide the first activation signal. The second activation signal is
provided by costimulatory molecules such as B7.1, B7.2 and ICAM-1 that are expressed
at high levels on the APC. Thus the CD4+ T helper cell becomes an activated effector
cell and leave the lymph node to return to the blood stream via the thoracic duct.
The activated CD4+ T helper cell leave the blood stream in areas of inflammation
and new vessel formation such as in a tumor or metastases. The cell now becomes
a tumor infiltrating lymphocyte (TIL). However, due lo the local hostile environment
in the tumor and possibly by immunosuppressive cytokines released from the tumor
the TIL cells frequently become immunosuppressed and unresponsive (anergic).
Dukes C (B): In the presence of metastatic cells in a sentinel node we find that
lymphocytes are unable to proliferate against tumor extract nor against an unspecific
activator such as Concanavalin A. APCs with phagocytosed tumor debris are likely
to be present and the cells seem to be activated. One explanation is that metastatic
cells produce immunosuppressive factors. Another explanation is that Dukes C (presence
of lymph node metastases) only occurs in patients with an immune failure to recognize
the tumor as foreign. Thus a similar situation as seen in different mice strains
challenged with Leishmaniasis where some strains clear the infection whereas others
succumb due to genetical background differences.
During the extensive studies leading to the present invention
the inventors analyzed the immune profile of patients suffering from colorectal
cancer and surprisingly found that even though lymphocytes isolated from peripheral
blood or generically from the lymph system of the patients were unresponsive towards
the tumor, sentinel lymph nodes of said patients harbor lymphocytes showing activity
against tumor cells. Based on this finding the invention was accomplished, and a
useful tool in cellular immuno-therapy is provided.
When carrying out the method as disclosed herein, first
the sentinel nodes have to be located. Sentinel lymph nodes are generically defined
as the first lymph node(s) in the lymphatic system that receive(s) lymphatic drainage
from a primary tumor area. This may conveniently be achieved during surgery of the
primary tumor(s), e.g. by introducing a tracer substance around or in the circumference
of the tumor site, preferably prior to the surgical removal of the primary tumor.
The tracer is transported in the lymph capillaries and accumulates via phagocytosis
by macrophages in the lymph nodes located downstream. Usually, the sentinel lymph
nodes may be determined visually some minutes after applying the tracer substance
by checking for lymph nodes coloured first or more intense.
As a tracer substance e.g. patent blue dye, lymphazurine
blue or 99 Tc labelled albumin may be utilized.
Once the sentinel lymph nodes have been identified, they
are isolated by surgical means and the histological status thereof may be assessed
in representative slices from the nodes.
In a next step lymphocytes present in the remaining sentinel
node material are collected and transferred to an in vitro culture. This may e.g.
be achieved by pressing the lymph nodes so that they release their content with
our without the presence of collagenase.
The cells thus obtained are subsequently subjected to an
in vitro culture.
In case of removing unwanted cells from the culture and/or
selecting for a specific sub-population of T-cells, e.g. CD4, CD8, CD69, CD62L positive
or negative selection techniques may be applied, such as antibodies directed to
surface markers unique to the cells selected. An example for such techniques is
magnetic immuno-adherence wherein monoclonal antibodies directed to cell surface
markers present on the cells to be selected are bound to a carrier. Selection of
cells can also be achieved by flow cytometry assisted cell sorting under sterile
The cells may be cultured under conventional conditions
in any medium suitable for growing lymphocytes cells including a Minimal Essential
Media or RPMI Media 1640. In order to promote growth of the cells, factors necessary
for proliferation and viability thereof may be added, including serum, e.g. fetal
calf serum or human serum and antibiotics, e.g., penicillin streptomycin. The lymphocytes
are maintained under conditions necessary to support growth, e.g. an appropriate
temperature of around 37 °C and an atmosphere, e.g. air plus 5% CO2.
During culture the lymphocytes are exposed to stimulating
agents for expansion and optionally also to additional activation signals.
Expansion of lymphocytes may be achieved by contacting
the cells with anti-CD3 antibodies, or contacting them with a protein kinase C activator
(phorbol ester) in conjunction with a calcium ionophore. For a stimulation of an
accessory molecule on the surface of the T-cells, a ligand which binds the accessory
molecule may be employed. For example, T cells can be activated with an anti-CD3
antibody and an anti-CD28 antibody, under conditions appropriate for stimulating
proliferation of the T-cells.
The primary and the co-stimulatory signal may be provided
by different protocols. For example, the agents providing each signal can be in
solution or coupled to a solid phase surface, e.g. the culture container. When coupled
to a solid phase surface, the agents can be coupled to the same solid phase surface
(i.e., in "cis" formation) or to separate surfaces (i.e., in "trans" formation).
Also, one agent can be coupled to a solid phase surface and the other agent in solution.
To maintain long term stimulation of a population of T-cells
following stimulation, the T-cells are subsequently separated from the stimulus.
Yet, the T-cells may be maintained in contact with the co-stimulatory ligand throughout
the culture term. The rate of T-cell proliferation is monitored periodically (e.g.,
daily) by, for example, examining the size or measuring the volume of the T-cells.
When the mean T-cell diameter decreases after a peak they are reactivated and restimulated
to induce further proliferation. Alternatively, the rate of T-cell proliferation
and time for T-cell restimulation can be monitored by assaying for the presence
of cell surface molecules, such as CD25, CD69, CD62L and MHC class II which are
modulated on activated T-cells. For inducing long term stimulation of a population
of CD4 or CD8 T-cells, it may be necessary to reactivate and restimulate the T-cells
with a anti-CD3 antibody and an anti-CD28 antibody several times.
In addition, the T-cells obtained from the sentinel lymph
nodes may be stimulated by the addition of cytokines, such as IL-2 for maintenance
and expansion and IL-12, INF-&agr;, and anti IL-4 antibody in order to activate
CD4+ T helper cells towards IFN-&ggr; producing Thl effector cells. The amount
of cytokine that should be added to the T-cell culture to obtain expansion to a
sufficient extent may easily be determined by the skilled person. The cytokine is
added from the first day of the culture, and added every other day of the culture
in amounts sufficient to maintain proliferation of the T-cells. E.g., IL-2 can be
added to the cultures to obtain a final concentration of about 100 U/ml and is added
every other day to the culture, such as every second or third day, when new medium
is added to the cell culture.
Since according to the method of the present invention
lymphocytes are utilized, that have been primed already in vivo and have a specificity
against tumor antigens, no additional activation/priming is required in principle,
since clonal expansion has already has occurred.
However, in patients with tumors producing immuno-suppressive
substances the lymphocyte culture may be stimulated with tumor antigen in a form
suitable to trigger an additional activation signal in the T-cells, i.e., the antigen
is presented to the T-cell such that a signal is triggered in the T-cell through
the TCR/CD3 complex. For example, the antigen can be presented to the T-cell by
an antigen presenting cell, such as a B-cell, macrophage, monocyte, dendritic cell,
Langerhans cell in conjunction with an MHC molecule. To this end, tumor cells, an
autologous tumor extract and/or a recombinant tumor antigen are added to the culture
of lymphocytes and incubated for a time sufficient for additional priming. Similarly,
a cell infected with a pathogen, e.g., a virus, which presents antigens of the pathogen
can be incubated with the lymphocytes. Following antigen specific activation of
a population of T-cells, the cells can be further expanded according to the methods
described herein. In view of patient safety use of an autologous tumor extract is
preferred, since the step of removing tumor cells and or recombinant cells may be
It has further been found that when tumor cells are present
in the sentinel lymph nodes the lymphocytes obtained therefrom exhibit a lower activity
and/or specificity against the tumor cells as compared to lymphocytes obtained from
tumor cell free sentinel lymph node. Without wishing to be bound by any theory it
is presently believed that this finding is likely due to the tumor's capability
of immuno-suppression, i.e. a functional state of anergy due to the presence of
When the lymphocyte culture has been expanded and stimulated
to an extent desired, the lymphocytes are collected, optionally purged from any
material, detrimental to the patient's health and transferred back into the patient.
This may be achieved by intravenous infusion, during a period of from about 1 to
6 hours, with the number of mononuclear cells administered being dependent solely
on the number of cells generated during the proliferation step.
According to another embodiment the present invention also
provides a kit for carrying out the present method. The kit comprises a dye, preferably
patent blue dye and agents for stimulating proliferation and expansion of lymphocytes.
The method of the present invention provides clear advantages
over the prior art. Since the lymphocytes are collected from sentinel lymph nodes,
specifically those lymphocytes are expanded that already have an activity and specificity
directed against tumor antigens. This specificity may be enhanced in vitro, by culturing
the lymphocytes in the presence of an autologous tumor extract to promote clonal
selection and expansion. T-cells with specific reactivity towards the primary tumor
can be identified in sentinel nodes and these cells can be expanded specifically
for later use in cellular immuno-therapy.
The invention will now be explained by the following example
that is not to be construed to be limiting but are given for illustrative purpose
Collection and preparation of cells
Five patients with colon cancer, with no signs of distant
metastases or lymph node involvement prior to surgery, were included in the study
(Table 1). The study was approved by the local ethical committee and informed consent
was given by the patients.
The colonic tumor site was mobilized through division of
peritoneal adhesions to facilitate inspection. One ml Patent blue dye (Guerbet,
Paris) was injected superficially in the circumference of the tumor. Within five
minutes, one to three blue-coloured mesenteric lymph nodes were identified macroscopically
as sentinel nodes and they were marked with sutures.
The sentinel and non-sentinel nodes were cut in half. Slices
less than 1 mm thick were cut from the central and the peripheral part of the nodes
for flow cytometry and proliferation analysis. The rest of the node underwent routine
histopathological examination. Tumors were histopathologically classified as Dukes
stages A-C (11) (Table 1).
One piece of the primary tumor (including part of the invasive
margin) was removed for flow cytometry analyses and as an antigen source.
Venous blood, sentinel and non-sentinel lymph nodes and
tumors were immediately taken care of to minimize handling time. Peripheral blood
leukocytes (PBL) where purified by ficoll-hypaque (Pharmacia, Amersham). Single
cell suspensions of lymph node cells were obtained by gentle pressure using a loose
fit glass homogenizer. Cells were resuspended and washed twice in DMEM containing
2.5% fetal calf serum (FCS) (Life technologies). Finally, cells were resuspended
in RPMI proliferation media containing 10% human AB serum (Sigma), 1% penicillin-streptomycin
(Sigma) and 1% glutamin (Sigma).
Tumor samples were homogenized using a Dounce homogenizer
in 5 volumes (w/v) of phosphate buffered saline (PBS) followed by 5 minutes denaturation
at 97° C. No intact cells were visible under the microscope. Tumor homogenates
were diluted 1:10 and 1:100 in complete proliferation media. Purified PBL and lymph
node cells were used at 3x105 cells/well in a proliferation assay against
diluted tumor homogenate, concanavalin A 10 µg/ml (Sigma) or carcinoembryonic
antigen 100 µg/ml (Sigma) in triplicate. Proliferation was measured on day
5, 6 and 7 by adding 1 µCi of 3H-Thymidine/well (Amersham) 16 hours
prior to harvesting. Samples were subjected to scintillation counting.
PBL, lymph node cells and tumor cell suspensions at 1x106
cells/sample were subjected to investigation using flow cytometry (FACS). Cells
were washed in PBS containing 2% FCS and 0.02% NaN3 (FACS buffer) and
directly triple labeled using fluorescent cell surface markers against CD4 PE, CD8
PerCp and the very early activation marker CD 69 FITC (Pharmingen). For intracellular
FACS, cells were permeabilized with 0.3% saponin (Sigma) in FACS buffer for 15 minutes
at room temperature followed by a 30 minute incubation with cytokeratin-20 antibody
(Dakopatts). After washes, cells were incubated with an anti-mouse IgG FITC (Jackson)
conjugated antibody for 30 minutes. Cells stained without the primary antibody were
used as controls. After the staining, cells were investigated using a FACSscan (Becton
Dickinson) and data was analyzed using the cellquest computer software (Becton Dickinson).
Peroperative identification of sentinel node and pathological classification
characteristics, locations of tumors, staging, investigated lymph nodes and proliferative
Positive nodes/ Harvest nodes
Mean CPM proliferation
a. Peak at day 6 of mean proliferate
responses where seen at tumor extract dilution 1/100
One to three sentinel nodes were detected intraoperatively
using Patent blue injection in the circumference of the tumor (Fig. 1). Patient
characteristics and location of the tumors are presented in table 1. Upon macroscopical
dissection of the removed specimens, between 18 and 29 lymph nodes (average 22)
were identified and embedded for histopathological evaluation (Fig. 2A, B left panels).
Patients 2 and 3 (Table 1) had metastatic spread to the sentinel node and were histopathologically
classified as Dukes C (Fig. 2B left panel). The other 3 patients did not show any
signs of metastatic spread to sentinel node(s) nor to other lymph nodes despite
tumors grew through the bowel muscular wall (Fig. 2A left panel). They were classified
as Dukes B.
Single cell suspensions of lymphocytes collected separately
from peripheral blood (PBL) (not shown), the tumor, draining sentinel lymph nodes
and non-draining lymph nodes were triple stained with antibodies recognizing the
very early activation marker CD69 and the T-cell markers CD4 (Fig. 2A, B middle
panel) and CD8 (not shown) followed by flow cytometry analyses (FACS). Similar numbers
of activated CD4+ lymphocytes where found both in sentinel and non-sentinel
nodes regardless of presence (Fig. 2A middle panel) or absence of metastases (Fig.
2B middle panel). All investigated tumors contained tumor infiltrating lymphocytes
(TILs) to a various extent and the majority of these TILs, which were both CD4+
and CD8+ T-cells, presented and activated CD69+ phenotype.
In peripheral blood activated CD4+CD69+ lymphocytes were absent
The functional status of the lymphocytes was further characterized
in time course proliferation assays using homogenized tumor cell extracts as antigen
source (Fig. 2A, B right panels). Stimulation with Patent blue dye did not cause
proliferation in any case (data not shown). In all sentinel lymph nodes without
metastasis (Dukes B cases), the lymphocytes proliferated in a dose dependent manner
against autologous tumor extract (Fig. 2A, right panel). Peak proliferation was
regularly seen at day 6 at the highest amount of antigen (Table 1). No antigen dependent
proliferation was recognized among lymphocytes from non-sentinel nodes or from TILs.
In the Dukes C cases (Table 1, Fig. 2B right panel), where the sentinel nodes contained
metastatic cells, none or a very weak proliferative response was identified. To
further investigate the functional state of the lymphocytes, T-cells were non-specifically
stimulated with Concanavalin A which by-pass the activation through the T-cell receptor.
All PBLs and non-sentinel node lymphocytes responded with strong proliferation as
well as sentinel node lymphocytes from the Dukes B patients (Fig. 3A). However,
sentinel node lymphocytes from the Dukes C patients did not respond with proliferation
against Concanavalin A, nor did the TILs from Duke B or C cases. Stimulation of
sentinel node lymphocytes from a Dukes B patient with 100 µg/ml of carcinoembryonic
antigen did not result in any proliferative activity (data not shown).
Lymphocytes with specific activity towards autologous tumor
extract were selected and put in long term cultures. The stimulated cells consisted
of predominantly CD4+ T lymphocytes and they were expanded in the presence
of IL-2 and survived in vitro for several weeks.
Cells from the tumors, sentinel and non-sentinel nodes
were investigated by FACS after intracellular staining with cytokeratin-20, a marker
of epithelial cancers. We found that permeabilization of cells with saponin permitted
detection of a large proportion of cytokeratin-20 positive cells in the tumors (Fig.
3B). Interestingly, we were able to detect a small number of cytokeratin-20 positive
cells in a sentinel node with micrometastasis (Fig. 3B, Fig. 2B left panel).