WO2007071409A1

WO2007071409A1 - Method for treating colon cancer

Info

Publication number: WO2007071409A1
Application number: PCT/EP2006/012338
Authority: WO
Inventors: Ola Winqvist; Magnus Thörn
Original assignee: Sentoclone Ab
Priority date: 2005-12-21
Filing date: 2006-12-20
Publication date: 2007-06-28
Also published as: US20090123443A1; WO2007071409A8; US8007785B2

Abstract

The present invention discloses an immunotherapeutic method for treating patients suffering from colon cancer, by administering expanded tumour-reactive CD4+ helper and/or CD8+ T-lymphocytes obtainable from one or more sentinel or metinel lymph nodes draining a tumour in the colon or a metastasis arising from a tumour in the colon. The present invention provides a new effective method for treating colon cancer and metastatic colon cancer, without adverse side effects associated with the known treatments. The method comprises identification of in a patient one or more sentinel and/or metinel lymph nodes draining a tumour in the colon or a metastasis from a tumour in the colon, resection of the one or more nodes and, optionally all or part of the tumour or metastasis, isolation of tumour-reactive T-lymphocytes from said lymph nodes, in vitro expansion of said tumour-reactive T-lymphocytes, and administration of the thus obtained tumour-reactive T-lymphocytes to the patient, wherein the T- lymphocytes are CD4+ helper and/or CD8+ T-lymphocytes.

Description

Method for treating colon cancer

Field of the invention

The invention relates to an immuno-therapeutically method for treating patients suffering from colon cancer, by administering expanded tumour-reactive CD4+ helper and/or CD8+ T-lymphocytes obtainable from one or more sentinel or metinel lymph nodes draining a tumour in the colon or a metastasis arising from a tumour in the colon, wherein the T-lymphocytes are not CD4+ CD25+^H| lymphocytes, i.e. the present invention does not cover regulatory T-lymphocytes.

Background of the invention

Worldwide, colorectal cancer is the third most common cause of death in cancer and approximately one million individuals are diagnosed each year. Despite optimal surgical treatment and modern adjuvant chemotherapy half of patients have recurrences and ultimately die within 5 years from diagnosis. The most important prognostic factors are presence of lymph node or distant metastases, being found in about 50% of all cases. Patients with locoregional lymph node metastases, Duke^'s C/stage III, have about 60% 5-year survival after surgery, and when distant metastases are present, the 5-year survival rate is less than 10 %. The established, adjuvant treatment in stage III patients is 5-FluorouraciI (5-FU)-based chemotherapy, improving absolute 5-year survival by approximately 10%. There is no evidence for a statistically significant survival benefit for chemotherapy in Duke's B/Stage Il patients. However, 20% of patients being classified as lymph node negative will suffer from recurrent disease. Some of these patients are likely to be understaged, since it is widely accepted that accurate staging of colon cancer is difficult and heavily dependent on the number of lymph nodes analysed by the pathologist. The sentinel node, originally defined in penile carcinoma, is the first lymph node to receive lymphatic drainage from a tumour. Sentinel node detection and analysis has recently been applied in colon cancer, improving staging considerably.

The immune system often appears informed about tumours, as shown by an accumulation of immune cells at tumour sites, which correlates with improved prognosis, lmmuno competent cells respond to "danger" signals, which can be provided of growing tumours as a consequence of the genotoxic stress of cell transformation and disruption of the surrounding microenvironment. Under ideal conditions, these signals will induce inflammation, activate innate effector cells with antitumour activity, and stimulate professional antigen-presenting cells (APCs), particularly dendritic cells (DCs), to engulf tumour-derived antigens and migrate to draining lymph nodes to trigger an adaptive response by T and B lymphocytes. Thus, the immune system is capable of recognizing and eliminating tumour cells but unfortunately tumours often interfere with the development and function of immune responses. However, recent advances in cellular and molecular immunology suggest strategies, which may prevent antitumour responses. Briefly, the presence of a tumour indicates that the developing cancer was able to avoid detection or to escape or to overwhelm the immune response. Progressing tumours often exhibit strategies that promote evasion from immune recognition. Examples are physical exclusion of immune cells from tumour sites, poor immunogenicity due to reduced expression of major histocompatibility complex (MHC) or costimulatory proteins, and disruption of natural killer (NK) and natural killer T (NKT) cell recognition. Further, some tumours prevent triggering of an inflammatory response by secreting proteins such as interleukin 10 (IL- 10) or vascular endothelial growth factor (VEGF) that interfere with DC activation and differentiation, or by blocking the production of proinflammatory molecules by increasing expression of the STAT3 protein. Even if a response is induced, tumour cells may escape elimination by losing targeted antigens, rendering tumour-reactive T cells anergic, inducing regulatory T cells, or specifically deleting responding T cells. The tumour that finally develops reflects selection of poorly immunogenic and/or immune-resistant malignant cells.

In the adjuvant setting, tumour immunotherapy offers an appealing alternative to traditional cytostatics. One strategy has been to expand and activate NK cells in vitro with out specific antigen by culture with IL-2 followed by infusion of large numbers of these NK cells back into patients alone or with high doses of IL-2. This approach, or administration of high doses of IL-2 to expand and activate NK cells entirely in vivo, has yielded antitumour activity and remission in a subset of patients (Rosenberg SA et al., J Natl Cancer Inst 85, 622, 1993). However, life-threatening toxicity often develops, largey due to the release of tumour necrosis factor (TNF) from activated NK cells.

Other attempts to stimulate the innate specific T cell immunity have been done by different types of vaccines. Promising results from animal studies entailed a study in which autologous tumour cells and an adjuvant immunomodulating agent, bacillus Calmette-Guerin (BCG) was given in combination several times to 98 patients with colorectal cancer in a prospectively randomized study. No statistically significant differences were detected in survival but maybe a small decrease in recurrence rate in stage Il colon cancer patients. Further studies were done but no statistical clinical benefit in disease-free interval or survival could be seen, not even when combined with 5-FU and Leucovorin. Carcinoembryonic antigen (CEA) is expressed by colon cancers and trials have been done to create a monoclonal antibody against CEA. In a study with 23 patients with advanced colorectal cancer 13 true anti-CEA responses were generated, but without clinical responses. Vaccines based on CEA antibodies in combination with 5-FU or based on CEA proteins have been done, but without any clinical responses. The first results with the murine anti-17-1A monoclonal antibody in stage III colon cancer seemed very promising with a 5 year reduction of overall death rate by 30 % and a decrease in recurrence rate by 27%, but later analysis of a large randomized study showed no effectiveness superior to standard adjuvant chemotherapy treatment.

Recently, two targeted therapies have been registered for the use in metastatic colorectal cancer. Cetuximab is a monoclonal antibody blocking the epidermal growth factor receptor-EGFR) and Bevacizumab is the first registered monoclonal antibody targeting angiogenesis. Studies, so far, have shown a limited but significant additive increase in survival of metastatic colorectal cancer using a successive combination of these drugs. When comparing 5-FU/LV+irinotecan with 5-

FU/LV+irinotecan+bevacizumab (including post-progression chemotherapy in 55% of both groups), the overall survival was 15.6 months and 20.3 months, respectively. However, side effects were very common, with a proportion of patients having severe adverse effects of 85%. Patients treated with bevacizumab developed hypertension in 22% of cases, arterial thrombothic event (10%) and gastrointestinal perforation (1.5%). At least 50% of the patients had hypertension, thrombosis or minor bleedings.

Thus, it is obvious that there is still a need for an effective and at the same time safe treatment of colon cancer and metastases of colon cancer.

Disclosure of the invention

Accordingly, the present invention discloses a new effective method for treating colon cancer and metastatic colon cancer, without adverse side effects associated with the known treatments. Adoptive immunotherapy, i.e. collection and in vitro expansion of autologous tumour- reactive lymphocytes, followed by transfusion back to the patient has never been done for treating colon cancer. The present inventors described herein a very successful attempt to use sentinel or metinel node acquired lymphocytes for immunotherapy of colon cancer. In short, the tumour or metastasis draining lymph node(s) is identified by injection of a lymph node locator. The nodes contain T-lymphocyte clones that have been naturally activated against tumour-derived antigens in vivo. This specific T lymphocyte population is further expanded using an improved method for expansion of tumour-reactive T-lymphocytes, wherein specific culturing conditions have been determined and optimized, and wherein specific markers on the T-lymphocytes and in the culture medium are monitored throughout the expansion phase, in order to obtain high numbers of tumour-reactive T-lymphocytes in the shortest possible time span. The inventors have returned such expanded tumour-reactive T-lymphocytes to patients with colon cancer, and can conclude that side effects were minimal and efficacy seems evident with total or marked regression of the disease in some patients with liver and lung metastases, while others displayed a partial regression or at least stable disease.

Accordingly, the present invention relates to a method for treating patients suffering from colon cancer, the method comprising i) identifying in a patient one or more sentinel and/or metinel lymph nodes draining a tumour in the colon or a metastasis from a tumour in the colon, ii) resecting the one or more nodes and, optionally, all or part of the tumour or metastasis, iii) isolating tumour-reactive T-lymphocytes from said lymph nodes, iv) in vitro expanding said tumour-reactive T-lymphocytes, v) administering the thus obtained tumour-reactive T-lymphocytes to the patient, wherein the T-lymphocytes are CD4+ helper and/or CD8+ T-lymphocytes and not CD4+ CD25+^H| lymphocytes, i.e. the present invention does not cover regulatory T- lymphocytes.

Before going in to further details with the steps of the method of the invention, the following terms will be defined:

By the term "tumour-reactive T-lymphocytes" is intended to mean T-lymphocytes carrying a T cell receptor (TCR) specific for and recognizing a tumour antigen. Herein the term tumour-reactive T-lymphocytes also tends to cover T-lymphocytes carrying a TCR specific for and recognizing metastasis antigens. I.e. the terms tumour-reactive T- lymphocytes and metastasis-reactive T-lymphocytes may be used interchangeable.

By the term "tumour-reactive T-lymphocytes" is intended to mean T-lymphocytes carrying a T cell receptor specific for and recognizing a tumour antigen.

By the term "T helper cells" is intended to mean T-lymphocytes that promote adaptive immune responses when activated.

By the term "Th1 cells" is intended to mean T helper cells that promote cell mediated immune responses when activated, using cytokines such as IFN-gamma.

By the term "Th2 cells" is intended to mean T helper cells promoting humoral immune responses when activated, using cytokines such as IL-4.

By the term "CD4+ helper T-lymphocytes" is intended to mean T-lymphocytes that express CD4 but not the transcription factor FoxP3.

By the term "CD8+ T-lymphocytes" is intended to mean T-lymphocytes that express CD8.

By the term "regulatory T-lymphocyte" is intended to mean T-lymphocytes that suppress adaptive immune responses, expressing transcription factor FoxP3.

By the term "specific activation" of T-lymphocytes is intended to mean antigen specific and MHC restricted T-cell receptor mediated activation. In contrast the term "unspecific activation" of T-lymphocytes is intended to mean a general activation of all T-cells, regardless of T-cell receptor specificity.

The term "tumour-derived antigen" intends to cover tumour cells, a homogenate of a tumour, which homogenate may be denatured, or tumour proteins, polypeptides or peptides, e.g. in the form of purified, natural, synthetic and/or recombinant protein, polypeptide or peptide. The tumour-derived antigen may be intact molecules, fragments thereof or multimers or aggregates of intact molecules and/or fragments. Examples of suitable polypeptides and peptides are such that comprises from about 5 to about 30 amino acids, such as, e.g. from about 10 to 25 amino acids, from about 10 to 20 amino acids or from about 12 to 18 amino acids. If peptides are used, a final molar concentration in the culture of from about 0.1 to about 5.0 μM, such as, e.g., from about 0.1 to about 4.0 μM, from about 0.2 to about 3.0 μM, from about 0.3 to about 2.0 μM or from about 0.3 to about 1.0 μM may be used. The tumour-derived antigen may be autologous or heterologous, i.e. arise from the patient to be treated or be obtained from another subject suffering from cancer. In the present Examples the inventors uses an autologous denatured tumour extract, however, as mentioned above, other sources of the tumour-derived antigen may also be feasible for use in a method according to the invention.

By the term "day 1 of the first phase" or e.g. "day 5 of the second phase" is to be understood the following: The day on which the lymphocytes are harvested is denoted day 0 (zero). Day 1 of the first phase is defined as the day where the expansion is initiated by addition of at least one substance having agonistic activity towards the IL-2 receptor, and maybe culture medium and/or tumour-derived antigen. The expansion phase i) may be initiated on day 0 (zero) or up till 2 days after harvest of the lymphocytes. The day on which the second phase is initiated by addition of tumour- derived antigen is throughout the text described as "day 1 of the second phase".

By the term "sentinel lymph node" is intended to mean the first lymph node(s) to receive lymphatic drainage from a tumour. Primary tumours or primary tumour areas drain to one or more so-called sentinel lymph nodes. The sentinel nodes are also the first site of metastasis and it has been shown in several solid tumour types that the risk of lymph node metastases is almost negligible if the sentinel node is free of tumour cells. The term "metinel lymph node" refers to the first lymph node(s) to receive lymphatic drainage from a metastasis or a metastases area.

The first step in the present method is the identification of one or more sentinel or metinel lymph nodes draining a tumour in the colon or a metastasis from a tumour in the colon. The step i) of identifying one or more sentinel or metinel nodes are crucial for the method according to the invention, as the present inventors have shown that these nodes comprises a high amount of T-lymphocytes activated against tumour-derived antigens in vivo. One way of identifying the sentinel or metinel lymph node is by injecting one or more lymph node locators into the patient, i.e. any substances suitable for locating a lymph node. Such locators are preferably pharmaceutically acceptable and/or biocompatible. The locators can either be affinity based or non-affinity based. Examples of affinity based lymph node locators are antibodies in whole or fragments, nanobodies, nucleic acids such as RNA, DNA, and PNA all of which can be in turn labeled using various detection modalities. Detection of affinity based lymph node locators can be done by labeling with tracers and dyes, such as, e.g., the ones mentioned below. Visualization is then made by i) radiological methods such as x-ray, computerized tomography, scintigraphy, positron emission technique after labeling with contrast generating substances, such as, e.g., iodine containing substances or radioactive substances such as, e.g. technetium-99m, ii) magnetic resonance imaging after labeling with magnetic or paramagnetic substances, such as e.g., gadolinium, magnetodendromers or iron oxide containing particle; iii) light in the IR-visible- UV spectra by labeling with dyes, fluorescent dyes or luminescent dyes for detection by the naked eye or photon detecting devices such as CCD or CMOS sensors.

Examples of non-affinity based lymph node locators encompass tracers and dyes. These substances are transported in the lymph capillaries and accumulate through phagocytosis by macrophages in the sentinel or metinel node(s), thus identifying the tumour or metastasis draining lymph node(s).

Examples of tracers are radioactive substances such as, e.g., technetium-99 for radioactive decay based detection with photon sensitive films or sensors such as PET detectors. Further on magnetic, paramagnetic or superparamagnetic substances, such as, e.g., gadolinium containing contrast agents, iron oxide particles, magnetic oxide particles, magnetodendrimers for magnetic resonance based detection, contrast agents, such as, e.g., iodine for radiological based detection such as, e.g., computerized tomography or regular X-ray may be used.

Examples of dyes encompasses e.g., azo dyes, bisazo dyes, triazo dyes, diaryl methan dye, triaryl methan dye, anthrachino dye, polycyclic aromatic carbonyl dyes, indigo dyes for visualization by luminescence, near infrared, fluorescence, UV and visible light. Further on dyes also encompass luminescent substances for luminescence based detection and fluorescent substances, such as, e.g., pico green, sybr green, red O oil, texas red for fluorescence based detection. Detection can depending on the chosen wavelengths be made either by the naked eyes or photon detecting devices such as CCD or CMOS sensors.

In one embodiment the dye has an emission maximum that permits visualization by the naked eye in normal light. In another embodiment the dye has an emission maximum that permits visualization by the naked eye in UV light.

Other examples of suitable dyes or tracers appear from WO 04/045650, which is hereby incorporated by reference.

Another, but far more time-consuming way to identify sentinel or metinel nodes is to remove and investigate a selection of lymph nodes in the presumed tumour or metastasis area. A tumour extract from the tumour or metastasis of the actual patient could then be used to identify lymph nodes containing tumour-reactive T-lymphocytes by proliferating assays.

The lymph node locators are injected into the patient into, above, around, adjacent and/or under the tumour or metastasis. The locator will then spread through lymph vessels leading into the metinel lymph node(s), and the one or more nodes will start to get stained within a certain period of time, such as, e.g. within 5 min to 30 min, such as, e.g. within 5 min to 15 min after injection of the locator substance, where after the locator substance is imaged. As described above, imaging of the locator is of course dependent on the locator substance used.

If a dye having an emission maximum that allows visualization by the naked eye in normal light is used, such as, e.g. Patent Blue, the one or more sentinel or metinel nodes are simply identified as the nodes, which are first to accumulate the coloured dye, i.e. if Patent Blue is used, the surgeon will look for the lymph nodes first to accumulate a blue colour.

The locators may be injected by a single injection or by multiple injections, such as, e.g., by two or more injections, by three or more injections, by four or more injections, by five or more injections or by six or more injections.

How to perform the injections of the lymph node locators is dependent on the location of the tumour or metastasis. In case of a tumour in the colon, the lymph node locators may be injected involving a surgical procedure, i.e. a procedure that includes an incision. In such cases, the surgeon will perform an incision in the area of the tumour and subsequently, a lymph node locator may be injected directly into, above, around, adjacent and/or under the metastasis in order to identify the one or more sentinel lymph nodes.

The lymph node locators may also be injected by a non-surgical procedure, i.e. a procedure that does not involve a surgical step, wherein a surgical step is defined a one including surgical operative procedures, i.e. involving incisions with an instrument. In the present context, an injection, i.e. the punctuation of the skin with a needle, is not considered a surgical step. Accordingly, by the statement that the lymph node locator may be injected by a non-surgical procedure is intended to mean that the lymph node locator may be injected into, above, around, adjacent and/or under the metastasis directly into or through the skin.

Examples of situations wherein the lymph node locators may be injected into or through the skin, is e.g. cases where metastasis of colon tumours are located in the skin of the patient. In such situations the lymph node locator should preferentially be injected into the skin above the metastasis, or through the skin into, around, adjacent and/or under the metastasis. If the metastasis is located in the breast area of the patient, the lymph node locator should preferentially be injected into the skin above the metastasis, or through the skin into, around, adjacent and/or under the metastasis.

In case of a metastasis of a colon tumour, the metinel lymph nodes may not always be placed at the shortest or most logical anatomical distance from the metastasis. Accordingly, as a metinel lymph node may be placed distant from the metastasis, it may in some cases be very beneficial to inject a lymph node locator without the need for surgery, as it may be very difficult to predict the position of such a metinel lymph node, and there by difficult to predict the place in the body to perform the surgery to remove the metinel node. In a specific embodiment of the invention, the lymph node locator is a radioactive substance, such as, e.g. technetium-99m, which may be injected by a non-surgical procedure, and later imaged by performing a lymphoscintigraphy. Some times the identification of the metinel lymph nodes may involve injection of lymph node locators by a combination of a non-surgical and a surgical step. As an example of this a radioactive lymph node locator, such as, e.g., technetium-99m may be injected using a needle, i.e. without the need for surgery, and the accumulation of the locator, i.e. identification of the metinel node(s) may be performed using a gamma detector. This gives the surgeon an indication towards where the metinel nodes are located. Later on, when the patient is undergoing surgery to have the metinel lymph nodes and at least part of metastasis removed, a coloured dye such as, e.g., Patent Blue Dye may be injected. Furthermore, if there is a lapse of more than about 18 to 24 hours after the first injection, it might be beneficial to add one or more extra injections with radioactive tracer dependent on the half-life of the radioactive tracer (usually about 6 hours) in order to identify the metinel nodes during surgery.

After having located the one or more sentinel or metinel lymph nodes by one or the other method, the surgeon will remove these in order to investigate whether the sentinel or metinel lymph nodes contain any tumour cells, and in order to obtain a culture of tumour-reactive T-lymphocytes.

The harvesting of lymphocytes from the one or more sentinel or metinel lymph nodes may be performed by homogenizing the sentinel or metinel lymph node material in order to obtain single cell suspensions of lymphocytes. The single cell suspensions may then be subjected to in vitro expansion in order to obtain tumour-reactive T- lymphocytes.

In vitro expansion

The in vitro expansion step iv) of the method according to the invention comprises i) a first phase of stimulating tumour-reactive CD4+ helper and/or CD8+ T-lymphocytes with tumour-derived antigen together with at least one substance having agonistic activity towards the IL-2 receptor, to promote survival of tumour-reactive CD4+ helper and/or CD8+ T-lymphocytes, and ii) a second phase of activating and promoting growth of tumour-reactive CD4+ helper and/or CD8+ T-lymphocytes, wherein the second phase ii) is initiated when the CD25 cell surface marker (IL-2R marker) is down-regulated on T-lymphocytes.

Phase i) The purpose of the first phase i) is to obtain a culture comprising a substantially high ratio of tumour-reactive CD4+ helper and/or CD8+ T-lymphocytes. The first phase is to be considered a "nursing phase" where the tumour-reactive T-lymphocytes are brought to survive and divide. Depending on the source of the T-lymphocytes (starting material for the in vitro expansion method), they may have phased relatively harsh conditions, such as, e.g., suppression and inhibition by factors secreted by cancer cells.

The starting material for use in the expansion method is a mixture of lymphocytes obtained from lymph nodes draining a metastasis.

The T-lymphocytes to be expanded in culture can be obtained from the subject to be treated, i.e. the resulting specific tumour-reactive T-lymphocytes for administering may be autologous. However, the T-lymphocytes can also be obtained from a source other than the subject to be treated, such as, e.g. another subject suffering from a cancer. In such case the recipient and the expanded tumour-reactive T-lymphocytes are preferably immunologically compatible (or the recipient is otherwise made immuno- tolerant of the expanded tumour-reactive T-lymphocytes).

The starting material will most likely comprise a mixture of various lymphocytes, such as, e.g., T-lymphocytes, B-lymphocytes, antigen presenting cells, tumour-reactive T- lymphocytes and non-activated/non-reactive T-lymphocytes. In order to promote survival specifically of the tumour-reactive T-lymphocytes, tumour-derived antigen and one or more substances having agonistic activity towards the IL-2 receptor are added.

As mentioned above the first phase i) is initiated by adding at least one substance having agonistic activity towards the IL-2 receptor. The function of such substances is to stimulate T-lymphocytes via the IL-2 receptor to promote cell division of T- lymphocytes, thereby preventing cell death.

Antigen specific MHC restricted activation of T-lymphocytes promotes clonal expansion of the useful T-lymphocyte population specific for the recognition of tumour cells. On the contrary, unspecific activation of T lymphocytes will lead to the expansion of T lymphocyte clones recognizing irrelevant peptides without any relation to the recognition of tumour cells, thus the majority of unspecifically expanded T lymphocytes will not recognize the tumour. The invention aims to promote specific activation and growth of tumour-reactive CD4+ helper and CD8+ T-lymphocytes. A specific activation against a certain tumour antigen enables the T-lymphocytes to have therapeutic effect when administered to a cancer patient with the same tumour type as the T-lymphocytes are activated against.

In one embodiment of the invention the substances having agonistic activity towards the IL-2 receptor are agonists. Examples of such substances include proteins, polypeptides, peptides, antibodies, affibodies, and fragments thereof, fusion proteins, synthetic and/or organic molecules, such as, e.g., small molecules, and natural ligands. In a preferred embodiment the substance is the natural ligand of the IL-2 receptor, namely IL-2.

If IL-2 is used it is preferentially added in a low dose in order to reduce lymphocyte apoptosis and to increase the population of CD4 positive tumour-reactive T- lymphocytes. In a specific embodiment of the invention, the low dose of IL-2 is from about 100 IU/ml culture medium to about 700 IU/ml culture medium, such as, e.g., from about 100 IU/ml culture medium to about 600 IU/ml culture medium, from about 100 IU/ml culture medium to about 500 IU/ml culture medium, from about 100 IU/ml culture medium to about 400 IU/ml culture medium, from about 100 IU/ml culture medium to about 300 IU/ml culture medium and from about 100 IU/ml culture medium to about 200 IU/ml culture medium. In a specific embodiment, the amount of IL-2 added is 240 IU/ml.

In case other substances, than IL-2, having agonistic activity towards the IL-2 receptor are used the specific doses of these should be such that lead to an effect corresponding to the effect obtained by the above-mentioned doses of IL-2.

A further amount of the at least one substance having agonistic activity towards the IL- 2 receptor may be added regularly throughout phase i), such as, e.g., every 2^nd, 3^rd or 4^th day of phase i), in order to maintain optimal conditions for promoting cell division. By the term every 2^nd, 3^rd or 4^th is intended to mean that at least one substance having agonistic activity towards the IL-2 receptor is added throughout phase i) every 2^nd, 3^rd or 4^th day, starting at the 2^nd, 3^rd or 4^th day after the first addition of the at least one substances having agonistic activity towards the IL-2 receptor, i.e. after initiating phase i). In one embodiment the substance to be added regularly throughout phase i) is an agonist of IL-2. In a preferred embodiment the substance is IL-2.

The further dose of substances having agonistic activity towards the IL-2 receptor, such as, e.g., IL-2, to be added regularly, such as, e.g. every 2^nd, 3^rd, or 4^th day lies within the ranges mentioned above.

A further important step in the first phase i) of expansion is the addition of tumour- derived antigen in order to promote cell division of T-lymphocytes expressing T lymphocyte receptors recognizing tumour antigens, i.e. tumour-reactive T-lymphocytes.

The optimal point of time to add the tumour-antigen is depending on the source of lymphocytes. When the lymphocytes originates from lymph nodes the lymphocytes may have been subjected to close proximity and immuno-suppression by tumour cells, and need incubation with a substance having agonistic activity towards the IL-2 receptor, such as, e.g., IL-2 for some days in order to promote the ability of the T- lymphocytes to respond with proliferation upon tumour antigen presentation. Accordingly, in such case the tumour-derived antigen is preferentially added from day 2 to and including day 5 of the first phase i), such as, e.g., on day 2, on day 3, on day 4 or on day 5.

The tumour-derived antigen, such as, e.g., a tumour homogenate, is likely to be endocytosed and processed by antigen presenting cells present in the starting material, such as, e.g., B-lymphocytes, dendritic cells and macrophages. In most cases the tumour-derived antigen will be presented by class Il MCH molecules leading to cell division of CD4⁺ tumour-reactive T-lymphocytes. However, by cross presentation antigens taken up by endocytosis may be processed and presented in the class I pocket resulting in activation of CD8⁺ T lymphocytes. As stated above, one of the objects of the expansion method is to in some respect imitate the natural pathway of the patients own immune system, and to a certain degree let the components of the patients immune system determine whether CD4⁺ or CD8⁺ lymphocytes are generated, depending on whether antigen is presented by MCHI or MCHII. In most cases, the antigens will be presented by the class Il MCH molecule leading to generation of CD4⁺ T-lymphocytes, however, in some cases CD8⁺ T-lymphocytes are generated.

Phase H) The purpose of the second phase ii) is to activate and expand the tumour-reactive CD4+ helper and/or CD8+ T-lymphocytes obtained by phase i) and to obtain a specific sub-population of tumour-reactive CD4+ helper and/or CD8+ T-lymphocytes by directing them into a desired pathway.

The present inventors have found, that one way of determining the optimal point in time to initiate phase ii) is by monitoring the expression of the CD25 cell surface marker on the T-lymphocytes, in order to determine specifically when the T-lymphocytes are susceptible to re-stimulation. The present inventors have found that the second phase ii) should preferably be initiated when the expression of CD25 on T-lymphocytes is down-regulated. CD25 is an activation marker, indicating that the lymphocytes have received an activating signal. If the second phase is initiated when the expression of CD25 on the T-lymphocytes is high, meaning that the lymphocytes have already received a signal, cell death would occur.

The down-regulation of CD25 is defined as that a substantial part of the T-lymphocyte population express very few or essentially none CD25 markers. In a preferred embodiment the down-regulation of CD25 is defined as that less than 5% of the T- lymphocyte population expresses CD25, i.e. 95% or more of the T-lymphocytes in the culture does not express CD25 at all. The 5% or less of the T-lymphocytes expressing CD25 is most likely regulatory CD4+ T-lymphocytes which have a high permanent expression of CD25. In addition the T-lymphocyte population should preferably express very few or essentially none FoxP3 markers, which are specific markers of regulatory T-lymphocytes. In a preferred embodiment the down-regulation of FoxP3 is defined as that less than 5% of the T-lymphocyte population expresses FoxP3, i.e. 95% or more of the T-lymphocytes in the culture do not express FoxP3 at all.

Besides CD25, there are also other markers, the expression of which is relevant to monitor in order to determine the optimal point in time to initiate the second phase. Examples of such markers are the early activation marker CD69, and MCHII, which is an activation marker for T-lymphocytes. As the expression of CD69 and MCHII indicates that the "activation program" of the T-lymphocytes is already turned on, meaning that the cells are not able to respond to additional stimuli, both of these markers should preferably be down-regulated before the second phase is initiated. The term down regulation may be defined as that less than 5-10% of the T-lymphocyte population expresses CD69 and/or MCHII. In another embodiment of the present invention, anti-CD4 antibodies are used to separate T helper cells from possible tumour cells in the culture in the expansion in phase ii) of the expansion method.

In a further or yet another embodiment of the present invention, products such as Dynabeads® with anti-CD3 and anti-CD28 antibodies are used to promote the expansion in phase ii) of the expansion method. Use of Dynabeads® CD3/CD28 will provide lymphocytes with activation signals and could also be used for separation from possible tumour cells in the culture. Dynabeads® CD3/CD28 will bind to T lymphocytes expanded antigen specifically during phase i), where these cells now can be enriched magnetically. Since the initial antigen specific activation has initiated and led to clonal T lymphocyte expansion the Dynabeads® CD3/CD28 restimulation will further promote clonal expansion since phase i) does not support activation of unspecific T lymphocyte clones.

Even though the exact starting point of phase ii) will vary depending on when the lymphocytes has acquired the preferred expression of specific markers, the second phase ii) is most often initiated from day 17 to and including day 23 of the first phase i), such as, e.g. on day 17, on day 18, on day 19, on day 20, on day 21 , on day 22 or on day 23. In other words, the point in time, where the lymphocytes expresses the preferred amount and combination of markers, is most often seen as being from day 17 to day 23 of the first phase i).

The expansion of the T-lymphocytes, i.e. phase i) and ii) will most often take place in a suitable culture medium. Preferably a serum-free medium or autologous serum is used in order to avoid the risk of transmitting diseases to the patient. Examples of suitable standard media include AIMV medium, RPM1 1640, DMEM and MEM. However, other media may also be used, comprising a suitable blend of amino acids, steroids, vitamins, growth factors, cytokines and minerals.

During the two phases of the expansion, the cells may be split into several culture vessels in order to maintain a suitable cell density in the cultures. The density of the T- lymphocytes in the expansion phases should preferably be from about 3 to about 6 million cells/ml of culture medium. During expansion an exchange of culture medium with fresh medium, a step, which is denominated conditioning of the medium, may also be needed. The point of time to split cultures and to condition the medium may be determined based on the morphology of the cells and the cell culture density (which should not exceed about 6 million cells/ml), or the medium may contain a suitable indicator, such as, e.g., a phenol indicator. In case an indicator is included in the medium, the point of time to split cultures or condition medium may be based on the color of the medium. If a phenol red indicator is used, the cells should be split or conditioned, when the medium turns yellow, indicating that the pH of the culture is turning acidic. A suitable schedule for conditioning the medium used in the present invention may be to exchange from 1/4 to 1/2, such as, e.g., 1/3 of the medium every 3-9 days, such as, e.g. once a week.

Except for the specific conditions mentioned herein, for other parameters standard conditions for growth of lymphocyte cultures will be used, such as, e.g. a temperature of 37°C and 5% CO₂.

As mentioned above, the second phase ii) is initiated by the addition of tumour-derived antigen as defined above to the T-lymphocytes for activating the tumour-reactive CD25-negative T-lymphocytes, in order to promote clonal expansion of tumour-reactive T-lymphocytes.

In a specific embodiment of the invention antigen presenting cells (APCs) are added to the T-lymphocytes together with the tumour-derived antigen. Antigen presenting cells (APCs) include leukocytes such as, e.g., monocytes, macrophages and lymphocytes, such as, e.g., B cells. These diverse cell types have in common the ability to present antigen in a form that is recognized by specific T lymphocyte receptors. The leukocyte preparation is isolated from, for example, blood, lymph fluid, bone marrow, lymphatic organ tissue or tissue culture fluid obtained from the patient to be treated. In a preferred embodiment the APCs cells are irradiated peripheral blood leucocytes containing antigen-presenting B-cells and/or monocytes. The amount of APCs added lies within the range of from about 0.5 million APCs/ml lymphocyte culture to about 5 million APC/ml lymphocyte culture, such as, e.g., from about 1 million APCs/ml lymphocyte culture to about 4 million APC/ml lymphocyte culture, from about 1 million APCs/ml lymphocyte culture to about 3 million APC/ml lymphocyte culture, or from about 1 million APCs/ml lymphocyte culture to about 2 million APC/ml lymphocyte culture. Besides the addition of tumour-derived antigen to the T-lymphocytes in order to promote clonal expansion of tumour-reactive T-lymphocytes, the second phase ii) comprises the addition of specific components the function of which are to direct the expansion of the tumour-reactive T-lymphocytes towards the desired sub-population.

As mentioned above, the present invention provides a method for the generation of tumour-reactive CD4+ T-lymphocytes. CD4+ T-lymphocytes recognizes and binds tumour antigen when the antigen is associated with a major histocompatibility complex class Il molecule. Activated CD4+ T lymphocytes secrete cytokines, proteins and/or peptides that stimulate other cells of the immune system, such as other lymphocytes. The most common cytokine secreted is interleukin-2 (IL-2), which is a potent T lymphocyte growth factor. Activated, proliferating CD4+T-lymphocytes can differentiate into two major subtypes of cells, Th1 and Th2 cells, which are defined on the basis of specific cytokines produced. TM cells produce interferon-gamma and interleukin 12 (IL-12), while Th2 cells produce interleukin-4, interleukin-5 and interleukin-13. Th1 T- lymphocytes are believed to promote activation of cytotoxic T lymphocytes (Tc), NK cells, macrophages, and monocytes, all of which can attack cancer cells and generally defend against tumours.

T-helper (CD4+) lymphocytes of type Th1 and Th2 can differentiate into memory cells and effector cells. Memory T-helper (CD4+) lymphocytes are specific to the antigen they first encountered and can be called upon during a secondary immune response, calling forth a more rapid and larger response to the tumour-antigens. There is evidence in humans that lymphocytes survive at least 20 years; perhaps for life.

Effector CD4+ T-lymphocytes are active cells producing cytokines and INF-gamma.

For an effective treatment of cancer, administration of tumour-reactive T-lymphocytes of the Th1 type is especially beneficial, as this type is believed to promote activation of cytotoxic T lymphocytes (Tc), NK cells, macrophages, and monocytes, all of which can attack cancer cells and generally defend against tumours. I.e. in a specific embodiment the invention relates to a method for generating tumour-reactive CD4+ T-lymphocytes, and in a further embodiment, the percentage of T-lymphocytes of the Th2 type generated by the present method is 30% or less, such as, e.g., 25% or less, 20% or less, 15% or less, 10% or less, 5& or less or 0%, i.e. at least 70% of the tumour- reactive CD4+ T-lymphocytes are of the Th1 type, such as, e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100%.

Accordingly, the second phase may comprise the addition of a substance capable of up-regulating IL-12R on the T-lymphocytes. Up regulation of the IL-12R will increase the readiness of the T cell to receive and optimize the IL-12 cytokine activation resulting in maximal STAT-4 signaling and thus skewing the lymphocytes towards Th1 cells and IFN-γ production.

The substance(s) capable of up-regulating IL-12R on the T-lymphocytes may be substance(s) having agonistic activity towards an interferon receptor. In one embodiment of the invention the substances having agonistic activity towards the interferon receptor are agonists. Examples of such substances include proteins, polypeptides, peptides, antibodies, affibodies, and fragments thereof, fusion proteins, synthetic and/or organic molecules, such as, e.g., small molecules, and natural ligands. In a specific embodiment the substance is the natural ligand of the interferon receptor, namely an interferon, such as interferon-α.

The optimal point of time to add the substance(s) capable of up-regulating IL-12R on the T-lymphocytes, such as, e.g. a substance having agonistic activity towards an interferon receptor may be determined by measuring the level of IL-12 in the culture medium. The substance(s) should preferably be added when the level of IL-12 is at least 1 fold, such as, e.g., at least 2, at least 3 fold, at least 4 fold, or at least 5 fold increased as compared to the level of IL-12 on day 1 of phase ii). In most cases, such an increase in the level of IL-12 will be seen from day 2 to and including day 4 after initiating the second phase ii), such as, e.g. on day 2, on day 3 or on day 4.

In order to substantially avoid the generation of tumour-reactive T-lymphocytes of the Th2 type, the second phase may further comprise the addition of one or more substances capable of antagonizing development of Th2 type T-lymphocytes.

Examples of such substances are substances capable of neutralizing the interleukins IL-4, IL-5, IL-10, and/or TGF-beta (the latter not being an interleukin) all four of which are required for the establishment of the Th2 cytokine profile and for down regulation of Th1 cytokine production. Examples of such substances include proteins, polypeptides, peptides, soluble receptors, antibodies, affibodies, and fragments thereof, fusion proteins, synthetic and/or organic molecules, such as, e.g., small molecules, and natural ligands. In a specific embodiment the substances are selected from antibodies that binds to the interleukins, thereby neutralizing them, such as, e.g. anti IL-4 antibody, anti IL-5 antibody and/or anti IL-10 antibody, together with soluble receptors (such as, e.g. TGF- beta receptor I and II) and binding proteins for TGF-beta (such as, e.g. LAP and/or LTBP).

The one or more substances capable of antagonizing development of Th2 type T- lymphocytes, such as, e.g., one or more substances capable of neutralizing IL-4, IL-5, IL-10 and/or TGF-beta may be added on day 1 of the second phase ii). However, as antibodies are expensive, the addition of antibodies can also be performed in a subsequent step after addition of the substance capable of up-regulating IL-12R on the T-lymphocytes, such as, e.g., one day, two days or three days after addition of the substance capable of up-regulating IL-12R on the T-lymphocytes.

The neutralizing substances should be added in an amount sufficient to neutralize the interleukins, such as, e.g., in a 10-100 fold (molar) excess of the amount of interleukin to be neutralized. When using antibodies, a final concentration of from about 2 to about 4 ng/ml culture medium will normally be needed. For other types of neutralizing substances, a final concentration, giving the same effect as the concentration mentioned for antibodies, should be used.

In order to maintain the suppression of the development of Th2 type T-lymphocytes a further amount of the one or more substance capable of antagonizing development of Th2 type T-lymphocytes, such as, e.g., one or more substance capable of neutralizing IL-4, IL-5, IL-10 and/or TGF-beta may be added regularly throughout phase ii), such as, e.g. every 2^nd, 3^rd or 4^th day of phase ii). It is to be understood that by the term every 2^nd, 3^rd or 4^th is intended to mean that at least one substance capable of antagonizing development of Th2 type T-lymphocytes is added throughout phase i) every 2^nd, 3^rd or 4^th day, starting at the 2^nd, 3^rd or 4^th day after the first addition of the at least one substance capable of antagonizing development of Th2 type T-lymphocytes.

Furthermore, as for phase i) a further amount of a substance having agonistic activity towards the IL-2 receptor, such as, e.g., an agonist may be added regularly throughout phase ii) such as, e.g., every 2^nd to 4^th day of phase N), i.e. on the 2^nd, 3^rd or 4^th day in order to maintain optimal conditions promoting cell division. The dose of the substance to be added regularly lies within the optimal ranges mentioned under phase i) for addition of substances having agonistic activity towards the IL-2 receptor, such as, e.g., IL-2.

In order to favour the generation of Th1 type tumour-reactive T-lymphocytes, the second phase ii) may comprise adding one or more substances promoting the development of Th1 type T-lymphocytes. Examples of such substances are substances having agonistic activity towards the IL-7, IL-12, IL-15 and/or IL-21 receptor. More specific, the substances may be agonists for the IL-7, IL-12, IL-15 and/or IL-21 receptor. Examples of such agonists include proteins, polypeptides, peptides, antibodies, affibodies, and fragments thereof, fusion proteins, synthetic and/or organic molecules, such as, e.g., small molecules, and natural ligands. In a specific embodiment the substances are the natural ligands of the IL-7, IL-12, IL-15 and/or IL- 21 receptor, respectively, such as IL-7, IL-12, IL-15 and/or IL-21.

The effect of IL-12 is activating the IFN-gamma inducing STAT pathway by stimulating the IL-12R thereby promoting activation of Th1 lymphocytes. The function of IL-21 is to enhance proliferation, activation and development towards a Th1 type of T- lymphocytes.

Both IL-7 and IL-15 work by promoting homeostatic expansion of the T-lymphocytes, enhancing the enumeration of activated Th1 programmed T-lymphocytes.

The optimal point of time to add one or more substances promoting development of Th1 type T-lymphocytes is when the T-lymphocytes are susceptible to modification. If the substances are added when the T-lymphocytes are not susceptible to modification, the addition will have no effect, i.e. the development of Th1 type T-lymphocytes will not be favoured. In order to determine the optimal point in time for adding substances promoting development of Th1 type T-lymphocytes, such as, e.g., substances having agonistic activity towards the IL-7, IL-12, IL-15 and/or IL-21 receptor, the production of INF-Y by the T-lymphocytes, may be monitored. In a preferred embodiment, the one or more substances promoting the development of Th1 type T-lymphocytes, such as, e.g., substances having agonistic activity towards the IL-7, IL-12, IL-15 and/or IL-21 receptor should be added when the level of IFN-gamma is increased as compared to the level of IFN-gamma on initiation of second phase ii).

In a specific embodiment, the increase in IFN-gamma level may be determined as at least a 1 fold increase in IFN-gamma level, such as, e.g., at least a 2 fold, at least a 3 fold, at least a 4 fold increase as compared to the level of IFN-gamma on initiation of the second phase ii). Often will such an increase can be correlated to that the content IFN-gamma in the culture medium should be at least 100 picogram/ml culture medium, such as, e.g. at least 150 picogram/ml culture medium, at least 200 picogram/ml culture medium or at least 250 picogram/ml culture medium.

When determining the optimal point in time to add substances promoting development of Th1 type T-lymphocytes, such as, e.g., substances having agonistic activity towards the IL-7, IL-12, IL-15 and/or IL-21 receptor, one may further look at the expression of the activation markers CD25 and CD69 on CD4+ T-lymphocytes, which markers should preferentially be up-regulated. By up-regulation is understood that at least about 40% to about 60% or more of the CD4+ T-lymphocytes should express CD25 and CD69 as compared to the expression of CD25 and CD69 on T-lymphocytes on day 1 of phase ii), showing that the T-lymphocytes have received an activating signal.

Normally the optimal point of time for adding the substances promoting development of Th1 type T-lymphocytes will fall subsequent to the steps of adding the substances capable of up-regulating IL-12R on the T-lymphocytes and the substances capable of antagonizing development of Th2 type T-lymphocytes. More specific the optimal point in time to add the substances promoting development of Th1 type T-lymphocytes will fall between day 5 to day 8 after initiating the second phase ii), such as, on day 5, day 6, day 7 or day 8.

In case IL-7, IL-12, IL-15 and/or IL-21 are added the concentration of each of these substances in the culture medium should lie within the range from about 150 IU/ml culture medium to about 300 IU/ml culture medium, such as, e.g. 250 IU/ml culture medium. When other substances than the specific ones mentioned is used, they should be added to the culture in final concentration, which leads to the same effect as the addition of IL-7, IL-12, IL-15 and/or IL-21 within the specific ranges mentioned will give. As mentioned above, the present method if preferentially used for the expansion of T- lymphocytes in order to achieve CD4+ tumour-reactive T-lymphocytes of the Th1 type. One further aspect of the invention is that by using the method described herein for expanding tumour-reactive T-lymphocytes, a relatively high amount of T-lymphocytes of the memory type will be obtained. In treating cancer it is of course important that the patient to be treated receive a high amount of effector tumour-reactive CD4+ T- lymphocytes, as these - as mentioned above - promote activation of cytotoxic T lymphocytes (Tc), NK cells, macrophages, and monocytes, all of which can attack cancer cells and generally defend against tumours.

However, by at the same time administering a substantial amount of memory tumour- reactive CD4+ T-lymphocytes, the patient achieve up to life long protection towards recurrence of the tumour or metastasis of the primary tumour.

Accordingly, the present invention relates to a method for the preparation of memory T- lymphocytes. Normally, when a culture of tumour-reactive T-lymphocytes are expanded according to the present invention from about 35% to about 90% of tumour-reactive T- lymphocytes of the memory type, such as, e.g. from about 40% to about 90%, from about 50% to about 80% or from about 60% to about 70%, will be obtained. The present inventors speculates that the fact that the lymphocytes in phase i) are allowed to regenerated before tumour antigen is added, together with the relatively slow expansion phase leads to formation of a high ratio of memory lymphocytes to effector lymphocytes.

As mentioned above the expression of the cell surface activation markers CD25 and CD69 on the T-lymphocytes may be used for determining when to initiate important steps of the present method, such as, e.g., when to initiate the second phase ii). Accordingly, it may be beneficial to continuously monitor the expression of CD25 and CD69 throughout phase i) and phase ii), such as, e.g., every 2^nd, every 3^rd or every 4^th day.

As one of the purposes of the present method is to obtain a high number of specific CD4+ tumour-reactive T-lymphocytes, which may be used for administering to a patient, the tumour-reactive T-lymphocytes may be harvested at some point, leading to the termination of the expansion step. The optimal point of time to harvest the tumour- reactive T-lymphocytes is when the expression of CD25 on the T-lymphocytes is down- regulated, where the down-regulation is defined as that 5% or less of the CD4+ T- lymphocyte population expresses CD25. The optimal point in time to harvest may also be determined based on measurement of the amount of IFN-gamma produced. The IFN-gamma production should be at least 2 fold increased, such as, e.g., at least 3 fold, at least 4 fold or at lest 5 fold increased as compared to initial IFN-gamma production, which normally correspond to a level of IFN-gamma of at least 100 pg/ml of culture medium.

Normally, this event will occur from day 10 to and including day 14 after initiating the second phase ii), i.e. normally the cells will be harvested from day 10 to and including day 14 after initiating the second phase ii).

Accordingly, the entire process for expansion of tumour-reactive T-lymphocytes according to the invention may in general take from about 25 days to and including about 45 days, such as, e.g. from about 26 days to and including about 44 days, from about 27 days to and including 43 days, from about 27 days, to and including 42 days, from about 27 days to and including 41 days, and from about 27 days to and including about 40 days.

Instead of harvesting the tumour-reactive T-lymphocytes when the CD25 marker is down regulated, they may be subjected to one or more additional rounds of phase ii). This could be beneficial to do if the amount of tumour-reactive T-lymphocytes obtained by the expression method is not considered an effective amount to be administered to a patient suffering from cancer, or if the patient is in a chemo-therapy treatment regimen, where it may be considered beneficial to postpone the administration of T- lymphocytes until the chemo-therapy treatment is finished. In order to determine whether the tumour-reactive T-lymphocytes should be subjected to one or more additional rounds of phase ii) one may look at the level of IFN-gamma produced, and/or the total number of tumour-reactive T-lymphocytes obtained and/or the expression of CD25. In the case, the IFN-γ levels is 30 pg/ml culture medium or less, such as, e.g. 20 pg/ml culture medium or less, and/or the total number of T cells are unsatisfactory, additional rounds of phase ii) may be initiated beginning when the majority of T cells are CD25 negative (i.e. less than 5% of the T-lymphocytes population express CD25) and thereby susceptible to restimulation. After harvest the tumour-reactive T-lymphocytes may be purified by any conventional means, such as, e.g. by using density gradient, such as, e.g., a Ficoll medium. A portion of the tumour-reactive T-lymphocytes may be stored by freezing in a suitable freezing medium after harvesting and purifying the tumour-reactive T-lymphocytes.

Administration

The tumour-reactive T-lymphocytes obtained by an improved expansion method are used herein for treating patients suffering from colon cancer or metastasized colon cancer.

The definition of an effective amount of tumour-reactive T-lymphocytes to be administered is depending on the specific type of lymphocytes, the ratio of memory to effector T-lymphocytes and on the severity of the disease. However, in average a minimum of at least 10 million, such as, e.g. at least 20 million, at least 30 million, at least 40 million, at least 50 million, at least 60 million, at least 70 million or at least 80 million tumour-reactive T-lymphocytes may be administered. The present inventors have not identified any upper limit with respect to the amount of tumour-reactive T- lymphocytes to be administered in a single dose.

In a preferred embodiment the tumour-reactive T-lymphocytes for administration comprises a combination of effector T-lymphocytes and memory T-lymphocytes. More specific the amount of tumour-reactive T-lymphocytes of the memory type may be from about 35% to about 90%, such as, e.g. from about 40% to about 90%, from about 50% to about 80% or from about 60% to about 70%, and a percentage of effector T- lymphocytes from about 10% to about 65%, such as, e.g., from about 20% to about 50% or from about 30% to about 40%.

The tumour-reactive T-lymphocytes may be formulated as a pharmaceutical composition suitable for parenteral administration to the patient such as, e.g., intravenous, intraarterial, intrathecal, or intraperitonal administration.

When the tumour-reactive T-lymphocytes are administered parenterally, they may be formulated in an isotonic medium, i.e. in a medium having the same tonicity as blood, and comprising one or more substances preventing aggregation of the cells. A specific example of a suitable medium is a 0.9% NaCI solution comprising up to 3% human serum albumin such as, e.g. up to 2% human serum albumin or up to 1% human serum albumin. For intravenously administration the concentration of tumour-reactive T-lymphocytes in the composition to be administered normally lies within the range from about 0.5 million lymphocytes/ml medium to about 4 million lymphocytes/ml medium, such as, e.g., from about 0.5 million lymphocytes/ml medium to about 3 million lymphocytes/ml medium, from about 0.5 million lymphocytes/ml medium to about 2 million lymphocytes/ml medium or from about 1 million lymphocytes/ml medium to about 2 million lymphocytes/ml medium.

The composition comprising tumour-reactive T-lymphocytes may be administered as a single dose or multiple doses. It may be infused over 1 to 2 hours.

The treatment method may be performed once or repeated depending on the severity of the disease. Furthermore, the treatment may be reiterated upon recurrence of the disease.

The treatment according to the present invention may be supplemented with any other relevant treatment for colon cancer. Such supplemental treatment may be given before, at the same time or after the administration of the lymphocytes and it may be given at frequencies normally used for such treatments. A suitable example of supplemental treatment is chemotherapy and the like.

Kits

The invention further relates to kits for use in a method according to the invention, the kit comprising a medium for cultivation of T-lymphocytes. The medium may be any suitable serum-free medium, such as, e.g., AIMV, RPMI 1640, DMEM or MEM.

The kit may further comprise one or more substances for stimulating, activating and directing tumour-reactive T-lymphocytes. Examples of such substances may be tumour-derived antigen, substances having agonistic activity towards the IL-2 receptor, substances capable of up-regulating IL-12R on the T-lymphocytes, substances capable of antagonizing development of Th2 type T-lymphocytes and/or substances promoting the development of Th1 type T-lymphocytes.

More specific, such substances may be IL-2, interferon-alpha, anti-IL-4 antibody, anti- IL-5 antibody, anti-IL-10 antibody, IL-7, IL-12, IL-15 and/or IL-21. The kit may also comprise a pharmaceutical composition suitable for intravenous administration. The pharmaceutical composition may be mixed with the population of tumour-reactive T-lymphocytes before administration.

The invention also relates to a kit for identification of sentinel or metinel lymph nodes, the kit comprising one or more syringes and a lymph node locator. In one embodiment, the syringes are prefilled with a lymph node locator.

The kits may also comprise instructions for use, such as, e.g. instructions in the form of computer software.

Figure legends

Figure 1 illustrates that the sentinel node is the natural primary site for the presentation and activation of T cell reactivity towards tumour antigen.

Figure 2 shows that initially sentinel node lymphocytes are activated with tumour antigen and low dose IL-2 resulting in activation and expression of the activation marker CD25 (Top panel). The end of phase I activation phase is defined by the decreased number of CD4⁺ T cells expressing CD25 (Bottom panel). When less than 5% of the CD4⁺ T cells express CD25 phase Il is initiated with restimulation with antigen.

Figure 3 illustrates that Phase I and Phase Il activation results in expansion and enrichment of CD4⁺ T helper cells.

Figure 4 illustrates that in Phase I the majority of cells are naive CD62L+ cells or activated CD69+CD62L+ cells. After Phase Il the majority of the cells are CD62L- and are composed of memory and effector CD4+ T helper cells. CD62L- T cells are not expressing the preferred lymph node homing molecule, thus they are seeking inflammatory areas in non-lymphatic organs.

Figure 5 shows primary cells stimulated in Phase I from the tumour (Tumour infiltrating lymphocytes), sentinel nodes (SN) and an irrelevant lymph node (LN) results in no little IFN-γ production. Figure 6 illustrates that after expansion after phase ii) there is a dose dependent increase in antigen dependent IFN-γ production.

Figure 7 illustrates that the expansion and activation protocol promotes the expansion of antigen specific T cell clones as investigated by the selective enrichment of TCR Vβ expression.

Figures 8 A-D are CT scans of patient # 5. After transfusion of tumour-reactive lymphocytes the patient had total regress of liver metastases located in both lobes (which had been declared incurable by liver surgery), normalisation of CEA levels, disappearance of ascites and was physically well fit, exercising regularly.

Figures 9 A-F are CT scans of patient # 10. After transfusion the patient had regress of liver metastases and ascitic fluid. He was in fairly good health and further imaging showed stable disease.

Figures 10 A-H are CT scans of patient # 12. Three months after transfusion he had regress of metastases in the liver and lungs with almost a normalised CEA level at 5.9 (Normal < 4.0), disappearance of ascites and he appears clinically healthy.

Figure 11 is dose-dependent responses in the Dukes' D patients. Complete response was seen in four of the nine Dukes' D patients. These patients were transfused with a significantly (p<0.05) larger number of cells (average 140 million cells), then patients with partial response or stable disease (average 36 million cells).

The following examples tend to illustrate the invention without limiting it in any way.

Examples

Example 1

Treatment of patients being diagnosed with colon cancer or metastatic colon cancer

Methods

Identification and removal of sentinel and metinel lymph nodes from colon cancer patients: Sixteen patients diagnosed with colon cancer, six woman and ten men with an average age of 62 years were studied. Patients were histopathologically classified as Duke's C or D. There were also 5 patients with Duke's B with aggressive tumour characteristics such as ulcerations, vascular or perineural invasion. Patients 7 and 14 however had earlier been surgically treated due to colon cancer and now had recurrent disease with metastases to the liver. The local ethical committee approved the study and each patient gave informed consent.

Identification of sentinel or metinel nodes was done intraoperative^ using the sentinel node technique described herein. Mobilisation of the colonic tumour site was achieved by division of peritoneal adhesions in order to facilitate inspection of tumour and mesentery. Injections of Patent blue dye (Guerbet, Paris) were distributed superficially in the serosa around the tumour. Within five minutes, one to three mesenteric lymph nodes were coloured blue, these sentinel nodes were marked with sutures and removed when the resection was complete. One non-sentinel mesenteric lymph node, distant from the tumour, was handled the same way.

The sentinel- and non-sentinel lymph nodes were cut in half and 1 mm thick slices were taken from the center and the periphery. The rest of the lymph nodes were sent for histopathological examination according to routine procedure. A piece of the tumour, including a part of the invasive margin, was used for antigen preparation.

Expansion of tumour-reactive T-lymphocytes Cell culture Phase I, initial activation

The node material was kept on ice and immediately taken care of using AIM V® Media (Invitrogen) at all times. Single cell suspensions of sentinel or metinel node lymphocytes was obtained through gentle homogenisation in a loose fit glass homogenisator, and following homogenisation cells were washed twice in medium. The metinel node lymphocytes were put in cell culture flasks at 4 million cells/ ml and interleukin-2 (IL-2) (Proleukin®, Chiron) was added to a concentration of 240 IU/ml medium.

Autologous metastasis extract was prepared by homogenisation with an Ultra Turrax in 5 volumes (w/v) 2 x PBS followed by denaturation for 5 minutes at 97°C. Three to four days after initiation of the cell culture autologous tumour extract was added at a concentration of 1/100. For long-term culture the cells were kept in a cell incubator at 37^"C and 5% CO₂ and 240 IU IL-2/mL media added every 3-4 days.

Phase II, activation and expansion After 18-22 days the cell cultures were monitored for the expression of CD25. When the number of CD25 expressing cells was diminished below 5% the cells were restimulated in Phase Il (Figure 2) by the addition of autologus metastasis extract at a concentration of 1/100. For efficient antigen presentation autologous PBMC were collected using Ficoll-Paque PLUS (Amersham Biosciences, GE Healthcare), radiated with 2500 rad and added to the cell cultures. Three days after restimulation interferon-α (Introna) in cone. 100-500 IU/ml and anti IL-4 antibody was added to a concentration of 2 μg/ml. After 5 to 8 days IL-12 (4 ng/ml) was added to the expansion in order to promote induction of IFN-γ producing Th1 cells.

The day before transfusion to the patient the cell cultures were subject to purification using a Ficoll-Paque PLUS (Amersham Biosciences, GE Healthcare) in order to retrieve the viable cells in the culture. On the day of transfusion the cells were washed twice in Saline solution (Natriumklorid Baxter Viaflo 9 mg/ml, Baxter) and then transferred to a transfer bag containing 100-200 ml of saline solution and 1% Human Serum Albumin (Baxter). Investigations for microbial presence were performed prior to transfusion. Infusions of the cells were performed during 1-2 hours under professional medical supervision.

Immunological evaluation Further immunological evaluation was performed using tritium labelled thymidine incorporation proliferation assays. An aliquot of metinel node lymphocytes was set aside for this purpose, a single cell suspension of non-metinel node lymphocytes was obtained by gentle pressure in a loose fit glass homogenisator and peripheral blood leukocytes were purified by Ficoll-Paque PLUS (Amersham Biosciences, GE Healthcare).

Cells were resuspended and washed twice in RPMI 1640 (Life technologies) containing 2.5% fetal calf serum (FCS) (Life technologies). Finally, cells were resuspended in RPMI 1640 proliferation media containing 10% human AB serum (Sigma), 1% penicillin-streptomycin (Sigma) and 1% glutamine (Sigma). Lymph node cells and purified PBL were used at 3x10⁵ cells/well in a 96-well plate and stimulated with metastasis homogenate diluted 1/100, 1/10 or Con A 10 μg/ml (Sigma) in triplicates. Proliferation was measured on day 5, 6 and 7 by adding 1 μCi of ³H-Thymidine/well (Amersham) 18 hours prior to harvesting. Samples were subjected to scintillation counting.

At the start of cell culture, stimulations of lymph node cells and PBL, for the measurement of IFN-γ secretion, were performed in 96-well plates with 3x10⁵ cells/well in triplicate with tumour homogenate diluted 1/10 and 1/100, or Con A 10 μg/ml (Sigma). The amount of secreted IFN- y was measured with ELISA (Human IFN- Y Duoset, R&D Systems) on culture supernatants in pooled samples of the triplicates (Figure 5). At the end of cell cultures samples of the supernatant was removed and IFN- Y and IL-4 secretion measured in triplicates with ELISA (Human IFN- Duoset and Human IL-4 Duoset, R&D Systems) (Figure 6 A and 6B).

Flow cytometry analyses

Characterisation of cells was performed using flow cytometry initially on cells from the metinel node, non-metinel node, PBMC and from the metastasis. From the metinel node acquired lymphocytes in culture samples were taken every two to three weeks for flow cytometry analyses. Cells were incubated for 30 minutes in PBS supplemented with 2% FCS and 0.05% NaN₃ (FACS buffer) with antibodies against markers for immune cell subpopulations and for lymphocyte activation (Figure 3, 4 and 5). Antibodies conjugated with Fluorescein isothiocyanate (FITC) against the following markers were used: CD69, HLA-DR, CD45RA, CD25, conjugated with phycoerythrin (PE): CD62L, CD19, CD45RO, CD56, conjugated with Peridinin-Chlorophyll-Protein (PerCP): CD8, CD3, conjugated with allophycocyanin (APC): CD4, CD14, CD8.

The Vβ-repertoire was examined using the Beta mark kit (Beckman Coulter), 5x10⁵ cells/tube was stained in 10 μl of the 8 different vials containing mixtures of FITC, PE and dual-colour FITC-PE conjugated TCR Vβ antibodies and with the addition of CD8 PerCP and CD4 APC to each tube. (Figure 7).

Administration of tumour-reactive T-lymphocytes

In average 74.7 million activated and clonally expanded T lymphocytes were administered per patient as a transfusion. No toxic side effects like fever, chills, malaise, severe fluid retention, pulmonary oedema or respiratory distress were observed. Follow-up evaluations

Patients were clinically evaluated according to the regular follow-up program after diagnosis and treatment of colon cancer in Sweden. Usually follow-up visits took place 3, 6, 12 and 18 months after the cell transfusion, including clinical examination and control of CEA levels. All stage III and IV patients were in addition investigated with computer tomography of the thorax and abdomen. The patients were followed at regular visits on average for 13 months (range 5-20), median follow-up time was 13¹/4 months. Out of the 16 patients who had been treated with infusion of autologous lymphocytes eight had known distant metastases at diagnosis. Four patients received their transfusions due to known recurrences and out of them three are still without signs of recurrences. One patient was operated due to a solitary liver metastases and has since then been without relaps. As it appears from Figure 8 and Figure 9, one patient with liver metastases located in both lobes (which had been declared incurable by liver surgery) had total regress of liver metastases after transfusion of tumour- reactive lymphocytes, and furthermore had normalisation of CEA levels, disappearance of ascites and is physically well fit, and exercising regularly. One further patient with liver metastases had regress of liver metastases and ascetic fluid after transfusion (see Figure 10, 11 and 12). One patient had three months after transfusion regress of metastases in the liver and lungs (see Figure 13, 14, 15, and 16) with almost a normalised CEA level at 5.9 (Normal < 4.0), disappearance of ascites and he appears clinically healthy.

Results During the period of October 2003 to March 2005, sixteen patients with colon cancer or solitary colorectal liver metastases were operated on at the South Stockholm General Hospital and included in the study. The primary locations of the tumours were three in caecum, 4 in colon ascendens, 1 in colon descendens, 7 in the sigmoid colon and 1 in rectum. Seven right-sided hemicolectomies, 1 left-sided hemicolectomy, 7 sigmoid resections and 1 rectumamputation were performed. Two patients had earlier been operated on with rectumamputation and sigmoid resection, they now underwent partial liver resections due to liver metastases. One patient had recurrences at two abdominal locations and had earlier been operated due to a tumour in the caecum. At our operation two sentinel nodes draining the metastasis were identified, one in the colonic mesentery and one in the mesentery of the small intestine. An extended resection of the anastomotic ileocolonic region with mesentery was done. In all patients, one to three (average 2.1) sentinel node(s) were identified intraoperatively by peritumoural patent blue injections. Among the patients with primary colonic resection on average 15.8 lymph nodes were retrieved from each specimen. After histopathological investigation of these lymph nodes five patients were classified as Duke's C and 5 patients as Duke^'s B, all of them were classified as high-risk tumours due to growth of tumour cells along nerves and in vessels at pathological anatomical investigation. Five patients had distant metastases and were at time of metastatic resection classified as Duke^'s D. Two patients of them had solitary liver metastases. In addition sentinel nodes were also analysed by FACS (Fluorescens activated cell sorter) and antibodies against cytokeratin 20, which is expressed by colon cancer tumours, for the purpose to detect micrometastases. The cytokeratin 20 assessments of lymph nodes by flow cytometry were in agreement with the pathological anatomical diagnosis (not shown) except in one case where a false negative sentinel node (according to histopathological analysis) was positive in the cytokeratin 20 FACS analysis.

The sentinel node is the first lymph node draining the tumour and is therefore the first site of lymph node metastasis (Dahl et al), but the sentinel node is also the primary site for the activation of the immune system. Tumour cells, debris, necrotic cells and antigen presenting cells accumulate in the sentinel node where presentation, activation and clonal expansion of T cells directed against the tumour occur. The present inventors took advantage of this population of in vivo expanded T cell population of sentinel node acquired lymphocytes for in vitro cell culture, expansion and transfusion.

Sentinel node acquired lymphocytes is a population of T cells activated and clonally expanded against tumour antigens that can efficiently be harvested during the surgical procedure. In contrast to recent immunotherapy trials focusing on cytotoxic T cells, the aim of the present inventors was to create a protocol for in vitro enhancement of the in vivo initiated clonal expansion of T helper cells. T helper cells seem to be necessary for the effective function of cytotoxic T cells and for the creation of memory cells. Furthermore, in a T cell receptor transgenic system targeting an islet cell antigen, the transfusion of Th1 cells was found to be sufficient for the β cell destruction and development of diabetes mellitus (Webb S Nature Immunol). In vitro culture of sentinel node acquired lymphocytes resulted in a Th1 activation and clonal expansion of T helper cells as indicated by the dominant production of the hallmark Th1 cytokine IFN-γ (figure 2) and the enrichment of a restricted TCR Vβ repertoire (figure 2). The tumour homogenate used to expand the T cells is likely to be endocytosed and processed by antigen presenting cells for class Il presentation leading to activation of CD4⁺ T helper cells resulting in expansion favouring T helper cells. By cross presentation antigens taken up by endocytosis may be processed and presented in the class I pocket resulting in activation of CD8⁺cytotoxic T cells. Interestingly, in some cases the inventors found clonal expansion of both CD4⁺ and CD8⁺ T cells.

The average number sentinel node acquired lymphocytes at start of expansion was 107.4 million cells (range 3.6-509 millions, median 70 millions). Cells were characterised by flow cytometry. The ratio between CD4⁺ and CD8⁺ cells at start was in average 4,9 (range 0.36-10, median 5.4) indicating an expansion CD4⁺ T helper cells in sentinel nodes compared to the CD4/CD8 ratio in peripheral blood (normal range 1.0- 2.5) (figure 2A). In addition B lymphocytes (CD 19) and natural killer (NK) cells (CD 56) were present in sentinel nodes (not shown). The cells were held in culture in average 36.1 days (range 23-58 days), median 33 days. Cells were monitored closely by flow cytometry at least weekly. Initially the total number of cells decreased. B cells and NK cells disappeared almost completely and the number of CD8⁺ T killer cells was diminished. The culture procedure used promoted mainly the expansion of T helper cells since the average CD4/CD8 ratio was 92.5. Restimulation with autologous tumour antigen resulted in clonal expansion of tumour reactive T cells as assessed by investigating the T cell receptor Vβ repertoire of sentinel node acquired lymphocytes before and after in vitro culture.

Before transfusion expanded T cells were functionally tested against autologous tumour antigens by measuring activation and cytokine production of the Th1 cytokine IFN-γ and the Th2 cytokine IL-4. In vitro expanded sentinel node acquired lymphocytes responded upon restimulation with tumour antigen with the production of IFN-g and no or very little IL-4 indicating that the expanded T cells were functional and Th1 responsive.

Six patients with Duke's D were treated in the study. Two patients staged as Duke^'s D at surgery with metastases to the liver and to the lungs and liver, respectively displayed marked regression of disease (pat 5 and 12). After transfusion of lymphocytes the first patient had total regress of liver metastases located in both lobes (which had been declared incurable by liver surgery normalisation of CEA levels, disappearance of ascites and appear healthy. Patient 12 shows regress of metastases in the liver and lungs with almost a normalised CEA level at 5.9 (Normal < 4.0), disappearance of ascites and he appears clinically healthy. Patient 1 displayed a regression of the size of liver metastasis, and initially a decrease in CEA levels, disappearance of ascites and she was in excellent shape when she suddenly died (day 191), what appears to have been a lung embolus. Two Duke's D patients display stable disease without progression of metastasis or increase in CEA levels. The oldest patient no 7 in the study displayed stable disease for five months, but thereafter CEA levels started to increase and she died at age 83. No autopsy was performed. One patient was staged as Duke^'s C at surgery but soon developed metastases to the liver and lungs (Duke^'s D), but following transfusion and chemotherapy a regress of the lung and liver metastases were seen with only slightly elevated CEA levels. The patients classified as Duke's C all have normal CEA levels and appear without any signs of radiological or clinical recurrence of disease. Four of the Duke's B patients are healthy with normal CEA levels and have no signs of recurrent disease. Patient no 9 classified as Duke's B, but with an aggressive growing tumour shows signs of recurrent disease with elevated CEA levels (67) and signs of liver metastases.

To investigate the fate of transfused T cells the present inventors analysed T cell proliferation against tumour extract in peripheral blood. As mentioned before, they could not demonstrate any T cell reactivity in peripheral blood against autologous tumour antigens in any of the patients prior to transfusion. However, we were able to detect T cell proliferation against autologous tumour antigens in peripheral blood in all investigated patients up to 10 months after transfusion indicating the presence of clonally expanded circulating tumour-reactive T cells.

Summary of patient characteristics

Below is a table of all participants in the study, sorted after Duke's classification at surgery:

Participant characteristics

Age/ Duke's Infused CD4/ IFN-v Overall survival Response

Sex Classification cells (x10⁶) CD8 ^a (pg/ml) (months)

67/M B 4 92 / 0.2 ND 31 SD 67/F B 8 15 / 51 ND 30 SD

71/M B 50 74 / 15 2091 29 SD

74/M B 63 64 / 22 ND 29 SD

66/M B 152 82 / 1.5 1411 27 SD

64/F C 110 64 / 25 ND 34 SD

58/F C 16 77 / 18 417 23 SD

61/F D 1 3.7 / 35 ND 6 SD

47/M D 80 24 / 16 ND 36 CR

54/M D 40 37 / 24 ND 36 SD

65/M D 270 82 / 15 ND 36 CR

42/F D 80 66 / 11 ND 33 CR

82/F D 40 98 / 0.1 ND 6 SD

74/M D 130 73 / 22 142 30 CR

33/M D 72 72 / 1.5 908 12 PR

66/M D 25 37 / 27 764 26 PR ^a) The numbers represent the percentage of CD4 and CD8 positive cells detected with FACS.

Discussion

To the knowledge of the present inventors, sentinel or metinel node-based immunotherapy in patients with colon cancer has never been presented before. Thus, this is the first attempt to use lymphocytes acquired from sentinel or metinel nodes for therapy. There are some major differences between the present study and e.g. treatment with high-dose IL-2 (Rosenberg). Firstly the use of sentinel node acquired lymphocytes that have been in vitro stimulated by autologous tumour homogenate and APCs, causes a highly specific cellular immune response towards the tumour. Only T cells with high affinity to the primary tumour will survive until transfusion. In a systemic generalized treatment with high-dose IL-2 intravenously to patients all lymphocytes will be equally stimulated and reasonably only a very small fraction of them are tumour specific. The present inventors believe that since the sentinel node(s) are the first draining lymph nodes to a tumour there will be an excessive accumulation of tumour specific lymphocytes. The proliferation and transfusion of true tumour recognising T cells should create a massive tumour specific reaction. Secondly the high-dose IL-2 regimen causes high-toxicity and severe complications, long treatment periods and high costs. The transfusions according to the present method have been given without complications during about one hour and the patients are often discharged the same day. Thirdly, the present protocol aim towards expansion of T helper cells from sentinel nodes, in contrast to expansion of cytotoxic T cells harvested as tumour infiltrating lymphocytes.

This study shows that freshly isolated sentinel node acquired lymphocytes possesses a proliferative ability in vitro against autologous tumour homogenate and can without complications be transfused to the patient as adoptive immunotherapy. There is a strong indication to that treatment with expanded sentinel node acquired lymphocytes may improve the outcome of patients with high-risk or disseminated colon cancer.

Claims

1. A method for treating a patient suffering from colon cancer, the method comprising i) identifying in a patient one or more sentinel and/or metinel lymph nodes draining a tumour in the colon or a metastasis from a tumour in the colon, ii) resecting the one or more nodes and, optionally all or part of the tumour or metastasis, iii) isolating tumour-reactive T-lymphocytes from said lymph nodes, iv) in vitro expanding said tumour-reactive T-lymphocytes, v) administering the thus obtained tumour-reactive T-lymphocytes to the patient, wherein the T-lymphocytes are CD4+ helper and/or CD8+ T-lymphocytes.

2. A method according to claim 1 or 2, wherein the one or more lymph nodes are identified in step i) by injecting one or more lymph node locators into the patient.

3. A method according to any of the preceding claims, wherein the one or more lymph node locators are affinity based.

4. A method according to any of the preceding claims, wherein the one or more lymph node locators are non-affinity based.

5. A method according to any of the preceding claims wherein one or more lymph node locators are injected into, above, around, adjacent and/or under the tumour or metastasis.

6. A method according to any of the preceding claims, wherein the one or more lymph node locators are injected by a single injection.

7. A method according to any of claims 1-5, wherein the one or more lymph node locators are injected by multiple injections.

8. A method according to any of the preceding claims, wherein the one or more lymph node locators are injected by a non-surgical procedure.

9. A method according to any of claims 1-79, wherein the one or more lymph node locators are injected as part of a surgical procedure.

10. A method according to any of the preceding claims, wherein all or part of the tumour or metastasis is removed from the patient.

11. A method according to any of the preceding claims wherein the in vitro expansion step iv) comprises i) a first phase of stimulating tumour-reactive CD4+ helper and/or CD8+ T-lymphocytes with tumour-derived antigen together with at least one substance having agonistic activity towards the IL-2 receptor to promote survival of tumour-reactive CD4+ helper and/or CD8+ T-lymphocytes, and ii) a second phase of activating and promoting growth of tumour-reactive CD4+ helper and/or CD8+ T-lymphocytes, wherein the second phase ii) is initiated when the CD25 cell surface marker (or IL-2R marker) is down-regulated on T-lymphocytes.

12. A method according to claim 11 , wherein the down-regulation is defined as that 5% or less of the T- lymphocyte population expresses CD25.

13. A method according to claim 11 or 12, wherein the T-lymphocytes are present in a culture medium.

14. A method according to claim 13, wherein the culture medium is a serum-free medium, such as, e.g. AIMV medium.

15. A method according to any of the preceding claims, wherein the first phase i) is initiated by adding the at least one substance having agonistic activity towards the IL-2 receptor.

16. A method according to claim 15, wherein the substance having agonistic activity towards the IL-2 receptor is IL-2.

17. A method according to claim 16, wherein IL-2 is added in a low dose, such as, e.g., from about 100 IU/ml culture medium to about 700 IU/ml culture medium, from about 100 IU/ml culture medium to about 600 IU/ml culture medium, from about 100 IU/ml culture medium to about 500 IU/ml culture medium, from about 100 IU/ml culture medium to about 400 IU/ml culture medium, from about 100 IU/ml culture medium to about 300 IU/ml culture medium and from about 100 IU/ml culture medium to about 200 IU/ml culture medium.

18. A method according to any of the preceding claims, wherein a further amount of the at least one substance having agonistic activity towards the IL-2 receptor is added regularly throughout phase i), such as, e.g., every 2^nd, 3^rd or 4^th day of phase i).

19. A method according to claim 18, wherein the substance having agonistic activity towards the IL-2 receptor is IL-2.

20. A method according to claim 19, wherein the concentration of IL-2 added is from about 100 IU/ml culture medium to about 700 IU/ml culture medium, from about 100 IU/ml culture medium to about 600 IU/ml culture medium, from about 100 IU/ml culture medium to about 500 IU/ml culture medium, from about 100 IU/ml culture medium to about 400 IU/ml culture medium, from about 100 IU/ml culture medium to about 300 IU/ml culture medium and from about 100 IU/ml culture medium to about 200 IU/ml culture medium.

21. A method according to any of the preceding claims, wherein the tumour-derived antigen is added from day 2 to and including day 5 of the first phase i), such as, e.g., on day 2, on day 3, on day 4 or on day 5.

22. A method according to any of claims 11-20, wherein the tumour-derived antigen is added essentially at the same time as when phase i) is initiated or at the most up to 3 days thereafter.

23. A method according to any of the preceding claims, wherein the tumour-derived antigen is a denatured homogenate of a tumour.

24. A method according to any of the preceding claims, wherein the tumour-derived antigen is a protein, polypeptide or peptide.

25. A method according to any of the preceding claims, wherein the second phase ii) is initiated from day 17 to and including day 23 of the first phase i), such as, e.g. on day

17, on day 18, on day 19, on day 20, on day 21 , on day 22 or on day 23.

26. A method according to any of the preceding claims, wherein the second phase is initiated by the addition of tumour-derived antigen to the T-lymphocytes for activating tumour-reactive CD25-negative T-lymphocytes.

27. A method according to claim 26, wherein the tumour-derived antigen is a denatured homogenate of a tumour.

28. A method according to claim 26, wherein the tumour-derived antigen is a tumour protein, polypeptide or peptide.

29. A method according to any of claims 26-28, which further comprises addition to the T-lymphocytes of antigen presenting cells together with the tumour-derived antigen.

30. A method according to claim 20, wherein the antigen presenting cells are irradiated peripheral blood leucocytes containing antigen-presenting B-cells and/or monocytes.

31. A method according to any of the preceding claims, wherein the second phase ii) comprises adding at least one substance capable of up-regulating IL-12R on the T- lymphocytes.

32. A method according to claim 31 , wherein the substance(s) capable of up-regulating IL-12R on the T-lymphocytes is substance(s) having agonistic activity towards an interferon receptor.

33. A method according to claim 32, wherein the substance(s) having agonistic activity towards an interferon receptor is an interferon.

34. A method according to claim 33, wherein the substance(s) having agonistic activity towards an interferon receptor is interferon-α.

35. A method according to any of claims 31-34, wherein the substance(s) capable of up-regulating IL-12R on the T-lymphocytes, such as, e.g. a substance having agonistic activity towards an interferon receptor, is added when the level of IL-12 is at least 1 fold, such as, e.g., at least 2, at least 3 fold, at least 4 fold, or at least 5 fold increased as compared to the level of IL-12 on day 1 of phase ii).

36. A method according to any of claims 31-35, wherein the substance capable of up- regulating IL-12R on the T-lymphocytes, such as, e.g. a substance having agonistic activity towards an interferon receptor is added from day 2 to and including day 4 after initiating the second phase ii), such as, e.g. on day 2, on day 3 or on day 4.

37. A method according to any of the preceding claims, wherein the second phase ii) comprises adding one or more substances capable of antagonizing development of Th2 type T-lymphocytes.

38. A method according to claim 37, wherein the one or more substances capable of antagonizing development of Th2 type T-lymphocytes are one or more substances capable of neutralizing IL-4, IL-5, IL-10, and/or TGF-beta.

39. A method according to claim 38, wherein the one or more substances capable of neutralizing IL-4, IL-5, IL-10, and/or TGF-beta are anti IL-4 antibody, anti IL-5 antibody and/or anti IL-10 antibody.

40. A method according to any of claims 37-39, wherein the one or more substances capable of antagonizing development of Th2 type T-lymphocytes, such as, e.g., one or more substances capable of neutralizing IL-4, IL-5, IL-10, and/or TGF-beta is added on day 1 of the second phase ii).

41. A method according to any of claims 37-39, wherein the one or more substances capable of antagonizing development of Th2 type T-lymphocytes, such as, e.g., one or more substance capable of neutralizing IL-4, IL-5, IL-10, and/or TGF-beta is added in a subsequent step after addition of the substance capable of up-regulating IL-12R on the T-lymphocytes.

42. A method according to claim 41 , wherein the one or more substances capable of antagonizing development of Th2 type T-lymphocytes, such as, e.g., one or more substance capable of neutralizing IL-4, IL-5, IL-10, and/or TGF-beta is added one day after addition of the substance capable of up-regulating IL-12R on the T-lymphocytes.

43. A method according to any of the preceding claims, wherein a further amount of the one or more substance capable of antagonizing development of Th2 type T- lymphocytes, such as, e.g., one or more substance capable of neutralizing IL-4, IL-5, IL-10 and/or TGF-beta is added regularly throughout phase ii).

44. A method according to claim 43, wherein a further amount of the one or more substance capable of antagonizing development of Th2 type T-lymphocytes, such as, e.g., one or more substance capable of neutralizing IL-4, IL-5, IL-10 and/or TGF-beta is added every 2^nd, 3^rd or 4^th day of phase ii).

45. A method according to any of the preceding claims, wherein a further amount of a substance having agonistic activity towards the IL-2 receptor is added regularly throughout phase ii).

46. A method according to claim 45, wherein the substance having agonistic activity towards the IL-2 receptor is added every 2^nd, 3^rd or 4^th day of phase ii), such as, e.g., every 3^rd day.

47. A method according to claim 45 or 46, wherein the substance having agonistic activity towards the IL-2 receptor is IL-2.

48. A method according to any of the preceding claims, wherein the second phase ii) comprises adding one or more substances promoting the development of TM type T- lymphocytes.

49. A method according to claim 48, wherein the one or more substances promoting the development of TM type T-lymphocytes is substances having agonistic activity towards the IL-7, IL-12, IL-15 and/or IL-21 receptor.

50 . A method according to claim 49, wherein the one or more substances is selected from IL-7, IL-12, IL-15 and IL-21.

51. A method according to any of claims 48-50, wherein one or more substances promoting the development of Th1 type T-lymphocytes, such as, e.g., substances having agonistic activity towards the IL-7, IL-12, IL-15 and/or IL-21 receptor is added when the level of IFN-gamma is increased as compared to the level of IFN-gamma on initiation of second phase ii).

52. A method according to claim 51 , wherein the increased level of IFN-gamma is determined as at least a 1 fold increase in IFN-gamma level, such as, e.g., at least a 2 fold, at least a 3 fold, at least a 4 fold increase as compared to the level of IFN-gamma on initiation of the second phase ii).

53. A method according to any of claims 48-52, wherein the one or more substances promoting the development of TM type T-lymphocytes, such as, e.g., substances having agonistic activity towards the IL-12, IL-15 and/or IL-21 receptor is added when CD25 and/or CD69 are down-regulated.

54. A method according to any of claims 48-53, wherein the concentration of each of the one or more substances promoting the development of Th1 type T-lymphocytes, such as, e.g., substances having agonistic activity towards the IL-7, IL-12, IL-15 and/or IL-21 receptor added is from about 150 IU/ml culture medium to about 300 IU/ml culture medium, such as, e.g. 250 IU/ml culture medium.

55. A method according to any of claim 48-54, wherein the one or more substances promoting the development of Th1 type T-lymphocytes, such as, e.g., substances having agonistic activity towards the IL-12, IL-15 and/or IL-21 receptor is added from day 5 to and including day 8 after initiating the second phase ii), such as, on day 5, day 6, day 7 or day 8.

56. A method according to any of the preceding claim for the preparation of CD4+ helper T lymphocytes.

57. A method according to any of the preceding claims for the preparation of effector T- lymphocytes.

58. A method according to any of the preceding claims for the preparation of memory T-lymphocytes.

59. A method according to any of the preceding claims for the preparation of Th1 type T-lymphocytes.

60. A method according to any of the preceding claims, which further comprises monitoring the expression of cell surface markers, such as, e.g., CD25 and/or CD69 on the T-lymphocytes continuously during the first phase i) and second phase ii).

61. A method according to claim 60, wherein the T-lymphocytes are harvested when CD25 on T-lymphocytes in the second phase ii) is down-regulated.

62. A method according to claim 61 , wherein the T-lymphocytes are subjected to at least one additional round of phase ii), when CD25 on T-lymphocytes is down- regulated.

63. A method according to claim 61 or 62, wherein the down-regulation is defined as that 5% or less of the CD4 positive T- lymphocyte population expresses CD25.

64. A method according to any of the preceding claims, wherein the tumour-reactive T- lymphocytes are harvested from day 10 to and including day 14 after initiating the second phase ii).

65. A method according to claim 64, wherein the tumour-reactive T-lymphocytes are purified after harvest.

66. A method according to any of the preceding claims further comprising a step of freezing the tumour-reactive T-lymphocytes obtained in the second phase ii).

67. A method according to any of the preceding claims, wherein the T-lymphocytes are derived from lymph nodes draining a primary tumour and/or a metastasis, or they are derived from blood.

68. A method according to any of the preceding claims, wherein the tumour-reactive T- lymphocytes in step iv) are administered intravenously, intraarterially or intrathecal^, intraperitonally.

69. A method according to any of the preceding claims, wherein the amount of tumour- reactive T-lymphocytes administered is at least 10 million, such as, e.g. at least 20 million, at least 30 million, at least 40 million, at least 50 million, at least 60 million, at least 70 million or at least 80 million.

70. A method according to any the preceding claims, wherein the tumour-reactive T- lymphocytes administered are a combination of effector T-lymphocytes and memory T- lymphocytes.

71. A method according to claim 70, wherein the percentage of effector T-lymphocytes is from about 10% to about 65%, such as, e.g., from about 20% to about 50% or from about 30% to about 40%.

72. A method according to any of the preceding claims, wherein the tumour-reactive T- lymphocytes are autologous.

73. A method according to any of the preceding claims, wherein the tumour-reactive T- lymphocytes are non-autologous.

T cells

74. A tumour-reactive T-lymphocyte prepared according to the method defined in any of claims 1-73.

75. A tumour-reactive T-lymphocyte according to claim 74, which is a CD4+ T- lymphocyte.

76. A tumour-reactive T-lymphocyte according to claim 74 or 75, which is an effector T- lymphocyte.

77. A tumour-reactive T-lymphocyte according to any of claims 74-76, which is a memory T-lymphocyte.

78. A tumour-reactive T-lymphocyte according to any of claim 74-77, which is a Th1 type T-lymphocyte.

79. Use of tumour-reactive T-lymphocytes according to any of claims 74-78, for the preparation of a medicament for the treatment of disseminated cancer.

Kits 80. Kit for use in a method according to any of claims 1-73, the kit comprising a media for cultivation of T-lymphocytes.

81. Kit according to claim 80 further comprising one or more substances for stimulating, activating and directing tumour-reactive T-lymphocytes.

82. Kit according to claim 80 or 81 , wherein the media a serum free medium, such as, e.g. AIMV, RPMI 1640, DMEM or MEM.

83. Kit according to any of claims 80-82, wherein the one or more substances for stimulating, activating an directing tumour-reactive T-lymphocytes are selected from tumour-derived antigen, substances having agonistic activity towards the IL-2 receptor, substances capable of up-regulating IL-12R on the T-lymphocytes, substances capable of antagonizing development of Th2 type T-lymphocytes and substances promoting the development of Th1 type T-lymphocytes.

84. Kit according to any of claims 80-83, wherein the one or more substances for stimulating, activating and directing tumour-reactive T-lymphocytes are selected from the group comprising IL-2, interferon-alpha, anti-IL-4 antibody, anti-IL-5 antibody, anti- IL-10 antibody, IL-7, IL-12, IL-15 and IL-21.

85. Kit according to any of claims 80-84, comprising a pharmaceutical composition suitable for intravenous administration.

86. A kit for detection of sentinel or metinel lymph nodes, the kit comprising a syringe and a lymph node locator.

87. A kit for detection of sentinel or metinel lymph nodes, the kit comprising a syringe prefilled with a lymph node locator.

88. Kit according to any of claims 80-87 further comprising instructions for use.

89. Kit according to claim 88, wherein the instructions are in the form of computer software.