WO2013167136A1

WO2013167136A1 - Improving adoptive cell therapy with interferon gamma

Info

Publication number: WO2013167136A1
Application number: PCT/DK2013/050133
Authority: WO
Inventors: Marco DONIA; Inge Marie SVANE
Original assignee: Herlev Hospital
Priority date: 2012-05-08
Filing date: 2013-05-08
Publication date: 2013-11-14

Abstract

The present invention relates to a method for improving the clinical efficacy of adoptive cell therapy with Interferon-gamma (IFN-γ). The invention further relates to Interferon γ for use as a medicament in a method of promoting regression of a cancer in a mammal, wherein said method comprises autologous adoptive cell therapy of tumour-infiltrating lymphocytes. Also provided is a method of treating a mammal with a recurrent cancer, said mammal having previously received immunotherapy.

Description

IMPROVING ADOPTIVE CELL THERAPY WITH INTERFERON GAMMA Field of invention The present invention relates to a method for improving adoptive cell therapy using with interferon-gamma (IFN-y).

Background of invention Cancer immunotherapy may be divided into three major categories: non-specific stimulation of the immune system, active immunization using cancer vaccines, and adoptive cell transfer immunotherapy.

Adoptive cell transfer (ACT) is a very effective form of immunotherapy and involves the transfer of immune cells with antitumour activity into cancer patients. ACT is a treatment approach that involves the identification, in vitro, of lymphocytes with antitumour activity, the in vitro expansion of these cells to large numbers and their infusion into the cancer-bearing host. Lymphocytes used for adoptive transfer can either be derived from the stroma of resected tumours (tumour infiltrating lymphocytes or TILs), or from blood and: genetically engineered to express antitumour T cell receptors (TCRs) or chimeric antigen receptors (CARs) as described previously by Rosenberg (Nat Rev Clin Oncol. 2011 Aug 2;8(10):577-85);, enriched with mixed lymphocyte tumour cell cultures (MLTCs) as described by Mazzarella (Mazzarella et al., 2012) or cloned using autologous antigen presenting cells and tumor derived peptides as described by Yee (Yee et al., 2002). The lymphocytes used for infusion can be isolated from a donor, or from the cancer-bearing host himself. ACT in which the lymphocytes originate from the cancer-bearing host to be infused is termed autologous ACT. US 201 1/0052530 relates to a method for performing adoptive cell therapy to promote cancer regression, primarily for treatment of patients suffering from metastatic melanoma. US 201 1/0052530 is incorporated by reference in its entirety. Verdegaal et al., 2011 , disclose autologous transfer of tumour-derived lymphocytes in conjunction with daily IFN alpha injections (3 million IU, 7 days before infusion, for a total of 12 weeks) for treating melanomas. Li et al., 201 1 , disclose the expansion of γδ T cells isolated from the peripheral blood from healthy donors. Cytotoxic activity of γδ T cells on osteosarcoma cell lines was measured, and the authors find that IFN-γ sensitizes osteosarcoma cell lines to γδ T cells cytotoxicity. Queirolo et al., 1999, disclose the adoptive cell transfer of autologous TILs for treating melanoma, in conjunction with administration of IFN alpha to the patients. The authors conclude that a treatment combining IFN alpha and IL2 is feasible but presents no advantage, since they observe no significant improvement compared to a treatment based on each compound alone.

Dubinett, 1989, use a murine model. Lymphocytes isolated from healthy mice are infused in cancer-injected mice, in conjunction with IFN-γ.

WO9637208 discloses a method for allogeneic adoptive cell transfer in patients having undergone allogeneic stem cell transplantation. The allogeneic infused lymphocytes

(which are not tumour-derived) and/or the patients can be treated with T-cell activators including IFN gamma and IL2.

WO9740156 discloses a method of generating cytotoxic T lymphocytes specific against tumors expressing a ras mutation for treating cancers or inhibiting growth of tumours expressing a ras mutation.

US2004/0071671 regards a method of expanding lymphocytes isolated from patients, e.g. cancer patients, and stimulating those lymphocytes by cancer-specific antigens displayed at the surface of antigen-presenting insect cells.

It has previously been shown that only 0.5-50 % of the total CD8⁺ TIL population is tumour-reactive ex vivo (Donia et al., 2011). Thus, the frequency of tumour-reactive CD8⁺ T cells in currently applied clinical grade TIL cultures is low. Further development of adoptive cell therapy with e.g. autologous tumour infiltrating lymphocytes has the potential to markedly change the long-term prognosis of cancer patients and modifications of the original protocol that can improve its clinical efficacy are highly desirable.

Summary of invention

The present invention relates to a method of promoting regression of a cancer in a mammal comprising the steps of:

a) obtaining autologous tumour-infiltrating lymphocytes from the mammal, b) culturing the lymphocytes,

c) expanding the lymphocytes,

d) administering Interferon γ to the mammal, and

e) administering the expanded lymphocytes to the mammal,

whereupon regression of the cancer is promoted.

The invention further relates to IFN-γ for use as a medicament in a method of promoting regression of a cancer in a mammal, wherein said method comprises autologous adoptive cell transfer of tumour-infiltrating lymphocytes.

Definitions

Interferons (IFNs) are proteins made and released by host cells in response to the presence of pathogens such as viruses, bacteria, parasites or tumour cells. They allow for communication between cells to trigger the protective defenses of the immune system that eradicates pathogens or tumours. IFNs belong to the large class of glycoproteins known as cytokines. Interferons are named after their ability to "interfere" with viral replication within host cells. IFNs have other functions: they activate immune cells, such as natural killer cells and macrophages; they increase recognition of infection or tumour cells by up-regulating antigen presentation to T lymphocytes; and they increase the ability of uninfected host cells to resist new infection by virus. About ten distinct IFNs have been identified in mammals; seven of these have been described for humans. They are typically divided among three IFN classes: Type I IFN, Type II IFN, and Type III IFN. Type I IFNs bind to a specific cell surface receptor complex known as the IFN-a receptor (IFNAR) that consists of IFNAR1 and IFNAR2 chains. The type I interferons present in humans are IFN-a, IFN-β and IFN-ω.

Interferon type II (IFN-γ in humans) binds to IFNGR that consists of IFNGR1 and IFNGR2 chains. Interferon type III signal through a receptor complex consisting of IL10R2 (also called CRF2-4) and IFNLR1 (also called CRF2-12).

Interferon-gamma, abbreviated herein as IFN-γ, IFN-γ, or IFN-γ, is a dimerized soluble cytokine that is the only member of the type II class of interferons. This interferon was originally called macrophage-activating factor, a term now used to describe a larger family of proteins to which IFN-γ belongs. In humans, the IFN-γ protein is encoded by the IFNG gene. IFN-γ, or type II interferon, is a cytokine that is critical for innate and adaptive immunity against viral and intracellular bacterial infections and for tumour control. Aberrant IFN-γ expression is associated with a number of autoinflammatory and autoimmune diseases. The importance of IFN-γ in the immune system stems in part from its ability to inhibit viral replication directly, and most importantly from its immunostimulatory and immunomodulatory effects. IFN-γ is produced predominantly by natural killer (NK) and natural killer T (NKT) cells as part of the innate immune response, and by CD4 Th1 and CD8 cytotoxic T lymphocyte (CTL) effector T cells once antigen-specific immunity develops. Adoptive cell therapy, abbreviated ACT is herein used interchangeably with the term adoptive cell transfer. ACT involves the transfer of immune cells with antitumour activity into cancer patients. ACT is a treatment approach that involves the identification, in vitro, of lymphocytes with antitumour activity, the in vitro expansion of these cells to large numbers and their infusion into the cancer-bearing host. Of particular interest for the invention is autologous ACT, i.e. adoptive cell therapy in which the immune cells with antitumour activity to be transferred into cancer patients originate from the patient himself.

Tumor infiltrating lymphocytes, abbreviated as TILs, are white blood cells that have left the bloodstream and migrated into a tumour.

Autologous: a situation in which the donor and recipient of e.g. lymphocytes are the same person. Immunogenicity is the ability to induce humoral and/or cell-mediated immune responses.

Recurrent or relapsing is a term used to designate a disease which is returning after a period of time during which could not be detected, i.e. a period of remission. In the context of cancer, recurrence is characterised by the reappearance of cancer cells at the same site or at a different site than the original cancer.

Description of Drawings

Figure 1. Adoptive cell transfer immunotherapy using either autologous TILs obtained from resected tumours or using peripheral lymphocytes genetically transduced with retroviruses to express antitumour T-cell receptors. Cells are expanded in vitro to large numbers (up to 10¹¹) and infused after patients have received a preparative

lymphodepleting regimen. Abbreviations: TCR, T-cell receptor; TIL, tumour-infiltrating lymphocyte. (From: Nat Rev Clin Oncol. 2011 Aug 2;8(10):577-85)

Figure 2. Association of clinical response and absolute number of ex vivo tumour reactive CD8⁺ T cells in infusion products for ACT. Infusion products were tested for reactivity against autologous melanoma cell lines (indicated with the arrow) or, when an autologous cell line was not available against a panel of four to eight HLA-A matched allogeneic melanoma cell lines (only the highest reactivity is shown). Reactivity was evaluated by defining the frequency of cells expressing at least the two type 1 cytokines TNF-a and IFN- γ. Data show that clinical response is associated with a higher absolute number of infused ex vivo tumour-reactive CD8⁺ TILs. CR/PR:

Complete Response or Partial Response. SD/PD: Stable Disease or Progressive Disease.

Figure 3. IFN- γ increased autologous tumour recognition by CD8⁺ TILs. Matched TILs were stimulated with autologous target melanoma cells as described in Materials and Methods. (A) Figure shows the frequency of cytokine-producing CD8⁺ TILs upon stimulation with autologous tumours in the left panel, and the difference for each sample (IFN-γ treated minus control) in the right panel. Reactivity was evaluated by defining the frequency of cells expressing at least the two type 1 cytokines TNF-a and IFN-γ. CI: confidence interval. (B) Curves and plots show respectively class I MHC expression cytokine production by CD8⁺ TILs. Two TIL/Autologous tumour pairs are depicted, representing an increase of tumour recognition associated with significant upregulation of class I MHC (upper panel) in a constitutively MHC 1+ tumour and (lower panel) in a tumour with very low MHC I expression. Plots are gated on live CD8⁺ TILs. Pt.: Patient. Dotted Grey Line: Isotype. Solid Grey Line: Untreated cell line. Solid Black Line: IFN-γ treated cell line.

Figure 4. IFN-γ increased autologous tumour recognition by CD4⁺ TILs. Matched TILs were stimulated with autologous target melanoma cells as described in Materials and Methods. (A) Figure shows the frequency of cytokine-producing CD8⁺ TILs upon stimulation with autologous tumours in the left panel, and difference for each sample (IFN-γ treated minus control) in the right panel. Reactivity was evaluated by defining the frequency of cells expressing at least the two type 1 cytokines TNF-a and IFN-γ. CI: confidence interval (B) Curves and plots show respectively class II MHC expression and cytokine production by CD4⁺ TILs. Two TIL/Autologous tumour pairs are depicted, representing an increase of tumour recognition associated with significant upregulation of class II MHC molecules of tumours (upper panel) constitutively MHC II+ or (lower panel) MHC II-. Plots are gated on live CD4⁺ TILs.

Pt.: Patient. Dotted Grey Line: Isotype. Solid Grey Line: Untreated cell line. Solid Black Line: IFN-γ treated cell line.

Figure 5. Effects of IFN-γ on the expression of MHC molecules in melanoma. Short- term cultured melanoma cell lines were treated with IFN-γ 100 lU/ml (+ IFN-γ) or left untreated (- IFN-γ) and then collected after standard trypsinization. They were stained with fluorochrome conjugated anti H LA- ABC (left panels) or anti HLA- DR,DQ,DP antibodies (right panels). Dotted Grey Line: Isotype. Solid Grey Line: Untreated cell line. Solid Black Line: IFN-γ treated cell line.

Figure 6. IFN- γ increased tumour recognition by multifunctional CD8⁺ TILs. TILs were stimulated with autologous tumours as described in Materials and Methods. Figure shows (A) the percentage of CD8⁺ TILs co-expressing TNF-a/IFN-Y/CD107a upon stimulation with autologous tumour cells and (B) one representative TIL culture with a strong increase in frequency of CD8⁺ TILs expressing markers of cytotoxicity. Figure 7. Direct effects of IFN-γ on melanoma growth, apoptosis and cell cycle. Short- term cultured melanoma cell lines were treated with IFN-γ 100 lU/ml as indicated in Materials and Methods. (A) Growth inhibition was evaluated after 72 hours of incubation. IFN-γ significantly reduced cell growth in 5/9 cell lines. Pooled data from two experiments are shown (B) Cell cycle distribution and frequency of subG events, considered apoptotic bodies, were evaluated after 24 hours of incubation. IFN-γ did not significantly modify the parameters considered. Pooled data from two experiments performed with all the 9 cell lines are shown. * p<0.05 Figure 8. The figure shows an exemplary timeline for adoptive cell therapy treatment of a patient utilising TILs obtained from a resected tumour (day 0). TILs are obtained from the patient by isolation of T cells from resected tumour tissue. The isolated T cells are cultured and expanded for approximately 26 days, before re-introduction into the patient (infusion). Approximately one week prior to infusion of the expanded TILs, nonmyeoloablative chemotherapy (lymphodepletion) is initiated (day 19). IFN-γ is administered to the patient for approximately 7 days before infusion of the TILs.

Figure 9. Schematic representation of the process of TIL expansion and TIL therapy for metastatic melanoma starting from tumour fragments. Suitable tumours from eligible stage lllc-IV patients undergo a resection and are taken to the laboratory under sterile conditions where tumours are cut up into small 3- to 5-mm² fragments and placed in culture plates or small culture flasks with growth medium and high-dose (HD) IL-2. The TILs are initially expanded for 3 to 5 weeks during this pre-REP phase to at least 50 x 10⁶ cells. The cells are than subjected to a REP over 2 weeks by stimulating the T cells using anti-CD3 in the presence of PBMC feeder cells and IL-2. The expanded TILs

(now billions of cells) are washed, pooled, and infused into the patient followed by 1 or 2 cycles of HD IL-2 therapy. Before TIL transfer, the patient is treated with a

preparative regimen using cyclophosphamide (Cy) and fludarabine (Flu) that transiently depletes host lymphocytes, "making room" for the infused TILs and removing cytokine sinks and regulatory T cells to facilitate TIL persistence (from Wu et al., Cancer J. 2012; 18: 160-175).

Figure 10. IFN-γ to improve pre-existing T-cell responses to relapsing tumours. Clinical complete response upon TIL treatment was associated with induction of peripheral (blood) ex vivo antitumor responses against a melanoma cell line generated from the same metastasis used for TIL generation (pre-TIL tumor). A tumor recurrence was diagnosed 13 months after TIL treatment, despite persistence of peripheral antitumor responses (A). A cell line generated from tumor recurrence (recurrent tumor) showed reduced immunosensitivity, as demonstrated by reduced recognition of both TILs obtained from the pre-TIL treatment lesion and used for treatment (OLD TILs) as well as TILs obtained from the recurrent tumor lesion (NEW TILs) (B). Reduced recognition is shown also for T-cells specific for the cancer testis antigen TAG (which were present in large numbers in the OLD TILs) and for PBMCs obtained 13 months after treatment (C). The recurrent tumor did not show increased expression of immunosuppressive molecules such as PD-L1 neither before or after exposure to IFN-γ. Black: pre-TIL tumor; Grey: recurrent tumor; Dotted lines: isotype stained; Solid thick lines: baseline level; Solid thin lines: after treatment with 100 lU/ml IFN-y for 72 hours (D). No evidence of target antigen downregulation was found in semiquantitative PCR (E). The recurrent tumor displayed impaired expression of MHC class I molecules, which could be reverted by treatment with IFN-γ. Colour code is shown as in Figure XD. (F). While responses to pre-TIL tumor did not increase with IFN-γ, responses of either the NEW TILs as well as the OLD TILs towards the recurrent tumor were increased.

Detailed description of the invention

The present invention provides a method for improving the clinical efficacy of autologous ACT by performing autologous ACT using tumour-infiltrating lymphocytes and administering IFN-γ. Hence, the present invention relates to a method of promoting regression of a cancer in a mammal comprising the steps of:

c) expanding the lymphocytes,

d) administering Interferon γ to the mammal, and

e) administering the expanded lymphocytes to the mammal,

whereupon regression of the cancer is promoted.

The IFN-γ treatment is believed to stimulate tumour cell immunogenicity, thereby augmenting tumour reactivity of tumour infiltrating lymphocytes (TILs). An increased proportion of tumour-reactive TILs will result in enhanced clinical efficacy of adoptive cell therapy (ACT). ACT involves the transfer of lymphocytes with antitumour activity into cancer patients. ACT is a treatment approach that involves the identification, in vitro, of lymphocytes with antitumour activity, the in vitro expansion of these cells to large numbers and their infusion into the cancer-bearing host. Lymphocytes used for adoptive transfer can either be derived from the stroma of resected tumours (tumour infiltrating lymphocytes or TILs), or from blood: genetically engineered to express antitumour T cell receptors (TCRs) or chimeric antigen receptors (CARs) as described previously by Rosenberg (Nat Rev Clin Oncol. 2011 Aug 2;8(10):577-85); enriched with mixed lymphocyte tumour cell cultures (MLTCs) as described by Mazzarella (Mazzarella et al., 2012) or cloned using autologous antigen presenting cells and tumor derived peptides as described by Yee (Yee et al., 2002). The lymphocytes used for infusion can be isolated from a donor, or from the cancer-bearing host himself. ACT in which the lymphocytes originate from the cancer-bearing host to be infused is termed autologous ACT.

According to the present invention, ACT may be performed by (i) obtaining autologous lymphocytes from a mammal, (ii) culturing said autologous lymphocytes, (iii) expanding the cultured lymphocytes, and (iv) administering the expanded lymphocytes to the mammal. Preferably, the lymphocytes are tumour-derived, i.e. they are TILs, and are isolated from the mammal to be treated, i.e. autologous transfer.

The IFN-γ administration may be performed at any time before and after autologous ACT, such as before obtaining the autologous lymphocytes, before administering to the mammal the expanded lymphocytes and after administering the expanded lymphocytes to the mammal. In a preferred embodiment of the present invention, the IFN-

Y administration takes place before the lymphocytes are administered to the mammal, i.e. before step (iv) above.

Autologous ACT as described herein may also be performed by (i) culturing autologous lymphocytes from a mammal; (ii) expanding the cultured lymphocytes; (iii)

administering nonmyeloablative lymphodepleting chemotherapy to the mammal; and (iv) after administering nonmyeloablative lymphodepleting chemotherapy, administering the expanded lymphocytes to the mammal, essentially as previously described in US 201 1/0052530. Autologous TILs may be obtained from the stroma of resected tumours. Tumour samples are obtained from patients and a single cell suspension is obtained. The single cell suspension can be obtained in any suitable manner, e.g., mechanically (disaggregating the tumour using, e.g., a gentleMACS(TM) Dissociator, Miltenyi Biotec, Auburn, Calif.) or enzymatically (e.g., collagenase or DNase).

Expansion of lymphocytes, including tumour-infiltrating lymphocytes, such as T cells can be accomplished by any of a number of methods as are known in the art. For example, T cells can be rapidly expanded using non-specific T-cell receptor stimulation in the presence of feeder lymphocytes and interleukin-2 (IL-2), IL-7, IL-15 and IL-21 , with IL-2 being preferred. The non-specific T-cell receptor stimulus can e.g. include around 30 ng/ml of OKT3, a mouse monoclonal anti-CD3 antibody (available from Ortho-McNeil(R), Raritan, N.J. or Miltenyi Biotec, Bergisch Gladbach, Germany). Alternatively, T cells can be rapidly expanded by stimulation of peripheral blood mononuclear cells (PBMC) in vitro with one or more antigens (including antigenic portions thereof, such as epitope(s), or a cell of the cancer, which can be optionally expressed from a vector, such as an human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., approximately 0.3 μΜ MART-1 :26-35 (27 L) or gp100:209-217 (210M)), in the presence of a T-cell growth factor, such as around 200-400 Ill/ml, such as 300 lU/ml IL-2 or IL-15, with IL-2 being preferred. The in vitro-induced T-cells are rapidly expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto HLA- A2-expressing antigen-presenting cells. Alternatively, the T-cells can be re-stimulated with irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2, for example.

In one embodiment, nonmyeloablative lymphodepleting chemotherapy is administered to the mammal prior to administering to the mammal the expanded tumour-infiltrating lymphocytes. The purpose of lymphodepletion is to make room for the infused lymphocytes, in particular by eliminating regulatory T cells and other non-specific T cells which compete for homeostatic cytokines.

Nonmyeloablative lymphodepleting chemotherapy can be any suitable such therapy, which can be administered by any suitable route known to a person of skill. The nonmyeloablative lymphodepleting chemotherapy can comprise, for example, the administration of cyclophosphamide and fludarabine, particularly if the cancer is melanoma, which can be metastatic. A preferred route of administering

cyclophosphamide and fludarabine is intravenously. Likewise, any suitable dose of cyclophosphamide and fludarabine can be administered. Preferably, around 40-80 mg/kg, such as around 60 mg/kg of cyclophosphamide is administered for

approximately two days after which around 15-35 mg/m², such as around 25 mg/m² fludarabine is administered for around five days, particularly if the cancer is melanoma.

An embodiment of the method comprises administering the expanded TILs to the mammal, wherein the TILs administered to the mammal are about 14 to about 40 days old, such as about 15 to about 39 days, for example about 16 to about 38 days, such as about 17 to about 37 days, for example about 18 to about 36 days, such as about 19 to about 35 days, for example about 20 to about 34 days, such as about 21 to about 33 days, for example about 22 to about 32 days, such as about 23 to about 31 days, for example about 24 to about 30 days, such as about 25 to about 29 days, for example about 26 to about 28 days, such as about 27 days old.

In one embodiment, the TILs administered to the mammal are about 19 to about 35 days old. In some embodiments, the TILs administered to the mammal are about 19 to about 29 or about 23 to about 29 days old, or about 26 days old. In this regard, the lymphocytes that are administered to the mammal according to an embodiment of the invention are "young" lymphocytes, i.e., minimally cultured lymphocytes. Young lymphocyte cultures advantageously have features associated with in vivo persistence, proliferation, and antitumour activity.

In other embodiments of the invention, the administered lymphocytes are younger than about 14 days and in yet other embodiments, the lymphocytes are older than about 40 days. The lymphocytes can be administered by any suitable route as known in the art.

Preferably, the lymphocytes are administered as an intra-arterial or intravenous infusion, which preferably lasts about 30 to about 60 minutes. Other examples of routes of administration include intraperitoneal, intrathecal and intralymphatic. Likewise, any suitable dose of lymphocytes can be administered. In one embodiment, about 1 x 10¹⁰ lymphocytes to about 15 x 10¹⁰ lymphocytes are administered, such as about 1 x 10¹⁰ lymphocytes to about 13 x 10¹⁰ lymphocytes, for example about 1 x 10¹⁰ lymphocytes to about 1 1 x 10¹⁰ lymphocytes, such as about 1 x 10¹⁰ lymphocytes to about 9 x 10¹⁰ lymphocytes, for example about 1 x 10¹⁰ lymphocytes to about 8 x 10¹⁰ lymphocytes, such as about 1 x 10¹⁰ lymphocytes to about 7 x 10¹⁰ lymphocytes, for example about 1 x 10¹⁰ lymphocytes to about 6 x 10¹⁰ lymphocytes, such as about 1 x 10¹⁰ lymphocytes to about 5 x 10¹⁰ lymphocytes, for example about 1 x 10¹⁰ lymphocytes to about 4 x 10¹⁰ lymphocytes, such as about 1 x 10¹⁰ lymphocytes to about 3 x 10¹⁰ lymphocytes, for example about 2 x 10¹⁰ lymphocytes.

In other embodiments about 4 x 10¹⁰ to about 6 x 10¹⁰ lymphocytes, such as about 5 x 10¹⁰ lymphocytes are administered, particularly if the cancer is melanoma. In yet other embodiments, less than 5 x 10¹⁰ lymphocytes are administered, such as less than 4 x 10¹⁰ lymphocytes, for example less than 3 x 10¹⁰ lymphocytes, such as less than 2 x 10¹⁰ lymphocytes.

In one embodiment, the lymphocytes are not tested for specific tumour reactivity to identify tumour reactive lymphocytes prior to administration to the patient. Specific tumour reactivity can however be tested by any method known in the art, e.g., by measuring cytokine release (e.g., interferon-gamma) following co-culture with tumour cells. In one embodiment, the autologous ACT method comprises enriching cultured TILs for CD8⁺ T cells prior to rapid expansion of the cells. Following culture of the TILs in IL-2, the T cells are depleted of CD4⁺ cells and enriched for CD8⁺ cells using, for example, a CD8 microbead separation (e.g., using a CliniMACS<plus >CD8 microbead system (Miltenyi Biotec)). Without being bound to a particular theory, it is believed that CD4⁺, CD25⁺ regulatory T-cells can impede anti-tumour responses. Accordingly, it is believed that enriching cultured T cells for CD8⁺ T cells and reducing or eliminating CD4⁺ cells may improve the impact of adoptively transferred anti-tumour CD8⁺ cells, improve the response rates in patients, and/or reduce the toxicities seen by production of cytokines by CD4⁺ cells. Moreover, it is believed that CD8⁺ enrichment of some T cell cultures reveals in vitro tumour recognition that may not be evident in the bulk culture, and improved in vitro recognition of tumour in other cultures. Additionally, the enriched CD8⁺ young T cells are believed to behave more reliably and predictably in clinical scale rapid expansions than the bulk T cells. In an embodiment of the method, a T-cell growth factor that promotes the growth and activation of the autologous T cells is administered to the mammal either concomitantly with the autologous T cells or subsequently to the autologous T cells. The T-cell growth factor can be any suitable growth factor that promotes the growth and activation of the autologous T-cells. Examples of suitable T-cell growth factors include interleukin (IL)-2, IL-7, IL-15, IL-12 and IL-21 , which can be used alone or in various combinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL- 12 and IL-15, or IL-12 and IL2. IL-12 is a preferred T-cell growth factor.

In an embodiment of the method, the autologous T-cells are modified to express a T cell receptor (TCR) having antigenic specificity for a cancer antigen.

The cancer treated by the present invention can be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, cervical cancer, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, soft tissue cancer, testicular cancer, thyroid cancer, ureter cancer, urinary bladder cancer, and digestive tract cancer such as, e.g., esophageal cancer, gastric cancer, pancreatic cancer, stomach cancer, small intestine cancer, gastrointestinal carcinoid tumor, cancer of the oral cavity, colorectal cancer, and hepatobiliary cancer.

The cancer can be a recurrent cancer. Preferably, the cancer is a solid cancer. Preferably, the cancer is melanoma, ovarian, breast and colorectal cancer, even more preferred is melanoma, in particular metastatic melanoma. As used herein, the term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.

The term "regression," as well as words stemming therefrom, as used herein, does not necessarily imply 100% or complete regression. Rather, there are varying degrees of regression of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of regression of cancer in a mammal. Furthermore, the regression provided by the inventive method can include regression of one or more conditions or symptoms of the disease, e.g., cancer. Also, for purposes herein, "regression" can encompass delaying the onset of the disease, or a symptom or condition thereof.

Interferon gamma The IFN-γ administration according to the present invention may be performed at any time before, during or after ACT, such as before obtaining the autologous TILs, before administering to the mammal the expanded TILs and after administering to the mammal the expanded TILs. In a preferred embodiment of the present invention, the IFN-γ administration takes place before the TILs are administered to the mammal.

Previous clinical experience with IFN-γ monotherapy for melanoma has been largely disappointing (Kirkwood et al., 1990; Maluish et al., 1988; Propper et al., 2003; Schiller et al., 1996). Notably, IFN-γ (interferon gamma-1 b, marketed as: Actimmune,

Intermune Inc., Brisbane, CA, USA or Imukin, Boehringer Ingelheim) has been used safely for over 10 years in the treatment of chronic granulomatous disease and severe, malignant osteopetrosis. Previous clinical trials in melanoma have safely used doses of IFN-Y of up to 3000 μg/m2/day (Kirkwood et al., 1990).

According to the present invention, the IFN-γ may be administered in any suitable route known to a person of skill. The IFN-γ may e.g. be administered subcutaneously, intravenously, intramuscularly or intraarterially. In a preferred embodiment, the IFN-γ is administered subcutaneously.

The IFN-γ of the present invention is preferably administered in a low dose, for example from about 25 μg/m² to about 1000 μg/m² per dose, such as from about 25 μg/m² to about 50 μg/m² per dose, for example from about 50 μg/m² to about 100 μg/m² per dose, such as from about 100 μg/m² to about 200 μg/m² per dose, for example from about 200 μg/m² to about 300 μg/m² per dose, such as from about 300 μg/m² to about 400 μg/m² per dose, for example from about 400 μg/m² to about 500 μg/m² per dose, such as from about 500 μg/m² to about 600 μg/m² per dose, for example from about 600 μg/m² to about 700 μg/m² per dose, such as from about 700 μg/m² to about 800 μg/m² per dose, for example from about 800 μg/m² to about 900 μg/m² per dose, such as from about 900 μg/m² to about 1000 μg/m² per dose. In other embodiments, less than 25 μg/m² IFN-γ is administered per dose and in yet other embodiments, more than 1000 μg/m² IFN-γ is administered per dose.

The IFN-γ of the present invention may be administered at about 1 μg/m² to about 1000 μg/m² per day, such as from about 1 μg/m² to about 10 μg/m² per day, for example from about 10 μg/m² to about 20 μg/m² per day, such as from about 20 μg/m² to about 50 μg/m² per day, for example from about 50 μg/m² to about 100 μg/m² per day, such as from about 100 μg/m² to about 200 μg/m² per day, for example from about 200 μg/m² to about 300 μg/m² per day, such as from about 300 μg/m² to about 400 μg/m² per day, for example from about 400 μg/m² to about 500 μg/m² per day, such as from about 500 μg/m² to about 600 μg/m² per day, for example from about 600 μg/m² to about 700 μg/m² per day, such as from about 700 μg/m² to about 800 μg/m² per day, for example from about 800 μg/m² to about 900 μg/m² per day, such as from about 900 μg/m² to about 1000 μg/m² per day, for example from about 600 μg/m² to about 700 μg/m² per day, such as from about 700 μg/m² to about 800 μg/m² per day, for example from about 800 μςΛτι² to about 900 μςΛτι² per day, such as from about 900 μςΛτι² to about 1000 μςΛτι² per day.

In one embodiment the IFN-γ is administered at about 50 μςΛτι² to about 1000 μςΛτι² per day.

In one embodiment, the IFN-γ is administered at about 1000 μςΛτι² to about 5000 μςΛτι² per day, such as from about 1000 μςΛτι² to about 1500 μςΛτι² per day, for example from about 1500 μςΛτι² to about 2000 μςΑτι² per day, such as from about 2000 μςΛτι² to about 2500 μςΛτι² per day, for example from about 2500 μςΛτι² to about 3000 μςΛτι² per day, such as from about 3000 μςΑτι² to about 3500 μςΛτι² per day, for example from about 3500 μςΛτι² to about 4000 μςΑτι² per day, such as from about 4000 μςΛτι² to about 4500 μςΛτι² per day, for example from about 4500 μςΛτι² to about 5000 μςΛτι² per day.

In some embodiments, more than about 1 mg/m² IFN-γ per day is administered, such as about 1.1 mg/m² IFN-γ per day, for example about 1.2 mg/m² IFN-γ per day, such as about 1.3 mg/m² IFN-γ per day, for example about 1.4 mg/m² IFN-γ per day, such as about 1.5 mg/m² IFN-γ per day, or more.

The IFN-γ may for example be administered to the mammal as a single daily dose or as multiple daily doses. Alternatively, the IFN- γ may be administered more than once a week, such as twice a week, for example three times a week, such as four times a week, for example five times a week, such as six times a week or more.

In one embodiment, the IFN-γ treatment is initiated before administration of TILs during ACT, such as from about 1 day before to about 30 days before, for example from about 2 days before to about 30 days before, such as from about 3 days before to about 30 days before, for example from about 4 days before to about 30 days before, such as from about 5 days before to about 30 days before, for example from about 5 days before to about 30 days before, such as from about 6 days before to about 30 days before, for example from about 7 days before to about 30 days before.

In one embodiment, the IFN-γ treatment is initiated approximately 7 days before administration of the TILs during ACT. In one embodiment, the IFN-γ treatment is initiated more than approximately 7 days before administration of the TILs during ACT. In one embodiment of the present invention, the IFN-γ is administered before obtaining the autologous TILs from the mammal.

In another embodiment, IFN-γ is administered after administration of the TILs during ACT, such as for approximately one week, for example two weeks or more.

The IFN-γ may be administered continuously or discontinuously, i.e. with one or more pauses.

In a preferred embodiment, the IFN-γ is administered as a single subcutaneous daily dose for about one week before administration of the Tl Ls.

Recurrent cancer

The present invention also relates to a method of promoting regression of a recurrent cancer in a mammal, comprising administration of Interferon γ to the mammal, wherein said mammal has previously been treated with immunotherapy.

Thus, in one embodiment, the mammal has already received a previous treatment against a cancer, said treatment comprising immunotherapy capable of inducing effective cellular immune responses. In one embodiment, the previous treatment is adoptive cell therapy.

Anectodal data from other groups indicate that relapse after an initial response to T-cell therapy can be caused by reduced or loss of class I MHC expression in recurrent tumors (Dudley, M. E. et al., 2005). When the previous treatment received by the mammal is ACT, the ACT may be either autologous ACT, i.e. infusion of autologous TILs combined with administration of IFN- γ, or allogeneic ACT, i.e. infusion of allogeneic T cells to the patient, or ACT performed in any other way known to the skilled person. The response to the said treatment may have been a complete response (CR) or a partial response (PR) according to the Response Evaluation Criteria In Solid Tumors (RECIST). If the cancer relapses after a period of remission, IFN-γ may be administered to the mammal to activate or reactivate the circulating T lymphocytes.

Administration of IFN-γ to the mammal for treating a recurrent cancer may be performed alone or in combination with another round of ACT. For example, new TILs may be isolated from the mammal at the time of treating the recurrent cancer; the TILs are expanded in vitro; IFN-γ is administered prior to, concurrently with and/or after the new TILs are infused into the mammal.

In another embodiment, autologous TILs infused during a previous ACT may be reexpanded and reinfused into the mammal for a second ACT, in combination with IFN- Y treatment.

Items

A method of promoting regression of a cancer in a mammal comprising the steps of:

a) administering to the mammal Interferon, and

b) treating said mammal with adoptive cell therapy,

whereupon regression of the cancer is promoted.

The method according to item 1 , wherein the Interferon is Interferon-gamma.

The method according to any of the preceding items, wherein the adoptive cell therapy comprises the use of tumour infiltrating lymphocytes.

The method according to any of the preceding items, wherein the Interferon is administered subcutaneously, intravenously, intramuscularly or intraarterially.

The method according to item 2, wherein the Interferon is administered subcutaneously.

The method according to any of the preceding items, wherein the Interferon is administered to the mammal as a single daily dose or as multiple daily doses.

The method according to item 4, wherein the Interferon is administered to the mammal as a single daily dose.

The method according to any of the preceding items, wherein the Interferon is administered at about 50 μg/m² to about 1000 μg/m² per day.

The method according to any of the preceding items, wherein the Interferon is administered to the mammal for approximately one week before infusion of lymphocytes, such as more than 3 days, for example more than 4 days, such as more than 5 days, for example more than 6 days, such as about 7 days.

0. The method according to any of the preceding items, wherein the cancer is selected from the group consisting of melanoma, ovarian, breast and colorectal cancer The method according to item 9, wherein the cancer is melanoma. The method according to any of the preceding items, wherein the cancer is metastatic. The method according to any of the preceding items, wherein the mammal is a human. Interferon for use in a method of promoting regression of a cancer in a mammal, wherein said method comprises adoptive cell therapy. The Interferon for use according to item 14, wherein said Interferon is Interferon- gamma. The Interferon for use according to item 14, wherein the adoptive cell therapy comprises the use of tumour infiltrating lymphocytes. The Interferon for use according to any of items 14-16, wherein the Interferon is administered subcutaneously, intravenously, intramuscularly or intraarterially. The Interferon for use according to item 17, wherein the Interferon is administered subcutaneously. The Interferon for use according to any of items 14-18, wherein the Interferon is administered to the mammal as a single daily dose or as multiple daily doses. The Interferon for use according to item 19, wherein the Interferon is administered to the mammal as a single daily dose. The Interferon for use according to any of items 14-20, wherein the Interferon is administered at about 50 μg/m² to about 1000 μg/m² per day. The Interferon for use according to any of items 14-21 , wherein the Interferon administration is initiated approximately one week before infusion of lymphocytes, such as more than 3 days, for example more than 4 days, such as more than 5 days, for example more than 6 days, such as 7 days. The Interferon for use according to any of items 14-22, wherein the cancer is selected from the group consisting of melanoma, ovarian, breast and colorectal cancer The Interferon for use according to item 23, wherein the cancer is melanoma. The Interferon for use according to any of items 14-24, wherein the cancer is metastatic. The Interferon for use according to any of items 14-25, wherein the mammal is a human. Use of Interferon for the manufacture of a medicament for the treatment of cancer, wherein said treatment comprises adoptive cell therapy. Interferon for use in a method of promoting regression of a cancer in a mammal, wherein said method comprises the steps of: a) obtaining autologous lymphocytes from the mammal,

b) culturing the lymphocytes,

c) expanding the cultured lymphocytes,

d) administering Interferon to the mammal, and

e) administering to the mammal the expanded lymphocytes. A method of promoting regression of a cancer in a mammal comprising the steps of:

a) obtaining autologous lymphocytes from the mammal,

b) culturing the lymphocytes,

c) expanding the cultured lymphocytes,

d) administering Interferon to the mammal, and

e) administering to the mammal the expanded lymphocytes. Examples

Materials and Methods Clinical Trial and Tumour Specimens

All the procedures were approved by the Scientific Ethics Committee for the Capital Region of Denmark. Written informed consent was obtained from patients before any procedure according to the Declaration of Helsinki. Response evaluation in the ACT clinical trial (clinicaltrials.gov identifier: NCT 00937625) was carried out according to standard criteria for response evaluation in solid tumours (RECIST), as described previously.

Tumour specimens of at least 1cm³ were obtained from patients with melanoma stage III or IV undergoing standard-of-care surgical procedures or specimen collection for enrolment in a clinical trial (clinicaltrials.gov identifier : NCT 00937625). A standard two step protocol was applied for the generation of clinical grade TIL cultures from tumour fragments, as previously described (Donia et al., 2011).

Clinical grade TIL cultures comprise a vast majority of effector memory (CCR7- CD45RA-) CD3+ cells (>95 %) (Donia et al., 201 1). TILs object of this study had highly variable CD8⁺/ CD4⁺ ratios but, in general, a predominance of CD8⁺ T cells (mean value CD8⁺/CD4⁺ ratio 26±53, range 0.01-170).

Autologous short term (<15 in vitro passages) melanoma cell lines were established from additional tumour fragments originated from the same lesions used for TIL generation, by serial passage of adherent cells, as previously described (Donia et al., 201 1).

Antitumour Activity of TILs

The following fluorochrome-conjugated antibodies were used for flow cytometry: anti CD8-PerCP, CD4-FITC, CD107a-PE, IFN-y-PeCy7; TNF-a-APC; IL-4-PE (all from BD, Br0ndby, Denmark). Fixation/Permeabilization Buffer, Permeabilization Buffer and Fixable Viability Dye eFluor® 450 were from Ebiosciences, GolgiPlug from BD, and Staphylococcal Enterotoxin B (SEB) from Sigma-Aldrich. For simultaneous CD107a and intracellular cytokine analysis, TILs were cultured for 5 hours at 37°C with 5 % C02 in air in the presence or absence (negative control) of autologous cancer cells or four to eight HLA-A matched allogeneic melanoma cell lines (only for infusion products from the clinical trial) at an effector/target (E:T) ratio of 3: 1 , as previously described (Donia et al., 201 1). Autologous cancer cells used as target were pretreated with IFN-y 100 lU/ml for 72 hours or left untreated. Cells were acquired using a BD FACSCanto II flow cytometer. At least 200 000 live TILs were acquired. Analysis was performed with BD FACSDiva Software or FlowJo (Tree Star, Ashland, OR, USA).

Criteria to define a positive antitumour response were: at least twice the frequency of the background and at least 50 positive events; otherwise, at least 10 times the background. When the frequency was between twice and 10 times the background but less than 50 positive events were acquired, the response was defined as "borderline".

D'Agostino-Pearson normality test was performed to check for normal distribution of the values. Results were compared between different groups with two-tailed Mann-Whitney test (comparison between responders/non responders of the clinical trial), or with two- tailed t-test for paired data and Wlcoxon matched pairs test (comparison with IFN-y treated or untreated) respectively for normally or non-normally distributed sets of data. Statistical Analysis was performed with GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA).

FACS analysis of melanoma cells

After 72 hours of incubation with or without 100 lU/ml of IFN-γ, melanoma cells were collected by standard trypsinization, washed twice with cold PBS and stained with anti- HLA-ABC(class I MHC)-APC, HLA-DR,DQ,DP (class II MHC)-FITC, PD-L1-PECy7 (from BD). 7-actinomycin D (from Sigma-Aldrich) was added in each tube to exclude dead cells. Additional controls with isotype-matched antibodies were set up to define background staining. At least 10 000 melanoma cells were acquired using a BD FACSCanto II flow cytometer. Analysis was performed with BD FacsDiva Software or FlowJo.

Proliferation Assay and analysis of cell cycle

Cell proliferation was evaluated using a flow cytometry-based counting method, as previously described. Growth inhibition of cells treated with IFN-y 100 lU/ml was calculated using the following formula: (T72-T0)/(K72-T0) ^χ 100 , where T72 is the cell count after 72 hours, TO is the cell count of the control well at time zero and K72 is the cell count of the control well (medium) after 72 hours.

Cell cycle analysis was performed by quantifying the DNA content of melanoma cells with a PI based method, as previously described. Briefly, the cells were incubated in the presence or absence of IFN-y for 24 h and after collection were stained with PI. DNA quantity and cell cycle distribution including frequency of subG (apoptotic) cells was determined by flow cytometry. Reagents and cells for TIL and autologous tumour cell generation

Human AB serum (HS) was purchased from Sigma-Aldrich (Br0ndby, Denmark), RPMI-1640 with GlutaMAX, AIM-V medium and Fetal Bovine Serum (FBS) were obtained from Invitrogen (Naarum, Denmark). rhlL-2 (Proleukin) was from Novartis (Basel, Switzerland), OKT3 (anti-CD3) antibody was from Cilag AG (Schaffausen, Switzerland). Pulmozyme was purchased from Roche (Basel, Switzerland). Allogeneic peripheral blood mononuclear cells (PBMCs, or feeder cells) were obtained from buffy coats from healthy donors. Solu Cortef (hydrocortisone sodium succinate) was obtained from the local hospital pharmacy. rhIFN-y (interferon gamma-1 b, Imukin) was from Boehringer-lngelheim GmbH (Ingelheim am Main, Germany). Complete medium (CM) consisted of RPMI-1640 with GlutaMAX, 25 mmol/L HEPES pH 7.2, 100 U/mL penicillin, 100 μg/mL streptomycin, Fungizone® (Bristol-Myers Squibb, New York, NY, USA) 1 ,25 g/ml supplemented with 10% HS and 6000 IU per ml_ of rhlL-2. Rapid expansion medium (RM) consisted of AIM-V medium, Fungizone® 1 ,25 μg/ml supplemented with 6000 lU/mL rhlL-2.

HLA-typing

Typing at the HLA-A locus was performed at the Copenhagen University Hospital at Herlev by PCR. Proliferation Assay for melanoma cells

In detail, after standard trypsinization, at day -1 melanoma cells were seeded at 5 x 104/well into 24 well plates and grown for 24 hours into standard medium. Thereafter, medium was exchanged with fresh standard medium +/- 100 lU/ml of IFN-y, for a total volume of 2 ml/well. Baseline control wells were trypsinized at day 0 with 50 μΙ_ of trypsin solution/well, 250 μΙ_ of standard medium with 0.05 μg/ml of propidium iodide (PI; from Sigma-Aldrich) to exclude dead cells was added into each well was added and the obtained suspension was counted under a standard rate for a constant amount of time (for 90 seconds at high flow rate) in a BD FACSCanto II flow cytometer equipped with BD FACS Loader carousel. After 72 hours of drug exposure, the other wells were trypsinized and the cells were counted after identical working conditions of the baseline control wells. Growth inhibition was calculated using the following formula: (T72-T0)/(K72-T0) 100 , where T72 is the cell count after 72 hours, TO is the cell count of the control well at time zero and K72 is the cell count of the control well (medium) after 72 hours. Control values were arbitrarily set to 100. Values below 0 indicate net cell loss while values between 0 and 100 indicated growth inhibition.

Example 1. Association of absolute number of tumour-reactive T cells and clinical response We analyzed the number of tumour-reactive CD8⁺ T cells contained in TIL products for patient infusion in a pilot ACT clinical trial initiated at our institution (clinicaltrials.gov identifier: NCT937625). Infusion products from four patients were tested against autologous melanoma cell lines, while from four additional patients an autologous cell line was not available. Since previous studies have shown that allogeneic cell lines are suitable for the analysis of TIL reactivity, for this last group of patients the reactivity of infusion products were tested against a panel of four to eight HLA-A matched allogeneic melanoma cell lines.

Previously, the analysis of infusion products for ACT from the first six patients treated in this trial had suggested a possible association between tumour-reactivity of TILs and achievement of a clinical response (Eva Ellebask et al., manuscript submitted). An updated analysis from eight patients evaluated to date confirmed the association of clinical response and higher absolute numbers of tumour-reactive CD8⁺ T cells infused (Figure 2).

These data point out the importance of tumour recognition as a prerequisite for clinical efficacy. Example 2. Generation of matched melanoma and TIL cultures

Tumour specimens obtained from subcutaneous or lymph node metastasis of twenty- nine patients with melanoma stage III or IV were received for processing. From twelve patients (41 %), we were able to generate a short term melanoma cell line and at least one clinical grade TIL culture from the same metastatic lesion. The mean age of these twelve patients at time of tissue acquisition was 52±21 years. Six specimens were from male patients, and six from female patients. Four patients were HLA-A2+. Example 3. MHC expression by short-term cultured melanoma cell lines

Previous studies have demonstrated a close correlation between melanoma lesions and corresponding expression of antigen-processing machinery components and class I and II MHC molecules on short-term cultured cell lines. Although all cell lines generated in our study expressed class I MHC molecules, their level of expression was not uniform and very low in one case (patient 3). Importantly, increased expression was achieved in all the cell lines by treatment with low dose IFN- γ although the increase was not uniform (Figure 5). 6 out of 12 (50 %) melanoma cell lines constitutively expressed class II MHC molecules. Treatment with IFN-γ led to increased expression of MHC II in 11 out of 12 (92 %) melanoma cell lines (Figure 5). One cell line (from patient 4) did not express class II MHC neither before nor after IFN- γ (Figure 5).

Hence, IFN- γ was able to induce expression of class I and II MHC molecules in short- term cultured melanoma cell lines.

Example 4. Ex vivo antitumour activity of CD8⁺ T cells in clinical grade TILs

The antitumour activity of CD8⁺ T cells expanded from TILs was evaluated by defining the frequency of cells expressing at least the two type 1 cytokines TNF-a and IFN-γ (Double Positive, DP) or these two cytokines and CD107a (Triple Positive, TP) upon stimulation with autologous cancer cells treated with or without low dose IFN- γ.

Stimulation with untreated target cells defined the "constitutive" response of TILs.

An increased antitumour response following IFN-γ treatment was detected in all the TILs analyzed, with frequency of responses varying greatly between different pairs (mean DP cells: 5.5±10.5 % , range 0.27-37.6 % of CD8⁺ TILs) (Figure 3a and Table 1). Interestingly, co-incubation with target cells pretreated with IFN- γ led to an increased frequency of DP cells in 10 out of 12 pairs, with a mean increase of 4.9±4.3 % (range 0.2-12.9 %) (Figure 3 and Table 1). Thus, in TIL products from most patients, pre-treatment of target cells with IFN- γ unleashed the responses of a large quantity of cells that were not able to recognize tumour/exert effector functions constitutively.

A similar trend was observed for production of CD107a, with increased frequency of TP cells in 9 out of 12 (mean TP cells in CD8⁺ TILs: 2.1±5.6 % versus 2.9±5.6 %, respectively for untreated cells or treated with IFN-γ (Figure 6), confirming the multifunctionality of these antitumour CD8⁺ T cells.

Of particular interest, responses towards a tumour cell line which showed very low constitutive expression of class I MHC increased from almost undetectable to high levels (patient 3, Figure 3b lower panel and Table 1).

In light of our data, an in vivo sensitization step of the cancer cells by IFN-γ

administration to cancer patients could increase clinically significant antitumour response upon adoptive transfer of cancer-specific CD8⁺ TILs. Especially for those patients bearing tumours with strongly impaired class I MHC expression.

CD4⁺ T cells recognize antigens presented in association with class II MHC molecules, whose expression is mostly restricted to professional antigen presenting cells which process exogenous tumour antigens and display them on their surface. However, a significant fraction (over 40 %) of human melanoma cells constitutively express class II MHC molecules (Mendez et al., 2009), and class II MHC-restricted peptides can also be processed endogenously via autophagy and displayed on the surface of the tumour cells allowing direct recognition of tumour antigens (Muranski and Restifo, 2008; Nuchtern et al., 1990).

Our data point out that clinical grade TIL cultures may actually contain a significant fraction of tumour specific Th1 polarized CD4⁺ T cells, and a combination of ACT with administration of IFN-γ could increase clinical responses at least in part based on a more proper exploitation of the anti-tumour functionality of CD4⁺ T cells comprised in unselected cellular products. The data suggest a synergism between IFN-γ and ACT.

Table 1 : Summary of antitumour responses of TILs stimulated with autologous tumour cells treated IFN-γ (+ IFN-γ) or left untreated (- IFN-γ)

Responses are expressed as percentage of TILs co-expressing TNF-a and INF-y. Limit of sensitivity was set at 0.01. NR: No Response; BR: Borderline Response. * more than one independent TIL culture was analysed, mean ± SD is shown.

Example 5. Ex vivo antitumour activity of CD4⁺ T cells in clinical grade TILs

The antitumour activity of CD4⁺ T cells expanded from TILs was evaluated by defining the frequency of DP cells in the same setting described earlier. A positive constitutive antitumour CD4⁺ Th1 response was detected in 4 out of 12 TILs analyzed (mean number of DP in responding cultures: 1.5±1.3 % , range 0.08-3.3 % of CD4⁺ TILs) (Figure 4 and Table 1). As expected, constitutive responses were detected only in pairs with MHC II+ tumours (Table 1 and Figure 5). IFN-γ pre-treatment induced an increase in frequency of CD4⁺ DP TILs in 6 out 12 pairs with a mean increase of 4.0±7.0 % (range 0.21±17.9 %). Of note, four of these pairs displayed responses at baseline (patient 1 , 2, 5 and 11) and two additional borderline responses (patient 8 and 9) (Figure 4 and Table 1). All the increased responses matched with increased expression of class II MHC (Figure 5). As expected, we did not detect any expression of CD107a by CD4⁺ T cells (representative CD8- population in Figure 6b).

Two pairs selected for high frequency of CD4⁺ DP TILs (patients 1 and 5, Table 1) were tested for IL-4 production by FACS along with TNF-a and IFN-γ. Production of IL- 4 could not be detected under these conditions, confirming the Th1 profile of these tumour specific cells (data not shown).

Example 6. Direct effects of IFN-γ on short-term cultured melanoma cell cultures

Previous reports have shown that IFN-γ treatment, especially when delivered at doses exceeding 1000 Ill/ml, might directly influence melanoma cell growth by inhibition of proliferation and induction of cell cycle arrest followed by a marginal induction of apoptosis (Kortylevski et al., 2004). To check whether the applied low dose of 100 lU/ml IFN-γ, able to increase tumour cell recognition, may also influence growth and viability of tumour cells, 9 short-term cultured melanoma cell lines were exposed to this dose for 72 hours. Analysis of cell growth revealed significant growth inhibition in 5/9 cell lines, with various degrees of inhibition observed and in one case (cell line from patient 3), cell loss was observed (Figure 7a). An increased frequency of apoptotic cells after 24 hours of exposure to the drug (higher percentage of subG events) was observed in only one cell line (from patient 3) (data not shown), corresponding to the same cell line where IFN-γ had shown lethal effects.

Under the same experimental conditions, the cell cycle distribution was not significantly influenced (Figure 7b). Thus, we concluded that short-term cultured melanoma cells exposed to 100 lU/ml may in some cases induce inhibition of cell growth, without significant induction of apoptosis. Example 7. Effects of IFN-γ on the cytotoxicity of TILs isolated from a recurrent tumour.

In one patient of our cohort, despite evidence of anticancer T cells persistence in blood, a relapsing tumor emerged more than a year after complete response to TILs (Figure 10A). In the cell line generated from the recurrent tumour, we observed a reduced tumour immunogenicity against the pre-infusion tumour, illustrated by MHC I down- regulation and reduced recognition by TILs, tumour antigen-specific T cells and peripheral blood mononuclear cells (PBMCs) (Figures 10B and 10C), which was reverted with IFN-γ (Figure 10F and 10G). Reduced recognition was not apparently due to increased tumour immune suppression (Figure 10D) or loss of target antigen (Figure 10E). This was reverted by addition of IFN-γ to the new TILs (Figure 10E). These data indicate that IFN-γ might be useful as rescue treatment to reactivate circulating anticancer T-cells.

References

Donia M, Junker N, Ellebaek E et al. (201 1) Characterization and comparison of "Standard" and "Young" tumour infiltrating lymphocytes for adoptive cell therapy at a Danish Translational Research Institution. Scand J Immunol. 201 1 Sep 28.

Dubinett SM, Kurnick JT, Kradin RL. (1989). Adoptive immunotherapy of murine pulmonary metastases with interleukin 2 and interferon-gamma. Am J Respir Cell Mol Biol. 1 (5):361-9 Dudley ME, Wunderlich JR, Yang JC et al. (2005). Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. J Clin Oncol. 23, 2346-57.

Kirkwood JM, Ernstoff MS, Trautman T et al. (1990). In vivo biological response to recombinant interferon-gamma during a phase I dose-response trial in patients with metastatic melanoma. J Clin Oncol 8: 1070-82.

Kortylewski M, Komyod W, Kauffmann ME et al. (2004) Interferon-gamma-mediated growth regulation of melanoma cells: involvement of STAT1 -dependent and STAT1- independent signals. J Invest Dermatol 122:414-22.

Li Z, Xu Q, Peng H et al. (2011). IFN-γ enhances HOS and U20S cell lines susceptibility to γδ T cell-mediated killing through the Fas/Fas ligand pathway. Int Immunopharmacol. 1 1 (4):496-503.

Maluish AE, Urba WJ, Longo, DL et al. (1988). The determination of an

immunologically active dose of interferon-gamma in patients with melanoma. J Clin Oncol 6:434-45. Mazzarella T, Cambiaghi V, Rizzo N et al. (2012). Ex vivo enrichment of circulating anti-tumor T cells from both cutaneous and ocular melanoma patients: clinical implications for adoptive cell transfer therapy. Cancer Immunol Immunother

;61 (8): 1 169-82 Mendez R, Aptsiauri N, Del Campo A et al. (2009) HLA and melanoma: multiple alterations in HLA class I and II expression in human melanoma cell lines from

ESTDAB cell bank. Cancer Immunol Immunother 58: 1507-15. Muranski P, Restifo NP (2009) Adoptive immunotherapy of cancer using CD4(+) T cells. Curr Opin Immunol 21 :200-08

Nuchtern JG, Biddison WE, Klausner RD (1990) Class II MHC molecules can use the endogenous pathway of antigen presentation. Nature 343:74-76.

Propper DJ, Chao D, Braybrooke JP et al. (2003) Low-dose IFN-gamma induces tumour MHC expression in metastatic malignant melanoma. Clin Cancer Res 9:84-92.

Queirolo P, Ponte M, Gipponi M et al. (1999). Adoptive immunotherapy with tumor- infiltrating lymphocytes and subcutaneous recombinant interleukin-2 plus interferon alfa-2a for melanoma patients with nonresectable distant disease: a phase l/ll pilot trial. Melanoma Istituto Scientifico Tumori Group. Ann Surg Oncol. 6(3):272-8.

Schiller JH, Pugh M, Kirkwood JM et al. (1996) Eastern cooperative group trial of interferon gamma in metastatic melanoma: an innovative study design. Clin Cancer Res 2:29-36.

Verdegaal EME, Visser M, Ramwadhdoebe TH et al. (201 1) Successful treatment of metastatic melanoma by adoptive transfer of blood-derived polyclonal tumor-specific CD4+ and CD8+ T cells in combination with low-dose interferon-alpha. Cancer

Immunol Immunother. 60(7):953-63.

Yee C, Thompson JA, Byrd D et al., (2002). Adoptive T cell therapy using antigen- specific CD8+ T cell clones for the treatment of patients with metastatic melanoma: in vivo persistence, migration, and antitumor effect of transferred T cells. Proc Natl Acad Sci U S A;99(25):16168-73.

Wu R, Forget MA, Chacon J, Bernatchez C, Haymaker C, Chen JQ, Hwu P, Radvanyi LG. (2012) Adoptive T-cell therapy using autologous tumour-infiltrating lymphocytes for metastatic melanoma: current status and future outlook. Cancer J. 18(2): 160-75.

Claims

c) expanding the lymphocytes,

d) administering Interferon γ to the mammal, and

e) administering the expanded lymphocytes to the mammal,

whereupon regression of the cancer is promoted.

The method according to claim 1 , wherein the Interferon γ is administered subcutaneously, intravenously, intramuscularly or intraarterially.

The method according to claim 2, wherein the Interferon γ is administered subcutaneously.

The method according to any of the preceding claims, wherein the Interferon γ is administered to the mammal as a single daily dose or as multiple daily doses.

The method according to claim 4, wherein the Interferon γ is administered to the mammal as a single daily dose.

The method according to any of the preceding claims, wherein the Interferon γ is administered at about 50 μg/m² to about 1000 μg/m² per day.

7. The method according to any of the preceding claims, wherein the Interferon γ is administered to the mammal for approximately one week before administration of the lymphocytes, such as more than 3 days, for example more than 4 days, such as more than 5 days, for example more than 6 days, such as about 7 days.

8. The method according to any of the preceding claims, wherein the cancer is a solid cancer.

9. The method according to any of the preceding claims, wherein the cancer is

selected from the group consisting of melanoma, ovarian, breast and colorectal cancer.

10. The method according to claim 9, wherein the cancer is melanoma.

1 1. The method according to any of the preceding claims, wherein the cancer is

metastatic.

12. The method according to any of the preceding claims, wherein the cancer is

recurrent.

13. The method according to any of the preceding claims, wherein the mammal is a human.

14. Interferon γ for use in a method of promoting regression of a cancer in a mammal, wherein said method comprises the steps of:

c) expanding the lymphocytes,

d) administering Interferon γ to the mammal, and

e) administering the expanded lymphocytes to the mammal.

15. The interferon γ for use according to claim 14 in a method according to any of claims 1 to 13.

16. A method of promoting regression of a recurrent cancer in a mammal, comprising administration of Interferon γ to the mammal, wherein said mammal has previously received immunotherapy treatment capable of inducing a cellular immune response.

17. The method according to claim 16, wherein said immunotherapy treatment

comprises adoptive cell therapy, such as autologous adoptive cell therapy.

18. Interferon γ for use in a method of promoting regression of a recurrent cancer in a mammal, wherein said mammal has previously received immunotherapy treatment capable of inducing a cellular immune response, such as adoptive cell therapy.

19. Use of Interferon γ for the manufacture of a medicament for the treatment of

cancer, wherein said treatment comprises autologous adoptive cell therapy of tumour-infiltrating lymphocytes.