Dosing regimens comprising administration of tumor infiltrating lymphocytes and checkpoint inhibitors

Abstract

A dosing regimen for the treatment of cancer comprising administering an in vitro amplified tumor infiltrating lymphocyte (TIL) to a mammal suffering from cancer on the 0 (TIL) day followed by administering a checkpoint inhibitor on the 0 (CI) day wherein the 0 (CI) day is in the range of 1 month to 5 months later than the 0 (TIL) day, for example, 6 weeks to 4 months later than the 0th (TIL) day, 8 weeks to 3 months later than the 0th (TIL) day, or about 2 months later than the 0th (TIL) day.

Description

Technical Field

[0001] The present invention relates to the use of a combination of tumor infiltrating lymphocytes and one or more checkpoint inhibitors for treating cancer (e.g., prostate cancer). Background Art

[0002] Cancer cells are characterized by numerous genetic mutations and epigenetic alterations that result in the production of a variety of cancer-specific antigens. These antigens are detected by T cells, which use them to distinguish precancerous and / or cancer cells from normal cells and to elicit a cancer-specific immune response. The magnitude and quality of the T cell-mediated immune response are often regulated by immune checkpoints, which can be defined as stimulatory and inhibitory molecules and / or molecular pathways that act to increase or decrease the magnitude of the response, respectively. Under normal physiological conditions, immune checkpoints are essential for preventing autoimmunity and protecting against tissue damage caused by pathogenic infections. However, cancer cells can exploit the dysregulation of immune checkpoint proteins to gain immune resistance.

[0003] One approach to triggering a T cell-mediated anti-tumor immune response is known as "checkpoint blockade," which refers to blocking or inhibiting immunosuppressive checkpoints utilized by cancer cells. Since many immune checkpoints are triggered by ligand-receptor interactions, these checkpoints may be blocked by antibodies or modulated by recombinant forms of the ligands and / or receptors.

[0004] Advanced prostate cancer is associated with a poor prognosis and remains incurable. With the exception of the FDA-approved therapeutic cancer vaccine Sipuleucel-T, effective immunotherapeutic approaches for prostate cancer remain challenging.

[0005] Immune checkpoint inhibitors (anti-PD-1 / anti-PD-L1 / anti-CTLA4), which are effective in different malignancies, have shown only limited activity in prostate cancer, in part because of the relatively low mutational burden of this disease. For example, response rates with pembrolizumab were only 5% and 3%.

[0006] Therefore, new immunotherapeutic strategies are urgently needed.

[0007] Over the past several decades, adoptive therapy using tumor-infiltrating T lymphocytes (TILs) has demonstrated remarkable efficacy in treating individual patients with different types of cancer, with the latest successful clinical case reported in 2018. Recognition of multiple individual neoantigens as well as shared tumor-associated antigens (TAAs) associated with enhanced tissue homing ability and potent immune effector functions contribute to the clinical efficacy of TILs in patients with solid tumors. TIL therapy has shown clinical benefit in patients with chemotherapy-refractory cancers such as metastatic melanoma, cholangiocarcinoma, renal cell carcinoma, colorectal cancer, cervical cancer, breast cancer, and ovarian cancer. Currently, only a few clinical studies of adoptive transfer of T cells for metastatic hormone-refractory prostate cancer (mHRPC) are listed on www.clinicaltrials.gov. Summary of the invention

[0008] The present invention provides a novel dosing regimen for treating cancer. The cancer is typically a cancer that has not responded effectively to monotherapy with a checkpoint inhibitor and / or a cancer with a relatively low mutation load. An example of such a cancer is prostate cancer.

[0009] In its broadest aspect, the present invention relates to a composition for use in treating cancer, and to a specific dosing regimen comprising administering to a mammal having cancer a composition comprising in vitro expanded tumor infiltrating lymphocytes (TIL) on day 0 (TIL), followed by administering a composition comprising a checkpoint inhibitor on day 0 (CI), wherein day 0 (CI) is in the range of 1 month to 5 months later than day 0 (TIL), such as 6 weeks to 4 months later than day 0 (TIL), 8 weeks to 3 months later than day 0 (TIL), or about 2 months later than day 0 (TIL).

[0010] In this context, use of the term "in vitro expanded tumor infiltrating lymphocytes" is meant to include compositions comprising in vitro expanded tumor infiltrating lymphocytes. Similarly, the term "check point inhibitor" includes compositions comprising a check point inhibitor.

[0011] TILs may be administered one or more times before treatment, and TILs may be administered one or more times and / or a checkpoint inhibitor may be administered one or more times after treatment.

[0012] Specifically, the present invention relates to a composition for treating cancer, comprising administering in vitro expanded tumor infiltrating lymphocytes (TIL-2) to a mammal suffering from cancer on day 0 (TIL-2), followed by administering a checkpoint inhibitor on day 0 (CI), wherein day 0 (CI) is in the range of 1 month to 5 months later than day 0 (TIL-2), for example, 6 weeks to 4 months later than day 0 (TIL-2), 8 weeks to 3 months later than day 0 (TIL-2), or about 2 months later than day 0 (TIL-2).

[0013] In the present context, the term "tumor infiltrating lymphocytes" or "TILs" or "TIL" is intended to refer to T lymphocytes that carry T cell receptors (TCRs) that are specific for and recognize tumor antigens. The term is similar to "tumor-reactive T lymphocytes". The term also encompasses T lymphocytes that carry TCRs that are specific for and recognize metastatic antigens, i.e., the term "tumor infiltrating lymphocytes" / "TILs" / "TIL" includes "metastatic infiltrating lymphocytes".

[0014] In the dosing regimen, it is important to administer TILs before the checkpoint inhibitors. It is contemplated that the immunotherapeutic effect elicited by TILs makes the cancer susceptible to treatment with checkpoint inhibitors. As seen in the examples herein, significant effects were achieved in patients with prostate cancer who underwent the dosing regimen described herein (see Figure 1 b and 1c).

[0015] The present invention also relates to a kit for treating cancer, the kit comprising

[0016] i) in vitro expanded tumor infiltrating lymphocytes, such as TIL-1, TIL-2 and / or TIL-3, and

[0017] ii) one or more checkpoint inhibitors,

[0018] Therein, the kit comprises two or more separate components, wherein the first component comprises one or more compositions comprising in vitro expanded tumor infiltrating lymphocytes, and the second component comprises one or more compositions comprising checkpoint inhibitors. DETAILED DESCRIPTION

[0019] The present invention provides a novel dosing regimen for treating cancer. The dosing regimen includes sequential administration of tumor infiltrating lymphocytes (TIL), in some cases after administration of checkpoint inhibitors, and in some cases before administration of checkpoint inhibitors. Tumor infiltrating lymphocytes are obtained by in vitro culture of lymphocytes from biopsies or body samples containing TIL from patients with cancer, and the tumor infiltrating lymphocytes (TIL) thus obtained are applied to the patient and obtained. The in vitro expansion of tumor infiltrating lymphocytes can be carried out in a culture medium containing one or more of IL-2, IL-15 and IL-21.

[0020] The simplest form of the dosing regimen involves the following steps:

[0021] i) administering in vitro expanded tumor infiltrating lymphocytes (TIL) to a mammal having cancer on day 0 (TIL), then

[0022] ii) administering a checkpoint inhibitor on day 0(CI), wherein day 0(Cl) is in the range of 1 month to 5 months later than day 0(TIL), such as 6 weeks to 4 months later than day 0(TIL), 8 weeks to 3 months later than day 0(TIL-2), or about 2 months later than day 0(TIL-2).

[0023] As mentioned above, it is important to administer TIL to a subject with cancer before starting treatment with a checkpoint inhibitor. The mutation frequency of many cancer types is relatively low, such as a somatic mutation frequency ( / Mb) less than 10 (Lawrence, MS, "Nature (Natrue)" 2013, 499, 214-8). Such cancers include rhabdoid tumors, Ewing's sarcoma, thyroid cancer, AML, medulloblastoma, glioblastoma, neuroblastoma, carcinoid tumors, prostate cancer, breast cancer and pancreatic cancer. The inventors have observed that such cancer types generally do not respond effectively to treatment with checkpoint inhibitors, but - as described herein - by involving pre-treatment of cancer with TIL, followed by treatment with a checkpoint inhibitor, an effective treatment regimen can be obtained. Treatment can be repeated as needed, and checkpoint inhibitors can be administered as needed as many times as needed, even after the last treatment with TIL.

[0024] As an example of the dosing regimen of the present invention, a dosing regimen comprising three administrations of TIL is given. It will be appreciated by those skilled in the art that more TILs may be administered before administration of TIL-1, and more TILs may be administered after administration of TIL-3.

[0025] Non-limiting examples of dosing schedules are given below. The time intervals for administration of TILs and checkpoint inhibitors and the time intervals for administration of TIL doses are universal and apply regardless of the number of doses of TILs or checkpoint inhibitors administered:

[0026] In vitro expanded tumor infiltrating lymphocytes (TIL-2) are administered to a mammal having cancer on day 0 (TIL-2), and then

[0027] ii) administering a checkpoint inhibitor on day 0(CI), wherein day 0(CI) is in the range of 1 month to 5 months later than day 0(TIL-2), such as 6 weeks to 4 months later than day 0(TIL-2), 8 weeks to 3 months later than day 0(TIL-2), or about 2 months later than day 0(TIL-2).

[0028] The dosing regimen may also include administering in vitro expanded tumor infiltrating lymphocytes (TIL-3) to the mammal on day 0 (TIL-3), wherein day 0 (TIL-3) is within the range of 1 to 14 days after day 0 (CI), such as 2 to 12 days after day 0 (CI), 5 to 12 days after day 0 (CI), or 7 days after day 0 (CI). Therefore, after the administration of the checkpoint inhibitor, it may be necessary to further treat with TIL. TIL can be administered even before the administration of TIL-2. Therefore, the dosing regimen may also include administering in vitro expanded tumor infiltrating lymphocytes (TIL1-1) to the mammal on day 0 (TIL-1), wherein day 0 (TIL-1) is within the range of 1 month to 4 months before day 0 (TIL-2), such as 6 weeks to 4 months before day 0 (TIL-2), 8 weeks to 3 months before day 0 (TIL-2), or about 2 months before day 0 (TIL-2).

[0029] The frequency of administration and the number of cells (TIL) administered depend on the effectiveness of the treatment. After treatment, markers specific to cancer types, such as PSA for prostate cancer, can be monitored. With respect to prostate cancer, PSA increases indicate the progression of cancer, and indicate that TIL and / or checkpoint inhibitors should be further used for treatment. Typically, TIL can be administered 1 to 10 times, such as 2 to 8 times, 2 to 6 times, 2 to 5 times, 3 to 4 times, such as 3 times. The time interval for TIL administration is 1 month to 6 months, such as 6 weeks to 6 months, 8 weeks to 5 months, 8 weeks to 4 months, 8 weeks to 3 months or about 2 months.

[0030] Typically, the date of administration of the second (third, fourth, etc.) dose of TIL can be determined by monitoring the plasma levels of markers specific to the cancer, tumor-associated antigens specific to the cancer, or neoantigens specific to the cancer, wherein the monitored time period begins after the administration of the first portion of TIL. When the level changes by 4 times or more, the second (third, fourth, etc.) dose of TIL is administered 1 day to 1 month (e.g., 1 day to 3 weeks, 1 day to 2 weeks, or 1 day to 7 days) after the change is observed. The change can be an increase or decrease in the level of the monitored marker, which depends on the type of cancer. In the case of prostate cancer, PSA levels are monitored, and an increase in PSA levels indicates progression of the disease, so the above change is an increase in PSA levels. A generally applicable marker is NY-ESO-1, and here, the change is also an increase in NY-ESO-1 antibody levels.

[0031] The number of TILs administered is usually 1x 10 8 Up to 1x 10 11 In the range of, for example, 1x 10 8 Up to 1x 10 10 , for example 1x10 9 Up to 1x 10 10 .

[0032] In the dosing regimen, the number of TILs administered can be increased during the treatment period, for example, the number can be increased from TIL-1 to TIL-2, and to TIL-3.

[0033] In addition to administering TIL, chemotherapeutic agents can also be administered. Such chemotherapeutic agents are usually administered 7 days to 1 day before administering TIL (e.g., TIL-1, TIL-2 and / or TIL-3). Examples of chemotherapeutic agents suitable for use include cyclophosphamide (CTX) and fludarabine. Such chemotherapeutic agents are considered to have a Treg down-regulation effect, which can down-regulate the cytotoxic potential of TIL.

[0034] In certain aspects, the chemotherapeutic agent is an agent that is normally used to treat the cancer, but is administered at a reduced dose compared to the dose normally used to treat the cancer.

[0035] The dosing regimen may also include administration of, for example, IL-2. IL-2 is typically administered 1 to 5 days after administration of TIL, for example, 1 to 3 days after administration of TIL. The dose of IL-2 is typically between 6 x 10 5 IU / kg body weight up to 5x 6x 10 6 IU / kg body weight, for example 2x 6x 10 5IU / kg body weight up to 8x 6x 10 5 IU / kg body weight, 3x 6x 10 5 IU / kg body weight up to 7x 6x10 5 IU / kg body weight, 4x 6x 10 5 IU / kg body weight up to 6x 6x 10 5 IU / kg body weight or 4x 6x 10 5 IU / kg body weight up to 5x6x10 5 IU / kg body weight.

[0036] In aspects of the invention, the subject suffering from cancer has not undergone chemotherapy for a period of at least 6 months prior to the initiation of the dosing regimen of the invention.

[0037] As demonstrated in the Examples, repeated administration of TILs can improve clinical outcomes by prolonging the phase of TIL-tumor interactions in vivo and reducing treatment-related toxicities compared to a single infusion.

[0038] like Figure 1 As shown in a, the novel dosing regimen may include one or more infusions of TIL. Typically, three infusions of TIL have achieved significant results. Therefore, the method of the present invention may include TIL-1, TIL-2 and TIL-3, wherein TIL-1 is the first part of the TIL to be infused, TIL-2 is the second part, and TIL-3 is the third part. Depending on the development (progress, regression or recurrence) of the disease, fewer infusions may be required, or more infusions may be required. In the case illustrated by the example, the presence of cancer disease markers (PSA for prostate cancer) in plasma or serum and / or the presence of specific tumor-associated antigens (e.g., NY-ESO-1) or new antigens is used to track the development of the disease.

[0039] Based on the relevant parameters for the particular cancer type, the progression of the disease can be followed and one skilled in the art will be able to determine when to stop treatment or when to repeat it.

[0040] The new dosing regimen is based on the observation that initial administration of TILs has some effect, but after a period of time the disease relapses (see Figure 1b), that is, only a short-term effect can be obtained. The short-term response may be due to too few TILs, impaired persistence of transferred T cells, or insufficient migration of transferred T cells to the tumor microenvironment. In order to improve the results and achieve a lasting response, more cells (TIL-2 and / or TIL-3) can be further infused. In addition, it seems that the dosing regimen involving first administering TIL (optionally repeated once or multiple times) and then administering checkpoint inhibitors is effective. It is considered that before starting treatment with checkpoint inhibitors, it is beneficial to treat with tumor-infiltrating lymphocytes with homing markers of cancer / tumors (e.g., NY-ESO-1). As described herein, the use of checkpoint inhibitors to treat, for example, prostate cancer has not been successful, but as reported herein, the treatment involving initial treatment with TIL and then administering checkpoint inhibitors is successful, and the patient has been symptom-free for 3 years.

[0041] The TILs used according to the present invention may be autologous or allogeneic.

[0042] Checkpoint inhibitors

[0043] As mentioned above, in the novel dosing regimen, TILs will be used in combination with checkpoint inhibitors.

[0044] Checkpoint inhibitor therapy is a type of cancer immunotherapy. Checkpoint inhibitors target immune checkpoints, key regulators of the immune system that, when stimulated, can suppress the immune response to immune stimulation. Checkpoint therapy can block inhibitory checkpoints, thereby restoring immune system function. The first anticancer drug to target immune checkpoints was ipilimumab, a CTLA4 blocker.

[0045] Currently approved checkpoint inhibitors target CTLA4, PD1, and PD-L1 molecules. PD-1 is a transmembrane programmed cell death 1 protein (also known as PDCD1 or CD279), which interacts with PD-L1, PD-1 ligand 1. PD-L1 on the surface of cells binds to PD-1 on the surface of immune cells, thereby inhibiting immune cell activity. One of the functions of PD-L1 is to play a key regulatory role in T cell activity. It seems that cancer-mediated upregulation of cell surface PD-L1 can inhibit T cells that might otherwise attack. Antibodies that bind to PD-1 or PD-L1 and thus block the interaction can allow T cells to attack tumors.

[0046] Several immune checkpoints, either alone or in combination, are relevant in enhancing T cell-mediated anti-tumor immune responses. These include, but are not limited to, cytotoxic T lymphocyte-associated antigen 4 (CTLA4, also known as CD152), programmed cell death protein 1 (PD-1, also known as CD279), PD-1 ligand 1 (PD-L1, also known as B7-H1 and CD274), PD-1 ligand 2 (PD-L2, also known as B7-DC and CD-273), T cell membrane protein 3 (TIM3, also known as HAVcr2), adenosine A2a receptor (A2aR), lymphocyte activation gene 3 (LAG3, also known as CD 223), B7-H3 (also known as CD276), B7-H4 (also known as B7-S1, B7X and VCTN1), 2B4 (also known as CD244), and B and T lymphocyte attenuator (BTLA, also known as CD272).

[0047] Of particular interest are immune checkpoint inhibitors selected from PD-1 inhibitors (e.g. nivolumab, pembrolizumab or cemiplimab), PD-L1 inhibitors (e.g. atezolizumab, avelumab or durvalumab) and CTLA-4 inhibitors (e.g. ipilimumab).

[0048] Furthermore, other examples of relevant immune checkpoints can be found in the scientific and patent literature and are also within the scope of the present invention.

[0049] Although immune checkpoint inhibition can be used to enhance T cell-mediated anti-tumor immunity, the inventors believe that combining immune checkpoint inhibition with one or more complementary mechanisms to further enhance T cell activation will produce better anti-tumor effects.

[0050] In vitro expansion of TILs

[0051] Generally, TILs obtained from a subject suffering from cancer can be expanded using a variety of different methods.

[0052] A method involves the use of three different cytokines, IL-2, IL-15 and IL-21. A method may also involve the use of IL-15 and IL-21, i.e. without the use of IL-2, or a method may involve the use of IL-2. When using IL-2, IL-15 and IL-21, the method involves two stages, wherein the first stage i) is to obtain a culture containing a relatively high proportion of TIL (particularly CD8+T lymphocytes). In this stage, TIL survives and divides. The starting material of the first stage can be a mixture of lymphocytes and antigen presenting cells provided by a biopsy or body sample from a subject suffering from cancer.

[0053] T lymphocytes to be expanded in culture can be obtained from the subject to be treated, i.e. the resulting TIL for administration can be autologous. However, T lymphocytes can also be obtained from a source other than the subject to be treated, for example, another subject with cancer. In this case, the receptor and the amplified TIL are preferably immunocompatible, or the receptor has immune tolerance to the amplified TIL. In one method, the first stage is started by adding a cytokine mixture containing IL-2, IL-15 and IL-21 to promote cell division and prevent cell death by cytokine receptor stimulation of TIL. In another method, only IL-2 is used to stimulate TIL by IL2 receptor. Cytokine mixtures or IL-2 are used alone to promote the specific activation and growth of CD4+ helper lymphocytes and CD8+ lymphocytes. This specific activation for a certain tumor antigen enables TIL to have a therapeutic effect when applied to a cancer patient with the same tumor type as that activated by TIL.

[0054] The second stage ii) aims to clone the TILs from this stage. In the second stage, the culture medium containing the cytokine mixture or IL-2 is supplemented with human serum, feeder cells, anti-human CD3 or CD28 antibodies.

[0055] The amplification of TIL in stage i) and stage ii) will usually be carried out in a suitable culture medium. Preferably, serum-free culture medium or autologous serum is used to avoid the risk of disease transmission to the patient. Examples of suitable standard culture medium include AIMV culture medium, RPMI 1640, DMEM and MEM. However, other culture mediums may also be used, including a suitable mixture of amino acids, steroids, vitamins, growth factors, other cytokines and minerals.

[0056] In the example of this article, TIL-1 was obtained by expanding TIL using IL-2, IL-15 and IL-21. The combination of these three cytokines resulted in a high percentage of CD8+ cytotoxic T cells (78%-95%) and a relatively low number of CD4+ T cells (11%-17%) expansion, most of which were effector memory T cells (CD45RA-CCR7). In addition, this culture method does not promote the growth of Tregs.

[0057] Therefore, the characteristics of TIL cell populations are:

[0058] i) The content of CD8+ cytotoxic T cells is in the range of 70% to 98%, such as in the range of 75% to 97% or in the range of 78% to 95%, based on the total number of T cells.

[0059] ii) The content of CD4+ T cells is in the range of 0.5% to 20%, such as in the range of 1% to 15%, or in the range of 1.5% to 12%, based on the total number of T cells.

[0060] iii) the content of CD8-positive effector memory T cells (CD45RA-CCR7+) is in the range of 45% to 80%, such as 45% to 78%.

[0061] IL-2, IL-15 and IL-21 are members of the cytokine family, each with four alpha-helical bundles. IL-2 has a key role in key functions of the immune system, tolerance and immunity, primarily through its direct effects on T cells. IL-2 induces T cell proliferation and differentiation into effector and memory T cells.

[0062] IL-15 is a cytokine that is structurally similar to IL-2. Like IL-2, IL-15 binds to and signals through a complex composed of the IL-2 / IL-15 receptor β chain. IL-15 induces T cell activation and proliferation, particularly CD8+ T cells (30), and also provides survival signals to maintain memory cells in the absence of antigen, promotes CD8+ T cells, and activates monocytes. IL-15 appears to drive the proliferation of immune effector T cells to some extent while protecting them from tumor-associated immunosuppression.

[0063] IL-21 is a cytokine with a powerful regulatory effect on cells of the immune system, including natural killer (NK) cells and cytotoxic T cells. IL-21 enriches central memory T cells with a CD28+Cd127hiCD45RO+ phenotype and enhances the cytotoxicity of cytotoxic T cells. IL-21 can activate T cells in the early stages of their differentiation and maturation.

[0064] As used herein, "interleukin 2" or "IL-2" refers to human IL-2 as defined in SEQ ID NO:9 and functional equivalents thereof. Functional equivalents of IL-2 include related substructures or fusion proteins of IL-2 that retain the function of IL-2. Thus, the definition of IL-2 includes any protein having a sequence identity of at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% to SEQ ID NO:9. Recombinant human IL-2 produced in E. coli as a single non-glycosylated polypeptide chain of 134 amino acids and having a molecular weight of 15 kDa is commercially available from Prospec in lyophilized form under the catalog number CYT-209.

[0065] As used herein, "interleukin 15" or "IL-15" refers to human IL-15 and its functional equivalents. Functional equivalents of IL-15 include related substructures or fusion proteins of IL-15 that retain the function of IL-15. Therefore, the definition of IL-15 includes any protein having a sequence identity of at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% with SEQ ID NO: 10. Recombinant human IL-15 produced in Escherichia coli as a single non-glycosylated polypeptide chain with 114 amino acids (and one N-terminal methionine) and a molecular weight of 12.8 kDa is commercially available from Prospec in lyophilized form under the catalog number CYT-230.

[0066] As used herein, "interleukin 21" or "IL-21" refers to human IL-21 and its functional equivalents. Functional equivalents of IL-21 include related substructures or fusion proteins of IL-21 that retain the function of IL-21. Therefore, the definition of IL-21 includes any protein having a sequence identity of at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% with SEQ ID NO: 11. Recombinant human IL-21 produced in Escherichia coli with a single non-glycosylated polypeptide chain of 132 amino acids and a molecular weight of 15 kDa is commercially available from Prospec in lyophilized form under the catalog number CYT-408.

[0067] As used herein, a "peptide" may consist of any number of any type of amino acids, preferably naturally occurring amino acids, which are preferably linked by peptide bonds. In particular, the peptide comprises at least 3 amino acids, preferably at least 5, at least 7, at least 9, at least 12 or at least 15 amino acids. However, preferably, the peptide used according to the present invention has a length of no more than 500 amino acids, more preferably, a length of no more than 300 amino acids; even more preferably, a length of no more than 250 amino acids.

[0068] In addition to cytokines, the culture medium for expanding TILs may contain various factors, such as growth factors, such as those commonly used for lymphocyte expansion.

[0069] TILs expanded by one / more of the above methods all show reactivity against tumor-associated antigens. In particular, as shown in the examples therein, TILs expanded from cells obtained from a biopsy of a subject with prostate cancer show reactivity against NY-ESO-1. As described herein, the peptide group used to identify TIL specificity does not represent the full range of TAAs and new antigens expressed in tumors. T cells that respond to other target antigens expressed on autologous tumors (e.g., MAGE) may contribute to clinical outcomes.

[0070] Tumor-associated antigens (TAA)

[0071] Tumor-associated antigens are antigenic substances produced in tumor cells that elicit an immune response in the host. TAAs are useful tumor markers for identifying tumor cells by diagnostic tests and are potential candidates for cancer therapy.

[0072] In this context, TAA is an antigen presented by MHC I or MHC II molecules or non-classical MHC molecules on the surface of tumor cells. As used herein, TAA includes tumor-specific antigens, which are present only on the surface of tumor cells and not on the surface of normal cells.

[0073] Tumor-associated antigens are antigens that are associated with cancer diseases.

[0074] In this article, antigen is any structural substance, which is used as a target for adaptive immune response receptors, TCR or antibodies, respectively. In particular, antigens are proteins, polysaccharides, lipids or their substructures, such as peptides. Lipids and nucleic acids can have antigenicity when combined with proteins or polysaccharides.

[0075] TAAs useful in the present invention include, for example, cancer-testis antigens, such as MAGE-A1, MAGE-A3, MAGE-A4, NY-ESO-1-, PRAME, CT83, SSX3, NY-ESO-1.

[0076] Cancer-testis antigens are a group of proteins united by their importance in development and cancer immunotherapy. Normally, the expression of these proteins is restricted to male germ cells in adult animals. However, in cancer, these developmental antigens are frequently re-expressed and can serve as loci for immune activation.

[0077] Other TAAs for use according to the present invention include survivin, GPI, EGRvrlll, tyrosinase tumor antigen, tyrosinase related protein (TRP)-I, TRP-2, VEGFR-2, MAGE protein family members (can be found in melanoma), telomerase, p53, HER2 / neu, mesothelin, carcinoembryonic, VEGF, CAMPATH 1-antigen, CD22, CA-125 (can be found in ovarian cancer), MUC-1 (can be found in breast cancer), epithelial tumor antigen (can be found in breast cancer), mucin-1, alpha-1-fetoprotein (can be found in germ cell tumors or hepatocellular carcinoma), PSMA, RAS or substructures or fragments thereof. Other examples are peptides derived from single tumor mutant proteins PRPF8, TRPS1 and androgen receptor splice variant-12.

[0078] The particular suitable TAA for use according to the present invention depends on the type of cancer to be treated.

[0079] The fragment of TAA is in particular a peptide. Such a peptide may be, for example, a peptide comprising at least eight consecutive amino acids having an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.

[0080] SEQ ID NO: 5 is the amino acid sequence of the known tumor-associated antigen NY-ESO-1. SEQ ID NO: 6 is the amino acid sequence of the known tumor-associated antigen survivin. SEQ ID NO: 7 is the amino acid sequence of the known tumor-associated antigen mesothelin.

[0081]

[0082]

[0083]

[0084] NY-ESO-1 is a highly immunogenic cancer-testis antigen that induces both cellular and humoral immune responses in a high percentage of patients with advanced NY-ESO1-expressing tumors and is therefore considered a promising target for cancer immunotherapy. Antibodies against NY-ESO-1 in serum have been found to increase with disease progression and decrease with disease regression, making this antibody a useful biomarker for monitoring disease activity and response to treatment. Multiple studies have also shown that patients with pre-existing NY-ESO-1 antibodies gain additional clinical benefit from immune checkpoint blockade. Adoptive transfer of NY-ESO-1-specific T cells has improved clinical response rates in patients with melanoma, synovial cell sarcoma, and multiple myeloma.

[0085] Here we report complete and durable remissions in patients with advanced prostate cancer treated with ex vivo expanded tumor-infiltrating lymphocytes (TILs) that recognized NY-ESO-1 and multiple mutant tumor antigens and may be associated with antitumor immunity.

[0086] Other aspects of the invention

[0087] The present invention also provides a dosage regimen for use according to any one of the preceding claims, wherein the TIL has been obtained in vitro by the following steps:

[0088] i) providing a tumor or tumor metastasis sample obtained from the mammal;

[0089] ii) isolating TILs from the sample;

[0090] iii) culturing the TILs in a medium containing IL-2, IL-15 and / or IL-21,

[0091] iv) Add CD3 antibody and feeder cells to the culture medium.

[0092] Also disclosed is a kit for treating cancer, the kit comprising

[0093] i) in vitro expanded tumor infiltrating lymphocytes (TIL), and

[0094] ii) one or more checkpoint inhibitors,

[0095] Therein, the kit comprises two or more separate components, wherein the first component comprises one or more compositions comprising in vitro expanded tumor infiltrating lymphocytes, and the second component comprises one or more compositions comprising checkpoint inhibitors.

[0096] The kit may further comprise a third component comprising a composition comprising a chemotherapeutic agent.

[0097] The first component may include a composition comprising TIL-1, TIL-2 or TIL-3, or may include two compositions, a first composition comprising TIL-1 and a second composition comprising TIL-2.

[0098] The first component may include two compositions, the second composition comprising TIL-2 and the third composition comprising TIL-3, or it may include three compositions, the first composition comprising TIL-1, the second composition comprising TIL-2 and the third composition comprising TIL-3.

[0099] The kit may also include instructions for use.

[0100] Discussion of Example Results

[0101] Cell-based cancer immunotherapy is a viable approach for the treatment of patients with advanced malignancies, which can produce durable clinical responses. Here we report the clinical and immunological efficacy of ex vivo expanded TILs in patients with metastatic prostate cancer. Three administrations of TILs with relatively low cell numbers resulted in complete tumor regressions that are currently persisting for 3 years. Compared with other groups working on TIL therapy, we used a different strategy in TIL expansion, application mode and amount, and conditioning regimen, which may have contributed to this remarkable result.

[0102] First, we used a modified culture method for the in vitro isolation and expansion of TILs, using a culture medium containing IL-2, IL-15, and IL-21 and a closed perfusion bioreactor system. We infer that these cytokines combined with the culture procedure resulted in the expansion of a high percentage of CD8+ cytotoxic T cells (78%-96%) and a relatively low number of CD4+ T cells (11%-1.7%), with most of the TILs being effector memory T cells (CD45RA-CCR7+). In addition, this culture method did not promote the growth of Tregs (<0.13%).

[0103] Second, a modified preparatory chemotherapy regimen, different from that used in the TIL study reported by the National Cancer Institute, used a single low-dose infusion of cyclophosphamide, which was designed to reduce regulatory T cells without having a greater impact on overall lymphocyte depletion. A single low dose was given one day (TIL-1) and four days (TIL-2 and TIL-3) after the infusion of TIL, which helped to significantly reduce treatment-related toxicity compared with the widely used high-dose cyclophosphamide and fludarabine regimen with considerable side effects. A single dose of cyclophosphamide mediated mild lymphopenia and moderate neutropenia, but did not cause complete lymphocyte depletion in patients. To protect the infused TIL from toxic effects, we administered cyclophosphamide 4 days before TIL-2 and TIL-3 transfer.

[0104] We decided to use three escalating doses of relatively small numbers of TILs (TIL-1: 1.4x10 9 T cells; TIL-2: 2.0x 10 9 T cells; TIL-3: 8x 10 9 T cells) are administered repeatedly, rather than a single high-dose transplant of several times 10 10 Our therapeutic approach using repeated transfers of TIL preparations may improve clinical outcomes by prolonging TIL-tumor interactions in vivo compared with a single infusion.

[0105] We observed PSA and radiological responses after the first and second TIL treatments, which lasted for 5 and 7 weeks, respectively, indicating that TIL treatment had a transient efficacy. This partial and non-durable response could be due to the low number of TILs, impaired persistence of the transferred T cells, or insufficient migratory potential of the transferred T cells to the tumor microenvironment. To improve the results, a significantly higher number of cells was infused in TIL-3 (4 times that of TIL-2). In addition, we combined TIL-3 treatment with peri-interventional pembrolizumab. Compared with the first and second TIL treatments, PSA values ​​completely decreased to <0.01 μg / L and remained negative for 3 years so far. Closely correlated with the decrease in PSA, the high NY-ESO-1 serum antibody titers before TIL continued to decrease during TIL treatment, accompanied by disease regression.

[0106] We were able to identify specificity for NY-ESO-1 in each of the three TIL transplants. Additional TIL specificity for peptides derived from individual tumor mutant proteins, PRPF8, TRPS1, and androgen receptor splice variant-12, was identified in all three TIL preparations. AR-V12 induced ligand-independent expression of prostate-specific antigens (KLK-3, KLK-2, FOLH1), overexpression of which was observed at the mRNA level. The corresponding proteins represent potential immune targets, and their targeting may contribute to the durable clinical response to treatment in this patient. The peptide panels used to identify TIL specificity do not represent the full range of TAAs and neoantigens expressed in the tumor. T cells reactive to other target antigens, such as MAGE, which are also expressed in the tumor, may also contribute to the clinical outcome. Nevertheless, it seems unlikely that the quantity, quality, and specificity of TIL-3 solely contributed to the durable outcome. It is also unlikely that the tumor response and clinical improvement can be attributed solely to immune checkpoint blockade with pembrolizumab for the following reasons. First, we looked at clinical and radiological responses after TIL-1 and TIL-2 treatment without pembrolizumab. Second, the patients' tumors were classified as exhibiting MSS (microsatellite stability) and low TMB, and third, low PD-L1, making them unlikely to respond to immune checkpoint blockade alone.

[0107] In this study, we demonstrated that prostate cancer-derived TILs can be successfully isolated and efficiently expanded in vitro. TILs are reactive to NY-ESO-1 and multiple individual tumor antigens. TIL therapy combined with immune checkpoint blockade may help overcome PD-1 / PD-L1-mediated T cell suppression in the tumor microenvironment, resulting in complete and durable tumor responses. With regard to different immune escape mechanisms, TIL therapy targeting multiple tumor antigens may have advantages over monospecific immunotherapy strategies such as cancer vaccines or CAR-T cell therapy.

[0108] In conclusion, adoptive transfer of TILs is a promising immunotherapeutic approach also for tumors with low TMB, such as mHRPC. Further investigation in larger patient cohorts in clinical studies, including precise analysis of the tumor antigen-and T-cell repertoire, is warranted.

[0109] Overview

[0110] It will be appreciated that any features and / or aspects discussed above in conjunction with the compounds according to the invention apply by analogy to the methods described herein.

[0111] The following figures and examples are provided below to illustrate the present invention. They are intended to be illustrative and should not be construed as limiting in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0112] Figure 1.TIL treatment and its correlation with immune and clinical responses. a: Treatment plan. The time point of each TIL administration dose was defined as day 0. Patients received conditioning restorative non-myeloablative lymphodepleting chemotherapy (CTX) with cyclophosphamide (60 mg / kg) on ​​day 1 (TIL-1) and cyclophosphamide (30 mg / kg) on ​​day 4 (TIL-2 and TIL-3), followed by intravenous (iv) infusion of TIL and administration of IL-2 (aldesleukin) at 600.000 IU / kg. IL-2 was infused 5 times every 12 hours starting 16 hours after each TIL infusion. In the third TIL treatment (TIL-3), four doses of pembrolizumab (1 mg / kg) were administered on days -7, +15, +59 and +80, respectively. The first and second TIL infusions expanded TILs with IL-2, IL-15, and IL-21, while the third TIL infusion was expanded only with IL-2. b: PSA values: Shortly after the first TIL treatment, PSA dropped from 33μg / l to 18μg / l within 5 weeks and then rose rapidly to 34μg / l. After the second TIL treatment, PSA dropped to 5μg / l within 7 weeks and then rose again to a maximum level of 35μg / l. After the third TIL treatment, PSA levels continued to drop to <0.01μg / l within 8 weeks and have been undetectable since then. c: NY-ESO-1 serum antibodies. The patient showed high pre-TIL treatment antibodies. During and after TIL treatment, the antibodies continued to decrease and are now only detectable at residual levels. OD values ​​are shown as 1:400 serum dilution. () Cut-off OD in healthy donor serum. d: Imaging. MRI / CT images were obtained 3 months and 1 day before TIL-1 treatment (Pre-TIL), 2 weeks and 6 weeks after TIL-1 and TIL-2, and 1 day before TIL-3 treatment (Pre-TIL-3). Eight weeks after TIL-3, the tumors regressed significantly. PET-CT / CT showed complete regression of the tumor mass 1 year after TIL-3 (PET / CT). To date, the patient continues to show a complete response 3 years after TIL-3 treatment.

[0113] Figure 2 : TIL specificity. IFN-γ production by TIL was measured by ELISA. a: Specific reactivity to NY-ESO-1 was detected in all three TIL infusions, especially to the HLA-A2 restricted epitope NY-ESO-1 p157-165, NY-ESO-1 20mer peptides p81-100 and p91-110, and NY-ESO-1 24mer peptide p119-143 ( Figure 2 a). In standard chromium-51 (Cr 51) release assay, TIL showed specific cytotoxicity against tumor cells expressing NY-ESO-1 / HLA-A2 ( Figure 2 b). The reactivity of several mutant peptides was also demonstrated: KKRCLFRSF (SEQ ID NO: 12) derived from the gene PRPF8 (TIL-1, TIL-2, TIL-3), YLGEITYPF (SEQ ID NO: 15) derived from the gene TRPS1 (TIL-1, TIL-3), FVSGKYKCL (SEQ ID NO: 17) derived from the gene ZNF512 (TIL1, 3), NQVVQASMEK (SEQ ID NO: 18) derived from the gene GFM1 (TIL 2, 3), CPSKPSPLV (SEQ ID NO: 13) derived from the gene SGIP1 (TIL 3), and the peptide VKWAKALPDCERAAS (SEQ ID NO: 21) derived from a portion of the unique C-terminus of the V12 splice variant AR-V12 (TIL1, 2, 3). Given that TIL-1 and TIL-2 infusions were generated from the same pre-expansion cultures, these results suggest that the immunophenotype of TILs consisted of NY-ESO-1-specific T cells in all three TIL infusions and of four and six mutation-specific T cells in TIL1 / 2 and TIL3, respectively ( Figure 2 c). TIL-3-infused T cells were also analyzed for recognition of wild-type peptides derived from genes PRPF8 and TRPS1, respectively. The mutant peptides produced significantly more IFN-γ than the wild-type peptides ( Figure 2 d).

[0114] Figure 3 :TIL phenotype and function. TIL are mainly CD8 (a), and most of the CD8 are effector memory (EM) T cells (b). IFN-γ release of TIL after 24 hours of stimulation with OKT3 (c). PMA induces CD107a expression in TIL, showing cytotoxic potential (d). CM = central memory T cells, EMRA = terminal effector memory T cells.

[0115] Materials and methods

[0116] TIL preparation: isolation, culture and expansion

[0117] Surgical tumor biopsies were performed in June 2018 (TIL-1 and TIL-2 starting material) and January 2019 (TIL-3). Tissue samples were cut into approximately 1 mm 3The cells are cultured in small pieces and placed in a GMP-compliant and fully closed perfusion bioreactor system, which runs on a platform consisting of a GMP Bredder, a control unit and the associated software. TIL expansion begins in a 30MM perfusion bioreactor (stage 1) and continues in a 500MM perfusion bioreactor (stage 2). The culture process is automatically controlled by an algorithm based on pH, hyperoxia pO2 and temperature, providing fresh medium to the growing cells. Cellgenix medium containing 10% AB human serum and provided growing TILs isolated from tumor samples were cultured in medium containing IL-2 (1000 IU / mL), IL-15 (180 IU / mL) and IL-21 (1 IU / mL) (Miltenyi, Bergisch Gladbach, Germany), first supplemented with human serum (10%), anti-human CD3 antibody (clone OKT3, Miltenyi) and allogeneic 55 Gy irradiated feeder cells (1x10 6 Cellgro medium containing 100 μg / mL of ... Count the cells routinely and estimate the glucose and lactate concentrations. When the density is approximately 1-3 x 10 9When TIL, TIL is transferred to a disposable 500M perfusion bioreactor through a mesh port by gravity to separate TIL from tissue material. At this stage, continue to feed automatically with culture medium supplemented with 10% AB human serum and interleukin until TIL is harvested. Therefore, there is no additional stimulation to cause changes in the phenotype formed in stage 1. Wash the cells twice with 0.9% saline solution, resuspend in 5% albumin solution, and transfer to an infusion bag. The bag is transported to the patient by a biological sample processing device at an ambient temperature of 15°C-22°C. The spare TIL sample (if any) is suspended in 90% human AB serum containing 10% dimethyl sulfoxide (DMSO) for storage in liquid nitrogen.

[0118] Flow cytometry and functional characterization of TILs

[0119] TIL phenotype, Treg cells, and CD107a induction were determined using Beckman Coulter DURAClone IM T cell subsets and DuraClone IM Treg tube kits. CD107a induction was performed using Beckman Coulter DURActive 1 to activate cells and stain with anti-human CD107a PE antibody (clone H4A3), anti-human CD3 PE-Cy7, anti-human CD4 V450, and anti-human CD8α APC-Cy7 antibodies. Event acquisition was performed in Beckman Coulter's CytoFlex flow cytometer. IFN-γ production was measured after 24 hours of stimulation of TIL with OKT3, and cytokine quantification of culture supernatants was performed by enzyme-linked immunosorbent assay (ELISA).

[0120] Immunohistochemistry (IHC)

[0121] Tumor tissue sections were cut, dewaxed and treated with 3% H2O2 to block endogenous peroxidase activity and "demask" for antigen retrieval. Primary monoclonal mouse anti-NY-ESO-1 (clone E978) provided by the Monoclonal Antibody Core Facility (MACF) of the Sloan Kettering Institute for Cancer Research diluted in antibody diluent was added for 30 minutes. The sections were then incubated with horseradish peroxide-conjugated ENVision for 30 minutes. The final reaction can be seen by incubating with diaminobenzidine + substrate-chromogen and then counterstaining the sections with hematoxylin 26 Tissue from testis was used as positive control.

[0122] IFN-γ ELISpot

[0123] PBMCs were isolated by Ficoll / Hypaque density-gradient centrifugation (GE Healthcare) and stored frozen in FBS with 10% DMSO until analysis. NY-ESO-1, neoantigen short and long peptides, and a CEF-MHC class I control peptide pool (PANATEcs) were plated at 1.2 x 10 6 PBMCs were pre-stimulated in vitro with 10 cells / well. After a round of restimulation on day 7, specific recognition of peptides pulsed on autologous or HLA-matched allogeneic antigen presenting cells (APCs) (e.g., autologous DCs and / or EBV-transformed B cells, T2) by pre-sensitized T cells was analyzed in IFN-γ ELISpot between days 10 and 12. The reaction was considered positive if the number of spots in the peptide-exposed wells was twice or more the number of spots in the unstimulated control wells and there were at least 10 peptide-specific spots / 25,000 T cells after subtracting the background spots.

[0124] IFN-γ ELISA

[0125] TIL or PBMC were incubated with peptides (1 μg / ml) in 200 μl TCM in round-bottom 96-well microtiter plates at 37°C, 5% CO2 for 3-7 days. Culture supernatants (100 μl / well) were harvested and analyzed for IFN-γ production in flat-bottom 96-well plates using the IFN-γ ELISA kit (#3420-1A-6) from Mabtech AB according to the manufacturer's instructions.

[0126] Nucleic acid isolation, whole exome sequencing, and neoepitope selection

[0127] Genomic DNA from tumor tissue and corresponding normal blood was purified by DNeasy Blood and Tissue Kit (Qiagen, Cat#: 69504). All DNA isolations included an optional RNAse-A digestion step. Ion TorrentAmpliSeq TM Exome RDY Kit (Thermo Fisher Scientific, Carlsbad, CA, USA, Cat#: A38264) and Ion AmpliSeq TM Library Preparation Kit plus (Thermo Fisher Scientific, Cat#: 4488990) was used to enrich DNA exome sequences from tumor and normal DNA. TMThe barcoded library was quantified using a quantification kit (Thermo Fisher Scientific, Cat# 4468802). Tumor and normal DNA derived libraries were adjusted at a molecular ratio of 2:1. TM Software 5.6 and 200bp OT2 kit in IonGeneStudio using Chip 540 TMSequencing was performed on the S5 System, providing 80 to 90 million reads per exome pair. Reads were aligned to the human genome reference sequence (hg19) using the integrated torrent suite algorithm. Typical average coverage was 120× for tumors and 60× for normal DNA. Output BAM files were screened with Ion Reporter software using the AmpliSeq Exome Tumor-Normal Pairs workflow to identify point mutations, small insertions, deletions, and stop codons, with filters focused on Ion AmpliSeqExome HiQ regions. Minimum requirements for mutations to be included in further analysis were: more than 49× coverage in tumor tissue, more than 19× coverage in normal DNA, mutation frequency >10% in tumor, and no single affected read in the accompanying normal DNA sample. The corresponding protein sequence was translated by an algorithm developed in-house for SNPs, which allowed the construction of 31-mer wild-type and mutant peptides, respectively, by placing the mutation in the center of the sequence. Indels were translated individually using the "codong_change.pl" function in the ANNOVAR software, which was also used to annotate mutations and exclude variants that were significantly present in the human genome database (kaviar_20150923, genomic variations in 64K exomes). To evaluate expressed genes, total RNA was extracted from tumor sections using the RNeasy FFPE kit (Qiagen, Cat#: 73504), and transcribed, amplified, and cyanine-3 stained according to the Gene Expression FFPE Workflow Guide (Agilent Technologies, Cat#: G4112-90000). The stained cDNA was hybridized with a human GE 4x44 v2 microarray slide (Agilent, Cat#: G4845) and scanned on a DNA microarray scanner (Agilent, Cat#: G2505C). Peptides carrying mutations corresponding to higher mRNA expression are conducive to downstream selection. HLA typing (HLA A / B / C and HLA-DRB1 loci, at least 4-bit resolution) was performed using EDTA blood at the Institute for Medical Diagnostics (IMD) in Berlin-Potsdam, Germany. 9-mer peptides carrying putative HLA class I neoepitopes were selected by prediction scores using netMHC-4.0 and IEDB MHC-I--2.17 software packages.For HLA class II loci, 15-mer to 17-mer peptides were selected using netMHCIIpan-3.1 and IEDB MHCII-2.17.3) (ranking scores using the "IEDB recommended" method in the IEDB algorithm, respectively). Peptides carrying mutations in the predicted HLA class II core region were preferred. Peptides were synthesized by Intavis Bioanalytical Instruments and Peptide Services (Cologne, Germany) using MALDI-MS and RP-HPLC (214 nm) and HPLC purified with a purity threshold of at least 90% (mostly >98%).

[0128] Digital PCR for detecting residual disease

[0129] To detect residual disease with high specificity, we analyzed mutation frequencies in circulating free DNA. We selected a single nucleotide variant (SNV) in the RFPL1 gene (RFPL1, chr22:29835153G>T, NM_021026, p.Val125Leu, c.G373T) for deep mutation analysis and found this SNV to be one of the most prevalent SNPs in the patient's tumor DNA with a frequency of 64% by whole exome sequencing. Comparison with the patient's PBL DNA demonstrated that the SNV was somatic. Primers (SEQ ID NO:1 and 2) and a dual-labeled LNA probe (SEQ ID NO:3 and SEQ ID NO:4) were used. Designed by Sigma-Aldrich and synthesized by Integrated DNA Technologies Belgium. CE tube (STRECK, La Vista, NE, USA) and Cf DNA was extracted from 20 ml of blood using the Circulating Nucleic Acids Kit (QIAGEN, Hilden, Germany). Digital PCR was performed on a QIAquite Cube system using a 26k nanoplate. Amplification parameters were (2 min 95°C, 40x (15 sec 95°C, 15 sec 58°C, 30 sec 72°C).

[0130] Example

[0131] Example 1 - Clinical Study

[0132] Case

[0133] A now 72-year-old man was first diagnosed with prostate cancer in December 2004. He underwent a complete prostate capsulectomy and pelvic lymph node dissection. The disease was classified as pT2c, N0 Mx G3 R1, Gleason score 9 (=4+5). Postoperative radiotherapy was administered to the bone metastases with a total dose of 67 Gy. He received various anti-hormonal therapies until May 2018 when the disease progressed with infiltration of the urethra, base of penis, and lymph nodes. At that time, the patient refused standard chemotherapy, so he was evaluated for experimental TIL therapy on a single patient compassionate use basis. As a cytoreductive approach and to isolate TILs, a subtotal resection of the soft tissue tumor metastasis was performed in June 2018. NY-ESO-1 expression of the tumor was confirmed by RT-PCR and immunohistochemistry. Twelve weeks later, the first TIL infusion (TIL-1) consisting of 1400Mill T cells was administered, and two months later the second TIL infusion (TIL-2) consisting of 2000Mill T cells was performed. The tumor was re-biopsied in January 2019 to generate a new batch of TILs. This biopsy also confirmed the expression of NY-ESO-1. The third TIL infusion (TIL-3) was administered in March 2019 at a dose of 8000Mill T cells. Before each TIL infusion, the patient received conditioning chemotherapy with cyclophosphamide, 60 mg / kg on day 1 (TIL-1) and 30 mg / kg on day 4 (TIL-2, TIL-3). TIL infusions were supported by interleukin (IL-2), which was given 5 times every 12 hours starting 12 hours after TIL infusion at 600,000U / kg. One week before TIL-3 infusion, checkpoint blockade with pembrolizumab (1 mg / kg) was initiated and given every 3 weeks for a total of 4 doses. Figure 1 a provides an overview of the treatment plan. After three TIL infusions, PSA levels decreased. Two months after TIL-3 infusion, PSA dropped to 0.01 μg / ml ( Figure 1 b), and all tumor manifestations also completely remitted, thus recognizing complete remission for the first time. At the same time, the high NY-ESO-1 serum antibody titer before TIL continued to decrease during TIL treatment ( Figure 1 c). MRT images show a significant reduction in tumor burden 8 weeks after TIL-3 administration. The patient’s most recent imaging shows a complete and sustained tumor remission, which has now lasted 3 years ( Figure 1 d). Corresponding to the clinical manifestation of complete remission, the mutant variant of the tumor RFPL1 gene was undetectable in the patient's current plasma using circulating free DNA (cfDNA) digital PCR analysis.

[0134] NY-ESO-1 immunization

[0135] NY-ESO-1 expression by the tumor was confirmed by RT-PCR and immunohistochemistry, showing strong positivity in both tumor biopsies used for TIL isolation. Serum samples were collected before the first TIL treatment and continuously throughout the treatment period until today. NY-ESO-1 antibodies were measured longitudinally by standard Elisa. The first serum obtained in 2006 was negative for NY-ESO-1 antibodies. During the first three months of TIL treatment, the patient developed strong NY-ESO-1 antibody titers as well as significant disease progression. During and after TIL treatment, antibodies continued to decrease in parallel with the reduction in tumor burden and are currently detectable only at residual titer levels ( Figure 2 a). PSA values ​​were routinely measured during the course of the disease to monitor clinical development and response to treatment. Shortly after the first TIL treatment, PSA decreased from 33 μg / l to 18 μg / l within 5 weeks and then rapidly increased to 34 μg / l. After the second TIL treatment, PSA decreased to 5 μg / l within 7 weeks and then increased again to a maximum level of 35 μg / l. After the third TIL treatment, PSA levels continued to decrease to < 0.01 μg / l within 8 weeks and have been undetectable since then. ( Figure 2 b) After the first and second TIL treatments, the tumor partially regressed; 2 months after the third TIL treatment, the tumor completely regressed, and the tumor has been confirmed to have regressed for 18 months ( Figure 2 c).

[0136] Phenotype and functional activity of TILs

[0137] Flow cytometric phenotyping showed that the proportion of CD8+ T cells was approximately 78%, 81%, and 96% in the first (TIL-1), second (TIL-2), and third (TIL-3) infusions, respectively. CD4+ T cells decreased from 18% in TIL-1 to 11% in TIL-2 and 1.7% in TIL-3 ( Figure 3 a). TILs were further characterized for the expression of co-stimulatory molecules (CD25 / CD28) and differentiation markers (CD45RA, CCR7). The majority of TILs were effector memory T cells, of which 24%, 54% and 39% were terminal effector memory T cells (EMRA) and <10% were central memory T cells ( Figure 3 b) Stimulation with OKT3 1x 10 5 The functional activity of each T cell was evaluated after 24 hours. The IFN-γ release of TIL in TIL-1, TIL-2 and TIL-3 reached approximately 1298 pg, 1095 pg and 2184 pg, respectively. Figure 3c). Cytotoxic potential was confirmed by the expression of CD107a, which is known to be upregulated upon activation. 9.1% of TIL-1, 15.4% of TIL-2, and 23.8% of TIL-3 (mainly CD8+ T cells) were CD107a+, showing cytotoxic potential ( Figure 3 d). In all TIL preparations, CD25hiCD127-FoxP3+CD4+ regulatory T cells (Treg) were <0.13%.

[0138] Specificity of TILs

[0139] Based on the antigenic profile and strong expression of NY-ESO-1 in patient tumors, we used a panel of immunogenic “hot spot” NY-ESO-1 peptides known to be recognized by T cells from patients with NY-ESO-1-expressing tumors. In addition, we used 10 single HLA-matched peptide panels derived from mutant antigens identified by WES in patient tumors (Table 1). This peptide panel represented 9 peptides selected from 77 non-synonymous encoded neoantigens detected in tumors by exome sequencing and the frameshifted androgen receptor (AR) alternative splice variant AR-V12 discovered by Oncomine RNA sequencing. TILs were incubated with these peptides, and IFN-γ in cell culture supernatants was then analyzed by ELISA assay. Specific reactivity to NY-ESO-1 was found in all three TIL infusions, particularly to the HLA-A2 restricted peptide NY-ESO-1 p157-165, the NY-ESO-1 20mer peptides p81-100 and p91-110 including the 9mer peptides p92-100 and p96-104 known to be present on HLA-Cw3, and the NY-ESO-1 24mer peptide p119-143 ( Figure 2 a). In addition, in the standard chromium-51 (Cr 51 ) release assay, TIL showed specific cytotoxicity against tumor cells expressing NY-ESO-1 / HLA-A2 ( Figure 2b). The reactivity of several mutant peptides was also demonstrated: KKRCLFRSF (SEQ ID NO: 12) derived from the gene PRPF8 (TIL-1, TIL-2, TIL-3), YLGEITYPF (SEQ ID NO: 15) derived from the gene TRPS1 (TIL-1, TIL-3), FVSGKYKCL (SEQ ID NO: 17) derived from the gene ZNF512 (TIL 1, 3), NQVVQASMEK (SEQ ID NO: 18) derived from the gene GFM1 (TIL 2, 3), CPSKPSPLV (SEQ ID NO: 13) derived from the gene SGIP1 (TIL-3), and the peptide VKWAKALPDCERAAS (SEQ ID NO: 21) derived from a portion of the unique C-terminus of the V12 splice variant AR-V12 (TIL-1, TIL-2, TIL-3) ( Figure 2 c). Given that TIL-1 and TIL-2 infusions were generated from the same pre-expansion cultures, whereas TIL-3 was generated from a separate pre-expansion culture, these results suggest that the immunophenotype of the TILs consisted of NY-ESO-1-specific T cells in all three TIL infusions and of four and six mutation-specific T cells in TIL1, IL2, and TIL3, respectively. TIL-3-infused T cells were also analyzed for recognition of wild-type peptides derived from genes PRPF8 and TRPS1, respectively. IFN-γ production was significantly higher with the mutant peptides than with the wild-type peptide ( Figure 2 d).

Claims

1. A composition for treating cancer, comprising i) administering a composition (TIL-1) comprising in vitro expanded tumor infiltrating lymphocytes (TIL) to a mammal having cancer on day 0 (TIL-1), ii) administering a composition (TIL-2) comprising in vitro expanded tumor infiltrating lymphocytes (TIL) to a mammal having cancer on day 0 (TIL-2), and then iii) administering a composition comprising a checkpoint inhibitor on day 0 (CI), Wherein Day 0 (CI) is within the range of 1 month to 5 months later than Day 0 (TIL-1), such as 6 weeks to 4 months later than Day 0 (TIL-1), 8 weeks to 3 months later than Day 0 (TIL-1), or about 2 months later than Day 0 (TIL-1), and Day 0 (CI) is within the range of 1 month to 5 months later than Day 0 (TIL-2), such as 6 weeks to 4 months later than Day 0 (TIL-2), 8 weeks to 3 months later than Day 0 (TIL-2), or about 2 months later than Day 0 (TIL-2).

2. A composition for use according to claim 1, further comprising administering to the mammal a composition (TIL-3) comprising in vitro expanded tumor infiltrating lymphocytes (TIL) on day 0 (TIL-3), wherein day 0 (TIL-3) is in the range of 1 to 14 days after day 0 (CI), for example, 2 to 12 days after day 0 (CI), 5 to 12 days after day 0 (CI), or 7 days after day 0 (CI).

3. A composition for use according to any preceding claim, comprising administering to the mammal a composition (TIL-1) comprising in vitro expanded tumor infiltrating lymphocytes (TIL) on day 0 (TIL-1), wherein day 0 (TIL-1) is in the range of 1 month to 4 months before day 0 (TIL-2), for example 6 weeks to 4 months before day 0 (TIL-2), 8 weeks to 3 months before day 0 (TIL-2), or about 2 months before day 0 (TIL-2).

4. A composition for use according to any one of the preceding claims, further comprising administering a chemotherapeutic agent to the mammal 7 to 1 day prior to administering TIL-1, TIL-2 and / or TIL-3.

5. The composition for use according to claim 4, wherein The chemotherapeutic agent is an agent normally used to treat the cancer, but wherein the dosage is reduced compared to the dosage normally used to treat the cancer, such that the dosage of the chemotherapeutic agent is at most 70%, e.g., at least 65%, at least 60%, at least 55%, at least 50%, or 65%, 60%, 55% or 50% of the dosage normally used to treat the cancer.

6. A composition for use according to any one of the preceding claims, wherein The number of TILs increased from TIL-1 to TIL-2 and to TIL-3.

7. A composition for use according to any one of the preceding claims, wherein Said mammal having cancer has not undergone chemotherapy for a period of at least 6 months prior to initiation of said cancer treatment.

8. A composition for use according to any one of claims 2 to 7, wherein The 0 (TIL-3) day is determined by monitoring the plasma level of a marker specific to the cancer, a tumor-associated antigen specific to the cancer, or a neoantigen specific to the cancer, wherein the monitoring period begins after administration of TIL-2, and when the level increases 4-fold or more, the 0 (TIL-3) day is 1 day to 7 days after such an increase has been observed.

9. A composition for use according to any one of the preceding claims, wherein The 0 (TIL-2) day is determined by monitoring the plasma level of a marker specific to the cancer, a tumor-associated antigen specific to the cancer, or a neoantigen specific to the cancer, wherein the monitoring period begins after administration of TIL-1, and when the level increases 4-fold or more, the 0 (TIL-2) day is 1 day to 7 days after such an increase has been observed.

10. A composition for use according to any one of the preceding claims, wherein The cancer is selected from a cancer that does not effectively respond to monotherapy with a checkpoint inhibitor, such as rhabdoid tumor, Ewing's sarcoma, thyroid cancer, AML, medulloblastoma, glioblastoma, neuroblastoma, carcinoid tumor, prostate cancer, breast cancer and pancreatic cancer.

11. A composition for use according to any one of the preceding claims, wherein The cancer is prostate cancer.

12. The composition for use according to claim 11, wherein Levels of PSA and / or NY-ESO-1 are monitored.

13. A composition for treating a mammal suffering from cancer, comprising i) administering to said mammalian patient a composition comprising in vitro expanded tumor infiltrating lymphocytes on day 0 (TIL-1), ii) administering to the mammalian patient a composition comprising in vitro expanded tumor infiltrating lymphocytes on day 0(TIL-2), wherein day 0(TIL-2) is in the range of 1 month to 4 months after day 0(TIL-1), such as 6 weeks to 4 months after day 0(TIL-1), 8 weeks to 3 months after day 0(TIL-1), or about 2 months after day 0(TIL-1), iii) administering to the mammal a composition comprising a checkpoint inhibitor on day 0(CI), wherein day 0(CI) is in the range of 1 month to 5 months later than day 0(TIL-2), such as 6 weeks to 4 months later than day 0(TIL-2), 8 weeks to 3 months later than day 0(TIL-2), or about 2 months later than day 0(TIL-2), iv) administering to the mammal a composition (TIL-3) comprising in vitro expanded tumor infiltrating lymphocytes (TIL) on day 0 (TIL-3), wherein day 0 (TIL-3) is in the range of 1 day to 14 days after day 0 (CI), for example, 2 days to 12 days after day 0 (CI), 5 days to 12 days after day 0 (CI), or 7 days after day 0 (CI).

14. A composition for use according to any one of the preceding claims, wherein The composition comprising in vitro expanded tumor infiltrating lymphocytes is obtained by providing a tumor or tumor metastasis sample from the mammal and subjecting the sample to in vitro culture.

15. A composition for use according to any one of the preceding claims, wherein At least 80% of the TIL-1, TIL-2 and / or TIL-3 are CD8 positive, and at least 20% of the CD8 positive TILs are effector memory cells.

16. A composition for use according to any one of the preceding claims, wherein The checkpoint inhibitor is administered one or more times, such as 1, 2, 3, 4, 5 or more times, after administration of TIL-3.

17. The composition for use according to claim 16, wherein The check point inhibitor is administered three times after administration of TIL-3, and the time interval between the administration of TIL-3 and the administration of the check point inhibitor is 10 to 100 days, for example, the first administration of the check point inhibitor is 10 to 20 days after administration of TIL-3, the second administration of the check point inhibitor is 40 to 70 days after administration of TIL-3, and the third administration of the check point inhibitor is 70 to 120 days after administration of TIL-3.

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