A dosing regimen comprising administration of tumor infiltrating lymphocytes and a checkpoint inhibitor

Abstract

A dosing regimen for treating cancer, comprising administering in vitro - propagated tumor - infiltrating lymphocytes (TIL) to a mammal suffering from cancer on day 0 (TIL), and subsequently administering a checkpoint inhibitor on day 0 (CI), wherein day 0 (CI) is within the range of 1 month to 5 months after day 0 (TIL), such as 6 weeks to 4 months, 8 weeks to 3 months, or about 2 months after day 0 (TIL).

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 use in the treatment of cancers such as prostate cancer.

Background Art

[0002] Cancer cells are characterized by innumerable genetic mutations and epigenetic changes that give rise to a wide variety of cancer-specific antigens. These antigens are detected by T cells, which utilize the antigens to distinguish pre-cancerous and / or cancer cells from normal cells and trigger a cancer-specific immune response. The amplitude and quality of the immune response mediated by T cells are usually 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 the prevention of autoimmunity and protection from tissue damage caused by pathogen infection. However, cancer cells may exploit dysregulation of immune checkpoint proteins as a means of acquiring immune resistance.

[0003] One approach to inducing an anti-tumor immune response mediated by T cells is referred to as "checkpoint blockade" and refers to the blockade or inhibition of immunosuppressive checkpoints utilized by cancer cells. Since many immune checkpoints are initiated by ligand-receptor interactions, these checkpoints may be blocked by antibodies or modulated by recombinant forms of the ligand and / or receptor in question.

[0004] Advanced prostate cancer has a poor prognosis and is in an incurable state. Except for Sipuleucel-T, an FDA-approved cancer vaccine for the treatment of prostate cancer, there are still challenges remaining for effective immunotherapy approaches for prostate cancer.

[0005] Immune checkpoint inhibitors (anti-PD-1 / anti-PD-L1 / anti-CTLA4), which are effective against various cancers, show limited activity in prostate cancer, possibly due to its relatively low mutational burden. For example, the response rates when using pembrolizumab have only been reported to be 5% and 3%.

[0006] Therefore, new immunotherapy strategies are urgently needed. Adoptive therapy using tumor-infiltrating T lymphocytes (TILs) has shown remarkable results in the treatment of individual patients with various types of cancer over the past few decades, and recent successful clinical cases were reported in 2018. Recognition of multiple individual neoantigens and common tumor-associated antigens (TAAs) related to enhanced tissue homing and potent immune effector functions both contribute to the clinical efficacy of TILs in solid tumor patients. TIL therapy has been shown to have clinical effects in patients with chemotherapy-resistant cancers such as metastatic melanoma, cholangiocarcinoma, renal cell carcinoma, colorectal cancer, cervical cancer, breast cancer, and ovarian cancer. Currently, there are only a few clinical studies researching adoptive transfer of T cells for metastatic hormone-refractory prostate cancer (mHRPC) listed on www.clinicaltrials.gov.

Summary of the Invention

Problems to be Solved by the Invention

[0007] The present invention provides a novel dosing schedule for the treatment of cancer. This cancer is typically a cancer that does not effectively respond to monotherapy with checkpoint inhibitors and / or a cancer with a relatively low mutational burden. An example of such a cancer is prostate cancer.

Means for Solving the Problems

[0008] In its broadest aspect, the present invention is a composition for use in the treatment of cancer with a specific dosing schedule, comprising administering to a mammal suffering from cancer tumor infiltrating lymphocytes (TIL) grown in vitro on day 0 (TIL), and subsequently administering on day 0 (CI) a composition comprising a checkpoint inhibitor (CI), wherein day 0 (CI) is within the range of 1 to 5 months after day 0 (TIL) (e.g., 6 weeks to 4 months, 8 weeks to 3 months, or about 2 months after day 0 (TIL)).

[0009] In the context of this specification, the use of the term "tumor infiltrating lymphocytes grown in vitro" means including compositions containing tumor infiltrating lymphocytes grown in vitro. Similarly, the term "checkpoint inhibitor" includes compositions containing a checkpoint inhibitor.

[0010] Before treatment, TIL may be administered one or more times, and thereafter TIL may be administered one or more times, and / or a checkpoint inhibitor may be administered one or more times.

[0011] Specifically, the present invention relates to a composition for use in the treatment of cancer, comprising administering to a mammal suffering from cancer tumor infiltrating lymphocytes (TIL - 2) grown in vitro on day 0 (TIL - 2), and subsequently administering a checkpoint inhibitor on day 0 (CI), wherein day 0 (CI) is within the range of 1 to 5 months after day 0 (TIL - 2) (e.g., 6 weeks to 4 months, 8 weeks to 3 months, or about 2 months after day 0 (TIL - 2)).

[0012] In the context of this specification, the term "tumor-infiltrating lymphocyte" or "TIL" or "TIL" shall mean a T lymphocyte that is specific for a tumor antigen and possesses a T cell receptor (TCR) that recognizes the tumor antigen. This term is similar to the term "tumor-reactive T lymphocyte". This term also includes T lymphocytes that are specific for a metastatic antigen and have a TCR that recognizes the translocation antigen. That is, the term "tumor-infiltrating lymphocyte" / "TIL" / "TIL" includes "metastasis-infiltrating lymphocyte".

[0013] In the said administration plan, it is important to administer TIL before the checkpoint inhibitor. Due to the immunotherapeutic effect initiated by TIL, cancer is considered to be sensitive to treatment with the checkpoint inhibitor. As can be seen from the examples in this specification, significant effects are obtained in patients suffering from prostate cancer who have received the administration plan described in this specification (see FIGS. 1b and 1c). The present invention also relates to a kit for use in the treatment of cancer, the kit comprising i) tumor-infiltrating lymphocytes such as TIL-1, TIL-2, TIL-3, etc. grown in vitro, and ii) one or more checkpoint inhibitors, The kit includes two or more separate components, the first component includes one or more compositions containing tumor-infiltrating lymphocytes grown in vitro, and the second component includes one or more compositions containing checkpoint inhibitors.

Mode for Carrying Out the Invention

[0014] The present invention provides a novel dosing schedule for the treatment of cancer. The dosing schedule includes the continuous administration of tumor-infiltrating lymphocytes (TIL), and in certain cases, the administration of a checkpoint inhibitor precedes and in certain cases, the administration of a checkpoint inhibitor follows. Tumor-infiltrating lymphocytes are obtained by culturing lymphocytes obtained from a biopsy or body sample containing TIL of a patient suffering from cancer in vitro and administering the thus obtained tumor-infiltrating lymphocytes (TIL) to the patient. The in vitro proliferation of tumor-infiltrating lymphocytes can be carried out in a culture medium containing one or more of IL-2, IL-15, and IL-21.

[0015] The simplest dosing schedule includes the following steps. i) Administering to a mammal suffering from cancer on day 0 (TIL) tumor-infiltrating lymphocytes (TIL) that have been grown in vitro, and then ii) Administering a checkpoint inhibitor on day 0 (CI), where day 0 (CI) is within the range of 1 to 5 months (e.g., 6 weeks to 4 months, 8 weeks to 3 months, or about 2 months) after day 0 (TIL).

[0016] As described above, it is important to administer TIL to a subject suffering from cancer before starting treatment with a checkpoint inhibitor. In many cancer types, the somatic mutation frequency ( / Mb) is relatively low, such as less than 10 (Lawrence, M.S., Nature 2013, 499, 214-8). Such cancers include rhabdoid tumors, Ewing sarcoma, thyroid cancer, AML, medulloblastoma, glioblastoma, neuroblastoma, carcinoid tumors, prostate cancer, breast cancer, and pancreatic cancer. The inventors have observed that such cancer types typically do not respond effectively to treatment with checkpoint inhibitors, but as described herein, an effective treatment plan can be obtained by a dosing schedule in which the cancer is pretreated with TIL and then treated with a checkpoint inhibitor. The treatment can be repeated as many times as necessary, and the checkpoint inhibitor can be administered as many times as necessary even after the last treatment with TIL.

[0017] As an example of the administration schedule of the present invention, an administration schedule including three administrations of TIL is shown. Those skilled in the art will understand that additional administrations of TIL may be performed before the administration of TIL-1 or after the administration of TIL-3.

[0018] Non-limiting examples of the administration schedule are shown below. The administration intervals of TIL and checkpoint inhibitors and the administration intervals of TIL dosages are representative and apply regardless of the number of administrations of TIL or checkpoint inhibitors administered. Tumor-infiltrating lymphocytes (TIL) (TIL-2) grown in vitro are administered to a mammal suffering from cancer on day 0, and then, ii) A checkpoint inhibitor is administered on day 0 (CI), where day 0 (CI) is within the range of 1 to 5 months (e.g., 6 weeks to 4 months, 8 weeks to 3 months, or about 2 months) after day 0 (TIL-2).

[0019] The administration schedule may further include administering to the mammal a composition comprising tumor-infiltrating lymphocytes (TIL) (TIL-3) grown in vitro on day 0 (TIL-3), where day 0 (TIL-3) is within the range of 1 to 14 days (e.g., 2 to 12 days, 5 to 12 days, or 7 days) after day 0 (CI). Thus, additional treatment with TIL may be required after the administration of the checkpoint inhibitor. TIL may also be administered even before the administration of TIL-2. Thus, the administration schedule may further include administering to the mammal a composition comprising tumor-infiltrating lymphocytes (TIL) (TIL-1) grown in vitro on day 0 (TIL-1), where day 0 (TIL-1) is within the range of 1 to 4 months (e.g., 6 weeks to 4 months, 8 weeks to 3 months, or about 2 months) before day 0 (TIL-2).

[0020] The dosing frequency and the number of administered cells (TIL) vary depending on the treatment effect. After treatment, markers specific to the type of cancer, such as PSA in the case of prostate cancer, may be monitored. In the case of prostate cancer, an increase in PSA indicates cancer progression and suggests the need for further treatment with TIL and / or checkpoint inhibitors. Usually, TIL can be administered 1 to 10 times (e.g., 2 to 8 times, 2 to 6 times, 2 to 5 times, 3 to 4 times, e.g., 3 times). The dosing interval of TIL is 1 to 6 months (e.g., 6 weeks to 6 months, 8 weeks to 5 months, 8 weeks to 4 months, 8 weeks to 3 months, or about 2 months).

[0021] Typically, the dosing date of the second (third, fourth, etc.) administration 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. The monitoring period starts after the first administration of TIL. If the level changes by 4-fold 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 such a change is observed. The change can be an increase or decrease in the level of the marker being monitored depending on the type of cancer. In the case of prostate cancer, the PSA level is monitored, and an increase in the PSA level indicates disease progression, so the above change is an increase in the PSA level. A commonly applicable marker is NY-ESO-1, and this change is also an increase in the NY-ESO-1 antibody level.

[0022] The number of TIL administered is usually in the range of 1×10 8 ~1×10 11 (e.g., 1×10 8 ~1×10 10 , e.g., 1×10 9 ~1×10 10 ).

[0023] In the dosing plan, the number of TIL administered may be increased during the treatment period. For example, the number may increase from TIL-1 to TIL-2 and then to TIL-3.

[0024] Separate from the administration of TIL, a chemotherapeutic agent may be administered. Such a chemotherapeutic agent is usually administered 7 days to 1 day before the administration of TIL (e.g., TIL-1, TIL-2, and / or TIL-3, etc.). Examples of chemotherapeutic agents suitable for use include Cyclophosphamide (CTX) and Fludarabine. Such chemotherapeutic agents are considered to have a Treg down-regulation effect that can down-regulate the cytotoxic performance of TIL.

[0025] In some embodiments, the chemotherapeutic agent is a drug commonly used for the treatment of the cancer, but its dosage is lower compared to the dosage commonly used for the treatment of the cancer.

[0026] The administration plan may include, for example, the administration of IL-2. IL-2 is usually administered 1 day to 5 days after the administration of TIL, for example, 1 day to 3 days after the administration of TIL. The dosage of IL-2 is usually in the range of 6×10 5 IU / kg body weight to 5×6×10 6 IU / kg body weight, for example, 2×6×10 5 IU / kg body weight to 8×6×10 5 IU / kg body weight, 3×6×10 5 IU / kg body weight to 7×6×10 5 IU / kg body weight, 4×6×10 5 IU / kg body weight to 6×6×10 5 IU / kg body weight, or 4×6×10 5 IU / kg body weight to 5×6×10 5 IU / kg body weight.

[0027] In an embodiment of the present invention, a subject suffering from cancer has not received chemotherapy within at least 6 months before the start of the administration plan of the present invention.

[0028] As demonstrated in the examples, in contrast to single injection, repeated administration of TILs has been found to improve clinical outcomes by extending the phase of TIL-tumor interaction in vivo and reducing treatment-related toxicities.

[0029] As shown in Figure 1a, the novel dosing regimen involves injecting TILs one or more times. Typically, significant results are obtained when TILs are injected three times. Thus, the method of the invention includes TIL-1, TIL-2, and TIL-3, where TIL-1 is the first portion of TILs injected, TIL-2 is the second portion, and TIL-3 is the third portion. Depending on the progression of the disease state (progression, regression, or recurrence), fewer or more injections may be required. In the examples illustrated, the presence of a cancer disease marker (PSA for prostate cancer) in plasma or serum and / or the presence of a specific tumor-associated antigen (e.g., NY-ESO-1, etc.) or neoantigen are used to track the progression of the disease.

[0030] Based on parameters related to a particular cancer type, the progression of the disease can be tracked, and one of ordinary skill in the art can determine when to discontinue treatment or when to repeat treatment.

[0031] This new dosing regimen is based on the observation that while the initial administration of TILs had some effect, the disease soon recurred, i.e., only a transient effect was obtained (see Figure 1b). This transient response can be explained by reasons such as too few TILs, impaired persistence of the transplanted T cells, or insufficient migration of the transplanted T cells into the tumor microenvironment. To improve the results and achieve a durable response, more cells (TIL-2 and / or TIL-3) can be further injected. Furthermore, a dosing regimen in which TILs are first administered (repeated one or more times if necessary) and then checkpoint inhibitors are administered is considered effective. It is considered beneficial to initiate treatment with tumor-infiltrating lymphocytes having a homing marker (e.g., NY-ESO-1) for the cancer / tumor before initiating treatment with checkpoint inhibitors. As described herein, for example, the use of checkpoint inhibitors in the treatment of prostate cancer has not been successful, but as described herein, treatment with checkpoint inhibitors following initial treatment with TILs has been successful, and the patient has been symptom-free for 3 years now.

[0032] The TILs used in the present invention may be autologous or allogeneic.

[0033] Checkpoint inhibitor As described above, in the new dosing regimen, TILs are used in combination with checkpoint inhibitors.

[0034] Checkpoint inhibitor therapy is a type of cancer immunotherapy. Checkpoint inhibitors target immune checkpoints, which are key regulators of the immune system, and when stimulated, can weaken the immune response to immune stimulation. Checkpoint therapy blocks inhibitory checkpoints and restores the function of the immune system. The first anticancer agent targeting an immune checkpoint was ipilimumab, a CTLA4 inhibitor.

[0035] Currently approved checkpoint inhibitors target the CTLA4 molecule, the PD1 molecule, and the PD-L1 molecule. PD-1 is a transmembrane programmed cell death 1 protein (also called PDCD1 or CD279) and interacts with PD-L1 (PD-1 ligand 1). PD-L1 on the cell surface binds to PD-1 on the surface of immune cells and inhibits the activity of immune cells. Among the functions of PD-L1 is an important role in regulating the activity of T cells. Upregulation of cancer-mediated PD-L1 on the cell surface can inhibit T cells that may attack. Antibodies that bind to either PD-1 or PD-L1 and block the interaction may enable T cells to attack tumors.

[0036] Some immune checkpoints are related in that they enhance the anti-tumor immune response mediated by T cells, either alone or in combination. 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 CD223), B7-H3 (also known as CD276), B7-H4 (also known as B7-S1, B7X, VCTN1), 2B4 (also known as CD244), B lymphocyte and T lymphocyte attenuator (BTLA, also known as CD272).

[0037] Of particular interest are immune checkpoint inhibitors selected from inhibitors of PD-1 (such as Nivolumab, Pembrolizumab, or Cemiplimab), PD-L1 (such as Atezolizumab, Avelumab, or Durvalumab), and CTLA-4 (such as Ipilimumab).

[0038] Furthermore, other examples of relevant immune checkpoints are described in the scientific and patent literature, and these are also within the scope of the present invention.

[0039] Immune checkpoint inhibition is useful for enhancing anti-tumor immunity mediated by T cells. However, the inventors believe that even more excellent anti-tumor effects can be obtained by further enhancing T cell activation by combining immune checkpoint inhibition with one or more complementary mechanisms.

[0040] In vitro expansion of TIL Generally, various different methods can be used to expand TIL obtained from a subject suffering from cancer.

[0041] In one method, three different cytokines, IL-2, IL-15, and IL-21, are used. The use of IL-15 and IL-21, i.e., a method without using IL-2, or a method with the use of IL-2, may be employed. When using IL-2, IL-15, and IL-21, this method has two phases. In the first phase (i), cultures with a relatively high ratio of TIL, particularly CD8 + T lymphocytes are obtained. In this phase, the TIL will survive and divide. The starting material for the first phase may be a mixture of lymphocytes and antigen-presenting cells provided from a biopsy or body sample of a subject suffering from cancer.

[0042] T lymphocytes to be expanded by culture can be obtained from the subject to be treated, i.e., the TILs obtained for administration may be autologous. However, T lymphocytes can also be obtained from sources other than the subject to be treated, such as another subject suffering from cancer, for example. In such cases, it is preferred that the recipient and the expanded TILs are immunologically compatible, or that the recipient is made immunotolerant to the expanded TILs. In one method, a cytokine cocktail containing IL-2, IL-15, and IL-21 is added to initiate the first phase, stimulating the TILs via cytokine receptors to promote cell division and prevent cell death. In another method, only IL-2 is used to stimulate the TILs via the IL2 receptor. The use of the cytokine cocktail or IL-2 alone is + aimed at promoting the specific activation and expansion of CD4 + helper lymphocytes and CD8

[0043] lymphocytes. Such specific activation against specific tumor antigens enables the TILs to exert a therapeutic effect when administered to cancer patients of the same type as the tumor in which the TILs are activated.

[0044] In the second phase ii), the aim is the clonal expansion of the TILs from phase i). In the second phase, human serum, feeder cells, and human CD3 or CD28 antibodies are added to the culture medium containing either the cytokine cocktail or IL-2.

[0045] In the examples of this specification, TIL-1 is obtained by growing TIL using IL-2, IL-15, and IL-21. The combination of the three cytokines results in a high proportion of CD8 + cytotoxic T cells (78% - 95%), a relatively small proportion of CD4 + T cells (11% - 1.7%), and most of the TIL being effector memory T cells (CD45RA - CCR7 + ). Furthermore, the growth of Treg was not promoted by this culture method.

[0046] Therefore, the characteristics of the TIL cell population are as follows. i) The content of CD8 + cytotoxic T cells is within the range of 70% - 98% (e.g., within the range of 75% - 97% or within the range of 78% - 95%) based on the total number of T cells. ii) The content of CD4 + T cells is within the range of 0.5% - 20% (e.g., 1% - 15%, 1.5% - 12%) based on the total number of T cells. iii) The content of effector memory T cells that are CD8 positive (CD45RA - CCR7 + ) is within the range of 45% - 80% (e.g., 45% - 78%).

[0047] IL-2, IL-15, and IL-21 are members of the cytokine family that each have four alpha helix bundles. IL-2 plays an important role in the main functions of the immune system, tolerance, and immunity mainly through its direct action on T cells. IL-2 induces the proliferation of T cells and the differentiation into effector T cells and memory T cells.

[0048] IL-15 is a cytokine that is structurally similar to IL-2. Similar to IL-2, IL-15 binds to a complex composed of the IL-2 / IL-15 receptor beta chain and transmits signals through it. IL-15 is involved in the activation of T cells and especially CD8 +Induce the proliferation of T cells, present survival signals to maintain memory cells in the absence of antigen, and preferentially activate monocytes over CD8 + T cells. IL-15 is thought to promote the proliferation of immune effector T cells, along with protection from the inhibition of tumor-associated immunosuppression.

[0049] IL-21 is a cytokine that has a strong regulatory effect on cells of the immune system such as natural killer (NK) cells and cytotoxic T cells. IL-21 enriches central memory-type T cells with the CD28 + CD127hi CD45RO + phenotype and enhances the cytotoxicity of cytotoxic T cells. IL-21 can maintain T cells at the early stages of differentiation and maturation.

[0050] As used herein, "interleukin 2" or "IL-2" refers to human IL-2 as defined by SEQ ID NO: 9 and its functional equivalents. Functional equivalents of IL-2 include related substructures or fusion proteins of IL-2 that maintain the function of IL-2. Accordingly, the definition of IL-2 includes any protein that has at least 80% sequence identity to SEQ ID NO: 9, preferably at least 90%, more preferably at least 95%, and most preferably at least 98%. Recombinant human IL-2, which is a single non-glycosylated polypeptide chain produced in E. coli, has 134 amino acids and a molecular weight of 15 kDa, and is commercially available as CYT-209 from Prospec in lyophilized form.

[0051] As used herein, "Interleukin 15" or "IL-15" refers to human IL-15 and its functional equivalents. The functional equivalents of IL-15 include related substructures or fusion proteins of IL-15 that maintain the function of IL-15. Accordingly, the definition of IL-15 includes any protein having a sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more to SEQ ID NO: 10. Recombinant human IL-15, which is a single non-glycosylated polypeptide chain produced in E. coli, having 114 amino acids (and an N-terminal methionine) and a molecular weight of 12.8 kDa, is commercially available from Prospec as CYT-230 in lyophilized form.

[0052] As used herein, "Interleukin 21" or "IL-21" refers to human IL-21 and its functional equivalents. The functional equivalents of IL-21 include related substructures or fusion proteins of IL-21 that maintain the function of IL-21. Accordingly, the definition of IL-21 includes any protein having a sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more to SEQ ID NO: 11. Recombinant human IL-21, which is a single non-glycosylated polypeptide chain produced in E. coli, having 132 amino acids and a molecular weight of 15 kDa, is commercially available from Prospec as CYT-408 in lyophilized form.

[0053] As used herein, "peptide" is composed of any number of any type of amino acids, preferably naturally occurring amino acids, and is preferably linked by peptide bonds. In particular, a peptide contains 3 or more amino acids, preferably 5 or more, 7 or more, 9 or more, 12 or more, or 15 or more amino acids. However, preferably, the peptides for use according to the present invention do not exceed 500 amino acids in length, more preferably do not exceed 300 amino acids in length, and even more preferably are not longer than 250 amino acids.

[0054] In addition to cytokines, the culture medium for growing TIL may contain various factors such as growth factors commonly used for lymphocyte growth.

[0055] All of the TIL grown by the above-described one or more methods are reactive against tumor-associated antigens. In particular, as can be seen from the examples thereof, TIL grown from cells obtained from a biopsy of a subject suffering from prostate cancer showed reactivity against NY-ESO-1. As described herein, the peptide panel used for the identification of TIL specificity did not represent the entire range of TAAs and neoantigens expressed in tumors. T cells reactive against other target antigens such as MAGE expressed on autologous tumors may contribute to clinical outcomes.

[0056] Tumor-associated antigen (TAA) Tumor-associated antigens are antigenic substances produced within tumor cells and elicit an immune response in the host. TAAs are tumor markers useful for identifying tumor cells in diagnostic tests and are promising candidates for use in cancer treatment.

[0057] In the context of this specification, a TAA is an antigen presented by MHC I molecules or MHC II molecules on the surface of tumor cells, or non-classical MHC molecules. TAAs used herein include tumor-specific antigens that are present only on the surface of tumor cells and not on the surface of normal cells.

[0058] Tumor-associated antigens are antigens involved in cancer diseases.

[0059] In the context of this specification, an antigen is a structural substance that functions as a target for a TCR or an antibody, which is a receptor of an adaptive immune response. In particular, an antigen is a partial structure thereof such as a protein, a polysaccharide, a lipid, or a peptide. Lipids and nucleic acids may have antigenicity when bound to a protein or a polysaccharide.

[0060] Examples of TAAs for use in the present invention include cancer testicular antigens such as MAGE-A1, MAGE-A3, MAGE-A4, NY-ESO-1, PRAME, CT83, SSX3, NY-ESO-1.

[0061] Cancer testicular antigens are a group of proteins that are important in development and cancer immunotherapy. Usually, the expression of these proteins is limited to male germ cells in adult animals. However, in cancer, these developmental antigens are often re-expressed and may function as sites of immune activation.

[0062] Other TAAs for use in the present invention include survivin, GPI, EGRvrlll, tyrosinase tumor antigen, tyrosinase-related protein (TRP)-I, TRP-2, VEGFR-2, MAGE family proteins (present in melanoma), telomerase, p53, HER2 / neu, mesothelin, cancer fetal, VEGF, CAMPATH 1 antigen, CD22, CA-125 (detectable in ovarian cancer), MUC-1 (detectable in breast cancer), epithelial tumor antigen (detectable in breast cancer), mucin-1, α-1-fetoprotein (detectable in germ cell tumor or hepatocellular cancer), PSMA, RAS, or their substructures or fragments. Other examples include peptides derived from the individual tumor variant proteins PRPF8, TRPS1, and androgen receptor splice variant 12.

[0063] TAAs particularly suitable for use in the present invention vary depending on the type of cancer to be treated.

[0064] Fragments of TAAs are particularly peptides. Such peptides can be, for example, peptides containing eight or more consecutive amino acids with an amino acid sequence that is 80% or more identical to the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

[0065] 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.

[0066]

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

[0067] NY-ESO-1 is a cancer testis antigen with very high immunogenicity and has been considered promising as an effective target for cancer immunotherapy because it induces both cellular and humoral immune responses in many patients with advanced NY-ESO-1-expressing tumors. Serum antibodies against NY-ESO-1 have been found to increase with disease progression and decrease with disease regression, and this antibody has become a useful biomarker for monitoring disease activity and response to treatment. Some studies have also shown that patients who already have NY-ESO-1 antibodies have an increased clinical benefit from immune checkpoint blockade. The adoptive transfer of NY-ESO-1-specific T cells has improved the clinical response rate in patients with melanoma, synovial cell sarcoma, and multiple myeloma.

[0068] In this specification, we report on a patient with advanced prostate cancer who experienced a complete and durable tumor remission after treatment with tumor-infiltrating lymphocytes (TILs) that were expanded in vitro and recognized NY-ESO-1 and multiple mutant tumor antigens that may be related to anti-tumor immunity.

[0069] Other aspects of the invention The present invention also provides a dosing schedule for use according to any one of the preceding claims. The aforementioned TILs are obtained in vitro by the following steps: i) Preparing a tumor sample or tumor metastasis sample obtained from the mammal; ii) Isolating TILs from the sample; iii) Culturing the TILs in a medium containing IL-2, IL-15, and / or IL-21; and iv) Adding a CD3 antibody and feeder cells to the culture medium.

[0070] Also disclosed is a kit for use in the treatment of cancer, the kit comprising: i) Tumor-infiltrating lymphocytes (TILs) that have been expanded in vitro; and ii) One or more checkpoint inhibitors. The kit comprises two or more separate components. The first component comprises one or more compositions containing tumor-infiltrating lymphocytes expanded in vitro, and the second component comprises one or more compositions containing a checkpoint inhibitor.

[0071] The kit may further comprise a third component comprising a composition containing a chemotherapeutic agent.

[0072] The first component may comprise one composition containing TIL-1, TIL-2, or TIL-3, or may comprise two compositions, a first composition containing TIL-1 and a second composition containing TIL-2.

[0073] The first component may include two compositions, namely, a second composition containing TIL-2 and a third composition containing TIL-3, or may include three compositions, namely, a first composition containing TIL-1, a second composition containing TIL-2, and a third composition containing TIL-3.

[0074] The kit also includes instructions for use.

[0075] Examination of the results of the examples Cell-based cancer immunotherapy is a feasible method for treating patients with advanced malignancies and results in durable clinical responses. Here, we report the clinical and immunological efficacy of in vitro-expanded tumor-infiltrating lymphocytes (TILs) in patients with metastatic prostate cancer. Administration of TILs three times with a relatively low cell number resulted in complete tumor regression, which has persisted for 3 years now. In contrast to other groups working on TIL therapy, we have adopted different strategies regarding TIL expansion, mode and number of applications, and conditioning regimens, which may have contributed to this surprising result.

[0076] First, we used an improved culture method for the isolation and expansion of TILs in vitro using a medium containing IL-2, IL-15, and IL-21 and a closed-loop perfusion bioreactor system. By combining these cytokines with the culture procedure, the proportion of CD8 + cytotoxic T cells was high (78% - 96%), the proportion of CD4 + T cells was relatively low (11% - 1.7%), and most of the TILs were effector memory T cells (CD45RA - CCR7 + ). Furthermore, the growth of Tregs was not promoted (<0.13%) in this culture method.

[0077] The second is a modified pre - chemotherapy regimen that is different from that employed in the TIL studies reported by the National Cancer Institute (NCI) in the United States, and aims to reduce regulatory T cells without significantly affecting normal lymphodepletion by using a single low - dose injection of cyclophosphamide. The single low - dose administrations were given 1 day (TIL - 1) and 4 days (TIL - 2 and TIL - 3) prior to TIL injection. As a result, side effects were prominent, and treatment - related toxicity was significantly reduced compared with the administration schedules of high - dose cyclophosphamide and fludarabine, which are widely used. Single - dose administration of cyclophosphamide induced mild lymphopenia and moderate neutropenia, but did not completely cause lymphodepletion of the patients. To protect the injected TIL from toxic effects, cyclophosphamide was administered 4 days before the transplantation of TIL - 2 and TIL - 3.

[0078] The inventors did not use a single high - dose transplantation that multiplied 10 10 T cells several times, but rather administered relatively low - dose TIL (TIL - 1: 1.4×10 9 cells, TIL - 2: 2.0×10 9 cells, TIL - 3: 8×10 9 T cells) in three divided steps with incremental increases and performed repeated transplantation. In contrast to single - injection, the modification of the treatment method by the inventors using repeated transplantation of TIL preparations may improve clinical outcomes by extending the in - vivo TIL - tumor interaction.

[0079] After the first and second TIL treatments, PSA and radiological responses were observed over 5 weeks and 7 weeks, respectively, indicating that the effectiveness of TIL treatment was temporary. This partial and non-sustained response is thought to be explained by the low number of TILs, impaired persistence of the transplanted T cells, or insufficient ability of the transplanted T cells to migrate into the tumor microenvironment. To improve the results, a significantly higher cell number (4 times that of TIL-2) was infused in TIL-3. Furthermore, pembrolizumab was used in combination with TIL-3 treatment before and after the intervention. In contrast to the first and second TIL treatments, the PSA value decreased completely to less than 0.01 μg / L and has remained negative for 3 years to date. Closely correlated with the decrease in PSA, the high pre-TIL NY-ESO-1 serum antibody titer decreased continuously in parallel with the regression of the disease during the TIL treatment process.

[0080] The specificity for NY-ESO-1 could be identified in each of the three TIL transfers. Additional TIL specificities for the individual tumor variant proteins PRPF8 and TRPS1, and peptides derived from androgen receptor splice variant 12 were identified in all three TIL preparations. AR-V12 induces ligand-independent expression of prostate-specific antigen (KLK-3, KLK-2, FOLH1), for which overexpression was observed at the mRNA level. The corresponding proteins represent potential immune targets, and targeting them may have contributed to the sustained clinical response to the treatment of this patient. The peptide panel used to identify TIL specificities did not represent the full range of TAAs and neoantigens expressed in the tumor. T cells reactive against other target antigens, such as MAGE, which were also expressed within the tumor, may also have contributed to the clinical outcome. However, it seems unlikely that only the quantity, quality, and specificity of TIL-3 influenced the durable results. Also, for the following reasons, it is considered unlikely that tumor response and clinical improvement were due solely to immune checkpoint blockade with pembrolizumab. First, clinical and radiological responses were observed after TIL-1 and TIL-2 treatments without the use of pembrolizumab. Second, the patient's tumor was classified as MSS (microsatellite stability) and had a low TMB, and third, PD-L1 was low, making a response to immune checkpoint blockade alone less likely.

[0081] In this study, we demonstrated that TILs derived from prostate cancer can be isolated and effectively expanded in vitro. The TILs showed reactivity against NY-ESO-1 and several individual tumor antigens. A complete and sustained tumor response may have been brought about by the combination of TIL therapy and immune checkpoint blockade, and may have helped overcome T cell inhibition via PD-1 / PD-L1 in the tumor microenvironment. With regard to various immune evasion mechanisms, TIL therapy targeting several tumor antigens may be more advantageous than single-specificity immunotherapy strategies such as cancer vaccines or CAR-T cell therapy.

[0082] In conclusion, adoptive transfer of TILs represents a promising immunotherapy approach even for tumors with low TMB, such as mHRPC. Further investigation in larger patient populations in clinical studies is needed, including accurate analysis of tumor antigens and T cell repertoires.

[0083] General rule It should be understood that the features and / or aspects described above in connection with the compounds according to the invention apply equally to the methods described herein.

[0084] To illustrate the present invention, the following figures and examples are provided. These are for illustrative purposes only and should not be construed as limiting in any form.

Brief Description of the Drawings

[0085]

Figure 1a

Figure 1b

Figure 1c

Figure 1d

Figure 2a

Figure 2b

Figure 2c

Figure 2d

Figure 3a

Figure 3b

Figure 3c

Figure 3d

[0086] Materials and Methods TIL Production: Isolation, Culture, and Expansion Surgical tumor biopsies were performed in June 2018 (starting materials were TIL-1 and TIL-2) and in January 2019 (TIL-3). Tissue samples were about 1 mm 3It was sliced into fragments and placed in a GMP-compliant, fully closed perfusion bioreactor system that is operated on the platform of the GMP Bredder Control Unit and its attached software. The growth of TILs was initiated in a 30MM perfusion bioreactor (Phase 1) and continued in a 500MM perfusion bioreactor (Phase 2). The culture process was automatically controlled by an algorithm based on pH, high oxygen pO2, and temperature, and fresh medium was supplied to the growing cells. The Cellgenix medium contains 10% AB human serum and promotes the growth of TILs. TILs were isolated from tumor specimens and cultured in a medium containing IL-2 (1000 IU / mL), IL-15 (180 IU / mL), and IL-21 (1 IU / mL) (Miltenyi, Bergisch Gladbach, Germany). First, human serum (10%), anti-human CD3 antibody (clone OKT3, Miltenyi), and allogeneic 55Gy irradiated feeder cells (added on day 3 (1×10 6 cells)) were added to Cellgro medium in a 24-well plate and then rapidly grown using OKT3 (30 μg / mL) and allogeneic 55Gy irradiated feeder cells. The GMP-scale production of TILs for clinical use was carried out by Zellwerk (Berlin, Germany) using an ISO 13485-certified closed perfusion bioreactor cell culture platform for advanced therapy medicinal products (ATMP). IL-2, IL-15, and IL-21 (Miltenyi, Bergisch Gladbach, Germany) were added to the culture medium for the growth of TIL-1 and TIL-2, and the growth of TIL-3 was carried out using only IL-2 medium. In the short initiation phase, the anti-human CD3 antibody (clone OKT3, Miltenyi) and a small amount of allogeneic feeder cells irradiated at 55Gy were added once. The growth of TILs from tumor sections was carried out under the controlled circulation of the medium in the bioreactor vessel and the maintenance of a proven ratio of the circulating medium to the newly supplied medium. The cell count was periodically performed, and the concentrations of glucose and lactate were estimated. The density of TILs in the 30MM bioreactor was approximately 1 - 3×10 9Upon dissociation into single cells, the TILs were transferred by gravity through a mesh port into a disposable 500M perfusion bioreactor and isolated from the tissue material. During this phase, continuous automated supply with medium supplemented with 10% AB human serum and interleukins was maintained until TIL collection. Thus, no additional stimuli that would cause changes to the phenotypes formed during Phase 1 occurred. The cells were washed twice with 0.9% saline, resuspended in 5% albumin solution, and transferred to an infusion bag. The bag was transported to the patient in a biological sample processing unit at an ambient temperature of 15°C to 22°C. If there was a backup TIL sample, it was suspended in 90% human AB serum containing 10% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen.

[0087] Flow Cytometry and Functional Characterization of TILs TIL phenotypes, Treg cells, and CD107a induction were determined using the Beckman Coulter DURAClone IM T-cell subset and DuraClone IM Treg tube kits. CD107a induction was performed using Beckman Coulter DURActive 1 for cell activation and stained 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 on a Beckman Coulter CytoFlex flow cytometer. After stimulating the TILs with OKT3 for 24 hours, cytokines in the culture supernatant were quantified by enzyme-linked immunosorbent assay (ELISA) and IFN-γ production was measured.

[0088] Immunohistochemistry (IHC) Tumor tissue sections were cut, deparaffinized, treated with 3% H2O2 to inhibit endogenous peroxidase activity, and "demasked" for antigen retrieval. Primary monoclonal mouse anti-NY-ESO-1 (clone E978) provided by the Monoclonal Antibody Core Facility (MACF) of the Memorial Sloan Kettering Institute for Cancer Research was diluted with antibody diluent and added for 30 minutes. Subsequently, the sections were incubated with horseradish peroxidase-conjugated EnVision for 30 minutes. The final reaction was visualized by incubation with diaminobenzidine + substrate chromogen, followed by hematoxylin 26 to counterstain the sections. Testicular tissue was used as a positive control.

[0089] IFN-γ ELISpot PBMCs were separated by Ficoll / Hypaque density gradient centrifugation (GE Healthcare) and cryopreserved in 10% DMSO-containing FBS until analysis. PBMCs were pre-stimulated in vitro at 1.2 × 10 6 cells / well in 48-well plates using NY-ESO-1, neoantigen short-chain peptides and long-chain peptides, and CEF-MHC class I control peptide pool (PANATecs). After a single restimulation on day 7, pre-sensitization T cells were analyzed by IFN-α ELISpot between days 10 and 12 to examine specific recognition of peptides pulsed with autologous antigen-presenting cells or HLA-matched allogeneic antigen-presenting cells (APCs), such as autologous DCs and / or T2, which are EBV-converted B cells. The number of spots in wells exposed to the peptide was at least twice the number of spots in unstimulated control wells, and the peptide-specific spots after subtracting background spots were at least 10 per 25,000 T cells, the response was considered positive.

[0090] IFN-γ ELISA TIL or PBMC was incubated with the peptide (1 μg / mL) in 200 μL of TCM in a round-bottom 96-well microtiter plate at 37 °C with 5% CO2 for 3 to 7 days. The culture supernatant (100 μL / well) was collected and IFN-production was analyzed using the IFN-ELISA kit (No.: 3420-1A-6) from Mabtech AB in a flat-bottom 96-well multiplate according to the manufacturer's instructions.

[0091] Isolation of nucleic acids, whole exome sequencing, and selection of neoepitopes Genomic DNA from tumor tissue and corresponding normal blood was purified using the DNeasy Blood & Tissue Kit (Qiagen, catalog number: 69504). Total DNA isolation optionally included an RNase-A digestion step. DNA exome sequencing was enriched from tumor DNA and normal DNA using the Ion Torrent AmpliSeq™ Exome RDY Kit (Thermo Fisher Scientific, Carlsbad, California, catalog number: A38264) and the Ion AmpliSeq™ Library Preparation Kit plus (Thermo Fisher Scientific, catalog number: 4488990). Barcoded libraries were quantified using the Ion Library TaqMan™ Quantification Kit (Thermo Fisher Scientific, catalog number: 4468802). Tumor-derived libraries and normal DNA-derived libraries were adjusted to a molar ratio of 2:1. Sequencing was performed on an Ion GeneStudio™ S5 System using a Chip 540 to achieve 80-90 million reads per exome pair, with Torrent Suite™ Software 5.6 and the 200bp OT2 kit. Reads were aligned to the human genome reference sequence (hg19) using the integrated Torrent Suite algorithm. The typical average coverage was 120x in tumors versus 60x in normal DNA. The output BAM files were screened using the AmpliSeq Exome Tumor-Normal Pair Workflow with filters applied to the Ion AmpliSeq Exome HiQ regions by the Ion Reporter Software to identify point mutations, small insertions, deletions, and stop codons. The minimum requirements for mutations to be included in further analysis were coverage greater than 49x in tumor tissue and greater than 19x in normal DNA, a mutation frequency greater than 10% in the tumor, and no affected reads in the accompanying normal DNA sample. The corresponding protein sequences were translated using an in-house developed algorithm for SNPs.By placing the mutations in the center of the array, wild-type and mutant peptides of 31-mers can be constructed respectively. Indels were translated individually using the "codong_change.pl" function of ANNOVAR software. This function was also used to annotate the mutations and exclude mutations that significantly exist in the human genomic database (kaviar_20150923, including genomic mutations of 64K exomes). For the evaluation of expressed genes, total RNA was extracted from tumor sections using the RNeasy FFPE Kit (Qiagen, catalog number: 73504), and transcription, amplification, and cyanine-3 staining were performed according to the Gene Expression FFPE Workflow Guide (Agilent Technologies, catalog number: G4112-90000). The stained cDNA was hybridized to a Human GE 4×44 v2 microarray slide (Agilent, catalog number: G4845) and scanned with a DNA microarray scanner (Agilent, catalog number: G2505C). Mutant-containing peptides corresponding to higher mRNA expression were prioritized for downstream selection. HLA typing (HLA A / B / C and HLA-DRB1 loci, resolution of 4 digits or more) was performed using EDTA blood at the Institute for Medical Diagnostics (IMD) in Berlin-Potsdam, Germany. Nonamer peptides containing putative HLA class I neoepitopes were selected by the prediction scores from the netMHC-4.0 software package and the IEDB MHC-I--2.17 software package. For HLA class II loci, 15-mer to 17-mer peptides were selected using the netMHCIIpan-3.1 program and the IEDB MHCII-2.17.3 program (using the rank scores of the "IEDB recommended" method of the IEDB algorithm respectively). Peptides with mutations in the predicted core region of HLA class II are preferred.The peptides were synthesized by Intavis Bioanalytical Instruments and Peptide Services (Cologne, Germany) and purified by HPLC with a purity threshold of 90% or higher (most >98%) using MALDI-MS and RP-HPLC (214 nm).

[0092] Digital PCR for residual disease detection For highly specific detection of residual disease, the mutation frequency in circulating free DNA was analyzed. For deep mutation analysis, a single nucleotide variant (SNV) of the RFPL1 gene (RFPL1, chr22:29835153G>T, NM_021026, p.Val125Leu, c.G373T) was selected. This mutation was found to be one of the most frequently seen SNPs in the patient's tumor DNA by whole exome sequencing, with a frequency of 64%. The SNV was demonstrated to be somatic by comparison with the patient's PBL DNA. Primers (SEQ ID NO: 1 and SEQ ID NO: 2) and dual-labeled LNA probes (SEQ ID NO: 3 and SEQ ID NO: 4) were designed using OligoArchitect® Online (Sigma-Aldrich) and synthesized by Integrated DNA Technologies Belgium. Cf DNA was extracted from 20 mL of blood collected in cfDNA BCT® CE tubes (STRECK La Vista, Nebraska, USA) and the QIAamp® Circulating Nucleic Acid Kit (QIAGEN, Hilden, Germany). Digital PCR was performed on a QIAquite Cube system using 26k nanoplates. The amplification parameters were 2 minutes at 95°C, 40 cycles (15 seconds at 95°C, 15 seconds at 58°C, 30 seconds at 72°C).

Example

[0093] Example 1 - Clinical study Case A 72-year-old male was first diagnosed with prostate cancer in December 2004. The male underwent radical prostatectomy and pelvic lymph node dissection. The disease was classified as pT2c, N0 Mx G3 R1, Gleason score 9 (=4+5). After surgery, radiotherapy with a total dose of 67 Gy was performed for bone metastases. He received various anti-hormonal therapies, but the disease progressed in May 2018 and invaded the urethra, the root of the penis, and the lymph nodes. At this point, since the patient refused standard chemotherapy, an evaluation of experimental TIL therapy was conducted based on compassionate use treatment for a single patient. For the purpose of tumor debulking surgery and TIL isolation, a soft tissue tumor metastasis was subtotally resected in June 2018. The 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 1,400×10 6 cells (1400 Mill T cells) was administered, and two months later, the second TIL infusion (TIL-2) consisting of 2,000×10 6 cells was administered. In January 2019, a tumor re-biopsy was performed and a new TIL batch was generated. The expression of NY-ESO-1 was also confirmed in this biopsy. The third TIL infusion (TIL-3) was 8,000×10 6administered at individual T cell doses. Prior to each TIL infusion, the patient received pretreatment chemotherapy with cyclophosphamide at 60 mg / kg on day 1 (TIL-1) and 30 mg / kg on days 4 (TIL-2, TIL-3). TIL infusions were supported by interleukin (IL-2), and 600,000 U / kg was administered 5 times every 12 hours starting 12 hours after TIL infusion. One week prior to the TIL-3 infusion, checkpoint blockade with pembrolizumab (1 mg / kg) was initiated and administered a total of 4 times every 3 weeks. An overview of the treatment schedule is shown in Figure 1a. After each of the 3 TIL infusions, the PSA level decreased. Complete remission was first recognized when PSA decreased to 0.01 μg / mL 2 months after the TIL-3 infusion (Figure 1b), and concurrently, complete remission of all tumor symptoms progressed. At the same time, the high pre-TIL NY-ESO-1 serum antibody titer continuously decreased during the course of TIL treatment (Figure 1c). MRT images showed that the tumor volume significantly decreased 8 weeks after the TIL-3 administration. The latest images of the patient showed that the tumor had completely remitted and has persisted for 3 years now (Figure 1d). Consistent with the clinical picture of complete remission, using circulating free DNA (cfDNA) digital PCR analysis, the tumor variant RFPL1 gene was not detected in the patient's current plasma.

[0094] NY-ESO-1 immunity The NY-ESO-1 expression of the tumor was confirmed by RT-PCR and immunohistochemistry, showing strong positive reactions in both tumor biopsies used for TIL isolation. Serum samples were continuously collected before the first TIL treatment and throughout the treatment period until today. The long-term measurement of NY-ESO-1 antibody was performed by the standard ELISA method. The first serum available since 2006 was negative for the NY-ESO-1 antibody. Three months before TIL treatment, the patient had a strong NY-ESO-1 antibody titer and the disease was progressing significantly. During and after TIL treatment, the antibody continuously decreased in parallel with the reduction of the tumor mass and is now detectable only at the residual titer level (Figure 2a). PSA values were measured regularly throughout the course of the disease to monitor clinical progression 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, the PSA level continuously decreased to less than 0.01 μg / L within 8 weeks and has not been detected since then (Figure 2b). Partial tumor regression was demonstrated after the first and second TIL treatments, and complete tumor regression was achieved 2 months after the third TIL treatment and the state has been confirmed even after 18 months have passed (Figure 2c).

[0095] Phenotype and functional activity of TIL By flow cytometry phenotype analysis, approximately 78%, approximately 81%, and approximately 96% of CD8 + T cells were revealed in the first (TIL-1), second (TIL-2), and third (TIL-3) infusions, respectively. CD4 +The T cells were 18% in TIL-1, but decreased to 11% in TIL-2 and 1.7% in TIL-3 (Figure 3a). TILs were further characterized by the expression of costimulatory molecules (CD25 / CD28) and differentiation markers (CD45RA, CCR7). Most of the TILs were effector memory T cells, with terminal effector memory (EMRA) T cells being 24%, 54%, and 39%, and central memory T cells being less than 10% (Figure 3b). Functional activity was evaluated after stimulating 1×10 5 T cells with OKT3 for 24 hours. The IFN-γ release of TILs reached approximately 1298 pg in TIL-1, approximately 1095 pg in TIL-2, and approximately 2184 pg in TIL-3, respectively (Figure 3c). Cytotoxicity was confirmed by the expression of CD107a, which is known to be upregulated after activation. 9.1% of TIL-1, 15.4% of TIL-2, and 23.8% of TIL-3 (most of which were CD8 + T cells) were CD107a + and showed cytotoxicity (Figure 3d). CD25hi CD127 - FoxP3 + regulatory CD4 + T cells (Tregs) were less than 0.13% in all TIL preparations.

[0096] Specificity of TILs Based on the antigen profile and strong expression of NY-ESO-1 in the patient's tumor, a panel of immunogenic "hot spot" NY-ESO-1 peptides known to be recognized by T cells of patients with NY-ESO-1-expressing tumors was used. Additionally, a panel of 10 individual HLA-matched peptides derived from mutant antigens identified by WES within the patient's tumor was used (Table 1). This peptide panel represents the selection of 9 peptides out of 77 non-synonymous coding neoantigens detected within the tumor by exome sequencing and the frame shift androgen receptor (AR) alternative splice variant AR-V12 discovered by Oncomine RNA sequencing. TILs were incubated with these peptides and the cell culture supernatants were analyzed for IFN-γ by ELISA assay. Specific reactivity against NY-ESO-1 was seen in all three TIL infusions. In particular, reactivity was seen against the HLA-A2-restricted peptide NY-ESO-1 p157-165, the nonamer peptides p92-100 and p96-104 known to be presented on HLA-Cw3, the NY-ESO-1 20-mer peptides p81-100 and p91-110 containing them, and the NY-ESO-1 24-mer peptide p119-143 (Figure 2a). Additionally, the TILs were standard chromium 51 (Cr 51)It showed specific cytotoxicity against NY-ESO-1 / HLA-A2-expressing tumor cells in the release assay (Figure 2b). Reactivity was also demonstrated for several mutant peptides: KKRCLFRSF (SEQ ID NO: 12) is derived from the gene PRPF8 (TIL-1, TIL-2, TIL-3), YLGEITYPF (SEQ ID NO: 15) is derived from the gene TRPS1 (TIL-1, TIL-3), FVSGKYKCL (SEQ ID NO: 17) is derived from the gene ZNF512 (TIL-1, TIL-3), NQVVQASMEK (SEQ ID NO: 18) is derived from the gene GFM1 (TIL-2, TIL-3), CPSKPSPLV (SEQ ID NO: 13) is derived from the gene SGIP1 (TIL-3), and the peptide VKWAKALPDCERAAS (SEQ ID NO: 21) is derived from a unique C-terminal part of the V12 splice variant AR-V12 (TIL-1, TIL-2, TIL-3) (Figure 2c). Considering that the infusates of TIL-1 and TIL-2 were generated from the same pre-propagation culture and that of TIL-3 was generated from a different culture, these results indicate that the immunological phenotype of TILs was composed of NY-ESO-1-specific T cells in all three TIL infusates and was composed of 4 and 6 mutant-specific T cells in TIL-1, TIL-2, and TIL-3, respectively. The T cells of the TIL-3 infusate were also analyzed for specific recognition of wild-type peptides derived from the PRPF8 gene and the TRPS1 gene, respectively. IFN-γ production was significantly higher with the mutant peptides compared to the wild-type peptides (Figure 2d).

Claims

1. A composition for use in the treatment of cancer, wherein the treatment comprises: i) administering to a mammal suffering from cancer, on day 0 (TIL-1), a composition comprising tumor-infiltrating lymphocytes (TIL-1) that have been expanded in vitro; ii) administering to a mammal suffering from cancer, on day 0 (TIL-2), a composition comprising tumor-infiltrating lymphocytes (TIL-2) that have been expanded in vitro, and then iii) administering a composition comprising a checkpoint inhibitor on day 0 (CI), wherein day 0 (CI) is within the range of 1 to 5 months (e.g., 6 weeks to 4 months, 8 weeks to 3 months, or about 2 months) after day 0 (TIL-1), and day 0 (CI) is within the range of 1 to 5 months (e.g., 6 weeks to 4 months, 8 weeks to 3 months, or about 2 months) after day 0 (TIL-2).

2. The composition according to claim 1, further comprising administering to the mammal, on day 0 (TIL-3), a composition comprising tumor-infiltrating lymphocytes (TIL-3) that have been expanded in vitro, wherein day 0 (TIL-3) is within the range of 1 to 14 days (e.g., 2 to 12 days, 5 to 12 days, or 7 days) after day 0 (CI).

3. The composition according to claim 1 or claim 2, comprising administering to the mammal, on day 0 (TIL-1), a composition comprising tumor-infiltrating lymphocytes (TIL-1) that have been expanded in vitro, wherein day 0 (TIL-1) is within the range of 1 to 4 months (e.g., 6 weeks to 4 months, 8 weeks to 3 months, or about 2 months) before day 0 (TIL-2).

4. The composition according to any one of claims 1 to 3, further comprising administering a chemotherapeutic agent to the mammal 7 to 1 days before the administration of TIL-1, TIL-2, and / or TIL-3.

5. The composition according to claim 4, wherein the chemotherapeutic agent is a drug commonly used in the treatment of the cancer, but the dosage is reduced compared to the dosage commonly used in the treatment of the cancer, and the dosage of the chemotherapeutic agent is at most 70% (e.g., 65% or more, 60% or more, 55% or more, 50% or more, or 65%, 60%, 55%, or 50%) of the dosage commonly used in the treatment of the cancer.

6. The composition according to any one of claims 1 to 5, wherein the number of TILs increases from TIL-1 to TIL-2 and then to TIL-3.

7. The composition according to any one of claims 1 to 6, wherein the mammal suffering from cancer has not received chemotherapy within at least 6 months before the start of treatment of the cancer.

8. The composition according to any one of claims 2 to 7, wherein the 0th day (TIL-3) 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, the monitoring period starts from the time when the level increases by 4 times or more after the administration of TIL-2, and the 0th day (TIL-3) is 1 to 7 days after such an increase is observed.

9. The composition according to any one of claims 1 to 8, wherein the 0th day (TIL-2) 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 in the mammal, the monitoring period starts from the time when the level increases by 4 times or more after the administration of TIL-1, and the 0th day (TIL-2) is 1 to 7 days after such an increase is observed.

10. The composition according to any one of claims 1 to 9, wherein the cancer is selected from cancers that do not respond effectively to monotherapy with checkpoint inhibitors, such as rhabdoid tumors, Ewing sarcoma, thyroid cancer, AML, medulloblastoma, glioblastoma, neuroblastoma, carcinoid tumors, prostate cancer, breast cancer, and pancreatic cancer.

11. The composition according to any one of claims 1 to 10, wherein the cancer is prostate cancer.

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

13. A composition for use in the treatment of a mammal suffering from cancer, i) administering to the mammal on the 0th day (TIL-1) a composition containing tumor-infiltrating lymphocytes proliferated in vitro; ii) Administering a composition comprising tumor-infiltrating lymphocytes grown in vitro to the mammal on day 0 (TIL-2), wherein day 0 (TIL-2) is within the range of 1 to 4 months (e.g., 6 weeks to 4 months, 8 weeks to 3 months, or about 2 months) after day 0 (TIL-1). iii) Administering a composition comprising a checkpoint inhibitor to the mammal on day 0 (CI), wherein day 0 (CI) is within the range of 1 to 5 months (e.g., 6 weeks to 4 months, 8 weeks to 3 months, or about 2 months) after day 0 (TIL-2), and iv) Administering a composition comprising TIL (TIL-3), which are tumor-infiltrating lymphocytes grown in vitro, to the mammal on day 0 (TIL-3), wherein day 0 (TIL-3) is within the range of 1 to 14 days (e.g., 2 to 12 days, 5 to 12 days, or 7 days) after day 0 (CI). A composition comprising the above.

14. The composition according to any one of claims 1 to 13, wherein the composition comprising tumor-infiltrating lymphocytes grown in vitro is obtained by preparing a tumor sample or a tumor metastasis sample from the mammal and culturing the sample in vitro.

15. The composition according to any one of claims 1 to 14, wherein 80% or more of the TIL-1, TIL-2, and / or TIL-3 are CD8 positive, and 20% or more of the CD8-positive TIL are effector memory cells.

16. The composition according to any one of claims 1 to 15, wherein the checkpoint inhibitor is administered once or multiple times (e.g., 1, 2, 3, 4, 5, or more times) after the administration of TIL-3.

17. The composition according to claim 16, wherein the checkpoint inhibitor is administered 3 times after the administration of TIL-3, and the interval between the administration of TIL-3 and the administration of the checkpoint inhibitor is 10 to 100 days. For example, in the case of the first administration of the checkpoint inhibitor after TIL-3 administration, it is 10 to 20 days; in the case of the second administration of the checkpoint inhibitor after TIL-3 administration, it is 40 to 70 days; and after the third administration of the checkpoint inhibitor after TIL-3 administration, it is 70 to 120 days.

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