KR20240170059A - A method for activating and proliferating a tumor-reactive T-cell population for immune cell therapy - Google Patents

Abstract

A method of obtaining an enriched cell population for tumor-reactive T cells. The method comprises: (a) obtaining a bulk population of peripheral blood mononuclear cells (PBMCs) from a sample of peripheral blood; (b) selecting CD8+ T cells, particularly those expressing PD-1 and/or TIM-3, from the bulk population; and (c) separating the cells selected in (b) from unselected cells to obtain a cell population enriched for tumor-reactive T cells. Using the above method, the present invention discloses a method of obtaining a pharmaceutical composition comprising a cell population enriched for tumor-reactive T cells, a method comprising a pharmaceutical composition comprising a cell population enriched for tumor-reactive T cells, and an isolated or purified cell population.

Description

{A method for activating and proliferating a tumor-reactive T-cell population for immune cell therapy}

The present invention relates to a method for activating and proliferating a tumor-reactive T cell population for immune cell therapy.

Adoptive cell therapy (ACT) using tumor-reactive T cells can produce positive clinical responses in some cancer patients. Nevertheless, several obstacles remain to the successful use of ACT in the treatment of cancer and other diseases. For example, T cells isolated from peripheral blood may not exhibit sufficient tumor-specific reactivity. Therefore, improved methods for obtaining tumor-reactive T cell populations from peripheral blood are needed.

The present invention discloses a method for obtaining an enriched cell population by selectively isolating CD8+ T cells from a peripheral blood sample, a method for using the same to obtain a pharmaceutical composition comprising a cell population enriched in tumor-reactive T cells, a method comprising a pharmaceutical composition comprising a cell population enriched in tumor-reactive T cells, and an independent or purified cell population.

An embodiment of the present invention comprises the steps of: (a) obtaining a bulk population of peripheral blood mononuclear cells (PBMCs) from a peripheral blood sample; (b) specifically selecting CD8+ T cells that also express PD-1 and/or TIM-3 from the bulk population; and (c) separating the cells selected in (b) from unselected cells, thereby obtaining a cell population enriched for tumor-reactive T cells.

Another embodiment of the present invention comprises the steps of: (a) obtaining a bulk population of PBMCs from a peripheral blood sample; (b) specifically selecting CD8+ T cells that also express PD-1 and/or TIM-3 from the bulk population; (c) separating the selected cells in (b) from the unselected cells to obtain a cell population enriched for tumor-reactive T cells; and (d) administering the cell population enriched for tumor-reactive T cells to a mammal.

Another embodiment of the present invention comprises the steps of: (a) obtaining a bulk population of PBMCs from a peripheral blood sample; (b) specifically selecting CD8+ T cells that also express PD-1 and/or TIM-3 from the bulk population; (c) separating the selected cells in (b) from the unselected cells to obtain a cell population enriched for tumor-reactive T cells; and (d) combining the cell population enriched for tumor-reactive T cells with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition comprising the cell population enriched for tumor-reactive T cells.

Additional embodiments of the present invention provide methods of treating or preventing related cell populations and cancers.

The present invention has the effect of providing a method for obtaining a cell population enriched in tumor-reactive T cells, a method for obtaining a pharmaceutical composition comprising a cell population enriched in tumor-reactive T cells using the same, a method comprising a pharmaceutical composition comprising a cell population enriched in tumor-reactive T cells, and an independent or purified cell population.

Figures 1A-1B are graphs showing interferon (IFN)-gamma secretion (pg/ml) (black bars) and the percentage of CD8+ cells expressing 4-1BB (gray bars) in cells that were expanded in vitro, isolated from the peripheral blood of melanoma patient 1913 (A) or melanoma patient 3713 (B) and sorted by fluorescence-activated cell sorting (FACS) for expression or lack of expression of CD8, PD-1 or TIM-3, and cocultured with autologous tumor cell lines.
Figure 1C is a graph showing the percentage (gray bars) of CD8+ cells expressing 4-1BB ifn-gamma secretion (pg/ml) (black bars) and the percentage (gray bars) of cells isolated from the peripheral blood of melanoma patient 3289 and cocultured with autologous tumor, sorted by FACS for expression or lack of expression of CD8, PD-1, LAG-3 or TIM-3.
Figures 2A-2C are graphs showing the percent specific lysis of effector cells from the target autologous tumor cell line TC1913 (A), the allogeneic (allo.) tumor cell line TC3289 (B), or the HLA-A0201-matched tumor cell line TC2448 (C) isolated from the peripheral blood of patient 1913, classified for expression as follows: CD8+ (open circles), PD-1+ (black circles), PD-1- (gray circles), TIM-3+ (black diamonds), or TIM-3? (gray diamonds), and expanded in vitro.
Figures 2D-2F are graphs showing the percent specific lysis of effector cells from the patient's peripheral blood, the target autologous tumor cell line TC3289 (D), the allogeneic tumor cell line TC1913 (E) or the allogeneic tumor cell line TC624 (F) and 3289 (D-F) and classified for expression as: CD8+ (open circles), PD-1+ (black circles), PD-1? (grey circles), TIM-3+ (black diamonds) or TIM-3? (grey diamonds). Target cell lysis following expansion in vitro is depicted.

Selecting CD8+ cells expressing PD-1 (PD-1; CD279) and/or T-cells was found to enrich for the immunoglobulin and mucin domain 3 (TIM-3) biomarkers that enrich for tumor-reactive T cells present in the peripheral blood. Usefully, selecting CD8+ cells expressing PD-1 and/or TIM-3 enriches for a greater number of tumor-reactive T cells compared to CD8+ cells that do not express these markers.

In this regard, embodiments of the present invention provide a method for obtaining a cell population enriched for tumor-reactive T cells, comprising: (a) obtaining a bulk population of peripheral blood mononuclear cells (PBMCs) from a peripheral blood sample; (b) specifically selecting CD8+ T cells that also express PD-1 and/or TIM-3 from the bulk population; and (c) separating the cells selected in (b) from the unselected cells, thereby obtaining a cell population enriched for tumor-reactive T cells. The method of the present invention advantageously enables shortening the in vitro culture time and selecting tumor-reactive T cells without the need for screening for autologous tumor recognition.

The method of the present invention may comprise obtaining a bulk population of PBMCs from a peripheral blood sample by any suitable method known in the art. Suitable methods of obtaining a bulk population of PBMCs may include, but are not limited to, hematopoiesis and/or leukapheresis. The bulk population of PBMCs obtained from a tumor sample may comprise T cells comprising tumor-reactive T cells.

Peripheral blood can be obtained from any mammal. Unless otherwise stated, the term "mammal" as used herein refers to a mammal of the order Logomorpha, such as rabbits; the order Carnivora, which includes Felines (cats) and Canines (dogs); the order Artiodactyla, which includes bovines (cattle) and pigs (swine); or the order Perssodactyla, which includes Equines (horses). Preferably, the mammal is a non-human primate, such as an order Primate, Ceboids or Simoids (monkeys) or an order Apes (humans and apes). In some embodiments, the mammal can be a mammal of the order Rodentia, such as a mouse and a hamster. Preferably, the mammal is a non-human primate or a human. A particularly preferred mammal is a human.

The method of the present invention may comprise specifically selecting CD8+ T cells that also express PD-1 and/or TIM-3 from the bulk population. In a preferred embodiment, the method further comprises selecting cells that express CD3. The method may comprise specifically selecting cells in any suitable manner. Preferably, the selection is performed using flow cytometry. The flow cytometry may be performed using any suitable method known in the art. The flow cytometry may utilize any suitable antibody and stain. For example, specific selection of CD3, CD8, TIM-3 or PD-1 may be performed using anti-CD3, anti-CD8, anti-TIM-3 or anti-PD-1 antibodies, respectively. Preferably, the antibodies are selected to specifically recognize and bind to the particular biomarker being selected. The antibody or antibodies may be conjugated to beads (e.g., magnetic beads) or fluorochromes. Preferably, the flow cytometry is fluorescence activated cell sorting (FACS).

In one embodiment of the invention, specifically selecting can comprise specifically selecting CD8+ T cells that are positive for expression of TIM-3, PD-1, or both TIM-3 and PD-1. In this regard, specifically selecting can comprise specifically selecting T cells that are single positive for expression of TIM-3 or PD-1, or double positive for simultaneous co-expression of TIM-3 and PD-1. In an embodiment of the invention, the method comprises specifically selecting CD8+ T cells that express TIM-3 from the bulk population. In another embodiment of the invention, the method comprises specifically selecting CD8+ T cells that express PD-1 from the bulk population. Another embodiment of the invention comprises specifically selecting CD8+ T cells that are (i) TIM-3+/PD-1+, (ii) TIM-3−/PD-1+, or (iii) TIM-3+/PD-1? from the bulk population. In another embodiment of the present invention, any of the methods described herein may further comprise selecting cells that express CD3+.

In an embodiment of the present invention, specifically selecting can comprise specifically selecting a combination of CD8+ cells expressing any of the markers described herein. In this regard, the method can generate a cell population enriched for tumor-reactive cells comprising a mixture of cells expressing any two biomarkers described herein. In an embodiment of the present invention, specifically selecting comprises specifically selecting a combination of PD-1+ cells and TIM-3+ cells. In another embodiment of the present invention, any of the methods described herein can further comprise selecting cells expressing CD8+ and/or CD3+.

The method of the present invention may comprise separating the selected cells from the unselected cells to obtain a cell population enriched for tumor-reactive T cells. The selected cells may be physically separated from the unselected cells. The selected cells may be separated from the unselected cells by any suitable method, such as, for example, sorting. Separating the selected cells from the unselected cells preferably produces a cell population enriched for tumor-reactive T cells.

The cell population obtained by the method of the present invention is advantageously enriched for tumor-reactive T cells. In this regard, the cell population obtained by the method of the present invention may contain a higher proportion of tumor-reactive T cells compared to a cell population that is not obtained by sorting for expression of TIM-3 and/or PD-1.

In an embodiment of the present invention, the method comprises obtaining a cell population enriched for tumor-reactive T cells without screening for self-tumor recognition. In this regard, the method of the present invention advantageously provides a cell population enriched for cells having tumor reactivity without the need to screen the cells for self-tumor recognition.

In an embodiment of the present invention, the method does not comprise non-specifically stimulating the bulk population of T cells prior to specifically selecting the cells. In this regard, the method of the present invention comprises non-specifically stimulating the bulk population of T cells (e.g., with an anti-4-1BB antibody, an anti-CD3 antibody, and/or an anti-CD28 antibody).

In an embodiment of the present invention, the method further comprises expanding the number of T cells in the enriched cell population obtained in vitro by the method of the present invention. The number of T cells can be increased by at least about 3-fold (or 4-fold, 5-fold, 6-fold, 7-fold, 8-fold or 9-fold), more preferably by at least about 10-fold (or 20-fold), more preferably by at least about 100-fold, more preferably by at least about 1,000-fold, and most preferably by at least about 100,000-fold. The number of T cells can be expanded using any suitable method known in the art. Exemplary methods of expanding the number of cells are described in U.S. Pat. No. 8,034,334 and U.S. Patent Application Publication No. 2012/0244133, each of which is incorporated herein by reference.

In an embodiment of the present invention, the method further comprises culturing the enriched cell population obtained by the method of the present invention in the presence of any one or more of TWS119, interleukin (IL-21), IL-12, IL-15, IL-7, transforming growth factor (TGF) beta and an AKT inhibitor (AKTi). Without being bound by a particular theory, culturing the enriched cell population in the presence of TWS119, IL-21 and/or IL-12 advantageously enhances the anti-tumor response of the enriched cell population or delays differentiation of the enriched cell population.

In an embodiment of the present invention, the method further comprises the step of transducing or transfecting cells of the enriched population obtained by any of the methods of the present invention described herein with a nucleotide sequence encoding any one or more of IL-12, IL-7, IL-15 IL-2, IL-21, mir155 and anti-PD-1 siRNA.

In an embodiment of the present invention, the method further comprises stimulating the enriched cell population obtained by the method of the present invention with a cancer antigen and/or autologous tumor cells. Stimulating the enriched cell population with a cancer antigen and/or autologous tumor cells can be accomplished by any suitable method. For example, stimulating the enriched cell population can be accomplished by physically contacting the enriched cell population with the cancer antigen and/or autologous tumor cells. Without being bound by a particular theory, it is believed that stimulating the enriched cell population with a cancer antigen and/or autologous tumor cells can advantageously enhance the anti-tumor response of the enriched cell population.

The term "cancer antigen" as used herein means an antigen that is expressed by a tumor or cancer. A cancer antigen may be additionally expressed by normal, non-tumor, or non-cancerous cells. However, in such cases, the expression of the cancer antigen by the normal, non-tumor, or non-cancerous cells is not as strong as the expression by the tumor or cancer cells. In this regard, the tumor or cancer cells may overexpress the antigen or express the antigen at a significantly higher level compared to the expression of the antigen by the normal, non-tumor, or non-cancerous cells. In addition, the cancer antigen may be additionally expressed by cells in other developmental or maturational states. For example, the cancer antigen may be additionally expressed by cells in embryonic or fetal stages that are not generally found in the adult host. Alternatively, the cancer antigen may be additionally expressed by stem cells or progenitor cells, which are not generally found in the adult host.

A cancer antigen can be an antigen expressed by any cell of any cancer or tumor, including the cancers and tumors described herein. The cancer antigen can be a cancer antigen of only one type of cancer or tumor, such that the cancer antigen is associated with or characteristic of only one type of cancer or tumor. Alternatively, the cancer antigen can be a cancer antigen (e.g., characteristic) of more than one type of cancer or tumor. For example, the cancer antigen can be expressed on both breast cancer and prostate cancer cells and not expressed at all on normal, non-tumor, or non-cancerous cells. Exemplary cancer antigens can include any one or more of gp100, MART-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A7. A9, MAGE-A10, MAGE-A11, MAGE-A12, NY-ESO-1, vascular endothelial growth factor receptor-2 (VEGFR-2), HER-2, mesothelin, and epidermal growth factor receptor variant III (EGFR III).

The method of the present invention advantageously produces a cell population enriched for tumor-reactive T cells. The T cells may be tumor-reactive so as to specifically recognize, lyse, and/or kill tumor cells.

In this regard, embodiments of the present invention provide an isolated or purified cell population enriched for tumor-reactive T cells obtained by any of the methods of the present invention described herein. In one embodiment, the isolated or purified cell population comprises (a) CD8+/TIM-3+/PD-1+ T cells, (b) CD8+/TIM-3−/PD-1+ T cells, and (c) CD8+/TIM-3+/PD-1− T cells, wherein the cell population is enriched for tumor-reactive T cells.

In another embodiment of the invention, the isolated or purified cell population is (a) CD8+/TIM-3+/PD-1+ T cells, (b) CD8+/TIM-3-/PD-1+ T cells, or (c) CD8+/TIM-3+/PD-1- T cells. In another embodiment of the invention, any of the cell populations described herein can also be CD3+.

In one embodiment of the invention, the isolated or purified cell population comprises a mixture of cells expressing any of the biomarkers described herein. For example, the isolated or purified cell population may comprise a combination of PD-1+ cells and TIM-3+ cells. In another embodiment of the invention, any of the cell populations described herein may also be CD8+ and/or CD3+.

The term "isolated" as used herein means removed from its natural environment. The term "purified" as used herein means increased purity, where "purity" is a relative term and should not necessarily be construed as absolute purity. For example, the purity can be at least about 50%, can be 60%, 70%, or 80%, can be greater than 90%, or can be 100%.

Another embodiment of the present invention comprises the steps of: (a) obtaining a bulk population of PBMCs from a peripheral blood sample; (b) specifically selecting CD8+ T cells from the bulk population that also express PD-1 and/or TIM-3; (c) separating the cells selected in (b) from the unselected cells, thereby obtaining a cell population enriched for tumor-reactive T cells; and (d) administering the cell population enriched for tumor-reactive T cells to a mammal. Obtaining a bulk population of PBMCs from a peripheral blood sample, and particularly selecting CD8+ T cells from the bulk population that also express PD-1 and/or TIM-3, and separating the selected cells from the unselected cells, thereby obtaining a cell population enriched for tumor-reactive T cells. The tumor-reactive T cells can be performed as described herein in connection with other aspects of the present invention.

The method of the present invention may further comprise administering to the mammal a cell population enriched for tumor-reactive T cells. The cell population enriched for tumor-reactive T cells may be administered in any suitable manner. Preferably, the cell population enriched for tumor-reactive T cells is administered by injection, for example, intravenously.

The cell populations of the present invention enriched for tumor-reactive T cells may be included in a composition, such as a pharmaceutical composition. In this regard, the present invention provides a pharmaceutical composition comprising any of the cell populations described herein and a pharmaceutically acceptable carrier.

Another embodiment of the present invention comprises the steps of: (a) obtaining a bulk population of PBMCs from a peripheral blood sample; (b) specifically selecting CD8+ T cells from the bulk population that also express PD-1 and/or TIM-3; (c) separating the cells selected in (b) from the unselected cells, thereby obtaining a cell population enriched for tumor-reactive T cells; and (d) combining the cell population enriched for tumor-reactive T cells with a pharmaceutically acceptable carrier, thereby obtaining a pharmaceutical composition comprising the cell population enriched for tumor-reactive T cells. Obtaining a bulk population of PBMCs from a peripheral blood sample, and particularly selecting CD8+ T cells from the bulk population that also express PD-1 and/or TIM-3, and separating the selected cells from the unselected cells, thereby obtaining a cell population enriched for tumor-reactive T cells, can be performed as described herein in connection with other aspects of the present invention.

The method may comprise combining the cell population enriched for tumor-reactive T cells with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition comprising the cell population enriched for tumor-reactive T cells. Preferably, the carrier is a pharmaceutically acceptable carrier. In relation to the pharmaceutical composition, the carrier may be any one commonly used for cell administration. Such pharmaceutically acceptable carriers are well known to those skilled in the art and are readily available to the public. Preferably, the pharmaceutically acceptable carrier is free of harmful side effects or toxicity under the conditions of use. Pharmaceutically acceptable carriers suitable for injection include any isotonic carrier, such as saline (about 0.90% w/v NaCl in water, about 300 mOsm/L NaCl in water or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, Ill.), PLASMA-LYTE A (Baxter, Deerfield, Ill.), about 5% dextrose in water, or Ringer's lactate. In one embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

For purposes of the present invention, the dose administered, e.g., the number of cells of the cell population of the present invention enriched for tumor-reactive T cells, should be sufficient to elicit, e.g., a therapeutic or preventative response in a mammal over a reasonable time frame. For example, the number of cells should be sufficient to bind to a cancer antigen or to detect, treat, or prevent a cancer within a period of at least about 2 hours, e.g., at least 12 to 24 hours, from the time of administration. In certain embodiments, the period of time may be much longer. The number of cells will depend on, e.g., the potency of the particular cells and the condition of the mammal (e.g., human), as well as the weight of the mammal (e.g., human) to be treated.

Many assays are known in the art for determining the number of cells administered from a cell population of the present invention enriched for tumor-reactive T cells. For purposes of the present invention, assays that involve comparing the extent to which target cells are lysed or the extent to which one or more cytokines, such as, for example, IFN-γ and IL-2, are secreted upon administration of a given number of such cells to the target cells can be used to determine a starting number of cells to be administered to a mammal from a set of mammals given a given number of cells, respectively. The extent to which target cells are lysed or the extent to which cytokines, such as, for example, IFN-γ and IL-2, are secreted upon administration of a given number of cells can be assayed by methods known in the art. For example, secretion of a cytokine, such as, for example, IL-2, can also provide an indication of the quality (e.g., phenotype and/or efficacy) of the cell preparation.

The number of cells from the cell population of the present invention enriched for tumor-reactive T cells will also be determined by the presence, nature and extent of any adverse side effects that may accompany administration of a particular cell population. Typically, the attending physician will determine the number of cells to treat each individual patient, taking into account various factors such as age, weight, general health, diet, sex, route of administration and severity of the condition being treated. By way of example and not intended to limit the invention, the number of cells can be from about 10×106 to about 10×1011 cells per infusion, from about 10×109 to about 10×1011 cells per infusion, or from 10×107 to about 10×1011 cells per infusion. Cell populations obtained by the methods of the present invention can advantageously enable effective treatment or prevention of cancer.

It is contemplated that a cell population obtained by the methods of the present invention may be used in a method of treating or preventing cancer. In this regard, the present invention provides a method of treating or preventing cancer in a mammal, comprising administering to the mammal a pharmaceutical composition or cell population obtained by any of the methods of the present invention described herein in an amount effective to treat or prevent cancer in the mammal. Another embodiment of the present invention provides a method of treating or preventing cancer in a mammal, comprising administering to the mammal a cell population enriched for tumor-reactive T cells in an amount effective to treat or prevent cancer in the mammal by any of the methods of the present invention described herein.

The terms "treat" and "prevent" and words derived therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that those skilled in the art would recognize as having potential benefit or therapeutic effects. In this regard, the methods of the present invention can provide any amount or level of cancer treatment or prevention in a mammal. Furthermore, the treatment or prevention provided by the methods of the present invention can include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented. Furthermore, for purposes herein, "prevention" can include delaying the onset of the disease, or a symptom or condition thereof.

For purposes of the methods of the present invention in which a population of cells is administered, the cells may be allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal.

A specific embodiment of the present invention further comprises lymphodepleting the mammal prior to administering any of the enhanced cell populations obtained by any of the inventive methods described herein. Examples of lymphodepletion include, but are not limited to, non-myeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, and the like.

In connection with the methods of the present invention, the cancer can be any cancer, including any sarcoma (e.g., synovial sarcoma, osteosarcoma, uterine leiomyosarcoma, and alveolar rhabdomyosarcoma), lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), and hepatocellular carcinoma. By way of example, the cancer may be any one of glioma, head and neck cancer, acute lymphoblastic cancer, acute myeloid leukemia, bone cancer, brain cancer, breast cancer, anal cancer, anorectal cancer, eye cancer, intrahepatic bile duct cancer, cancer of the joints, neck, gallbladder or pleura, cancer of the nose, nasal cavity or middle ear, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic bone marrow cancer, colon cancer (e.g., colon cancer), esophageal cancer, cervical cancer, gastrointestinal cancer (e.g., gastrointestinal carcinoid tumor), hypopharyngeal cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer, mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureter cancer and bladder cancer.

The following examples are intended to illustrate the present invention but do not limit its scope.

Example 1

This example demonstrates in vitro autologous tumor recognition of T cells isolated from the peripheral blood of melanoma patients, after expansion of the cells in large numbers of test tubes, depending on expression of PD-1, TIM-3, or LAG-3.

4-1BB up-regulation is an indicator of TCR stimulation. After the numerous cells in which it is observed are expanded and in the absence of TCR stimulation, the expression of the 4-1BB branch is lost. It is also observed that after the numerous cells are expanded and the communication range is co-cultured with autologous tumor cell lines, previously lost expression of the 4-1BB branch and T cells stimulated by the cell line will express 4-1BB again. Therefore, the expression of the 4-1BB branch is measured as a marker of TCR stimulation for autologous tumor cell lines after 24 hours of co-culture with autologous tumors.

Cell apheresis from peripheral blood of three separate melanoma patients (1913, 3713, and 3289) was placed overnight at 4 °C in the absence of cytokines and stained. Cells from patients 1913 and 3713 were sorted into the following CD3+ populations using anti-CD3, anti-CD8, anti-PD-1, and TIM-3 antibodies: CD8+, CD8+/PD-1+, CD8+/TIM-3+, CD8+/PD-1?, or CD8+/TIM-3? by fluorescence-activated cell sorting (FACS). Cells from well-tolerated 3289 were sorted into the following CD3+ populations using anti-CD3, anti-CD8, anti-PD-1, TIM-3 and LAG-3 antibodies: CD8+ by fac, CD8+/PD-1+, CD8+/LAG3+, CD8+/TIM-3+, CD8+/PD-1?, CD8+/LAG3?, or CD8+/TIM-3?. The cells were expanded in vitro for 14 days. On day 14, the communication range was washed and co-cultured with the corresponding autologous tumor cell line (1 × 105 effectors: 1 × 105 target cells) and the reactivity was assessed by measuring ifn-gamma release and the percent of CD8+ cells expressing 41 BB after 24 h of co-culture. The results are shown in Figures 1A-1C and Tables 1-3. As illustrated in Figures 1A-1C and Tables 1-3, cells were classified according to the expression of PD-1 or TIM-3 and enriched for tumor-responsive cells compared to negative cells for PD-1 or TIM-3 expression, respectively.

An in vitro expanded effector population isolated from the peripheral blood of patient 1913 was co-cultured against autologous (Aut.) tumor cell lines (TC1913) and allogeneic (Allo.) tumor cell lines based on expression of indicated cell surface markers.

Reactivity to IFN-gamma (pg/ml) was shown.

Values in parentheses are the percentages of CD3+ CD8+ cells that upregulated CD137 (41BB) at 24 hours (h) after co-culture.

Tumor cell lines (TC) 624 CIITA, 2119, 2448, and 1865 share the HLA A*0201 allele with TC1913, and TC 1379 shares A*11 with TC1913.

TC2301 is an isogeneic control (mismatched for all HLA) used as a negative control.

Values >300 pg/ml and greater than twice the background are considered positive and are underlined and displayed in bold.

An in vitro expanded effector population isolated from the peripheral blood of patient 3713 was co-cultured against an autologous tumor cell line (TC3713) and an allogeneic tumor cell line based on expression of indicated cell surface markers.

Reactivity to IFN-gamma (pg/ml) was shown.

Values in parentheses are the percentage of CD3+ CD8+ cells that upregulated CD137 (41BB) after 24 h of co-culture.

Tumor cell lines (TC) 624 CIITA and 2119 share the HLA A*0201 allele with TC3713. TC 1379 and TC 3460 are isogenic target cell lines (mismatched for all HLA) used as negative controls.

Values >300 pg/ml and greater than twice the background are considered positive and are underlined and displayed in bold.

An in vitro expanded effector population isolated from the peripheral blood of patient 3289 was co-cultured against autologous tumor cell lines (3289) and allogeneic tumor cell lines based on expression of indicated cell surface markers.

Reactivity to IFN-gamma (pg/ml) was shown.

Values in parentheses are the percentage of CD3+ CD8+ cells that upregulated CD137 (41BB) after 24 h of co-culture.

Tumor cell lines (TC) 1379 and TC526 are allogeneic cell lines (mismatched for all HLA) and autologous CD4+ CD25- cells isolated from peripheral blood used as negative controls.

Values >300 pg/ml and greater than twice the background are considered positive and are underlined and displayed in bold.

Example 2

This example demonstrates in vitro autologous tumor recognition of T cells isolated from peripheral blood of matched melanoma patients and expressed either PD-1 or TIM-3 after expansion of the cells in large numbers of test tubes.

The communication-capable cells were obtained from peripheral blood of patient 1913 or patient 3289 as described in Example 1 and sorted by FACS for expression of PD-1 or TIM-3. The sorted cells were expanded for 14 days in vitro. On day 15, the target tumor cell lines (autologous and allogeneic) were co-cultured with the sorted population of communication-capable cells (effector cells) at the rates shown in FIGS. 2A-2F. 51Cr release was determined in triplicate by γ-counting for 4 hours and the percent specific inhibition was calculated using the formula: [(spontaneous experimental counts per minute (cpm) cpm)/(maximal cpm spontaneous cpm)] × 100. The results are shown in FIGS. 2A-2F.

As illustrated in Figures 2A-2F, the communication range obtained from peripheral blood and sorted for FD-1+ or TIM-3+ expression can be used to sequence autologous tumor cell lines.

All references, including publications, patent applications and patents, cited herein are hereby specifically incorporated by reference into and are intended to be incorporated by reference to the same extent as if each reference were individually set forth in its entirety herein.

Unless otherwise indicated herein or clearly contradicted by context, the use of the terms “a” and “an” and “the” and “at least one” and similar references in connection with describing the present invention (especially in connection with the following claims) are to be construed to cover both the singular and the plural. Unless otherwise indicated herein or clearly contradicted by context, the use of the term “at least one” followed by a list of one or more items (e.g., “at least one of A and B”) is to be construed to mean one item selected from two or more of the listed items (A or B) or a combination of the listed items (A and B). The terms “comprising,” “having,” “including,” and “containing” are to be construed as flexible terms (i.e., “including” means “including”) unless otherwise indicated herein. Unless otherwise indicated herein, the recitation of ranges of values in this specification is merely intended to serve as a shorthand way of referring individually to each separate value included in the range, and each and every value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language provided herein (e.g., “such as”) is intended merely to illuminate the invention, and not to place a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of the present invention are described herein, including the best mode discovered by the inventors for carrying out the invention. Variations of the preferred embodiments will become apparent to those skilled in the art upon reading the above description. The inventors expect that those skilled in the art will employ such variations as they deem appropriate, and the inventors intend the invention to be practiced better than otherwise because it is specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the material set forth in the appended claims as permitted by the law applicable hereto. Moreover, any combination of the elements described above in all possible variations thereof is encompassed by the present invention unless otherwise indicated herein or otherwise clearly contradicted by the context.

Claims (1)

A method of administering to a mammal a cell population enriched in tumor-reactive T cells, comprising:
(a) obtaining a bulk population of peripheral blood mononuclear cells (PBMCs) from a peripheral blood sample;
(b) specifically selecting CD8+ T cells that also express PD-1 and/or TIM-3 from the bulk population;
(c) a step of separating the cells selected in (b) from the unselected cells to obtain a cell population enriched in tumor-reactive T cells; and
(d) a step of inducing overexpression of Klf4 protein in a cell selected from the group consisting of CAR-CD8 T cells transduced with a gene encoding a chimeric antigen receptor (CAR) into the CD8 T cells;
A method for activating and proliferating a tumor-reactive T cell population for immune cell therapy.