JP2018510644A - In vitro artificial lymph nodes for sensitization and proliferation of T cells for therapy and epitope mapping - Google Patents

Abstract

HER2 + invasive breast cancer (IBC) patients with residual disease after neoadjuvant chemotherapy have a significant risk of anti-HER2 type 1 T helper (Th1) cell immunodeficiency and recurrent disease. Anti-HER2 CD4 + T cell responses have been shown to decrease gradually along the breast cancer continuum-a robust response in healthy donors and patients with benign disease, an inhibitory response in patients with HER2 + non-invasive breast cancer, and In HER2 + IBC patients, there was little response. The present invention relates to a method for creating a microenvironment for T cell culture growth. Proliferated T cells can be used for various therapeutic and research purposes.

Description

Cross-reference of related applications

  This application claims the priority of Patent Document 1 filed on March 26, 2015 and Patent Document 2 filed on March 26, 2015, the contents of each of which are hereby incorporated by reference in their entirety. Incorporated in the description.

HER2 ⁺ invasive breast cancer (IBC) patients with residual disease after neoadjuvant chemotherapy have a significant risk of anti-HER2 type 1 T helper (Th1) cell immunodeficiency and recurrent disease. The anti-HER2 CD4 ⁺ T cell response was shown to gradually decrease along the breast cancer continuum-a tonic response in healthy donors and benign disease patients, an inhibitory response in HER2 ⁺ noninvasive breast cancer patients, As well as HER2 ⁺ IBC patients, there was little response.

The lifetime risk of developing breast cancer is about 1 in 8 people. The erb-B2 oncogene (HER-2 / neu) is a molecular driver that is overexpressed in the majority of breast, ovarian, gastroesophageal, lung, pancreatic, prostate, and other solid tumors . Overexpression of HER2, a molecular carcinogenic driver in several tumor types, including about 20-25% breast cancer (“HER2 ^pos ”) (Non-Patent Document 1), has an aggressive clinical course, resistance to chemotherapy, and Associated with overall poor prognosis in BC. See Non-Patent Document 2 (“Henson et al.”) And Non-Patent Document 3. In early BC, overexpression of HER2 is associated with enhanced invasiveness (Non-patent document 4), tumor cell migration (Non-patent document 5), and expression of angiogenic factors (Non-patent document 6). It suggests an important role in promoting the tumorigenic environment. A retrospective analysis of patients with non-invasive breast cancer (“DCIS”) found that DCIS lesions that overexpress HER2 were more than 6 times more likely to be associated with invasive breast cancer than DCIS lesions that did not overexpress HER2. Met.

Molecular targeted therapeutics that target HER2 (ie, HERCEPTIN® / trastuzumab) significantly improve survival in patients with HER2 ^pos BC when combined with chemotherapy (7) Of patients become resistant to such treatment (Non-Patent Document 8). Strategies to identify patient subgroups at high risk of treatment failure are needed, as are new approaches to improve response rates to treatments targeting HER2. A molecular-targeted therapeutic that targets HER2, trastuzumab, has had a surprisingly positive clinical effect in this type of breast cancer, but almost full resistance to existing HER2 therapies in advanced disease states, plus a target A significant proportion of disease recurrences in women receiving therapy demonstrates the need for additional strategies targeting HER2. The expectation of a vaccine that activates and works against the HER2 immune system to mitigate tumor progression and prevents recurrence has not been fully realized. There is a need for additional tests and therapies to diagnose and treat HER2 breast cancer.

The role of systemic anti-HER2 CD4 ⁺ Th1 response in breast cancer formation by HER2 has been elucidated in recent years. A progressive loss of anti-HER2 CD4 ⁺ Th1 response across the tumorigenic continuum in HER2 ^pos breast cancer, which appears to be HER2-specific and regulatory T cell (T _reg ) independent, has been identified. Specifically, there is an inverse correlation between anti-HER2 CD4 ⁺ Th1 response and HER2 expression and disease progression. The Th1 reactivity profile shows a significant gradual decline in anti-HER2 Th1 immunity, continuation in HER2 ^pos breast cancer formation (HD (healthy donor) → BD (benign breast biopsy) → HER2 ^neg -DCIS (non-invasive milk) Tube cancer) → HER2 ^neg -IBC (invasive breast cancer) → HER2 ^pos -DCIS → HER2 ^pos -IBC (invasive breast cancer)). See Non-Patent Document 9 and Patent Document 3 filed on March 13, 2015 (collectively, “Datta et al.”). Suppression of the anti-HER2 Th1 response in HER2 ^pos -invasive breast cancer recovered differentially after HER2-pulse type 1 polarized dendritic cell ("DC1") vaccination, but trastuzumab and chemotherapy ("T / C )) After HER2-targeted therapy or by other standard therapies such as surgical excision or radiation did not restore the suppressive response. Id. The recovered anti-HER2 Th1 response also seems to persist for at least about 6 months or more.

Proliferation of T lymphocyte subsets (CD4 ⁺ or CD8 ⁺ ) is an essential step to acquire sufficient T cells to perform adoptive therapy or to identify epitopes on target antigens of peptide-based vaccines It is. The proliferation of T cells is a simple process in principle. In practice, however, there are many technical problems including low levels of proliferation, premature activation-induced cell death (apoptosis), or loss of antigen specificity and / or function.

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  Part of the problem is the inability to replicate in the body environment, ie lymph nodes, where antigen-specific T cell proliferation occurs in vitro. These are specialized tissues containing many different cell types apart from T lymphocytes containing stromal cells such as antigen presenting dendritic cells and epithelial cells. By providing contact-dependent signals (surface receptors) and water-soluble signals (cytokines) that are important for T cell proliferation and maintenance of cell function, each of these cell types plays a different role (both are currently defined). But the characterization is still incomplete).

  There is a need for new cancer treatments. Therefore, there is a need in the art for additional immunotherapy approaches to treat or prevent breast cancer and other malignancies. The present invention satisfies this need.

Means for solving the solution

  In one embodiment, the method provides a method of expanding T cells. In one embodiment, the method comprises contacting a T cell with one or more dendritic cells, at least two cytokines, and a T cell growth factor.

  In one embodiment, T cells are contacted with an antigen, thereby generating antigen-specific T cells.

  In one embodiment, the dendritic cell is a type I dendritic cell.

  In one embodiment, the at least two cytokines are interleukin-7 (IL-7) and interleukin-15 (IL-15).

  In one embodiment, the T cell growth factor is interleukin-2 (IL-2).

  In one embodiment, the method comprises the steps of a) contacting a T cell with an autologous type I dendritic cell DC in vitro, thereby generating an antigen-specific T cell; b) an antigen-specific T cell; Contacting the cells with IL-7 and IL-5 to generate stimulated antigen-specific T cells; c) contacting the stimulated antigen-specific T cells with IL-2, thereby antigen specificity And generating a proliferated antigen-specific T cell population that maintains cellular function.

  In one embodiment, the method provides T cells generated by the method of the method.

  In one embodiment, the method is a population of cultured and expanded antigen-specific T cells that exhibit anti-tumor activity generated by the method of any of claims 1-6, wherein the cells are mammalian Provide a population that has grown to a sufficient number for effective treatment in

  In one embodiment, the method is a population of cultured and expanded antigen-specific T cells that exhibit anti-tumor activity generated by the method of the invention, wherein the cells are expanded to a number sufficient for effective epitope mapping. Provide a group.

  In one embodiment, the method is an isolated polynucleotide encoding a T cell receptor (TCR) derived from an antigen specific T cell, wherein the antigen specific T cell is generated by the method of the invention. Isolated polynucleotides are provided.

  In one embodiment, the method provides an immunotherapeutic method comprising administering a T cell to a subject in need thereof, wherein the T cell is generated by the method of the invention.

  In one embodiment, the method is a method of expanding a T cell population comprising at least one T cell obtained from a blood sample from a subject vaccinated against an antigen, the T cell comprising one T cell. There is provided a method comprising the step of contacting a dendritic cell or a precursor thereof, at least two cytokines, and a T cell growth factor.

  In one embodiment, the blood sample comprises at least one T cell and at least one DC precursor of a population specific for a vaccine antigen.

  In one embodiment, DC precursors are pulsed with antigen, activated to antigen-specific type I dendritic cells (DC1), and then co-cultured with T cells to produce antigen-specific DC1.

  In one embodiment, the at least two cytokines are interleukin-7 (“IL-7”) and interleukin-15 (“IL-15”).

  In one embodiment, the T cell growth factor is interleukin-2 (“IL-2”).

  In one embodiment, the method comprises a) co-culturing T cells from a patient sample with antigen-specific T cell autologous type I dendritic cells (DC1) in vitro; b) from step a) Contacting the cells with IL-7 and IL-5 to generate stimulated antigen-specific T cells; c) then contacting the stimulated antigen-specific T cells with IL-2, thereby producing an antigen Generating an expanded antigen-specific T cell population that maintains specificity and cellular function.

  In one embodiment, the method further comprises repeating steps a) -c) from one to at least three more times to generate a further expanded antigen-specific T cell population.

In one embodiment, the T cell is CD4 ⁺ .

  In one embodiment, the antigen is HER2.

In one embodiment, the method is a method of expanding a CD4 ⁺ T cell population comprising at least one CD4 ⁺ T cell obtained from a blood sample from a breast cancer patient vaccinated against HER2, comprising CD4 ⁺ Provided is a method comprising contacting a T cell with one or more dendritic cells or precursors thereof, at least two cytokines, and a T cell growth factor.

In one embodiment, at least one DC precursor in the sample is pulsed with MHC class II HER2 peptide and contacted with CD4 ⁺ T cells.

  In one embodiment, the method comprises: a) co-culturing T cells from claim 11 with HER2 pulsed DC1; b) contacting the cells from step a) with IL-7 and IL-5 and stimulating C) subsequently contacting the stimulated antigen-specific T cells with IL-2, thereby maintaining the antigen specificity and cellular function, and the expanded antigen-specific T cells Generating a cell population.

  In one embodiment, the method further comprises repeating steps a) -c) from one to at least three more times to generate a further expanded antigen-specific T cell population.

  The following preferred embodiments for carrying out the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings preferred embodiments of the invention. However, it should be understood that the invention is not limited to the precise configuration and instrumentalities of the embodiments shown in the drawings.

FIG. 1 shows the anti-HER2 Th1 response repertoire of 4 HER2 ⁺ IBC patients who received adjuvant HER2 pulsed DC1 vaccine and have residual disease after neoadjuvant therapy. Each patient is depicted in a different color and indicates the number of reactive peptides (n) (also called “response repertoire”) before vaccination, 3 months after vaccination, and 6 months after vaccination. The average response repertoire increased from 0.5 ± 1 peptide before vaccination to 3.25 ± 0.96 peptide (p = 0.01) 3 months after vaccination and 4 ± 0.8 peptide (p = 0.01) 6 months after vaccination . FIG. 2 shows the anti-HER2 Th1 cumulative response of 4 HER2 ⁺ IBC patients who received adjuvant HER2 pulsed DC1 vaccine and had residual disease after neoadjuvant therapy. Each patient is depicted in a different color and shows a cumulative response (SFC / 10 ⁶ cells) before vaccination, 3 months after vaccination, and 6 months after vaccination. The mean patient cumulative response was 36.5 ± 38.3 SFC / 10 ⁶ before vaccination, 151.0 ± 60.0 SFC / 10 ⁶ (p = 0.04) 3 months after vaccination, and 198.4 ± 39.7 SFC / 6 months after vaccination. Improved to 10 ⁶ (p = 0.02). Figures 3 and 4 show HER2 peptide-pulsed DC1 vaccine stimulated with IL-2 vs. CD4 ⁺ T cells co-cultured with HER2-specific DC1 from a patient vaccinated with IL-2 / 7/15 A direct comparison of is shown for two different patients each. Immature DC ("iDC") from each patient was categorized into the following MHC class II peptides: peptide 42-56 (SEQ ID NO: 1), peptide 98-114 (SEQ ID NO: 2), peptide 328-345 (SEQ ID NO: 3). ), And peptide 776-790 (SEQ ID NO: 4) and matured to DC1. The resulting HER2 pulsed DC1 was then co-cultured with CD4 ⁺ T cells and stimulated with IL-2 alone or IL-2 / 7/15 as indicated. The red outline represents specific peptides and stimulation protocols that showed specificity (specific antigen: control antigen IFN-γ production ratio greater than 2: 1). For each set of peptide / stimulation protocols: “control antigen” indicates non-specific iDC co-cultured with control antigen; “specific antigen” is anti-HER2 CD4 co-cultured with iDC pulsed with HER2 antigen / peptide ⁺ Represents T cells; and “T cells” represent anti-HER2 CD4 ⁺ T cells in culture medium. Graphs showing the multiplication factor (defined as the number of T cells after proliferation / the number of T cells before proliferation) are shown on the right side, respectively. Specificity was measured by ELISA by antigen-specific IFN-γ production. Figures 3 and 4 show HER2 peptide-pulsed DC1 vaccine stimulated with IL-2 vs. CD4 ⁺ T cells co-cultured with HER2-specific DC1 from a patient vaccinated with IL-2 / 7/15 A direct comparison of is shown for two different patients each. 5 and 6 show a specific response followed by a non-specific immune response: FIG. 5 shows a specific response following the first stimulation / proliferation with HER2-specific DC1, and FIG. The subsequent disappearance of that specific response after a second stimulation / proliferation with specific anti-CD3 / CD28 is shown. The first stimulation of CD4 ⁺ T cells with HER2-specific DC1 resulted in multiple specific immune responses, as shown by the red outline in FIG. Figure 6 shows that the second stimulation of HER2-specific CD4 ⁺ T cells with non-specific anti-CD3 / CD28 stimulation resulted in a 4-fold proliferation (horizontal graph), but 3/4 of the peptide group had specificity. Indicates lost. Patient-derived iDCs were divided into the following MHC class II peptides: peptide 42-56 (SEQ ID NO: 1), peptide 98-114 (SEQ ID NO: 2), peptide 328-345 (SEQ ID NO: 3), and peptide 776-790 (sequence) Pulsed with number 4) and matured to DC1. The resulting HER2 pulsed DC1 was then co-cultured with CD4 ⁺ T cells and stimulated with IL-2 alone or IL-2 / 7/15 as indicated. Figures 5 and 6 show a specific response followed by a non-specific immune response: Figure 6 shows the subsequent disappearance of that specific response after a second stimulation / proliferation with non-specific anti-CD3 / CD28. Indicates. Figures 7 and 8 show a non-specific immune response followed by a specific immune response: Figure 7 shows non-specific proliferation of CD4 ⁺ T cells. FIG. 8 shows that specificity cannot be obtained after subsequent stimulation with HER2-specific DC1. The first stimulation of CD4 ⁺ T cells with non-specific anti-CD3 / CD28 resulted in 3.8-fold proliferation (FIG. 7). Second stimulation of non-specific CD4 ⁺ T cells with HER2-specific DC1 did not result in a specific immune response (FIG. 8). Patient-derived iDCs were divided into the following MHC class II peptides: peptide 42-56 (SEQ ID NO: 1), peptide 98-114 (SEQ ID NO: 2), peptide 328-345 (SEQ ID NO: 3), and peptide 776-790 (sequence) Pulsed with number 4) and matured to DC1. The resulting HER2 pulsed DC1 was then co-cultured with CD4 ⁺ T cells and stimulated with IL-2 alone or IL-2 / 7/15 as indicated. 7 and 8 show a specific immune response following a non-specific immune response: FIG. 8 shows that specificity cannot be obtained after subsequent stimulation with HER2-specific DC1. FIGS. 9A and 9B show in vitro primary / first growth of HER2-specific Th1 cells in vitro, with HER2-specific DC1 grown on IL-2 versus IL-2 / 7/15. CD4 ⁺ T cells co-cultured with. Immature DC ("iDC") was converted into the following MHC class II peptides: peptide 42-56 (SEQ ID NO: 1), peptide 98-114 (SEQ ID NO: 2), peptide 776-790 (SEQ ID NO: 4), and peptide 927 Pulsed with -941 (SEQ ID NO: 5) and matured to DC1. The resulting HER2 pulsed DC1 was then co-cultured with CD4 ⁺ T cells and stimulated with IL-2 alone or IL-2 / 7/15 as indicated. The red outline (FIG. 9B) represents specific peptides and stimulation protocols that showed specificity (specific antigen: control antigen IFN-γ production ratio greater than 2: 1). For each set of peptide / stimulation protocols: “control antigen” indicates non-specific iDC co-cultured with control antigen; “specific antigen” is anti-HER2 CD4 co-cultured with iDC pulsed with HER2 antigen / peptide ⁺ Represents T cells; and “T cells” represent anti-HER2 CD4 ⁺ T cells in culture medium. FIG. 9A shows that when the average proliferation ratio of Th1 cells (defined as number of T cells after proliferation / number of T cells before proliferation) was stimulated with IL-2, IL-7, and IL-15, IL -2 represents significantly better than alone (2.6 ± 0.75 vs 1.0 ± 0.12; p = 0.001). FIG. 9B shows the specificity of various peptide / growth protocols as measured by ELISA by antigen-specific IFN-γ production. Stimulation with IL-2, IL-7, and IL-15 and stimulation with IL-2 alone both resulted in specific Th1 responses in the same HER2 peptide 776-790. FIGS. 10A and 10B show in vitro secondary / second proliferation of HER2 pulsed DC1 versus anti-CD3 / CD28. Restimulation of Th1 cells with HER2-peptide pulse DC1 and anti-CD3 / CD28 resulted in similar proliferation rates (3.9 ± 1.0 vs 4.3 ± 2.0, p = 0.7), respectively (FIG. 10A). However, FIG. 10B shows that stimulation of Th1 cells with HER2-specific DC1 enhanced the specific Th1 response; whereas nonspecific stimulation with anti-CD3 / CD28 resulted in an overall loss of HER2 peptide specificity. To show that The following MHC class II peptides were used: peptide 42-56 (SEQ ID NO: 1), peptide 98-114 (SEQ ID NO: 2), peptide 776-790 (SEQ ID NO: 4), and peptide 927-941 (SEQ ID NO: 5) . The red outline (FIG. 10B) represents specific peptides and stimulation protocols that showed specificity (specific antigen: control antigen IFN-γ production ratio greater than 2: 1) (ie, peptides 42-56 And DC1 restimulation of peptide 776-790 specific Th1 cells). For each set of peptide / stimulation protocols: “control antigen” indicates non-specific iDC co-cultured with control antigen; “specific antigen” is anti-HER2 CD4 co-cultured with iDC pulsed with HER2 antigen / peptide ⁺ Represents T cells; and “T cells” represent anti-HER2 CD4 ⁺ T cells in culture medium. 11A and 11B show the third / third proliferation of Th1 cells with HER2 pulsed DC1 (peptide 42-56 (SEQ ID NO: 1), peptide 98-114 (SEQ ID NO: 2), peptide 776-790 (sequence). No. 4) and peptide 927-941 (SEQ ID NO: 5) were used). After the third stimulation with the indicated HER2-specific DC1, both the mean growth fold (4.32 ± 0.5, 43.7-fold cumulative growth (FIG. 11A) and antigen specificity (FIG. 11B) increased again, in particular 4 All peptides showed specificity and increased IFN-γ production. Figures 12-15 show the sequential results of repeated in vitro stimulation (4 times) of HER2-specific CD4 ⁺ Th1 cells with IL-2 / 7/15. For all FIGS. 12-15, each left panel shows peptide specificity by IFN-γ production (“Tet” is a tetanus patient control); each right panel shows the specific HER2 used -Indicates the growth rate of the peptide. In FIG. 12, in addition to the other four used in the above figure, iDC was pulsed with two additional MHC class II peptides: peptide 927-941 (SEQ ID NO: 5); and peptides 1166-1180 (sequence) Number 6). However, as seen in the growth factor results (FIG. 12, right panel), peptide 328-345 specific and peptide 1166-1180 specific Th1 cells did not produce enough cells for further growth, and therefore Only HER2 Th1 cells specific for the four peptides were used as such. Subsequently, FIG. 12 for the first stimulation shows specificity only for peptide 776-790 specific Th1 cells; in FIG. 13 for the second stimulation, peptide 42-56- and peptide 776-790 specific Shows increased specificity for Th1 cells; FIG. 14 for the third growth shows specificity for all four peptides and FIG. 15 for the fourth growth shows one of the peptides (peptide 927-941) Shows the loss of specificity for, and the remaining three HER2-specific peptides remain. Figures 12-15 show the sequential results of repeated in vitro stimulation (4 times) of HER2-specific CD4 ⁺ Th1 cells with IL-2 / 7/15. Figures 12-15 show the sequential results of repeated in vitro stimulation (4 times) of HER2-specific CD4 ⁺ Th1 cells with IL-2 / 7/15. Figures 12-15 show the sequential results of repeated in vitro stimulation (4 times) of HER2-specific CD4 ⁺ Th1 cells with IL-2 / 7/15. FIG. 16 shows the cumulative growth rate of the four growths shown in FIGS. 12 to 15 for all HER2-specific Th1 cells, and the last bar (dot) in each group shows the cumulative growth rate.

  Embodiments of the present invention relate to a method for creating in vitro a microenvironment for culture growth of antigen-specific helper T cells. Proliferated antigen-specific T cells can be used on target antigens for various therapeutic and research purposes, eg, to promote the adoption of cancer or other conditions of adoptive T cell therapy, and / or peptide-based vaccines. It can be used for identification of epitopes.

  In one embodiment, the invention includes the use of autologous type I dendritic cells (DCs) in combination with protein or peptide antigens to stimulate T cells in vitro. After stimulation, at least two soluble factors (eg, cytokines) are added to the T cells. In some examples, the at least two soluble factors are interleukin-7 (“IL-7”) and interleukin-15 (“IL-15”). A soluble factor is added to the T cells followed by a T cell growth factor. In some cases, the T cell growth factor is interleukin-2 ("IL-2"). Soluble factors aid in the growth and acquisition / maintenance of T cell function in addition to those naturally produced by type I DCs. This stimulation process can be repeated on a weekly cycle until T cells are sufficient for therapy or epitope scanning / mapping. In one embodiment, T cells are expanded to the level required for adoptive immunotherapy and epitope mapping studies while maintaining antigen specificity and cellular function.

(Definition)
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

  In general, the nomenclature and experimental procedures used herein in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization are well known and commonly used in the art. Is.

  Standard techniques are used for nucleic acid and peptide synthesis. The techniques and procedures are generally performed according to conventional methods in the art and various general references (eg, Non-Patent Document 10 and Non-Patent Document 11), which are provided throughout this document.

  The nomenclature used herein and the experimental procedures used in analytical chemistry and organic synthesis described below are those well known and commonly used in the art. Standard techniques or modifications thereof are used for chemical synthesis and chemical analysis.

  As used herein, each of the following terms has the meaning associated with it in this section.

  The article “one” (“a” and “an”) is used herein to refer to one or more (ie, at least one) grammatical objects. By way of example, “one element” means one element or more than one element.

  As used herein, “about” when used to refer to a measurable value, such as an amount, a time period, etc., is ± 20%, or ± 10%, or ± 5%, or ± It is meant to encompass variations of 1%, or ± 0.1%, so that such variations are suitable for carrying out the disclosed method.

  The term “abnormal”, when used in the context of an organism, tissue, cell or component thereof, “normal” in at least one observable or detectable property (eg, age, treatment, time of day, etc.) "Refers to organisms, tissues, cells or components that are different from the organisms, tissues, cells or components thereof that exhibit the respective properties (expected) Properties that are normal or expected for one cell or tissue type may be abnormal for a different cell or tissue type.

  The term “activation” as used herein refers to the state of a cell after cell surface site ligation sufficient to induce significant biochemical or morphological changes. In the context of T cells, such activation refers to a state of T cells that is sufficiently stimulated to induce cell proliferation. T cell activation can also induce cytokine production and performance of regulatory or cytolytic effector functions. In the context of other cells, this term presumes either up- or down-regulation of specific physicochemical processes. The term “activated T cells” refers to T cells undergoing cell division, cytokine production, regulatory or cytolytic effector functions, and / or T cells that have recently undergone the process of “activation”. Indicates.

  As used herein, “adjuvant therapy” for breast cancer refers to any treatment given after primary treatment (ie, surgery) to increase the likelihood of long-term survival. “Neoadjuvant therapy” is a treatment given before primary therapy.

  The term “antigen” or “ag” as used herein is defined as a molecule that causes an immune response. The immune response can include either antibody production or activation of specific immune competent cells, or both. One skilled in the art will understand that any macromolecule, including substantially any protein or peptide, can act as an antigen. Furthermore, the antigen can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA includes a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response, and thus encodes an “antigen” as that term is used herein. You will understand. Furthermore, one skilled in the art will understand that the antigen need not be encoded solely by the full-length nucleotide sequence of the gene. The present invention includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, and it is readily apparent that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that the antigen can be synthesized and produced or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells or body fluids.

  The term “agent”, “ligand” or “agent that binds to a cell surface site” as used herein refers to a molecule that binds to a defined population of cells. The agent can bind to any cell surface site, such as a receptor, antigenic determinant, or other binding site present on the target cell population. The agent may be a protein, peptide, antibody and antibody fragment thereof, fusion protein, synthetic molecule, organic molecule (eg, small molecule), carbohydrate, and the like. In the present specification and in the context of T cell stimulation, antibodies and natural ligands are used as typical examples of such agents.

  As used herein, the terms “agent that binds to a cell surface site” and “cell surface site” are used in the context of a ligand / anti-ligand pair. Therefore, these molecules should generally be considered as a set of complement / anti-complement molecules that exhibit specific binding with relatively high affinity.

  An “antigen-presenting cell” (APC) is a cell capable of activating T cells, including but not limited to monocytes / macrophages, B cells and dendritic cells (DC).

  “Antigen-loaded APC” or “antigen-pulsed APC” includes APC that has been exposed to and activated by an antigen. For example, APC can be Ag loaded in vitro, eg, during culture in the presence of antigen. APC may also be loaded in vivo by exposure to an antigen. “Antigen-loaded APC” is conventionally prepared in one of two ways: (1) Small peptide fragments known as antigenic peptides are “pulsed” directly outside the APC; or (2) Incubate APC with total protein or protein particles ingested by APC. These proteins are digested into small peptide fragments by APC and finally transported to the APC surface for presentation. Furthermore, antigen-loaded APC can be generated by introducing a polynucleotide encoding an antigen into a cell.

  An “anti-HER2 response” is an immune response specific for HER2 protein.

  As used herein, the term “anti-tumor effect” refers to a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancer conditions Refers to a biological effect that can be revealed by. An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the present invention to initially prevent tumor development.

  The term “autoimmune disease” as used herein is defined as a disease resulting from an autoimmune response. Autoimmune diseases are the result of an inappropriate and excessive response to self antigens. Examples of autoimmune diseases include, but are not limited to, Addis disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (type I), Dystrophy epidermolysis bullosa, epididymis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis , Rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxoma, pernicious anemia, ulcerative colitis.

  As used herein, the term “self” refers to any substance derived from the same individual that is subsequently reintroduced.

  “Homologous” refers to a graft derived from a different animal of the same species.

  The term “B cell” as used herein is defined as a cell derived from bone marrow and / or spleen. B cells can grow into plasma cells that produce antibodies.

"CD4 ⁺ Th1 cells", "Th1 cells", "CD4 ⁺ T helper type 1 cells", "CD4 ⁺ T cells", etc. express the surface protein CD4 and produce high levels of the cytokine IFN-γ Defined as a subtype of cell. See also T helper cell.

  As used herein, the term "cancer" refers to cell overgrowth whose unique property-loss of normal control-results in disordered growth, lack of differentiation, local tissue invasion, and / or metastasis. Defined. Examples include but are not limited to breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain tumor, lymphoma, leukemia, Examples include lung cancer and germ cell tumor.

  As used herein, “costimulatory signal” refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or upregulation or downregulation of major molecules.

  As used herein, “costimulatory signal” refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or upregulation or downregulation of major molecules.

  A “costimulatory molecule” refers to an allogeneic binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response, such as, but not limited to, a T cell.

  As used herein, “costimulatory ligand” includes molecules on antigen presenting cells (eg, dendritic cells, B cells, etc.) that specifically bind to allogeneic costimulatory molecules on T cells, thereby For example, in addition to primary signals provided by binding of TCR / CD3 complexes to peptide-loaded MHC molecules, signals that mediate T cell responses including but not limited to proliferation, activation, differentiation, etc. provide. Costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L) Binds to intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, Toll ligand receptor And ligands that specifically bind to B7-H3.

“Cumulative response” refers to the combined immune response of a group of patients, summing the reactive spots from all six MHC class II binding peptides from a given group of patients (per 10 ⁶ cells from IFN-γ ELISPOT analysis) Spot forming cells "SFC").

  The term “dendritic cell” (DC) is an antigen presenting cell that may be present in vivo, in vitro, ex vivo, or in a host or subject, or derived from a hematopoietic stem cell or monocyte. Dendritic cells and their precursors can be isolated from various lymphoid organs such as the spleen, lymph nodes, and from bone marrow and peripheral blood. DC has a characteristic form having a thin film (foliate foot) extending in multiple directions from dendritic cell bodies. Typically, dendritic cells express high levels of MHC and costimulatory (eg, B7-1 and B7-2) molecules. Dendritic cells can induce antigen-specific differentiation of T cells in vitro and can initiate primary T cell responses in vitro and in vivo.

  “DC vaccination”, “DC immunization”, “DC1 immunization”, etc. are strategies that use autologous dendritic cells that use the immune system to recognize specific molecules and initiate specific responses to them. Point to.

  “DC-1 polarized dendritic cells”, “DC1” and “type 1 polarized DC” refer to mature DCs that secrete Thr-driven cytokines such as IL-12, IL-18 and IL-23. DC1 can completely promote cellular immunity. DC1 is pulsed with a HER2 MHC class II binding peptide in a preferred embodiment herein.

  As used herein, “activated DC” is DC that has been exposed to a Toll-like receptor agonist. Activated DCs may or may not be antigen loaded.

  The term “mature DC” as used herein is defined as dendritic cells that express molecules including high levels of MHC class II, CD80 (B7.1) and CD86 (B7.2). In contrast, immature dendritic cells express low levels of MHC class II, CD80 (B7.1) and CD86 (B7.2) molecules, but can still take up antigen. “Mature DC” also refers to antigen presenting cells present in a host or subject that are in vivo, in vitro, ex vivo, or DC1 polarized (ie, can fully promote cellular immunity). .

  A “disease” is an animal health condition in which the animal cannot maintain homeostasis and the animal's health continues to deteriorate if the disease is not ameliorated.

  A “disorder” in an animal is one in which the animal can maintain homeostasis, but the health of the animal is a less favorable health than in the absence of the disorder. If left untreated, the disorder does not necessarily further reduce the animal's health.

  A disease or disorder is “ameliorated” when the severity or frequency of at least one sign or symptom of the disease or disorder experienced by the patient decreases.

  “Effective amount” or “therapeutically effective amount” is used interchangeably herein and, as described herein, is a compound, formulation, substance that is effective to produce a particular biological result. Or refers to the amount of the composition. Such results can include, but are not limited to, inhibition of viral infection as determined by any means appropriate in the art.

  As used herein, “endogenous” refers to any substance that originates from or is produced within an organism, cell, tissue or system.

  As used herein, the term “exogenous” refers to any substance introduced from or produced outside an organism, cell, tissue or system.

  The term “epitope” as used herein is defined as a small chemical molecule on an antigen that can elicit an immune response that induces a B and / or T cell response. An antigen can have one or more epitopes. Most antigens have many epitopes; that is, they are multivalent. In general, an epitope is approximately 5 amino acids and / or the size of a sugar. One skilled in the art understands that generally the overall three-dimensional structure, rather than a specific linear sequence of molecules, is the primary criterion for antigen specificity, thus distinguishing one epitope from another.

  “HER2” is a member of the human epidermal growth factor receptor (“EGFR”) family. HER2 is overexpressed in about 20-25% of human breast cancers and is expressed in many other cancers.

  As used herein, "HER2 binding peptide", "HER2 MHC class II binding peptide", "binding peptide", "peptide antigen", "HER2 peptide", "immunogenic MHC class II binding peptide", "antigen `` Binding peptide '', `` HER2 epitope '', `` reactive peptide '', etc. are MHC class II peptides derived from or based on the sequence of the HER2 / neu protein, which is a target found in about 20-25% of all human breast cancers, and Refers to the equivalent. The HER2 extracellular domain “ECD” refers to a domain of HER2 that is extracellular and either anchored to the cell membrane or circulating, including fragments thereof. The HER2 intracellular domain “ICD” refers to the domain of the HER2 / neu protein in the cytoplasm of the cell. According to a preferred embodiment, the HER2 epitope or otherwise binding peptide comprises 6 HER2 binding peptides containing 3 HER2 ECD peptides and 3 HER2 ICD peptides. Preferred HER2 ECD peptides include: peptide 42-56: HLDMLRHLYQGCQVV (SEQ ID NO: 1); peptide 98-114: RLRIVRGTQLFEDNYAL (SEQ ID NO: 2); and peptide 328-345: TQRCEKCSKPCARVCYGL (SEQ ID NO: 3); Includes peptide 776-790: GVGSPYVSRLLGICL (SEQ ID NO: 4); peptide 927-941: PAREIPDLLEKGERL (SEQ ID NO: 5); and peptide 1166-1180: TLERPKTLSPGKNGV (SEQ ID NO: 6).

“HER2 ^pos ” is a classification or molecular subtype of one type of breast cancer as well as many other types of cancer. HER2 positivity is currently defined by gene amplification by FISH (fluorescence in situ hybridization) assay and 2+ or 3+ pathological staining intensity.

“HER2 ^neg ” is defined by the lack of gene amplification by FISH, and in most cases can encompass a range of pathological staining from 0 to 2+.

  As used herein, “homologous” refers to subunit sequences between two polymer molecules, eg, between two nucleic acid molecules, eg, between two DNA molecules or between two RNA molecules, or between two polypeptide molecules. Similarity. If both subunit positions of two molecules are occupied by the same monomer subunit, for example, if one position of each of the two DNA molecules is occupied by adenine, they are completely or 100% homologous at that position It is. The percent homology between two sequences is a linear function of the number of matches or homologous positions, for example, half of the positions in two compound sequences (eg, 5 positions in a 10 subunit long polymer) If homologous, the two sequences are 50% identical, and if 90% of the positions, for example 9 out of 10, are identical or homologous, the two sequences share 90% homology. As an example, the DNA sequences 5′ATTGCC3 ′ and 5′TATGGC3 ′ share 50% homology.

  Further, when the term “homology” or “identity” is used herein to refer to nucleic acids and proteins, it is to be understood that it applies to homology or identity both at the nucleic acid and amino acid sequence level. Should.

  As used herein, the term “inhibit” means to inhibit or block activity or function, eg, about 10 percent compared to a control value. Preferably, the activity is inhibited or prevented by 50%, more preferably 75%, even more preferably 95% compared to the control value. As used herein, “inhibit” reduces or completely prevents a molecule, reaction, interaction, gene, mRNA and / or protein expression, stability, function or activity by a measurable amount. It also means to do. Inhibitors bind and partially or completely block stimulation, reduce, block, delay activate, inactivate proteins, genes, and mRNA stability, expression, function and activity A compound that desensitizes or down-regulates, for example an antagonist.

  As used herein, “handling material” includes publications, records, figures or any other representation medium that can be used to convey the usefulness of the compositions and methods of the present invention. The handling material of the kit of the present invention may be attached to, for example, a container containing the nucleic acid, peptide and / or composition of the present invention, or shipped together with a container containing the nucleic acid, peptide and / or composition. May be. Alternatively, the handling material may be shipped separately from the container with the intention that the handling material and the compound be used cooperatively by the recipient.

  Interleukin 2 (“IL-2” or “IL2”) is an interleukin that is a type of cytokine signaling molecule in the immune system. IL-2 is a major T cell growth and growth factor.

  Interleukin 7 (“IL-7” or “IL2”) is a hematopoietic growth factor produced by stromal epithelial cells of lymph nodes. IL-7 is essential for lymphocyte proliferation and survival.

  Interleukin-15 ("IL-15" or "IL2")) is a T cell proliferation activation and survival factor. IL-15 is produced by fibroblasts, dendritic cells and macrophages.

  “Isolated” means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not “isolated”, but an identical nucleic acid or peptide that is partially or completely separated from its naturally coexisting material is “isolated. " An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment, such as in a host cell.

  As used herein, “loaded” with a peptide refers to the presentation of an antigen in the context of an MHC molecule.

As used herein, the term “major histocompatibility complex” or “MHC” is defined as a specific cluster of genes that encode evolution-related surface proteins, many of which are involved in antigen presentation and are histocompatible. It is the most important determinant. Class I MHC, or MHC class I, functions primarily in antigen presentation to CD8 T lymphocytes. Class II MHC, or MHC class II, functions primarily in antigen presentation to CD4 ⁺ T lymphocytes (T helper cells).

  “Mature DC” as used herein refers to dendritic cells that express molecules including high levels of MHC class II, CD80 (B7.1) and CD86 (B7.2) molecules. In contrast, immature DC ("iDC") express low levels of MHC class II, CD80 (B7.1) and CD86 (B7.2) molecules, but can still take up antigen. “Mature DC” can also be antigen presenting cells present in a host or subject that can be in vivo, in vitro, ex vivo, or DC1 polarized (ie, can fully promote cellular immunity). Point to.

For each group of subjects analyzed for anti-HER2 CD4 ⁺ Th1 immune response, a “metric” of CD4 ⁺ Th1 response (or Th1 response) is defined: (a) overall anti-HER2 responsiveness (≧ 1 Expressed as a percentage of subjects responding to reactive peptides); (b) response repertoire (expressed as the average number of reactive peptides recognized by each subject group (n)); and (c) cumulative Response (represented as the sum of reactive spots from 6 MHC class II binding peptides from each subject group (spot forming cells “SFC” per 10 ⁶ cells from IFN-γ ELISPOT analysis)).

  As used herein, the term “modulate” in a subject that is the same except compared to the level of response in the subject in the absence of treatment or compound and / or not receiving treatment. Means to mediate a detectable increase or decrease in the level of response in a subject compared to the level of response. The term encompasses perturbing and / or affecting a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

“Uncertain HER2 ^neg ” is not gene amplified and is defined as 0 or 1+ in pathological staining. “Uncertain HER2 ^neg ” is not gene amplified but is defined as 2+ in pathological staining.

  As used herein, “population” includes reference to an isolated culture comprising a homogeneous, substantially homogeneous, or heterogeneous cell culture. In general, a “population” may be considered an “isolated” cell culture.

  As used herein, a “recombinant cell” is a host cell that contains a recombinant polynucleotide.

  “Responsiveness” or “anti-HER2 responsiveness” are used interchangeably herein and mean the percentage of subjects that respond to at least one of the six binding peptides.

  “Response repertoire” is defined as the average number of reactive peptides (“n”) recognized by each group of subjects.

  As used herein, “sample” or “biological sample” refers to biological material from a subject, including but not limited to organs, tissues, exosomes, blood, plasma, saliva, urine and other bodily fluids. To do. A sample can be any source of material obtained from a subject.

  As used herein, the term “specifically binds” refers to an antibody that recognizes and binds to another molecule or feature, but does not substantially recognize or bind to another molecule or feature in the sample. Means a molecule.

  The term “stimulation” refers to a primary response that is induced by the binding of a stimulatory molecule to its homologous ligand and thereby mediates a signaling event, such as but not limited to signaling by a TCR / CD3 complex. Stimulation can mediate altered expression of specific molecules, such as downregulation of TGF-β and / or reorganization of cytoskeletal structures.

  As used herein, “stimulatory ligand” refers to an allogeneic binding partner (herein referred to as “stimulatory molecule”) on a T cell when present on an antigen presenting cell (eg, a dendritic cell, a B cell, etc.). Refers to a ligand capable of specifically binding a primary response by T cells, including but not limited to activation, initiation of an immune response, proliferation, and the like.

  As used herein, the term “stimulatory molecule” means a molecule on a T cell that specifically binds to an allogeneic stimulating ligand present on an antigen presenting cell.

  The terms “subject”, “patient”, “individual” and the like are used interchangeably herein and are suitable for any of the methods described herein, whether in vitro or in situ. Refers to an animal or its cells. In certain non-limiting embodiments, the patient, subject or individual is a human.

  As used herein, a “substantially purified” cell is a cell that is essentially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types with which they are normally associated in their natural state. In some examples, a substantially purified cell population refers to a homogeneous cell population. In other examples, the term simply refers to a cell that has been isolated from a cell that naturally associates in its natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

  As used herein, the term “targeted therapy” uses drugs or other substances that interfere with specific target molecules involved in cancer cell growth, usually with little damage to normal cells. Refers to cancer treatment that achieves a tumor effect. In contrast, conventional cytotoxic chemotherapeutic drugs act on all actively dividing cells. Breast cancer therapeutic monoclonal antibodies, particularly trastuzumab / HERCEPTIN®, target the HER2 / neu receptor.

  “T / C” is defined as trastuzumab and chemotherapy. This refers to patients who receive both trastuzumab and chemotherapy before and after breast cancer surgery.

  The term “T cell” as used herein is defined as a thymus-derived cell involved in various cellular immune responses.

The terms “T helper cell”, “helper T cell”, “Th cell” and the like are used herein with respect to a cell and are a subgroup of lymphocytes (a type of white blood cell) comprising different cell types that are distinguishable by those skilled in the art. Represents. In particular, T helper cells are effector T cells whose primary function is to promote the activation and function of other B and T lymphocytes and / or macrophages. Helper T cells differentiate into two major subtypes of cells known as “Th1” or “type 1” and “Th2” or “type 2” phenotypes. These Th cells secrete cytokines, proteins or peptides that stimulate or interact with other leukocytes. As used herein, “Th1 cells”, “CD4 ⁺ Th1 cells”, “CD4 ⁺ T helper type 1 cells”, “CD4 ⁺ T cells” and the like refer to mature T cells that express the surface glycoprotein CD4. Used for. CD4 ⁺ T helper cells are activated when peptide antigens are presented by MHC class II molecules expressed on the surface of antigen presenting peptides (“APC”) such as dendritic cells. Upon activation of CD4 ⁺ T helper cells by the MHC antigen complex, it secretes high levels of cytokines such as interferon gamma (“IFN-γ”). Such cells are found inside host cells and are considered to be very effective against microorganisms that cause certain diseases and are critical in the anti-tumor response in human cancer.

The term “cytotoxic T cell” or “CD8 ⁺ T cell” or “killer T cell” refers to a T lymphocyte that kills a target cell, such as a cancer cell, an infected cell, or a cell otherwise damaged. It is.

“Treg”, “T _reg ” and “regulatory T cell” are used herein to refer to cells that are police officers of the immune system and act to control the anticancer activity of the immune system. Is done. They are increased in some cancers and are mediators resistant to immunotherapy in these cancer types.

  The term “treatment” as used herein means treatment and / or prevention. The therapeutic effect is obtained by suppression, remission or eradication of the disease state.

  A “therapeutically effective amount” is the amount of a compound of the invention that, when administered to a patient, ameliorates the symptoms of the disease. The amount of a compound of the invention that constitutes a “therapeutically effective amount” will vary depending on the compound, the condition and severity of the disease, the age of the patient being treated, and the like. A therapeutically effective amount can be routinely determined by one of ordinary skill in the art in view of his own knowledge and the present disclosure.

  The terms “treat”, “treating”, and “treatment” refer to the treatment or prophylactic measures described herein. A “treatment” method is to apply a composition of the invention to a subject in need of such treatment, eg, to a subject suffering from a disease or disorder, or such a disease or disorder. To a subject who can ultimately acquire one or more symptoms of a disorder or recurrent disease, such as to prevent, cure, delay, reduce severity, or ameliorate the severity of a subject or such Give to extend more than expected in the absence of treatment.

  The term “vaccine” as used herein is defined as a substance used to elicit an immune response after the substance has been administered to an animal, preferably a mammal, more preferably a human. When introduced into a subject, a vaccine can elicit an immune response including, but not limited to, antibody, cytokine production and / or other cellular responses.

  Scope: Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and sake of brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, a description of a range, such as 1-6, is an individual number within the range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6 as well as 1-3, 1-4, 1 Partial ranges such as -5, 2-4, 2-6, 3-6, etc. should also be considered as specifically disclosed. This applies regardless of the width of the range.

(Description)
The present invention relates to replicating the lymph node environment for generating therapeutic doses of T cells for adoptive therapy. T cells can also be used to identify epitopes on target antigens to facilitate the production of peptide-based vaccines.

  In one embodiment, the present invention provides an in vitro environment that replicates the lymph node environment. In one embodiment, lymph node replication comprises supplying one or more of the following elements to the culture conditions: type 1 dendritic cells, IL-15, IL-7 and IL-2.

  Type 1 dendritic cells process and present peptide antigens to T cells, surface expressed CD80 and CD86 (binding to the CD28 counterreceptor on T cells), and CD40 (interacting with CD40L on T cells) So-called "costimulatory molecules". In addition, DCs produce not only interferon-gamma production (T cell function) but also soluble factors such as interleukin 12 ("IL-12") that aid in longevity (anti-apoptotic factor). DC produces many other factors that are important for T cell growth. DCs are usually found in lymph nodes and are known to be important for T cell activation and proliferation.

  IL-15 is a T cell proliferation activation and survival factor. IL-15 is produced by fibroblasts, dendritic cells and macrophages.

  IL-7 is a factor produced by stromal epithelial cells of lymph nodes. IL-7 is essential for lymphocyte proliferation and survival.

  IL2 is a major T cell growth and growth factor.

  Thus, the present invention provides compositions and methods for combining cytokines and certain dendritic cells to generate desired T cells. In one embodiment, T cells are expanded to the level required for adoptive immunotherapy and epitope mapping studies while maintaining antigen specificity and cellular function.

In one embodiment, the present invention provides that HER2 ⁺ invasive breast cancer (“IBC”) patients with residual disease after neoadjuvant chemotherapy are prone to anti-HER2 type 1 T helper (Th1) cell immunodeficiency and recurrent disease. It relates to the discovery of having a risk. Datta et al. Showed that the anti-HER2 CD4 ⁺ T cell response gradually decreased along the breast cancer continuum-a robust response in healthy donors and patients with benign disease, HER2 ⁺ noninvasive breast cancer patients Showed a suppressive response and little response in HER2 ⁺ IBC patients. Proliferating antigen-specific t cells for (A) Adjuvant type 1 polarized dendritic cell ("DC1") vaccination and (B) adoptive T cell transplantation to restore anti-HER2 Th1 immunity Examine the role of the method.

Embodiments of the present invention also relate to methods for creating in vitro a microenvironment for the culture growth of antigen-specific CD4 ⁺ or CD8 ⁺ T cells. Proliferated antigen-specific T cells can be used for various therapeutic and research purposes, for example, infectious diseases such as cancer or chronic viral infections, adoptive T cell therapy of other conditions, and / or peptide-based vaccines It can be used for identification of epitopes on the target antigen to promote the production of.

  In one embodiment, the invention includes the use of autologous type I dendritic cells (“DC1”) in combination with protein or peptide antigens to stimulate T cells in vitro. After stimulation, at least two soluble factors (eg, cytokines) are added to the T cells. In some examples, the at least two soluble factors are interleukin-7 (“IL-7”) and interleukin-15 (“IL-15”). A soluble factor is added to the T cells followed by a T cell growth factor. In some cases, the T cell growth factor is interleukin-2 ("IL-2"). Soluble factors aid in the proliferation and acquisition / maintenance of T cell function in addition to those naturally produced by DC1. This stimulation process can be repeated on a weekly cycle until T cells are sufficient for therapy or epitope scanning / mapping. In certain embodiments, T cells are expanded to the level required for adoptive immunotherapy and epitope mapping studies while maintaining antigen specificity and cellular function.

(Th1 response in vivo to HER-2 pulsed DC1 vaccination: recovery of anti-HER2CD4 ⁺ Th1 response in IBC patients)
In addition to identifying the progressive loss of anti-HER2 CD4 ⁺ Th1 response across the tumorigenic continuum in HER2 ^{pos -breast} cancer, as taught by Datta et al., Suppression of anti-HER2 Th1 response in HER2 ^{pos -invasive} breast cancer is HER2-pulse type 1 polarized dendritic cells ("DC1") recovered differentially after vaccination. After HER2-targeted therapy with trastuzumab and chemotherapy (“T / C”) or with other standard treatments such as surgical excision or radiation, the suppressive response was not restored. The recovered anti-HER2 Th1 response appears to persist for at least about 6 months or considerably longer.

HER2 ⁺ IBC patients with residual disease after neoadjuvant therapy were vaccinated with adjuvant HER2 pulsed DC1. Immune responses were generated from PBMC pulsed with HER2 class II peptides by measuring IFN-γ production with ELISPOT. Response was assessed with three metrics of CD4 ⁺ Th1 response: (1) overall anti-HER2 responsiveness (responsive to> 1 peptide), (2) number of reactive peptides (response repertoire), and (3) Cumulative response across 6 HER2 peptides. The Th1 response before vaccination was compared with the responses at 3 and 6 months after vaccination.

Datta et al. Describe a method of making a DC1 vaccine. See also Non-patent document 12 (Koski et al.); Non-patent document 13 (Sharma et al.); Non-patent document 14; Non-patent document 15; Non-patent document 16; Briefly, patient monocytes are first separated from other leukocytes by leukapheresis and elution. These monocytes are then placed in immature dendritic cells (“SFM”) in serum-free medium (“SFM”) containing granulocyte macrophage colony stimulating factor (“GM-CSF”) and interleukin (“IL”)-4. Incubate until “iDC”). These cells are then preferably pulsed with six HER2 MHC class II binding peptides, in which case the binding peptide identified by SEQ ID NOs: 1-6, then interferon (“IFN”)-γ and lipopolysaccharide ("LPS") is added to complete the maturation and activation process and achieve full DC activation to DC1 before being injected back into the patient. See Non-Patent Document 14. For HLA-A2 ^pos patients, half of the cells are pulsed with MHC class I binding peptides and the other half are pulsed with different MHC class 1 binding peptides.

Datta et al. Also describe a blood test / assay that generates a circulating anti-cancer CD4 ⁺ Th1 response (ie, IFN-γ secretion) and detects and measures the resulting IFN-γ production. Such blood tests were performed on patients before DC1 vaccination and on patients 3 and 6 months after vaccination. In a preferred embodiment, a subject blood sample containing CD4 ⁺ Th1 cells and antigen presenting cells or precursors thereof is capable of inducing an immune response in said subject based on the type of cancer the subject is suffering from. There are pulses with MHC class II immunogenic peptides. Preferably, the antigen presenting cell or precursor thereof is a mature or immature dendritic cell or monocyte precursor thereof. In particularly preferred embodiments, the cancer is preferably HER2-expressing, the mammalian subject is preferably a human, more preferably the cancer is HER2 ^pos breast cancer, and the human subject is female.

A preferred embodiment for generating a circulating anti-HER2 CD4 ⁺ Th1 response in a mammalian subject is to isolate non-proliferating peripheral blood mononuclear cells (“PBMC”) from the subject and generate an immune response in the subject Provided by pulsing PBMC with a composition comprising a HER2-derived MHC class II antigenic binding peptide capable of causing Without wishing to be bound by any particular theory, when binding peptides are presented to CD4 ⁺ Th1 cells present in PBMC samples, they activate CD4 ⁺ Th1 cells, and activated CD4 ⁺ Th1 cells Interferon gamma (“IFN-γ”) is produced. DC1 (type 1 polarized dendritic cells) derived from progenitor pluripotent monocytes contained in a subject's PBMC sample is an antigen-presenting cell (“APC”), and antigen-loaded APC by exposure to the binding peptide Thus, the MHC class II antigen-binding peptide is presented to the subject's CD4 ⁺ Thl cells in the sample, thereby activating the CD4 ⁺ Th1 cells and producing / secreting IFN-γ. The so produced IFN-γ is subsequently measured for analysis.

In this case, according to this preferred embodiment, the PBMC of each patient was pulsed with six HER2-specific MHC class II peptides, particularly those having the sequences identified in SEQ ID NOs: 1-6. IFN-γ produced by anti-HER2 CD4 ⁺ Th1 cells was detected and measured by an IFN-γ enzyme-linked immunospot (“ELISPOT”) assay.

In a particularly preferred embodiment for HER2 ^pos cancer, a composition comprising 6 MHC class II binding peptides derived from or based on DC, immature or type 1 polarized DC1 capable of causing an immune response in a patient Pulse with an object. HER2 MHC class II binding peptide or epitope is
Peptide 42-56: HLDMLRHLYQGCQVV (SEQ ID NO: 1);
Peptide 98-114: RLRIVRGTQLFEDNYAL (SEQ ID NO: 2);
Peptide 328-345: TQRCEKCSKPCARVCYGL (SEQ ID NO: 3);
Peptide 776-790: GVGSPYVSRLLGICL (SEQ ID NO: 4);
Peptide 927-941: PAREIPDLLEKGERL (SEQ ID NO: 5); and Peptide 1166-1180: TLERPKTLSPGKNGV (SEQ ID NO: 6);
including.

In embodiments where the donor is an A2.1 blood group, the HER2 MHC class I peptide or epitope is
Peptide 369-377: KIFGSLAFL (SEQ ID NO: 7); and Peptide 689-697: RLLQETELV (SEQ ID NO: 8)
including.

Autologous immature or mature pulsed pre-stimulated CD4 ⁺ T cells from a subject blood sample with a composition comprising a HER2-derived MHC class II antigenic binding peptide capable of eliciting an immune response in the subject Alternative preferred embodiments that cause a circulating anti-HER2 CD4 ⁺ Th1 response in a mammalian subject by co-culture with dendritic cells (“iDC” or mature “DC”) have also been described. Without wishing to be bound by any particular theory, when binding peptides are presented to CD4 ⁺ Th1 cells present in T cell samples, they activate CD4 ⁺ Th1 cells and activate CD4 ⁺ Th1 cells produce / secrete IFN-γ. Immature DC matures to DC1, which presents MHC class II binding peptide to the subject's CD4 ⁺ Th1 cells present in the sample, which activates CD4 ⁺ Th1 cells and produces IFN-γ, It is then measured for analysis.

In any of the alternative preferred embodiments for eliciting an anti-HER2 immune response in a subject, IFN-γ produced by anti-HER2 CD4 ⁺ Th1 cells is measured by an IFN-γ enzyme-linked immunospot (“ELISPOT”) assay. Although detected and measured, it should be understood by those skilled in the art that other detection methods may be used. For example, flow cytometry, enzyme-linked immunosorbent assay (“ELISA”), and immunofluorescence (“IF”) can be used to monitor immune responses. Alternatively, for patient immune monitoring, it may be advantageous to measure the ratio of IFN-γ to IL-10 as compared to or in addition to a straight IFN-γ test such as ELISPOT, which shows the total CD4 ⁺ cell spot. possible. Such a test is particularly advantageous for patients at risk. Furthermore, the use of immunofluorescence provides another way to measure and visualize the immune response by using an ELISPOT reader that reads the results by fluorescence. In such cases, the results can be arranged to show two, three, or more cytokines / other secreted immune molecules, each shown in a different color, in the same patient sample. In this case, IFN-γ ELISPOT was used.

  A preferred embodiment of the invention features six HER2 MHC class II binding peptides / epitopes, but as long as it is sufficiently immunologically active in the patient, any component of the entire HER2 molecule can be combined with other binding peptides. Other possible MHC class II HER2 peptides can be used in embodiments of the present invention.

Responsiveness: Only one IBC patient showed an immune response defined as> 20 SFC / 10 ⁶ cells in experimental wells after subtracting unstimulated background before vaccination. Compared to the pre-vaccination results, all vaccinated IBC patients showed an immune response defined as a> 2-fold increase in anti-HER2 IFN-γ ^pos Thl response.

  Response repertoire: FIG. 1 shows from 0.5 ± 1 peptide before vaccination to 3.25 ± 0.96 peptide (p = 0.01) after 3 months and 4 ± 0.8 peptide (p = 0.01) after 6 months in IBC patients. Shows increased average repertoire.

Cumulative response: Figure 2 shows that from 36.5 ± 38.3 SFC / 10 ⁶ before vaccination, 151.0 ± 60.0 SFC / 10 ⁶ (p = 0.04) after 3 months, and 198.4 ± 39.7 SFC / 10 ⁶ (6 months later) p = 0.02) shows the mean cumulative response in the improved patients.

In addition to breast cancer, there are many other HER2 ^pos solid cancers, such as brain, bladder, esophagus, lung, pancreas, liver, prostate, ovary, colorectal and stomach, for which they are described herein. The materials and methods of the embodiments can be used for diagnosis and treatment. Thus, the six anti-HER2 binding peptides described above can be used in accordance with embodiments herein to detect these and other HER2-expressing cancers and can elicit immune responses useful for diagnosis. .

  The vaccine may be developed to target HER2-expressing tumors using the same anti-HER2-binding peptides described above, or any composition of HER2 that is immunogenic, such as DNA, RNA, peptides, or Proteins etc. or their components such as ICD and ECD domains may be used. For example, a subject can be vaccinated against total HER2 protein and the 6 binding peptides described above can be used to monitor a patient's immune response. Similarly, vaccines can be developed for other types of cancer, such as other members of the HER2 family, including HER1, HER3, and c-MET.

While preferred embodiments of the present invention are directed to the treatment and diagnosis of female HER2 ^pos breast cancer, it should be readily appreciated by those skilled in the art that embodiments of the present invention are not limited to female humans. is there. Preferred embodiments of the invention include male humans, such as HER2-expressing prostate cancer, as well as other mammalian subjects.

The identified decrease in anti-HER2 CD4 ⁺ Th1 response occurs in such a blood test and the detected immune response is used exclusively as a cancer diagnosis / response predictor, or as in this example In parallel with the use of special vaccines, it is possible to restore the patient's immune response. Thus, preferred embodiments described herein change the focus of cancer diagnosis and treatment to the use of patient immunity and blood tests to determine and / or predict immune responses to cancer, which is a risk of recurrence. In contrast to diagnostics and treatments that involve certain patients and rely on tumor cell identification.

(In vitro growth of HER2-specific TH1 cells)
In vitro, HER2-specific Th1 cells were generated by co-culture with HER2 peptide pulsed DC1 and grown with IL-2 alone or IL-2, IL-7, and IL-15. Subsequently, Th1 cells were grown either by repeated HER2-peptide pulsed DC1 co-culture or anti-CD3 / CD28 stimulation. The fold proliferation was defined as follows: (#T cells after proliferation / # T cells before proliferation); specificity was measured by ELISA by antigen-specific IFN-γ production.

Embodiments of the invention relating to T cell proliferation are in no way limited to CD4 ⁺ T cells. Thus, embodiments of the invention provide methods for growing chimeric antigen receptor T cells (“CART cells”), cytotoxic T lymphocytes (CD8 ⁺ ), as well as all other types of T cells. For example, see Non-Patent Document 18.

Embodiments of the lymph node environment for generating therapeutic doses of antigen-specific T cells, either helper (CD4 ⁺ ) or cytotoxic (CD8 ⁺ ), for adoptive therapy of cancer or other conditions Relating to duplicating. Proliferated antigen-specific T lymphocytes can also be used to identify epitopes on target antigens to facilitate the production of peptide-based vaccines.

  Embodiments of the invention provide an in vitro environment that replicates the lymph node environment. In that embodiment, lymph node replication comprises supplying one or more of the following elements to the culture conditions: type 1 dendritic cells, IL-15, IL-7 and IL-2.

  Type 1 dendritic cells process and present peptide antigens to T cells, surface expressed CD80 and CD86 (binding to the CD28 counterreceptor on T cells), and CD40 (interacting with CD40L on T cells) So-called "costimulatory molecules". In addition, DCs produce not only IFN-γ production (T cell function) but also soluble factors such as interleukin 12 (“IL-12”) that aid in longevity (anti-apoptotic factor). DC produces many other factors that are important for T cell growth. DCs are usually found in lymph nodes and are known to be important for T cell activation and proliferation.

  IL-15 is a T cell proliferation activation and survival factor. IL-15 is produced by fibroblasts, dendritic cells and macrophages.

  IL-7 is a factor produced by stromal epithelial cells of lymph nodes. IL-7 is essential for lymphocyte proliferation and survival.

  IL-2 is a major T cell growth and growth factor.

  Thus, the present embodiments combine compositions of specific cytokines and specific types of dendritic cells, while generating specific T cells using specific timing and specific sequences of lymphocyte addition. And providing a method. In preferred embodiments, T cells are expanded to the level required for adoptive immunotherapy and epitope mapping studies while maintaining antigen specificity and cellular function.

(T cell source)
Prior to expansion, a source of T cells is obtained from the subject. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subject is a human. T cells can be obtained from a number of sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, and tumors. In certain embodiments of the invention, any number of T cell lines available in the art can be used. In certain embodiments of the invention, T cells can be obtained from blood units taken from a subject using any number of techniques known to those skilled in the art, such as Ficoll separation. In a preferred embodiment, the cells from the individual's circulating blood are obtained by apheresis or leukapheresis. Apheresis products typically contain lymphocytes including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes and platelets. In one embodiment, the cells collected by apheresis can be washed to remove the plasma fraction and the cells can be placed in an appropriate buffer or medium for subsequent processing steps. In one embodiment of the invention, the cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the cleaning solution may lack calcium, may lack magnesium, or may lack many, if not all, divalent cations. After washing, the cells may be resuspended in various biocompatible buffers such as Ca-free, Mg-free PBS. Alternatively, undesired components of the apheresis sample may be removed and the cells resuspended directly in the medium.

  In another embodiment, T cells are isolated from peripheral blood by lysing red blood cells and depleting monocytes, for example by centrifugation on a PERCOLL ™ gradient. Alternatively, T cells can be isolated from the umbilical cord. In any case, a particular subpopulation of T cells can be further isolated by positive or negative selection techniques.

Enrichment of T cell populations by negative selection can be achieved using a combination of antibodies directed against surface markers characteristic of negative selected cells. A preferred method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on negatively selected cells. For example, to enrich CD4 ⁺ cells by negative selection, monoclonal antibody cocktails typically include antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

  In order to isolate a desired cell population by positive or negative selection, the concentration of cells and surfaces (eg, particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume at which the beads and cells are mixed together (ie, increase the concentration of cells) to ensure maximum contact of the cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, more than 100 million cells / ml are used. In further embodiments, cell concentrations of 10 million, 1.5 million, 20 million, 2.5 million, 30 million, 3.5 million, 40 million, 45 million, or 50 million cells / ml are used. Is done. In yet another embodiment, cell concentrations of 75 million, 80 million, 850 million, 90 million, 95 million, or 100 million cells / ml are used. In further embodiments, a concentration of 125 billion or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell proliferation.

  T cells for stimulation can also be frozen after the washing step, which does not require a monocyte removal step. While not wishing to be bound by theory, the freezing and subsequent thawing steps provide a more uniform product by removing granulocytes and some monocytes from the cell population. After a washing step that removes plasma and platelets, the cells may be suspended in a frozen solution. Many freezing solutions and parameters are known in the art and useful in this context, but in a non-limiting example, one method is PBS containing 20% DMSO and 8% human serum albumin, or With the use of other suitable cell freezing media. The cells are then frozen at -80 ° C at a rate of 1 ° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Of course, a method of freezing in ice temperature may be used as well as a method of freezing immediately at -20 ° C. or in liquid nitrogen.

(T cell activation and proliferation)
In general, the T cells of the present invention are grown under conditions that replicate lymph nodes. In one embodiment, lymph node replication comprises supplying one or more of the following elements to the culture conditions: type 1 dendritic cells, IL-15, IL-7 and IL-2. In one embodiment, antigen-specific T cells can be grown in the presence of one or more type 1 dendritic cells, IL-15, IL-7 and IL-2.

  In one embodiment, T cells can be stimulated as described herein, such as by contact with DC. DC can supply costimulatory molecules to T cells. After contacting the T cells with DC, the T cells are cultured in the presence of IL-15, IL-7 and IL-2.

  In some examples, T cells are cultured with a mixture comprising one or more DCs, IL-15, IL-7, and IL-2. In one embodiment of the invention, the mixture may be incubated for several hours (about 3 hours) to about 14 days, or any integer value in between. In another embodiment, the mixture may be cultured for 21 days. In one embodiment of the invention, the T cells are cultured for about 8 days. In another embodiment, T cells are cultured together for 2-3 days. Several stimulation cycles may be desired so that the T cell culture time is 60 days or longer. Suitable conditions for T cell culture include serum (eg, fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL Appropriate to contain factors necessary for growth and survival, including -10, IL-12, IL-15, TGFβ and TNF-α or any other additive for cell growth known to those skilled in the art Medium (for example, minimal essential medium or RPMI medium 1640 or X-vivo 15 (Lonza)). Other additives for cell growth include, but are not limited to, surfactants, plasmanates, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol.

The medium is supplemented with amino acids, sodium pyruvate, and vitamins and is serum-free or adequate for serum (or plasma) or a defined set of hormones and / or T cell growth and proliferation RPMI1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15 and X-Vivo 20, supplemented with an amount of cytokine (s). Antibiotics such as penicillin and streptomycin are included only in experimental cultures, not in cultures of cells injected into a subject. Target cells are maintained under conditions necessary to support growth, eg, at an appropriate temperature (eg, 37 ° C.) and atmosphere (eg, air + 5% CO ₂ ).

  In one embodiment, T cells are expanded to the level required for adoptive immunotherapy and epitope mapping studies while maintaining antigen specificity and cellular function. Thus, any number of cells is within the context of the present invention. Cells stimulated by the methods of the invention are activated as indicated by induction of signal transduction, expression of cell surface markers and / or proliferation. One such marker suitable for T cells is the production of IFN-γ, an important immunoregulatory molecule. The production of IFN-γ is extremely beneficial for the amplification of immune responses.

  With respect to T cells, the T cell population resulting from the various growth methods described herein may have various specific phenotypic characteristics depending on the conditions used. Such phenotypic characteristics include enhanced expression of CD25, CD154, IFN-γ and GM-CSF, and altered expression of CD137, CD134, CD62L and CD49d. The ability to differentially control the expression of these sites can be very important. For example, higher level surface expression of CD154 on “tailored T cells” via contact with CD40 molecules expressed on antigen presenting cells (such as dendritic cells, monocytes, and even leukemia B cells or lymphomas) Enhances antigen presentation and immune function. Such strategies are currently employed by various companies to ligate CD40 via antibodies or recombinant CD40L. The approach described herein allows this same signal to be delivered in a more physiological manner, for example by T cells. The ability to increase IFN-γ secretion by modulating the T cell activation process may help promote the generation of Th1-type immune responses that are important for anti-tumor and anti-viral responses. Similar to CD154, increased expression of GM-CSF enhances APC function, particularly through its effect of promoting the maturation of APC progenitor cells into APC, such as more functionally qualified dendritic cells Can help. Altering the expression of CD137 and CD134 can affect the ability of T cells to resist or be sensitive to apoptotic signals. Control of the expression of adhesion / homing receptors such as CD62L and / or CD49d and / or CCR7 can determine the ability of injected T cells to result in lymphoid organs, infection sites, or tumor sites.

  The phenotypic characteristics of the T cell populations of the present invention can be observed by a variety of methods including standard flow cytometry and ELISA methods known to those skilled in the art.

  One skilled in the art will readily appreciate that the cell stimulation methods described herein can be performed in a variety of environments (ie, containers). For example, such a container may be a culture flask, culture bag, or any container that can hold cells, and is preferably a sterile environment. In one embodiment of the invention, a bioreactor is also useful. For example, some manufacturers are now making devices that can be used to grow cells and can be used in combination with the methods of the present invention. See, for example, Celdyne, Houston, Texas; Unisyn Technologies, Hopkinton, Massachusetts; Synthecon, Houston, Texas; Aastrom Biosciences, Ann Arbor, Michigan; Wave Biotech, Bedminster, NJ. In addition, patents dealing with such bioreactors include US Pat. Nos. 5,099,086; 5,637; 5,637;

  In one embodiment, a bioreactor with a base rocker platform that allows varying rocking speeds and a variety of different rocking angles, such as “The Wave” (Wave Biotech, Bedminster, NJ) is used. Is done. Those skilled in the art will recognize that any platform that allows proper operation for optimal growth of cells is within the context of the present invention. In certain embodiments, the stimulation and growth methods of the present invention comprise culturing vessels at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, Prepare for rocking 13, 14, 15, 16, 17, 18, 19, or 20 times. In certain embodiments, the stimulation and proliferation methods of the present invention provide the swing platform angles of 1.5 °, 2 °, 2.5 °, 3 °, 3.5 °, 4 °, 4.5 °, 5 °, 5.5 °, 6 °. , 6.5 °, 7 °, 7.5 °, 8 °, 8.5 ° or 9.0 °.

In certain embodiments, the volume of the bioreactor vessel ranges from about 0.1 liter to about 200 liters of media. One skilled in the art will readily appreciate that the volume used for culture will vary depending on the starting cell number and the desired final cell number. In certain embodiments, cells of the invention, such as T cells, are seeded at an initial concentration of about 0.2 × 10 ⁶ cells / ml to about 5 × 10 ⁶ cells / ml, and any concentration therebetween. In one particular embodiment, the cells are first cultured in a static environment and bios on a rocking platform on days 1, 2, 3, 4, 5, 6, 7, 8 or later in culture. It may be transferred to a reactor. In a related embodiment, as described above, the entire stimulation, activation, and proliferation process is performed in a bioreactor that includes a rocking platform and an integral magnet. Exemplary bioreactors include, but are not limited to, “The Wave”.

In one particular embodiment, the cell stimulation method of the present invention is performed in a closed system, such as a bioreactor, that allows perfusion of the medium at various rates, such as from about 0.1 ml / min to about 10 ml / min. . Thus, in certain embodiments, such a closed system container comprises an outlet filter, an inlet filter, and a sample port for sterile transfer to and from the closed system. In other embodiments, such closed system containers include a syringe pump and controls for sterile transfer to and from the closed system. A further embodiment comprises a device such as a load cell for controlling the input and output of media by continuous monitoring of the weight of the bioreactor vessel. In one embodiment, the system includes a gas manifold. In another embodiment, the bioreactor of the present invention includes a CO ₂ gas mixing rack that supplies a mixture of ambient air and CO _{2 to} the bioreactor vessel and maintains the vessel at a positive pressure. In another embodiment, the bioreactor of the present invention includes a variable heater.

  In one embodiment, the medium can be placed in a container from day 2, 3, 4, 5 or 6 at about 0.5-5.0 liters a day until the desired final volume is obtained. In a preferred embodiment, the medium is placed in a container at 2 liters per day from day 4 until the volume reaches 10 liters. Once the desired volume is reached, media perfusion can begin. In certain embodiments, perfusion of media through the system begins at about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days in culture. In one embodiment, perfusion is initiated when the volume is about 0.1 liters to about 200 liters of media. In one particular embodiment, perfusion is initiated when the final volume is 4, 5, 6, 7, 8, 9, 10 or 20 liters or more. The rate of perfusion can be from about 0.5 ml / min to about 10 ml / min. In certain embodiments, the perfusion rate is about 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8.0 mls / min.

  In a further embodiment of the invention, cells such as T cells are cultured for up to 5 days in a closed quiescent system and then transferred to a closed system that includes a rocking body that allows rocking of the culture vessel at various rates.

In certain embodiments, the methodologies of the invention provide for the growth of cells, such as T cells, to a concentration of about 6 × 10 ⁶ cells / ml to about 90 × 10 ⁶ cells / ml in less than about 2 weeks. In particular, the methodology herein describes T cells at about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85 × 10 ⁶ Providing growth to a concentration of cells / ml and all concentrations therein. In certain embodiments, the cells reach a desired concentration, such as any of those described above, by about 5, 6, 7, 8, 9, 10, 11, or 12 days in culture. In one embodiment, T cells proliferate at least about 1.5 fold in about 24 hours on about day 4 to about day 12 of culture. In one embodiment, cells such as T cells grow from a starting cell number of about 100 × 10 ⁶ to a total of about 500 × 10 ⁹ cells in less than about 2 weeks. In a further embodiment, T cells grow from a starting cell number of about 500 × 10 ⁶ to a total of about 500 × 10 ⁹ cells in less than about 2 weeks. In related embodiments, the cells grow from a starting cell number of about 100-500 × 10 ⁶ to a total of about 200, 300, or 400 × 10 ⁹ cells in less than about 2 weeks.

(Treatment)
In certain embodiments, a population of T cells is first contacted with an antigen and then exposed to a mixture of the invention comprising one or more DCs, IL-15, IL-7, and IL-2. In one particular embodiment, a specific antigen, alone or in combination with an adjuvant, or pulsed dendritic cells, induces antigen-specific T cells by vaccinating the patient. Antigen-specific cells for use in proliferation using the stimulation methods of the present invention may be generated in vitro.

  Another aspect of the present invention is a method for expanding antigen-specific T cells, wherein the population of T cells is treated with the antigen for a time sufficient to induce activation of T cells specific for said antigen. Contacting said population of antigen-specific T cells ex vivo with a mixture comprising one or more DCs, IL-15, IL-7 and IL-2, and said antigen-specific T cells Contacting the cells under conditions and for a time sufficient to induce proliferation, thereby expanding antigen-specific T cells. In one embodiment, the antigen is a tumor antigen. In another embodiment, the antigen is pulsed on or expressed by the antigen presenting cell. In another embodiment, a population of T cells is contacted with the antigen ex vivo. In another embodiment, the method comprises at least one peptide-MHC tetramer sorting of said antigen-specific T cells. In certain embodiments, the methods of the invention further comprise at least one peptide-MHC tetramer magnetic sorting of said antigen-specific T cells.

  Another aspect of the present invention provides a method for treating cancer comprising administering to a cancer patient antigen-specific T cells expanded according to the methods provided herein.

  T cells generated according to the present invention can also be used to treat autoimmune diseases. Examples of autoimmune diseases include, but are not limited to, acquired immune deficiency syndrome (AIDS, a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, Autoimmune Addison disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, celiac disease-herpetic dermatitis; chronic fatigue immunodeficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), scarring pemphigus, cold agglutinin disease, crest syndrome , Crohn's disease, Dogo's disease, dermatomyositis-juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic lung fiber Disease, idiopathic Thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis Disease, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis , Psoriatic arthritis, Raynaud's phenomenon, Reiter syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (also known as progressive systemic sclerosis (PSS), systemic sclerosis (SS)), Sjogren Syndrome, stiff man syndrome, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vitiligo and Wegener's granulomatosis Can be mentioned.

  Cells produced according to the present invention can also be used to treat inflammatory disorders. Examples of inflammatory diseases include, but are not limited to, chronic and acute inflammatory disorders. Examples of inflammatory diseases include Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft-versus-host disease, hemolytic anemia, osteoarthritis, sepsis Stroke, tissue and organ transplantation, vasculitis, diabetic retinopathy and ventilator-induced lung injury.

  The present invention also relates to a method for preventing, inhibiting or reducing the presence of cancer cells or malignant cells in an animal, comprising administering to the animal an anticancer effective amount of the antitumor cells of the present invention. provide.

  Cancers contemplated by the present invention are not limited to those in which an immune response is induced or should be prevented, inhibited or reduced in the presence thereof, Melanoma, non-Hodgkin lymphoma, Hodgkin disease, leukemia, plasmacytoma, sarcoma, glioma, thymoma, breast cancer, prostate cancer, colorectal cancer, kidney cancer, renal cell cancer, pancreatic cancer, esophagus Cancer, brain cancer, lung cancer, ovarian cancer, cervical cancer, multiple myeloma, hepatocellular carcinoma, nasopharyngeal carcinoma, ALL, AML, CML, CLL, and other new known in the art Examples include living organisms.

  Alternatively, using the compositions described herein, pathogenic organisms such as viruses (eg, single stranded RNA viruses, single stranded DNA viruses, double stranded DNA viruses, HIV, type A, type B and Hepatitis C virus, HSV, CMV, EBV, HPV), parasites (eg protozoan and metazoans such as Plasmodium species, Leishmania species, Schistosoma species, Trypanosoma species), bacteria (eg, mycobacteria, Salmonella , Streptococci, E. coli, staphylococci), fungi (eg Candida spp., Aspergillus spp.), Pneumocystis carini, etc. can be induced or enhanced.

The immune response induced in an animal by administering the composition of the present subject matter includes a cellular immune response mediated by CD8 ⁺ T cells that can kill tumors and infected cells, and CD4 ⁺ T cells. A response. A humoral immune response mediated mainly by B cells producing antibodies after activation by CD4 ⁺ T cells can also be induced. Various techniques well described in the art can be used to analyze the type of immune response induced by the composition of the present invention;

  Where an “immunologically effective amount”, “antitumor effective amount”, “tumor inhibitory effective amount” or “therapeutic amount” is indicated, the exact amount of the composition of the invention administered will be determined by the physician, It can be determined taking into account individual differences in body weight, tumor size, degree of infection or metastasis, and patient condition. It is generally defined that pharmaceutical compositions comprising the subject cells of the invention can be administered at doses determined during appropriate clinical trials. The cells of the invention may be administered multiple times at these doses. Optimal doses and treatment regimens for a particular patient can be readily determined by one of ordinary skill in the medical arts by monitoring the patient for signs of disease and adjusting treatment accordingly.

  The cells of the invention can be administered at a dose and route and time determined in an appropriate clinical trial. The cell composition may be administered multiple times at doses within these ranges. The cells of the present invention may be combined with other methods. The cells of the invention for administration can be autologous, allogeneic or xenogeneic to the patient being treated. If desired, treatment may also include mitogens (eg, PHA) or lymphokines, cytokines, and / or chemokines (eg, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, M1P1-α, etc.) And may enhance the induction of an immune response as described herein.

  Administration of the cells of the invention may be performed in any convenient manner. The cells of the invention may also be administered to a patient subcutaneously, intradermally, intramuscularly, intravenously (i.v.) or intraperitoneally. In some examples, the cells of the invention are administered to a patient by intradermal or subcutaneous injection. In other examples, the cells of the invention are administered by i.v. injection. In other examples, the cells of the invention are injected directly into a tumor or lymph node.

  The cells of the invention can also be administered using any number of matrices. The present invention utilizes such a matrix in the novel context of acting as an artificial lymphoid organ to assist, maintain, or regulate the immune system, typically through modulation of T cells. Thus, the present invention can utilize matrix compositions and formulations that have proven useful in tissue engineering. Thus, the types of matrices that can be used in the compositions, devices and methods of the present invention are virtually limitless and can include both biological and synthetic matrices. In one particular embodiment, the compositions and devices specified by US Pat. Nos. 6,099,637; 5,637; 5,637; 6,412; The entirety of which is incorporated herein by reference. The matrix generally includes features associated with being biocompatible when administered to a mammalian host. The matrix may be formed from natural and / or synthetic materials. The matrix may be non-biodegradable if it is desired to leave a permanent or removable structure in the animal's body, such as an implant; or it may be biodegradable. The matrix can take the form of sponges, implants, tubes, terfa pads, fibers, hollow fibers, lyophilized ingredients, gels, powders, porous compositions, or nanoparticles. In addition, the matrix can be designed to allow sustained release of seeded cells or produced cytokines or other active agents. In certain embodiments, the matrix of the present invention can be described as a semi-solid framework that is flexible and elastic and permeable to substances such as dissolved gaseous drugs including inorganic salts, aqueous fluids and oxygen.

  Herein, the matrix is used as an example of a biocompatible material. However, the current invention is not limited to matrices, and therefore where the term matrix (“matrix” or “matrics”) appears, these terms allow cell retention or cell crossing and are biocompatible. , Devices and other materials that are capable of crossing macromolecules by allowing the material to pass directly, such as the material itself is a semi-permeable membrane, or used in combination with certain semi-permeable materials Must be read to include.

  In one aspect of the invention, the cells of the invention can be used as an adjuvant in vivo, as described in US Pat. In further embodiments, the cells of the invention are used as vaccines that induce an immune response in vivo against an antigen of interest, such as an antigen described herein (eg, a tumor antigen, viral antigen, autoantigen, etc.). Can be used. In one embodiment, the cells of the invention can be administered alone or in combination with other immune modulators and in combination with other known therapies to generate an immune response in vivo.

(Identification of epitope)
In one embodiment, the present invention provides compositions and methods for expanding antigen-specific T cells. Antigen-specific T cells can be expanded in accordance with the present invention comprising contacting the T cells with one or more DCs, IL-15, IL-7, and IL-2. Proliferated T cells can be used to identify antigen-specific T cell receptors (TCRs) and epitopes derived therefrom. For example, TCRs derived from expanded T cells can be cloned. Cloned TCRs represent a promising tool for the development of specific adoptive T cell therapies to treat desired diseases or disorders. For example, a cloned TCR can be used to generate peptides / antigens useful for vaccines.

  In addition to their role in combating infection, T cells are also involved in the destruction of cancerous cells. Autoimmune diseases are also associated with antigen-specific T cell attacks on various parts of the body. One of the major problems that hinders understanding and intervention of the mechanisms involved in these diseases is the difficulty in identifying T cells specific for the antigen to be studied.

  TCR is closely related to the antibody molecule in the structure and is involved in antigen binding, but unlike antibodies, it does not recognize free antigen; instead, it binds to antigen fragments that are bound and presented by antigen presenting molecules. Join. An important group of antigen presenting molecules are MHC class I and class II molecules that present antigenic peptides and protein fragments to T cells.

  Variability in the antigen binding site of TCR is created in a manner similar to that of an antibody, and also provides specificity for a vast number of different antigens. Diversity occurs in the complementarity determining regions (CDRs) of the N-terminal domain of TCR disulfide bond alpha (α) and beta (β), or gamma (γ) and delta (Δ) polypeptides. The CDR loops are clustered together to form an MHC antigen binding site similar to the antibody antigen binding site, but in the TCR, each of the various chains contains two more hypervariable loops compared to the antibody. TCR diversity for specific antigens is also directly related to MHC molecules on the surface of the APC where the antigen binds and is presented to the TCR.

In embodiments described elsewhere herein, the peptides of the invention can be located within the MHC molecule of dendritic cells in order to generate suitable T cells. In some embodiments, cells are extracellular peptide loaded with MHC molecules by incubating with various concentrations of peptides for 4 hours at 37 ° C., 5% CO ₂ and then washed once with serum-free RPMI. However, in another embodiment, antigen presenting cells are transfected with a polynucleotide encoding a fusion protein comprising a peptide linked to at least the MHC class I molecule alpha chain by a flexible linker peptide. Thus, when expressed, the fusion protein yields a peptide that occupies the MHC class I binding groove. Suitable MHC class I molecules and costimulatory molecules are available from public databases. Further details of the synthesis of such fusion molecules can be found in Non-Patent Document 20, which is incorporated herein by reference. The advantage of expressing a fusion protein of peptide and MHC molecule is that a much higher concentration of peptide is presented on the surface of antigen presenting cells.

  In some situations in the preparation of T cells, it is preferred that there is an HLA match between antigen presenting cells and T cells. That is, antigen presenting cells present allelic MHC molecules whose T cell donor is HLA positive. In some embodiments, this is achieved by obtaining antigen-presenting cells from a first individual and T cells from a second individual, wherein the first and second individuals are HLA compatible.

  However, in an alternative embodiment, the antigen-presenting cells and T cells are obtained from the same individual, but the antigen-presenting cells are transfected with a polynucleotide encoding an MHC molecule of a similar HLA allele. In some embodiments, the polynucleotide encodes a protein that encodes an MHC molecule linked to a peptide via a linker. There are numerous HLA class I alleles in humans, and MHC molecules presented by antigen-presenting cells can in principle be any of these alleles. However, since the HLA-A * 0201 allele is particularly common, the MHC molecule is preferably this allele. However, any HLA-A2 allele can be used, or other alleles such as HLA-A1, HLA-A3, HLA-A11 and HLA-A24 may be used instead.

  In a further embodiment of the invention, a method is provided for preparing T cells suitable for delivery to a patient suffering from cancer. The method includes providing dendritic cells that express the HLA molecule of the first HLA allele and placing a peptide in the binding groove of the HLA molecule. The peptide may or may not be the peptide of the present invention. The T cells are then stimulated with dendritic cells, which are obtained or obtainable from an individual that is HLA matched for the first HLA allele. As described elsewhere herein, dendritic cells are either obtained from a first donor individual and T cells are obtained from a second donor individual, where the first and second donor individuals are HLA. Fits. The advantage of using dendritic cells rather than non-professional antigen presenting cells is that it results in a much stronger stimulation of T cells.

  In these embodiments, the peptide is a cell type specific peptide, ie, only in certain cells or at a much higher level (eg, at least 10 times higher concentration) than other cell types in certain cells. Peptides obtained from the expressed protein are preferred.

  T cells prepared according to the present invention are administered to a patient to treat the patient's cancer. In principle, the T cells of the present invention can be used to treat many different types of cancer, including leukemias, lymphomas such as non-Hodgkin lymphoma, multiple myeloma, and the like.

  Accordingly, some embodiments of the present invention provide pharmaceutical preparations comprising the T cells of the present invention and a pharmaceutically acceptable carrier, diluent or excipient, the details of which are described in Can see.

  As discussed elsewhere herein, the HLA allele of the MHC molecule used to present the peptide to T cells is an HLA allele that is also expressed by the patient, so that the T cell is the patient. When they are administered, they recognize peptides presented on MHC molecules of the HLA allele.

  In some alternative embodiments, multiple sets (eg, 2 or 3 sets) of T cells are provided, each T cell being specific for a different peptide. In each case, as described elsewhere herein, the T cell is allogeneic, i.e., the HLA allele of the MHC molecule to which the peptide is presented during T cell preparation is An HLA allele that is not expressed in the resulting donor individual. The peptides may all be derived from the same cell specific protein, or may be derived from a protein that is specific for the same cell type but different. The peptide may or may not be the peptide of the present invention. In some embodiments, multiple sets of T cells are co-administered, while in other embodiments they are administered sequentially.

  Reference is made to T cell preparation and preparation. However, an important feature of T cells is the T cell receptor (TCR) displayed on the T cell, more specifically, the specificity of the T cell receptor for the complex of peptide and MHC molecule It should be understood that Thus, in some alternative embodiments of the invention, following preparation of T cells as described elsewhere herein, T cell receptors for T cells specific for a particular peptide are identified. Collect and sequence when complexed with MHC molecules of alleles. A cDNA sequence encoding the T cell receptor can then be generated and used to recombinantly express the T cell receptor in a T cell (eg, a patient's own T cell or a T cell from a donor). For example, the cDNA may be incorporated into a vector such as a viral vector (eg, a retroviral vector), a lentiviral vector, an adenoviral vector, or a vaccinia vector. Alternatively, non-viral approaches such as using naked DNA or lipoplexes and polyplexes or mRNA may be continued to transfect T cells.

  Thus, because recombinantly expressed TCRs are produced naturally, T cells “obtainable” from a donor individual include T cells that are recombinantly obtained in the manner described elsewhere herein.

  Since transfected T cells also display their endogenous TCR, it is preferable to pre-select T cells prior to transfection to eliminate T cells that can cause graft-versus-host disease. In some embodiments, T cells are pre-selected such that their endogenous TCR specificity is known. For example, T cells that react with glypican-3 are selected. In other embodiments, T cells are obtained from the patient and are naturally tolerant to the patient. This approach can only be taken if the patient's T cells are healthy.

  In some other embodiments, the T cell receptor is not expressed recombinantly as a whole, but incorporates regions of the T cell receptor responsible for its binding specificity into structures that maintain the definition of these regions. More specifically, the complementarity determining regions (CDRs) 1-3 of the T cell receptor are sequenced and these sequences are maintained in the same structure in the recombinant protein.

  In one embodiment, expanded T cells provide a source for cloning TCRs and epitopes / antigens associated therewith. The identified epitope / antigen can be used to generate a vaccine. In one embodiment, a vaccinated antigen can be constructed by modifying a polypeptide (eg, a target antigen) at a particular amino acid position identified by epitope mapping. Accordingly, the methods of the present invention include a method for identifying relevant positions for modification in a target antigen by epitope mapping, a method for modifying a target antigen at a relevant position to generate variants, and a variant as a separate candidate preparation. Including methods of inclusion.

  Vaccinated antigen polypeptides can be epitope mapped by a number of methods, including those disclosed in detail in US Pat. In summary, these methods involve a database of epitope patterns (determined from the input of peptide sequences known to specifically bind to anti-protein antibodies) and the 3-D structure of a given protein as an epitope. And an algorithm for analyzing the pattern database. This determines the possible epitopes on the protein and the preference of each amino acid in the protein sequence that becomes part of the epitope.

  In another aspect, a method for identifying candidate MHC class II epitopes is provided. In certain embodiments, candidate epitopes can be identified using a computer implemented algorithm for candidate epitope identification. Such computer programs include, for example, the TEPITOPE program (see, eg, Non-Patent Document 22; Non-Patent Document 23; Non-Patent Document 24; Non-Patent Document 25; Non-Patent Document 26; the disclosure of which is hereby incorporated by reference) As well as other computer-implemented algorithms.

  Computer-implemented algorithms for identifying candidate epitopes include, for example, in a single protein, in very large proteins, in related protein groups (eg, homologues, orthologs, or polymorphic variants), in a mixture of unrelated proteins, in tissue or Candidate epitopes can be identified in organ proteins or in the proteome of an organism. This approach can be used as an efficient, sensitive and specific approach for the identification of T cell epitopes in addition to analysis of known candidate molecular targets, and sequence information of expressed proteins (eg, putative open Complex tissues or organisms can be examined on the basis of reading frames or cDNA libraries.

  Following identification of candidate epitopes, peptides or pools of peptides corresponding to the candidate epitope (s) can be formed. For example, once a candidate epitope is identified, prepare an overlapping peptide that spans the candidate epitope or part of it, confirms the binding of the epitope by MHC class II molecules, and improves the identification of that epitope if necessary Can do. Alternatively, a pool of peptides can be prepared to include multiple candidate epitopes identified using a computer implemented algorithm for candidate epitope identification.

(T cell epitope peptide)
The T cell epitope of the present invention is a short peptide that can be derived from a protein antigen. Antigen presenting cells can bind directly to the antigen via surface MHC molecules and / or can be processed into short fragments capable of internalizing the antigen and binding it to the MHC molecule. The specificity of a peptide that binds to MHC depends on the specific interaction between the peptide and the peptide binding groove of a particular MHC molecule.

  Peptides that bind to MHC class I molecules are usually 6-30 amino acids, more commonly 7-20 amino acids or 8-15 amino acids in length. The amino terminal amine group of the peptide contacts an invariant site at one end of the peptide groove, and the carboxylate group at the carboxy terminus binds to the invariant site at the other end of the groove. Thus, typically such peptides have a hydrophobic or basic carboxy terminus and no proline at the extreme amino terminus. The peptide is in a definite state extended along the groove and has further contact between the main chain atoms and the conserved amino acid side chains that line the groove. Changes in peptide length are often accommodated by twisting of the peptide backbone at proline or glycine residues.

  Peptides that bind to MHC class II molecules are usually at least 10 amino acids in length, for example about 13-18 amino acids, and can be much longer. These peptides are in a defined state extending along the MHCII peptide binding groove open at both ends. Peptides are held in place primarily by main chain atom contacts with conserved residues that line up in the peptide binding groove.

  The peptides of the present invention may be made using chemical methods. For example, the peptide can be synthesized by solid phase technology (Non-patent Document 27), cleaved from the resin, and purified by preparative high performance liquid chromatography. Automated synthesis can be completed, for example, using an ABI 431A peptide synthesizer (Perkin Elmer) and following the instructions provided by the manufacturer.

  Alternatively, the peptide may be made by recombinant means or by cleavage from a longer polypeptide. For example, the peptide may be obtained by cleavage from full-length glypican-3 protein. The composition of the peptide can be confirmed by amino acid analysis or sequencing.

  The peptides of the present invention can be tested in an antigen presenting system comprising antigen presenting cells and T cells. For example, the antigen presentation system may be a murine spleen cell preparation, a human cell derived from tonsils or a preparation of PBMC. Alternatively, the antigen presentation system can include specific T cell lines / clones and / or specific antigen presenting cell types.

T cell activation can be measured via T cell proliferation (eg, using ³ H thymidine incorporation) or cytokine production. Activation of TH1-type CD4 ⁺ T cells can be detected via IFN-γ production, which can be detected by standard techniques such as, for example, ELISPOT assay.

  Measurement of antigen-specific T cells during an immune response is an important parameter in vaccine development, autologous cancer therapy, transplantation, infection, inflammation, autoimmunity, toxicity studies, and so on. Peptide libraries are important reagents in monitoring antigen-specific T cells. The present invention provides improved methods for the use of peptide libraries in the analysis of T cells in a sample, including diagnostic, prognostic and immune monitoring methods. In addition, the use of peptide libraries in anti-tumor therapy is described elsewhere in this specification and is intended to isolate antigen-specific T cells that can inactivate or eliminate unwanted target cells, or otherwise. Isolation of specific T cells capable of regulating the immune cells. The present invention also relates to MHC multimers comprising one or more tumor-derived peptides.

  The identification of specific antigenic peptides provides new opportunities for the development of diagnostic and therapeutic strategies for cancer. Advantageously, the identification of novel T cell epitopes enables the production of MHC class I and class II multimers, tetramers and pentamers, both useful as analytical tools for achieving high sensitivity of immune monitoring To do. Furthermore, detection of antigen-specific CTLs in the periphery of individuals at risk for disease recurrence is a useful diagnostic tool.

  Accordingly, in a further aspect, the present invention provides MHC multimers, tetramers or pentamers comprising at least one MHC class I or II glypican-3 peptide epitope described herein.

  The present invention also provides compositions and methods for identifying peptides useful for cancer treatment. A peptide sequence derived from a candidate protein predicted to bind to HLA-A * 0201 can be identified by a computer algorithm. Peptides are selected for synthesis according to their predicted affinity with a cut-off value of 500 nM or lower, although higher values can also be selected. Peptides can be synthesized and binding to HLA-A * 0201 can be confirmed using biochemical assays. Peptide binding is compared to binding obtained with a pass / fail control peptide designated 100% and a positive control peptide. Corresponding HLA-A * 0201-peptide multimers are also synthesized for peptides with higher binding affinity than pass / fail control peptides. These peptides are tested for the ability to generate T cell lines that react specifically with specific peptide-HLA-A * 0201 complexes. Cell lines can be referred to as multimers, tetramers, or pentamer positive T cells. Multimer positive cells show high immunogenicity against the corresponding peptide. Further responses can be measured to assess cytokine interferon-γ production, target cell degranulation and death.

  In some embodiments, the peptides of the invention can be administered directly to a patient as a vaccine. Accordingly, the peptides of the present invention are immunogenic epitopes of specific proteins and are used to elicit T cell responses against their respective proteins. In some embodiments, a polypeptide of the invention is administered directly to a patient as a vaccine. For example, in leukemia patients, a polypeptide comprising a peptide derived from a hematopoietic cell-specific protein is administered to the patient to elicit a T cell response to that protein. T cell responses result in the death of hematopoietic cells, including cancerous cells, but are specific for these cells and do not result in an immune response against other cell types.

  In many patients, even if such a peptide is administered directly, any T cell whose cell-specific protein is a “self-protein” and capable of binding to the polypeptide will remain on the MHC molecule of the patient's HLA allele. It should also be noted that no T cell response is elicited because it is tolerated when presented in. That is, T cells that may be reactive are destroyed in the patient's thymus during the selection process or inactivated by central or peripheral resistance mechanisms. Accordingly, it is preferred to use the peptides of the present invention to generate T cells obtained or obtainable from allogeneic donor individuals. This individual should preferably be HLA negative for an HLA allele for which the patient is HLA positive. For example, if a patient is HLA positive for the HLA allele HLA-A * 0201, T cells are obtained from individuals who are negative for HLA-A * 0201. It is generally preferred that the donor individual is otherwise HLA-matched with the patient. The HLA-A * 0201 allele MHC molecule is then presented, and a peptide-loaded antigen presenting cell (APC) is provided. The donor individual's T cells are then stimulated with APC and the resulting cells are expanded.

  The expanded T cells that are capable of binding to the peptides of the invention when complexed with the HLA-A * 0201 antigen are then combined with a plurality of peptide-MHC molecules (eg, pentamers or tetramers). ) To concentrate using an artificial structure containing T cells specific for certain peptide-HLA-A * 0201 complexes in a mixture of T cells have the ability to bind to these structures when mixed with them. Subsequently, the T cells are mixed with magnetic beads that have the ability to bind to the artificial structure. The artificial structures and T cells bound to them are then removed from the remaining mixture by the magnetic attraction of the beads.

(Treatment)
Antigen-specific T cells can be administered to animals several times daily, or less frequently, such as once a day, once a week, once every two weeks, once a month, etc. Alternatively, it may be administered more frequently, such as once every few months, even once a year or less. The frequency of dosing will be readily apparent to those skilled in the art and will depend on any number of factors such as, but not limited to, the type and severity of the disease being treated, the type of animal and the age.

  Antigen-specific T cells may be co-administered with a variety of other compounds, such as cytokines, chemotherapeutic agents and / or antiviral agents, among others. Alternatively, the compound (s) may be administered 1 hour, 1 day, 1 week, 1 month or more before or in any permutation of the antigen-specific T cells. Also good. In addition, the compound (s) may be administered 1 hour, 1 day, 1 week or more after administration of antigen-specific T cells, or in any permutation thereof. The frequency and dosing schedule will be readily apparent to those skilled in the art and include, but are not limited to, the type and severity of the disease being treated, the age and health of the animal, the compound or compounds administered Depending on any number of factors such as the identity of the various compounds and the route of administration of the various compounds and antigen specific T cells.

  In therapeutic methods, administration of the compositions of the invention may be for “prevention” or “treatment” purposes. When provided prophylactically, the compositions of the invention are provided before any symptoms, but in certain embodiments, the vaccine is provided after the onset of one or more symptoms to prevent further symptoms from developing. Or prevent the current symptoms from getting worse. Prophylactic administration of the composition contributes to preventing or ameliorating subsequent infection or disease. When provided therapeutically, the pharmaceutical composition is provided at or after the onset of symptoms of the infection or disease. Thus, the present invention may be provided either prior to anticipated exposure to a disease inducing agent or prior to a disease state, or after onset of an infection or disease.

  An effective amount of the composition is that amount that produces this selected result that enhances the immune response, and such amount can be routinely determined by one of ordinary skill in the art. For example, an amount effective to treat an immune system failure against a cancer or pathogen is that amount necessary to cause activation of the immune system and result in the generation of an antigen-specific immune response upon exposure to the antigen. . The term is also synonymous with “sufficient amount”.

  The effective amount for any particular application will depend on factors such as the disease or condition being treated, the particular composition being administered, the size of the subject, and / or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular composition of the present invention without necessitating undue experimentation.

  The invention will now be described in further detail with reference to the following examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Accordingly, the present invention should in no way be construed as limited to the following examples, but rather is construed to encompass any and all variations that become apparent as a result of the teaching provided herein. It should be.

  Unless further explained, one of ordinary skill in the art will be able to make and use the invention and practice the claimed methods using the foregoing description and the following examples. Accordingly, the following examples specifically point out preferred embodiments of the invention and should not be construed as limiting the remainder of the disclosure in any way.

(Example 1: Recovery of anti-HER2 CD4 ⁺ Th1 response in breast cancer patients receiving DC1 vaccination)
The following studies were designed to investigate the role of adjuvant type 1 polarized dendritic cell ("DC1") vaccination.

(DC1 vaccination of HER2 ⁺ IBC patients with residual disease after neoadjuvant therapy)
Four HER2 ⁺ IBC patients with residual disease after neoadjuvant therapy received adjuvant HER2 pulsed DC1 vaccine. Each patient's Th1 immune response was determined before DC1 vaccination, 3 months after DC vaccination, and 6 months after vaccination and from patient PBMC pulsed with 6 HER2 class II peptides (SEQ ID NOs: 1-6) Was generated by measuring IFNγ production via ELISPOT. Autologous DC1 vaccine was prepared as described above. Responses were assessed by (1) overall anti-HER2 responsiveness (responding to> 1 peptide), (2) number of reactive peptides (response repertoire), and (3) cumulative response across 6 HER2 peptides .

(result:)
Responsiveness: Only one IBC patient showed an immune response defined as> 20 SFC / 10 ⁶ cells in experimental wells after subtracting unstimulated background before vaccination. Compared to the pre-vaccination results, all vaccinated IBC patients showed an immune response defined as a> 2-fold increase in anti-HER2 IFN-γ ^pos Thl response.

  Response repertoire: average repertoire increased from 0.5 ± 1 peptide before vaccination to 3.25 ± 0.96 peptide (p = 0.01) after 3 months and 4 ± 0.8 peptide (p = 0.01) after 6 months (FIG. 1) .

Cumulative response: 36.5 ± 38.3 SFC / 10 ⁶ before vaccination, 151.0 ± 60.0 SFC / 10 ⁶ (p = 0.04) after 3 months, and 198.4 ± 39.7 SFC / 10 ⁶ (p = 0.02) after 6 months The average cumulative response improved (Figure 2).

(Conclusion :)
HER2-pulsed DC1 vaccination of HER2 ⁺ IBC patients with residual disease after treatment with neoadjuvant chemotherapy enhances anti-HER2 Th1 immune response.

  The anti-HER2 Th1 immune response increases in both width (response repertoire) and depth (cumulative response).

(Example 2: Proliferation of HER2-specific Th1 cells in vitro)
The following studies were designed to investigate the role of adoptive T cell transplantation in restoring anti-HER2 Th1 immunity. T cells are expanded to the level required for adoptive therapy and epitope mapping studies while maintaining antigen specificity and cell function.

  Briefly, in vitro, HER2-specific Th1 cells were generated by co-culture with HER2 peptide pulsed DC1 and grown using IL-2 alone or IL-2, IL-7 and IL-15. Subsequently, Th1 cells were grown either by repeated HER2-peptide pulsed DC1 co-culture or anti-CD3 / CD28 stimulation. The fold proliferation was defined as follows: (#T cells after proliferation / # T cells before proliferation); specificity was measured by antigen-specific IFNγ production by ELISA.

As shown herein, repeated co-culture of CD4 ⁺ T cells with HER2 peptide-pulsed DC1 stimulated with IL-2, IL-7 and IL-15 is highly specific for anti-HER2 Th1 cells. Provides significant growth and provides a potential population of cells for adoptive transfer. Co-culture with specific peptide-specific DC1 and IL-2, IL-7, and IL-15 stimuli mimics the lymph node environment and is used to significantly expand any population of antigen-specific Th1 cells Can be done.

Without wishing to be bound by any particular theory, if a subject is vaccinated against a protein antigen (eg, a tumor target antigen), blood may be removed from the subject and collected after vaccination. It is considered possible. The collected blood contains dendritic cell precursors as well as low levels of T cells specific for the tumor target antigen. DC precursors and T cells are separated from each other. DC precursors can be loaded / pulsed with tumor target protein / antigen and then activated to the DC1 state. Antigen-specific DC1 is then co-cultured with T cells and cytokines (IL-15, IL-7 and IL-2) are added to the co-culture in the appropriate order. This cycle can be repeated weekly until T cells proliferate to a sufficient number (eg, 1 × 10 ⁹ ). T cells are supplied to the original subject, and a large amount of T cells that the subject's body cannot naturally produce are injected into the subject. This large unit of antigen-specific T cells can have strong anti-tumor activity.

(Methods for proliferating T cells)
(Preparation of HER2-specific DC1 :)
As described above, DC precursors were obtained from HER2 breast cancer patients (DCIS) vaccinated with HER2 peptide pulsed DC1 vaccine. DC precursors were obtained by tandem leukapheresis / countercurrent centrifugal elution. DC were incubated at 37 ° C. with 3 × 10 ⁶ cells in 1 ml macrophage serum-free medium (SFM-Gibco Life Technologies, Carlsbad, Calif.) With GM-CSF 50 ng / ml (Berlex, Richmond, Calif.) did. 48-72 hours after initial seeding of the cells, DCs were isolated from a single HER2 peptide antigen (42-56,98-114,328-345,776-790,927-941,1166-1180 (SEQ ID NO: 1-6)); 20 μg / ml ) Pulsed. For maturation, DC were further activated 6 hours later with IFN-γ (1000 U / ml) and the next day with lipopolysaccharide (“LPS”) (10 ng / ml). HER2-specific DC1 was harvested 6 hours after LPS administration at the time of maximum IL-12 production.

(CD4 ⁺ T cell preparation :)
Lymphocytes were also obtained from breast cancer patients who had been previously vaccinated (HER2-pulse DC1 vaccination) by tandem leukapheresis / countercurrent centrifugal elution. CD4 ⁺ T cells were purified by negative selection using a human CD4 + T cell enrichment kit (Stemcell Technologies; Vancouver BC, Canada). CD4 ⁺ T cells at 2x10 ⁶ cells / ml in culture medium (Iskov medium, 1% L-glutamine, 1% penicillin / streptomycin, 1% sodium pyruvate, 1% non-essential amino acids, intervening; Manassas, VA , And 5% heat inactivated human AB serum).

(DC1-CD4 ⁺ co-culture :)
Seed DC1 with CD4 ⁺ T cells in a 1:10 ratio (2 × 10 ⁵ DC1 / ml and 2 × 10 ⁶ CD4 ⁺ T cells / ml) in a 24-well plate and incubate at 37 ° C. did. Recombinant human IL-7 (10 ng / ml) and IL-15 (10 ng / ml) (BioLegend; San Diego, Calif.) Were added 48-72 hours after co-culture. Recombinant human IL-2 (5 U / ml) was added 24 hours after adding IL-7 and IL-15.

(HER2-specific iDC for testing and HER2-specific DC1 for restimulation :)
Two additional groups of DCs were prepared as described above. In one group, each well was pulsed with a single peptide antigen (20 ug / ml) and considered immature DC (“iDC”). In the second group, each well was pulsed with a single peptide antigen (20 μg / ml) and matured to DC1 as described above. HER2-specific CD4 ⁺ T cells were harvested 7-9 days after the previous DC1 co-culture. T cells were co-cultured with iDC for ELISA testing. Interferon gamma production was measured by ELISA assay according to manufacturer recommendations and protocols. T cells were also co-cultured with DC1 and stimulated with IL-7 / 15 and IL-2 as described above. This cycle was repeated a total of 4 times with CD4 ⁺ T cells and HER2-specific DC1 co-culture.

(Overview of breeding method :)
Develop HER2-specific DC1 as described above
Day 1: Incubate monocytes (one well for each peptide)
Day 4: Mature HER2-specific DC1
AM: Pulse with antigen (20 μg / ml).
PM (after 6 hours): Add IFN-γ
Day 5: Purify CD4 ⁺ T cells and DC1 co-culture.
AM: LPS
6 hours later: co-culture: 2x10 ⁶ CD4 ⁺ T cells and 2x10 ⁵ DC1
* 48-72 hours after co-culture: add IL-7 (10 ng / ml) and IL-15 (10 ng / ml);
24 hours after adding IL-7 / 15: add IL-2 (5 U / ml);
(In IL-2 alone conditions, IL-2 was added 72-96 hours after co-culture (simultaneously with IL-2 being added to the IL2 / 7/15 group).

Make both HER2-specific iDC for ELISA testing and HER2-specific DC1 for restimulation
Day 9-11: Monocytes are cultured (2 wells for each peptide)
48 hours after monocyte culture: treat HER2-specific immature DCs to mature DC1
iDC: Pulse with antigen (20 μg / ml) (given to future iDC and DC1 simultaneously)
Pulse with DC1: AM: antigen (20 μg / ml).
PM (6 hours later): Add IFN-g Next day: iDC co-culture and DC1 co-culture (ie, 7-9 days after DC1 co-culture)
DC1: AM: LPS
Collect HER2-specific CD4 ⁺ T cells
Collect HER2-specific iDC
→ Co-culture: 2x10 ⁶ CD4 ⁺ T cells and 2x10 ⁵ iDC
Harvest HER2-specific DC1
→ Co-culture: 2x10 ⁶ CD4 ⁺ T cells and 2x10 ⁵ DC1
The next day: Repeat the above steps to perform ELISA on iDC co-culture; resume upon stimulation with IL7 / 15 (*)

(result:)
In the experiments and studies whose results are shown in FIGS. 3-16, the method described above was used:

Figures 3 and 4 show a direct comparison between HER2-specific DC1 stimulated with IL-2 versus CD4 ⁺ T cells co-cultured with those stimulated with IL-2 / 7/15 and received HER2 pulsed DC1 vaccination. Two different patients are shown respectively.

Briefly, immature DCs from each patient (“iDC”) are divided into the following MHC class II peptides: peptide 42-56 (SEQ ID NO: 1), peptide 98-114 (SEQ ID NO: 2), peptide 328-345. (SEQ ID NO: 3), and pulsed with peptide 776-790 (SEQ ID NO: 4) and matured to DC1. The resulting HER2 pulsed DC1 was then co-cultured with CD4 ⁺ T cells and stimulated with IL-2 alone or IL-2 / 7/15 as indicated. The red outline represents specific peptides and stimulation protocols that showed specificity (specific antigen: control antigen IFN-γ production ratio greater than 2: 1). Thus, in FIG. 3, the specificity is expressed as peptide 42-56-IL2 / 7/15 protocol; peptide 98-114-IL-2 protocol and IL-2 / 7/15 protocol, both peptides 328-345 protocol, and peptide 776-790, shown in both protocols. In FIG. 4, specificity was shown with peptide 776-790, both protocols only. A graph showing the multiplication factor (defined as T cell number after proliferation / T cell number before proliferation) is shown on the right side of each figure. In general, as indicated by the respective growth rate data, the growth rate of IL-2 / 7/15 stimulation was greater than that of IL-2 stimulation alone. Specificity was measured by ELISA by antigen-specific IFN-γ production.

  5 and 6 show specific to non-specific immune responses: FIG. 5 shows the specific response following the first stimulation / proliferation with HER2-specific DC1, and FIG. The subsequent disappearance of that specific response after a second stimulation / proliferation with CD3 / CD28 is shown.

The first stimulation of CD4 ⁺ T cells with HER2-specific DC1 resulted in multiple specific immune responses, as shown by the red outline in FIG. 5: Peptide 42-56, IL2 / 7/15 protocol Peptide 98-114, both protocols, peptide 328-345, both protocols, and peptide 776-790, both protocols. Figure 6 shows that the second stimulation of HER2-specific CD4 ⁺ T cells with non-specific anti-CD3 / CD28 stimulation resulted in a 4-fold proliferation (horizontal graph), but 3/4 of the peptide group had specificity. Shown lost (only peptide 328-345 showed specificity after CD3 / 28 growth). Patient-derived iDCs were divided into the following MHC class II peptides: peptide 42-56 (SEQ ID NO: 1), peptide 98-114 (SEQ ID NO: 2), peptide 328-345 (SEQ ID NO: 3), and peptide 776-790 (sequence) Pulsed with number 4) and matured to DC1 as described above. The resulting HER2 pulsed DC1 was then co-cultured with CD4 ⁺ T cells and stimulated with IL-2 alone or IL-2 / 7/15 as indicated.

Figures 7 and 8 show a non-specific immune response followed by a specific immune response: Figure 7 shows non-specific proliferation of CD4 ⁺ T cells. FIG. 8 shows that specificity cannot be obtained after subsequent stimulation with HER2-specific DC1. The first stimulation of CD4 ⁺ T cells with non-specific anti-CD3 / CD28 resulted in 3.8-fold proliferation (FIG. 7). Second stimulation of non-specific CD4 ⁺ T cells with HER2-specific DC1 did not result in a specific immune response (FIG. 8). Patient-derived iDCs were divided into the following MHC class II peptides: peptide 42-56 (SEQ ID NO: 1), peptide 98-114 (SEQ ID NO: 2), peptide 328-345 (SEQ ID NO: 3), and peptide 776-790 (sequence) Pulsed with number 4) and matured to DC1 as described above. The resulting HER2 pulsed DC1 was then co-cultured with CD4 ⁺ T cells and stimulated with IL-2 alone or IL-2 / 7/15 as indicated.

Figures 9A and 9B show in vitro primary / first growth of HER2-specific Th1 cells in vitro, with HER2-specific DC1 grown on IL-2 versus IL-2 / 7/15 CD4 ⁺ T cells co-cultured with. iDCs were synthesized from the following MHC class II peptides: peptide 42-56 (SEQ ID NO: 1), peptide 98-114 (SEQ ID NO: 2), peptide 776-790 (SEQ ID NO: 4), and peptide 927-941 (SEQ ID NO: 5). Pulsed and matured to DC1. The resulting HER2 pulsed DC1 was then co-cultured with CD4 ⁺ T cells and stimulated with IL-2 alone or IL-2 / 7/15 as indicated. The red outline (FIG. 9B) represents specific peptides and stimulation protocols that showed specificity (specific antigen: control antigen IFN-γ production ratio greater than 2: 1). FIG. 9A shows that when the average proliferation ratio of Th1 cells (defined as number of T cells after proliferation / number of T cells before proliferation) was stimulated with IL-2, IL-7, and IL-15, IL -2 represents significantly better than alone (2.6 ± 0.75 vs 1.0 ± 0.12; p = 0.001). FIG. 9B shows the specificity of various peptide / growth protocols as measured by ELISA by antigen-specific IFN-γ production. Stimulation with IL-2, IL-7, and IL-15 and stimulation with IL-2 alone both resulted in specific Th1 responses in the same HER2 peptide 776-790.

  Summary of primary growth: IL-2 vs. IL-2 / 7/15. The average growth rate of Th1 cells was significantly better than IL-2 alone when stimulated with IL-2, IL-7, and IL-15 (2.6 ± 0.75 vs 1.0 ± 0.12; p = 0.001) (FIG. 9A). Stimulation with IL-2, IL-7, and IL-15 and stimulation with IL-2 alone both resulted in a specific Th1 response in the same HER2 peptide (peptide 776-790) (FIG. 9B).

  FIGS. 10A and 10B show in vitro secondary / second proliferation of HER2 pulsed DC1 versus anti-CD3 / CD28. Restimulation of Th1 cells with HER2-peptide pulse DC1 and anti-CD3 / CD28 resulted in similar proliferation rates (3.9 ± 1.0 vs 4.3 ± 2.0, p = 0.7), respectively (FIG. 10A). However, FIG. 10B shows that stimulation of Th1 cells with HER2-specific DC1 enhanced the specific Th1 response; whereas nonspecific stimulation with anti-CD3 / CD28 resulted in an overall loss of HER2 peptide specificity. To show that The following MHC class II peptides were used: peptide 42-56 (SEQ ID NO: 1), peptide 98-114 (SEQ ID NO: 2), peptide 776-790 (SEQ ID NO: 4), and peptide 927-941 (SEQ ID NO: 5) . The red outline (FIG. 10B) represents specific peptides and stimulation protocols that showed specificity (specific antigen: control antigen IFN-γ production ratio greater than 2: 1) (ie, peptides 42-56 DC1 restimulation of specific Th1 cells and peptide 776-790 specific Th1 cells).

  Summary of secondary growth: HER2 peptide pulsed DC1 vs. anti-CD3 / CD28. Restimulation of Th1 cells with HER2-peptide pulse DC1 and anti-CD3 / CD28 resulted in similar proliferation rates (3.9 ± 1.0 vs 4.3 ± 2.0, p = 0.7), respectively (FIG. 10A). However, stimulation of Th1 cells with HER2-specific DC1 enhanced the specific Th1 response, whereas non-specific stimulation with anti-CD3 / CD28 resulted in an overall loss of HER2 peptide specificity ( FIG. 10B).

  Tertiary proliferation of Th1 cells with HER2 pulsed DC1. After the third stimulation with the indicated HER2-specific DC1, both the mean growth fold (4.32 ± 0.5, 43.7-fold cumulative growth (FIG. 11A) and antigen specificity (FIG. 11B) increased again, in particular 4 All of the peptides (peptide 42-56 (SEQ ID NO: 1), peptide 98-114 (SEQ ID NO: 2), peptide 776-790 (SEQ ID NO: 4), and peptide 927-941 (SEQ ID NO: 5)) show specificity, Increased IFN-γ production.

  Summary of tertiary growth: HER2 peptide pulsed DC1. After the third stimulation with HER2-specific DC1, mean growth rate (4.32 ± 0.5, 43.7-fold cumulative growth (FIG. 11A) and both specificity-specific peptide number and IFN-γ production (FIG. 11B) Both increased again.

  Overall, in FIGS. 9B, 10B and 11B with the same number of T cells, IFN-γ production was seen to increase logarithmically with each proliferation. Non-specific CD3 / CD28 of the second stimulation (FIG. 10B) also showed an overall loss of specificity. Cells proliferated as a Th1 phenotype, with 50-200 fold proliferation (FIGS. 9A, 10A, and 11A) that became more specific and stronger with each stimulus.

Figures 12-15 show the sequential results of repeated in vitro stimulation (4 times) of HER2-specific CD4 ⁺ Th1 cells with IL-2 / 7/15. For all FIGS. 12-15, each left panel shows peptide specificity by IFN-γ production (“Tet” is a tetanus patient control); each right panel shows the specific HER2 used -Indicates the growth rate of the peptide. In FIG. 12, iDC was pulsed with an additional MHC class II peptide in addition to the other five used in the above study: Peptides 1166-1180 (SEQ ID NO: 6). However, as seen in the growth factor results (FIG. 12, right panel), peptide 328-345 specific Th1 cells and peptide 1166-1180 specific Th1 cells did not produce enough cells for further growth, and therefore Only the HER2 Th1 cells specific for the last four peptides were used.

  Subsequently, FIG. 12 for the first stimulation shows specificity only for peptide 776-790-specific Th1 cells; in FIG. 13 for the second stimulation, in addition to peptide 776-790-specific Th1 cells 14 shows an increase in specificity for peptide 42-56; in FIG. 14 for the third growth, all four peptide specific Th1 cells (peptide 42-56, peptide 98-114, peptide 776-790, and Figure 15 for the fourth round of growth shows the loss of specificity for one of the peptides (peptide 927-941) and the remaining three HER2-specific peptides remain (Peptide 42-56, peptide 98-114, peptide 776-790).

  FIG. 16 shows the cumulative growth rate of the four growths shown in FIGS. 12 to 15 for all HER2-specific Th1 cells, and the last bar (dot) in each group shows the cumulative growth rate. The average cumulative magnification exceeded 100 times.

(Conclusion :)
Repeated co-culture of CD4 ⁺ T cells with HER2-peptide pulsed DC1 stimulated with IL-2, HL-7, and IL-15 resulted in significant proliferation of highly specific anti-HER2 Th1 cells and adopted Provides a potential population of cells for transplantation.

  Each stimulus of all four stimuli resulted in both increased fold growth and increased antigen specificity without reaching either limit. In fact, 100-400 fold growth was shown.

Co-culture with peptide-specific DC1 and IL-2, IL-7, and IL-15 stimuli mimics the lymph node environment and can be used to significantly expand any population of antigen-specific Th1 cells . One skilled in the art will readily recognize that embodiments of the invention relating to T cell proliferation are in no way limited to CD4 ⁺ t cells. Thus, embodiments of the present invention provide methods for growing CART cells, cytotoxic T lymphocytes (CD8 ⁺ ) as well as all other types of T cells.

The results presented herein demonstrate that HER2 pulsed DC1 vaccination of HER2 ⁺ IBC patients with residual disease after treatment with neoadjuvant chemotherapy enhances anti-HER2 Th1 immune response. The anti-HER2 Th1 immune response increases in both width (response repertoire) and depth (cumulative response). Adoptive transplantation of HER2-specific Th1 cells may play a role in restoring the CD4 ⁺ Th1 immune response. Repeated co-culture with HER2-peptide pulsed DC1 stimulated with IL-2, IL-7 and IL-15 can result in significant proliferation of highly specific anti-HER2 Th1 cells.

(Example 3: Anti-HER2 CD4 ⁺ Th1 response can be restored in breast cancer patients receiving DC1 vaccination)
Proliferation of T cell subsets is an essential step for obtaining sufficient T cells to perform adoptive therapy or to identify epitopes on target antigens of peptide-based vaccines. The proliferation of T cells is a simple process in principle. In practice, however, there are many technical problems including low levels of proliferation, premature activation-induced cell death (apoptosis), or loss of antigen specificity and / or function.

  Part of the problem is the inability to replicate in the body environment, ie lymph nodes, where antigen-specific T cell proliferation occurs in vitro. These are specialized tissues containing many different cell types apart from T cells containing stromal cells such as antigen presenting dendritic cells and epithelial cells. Each of these cell types plays a different role by providing contact-dependent signals (surface receptors) and water-soluble signals (cytokines) that are important for T cell proliferation and maintenance of cell function.

  Experiments were designed to investigate (1) the role of adjuvant type 1 polarized dendritic cells (DC1) vaccination and (2) the role of adoptive T cell transplantation in restoring anti-HER2 Th1 immunity. T cells are expanded to the level required for adoptive therapy and epitope mapping studies while maintaining antigen specificity and cell function.

  The materials and methods used in these experiments are now described.

(Preparation of fully activated DC)
Freshly eluted bone marrow monocytes were cultured in 6-well microplates (12 × 10 ⁶ cells / well). The culture medium consisted of serum-free medium (SFM Invitrogen, Carlsbad, CA). The final concentration of GMCSF added was 50 ng / ml and the final concentration of IL4 was 1000 U / ml. Cells were cultured overnight at 37 ° C., 5% CO ₂ . In some batches, cells were pulsed with the appropriate peptide after 16-20 hours and cultured for an additional 6-8 hours, after which 1000 U / ml IFN-γ was added. Dendritic cells were matured with the TLR agonist LPS (TLR4, 10 ng / ml) or R848 (TLR8, 1 μg / ml).

  To induce the production of the Th1 polarized cytokine IL-12, DCs are activated using the cytokine IFN-γ, or a combination of the TLR agonist bacteria LPS and / or R848. This should induce T cells that produce IFN-γ. Alternatively, DC can be activated with a combination of ATP, bacterial LTA, LPS and prostaglandin E2 (PGE2). This can amplify IL-23, IL-6 and IL-1β, leading to an immune response dominated by IL-17 and IL-22 secreting Th17 cells.

(Proliferation method)
HER2-specific DC1 as described elsewhere herein
Day 1: Incubate monocytes (one well for each peptide)
Day 4: Mature HER2-specific DC1
AM: Pulse with antigen (20ug / ml)
PM (after 6 hours): Add IFN-γ
Day 5: Purify CD4 ⁺ T cells and DC1 co-culture.
AM: LPS
6 hours later: co-culture: 2x10 ⁶ CD4 ⁺ T cells and 2x10 ⁵ DC1
* 48-72 hours after co-culture: Add IL-7 (10 ng / ml) and IL-15 (10 ng / ml)
24 hours after adding IL-7 / 15: Add IL-2 (5 U / ml) (In the IL-2 alone condition, IL-2 was added 72 to 96 hours after co-culture (IL- At the same time 2 was added to the IL2 / 7/15 group)

(Vaccination)
HER2 ⁺ IBC patients with residual disease after neoadjuvant therapy were vaccinated with adjuvant HER2 pulsed DC1. Immune responses were generated from PBMC pulsed with HER2 class II peptides by measuring IFN-γ production with ELISPOT. Responses were evaluated as follows: (1) overall anti-HER2 responsiveness (responding to> 1 peptide), (2) number of reactive peptides (response repertoire), and (3) spanning 6 HER2 peptides Cumulative response. The Th1 response before vaccination was compared with the responses after 3 and 6 months.

(Production of HER2-specific Th1 cells)
In vitro, HER2-specific Th1 cells were generated by co-culture with HER2 peptide pulsed DC1 and grown with IL-2 alone or IL-2, IL-7, and IL-15. Subsequently, Th1 cells were grown either by repeated HER2-peptide pulsed DC1 co-culture or anti-CD3 / CD28 stimulation. The fold proliferation was defined as follows: (#T cells after proliferation / # T cells before proliferation); specificity was measured by ELISA by antigen-specific IFN-γ production.

  Here, the result of the experiment disclosed in this specification will be described.

(In vivo-Th1 response to HER2 pulsed DC1 vaccination :)
Responsiveness: Only one IBC patient showed an immune response defined as> 20 SFC / 2 * 10 ⁵ cells in experimental wells after subtracting unstimulated background before vaccination. Compared to the pre-vaccination results, all vaccinated IBC patients showed an immune response defined as a> 2-fold increase in anti-HER2 IFN-γ ^pos Thl response.

  Response repertoire: average repertoire increased from 0.5 ± 1 peptide before vaccination to 3.25 ± 0.96 peptide (p = 0.01) after 3 months and 4 ± 0.8 peptide (p = 0.01) after 6 months (FIG. 1) .

Cumulative response: 36.5 ± 38.3 SFC / 2 * 10 ⁵ before vaccination, 151.0 ± 60.0 SFC / 2 * 10 ⁵ (p = 0.04) after 3 months, and 198.4 ± 39.7 SFC / 2 * 10 after 6 months Average cumulative response improved to ⁵ (p = 0.02) (Figure 2).

(In vitro-HER2-specific Th1 cell proliferation :)
Primary growth: IL-2 vs. IL-2 / 7/15. The average growth rate of Th1 cells was significantly better than IL-2 alone when stimulated with IL-2, IL-7, and IL-15 (2.6 ± 0.75 vs 1.0 ± 0.12; p = 0.001) (FIG. 9A). Stimulation with IL-2, IL-7, and IL-15 and stimulation with IL-2 alone both resulted in a specific Th1 response in the same HER2 peptide (peptide 776-790) (FIG. 9B).

  Secondary proliferation: HER2 peptide pulsed DC1 anti-CD3 / CD28. Restimulation of Th1 cells with HER2-peptide pulse DC1 resulted in 3.9-fold proliferation and 10.1-fold cumulative proliferation; similarly, subsequent stimulation with anti-CD3 / CD28 resulted in 4.4-fold proliferation and 11.5-fold proliferation. Resulting in double cumulative growth (p = 0.5, FIG. 10A). Restimulation of Th1 cells with HER2-specific DC1 enhanced the specific Th1 response; whereas non-specific stimulation with anti-CD3 / CD28 resulted in an overall loss of HER2 peptide specificity (FIG. 10B). ).

  Tertiary growth: HER2 peptide pulse DC1. After the third stimulation with HER2-specific DC1, both the mean growth factor (4.32 ± 0.5; FIG. 11A) and specificity (FIG. 11B) increased again.

  Quaternary proliferation: T cells can proliferate with a fourth stimulation. It was observed that T cells continued to proliferate and maintained T cell function (Figure 12).

The results presented herein demonstrate that HER2 pulsed DC1 vaccination of HER2 ⁺ IBC patients with residual disease after treatment with neoadjuvant chemotherapy enhances anti-HER2 Th1 immune response. The anti-HER2 Th1 immune response increases in both width (response repertoire) and depth (cumulative response). Adoptive transplantation of HER2-specific Th1 cells may play a role in restoring the CD4 ⁺ Th1 immune response. Repeated co-culture with HER2-peptide pulsed DC1 stimulated with IL-2, IL-7 and IL-15 can result in significant proliferation of highly specific anti-HER2 Th1 cells.

Without wishing to be bound by any particular theory, it is believed that if a subject is vaccinated against a protein antigen (eg, a tumor target antigen), blood can be removed from the subject after vaccination. It is done. Blood containing dendritic cell precursors as well as low levels of T cells specific for tumor target antigens can be collected. DC precursors and T cells are separated from each other. DC precursors can be loaded with a tumor target protein and then activated. They are then co-cultured with T cells and cytokines (IL-15, IL-7 and IL-2) are added to the co-culture in the appropriate order. This cycle can be repeated weekly until T cells proliferate to a sufficient number (eg, 1 × 10 ⁹ ). T cells are supplied to the original subject, and a large amount of T cells that the subject's body cannot naturally produce are injected into the subject. This large unit of antigen-specific T cells can have strong anti-tumor activity.

  The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety. Although the invention has been disclosed with reference to particular embodiments, it is to be understood that other embodiments and variations of the invention may be devised by those skilled in the art without departing from the true spirit and scope of the invention. it is obvious. It is intended that the appended claims be construed to include all such embodiments and equivalent variations.

Claims (26)

  A method of proliferating T cells, comprising contacting the T cells with one or more dendritic cells, at least two cytokines, and a T cell growth factor.   2. The method of claim 1, wherein the T cell is contacted with an antigen, thereby generating an antigen-specific T cell.   The method of claim 1, wherein the dendritic cell is a type I dendritic cell.   2. The method of claim 1, wherein the at least two cytokines are interleukin-7 ("IL-7") and interleukin-15 ("IL-15").   The method of claim 1, wherein the T cell growth factor is interleukin-2 (“IL-2”). The method of claim 1, comprising:
a) contacting said T cells in vitro with autologous type I dendritic cells DC together with antigen, thereby generating antigen-specific T cells;
b) contacting said antigen-specific T cells with IL-7 and IL-5 to generate stimulated antigen-specific T cells;
c) contacting the stimulated antigen-specific T cells with IL-2, thereby generating a proliferated antigen-specific T cell population that maintains antigen specificity and cell function.   The T cell produced | generated by the method in any one of Claims 1-6.   A population of cultured and expanded antigen-specific T cells exhibiting anti-tumor activity produced by the method of any of claims 1-6, wherein said cells are sufficient for effective treatment in a mammal. Population that has grown to.   A population of cultured and expanded antigen-specific T cells exhibiting anti-tumor activity generated by the method of any of claims 1-6, wherein said cells have grown to a sufficient number for effective epitope mapping Group.   An isolated polynucleotide encoding a T cell receptor (TCR) derived from an antigen-specific T cell, wherein the antigen-specific T cell is generated by the method of any of claims 1-6. An isolated polynucleotide.   7. A method of immunotherapy comprising administering a T cell to a subject in need thereof, wherein the T cell is generated by the method of any of claims 1-6.   A method of expanding a T cell population comprising at least one T cell obtained from a blood sample from a subject vaccinated against an antigen, said T cell comprising one or more dendritic cells or precursors thereof Contacting the body with at least two cytokines and a T cell growth factor.   13. The method of claim 12, wherein the blood sample comprises at least one T cell and at least one DC precursor of the population specific for the vaccine antigen.   13. The DC precursor is pulsed with the antigen, activated to antigen-specific type I dendritic cells (“DC1”), and then co-cultured with the T cells to produce antigen-specific DC1. The method described.   13. The method of claim 12, wherein the at least two cytokines are interleukin-7 (“IL-7”) and interleukin-15 (“IL-15”).   13. The method of claim 12, wherein the T cell growth factor is interleukin-2 ("IL-2"). The method of claim 12, comprising:
a) co-culturing said T cells from said patient sample with antigen-specific T cell autologous type I dendritic cells (DC1) in vitro;
b) contacting the cells from step a) with IL-7 and IL-5 to generate stimulated antigen-specific T cells;
c) subsequently contacting the stimulated antigen-specific T cells with IL-2, thereby generating a proliferated antigen-specific T cell population that maintains antigen specificity and cell function.   18. The method of claim 17, further comprising repeating steps a) -c) from one to at least three more times to generate a further expanded antigen-specific T cell population. 13. The method of claim 12, wherein the T cell is CD4 ⁺ .   13. The method of claim 12, wherein the antigen is HER2. A method of expanding a CD4 ⁺ T cell population comprising at least one CD4 ⁺ T cell obtained from a blood sample from a breast cancer patient vaccinated against HER2, comprising said CD4 ⁺ T cell comprising one or more CD4 ⁺ T cells A method comprising the step of contacting a dendritic cell or a precursor thereof, at least two cytokines, and a T cell growth factor. 23. The method of claim 21, wherein at least one DC precursor in the sample is pulsed with MHC class II HER2 peptide and contacted with the CD4 ⁺ T cells.   24. The method of claim 21, wherein the at least two cytokines are interleukin-7 ("IL-7") and interleukin-15 ("IL-15").   23. The method of claim 21, wherein the T cell growth factor is interleukin-2 ("IL-2"). The method of claim 21, comprising:
a) co-culturing said T cells from claim 11 with said HER2 pulsed DC1;
b) contacting said cells from step a) with IL-7 and IL-15 to generate stimulated antigen-specific T cells;
c) subsequently contacting the stimulated antigen-specific T cells with IL-2, thereby generating a proliferated antigen-specific T cell population that maintains antigen specificity and cell function.   26. The method of claim 25, further comprising repeating steps a) -c) from one to at least three more times to generate a further expanded antigen-specific T cell population.

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