HK1168869B - Method for proliferation of antigen-specific t cells - Google Patents

Description

Method for proliferating antigen-specific T cells

Technical Field

The present invention relates to the fields of immunology and cancer therapy, and more specifically to a method of activating antigen-specific T cells and T cells prepared by said method.

Background

T cells recognize tumors or infected cells and prevent disease by killing these target cells. However, the interaction of tumors or pathogens with the immune system is complex, which is evidenced by the development of cancer or chronic infectious disease in the presence of specific T cells, so that the pathogen or tumor can apparently evade T cell surveillance.

The ability of T cells to detect virtually any pathogenic invader is the result of its very diverse receptor repertoire, which allows the T cell pool to recognize a large number of peptides presented by Major Histocompatibility Complex (MHC) molecules. Moreover, signal transduction through the T Cell Receptor (TCR) (signal 1) is insufficient to produce adequate T cell activation, as the costimulatory molecules provide the indispensable signals for proliferation, survival and differentiation (signal 2). In fact, naive T cells that received only signal 1 and no signal 2 were considered anergic (non-responsive) or dead by apoptosis. Full T cell activation requires signals 1 and 2 to work together, and the intensity of these signals determines the size of the ensuing T cell pool. Moreover, complete differentiation into effector T cells generally relies on a third signal, which is provided by Antigen Presenting Cells (APCs) in soluble form and provides an instructional signal for the desired effector T cell type. This concept of "three signals" describes a model for the activation of naive T cells and the subsequent formation of effector T cells. However, the immune system provides a large variety of co-stimulatory molecules and these various types of signals 2 and 3 all contribute to the quality of the T cell response in its unique way. Costimulatory molecules and soluble forms of signal 3 can act on T cell activation, such as survival, cell cycle progression, the type of effector cell to be developed, and specific aspects of differentiation of effector or memory cells.

It is now generally accepted that maturation of antigen-presenting Dendritic Cells (DCs) requires the maturation of other lymphocytes, including CD4⁺"helper" of T cells, NK cells and NKT cells to induce long-lived memory CD8⁺T cells. This "help" induces the mature DCs to further differentiate, a process called restriction (licensing). "helper" signals have a variety of effects on DCs, including upregulation of costimulatory molecules, secretion of cytokines, and upregulation of a variety of anti-apoptotic molecules, which cumulatively enhance optimal activation of cognate T cells by DCs, particularly CD8⁺The capacity of T cells. Furthermore, "helper" lymphocytes may also express or secrete factors that directly affect T cell survival, cell cycle progression, the type of effector cell to be developed, and differentiation of effector or memory cells.

One strategy for combating chronic infectious diseases or aggressive cancers is adoptive T cell therapy, which involves the transfer of effector T cells to restore a specific T cell response in the host. Recent technological developments to obtain the required specificity of T cells have led to an increasing interest in the use of adoptive T cell therapy in different clinical settings. Adoptive cell transfer therapy is the administration of autologous tumor-reactive T cells that are activated and expanded in vitro. There are several potential advantages to using adoptive cell transfer therapy in cancer therapy. Tumor-specific T cells can be activated and expanded to large numbers in vitro, independent of the immunogenicity of the tumor, and the functional and phenotypic properties of the T cells can be selected prior to adoptive transfer.

After adoptive transfer, several events must occur for T cells to cause regression of the established tumor. More specifically: t cells must be activated in vivo by antigen-specific restimulation, -then T cells must be expanded to a level that can cause destruction of a significant tumor burden, -anti-tumor cells must survive long enough to complete elimination of all tumor cells.

Previously, the criterion for selecting cells for adoptive transfer to patients with solid tumors was the ability of anti-tumor T cells to release IFN- γ and kill tumor cells after co-culture. However, it is now clear that these criteria alone are not predictive of in vivo efficacy. Gattinoni et al, j.clin.invest.115: 1616-1626(2005) found CD8 that achieved full effector properties and showed enhanced anti-tumor reactivity in vitro⁺T cells, have poor efficacy in initiating tumor regression and healing in vivo.

The methods according to the prior art require one or more restimulations to reach clinically relevant levels of tumor-specific cytotoxic T cells. See, e.g., Ho et al (Journal of immunological methods, 310(2006), 40-50) and Gritzapis et al (J.Immunol., 2008; 181; 146-154) where re-stimulation 1-2 times is required to achieve about 19% tumor-specific CD8⁺T cell level. Restimulation results in decreased cell activity and approaches apoptosis.

Therefore, there is a need for methods of preparing T cell populations for adoptive immunotherapy that increase antigen-specific T cell proliferation and survival upon activation.

Disclosure of Invention

The present invention relates to a method for in vitro priming of antigen-specific T helper 1(Th1) cells or cytotoxic T Cells (CTLs) suitable for administration to a tumor patient. The method comprises co-culturing target T cells, autologous monocyte-derived dendritic cells, autologous or allogeneic tumor material or tumor-associated proteins or peptides and allogeneic lymphocytes from the patient to be treated. The allogeneic lymphocytes are sensitized to MHC class I and/or MHC class II antigens on Antigen Presenting Cells (APCs) from the patient, or to APCs from a non-relevant blood donor expressing at least one MHC class II antigen that is the same as the MHC class II antigen expressed on the APCs from the patient to be treated.

The invention also relates to antigen-specific TH1 cells and/or CTLs obtainable by the method and uses thereof.

Drawings

Figure 1 illustrates that MHC antigen-sensitized lymphocytes expressed on allogeneic Peripheral Blood Mononuclear Cells (PBMCs) irradiated in conventional MLRs (allo-sensitized allo-allogeneic lymphocytes; ASALs) significantly enhanced the expression of CD70 on co-cultured mature monocyte-derived DCs autologous to the irradiated PBMCs used to excite the ASALs.

Figure 2 illustrates that gamma-irradiated ASALs enhance the expression of CD70 on co-cultured mature monocyte-derived DCs that are autologous relative to irradiated PBMCs used to prime the ASALs.

FIG. 3 illustrates that co-culture of ASAL with monocyte-derived DCs autologous to the PBMCs used to excite the ASAL induces large amounts of IL-12 production.

Figure 4 illustrates that co-culture of ASALs with monocyte-derived DC autologous to PBMCs used to excite the ASALs induces large amounts of IFN- γ production.

Figure 5 illustrates that co-culture of ASALs with monocyte-derived DC autologous to the PBMCs used to excite the ASALs induces large amounts of IL-2 production.

FIG. 6 illustrates the production of IL-2, IL-12 and IFN- γ, as well as mature monocyte-derived DCs and CD4 depletion⁺、CD8⁺Or CD56⁺Results of ASAL co-culture of lymphocytes.

FIG. 7 illustrates ASAL versus ASALCo-culture of autologous monocyte-derived DCs in irradiated PBMC for priming of ASAL increased non-sensitized allogeneic CD8⁺Proliferative responses of T cells.

FIG. 8 shows tumor specific CD8 as determined by flow cytometry⁺Percentage of T lymphocytes. A: the upper right corner shows the induction of HER-2 specific cytotoxic lymphocytes from HER-2 positive breast cancer patients. Self-loading with antigen (as opposed to CD 8)⁺Target cells) DCs were co-cultured with irradiated ASAL for 9 days, 25.2% of total CD8⁺The target cells become tumor-specific CTLs. B: the upper right corner shows the frequency of Her 2-specific cytotoxic lymphocytes in control experiments without DC-loaded Her2 peptide. After 9 days of culture, only 0.4% of total CD8⁺T cells become tumor-specific CTLs. (Upper right corner plus lower right corner represents total CD8T cell population.)

FIG. 9 illustrates these CD8 when restimulated with B cells autologous to the DC used for primary target cell stimulation⁺Targeting cells, in contrast to primary stimulation of allogeneic CD8⁺Irradiation of PBMCs for excitation of ASAL during target cell addition of irradiated ASAL to autologous monocyte-derived DCs, resulting in CD27 expressing alloresponse CD8⁺The number of target cells increases.

FIG. 10 illustrates these CD8 when restimulated with B cells autologous to the DC used for primary target cell stimulation⁺Targeting cells, in contrast to primary stimulation of allogeneic CD8⁺Irradiated PBMC used to prime ASAL during target cells irradiated PBMC that is autologous to the irradiated ASAL results in a reduction in the number of apoptotic (annexin-V-positive) target cells.

FIG. 11 illustrates these alloresponse CD8 when restimulated with B cells autologous to the DC used for primary target cell stimulation⁺Targeting cells, in contrast to primary stimulation of allogeneic CD8⁺Irradiated PBMC for priming of ASALs during target cells irradiated ASALs were added to autologous irradiated monocyte-derived DC, resulting in a stronger (6-fold) secondary proliferative response.

FIG. 12 illustrates these alloresponse CD8 when restimulated with B cells autologous to the DC used for primary target cell stimulation⁺Targeting cells, in contrast to primary stimulation of allogeneic CD8⁺Irradiated PBMC for priming of ASALs during target cells added irradiated ASALs to autologous irradiated monocyte-derived DCs, resulting in a substantial increase in IFN- γ production.

Definition of

Before describing the present invention, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Also, the term "about" is used to refer to a numerical value of +/-2% of a given value, preferably +/-5%, and most preferably +/-10% where appropriate.

In the context of the present invention, the term "antigen specificity" relates to the specific recognition/binding of a distinct T Cell Receptor (TCR) by a short distinct peptide sequence present on a self MHC molecule.

In the context of the present invention, the terms "priming" and "activation" relate to the programmed activation process that occurs in naive antigen-specific T cells stimulated by antigen presenting cells with or without concomitant presence of "helper" cells.

In the context of the present invention, the term "responsive cells" relates to different subpopulations of lymphocytes, including but not limited to T cells, NK cells and NKT cells of allogeneic PBMCs co-cultured in response to activation and/or proliferation.

In the context of the present invention, the term "primed cells" relates to different subpopulations of lymphocytes, including allogeneic cells by co-culture, including preactivated T cells, NK cells and NKT cells of PBMCs.

In the context of the present invention, the term "target cell" relates to CD4 activated/activated by allogeneic or antigen-presenting autologous APC⁺Or CD8⁺T cells. The site of collection of lymphocytes (target cells) from the patient may, for example, be peripheral blood, a tumor draining lymph node or bone marrow.

In the context of the present invention, the term "maturation" in relation to monocyte-derived DCs relates to the expression of "maturation markers" (including but not limited to CD40, CD86, CD83 and CCR7) induced by stimulation of immature DCs with microbial products (such as LPS) or inflammatory mediators (such as TNF- α and/or IL-1 β).

Immature DCs are cells characterized by high endocytic activity and low T cell activation potential. Immature DCs are often sampled (sample) for pathogens such as viruses and bacteria in the surrounding environment. Immature DCs phagocytose pathogens and degrade their proteins into small fragments, which are presented to their cell surface using MHC molecules upon maturation. At the same time, they upregulate cell surface receptors that act as co-receptors in T cell activation, such as CD80, CD86, and CD40, greatly enhancing their ability to activate T cells. They also up-regulate CCR7, which CCR7 is a chemokine receptor that induces dendritic cells to enter the spleen via the bloodstream or lymph system into lymph nodes. Here, they act as antigen presenting cells: by presenting them with non-antigen specific co-stimulatory signals, antigens derived from pathogens that activate helper and killer T cells as well as B cells. Mature DCs may be produced by monocytes, which are circulating leukocytes in the body and, depending on the correct signals, may be converted into DCs or macrophages. Whereas monocytes are formed from stem cells in the bone marrow. Monocyte-derived DCs can be generated in vitro from peripheral blood monocytes.

In the context of the present invention, the term "inactivation" of a cell is used to refer to the inability of a cell to undergo cell division to form progeny. However, cells are capable of responding to stimuli, or biosynthesizing and/or secreting cellular products (e.g., cytokines). Methods of inactivation are known in the art. Preferred methods of inactivation are treatment with a toxin (e.g., mitomycin C) or irradiation, such as gamma irradiation. Cells that are fixed or permeabilized and unable to divide are also examples of inactivated cells.

In the context of the present invention, the terms "mixed lymphocyte reaction", "mixed lymphocyte culture", "MLR" and MLC are used interchangeably and refer to a mixture comprising a minimum of two different cell populations that differ allotypically. At least one of the allotypically distinct cells is a lymphocyte. The cells are cultured together under suitable conditions for a period of time to produce stimulation of the lymphocytes. The usual purpose of MLRs is to provide allogeneic stimulation, such as to initiate proliferation of lymphocytes; however, unless otherwise specified, proliferation during culture is not required. In the appropriate context, these terms may alternatively refer to a mixture of cells derived from such a culture.

As used herein, the term "treatment" refers to clinical intervention in an attempt to alter the natural course of the individual or cell to be treated, and may be used prophylactically or during clinical pathology. Desirable effects include preventing the onset or recurrence of disease, alleviating symptoms and reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis.

The terms "antigen presenting cell", "APC" include whole, complete cells and other molecules (all allogeneic sources) capable of inducing one or more antigen presentations, preferably associated with MHC class I molecules, as well as all types of monocytes capable of inducing an allogeneic immune response. Preferably, fully viable cells are used as APCs. Examples of suitable APCs include, but are not limited to, whole cells such as monocytes, macrophages, DCs, monocyte-derived DCs, macrophage-derived DCs, B cells and myeloid leukemia cells, for example the cell lines THP-1, U937, HL-60 or CEM-CM 3. Myeloid leukemia cells are said to provide so-called promonocytes.

The terms "cancer", "neoplasm" and "tumor" are used interchangeably and in the singular or plural form, as appearing in the specification and claims, refer to a cell that is maliciously transformed to be pathogenic to a host organism. Primary cancer cells (i.e., cells obtained from the vicinity of a malignant transformation site) can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. As used herein, the definition of cancer cell includes not only primary cancer cells, but also any cells derived from a cancer cell progenitor. This includes metastatic cancer cells, and in vitro cultures and cell lines derived from cancer cells. When referring to a type of cancer that usually manifests as a solid tumor, a "clinically detectable" tumor is one that is detectable on the basis of a tumor mass, for example, by procedures such as CAT scanning, Magnetic Resonance Imaging (MRI), X-ray, ultrasound, or palpation. Non-limiting examples of tumors/cancers associated with the present invention are breast cancer, glioma, glioblastoma, fibroblast tumor, neurosarcoma, lung cancer, uterine cancer, lymphoma, prostate cancer, melanoma, testicular tumor, astrocytoma, ectopic hormone-producing tumor, ovarian cancer, bladder cancer, wemson's tumor, pancreatic cancer, bone cancer, lung cancer, colorectal cancer, cervical cancer, vaginal cancer, synovial sarcoma, vasoactive intestinal peptide-secreting tumor, glioblastoma, medulloblastoma, head and neck squamous cell carcinoma, oral cancer, oral leukoplakia, esophageal cancer, gastric cancer or metastatic cancer, leukemia. Prostate and breast cancer are particularly preferred.

In the context of the present invention, the term "culturing" refers to the propagation of cells or organisms in vitro in various media. It is understood that progeny of a cell grown in culture may not be identical (morphological, genetic, or phenotypic) to the parent cell. Suitable media can be selected by those skilled in the art, and examples of such media are RPMI medium or Eagles minimal basal medium (EMEM).

The terms "major histocompatibility complex" and "MHC" refer to a gene complex encoding a cell surface molecule required for antigen presentation to T cells and for rapid graft rejection. In humans, MHC complexes are also known as HLA complexes. Proteins encoded by MHC complexes are referred to as "MHC molecules" and are classified as MHC class I and class II molecules. MHC class I molecules include membrane heterodimeric proteins consisting of chains encoded in the MHC non-covalently linked to β 2-microglobulin. MHC class I molecules are expressed by almost all nucleated cells and are shown to be presented on antigen to CD8⁺Plays a role in T cells. Class I molecules include HLA-A, -B and-C in humans. Class I molecules typically bind peptides 8-10 amino acids in length. MHC class II molecules also include membrane heterodimeric proteins.

Class II MHC molecules are also known to be involved in antigen presentation to CD4⁺T cells, and in humans, HLA-DP, -DQ, and DR. Class II molecules are formed by binding peptides that are 12-20 amino acids in length. The term "MHC restricted" refers to the property of allowing T cells to recognize only antigen after being processed and the resulting antigenic peptide to be displayed in association with self class I or self class II MHC molecules.

The terms "vaccine", "immunogen" or "immunogenic composition" as used herein refer to a compound or composition that confers a degree of specific immunity when administered to a human or animal subject. As used in this disclosure, "cell vaccine" or "cell immunogen" refers to a composition comprising at least one cell population, optionally an inactivated cell population, as an active ingredient. The immunogens and immunogenic compositions of the present invention are active, by which is meant that they are capable of stimulating a specific immunological response (e.g., an anti-tumor antigen or an anti-cancer cell response) that is at least partially mediated by the host's immune system. The immunological response may comprise antibodies, immunoreactive cells (such as helper/inducer or cytotoxic cells) or any combination thereof, and is preferably directed against an antigen present on the tumor against which the treatment is directed. The response may be elicited or restimulated in a subject by administering a single dose or multiple doses.

If it is possible a) to generate an immune response against an antigen (e.g.a tumour antigen) in the individual to be tested for the first time; or b) a compound or composition is "immunogenic" if no recombination, enhancement or maintenance of an immune response in an individual will occur without administration of the compound or composition. When administered in single or multiple doses, the compositions are immunogenic if any of these criteria are met.

Detailed Description

The present invention relates to the preparation of allogeneic primed allogeneic lymphocytes (ASALs) to promote increased proliferation and survival of antigen-specific T cells during their activation by autologous antigen presenting cells, including Dendritic Cells (DCs).

The present invention is based on in vitro studies using PBMCs and subpopulations thereof from human healthy blood donors and breast cancer patients, demonstrated in the induction of antigen-specific human CD8⁺Positive regulation of ASAL in T cell responses. Alloreactivity CD8 in the presence of ASAL was followed using allogens in an in vitro model⁺T cell proliferation and survival, proliferation capacity increased more than 5 times, and apoptotic cell death decreased by 10-5%.

Addition of ASALs resulted in a strong up-regulation of the expression of the co-stimulatory molecule CD70 on antigen-presenting DCs and the production of IL-12 and IFN- γ, two factors that had a well-known positive effect on T cell development into type 1 CD4+ and CD8+ T cells. In addition, the addition of ASAL also results in the production of the well-known growth factor IL-2 for T cells. In particular, it has recently been shown that CD 70-mediated interactions promote survival of activated T cells throughout successive rounds of division, thereby contributing to the accumulation of effector T cells.

The present invention relates to a method for in vitro priming of antigen-specific T helper 1(Th1) cells or cytotoxic T Cells (CTLs) suitable for administration to a tumor patient. The method comprises co-culturing target T cells from the patient to be treated with autologous monocyte-derived DCs, autologous or allogeneic tumor material or tumor-associated proteins or peptides and allogeneic lymphocytes sensitized against MHC class I and/or MHC class II antigens on antigen-presenting cells (APCs) from the patient, or against APCs from a non-relevant blood donor expressing at least one MHC class II antigen identical to the MHC class II antigen expressed on the APCs from the patient to be treated.

ASALs are responder cells obtained from mixed lymphocyte reactions and cultured with monocyte-derived DCs and target cells. Said ASAL is allogeneic to said patient and is selected from the group consisting of peripheral blood lymphocytes, including CD4⁺T cell, CD8⁺T cells and Natural Killer (NK) cells. CD4 of DC self body with target cell being monocyte source⁺And/or CD8⁺T cells. Monocytes-derived DCs are loaded with tumor material or tumor-associated proteins or polypeptides or virus-derived antigens.

Further addition of ASAL resulted in target CD8 expressing high levels of CD27⁺Enrichment of T cell populations. CD27⁺CD8⁺T cells represent potentially more potent CTLs (cytotoxic T cells) for adoptive immunotherapy because they provide antigen-driven autocrine signals for proliferation. Such helper independent CD8T cells do not require exogenous help in the form of IL-2 or CD4⁺T cells survive and expand. Thus, the present invention provides CD8 to a subject⁺T cell populations providing a method for treating immune mediated diseases, said CD8⁺T cell population with no or low amount of additional cytokine (such as IL-2) or CD4⁺T cells, are programmed for strong cytotoxic activity. The method is particularly useful for in vivo expansion of cytolytic, antigen-specific CD8⁺T cells, but also for expansion of tumor-specific CD4⁺T cells.

With CD8⁺Cytolytic antigen-specific CD8 as a percentage of the total number of T lymphocytes⁺The percentage of T cells is preferably at least about 5%, more preferably at least about 10%, more preferably at least about 15%, more preferably at least about 20% and most preferably at least about 25%.

More specifically, the method of the present invention relates to a method for priming in vitro antigen-specific Th1 cells or CTLs suitable for administration to a tumor patient, the method comprising the steps of:

a) the inactivated antigen-presenting cells from the patient are cultured with peripheral blood mononuclear cells from a healthy donor,

b) culturing monocytes from the patient (said composition being further described below) in a composition that allows the monocytes to mature into mature DCs, and

c) sensitizing allogeneic lymphocytes, including but not limited to CD4 from step a)⁺T cell, CD8⁺T cells and/or Natural Killer (NK) cells are cultured with the mature DCs from step b).

Monocyte-derived DCs are obtained by first culturing monocytes in a composition comprising GM-CSF and IL-4 for about 2-7 days, preferably for about 5 days, to obtain immature DCs, followed by addition of a second composition capable of making the immature DCs mature DCs, by culturing for at least about 12-72 hours, and preferably for about 24-48 hours. The second composition comprises a component that enables immature DCs to become mature monocyte-derived DCs that can be used to activate CD4⁺And CD8⁺T cells. In one embodiment, the second composition comprises TNF α, IL-1 β, interferon γ, interferon β and a TLR3 ligand, such as poly-I: C (Maillard et al, Alpha-type-apolarized DCs: a novel immunization tool with optimized CTL-induced activity. cancer Res. 2004; 64: 5934-. In another embodiment, the second composition comprises interferon gamma, TLR3 and/or TLR4 ligand and TLR7 and/or TLR8 ligand and/or TLR9 ligand. A non-limiting example of a TLR3 ligand is poly-I: C, a non-limiting example of a TLR7/8 ligand is R848, and a non-limiting example of a TLR9 ligand is CpG.

Sensitization of allogeneic lymphocytes is induced by a classical mixed lymphocyte reaction (MLR or MLC-mixed lymphocyte culture) comprising culturing inactivated allogeneic antigen-presenting cells with Peripheral Blood Mononuclear Cells (PBMCs) from healthy donors. The implementation of MLR is well known to those skilled in the art (Jordan WJ, RitterMA. optimal analysis of composite cytokine responses. jimmunol Methods 2002; 260: 1-14). In MLR, PBMCs (predominantly lymphocytes) from two individuals are mixed together in tissue culture for several days. Lymphocytes from incompatible individuals will mutually stimulate to significantly proliferate (as measured by tritium thymidine uptake), while lymphocytes from incompatible individuals will not mutually stimulate. In one-way MLC lymphocytes from one of the individuals are inactivated (by treatment with a toxin, such as mitomycin, or irradiation, such as gamma irradiation) so that only the untreated remaining cell population proliferates in response to the foreign histocompatibility antigen.

The antigen-presenting cells used in MLR are selected from PBMC and monocyte-derived DCs. Monocyte-derived DCs are derived from a patient or from a healthy donor having mhc class ii (HLA-DR) antigens that match the patient's HLA-DR antigens.

The tumor material or tumor-associated protein or peptide is selected from the group consisting of killed tumor cells from the patient, allogeneic tumor cells of the same type as the patient's tumor, and isolated and purified tumor proteins or peptides. Isolated and purified tumor proteins or peptides are well known to those skilled in the art. In one embodiment, the tumor material is a tumor protein loaded into monocyte-derived DCs by transfection of mrnas encoding the tumor protein.

Examples of tumor-associated peptides are peptides derived from the HER-2 protein (associated with breast cancer), PSA (prostate specific antigen associated with prostate cancer), MART-1 protein (associated with malignant melanoma) and peptides derived from the "universal" tumor-associated proteins survivin and p53, further examples of tumor-associated peptides/proteins being well known to the person skilled in the art.

In the method of the present invention, the cells are co-cultured for about 20 days, preferably about 4 to 20 days, preferably 6 to 20 days, more preferably 7 to 14 days, and most preferably about 9 to 14 days.

In one embodiment of the invention, exogenous IL-2, IL-7, IL-15, anti-IL-4 and/or IL-21 are added to the cell culture to optimize cell proliferation and survival.

Stimulated antigen-specific Th1 cells or CTLs may also be restimulated by culturing the cells with new monocyte-derived DCs, newly sensitized allogeneic lymphocytes and optionally adding exogenous IL-2, IL-7, IL-15, anti-IL-4 and/or IL-21 to the cell culture.

The invention also relates to immunogenic compositions obtainable by the above method and to antigen-specific Th1 cells and/or CTLs obtainable by the above method.

The antigen-specific TH1 cells and/or CTLs are suitable for administration to a patient, and preferably have at least one of the following characteristics:

-ability to proliferate

Expression of the memory marker CD45RO

Expression of the low level of the apoptosis marker annexin-V (i.e. up to 40%, preferably up to 20%, of the cells show positive staining for annexin-V as determined by FACS)

Expression of CD27 and/or CD28 on the cell surface

A further capability of the antigen-specific TH1 cells and/or CTLs obtainable by the method of the invention is the ability to kill tumor cells in vitro.

Furthermore, the present invention relates to the use of the antigen-specific TH1 cells and/or CTLs obtainable by the present invention or the method as defined above for the treatment of tumors or for eliciting anti-tumor immunological responses in humans, and for the preparation of a medicament for the treatment of tumors or for eliciting anti-tumor immunological responses in humans. The TH1 cells and/or CTLs can be administered after a first stimulation or, alternatively, after restimulation. In one embodiment, TH1 cells and/or CTLs are administered in combination with a therapeutic cancer vaccine.

Methods of using T cell populations for adoptive cell therapy in the treatment of human subjects are known to clinicians in the art. T cell populations prepared according to the methods described herein and known in the art can be used in such methods. For example, adoptive cell therapy using tumor-infiltrating lymphocytes with MART-1 antigen-specific T cells has been tested clinically (Powell et al, Blood 105: 241-250, 2005). Renal cell carcinoma patients have been inoculated with irradiated autologous tumor cells. The harvested cells were then activated with anti-CD 3 monoclonal antibody and IL-2 and then re-administered to the patient (Chang et al, J.clinical Oncology 21: 884-890, 2003).

Upon exposure to ASALs during initial in vitro DC-mediated priming, antigen-primed T cells undergo increased proliferation and decreased apoptosis following restimulation. Thus, the invention also relates to a method for enhancing a secondary T cell response after vaccination if adoptively transferred back to a patient before and/or during vaccination.

The invention also provides methods of preparing a population of T cells for adoptive immunotherapy comprising engineering (by viral transduction, transfection, electroporation or other methods of introducing genetic material) T cells to express a T cell receptor or chimeric T cell receptor that recognizes a target antigen; activating the engineered T cells with antigen-loaded DCs in the presence of sensitized allogeneic lymphocytes; expanding the cells in culture, and reintroducing the cells back into the patient.

The invention also provides methods for improving cancer vaccine therapy. Many tumors express foreign antigens that can potentially serve as targets for destruction by the immune system. Cancer vaccines generate a systemic tumor-specific immune response in a subject comprising both humoral and cellular components. The response is elicited by the subject's own immune system by administering the vaccine composition at a site remote from the tumor or at a site local to the tumor. The antibody or immune cell binds to the tumor antigen and lyses the tumor cells. However, there is still a need to increase the T cell response when vaccinating cancer patients. Thus, adoptive transfer of preactivated apoptosis-resistant tumor-specific T cells with high proliferative potential prior to or at the time of vaccination can enhance vaccine-mediated immune responses in vivo.

The composition according to the invention may be administered in combination with a therapeutic cancer vaccine. Non-limiting examples of such therapeutic cancer vaccines are ex vivo proliferated and tumor-bearing DCs, cytokine-producing tumor cells, DNA vaccines and vaccines using TLR-ligands in combination with tumor antigens.

The cells obtainable by the method of the invention may be administered directly to an organism, such as a human, to increase the proliferation and survival of antigen-specific T cells upon their activation. These cells, often with a pharmaceutically acceptable carrier, are generally administered by any route useful for introducing the cells into ultimate contact with mammalian blood or tissue cells.

Formulations and carriers suitable for parenteral administration, such as, for example, by intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, and intratumoral routes, include aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes of the formulation which render the formulation isotonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Intravenous administration is a preferred method of administering the TH1 cells and CTLs of the invention.

In the context of the present invention, the dose of TH1 cells and CTL administered to a patient should be sufficient to enhance the immune response of the patient. Thus, the cells are administered to the patient in an amount sufficient to elicit an effective immune response to the tumor antigen and/or to alleviate, reduce, cure or at least partially arrest the symptoms and/or complications of the disease. An amount sufficient to achieve the effect is defined as a "therapeutically effective amount". The dose is determined by the activity of the cells produced and the condition of the patient, as well as the weight or surface area of the patient to be treated. In determining the effective amount of cells to be administered in the treatment or prevention of a disease (e.g., cancer), a physician needs to assess the progression of the disease and the induction of an immune response against any relevant tumor antigen.

The present invention has several major advantages over prior art methods. The present invention provides high levels of tumor specific CD8 without the need for restimulation⁺T cells. Restimulation decreases the activity of the cell and brings it closer to apoptosis. Therefore, a method that efficiently expands tumor-specific T cells without the need for restimulation would be advantageous. Furthermore, tumor specific T cells can allow patients to recover in a shorter time, and therefore more cost effective, without the need to restimulate the cells. Furthermore, with the method according to the invention, there is no need to consume suppressor cells or to add exogenous growth factors (which is a very expensive process).

The invention is further illustrated by the following non-limiting examples.

Examples

Example 1

Materials and methods: allogeneic Sensitized Allogeneic Lymphocytes (ASALs) were generated in a standard one-way Mixed Lymphocyte Reaction (MLR) in tissue culture flasks by co-culturing gamma-irradiated PBMCs from healthy blood donors with non-irradiated PBMCs from allogeneic donors (relative to healthy donors) in a 1: 1 ratio in serum-free X-VIVO15 medium for 5-7 days. For proliferation of immature DCs, Peripheral Blood Mononuclear Cells (PBMCs) obtained from healthy blood donors were isolated on a density gradient (Lymphoprep, nychomed, oslo, norway). The isolated PBMCs were resuspended in AIM-V (Invitrogen, Persley, UK) medium at 2.5X 10⁶Each cell was plated in a 24-well plastic culture plate and allowed to adhere for 2 hours. Removing non-adherent cells, and adding the remaining adherent monocytes with recombinant human GM-CSF and IL-4 (R)&D Systems, Abingdon, UK; all 1,000U/mL) in AIM-V medium for 4-6 days. At the last 24 hours of incubation, the cells were incubated by adding IFN-. alpha.s (3,000U/mL), IFN-. gamma.s (1,000U/mL), TNF-. alpha.s (50ng/mL),IL-1 β (25ng/mL) (both from R&D Systems, Inc.) and p-I: C (Sigma-Aldrich, Inc.; 20 μ g/mL) of the medium induced maturation of immature DCs.

Mature DC populations all contained more than 70% CD83+ DCs as determined by FACS analysis.

After washing, the mature DCs were co-cultured with either unirradiated or gamma irradiated (25Grey) ASAL in X-VIVO15 medium for 24 hours and analyzed by FACS.

Sensitization of allogeneic lymphocytes was performed by primary unidirectional MLR in serum-free medium (X-VIVO 15) with gamma-irradiated PBMC as stimulating cells and unirradiated PBMC as responding cells. PE-bound anti-human CD70 was used for FACS studies.

Results: as shown in figure 1, ASALs significantly enhanced the expression of CD70 on mature monocyte-derived DCs that were autologous relative to irradiated PBMCs used to prime the ASALs.

As shown in figure 2, similarly, gamma-irradiated ASALs enhanced the expression of CD70 on mature monocyte-derived DC that were autologous relative to irradiated PBMCs used to prime the ASALs.

As shown in FIGS. 3, 4 and 5, ASAL co-cultured with mature DCs autologous to irradiated PBMC used to prime the ASAL induced massive production of IL-12, IFN- γ and IL-2.

Example 2

Materials and methods: ASALs were produced during conventional MLR7 days using irradiated allogeneic PBMC as a stimulus (see example 1). After harvesting and irradiation, ASAL ("MLR") or CD4 is removed⁺、CD8⁺Or CD56⁺The mixed population of ASALs (NK/NKT) was co-cultured with mature allogeneic monocyte-derived DC (autologous with respect to PBMCs used to prime the ASALs). After 24 hours, the co-cultured supernatants were collected and then assayed for IL-2, IL-2 and IFN- γ production.

Results: IL-2 production was found to be strictly CD 4-dependent (FIG. 6A), while IL-12 production (FIG. 6B) showed no ASAL-dependence at all, and IFN- γ production (FIG. 6C) showed partial dependence on co-cultured and allo-stimulated CD4 in the ASAL population⁺、CD8⁺And CD56⁺(NK/NKT)。

Example 3

Materials and methods: immature DCs were generated by plastic adhesion of monocytes. After addition of IL-4 and GM-CFS, both 1000U/mLMonocytes were cultured in DC for 7 days. Maturation of DCs was induced during the last 2 days of incubation by the addition of 50ng/mL TNF- α, 25ng/mL IL-1 β, 50ng/mLIFN- γ, 3000U/mL IFN- α and 20 μ g/mL poly I: C.

ASALs were produced in a one-way mixed lymphocyte reaction by co-culturing gamma-irradiated PBMC with unirradiated autologous PBMC against DC donors for 7 days at a ratio of 1: 1 in X-VIVO 15.

From the addition of 50ng/mL IL-15 (final concentration of 0.5X 10)⁶lymphocytes/mL) in autologous PBMC cultured for 7 days in X-VIVO15 CD8 was isolated by positive selection⁺T lymphocytes. PBMC were centrifuged and resuspended in PBS-0.5% BSA-2M EDTA to a final concentration of 1X 10⁷And 80 mu L. Mixing PBMC with CD8⁺The microbeads (Miltenyi Biotec Inc.) were incubated at 4 ℃ for 15 minutes, washed, resuspended and placed on a LS MACS column. Unlabeled cells were washed through and collected containing CD8⁺The effluent of lymphocytes. Resuspending isolated CD8 in prewarmed PBS-1% BSA⁺T lymphocytes to a concentration of 1X 10⁶mL, and stained with 10. mu.M CFSE (Molecular probes Invitrogen) at 37 ℃ for 10 minutes. The staining was stopped by adding 5ml ice-cold X-VIVO15 medium and incubated on ice for 5 min. Cells were washed 2 times in culture medium and resuspended to a final concentration of 1X 10⁶And/ml. In the ratio of 4 to4: 1 ratio of stained CD8⁺T lymphocytes were co-cultured with irradiated allogeneic sensitized allogeneic PBMC and mature autologous DCs for 4-7 days. After culture, lymphocytes were harvested and stained with CD3-APC-H7, CD8-PerCP, CD27-APC and annexin V. Determination of proliferating CD8 by flow cytometry⁺Percentage of T lymphocytes and expressed as percentage of total lymphocytes.

Results: as illustrated in fig. 7, the addition of irradiated "allo-assist" (═ ASAL) strongly increased CD8⁺Division of T cells (more cells with low fluorescence intensity). Thus, ASALs increased monocyte-derived DC-induced allogeneic CD8⁺The ability of T cells to proliferate in response.

Example 4

Materials and methods: immature DCs were generated by plastic adhesion of monocytes. After addition of IL-4 and GM-CFS, both 1000U/mLMonocytes were cultured in DC for 7 days. Maturation of DCs was induced during the last 2 days of incubation by the addition of 50ng/mL TNF- α, 25ng/mL IL-1 β, 50ng/mLIFN- γ, 3000U/mL IFN- α and 20 μ g/mL poly I: C. Washing of non-adherent cells, i.e. CD8⁺Lymphocytes, and at 0.5X 10⁶The final concentration was cultured in X-VIVO15 supplemented with 50ng/mLIL-15 for 7 days. Gamma-irradiated autologous PBMC were co-cultured with unirradiated allogeneic PBMC to the DC donor for 7 days in a 1: 1 ratio in X-VIVO15 to generate allogeneic sensitized allogeneic lymphocytes in a one-way Mixed Lymphocyte Reaction (MLR).

Mature DCs were harvested and loaded with 20. mu.g/mL HER-2 peptide (KIFGSLAFL) in X-VIVO15 for 1 hour at 37 ℃. Peptide-loaded mature DCs were used to induce autologous HER 2-specific cytotoxic T lymphocytes by co-culturing the DCs with irradiated MLR and non-adherent PBMC at a ratio of 1: 4. Cells in the presence or absence of 50U/mL IL-2 and 10ng/mL IL-7The culture was carried out in DC for 9 days. After culture, lymphocytes were harvested, washed, and HER-2-specific PE-binding pentamer (A)^*0201KIFGSLAFL) was incubated in the dark at room temperature for 10 minutes. Cells were washed and then stained with CD3-FITC and CD 8-APC. The percentage of HER-2 positive cytotoxic T lymphocytes was determined by flow cytometry and was assessed using CD8⁺The percentage of the total number of T lymphocytes is expressed.

Results: FIG. 8 shows the expansion of tumor-specific CTLs stimulated with autologous DCs, allogeneic lymphocytes of the invention, and with Her-2 peptide (A) and without HER-2 peptide B. Her2 is shown in the upper right part of each scatter plot⁺/CD8⁺Cells (0.4% for allogeneic primed PBMC without Her2 peptide-loaded DCs and 25.2% for Her2 peptide-loaded DCs).

It is very unusual to obtain this level of amplification after only one stimulation compared to the amplification of tumor-specific CTLs of the prior art. See, e.g., Ho et al (Journal of immunologicalcalemethods, 310(2006), 40-50), where two restimulations are required to obtain 18.8% tumor-specific CD8⁺And (4) expanding the cells. Similarly, Gritzapis et al (J.Immunol., 2008; 181; 146-⁺A cell.

Example 5

Materials and methods: see example 1 for materials and methods.

CD8 was isolated 6 days after co-culturing of DCs, irradiated ASAL (allogenic for DCs)⁺Lymphocytes (negative selection using antibody-coated magnetic beads), were subsequently re-stimulated with B cells (autologous to the DC used during primary stimulation) and stained for expression of CD27 and annexin-V. Subsequent analyses were performed with FACS.

Results: as shown in FIG. 9, CD8 was re-stimulated with B cells⁺When cells were used, the addition of ASALs during primary stimulation greatly increased the expression of CD 27.

Restimulation of CD8 with B cells⁺When cells were stimulated, addition of ASAL greatly reduced annexin-V expression during the primary stimulation period (see figure 10).

Example 6

Materials and methods: see materials and methods in example 5.

Sensitized and isolated CD8 before restimulation with B cells⁺Cells were pulsed with 3H-thymidine.

Results: as shown in figure 11, addition of ASAL during primary stimulation after restimulation strongly increased alloreactivity CD8⁺Cell proliferation response (measured by incorporation of 3H-thymidine cpm/min, day 3).

Example 7

Materials and methods: see example 5 for materials and methods.

In the process of mixing B cells with pre-activated CD8⁺After 2 days of cell co-culture, culture supernatants were collected and assayed by conventional ELISA (R)&D Systems company) was analyzed for IFN- γ production.

Results: FIG. 12 shows that addition of ASAL during primary stimulation after restimulation greatly increased the conversion of alloreactive CD8⁺Increased production of IFN- γ by the cell.

Although specific embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to limit the scope of the claims that follow. In particular, the inventors contemplate that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims.

Claims (16)

1. A method for ex vivo priming of antigen-specific T helper 1 cells or cytotoxic T cells suitable for administration to a tumour patient, the method comprising co-culturing target T cells, autologous monocyte-derived dendritic cells, autologous or allogeneic tumour material or tumour-associated proteins or peptides from the patient to be treated, and irradiated allogeneic lymphocytes primed against MHC class I and/or II antigens on antigen-presenting cells from the patient or against APC from an unrelated blood donor expressing at least one MHC class II antigen identical to the MHC class II antigen expressed on APC from the patient to be treated, wherein the tumour material or tumour-associated proteins or peptides are selected from the group consisting of killed tumour cells from the patient, allogeneic tumour cells of the same type as the tumour of the patient and known isolated and purified tumour proteins or peptides In the group consisting of.

2. The method of claim 1, wherein the sensitization is induced by a mixed lymphocyte reaction comprising culturing irradiated allogeneic antigen presenting cells with peripheral blood mononuclear cells from a healthy donor.

3. The method of claim 2, wherein the antigen presenting cells are selected from the group consisting of PBMCs and monocyte-derived dendritic cells.

4. The method according to claim 3, wherein the monocyte-derived dendritic cell is a dendritic cell having an MHC class II antigen that matches the patient HLA-DR antigen.

5. The method according to claim 1, wherein the monocyte-derived dendritic cells are obtained by first culturing monocytes in a composition comprising GM-CSF and IL-4 for 1-7 days to obtain immature dendritic cells and then adding a second composition capable of making the immature dendritic cells mature dendritic cells by culturing for at least 12 hours.

6. The method of claim 5, wherein the second composition comprises TNF α, IL-1 β, interferon γ, interferon α or β, a TLR3 ligand, and/or a TLR4 ligand.

7. The method of claim 6, wherein the TLR3 ligand is poly-I: C.

8. The method of claim 5, wherein the second composition comprises TNF α, interferon γ, a TLR3 ligand and/or TLR4 ligand, TLR7 and/or TLR8 agonist.

9. The method of claim 8, wherein the TLR3 ligand is poly-I: C and the TLR8 agonist is R848.

10. The method of claim 1, wherein said tumor-associated peptide is a peptide derived from the HER-2 protein, PSA protein, MART-1 protein, p53 protein, and/or survivin.

11. The method of claim 1, wherein the tumor material is a tumor protein loaded in mature dendritic cells transfected with mRNA encoding the tumor protein.

12. The method of claim 1, wherein the cells are cultured for 4-20 days.

13. The method of claim 1, wherein exogenous IL-2, IL-7, IL-15, anti-IL-4 and/or IL-21 is added to the cell culture.

14. The method according to claim 1, wherein the antigen-specific Th1 cells or CTLs are activated by culturing the cells with new monocyte-derived dendritic cells, newly sensitized allogeneic lymphocytes and optionally restimulating the cell culture by adding exogenous IL-2, IL-7, IL-15, anti-IL-4 and/or IL-21.

15. Antigen-specific TH1 cells and/or CTLs obtained according to the method of any one of claims 1-14.

16. Use of the antigen-specific TH1 cells and/or CTLs of claim 15 in the manufacture of a medicament for treating a tumor or for eliciting an anti-tumor immunological response in a human.