Redirected, genetically-engineered t regulatory cells and their use in suppression of autoimmune and inflammatory disease

Abstract

A redirected Treg cell is endowed with specificity toward a selected target antigen or ligand. The cell contains a chimeric receptor polypeptide that is expressed in a single, continuous chain, with an extracellular recognition region displayed on the surface of the cell, a transmembrane region and an intracellular signaling region. The extracellular recognition region is specific for the selected target antigen or ligand. The intracellular signaling region includes a combination of T-cell signaling polypeptide moieties, which combination, upon binding of the extracellular recognition region to the selected target antigen or ligand, triggers activation of the redirected Treg cells to cause suppression of T-cell mediated immunity. Such redirected Treg cells may be used to suppress undesired activity of T effector cells thereby mediating an immune or inflammatory response. They are particularly useful in treating T effector cell-mediated diseases, such as inflammatory bowel disease, transplant rejection and GVH disease.

Description

BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]The invention in the field of immunology and medicine relates to genetic modification of T regulatory cells with chimeric receptors with antibody-type specificity, and the use of such cells to suppress the action of T effector cells and treat any of a number of diseases and conditions in which such suppression is beneficial, primarily autoimmune and inflammatory diseases such as inflammatory bowel diseases (IBD), organ-specific autoimmune diseases, allograft rejection and Graft-vs. Host disease.[0003]2. Description of the Background Art[0004]Regulatory T-Cells (Tregs)[0005]One line of research that led to discovery of Treg cells was the observation that thymectomy of mice of certain susceptible strains on postnatal day 3 results in a spectrum of organ-specific autoimmune effects, which were preventable by “reconstitution” of these animals early in life with normal adult lymphocytes (Asano M et al., J Exp Med 1996; 184:3...

AI Technical Summary

Benefits of technology

[0048]The present therapeutic approach has several unique advantages. In contrast to other immunotherapeutic models, it involves T-bodies redirected with a CR, and preferably a TpCR, that combines antibody / antigen or ligand / receptor recognition with stimulatory and costimulatory motifs. Thus, T-bodies can be fully activated in a way that is not restricted by the MHC and is independent of a requirement for costimulation. The second advantage of the present invention stems from the fact that, although Treg activation is antigen-dependent, the suppressive action of these cells is antigen-, TCR-, and MHC-independent. By exploiting this property, one can construct a chimeric receptor that is specific for one or more tissue-associated antigens rather than requiring specificity for an unknown number of yet undefined autoimmune disease-specific antigens. Expression of such chimeric receptors in Tregs redirects these cells and their activation to the appropriate target tissue (in a preferred embodiment, the colon) so that they are activated in an antigen-specific manner, where their potent suppressive effects take place without a need for further recognition of disease-associated-antigens (the “bystander effect”). By using specifically-activated Tregs, many fewer cells are required to treat autoimmune inflammatory conditions; such as IBD, or allograft-associated reactions in patients than would have been possible prior to this invention when much higher numbers of non-specific Tregs would have been needed.

[0049]The present inventors have constructed strains of transgenic (Tg) mice whose T-cells and natural killer (NK) cells express an antigen-specific TpCR. For exemplification of the invention, the inventors selected the trinitrophenyl (TNP) hapten as the specific target of the TpCRs. TNP specific Tregs isolated from these Tg mice suppressed TNP-specific effector T-cells in vitro and in vivo and were able to suppress trinitrobenzenesulfonic acid (TNBS)-induced colitis in mice. In this embodiment, the target antigen for Treg and the pathogenic antigen—the hapten TNP—are the same. In another example, TNP-specific Tregs suppressed oxazolone-induced colitis in mice in which a low dose of TNP was introduced into the colon together with the oxazolone challenge. However, the TNP-specific Tregs had no effect on the oxazolone-induced colitis in the absence of TNP introduction. This establishes the “bystander” effect of the present invention, i.e., that the target antigen need not be the pathogenic antigen, as long as the redirected Tregs are activated in the vicinity of the pathogenic or undesired immune response.

[0050]One distinct advantage of the present invention is that it provides cells and methods that permit antigen-specific activation and antigen-nonspecific action of Treg cells used to suppress effector T cell responses (and treat consequent pathologies) in a way that does not require identity between the ligand (e.g., the antigen) recognized by the TpCR (e.g., by its target recognition portion) and the ligand / antigen that plays a pathogenic role in the disease process. Thus, the antigen that is pathogenic does not have to be recognized by the T effector cells being suppressed, and, indeed, may be unrelated to the disease or condition being treated. Thus, the invention exploits the “bystander” effect. As long as the Treg is in the correct vicinity where T effector cells are located and mediating their undesired effects, the redirected Tregs of the present invention can be triggered or activated at that location to release of suppressive cytokines (e.g., IL-10 and TGF-β), that will result in suppression of any “bystander” effector T-cells, and by this mechanism, quell an ongoing inflammatory / autoimmune response.

[0051]The unique characteristics of the Tg system used in the present examples enables evaluation of the suppressive effect of antigen-specific Tregs in IBD both in vitro and in vivo. Different means are used to induce Tregs, allowing those of skill in the art to select the optimal method for generating efficient numbers of antigen-specific redirected Tregs for a desired antigen or disease / condition. According to this invention, redirected human Tregs constitute an effective cell-based therapeutic modality for IBD or ulcerative colitis and, more broadly, for any T effector cell-mediated disease or condition.

[0052]To overcome the scarcity of antigen-specific Tregs, the present invention includes methods to induce these cells using cytokines (e.g. TGF-β) or by expression of transgenes (e.g. encoding the Foxp3 transcription factor) that will, together with the TpCR, allow antigen-specific Treg expansion.

[0053]According to the present invention, human Treg cells, derived from either the subject with the autoimmune / inflammatory disease or condition to be treated, or from an HLA-matched healthy donor (or a universal cell that is not recognized by the recipient's immune system), are endowed with antigen / ligand-specificity, by transduction with the antigen / ligand-specific TpCR as disclosed herein. Alternatively, the cells being endowed with antigen-specificity are the entire T-cell population and the nucleic acid construct including the sequence encoding the TpCR further includes a Foxp3 transgene that is present as an independently transcribed cistron. In another such alternative, a Foxp3 transgene is separately transfected into the T-cell population, to turn the T-cells into Treg cells. Examples provided below include studies using murine colonoscopy, in vivo imaging and immunofluorescence, and provide the basis for a novel cell-based therapeutic modality for IBD, and, by extension, for other pathologic and undesired immune responses mediated by antigen specific T effector cells.

Problems solved by technology

The lack of success in vaccinating tumor patients with a large variety of tumor-associated antigen vaccines were thus frustrating (Rosenberg S A et al., Nat Med 2004; 10:909-15).

Passive vaccination with anti-tumor antibodies (Abs), tumor infiltrating lymphocytes (TILs), or lymphokine activated killer (LAK) cells showed very limited effectiveness (primarily for vascular tumors).

More recent successes with the adoptive transfer of TILs into stage 1V melanoma patients ((Dudley M E, et al., J Immunother 2001; 24:363-73; Dudley M E, et al., Science 2002; 298:850-4) were still burdened by the fact that the results were limited to a few cancers and to individuals from whom it is possible to derive specific TILs.

Patients with IBD are also at increased risk for the development of intestinal cancer.

Unfortunately, new therapies for IBD are few, and both diagnosis and treatment have been hampered by a lack of detailed knowledge of the etiology.

It has therefore been difficult to design effective therapies to block induction of disease.

In the case of IBD this development has not been uniform.

Normal mucosal homeostasis was disrupted by cytokine imbalance, abrogation of oral tolerance, breach of epithelial barriers, and loss of immunoregulatory cells.

The progression from the acute to the chronic phase of IBD has not been well characterized in animal models and cannot be easily evaluated in patients.

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