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EP-3452062-B1 - INTRACELLULAR DELIVERY OF BIOMOLECULES TO INDUCE TOLERANCE

EP3452062B1EP 3452062 B1EP3452062 B1EP 3452062B1EP-3452062-B1

Inventors

  • LOUGHHEAD, Scott
  • GILBERT, Jonathan, B.
  • BERNSTEIN, HOWARD
  • SHAREI, Armon, R.
  • MOORE, Finola

Dates

Publication Date
20260506
Application Date
20170503

Claims (14)

  1. A method for delivering an antigen into a tolerogenic immune cell or an immunosuppressive immune cell, the method comprising passing a cell suspension comprising the tolerogenic immune cell or the immunosuppressive immune cell through a constriction, wherein said constriction deforms the tolerogenic immune cell or the immunosuppressive immune cell, causing a perturbation of the cell such that the antigen can enter the cell, and contacting said cell suspension with the antigen, wherein the immune cell is a B cell or a T cell, wherein the antigen is: (i) a polypeptide antigen; (ii) a disease-associated antigen; (iii) a foreign antigen; (iv) a self-antigen; (v) a therapeutic agent; (vi) an allograft transplantation antigen; (vii) a non-protein antigen; (viii) an antigen associated with a virus; or (ix) an adenovirus, adeno-associated virus, baculovirus, herpes virus, or retrovirus, wherein an immune response is suppressed by at least about 10% and wherein the suppressed immune response comprises decreased production and/or secretion of one or more inflammatory cytokines, wherein the tolerogenic immune cell or the immunosuppressive immune cell comprises intracellularly a tolerogenic factor comprising a nucleic acid encoding an arginase-1 (ARG1), a nitric-oxide synthase 2 (NOS2), an indoleamine 2,3- dioxygenase (IDO), an IL-4, an IL-10, an IL-13, a transforming growth factor-beta (TGF-β), an IL-35, or an IFN-α, and wherein the tolerogenic immune cell or the immunosuppressive immune cell induces an immunological unresponsiveness or decreased responsiveness to challenge with the antigen.
  2. The method of claim 1, wherein the tolerogenic immune cell or the immunosuppressive immune cell has not been contacted with an adjuvant.
  3. The method of claim 2, wherein the adjuvant is selected from the group consisting of TLR3 and RLR ligands, TLR4 ligands, TLR5 ligands, TLR7/8 ligands, TLR9 ligands, NOD2 ligands, alum, water-in-oil emulsions, rhIL-2, anti-CD40, CD40L, IL-12, and cyclic dinucleotides.
  4. The method of claim 1 or 2, wherein the tolerogenic immune cell or the immunosuppressive immune cell comprises a reduced ability to provide one or more costimulatory signals as compared to a non-tolerogenic precursor of the tolerogenic immune cell or a non-immunosuppressive precursor of the immunosuppressive immune cell.
  5. The method of claim 4, wherein the one or more costimulatory signals are mediated by a molecule selected from the group consisting of CD40, CD80, CD86, CD54, CD83, CD79, and ICOS ligand.
  6. The method of claim 1, 2 or 4, wherein the tolerogenic immune cell or the immunosuppressive immune cell comprises a reduced ability to provide one or more inflammatory signals as compared to a non-tolerogenic precursor of the tolerogenic immune cell or a non-immunosuppressive precursor of the immunosuppressive immune cell.
  7. The method of claim 6, wherein the one or more inflammatory signals are mediated by a molecule selected from the group consisting of interleukin-1 (IL-1), IL-12, IL-18, tumor necrosis factor (TNF), interferon gamma (IFN-gamma), granulocyte-macrophage colony stimulating factor (GM-CSF), NF-κB, an interferon regulatory factor (IRF), and a molecule associated with the JAK-STAT signaling pathway.
  8. The method of any one of claims 1, 2 or 4, wherein the antigen is presented by the tolerogenic immune cell or the immunosuppressive immune cell.
  9. The method of any one of claims 1 to 8, wherein the cell suspension is contacted with the antigen before, concurrently, or after passing through the constriction.
  10. The method of any one of claims 1 to 9, wherein the immune response is suppressed by at least about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 90%, or about 100%.
  11. The method of any one of claims 1 to 10, wherein the one or more inflammatory cytokines are selected from the group consisting of interleukin-1 (IL-1), IL-12, and IL-18, tumor necrosis factor (TNF), interferon gamma (IFN-gamma), and granulocyte-macrophage colony stimulating factor (GM-CSF).
  12. The method of any one of claims 1 to 11, wherein the suppressed immune response comprises a decreased T cell response, an enhanced Treg response, a decreased B cell response, decreased cytokine production, a decreased autoimmune response or a decreased allergic response.
  13. The method of claim 12, wherein the decreased T cell response comprises decreased T cell activation, decreased T cell survival, decreased T cell proliferation or decreased T cell functionality and wherein the decreased B cell response comprises decreased antibody production.
  14. The method of any one of claims 1-13, wherein the method is repeated at least 1, 2, 3, 4, 5, or 6 times.

Description

FIELD OF THE INVENTION The present disclosure relates generally to methods for suppressing an immune response or inducing tolerance by delivering an antigen into a tolerogenic cell by passing a cell suspension through a cell-deforming constriction. BACKGROUND Undesired immune responses contribute to autoimmunity, transplant rejection, allergy, and anti-drug responses. Autoimmunity develops when an organism mounts an anti-self response, usually as a result of a dysregulated immune response against self-antigens. Autoimmune diseases include, for example, type I diabetes, systemic lupus erythematosus, rheumatoid arthritis, autoimmune hemolytic anemia, and multiple sclerosis. Pathogenic immune responses after transplantation of a donor organ in a receiving organism can lead to rejection of the transplant and decreased patient survival. In addition, unwanted immune responses against food and environmental antigens drive allergic diseases such as asthma, food allergy, and atopic dermatitis. Therefore, approaches to establish immunological tolerance to an antigen are a focus of intense therapeutic development. Current intracellular delivery methods are not effective at modulating cell phenotype or function in order to induce antigen-specific tolerance. Thus, there is an unmet need for intracellular delivery techniques that can load antigen and tolerogenic factors into the cytoplasm of cells and drive a powerful immunosuppression response for the treatment of pathogenic immune responses underlying autoimmune diseases and transplant rejection. References that describe methods of using microfluidic constrictions to deliver compounds to cells include WO2013059343, WO2015023982, WO2016070136, WO2016077761, and PCT/US2016/13113. WO 2016/070136 A1 describes a method and device for preferentially delivering a compound such as an antigen to the cytosol of an immune cell by passing a cell suspension comprising the target immune cell through a microfluidic device and contacting the suspension with the compound(s) or payload to be delivered. WO 2017/041050 A1 describes methods and devices for delivering a compound into a cell by passing a cell suspension through a surface containing pores, wherein the pores deform the cell thereby causing a perturbation of the cell such that the compound enters the cell, wherein the cell suspension is contacted with the compound. Stewart MP et al. ("In vitro and ex vivo strategies for intracellular delivery", NATURE, vol. 538, no. 7624, 12 October 2016 (2016-10-12), pages 183-192) describes in vitro and ex vivo intracellular delivery approaches with a focus on membrane-disruption-based delivery methods. WO 2013/059343 A1 describes a microfluidic system for causing perturbations in a cell membrane that includes a microfluidic channel defining a lumen and a cell-deforming constriction. WO 03/020039 A1 describes methods for inducing immune tolerance to one or more specific antigens in a host mammal by engineering white blood cells, in vitro, to express an antigen which is not native to the host mammal. Steinman RM et al. ("Tolerogenic dendritic cells", ANNUAL REVIEW OF IMMUNOLOGY, ANNUAL REVIEWS INC, US, vol. 21, 1 January 2003 (2003-01-01), pages 685-711) describes the role of natural dendritic cells (DCs) in T cell tolerance, covering aspects such as T cell deletion and control of suppressor and regulatory T cells. Rutella S et al. ("Tolerogenic dendritic cells: cytokine modulation comes of age", BLOOD, vol. 108, no. 5, 1 September 2006 (2006-09-01), pages 1435-1440) describes natural tolerogenic dendritic cells and teaches growth factors that modulate DC maturation and favor differentiation of tolerogenic DCs. WO 02/067863 A2 describes a method of altering the reactivity of a patient's own defense cells by treating antigen presenting cells (APCs) from the patient so as to make them tolerogenic to specific antigen, and describes incubating APCs with an inhibitory cytokine or an antigen to generate tolerogenic APCs. US 2008/311140 A1 describes genetically modified dendritic cells expressing at least two immunosuppressive molecules which have the ability to induce tolerance. Sim WJ et al. ("Metabolism Is Central to Tolerogenic Dendritic Cell Function", MEDIATORS OF INFLAMMATION., vol. 2016, 1 January 2016 (2016-01-01), pages 1-10) describes natural immune tolerance of DCs and teaches the role of metabolic reprogramming in making DCs immunogenic or tolerogenic. BRIEF SUMMARY OF THE INVENTION The present invention provides a method for delivering an antigen into a tolerogenic immune cell or an immunosuppressive immune cell, the method comprising passing a cell suspension comprising the tolerogenic immune cell or the immunosuppressive immune cell through a constriction, wherein said constriction deforms the tolerogenic immune cell or the immunosuppressive immune cell, causing a perturbation of the cell such that the antigen can enter the cell, and contacting said cell suspension with the antigen, w