WO2021252986A1 - Tlr7/8-mediated tissue repair by regulatory t cells - Google Patents
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Abstract
Human regulatory T cells (hTregs) proliferation or amphiregulin production is induced by treatment with a TLR 7/8 agonist, and formulations of the treated hTreg cell may be introduced into persons in need thereof as ex vivo cell therapy to promote tissue repair.
Description
TLR7 /8-mediated tissue repair by regulatory T cells
[001] Introduction
[002] Following infection, inflammation, or injury, tissues (e.g., lung, muscle) sustain damage that can result in impaired function. A current prominent example is patients who suffer from severe COVID-19 infection, characterized by lung damage and resulting loss of blood oxygen saturation. Clinical recovery occurs not necessarily when the source of an injury is resolved, such as the clearance of virus, but rather when the tissue is fully repaired and able to function normally. Therefore, therapies that are able to boost repair of damaged tissue are highly desirable.
[003] Regulatory T cells ("Tregs"), an immune cell type generally associated with prevention of autoimmunity, have recently been shown to play an important role in repair of tissue damage. Tregs are recruited to the site of damage and produce a critical molecule called amphiregulin, which binds to its receptor and drives regeneration of the tissue. This mechanism has been solidly established in mice, and there has been significant interest in translational applications for humans. However, efforts were hindered because it was unknown which molecular signals cause Tregs to adopt this "repair" function characterized by production of amphiregulin. Studies targeting the most likely candidates all showed results that were mediocre, at best.
[004] Relevant Literature
[005] Human Tregs have been treated with TLR 7/8 agonists to reduce suppressive capacity by restraining glycolysis, e.g. Li et al, Cell Metabolism 29, 103-123, Jan 8, 2019; Caron et ak, J Immunol 2005; 175:1551-1557; Peng et ak, Science 309, 1380, Aug 26, 2005. What is unprecedented and inventive is treating hTregs of a person in need thereof with a TLR 7/8 agonist to promote amphiregulin expression, tissue repair, or proliferation of the hTregs.
[006] Summary of the Invention
[007] In an aspect the invention provides a method of inducing or increasing proliferation of, or production of amphiregulin by human regulatory T cells (hTregs), which comprises: (a) treating hTregs with a TLR 7/8 agonist under conditions wherein the treatment increases amphiregulin expression by, or proliferation of the hTreg cells, and (b) providing a formulation of the treated hTreg cells configured and suitable for introduction into a person in need thereof. [008] In an aspect the invention provides a method of introducing human regulatory T cells (hTregs) into a person in need thereof, as ex vivo cell therapy, which comprises: introducing into the person a formulation of hTregs cells made by a disclosed method of inducing or increasing proliferation of, or production of amphiregulin by hTregs, i.e. hTregs treated with a TLR 7/8
agonist under conditions wherein the treatment increased amphiregulin expression or proliferation of the hTreg cells.
[009] In embodiments:
[010] the hTregs comprise a T cell receptor (TCR) or chimeric antigen receptor (CAR), of predetermined antigen specificity;
[Oil] the method further comprising engineering the hTgregs to comprise a T cell receptor (TCR) or chimeric antigen receptor (CAR), of predetermined antigen specificity;
[012] introduction of the hTregs promotes tissue repair in the person;
[013] the method further comprises detecting a resultant increased amphiregulin expression by the hTregs;
[014] the method further comprises detecting a resultant increased proliferation of the Tregs; [015] the method further comprises detecting a resultant increased tissue repair;
[016] the method further comprises comprising the prior step of extracting the Tregs from the patient; and/or
[017] the agonist is selected from: compound 3 (N-4-butyl-6-methyl-5-(3-morpholinopropyl)- pyrimidine-2, 4-diamine; SM-360320, SM-276001; TMX101, TMX-202, TMX-302 and TMX- 306; SZU-101, CL264, CL307, CL347 (AdiFectin), CL413 (Adilipoline), CL572; GSK2245035, DSR-6434, DSR- 29133; Loxoribine, isatoribine; Selgantolimod, GS-9620; 52al, SCI (Pluripotin); VTX-2337 (Motolimod), VTX-1463, VTX-294, VTX763, VTX463; TL8-506; Imiquimod (R-837, Aldara), Resiquimod (R-848); 3M-001, 3M-002, 3M-003, 3M-052 (Telratolimod), Gardiquimod; CL075, and LHC165. Suitable TLR7/8 agonists are known in the art, e.g. Patintote, et al., European J. Med Chem 193 (2020) 112238.
[018] The invention encompasses all combinations of the particular embodiments recited herein, as if each combination had been laboriously recited.
[019] Brief Description of the Drawings
[020] Fig. 1. Stimulation of proliferation of human Tregs.
[021] Fig. 2. Stimulation of Amphiregulin production by human Tregs.
[022] Description of Particular Embodiments of the Invention
[023] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and/or and polypeptide sequences are understood to encompass opposite strands as well as alternative backbones described herein. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light
thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.
[024] We disclose toll-like receptor 7/8 mediated tissue repair by regulatory T cells. We demonstrate that signaling through Toll-like receptors (TLR) 7 and 8 strongly induces Tregs to divide and produce amphiregulin (Figure 1). This invention is highly unexpected - the field is not aware that Tregs express TLR7/8, and there has been no suggestion that these receptors can trigger a response to tissue damage. We disclose that 1) Tregs express TLR7, 2) TLR7 causes mouse Tregs to adopt the repair phenotype, 3) this pathway is required for proper recovery from lung damage, and 4) the same pathway is active and robust in human cells. These findings are all novel and nonob vious: no other groups have been successful in boosting the amphiregulin production of unaltered human Tregs, and nobody has suggested using TLR ligands to achieve this result.
[025] The invention provides a method for facilitating repair of damaged tissue by regulatory T cells. We have developed a way to enhance Treg production of amphiregulin, a key molecule involved in repair of damaged tissues such as lung and muscle, by stimulating these cells with ligands for TLR7 and/or TLR8. Regulatory T cells can be removed from a patient and treated with TLR7/8 ligands, or engineered to have greater sensitivity to TLR7/8 ligands, and then reimplanted, for the purpose of increasing the number of Tregs and also inducing their production of amphiregulin to facilitate repair of damaged tissue. This result can be achieved either by ex vivo cell therapy, or by direct treatment of patients. The invention can also be paired with extant CAR-Treg therapies.
[026] The applications for such treatments are wide-ranging, including treating patients who suffer acute lung damage from viral infections, or any type of tissue damage, including chronic obstructive pulmonary disease and muscle wasting diseases. The invention can also be used to expand conventional Treg cells for cell-based therapies designed to blunt autoimmune diseases or transplant rejection.
[027] Examples
[028] 1. Induction of Human Treg Proliferation and Amphiregulin Production [029] 1. Isolation of donor PBMCs followed by purification of CD4+ T cells.
[030] 2. Stain purified cells with a dye used to track proliferation (v450).
[031] 3. Culture for 3, 4 or 6 days under the following conditions: unstimulated control; lug/ml R848 (TLR7/8 ligand); 5uM CpG (TLR9 ligand).
[032] 4. All conditions receive lOOU/ml IL-2 for Treg maintenance.
[033] 5. At the specified timepoints, analyze samples by flow cytometry for: amphiregulin production; dilution of the proliferation dye; classic Treg markers (Foxp3, CD25, CD127).
[034] Human CD4+ T cells were isolated from donor blood and cultured in the presence of IL- 2, R848 (ligand for TLR7/8) and CpG (ligand for TLR9, which is not expressed by Tregs). Production of amphiregulin (Areg) was measured by intracellular cytokine staining followed by flow cytometry. Results demonstrate treatment of hTregs with a TLR 7/8 agonist effects increased amphiregulin expression by, and proliferation of the hTreg cells.
[035] Figs. 1 and 2 show stimulation of proliferation of human Tregs, and amphiregulin production by human Tregs: CD4+ T cells isolated from blood of 8 human donors; 6 days stimulation in vitro with the indicated ligand; division and amphiregulin production measured by flow cytometry; NR=non-responder.
[036] 2. Tissue Repair: Lung Infection Injury Repair Model
[037] An established murine influenza model was adapted to demonstrate efficacy of our TLR 7/8 treated Tregs to promote lung repair; for model details, see, e.g. Arpaia et al, Cell. 2015 Aug 27;162(5):1078-89. doi: 10.1016/j.cell.2015.08.021., A Distinct Function of Regulatory T Cells in Tissue Protection.
[038] Experimental conditions. Subject mice are infected with approximately 10-100 PFXJ of influenza. Tregs are isolated from donor mice and treated with TLR7/8 ligand for at least 1 day to allow for enhanced production of amphiregulin. 1 x 105 treated Tregs in lOOul PBS are then injected I.V., using an insulin syringe, into subject mice between days 2 and 4 post-infection. Blood oxygen saturation (%Sp02) of subject mice is monitored daily using pulse oximetry as a readout of lung function. In the repair phase, approximately 6-8 days post-infection, mice are sacrificed and lungs isolated to measure tissue damage by histology or immunofluorescence. In another cohort lungs are homogenized to measure cellular infiltration by flow cytometry as a proxy for resolution of lung damage.
[039] Results. Clinical examinations and histological analysis reveal an improved resolution of lung damage following I.V. injection of our TLR 7/8 treated Tregs.
[040] 3. Tissue Repair: Cornea Injury Repair Model
[041] An established murine comeal alkali-bum model was adapted to demonstrate efficacy of our TLR 7/8 treated Tregs to promote tissue repair; for model details, see, e.g. Yan et al, Invest Ophthalmol Vis Sci. 2020 Dec 1;61(14):22. doi: 10.1167/iovs.61.14.22., Subconjunctival Injection of Regulatory T Cells Potentiates Corneal Healing Via Orchestrating Inflammation and Tissue Repair After Acute Alkali Burn.
[042] Experimental conditions. TLR 7/8 treated Tregs are isolated and labeled with CFDA-SE. 1 x 105 Tregs/10 pL of PBS are injected subconjunctivally immediately after the corneal alkali bum, using a 33-gauge metal needle and a 50-pL syringe. The control group is treated with 10 pL of PBS. The injured cornea and ipsilateral conjunctiva are digested and analyzed for labeled Tregs using flow cytometry. Flow cytometry dot plots show frequencies of Treg cells stained with CFDA at different time points after injection.
[043] A 2-mm wound is made in the center of the cornea, and the healing process visualized with fluorescein staining to highlight the denuded area.
[044] Imaging: Images during the acute stage of comeal alkali burn injury (0-16 h) are recorded, and the percentage of corneal epithelial defective area in both groups at each time point analyzed. Ocular surface fluorescein staining images of mice with comeal alkali bum during the latter recovery and reconstmction stage of injury (days 1-16) are also recorded. The corneal epitheliopathy evaluated by the National Eye Institute score in both groups at each time point are analyzed. Optical coherence tomography (OCT) images show the comeal thickness at various time points. Bright-field comeal images show corneal opacity at various time points. Hematoxylin and eosin staining images show alkali-injured corneas at serial time points in Treg- treated and PBS-treated groups. Masson's Goldner (MG) staining images of normal and alkali- injured corneas are recorded.
[045] Results. Clinical examinations and histological analysis reveal an improved corneal restoration by treatment with subconjunctival injection of our TLR 7/8 treated Tregs.
Claims
1. A method of preparing human regulatory T cells (hTregs) for ex vivo cell therapy, which comprises:
(a) treating the hTregs with a Toll-like receptor (TLR) 7/8 agonist under conditions wherein the treatment increases amphiregulin expression by, and proliferation of the hTreg cells; and
(b) formulating the treated hTreg cells in a therapeutic formulation configured and suitable for introduction into a person in need thereof.
2. The method of claim 1 further comprising detecting a resultant increased amphiregulin expression by the hTregs, or a resultant increased proliferation of the Tregs.
3. The method of claim 1 further comprising engineering the hTregs to comprise a T cell receptor (TCR) or chimeric antigen receptor (CAR), of predetermined antigen specificity.
4. The method of claim 1 further comprising the prior step of extracting the Tregs from the person.
5. The method of claim 1, 2, 3 or 4 further comprising the step of introducing into the person the therapeutic formulation.
6. The method of claim 5 wherein the person is in need of tissue repair, and the introduction of the hTregs promotes tissue repair in the person.
7. The method of claim 6 wherein the tissue repair is lung infection injury repair.
8. The method of claim 6 wherein the tissue repair is cornea injury repair.
9. The method of claim 6 further comprising detecting a resultant increased tissue repair.
10. The method of claim 1 or wherein the hTregs comprise a T cell receptor (TCR) or chimeric antigen receptor (CAR), of predetermined antigen specificity.
11. The method of claim 5 or wherein the hTregs comprise a T cell receptor (TCR) or chimeric antigen receptor (CAR), of predetermined antigen specificity.
12. The method of claim 9 or wherein the hTregs comprise a T cell receptor (TCR) or chimeric antigen receptor (CAR), of predetermined antigen specificity.
13. The method of claim 1 wherein the agonist is selected from: compound 3 (N-4-butyl-6- methyl-5-(3-morpholinopropyl)-pyrimidine-2, 4-diamine; SM-360320, SM-276001; TMX101, TMX-202, TMX-302 and TMX-306; SZU-101, CL264, CL307, CL347 (AdiFectin), CL413 (Adilipoline), CL572; GSK2245035, DSR-6434, DSR- 29133; Loxoribine, isatoribine; Selgantolimod, GS-9620; 52al, SCI (Pluripotin); VTX-2337 (Motolimod), VTX-1463, VTX- 294, VTX763, VTX463; TL8-506; Imiquimod (R-837, Aldara), Resiquimod (R-848); 3M-001, 3M-002, 3M-003, 3M-052 (Telratolimod), Gardiquimod; CL075, and LHC165.
14. The method of claim 5 wherein the agonist is selected from: compound 3 (N-4-butyl-6- methyl-5-(3-morpholinopropyl)-pyrimidine-2, 4-diamine; SM-360320, SM-276001; TMX101, TMX-202, TMX-302 and TMX-306; SZU-101, CL264, CL307, CL347 (AdiFectin), CL413 (Adilipoline), CL572; GSK2245035, DSR-6434, DSR- 29133; Loxoribine, isatoribine; Selgantolimod, GS-9620; 52al, SCI (Pluripotin); VTX-2337 (Motolimod), VTX-1463, VTX- 294, VTX763, VTX463; TL8-506; Imiquimod (R-837, Aldara), Resiquimod (R-848); 3M-001, 3M-002, 3M-003, 3M-052 (Telratolimod), Gardiquimod; CL075, and LHC165.
15. The method of claim 9 wherein the agonist is selected from: compound 3 (N-4-butyl-6- methyl-5-(3-morpholinopropyl)-pyrimidine-2, 4-diamine; SM-360320, SM-276001; TMX101, TMX-202, TMX-302 and TMX-306; SZU-101, CL264, CL307, CL347 (AdiFectin), CL413 (Adilipoline), CL572; GSK2245035, DSR-6434, DSR- 29133; Loxoribine, isatoribine; Selgantolimod, GS-9620; 52al, SCI (Pluripotin); VTX-2337 (Motolimod), VTX-1463, VTX- 294, VTX763, VTX463; TL8-506; Imiquimod (R-837, Aldara), Resiquimod (R-848); 3M-001, 3M-002, 3M-003, 3M-052 (Telratolimod), Gardiquimod; CL075, and LHC165.
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| ARPAIA NICHOLAS, GREEN JESSE A., MOLTEDO BRUNO, ARVEY AARON, HEMMERS SASKIA, YUAN SHAOPENG, TREUTING PIPER M., RUDENSKY ALEXANDER : "A Distinct Function of Regulatory T Cells in Tissue Protection", CELL, vol. 162, no. 5, 27 August 2015 (2015-08-27), pages 1078 - 1089, XP055879564 * |
| CARON ET AL.: "Direct stimulation of human T cells via TLR5 and TLR7/8: flagellin and R-848 up- regulate proliferation and IFN-gamma production by memory CD 4+ T cells", JOURNAL OF IMMUNOLOGY, vol. 175, no. 3, 1 August 2005 (2005-08-01), pages 1551 - 1557, XP002712773, DOI: 10.4049/jimmunol.175.3.1551 * |
| QUNFANG ZHANG; WEIHUI LU; CHUN-LING LIANG; YUCHAO CHEN; HUAZHEN LIU; FEIFEI QIU; ZHENHUA DAI: "Chimeric Antigen Receptor (CAR) Treg: A Promising Approach to Inducing Immunological Tolecance", FRONTIERS IN IMMUNOLOGY, vol. 9, no. 2359, 12 October 2018 (2018-10-12), pages 1 - 8, XP055591405 * |
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