EP-4735012-A1 - METHODS FOR EXTRACORPOREAL DEVELOPMENT OF AUTOLOGOUS CAR-X CELLS FOR THE TREATMENT OF NON-MALIGNANT DISEASES

Abstract

The present disclosure relates to methods of treating autoimmune diseases and allergic disorders by generating chimeric antigen receptor (CAR) cells (e.g., T cells, NK cells, and/or myeloid cells) that target and promote clearance of, e.g., by killing, pathogenic cells. The methods described herein can be performed at the bedside in a subject-connected, closed-loop continuous-flow manner.

Inventors

• PERITT, DAVID
• DAS, Nripendra
• THILL, Melissa

Assignees

• Lupagen, Inc.

Dates

Publication Date

20260506

Application Date

20240627

Claims (1)

1. CLAIMS 1. A method of providing a subject with a population of payload-associated cell complexes (PACCs) for the treatment of a non-malignant disease, comprising: (i) providing a population of peripheral blood cells, e.g., a population of peripheral blood cells from the subject, wherein a peripheral blood cell in the population of peripheral blood cells comprises a binding target; (ii) extracorporeally contacting the population of peripheral blood cells with a payload comprising a chimeric antigen receptor (CAR) construct under conditions (e.g., time, temperature) sufficient for association of the payload with the cell comprising the binding target, wherein the conditions are not sufficient for entry of the payload into the cell with which it is associated, thus forming a population of PACCs; and (iii) introducing the population of PACCs into the subject, thereby providing subject with a population of PACCs for the treatment a non-malignant disease. 2. The method of any of claim 1, wherein the population comprises at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, 750, 1,000, 5,000, 7,500, 10,000, 25,000, 50,000, 75,000, 100,000, 250,000, 500,000, or more PACCs. 3. The method of claim 1, wherein the population comprises at least 100,000 or more PACCs. 4. The method of claim 1, wherein the population comprises at least 1 million or more PACCs. 5. The method of claim 1, wherein the population comprises at least 10 million or more PACCs. 6. The method of claim 1, wherein the population comprises at least 100 million or more PACCs. 7. The method of claim 1 , wherein the percentage of peripheral blood cells associated with the payload in the population is greater than 0.5%, 1%, 2%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the total cells in the population 8. The method of claim 1, wherein the percentage of peripheral blood cells associated with the payload in the population is greater than 50%, 60%, 70%, 80%, 90%, 95%, or more, or more of the total cells in the population. 9. The method of claim 1, wherein the percentage of peripheral blood cells associated with the payload in the population is between 30%-90% total cells, e.g., 50%-80% total cells in the population. 10. The method of claim 1, wherein the conditions sufficient for association of the payload with a peripheral blood cell within the population comprise: (i) contacting the peripheral blood cells with the payload for between about 0 to about 10 hours, e.g., about 1 hour to about 9 hours, about 2 hours to about 8 hours. 11. The method of claim 10, wherein contacting the peripheral blood cells with the payload is between about 5 minutes and 30 minutes. 12. The method of claim 10, wherein contacting the peripheral blood cells with the pay load is between about 30 minutes and 1 hour. 13. The method of claim 10, wherein contacting the peripheral blood cells with the pay load is between about 1 hour and 1.5 hours. 14. The method of claim 1, wherein the PACCs are formulated in or on a delivery vehicle. 15. The method of claim 14, wherein the delivery vehicle comprises a lipid nanoparticle, viral vector, vesicle, or a liposome in which the payload is disposed. 16. The method of claim 14, wherein the concentration of the delivery vehicle is higher than the concentration of peripheral blood cells in the sample. 17. The method of claim 14, wherein the concentration of the delivery vehicle is lower than the concentration of peripheral blood cells in the sample. 18. The method of claim 14, wherein the concentration of the delivery vehicle is optimized for binding to peripheral blood cells in the subject sample. 19. The method of claim 1, wherein the binding of the pay load to the peripheral blood cells occurs at a selected temperature 0 to 40 degrees Celsius (e.g., 4 °C, 37 °C). 20. The method of claim 1, wherein the pay load is disposed on the surface of a peripheral blood cell within the PACC. 21. The method of claim 14, wherein the delivery vehicle is a viral vector. 22. The method of claim 21, wherein the viral vector is a lentiviral vector or an adeno- associated viral (AAV) vector. 23. The method of claim 14, wherein the delivery vehicle is a lipid nanoparticle. 24. The method of claim 23, wherein the lipid nanoparticle is a cationic lipid nanoparticle. 25. The method of claim 14, wherein the delivery vehicle is a vesicle. 26. The method of claim 25, wherein the vesicle is an extracellular vesicle, exo some, a nanovesicle, or a microvesicle. 27. The method of claim 14, wherein the delivery vehicle is disposed on the surface of a cell within the PACC. 28. The method of claim 14, wherein the delivery vehicle is targeted to a surface molecule. 29. The method of claim 14, wherein the delivery vehicle comprises a targeting moiety to a surface molecule. 30. The method of any one of claims 28 or 29, wherein the surface molecule is a cell-specific surface protein. 31. The method of claim 30, wherein the targeting moiety is an antibody. 32. The method of claim 1, wherein the method comprises creating an autologous cell capable of reducing the level of a pathogenic cell (e.g., an endogenous pathogenic cell) in the subject. 33. The method of claim 1, wherein the population of cells is selected from monocytes, macrophages, neutrophils, basophils, eosinophils, stem cells, mast cells, and dendritic cells. 34. The method of claim 1, wherein the population of cells is selected from B cells, T cells, effector or regulatory T cells, hematopoietic stem cells (HSCs), natural killer cells, NK T cells, g/d T cells, and plasma cells. 35. The method of claim 1, wherein the pay load comprises a nucleic acid, a peptide, a polypeptide, or a small molecule. 36. The method of claim 35, wherein the nucleic acid comprises DNA or RNA. 37. The method of claim 1, wherein the association of the pay load to the cell is covalent or non-covalent. 38. The method of claim 1, wherein the subject has or is diagnosed with having a disease. 39. The method of claim 1, wherein the method further comprises administering to the subject an additional agent, e.g., an immune-stimulatory agent. 40. The method of claim 1, wherein the introducing in (iii) is carried out by a subject- connected closed-loop device. 41. The method of claim 1, wherein between 1-10% of the cells in the population comprise the binding target. 42. The method of claim 1, further comprising: a) connecting a parenteral inlet to the subject, wherein the parenteral inlet is adapted to parenterally receive blood from the subject; b) permitting the blood, or a fraction thereof, from the subject to pass through the parenteral inlet to an extracorporeal cell binding (ECCB) module configured to allow extracorporeal formation of a PACC; c) maintaining conditions in the ECCB module such that cells from the subject’s blood and a payload form a PACC; and d) delivering the PACC to the subject via a parenteral outlet adapted to parenterally administer PACC to the subject. 43. The method of claim 42, wherein the parental inlet, the ECCB module, and the parental outlet are in fluid connection. 44. The method of claim 42, wherein each of the steps (a)-(d) occurs in a closed-loop system. 45. The method of claim 42, wherein: i) a subject cell is taken from the subject, ii) the subject cell is contacted with a payload to form a PACC, and iii) the PACC introduced into the subject, and i-iii occur in less than 0.5, 1, 2, 4, 6, or 8 hours. 46. The method of claim 42, wherein the peripheral blood cells may comprise T cells, B cells, natural killer cells, and/or myeloid cells. 47. The method of claim 1, wherein the non-malignant disease is an allergic, autoimmune, or fibrotic disease. 48. The method of claim 1, wherein the non-malignant disease comprises a disease selected from systemic lupus erythematosus (SLE), myasthenia gravis (MG), pemphigus vulgaris (PV), coeliac disease, Crohn’s disease, Grave’s disease, Hashimoto’s thyroiditis, multiple sclerosis, rheumatoid arthritis, Sjogren’s disease, ulcerative colitis, vasculitis, allergic asthma, atopic dermatitis (e.g., eczema), atopy, chronic rhinosinusitis, chronic sinusitis, eosinophil-associated diseases, eosinophilic esophagitis (atopic and non-atopic), hay fever, severe eosinophilic asthma (SEA), and fibrosis. 50. The method of claim 1, wherein the non-malignant disease is selected from systemic lupus erythematosus (SLE), an eosinophil-associated disease, and fibrosis. 51. The method of claim 1 , wherein the subject has a disease or disorder, wherein the disease or disorder is a non-malignant disease. 52. The method of claim 51, wherein the non-malignant disease is an allergic, autoimmune, or fibrotic disease. 53. The method of claim 52, wherein the non-malignant disease is selected from systemic lupus erythematosus, myasthenia gravis, pemphigus vulgaris, allergic asthma, atopic dermatitis, atopy, chronic rhinosinusitis, chronic sinusitis, eosinophil-associated diseases, and fibrosis. 54. The method of claim 1, wherein the subject is treatment naive, wherein treatment naive may comprise one or more of the following: (i) the subject is currently undergoing treatment for the non-malignant disease, but is not currently being treated according to the claimed method; (ii) the subject is currently being treated for one or more symptoms of the non-malignant disease, but is not being treated for the underlying mechanism of the disease; (iii) the subject is being treated for one or more symptoms of a non-malignant disease, but has not been diagnosed with the disease; (iv) the subject has previously received treatment for the non-malignant disease, but the treatment was insufficient to alleviate the symptom(s) it was administered to treat and/or the treatment did not inhibit or reduce the progression of the disease; (v) the subject has previously received treatment for the non-malignant disease, and the treatment was successful, but the symptom(s) has/have since recurred and/or the disease state has since progressed; or (vi) the subject has never been treated for the non-malignant disease or for any symptoms of the non-malignant disease. 55. The method of claim 1, wherein the subject has been previously treated with one or more therapeutics directed to non-malignant diseases. 56. A method of forming in a subject a cell that is transformed or transduced with a payload, comprising: introducing a population of extracorporeally formed PACCs into the subject under conditions sufficient for transformation or transfection of the cell of the PACC with the payload of the PACC in the subject; and allowing the transformation or transfection; thereby forming in a subject with a cell transformed or transfected with a payload. 57. A subject-connected closed- loop device for use with any one of the methods of any one of the preceding claims. 58. A viral vector for use with any one of the methods of any one of the preceding claims, wherein the viral vector may be selected from an adenoviral vector, an adenoviral-associated vector, a lentiviral vector, or a virus-like particle. 59. The viral vector of claim 58, wherein the viral vector is an adenoviral vector or an adenoviral-associated vector. 60. The viral vector of claim 58, wherein the viral vector is a lentiviral vector. 61. The viral vector of any one of claims 58-60, wherein the viral vector expresses a glycoprotein. 62. The viral vector of claim 61, wherein the glycoprotein is a Cocal glycoprotein.

Description

METHODS FOR EXTRACORPOREAL DEVELOPMENT OF AUTOLOGOUS CAR-X CELLS FOR THE TREATMENT OF NON-MALIGNANT DISEASES CLAIM OF PRIORITY The present application claims priority to U.S. Provisional Patent Application No. 63/523,434 filed on June 27, 2023. The entire contents of the foregoing application are incorporated herein by reference in their entirety. BACKGROUND Methods for introducing a genetic payload into a desired cell type derived from a subject are useful in a number of biomedical fields. The creation of autologous cells directed to reduce levels of endogenous pathogenic cells in subjects has been fairly well established in both animals and subjects particularly using Chimeric Antigen Receptor (CAR) gene modifications. While a variety of ex vivo methods have been developed for the introduction of payloads such as nucleic acids into peripheral blood derived mononuclear cells (PBMCs), the cost, complexity and safety of these methods limit broad access and use in all but the most serious diseases. Recent efforts to address these issues involve introduction of the payload, for example, a gene cargo, and creation of therapeutic cells in the subject (gene therapy). However, these methods must contend with many challenges including delivery of the payload, biodistribution, off target impact, accurate dosing, immunogenicity to the delivery vehicle, and limitations in the ability to re-dose. As such, there is a need for new devices and methods to improve the delivery of payloads to peripheral blood derived cells, particularly for non-malignant conditions with diminished banners relative to malignant conditions including lower costs, the ability to re-treat, the use of non-integrating gene editing or introduction, and limited or no lymphodepletion. SUMMARY The present disclosure features methods of providing a subject with a population of payload-associated cell complexes (PACCs) to treat a non-malignant disease in a subject, as well as related compositions thereof. In a first aspect, provided herein is a method of providing a subject with a population of payload-associated cell complexes (PACCs) to treat a non-malignant disease in the subject, comprising (i) providing a population of peripheral blood cells, e.g., a population of peripheral blood cells from the subject, wherein a peripheral blood cell in the population of peripheral blood cells includes a binding target; and (ii) cxtracorporcally contacting the population of peripheral blood cells with a payload including a chimeric antigen receptor (CAR) construct under conditions (e.g., time, temperature) sufficient for association of the payload with the cell including the binding target, wherein the conditions are not sufficient for entry of the payload into the cell with which it is associated, thus forming a population of PACCs; and (iii) introducing the population of PACCs into the subject, thereby providing subject with a population of PACCs for the treatment of a non-malignant disease. In an embodiment, the method comprises the creation of autologous cells useful for reducing the levels of endogenous pathogenic cells in the subject. In an embodiment, the non-malignant disease comprises an allergic, autoimmune, or fibrotic disease. In some embodiments of the foregoing aspect, the population includes at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, 750, 1,000, 5,000, 7,500, 10,000, 25,000, 50,000, 75,000, 100,000, 250,000, 500,000, or more PACCs. In some embodiments, the population includes at least 100,000 or more PACCs. In some embodiments, the population includes at least 1 million or more PACCs. In some embodiments, the population includes at least 10 million or more PACCs. In some embodiments, the population includes at least 100 million or more PACCs. In some embodiments of the foregoing aspect, the percentage of peripheral blood cells associated with the payload in the population is greater than 0.5%, 1%, 2%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the total cells in the population. In some embodiments, the percentage of peripheral blood cells associated with the payload in the population is greater than 50%, 60%, 70%, 80%, 90%, 95%, or more, or more of the total cells in the population. In some embodiments, the percentage of peripheral blood cells associated with the payload in the population is between 30%-90% total cells, e.g., 50%-80% total cells in the population. In some embodiments of the foregoing aspect, the population of cells is selected from monocytes, macrophages, neutrophils, basophils, eosinophils, stem cells, mast cells, and dendritic cells. In some embodiments, the population of cells is selected from B cells, T cells, effector or regulatory T cells, hematopoietic stem cells (HSCs), natural killer cells, NK T cells, g/d T cells, and plasma cells. In some embodiments, the peripheral blood cells may include T cells, B cells, natural killer cells, and/or myeloid cells. In some embodiments of the foregoing asp