Biologically relevant orthogonal cytokine / receptor pairs

Abstract

This invention provides engineered orthogonal cytokine receptor / ligand pairs and methods for using them.

Description

[Technical Field] This invention relates to biologically relevant orthogonal cytokine / receptor pairs. [Previous Technology] Manipulating cells, particularly immune cells, to differentiate, develop specialized functions, and increase their numbers has significant clinical benefits. Many protein factors, including, in particular, cytokines and chemokines, are known to influence these activities. However, these signaling molecules also have multiple effects on cells not intended as targets, thus necessitating methods for selectively activating signal transduction within target cell populations. In particular, engineering T cells for controlled behavior is beneficial. For example, in adoptive immunotherapy, T cells are isolated from the blood, processed in vitro, and then reinjected into the patient. T cells have been engineered for therapeutic applications, such as recognizing and killing cancer cells, intracellular pathogens, and participating in autoimmune processes. A key challenge in cell-based therapies is engineering adoptive cells into cells that block endogenous signaling pathways, do not affect non-targeted endogenous cells, and possess the desired behaviors (such as activation and expansion) that can be controlled once administered to the patient. This is particularly relevant to T-cell engineering due to the significant influence of developmental plasticity and environmental factors on the fate, function, and localization of T cells. The ability to manipulate the binding of proteins to modified ligands and their responses to modified ligands in a manner independent of orthogonal influence from native proteins or ligands constitutes a significant challenge in protein engineering. To date, numerous synthetic ligand-heterologous receptor pairs orthogonal to similar native interactions have been created. Proteins used in this work include nuclear hormone receptors and G protein-coupled receptors. Despite extensive work on engineering these receptors activated by synthetic small-molecule ligands, engineering biologically relevant protein pairs remains a major challenge. [Summary of the Invention] This invention provides engineered orthogonal cytokine receptor / ligand pairs and methods of using them. The engineered (orthogonal) cytokines specifically bind to their corresponding engineered (orthogonal) receptors. Upon binding, the orthogonal receptor activates signal transduction via natural cellular elements to provide a mimicking natural response, but exhibits the cell-specific bioactivity of the engineered orthogonal receptor. The orthogonal receptor exhibits significantly reduced binding to its endogenous counterpart (including the natural counterpart of the orthogonal cytokine), while the orthogonal cytokines exhibit significantly reduced binding to any endogenous receptor (including the natural counterpart of the orthogonal receptor). In some embodiments, the affinity of the orthogonal cytokine for the orthogonal receptor is comparable to the affinity of the natural cytokine for the natural receptor. A method for engineering orthogonal cytokine receptor pairs may include the following steps: (a) engineering to introduce amino acid alterations in the native receptor to disrupt binding to the native cytokine; (b) generating multiple cytokine analogs having selective amino acid alterations at the contact residues of the native cytokine used for receptor binding; (c) selecting cytokine heterohomosomes that bind to a heterologous receptor; (d) discarding heterologous cytokines that bind significantly to the native receptor, or performing steps (c) and (d); (e) selecting receptor heterohomosomes that bind to a heterologous cytokine; and (f) discarding heterologous receptors that bind to the native cytokine. In a preferred embodiment, structural knowledge of the cytokine / receptor complex is used to select amino acid positions for position-directed mutagenesis or error-prone mutagenesis. Conveniently, this selection process may use a yeast display system, but other display and selection methods may also be used. In some embodiments, engineered cells are provided, wherein the cells have been modified by introducing the orthogonal receptor of the present invention. Any cell may be used for this purpose. In some embodiments, the cells are T cells, including, but not limited to, naïve CD8+ T cells, cytotoxic CD8+ T cells, naïve CD4+ T cells, helper T cells such as TH1, TH2, TH9, TH11, TH22, TFH; regulatory T cells such as TR1, native TReg, induced TReg; memory T cells such as central memory T cells, effector memory T cells, NKT cells, γδT cells, and engineered variants of these T cells, including CAR-T cells. In other embodiments, the engineered cells are stem cells, such as hematopoietic stem cells, NK cells, macrophages, or dendritic cells. In some embodiments, the cell line is genetically modified in an in vitro procedure before being transferred into a subject. The engineered cells can be provided in a unit dose for use in therapy and can be allogeneic, autologous, or other types of recipients. In some embodiments, a vector is provided comprising a polynucleotide coding sequence encoding an orthogonal receptor, wherein the coding sequence is operatively linked to an active promoter in a desired cell. Various vectors are known in the art and can be used for this purpose, such as viral vectors, plasmid vectors, and small circular vectors, which can be integrated into the genome of a target cell or maintained in a cell-free form. The receptor-coding vector can be provided in a kit with a vector encoding an orthogonal cytokine that binds to and activates the receptor. In some embodiments, the coding sequence of the orthogonal cytokine is operatively linked to a high-performance promoter and can be optimized for production. In other embodiments, a kit is provided in which the vector encoding the orthogonal receptor and a purified composition of the orthogonal cytokine are provided, for example, in a unit dose (e.g., a pre-filled syringe) for administration to a patient. In some other embodiments, a kit is provided in which the carrier system encoding the orthogonal receptor is provided together with the carrier encoding the orthogonal cytokine, so that the orthogonal receptor can be expressed in the cell and also express the orthogonal cytokine expected to be secreted by the same cell, thereby enabling autologous secretion of orthogonal cytokine-receptor signal transduction. In some embodiments, a treatment method is provided that includes introducing an engineered cell population into a desired receptor, wherein the cell population has been modified by introducing a sequence encoding the orthogonal receptor of the present invention. The cell population may be engineered in vitro and is typically autologous or allogeneic relative to the receptor. In some embodiments, after administration of the engineered cells, the introduced cell population comes into contact in vivo with a homologous orthogonal cytokine. An advantage of the present invention is the absence of cross-reactivity between the orthogonal cytokine and the natural receptor.

Implementation Method

Claims

1. An engineered human IL-2 polypeptide, wherein (i) its binding to natural human CD122 is significantly reduced; and (ii) it contains at least one amino acid substitution at residues T51 and R81, which is an amino acid other than that of the natural protein, or contains amino acid substitution M23A; and (iii) it contains amino acid substitutions at each of E15, H16, L19, and D20.

2. The engineered human IL-2 polypeptide as claimed in claim 1, wherein the polypeptide comprises one or more amino acid substitutions selected from the following: [E15D, E15T, E15A, E15S], [H16N, H16Q], [L19V, L19I, L19A], [D20L, D20M], [Q22S, Q22T, Q22E, Q22K, Q22E], [M23A, M23W, M23H, M23Y, M23F, M23Q, M23Y], [G27K, G27S], [R81D, R81Y], [N88E, N88Q], [T51I].

3. The engineered human IL-2 polypeptide as claimed in claim 1 or 2, wherein the polypeptide comprises amino acid substitutions E15S; H16Q; L19V; and D20L.

4. The engineered human IL-2 polypeptide of any one of claims 1 to 3 further comprises an amino acid-substituted Q22K.

5. The engineered human IL-2 polypeptide, as described in any of claims 1 to 4, contains amino acid-substituted T51I.

6. The engineered human IL-2 polypeptide of any one of claims 1 to 5, comprising amino acid substitutions of R81D or R81Y.

7. The engineered human IL-2 polypeptide as claimed in claim 1, which contains a group of amino acid substitutions [E15D, H16N, L19V, D20L, Q22T, M23A].

8. The engineered human IL-2 polypeptide as claimed in claim 1, which contains a group of amino acid substitutions [E15D, H16N, L19V, D20L, Q22K, M23A].

9. The engineered human IL-2 polypeptide as claimed in claim 1, which contains a group of amino acid substitutions [E15S, H16Q, L19V, D20L, M23Q, R81D, T51I].

10. The engineered human IL-2 polypeptide as claimed in claim 1, which contains a group of amino acid substitutions [E15S, H16Q, L19V, D20L, M23Q, R81Y].

11. An engineered human IL-2 polypeptide as claimed in any of claims 1 to 10, wherein the polypeptide binds to and activates orthogonal human CD122 protein.

12. The engineered human IL-2 polypeptide of claim 11, wherein the orthogonal human CD122 protein is modified at one or more residues selected from the following: R41, R42, Q70, K71, T73, T74, V75, S132, H133, Y134, F135, E136, Q214.

13. The engineered human IL-2 polypeptide of claim 12, wherein the orthogonal human CD122 protein is modified at H133 and Y134.

14. The engineered human IL-2 polypeptide of claim 13, wherein the orthogonal human CD122 protein comprises amino acid substitutions for H133D and Y134F.

15. A system for selectively activating a receptor in a cell, the system comprising: (a) an orthogonal human CD122 receptor comprising amino acid substitutions at H133 and Y134; and (b) an engineered human IL-2 polypeptide as claimed in any one of claims 1 to 10.

16. The system of claim 15, wherein the orthogonal acceptor system is expressed by mammalian cells.

17. The system of claim 16, wherein the cell is an immune cell or a stem cell.

18. The system of claim 17, wherein the immune cell is a T cell.

19. The system of claim 18, wherein the T cell is a CAR-T cell.

20. A pharmaceutical composition comprising an engineered human IL-2 polypeptide as described in any one of claims 1 to 10; and a pharmaceutically acceptable excipient.

21. A nucleic acid encoding an engineered human IL-2 polypeptide as described in any one of claims 1 to 10 of the patent claims.

22. A presentation vector comprising the nucleic acid as claimed in claim 21.

23. A cell that has been genetically engineered to contain a vector as described in claim 22.

24. A method of treating an individual, the method comprising introducing immune effector cells expressing an orthogonal human CD122 receptor, the orthogonal human CD122 receptor comprising amino acid substitutions at H133 and Y134, and selectively activating the cells by contacting an engineered human IL-2 polypeptide as claimed in any one of claims 1 to 10.

25. The method of claim 24, wherein the immune effector cell is a T cell.

26. The method of claim 25, wherein the T cell is a CAR-T cell.

27. The method of any one of claims 24 to 26, wherein the individual receives cancer treatment.

28. The method of any one of claims 24 to 26, wherein the individual receives treatment for an autoimmune disease.

29. The method of any one of claims 24 to 26, wherein the individual receives treatment for an infection.

30. A kit comprising the system as described in claim 15.