Biologically related orthogonal cytokine / receptor pairs

Abstract

To provide an altered orthogonal cytokine receptor / ligand pair, and a method of using the pair.SOLUTION: Provided is a system that selectively activates a receptor in a cell, where the system includes (a) an orthogonal receptor which does not bind to an unmodified ligand thereof, and (b) an orthogonal cytokine (i) which does not bind to an unmodified receptor thereof, and (ii) binds to the orthogonal receptor to activate it. Further, also provided is a method of treating a disorder by using an altered cell population.SELECTED DRAWING: Figure 1

Description

[Background technology]

[0001] There is significant clinical interest in the manipulation of cells, specifically immune cells, to differentiate them, induce specialized functions, and increase their number. Numerous protein factors that influence such activity are known in this field, particularly cytokines and chemokines. However, because these signaling molecules also have multifaceted effects on cells other than the target, methods that selectively activate signaling within the target cell population are desirable. Particular interest lies in modifying T cells to exhibit controlled behavior. For example, in adoptive immunotherapy, T cells are isolated from blood, treated ex vivo, and then reinjected into the patient's body. These T cells are modified for therapeutic purposes such as identifying and killing cancer cells, intracellular pathogens, and autoimmune-related cells.

[0002] A key challenge in cell-based therapies is to introduce desired behaviors, such as activation and proliferation, into adoptive transplant cells that are protected from endogenous signaling pathways after administration to the patient, do not affect non-target endogenous cells, and are controllable. This is particularly relevant to T cell engineering due to the flexibility of development and the significant role that environmental factors play in determining T cell fate, function, and localization.

[0003] The ability to manipulate proteins to bind to and respond to modified ligands orthogonally, without being affected by undenatured proteins or ligands, is a major challenge in protein engineering. To date, many synthetic ligand-ortholog receptor pairs have been created that are orthogonal to similar native interactions. Among the proteins used in this work are nuclear hormone receptors and G protein-coupled receptors. While considerable effort has been spent creating receptors activated by synthetic small molecule ligands, creating bio-relevant protein pairs remains a challenge. [Overview of the project]

[0004] This invention provides modified orthogonal cytokine receptor / ligand pairs and methods for using them. The modified (orthogonal) cytokine specifically binds to the modified (orthogonal) receptor of its counterpart. Upon binding, the orthogonal receptor activates signaling, which is transmitted through undenatured cellular elements, resulting in biological activity that mimics a native response, but is specific to modified cells expressing the orthogonal receptor. The orthogonal receptor does not bind to endogenous counterpart cytokines, including the undenatured counterpart of the orthogonal cytokine, and the orthogonal cytokine does not bind to any endogenous receptor, including the undenatured counterpart of the orthogonal receptor. In some embodiments, the affinity of the orthogonal cytokine to the orthogonal receptor is comparable to the affinity of the undenatured cytokine to the undenatured receptor.

[0005] Methods for constructing orthogonal cytokine-receptor pairs may include the steps of (a) introducing amino acid modifications to the undenatured receptor to inhibit binding to the undenatured cytokine, (b) introducing amino acid modifications to the contact residue of the undenatured cytokine that binds to the receptor, (c) selecting an ortholog cytokine that binds to the orthologous receptor, (d) discarding an orthologous cytokine that binds to the undenatured receptor, or, as an alternative to steps (c) and (d), (e) selecting an orthologous receptor that binds to the orthologous cytokine, and (f) discarding an orthologous receptor that binds to the undenatured cytokine. In preferred embodiments, site-specific or error-prone mutagenesis amino acid positions are selected using knowledge of the structure of the cytokine / receptor complex. A yeast presentation system can be conveniently used for this selection method, but other presentation and selection methods are also possible. It is for use.

[0006] In some embodiments, modified cells are provided, which are modified by the introduction of an orthologous receptor of the present invention. Any cell can be used for this purpose. In some embodiments, the cells are T cells, including but not limited to naive CD8 ,

[0007] , FH , H , H , H , , Reg , R , Reg , T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, helper T cells, such as T H 1, T H 2, T H 9, T H ​​​​​​​​​​​​​​​​In some embodiments, a vector is provided containing a coding sequence encoding an orthologous receptor, the coding sequence functionally linked to a promoter that is active in the desired cell. Various vectors are known in the art and can be used for this purpose. For example, viral vectors, plasmid vectors, and small oval vectors are available, and these vectors can be incorporated into the target cell genome or maintained as episomes. The receptor-encoding vector may also be provided as a kit together with a vector encoding an orthologous cytokine that binds to and activates the receptor. In some embodiments, the orthologous cytokine coding sequence may be functionally linked to a high-expression promoter to optimize production. In other embodiments, a kit is provided in which the orthologous receptor-encoding vector is provided together with a purified composition of the orthologous cytokine and packaged, for example, in unit doses for administration to a patient. In several further embodiments, a kit is provided in which the orthologous receptor-encoding vector is provided together with a vector encoding an orthologous cytokine, enabling the expression of the orthologous receptor in the cell, and also the expression of the orthologous cytokine intended to be secreted by this same cell, thus enabling autocrine orthologous cytokine-receptor signaling.

[0008] In some embodiments, a therapeutic method is provided, which includes introducing a modified cell population into a recipient requiring the method, the cell population being modified by introducing a sequence encoding the orthologous receptor of the present invention. This cell population can be modified ex vivo and is usually homogeneous or autologous to the recipient. In some embodiments, after administration of the modified cells, the introduced cell population comes into contact in vivo with a congeneral orthologous cytokine. One advantage of the present invention is that there is no cross-reactivity between the orthologous cytokine and the undenatured receptor.

[0009] The present invention can be best understood when read in conjunction with the following detailed description and the accompanying drawings. It is emphasized that the various features of the drawings are not to scale in accordance with convention. Rather, the dimensions of the various features are enlarged or reduced as appropriate for clarity. The drawings include the following figures.

Brief Description of the Drawings

[0010] <关于用于控制T细胞增殖的正交性IL-2 / IL-2受体对的图。 <00关于制备正交性IL-2 / IL-2Rβ对的工作流程的图。<关于正交性小鼠IL-2Rβ变体序列的图。<关于显示抑制wt mIL-2结合的mIL-2Rβ H134D Y135F突变的图。<关于制备正交性IL-2 / IL-2Rβ对的工作流程的图。<关于表征的正交性小鼠IL-2变体序列的图。<图显示正交性(ortho)IL-2变体以与野生型IL-2和IL-2Rβ的相互作用同等或更高的亲和力与正交性(ortho)IL-2Rβ结合。<图显示正交性IL-2变体对野生型CD25阳性和CD25阴性脾细胞表现出钝化的活性(磷酸化STAT5)。<关于正交性IL-2R表达小鼠CTLL-2 T细胞生成的图。<图显示第一组正交性IL-2变体对正交性(ortho)T细胞具有选择性。<图显示正交性IL-2变体在正交性IL-2Rβ表达的CTLL-2细胞中诱导选择性STAT5磷酸化。 ​​​​​​​​​​​​​​​​​​​​​ [Figure 12] This is a diagram of primary lymph node-derived T cells (H134D Y135F) modified to express ortho-IL-2Rβ. [Figure 13] This figure shows that ortho-IL-2 variants induce selective STAT5 phosphorylation in ortho-IL-2Rβ expressing primary mouse T cells. [Figure 14] This figure shows that ortho-IL-2 variants induce selective cell proliferation of ortho-IL-2Rβ-expressing CTLL-2 cells compared to wild-type T cells. [Figure 15] This figure shows the alignment of mouse and human reference IL-2Rβ / IL-2 sequences. [Figure 16] This figure shows the yeast evolution of orthogonal human IL-2 pairs. (A) FACS analysis shows that yeast-presented wild-type human IL-2 binds to wild-type (blue histogram) but not to the ortho (red histogram) human IL-2Rβ H133D Y134F mutant tetramer. (B) A library of human IL-2 mutants (approximately 18 mutants) with randomized IL-2 residues predicted to be near or adjacent to the human IL-2Rβ HY mutant was presented on the yeast surface. By repeatedly selecting both positive (vs. ortho hIL-2Rβ) and negative (vs. wild-type hIL-2Rβ) mutants, yeast-presented human IL-2 mutants that bind to ortho (red histogram) but not to the wild-type (blue histogram) human IL-2Rβ tetramer were obtained. (C, D) Next, the ortho hIL-2 mutants were isolated from the yeast library and sequenced. We identified a consensus set of mutations that show convergence of ortho-hIL-2 sequences capable of binding to ortho-hIL-2Rβ. [Figure 17]This figure demonstrates the selective proliferation or increased viability of orthogonal IL-2Rb-expressing T cells in mice using an in vivo mouse model. Donor cells were isolated from the spleen of wild-type C57BL / 6J mice expressing CD45.2, activated ex vivo with CD3 / CD28, transduced with a retrovirus encoding orthogonal IL-2Rb-IRES-YFP, grown in 100 IU / mL mIL-2 for 2 days, and purified using a mouse CD8 T cell isolation kit (Miltenyi). An approximately 1:1 mixture of wild-type (CD45.2-positive, YFP-negative) and orthogonal IL-2Rb-expressing T cells (CD45.2-positive, YFP-positive) were adopted into recipient BL6.Rag2- / -xIL2rg- / -CD45.1 mice by post-orbital injection. PBS, wild-type mIL-2 (150,000 IU / mouse), or ortho-IL-2 clone 1G12 / 149 (1,000,000 IU / mouse) were administered via IP infusion every 24 hours for 5 consecutive days, starting immediately after T cell transplantation (day 0) until day 4. Mice were sacrificed on days 5 and 7, and the total number of donor T cells in the mice's blood and spleen was quantified by flow cytometry. [Figure 18] This figure shows the gating strategy used to quantify donor T cell proliferation in recipient mice. Single-cell suspensions were prepared from mouse blood and spleen and stained with CD45.2 Pacific Blue at 4C for 1 hour to identify donor T cells. Immediately before flow cytometry, cells were incubated diluted with propidium iodide (PI) at a ratio of 1:2000 to remove live / dead cells. Cells were gated based on forward and lateral scattering (SSC-A v FSC-A), and the total number of singlets (FSC-A v FSC-H), live cells (PI-negative), and wild-type T cells (CD45.2-positive, YFP-negative) and orthogonal T cells (CD45.2-positive, YFP-positive) was quantified by FACS. **p<0.01, ***p<0.001, ****p<0.0001, determined by one-way ANOVA using Prism. [Figure 19]This figure shows that ortho-IL-2 clone 1G12 / 149 selectively promotes the proliferation of orthogonal T cells in mice, while inhibiting the proliferation of wild-type T cells. The number of wild-type and orthogonal T cells in blood (103 cells / uL) and spleen (total number of cells per spleen) was quantified by flow cytometry as explained in Figure 18. The ratio of orthogonal T cells to wild-type T cells was calculated by dividing the total number of orthogonal T cells in blood and spleen by the total number of wild-type T cells. A ratio greater than 1 indicates selective proliferation of orthogonal T cells, which was obtained with ortho-IL-2 clone 1G12 / 149. The total number of viable cells in blood (left) and spleen (right) on day 5 (top) and day 7 (bottom) was quantified by flow cytometry. Treatment with wild-type IL-2 resulted in proliferation of both wild-type and ortho-T cells compared to the PBS control. However, treatment with ortho-IL-2 clone 1G12 / 149 selectively proliferated ortho-T cells, but its activity against wild-type T cells was limited. [Figure 20] This figure shows that orthogonal IL-2 has selective activity against orthogonal IL-2Rβ T cells. (A) FACS analysis of primary spleen-derived mouse T cells isolated from IL-2 KO NOD mice and transduced via virus to express ortho-IL-2Rβ, which can be confirmed by IRES-YFP reporter and surface staining of IL-2Rβ. The T cells also retain expression of wild-type IL-2Rβ. (B) Ortho-IL-2 induces selective STAT5 phosphorylation in ortho-IL-2Rβ-expressing T cells, but its activity is blunted or absent in wild-type T cells. [Figure 21]This figure shows that orthogonal IL-2 selectively proliferates orthogonal IL-2Rβ T cells in vitro. (A) FACS analysis of primary spleen-derived mouse T cells transduced by virus to express ortho-IL-2Rβ, confirmed by IRES-YFP. A mixture of transduced and untransduced T cells was cultured for 5 days in various concentrations of wild-type, ortho-IL-2 clone 1G12, or 3A10 and analyzed by FACS. IL-2 proliferated both wild-type and ortho-T cells, but when cultured in ortho-IL-2 3A10, only ortho-T cells proliferated. Ortho-IL-2 1G12 selectively proliferated ortho-T cells, but its activity against wild-type T cells was significantly reduced. The FACS plot shows the correspondence to culture in 100 nM IL-2, 64 pM ortho-IL-2 1G12, and 10 uM ortho-IL-2 3A10. (B) Proliferative dose-response of wild-type and ortho-IL-2 clones 1G12 and 3A10 to wild-type and ortho-IL-2 after 5 days of culture with gradually increasing cytokine concentrations. IL-2 promotes the proliferation of both wild-type and ortho-IL-2 cells with equivalent efficacy, ortho-IL-2 1G12 selectively promotes the proliferation of ortho-IL-2 cells, and ortho-IL-2 3A10 specifically promotes the proliferation of ortho-IL-2 cells. [Modes for carrying out the invention]

[0011] To facilitate understanding of this disclosure, certain terms and phrases are defined below and throughout this specification. The definitions provided herein are not limiting and should be read in terms of what a person skilled in the art would know at the time of the invention.

[0012] definition Before describing the methods and compositions described herein, it should be understood that the present invention is not limited to the specific methods and compositions described and is therefore naturally subject to change. Furthermore, the terminology used herein is for the sole purpose of describing specific embodiments and is not intended to be limiting, as the scope of the present invention is defined solely by the appended claims.

[0013] When a range of values ​​is given, each value between the upper and lower limits of that range should be understood to be specifically disclosed to the first decimal place of the lower limit, unless the context otherwise explicitly states otherwise. Each smaller range between any stated or intervening value within a given range and any other stated or intervening value within that range is included in the present invention. The upper and lower limits of these smaller ranges may or may not be included in the range independently, and each range that includes one or both (upper and lower limits) or does not include either is also included in the present invention, except for the upper and lower limits specifically excluded in the stated range. If the stated range includes one or both (upper and lower limits), the range excluding those included upper and lower limits is also included in the present invention.

[0014] Unless otherwise defined, all scientific and technical terms herein have the same meaning as those commonly understood by those skilled in the art in the industry to which this invention pertains. Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the present invention, but some possible preferred methods and materials are described below. All publications referenced herein are incorporated herein by reference in connection with the references to those publications to disclose and describe methods and / or materials. In the event of any conflict, this disclosure shall be understood to prevail over any disclosures of the referenced publications.

[0015] In this specification and the attached claims, the singular forms "a," "an," and "the" are also plural unless the context otherwise explicitly states. Therefore, for example, a reference to "a cell" includes multiple such cells, and a reference to "the peptide" includes one or more peptides and their equivalents, such as polypeptides known to those skilled in the art.

[0016] The publications described herein are listed only because they were disclosed prior to the filing date of this application. Nothing in this specification should be construed as acknowledging that the present invention cannot precede such publications by prior art. Furthermore, the publication dates listed may differ from the actual publication dates and may need to be verified on a case-by-case basis.

[0017] Cytokine receptor-ligand pairs include, but are not limited to, the following receptors:

[0018] [Table 1-1]

[0019] [Table 1-2]

[0020] An "ortholog," or "orthologous cytokine / receptor pair," refers to a pair of genetically modified proteins that have been modified by amino acid changes to (a) not bind to undenatured cytokines or their homologous receptors, and (b) specifically bind to a modified (orthogonal) ligand or receptor of their counterpart. When bound, the orthogonal receptor activates signaling, which is transmitted through undenatured cellular elements, resulting in biological activity that mimics the native response, but it is specific to the modified cells expressing the orthogonal receptor. The orthogonal receptor does not bind to endogenous counterpart cytokines, including the undenatured counterpart of the orthogonal cytokine, and the orthogonal cytokine does not bind to any endogenous receptor, including the undenatured counterpart of the orthogonal receptor. In some embodiments, the affinity of orthogonal cytokines to orthogonal receptors is comparable to the affinity of undenatured cytokines to undenatured receptors, for example, having an affinity of at least about 1%, at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100% of the affinity of an undenatured cytokine-receptor pair, and may be even higher, for example, having an affinity of 2, 3, 4, 5, or 10 times or more of the affinity of an undenatured cytokine to an undenatured receptor.

[0021] In this specification, “not binding” or “unable to bind” refers to the absence of detectable binding, i.e., non-significant binding, i.e., a binding affinity significantly lower than that of the native ligand. Affinity can be determined by competitive binding experiments, which measure the binding of the receptor to a single concentration of labeled ligand in the presence of various concentrations of unlabeled ligand. Generally, the concentrations of unlabeled ligand differ by at least sixfold. IC50 can be determined by competitive binding experiments. In this specification, “IC50” refers to the concentration of unlabeled ligand required to inhibit the binding of the receptor to the labeled ligand by 50%. IC50 is an indicator of ligand-receptor binding affinity. Low IC50 indicates high affinity, and high IC50 indicates low affinity.

[0022] Interleukin-2 (IL-2) is activated CD4 + IL-2 is a pluripotent cytokine primarily produced by T cells and plays a crucial role in the production of a normal immune response. IL-2 promotes the proliferation and increase of activated T lymphocytes, enhances B cell proliferation, and activates monocytes and natural killer cells. Due to these activities, IL-2 has been tested and approved as a cancer treatment (aldesleukin, proleukin®). Human IL-2 is synthesized in eukaryotic cells as a 153-amino acid precursor polypeptide, from which 20 amino acids are removed to produce mature, secretory IL-2.

[0023] In this specification, "IL-2" refers to undenatured, or wild-type, IL-2. Mature human IL-2 arises as a 133-amino acid sequence (lacking a signal peptide consisting of an additional 20 amino acids at the N-terminus), as described in Fujita, et al., PNAS USA, 80, 7437-7441 (1983). The amino acid sequence of human IL-2 can be confirmed in Genbank under contract locator NP_000577.2. The reference sequences of human and mouse IL-2 and IL-2Rβ are shown in Figure 15.

[0024] IL-2 supports T lymphocyte survival and differentiation by initiating cellular signaling pathways through interaction with the IL-2 receptor (IL-2R). IL-2 is clinically utilized in the treatment of numerous human diseases, including cancer and autoimmune diseases, and as an adjuvant in adoptive T cell therapy to promote the survival of transplanted T cells. However, IL-2 may also have parallel effects by activating untargeted cell types. To direct IL-2 activity to specific T cell subsets, the present invention provides a modified orthogonal IL-2 and IL-2 receptor pair. Orthogonal IL-2 replicates wild-type IL-2 activity by inducing potent STAT5 phosphorylation and in vitro proliferation of T cells modified to express orthogonal IL-2R beta. Orthogonal IL-2 exhibits limited or no activity, respectively, against wild-type CD25-positive or CD25-negative mouse T cells cultured ex vivo. These studies demonstrate that remodeling cytokine receptor interfaces to create interactions not found in nature is an effective strategy for targeting T cell subsets with indiscriminate cytokine activity, thereby allowing for precise control of T cell function through genetic engineering.

[0025] In addition to IL-2, IL-15 and IL-7 also regulate lymphatic homeostasis and are also used as adjuvants to enhance adoptive T-cell therapy. IL-2 and IL-15 share the same IL-2R-beta chain. Orthogonal IL-15 may be selected for the same orthogonal IL-2R-beta used for orthogonalization of IL-2. IL-7 uses a separate IL-7R-alpha chain as its orthogonalization target.

[0026] Orthogonal IL-2 can extend the half-lives of IgG's Fc domain, albumin, or other molecules by fusing them with methods known in this industry, such as pegylation and glycosylation. Fc fusion can also provide alternative Fc receptor-mediated properties in vivo. The "Fc region" may be a naturally occurring polypeptide or a synthetic polypeptide homologous to the C-terminal domain of IgG, resulting from papain digestion of IgG. The molecular weight of IgG Fc is approximately 50 kDa. The orthologous IL-2 polypeptide may contain the entire Fc region or a smaller portion that retains the ability to extend the cyclic half-life of the chimeric polypeptide in which it is part. Furthermore, the full-length or fragmentary Fc region may be a variant of the wild-type molecule. In other words, it may contain mutations that may or may not affect the function of the polypeptide, and as will be further discussed below, native activity is not always necessary or desirable.

[0027] In other embodiments, the orthologous polypeptide may include a polypeptide that functions as an antigen tag, such as a FLAG sequence. The FLAG sequence is recognized by a biotinylated highly specific anti-FLAG antibody, as described herein (see also Blanar et al., Science 256:1014, 1992 and LeClair et al., Proc. Natl. Acad. Sci. USA 89:8145, 1992). In some embodiments, this chimeric polypeptide further includes a C-terminal c-myc epitope tag.

[0028] As described above, the orthologous proteins of the present invention can exist as part of a chimeric polypeptide. In addition to, or instead of, the nucleic acid molecules of the present invention may include sequences encoding a “marker” or “reporter.” Examples of marker or reporter genes include β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo1, G418r), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacz (encoding β-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT). As with many standard techniques related to the implementation of the present invention, those skilled in the art will know of further useful reagents, such as further sequences that can perform the function of a marker or reporter.

[0029] Orthogonal cytokines and receptors may also include conserved modifications and substitutions at other cytokine locations (e.g., locations other than those involved in orthogonal modification). Such conserved substitutions are described in Dayhoff's The Atlas of Protein Sequence and Structure 5 (1978) and Argos' EMBO J., 8:779-785 (1989). For example, amino acids belonging to one of the following groups represent conserved modifications: Group I: ala, pro, gly, gin, asn, ser, thr; Group II: cys, ser, tyr, thr; Group III: val, ile, leu, met, ala, phe; Group IV: lys, arg, his; Group V: phe, tyr, trp, his; and Group VI: asp, glu.

[0030] The term "T cell" refers to mammalian immune effector cells that can be characterized by the expression of CD3 and / or T cell antigen receptors and can be modified to express orthologous cytokine receptors. In some embodiments, T cells express naive CD8+ T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, helper T cells, for example T H 1. T H 2, T H 9, T H 11, T H 22, T FH Regulatory T cells, for example, T R 1, Natural T Reg , inducible T Reg , memory T cells, for example, central memory T cells Cells are selected from cell cells, effector memory T cells, NKT cells, and γδT cells.

[0031] In some embodiments, T cells are brought into contact with orthologous IL-2 in vivo, i.e., modified T cells are transplanted into a recipient, and an effective amount of orthologous IL-2 is introduced. -2 is injected into the recipient and can come into contact with T cells and their specific environment, such as lymph nodes. In other embodiments, this contact is performed in vitro.

[0032] T cells collected from a subject can be separated from a cell mixture by techniques that enrich the desired cells. A suitable solution can be used for dispersion or suspension. Such solutions are generally equilibrium salt solutions, such as physiological saline, PBS, or Hank's equilibrium saline, combined with a low concentration of acceptable buffer (approximately 5–25 mM) and supplemented as appropriate with fetal bovine serum or other natural factors. Convenient buffers include HEPES, phosphate buffer, and lactate buffer.

[0033] Affinity separation techniques include magnetic separation using antibody-coated magnetic beads, affinity chromatography, "panning" using antibodies conjugated to a solid-phase matrix (e.g., a plate) with cytotoxic substances attached to monoclonal antibodies (e.g., complement and cytotoxic substances), or other convenient techniques. For precise separation, fluorescence-excited cell sorters are available, which can have varying degrees of sophistication, including multiple color channels, low-angle and obtuse-angle light scattering detection channels, and impedance channels. Cells can be selected for dead cells using dyes that bind to dead cells (e.g., propidium iodide). Any technique can be used as long as it does not impair the viability of selected cells. Affinity reagents may be receptors or ligands specific to the cell surface molecules mentioned above. In addition to antibody reagents, peptide-MHC antigen and T cell receptor pairs can also be used, such as peptide ligands and receptors, or effector and receptor molecules.

[0034] The isolated cells can be collected in any suitable medium that maintains cell viability, usually with a serum cushion at the bottom of the collection tube. Various media are commercially available, such as dMEM, HBSS, dPBS, RPMI, and Iscove medium, and can be used depending on the properties of the cells, often supplemented with fetal bovine serum.

[0035] The recovered and selectively enriched cell populations may be used immediately or lysed and cryopreserved at liquid nitrogen temperature for reuse. Cells are typically stored in 10% DMSO, 50% FCS, and 40% RPMI 1640 medium.

[0036] Modified T cells can be injected into a target in any physiologically acceptable culture medium, usually intravascularly, but may also be introduced into any other convenient site where the cells can find a suitable site for proliferation. Typically, at least 1 × 10⁶ cells are used. 6 Cells / kg are administered, and at least 1 × 10⁶ 7 cells / kg, at least 1 × 10⁶ 8cells / kg, at least 1 × 10⁶ 9 cells / kg, at least 1 × 10⁶ 10 The number of cells per kilogram or more, and is usually limited by the number of T cells obtained at the time of collection.

[0037] Expression construct: This method allows for the recombinant production of orthologous proteins, specifically orthologous cytokines. Orthologous receptors can be introduced into modified cells via expression vectors. The DNA encoding the orthologous proteins can be obtained from various sources designed during the modification process.

[0038] Amino acid sequence variants are prepared by introducing appropriate nucleotide alterations into the coding sequence, as described herein. Such variants represent the insertion, substitution, and / or deletion of the residues described herein. The insertions, substitutions, and / or deletions may be combined in any way to produce the final construct, provided that the final construct has the desired biological activity as defined herein.

[0039] Nucleic acids encoding orthologous proteins are inserted into a replicable vector for expression. Many such vectors are available. Common vector components include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Examples of vectors include viral vectors, plasmid vectors, and embedded vectors.

[0040] Orthologous proteins can be produced not only through direct recombination but also as fusion proteins with heterologous polypeptides, such as signal sequences, or with other polypeptides that have a specific cleavage site at the N-terminus of a mature protein or polypeptide. Generally, the signal sequence may be a component of the vector or part of a coding sequence inserted into the vector. The selected heterologous signal sequence is preferably one that is recognized and processed by the host cell (i.e., cleaved by a signal peptidase). For expression in mammalian cells, an undenatured signal sequence may be used, or other mammalian signal sequences, such as signal sequences from secretory polypeptides of the same or related species, as well as viral secretion leaders, such as the herpes simplex gD signal, may be preferred.

[0041] Expression vectors typically contain a selection gene, also known as a selectable marker. This gene encodes a protein necessary for the survival or proliferation of transformed host cells that grow in a selective culture medium. Host cells not transformed with a vector containing the selection gene cannot survive in the culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) compensate for nutritional deficiencies; or (c) supply essential nutrients not available in the combined medium.

[0042] An expression vector contains a promoter functionally linked to an orthologous protein-coding sequence that is recognized by the host organism. Promoters are untranslated sequences located upstream (5') of the start codon of a structural gene (usually within approximately 100–1000 bp) that control the transcription and translation of the specific nucleic acid sequence to which they are functionally linked. Such promoters are generally divided into two classifications: inducible and structural. Inducible promoters are those that, in response to some change in culture conditions, such as the presence or absence of nutrients or changes in temperature, initiate an increase in the transcription level from DNA under their control. Numerous promoters are well known that are recognized by a variety of possible host cells.

[0043] Transcription from vectors in mammalian host cells can be controlled by promoters obtained from the genomes of viruses such as polyomavirus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papillomavirus, aerosarcoma virus, cytomegalovirus, retrovirus (e.g., mouse stem cell virus), hepatitis B virus, most preferably simian virus 40 (SV40), and heterologous mammalian promoters such as actin promoters, PGK (phosphoglycerate kinase), or immunoglobulin promoters, as well as by promoters obtained from heat shock promoters, provided that such promoters are compatible with the host cell system. Early and late promoters of the SV40 virus are conveniently available as SV40 restriction fragments, which also contain the SV40 virus origin of replication.

[0044] Transcription in higher eukaryotic cells is often facilitated by inserting enhancer sequences into vectors. Enhancers are cis-acting elements of DNA, typically around 10-300 bp in length, and act on promoters to promote transcription. Enhancers are relatively independent of direction and position, found on the 5' and 3' ends of transcription units, and are present in introns and within the coding sequence itself. Currently, many enhancer sequences from mammalian genes are known. These include globin, elastase, albumin, alpha-fetoprotein, and insulin. However, generally, enhancers from eukaryotic cell viruses are used. Examples include the SV40 enhancer on the late side of the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the origin of replication, and the adenovirus enhancer. The enhancer may be ligated to the expression vector at the 5' or 3' end of the coding sequence, but it is preferable that it be located at the 5' end of the promoter.

[0045] Expression vectors used in eukaryotic host cells will also contain sequences necessary for transcription termination and mRNA stabilization. Such sequences are generally obtained from the 5', and sometimes 3', untranslated regions of eukaryotic cell or viral DNA or cDNA. Standard techniques are used to construct suitable vectors containing one or more of the aforementioned components.

[0046] In this specification, suitable host cells for cloning or expressing DNA in a vector are prokaryotic cells, yeast, or the higher eukaryotic cells described above. Examples of useful mammalian host cell lines include mouse L cells (LM[TK-], ATCC#CRL-2648), SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651), human fetal kidney cell line (293 or 293 cells subcloned for growth in suspension culture), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells / -DHFR (CHO), mouse Sertoli cells (TM4), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1 587), human cervical cancer cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), and buffalo rat liver cells (BRL 3A, ATCC The available cells include CRL 1442, human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumor cells (MMT 060562, ATCC CCL51), TRI cells, MRC5 cells, FS4 cells, and human hepatocellular carcinoma cell line (Hep G2).

[0047] Host cells, including modified T cells, can be transfected with the expression vectors described above to express orthologous IL-2 or IL-2R. Cells can be cultured in common nutrient media, modified as appropriate for promoter induction, selection of transformed cells, or amplification of genes encoding desired sequences. Mammalian host cells can be cultured in a variety of media. Commercial media such as Ham's F10 (Sigma), basal medium ((MEM), Sigma), RPMI 1640 (Sigma), and Dulbecco's modified Eagle medium ((DMEM), Sigma) are suitable for culturing host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphates), buffers (e.g., HEPES), nucleosides (e.g., adenosine and thymidine), antibiotics, tracking elements, and glucose or equivalent energy sources. Any other necessary adjuvants may be included in appropriate concentrations, as would be apparent to those skilled in the art. The culture conditions, such as temperature and pH, are those previously used with the host cells selected for expression and are therefore obvious to those skilled in the art.

[0048] Nucleic acids are "functionally linked" when they are in a functional relationship with another nucleic acid sequence. For example, the DNA of a signal sequence is functionally linked to the DNA of a polypeptide if it is expressed as a preprotein involved in the secretion of that polypeptide; a promoter or enhancer is functionally linked to a coding sequence if it affects the transcription of that sequence; or a ribosome binding site is functionally linked to a coding sequence if it is positioned to facilitate translation. In general, "functionally linked" means that the linked DNA sequences are continuous, so in the case of a secretion leader, they are continuous and readable. This means it is in the reading phase. However, the enhancers do not have to be consecutive.

[0049] Recombinantly produced orthologous cytokines can be recovered from culture media as secreted polypeptides, or from host cell lysates. Protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF), may be useful in preventing protein degradation during purification, and antibiotics that inhibit the growth of foreign contaminants may also be included. Various purification steps are known and used in this industry, such as affinity chromatography. Affinity chromatography separates molecules by their ability to bind to specific ligands, utilizing highly specific binding sites normally present on biological macromolecules. The ligand is covalently attached to an insoluble porous support medium so that the ligand is clearly presented to the protein sample, thereby separating and purifying a second species from the mixture using the natural biospecific binding of one molecular species. Antibodies are commonly used in affinity chromatography. Size selection steps may also be used, such as separating proteins by size using gel filtration chromatography (also known as size exclusion chromatography or molecular sieve chromatography). In gel filtration, the protein solution is passed through a column packed with semipermeable porous resin. Semipermeable resins have a range of pore sizes, which determines the size of proteins that can be separated by the column. Other methods, such as cation exchange chromatography, are also considered.

[0050] The final orthologous cytokine composition can be concentrated, filtered, dialyzed, etc., by methods known in the industry. For therapeutic purposes, the cytokine can be administered to mammals, including appropriately modified orthologous receptors. Administration can be intravenous, either as a bolus or by continuous infusion over a period of time. Alternative routes of administration include intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, subarachnoid, oral, topical, or inhalation. Orthologous cytokines are also suitable for administration via intratumoral, peritumoral, intralesional, or perilesional routes, or lymphatic administration, to exert local and systemic therapeutic effects.

[0051] Such dosage forms also include physiologically acceptable carriers that are essentially non-toxic and non-therapeutic. Examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, e.g., human serum albumin, buffers, e.g., phosphate buffer, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes, e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, and PEG. Carriers for topical or gel-based polypeptides include polysaccharides, e.g., sodium carboxymethylcellulose or methylcellulose, polyvinylpyrrolidone, polyacrylate, polyoxyethylene-polyoxypropylene-block polymers, PEG, and wood wax alcohol. All administrations are preferably in common depot forms. Such forms include, for example, microcapsules, nanocapsules, liposomes, plasters, inhalation forms, nasal sprays, sublingual tablets, and sustained-release formulations. Polypeptides are generally formulated in such vehicles at concentrations of approximately 0.1 μg / ml to 100 μg / ml.

[0052] If the ortholog IL-2 polypeptide of this disclosure is "substantially pure," it may be at least about 60% by weight (dry weight) of the polypeptide in question, for example, the polypeptide comprising the ortholog IL-2 amino acid sequence. For example, the polypeptide may be at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% (all by weight) of the polypeptide in question. Purity can be determined by any suitable standard method, for example. It can be measured by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0053] In another embodiment of the present invention, a manufactured article is provided that contains a substance useful for treating the above-described condition. The manufactured article includes a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. Containers can be made from a variety of materials, such as glass or plastic. The container contains a composition effective for treating the condition and may have a sterile access port (for example, the container may be an intravenous fluid bag or vial with a stopper that can be pierced with a subcutaneous needle). The activator in the composition is an orthologous cytokine. A label on or related to the container indicates that the composition is for the treatment of a selected condition. Further containers may be provided with the manufactured article that can contain, for example, pharmaceutically acceptable buffers, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. The manufactured article may further include other items that are commercially and user-desirable, such as other buffers, diluents, filters, needles, syringes, and accompanying documents describing instructions for use.

[0054] As used herein with respect to polypeptide or DNA sequences, the term “identity” refers to the identity of subunit sequences between two molecules. If, in both molecules, a certain subunit position is occupied by the same monomer subunit (e.g., the same amino acid residue or nucleotide), then those molecules are identical at that position. The similarity between two amino acid or nucleotide sequences is a direct function of the number of identical positions. Generally, sequences are aligned to obtain the highest degree of agreement. Identity can be calculated, if necessary, using published techniques and widely available computer programs, such as the GCS program package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990). Sequence identity can be measured using sequence analysis software, such as the sequence analysis software package from the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with its default parameters.

[0055] The terms "polypeptide," "protein," or "peptide" refer to any chain of amino acid residues, regardless of its length or post-translational modifications (e.g., glycosylation or phosphorylation).

[0056] In this specification, "protein variant," "variant protein," or "variant polypeptide" means a protein that differs from the wild-type protein by at least one amino acid modification. The parent polypeptide may be a naturally occurring or wild-type (WT) polypeptide, or a modified version of a WT polypeptide. The variant polypeptide may also refer to the polypeptide itself, a composition containing the polypeptide, or the amino sequence encoding it. Preferably, the variant polypeptide has at least one amino acid modification compared to the parent polypeptide, for example, about 1 to about 10 amino acid modifications compared to the parent, preferably about 1 to about 5 amino acid modifications.

[0057] In this specification, “parent polypeptide,” “parent protein,” “precursor polypeptide,” or “precursor protein” means an unmodified polypeptide that is later modified to generate a variant. The parent polypeptide may be a wild-type (or undenatured) polypeptide, or a variant or modified version of a wild-type polypeptide. The parent polypeptide may also refer to the polypeptide itself, a composition containing the parent polypeptide, or the amino acid sequence encoding it.

[0058] In this specification, "wild-type," "WT," or "unmodified (natural)" refers to the amino acid or nucleotide sequence found in nature, including allelic variations. WT proteins, polypeptides, antibodies, immunoglobulins, IgG, etc., have an amino acid or nucleotide sequence that has not been intentionally modified.

[0059] The terms “recipient,” “individual,” “subject,” “host,” and “patient” are interchangeable herein and refer to any mammalian subject, particularly humans, for which diagnosis, treatment, or treatment is desired. For therapeutic purposes, “mammal” refers to any animal classified as a mammal, such as humans, domesticated animals and livestock, and animals kept in zoos, for sporting or pets, such as dogs, horses, cats, cows, sheep, goats, pigs, etc. Preferably, the mammal is human.

[0060] In this specification, “therapeutic dose” refers to a sufficient amount of a therapeutic agent, such as a combination of adoptive T cells and orthogonal cytokines, to treat or manage a disease or disorder. A therapeutic dose may also refer to a sufficient amount of a therapeutic agent to delay or minimize the onset of a disease, such as slowing or minimizing the progression of cancer, or to reduce or increase signaling from a target receptor. A therapeutic dose may also refer to an amount of a therapeutic agent that provides a therapeutic effect for treating or managing a disease. Furthermore, with respect to the therapeutic agents of this invention, a therapeutic dose means an amount of the therapeutic agent alone or in combination with other therapeutic agents that provides a therapeutic effect for treating or managing a disease.

[0061] In this specification, the terms “prevent,” “prevent,” and “prevent” refer to preventing the recurrence or onset of one or more symptoms of a disorder in a subject as a result of the administration of a prophylactic or therapeutic agent.

[0062] In this specification, the term “in combination” refers to the use of two or more prophylactic and / or therapeutic agents. The use of the term “in combination” does not restrict the order in which the prophylactic and / or therapeutic agents are administered to the affected subject. The first prophylactic or therapeutic agent may be administered before (for example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously with, or after (for example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of the second prophylactic or therapeutic agent to the affected subject.

[0063] In this specification, the terms “cancer” (or “cancerous”), “hyperproliferative”, and “neoplastic” refer to cells capable of autonomous proliferation (e.g., abnormal conditions or situations characterized by rapid cell proliferation). Disease conditions of hyperproliferation and neoplasms may be classified as pathological (e.g., characterizing or constituting a disease condition) or non-pathological (e.g., deviating from normal but not associated with a disease condition). These terms include all types of cancerous proliferation or carcinogenic processes, metastatic tissues, or malignant cells, tissues, or organs, regardless of histopathological type or stage of invasion. “Pathologically hyperproliferative” cells occur in disease conditions characterized by malignant tumor proliferation. An example of non-pathologically hyperproliferative cells is wound-healing-related cell proliferation. The terms “cancer” or “neoplasm” are used to refer to malignant lesions of various organ systems, including those affecting the lungs, breasts, thyroid gland, lymph nodes and lymphoid tissue, digestive tract, and genitourinary tract, as well as adenocarcinomas, which are generally considered to include malignant lesions such as most colon cancers, renal cell carcinomas, prostate cancers and / or testicular tumors, non-small cell lung cancers, small intestine cancers, and esophageal cancers.

[0064] The term "cellular carcinoma" is recognized in this industry and includes respiratory cell carcinoma and gastrointestinal cell carcinoma. This refers to malignant lesions of epithelial or endocrine tissue, including urogenital cell carcinoma, testicular cell carcinoma, mammary cell carcinoma, prostate cell carcinoma, endocrine cell carcinoma, and melanoma. "Adenocarcinoma" refers to cancer of glandular origin or in which tumor cells form identifiable glandular structures.

[0065] Examples of tumor cells include, but are not limited to, AML, ALL, CML, adrenocortical carcinoma, anal cancer, aplastic anemia, cholangiocarcinoma, bladder cancer, bone cancer, bone metastases, brain cancer, central nervous system (CNS) cancer, peripheral nervous system (PNS) cancer, breast cancer, cervical cancer, pediatric non-Hodgkin lymphoma, colorectal cancer, endometrial cancer, esophageal cancer, Ewing tumor family (e.g., Ewing sarcoma), eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, Hodgkin lymphoma, Kaposi's sarcoma, kidney cancer, laryngopharyngeal cancer, liver cancer, lung cancer, pulmonary carcinoid tumors, and non-Hodgkin lymphoma. Examples of cancers include tumors, male breast cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, myeloproliferative disorders, nasal cavity and paranasal cancer, nasopharyngeal cancer, neuroblastoma, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, melanoma, non-melanoma, gastric cancer, testicular cancer, thymic cancer, thyroid cancer, uterine cancer (e.g., uterine sarcoma), transitional cell carcinoma, vaginal cancer, vulvar cancer, mesothelioma, squamous cell carcinoma or epidermoid carcinoma, bronchial adenoma, ciliary carcinoma, head and neck cancer, teratoma, or Waldenstrom hypergammaglobulinemia. Any cancer in which cancer cells show a higher CD47 expression rate compared to non-cancerous cells is suitable for treatment with this method and composition.

[0066] In other embodiments, the methods of the present invention are used to treat infectious diseases. Hereinafter, the term “infection” refers to any condition in which at least one cell of an organism (i.e., subject) is infected with an infectious pathogen (e.g., subject has an intracellular pathogen infection, e.g., chronic intracellular pathogen infection). Hereinafter, the term “infectious pathogen” refers to an alien organism (i.e., pathogen) that induces an increase in CD47 expression in at least one cell of an infected organism. Examples of infectious pathogens include, but are not limited to, bacteria, viruses, protists, and fungi. Intracellular pathogens are of particular importance. Infectious diseases are disorders caused by infectious pathogens. Some infectious pathogens do not cause recognizable symptoms or diseases under certain conditions, but have the potential to cause symptoms or diseases under different conditions. This method is used for chronic pathogen infections, such as, but not limited to, viral infections, such as retroviruses, lentiviruses, hepadnaviruses, herpesviruses, poxviruses, and human papillomaviruses; intracellular bacterial infections, such as Mycobacterium, Chlamydophila, Ehrlichia, Rickettsia, Brucella, Legionella, Francisella, Listeria, Coxiella, Neisseria, Salmonella, Yersinia sp., Helicobacter pylori, and intracellular protist pathogens, such as Plasmodium. It can be used to treat sp., Trypanosoma sp., Giardia sp., Toxoplasma sp., Leishmania sp., and other related conditions.

[0067] In further embodiments, regulatory T cells are modified for the treatment of autoimmune diseases. The range of infectious diseases and infection-associated diseases is broad and includes autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), and autoimmune hepatitis, insulin-dependent diabetes mellitus, and degenerative diseases such as osteoarthritis (OA), Alzheimer's disease (AD), and macular degeneration.

[0068] Many, if not all, autoimmune and inflammatory diseases involve multiple types of T cells, such as TH1, TH2, and TH17. Autoimmune diseases are characterized by T and B lymphocytes abnormally targeting self-proteins, self-polypeptides, and / or other self-molecules, resulting in damage and / or dysfunction of organs, tissues, or cell types in the body (e.g., pancreas, brain, thyroid, or digestive organs), leading to the clinical manifestation of the disease. Some autoimmune diseases affect specific tissues. Furthermore, this includes diseases that can affect multiple tissues, which in part depends on whether the response is limited to a specific tissue or to an antigen that is widely distributed throughout the body.

[0069] Compositions and methods This invention provides modified orthogonal cytokine receptor / ligand pairs and methods for using them. The modified (orthogonal) cytokine specifically binds to the modified (orthogonal) receptor of its counterpart. Upon binding, the orthogonal receptor activates signaling, which is transmitted through undenatured cellular elements, resulting in biological activity that mimics a native response, but is specific to modified cells expressing the orthogonal receptor. The orthogonal receptor does not bind to endogenous counterpart cytokines, including the undenatured counterpart of the orthogonal cytokine, and the orthogonal cytokine does not bind to any endogenous receptor, including the undenatured counterpart of the orthogonal receptor. In some embodiments, the affinity of the orthogonal cytokine to the orthogonal receptor is comparable to the affinity of the undenatured cytokine to the undenatured receptor.

[0070] Orthogonal cytokine-receptor pairs may be selected from any target cytokine. Methods for constructing orthogonal cytokine-receptor pairs may include the steps of (a) introducing amino acid modifications to the denatured receptor to inhibit binding to the denatured cytokine, (b) introducing amino acid modifications to the contact residue of the denatured cytokine that binds to the receptor, (c) selecting an ortholog of the cytokine that binds to an orthologous receptor, (d) discarding an orthologous cytokine that binds to the denatured receptor, or (e) selecting an orthologous receptor that binds to an orthologous cytokine, or (f) discarding an orthologous receptor that binds to an denatured cytokine. In preferred embodiments, site-specific or erer-prone mutagenic amino acid positions are selected using knowledge of the structure of the cytokine / receptor complex. A yeast presentation system can be conveniently used for this selection method, but other presentation and selection methods are also useful.

[0071] In some cases, amino acid modifications are obtained through affinity maturation. An "affinity-matured" polypeptide is a polypeptide having one or more modifications to one or more residues, resulting in improved affinity of the orthologous polypeptide to its congeneral orthologous receptor (or vice versa) compared to the parent polypeptide without such modifications. Affinity maturation may be performed to increase binding affinity by at least approximately 10%–50–100–150% or more, or by 1–5 times, compared to the "parent" polypeptide.

[0072] The modified orthologous cytokines of the present invention specifically bind to one or more residues or regions of the orthologous receptor but do not cross-react with the wild-type receptor. Generally, the lack of cross-reactivity means that, when evaluated by ELISA and / or FACS analysis using sufficient amounts of the molecule under suitable assay conditions, the relative competitive inhibition between molecules is less than approximately 5%.

[0073] In some embodiments of the present invention, the orthogonal receptor is a polypeptide selected from the IL-2 receptor chain, namely interleukin-2 receptor alpha (IL-2R-alpha, CD25), interleukin-2 receptor beta (IL-2R-β, CD122), and interleukin-2 receptor gamma (IL-2R-γ, CD132, common gamma chain). In some specific embodiments, the orthogonal receptor is CD132 and is involved in signaling from IL-2, IL-4, IL-7, and IL-15. In other specific embodiments, the orthogonal receptor is CD122 and is involved in signaling from IL-2 and IL-15. The orthogonal receptor is typically an orthogonal cytokine with a counterpart, e.g., IL-2. It is paired with IL-4, IL-7, IL-15, etc.

[0074] In some specific embodiments, the orthogonal receptor is CD122. In some such embodiments, the orthogonal receptor is introduced into T cells or NK cells that may also express CD25 and / or CD132. Nucleic acid coding sequences and protein compositions of modified CD122 proteins are provided. In the present invention, CD122 is modified to inhibit the binding of undenatured cytokines by substituting amino acids in the undenatured sequence with non-undenatured amino acids, or by deleting undenatured amino acids at positions involved in binding to undenatured IL-2. In some embodiments, amino acids are substituted with non-conservative changes. Positions that may be substituted or deleted include, but are not limited to, R41, R42, Q70, K71, T73, T74, V75, S132, H133, Y134, F135, E136, and Q214 in human CD122 (hCD122). While not limited to all, some of the locations that can be replaced or deleted in mouse CD122 (mCD122) include R42, F67, Q71, S72, T74, S75, V76, S133, H134, Y135, I136, E137, and R215.

[0075] In some embodiments, CD122 is substituted at one or a combination of positions selected from Q71, T74, H134, and Y135 in mouse proteins, or at one or a combination of positions selected from Q70, T73, H133, and Y134 in human proteins. In some embodiments, the modified protein includes amino acid substitutions in mCD122 H134 and Y135, or hCD122 H133 and Y134. In some embodiments, the amino acid substitution is a substitution to an acidic amino acid, such as aspartic acid and / or glutamic acid. Specific amino acid substitutions, though not limited to them, include substitutions of mCD122 (Q71Y, T74D, T74Y, H134D, H134E, H134K, Y135F, Y135E, Y135R) and changes of hCD122 (Q70Y, T73D, T73Y, H133D, H133E, H133K, Y134F, Y134E, Y134R). The selection of orthologous cytokines depends on the selection of orthologous receptors.

[0076] In some embodiments where the orthologous receptor is CD122, the orthologous cytokine is IL-2 or IL-15. Cytokines may be selected for binding to the orthologous receptor by means of, for example, yeast presentation evolution, erer-prone, or targeted mutagenesis. A representative set of selected orthologous sequences is shown in Figure 6.

[0077] In some embodiments, the orthogonal cytokine is IL-2. In some embodiments, for mouse IL-2 (mIL-2), one or more amino acid residues from any of H27, L28, E29, Q30, M33, D34, Q36, E37, R41, N103, and for human IL-2 (hIL-2), one or more of Q13, L14, E15, H16, L19, D20, Q22, M23, G27, N88 are substituted with amino acids not present in the undenatured protein, or deleted at that position. In some such embodiments, the set of amino acid substitutions is selected from one or more of E29, Q30, M33, D34, Q36, and E37 (for mIL-2), and from one or more of E15, H16, L19, D20, Q22, and M23 (for hIL-2).

[0078] In some embodiments, the amino acid substitutions of mRNA are one or more of the following: [H27W], [L28M, L28W], [E29D, E29T, E29A], [Q30N], [M33V, M33I, M33A], [D34L, D34M], [Q36S, Q36T, Q36E, Q36K, Q36E], [E37A, E37W, E37H, E37Y, E37F, E37A, E37Y], [R41K, R41S], [N103E, N103Q], and the amino acid substitutions of hIL-2 are [Q13W], [L14M, L14W], [E15D, E It is one or more of the following: 15T, E15A, H16N, L19V, L19I, L19A, D20L, D20M, Q22S, Q22T, Q22E, Q22K, Q22E, M23A, M23W, M23H, M23Y, M23F, M23A, M23Y, G27K, G27S, N88E, N88Q. In some embodiments, the set of amino acid substitutions for mRNA is one of the substitution sets [Q30N, M33V, D34N, Q36T, E37H, R41K], [E29D, Q30N, M33V, D34L, Q36T, E37H], [E29D, Q30N, M33V, D34L, Q36T, E37A], and [E29D, Q30N, M33V, D34L, Q36K, E37A], or the set of amino acid substitutions for mRNA. It includes a conservative variant, and for hIL-2, it includes one of the substitution sets of [H16N, L19V, D20N, Q22T, M23H, G27K], [E15D, H16N, L19V, D20L, Q22T, M23H], [E15D, H16N, L19V, D20L, Q22T, M23A], and [E15D, H16N, L19V, D20L, Q22K, M23A], or its conservative variant.

[0079] In some embodiments, the amino acid substitutions of hIL-2 are one or more of the following: [E15S, E15T, E15Q, E15H], [H16Q], [L19V, L19I], [D20T, D20S, D20M, D20L], [Q22K, Q22N], [M23L, M23S, M23V, M23T]. In some embodiments, the consensus set of mutations for hIL-2 is [E15S, H16Q, L19V, D20T / S / M, Q22K, M23L / S]. In some embodiments, the set of amino acid substitutions for hIL-2 is [E15S, H16Q, L19V, D20T / S, Q22K, M23L / S], [E15S, H16Q, L19I, D20S, Q22K, M23L], [E15S, L19V, D20M, Q22K, M23S], [E15T, H16Q, L19V, D20S, M23S], [ Includes one of the following substitution sets: E15Q, L19V, D20M, Q22K, M23S], E15Q, H16Q, L19V, D20T, Q22K, M23V], E15H, H16Q, L19I, D20S, Q22K, M23L], E15H, H16Q, L19I, D20L, Q22K, M23T], L19V, D20M, Q22N, M23S].

[0080] Treatment method The present invention provides a method for enhancing cellular responses by modifying cells from a recipient or donor by introducing the orthologous receptor of the present invention, and stimulating the orthologous receptor by contacting the modified cells with a congeneral orthologous cytokine. The method includes a step of obtaining target cells, such as T cells or hematopoietic stem cells, which may be isolated from a biological sample or induced in vitro from a progenitor cell source. The cells are transfected or transfected with an expression vector containing a sequence encoding the orthologous receptor, and this step can be carried out in any suitable culture medium.

[0081] In some embodiments, modified cells are provided, which are modified by the introduction of the orthologous receptor of the present invention. Any cells can be used for this purpose. In some embodiments, the cells are T cells, and not limited to naive CD8 + T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, helper T cells, for example T H 1. T H 2, T H 9, T H 11, T H 22, T FH Regulatory T cells, for example, T R 1, Natural T Reg , inducible T Reg , memory T cells, for example, central memory T cells, ephe Examples include Co-receptor memory T cells, NKT cells, and γδT cells. In other embodiments, the modified cells are stem cells, such as hematopoietic stem cells or NK cells. In some embodiments, the cells are genetically modified by an ex vivo procedure before being transplanted into the subject. The modified cells can be supplied in unit doses for therapeutic purposes and may be allogeneic, autologous, or otherwise derived from the intended recipient.

[0082] In some embodiments, a vector is provided containing a coding sequence encoding an orthogonal receptor, the coding sequence functionally ligated to a promoter that is active in the desired cell. Various vectors are known in the art and can be used for this purpose. For example, viral vectors, plasmid vectors, and small oval vectors can be incorporated into the target cell genome or maintained as episomes. The receptor-encoding vector may also be provided as a kit together with a vector encoding an orthologous cytokine that binds to and activates the receptor. In some embodiments, the orthologous cytokine coding sequence may be functionally ligated to a high-expression promoter to optimize production. In other embodiments, a kit is provided in which the orthologous receptor-encoding vector is provided together with a purified composition of the orthologous cytokine and packaged, for example, in unit doses for administration to a patient.

[0083] In some embodiments, a therapeutic method is provided, which includes introducing a modified cell population into a recipient requiring the method, the cell population being modified by introducing a sequence encoding the orthologous receptor of the present invention. This cell population can be modified ex vivo and is usually homogeneous or autologous to the recipient. In some embodiments, after administration of the modified cells, the introduced cell population comes into contact in vivo with a congeneral orthologous cytokine. One advantage of the present invention is that there is no cross-reactivity between the orthologous cytokine and the undenatured receptor.

[0084] When cells are brought into contact with orthologous cytokines in vitro, the cytokines are added to the modified cells in an amount and for a sufficient duration to activate signal transduction from the receptor. This can include unmodified cellular mechanisms, such as accessory proteins and co-receptors. Any suitable culture medium can be used. The thus activated cells can be used for any desired purpose, including experimental purposes related to antigen specificity determination and cytokine profiling, as well as for in vivo delivery.

[0085] When contact is performed in vivo, an effective amount of modified cells is injected into the recipient together with or prior to the administration of orthologous cytokines. Dosage and frequency may vary depending on the agent, mode of administration, and properties of the cytokines. Those skilled in the art will understand that such guidelines should be adjusted to individual circumstances. Dosage may also vary depending on whether it is administered locally, e.g., intranasally, by inhalation, or systemically, e.g., im, iP, IV. Generally, at least about 10 4 Individual modified cells / kg were administered, and at least approximately 10 5 modified cells / kg, at least about 10 6 modified cells / kg, at least about 10 7 Individual modified cells / kg, or more, are administered.

[0086] If the modified cells are T cells, the enhancement of the immune response may manifest as an increased cytolytic response of T cells against target cells present in the recipient (e.g., toward the elimination of tumor cells or infected cells), and a reduction in the symptoms of autoimmune diseases.

[0087] cell composition Modified T cells can be provided in pharmaceutical compositions suitable for therapeutic use, for example, in the treatment of humans. Therapeutic formulations containing such cells may be frozen or prepared and administered as aqueous solutions with physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). The cells are formulated, prescribed, and administered in accordance with the appropriate medical practice. Factors to be considered in this case include: These factors include the specific disorder being treated, the specific mammal being treated, the patient's individual clinical condition, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to healthcare professionals.

[0088] Cells can be administered by any suitable means, usually parenterally. Parenteral administration methods include intramuscular, intravenous (bolus or slow drip), intra-arterial, intraperitoneal, subarachnoid, or subcutaneous administration.

[0089] The preferred form depends on the intended mode of administration and therapeutic use. The composition may also contain pharmaceutically acceptable non-toxic carriers or diluents, depending on the desired formulation, which are defined as vehicles widely used in the formulation of pharmaceutical compositions administered to animals or humans. The diluent is selected so as not to affect the biological activity of the mixture. Examples of such diluents include distilled water, phosphate-buffered saline, Ringer's solution, dextrose solution, and Hank's solution. Furthermore, the pharmaceutical composition or formulation may also contain other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc.

[0090] In some other embodiments, the pharmaceutical composition may also include large, slowly metabolizing macromolecules such as proteins, polysaccharides such as chitosan, polylactic acid, polyglycolic acid, and copolymers (e.g., latex-functionalized Sepharose®, agarose, cellulose, etc.), polymerized amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes).

[0091] Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dose and concentration used, and include buffers such as phosphate buffer, citrate buffer, and other organic acids, antioxidants such as ascorbic acid and methionine, preservatives (e.g., ammonium octadecydimethylbenzyl chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins, and serum albumin. Examples include gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other sugars such as glucose, mannose, or dextrin, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, counterions that form salts such as sodium, metal complexes (e.g., Zn-protein complexes), and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG). Formulations for in vivo administration must be sterile. This can be easily achieved by filtration with a sterile filtration membrane.

[0092] Generally, the compositions are prepared as liquid solutions or suspensions for injection, but they can also be prepared as solids suitable for use as solutions or suspensions in a liquid vehicle before injection. The preparations can also be made into emulsions or encapsulated in liposomes or microparticles, such as polylactic acid, polyglycolide, or copolymers, to enhance the adjuvant effect as described above. Langer, Science 249:1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997. The agents of the present invention can be administered as depot injections or as implantation preparations, in which case they can be formulated so that the active ingredient is released continuously or pulsatilely. The pharmaceutical compositions are generally sterile, substantially isotonic, and comply with all the rules of the U.S. Food and Drug Administration's Good Manufacturing Practice (GMP). It is formulated accordingly.

[0093] kit A kit used in this method is also provided. This kit includes an expression vector encoding an orthologous cytokine receptor, or cells containing said expression vector. The kit may further include homologous orthologous cytokines. In some embodiments, these components are provided in liquid or solid dosage forms (e.g., therapeutically effective dosage forms) in any convenient packaging (e.g., stick packaging, dose packaging, etc.). Reagents for cell selection or in vitro induction, such as growth factors, differentiation factors, and tissue culture reagents, may also be provided.

[0094] In addition to the components described above, the kit may further include (in certain embodiments) instructions for carrying out the method. These instructions may exist in various forms within the kit, and the kit may contain one or more instructions. One possible form of such instructions is information printed on a suitable medium or support, for example, one or more sheets of paper with the information printed on them included in the kit packaging as an accompanying document. Yet another form of such instructions is a computer-readable medium, such as a diskette, compact disc (CD), or flash drive, on which the information is recorded. Yet another possible form of such instructions is a website address, which can be used via the internet to access the information remotely.

[0095] In some embodiments, the composition, method, and kit are used to enhance a T cell-mediated immune response. In some embodiments, the immune response is directed towards a state where it is desirable to deplete or control target cells, such as cancer cells, infected cells, or immune cells associated with autoimmune diseases.

[0096] In some embodiments, the condition is a chronic infection, i.e., an infection that is not cleared by the host's immune system within a week or two. In some cases, chronic infections may involve the integration of genetic elements of pathogens, such as retroviruses, lentiviruses, or hepatitis B viruses, into the host genome. In other cases, chronic infections are caused by pathogenic cells residing within host cells, such as certain intracellular bacteria or protist pathogens. Furthermore, in some embodiments, the infection is in a latent stage, such as herpesviruses or human papillomaviruses.

[0097] The target viral pathogens are not limited to retroviruses and lentiviral pathogens, such as HIV-1, HIV-2, HTLV, FIV, and SIV, as well as hepatitis B virus, etc. The target microorganisms are not limited to, but include Yersinia sp., e.g., Y. pestis, Y. pseudotuberculosis, Y. enterocolitica, Franciscella sp., Pasturella sp., Vibrio sp., e.g., V. cholerae, V. parahemolyticus, Legionella sp., e.g., L. pneumophila, Listeria sp., e.g., L. monocytogenes, Mycoplasma sp., e.g., M. hominis, M. pneumoniae, Mycobacterium sp., e.g., M. tuberculosis, M. leprae, Rickettsia sp., e.g., R. rickettsii, R. typhi, Chlamydia sp., e.g., C. trachomatis, C. pneumoniae, C. psittaci, Helicobacter sp., e.g., H. pylori. Also, intracellular protist pathogens, e.g., Plasmodium sp., Trypanosoma sp. Other examples include Giardia sp., Toxoplasma sp., and Leishmania sp.

[0098] Infections treated by the method of the present invention generally involve pathogens in which at least part of their life cycle is within host cells, i.e., intracellular phase. The method of the present invention makes the killing of infected cells by T effector cells of the host organism more effective than removal in the absence of treatment, and is therefore directed towards the intracellular phase of the pathogen's life cycle. The method may further include monitoring the patient for the effectiveness of the treatment. Monitoring may involve measuring clinical signs of infection, such as fever and white blood cell count, and / or direct monitoring of the presence of the pathogen.

[0099] Treatment can be carried out in combination with other active agents. Antibiotics include penicillins (e.g., penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafcillin, ampicillin, etc.), penicillins used in combination with β-lactamase inhibitors (e.g., seraphosporins such as cefaclor, cefazolin, cefuroxime, moxalactam), carbapenems, monobactams, aminoglycosides, tetracyclines, macrolides, lincomycin, polymyxin, sulfonamides, quinolones, chloramphenicol, metronidazole, spectinomycin, trimethoprim, vancomycin, etc.). Cytokines (e.g., interferon-gamma, tumor necrosis factor-alpha, interleukin-12, etc.) may also be included. Antiviral agents (e.g., acyclovir, ganciclovir, etc.) can also be used in treatment.

[0100] In some embodiments, the condition is cancer. Hereinafter, the term “cancer” refers to a range of conditions resulting from abnormal, uncontrolled cell proliferation. Cells that may cause cancer are called “cancer cells” and possess characteristic properties such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and / or specific typical morphological features. Cancer may be detected by a number of methods, including, but not limited to, detection of the presence of one or more tumors (e.g., by clinical or radiological means), examination of cells from tumor cells or other biological specimens (e.g., from tissue biopsy), measurement of cancer-indicating blood markers, and detection of cancer-indicating genotypes. However, a negative result from one or more of these detection methods does not necessarily indicate the absence of cancer. For example, cancer may still be present in patients who have responded completely to cancer treatment, and there may be evidence of recurrence later.

[0101] In this specification, the term “cancer” includes cellular carcinomas (e.g., Insights' cellular carcinoma, invasive cellular carcinoma, metastatic cellular carcinoma) and pre-malignant conditions, i.e., new morphological changes unrelated to a histological origin. The term “cancer” is not limited to any stage, grade, histological characteristics, invasiveness, progression, or degree of malignancy of the affected tissue or cell aggregate. In particular, it includes stage 0 cancer, stage I cancer, stage II cancer, stage III cancer, stage IV cancer, grade I cancer, grade II cancer, grade III cancer, malignant cancer, and primary cellular carcinoma.

[0102] Having carefully described the present invention, it will be clear to those skilled in the art that various modifications and variations can be made without departing from the spirit or scope of the invention.

[0103] experiment Orthologous IL-2 and IL-2Rβ This invention describes an invention involving modified cytokines and receptors that enable the selective proliferation of a desired cell subset in the setting of ex vivo adoptive cell therapy. This particular invention describes the cytokine interleukin-2 (IL-2) and its receptor IL-2Rβ chain (IL-2Rβ), which enable T cell proliferation in adoptive cell therapy and thus address an unmet need in immunotherapy. The method described herein can be generalized to adoptive cell therapy in any setting in which cells are stimulated by a specific receptor-ligand pair, such as bone marrow transplantation, stem cell transplantation, and many other modalities.

[0104] This paper specifically describes the ligand-receptor pair of orthologous IL-2 and IL-2Rβ. Orthologous versions of IL-2 and IL-2Rβ bind specifically to each other but not to their respective wild-type counterparts. Multiple orthologous IL-2 variant sequences exhibiting varying degrees of affinity for orthologous IL-2Rβ are provided. Orthologous IL-2-dependent signaling and T cell proliferation in T cells modified to express orthologous IL-2Rβ are demonstrated.

[0105] IL-2 is an attractive biologic for treating cancer and autoimmune diseases due to its ability to promote the proliferation of effector T cells and regulatory T cells, respectively. However, its multifaceted nature and off-target toxicity limit its clinical use. If the immunostimulatory and immunoinhibitory isomers of IL-2 can be separated, a superior form of IL-2 immunotherapy could be provided.

[0106] This specification demonstrates the ability to modify T cells to express orthogonal IL-2Rβ. These modified T cells are shown to respond to orthogonal IL-2, resulting in phosphorylation of downstream signaling molecules (e.g., STAT5) and T cell proliferation. The activity of orthogonal IL-2 on wild-type T cells is either completely suppressed or significantly blunted compared to the activity of wild-type IL-2. Thus, selective T cell proliferation using the orthogonal IL-2 / IL-2 receptor pair is demonstrated.

[0107] Applications of orthogonal IL-2 / IL-2 receptor pairs include, but are not limited to, the selective proliferation of tumor-reactive cytotoxic T cells for cancer treatment, NK cells for infectious diseases and / or cancer, and regulatory T cells for autoimmune diseases.

[0108] IL-2 variants that inhibit binding to IL-2Rβ but do not completely eliminate it, resulting in blunted affinity for intermediates (IL-2Rβ and IL-2Rγ) or high-affinity wild-type IL-2 receptors (IL-2Rα, Rβ, Rγ), are also useful for selectively targeting the activity of ortholog IL-2 against IL-2Rα hypercellularity, for example, in the treatment of autoimmune diseases. IL-2 variants that lack affinity for the IL-2Rβ chain but retain binding to IL-2Rα, and thus act as antagonists competing with wild-type IL-2 by inhibiting high-affinity IL-2R formation, are useful in the treatment of autoimmune diseases or graft-versus-host diseases.

[0109] Figure 1 schematically illustrates the overall concept of generating and utilizing orthogonal IL-2 / IL-2 receptor pairs to control T cell proliferation. Figure 2 provides a workflow that includes a step of generating IL-2Rβ orthologues that do not bind to wild-type IL-2 using structural information-based mutagenesis. Mutations predicted to inhibit IL-2Rβ binding to wild-type IL-2 are experimentally confirmed by yeast-based screening assays and further demonstrated by surface plasmon resonance using purified recombinant proteins. This method explains a number of IL-2Rβ point mutations that inhibit binding to wild-type IL-2, and each of these receptor variants can function as an orthologous receptor. Single point mutations can also be combined with three or more further point mutations to create a larger library of IL-2Rβ orthologues.

[0110] The sequences of orthogonal mouse IL-2Rβ variants are shown in Figure 3. These mutations generate IL-2Rβ orthologues with one, two, three, or more point mutations. It can be used as a single point mutation, or in any combination of mutations that inhibits the binding of wild-type IL-2.

[0111] Figure 4 shows the characteristics of the mRNA-2Rβ variant, including amino acid modifications H134D and Y135F that suppress the binding of wild-type mRNA. These two residues are known IL-2 interaction hotspots (Ring A et al, Nat Immunol (2012) 13:1187-95), and the inventors confirmed by surface plasmon resonance (SPR) that these mutations inhibit the binding of wild-type mRNA.

[0112] Figure 5 shows the workflow for creating orthogonal IL-2 / IL-2Rβ pairs. An ortholog library of IL-2 is created by randomizing the residues adjacent to or touching the orthologous IL-2Rβ orthologous amino acid residues. By yeast presentation, IL-2 variants that bind to orthologous IL-2Rβ are selected, and clones that bind to wild-type IL-2Rβ are discarded. This method can be repeated using site-directed or error-prone mutagenesis to generate IL-2 variants that have different binding characteristics to orthologs but not to wild-type IL-2Rβ. Using this method, the inventors created a library of IL-2 orthologs that 1) retain binding to the IL-2Rα chain, showing the intact structural integrity of the yeast-presented orthologous IL-2 variant (green curve), 2) bind to orthogonal IL-2Rβ (orange curve), but 3) do not bind to wild-type IL-2Rβ (blue curve).

[0113] The sequences of the characterized orthogonal mouse IL-2 variants are shown in Figure 6. Alignments of mouse IL-2 and IL-2Rβ, as well as their human counterparts, are shown in Figure 15. These four sequences provide a reference for the undenatured, or wild-type, sequence. The amino acid residues modified to create the orthogonal mouse IL-2 / IL-2Rβ pair are largely conserved in humans. Therefore, the sequences of orthogonal mouse IL-2 and IL-2Rβ can be readily translated into human IL-2 and IL-2Rβ proteins.

[0114] As shown in Figure 7, the ortho-IL-2 variant binds to ortho-IL-2Rβ with the same or higher affinity as the interaction between wild-type IL-2 and IL-2Rβ. Soluble ortho-IL-2 or wild-type IL-2 protein was flowed onto sensor chips coated with wild-type ortho-IL-2Rβ. Binding was determined by surface plasmon resonance (SPR), and curves were fitted using a 1:1 binding model. As shown in Figure 8, the ortho-IL-2 variant showed blunted activity against wild-type CD25-positive and CD25-negative splenocytes (phosphoSTAT5).

[0115] Figure 9 shows the generation of ortho-IL-2Rβ expressing mouse CTLL-2 T cells. The inventors created an immortalized mouse T cell line (CTLL-2) expressing ortho-IL-2Rβ (ortho-CTLL-2) by introducing the gene encoding the full-length ortho-ortho-receptor using a lentivirus. Transduced cells were selected with puromycin, a toxin for non-transduced cells, to obtain a stable CTLL-2 cell line expressing both wild-type and ortho-IL-2Rβ. This cell line is also positive for CD25 and CD132, thus representing T cells expressing a high-affinity IL-2 receptor complex. The antibody used to detect cell surface IL-2Rβ (CD122) does not distinguish between wild-type and ortho-IL-2Rβ. Therefore, the increase in average fluorescence intensity between wild-type and ortho-IL-2Rβ CTLL-2 cells suggests that these cells express the ortho-receptor. This is further supported by the fact that these cells are resistant to puromycin encoded by the same vector used to express ortho-IL-2Rβ.

[0116] As shown in Figure 10, the first set of ortho IL-2 variants against ortho T cells It is selective. To investigate orthogonal IL-2 signaling, the inventors utilized their CTLL-2 cell model, which was either unmodified (wild-type) or transduced to express orthogonal IL-2Rβ (ortho). They then determined the ability of wild-type or various orthogonal IL-2 clones to induce STAT5 phosphorylation (quantitative readout of IL-2-dependent signaling). The inventors identified several orthogonal IL-2 variants that induced selective STAT5 phosphorylation in orthogonal IL-2Rβ-expressing cells compared to wild-type cells. Dose-response curves for the selected clones are shown in Figure 11.

[0117] Primary lymph node-derived T cells modified to express ortho-IL-2Rβ (H134D Y135F). In addition to the inventors' immortalized mouse T cell model, mouse lymph node and spleen cells were isolated, activated with CD3 / CD28, and then a gene encoding the full-length ortho-orthogonal receptor was introduced via retrovirus to generate primary mouse T cells expressing ortho-IL-2Rβ. This construct also includes IRES and the subsequent fluorescent protein YFP, so transduction can be confirmed by analyzing YFP expression using FACS. As shown in Figure 12, the mouse T cells also express high-affinity IL-2 receptor complexes (e.g., CD25, CD122, and CD132).

[0118] As shown in Figure 13, the ortho-IL-2 variant induces selective STAT5 phosphorylation in ortho-IL-2Rβ expressing primary mouse T cells.

[0119] Ortho-IL-2 variants that selectively signal via ortho-IL-2Rβ (Figure 11) also induce selective proliferation of ortho-IL-2Rβ-expressing CTLL-2 cells compared to wild-type CTLL-2 cells (Figure 14).

[0120] The engineering techniques for orthogonal IL-2 were also applied to human IL-2 and human IL-2Rβ. The inventors introduced the H133D Y134F mutation, which was used to create mouse ortho-IL-2Rβ, into human IL-2Rβ because these residues are highly conserved between mice and humans. Indeed, wild-type hIL-2Rβ binds to yeast-presented wild-type IL-2, but the hIL-2Rβ H133D Y134F mutant (ortho-hIL-2Rβ) does not show detectable binding to wild-type IL-2 (Figure 15). The inventors created a library of human IL-2 mutants presented on the yeast surface by randomizing residues predicted to be in contact with or near the H133D Y134F mutation, and selected IL-2 variants that bind to ortho-IL-2 but not to wild-type human IL-2Rβ. This scheme is the same as that used to create mouse orthogonal pairs and was successful for human pairs as well. This strategy is shown in Figure 16. A consensus set of mutations that shows the convergence of ortho-hIL-2 sequences capable of binding to ortho-hIL-2Rβ is identified and shown in Figure 16C.

[0121] The polypeptide of the present invention is active in vivo. Using a mouse model, we demonstrated selective proliferation or increased viability of orthogonal IL-2Rb-expressing T cells in mice. This is shown in Figures 17-19. Ortho-IL-2 clone 1G12 / 149 was shown to selectively proliferate orthogonal T cells in mice, but not wild-type T cells. Treatment with wild-type IL-2 resulted in proliferation of both wild-type and ortho-T cells compared to the PBS control, but treatment with ortho-IL-2 clone 1G12 / 149 selectively proliferated ortho-T cells, with limited activity against wild-type T cells.

[0122] Cross-reference with related applications This application asserts the interests of U.S. Provisional Patent Application No. 62 / 217,364, filed September 11, 2016, and U.S. Provisional Patent Application No. 62 / 375,089, filed August 15, 2016, which are incorporated herein by reference in their entirety.

[0123] Federal government grants for research and development This invention was made with government funding provided by the National Institutes of Health under contract AI513210. The government has certain rights to this invention.

Claims

1. A system for selectively activating receptors within cells that express them, (a) An orthogonal human CD122 receptor comprising amino acid substitutions H133D and Y134F, wherein the amino acid substitution inhibits the binding of undenatured human interleukin-2 (IL-2) to the CD122 polypeptide, and is expressed by cells, and (b) An orthogonal human IL-2 cytokine comprising at least four amino acid substitutions selected from the group consisting of an amino acid substitution at position 15 selected from the group consisting of E15S, E15T, E15Q and E15H, an amino acid substitution at position 16 selected from the group consisting of H16Q, an amino acid substitution at position 19 selected from the group consisting of L19V and L19I, an amino acid substitution at position 20 selected from the group consisting of D20T, D20S, D20M and D20L, an amino acid substitution at position 22 selected from the group consisting of Q22K and Q22N, and an amino acid substitution at position 23 selected from the group consisting of M23L, M23S, M23V, M23A and M23T, which binds to the orthogonal human CD122 receptor and activates the orthogonal human CD122 receptor. A system that includes this.

2. The system according to claim 1, wherein the cells are immune cells or stem cells.

3. The system according to claim 2, wherein the immune cells are T cells.

4. The system according to claim 1, wherein the cells are human cells or mouse cells.

5. The orthogonal human IL-2 cytokine is [E15S; H16Q; L19V; D20S; Q22K; M23L], [E15S; H16Q; L19V; D20T; Q22K; M23L], [E15S; H16Q; L19V; D20T; Q22K; M23S], [E15S; H16Q; L19V; D20S; Q22K; M23S], [E15S; H16Q; L19I; D20S; Q22K; M23L], [E15S; L19V; D20M; Q22K; M23S], [E15T; H16Q; L19V; D20S; M23S], [E15Q; L19V; D20M; Q22K; M23S], [E15Q; H16Q; L19V; D20T; Q22K; M23V], [E15H; H16Q; L19I; D20S; Q22K; M23L], [E15H; H16Q; L19I; D20L; Q22K; M23T], and [L19V; D20M; Q22N; M23S] The system according to claim 1, comprising a set of amino acid substitutions selected from the group consisting of the following.

6. A composition comprising the orthogonal human CD122 receptor according to claim 1.

7. A system according to any one of claims 1 to 5, used in a method for treating an individual, comprising introducing cells genetically modified to express the orthogonal human CD122 receptor according to claim 1 into the individual, and selectively activating the cells relating to the orthogonal human CD122 receptor by contacting the cells with the orthogonal human IL-2 cytokine according to claim 5.

8. The system for use according to claim 7, wherein the cells are T cells.

9. The system for use according to claim 8, wherein the individual is used to treat cancer.

10. The system for use according to claim 7, wherein the individual is treated for an autoimmune disease.

11. The system for use according to claim 10, wherein the autoimmune disease is systemic lupus erythematosus.

12. The system for use according to claim 7, wherein the individual is treated for an infectious disease.

13. A kit comprising the system described in any one of claims 1 to 5.

14. A nucleic acid encoding the orthogonal human CD122 receptor according to claim 1, or an expression vector comprising the nucleic acid encoding the orthogonal human CD122 receptor according to claim 1, A composition comprising orthogonal human IL-2 cytokines according to claim 5 and The kit according to claim 13, comprising:

15. A pharmaceutical composition for use in treating an individual, It contains mammalian cells expressing an orthogonal human IL-2Rβ (CD122) receptor subunit in which residues H133 and Y134 are modified. The orthogonal human IL-2Rβ (CD122) receptor subunit is human CD122 containing amino acid substitutions H133D and Y134F. The pharmaceutical composition is administered in combination with an orthogonal human IL-2 cytokine that binds to the orthogonal human CD122 receptor and activates it, comprising at least four amino acid substitutions selected from the group consisting of E15S, E15T, E15Q, and E15H at position 15, an amino acid substitution at position 16 selected from the group consisting of H16Q, an amino acid substitution at position 19 selected from the group consisting of L19V and L19I, an amino acid substitution at position 20 selected from the group consisting of D20T, D20S, D20M, and D20L, an amino acid substitution at position 22 selected from the group consisting of Q22K and Q22N, and an amino acid substitution at position 23 selected from the group consisting of M23L, M23S, M23V, M23A, and M23T. Pharmaceutical composition.

16. The pharmaceutical composition according to claim 15, wherein the mammalian cell is a T cell.

17. The pharmaceutical composition according to claim 15 or 16, wherein the individual is treated for cancer.

18. The pharmaceutical composition according to claim 15 or 16, wherein the individual is treated for an autoimmune disease.

19. The pharmaceutical composition according to claim 18, wherein the autoimmune disease is systemic lupus erythematosus.

20. The pharmaceutical composition according to claim 15 or 16, wherein the individual is treated for an infectious disease.

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