Biologically relevant orthogonal cytokine / receptor pairs

Abstract

The present invention provides engineered orthogonal cytokine receptor / ligand pairs and methods of using the same.

Description

[0001] Cross-references

[0002] This patent application claims priority to U.S. Provisional Patent Continuation Application No. 15 / 916,689, filed on March 9, 2018, which is incorporated herein by reference in its entirety.

[0003] Federally Funded Research and Development

[0004] This invention was made with government support, including from the National Institutes of Health under grant AI513210. The government has certain rights in this invention. Background Art

[0005] Manipulating cells, especially immune cells, to differentiate, develop specific functions, and expand cell numbers is of great clinical significance. Many protein factors that affect these activities are known in the art, especially cytokines and chemokines. However, these signaling molecules also have pleiotropic effects on cells that are not targeted for manipulation, so methods for selectively activating signaling in target cell populations are needed. In particular, engineering T cells for controlled behavior is of interest. For example, in adoptive immunotherapy, T cells are isolated from the blood, processed ex vivo, and re-injected into the patient. T cells have been engineered for therapeutic applications, such as recognizing and killing cancer cells, intracellular pathogens, and cells involved in autoimmunity.

[0006] A key challenge in cell-based therapies is to engineer desired behaviors, such as activation, expansion, etc., in adoptively transferred cells that are immune to endogenous signaling pathways, do not affect non-targeted endogenous cells, and can be controlled in the patient to whom they are administered. This is particularly relevant to T cell engineering, as developmental plasticity and environmental factors play a huge role in determining T cell fate, function, and localization.

[0007] The ability to manipulate proteins to bind and respond to modified ligands in a manner that is independent of or orthogonal to the effects of the native protein or ligand constitutes a major challenge in protein engineering. To date, many synthetic ligand-ortholog-receptor pairs have been generated that are orthogonal to similar natural interactions. Proteins used for this research include nuclear hormone receptors and G protein-coupled receptors. Despite extensive work to design receptors activated by synthetic small molecule ligands, the engineering of biologically relevant protein pairs remains a major challenge. Summary of the invention

[0008] The present invention provides engineered orthogonal cytokine receptor / ligand pairs and methods of use thereof. Engineered (orthogonal) cytokines specifically bind to corresponding engineered (orthogonal) receptors. After binding, orthogonal receptors activate signal transduction by natural cell elements to provide biological activity that simulates the natural response but is specific to engineered cells expressing orthogonal receptors. Orthogonal receptors exhibit a significant reduction in binding to endogenous corresponding cytokines (including natural counterparts of orthogonal cytokines), while orthogonal cytokines exhibit a significant reduction in binding to any endogenous receptor (including natural counterparts of orthogonal receptors). In some embodiments, the affinity of orthogonal cytokines to orthogonal receptors is comparable to the affinity of natural cytokines to natural receptors.

[0009] The method of designing an orthogonal cytokine-receptor pair may comprise the steps of: (a) engineering amino acid changes to a native receptor to disrupt binding to the native cytokine; (b) generating a plurality of cytokine analogs having selective amino acid changes to the native cytokine at contact residues to which the receptor binds, (c) selecting cytokine orthologs that bind to the orthologous receptor; (d) discarding orthologous cytokines that bind significantly to the native receptor, or alternatively discarding steps (c) and (d); (e) selecting receptor orthologs that bind to the orthologous cytokine; (f) discarding orthologous receptors that bind to the native cytokine. In a preferred embodiment, knowledge of the structure of the cytokine / receptor complex is used to select amino acid positions for site-directed or error-prone mutations. A yeast display system may be conveniently used for the selection process, although other display and selection methods are also useful.

[0010] In some embodiments, an engineered cell is provided wherein the cell is modified by introducing an orthogonal receptor of the invention. Any cell may be used for this purpose. In some embodiments, the cell is a T cell, including but not limited to a naive CD8 T cell. + 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 11. T H 22. T FH Regulatory T cells, such as T R 1. Natural R 1. Natural T Reg , inducible T Reg; Memory T cells, such as central memory T cells, effector memory T cells, NKT cells, γδT cells and engineered variants of such T cells including CAR-T cells, etc. In other embodiments, the engineered cells are stem cells, such as hematopoietic stem cells, NK cells, macrophages or dendritic cells. In some embodiments, the cells are genetically modified in an ex vivo treatment before being transferred to a subject. The engineered cells can be provided in a unit dose for treatment, which can be allogeneic, autologous, etc. relative to the intended recipient.

[0011] In some embodiments, there is provided a carrier comprising a polynucleotide coding sequence encoding an orthogonal receptor, wherein the coding sequence is operably connected to a promoter active in a desired cell. Various carriers are known in the art and can be used for this purpose, such as viral vectors, plasmid vectors, microcirculation vectors, and their carriers can be integrated into the target cell genome, or episomes can be maintained. The receptor encoding carrier can be provided in a test kit, combined with a carrier encoding an orthogonal cytokine that binds and activates a receptor. In certain embodiments, the coding sequence of the orthogonal cytokine is operably connected to a high expression promoter, and can be optimized for production. In other embodiments, there is provided a test kit, wherein the carrier encoding the orthogonal receptor provides a purified composition of the orthogonal cytokine, such as packaged in unit doses for patient administration (such as pre-filled syringes). In some other embodiments, there is provided a test kit, wherein the carrier encoding the orthogonal receptor is provided to encode the orthogonal cytokine, so that the orthogonal receptor can be expressed in cells, and the orthogonal cytokine intended to be secreted by the same cell is also expressed to realize autocrine orthogonal cytokine receptor signal transmission.

[0012] In certain embodiments, a method of treatment is provided, the method comprising introducing an engineered cell population into a receptor in need thereof, wherein the cell population is modified by introducing a sequence encoding an orthogonal receptor of the present invention. The cell population can be engineered in vitro, and is generally autologous or allogeneic relative to the receptor. In certain embodiments, after administering the engineered cells, the introduced cell population is contacted with a homologous orthogonal cytokine in vivo. An advantage of the present invention is the lack of cross-reactivity between the orthogonal cytokine and the natural receptor. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The most comprehensive understanding of the present invention can be obtained by reading the following detailed description in conjunction with the accompanying drawings. It should be emphasized that, according to common practice, the various features of the drawings are not drawn to scale. On the contrary, the size of each feature may be arbitrarily expanded or reduced for the sake of clarity. The accompanying drawings include the following illustrations:

[0014] Figure 1-1 (ii). Orthogonal IL-2 / IL-2 receptor control of T cell expansion.

[0015] Figure 2 .Workflow for engineering orthogonal IL-2 / IL-2Rβ pairs.

[0016] Figure 3 .Sequences of orthogonal mouse IL-2Rβ variants.

[0017] Figure 4 The mIL-2RβH134D Y135F mutation abolished the binding of wild-type mIL-2.

[0018] Figure 5 .Workflow for engineering orthogonal IL-2 / IL-2Rβ pairs.

[0019] Figure 6 . Sequences of characterized orthogonal mouse 11-2 variants.

[0020] Figure 7 The orthogonal IL-2 variants bind to the orthogonal IL-2R with similar or greater affinity than the interaction between wild-type IL-2 and IL-2Rβ.

[0021] Figure 8 Orthogonal IL-2 variants exhibit inactivation activity (phosphorylation of STAT5) on wild-type CD25-positive and CD25-negative splenocytes

[0022] Fig. 9 .Generation of mouse CTLL-2 T cells expressing orthogonal IL-2R.

[0023] Fig.10 .The first group of orthogonal IL-2 variants are selective for orthogonal T cells.

[0024] Fig.11 Orthogonal IL-2 variants induce selective STAT5 phosphorylation on CTLL-2 cells expressing orthogonal IL-2Rβ

[0025] Fig.12 Primary lymph node-derived T cells were engineered to express orthogonal IL-2Rβ (H134D Y135F).

[0026] Fig.13 Orthogonal IL-2 variants induce selective STAT5 phosphorylation on orthogonal IL-2Rβ expressing primary mouse T cells.

[0027] Fig.14 Orthogonal IL-2 variants induce selective cell growth of CTLL-2 cells expressing orthogonal IL-2Rβ compared to wild-type T cells.

[0028] Fig.15. Alignment of mouse and human reference IL-2Rβ / IL-2 sequences. The partial sequence of human IL-2Rβ is provided as SEQ ID NO: 1, residues 1-235; the partial sequence of mouse IL-2Rβ is provided as SEQ ID NO: 2, residues 1-238. Mouse IL-2 is provided as SEQ ID NO: 3. Human IL-2 is provided as SEQ ID NO: 4.

[0029] Figures 16A-16D .Yeast evolution of orthogonal human IL-2 pairs. Fig.16A ) FACS analysis of yeast shows that wild-type human IL-2 binds to wild-type (blue histogram) but not to the orthogonal (red histogram) human IL-2RβH133D Y134F mutant tetramer. ( Fig. 16B ) A library of human IL-2 mutants (~1 8 mutants) were displayed on the yeast surface. After several consecutive rounds of positive (against orthogonal hIL-2Rβ) and negative (against wild-type hIL-2Rβ) selection, we obtained yeast-displayed human IL-2 mutants that bound to orthogonal (red histogram) but not wild-type (blue histogram) human IL-2Rβ tetramers. Fig. 16C , 16D ) Orthogonal hIL-2 mutants were subsequently isolated and sequenced in a yeast library. A set of common mutations was identified, indicating that the orthogonal hIL-2 sequence set was able to bind to orthogonal hIL-2Rβ.

[0030] Fig.17 In vivo mouse model used to demonstrate selective expansion or increased survival of orthogonal IL-2Rb expressing T cells in mice. Donor cells were isolated from the spleen of wild-type C57BL / 6J mice expressing CD45.2, activated ex vivo with CD3 / CD28, transduced with retrovirus encoding orthogonal IL-2Rb-IRES-YFP, expanded for 2 days in 100 IU / mL mIL-2, and purified using the Mouse CD8 T Cell Isolation Kit (Miltenyi). A ~1:1 mixture of wild-type (CD45.2 positive, YFP negative) and orthogonal IL-2Rb expressing T cells (CD45.2 positive, YFP positive) were adoptively transferred into recipient BL6.Rag2 mice by retro-orbital injection. - / - x IL2rg - / -CD45.1 mice. Immediately after T cell transfer (d0), daily intraperitoneal injections of PBS, wild-type mIL-2 (150,000 Iu / mouse) or orthogonal IL-2 clone 1G12 / 149 (1,000,000 Iu / mouse) were started and continued for 5 consecutive days (up to d4) at 24-hour intervals. Mice were sacrificed on d5 and d7, and total donor T cells in mouse blood and spleen were quantified by flow cytometry.

[0031] Figures 18A-18B Gating strategy for quantifying donor T cell expansion in recipient mice. Single cell suspensions from mouse blood and spleen were prepared and stained with Cd45.2-Pacific Blue for 1 hour at 4°C to identify donor T cells. Immediately prior to flow cytometry, cells were incubated with propidium iodide (PI) diluted 1:2000 for live / dead exclusion. Cells were gated by FACS quantification based on forward and side scatter (SSC-A v FSC-A), singlets (FSC-A v FSC-H), live cells (PI negative), and total wild-type T cells (CD45.2 positive, YFP negative) and orthotopic T cells (CD45.2 positive, YFP positive). **p<0.01, ***p<0.001, ****p<0.0001, determined using one-way ANOVA in Prism.

[0032] Fig.19 The orthogonal IL-2 clone 1G12 / 149 selectively expands orthogonal but not wild-type T cells in mice. Blood (10 3 The number of wild-type and orthogonal T cells in the blood (cells / uL) and spleen (total number of cells per spleen) is shown in the figure. 18. The ratio of orthogonal T cells to wild-type T cells was determined by dividing the total number of orthogonal T cells by the total number of wild-type T cells in the blood and spleen. A ratio greater than 1 indicates selective expansion of orthogonal T cells, achieved by orthogonal IL-2 clone 1G12 / 149. The total number of viable cells in the blood (left) and spleen (right) on day 5 (top) and day 7 (bottom) was quantified by flow cytometry. Compared with the PBS control, treatment with wild-type IL-2 resulted in expansion of wild-type and orthogonal T cells, while treatment with orthogonal IL-2 clone 1G12 / 149 selectively expanded orthogonal T cells that had limited activity against wild-type T cells.

[0033] Figures 20A-20B .Orthogonal IL-2 has selective activity against orthogonal IL-2Rβ T cells. ( Fig. 20A) Spleen-derived primary mouse T cells were isolated from IL-2KO NOD mice and subjected to FACS analysis of viral transfection to express orthogonal IL-2Rβ, as determined by surface staining with an IRES-YFP reporter and IL-2Rβ. T cells also retained expression of wild-type IL-2Rβ ( Fig. 20B ) Orthogonal IL-2 induces selective STAT5 phosphorylation on T cells expressing orthogonal IL-2Rβ and is inactive on wild-type T cells.

[0034] Figures 21A-21B . Orthogonal IL-2 selectively expands orthogonal IL-2RβT cells in vitro. ( Fig.21A )FACS analysis of spleen-derived primary mouse T cells virally transfected to express orthogonal IL-2Rβ, confirmed using IRES-YFP. Mixtures of transfected and untransfected T cells were cultured for 5 days in various concentrations of wild-type, orthogonal IL-2 clones 1G12 or 3A10 and analyzed by FACS. IL-2 amplified both wild-type and orthogonal T cells, while only orthogonal T cells expanded when cultured in orthogonal IL-2 3A10, and orthogonal IL-2 1G12 selectively amplified orthogonal T cells with significantly reduced activity against wild-type T cells. The FACS plots shown correspond to cultures in 100nM IL-2, 64pM orthogonal IL-2 1G12, and 10μM orthogonal IL-2 3A10. ( Fig. 21B ) Dose response of wild-type and orthogonal T cell proliferation to wild-type and orthogonal IL-2 clones 1G12 and 3A10 after 5 days of culture in increasing concentrations of cytokines. IL-2 expands wild-type and orthogonal T cells with equal potency, orthogonal IL-2 1G12 selectively expands orthogonal T cells, and orthogonal IL-2 3A10 specifically expands orthogonal T cells.

[0035] Figures 22A-22E Orthogonal human IL-2 signals in vitro through the orthogonal IL-2R expressed in YT cells. Dose response of STAT5 phosphorylation after 20 min stimulation. Stat5 phosphorylation was measured in the YT human NK cell line expressing human CD25 (YT+), without (YFP-, WT) or with (YFP+, orthogonal) human orthogonal IL-2Rb. Fig.22A ) fusion of human IL-2 or orthogonal variants to mouse serum albumin (MSA) ( Fig. 22B )1A1,( Fig. 22C )1C7,( Fig.22D )SQVLKA or ( Fig.22E)SQVKqA was titrated in RPMI complete medium and added to cells. The mean fluorescence intensity (MFI) of APC-pStat5 staining of WT (YFP-) and orthogonal Rb (YFP+) cells was plotted against the concentration of cytokines and modeled using Prism 5 (GraphPad) fitting logarithm (agonist) vs. response (three parameters). 1C7 was run on a separate day with other proteins and normalized to wild-type IL-2 staining from two runs. All data are presented as mean (n=3) ± standard deviation.

[0036] Figures 23A-23B Orthogonal human IL-2 preferentially expands human PBMC expressing orthogonal IL-2R. Human PBMC were isolated, activated and transformed with retrovirus containing orthogonal human IL-2Rβ containing IRES-YFP (YFP+). The initial ratio of YFP+ cells to total viable cells was 20%. 5x10 cells were plated on day 1 with the indicated concentrations of MSA-human IL-2 (circles) or orthotopic variants MSA-SQVLKA (diamonds), MSA-SQVLqA (squares) or MSA-1A1 (triangles). 5 cells and re-cultured at the same concentration on day 3. On day 5, the plates were read by flow cytometry. Fig.23A ) The ratio of YFP+ (orthogonal expression) cells to total viable cells was calculated and the mean (n=4) ± SD versus concentration was plotted (left). Fig. 23B ) Total viable cell counts (mean (n=4) ± SD) are plotted versus cytokine concentration (right). At the same concentration, the orthogonal cytokines were unable to support as much total cell growth as wild-type MSA-hIL-2. DETAILED DESCRIPTION

[0037] To make the present disclosure more readily understood, certain terms and phrases are defined below and throughout the specification. The definitions provided herein are non-limiting and should be considered as known by those skilled in the art at the time of the invention.

[0038] definition

[0039] Before further describing the present method and chapter, it should be understood that the present invention is not limited to the specific method or chapter described, because there will be differences in actual implementation. It should also be understood that the terms used in this patent are only used to describe specific embodiments and are not intended to limit the present invention. The scope of the present invention will be limited only by the appended claims.

[0040] Where a numerical range is provided, it is to be understood that each intermediate value between the upper and lower limits of the range and the intermediate values ​​within the range are included within the scope of the present invention. Unless the context clearly specifies otherwise, each intermediate value should be as low as one tenth of the lower limit unit. The present invention encompasses each smaller range between any of the values ​​or intervening values ​​within the range and any other values ​​or intervening values ​​within the range. The upper and lower limits of these smaller ranges may be independently included within the range or excluded within the range, and are also included within the present invention, subject to the requirements of any specifically excluded limits within the range. Where the range includes one or two limits, the range excluding any one or two of those included limits is also included within the present invention.

[0041] Unless otherwise defined, all technical and scientific terms used in this patent have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described in this patent can also be used in the implementation or testing of the present invention, some potential and preferred methods and materials are described below. All publications mentioned in this patent are incorporated herein by reference to disclose and describe the methods and / or materials related to the cited publications. It should be understood that when there is a conflict, the present invention shall replace any disclosure in the cited publications.

[0042] It should be noted that, as used in this patent and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and "the peptide" refers to one or more agents and equivalents known to those skilled in the art, such as polypeptides, and so on.

[0043] The publications discussed in this patent are provided solely for their disclosure prior to the filing date of this patent. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0044] Cytokine receptor and ligand pairs include, but are not limited to, the following receptors:

[0045]

[0046]

[0047]

[0048] "Orthologs" or "orthogonal cytokine / receptor pairs" refer to genetically engineered protein pairs modified by amino acid changes that (a) exhibit significantly reduced affinity for a native cytokine or homologous receptor; and (b) specifically bind to a corresponding engineered (orthogonal) ligand or receptor. Upon orthogonal ligand binding, the orthogonal receptor activates signaling transduced through native cellular elements to provide a biological activity that mimics the native response but is specific to the engineered cells expressing the orthogonal receptor.

[0049] The orthogonal receptor exhibits significantly reduced binding to its cognate natural cytokine ligand, while the orthogonal cytokine exhibits significantly reduced binding to its cognate natural receptor. In some embodiments, the affinity of the orthogonal cytokine for the cognate orthogonal receptor is comparable to the affinity of the natural cytokine for the natural receptor, such as having an affinity of at least about 1% of the affinity of the natural cytokine-receptor pair, at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 100%, and can be higher, such as 2 times, 3 times, 4 times, 5 times, 10 times or more the affinity of the natural cytokine for the natural receptor.

[0050] As used herein, "does not bind" or "cannot bind" means no detectable binding or insignificant binding, i.e., with a binding affinity much lower than that of the native ligand. Affinity can be determined using competitive binding experiments that measure the binding of a receptor to a single concentration of labeled ligand in the presence of various concentrations of unlabeled ligand. Typically, the concentration of unlabeled ligand is varied over at least six orders of magnitude. By competitive binding experiments, the IC 50 As used herein, “IC 50 IC refers to the concentration of unlabeled ligand required to inhibit the binding of the receptor to the labeled ligand by 50%. 50 It is an indicator of ligand-receptor binding affinity. Low IC 50 represents high affinity, while high IC 50 Represents low affinity.

[0051] As used herein, the term "specific binding" refers to the degree of selectivity or affinity with which one molecule binds to another molecule. In a binding pair (e.g., ligand / receptor, antibody / antigen, antibody / ligand, antibody / receptor binding pairs), a first molecule of a binding pair specifically binds to a second molecule of the binding pair when the first molecule of the binding pair does not bind to other components present in a sample in significant amounts. A first molecule of a binding pair is said to specifically bind to a second molecule of the binding pair when the affinity of the first molecule for the second molecule is at least two times greater, at least ten times greater, at least 20 times greater, or at least 100 times greater than the affinity of the first molecule for other components present in the sample. In a specific embodiment, the first molecule of the binding pair is an antibody if the affinity of the antibody for the second molecule of the binding pair is greater than about 109 liters / mole, or greater than about 10 10 L / mol, greater than about 10 11 L / mol, greater than about 10 12 If the antibody specifically binds to the second molecule (e.g., protein, antigen, ligand or receptor) of the binding pair, the antibody specifically binds to the second molecule (e.g., protein, antigen, ligand or receptor) of the binding pair, as determined by Scatchard analysis (Munsen et al. 1980 Analyt. Biochem. 107: 220-239). Specific binding can be assessed using techniques known in the art, including but not limited to competition ELISA, Determination and / or Determination.

[0052] As used herein, the affinity of an orthogonal ligand binding to an orthogonal receptor relative to the binding of the orthogonal ligand to its cognate receptor in a naturally occurring form uses the term "exhibiting significantly reduced binding". In the practice of the present invention, the term "exhibiting significantly reduced binding" is used to describe the comparative binding and activity of an orthogonal ligand relative to a naturally occurring ligand relative to a naturally occurring receptor. If the orthogonal ligand binds to the native form of the receptor less than 20%, or less than about 10%, or less than about 8%, or less than about 6%, or less than about 4%, or less than about 2%, or less than about 1%, or less than about 0.5% of the naturally occurring ligand, the orthogonal ligand exhibits significantly reduced binding relative to the native form of the ligand. Similarly, if the native form of the ligand binds to the orthogonal form of the receptor at less than 20%, or less than about 10%, or less than about 8%, or less than about 6%, or less than about 4%, or less than about 2%, or less than about 1%, or less than about 0.5% of the naturally occurring receptor, the orthogonal receptor exhibits significantly reduced binding relative to the native form of the ligand.

[0053] Orthogonal IL-2 polypeptides exhibit significantly reduced activation by native IL-2Rβ. Activity can be measured in proliferation assays, for example, using proliferation assays of CTLL-2 mouse cytotoxic T cells, see Gearing, AJ Hand and CB Bird (1987) in Lymphokines and Interferons, A Practical Approach. Clemens, MJ et al. (eds.): IRL Press. 295. The specific activity of recombinant human IL-2 is approximately 2.1 x 10 4Iu / μg, which is for recombinant human, which is calibrated against the recombinant human IL-2 WHO International Standard (NIBSC code: 86 / 500). The orthogonal human IL-2 may have less than 20%, or less than about 10%, or less than about 8%, or less than about 6%, or less than about 4%, or less than about 2%, or less than about 1%, or less than about 0.5% of the human IL-2 polypeptide activity in the WHO International Standard (NIBSC code: 86 / 500) in a comparable assay.

[0054] As used herein with reference to polypeptides or DNA sequences, the term "identity" refers to the sequence identity between two molecules. The similarity between two amino acids or two nucleotide sequences is a direct function of the number of identical positions. Typically, the sequences are aligned to obtain the highest order matches. If necessary, identity can be calculated using disclosed techniques and widely available computer programs, such as the GCS program package (Devereux et al., Nucleic Acids Res. 12: 387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J. Molecular Biol. 215: 403, 1990). Sequence analysis software and its default parameters can be used, such as the sequence analysis software package of the genetics computer group of the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wisconsin, 53705), to measure sequence identity.

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

[0056] "Protein variant" or "variant protein" or "variant polypeptide" herein refers to a protein that differs from a wild-type protein by at least one amino acid modification. A parent polypeptide may be a naturally occurring or wild-type (WT) polypeptide, or may be a modified form of a WT polypeptide. The term variant polypeptide may refer to the polypeptide itself, a composition comprising the polypeptide, or a nucleic acid sequence encoding it. Compared to a parent polypeptide, a variant polypeptide ideally comprises at least one amino acid modification, such as about 1 to about 10 amino acid modifications compared to the parent, and preferably about 1 to about 5 amino acid modifications compared to the parent. A variant may be at least about 99% identical to a wild-type protein, at least about 98% identical, at least about 97% identical, at least about 95% identical, at least about 90% identical.

[0057] As used herein, "parent polypeptide", "parent protein", "precursor polypeptide" or "precursor protein" refers to an unmodified polypeptide that is subsequently modified to produce a variant polypeptide. The parent polypeptide may be a wild-type (or native) polypeptide. The parent polypeptide may refer to the polypeptide itself, a composition comprising the parent polypeptide, or an amino acid sequence encoding it.

[0058] "Wild type" or "WT" or "native" herein refers to an amino acid sequence or nucleotide sequence found in nature, including allelic variations. WT proteins, polypeptides, antibodies, immunoglobulins, IgG, etc. have an amino acid sequence or nucleotide sequence that has not been artificially modified.

[0059] The terms "recipient", "individual", "subject", "host" and "patient" are used interchangeably herein and refer to any mammalian subject, especially a human, in need of diagnosis, treatment or therapy. For therapeutic purposes, "mammal" refers to any animal classified as a mammal, including humans, livestock and farm animals, and zoo, sports or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc. The mammal is preferably a human.

[0060] As used herein, "therapeutically effective amount" refers to an amount of a therapeutic agent, such as adoptive T cells or orthogonal cytokines, sufficient to prevent, treat or control a disease or condition. A therapeutically effective amount may refer to an amount of a therapeutic agent sufficient to delay or minimize the onset of a disease, such as delaying or minimizing the spread of a cancer, or reducing or increasing the amount effect of signaling from a receptor of interest. A therapeutically effective amount may also refer to an amount of a therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease. In addition, a therapeutically effective amount with respect to a therapeutic agent of the present invention refers to an amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease.

[0061] As used herein, the terms "prevent," "prophylactic," and "prevention" refer to preventing the recurrence or onset of one or more disorders in a subject as a result of administration of a prophylactic or therapeutic agent.

[0062] As used herein, the term "combination" refers to the use of more than one preventive and / or therapeutic agent. The use of the term "combination" does not limit the order in which the subject suffering from the disease applies the preventive and / or therapeutic agent. The first preventive or therapeutic agent can be applied 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), followed by 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) to apply the second preventive or therapeutic agent to the subject suffering from the disease.

[0063] Interleukin 2 (IL-2) is a multipotent cytokine produced primarily by activated CD4+ T cells and plays a key role in generating a normal immune response. IL-2 promotes the proliferation and expansion of activated T lymphocytes, enhances B cell growth, and activates monocytes and natural killer cells. It is because of these activities that IL-2 has been tested and used as an approved cancer treatment (aldesleukin, ). Human IL-2 is synthesized as a 153 amino acid precursor polypeptide, from which 20 amino acids are removed to produce mature secreted IL-2. As used herein, "IL-2" refers to natural or wild-type IL-2. Mature human IL-2 exists as a 133 amino acid sequence (less a signal peptide, consisting of an additional 20 N-terminal amino acids) as described by Fujita et al., PNAS USA, 80, 7437-7441 (1983). The amino acid sequence of human IL-2 is retrieved in Genbank under accession number NPU000577.2. Reference sequences for human IL-2 (SEQ ID NO: 4) and mouse IL-2 (SEQ ID NO: 3), human IL-2Rβ (SEQ ID NO: 1) and mouse IL-2Rβ (SEQ ID NO: 2) are at Fig.15 Available in.

[0064] IL-2 supports the survival and differentiation of T lymphocytes by initiating cell signaling pathways upon interaction with the IL-2 receptor (IL-2R). IL-2 is used clinically to treat many human diseases, including cancer and autoimmunity, and as an adjuvant in adoptive T cell therapy to promote the survival of transplanted T cells. However, IL-2 can also have a corresponding effect by activating off-target cell types.

[0065] In order to direct the activity of IL-2 to specific T cell subsets, the present invention provides engineered orthogonal IL-2 and IL-2 receptor pairs. When bound to the orthogonal IL-2 receptor expressed by cells, the orthogonal IL-2 recapitulates the activity of wild-type IL-2 by inducing potent STAT5 phosphorylation and in vitro proliferation of T cells engineered to express orthogonal IL-2Rbeta. Orthogonal IL-2 has significantly reduced binding relative to wild-type CD25 positive or negative mouse T cells cultured in vitro. The studies disclosed herein indicate that remodeling the cytokine receptor interface to produce interactions that do not exist in nature is a viable strategy to direct the activity of promiscuous cytokines to T cell subsets of interest, thereby enabling precise control of T cell function through genetic engineering.

[0066] In addition to IL-2, IL-15 and IL-7 regulate lymphoid 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 can be selected against the same orthogonal IL-2R-beta used to orthogonalize IL-2. IL-7 utilizes a unique IL-7R-alpha chain, which is the target of orthogonalization.

[0067] In some embodiments, orthogonal cytokines, such as orthogonal IL2, can be conjugated to additional molecules to provide desired pharmacological properties, such as extended half-life. In one embodiment, orthogonal IL-2 can be fused to the Fc domain of IgG, albumin or other molecules to extend its half-life, such as by PEGylation, glycosylation, etc. as known in the art. In some embodiments, the orthogonal cytokine is conjugated to a polyethylene glycol molecule or "PEGylated". The molecular weight of the PEG conjugated to the orthogonal cytokine ligand includes, but is not limited to, PEG having a molecular weight between 5kDa and 80kDa, in some embodiments, PEG has a molecular weight of about 5kDa, in some embodiments, PEG has a molecular weight of about 10kDa, in some embodiments, PEG has a molecular weight of about 20kDa, in some embodiments, PEG has a molecular weight of about 30kDa, in some embodiments, PEG has a molecular weight of about 40kDa, in some embodiments, PEG has a molecular weight of about 50kDa, and in some embodiments, PEG has a molecular weight of about 60kDa. In some embodiments, PEG has a molecular weight of about 80kDa. In some embodiments, the molecular weight is from about 5 kDa to about 80 kDa, from about 5 kDa to about 60 kDa, from about 5 kDa to about 40 kDa, from about 5 kDa to about 20 kDa. In preferred embodiments, (wherein the polypeptides are named with reference to Table 1), the orthogonal ligand is a PEGylated form of 1A1, a PEGylated form of 1C7, a PEGylated form of SQVLKA and / or a PEGylated form of SQVLqA, in each case, the PEG has a molecular weight of about 5 kDa, or 10 kDa, 20 kDa, or 30 kDa, or 40 kDa, or 40 kDa, or 50 kDa, or 30 kDa. The PEG conjugated to the polypeptide sequence can be linear or branched. The PEG can be directly attached to the orthogonal polypeptide or attached through a linker molecule. The methods and chemical reactions necessary to achieve PEGylation of biological compounds are well known in the art.

[0068] Orthogonal IL-2 can be acetylated at the N-terminus using methods known in the art, such as by an enzymatic reaction with an N-terminal acetyltransferase and, for example, acetyl-CoA. Orthogonal IL-2 can be acetylated at one or more lysine residues, such as by an enzymatic reaction with a lysine acetyltransferase. See, e.g., Choudhary et al. (2009). Science. 325(5942):834-840.

[0069] Fc fusions can also confer alternative Fc receptor-mediated properties in vivo. An "Fc region" can be a naturally occurring or synthetic polypeptide that is homologous to the IgG C-terminal domain produced by digesting IgG with papain. The IgG Fc has a molecular weight of about 50 kDa. A direct homolog IL-2 polypeptide can include the entire Fc region or a smaller portion that retains the ability to extend the circulating half-life of the chimeric polypeptide to which it belongs. In addition, full-length or fragmented Fc regions can be variants of wild-type molecules. That is, they can contain mutations that may or may not affect the function of the polypeptide; natural activity is not required in the cases described below.

[0070] In other embodiments, the orthogonal polypeptide may comprise a polypeptide used as an antigen tag, such as a FLAG sequence. The FLAG sequence is recognized by a biotinylated, highly specific anti-FLAG antibody (see Blanar et al., Science 256:1014, 1992; LeClair et al., Proc. Natl. Acad. Sci. USA 89:8145, 1992). In some embodiments, the chimeric polypeptide further comprises a C-terminal C-myc epitope tag.

[0071] As mentioned above, orthogonal proteins of the present invention can exist as a part of chimeric polypeptides. In addition to or in place of the above-mentioned heterologous polypeptides, nucleic acid molecules of the present invention can include sequences encoding "markers" or "reporters". Examples of markers or reporter genes include β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neol, G418r), dihydrofolate reductase (DHFR), hygromycin B phosphotransferase (HPH), thymidine kinase (Tk), lacz (encoding β-galactosidase) and xanthine guanine phosphoribosyltransferase (XGPRT). As with many standard procedures associated with the practice of the present invention, those familiar with the art will know other useful reagents, for example, other sequences that can serve markers or reporter functions.

[0072] Orthogonal cytokines and receptors can also include conservative modifications and substitutions at other positions of the cytokine (e.g., positions other than those involved in orthogonal engineering). Such conservative substitutions include those described by Dayhoff in Atlas of Protein Sequence and Structure 5 (1978) and Argos in EMBO J., 8:779-785 (1989). For example, amino acids belonging to one of the following groups represent conservative changes: 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. In each case, the introduction of additional modifications can be evaluated to minimize any increase in the antigenicity of the modified polypeptide in the organism to which the modified polypeptide is to be administered.

[0073] The term "T cell" refers to a mammalian immune effector cell that can be characterized by expression of CD3 and / or T cell antigen receptor, which can be engineered to express orthogonal cytokine receptors. In some embodiments, the T cell is selected from naive CD8 + 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 11. T H 22. T FH Regulatory T cells, such as T R 1. Natural T reg , inducible T Reg , memory T cells, such as central memory T cells, effector memory T cells, NKT cells, and γδT cells.

[0074] In one embodiment of the present invention, the T cell expressing the orthogonal receptor is a T cell that has been modified to express a chimeric antigen receptor ("CAR-T" cell) on the surface. As used herein, the terms "chimeric antigen receptor T cell" and "CAR-T cell" are used interchangeably and refer to T cells that have been recombinantly modified to express a chimeric antigen receptor. As used herein, CAR-T cells can be modified to express orthogonal IL-2Rβ polypeptides. As used herein, the terms "chimeric antigen receptor" and "CAR" are used interchangeably to refer to a polypeptide comprising multiple functional domains arranged from amino to carboxyl terminus in a sequence: (a) antigen binding domain (ABD), (b) transmembrane domain (TD); (c) one or more cytoplasmic signaling domains (CSD), wherein the aforementioned domains may optionally be connected by one or more spacer domains. CAR may further include a signal peptide sequence that is routinely removed during post-translational processing and presentation of CAR on the cell surface. CAR useful in the practice of the present invention is prepared according to principles well known in the art. See the following references: Eshhaar et al., U.S. Patent No. 7,741,465B1, issued on June 22, 2010; Sadelain et al. (2013) Cancer Discovery 3(4):388-398; Jensen and Riddell (2015) Current Opinions in Immunology 33:9-15; Gross et al. (1989) PNAS (USA) 86(24):10024-10028; Curran et al. (2012) J Gene Med 14(6):405-15. Examples of commercially available CAR-T cell products that can be modified to incorporate the orthogonal receptors of the present invention include axicabtagene ciloleucel (Yescarta from Gilead Pharmaceuticals) and tisagenlecleucel (Kymriah from Novartis).

[0075] As used herein, the term antigen binding domain (ABD) refers to a polypeptide that specifically binds to an antigen expressed on the surface of a target cell. ABD can be any polypeptide that specifically binds to one or more antigens expressed on the surface of a target cell. In certain embodiments, the target cell antigen is a tumor antigen. Examples of tumor antigens that can be targeted by the ABD of CAR include one or more antigens selected from the group, including but not limited to CD19, CD20, HER2, NY-ESO-1, MUC1, CD123, FLT3, B7-H3, CD33, IL1RAP, CLL1 (CLEC12A) PSA, CEA, VEGF, VEGF-R2, CD22, ROR1, mesothelin, c-Met, glycolipid F77, FAP, EGFRvIII, MAGE A3, 5T4, WT1, KG2D ligands, folate receptor (Fra) and Wnt1 antigens.

[0076] In one embodiment, the ABD is a single-chain Fv (ScFv). ScFv is a polypeptide consisting of the variable regions of the immunoglobulin heavy and light chains of an antibody covalently linked by a peptide linker (Bird et al. (1988) Science 242: 423-426; Huston et al. (1988) PNAS (USA) 85: 5879-5883; Sz Hu et al. (1996) Cancer Research, 56, 3055-3061. The generation of ScFvs based on monoclonal antibody sequences is well known in the art. See, for example, The Protein Protocols Handbook, John M. Walker, Ed. (2002) Humana Press Section 150 "Bacterial Expression, Purification and Characterization of Single-Chain "Antibodies" Kipriyanov, S. Antibodies used to prepare scFvs can be optimized to select molecules with specific desired characteristics (e.g., enhanced affinity) by techniques well known in the art such as phage display and directed evolution. In some embodiments, the ABD comprises anti-CD19 scFv, anti-PSA scFv, anti-HER2 scFv, anti-CEA scFv, anti-EGFR scFv, anti-EGFRvIII scFv, anti-NY-ESO-1 scFv, anti-MAGE scFv, anti-5T4 scFv or anti-Wnt1 scFv. In another embodiment, the ABD is a single domain antibody obtained by immunizing a camel or llama with an antigen derived from a target cell, particularly a tumor antigen. See, e.g., Muyldermans, S. (2001) Reviews in Molecular Biotechnology 74:277-302. Alternatively, the ABD can be produced entirely synthetically by generating a peptide library and isolating compounds having the desired target cell antigen binding properties essentially as taught by or Wigler et al. U.S. Patent No. 6,303,313 B1 issued November 12, 1999; Knappik et al., U.S. Patent No. 6,696,248 B1 issued February 24, 2004, Binz et al. (2005) Nature Biotechnology 23:1257-1268, and Bradbury et al. (2011) Nature Biotechnology 29:245-254.

[0077] ABDs may have affinity for multiple target antigens. For example, the ABDs of the present invention may comprise chimeric bispecific binding members, i.e., capable of providing specific binding to an antigen expressed by a first target cell and an antigen expressed by a second target cell. Non-limiting examples of chimeric bispecific binding members include bispecific antibodies, bispecifically conjugated monoclonal antibodies (mab) 2, bispecific antibody fragments (e.g., F(ab) 2, bispecific scFv, bispecific diabodies, single-chain bispecific diabodies, etc.), bispecific T cell engagers (BiTEs), bispecifically conjugated single domain antibodies, mice and mutants thereof, etc. Non-limiting examples of chimeric bispecific binding members also include those chimeric bispecific agents described in Kontermann (2012) Mabs. 4(2):182–197; Stamova et al. (2012) Antibodies, 1(2), 172-198; Farhadfar et al. (2016) Leuk Res. 49:13-21; Benjamin et al. Ther Adv Hematol. (2016) 7(3):142-56; Kiefer et al. Immunol Rev. (2016) 270(1):178-92; Fan et al. (2015) J Hematol Oncol. 8:130; May et al. (2016) Am J Health Syst Pharm. 73(1):e6-e13. In some embodiments, the chimeric bispecific binding member is a bivalent single chain polypeptide. See, e.g., Thirion et al. (1996) European J. of Cancer Prevention 5(6):507-511; DeKruif and Logenberg (1996) J. Biol. Chem 271(13):7630-7634; and Kay et al. U.S. Patent Application Publication No. 2015 / 0315566, published on November 5, 2015. In some cases, the chimeric bispecific binding member can be a bispecific T cell engager (BiTE). Usually, the specific binding member (e.g., scFv) binding to the antigen is fused to the second binding domain of a specific binding T cell molecule such as CD3 to engage. In some cases, the chimeric bispecific binding member can be a CAR T cell adapter. As used herein, "CAR T cell adapter" refers to an expressed bispecific polypeptide that binds to the antigen recognition domain of CAR and redirects CAR to a second antigen. Typically, the CAR T cell adapter will have a binding region, one specific for the epitope on the CAR to which it is directed, and the other epitope is directed to a binding partner, and when combined, it transduces the binding signal of the activation CAR. Useful CAR T cell adapters include, but are not limited to, for example, those described by Kim et al.(2015) J Am Chem Soc. 137(8):2832-5; Ma et al. (2016) Proc Natl Acad Sci US A. 113(4):E450-8 and Cao et al. (2016) Angew Chem Int Ed Engl. 55(26):7520-4.

[0078] In some embodiments, a linker polypeptide molecule is optionally incorporated into the CAR between the antigen binding domain and the transmembrane domain to promote antigen binding. Moritz and Groner (1995) Gene Therapy 2 (8) 539-546. In one embodiment, the linker is a hinge region from an immunoglobulin, such as a hinge from any one of IgG1, IgG2a, IgG2b, IgG3, and IgG4, particularly a human protein sequence. Alternatives include the CH2CH3 region and part of CD3 of an immunoglobulin. In the case where the ABD is a scFv, an IgG hinge can be used. In some embodiments, the linker comprises the amino acid sequence (G4S) n , where n is 1, 2, 3, 4, 5, etc., and in some embodiments n is 3.

[0079] CAR useful in the practice of the present invention also includes a transmembrane domain that connects ABD (or a linker, if used) to the intracellular cytoplasmic domain of CAR. The transmembrane domain consists of any polypeptide sequence that is thermodynamically stable in the eukaryotic cell membrane. The transmembrane domain can be derived from the transmembrane domain of a naturally occurring transmembrane protein, or it can be synthetic. When designing a synthetic transmembrane domain, amino acids that are advantageous for alpha-helical structures are preferred. The transmembrane domain used to construct CARs is composed of approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 22, 23 or 24 amino acids, which are advantageous for forming an alpha-helical secondary structure. Amino acids that are advantageous for alpha-helical conformations are well known in the art. See, for example, Pace et al. (1998) Biophysical Journal 75: 422-427. Amino acids particularly favored in alpha helical conformations include methionine, alanine, leucine, glutamic acid and lysine. In some embodiments, the CAR transmembrane domain can be derived from the transmembrane domain of a type I transmembrane protein, such as CD3δ, CD4, CD8, CD28, etc.

[0080] The cytoplasmic domain of the CAR polypeptide includes one or more intracellular signaling domains. In one embodiment, the intracellular signaling domain includes the cytoplasmic sequence of the T cell receptor (TCR) and a co-receptor and its functional derivatives and subfragments thereof that initiate signal transduction after antigen receptor engagement. The cytoplasmic signaling domain derived from the zeta-chain of the T cell receptor is used as a part of CAR to generate a stimulation signal for T lymphocyte proliferation and effector function after the chimeric receptor is bound to the target antigen. Examples of cytoplasmic signaling domains include, but are not limited to, the cytoplasmic domain of CD27, the cytoplasmic domain of CD28, the cytoplasmic domain of CD137 (also known as 4-1BB and TNFRSF9), the cytoplasmic domain of CD278 (also known as ICOS), the p110α, β or δ catalytic subunit of PI3 kinase, the human Cd3δ-chain, the CD134 cytoplasmic region (also known as OX40 and TNFζRSF4), FcεR1γ and β chains, MB1 (Igα) chain, B29 (Igβ) chain, etc.), CD3 polypeptides (δ, Δ and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) and other molecules involved in T cell transduction, such as CD2, CD5 and CD28.

[0081] In some embodiments, CAR can also provide a costimulatory domain. The term "costimulatory domain" refers to the stimulatory domain of CAR, usually an internal domain, which provides a secondary nonspecific activation mechanism through which the primary specific stimulation is propagated. The costimulatory domain refers to the part of the CAR that enhances memory cell proliferation, survival or development. Examples of costimulation include antigen-nonspecific T cell costimulation after antigen-specific signaling through the T cell receptor and antigen-nonspecific B cell costimulation after signaling through the B cell receptor. Costimulation, such as T cell costimulation, and the factors involved have been described in Chen & Flies. (2013) Nat Rev Immunol 13(4):227-42. In some embodiments disclosed herein, the CSD comprises one or more members of the TNFR superfamily, CD28, CD137 (4-1BB), CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), Lck, TNFR-1, TNFR-II, Fas, CD30, CD40, or a combination thereof.

[0082] CARs are generally classified as first, second, third, or fourth generation. The term first-generation CAR refers to a CAR in which the cytoplasmic domain transmits signals from antigen binding only through a single signaling domain, such as a signaling domain derived from a high-affinity receptor for IgE FcεRIγ or Cd3δ chain. This domain contains one or three immunoreceptor tyrosine-based activation motifs [ITAMs] for antigen-dependent T cell activation. ITAM-based activation signals give T cells the ability to lyse target tumor cells and secrete cytokines in response to antigen binding. The second-generation CAR also includes a costimulatory signal in addition to the CD3δ signal. Coincidentally delivering a costimulatory signal enhances cytokine secretion and anti-tumor activity induced by T cells transduced by CART. The costimulatory domain is typically membrane-proximal relative to the CD3δ domain. The third-generation CARs include a tripartite signaling domain that includes, for example, CD28, CD3δ, OX40, or 4-1BB signaling regions. In the fourth generation, or “armored” CAR T cells are further genetically modified to express or block molecules and / or receptors to enhance immune activity.

[0083] Examples of intracellular signaling domains included in the CAR of the present invention include (amino to carboxyl): CD3δ; CD28–41BB-CD3δ; CD28–OX40–CD3δ; CD28–41BB–CD3δ; 41BB–CD-28--CD3δ and 41BB–CD3δ.

[0084] The term CAR includes CAR variants, including but not limited to split CAR, switch CAR, bispecific or tandem CAR, inhibitory CAR (iCARs) and induced pluripotent stem (iPS) CAR-T cells.

[0085] The term "split CAR" refers to the extracellular part of CAR, where the ABD and cytoplasmic signaling domain are present on two separate molecules. CAR variants also include switch CARs, which are conditionally activatable CARs, such as split CARs, wherein the conditional heterodimerization of the two parts of the split CAR is pharmacologically controlled. CAR molecules and their derivatives (i.e., CAR variants), for example, in PCT application numbers US2014 / 016527, US1996 / 017060, US2013 / 063083; Fedorov et al. Sci Transl Med (2013); 5 (215): 215ra172; Glienke et al. Front Pharmacol (2015) 6: 21; Kakarla & Gottschalk 52 Cancer J (2014) 20 (2): 151-5; Riddell et al. Cancer J (2014) 20 (2): 141-4; Pegram et al. Cancer J (2014) 20(2):127-33; Cheadle et al. Immunol Rev (2014) 257(1):91-106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entirety.

[0086] The term "bispecific or tandem CAR" refers to a CAR that comprises a secondary CAR binding domain that can amplify or inhibit the activity of a primary CAR.

[0087] The term "inhibitory chimeric antigen receptor" or "iCARs" is used interchangeably herein and refers to a CAR in which dual antigen targeting is used in conjunction with iCARs to shut down the activation of the active CAR by binding to a second inhibitory receptor equipped with an inhibitory signaling domain bound by a secondary CAR, resulting in inhibition of primary CAR activation. Inhibitory CARs (iCARs) are designed to regulate CAR-T cell activity by activation of an inhibitory receptor signaling module. This approach combines the activity of two CARs, one of which produces a dominant negative signal that limits the response of CAR-T cells activated by an activating receptor. When bound to a specific antigen expressed only by normal tissues, the iCAR can shut down the response of the counteracting activator CAR. In this way, iCARs-T cells can distinguish cancer cells from healthy cells and reversibly block the function of transduced T cells in an antigen-selective manner. The CTLA-4 or PD-1 intracellular domains in iCARs trigger inhibitory signals on T lymphocytes, resulting in less cytokine production, less efficient target cell lysis, and changes in lymphocyte motility.

[0088] The term "tandem CAR" or "TanCAR" refers to a CAR that mediates bispecific activation of T cells through engagement of two chimeric receptors designed to deliver stimulatory or co-stimulatory signals in response to independent engagement of two different tumor-associated antigens.

[0089] Typically, chimeric antigen receptor T cells (CAR-T cells) are T cells that have been recombinantly modified by transduction with an expression vector encoding a CAR essentially as taught above.

[0090] The patient's own T cells can be used to prepare cells for engineering. Therefore, the cell population to be administered is necessarily variable to the subject. In addition, since CAR-T cell agents are variable, the response to these agents can also vary, so it is related to the continuous monitoring and management of the treatment-related toxicity before the administration of CAR-T cell therapy with pharmacological immunosuppression or B cell depletion process. This immunosuppressive regimen. Examples include systemic corticosteroids (such as methylprednisolone). B cell depletion therapy includes intravenous immunoglobulin (IVIG) through established clinical dosage guidelines to restore normal levels of serum immunoglobulin levels. In some embodiments, before administering the CAR-T cell therapy of the present invention, the subject can choose a lymphatic depletion regimen. An example of this lymphatic clearance regimen includes administering fludarabine (30 mg / m per day) 2 intravenous [IV] for 4 days) and cyclophosphamide (500 mg / m daily starting with the first dose of fludarabine 2 intravenously for 2 days).

[0091] T cells for engineering with constructs described herein include naive T cells, central memory T cells, effector memory T cells or a combination thereof. T cells for engineering as described above are collected from subjects or donors, or the donors can be separated from the cell mixture by a technique for enriching the desired cells, or they can be engineered and cultured without separation. Appropriate solutions can be used for dispersion or suspension. The solution is typically a balanced salt solution, for example, saline, PBS, Hank's balanced salt solution, etc., wherein fetal bovine serum or other naturally occurring factors are appropriately supplemented, and an acceptable buffer at a low concentration (e.g., 5-25 mM). Convenient buffers include HEPES, phosphate buffer, lactate buffer, etc. Affinity separation techniques may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents such as complement and cytotoxins connected to or used in combination with monoclonal antibodies, and "panning" with antibodies attached to a solid matrix (e.g., a plate) or other convenient techniques. Technologies that provide accurate separation include fluorescence-activated cell sorters, which can have varying degrees of complexity, such as multicolor channels, low-angle and dull light scattering detection channels, impedance channels, etc. This can be accomplished by using dyes associated with dead cells (e.g., propidium iodide). Any technique that does not unduly damage the viability of the selected cells can be used. Affinity reagents can be specific receptors or ligands for the above-mentioned cell surface molecules. In addition to antibody reagents, peptide-MHC antigens and T cell receptor pairs; peptide ligands and receptors; effector and receptor molecules, etc. can also be used.

[0092] The isolated cells can be collected in any suitable culture medium that maintains cell viability, usually with serum buffer at the bottom of the collection tube. A variety of culture media can be purchased and used according to the nature of the cells, including dMEM, HBSS, DPBS, RPMI, Iscove's culture medium, etc., which are often supplemented with fetal calf serum (FCS). The collected and optionally enriched cell populations can be used immediately for genetic modification, or can be frozen and stored at liquid nitrogen temperatures, thawed and reused. Cells are usually stored in 10% DMSO, 50% FCS, 40% RPMI 1640 culture medium.

[0093] In some embodiments, the engineered cells comprise a complex mixture of immune cells, such as tumor infiltrating lymphocytes (TILs) isolated from an individual in need of treatment. See, e.g., Yang and Rosenberg (2016) Adv Immunol. 130: 279-94, "Adoptive T Cell Therapy for Cancer"; Feldman et al. (2015) Semin Oncol. 42 (4): 626-39 "Adoptive Cell Therapy-Tumor-Infiltrating Lymphocytes, T-Cell Receptors, and Chimeric Antigen Receptors"; Clinical Trial NCT01174121, "Immunotherapy Using Tumor Infiltrating Lymphocytes for Patients With Metastatic Cancer"; Tran et al. (2014) Science 344 (6184) 641-645, "Cancer immunotherapy based on mutation-specific CD4+T cells in a patient with epithelial cancer".

[0094] In some embodiments, the engineered T cells are allogeneic with respect to the individual being treated, e.g., see clinical trials NCT03121625; NCT03016377; NCT02476734; NCT02746952; NCT02808442. See review by Graham et al. (2018) Cells. 7(10) E155. In some embodiments, the allogeneic engineered T cells are fully HLA-matched. However, not all patients have fully matched donors, and cell products that are suitable for all patients independent of HLA type provide an alternative. Universal "off-the-shelf" T cell products have advantages in terms of uniformity of harvesting and manufacturing.

[0095] As described above, the T cells for engineering collected from the subject or donor can be separated from the cell mixture by the technology of enriching the desired cells, or can be engineered and cultured without separation. Appropriate solutions can be used for dispersion or suspension. The solution is usually a balanced salt solution, for example, saline, PBS, Hank's balanced salt solution, etc., wherein fetal bovine serum or other naturally occurring factors are appropriately supplemented, and an acceptable buffer at a low concentration (for example, 5-25mM). Convenient buffers include HEPES, phosphate buffer, lactate buffer, etc. Affinity separation techniques may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents such as complement and cytotoxins connected to or used in combination with monoclonal antibodies, and "panning" with antibodies attached to solid matrices (such as plates) or other convenient techniques. The technology that provides accurate separation includes a fluorescence-activated cell sorter, which can have varying degrees of complexity, such as multicolor channels, low-angle and dull light scattering detection channels, impedance channels, etc. It can be achieved by using dyes associated with dead cells (such as propidium iodide). Any technology that does not cause undue damage to the vitality of the selected cells can be used. Affinity reagents can be specific receptors or ligands for the above-mentioned cell surface molecules. In addition to antibody reagents, peptide-MHC antigens and T cell receptor pairs; peptide ligands and receptors; effector and receptor molecules, etc. can also be used. The separated cells can be collected in any suitable culture medium that maintains cell viability, usually with a serum buffer at the bottom of the collection tube. A variety of culture media can be purchased and can be used according to the nature of the cells, including dMEM, HBSS, DPBS, RPMI, Iscove's medium, etc., which are often supplemented with fetal calf serum (FCS). The collected and optionally enriched cell populations can be used immediately for genetic modification, or can be frozen and stored at liquid nitrogen temperatures, thawed and able to be reused. Cells are typically stored in 10% DMSO, 50% FCS, 40% RPMI 1640 culture medium. Engineered cells can be infused into the subject by any convenient route of administration (usually intravascularly) in any physiologically acceptable culture medium, although they can also be introduced by other routes where the cells can find a suitable growth site. Typically, at least 1×10 6 cells / kg, at least 1×10 7 cells / kg, at least 1×10 8 cells / kg, at least 1×10 9 cells / kg, at least 1×10 10 T cells obtained during collection are often limited in number, with the average number of cells / kg or more being 100 cells / kg.

[0096] The allogeneic T cells used in the practice of the present invention can be genetically modified to reduce graft-versus-host disease. For example, the engineered cells of the present invention can be TCRαβ receptor knockouts achieved by gene editing technology. TCRαβ is a heterodimer, and both α and β chains need to be present for expression. A single gene encodes the α chain (TRAC), while there are 2 genes encoding the β chain, so the TRAC gene site KO has been deleted for this purpose. Many different methods have been used to achieve this deletion, such as CRISPR / Cas9; large-range nucleases; engineered I-CreI homing nucleases, etc. See, for example, Eyquem et al. (2017) Nature 543:113–117, in which the TRAC coding sequence is replaced by the CAR coding sequence; Georgiadis et al. (2018) Mol.Ther.26:1215–1227, which links CAR expression to TRAC fragments through regularly interspaced short palindromic repeats (CRISPR) / Cas9 without directly integrating CAR into the TRAC gene site. An alternative strategy to prevent GVHD modifies T cells to express inhibitors of TCRαβ signaling, such as using a truncated form of Cd3δ as a TCR inhibitory molecule.

[0097] The preparation of T cells useful in the practice of the present invention is carried out by transforming isolated T cells with an expression vector comprising a nucleic acid sequence encoding an orthogonal receptor; optionally in combination with the nucleic acid sequence encoding CAR as described above. The nucleic acid sequences encoding CAR and orthogonal receptors can each be provided on a separate expression vector, each nucleic acid sequence operably linked to one or more expression control elements to achieve the expression of CAR and orthogonal receptors in target cells, and the vector is co-transfected into the target cell. Alternatively, the nucleic acid sequences encoding CAR and orthogonal receptors can each be provided on a single vector of each nucleic acid sequence under the control of one or more expression control elements to achieve the expression of the related nucleic acid sequence. Alternatively, both nucleic acid sequences can be under the control of a single promoter, wherein the insertion or downstream control element promotes the co-expression of the two sequences with the vector.

[0098] Ex vivo T cell activation can be achieved by procedures recognized in the art, including cell-based T cell activation, antibody-based activation, or activation using various bead-based activation reagents. Cell-based T cell activation can be achieved by exposing T cells to antigen presenting cells such as dendritic cells or artificial antigen presenting cells such as irradiated K562 cells. Antibody-based activation of T cell surface CD3 molecules with soluble anti-CD3 monoclonal antibodies also supports T cell activation in the presence of IL-2.

[0099] Typically, the T cells of the present invention are expanded by culturing cells in contact with a surface that provides reagents (e.g., anti-CD3 antibodies) that stimulate CD3TCR complex-related signals and reagents (e.g., anti-CD28 antibodies) that stimulate co-stimulatory molecules on the surface of T cells. Magnetic bead-based T cell activation has been accepted in the art for the preparation of CAR-T cells for clinical use. Magnetic bead-based T cell activation can be achieved using commercially available T cell activation reagents, including but not limited to CTS CD3 / 28 (Life Technologies, Inc. Carlsbad CA) or Miltenyi GMP ExpAct Treg Magnetic Beads or Miltenyi MACS GMP TransAct TM CD3 / 28 magnetic beads (Miltenyi Biotec, Inc). Conditions suitable for T cell culture are well known in the art. Lin et al. (2009) Cytotherapy 11(7):912-922; Smith et al. (2015) Clinical & Translational Immunology 4:e31 published online on January 16, 2015. Maintain target cells under conditions necessary to support growth, such as appropriate temperature (37°C) and environment (e.g., air and 5% CO2).

[0100] When the orthogonal receptor or CAR-T cells expressing the orthogonal receptor are growth factor receptors, the CAR-T cells expressing the orthogonal receptor can also be selectively expanded from background or mixed populations of transduced and untransduced cells by using a ligand for the orthogonal receptor. In one embodiment, the orthogonal receptor is an orthogonal IL-2 receptor, and the orthogonal IL-2 compound used to expand such cells is an orthogonal IL-2 selected from the group in Table 1.

[0101] In this method, orthogonal proteins, particularly orthogonal cytokines, can be produced by recombinant methods. Orthogonal receptors can be introduced into the cells to be engineered on expression vectors. DNA encoding orthogonal proteins can be obtained from various sources designed during the engineering process.

[0102] As described herein, amino acid sequence variants are prepared by introducing appropriate nucleotide changes in the coding sequence. Such variants represent insertions, substitutions and / or specified deletions of the residues described. Any combination of insertions, substitutions and / or specified deletions may be performed to obtain the final construct, as long as the final construct has the desired biological activity as defined herein.

[0103] In order to achieve the expression of the recombinant protein, the nucleic acid encoding the orthogonal protein (and / or CAR) is inserted into a replicable vector for expression. Many such vectors are available. The vector components generally 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. Vectors include viral vectors, plasmid vectors, integration vectors, and the like.

[0104] The expression vector used to express the orthogonal receptor and optionally the CAR in the T cell can be a viral vector or a non-viral vector. Plasmids are examples of non-viral vectors. In order to promote transfection of target cells, the target cells can be directly exposed to non-viral vectors under conditions that promote uptake of non-viral vectors. Examples of conditions that promote uptake of exogenous nucleic acids by mammalian cells are well known in the art and include, but are not limited to, chemical means (e.g., Thermo Fisher Scientific), high salt and magnetic field (electroporation).

[0105] In one embodiment, the non-viral vector can be provided in a non-viral delivery system. Non-viral delivery systems are generally complexes that facilitate transduction of target cells with nucleic acid cargo, wherein the nucleic acid is combined with a cationic lipid (DOTAP, DOTMA), a surfactant, a biologic (gelatin, chitosan), a metal (gold, magnetic iron), and a synthetic polymer (PLG, PEI, PAMAM). Many embodiments of non-viral delivery systems are well known in the art, including lipid carrier systems (Lee et al. (1997) Crit Rev Ther Drug Carrier Syst. 14: 173-206); polymer coated liposomes (Marin d et al., U.S. Patent No. 5213804 issued on May 25, 1993; Woodle et al., U.S. Patent No. 5013556 issued on May 7, 1991); cationic liposomes (Epand et al., U.S. Patent No. 5283185 issued on February 1, 1994; Jessee, JA, U.S. Patent No. 5578475 issued on November 26, 1996; Rose et al., U.S. Patent No. 5279833 issued on January 18, 1994; Gebeyehu et al., U.S. Patent No. 5334761 issued on August 2, 1994).

[0106] In another embodiment, the expression vector can be a viral vector. When a viral vector system is used for CAR and expresses an orthogonal receptor, a retroviral or lentiviral expression vector is preferred. In particular, the viral vector is a gamma retrovirus (Pule, et al. (2008) Nature Medicine 14 (11): 1264-1270), self-inactivating lentiviral vectors (June, et al. (2009) Nat Rev Immunol 9 (10): 704-716) and a retroviral vector, as previously described, Naldini, et al. (1996) Science 272: 263-267; Naldini, et al. (1996) Proc. Natl. Acad. Sci. USA Vol. 93, pp. 11382-11388; Dull, et al. (1998) J. Virology 72(11):8463–8471; Milone, et al. (2009) 17(8):1453-1464; Kingsman, et al., U.S. Patent No. 6096538, issued August 1, 2000, and Kingsman et al., U.S. Patent No. 6924123, issued August 2, 2005. In one embodiment of the present invention, the CAR expression vector is a lentiviral vector available from Oxford Biomedica.

[0107] Transduction of T cells with expression vectors can be accomplished using techniques well known in the art, including but not limited to co-incubation of host T cells with viral vectors, electroporation and / or chemically enhanced delivery.

[0108] Orthogonal proteins can be produced not only directly by recombination, but also as fusion polypeptides with heterologous polypeptides, such as signal sequences or other polypeptides having specific cleavage sites at the N-terminus of mature proteins or polypeptides. Typically, the signal sequence can be a component of the vector, or it can be part of the coding sequence inserted into the vector. The heterologous signal sequence preferably selected is a signal sequence that is recognized and processed by the host cell (i.e., cleaved by a signal peptidase). In mammalian cell expression, the native signal sequence can be used, or other mammalian signal sequences, such as signal sequences of secreted polypeptides from the same or related species, and viral secretory leaders, such as the herpes simplex gD signal.

[0109] Expression vectors usually contain selection genes, also known as selection markers. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective medium. Host cells not transformed with a vector containing a selection gene will not survive in the culture medium. Typical selection gene-encoded proteins (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, neomycin, methotrexate, or tetracycline), (b) supplement nutritional deficiencies, or (c) provide key nutrients that are not available in complex culture media.

[0110] Expression vector will contain a promoter that is recognized by the host organism and operably connected to an orthogonal protein coding sequence. Promoter is a non-translated sequence (usually in about 100 to 1000bp) located upstream (5') of the start codon of the structural gene, which controls the transcription and translation of the specific nucleic acid sequence that is operably connected to it. Such promoters are generally divided into inducible and constitutive types. Inducible promoters are promoters that initiate increased transcription levels from the DNA under their control in response to some changes in culture conditions (e.g., the presence or absence of nutrients or temperature changes). As is well known, a large number of promoters recognized by a variety of potential host cells.

[0111] Transcription from the vector in a mammalian host cell can be controlled, for example, by promoters obtained from viral genomes, such as polyoma virus, fowl pox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses (e.g., murine stem cell virus), hepatitis B virus, and most preferably, from heterologous mammalian promoters, such as the actin promoter, PGK (phosphoglycerate kinase) or the immunoglobulin promoter Simian virus 40 (SV40), provided that these promoters are compatible with the host cell systems. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment, which also contains the SV40 viral origin of replication.

[0112] Transcription of higher eukaryotic organisms is usually increased by inserting enhancer sequences into vectors. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp, which act on promoters to increase their transcription. Enhancers are relatively direction and position independent and have been found within the 5' and 3' of the transcription unit, within introns, and within the coding sequence itself. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). However, enhancers from eukaryotic cell viruses are commonly used. Examples include the SV40 enhancer at the late stage of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer at the late stage of the replication origin, and the adenovirus enhancer. The enhancer can be spliced ​​into the expression vector at the 5' or 3' position of the coding sequence, but is preferably located at a site 5' from the promoter.

[0113] Expression vectors for eukaryotic host cells will also contain sequences necessary for termination of transcription and stabilization of mRNA. These sequences are generally available from 5' and occasionally 3' untranslated regions of eukaryotic or viral DNA or cDNA. The construction of suitable vectors containing one or more of the above components employs standard techniques.

[0114] Suitable host cells for cloning or expressing the DNA in the vectors herein are the above-mentioned prokaryotes, yeast or higher eukaryotic cells. Useful mammalian host cell lines include, for example, mouse L cells (LM[TK-], ATCC CRL-2648), monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cell subclones 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-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); tri-cells; MRC 5 cells; FS4 cells; and human hepatoma cell line (HepG2).

[0115] Host cells, including engineered T cells, can be transfected with the above expression vectors for orthogonal IL-2 or IL-2R expression. Cells can be cultured in conventional nutrient media suitable for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. Mammalian host cells can be cultured in a variety of culture media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI 1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing host cells. Any of these media can be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics, trace elements, and glucose or an equivalent energy source. Any other necessary supplements can also be included at appropriate concentrations known to those skilled in the art. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

[0116] A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, if the DNA of a signal sequence is expressed as a preprotein that participates in the secretion of a polypeptide, it is operably linked to the DNA of a polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, it is operably linked to a coding sequence; or if a ribosome binding site is positioned to promote translation, it is operably linked to a coding sequence. Generally, "operably linked" means that the DNA sequences being linked are continuous, and in the case of a secretory leader sequence, continuous and in the reading phase. However, enhancers do not have to be continuous.

[0117] Recombinantly produced orthogonal polypeptides can be recovered from the culture medium as secreted polypeptides, although they can also be recovered from host cell lysates. Protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF), can also be used to inhibit proteolytic degradation during purification, and antibiotics can be included to prevent the growth of foreign contaminants. Various purification steps are known in the art and can be used, for example, in affinity chromatography. Affinity chromatography uses highly specific binding sites, which are often present in biomacromolecules, to separate molecules' ability to bind to specific ligands. Covalent bonds attach ligands to insoluble porous carrier media in a manner that visibly presents the ligand to the protein sample, thereby using the natural biospecific binding of one molecular substance to separate and purify a second substance from a mixture. Antibodies are often used in affinity chromatography. Size selection steps can also be used, such as using gel filtration chromatography (also known as size exclusion chromatography or molecular sieve chromatography) to separate proteins according to their size. In gel filtration, a protein solution is passed through a column filled with a semipermeable porous resin. The semipermeable resin has a range of pore sizes that determine the size of the protein that can be separated from the chromatographic column. The same is true for cation exchange chromatography.

[0118] Orthogonal cytokine compositions can be concentrated, filtered, dialyzed, etc. using methods known in the art. For therapeutic applications, the cytokines can be administered to mammals containing appropriately engineered orthogonal receptors. Intravenous administration, as a bolus or by continuous infusion for a period of time. Alternative routes of administration include intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical or inhalation routes. Orthogonal cytokines are also appropriately administered via intratumoral, peritumoral, intralesional or perilesional routes or lymphatic administration to exert local and systemic therapeutic effects.

[0119] This dosage form includes physiologically acceptable carriers, which are essentially nontoxic and non-therapeutic. The example of this type of carrier includes ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based materials and PEG. Carriers for local or gel-based polypeptide forms include polysaccharides, such as sodium carboxymethylcellulose or methylcellulose, polyvinyl pyrrolidone, polyacrylates, polyoxyethylene-polyoxyethylene block polymers, PEG and wood wax alcohol. For all administrations, traditional warehouse forms are appropriately used. These forms include, for example, microcapsules, nanocapsules, liposomes, plasters, inhalation formulations, nasal sprays, sublingual tablets and sustained-release preparations. The polypeptide is typically formulated in such a vehicle at a concentration of about 0.1 μg / ml to 100 μg / ml.

[0120] If the orthologous IL-2 polypeptides of the present disclosure are "substantially pure", they can be at least about 60% (dry weight) of the weight of the polypeptide of interest, such as a polypeptide containing an orthologous IL-2 amino acid sequence. For example, the polypeptide can be at least about 75%, about 80%, about 85%, about 90%, about 95% or about 99% of the weight of the polypeptide of interest. Purity can be measured by any suitable standard method, such as column chromatography, polyacrylamide gel electrophoresis or HPLC analysis.

[0121] In another embodiment of the present invention, an article of manufacture containing materials for treating the above conditions is provided. The article of manufacture includes a container and a label. Suitable containers include, for example, bottles, vials, syringes and test tubes. The container can be formed of various materials, such as glass or plastic. The container holds a composition that is effective in treating the condition and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The active agent in the composition is an orthogonal cytokine. The label on or associated with the container indicates that the composition is used to treat the selected condition. Additional containers can be provided with the article of manufacture, which can contain, for example, a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution or dextrose solution. The article of manufacture can further include other materials required from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes and package inserts with instructions for use.

[0122] As used herein, the terms "cancer" (or "cancerous"), "hyperproliferative" and "neoplastic" refer to cells that have the capacity for autonomous growth (e.g., an abnormal state or condition characterized by rapidly proliferating cell growth). Hyperproliferative and neoplastic disease states can be classified as pathological (e.g., characterizing or constituting a disease state), or they can be classified as non-pathological (e.g., as a deviation from normal but unrelated to a disease state). These terms are intended to include all types of cancerous growths or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. "Pathological hyperproliferative" cells occur in disease states characterized by malignant tumor growth. Examples of non-pathological hyperproliferative cells include cell proliferation associated with wound repair. The terms "cancer" or "tumor" refer to malignancies of various organ systems, including those affecting the lung, breast, thyroid, lymph glands and lymphoid tissue, gastrointestinal organs, and genitourinary tract, as well as adenocarcinomas generally considered to include malignancies, such as most colon cancers, renal cell carcinomas, prostate cancer and / or testicular tumors, non-small cell carcinoma of the lung, small intestine cancer, and esophageal cancer.

[0123] The term "carcinoma" is art-recognized and refers to malignant tumors of epithelial or endocrine tissue, including cancers of the respiratory system, gastrointestinal system, genitourinary system, testicular cancer, breast cancer, prostate cancer, endocrine system cancer, and melanoma. "Adenocarcinoma" refers to a cancer that originates from glandular tissue or in which the tumor cells form identifiable glandular structures.

[0124] Examples of tumor cells include, but are not limited to, AML, ALL, CML, adrenocortical carcinoma, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, brain cancer, central nervous system (CNS) cancer, peripheral nervous system (PNS) cancer, breast cancer, cervical cancer, childhood non-Hodgkin lymphoma, colon and rectal cancer, endometrial cancer, esophageal cancer, Ewing's family of tumors (e.g., Ewing's sarcoma), eye cancer, gallbladder cancer, gastrointestinal carcinoid, gastrointestinal stromal tumors, gestational trophoblastic disease, Hodgkin lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, lung cancer, non-Hodgkin lymphoma, male Breast cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, myeloproliferative disease, nasal and paranasal cancer, nasopharyngeal carcinoma, neuroblastoma, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, melanoma skin cancer, non-melanoma skin cancer, gastric cancer, testicular cancer, thymic cancer, thyroid cancer, uterine cancer (e.g., uterine sarcoma), transitional cell cancer, vaginal cancer, vulvar cancer, mesothelioma, squamous cell or epidermoid carcinoma, bronchial adenoma, choriocarcinoma, head and neck cancer, teratocarcinoma, or Waldenstrom's macroglobulinemia. Any cancer in which cancer cells exhibit increased expression of CD47 compared to non-cancerous cells is a suitable cancer to be treated by the subject methods and compositions.

[0125] The compositions and methods of the present invention can be combined with additional therapeutic agents. For example, when the disease, condition or disorder to be treated is a tumor disease (e.g., cancer). The methods of the present invention can be combined with conventional chemotherapeutic agents or other biological anticancer drugs such as checkpoint inhibitors (e.g., PD1 or PDL1 inhibitors) or therapeutic monoclonal antibodies (e.g., Avastin, Herceptin).

[0126] Examples of chemical agents identified in the art as useful for treating neoplastic diseases include, but are not limited to, abiraterone, doxorubicin, doxorubicin, ampicillin, asparaginase, anthracyclines, azacitidine, azathioprine, BiCNU, bleomycin sulfate, busulfan, bleomycin, camptothecin, camptothecin, carboplatin, carmustine, daunorubicin hydrochloride, chlorambucil, cisplatin, cladribine, dactinomycin, cytarabine, cedaxa, cyclophosphamide, cyclophosphamide, actinomycin, docetaxel, doxorubicin, daunorubicin, epirubicin, escitabine, epirubicin, etoposide, fludarabine, fluorouracil, fludarabine, gemcitabine, gizole, mesin, hydroxyurea, Aiz, idarubicin, idarubicin, ifosfamide, cyclophosphamide, irinotecan, lanvis, interleukin, leuprostatin, procarbazine, nitrogen mustard, mercaptopurine, methotrexate, mitomycin, mitoxantrone, mithramycin, mitomycin, malelan, atrocytidine, navelbine, pentostatin, mitoxantrone hydrochloride, vinblastine, oxaliplatin, paclitaxel, berdine, deoxycoformycin, cisplatin, plicamycin, procarbazine, leucoderma, raltitrexed, taxotere, taxol, teniposide, thioguanine, raltitrexed, topotecan, valrubicin, vinblastine, vanbiprofen, vinblastine, vindesine, vincristine, vinorelbine, VP-16 and tifenoxaline.

[0127] Targeted therapies that can be given in combination may include tyrosine kinase inhibitors such as imatinib mesylate (Gleevec, also known as STI-571), gefitinib (Iressa, also known as Zd1839), erlotinib (marketed as Tarceva), sorafenib (Nexavar), sunitinib (Sutent), dasatinib (Sprycel), lapatinib (Tykerb), nilotinib (Tasigna), and bortezomib (Velcade), ruxolitinib; Janus kinase inhibitors such as tofacitinib; ALK inhibitors such as crizotinib; Bcl-2 inhibitors such as imatinib, venetonola, and gossypol; FLT3 inhibitors such as midostaurin (Rydapt); IDH inhibitors such as AG-221 , PARP inhibitors such as Iniparib and Olaparib; PI3K inhibitors such as Perifosine; VEGF receptor 2 inhibitors such as Apatinib; AN-152 (AEZS-108) doxorubicin linked to [D-Lys(6)]-LHRH; Braf inhibitors such as Vemurafenib, Dabrafenib and LGX818; MEK inhibitors such as Trametinib; CDK inhibitors such as PD-0332991 and LEE011; Hsp90 inhibitors such as Salinomycin; and / or small molecule drug conjugates such as Vinorelbine; Serine / threonine kinase inhibitors such as Temsirolimus (Torisel), Everolimus (Afinitor), Vemurafenib (Zelboraf), Trametinib (Mekinist) and Dabrafenib (Tafinlar).

[0128] Examples of biologics identified in the art for treating neoplastic diseases include, but are not limited to, cytokines or cytokine antagonists such as IL-12, INFα or anti-epidermal growth factor receptor, radiation therapy, irinotecan; tetrahydrofolate antimetabolites such as pemetrexed; antibodies, monoclonal antibodies and toxins against tumor antigens, T cell adjuvants, bone marrow transplantation or antigen presenting cells (e.g., dendritic cell therapy), anti-tumor vaccines, replication-competent viruses, signal transduction inhibitors (e.g., or ) or immunomodulators to achieve additive or synergistic inhibition of tumor growth, cyclooxygenase 2 (COX-2) inhibitors, steroids, TNF antagonists (e.g. and ), interferon-β1a and interferon-β1b (Betaseron), as well as combinations of one or more of the above approaches, which are used in known chemotherapy regimens readily appreciated by the skilled clinician.

[0129] Tumor-specific monoclonal antibodies that can be administered in combination with anti-CD93 ABD polypeptides or engineered cells can include, but are not limited to, rituximab (marketed as MabThera or Rituxan), alemtuzumab, panitumumab, ipilimumab (Yervoy), and the like.

[0130] In some embodiments, the compositions and methods of the present invention can be combined with immune checkpoint therapy. Examples of immune checkpoint therapy include inhibitors that bind PD1 to PDL1 and / or PDL2. PD1 and PDL1 and / or PDL2 inhibitors are well known in the art. Examples of commercially available monoclonal antibodies that interfere with the binding of PD1 to PDL1 and / or PDL2 include nivolumab ( BMS-936558, MDX1106, purchased from BristolMyers Squibb, Princeton Nj), pembrolizumab ( MK-3475, lambrolizumab, purchased from Merck and Company, Kenilworth Nj), and atezolizumab ( Other examples of PD1 inhibitory antibodies include, but are not limited to, durvalumab (MEDI4736, Medimmune / AstraZeneca), pidilizumab (CT-011, CureTech), PDR001 (Novartis), BMS-936559 (MDX1105, Bristol-Myers Squibb), and avelumab (MSB0010718C, Merck Serono / Pfizer) and SHR-1210 (Incyte). Other PD1 pathway inhibitor antibodies are disclosed in U.S. Patent No. 8217149 (Genentech, Inc.), issued on July 10, 2012; U.S. Patent No. 8168757 (Merck Sharp and Dohme Corp.), issued on May 1, 2012; U.S. Patent No. 8008449 (Medarex), issued on August 30, 2011; and U.S. Patent No. 7943743 (Medarex, Inc.), issued on May 17, 2011. In addition, small molecule PD1 and PDL1 and / or PDL2 inhibitors are well known in the art. See Sasikumar, et al. WO2016142833A1 and Sasikumar, et al. WO2016142886A2, BMS-1166 and BMS-1001 (Skalniak, et al. (2017) Oncotarget 8(42):72167-72181).

[0131] In other embodiments, the methods of the present invention are used to treat infection. As used herein, the term "infection" refers to any state in which at least one cell of an organism (i.e., a subject) is infected with an infectious agent (e.g., a subject has an intracellular pathogen infection, such as a chronic intracellular pathogen infection). As used herein, the term "infectious agent" refers to a foreign biological entity (i.e., a pathogen) that induces increased CD47 expression in at least one cell of an infected organism. For example, infectious agents include, but are not limited to, bacteria, viruses, protozoa, and fungi. Intracellular pathogens are particularly interesting. Infectious diseases are diseases caused by infectious agents. Some infectious agents do not cause recognizable symptoms or diseases under certain conditions, but may cause symptoms or diseases under changing conditions. The subject methods can be used to treat chronic pathogen infections, for example, including but not limited to viral infections, such as retroviruses, lentiviruses, hepatitis viruses, herpes viruses, poxviruses, human papillomaviruses, etc.; intracellular bacterial infections, such as mycobacteria, chlamydia, ehrlichia, rickettsia, Brucella, Legionella, Francisella, Listeria, Coxsackie, Neisseria, Salmonella, Yersinia, Helicobacter pylori, etc.; and intracellular protozoan pathogens, such as Plasmodium, Trypanosoma, Giardia, Toxoplasma, Leishmania, etc.

[0132] Treatment can be combined with other active agents. Antibiotic classes include penicillins, such as penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafcillin, ampicillin, etc.; penicillins in combination with β-lactamase inhibitors, cephalosporins, such as cefaclor, cefazolin, cefuroxime, moxalactam, etc.; carbapenems; monocyclic amines; aminoglycosides; tetracyclines; macrolides; lincomycins; polymyxins; sulfonamides; quinolones; chloranil; metronidazole; spectinomycin; trimethoprim; vancomycin, etc. Cytokines, such as interferon gamma, tumor necrosis factor alpha, interleukin 12, etc., may also be included. Antiviral agents, such as acyclovir, ganciclovir, etc., may also be used for treatment.

[0133] In other embodiments, regulatory T cells are designed to treat autoimmune diseases. Inflammatory diseases and diseases associated with inflammation range widely, including autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), and autoimmune hepatitis; insulin-dependent diabetes, degenerative diseases such as osteoarthritis (Oa), Alzheimer's disease (AD), and macular degeneration.

[0134] Many, if not most, autoimmune and inflammatory diseases involve various types of T cells, such as TH1, TH2, TH17, etc. Autoimmune diseases are characterized by abnormal targeting of self-proteins, polypeptides, peptides, and / or other self-molecules by T and B lymphocytes, resulting in damage and / or dysfunction of organs, tissues, or cell types in the body (e.g., pancreas, brain, thyroid, or gastrointestinal tract) leading to the clinical manifestations of the disease. Autoimmune diseases include those that affect specific tissues as well as those that can affect multiple tissues, which can depend in part on whether the response is to an antigen that is confined to a specific tissue or to an antigen that is widely distributed throughout the body.

[0135] The present invention provides engineered orthogonal cytokine receptor / ligand pairs and methods of use thereof. Engineered (orthogonal) cytokines specifically bind to corresponding engineered (orthogonal) receptors. After binding, orthogonal receptors activate signal transduction by natural cell elements to provide biological activity that simulates the natural response but is specific to engineered cells expressing orthogonal receptors. Orthogonal receptors exhibit a significant reduction in binding to endogenous corresponding cytokines (including natural counterparts of orthogonal cytokines), while orthogonal cytokines exhibit a significant reduction in binding to any endogenous receptor (including natural counterparts of orthogonal receptors). In some embodiments, the affinity of orthogonal cytokines to orthogonal receptors is comparable to the affinity of natural cytokines to natural receptors.

[0136] Orthogonal cytokines and receptor pairs can be selected from any target cytokine. The process of designing orthogonal cytokine receptor pairs may include the following steps: (a) engineering amino acid changes to natural receptors to disrupt binding to natural cytokines; (b) engineering amino acids to natural cytokines at contact residues for receptor binding, (c) selecting cytokine orthologs that bind to orthogonal receptors; (d) discarding orthologous cytokines that bind to natural receptors, or (e) selecting receptor orthologs that bind to orthologous cytokines; (f) discarding orthologous receptors that bind to natural cytokines. In a preferred embodiment, knowledge of the cytokine / receptor complex structure is used to select amino acid positions for site-directed or error-prone mutations. Yeast display systems can be conveniently used for the selection process, although other display and selection methods are also useful.

[0137] In some cases, the amino acid changes are obtained by affinity maturation. An "affinity matured" polypeptide is a polypeptide having one or more changes in one or more residues that result in an improved affinity of the orthogonal polypeptide for the cognate orthogonal receptor compared to a parent polypeptide that does not have these changes, or vice versa. Affinity maturation can be performed to increase binding affinity by at least about 10% to 50-100-150% or more, or 1 to 5 times, compared to the "parent" polypeptide. As described above, the engineered orthogonal cytokines of the present invention activate orthogonal receptors, but have significantly reduced binding and activation of natural receptors, for example, the orthogonal cytokines may exhibit less than about 5% inhibition in competitive inhibition with the corresponding natural cytokines when evaluated by ELISA and / or FACS analysis using sufficient amounts of molecules under appropriate assay conditions.

[0138] In some embodiments of the present invention, the orthogonal receptor is an IL-2 receptor chain, i.e., a polypeptide selected from interleukin 2 receptor alpha (IL-2Rα; Cd25), interleukin 2 receptor beta (IL-2Rβ; CD122), and interleukin 2 receptor gamma (IL-2Rα; CD132; common gamma chain). In some specific embodiments, the orthogonal receptor is Cd132, which participates in signal transduction from IL-2, IL-4, IL-7, and IL-15. In other specific embodiments, the orthogonal receptor is Cd122, which participates in signal transduction from IL-2 and IL-15. Orthogonal receptors are typically paired with corresponding orthogonal cytokines, such as IL-2, IL-4, IL-7, IL-15, etc.

[0139] In some specific embodiments, orthogonal receptor is CD122. In some such embodiments, orthogonal receptor is introduced into T cells or NK cells that can also express CD25 and / or CD132. The nucleic acid coding sequence and protein composition of modified CD122 protein are provided. In the present invention, CD122 is engineered to replace the amino acid of the native sequence with non-natural amino acids at the position related to binding with natural IL-2 or to destroy the binding of natural cytokines by deleting natural amino acids. In certain embodiments, amino acid is replaced by non-conservative changes. The target position replaced or deleted includes but is not limited to human CD122 (hCD122) R41, R42, Q70, K71, T73, T74, V75, S132, H133, Y134, F135, E136, Q214. Target positions for substitution or deletion include, but are not limited to, mouse CD122 (mCD122) R42, F67, Q71, S72, T74, S75, V76, S133, H134, Y135, I136, E137, R215.

[0140] In some embodiments, CD122 is substituted at one or a combination of positions selected from Q71, T74, H134, Y135 in mouse protein or Q70, T73, H133, Y134 in human protein. In some embodiments, the engineered protein comprises amino acid substitutions at mCD122 H134 and Y135; or hCD122 H133 and Y134. In some embodiments, the amino acid substitution is an acidic amino acid, such as aspartic acid and / or glutamic acid. Specific amino acid substitutions include but are not limited to mCD122 substitutions Q71Y; T74D; T74Y; H134D, H134E; H134K; Y135F; Y135E; Y135R; and hCD122 changes Q70Y; T73D; T73Y; H133D, H133E; H133K; Y134F; Y134E; Y134R. The choice of orthogonal cytokine can vary with the choice of orthogonal receptor.

[0141] In some embodiments, the orthogonal receptor is CD122 and the orthogonal cytokine is IL-2 or IL-15. The cytokine can be selected to bind to the orthogonal receptor, for example, by yeast display evolution, error-prone or targeted mutagenesis, etc. A representative set of selected orthogonal sequences is shown in FIG. Figure 6 shown.

[0142] In some embodiments, the orthogonal cytokine is IL-2. In some embodiments, one or more of the following amino acid residues are substituted with an amino acid other than the native protein, or deleted at that position: mouse IL-2 (mIL-2), any of H27, L28, E29, Q30, M33, D34, Q36, E37, R41, N103; human IL-2 (hIL-2), Q13, L14, E15, H16, L19, D20, Q22, M23, G27, R81, N88. In some such embodiments, the amino acid substitution group is selected from (for mIL-2) one or more of E29, Q30, M33, D34, Q36 and E37; and for hIL-2, E15, H16, L19, D20, Q22, M23, R81.

[0143] In some embodiments, the amino acid substitutions of mIL-2 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 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]; Substitutes are one or more of the following: [Q13W], [L14M, L14W], [E15D, E15T, E15A, E15S], [H16N, H16Q], [L19V, L19I, L19A], [D20L, D20M], [Q22S, Q22T, Q22E, Q22K, Q22E], [M23A, M23W, M23H, M23Y, M23F, M23Q, M23Y], [G27K, G27S], [R81D, R81Y], [N88E, N88Q], [T51I]. In some embodiments, the set of amino acid substitutions comprises one of the following mIL-2 substitution groups: [Q30N, M33V, D34N, Q36T, E37H, R41K]; [E29D, Q30N, M33V, D34L, Q36T, E37H]; [E29D, Q30N, M33V, D34L, Q36T, E37A], and [E29D, Q30N, M33V, D34L, Q36T, E37A] 36K, E37A] and the following substitutions of hIL-2: [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 conservative variants thereof.

[0144] 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 mutation group of hIL-2 is [E15S, H16Q, L19V, D20T / S / M; Q22K; M23L / S]. In some embodiments, the consensus mutation group of hIL-2 is [E15S, H16Q, L19V, D20L, M23Q / a] and optionally Q22K.

[0145] In some embodiments, the amino acid substitution group comprises one of the following hIL-2 substitution groups: [E15S; H16Q; L19V, D20T / S; Q22K, M23L / S]; [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]; [L19V; D20M; Q22N; M23S]; [E15S,H16Q,L19V,D20L, M23Q,R81D,T51I],[E15S,H16Q,L19V,D20L,M23Q,R81Y],[E15S,H16Q,L19V,D20L,Q22K,M23A],[E15S,H16Q,L19V,D20L,M23A].

[0146] A method for enhancing cellular responses is provided by engineering cells from a recipient or donor by introducing an orthogonal receptor of the present invention and stimulating the orthogonal receptor by contacting the engineered cells with a cognate orthogonal cytokine. The subject method includes the step of obtaining target cells, such as T cells, hematopoietic stem cells, etc., which can be isolated from a biological sample or can be derived in vitro from a progenitor cell source. Cells are transduced or transfected with an expression vector comprising a sequence encoding an orthogonal receptor, which step can be performed in any suitable culture medium.

[0147] In some embodiments, an engineered cell is provided wherein the cell is modified by introducing an orthogonal receptor of the invention. Any cell may be used for this purpose. In some embodiments, the cell is a T cell, including but not limited to a naive CD8 T cell. + 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 11. T H 22. T FH Regulatory T cells, such as T R 1. Natural T reg , Inducible Reg , inducible T reg;Memory T cells, such as central memory T cells, effector memory T cells, NKT cells, γδT cells;etc.In other embodiments the engineered cell is a stem cell, e.g. a hematopoietic stem cell, or an NK cell.In some embodiments the cell is genetically modified in an ex vivo procedure before being transferred into a subject. The engineered cells can be provided in a unit dose for treatment, and can be allogeneic, autologous, etc. relative to the intended recipient.

[0148] Cells, such as cells collected from a subject, can be separated from mixed cells by techniques that enrich the desired cells. Appropriate solutions can be used for dispersion or suspension. The solution is typically a balanced salt solution, such as physiological saline, PBS, Hank's balanced salt solution, etc., which is appropriately supplemented with fetal bovine serum or other naturally occurring factors, and an acceptable buffer at a low concentration (e.g., 5-25 mM). Convenient buffers include HEPES, phosphate buffer, lactate buffer, etc. Alternatively, engineered cell lines, amplified allogeneic cells, etc. are used for engineering.

[0149] Affinity separation techniques may include magnetic separation, use of antibody-coated magnetic beads, affinity chromatography, cytotoxic agents linked to or used in conjunction with monoclonal antibodies (e.g., complement and cytotoxins), and "panning" with antibodies attached to a solid matrix (e.g., a plate), or other convenient techniques. Technologies that provide accurate separation include fluorescence-activated cell sorters, which may have varying degrees of complexity, such as multicolor channels, low-angle and dull light scattering detection channels, impedance channels, and the like. Cells may be selected for dead cells by using dyes associated with dead cells (e.g., dead cells) (e.g., propidium iodide). Any technique that does not cause undue damage to the viability of the selected cells may be employed. Affinity reagents may be specific receptors or ligands for the above-mentioned cell surface molecules. In addition to antibody reagents, peptide-MHC antigens and T cell receptor pairs; peptide ligands and receptors; effector and receptor molecules, and the like may also be used.

[0150] The isolated cells can be collected in any suitable culture medium that maintains cell viability, typically with a serum buffer at the bottom of the collection tube. A variety of culture media can be purchased and used depending on the nature of the cells, including dMEM, HBSS, DPBS, RPMI, Iscove's medium, etc., which are often supplemented with fetal calf serum (FCS).

[0151] The collected and optionally enriched cell population can be used immediately or can be frozen and stored at liquid nitrogen temperature, thawed and able to be reused. Cells are typically stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium.

[0152] In certain embodiments, a vector comprising a coding sequence encoding an orthogonal receptor is provided, wherein the coding sequence is operably connected to a promoter active in the desired cell. Various vectors are known in the art and can be used for this purpose, such as viral vectors, plasmid vectors, microcirculation vectors, and their vectors can be integrated into the target cell genome, or episomes can be maintained. The receptor encoding vector can be provided in a test kit, combined with a vector encoding an orthogonal cytokine that binds and activates the receptor. In certain embodiments, the coding sequence of the orthogonal cytokine is operably connected to a high expression promoter, and can be optimized for production. In other embodiments, a test kit is provided, wherein the vector encoding the orthogonal receptor provides a purified composition of the orthogonal cytokine, such as a unit dose, packaged for patient administration.

[0153] In certain embodiments, a method of treatment is provided, the method comprising introducing an engineered cell population into a receptor in need thereof, wherein the cell population is modified by introducing a sequence encoding an orthogonal receptor of the present invention. The cell population can be engineered in vitro, and is generally autologous or allogeneic relative to the receptor. In certain embodiments, after administering the engineered cells, the introduced cell population is contacted with a homologous orthogonal cytokine in vivo. An advantage of the present invention is the lack of cross-reactivity between the orthogonal cytokine and the natural receptor.

[0154] In the case of in vitro contact of cells with orthogonal cytokines, the cytokines are added to the engineered cells at a dose and for a period of time sufficient to activate signaling from receptors that may utilize native cellular machinery, such as accessory proteins, co-receptors, etc. Any suitable culture medium may be used. Thus, the activated cells may be used for any desired purpose, including experimental purposes related to determining antigen specificity, cytokine profiles, etc., as well as for in vivo delivery.

[0155] When contacting in vivo, an effective dose of engineered cells, including but not limited to CAR-T cells modified to express an orthogonal Il-2β receptor, is combined with the administration of an orthogonal cytokine (e.g., Il-2) and allowed to contact the T cells in their native environment, such as in lymph nodes, etc. The dose and frequency may vary depending on the agent, the mode of administration, the nature of the cytokine, etc. Those skilled in the art will understand that such guidance will be adjusted according to individual circumstances. For local administration, such as intranasal, inhalation, etc., or for systemic administration, such as intramuscular injection, peritoneal injection, intravenous injection, etc., the dose may also vary. Usually at least about 10 4engineered cells / kg, at least about 10 5 engineered cells / kg; at least about 10 6 engineered cells / kg, at least about 10 7 engineered cells / kg or more.

[0156] In the case where the engineered cells are T cells, the enhanced immune response can be manifested as an increase in the cytolytic response of the T cells to target cells present in the receptor, such as elimination of tumor cells, infected cells; reduction of symptoms of autoimmune diseases, etc.

[0157] Engineered T cells can be provided in the form of pharmaceutical compositions suitable for therapeutic use, for example, pharmaceutical compositions suitable for human treatment. The therapeutic preparations comprising these cells can be frozen, or prepared into physiologically acceptable carriers, excipients or stabilizers (Remington, Pharmacy, 16th edition, Osol, A.Ed. (1980)) in the form of aqueous solutions to prepare therapeutic preparations, which are stored in the form of aqueous solutions. The preparation, administration and mode of application of cells should comply with good medical management practices. Factors to be considered in this case include the specific disease being treated, the specific mammal being treated, the clinical condition of individual patients, the cause of the disease, the delivery site of the medicament, the method of administration, the dosing schedule, and other factors known to doctors.

[0158] The cells can be administered by any suitable means, typically parenterally. Parenteral administration includes intramuscular, intravenous (bolus or slow infusion), intraarterial, intraperitoneal, intrathecal or subcutaneous administration.

[0159] The engineered T cells can be infused into the subject in any physiologically acceptable medium, typically intravascularly, although they can also be introduced into any other convenient site where the cells can find a suitable growth site. Typically, at least 1x10 6 cells / kg, at least 1x10 7 cells / kg, at least 1x10 8 cells / kg, at least 1x10 9 cells / kg, at least 1x10 10 cells / kg or more, usually limited by the number of T cells obtained during collection.

[0160] For example, a typical range of cell administration for the practice of the present invention is about 1 x 10 cells per kg of subject body weight per treatment course. 5 Up to 5x10 8 Therefore, a typical range of viable cells administered to human subjects, adjusted for body weight, is approximately 1x10 6 About 1x10 13viable cells, or about 5x10 6 About 5x10 12 viable cells, or about 1x10 7 About 1x10 12 viable cells, or from approximately 5x10 7 to about 1x10 12 viable cells, or from approximately 1x10 8 to about 1x10 12 viable cells, or from approximately 5x10 8 to about 1x10 12 viable cells, or approximately 1x10 per treatment 9 to about 1x10 12 In one embodiment, the cell dose per treatment course is 2.5-5x10 9 within the range of living cells.

[0161] A course of treatment can be a single dose or multiple doses within a period of time. In certain embodiments, cells are administered in a single dose. In certain embodiments, cells are administered in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 60, 90, 120 or 180 days with two or more split doses. The amount of the engineering cells used in this fractionated dosing regimen can be the same in each administration, or can be provided at different levels. Considering the reaction of the subject to the treatment, including the adverse reactions of the treatment as described above and its adjustment, the technician (e.g., doctor) monitoring the administration of cells can provide a multi-day dosing regimen over time.

[0162] For example, in the current clinical practice of CAR-T cell therapy, CAR-T cells are often administered in combination with lymphocyte clearance (e.g., by administering alemtuzumab (monoclonal anti-Cd52), purine analogs, etc.) to promote CAR-T cell expansion until the host immune recovery. In some embodiments, CAR-T cells can be modified to resist alemtuzumab. In one aspect of the present invention, lymphocyte clearance currently used in conjunction with CAR-T therapy can be eliminated or reduced by expressing an orthogonal ligand of the CAR-Ts of the present invention. As described above, lymphocyte clearance is commonly used for CAR-T cell expansion. However, lymphocyte clearance is also associated with the major side effects of CAR-T cell therapy. Because orthogonal ligands provide a means for selectively amplifying specific T cell populations, the need for lymphocyte clearance before administering an orthogonal ligand expressing CAR-Ts can be reduced. The present invention is capable of implementing CAR-T cell therapy without or with reduced lymphocyte clearance before administering an orthogonal ligand expressing CAR-Ts.

[0163] In one embodiment, the present invention provides a method for treating a subject with a disease, and before administering orthogonal ligand CAR-Ts, by administering an orthogonal ligand expressing CAR-Ts in the absence of lymphocyte clearance, a condition or condition that can be treated with CAR-T cell therapy (e.g., cancer). In one embodiment, the present invention provides a method for treating a mammalian subject with a disease, the disease being associated with the presence of an abnormal cell population (e.g., a tumor), the cell population being characterized by the expression of one or more surface antigens (e.g., tumor antigens), the method comprising the following steps: (a) obtaining a biological sample containing T cells from an individual; (b) enriching the biological sample for the presence of T cells; (c) transfecting T cells with one or more expressions. The vector comprises a nucleic acid sequence encoding a CAR and a nucleic acid sequence encoding an orthogonal receptor, the antigen targeting domain of the CAR being able to bind to at least one antigen present on an abnormal cell population; (d) ex vivo amplification of a CAR-T cell population expressing an orthogonal receptor; administering (e) a pharmaceutically effective amount of CAR-T cells expressing an orthogonal receptor to a mammal; and (f) regulating the growth of CAR-T cells expressing an orthogonal receptor using a ligand that selectively binds to an orthogonal receptor expressed on a CAR-T cell. In one embodiment, the aforementioned method is related to lymphocyte clearance or immunosuppression of the mammal before the CAR-T cell therapy process begins. In another embodiment, the aforementioned method is implemented in the absence of lymphocyte clearance and / or immunosuppression in the mammal.

[0164] The preferred formulation depends on the intended mode of administration and therapeutic application. Depending on the desired formulation, the composition may also include a pharmaceutically acceptable non-toxic carrier or diluent, which is defined as a carrier commonly used to formulate a pharmaceutical composition for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate buffered saline, Ringer's solution, dextrose solution and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc.

[0165] In some other embodiments, the pharmaceutical composition may also include slowly metabolized macromolecules, such as proteins, polysaccharides such as chitosan, polylactic acid, polyglycolic acid, and copolymers (e.g., latex-functionalized Sepharose TM , agarose, cellulose, etc.), polymeric amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes).

[0166] Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, including buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride); hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN TM 、PLURONICS TM or polyethylene glycol (PEG).

[0167] Preparations for in vivo administration are generally sterile. Sterilization of the compositions of the present invention can be accomplished by filtration through sterile filtration membranes.

[0168] Typically, the composition is prepared as an injection, which can be a liquid solution or suspension; a solid form suitable for dissolution or suspension in a liquid carrier prior to injection can also be prepared. As described above, the preparation can also be emulsified or encapsulated in liposomes or microparticles such as polylactide, polyglycolide or copolymers to enhance the adjuvant effect. Langer, Science 249:1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997. The medicament of the present invention can be administered in the form of a reservoir injection or implant formulation, which can be formulated in a manner that allows sustained or pulsatile release of the active ingredient. The pharmaceutical composition is typically formulated to be sterile, substantially isotonic and in full compliance with all good manufacturing practice (GMP) regulations of the U.S. Food and Drug Administration.

[0169] Also provided are kits for methods. The subject kits include expression vectors encoding orthogonal cytokine receptors or cells comprising expression vectors. The kits may further include homologous orthogonal cytokines. In certain embodiments, the components are provided in dosage forms (e.g., therapeutically effective dosage forms) in liquid or solid form in any convenient packaging (e.g., stick packaging, dosage packaging, etc.). Reagents for selecting or in vitro derived cells, such as growth factors, differentiation agents, tissue culture reagents, etc., may also be provided.

[0170] In addition to the above components, the subject kit may further include (in some embodiments) instructions for implementing the subject method. These instructions may be present in the subject kit in various forms, one or more of which may be present in the kit. One form in which these instructions may exist is printed information printed on a suitable medium or substrate (e.g., a piece of paper or several pieces of paper with information printed thereon), kit packaging, package instructions, etc. Another form in which these instructions exist is a computer-readable medium with information recorded thereon, such as a floppy disk, a compact disk (CD), a portable flash drive, etc. Another form in which these instructions may exist is a website address, which can be used to access information on a remote website via the Internet.

[0171] In some embodiments, the subject compositions, methods and kits are used to enhance T cell-mediated immune responses. In some embodiments, the immune response is directed to conditions requiring the consumption or regulation of target cells, such as cancer cells, infected cells, immune cells involved in autoimmune diseases, and the like.

[0172] In some embodiments, the condition is a chronic infection, i.e., an infection that is not cleared by the host immune system for a period of up to 1 week, 2 weeks, etc. In some cases, chronic infections involve the integration of pathogen genetic elements into the host genome, such as retroviruses, lentiviruses, hepatitis B virus, etc. In other cases, chronic infections, such as certain intracellular bacterial or protozoan pathogens, are caused by pathogen cells within host cells. Additionally, in some embodiments, the infection is in a latent stage as with herpes virus or human papillomavirus.

[0173] The methods of the present invention provide that the T effector cells of the host organism kill infected cells more effectively relative to removal in the absence of treatment, and thus can target the intracellular stage of the pathogen life cycle. The method can further include monitoring the patient for treatment efficacy. Monitoring can measure clinical indicators of infection, such as fever, white blood cell count, etc., and / or directly monitor the presence of pathogens.

[0174] In some embodiments, the disease is cancer. As used herein, the term "cancer" refers to various diseases caused by abnormal, uncontrolled growth of cells. Cells that can cause cancer, called "cancer cells", have features such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate and / or certain typical morphological features. Cancer can be detected in a variety of ways, including but not limited to detecting the presence of a tumor or tumor (e.g., by clinical or radiological means), examining cells in a tumor or other biological sample (e.g., from a tissue biopsy), measuring blood markers indicating cancer, and detecting the genotype indicating cancer. However, a negative result in one or more of the above-mentioned detection methods does not necessarily indicate that there is no cancer, for example, as demonstrated by subsequent recurrence, a patient who has shown a complete response to cancer treatment may still have cancer.

[0175] The present invention is now fully described, it will be apparent to one skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.

[0176] experiment

[0177] Orthogonal IL-2 and IL-2Rβ

[0178] Here, we describe an invention involving engineered cytokines and receptors that enable the selective expansion of desired cell subsets in the setting of ex vivo adoptive cell therapy. The invention describes the cytokine interleukin-2 (IL-2) and its receptor IL-2Rβ chain (IL-2Rβ) that enable the specific expansion of T cells in adoptive cell therapy, thereby addressing unmet needs in immunotherapy. The methods described herein can be generalized to any setting of adoptive cell therapy in which cells are stimulated by specific receptor-ligand pairs, including bone marrow and stem cell transplantation, as well as many other modalities.

[0179] Specifically described are orthogonal IL-2 and IL-2Rβ ligand-receptor pairs. Ortholog versions of IL-2 and ortholog versions of IL-2Rβ that specifically bind to each other but not to the wild-type counterpart. Multiple orthogonal IL-2 variant sequences are provided, with varying degrees of affinity for orthogonal IL-2Rβ. Orthogonal IL-2-dependent signaling and T cell proliferation of T cells engineered to express orthogonal IL-2Rβ are shown.

[0180] IL-2 is an attractive biologic for the treatment of cancer and autoimmunity due to its ability to promote the expansion of effector T cells and regulatory T cells, respectively. However, this pleiotropic effect of IL-2 as well as off-target toxicity limits its use in the clinic. The ability to uncouple the immunostimulatory and immunosuppressive properties of IL-2 could provide an excellent form of IL-2 immunotherapy.

[0181] This article demonstrates the ability of engineered T cells to express orthogonal IL-2Rβ. These engineered T cells were 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 was completely abolished or significantly attenuated compared to the activity of wild-type IL-2. Thus, selective T cell expansion using an orthogonal IL-2 / IL-2 receptor pair was demonstrated.

[0182] Applications of orthogonal IL-2 / IL-2 receptor pairs include, but are not limited to, selective expansion of tumor-reactive cytotoxic T cells for cancer therapy, selective expansion of NK cells for infectious diseases and / or cancer, and selective expansion of regulatory T cells for autoimmune diseases.

[0183] IL-2 variants with blunted affinity for intermediate (IL-2Rβ and IL-2Rγ) or high affinity wild-type IL-2 receptors (IL-2Rα, Rβ, Rγ) due to mutations that disrupt but do not completely eliminate binding to IL-2Rβ may also be used to selectively target the activity of orthologous IL-2 to IL-2Rα high cells, for example to treat autoimmune diseases. IL-2 variants that have abolished affinity for the IL-2Rβ chain but retain binding to IL-2Rα and thus compete with wild-type IL-2 as antagonists by inhibiting the formation of high affinity IL-2Rs may be used to treat autoimmunity or graft-versus-host disease.

[0184] The overall concept of generating and utilizing orthogonal IL-2 / IL-2 receptor pairs to control T cell expansion is as follows Figure 1 As shown in the schematic diagram. Figure 2 A workflow is provided, including steps for generating IL-2Rβ orthologs that lack binding to wild-type IL-2 using structure-guided mutagenesis. Mutations predicted to disrupt IL-2Rβ binding to wild-type IL-2 were confirmed experimentally using a yeast-based screening assay and further validated using purified recombinant protein by surface plasmon resonance. Using this approach, a number of IL-2Rβ point mutations that disrupt binding to wild-type IL-2 were described, and each of these receptor variants can function as an orthogonal receptor. Single point mutations can also be combined with one, two, or more additional point mutations to generate a larger library of IL-2Rβ orthologs.

[0185] The sequence of the orthogonal mouse IL-2Rβ variant is shown in Figure 3 When the combined mutations disrupt wild-type IL-2 binding to these mutations, these mutations can be used as single point mutations or in any combination thereof to generate IL-2Rβ orthologs with 1, 2, 3 or more point mutations.

[0186] Figure 4Characterization of a mIL-2Rβ variant is shown, which contains amino acid changes H134D, Y135F that abolish wild-type mIL-2 binding. These two residues are known IL-2 interaction hotspots (Ring A et al., Nat Immunol (2012) 13: 1187-95), and we confirmed that this mutation disrupts wild-type IL-2 binding by surface plasmon resonance (SPR).

[0187] Figure 5 The workflow for engineering orthogonal IL-2 / IL-2Rβ pairs is illustrated. The orthologous library of IL-2 generated randomizes residues that are close to or in contact with the orthologous orthologous amino acid residues of IL-2Rβ. Using yeast display, IL-2 variants that bind to the ortholog IL-2Rβ and discard clones that bind to the wild-type IL-2Rβ are selected. The process can be repeated using site-directed mutagenesis or error-prone mutagenesis to generate IL-2 variants with different binding properties to the ortholog but not to the wild-type IL-2Rβ. Using this approach, we generated a library of IL-2 orthologs that: 1) retain binding to the IL-2Rα chain (green curve), indicating the full structural integrity of the orthogonal IL-2 variants displayed by yeast, 2) bind to the orthogonal IL-2Rβ (orange curve), but 3) do not bind to the wild-type IL-2Rβ (blue curve).

[0188] The sequences of the characterized orthogonal mouse IL-2 variants are shown in Figure 6 middle. Fig.15 An alignment of mouse IL-2 and IL-2Rβ to their human counterparts is shown. These four sequences provide a reference for the native or wild-type sequences. The amino acid residues that were altered to generate the orthogonal mouse IL-2 / IL-2Rβ pairs are primarily conserved in humans. Therefore, the orthogonal mouse IL-2 and IL-2Rβ sequences can be translated into human IL-2 and IL-2Rβ proteins.

[0189] like Figure 7 As shown, the orthogonal IL-2 variants bind to the orthogonal IL-2Rβ with similar or greater affinity than the interaction between wild-type IL-2 and IL-2Rβ. Soluble orthogonal IL-2 or wild-type IL-2 protein was flowed over a sensor chip coated with wild-type or orthogonal IL-2Rβ. Binding was determined by surface plasmon resonance (SPR) and the curves were fitted using a 1:1 binding model. Figure 8 As shown, orthogonal IL-2 variants exhibited inactivation activity (phosphoSTAT5) on wild-type CD25-positive and CD25-negative splenocytes.

[0190] Generation of mouse CTLL-2 T cells expressing primary IL-2Rβ was shown in Fig. 9We created an immortalized mouse T cell line (CTLL-2) that expresses an orthogonal IL-2Rβ (orthogonal CTLL-2) by lentiviral transduction with a gene encoding the full-length orthogonal receptor. Transduced cells were selected with puromycin, which is a toxin for untransduced cells, generating a stable CTLL-2 cell line that expresses both wild-type and orthogonal IL-2Rβ. This cell line is also positive for CD25 and CD132 and therefore represents T cells that express the high-affinity IL-2 receptor complex. The antibody used to detect cell surface IL-2Rβ (CD122) does not distinguish between wild-type and primary IL-2Rβ. Therefore, the increase in mean fluorescence intensity between wild-type and primary IL-2RβCTLL-2 cells indicates that these cells express the orthogonal receptor. This is further supported by their resistance to puromycin, which is encoded by the same vector used to express the orthogonal IL-2Rβ.

[0191] like Fig.10 As shown, the first set of orthogonal IL-2 variants were selective for orthogonal T cells. To interrogate orthogonal IL-2 signaling, we utilized the CTLL-2 cell model, either unmanipulated (wild type) or transduced to also express an orthogonal IL-2Rβ (orthogonal). We then determined the ability of wild type or various orthogonal IL-2 clones to induce STAT5 phosphorylation, a quantitative readout of IL-2-dependent signaling. We identified a number of orthogonal IL-2 variants that induced selective STAT5 phosphorylation on orthogonal IL-2Rβ expressing cells compared to wild type cells. Dose response curves for select clones are shown. Fig.11 shown.

[0192] Primary lymph node derived T cells were engineered to express an orthogonal IL-2Rβ (H134D Y135F). In addition to our immortalized mouse T cell models, we also generated an orthogonal IL-2Rβ expressing primary mouse T cells by isolating mouse lymph node and spleen cells, activating them with CD3 / CD28, and then retrovirally transducing the gene encoding the full-length orthogonal receptor. This construct also contains an IRES followed by the fluorescent protein YFP, allowing confirmation of transduction by FACS analysis of YFP expression. Mouse T cells also express the high affinity IL-2 receptor complex (e.g. CD25, CD122, and Cd132), as Fig.12 Shown

[0193] Orthogonal IL-2 variants induce selective STAT5 phosphorylation on orthogonal IL-2Rβ expressing primary mouse T cells, as Fig.13 shown.

[0194] Orthogonal IL-2 variants that selectively signal through orthogonal IL-2Rβ compared to wild-type CTLL-2 cells Fig.11) also induced the selective expansion of CTLL-2 cells expressing orthogonal IL-2Rβ ( Fig.14 ).

[0195] The orthogonal IL-2 engineering approach was also applied to human IL-2 and human IL-2Rβ. We introduced the H133D Y134F mutation used to create mouse orthogonal IL-2Rβ into human IL-2Rβ because these residues are highly conserved between mouse and human. Indeed, wild-type hIL-2Rβ binds yeast to wild-type IL-2, whereas the hIL-2RβH133D Y134F mutant (orthogonal-hIL-2Rβ) lacks detectable binding to wild-type IL-2 ( Fig.15 ). We created a library of human IL-2 mutants displayed on the yeast surface by randomizing residues predicted to contact or be close to the H133D Y134F mutation and selected IL-2 variants that bound orthotopically but not to wild-type human IL-2Rβ. This approach was identical to that used to design the mouse IL-2 orthogonal pair and was successful for the human pair. The approach is illustrated in Figure 16. A set of common mutations was identified, indicating a convergence of orthotopic hIL-2 sequences capable of binding orthotopic hIL-2Rβ, such as Fig. 16C shown.

[0196] The polypeptides of the invention are also active in vivo. Mouse models are used to demonstrate selective expansion or increased survival of T cells expressing orthogonal IL-2Rb in mice, such as Figure 17-19 As shown. Orthogonal IL-2 clone 1G12 / 149 was shown to selectively expand orthogonal but not wild-type mouse T cells. Compared to PBS control, treatment with wild-type IL-2 resulted in expansion of both wild-type and orthogonal T cells, whereas orthogonal IL-2 clone 1G12 / 149 treatment selectively expanded orthogonal T cells with limited activity against wild-type T cells.

[0197] Example 2

[0198] Human IL-2 ortholog

[0199] Materials and methods

[0200] Protein production. DNA encoding wild-type human IL-2 was cloned into the insect expression vector pAcGP67-A, which contains a C-terminal 8xHIS tag for affinity purification. DNA encoding mouse serum albumin (MSA) was purchased from Integrated DNA Technologies (IDT, Coralville, Iowa 52241) and cloned into pAcGP67-A as a fusion between the N-terminus of hIL-2 and the C-terminus of MSA. Variants of orthogonal human IL-2 isolated from activity screening were synthesized as Gblocks (IDT) and cloned into the pAcGP67-A-MSA vector by overlap extension.

[0201] use The insect expression DNA construct was transfected into Trichoplusia ni (High ) cells (Invitrogen), purified by Ni-NTA, followed by size exclusion chromatography using a Superdex-200 column, and sterilely prepared in phosphate-buffered saline (PBS). The protein was concentrated and stored at -80°C.

[0202] Mammalian expression vectors. Full-length human CD25 was cloned into the lentiviral vector pCDH-CMV-MSC-EF1-Puro (System Biosciences). The cDNA encoding full-length human IL-2Rβ was used as a template to clone the full-length orthogonal IL-2Rβ by overlap extension PCR using mutagenic primers that introduced the H133D and Y134F mutations. The resulting PCR product was cloned into the retroviral vector pMSCV-MCS-IRES-YFP.

[0203] Cell culture. The YT-NK-like cell line was generously provided by Dr. Yodoi Junji, Kyoto University. YT- cells were transduced with pCDH-CMV-MSC-EF1-Puro-hCD25 lentivirus, and YT cells stably expressing full-length human CD25 (YT+) were selected in 10 μg / mL puromycin. YT+ cells were transduced with retrovirus containing pMSCV-MCS-IRES-YFP-orthogonal-human-Rβ and sorted by FACS to enrich the YFP+ (orthogonal) population for purity. HEK293T cells were generously provided by Dr. Irving Weissman's laboratory at Stanford University. HEK293T cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine (L-glu), and 1% penicillin and streptomycin (P / S). YT cells were maintained in RPMI complete (RPMI+GlutaMax+10% FBS, 1% L-Glu, 1% NaPyr, 1% NEAA, 18 mM HEPES and 1% pen / strep).

[0204] Lentivirus and retrovirus production. Lentivirus was produced in HEK293T cells using third-generation packaging vectors. Briefly, HEK293T cells were plated at 5x10 cells per 10 cm tissue culture dish. 6 The cells were seeded at a density of 100 cells and allowed to adhere for 5-7 hours in complete medium (DMEM, 10% FBS, 1% L-Glu, 1% Pen / Strep). The supernatant was removed and supplemented with low FBS (5%) DMEM (10 mL) and the culture medium was maintained at 4 °C. Cells were transfected with pCDH:psPAX2:pMD2G at a ratio of 4:2:1 according to the manufacturer's instructions of HP DNA transfection reagent (Sigma-Aldrich) and cultured in complete medium overnight at 37°C. The medium was removed and supplemented with 7.5 mL of low FBS DMEM (DMEM, 5% FBS, 1% L-Glu, 1% P / S), and lentiviruses were collected from the supernatant after 24 and 48 hours, pooled, clarified through a 0.45 μm filter, precipitated with PEG-it virus precipitation solution (System Bio), resuspended in 1 / 100 volume of the original medium, snap-frozen in liquid nitrogen, and stored at -80°C.

[0205] Retrovirus was produced in HEK293T cells. Briefly, HEK293T cells were cultured at 5 × 10 cells per 10 cm tissue culture dish. 6Cells were plated at a density of 10 cells and allowed to adhere for 5-7 hours in complete medium (DMEM, 10% FBS, 1% L-Glu, 1% P / S). The supernatant was removed and supplemented with low FBS DMEM (10 mL), and the cells were transfected with a 1.5:1 ratio of pMSCV retroviral vector and pCL10A packaging vector (Novus Biologicals) (a gift from Dr. Melissa McCracken, Stanford University) according to HP manufacturer's recommendations and cultured overnight in low FBS DMEM. Remove the culture medium and supplement with 7.5 mL low FBS DMEM and culture for another 24 hours. Collect the culture medium, clarify using a 0.45 μm filter, and snap-freeze in liquid nitrogen for storage at -80°C. Supplement the culture medium (low FBS DMEM) and culture the cells for another 24 hours, and collect and store the virus as above.

[0206] Yeast Display of IL-2. Human IL-2 was displayed on the surface of the yeast Saccharomyces cerevisiae strain EBY100 along with an N-terminal cMyc epitope tag by fusion to the C-terminus of Aga2 using the pCT302 vector with a 3C protease cleavage site between the C-terminus of Aga2 and the N-terminus of IL-2. Briefly, competent EBY100 were electroporated with a plasmid encoding yeast displaying hIL-2 and recovered in SDCAA selection medium at 30°C overnight. The transformed yeast were passaged once in SDCAA, and the logarithmic phase yeast culture was pelleted and resuspended at an OD600 of 1.0 in SGCAA induction medium containing 10% SDCAA and cultured at 20°C for 24 h. 488-labeled anti-cMyc mAb (1:100 dilution; Cell Signaling) and Yeast were stained with streptavidin (SA) tetramers of 647-labeled wild-type hIL-2Rβ (500 nM SA) and surface expression of functional hIL-2 was confirmed by FACS.

[0207] Generation of a yeast display library of human IL-2 mutants.

[0208] Site-directed libraries were created by assembly PCR using primers with the following degenerate codons: Library 3 (E15, H16, L19, D20, Q22, M23): (SEQ ID NO: 10) 5'-CAAGTTCTACAAAGAAAACACAGCTACAACTGNHKNHKTTACTTNHKNHKTTANHKNHKATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTC-3' Library 4 (E15, H16, L19, D20, M23, N88): (SEQ ID NO: 11) 5'-GTTCTACAAAGAAAACACAGCTACAACTGNHK NHKTTACTTNHKNHKTTACAGNHKATTTTGAATGGAATTAATAATTACAAGAATCC-3', (SEQ ID NO: 12) 5'-CCCAGGGACTTAATCAGCNHKATCAACGTAATAGTTCTGGAACTAAAGGG-3'.

[0209] Sequence:(SEQ ID NO:13)5'-CGGTAGCGGTGGGGCGGTTCTCTGGAAGTTCTGTTCCAGGGTCCGAGCGGCGGA-3',(SEQ ID NO:14)5'-GTAGCTGCTGTAGTTGATTGTTGTTGTTGTTGGGGGCGGTTCTCTGGTTCCAGGTCCGAGCGGCGGA-3' CGCTCGGACCCTGG-3',(SEQ ID NO:15)5'-CTTAAATGTGAGCATCCTGGTGAGTTTGGGATTCTTGTAATTATTCCATTCAAAAT-3',(SEQ ID NO:16)5'-CCAGGATGCTCA CATTTAAGTTTTACATGCCCAAGAAGGCC,(SEQ ID NO:3' NO:17)5'-GAGGTTTGAGTT CTTCTTCTAGACTGAAGATGTTTCAGTTCTGTGGCCTTCTTGGGC-3',(SEQ ID NO:18)5'-CAGTGTCTAGAAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTAGCTCAAAGC-3',(SEQ ID NO:19)5'-GATTAAGTCCCTGGGTCTTAAGTGAAAGTTTTTGCTTTGAGCTAAATT TAGCACTTCCTC-3',(SEQ ID NO:20)5'-CAGCATATTCACACATGAATGTTGTTTCAGATC CCTTTAGTTCCAGAACTATTACGTTG-3',(SEQ ID 2:GAGTCAGATCAGATTCGATTG-3',(SEQ ID 2:GAGTCAGATCAGATTCGATTG-3',(SEQ ID NO:20). TATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAAC-3',(SEQ ID NO:22)5'-GAGATG ATGCTTTGACAAAAGGTAATCCATCTGTTCAGAAATTCTACAATGGTTGCTG-3',(SEQ IDNO:23)5'-GATTACCTTTTCAAACCATCCAACCTTGACCTTGAGCCTTGAGCCTTGGACCTTGGCTG-3',(SEQ IDNO:23). CGAAC-3',(SEQID NO:24)5'-GATCTCGAGCAAGTCTTCTTCGGAGATAAGCTTTTTGTTC GCCACCAGAAGCGG-3'.

[0210] The mutant IL-2 gene PCR product was assembled using an equimolar mixture of Pfu Ultra DNA polymerase (Agilent) and each primer. The DNA product was further PCR amplified using primers (SEQ ID NO: 25) 5'-CGGTAGCGGTGGGGGCGGTTC-3' and (SEQ ID NO: 26) 5'-CGAAGAAGACTTGCTCGAGATC-3' using Phusion DNA polymerase (NEB). The assembled PCR product was gel purified and electroporated into EBY-100 yeast with a linearized pCT302 vector to generate ~2x10 8 A library of transformants.

[0211] Evolution of orthogonal IL-2. Selection of yeast clones that specifically bind to orthogonal IL-2R was performed using a combination of magnetic activated cell sorting (MACS) and FACS. The first round of selection used 2x10 9 yeast, approximately 10-fold the diversity of the library, to ensure 100% coverage of all transformants. The overall strategy used was to first enrich the library for all full-length hIL-2 variants that bind to the orthogonal IL-2R (rounds 1-3), then use negative selection to remove IL-2 clones that bind to the wild-type IL-2R, reduce the concentration of the orthogonal IL-2R, and enrich for IL-2 clones that bind to the orthogonal IL-2R by affinity.

[0212] Yeast-based binding and functional screening. Single yeast clones were isolated by culture on SDCAA plates and single colony extraction or single cell FACS into 96-well round-bottom tissue culture plates containing 100 μL SDCAA and cultured overnight at 30°C in a shaking incubator. Yeast clones were further expanded in 1.5 mL SDCAA per well of a 96-well deep-well V-bottom plate at 30°C for 24 hours, and then induced in SGCAA medium containing 10% SDCAA in a shaking incubator at 20°C for 72 hours, with a starting OD600 of 1.0, also in 1.5 mL and 96-well deep-well V-bottom plates. The induced yeast was precipitated, washed once with PBS, and resuspended in 200 μL / well of cutting medium (RPMI containing 25 mM HEPES, 0.2 mM TCEP, 20 μg / mL 3C protease) and incubated at room temperature with agitation for 5 minutes and at 4°C overnight without agitation. Yeast was pelleted and passed through a 96-well 0.45 μm cellulose acetate filter plate (Cat. No. 7700-2808, GE Healthcare). Yt+ (wild type and ortho expression) and Yt- were plated as described in the IL-2R signaling method section, 50 μl of clarified yeast supernatant containing mutant IL-2 clones was added and incubated at 37°C for 20 minutes. The reaction was stopped and pSTAT5 was quantified as described below. Use The percentage of wild-type or Ortho-YT cells that were pSTAT5+ was quantified (TreeStar Inc., Ashland OR) and used to select clones with selective or specific activity against Ortho-YT+ cells.

[0213] Retroviral transduction of human peripheral blood mononuclear cells (PBMCs). Leukopenia chambers were obtained from the Stanford Blood Center. Blood was drained into sterile 50 ml conical tubes (~7 mL) and PBS + 2% FBS was added to a total of 34 mL. Density gradient medium (15 ml, Ficoll-Paque Plus, GE Healthcare, 17-1440-03) was loaded onto two tube (Stemcell, 15450) and gently pipette 17 mL of diluted cells to the top. The tubes were spun at 1200 x g for 15 minutes at room temperature. The top layer containing the PBMCs was poured into a new tube and RPMI was added to 50 mL. The cells were spun at 1200 rpm for 5 minutes to pellet. The pellet was resuspended in 10 mL ACK lysis buffer (Gibco A10492-01) for 4 minutes and quenched with RPMI complete to 40 mL. The cells were pelleted again, completely suspended in 15 mL RPMI and counted. The cells (1x10 6 ) were inoculated into each well of a 24-well tissue culture dish, and 25ul of Human T-Activator CD3 / CD28 (Cat. No. 11131D) and 100U / ml hIL-2. The cells were activated in a 37°C incubator for 48 hours.

[0214] Transduce activated human PBMCs by spinfection (see Berggren WT, Lutz M, Modesto V. General spinfection Protocol 10 Dec 2012. In: StemBook [Internet]. Cambridge (MA): Harvard Stem Cell Institute; 2008) using unconcentrated retroviral supernatant (approximately 2 mL per well) containing 10 μg / mL polytetracycline and 100 IU / mL hIL-2 at 32°C and 2500 RPM for 1.5 hours. Gently aspirate the viral supernatant and replace with fresh RPMI complete medium containing 100 IU / mL hIL-2 and incubate at 37°C for 24 hours. Collect the cells by gentle pipetting and remove with a magnet. The cells were pelleted by centrifugation and 1x10 6Cells were resuspended at a density of 10 cells / mL in fresh RPMI complete medium containing 100 IU / mL hIL-2 and expanded overnight at 37 °C before further downstream cell assays.

[0215] IL-2R signaling via phosphorylation of STAT5. Quantification of IL-2 and orthogonal IL-2 signaling via intracellular pSTAT5. Actively growing YT+ and Yt+ orthogonal cell pellets were combined in a 50 / 50 ratio and plated at 5x10 5 Ultra-low binding 96-well round-bottom plates (Cat. No. 7007; Costar) were seeded at a density of 10 cells / mL in 50 μL warm medium. Cells were stimulated by adding 50 μL medium containing serial dilutions of wild-type or orthogonal IL-2 for 20 min at 37°C and the reaction was terminated by fixation with 1.5% paraformaldehyde for 10 min at room temperature (RT) with agitation. Cells were pelleted, decanted and permeabilized with 200 μL of 100% ice-cold methanol on ice for at least 30 min or incubated overnight at –80°C. Fixed, permeabilized cells were washed three times with FACS buffer and fixed with 1:50 dilution of 1:50 in FACS buffer. Intracellular phosphorylated STAT5 was detected by 647-labeled anti-STAT5pY694 (BD Biosciences) and incubated at 4°C in the dark for 1 hour. The cells were washed and analyzed in a microplate reader equipped with a high-throughput autosampler (Beckman Coulter). The data represent the mean fluorescence intensity and are analyzed using (GraphPad) Points were fit to a log (agonist) versus response (three parameters) model. All data are presented as mean (n=3) ± SD.

[0216] In vitro primary human PBMC proliferation assay. Human peripheral blood mononuclear cells containing a mixture of wild-type and orthotopically transduced T cells were collected by centrifugation, resuspended in RPMI complete medium lacking hIL-2, and seeded at a density of 50,000 cells per well (50 μL) in 96-well round-bottom tissue culture plates (day 1). Cell growth was stimulated by adding serial dilutions of wild-type or orthotopic IL-2 (50 μL) to a total volume of 100 μL and cultured at 37°C for 2 days. On day 3, cells were additionally supplemented with fresh cytokines and cultured for another 2 days in a volume of 100 μL. On day 5, 50 μL of DAPI was added to a final concentration of 0.5 μg / mL and the cells were plated using a high-throughput sampler equipped with a Cell counts for each tested population were quantified by FACS. After gating live cells based on FSC and SSC and DAPI negativity, the total number of live cells in a set volume was derived. Data were analyzed by (Tree Star Inc.). Data represent total viable cell counts plotted against cytokine concentrations, or represent the ratio of neighboring cells to total viable cells plotted against cytokine concentrations. Data are expressed as mean (n=4) ± SD.

[0217] As shown in Figure 22, orthogonal human IL-2 signals in vitro through the orthogonal IL-2R expressed in YT cells. As shown in Figure 23, orthogonal human IL-2 preferentially amplifies human PBMCs expressing the orthogonal IL-2R. Human PBMCs were isolated, activated and transformed with retrovirus containing orthogonal human IL-2Rβwith an IRESYFP(YFP+). Initial ratio of YFP+cells to total live cells was 20%. 5x10 were plated on day 1 with the indicated concentrations of MSA-human IL-2 (circles) or orthotopic variants MSA-SQVLKA (diamonds), MSA-SQVLqA (squares) or MSA-1A1 (black triangles). 5 cells and re-fed at the same concentration on day 3. On day 5, the plates were read by flow cytometry. (A) The ratio of YFP+ (orthogonal expressing) cells to total viable cells was calculated and the mean (n=4) ± SD was plotted versus concentration (left). (B) Total viable cell counts (mean (n=4) ± SD) were plotted versus cytokine concentration (right). At the same concentration, the orthogonal cytokine did not support as much total cell growth as wild-type MSA-hIL-2, but was selective in strongly expanding orthotopic expressing T cells.

[0218] The amino acid substitutions in the orthogonal hIL-2 protein are shown in Table 1 below.

[0219] Table 1

[0220]

[0221] Sequence Listing <110> Leland Stanford Jr. Trusteeship Kenan Christopher Garcia Jonathan Shawkrosh Laura Pickton <120> Biologically relevant orthogonal cytokine / receptor pairs <130> STAN-1246WO2 <140> PCT / US2019 / 021451 <141> 2019-03-08 <150> US 15 / 916,689 <151> 2018-03-09 <160> 26 <170> PatentIn version 3.5 <210> 1 <211> 525 <212> PRT <213> Homo sapiens <400> 1 Ala Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser Arg Ala 1 5 10 15 Asn Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp Thr Ser 20 25 30 Cys Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln Thr Cys 35 40 45 Glu Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu 50 55 60 Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val Thr Leu 65 70 75 80 Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala Ile Gln 85 90 95 Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile Ser Leu 100 105 110 Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp Glu Ile 115 120 125 Ser Gln Ala Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu Ala Arg 130 135 140 Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu Thr Leu 145 150 155 160 Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro Asp Thr 165 170 175 Gln Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu Phe Thr 180 185 190 Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys Pro Ala 195 200 205 Ala Leu Gly Lys Asp Thr Ile Pro Trp Leu Gly His Leu Leu Val Gly 210 215 220 Leu Ser Gly Ala Phe Gly Phe Ile Ile Leu Val Tyr Leu Leu Ile Asn 225 230 235 240 Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys Asn Thr 245 250 255 Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His Gly Gly 260 265 270 Asp Val Gln Lys Trp Leu Ser Ser Pro Phe Pro Ser Ser Ser Phe Ser 275 280 285 Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu Arg 290 295 300 Asp Lys Val Thr Gln Leu Leu Leu Gln Gln Asp Lys Val Pro Glu Pro 305 310 315 320 Ala Ser Leu Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn Gln 325 330 335 Gly Tyr Phe Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys 340 345 350 Gln Val Tyr Phe Thr Tyr Asp Pro Tyr Ser Glu Glu Asp Pro Asp Glu 355 360 365 Gly Val Ala Gly Ala Pro Thr Gly Ser Ser Pro Gln Pro Leu Gln Pro 370 375 380 Leu Ser Gly Glu Asp Asp Ala Tyr Cys Thr Phe Pro Ser Arg Asp Asp 385 390 395 400 Leu Leu Leu Phe Ser Pro Ser Leu Leu Gly Gly Pro Ser Pro Pro Ser 405 410 415 Thr Ala Pro Gly Gly Ser Gly Ala Gly Glu Glu Arg Met Pro Pro Ser 420 425 430 Leu Gln Glu Arg Val Pro Arg Asp Trp Asp Pro Gln Pro Leu Gly Pro 435 440 445 Pro Thr Pro Gly Val Pro Asp Leu Val Asp Phe Gln Pro Pro Pro Glu 450 455 460 Leu Val Leu Arg Glu Ala Gly Glu Glu Val Pro Asp Ala Gly Pro Arg 465 470 475 480 Glu Gly Val Ser Phe Pro Trp Ser Arg Pro Pro Gly Gln Gly Glu Phe 485 490 495 Arg Ala Leu Asn Ala Arg Leu Pro Leu Asn Thr Asp Ala Tyr Leu Ser 500 505 510 Leu Gln Glu Leu Gln Gly Gln Asp Pro Thr His Leu Val 515 520 525 <210> 2 <211> 513 <212> PRT <213> mice <400> 2 Ala Val Lys Asn Cys Ser His Leu Glu Cys Phe Tyr Asn Ser Arg Ala 1 5 10 15 Asn Val Ser Cys Met Trp Ser His Glu Glu Ala Leu Asn Val Thr Thr 20 25 30 Cys His Val His Ala Lys Ser Asn Leu Arg His Trp Asn Lys Thr Cys 35 40 45 Glu Leu Thr Leu Val Arg Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu 50 55 60 Gly Ser Phe Pro Glu Ser Gln Ser Leu Thr Ser Val Asp Leu Leu Asp 65 70 75 80 Ile Asn Val Val Cys Trp Glu Glu Lys Gly Trp Arg Arg Val Lys Thr 85 90 95 Cys Asp Phe His Pro Phe Asp Asn Leu Arg Leu Val Ala Pro His Ser 100 105 110 Leu Gln Val Leu His Ile Asp Thr Gln Arg Cys Asn Ile Ser Trp Lys 115 120 125 Val Ser Gln Val Ser His Tyr Ile Glu Pro Tyr Leu Glu Phe Glu Ala 130 135 140 Arg Arg Arg Leu Leu Gly His Ser Trp Glu Asp Ala Ser Val Leu Ser 145 150 155 160 Leu Lys Gln Arg Gln Gln Trp Leu Phe Leu Glu Met Leu Ile Pro Ser 165 170 175 Thr Ser Tyr Glu Val Gln Val Arg Val Lys Ala Gln Arg Asn Asn Thr 180 185 190 Gly Thr Trp Ser Pro Trp Ser Gln Pro Leu Thr Phe Arg Thr Arg Pro 195 200 205 Ala Asp Pro Met Lys Glu Ile Leu Pro Met Ser Trp Leu Arg Tyr Leu 210 215 220 Leu Leu Val Leu Gly Cys Phe Ser Gly Phe Phe Ser Cys Val Tyr Ile 225 230 235 240 Leu Val Lys Cys Arg Tyr Leu Gly Pro Trp Leu Lys Thr Val Leu Lys 245 250 255 Cys His Ile Pro Asp Pro Ser Glu Phe Phe Ser Gln Leu Ser Ser Gln 260 265 270 His Gly Gly Asp Leu Gln Lys Trp Leu Ser Ser Pro Val Pro Leu Ser 275 280 285 Phe Phe Ser Pro Ser Gly Pro Ala Pro Glu Ile Ser Pro Leu Glu Val 290 295 300 Leu Asp Gly Asp Ser Lys Ala Val Gln Leu Leu Leu Leu Gln Lys Asp 305 310 315 320 Ser Ala Pro Leu Pro Ser Pro Ser Gly His Ser Gln Ala Ser Cys Phe 325 330 335 Thr Asn Gln Gly Tyr Phe Phe Phe His Leu Pro Asn Ala Leu Glu Ile 340 345 350 Glu Ser Cys Gln Val Tyr Phe Thr Tyr Asp Pro Cys Val Glu Glu Glu 355 360 365 Val Glu Glu Asp Gly Ser Arg Leu Pro Glu Gly Ser Pro His Pro Pro 370 375 380 Leu Leu Pro Leu Ala Gly Glu Gln Asp Asp Tyr Cys Ala Phe Pro Pro 385 390 395 400 Arg Asp Asp Leu Leu Leu Phe Ser Pro Ser Leu Ser Thr Pro Asn Thr 405 410 415 Ala Tyr Gly Gly Ser Arg Ala Pro Glu Glu Arg Ser Pro Leu Ser Leu 420 425 430 His Glu Gly Leu Pro Ser Leu Ala Ser Arg Asp Leu Met Gly Leu Gln 435 440 445 Arg Pro Leu Glu Arg Met Pro Glu Gly Asp Gly Glu Gly Leu Ser Ala 450 455 460 Asn Ser Ser Gly Glu Gln Ala Ser Val Pro Glu Gly Asn Leu His Gly 465 470 475 480 Gln Asp Gln Asp Arg Gly Gln Gly Pro Ile Leu Thr Leu Asn Thr Asp 485 490 495 Ala Tyr Leu Ser Leu Gln Glu Leu Gln Ala Gln Asp Ser Val His Leu 500 505 510 authorities <210> 3 <211> 149 <212> PRT <213> mice <400> 3 Ala Pro Thr Ser Ser Ser Thr Ser Ser Ser Thr Ala Glu Ala Gln Gln 1 5 10 15 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln His Leu Glu Gln Leu Leu 20 25 30 Met Asp Leu Gln Glu Leu Leu Ser Arg Met Glu Asn Tyr Arg Asn Leu 35 40 45 Lys Leu Pro Arg Met Leu Thr Phe Lys Phe Tyr Leu Pro Lys Gln Ala 50 55 60 Thr Glu Leu Lys Asp Leu Gln Cys Leu Glu Asp Glu Leu Gly Pro Leu 65 70 75 80 Arg His Val Leu Asp Leu Thr Gln Ser Lys Ser Phe Gln Leu Glu Asp 85 90 95 Ala Glu Asn Phe Ile Ser Asn Ile Arg Val Thr Val Val Lys Leu Lys 100 105 110 Gly Ser Asp Asn Thr Phe Glu Cys Gln Phe Asp Asp Glu Ser Ala Thr 115 120 125 Val Val Asp Phe Leu Arg Arg Trp Ile Ala Phe Cys Gln Ser Ile Ile 130 135 140 Ser Thr Ser Pro Gln 145 <210> 4 <211> 133 <212> PRT <213> Homo sapiens <400> 4 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 5 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 5 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Leu Val Leu Leu Gln Gln Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Ile Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Asp Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 6 <211> 133 <212> PRT <213> artificial sequence <220> <223> Synthesis of multiple peptides <400> 6 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Leu Val Leu Leu Gln Gln Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Tyr Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 7 <211> 133 <212> PRT <213> artificial sequence <220> <223> Synthesis of multiple peptides <400> 7 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Leu Val Leu Leu Lys Ala Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 8 <211> 133 <212> PRT <213> artificial sequence <220> <223> Synthesis of multiple peptides <400> 8 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln 1 5 10 15 Leu Leu Val Leu Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 9 <211> 525 <212> PRT <213> Homo sapiens <400> 9 Ala Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser Arg Ala 1 5 10 15 Asn Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp Thr Ser 20 25 30 Cys Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln Thr Cys 35 40 45 Glu Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu 50 55 60 Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val Thr Leu 65 70 75 80 Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala Ile Gln 85 90 95 Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile Ser Leu 100 105 110 Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp Glu Ile 115 120 125 Ser Gln Ala Ser Asp Phe Phe Glu Arg His Leu Glu Phe Glu Ala Arg 130 135 140 Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu Thr Leu 145 150 155 160 Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro Asp Thr 165 170 175 Gln Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu Phe Thr 180 185 190 Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys Pro Ala 195 200 205 Ala Leu Gly Lys Asp Thr Ile Pro Trp Leu Gly His Leu Leu Val Gly 210 215 220 Leu Ser Gly Ala Phe Gly Phe Ile Ile Leu Val Tyr Leu Leu Ile Asn 225 230 235 240 Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys Asn Thr 245 250 255 Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His Gly Gly 260 265 270 Asp Val Gln Lys Trp Leu Ser Ser Pro Phe Pro Ser Ser Ser Phe Ser 275 280 285 Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu Arg 290 295 300 Asp Lys Val Thr Gln Leu Leu Leu Gln Gln Asp Lys Val Pro Glu Pro 305 310 315 320 Ala Ser Leu Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn Gln 325 330 335 Gly Tyr Phe Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys 340 345 350 Gln Val Tyr Phe Thr Tyr Asp Pro Tyr Ser Glu Glu Asp Pro Asp Glu 355 360 365 Gly Val Ala Gly Ala Pro Thr Gly Ser Ser Pro Gln Pro Leu Gln Pro 370 375 380 Leu Ser Gly Glu Asp Asp Ala Tyr Cys Thr Phe Pro Ser Arg Asp Asp 385 390 395 400 Leu Leu Leu Phe Ser Pro Ser Leu Leu Gly Gly Pro Ser Pro Pro Ser 405 410 415 Thr Ala Pro Gly Gly Ser Gly Ala Gly Glu Glu Arg Met Pro Pro Ser 420 425 430 Leu Gln Glu Arg Val Pro Arg Asp Trp Asp Pro Gln Pro Leu Gly Pro 435 440 445 Pro Thr Pro Gly Val Pro Asp Leu Val Asp Phe Gln Pro Pro Pro Glu 450 455 460 Leu Val Leu Arg Glu Ala Gly Glu Glu Val Pro Asp Ala Gly Pro Arg 465 470 475 480 Glu Gly Val Ser Phe Pro Trp Ser Arg Pro Pro Gly Gln Gly Glu Phe 485 490 495 Arg Ala Leu Asn Ala Arg Leu Pro Leu Asn Thr Asp Ala Tyr Leu Ser 500 505 510 Leu Gln Glu Leu Gln Gly Gln Asp Pro Thr His Leu Val 515 520 525 <210> 10 <211> 98 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <220> <221> Other important biological function areas <222> (33)..(33) " <223> N at position 33 can be a, c, g or t" <220> <221> Other important biological function areas <222> (34)..(34) " <223> N at position 34 can be a, c or t" <220> <221> Other important biological function areas <222> (35)..(35) <223> N at position 35 can be g or t <220> <221> Other important biological function areas <222> (36)..(36) " <223> N at position 36 can be a, c, g or t" <220> <221> Other important biological function areas <222> (37)..(37) " <223> N at position 37 can be a, c or t" <220> <221> Other important biological function areas <222> (38)..(38) <223> N at position 38 can be g or t <220> <221> Other important biological function areas <222> (45)..(45) " <223> N at position 45 can be a, c, g or t" <220> <221> Other important biological function areas <222> (46)..(46) " <223> N at position 46 can be a, c or t" <220> <221> Other important biological function areas <222> (47)..(47) <223> N at position 47 can be either g or t <220> <221> Other important biological function areas <222> (48)..(48) " <223> N at position 48 can be a, c or t" <220> <221> Other important biological function areas <222> (49)..(49) " <223> N at position 49 can be a, c or t" <220> <221> Other important biological function areas <222> (50)..(50) <223> N at position 50 can be either g or t <220> <221> Other important biological function areas <222> (54)..(54) " <223> N at position 54 can be a, c, g or t" <220> <221> Other important biological function areas <222> (55)..(55) " <223> N at position 55 can be a, c or t" <220> <221> Other important biological function areas <222> (56)..(56) <223> N at position 56 can be either g or t <220> <221> Other important biological function areas <222> (57)..(59) " <223> n is a, c, g or t" <400> 10 caagttctac aaagaaaaca cagctacaac tgnnnnnntt acttnnnnnn ttannnnnna 60 ttttgaatgg aattaataat tacaagaatc ccaaactc 98 <210> 11 <211> 88 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <220> <221> Other important biological function areas <222> (30)..(30) " <223> N at position 30 is a, c, g or t" <220> <221> Other important biological function areas <222> (31)..(31) " <223> N at position 31 is a, c or t" <220> <221> Other important biological function areas <222> (32)..(32) <223> N at position 32 is g or t <220> <221> Other important biological function areas <222> (33)..(33) " <223> N at position 33 is a, c, g or t" <220> <221> Other important biological function areas <222> (34)..(34) " <223> N at position 34 is a, c or t" <220> <221> Other important biological function areas <222> (35)..(35) <223> N at position 35 is g or t <220> <221> Other important biological function areas <222> (42)..(42) " <223> N at position 42 is a, c, g or t" <220> <221> Other important biological function areas <222> (43)..(43) " <223> N at position 43 is a, c or t" <220> <221> Other important biological function areas <222> (44)..(44) <223> N at position 44 is g or t <220> <221> Other important biological function areas <222> (45)..(45) " <223> N at position 45 is a, c, g or t" <220> <221> Other important biological function areas <222> (46)..(46) " <223> N at position 46 is a, c or t" <220> <221> Other important biological function areas <222> (47)..(47) <223> N at position 47 is g or t <220> <221> Other important biological function areas <222> (54)..(54) " <223> N at position 54 is a, c, g or t" <220> <221> Other important biological function areas <222> (55)..(55) " <223> N at position 55 is a, c or t" <220> <221> Other important biological function areas <222> (56)..(56) <223> N at position 56 is g or t <400> 11 gttctacaaa gaaaacacag ctacaactgn nnnnnttact tnnnnnntta cagnnnattt 60 tgaatggaat taataattac aagaatcc 88 <210> 12 <211> 50 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <220> <221> Other important biological function areas <222> (19)..(19) " <223> N at position 19 is a, c, g or t" <220> <221> Other important biological function areas <222> (20)..(20) " <223> N at position 20 is a, c or t" <220> <221> Other important biological function areas <222> (21)..(21) <223> N at position 21 is g or t <400> 12 cccagggact taatcagcnn natcaacgta atagttctgg aactaaaggg 50 <210> 13 <211> 55 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 13 cggtagcggt gggggcggtt ctctggaagt tctgttccag ggtccgagcg gcgga 55 <210> 14 <211> 59 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 14 gtagctgtgt tttctttgta gaacttgaag taggtgcgga tccgccgctc ggaccctgg 59 <210> 15 <211> 60 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 15 cttaaatgtg agcatcctgg tgagtttggg attcttgtaa ttattaattc cattcaaaat 60 <210> 16 <211> 45 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 16 ccaggatgct cacatttaag ttttacatgc ccaagaaggc cacag 45 <210> 17 <211> 59 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 17 gaggtttgag ttcttcttct agacactgaa gatgtttcag ttctgtggcc ttcttgggc 59 <210> 18 <211> 57 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 18 cagtgtctag aagaagaact caaacctctg gaggaagtgc taaatttagc tcaaagc 57 <210> 19 <211> 60 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 19 gattaagtcc ctgggtctta agtgaaagtt tttgctttga gctaaattta gcacttcctc 60 <210> 20 <211> 59 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 20 cagcatattc acacatgaat gttgtttcag atccctttag ttccagaact attacgttg 59 <210> twenty one <211> 60 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> twenty one gaaacaacat tcatgtgtga atatgctgat gagacagcaa ccattgtaga atttctgaac 60 <210> twenty two <211> 58 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> twenty two gagatgatgc tttgacaaaa ggtaatccat ctgttcagaa attctacaat ggttgctg 58 <210> twenty three <211> 59 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> twenty three gattaccttt tgtcaaagca tcatctcaac actaactgcg gccgcttctg gtggcgaac 59 <210> twenty four <211> 53 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> twenty four gatctcgagc aagtcttctt cggagataag cttttgttcg ccaccagaag cgg 53 <210> 25 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 25 cggtagcggt gggggcggtt c 21 <210> 26 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 26 cgaagaagac ttgctcgaga tc 22

Claims

1. An engineered human IL-2 polypeptide having significantly reduced binding to native human CD122; and wherein an amino acid substitution selected from the following with reference to SEQ ID NO: 4 numbering is performed: (i) [E15S, H16Q, L19V, D20L, Q22K and M23A]: (ii) [E15S, H16Q, L19V, D20L, M23Q, R81Y]; (iii) [E15S, H16Q, L19V, D20L, M23Q, R81D and T51I]; or (iv) [E15S, H16Q, L19V, D20L and M23A].

2. The engineered human IL-2 polypeptide according to claim 1, wherein the polypeptide carries out the following amino acid substitutions: [E15S, H16Q, L19V, D20L, Q22K and M23A].

3. The engineered human IL-2 polypeptide according to claim 1, wherein the polypeptide undergoes the following amino acid substitutions: [E15S, H16Q, L19V, D20L, M23Q, R81Y].

4. The engineered human IL-2 polypeptide according to claim 1, wherein the polypeptide carries out the following amino acid substitutions: [E15S, H16Q, L19V, D20L, M23Q, R81D and T51I].

5. The engineered human IL-2 polypeptide according to claim 1, wherein the polypeptide carries out the following amino acid substitutions: [E15S, H16Q, L19V, D20L and M23A].

6. The engineered human IL-2 polypeptide according to any one of claims 1 to 5, wherein the engineered human IL-2 polypeptide is pegylated.

7. The engineered human IL-2 polypeptide according to claim 6, wherein the molecular weight of the polyethylene glycol is 5 kD to 80 kD.

8. The engineered human IL-2 polypeptide according to claim 6, wherein the molecular weight of the polyethylene glycol is 5 kD to 40 kD.

9. The engineered human IL-2 polypeptide according to claim 6, wherein the molecular weight of the polyethylene glycol is 40 kD.

10. The engineered human IL-2 polypeptide according to any one of claims 7 to 9, wherein the polyethylene glycol is a branched polyethylene glycol.

11. The engineered human IL-2 polypeptide according to any one of claims 7 to 9, wherein the polyethylene glycol is a linear polyethylene glycol.

12. The engineered human IL-2 polypeptide according to any one of claims 7 to 9, wherein polyethylene glycol is attached to the amino terminus of the engineered human IL-2 polypeptide via a linker.

13. The engineered human IL-2 polypeptide according to any one of claims 1-5 and 7-9, wherein the engineered human IL-2 polypeptide is fused to the Fc domain of IgG or albumin.

14. The engineered human IL-2 polypeptide of claim 10, wherein the engineered human IL-2 polypeptide is fused to the Fc domain of IgG or albumin.

15. The engineered human IL-2 polypeptide according to any one of claims 11, wherein the engineered human IL-2 polypeptide is fused to the Fc domain of IgG or albumin.

16. The engineered human IL-2 polypeptide of claim 12, wherein the engineered human IL-2 polypeptide is fused to the Fc domain of IgG or albumin.

17. The engineered human IL-2 polypeptide of any one of claims 1-5, 7-9 and 14-16, wherein the engineered human IL-2 polypeptide binds to and activates an orthogonal human CD122 protein, wherein the orthogonal human CD122 protein undergoes the following amino acid substitutions: H133D and Y134F, wherein the amino acid numbering is related to SEQ ID NO:

1.

18. A pharmaceutical composition comprising the engineered human IL-2 polypeptide according to any one of claims 1-17; and a pharmaceutically acceptable carrier.

19. A nucleic acid encoding the engineered human IL-2 polypeptide according to any one of claims 1-17.

20. An expression vector comprising the nucleic acid according to claim 19.

21. A cell genetically engineered to contain the expression vector according to claim 20.

22. A composition for use in a method of treating a mammalian subject suffering from a disease, the method comprising: administering to a mammalian subject a therapeutically effective amount of immune effector cells expressing an orthogonal human CD122 receptor having the following amino acid substitutions: H133D and Y134F, wherein the amino acid numbers are relative to SEQ ID NO: 1, and Administering to a mammalian subject a therapeutically effective amount of an engineered human IL-2 polypeptide according to any one of claims 1-17; or an expression vector according to claim 20.

23. The composition of claim 22, wherein the immune effector cells are T cells.

24. The composition of claim 23, wherein the T cells are CAR-T cells.

25. The composition of claim 24, wherein the antigen binding domain of the chimeric antigen receptor of the CAR-T cell specifically binds to one or more antigens expressed on the surface of the target cell.

26. The composition of claim 25, wherein the antigen binding domain of the chimeric antigen receptor of the CAR-T cell comprises a scFv.

27. The composition of claim 25, wherein the antigen binding domain of the chimeric antigen receptor of the CAR-T cell comprises a single domain antibody.

28. The composition of any one of claims 25-27, wherein at least one of the one or more antigens is a tumor antigen.

29. The composition of claim 28, wherein the tumor antigen is selected from CD19, CD20, HER2, NY-ESO-1, MUC1, CD123, FLT3, B7-H3, CD33, IL1RAP, CLL1 (CLEC12A), PSA, CEA, VEGF, VEGF-R2, CD22, ROR1, mesothelin, c-Met, glycolipid F77, FAP, EGFRvIII, MAGE A3, 5T4, WT1, KG2D ligand, folate receptor (FRa) and Wnt1 antigen.

30. The composition of claim 29, wherein the tumor antigen is CD19.

31. The composition of claim 30, wherein the CAR-T cell is axicabtagene ciloleucel or tisagenlecleucel.

32. The composition of any one of claims 22-27 and 29-31, wherein the method further comprises administering one or more additional therapeutic agents.

33. A prefilled syringe comprising the pharmaceutical composition of claim 18.

34. The expression vector of claim 20, wherein the vector is a viral vector.

35. The expression vector of claim 20, wherein the vector is a non-viral vector.

36. An in vitro method for recombinantly preparing a composition comprising an engineered human IL-2 polypeptide according to any one of claims 1 to 17, the method comprising: Conditionally culturing the cell genetically engineered to contain the expression vector according to claim 21 to result in expression of the recombinant human IL2 polypeptide; and The recombinant IL2 polypeptide is isolated.

37. The method of claim 36, further comprising: reacting the isolated recombinant IL2 polypeptide to chemically conjugate the polyethylene glycol molecule to the IL2 polypeptide; and Isolation of PEGylated IL2 polypeptide.

38. The composition of claim 23, wherein the T cells are selected from naive CD8 + T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, helper T cells; regulatory T cells; memory T cells, NK T cells, γδT cells, CAR-T cells, TIL and TCRαβ receptor knockout cells.

39. The composition of claim 24, wherein the antigen binding domain of the chimeric antigen receptor of the CAR-T cell specifically binds to one or more antigens expressed on the surface of a target cell.

40. The composition of claim 39, wherein the one or more antigens expressed on the surface of the target cell is CD19.

41. The composition of claim 24, wherein the disease is a neoplastic disease, and wherein at least one of the one or more antigens is a tumor antigen.

42. The composition of claim 41, wherein the tumor antigen is selected from CD19, CD20, HER2, NY-ESO-1, MUC1, CD123, FLT3, B7-H3, CD33, IL1RAP, CLL1 (CLEC12A), PSA, CEA, VEGF, VEGF-R2, CD22, ROR1, mesothelin, c-Met, glycolipid F77, FAP, EGFRvIII, MAGE A3, 5T4, WT1, KG2D ligand, folate receptor (FRa) and Wnt1 antigen.

43. An engineered human IL-2 polypeptide according to any one of claims 1-5, 7-9 and 14-16 for use in a method of treating a mammalian subject having a disease, disorder or condition associated with the presence of an abnormal cell population characterized by expression of one or more surface antigens, the method comprising the steps of: (a) obtaining a biological sample comprising T cells from the subject; (b) enriching T cells in a biological sample; (c) transfecting T cells with one or more expression vectors encoding a nucleic acid sequence encoding a CAR and a nucleic acid sequence encoding an orthogonal human CD122 receptor, wherein the antigen targeting domain of the CAR is capable of binding to at least one antigen present on the abnormal cell population, wherein the nucleic acid sequence encoding the orthogonal human CD122 receptor encodes a sequence with the following amino acid substitutions: H133D and Y134Y, wherein the amino acid numbering is related to SEQ ID NO: 1; (d) ex vivo expansion of CAR-T cell populations expressing orthogonal receptors; (e) administering a pharmaceutically effective amount of CAR-T cells expressing an orthogonal receptor to a mammal; and (f) regulating the growth of CAR-T cells expressing orthogonal receptors using a ligand that selectively binds to an orthogonal receptor expressed on CAR-T cells, wherein the ligand is an engineered human IL-2 polypeptide according to any one of claims 1-5, 7-9 and 14-16, or an expression vector according to any one of claims 20, 33-34.

44. The engineered human IL-2 polypeptide of claim 43, further comprising the step of lymphodepleting the subject prior to step (e).

45. The engineered human IL-2 polypeptide of claim 43, wherein the amplification step (d) is performed in the presence of the engineered human IL-2 polypeptide of any one of claims 1-5, 7-9, and 14-16.

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