Interleukine-2 muteins with enhanced specificity towards treg cells and improved production capacity

Abstract

The present invention is aimed at addressing the need for enhanced specificity of IL2 for Treg cell lineage responses while maintaining bioactivity and maintaining or improving production capacity to provide molecules useful as biological tools or therapeutics.

Description

[0001] Therapeutic molecules

[0002] Introduction

[0003] Interleukin 2 (IL2) is a highly potent cytokine, capable of both maintaining homeostasis and driving inflammatory reactions. These dual functions are based on the differential expression of the different receptor complexes. The high affinity receptor, a trimer of IL2RA, IL2RB and ILR2G (CD25, CD122, CD132, respectively), is dominantly expressed by regulatory T cells (Tregs), allowing IL2 to control Treg homeostasis through effects on Treg “fitness” and proliferation / apoptosis control. The intermediate affinity receptor, a dimer of IL2RB and ILR2G, is expressed by cells with more inflammatory potential, largely CD8 T cells and NK cells. Due to the ~100-fold lower affinity of the dimer for IL2, at normal levels of IL2 production the dominant role is to establish homeostasis. Elevated IL2 production is, however, among the earliest events following CD4 T cell activation, supporting the proliferation of CD8 T cells and NK cells during an immune reaction. This response is aided by transient low-level upregulation of IL2RA on responding cells, providing an inflammatory interlude before homeostasis is re-established through the Treg response to elevated IL2. This dominant IL2 network is further complicated by context-dependent IL2 circuits and alterations of receptor expression during pathology.

[0004] With the immunological capacity to both elevate tolerogenic (Treg) and immunogenic (CD8 T cell / NK) responses, IL2 is a highly attractive therapeutic target. The clinical utility is, however, limited by the concentration-dependent effects, especially as the halflife of IL2 is around 5 minutes following injection. Multiple approaches to engineer more stable concentrations have been developed, including modification of IL2 through post- translational modification, such as PEGylation, via fusion to protein carriers, such as the immunoglobulin Fc region or a soluble version of the IL2RA protein, or through sustained production using gene therapy vectors. An alternative approach has been to decouple this concentration-dependent effect by restricting function to either the trimeric or dimeric receptor, through either screening / / n vitro evolution approaches or rational design. IL2 muteins can be created with alterations to the IL2RA, IL2RB or IL2RG binding interfaces, including enhanced binding to IL2RA, reduced binding to IL2RA, increased binding to IL2RB; reduced IL2RB binding or reduced activation of the receptor complex via IL2RG. The net effects are designer IL2 variants with increased selectivity for Tregs, increased selectivity for CD8 T cells and NK cells, or antagonistic activity, depending on which binding structure is disrupted. IL2 variants combining both elevated longevity and altered specificity have also been generated, such as combining an IL2 mutein with an Fc fusion, or targeting PEGylation to a particular IL2 interface. Finally, more exotic approaches have been used, including the de novo design of protein capable of binding only one surface of the I L2R, or the creation of I L2 / IL2R orthogonal pairs that do not cross- react with the native system.

[0005] While I L2 muteins provide enhanced specificity for either the T reg or CD8 T cell lineage, depending on the mutation design, a feature commonly observed in muteins is reduced stability or bioactivity. As either of these attributes limit the utility of the mutein in biotechnology, we sought to design novel IL2 mutations using the SolubiS algorithm, which enables rational design of mutations to enhance stability. Using this system we designed and validated families of IL2 mutations with enhanced specificity for either Tregs or CD8 T cells, while maintaining total bioactivity and maintaining or even improving production capacity. This combination of features provides potential utility for these muteins in clinical therapeutics.

[0006] The present invention is aimed at addressing the need for enhanced specificity of IL2 for either the Treg or CD8 T cell lineage responses (thus promoting Treg or CD8 T cell respectively), in particular Treg responses, while maintaining total bioactivity and maintaining or improving production capacity to provide molecules useful as biological tools or therapeutics.

[0007] Summary of the invention

[0008] The pleotropic nature of interluekin-2 (IL2) has allowed it to be used as both a pro- inflammatory and anti-inflammatory therapeutic agent, through promotion of regulatory T cell (Treg) responses via the IL2RA receptor or promotion of CD8 T cell responses via the IL2RB receptor, respectively. However, the utility of IL2 as a treatment is limited by this same pleiotropy, and protein engineering to bias specificity towards either the regulatory T cell (Treg) or CD8 T cell lineage often requires a trade-off in protein production or total bioactivity.

[0009] The inventors have identified amino acid mutations within the IL2 structure to improve protein production yield, e.g. by reducing aggregation of the protein, while altering cellular selectivity, to generate a mutant protein (mutein) with elevated therapeutic potential. The inventors have identified Treg-enhancing modified IL2, creating a cation repulsion to inhibit primary binding to IL2RB, with a post-IL2RA confirmational shift enabling secondary IL2RB binding, and hence allowing the trimeric receptor complex to form. The inventors have also identified additional aggregation-protecting mutations to improve protein yield of the Treg-enhancing mutations.

[0010] The inventors have generated CD8 T cell-promoting mutations in the IL2 polypeptide. For example, the mutations identified prevented IL2RA binding. The inventors have also identified additional aggregation-protecting mutations to improve protein yield of the CD8 T cell-promoting mutations.

[0011] These muteins, designed with both cellular specificity for either T reg or CD8 T cells and advantageous protein production features, such as a reduction in aggregation, can be used as biological tools or therapeutics.

[0012] The invention therefore relates to isolated modified IL2 polypeptides comprising one or more amino acid substitution that promotes Treg responses or CD8 T cell responses and / or reduces aggregation of the IL2 polypeptide.

[0013] In one aspect, the invention relates to a modified interleukin-2 (IL2) polypeptide i. wherein the modified IL2 polypeptide is a modified human IL2 polypeptide and comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. an amino acid substitution at position 111 and one or more of: an R at position 110, an N at position 143 and / or an amino acid substitution at position 151 , ii. an amino acid substitution at position 151 , iii. a W at position 36 and / or iv. an R at position 110 and an N at position 143 or ii. wherein the modified IL2 polypeptide is a modified murine IL2 polypeptide and comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 2: i. an amino acid substitution at position 126, ii. an amino acid substitution at position 158, iii. an amino acid substitution at position 166, iv. an amino acid substitution at position 54, and / or v. an amino acid substitution at position 123.

[0014] In one embodiment, the modified human IL2 polypeptide comprises an amino acid substitution at positions 111 , 110, and 151 with reference to SEQ ID NO: 1 , wherein the amino acid substitution at position 110 comprises N110R.

[0015] In one embodiment, the modified human IL2 polypeptide comprises the following amino acid substitutions: V111 R, N110R and T151 R.

[0016] In another aspect, the invention relates to a nucleic acid construct comprising a nucleic acid encoding a modified IL2 polypeptide as described above. In another aspect, the invention relates to an isolated nucleic acid construct encoding a modified IL2 polypeptide as described above.

[0017] In another aspect, the invention relates to a viral vector comprising the construct as described above.

[0018] In another aspect, the invention relates to a pharmaceutical composition comprising the modified IL2 polypeptide, viral vector or construct as described above.

[0019] In another aspect, the invention relates to the modified IL2 polypeptide, viral vector or construct or a pharmaceutical composition as described above for use in the treatment of disease, e.g. a cancer, an immune disorder, neurological disease, inflammatory disorder, allergy, transplant rejection, viral infection, immune deficiency or other immune system-related disorder comprising administering a therapeutically effective amount of a modified IL2 polypeptide, viral vector or construct or a pharmaceutical composition as described above.

[0020] In another aspect, the invention relates to a method for treating a cancer, an immune disorder, neurological disease, inflammatory disorder, allergy, transplant rejection, viral infection, immune deficiency or other immune system-related disorder comprising administering a therapeutically effective amount of a modified IL2 polypeptide, viral vector, construct or a pharmaceutical composition as described above. In another aspect, the invention relates to a kit comprising a modified IL2 polypeptide, viral vector, construct or a pharmaceutical composition as described above optionally together with a reagent and / or instructions for use.

[0021] In another aspect, the invention relates to a host cell comprising a nucleic acid, nucleic acid construct or a vector as described above.

[0022] In another aspect, the invention relates to a method for producing a modified IL2 polypeptide as described above comprising expressing a nucleic acid encoding the modified IL2 polypeptide in a host cell and isolating the modified IL2 polypeptide from the host cell.

[0023] In another aspect, the invention relates to a method for making an IL2 modified polypeptide with reduced aggregation comprising the steps of

[0024] 1) predicting aggregation-prone regions from an IL2 polypeptide,

[0025] 2) identifying amino acid mutations that abolish the aggregation propensity of these segments without affecting the thermodynamic stability of the polypeptide and

[0026] 3) expressing a modified IL2 polypeptide with reduced aggregation.

[0027] In another aspect, the invention relates to a vector delivery system comprising a nucleic acid encoding an IL2 polypeptide as described above or a construct according as described above.

[0028] In another aspect, the invention relates to an in vivo, in vitro or ex vivo method of promoting Treg responses, comprising contacting a population of T cells with an effective amount of a modified IL2 polypeptide as defined above.

[0029] In another aspect, the invention relates to a method of promoting Treg responses, comprising introducing an amino acid substitution as defined above into an IL2 polypeptide.

[0030] In another aspect, the invention relates to a method of decreasing the aggregation of IL2 polypeptide, comprising introducing an amino acid substitution as defined above into an IL2 polypeptide. Figures

[0031] The invention is illustrated in the following non-limiting figures.

[0032] Figure 1. Screening of IL2 muteins identifies muteins with increased production capacity. A) Murine IL2 mutein production in mammalian expression system HEK293 detected via HisTag ELISA and normalised to transfection efficiency measured by qPCR (n=3). A158N, T166R relate to SolubiS monomer, Q50W, D54R, N123H, V126R relate to Kill IL2RB binding, V126R A158N, N123H T166R relate to SolubiS monomer + kill IL2RB binding, E96K, Y79K, Y79K E96K relate to Kill IL2RA binding, Y79K E96K A158 N, Y79K E96K T166R relate to Solubis monomer + kill IL2RA binding and Q50W Y79K relate to ad hoc combination.

[0033] B) Human IL2 mutein production in mammalian expression system HEK293 detected via HisTag ELISA and normalised to transfection efficiency measured by qPCR (n=3). Comparisons made using One-Way ANOVA (*P < 0.05, **P < 0.01 , ***P < 0.001 , **** P0.0001). N110R, N110E, T143N, T151 R, N110R T143N, N110R T151 R relate to SolubiS monomer, H36W, D40R, N108H, V111 R relate to Kill IL2RB binding, V111 R T143N, N108H T151 R relate to SolubiS monomer + kill IL2RB binding, E82K, Y65K, Y65K E82K, Y65K E82K N110E relate to Kill IL2RA binding, Y65K E82K N110R T151 relate to Solubis monomer + kill IL2RA binding, V111 R N110R T151 R relate to ad hoc combination.

[0034] Figure 2. Altered cellular responses to murine SolubiS IL2 muteins. A) Mouse splenocytes were incubated for 20 minutes with either media containing 200ng / mL IL2 or 200ng / mL murine mutein, prior to staining for pSTAT2. For muteins with poor production, where ELISA detected less than 200ng / ml, a 1 :2 dilution of the production supernatant was used instead. The percentage of pSTAT5 cells within the Treg or B) CD8 T cell population (n=3). Comparisons made using One-Way ANOVA (*P < 0.05, **P < 0.01 , ***P < 0.001 , **** P<0.0001). C) Mouse splenocytes were incubated for 20 minutes with either media containing IL2 or murine mutein, using a serial dilution of IL2 concentration, prior to staining for pSTAT5. The percentage of pSTAT5 cells within the Treg or D) CD8 T cell population (n=3).

[0035] Figure 3. Structural modelling of murine IL2 muteins with the trimeric IL2R complex. A) Structure of IL2 in complexwith the IL2RA / IL2RB / IL2RG trimeric complex, indicating the sites of the three mutated residues. A158N, T166R relate to SolubiS monomer, Q50W, D54R, N123H, relate to Kill IL2RB binding, V126R, V126R A158N, N123H T166R relate to SolubiS monomer + kill IL2RB binding, E96K, Y79K, Y79K E96K relate to Kill IL2RA binding, Y79K E96K A158N, Y79K E96K T166R relate to Solubis monomer + kill IL2RA binding. B) The Y79K IL2 mutation, with close proximity to the R25-R26 residues in IL2RA. A158N, T166R relate to SolubiS monomer, Q50W, D54R, N123H, relate to Kill IL2RB binding, V126R, V126R A158N, N123H T166R relate to SolubiS monomer + kill IL2RB binding, E96K, Y79K, Y79K E96K relate to Kill IL2RA binding, Y79K E96K A158N, Y79K E96K T166R relate to Solubis monomer + kill IL2RA binding. C) The V126R IL2 mutation, in relation to the R41-R42 residues in IL2RB. The structure of IL2 is shown based on both the unbound IL2 structure and the conformation of IL2 present in the IL2-IL2RA complex, indicating the rotation of the helix from which the V126R side-chain emerges. D) The Q50W IL2 mutation, in relation to the hydrophobic groove present between the F101 and F134 residues of IL2RB. Hydrophobicity in ILR2B represented by colour, with increasing intensity of green representing increasingly hydrophilic values.

[0036] Figure 4. In vivo testing of V126R IL2 enhances local Treg expansion. IL2 and IL2 mutein were delivered in vivo through AAV-mediated gene delivery. A. Schematic representation of the viral vector used. Expression is driven by the Scgblal (Secretoglobin 1A1) promoter and flanked by the inverted terminal repeats. The cargo was encoded in AAV6.2 for lung epithelial trophism and administrated through the intranasal route (n=5-7 mice / group). B. Available IL2 protein levels in the lung on days 4, 7, 14, 21 and 28 post-AAV delivery. C. Tissues were removed on day 14 and assessed via flow cytometry for the frequency of Foxp3+ Tregs within the CD4+ T cell population, and D. CD8 T cells within the viable cell population. All the data were analysed using Two Way Anova.

[0037] Figure 5. Combining mutations Q50W and Y79K enhances CD8 T cell selectivity of murine IL2. A) Mouse splenocytes were incubated for 20 minutes with either media containing IL2 or murine mutein, using a serial dilution of IL2 concentration, prior to staining for pSTAT5. The percentage of pSTAT5 cells within the Treg or B) CD8 T cell population (n=3).

[0038] Figure 6. Altered cellular responses to human SolubiS IL2 muteins. A) Human PBMCs were incubated for 20 minutes with either media containing 200ng / mL human mutein or 1 :2 dilution (where ELISA detected less than 200ng / ml), prior to staining for pSTAT5. The percentage of pSTAT5 cells within the Treg or B) CD8 T cell population (n=3). Comparisons made using One-Way ANOVA (*P < 0.05, **P < 0.01 , ***P < 0.001 , **** P0.0001). N110R, N110E, T143N, T151 R, N110R T143N, N110R T151 R relate to SolubiS monomer, H36W, D40R, N108H, V111 R relate to Kill IL2RB binding, V111 R T143 N, N108H T151 R relate to SolubiS monomer + kill IL2RB binding, E82K, Y65K, Y65K E82K relate to Kill IL2RA binding, Y65K E82K N110E, Y65K E82K N110R T151 R relate to Solubis monomer + kill IL2RA binding.

[0039] Figure 7. Cellular selectivity of Human IL2 muteins is maintained with addition of production boosting mutations N110R T151R. Human PBMCs were incubated for 20 minutes with either media containing IL2 or human Treg selective (A,B) or CD8 T cell selective (C,D) mutein, using a serial dilution of IL2 concentration, prior to staining for pSTAT5. A,C) The percentage of pSTAT5 cells within the Treg or B,D) CD8 T cell population (n=3). All muteins were tested in the same experiment using the same controls.

[0040] Figure 8: The capacity of the humanised AAV9.TetO-IL2 vector to expand the frequency of the brain-resident Treg population. A) ELISA results detecting levels of IL2 in the brain for animals treated with AAV9.GFP (white), AAV9.hulL2 (red), AAV9. mutein hulL2 (blue) at 1e10 (solid bars) and 5e10 (dashed bars). B) Percentage Treg expansion in animals treated with AAV9.GFP (white), AAV9.hulL2 (red), AAV9. mutein hulL2 (blue) in the brain and off-target tissues such as C) the liver at a dose of 1e10. D) The ratio of Treg to CD8 counts in the brain were increased in Treg specific mutein (blue) relative to wild type hulL2 (red). E) Intrathecal injection of AAV9. mutein hulL2 (dashed bars) boosts Treg counts in the brain above intravenous administration of AAV9. mutein IL2 (blue) and F) abrogates off-target Treg expansion in the liver. *** = p <.001 ; ** = p < .01 ; * = p < .05. G. shows a sketch of the injection.

[0041] Figure 9: EAE data showing delayed onset of disease and reduced disease severity in the AAV9. mutein IL2 treated mice at 5e9.

[0042] Detailed Description

[0043] The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012).

[0044] Modified IL2 polypeptides (muteins)

[0045] The invention relates to modified human IL2 polypeptides and to modified murine IL2 polypeptides.

[0046] According to a first aspect, the invention relates to a modified interleukin-2 (IL2) polypeptide a. wherein the modified IL2 polypeptide is a modified human IL2 polypeptide and comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. a W at position 36, ii. an R at position 110, iii. an N at position 143, iv. an amino acid substitution at position 151 ; and / or v. a K at position at position 65 and a K at position 82; or or b. wherein the modified IL2 polypeptide is a modified murine IL2 polypeptide and comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 2: i. an amino acid substitution at position 50, ii. an amino acid substitution at position 79, iii. an amino acid substitution at position 126, iv. an amino acid substitution at position 158, v. an amino acid substitution at position 166, vi. an amino acid substitution at position 54, vii. an amino acid substitution at position 123, viii. an amino acid substitution at position 96, and / or ix. an amino acid substitution at position 79.

[0047] In one embodiment, the invention relates to a modified interleukin-2 (IL2) polypeptide i. wherein the modified IL2 polypeptide is a modified human IL2 polypeptide and comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. an amino acid substitution at position 111 and one or more of: an R at position 110, an N at position 143 and / or an amino acid substitution at position 151 , ii. an amino acid substitution at position 151 , iii. a W at position 36 and / or iv. an R at position 110 and an N at position 143, or ii. wherein the modified IL2 polypeptide is a modified murine IL2 polypeptide and comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 2: i. an amino acid substitution at position 126, ii. an amino acid substitution at position 158, iii. an amino acid substitution at position 166, iv. an amino acid substitution at position 54, and / or v. an amino acid substitution at position 123.

[0048] In one embodiment, the modified human IL2 polypeptide comprises an amino acid substitution at positions 111 , 110, and 151 with reference to SEQ I D NO: 1 , and wherein the amino acid substitution at position 110 comprises N110R.

[0049] In one embodiment, the modified human IL2 polypeptide comprises the following amino acid substitutions: V111 R, N110R and T151 R. In one embodiment, the modified human IL2 polypeptide comprises the following amino acid substitutions: V111 R, N110R and T151 R.

[0050] A modified IL2 polypeptide or mutant IL2 polypeptide is a polypeptide with an amino acid sequence that differs from that of an IL2 wild type amino acid sequence. Such modified polypeptide is also referred to as a mutein herein. A “modified IL2 polypeptide” or “IL2 mutein” therefore means a polypeptide variant of a wild type IL2 polypeptide. A “modified human IL2 polypeptide” or “human IL2 mutein” means a polypeptide variant of a human wild type IL2.

[0051] The numbering of the amino acid residues in the modified human IL2 polypeptides as referred to herein is with reference to SEQ ID NO: 1. SEQ ID NO: 1 refers to full length human wild type IL2, comprising a human IL2 signal sequence (SEQ ID NO: 3) and human mature IL2 (SEQ ID NO: 4). Thus, the numbering system used herein is from the start of the unprocessed peptide. A skilled person would appreciate that the reference sequence does not limit the scope of the invention and alternatively, SEQ NO. 4 may be used as a reference sequence. In this case, for example, the amino acid substitution which corresponds to position 151 with reference to SEQ ID NO: 1 is an amino acid substitution at position 131 with reference to SEQ ID NO: 4.

[0052] In one embodiment, the modified human IL2 polypeptide comprises SEQ ID NO: 1 or a variant thereof, but comprising one or more amino acid substitution at the recited position(s). However, as will be appreciated by a person skilled in the art, variants of the reference sequence, e.g. SEQ ID NO: 1 exist, and the scope of the invention is not limited to a polypeptide comprising SEQ ID NO. 1 with the mutation(s) as described herein. For example, the invention also includes variants of SEQ ID NO: 1 with the corresponding amino acid substitutions as explained herein, for example truncated and allelic variants, fragments or human mature IL2 (SEQ ID NO: 4) after cleavage of the signal sequence (SEQ ID NO: 3). For example, such a variant may include one or more additional amino acid modification in the IL2 sequence or a deletion or addition of one or more amino acids. For example, such variants can have at least 75%, 80%, 85%, 90% or 95% sequence identity to the modified polypeptides. In another embodiment, the modified human IL2 polypeptide comprises SEQ ID NO. 4, but comprising one or more amino acid substitution corresponding to the position(s) recited herein with reference to position(s) for SEQ ID NO:1.

[0053] Similarly, a “modified murine IL2 polypeptide” or a “murine IL2 mutein” means a variant of murine wild type IL2 with reference to SEQ ID NO: 2. The numbering of the amino acid residues in the modified murine IL2 polypeptides as referred to herein is with reference to SEQ ID NO: 2. SEQ ID NO: 2 refers to full length murine wild type IL2, comprising a murine IL2 signal sequence (SEQ ID NO: 5) and murine mature IL2 (SEQ ID NO: 6). Thus, the numbering system used herein is from the start of the unprocessed peptide. A skilled person would appreciate that the reference sequence does not limit the scope of the invention and alternatively, SEQ NO: 6 may be used as a reference sequence. In this case, for example, the amino acid substitution which corresponds to position 126 with reference to SEQ ID NO: 2 is an amino acid substitution at position 106 with reference to SEQ ID NO: 6.

[0054] SEQ ID NO: 34 refers to the murine wild type IL2 nucleotide sequence. SEQ ID NO: 35 refers to the human wild type IL2 nucleotide sequence. The modified nucleic acid sequence for hl L2 (V111R, N110R, T151 R) is shown in SEQ ID NO: 36.

[0055] In one embodiment, the modified murine IL2 polypeptide comprises SEQ ID NO: 2 or a variant thereof, but comprising one or more amino acid substitution at the recited position(s). However, as will be appreciated by a person skilled in the art, variants of the reference sequence exist, and the scope of the invention is not limited to a polypeptide comprising SEQ ID NO: 2 with the mutation(s) as described herein. For example, the invention also includes variants of SEQ ID NO: 2 with the corresponding amino acid substitutions as explained herein, for example truncated and allelic variants, or murine mature IL2 (SEQ ID NO: 6) after cleavage of the signal sequence (SEQ ID NO: 5). For example, such a variant may include one or more additional amino acid modification in the IL2 sequence or a deletion or addition of one or more amino acids. For example, such variants can have at least 75%, 80%, 85%, 90% or 95% sequence identity to the modified polypeptides. In another embodiment, the modified murine IL2 polypeptide comprises SEQ ID NO: 6, but comprising one or more amino acid substitution corresponding to the position(s) recited herein with reference to position(s) for SEQ ID NO: 2.

[0056] In one embodiment, the murine IL2 polypeptide is a mouse IL2 polypeptide.

[0057] Interleukin-2 (IL2) is an interleukin, a type of cytokine signaling molecule in the immune system. IL2 is a highly potent cytokine, capable of both maintaining homeostasis and driving inflammatory reactions. The present inventors have introduced amino acid substitutions in human and murine IL2 polypeptides to generate muteins with altered function compared to wild type IL2. The altered function will be described further below.

[0058] Modified IL2 polypeptides according to the invention are preferably isolated polypeptides. The term "isolated" refers to a moiety that is isolated from its natural environment. For example, the term "isolated" refers to a polypeptide that is substantially free of other polypeptides and substantially free of other cellular material and / or chemicals. Modified IL2 polypeptides according to the invention are preferably recombinantly produced.

[0059] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length.

[0060] By "amino acid" herein is meant any one of the 20 naturally occurring amino acids or any non- natural analogues that may be present at a specific, defined position. Amino acid encompasses both naturally occurring and synthetic amino acids. Although in most cases, when the protein is to be produced recombinantly, only naturally occurring amino acids are used.

[0061] As used herein, a "substitution of an amino acid residue" with another amino acid residue in an amino acid sequence of a protein or polypeptide as described herein, is equivalent to "replacing an amino acid residue" with another amino acid residue and denotes that a particular amino acid residue at a specific position in the wild type amino acid sequence has been replaced by (or substituted for) by a different amino acid residue. This can be done using standard techniques available to the skilled person, e.g., using recombinant DNA technology.

[0062] By "wild type" or "WT" or "native" herein is meant a polypeptide, an amino acid sequence or a nucleotide sequence that is found in nature, including allelic variations. A WT IL2 protein, IL2 polypeptide, IL2 nucleic acid or IL2 molecule as used herein has an amino acid sequence or a nucleotide sequence that has not been intentionally and recombinantly modified.

[0063] Wild type residues are designated herein by the one letter amino acid code followed by the IL2 amino acid position, e.g., T143 is the threonine residue at position 143 of reference sequence SEQ ID NO: 1. Substitutions of amino acids are designated herein by the one letter amino acid code of the wild type amino acid followed by the IL2 amino acid position followed by the substituting one letter amino acid code., e.g., T143N is a substitution of the wild type threonine residue at position 143 of SEQ ID NO:1 with an asparagine residue at that position. Unless otherwise stated, the numbering of the residues is with reference to SEQ ID NO:1 for human IL2 and with reference to SEQ ID NO:2 for murine IL2.

[0064] As used herein, the term "homology" or “identity” generally refers to the percentage of amino acid residues in a sequence that are identical with the residues of the reference polypeptide with which it is compared, after aligning the sequences and in some embodiments after introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Thus, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. Neither N- or C-terminal extensions, tags or insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known. The percent identity between two amino acid sequences can be determined using well known mathematical algorithms.

[0065] In one embodiment, the modified human IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. an R at position 110 and / or an N at position 143; and / or ii. an R at position 110 and / or an amino acid substitution at position 151 ; and / or iii. an amino acid substitution at position 151 and further comprises an amino acid substitution at position 108; and / or iv. a K at position at position 65 and a K at position 82 and further comprises an amino acid substitution at position 108.

[0066] In one embodiment, the modified murine IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 2: i. an amino acid substitution at one or more of position 126 and / or 158; and / or ii. an amino acid substitution at one or more of position 50 and / or 79; iii. an amino acid substitution at one or more of position 123 and / or 166; and / or iv. an amino acid substitution at one or more of position 79 and / or 96; and / or v. an amino acid substitution at one or more of position 79, 96 and / or 158; and / or vi. an amino acid substitution at one or more of position 79, 96 and / or 166.

[0067] In one embodiment, the modified human IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. an amino acid substitution at positions 110 and 143, wherein the amino acid substitution at position 110 comprises N110R and the amino acid substitution at position 143 comprises T143N; and / or ii. an amino acid substitution at positions 110 and 151 , wherein the amino acid substitution at position 110 comprises N110R.

[0068] In one embodiment, the modified murine IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 2: i. an amino acid substitution at positions 126 and 158; and / or ii. an amino acid substitution at positions 50 and 79; and / or iii. an amino acid substitution at positions 123 and 166; and / or iv. an amino acid substitution at positions 79 and 96; and / or v. an amino acid substitution at positions 79, 96 and 158; and / or vi. an amino acid substitution at positions 79, 96 and 166.

[0069] In one embodiment, the modified human IL2 polypeptide further comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. an amino acid substitution at one or more of position 65 and / or 82; and / or. ii. an amino acid substitution at position 111.

[0070] In one embodiment, the modified human IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. an amino acid substitution at positions 65, 82, 110 and 151 and wherein the amino acid substitution at position 110 comprises N110R; and / or ii. an amino acid substitution at positions 111 , 110, and 151 , and wherein the amino acid substitution at position 110 comprises N110R.

[0071] In one embodiment, the modified human IL2 polypeptide comprises H36W, Y65K, E82K, N110R, T143N and / or T151 R. In one embodiment, the modified murine IL2 polypeptide comprises Q50W, D54R, Y79K, E96K, V126R, A158N, T166R, and / or N123H.

[0072] In one embodiment, the amino acid substitution at position 108 comprises N108H.

[0073] In one embodiment, the amino acid substitution at position 111 comprises V111 R.

[0074] In one embodiment, the modified human IL2 polypeptide comprises i. an R at position 110 and an R position 151 ; and / or ii. an R at position 110 and an N at position 143; and / or iii. an R at position 111 and an N at position 143; and / or iv. an H at position 108 and an R at position 151 ; and / or v. a K at position 65, a K at position 82, an R at position 110, an R at position 151 ; and / or vi. an R at position 111 , an R at position 110 and an R at position 151.

[0075] In one embodiment, the modified murine IL2 polypeptide comprises i. an R at position 126 and an N at position 158; and / or ii. an H at position 123 and an R at position 166; and / or iii. a K at position 79 and a K at position 96; and / or iv. a K at position 79, a K at position 96 and an N at position 158; and / or v. a K at position 79, a K at position 96 and an R at position 166; and / or vi. a W at position 50 and a K at position 79.

[0076] In one embodiment, the modified human IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. V111 R, N110R and T151 R; ii. H36W or iii. N108H and T151 R.

[0077] In one embodiment, the modified human IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. Y65K, E82K, N110R, and T151 R or ii. Y65K and E82K.

[0078] In one embodiment, the modified human polypeptide comprises the following amino acid substitutions with reference to SEQ I D NO: 1 : N 110R and T151 R.

[0079] In one embodiment, the modified murine IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 2: i. V126R or ii. V126R and A158N.

[0080] In one embodiment, the modified murine IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 2: i. Q50W and Y79K; ii. Q50W or iii. Y79K.

[0081] In one embodiment, the modified murine polypeptide comprises an amino acid substitution at T166R with reference to SEQ ID NO: 2:

[0082] In one embodiment, the modified IL2 polypeptide comprises SEQ ID NO: 7 to 11 or a sequence with at least 90% sequence identity thereto; or SEQ ID NO: 12 to 17, or a sequence with at least 90% sequence identity thereto, provided the modifications are retained.

[0083] In one embodiment, the modified polypeptides of the invention comprise one or more modification as shown herein and do not comprise any additional amino acid modification.

[0084] In one embodiment, the modified polypeptide further comprises at least one additional amino acid substitution. In one embodiment, the additional amino acid substitution further alters the function of the modified IL2 polypeptide. For example, the modified IL2 polypeptide may have at least one amino acid substitution that reduces or inhibits binding to IL2RB, such as D40R. In one embodiment, the modified IL2 polypeptide further comprises an amino acid substitution at position 40, wherein said substitution is optionally D40R. Alternatively, in one embodiment, the modification is a conservative sequence modification. As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the modified polypeptide containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into a modified polypeptide of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a modified polypeptide of the invention can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (using the functional assays described herein or known in the art.

[0085] Thus, these amino acid changes can typically be made without altering the biological activity, function, or other desired property of the polypeptide, such as its affinity or its specificity for receptor. In general, single amino acid substitutions in nonessential regions of a polypeptide do not substantially alter biological activity. Furthermore, substitutions of amino acids that are similar in structure or function are less likely to disrupt the polypeptides' biological activity. Abbreviations for the amino acid residues of the polypeptides and peptides described herein, and conservative substitutions for these amino acid residues are shown in Table A below.

[0086] Table A. Amino Acid Residues and Examples of Conservative Amino Acid Substitutions

[0087] As referred to above, IL2 is capable of both maintaining homeostasis and driving inflammatory reactions. These dual functions are based on the differential expression of the different receptor complexes. The high affinity receptor, a trimer of IL2RA, IL2RB and ILR2G (CD25, CD122, CD132, respectively), is dominantly expressed by regulatory T cells (Tregs), allowing IL2 to control Treg homeostasis through effects on Treg “fitness” and proliferation / apoptosis control. The intermediate affinity receptor, a dimer of IL2RB and ILR2G, is expressed by cells with more inflammatory potential, largely CD8 T cells and NK cells. The inventors have decoupled these dual roles by restricting function to either the trimeric or dimeric receptor, while maintaining total bioactivity and maintaining or even improving production capacity. The engineering of the IL2 polypeptide according to the invention has biassed specificity towards either Treg or CD8 T cell lineage. Thus, IL2 mutant proteins have been generated that promote Treg or CD8 T-cell responses whilst improving protein yield by decreasing aggregation of the IL2 polypeptide / protein. In one embodiment, the amino acid substitution promotes Treg responses. In one embodiment, the amino acid substitution promotes CD8 T cell responses. In one embodiment, the amino acid substitution improves protein production yield / production capacity, e.g. by reducing IL2 aggregation. Treg or CD8 response can be measured as shown in the examples or using methods known in the art.

[0088] In one embodiment, the amino acid substitution alters binding of IL2 to IL2RA or IL2RB, and / or reduces or prevents IL2 aggregation. In one embodiment, the term “alter” means “decreasing” or “reducing”. Altering the binding of IL2 to IL2RA or IL2RB in comparison to wild-type IL2 can be “decreasing” or “reducing” binding by at least 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In one embodiment, the term “alter” means “preventing”, “eliminating” or “abolishing.” In one embodiment, the reduction to binding is 100% or substantially 100%. In one embodiment, the amino substitution that alters binding of IL2 to IL2RA or IL2RB reduces or abolishes binding of IL2 to IL2RA or IL2R.

[0089] A problem in the art is that enhancing specificity for either the T reg or CD8 T cell lineage typically reduces protein stability (leading to increased aggregation and decreased protein production) or reduces bioactivity. The present invention addresses this problem.

[0090] Thus, in one embodiment, the term “reduces or prevents IL2 aggregation” means increasing IL2 production capacity. In one embodiment, increasing IL2 production capacity means increased IL2 production by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% in comparison to control wild type IL2. In one embodiment, increasing IL2 production capacity means increased IL2 production by at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold or more in comparison to control wild type IL2.

[0091] Protein aggregation can be caused by the mis-folding of polypeptides during transcription or translation. Reducing or prevent aggregation of the IL2 polypeptide means more functional IL2 is produced per cell. For use in biotechnology or therapeutics, proteins require production in recombinant incubators, typically in distinct cell types to the natural producer and frequently at concentrations that are several orders of magnitude above their physiological concentration. This often results in protein aggregation, reducing the total protein production yield and increasing costs, creating barriers in affordability and therefore availability of these drugs for patients. The inventors have identified aggregation-prone regions and designed amino acid substitutions which reduces or prevents said aggregation. In one embodiment, the amino acid substitution alters binding of IL2 to IL2RA or IL2RB, and / or increases IL2 production capacity. In one embodiment, the term “increases IL2 production capacity” includes mechanisms other than decreasing aggregation.

[0092] In one embodiment, the amino substitution does not alter or does not substantially alter the bioactivity of CD8+ T cells and / or Tregs.

[0093] For example, the inventors have shown that a modified human IL2 polypeptide with one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 has improved Treg responses: i. V111 R, N110R and T151 R; ii. H36W or iii. N108H and T151 R.

[0094] The inventors have also shown that a modified human IL2 polypeptide with one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 has improved CD8 T cell responses: i. Y65K, E82K, N110R, and T151 R or ii. Y65K and E82K.

[0095] The inventors have also shown that a modified human IL2 polypeptide with one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 has reduced aggregation: N 110R and T151 R.

[0096] The inventors have also shown that a modified murine IL2 polypeptide with one or more of the following amino acid substitutions with reference to SEQ ID NO: 2 has improved Treg responses:

[0097] V126R or

[0098] II. V126R and A158N. The inventors have also shown that a modified murine IL2 polypeptide with one or more of the following amino acid substitutions with reference to SEQ ID NO: 2 has improved CD8 T cell responses: i. Q50W and Y79K; ii. Q50W or iii. Y79K.

[0099] The inventors have also shown that a modified murine IL2 polypeptide with an amino acid substitution at T166R with reference to SEQ ID NO: 2 has reduced aggregation.

[0100] The term “Treg” means regulatory T cell. Tregs are a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. Treg cells are immunosuppressive and generally suppress or downregulate induction and proliferation of effector T cells. Methods to identify Tregs are routine in the art and would be identified by the skilled person. The scope of the invention is not limited to the choice of method to identify T regs. For example, a suitable method includes flow cytometry, where the presence and / or absence of protein markers such as CD4 and FoxP3 may be used to identify Treg populations within a heterogeneous population of cells. The skilled person would appreciate and select the relevant markers to identify Tregs. The choice of markers is not considered to be a limitation of the invention. Tregs may, for example, be identified as CD4+FoxP3+ cells, but the skilled person would appreciate that other markers could be used.

[0101] The term “CD8+ T cell” means cytotoxic T cell. CD8+ T cells are a subpopulation of T cells that play crucial roles in immune surveillance and defence against infections and cancer. After encountering antigenic stimulation, naive CD8 T cells differentiate and acquire effector functions, enabling them to eliminate infected or malignant cells. Methods to identify CD8+ T cells are routine in the art and would be identified by the skilled person. The scope of the invention is not limited to the choice of method to identify CD8+ T cells. For example, a suitable method includes flow cytometry, where the presence and / or absence of protein markers such as CD3 and CD8 may be used to identify CD8+ T cells populations within a heterogeneous population of cells. The skilled person would appreciate and select the relevant markers to identify CD8+ T cells. The choice of markers is not considered to be a limitation of the invention. CD8+ T cells may, for example, be identified as CD3+CD8+ cells, but the skilled person would appreciate that other markers could be used.

[0102] The “bioactivity” means biological effect. For example, the bioactivity of Tregs includes the ability to modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease by a variety of mechanisms including the production of inhibitory cytokines and the prevention of co-stimulation through CD28 on effector cells. For example, the bioactivity of CD8+ T cells includes the ability to carry out immune surveillance and defense against infections and cancer by a variety of mechanisms including the production of cytotoxins and via cell-surface interaction between the Tc and the infected cell. The bioactivation status of Tregs and CD8+ T cells can be measured by a number of different assays which can be identified by a person skilled in the art. The choice of assay is not intended to be a limitation of the scope of the invention. For example, differences in the bioactivity of Tregs and / or CD8+ T cells can be determined by measuring the signaling activity of Tregs and / or CD8+ T cells when incubated with a polypeptide of the invention in comparison to the signaling activity of Tregs and / or CD8+ T cells incubated with control wild type IL2 as described in the Examples below. The term “does not alter or does not substantially alter the bioactivity of CD8+ T cells and / or Tregs” therefore can include no difference or no substantial difference in the signaling activity of Tregs and / or CD8+ T cells when incubated with a modified polypeptide of the invention in comparison to the signaling activity of Tregs and / or CD8+ T cells incubated with control wild type IL2 as described in the Examples below.

[0103] In one embodiment, the amino acid substitution increases the bioactivity / responses of CD8 T cells and / or T regs. The term “increases the bioactivity / responses of CD8+ T cells and / or Tregs” therefore can include an increase in the signaling activity of Tregs and / or CD8+ T cells when incubated with a modified polypeptide of the invention in comparison to the signaling activity of Tregs and / or CD8+ T cells incubated with control wild type IL2 as described in the Examples below.

[0104] In one embodiment, the term “increases the bioactivity / responses of CD8+ T cells and / or Tregs” means an increase of at least 5%, an increase of at least 10%, an increase of at least 15%, an increase of at least 20%, an increase of at least 25%, an increase of at least 30%, an increase of at least 35%, an increase of at least 40%, an increase of at least 45%, an increase of at least 50%, an increase of at least 55%, an increase of at least 60%, an increase of at least 65%, an increase of at least 70%, an increase of at least 75%, an increase of at least 80%, an increase of at least 85%, an increase of at least 90%, an increase of at least 95%, an increase of at least 100%, an increase of at least 200%, an increase of at least 300%, an increase of at least 400%, an increase of at least 500%, an increase of at least 600%, an increase of at least 700%, at least an increase of 800%, an increase of at least 900%, or an increase of at least 1000% or more in comparison to control wild type IL2. In one embodiment, the term “increases the bioactivity / responses of CD8+ T cells and / or Tregs” means an increase of at least an increase of 10-fold, at least an increase of 20-fold, at least an increase of 50-fold, at least an increase of 100-fold, at least an increase of 500-fold, at least an increase of 1000-fold or more in comparison to control wild type IL2.

[0105] In one embodiment, the modified IL2 polypeptide is conjugated to one or more moiety selected from an enzyme, radioisotope, half-life extending moiety, label, therapeutic molecule or other chemical moiety. The term conjugated refers to any covalent or non- covalent linkage and includes fusion proteins. In one embodiment, the modified IL2 polypeptide is a modified human IL2 polypeptide as described herein.

[0106] In one embodiment, the half-life extending moiety is selected from the group consisting of an albumin binding moiety, a transferrin binding moiety, a polyethylene glycol molecule, a recombinant polyethylene glycol molecule, human or murine serum albumin, a fragment of human or murine serum albumin, and an albumin binding peptide or antibody or fragment thereof that binds to human or murine serum albumin or an antibody Fc domain.

[0107] The term "half-life" as used herein refers to the time taken for the serum concentration of the amino acid sequence, compound or polypeptide to be reduced by 50%, in vivo, for example due to degradation of the sequence or compound and / or clearance or sequestration of the sequence or compound by natural mechanisms. Half-life may be increased by at least 1 .5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding modified IL2 polypeptides of the invention. For example, increased halflife may be more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding modified IL2 polypeptides of the invention. The in vivo half-life of an amino acid sequence, compound or polypeptide of the invention can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art. Half life can for example be expressed using parameters such as the t1 / 2-alpha t1 / 2-beta and the area under the curve (AUG).

[0108] Constructs and vector delivery systems

[0109] According to another aspect, the invention relates to a construct comprising a nucleic acid encoding a modified IL2 polypeptide of the invention.

[0110] In one embodiment, the construct comprising a nucleic acid encoding a modified IL2 polypeptide is incorporated into a viral vector. Thus, the invention also relates to a viral vector comprising a construct of the invention. The construct is a nucleic acid construct.

[0111] As used herein the term “vector” refers to a nucleic acid sequence capable of transporting into a cell another nucleic acid to which the vector sequence has been linked.

[0112] The viral vector may also have additional features such as enhancer and promoter regions. In an embodiment, the vector comprises a constitutive promoter, examples include but are not limited to a CMV promoter, an RSV promoter, a human polypeptide chain elongation factor (EF1a) promoter, a ubiquitin C (UBC) promoter, a phosphoglycerate kinase (PGK) promoter. In one embodiment, the vector comprises a tissue specific promoter, examples include but are not limited to a glial fibrillary acidic protein (GFAP) promoter, a myosin light chain-2v (MLC) promoter, or the muscle creatine kinase (MCK) promoter. In one embodiment, the vector comprises an inducible promoter, examples include but are not limited to a tetracycline (Tet) - dependent system, or the use of small molecules to induce dimerization of promoter-activating TF domains, or histone deacetylase inhibitors to activate the stress-inducible Grp78 promoter.

[0113] In other embodiments, the promoter is GAG promoter, a CBA promoter, CMV promoter, EF1a promoter, PGK promoter, TRE promoter, U6 promoter, UAS promoter, EFS promoter, SFFV promoter, MSCV promoter, SV40 promoter, UBC promoter, Pro1A promoter, hRHO promoter, hBESTI promoter, Grm6 promoter, GJB2 promoter, a GJB6 promoter, a SLC26A4 promoter, a TECTA promoter, a DFNA5 promoter, a COCH promoter, a NDP promoter, a SYN1 promoter, a GFAP promoter, a PLP promoter, a TAK1 promoter, a SOX21 promoter, a SOX2 promoter, a FGFR3 promoter, a PROX1 promoter, a GLAST1 promoter, a LGR5 promoter, a HESI promoter, a HES5 promoter, a NOTCHI promoter, a JAG1 promoter, a CDKN1A promoter, a CDKN1 B promoter, a SOX10 promoter, a P75 promoter, a CD44 promoter, a HEY2 promoter, a LFNG promoter, a SlOOb promoter, a CLDN11 promoter, an NDP promoter, or synthetic modifications or combinations of these promoters.

[0114] In one embodiment, the viral vector is a viral vector from the list comprising, an adeno- associated viral virus vector (AAV), an adenoviral vector, a lentiviral vector or a retroviral vector. In one embodiment, the viral vector is an adeno-associated viral virus vector (AAV). In one embodiment the AAV comprises one or more of the following vectors: AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-rh8, AAV- rh10, AAV-rh39, AAV-rh43, AAVAnc80, AAV 2 / ShH10, AAV-S vector, AAV6.2, Adv5, oncolytic vectors. In an embodiment , the AAV vector comprises inverted terminal repeat (ITR) sequences. In an embodiment the AAV vector comprises a TATA box. In an embodiment the vector comprises a translation initiation sequence, for example a Kozak sequence.

[0115] According to another aspect, the invention relates to a vector delivery system comprising the nucleic acid or the construct above. In one embodiment, the vector comprises a viral vector. In one embodiment, is a viral vector from the list comprising, an adeno-associated viral virus vector (AAV), an adenoviral vector, a lentiviral vector or a retroviral vector. In one embodiment, the viral vector is an adeno-associated viral virus vector (AAV). In one embodiment, the AAV comprises one or more of the following vectors: AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV- rh8, AAV-rh10, AAV-rh39, AAV-rh43, AAVAnc80, AAV 2 / ShH10, AAV-S vector, AAV6.2, Adv5, oncolytic vectors. In an embodiment, the AAV vector comprises inverted terminal repeat (ITR) sequences. In an embodiment, the AAV vector comprises a TATA box. In an embodiment the vector comprises a translation initiation sequence, for example a Kozak sequence.

[0116] Pharmaceutical composition

[0117] According to another aspect, the invention relates to a pharmaceutical composition comprising the modified IL2 polypeptide or construct of the invention. In one embodiment, the modified IL2 polypeptide is a modified human IL2 polypeptide as described herein. The pharmaceutical composition of the invention can be administered by any convenient route, including but not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitreal, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, by inhalation, or topical, particularly to the ears, nose, eyes, or skin or by inhalation.

[0118] Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. Preferably, the compositions are administered parenterally.

[0119] The pharmaceutically acceptable carrier or vehicle can be particulate, so that the compositions are, for example, in tablet or powder form. The term "carrier" refers to a diluent, adjuvant or excipient, with which a drug antibody conjugate of the present invention is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. In one embodiment, when administered to an animal, the single domain antibody of the present invention or compositions and pharmaceutically acceptable carriers are sterile. Water is a preferred carrier when the drug antibody conjugates of the present invention are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0120] The pharmaceutical composition of the invention can be in the form of a liquid, e.g., a solution, emulsion or suspension. The liquid can be useful for delivery by injection, infusion (e.g., IV infusion) or sub-cutaneously. When intended for oral administration, the composition is preferably in solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.

[0121] As a solid composition for oral administration, the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition typically contains one or more inert diluents. In addition, one or more of the following can be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, corn starch and the like; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the composition is in the form of a capsule (e. g. a gelatin capsule), it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

[0122] The composition can be in the form of a liquid, e. g. an elixir, syrup, solution, emulsion or suspension. The liquid can be useful for oral administration or for delivery by injection. When intended for oral administration, a composition can comprise one or more of a sweetening agent, preservatives, dye / colorant and flavor enhancer. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included.

[0123] Compositions can take the form of one or more dosage units.

[0124] In specific embodiments, it can be desirable to administer the composition locally to the area in need of treatment, or by injection, intravenous injection or infusion. In one embodiment, the composition is part of a device which includes an injector pen. The composition may be provided as a pre-filled syringe or other self-administration device.

[0125] The amount of the therapeutic that is effective / active in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account.

[0126] Typically, the amount is at least about 0.01% of the modified IL2 polypeptide or construct of the invention of the present invention by weight of the composition. When intended for oral administration, this amount can be varied to range from about 0.1 % to about 80% by weight of the composition. Oral compositions can comprise from about 4% to about 50% of the the modified IL2 polypeptide or construct of the invention of the present invention by weight of the composition.

[0127] Preferred compositions of the present invention are prepared so that a parenteral dosage unit contains from about 0.01 % to about 2% by weight of the the modified IL2 polypeptide or construct of the invention of the present invention.

[0128] For administration by injection, the composition can comprise from about typically about 0.1 mg / kg to about 250 mg / kg of the subject’s body weight, preferably, between about 0.1 mg / kg and about 20 mg / kg of the animal's body weight, and more preferably about 1 mg / kg to about 10 mg / kg of the animal's body weight. In one embodiment, the composition is administered at a dose of about 1 to 30 mg / kg, e.g., about 5 to 25 mg / kg, about 10 to 20 mg / kg, about 1 to 5 mg / kg, or about 3 mg / kg. The dosing schedule can vary from e.g., once a week to once every 2, 3, or 4 weeks.

[0129] Exemplary therapeutic applications

[0130] According to another aspect, the invention relates to a modified IL2 polypeptide according to the invention, or a construct according to the invention, a viral vector or a pharmaceutical composition according to the invention for use in the treatment and / or prevention of disease. In one embodiment, the modified IL2 polypeptide is a modified human IL2 polypeptide as described herein.

[0131] As used herein, "treat", "treating" or "treatment" means inhibiting or relieving a disease or disorder. For example, treatment can include a postponement of development of the symptoms associated with a disease or disorder, and / or a reduction in the severity of such symptoms that will, or are expected, to develop with said disease. The terms include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result is being conferred on at least some of the mammals, e.g., human patients, being treated. Many medical treatments are effective for some, but not all, patients that undergo the treatment.

[0132] The term "subject" or "patient" refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline.

[0133] As used herein, the term "effective amount" means an amount of a modified IL2 polypeptide that when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective to achieve the desired therapeutic or prophylactic effect under the conditions of administration.

[0134] In one embodiment, said disease is selected from cancer, an immune disorder, neurological disease, inflammatory disorder, allergy, transplant rejection, viral infection, immune deficiency or other immune system-related disorder.

[0135] According to another aspect, the invention relates to a method for treating a cancer, an immune disorder, neurological disease, inflammatory disorder, allergy, transplant rejection, viral infection, immune deficiency or other immune system-related disorder comprising administering a therapeutically effective amount of a modified IL2 polypeptide according to the invention, a construct according to the invention, a vector according to the invention or a pharmaceutical composition according to the invention. In one embodiment, the modified IL2 polypeptide is a modified human IL2 polypeptide as described herein.

[0136] According to another aspect, the invention relates the use of a therapeutically effective amount of a modified IL2 polypeptide according to the invention, a construct according to the invention, a vector according to the invention or a pharmaceutical composition according to the invention in the manufacture of a medicament for to a method for treating a cancer, an immune disorder, neurological disease, inflammatory disorder, allergy, transplant rejection, viral infection, immune deficiency or other immune system- related disorder comprising administering. In one embodiment, the modified IL2 polypeptide is a modified human IL2 polypeptide as described herein.

[0137] The cancer can be selected from a solid or non-solid tumor. For example, the cancer may be selected from bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, breast cancer, brain cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, kidney cancer, sarcoma of soft tissue, cancer of the urethra, cancer of the bladder, renal cancer, lung cancer, non-small cell lung cancer, thymoma, urothelial carcinoma leukemia, prostate cancer, mesothelioma, adrenocortical carcinoma, lymphomas, such as such as Hodgkin's disease, non-Hodgkin's, gastric cancer, and multiple myelomas.

[0138] In one embodiment, the tumor is a solid tumor. Examples of solid tumors which may be accordingly treated include breast carcinoma, lung carcinoma, colorectal carcinoma, pancreatic carcinoma, glioma and lymphoma. Some examples of such tumors include epidermoid tumors, squamous tumors, such as head and neck tumors, colorectal tumors, prostate tumors, breast tumors, lung tumors, including small cell and non-small cell lung tumors, pancreatic tumors, thyroid tumors, ovarian tumors, and liver tumors. Other examples include Kaposi's sarcoma, CNS, neoplasms, neuroblastomas, capillary hemangioblastomas, meningiomas and cerebral metastases, melanoma, gastrointestinal and renal carcinomas and sarcomas, rhabdomyosarcoma, glioblastoma, preferably glioblastoma multiforme, and leiomyosarcoma. Examples of vascularized skin cancers for which the antagonists of this invention are effective include squamous cell carcinoma, basal cell carcinoma and skin cancers that can be treated by suppressing the growth of malignant keratinocytes, such as human malignant keratinocytes.

[0139] In one embodiment, the tumor is a non-solid tumor. Examples of non-solid tumors include leukemia, multiple myeloma and lymphoma.

[0140] In one embodiment, the cancer is locally advanced unresectable, metastatic, or recurrent cancer. In one embodiment, the immune disorder is an autoimmune disorder. In one embodiment, autoimmune disorder is autoimmune disease is alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison’s disease, autoimmune diseases of the adrenal glands, and autoimmune hemolytic anemia, Autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Bechet syndrome, bullous pemphigoid, cardiomyopathy, stomatitis diarrhea dermatitis, chronic fatigue immune dysfunction syndrome, Chronic inflammatory demyelinating polyneuropathy, Chusch's syndrome, scar pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, idiopathic mixed cold bulb Proteinemia, diabetes, eosinophilic fasciitis, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guerrilla-Barr syndrome, Hashimoto's thyroiditis, Hen-Sher's disease Purpura, idiopathic pulmonary fibrosis, idiopathic / autoimmune thrombocytopenic purpura, IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere syndrome, mixed connective tissue disease, multiple sclerosis, 1 Type or immune-mediated diabetes, myasthenia gravis, pemphigus-related diseases, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis, dermatomuscularis Inflammation, primary aglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Reye's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Jaglen syndrome, systemic myotonia syndrome, systemic lupus erythematosus, Svet's syndrome, Still's disease, lupus erythematosus, Gaoan's arteritis, transient arteritis / giant cell arteritis, ulcerative colitis, grapevine Meningitis, vasculitis, vitiligo, Wegener's granulomatosis.

[0141] The modified polypeptides, constructs, vectors or pharmaceutical composition of the invention may be administered as the sole active ingredient or in combination with one or more other therapeutic agent. A therapeutic agent is a compound or molecule which is useful in the treatment of a disease. Examples of therapeutic agents include antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, pro-apoptotic agents, anti-angiogenic agents, boron compounds, photoactive agents or dyes and radioisotopes. An antibody molecule includes a full antibody or fragment thereof e.g., a Fab, F(ab')2, Fv, a single chain Fv fragment (scFv) or a single domain antibody, for example a VH or VHH domain, or antibody mimetic protein. In yet another aspect, there is provided a method of modulating an immune response in a subject comprising administering to the subject the IL2 modified polypeptide, the construct, vector or pharmaceutical composition of the invention such that the immune response in the subject is modulated. In one embodiment, the modified IL2 polypeptide is a modified human IL2 polypeptide as described herein.

[0142] Exemplary kits

[0143] In another aspect, the invention provides a kit comprising a modified IL2 polypeptide according to the invention, or a construct according to the invention, or a pharmaceutical composition according to the invention optionally together with a reagent and / or instructions for use. In one embodiment, the modified IL2 polypeptide is a modified human IL2 polypeptide as described herein.

[0144] In one embodiment, the kit is suitable for the treatment or prevention of a disease for example as listed herein. Such a kit may contain other components, packaging, instructions, or material. The kit may include a labeled modified IL2 polypeptide and one or more compounds for detecting the label.

[0145] The invention in another aspect provides a modified IL2 polypeptide according to the invention, or a construct according to the invention, or a pharmaceutical composition according to the invention described herein packaged in lyophilized form, or packaged in an aqueous medium. In one embodiment, the modified IL2 polypeptide is a modified human IL2 polypeptide as described herein.

[0146] Exemplary nucleic acids, vectors and host cells

[0147] According to another aspect, the present invention further provides a nucleic acid encoding the modified IL2 polypeptide according to the invention. Nucleic acid may include DNA and / or RNA. In one embodiment, the modified IL2 polypeptide is a modified human IL2 polypeptide as described herein.

[0148] A nucleic acid according to the present invention may comprise DNA or RNA and may be wholly or partially synthetic or recombinantly produced. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which II is substituted for T, unless context requires otherwise.

[0149] Furthermore, the invention relates to a vector comprising at least one nucleic acid as defined above.

[0150] The invention also relates to a host cell comprising one or more nucleic acids or vectors described above. In one embodiment the host cell comprises an isolated recombinant host cell. The host cell may be a bacterial, viral, plant, mammalian or other suitable host cell. In one embodiment, the cell is an E. coli cell. In another embodiment, the cell is a yeast cell. In another embodiment, the cell is a Chinese Hamster Ovary (CHO) cell.

[0151] According to one aspect, the invention further comprises a method for producing a modified IL2 polypeptide according to the invention comprising expressing a nucleic acid encoding said modified IL2 polypeptide in a host cell and isolating the modified IL2 polypeptide from the host cell. The method may additionally comprise a step of culturing the host cell under conditions suitable for expression of the polynucleotide encoding the modified IL2 polypeptide.

[0152] Methods for identifying modified polypeptide (muteins)

[0153] According to one aspect, the invention further comprises a method for identifying a modified IL2 polypeptide comprising the steps of

[0154] 1) predicting aggregation-prone regions from an IL2 polypeptide,

[0155] 2) identifying amino acid mutations that abolish the aggregation propensity of these segments without affecting the thermodynamic stability of the polypeptide and

[0156] 3) expressing a modified IL2 polypeptide with reduced aggregation.

[0157] In one embodiment, the method for identifying a modified polypeptide comprises the SolubiS method (https: / / solubis.switchlab.org / about).

[0158] The term “aggregation-prone regions” means regions of the polypeptide sequence that are likely to aggregate and / or misfold. In one embodiment, the prediction of the aggregation-prone regions is based on a statistical mechanics algorithm, such as TANGO (https: / / tango.crg.es / ). The term “thermodynamic stability” means when a system is in its lowest energy state, or in chemical equilibrium with its environment. In one embodiment, the thermodynamic contribution of the aggregation-prone regions to the stability of the protein is predicted with a protein design algorithm. In one embodiment the protein design algorithm is FoldX (https: / / foldxsuite.crg.eu / ).

[0159] In one embodiment, the IL2 modified polypeptide is a modified human or modified murine IL2 polypeptide. In one embodiment, the polypeptide is a wild type IL2 molecule. In one embodiment, the IL2 molecule is a mammalian IL2 molecule, for example a human or murine IL2 molecule. In one embodiment the IL2 molecule is SEQ ID NO: 1 or SEQ ID NO: 2.

[0160] Exemplary methods for altering T cell responses, alter the ratio of non aggregated: aggregated IL2 production

[0161] According to one aspect, the invention further comprises an in vivo, in vitro or ex vivo method of promoting Treg responses within a population of T cells, comprising contacting the population of T cells with an effective amount of a modified IL2 polypeptide according to the invention. In one embodiment, the modified IL2 polypeptide is a modified human IL2 polypeptide as described herein.

[0162] According to one aspect, the invention further comprises a method of promoting Treg responses, comprising introducing an amino acid substitution as shown herein into an IL2 polypeptide, e.g. a wild type IL2 polypeptide, e.g. a human wild type IL2 polypeptide.

[0163] In one embodiment of these methods, the modified IL2 polypeptide is a modified human IL2 polypeptide and comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. an H at position 108 and an R at position 151 ; or ii. a W at position 36; or iii. an R at position 111 , an R at position 110 and an R at position 151.

[0164] In one embodiment, the modified IL2 polypeptide is a modified human IL2 polypeptide as described herein. According to one aspect, the invention further comprises an in vivo, in vitro or ex vivo method of promoting CD8+ T cell responses, comprising contacting the population of T cells with an effective amount of a modified IL2 polypeptide according to the invention. In one embodiment, the modified IL2 polypeptide is a modified human IL2 polypeptide as described herein.

[0165] According to one aspect, the invention further comprises a method of promoting CD8 responses, comprising introducing an amino acid substitution as shown herein into an IL2 polypeptide, e.g. a wild type IL2 polypeptide, e.g. a human wild type IL2 polypeptide.

[0166] In one embodiment of these methods, the modified IL2 polypeptide is a modified human IL2 polypeptide and comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. a K at position 65 and a K at position 82; or ii. a K at position 65, a K at position 82, an R at position 110 and an R at position 151.

[0167] In one embodiment, the modified IL2 polypeptide is a modified human IL2 polypeptide as described herein.

[0168] According to one aspect, the invention further comprises a method of decreasing the aggregation of IL2 polypeptide, comprising introducing an amino acid substitution as shown herein into an IL2 polypeptide, e.g. a wild type IL2 polypeptide, e.g. a human wild type IL2 polypeptide.

[0169] Thus, the invention further comprises a method of increasing the ratio non aggregated IL2 production to aggregated IL2 production within a population of cells, comprising introducing an amino acid substitution as described herein.

[0170] In one embodiment of these methods, the modified IL2 polypeptide is a modified human IL2 polypeptide and comprises i. an R at position 111 and an N at position 143; ii. an R at position 110 and an R at position 151 ; iii. an R at position 110 and an N at position 143; iv. a K at position 65, a K at position 82, an R at position 110, and an R at position

[0171] 151 ; or v. an R at position 111 , an R at position 110 and an R at position 151.

[0172] In one embodiment, the ratio of non-aggregated IL2 production to aggregated IL2 production increases at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% in comparison to control wild type IL2. In one embodiment, the ratio of non-aggregated IL2 production to aggregated IL2 production increases at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold or more in comparison to control wild type IL2.

[0173] The method of introducing the amino acid substitution of the invention and / or measuring the level or aggregation / IL2 protein production is not limited to any particular method. Suitable methods would be readily identified by the skilled person. Suitable methods for the introduction of the amino acid substitution include site-directed mutagenesis and PCR-mediated mutagenesis. The modified IL2 polypeptide can be tested for desired function (e.g. reduction in IL2 aggregation, increase in IL2 protein production) using the functional assays described herein, for example by a sandwich ELISA.

[0174] The above methods may be an in vitro, ex vivo or in vivo method for application in a subject, cell, tissue or cell culture.

[0175] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present disclosure, including methods, as well as the best mode thereof, of making and using this disclosure, the following examples are provided to further enable those skilled in the art to practice this disclosure. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present disclosure will be apparent to those skilled in the art in view of the present disclosure. All documents mentioned in this specification are incorporated herein by reference in their entirety, including references to gene accession numbers, scientific publications and references to patent publications.

[0176] "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and / or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[0177] The invention is now further described in the non-limiting examples.

[0178] Examples

[0179] The pleotropic nature of interluekin-2 (IL2) has allowed it to be used as both a pro- inflammatory and anti-inflammatory therapeutic agent, through promotion of regulatory T cell (Treg) responses via the IL2RA receptor or promotion of CD8 T cell responses via the IL2RB receptor, respectively. However, the utility of IL2 as a treatment is limited by this same pleiotropy, and protein engineering to bias specificity towards either the regulatory T cell (Treg) or CD8 T cell lineage often requires a trade-off in protein production or total bioactivity. Here we use SolubiS, a computational algorithm-based method, to predict mutations within the IL2 structure to improve protein production yield while altering cellular selectivity, to generate a mutein with elevated therapeutic potential. The design and testing process identified the V126R (murine) I V111 R (human) mutation as a Treg-enhancing mutein, creating a cation repulsion to inhibit primary binding to IL2RB, with a post-IL2RA confirmational shift enabling secondary IL2RB binding, and hence allowing the trimeric receptor complex to form. In human IL2, additional N110R T151 R aggregation-protecting mutations could improve protein yield of the V111 R mutation. The approach also generated novel CD8 T cell-promoting mutations. Y79K created a cation-cation repulsion with IL2RA, while Q50W enhanced CD8 T cell activity through potential TT-stacking enhancing binding to IL2RB, with the combination highly stimulatory for CD8 T cells. For human IL2, Y65K (homolog to murine Y79K) coupled with E82K prevented IL2RA binding, however it required the aggregation-protecting mutations of N110R T151 R to rescue production. These muteins, designed with both cellular specificity and protein production features, have potential as both biological tools and therapeutics.

[0180] Methods

[0181] !L2 mutation design

[0182] Mutations were designed based on four structures of human IL2: 1 m47 (IL2 alone, resolution of 1.99 A), 1z92 (IL2 with IL2RA, resolution of 2.8 A), 2b5i (IL2 with IL2RA / IL2RB / IL2RB, resolution of 2.3 A) and 2erj (IL2 with IL2RA / IL2RB / IL2RB, resolution of 3 A), and one structure of mouse IL2: 4yqx (IL2 bound to anti-l L2 single chain Fv, resolution of 2.8 A). Mutation design was based on the SolubiS method to predict aggregation-prone regions from the primary peptide sequence, and the thermodynamic contribution of these regions to the stability of the protein using FoldX, an empirical force field developed for the rapid evaluation of the effect of mutations, and TANGO, the statistical thermodynamic algorithm. TANGO calculates the intrinsic aggregation propensity of aggregation-prone regions as a Boltzmann distribution with competing secondary structural tendencies such as a-helical or p-hairpin structure. The benefit of this implementation is that TANGO predicts aggregation-prone regions with well-defined sequence boundaries, with high specificity and thus predicts few false positives. Each amino acid of the wildtype IL2 protein was mutated to every other amino acid in each structure. The effect of both the thermodynamic stability (ddG) as well as the stability of the interaction (ddGComplex) with each of the receptors was calculated using FoldX, and the effect of each mutation on the aggregation propensity was calculated using TANGO, based on sequence alone. This analysis resulted in a large table of FoldX energies and aggregation propensity (TANGO) scores. Mutations were selected using four strategies: SolubiS (reducing aggregation while retaining stability), kill IL2RA binding (reducing interaction energy with IL2RA, while retaining stability and preferably reducing aggregation), kill IL2RB binding (reducing interaction energy with IL2RB, while retaining stability and preferably reducing aggregation), and combinations (identifying synergies that combine aggregation reduction and IL2RA / IL2RB binding). The thresholds used were reduction of TANGO score by more than 100, for reduction in aggregation, while not destabilising h I L2 thermodynamically (ddG < 0.5 kcal / mol) and not affect any receptor interactions negatively (ddG_complex < 0.5 kcal / mol). These single point mutations were calculated in combination to allow for the previously described synergistic effect of SolubiS mutations on the aggregation resistance of proteins. These mutation combinations were selected in the same way as single SolubiS mutations, to ensure compatibility.

[0183] Structural models of IL2 in complex with the trimeric receptor was performed based off the 2erj structure of the human IL21 IL2R complex. Due to the high homology between mouse and human IL2 and IL2R complex, and the lack of a mouse structure, selected human mutations were directly imposed on the mouse sequence. Each introduced amino acid was the same for both human and mouse mutant. This resulted in some cases where a different wildtype amino acid was mutated to the same mutant amino acid. For example, in the case of the H36W mutation, where the human homologue has a histidine residue, the mouse homologue has a glutamine residue, making the mouse equivalent mutant Q50W. FoldX calculations were performed on the monomeric crystal structure 4yqx (mlL2 alone, resolution 2.8 A).

[0184] Cloning and molecular biology

[0185] IL2 muteins were ordered as Gene Fragments from GeneWiz, Azenta. Gene Fragments were cloned into pJet Blunt 1.2 (ThermoFisher, K1231) and sequenced via Sanger Sequencing (Genewiz, Azenta) to confirm correct sequence insertion. Correct insertions were amplified via PCR adding restriction enzyme sites to each end. PCR products were purified using GeneJET PCR purification kit (ThermoFisher, K0701) before being digested with Pfl23l I (ThermoFisher, FD0854) and Ndel (ThermoFisher, FD0583) and gel purified (GeneJET Gel Extraction Kit, ThermoFisher, K0691) to obtain the IL2 sequence. For low-expressing murine muteins V126R, N123H T166R and Y79K E96K T166R, codon-optimised sequences were used. Fragments were then cloned into an expression vector, downstream of an EF-1a promoter and in frame with a TEV protease target and a 6xHis-Tag. Finally, sequences were confirmed using Sanger sequencing (Genewiz, Azenta).

[0186] IL2 production and measurement

[0187] Expression plasmids were transfected into HEK293 cells using FuGENE HD Transfection reagent (Promega, E2311) in 24 well plates containing 500pL DMEM supplemented with 10% FBS. 24 hours following transfection, wells were topped with an additional 250pL of DMEM, 10% FBS. After a total of 48 hours, the culture supernatant containing IL2 protein was harvested and frozen at -80°C. All supernatants were aliquoted prior to freezing to prevent multiple freeze thaws cycles. The cells were washed with PBS and detached, and cell pellets were frozen at -80°C ready for DNA extraction.

[0188] DNA was extracted from frozen cell pellets using the DNeasy Blood and Tissue Kit (Qiagen, 69504) following manufacturer's instructions. DNA samples were analysed using QuantStudio 1 via qPCR using TaqMan Gene Expression Assays (FAM) for the presence of neomycin resistance cassette (plasmid)(custom), GAPDH (Hs02786624_g1) and PPIA (Hs04194521_s1) and normalised against transfection control to assess transfection efficiency. IL2 production was analysed via a modified sandwich ELISA. Plates were coated with anti-IL2 antibodies ((mouse: clone JES6- 1A12, BioLegend, 503701) (human: polyclonal, Bio-Techne, AF-202-NA)). The detection antibody was HRP-conjugated anti-Histag (BioLegend, 652503). Standard curves were created using his-tagged IL2 (GeneTex: Mouse; GTX00285-pro, Human; GTX00092-pro), from which concentrations of IL2 in cell culture supernatant were interpolated. To compare production efficiency of each IL2 mutein, final concentrations were normalised by transfection efficiency values calculated from aforementioned qPCR.

[0189] Phospho- STAT Flow cytometry

[0190] For murine phospho-STAT assays, spleens dissected from C57BI / 6 mice were mechanically disrupted between two glass slides, filtered through 100pm mesh and red blood cell lysed to form single cell suspensions. Cell suspensions were counted using CellDrop cell counter. Non-specific binding was blocked using 2.4G2 supernatant. Surface staining was performed with antibodies against CD4, CD127, CD62L, CD25, CD44 as well as an e780 fixable Live-Dead stain (eBioscience). Splenocytes were then stimulated with WT IL2 and mutated IL2 diluted to a range of concentrations (400- 0.02ng / ml) in complete DM EM. Immediately following stimulation cells were fixed with paraformaldehyde followed by methanol. An overnight stain was the preformed with antibodies against Foxp3, CD3, CD8, pSTAT5, CD122, CD132 and pSTAT3. Following staining, data was acquired on a Cytek Aurora, with data collection performed in SpectroFlo (Cytek) and analysed using FlowJo and GraphPad Prism. For human phospho-STAT assays, PBMCs were isolated from leukocytes cones from healthy donors (NHSBT NCI) using Ficoll following the manufacturer’s instructions. Cell suspensions were counted using CellDrop cell counter. Non-specific binding was blocked using FcX Trustain (1 :200) (BioLegend). Surface staining was performed with antibodies against CD4, CD3, CD8, CCR7, CD45RA, CD27, CD56 as well as an e780 fixable live dead stain (eBioscience). Cells were then stimulated with WT and mutant IL2, diluted to a range of concentrations (400-0.02 ng / mL) in complete DMEM. Immediately following stimulation cells were fixed with paraformaldehyde followed by methanol. An overnight stain {36373983} was the performed with antibodies against FOXP3, CD25, and pSTAT5. Following staining, data was acquired on a Cytek Aurora, with data collection performed in SpectroFlo (Cytek) and analysed using FlowJo and GraphPad Prism.

[0191] In vivo gene delivery and assessment

[0192] For gene therapy studies using adeno-associated vectors male and female C57BL / 6 wild-type mice were obtained from Babraham Institute Biological Support Unit. All mice were maintained in a specific pathogen free environment, with ambient temperature, humidity, and lighting maintained for the well-being. Before experimentation, mice were transferred to individually-ventilated cages, and grouped matching age and sex. Mice were fed with either wet or dry standard food pellets based on the animal food consumption preferences.

[0193] Wildtype (C57BL / 6) mice were intranasally inoculated with 50pL of 1x1011GC / ml of adeno-associated vectors (AAV) diluted in PBS. All mouse experimentation and procedures compiled with the UK Home Office guidelines observing the Animals Scientific Procedures Act 1986. Experiments were also approved by the Babraham Institute Animal Welfare and Ethical Review Body (Protocol study number PP3981824- Lymphocyte development and immune function in the tissues). rAAV constructs were created using AAV6.2 capsid and Scgblal promoter. The expressed gene was GFP-Myc-Cre (Addgene #49056), murine IL2 (NM_008366.3) or murine IL2 V126R (GTA to AGA in codon 126). The vectors were produced by Vector Builder, tested the vectors for sterility and mycoplasma contamination, quantified by quantitative PCR (qPCR) and confirmed above 90% purity using SDS PAGE and silver staining by Vector Builder.

[0194] At the indicated time points post-treatment, mice were injected intravenously with anti- CD45 antibody before euthanasia. The lung was harvested into PBS with 2.5% FCS and 2mM EDTA. Tissues were homogenized in (Protein Quant Sample Lysis kit Thermo Scientific™ buffer and a protein inhibitor (Complete Mini EDTA Sigma). The resultant supernatant was checked for IL2 levels using ProQuantum High-Sensitivity Immunoassays Kit from Thermo Fisher following manufacturers protocol. IL2 standard curves were done on ProQuantum platform (Thermo Scientific).

[0195] For flow cytometry analysis, tissues were processed as previously described. Samples were prepared by initially blocking Fc interactions with 2.4 G2 hybridoma, followed by viability staining, staining with the surface marker cocktail for 2 hours, and staining overnight for intracellular markers. 10,000 Precision Count beads (BioLegend) were added to sample before acquiring Cytek™ Aurora (5 Lasers N9-20001_Spectral). Data analysis, including flow cytometry gating, was performed with FlowJo v.10.0.7 and FlowJo version 10.8.1 (BD).

[0196] Results

[0197] Example 1 Directed disruption of IL2 aggregation sites designed by SolubiS

[0198] A key limitation in the production capacity of proteins is the aggregation of nascent polypeptides, mediated through aggregation-prone hydrophobic stretches. As proteinprotein binding sites are frequently hydrophobic, this feature allows the potential for the design of mutations that both disrupt a protein-protein interaction and also improve protein production through lowering the aggregation rates. In order to improve the properties of IL2, we designed a series of mutations in both human and mouse IL2. Mutation design was based on the structure of the human and murine IL2 structures, and used the SolubiS method to provide maximal improvements with minimal point mutations. Candidate mutations were selected for the ability to reduce the interaction energy of IL2 for IL2RA or IL2RB, while reducing the aggregation propensity of the polypeptide. (Table 1).

[0199] Table 1 IL2 muteins design strategy

[0200] Organism Mutation # of Human homolog Strategy

[0201] Example 2 Production screening of IL2 muteins identifies point mutations with improved production capacity To functionally test the designed IL2 mutations, we first screened for production capacity of each mutein, through cloning the IL2 muteins into a HEK293 expression system. Through normalisation of IL2 protein production (measured through ELISA for a His tag fused to the C terminus of each protein) to HEK293 transfection efficiency (measured through PCR quantification of expression plasmid), we could compare the production output of each mutein relative to the wildtype IL2. For the mutations in murine IL2, muteins T116R, V126R A158N, and Y79K resulted in a trend towards elevated IL2 production, up to ~3-fold higher than the wildtype IL2 levels, while other mutants were equivalent or worse in production (Figure 1A). For the mutation in human IL2, muteins N110R T143N, V111R T143N trended towards increased production but no mutants were significantly elevated and several had production defects (Figure 1B). The combination of N110R T151R with Y65K E82K resulted in a rescue of production (p=0.055) similar to that of WT whilst the Y65K E82K mutation on it’s own resulted in a markedly reduced production (Figure 1B).

[0202] Example 3 SolubiS murine IL2 muteins have altered cellular specificity

[0203] To determine the impact of these IL2 mutations on receptor specificity, we used a functional assay based on detection of signalling within murine Tregs or CD8 T cells. Using phospho-STAT5 (pSTAT5) as the readout for receptor triggering in both Tregs and CD8 T cells, following incubation in a mixed murine splenocyte population, activation was assessed by flow cytometry. Of the murine mutations designed to prevent binding to IL2RA (E96K and Y79K), both demonstrated reduced pSTAT5 activation in Tregs, alone and in combination (Figure 2A). In the case of Y79K, this indicated reduced bioactivity on Tregs. E96K, by contrast, also had production issues and was therefore tested at suboptimal doses (0.3ng / ml), with the poorer pSTAT5 activation potentially due to lower concentrations. Of the murine mutations designed to prevent binding to IL2RB (Q50W, D54R, N123H, V126R), Q50W and V126R had preserved activity on Tregs, while D54R and N123H had reduced activity on Tregs (Figure 2A), indicating the latter two mutations had off-target detrimental effects (although in the case of N123H, this may alternatively be attributed to suboptimal production (5.9ng / ml)). For the effects on CD8 T cells, V126R, V126R A158N and N123H T166R combined normal Treg stimulation with the predicted loss-of-signalling on CD8 T cells, while Q50W had an unexpected major gain-of-signalling effect on CD8 T cells (Figure 2B). The Y79K mutation retained CD8 T cell stimulation, while having impaired Treg stimulation (Figure 2A,B), consistent with impeded IL2RA binding. Residues designed to boost production, A158N and T166R, were neutral to cell selectivity (Figure 2A,B).

[0204] From the initial screen, three murine mutations warranted detailed functional analysis: Y79K, V126R and Q50W. To determine the relative bioactivity of these muteins on Tregs and CD8 T cells, we used the same pSTAT5 response assay, across a titrated dose range, providing enhanced sensitivity for detection of functional changes. Compared to both commercial IL2 and the wildtype IL2 control, Y79K demonstrated a >300 fold reduction in activity on Tregs (Figure 2C), while maintaining normal activity on CD8 T cells (Figure 2D), consistent with the designed defect in IL2RA binding. The reverse was observed for V126R (designed for defective IL2RB binding), with normal activity on Tregs (Figure 2C), but no detectable effect on CD8 T cells even at high doses (Figure 2D). As identified in the functional screening assay, Q50W demonstrated an unexpected effect: normal activity on Tregs (Figure 2C), but strongly elevated effects (

[0205] ~100-fold increased activity) on CD8 T cells (Figure 2D). Together, these results indicate Y79K results in CD8 T cell-specific signalling, V126R results in Treg-specific signalling, and Q50W acts as an unexpected CD8 T cell superkine.

[0206] In order to identify the potential mechanism by which Q50W displayed an unexpected increase in activity on CD8 T cells, we modelled the effects of the three mutations on murine IL2 1 IL2R binding, using a structure built on the human homologs (Figure 3A). First, for Y79K, when modelled as bound to the trimeric IL2R, the native Y79 residue of IL2 is in close proximity to positive charges on the R35 and R36 residues of IL2RA (Figure 3B), consistent with a cation-TT interaction. The replacement of this residue by Y79K creates a cation-cation repulsion, consistent with a loss of binding to IL2RA. For the V126R mutein, as the IL2RB interface of IL2 changes conformation after it binds IL2RA, we modelled the complex in the presence and absence of IL2RA. In the unbound state of IL2, the V126R residue would occupy the same space as R41 / R42 on IL2RB, creating a direct cation repulsion effect (Figure 3C). However following binding to IL2RA, IL2 changes confirmation in the helix C on which V126R lies, a confirmation change postulated to prime recruitment of IL2RB into the trimeric receptor complex {16293754}. This conformational change pulls back the V126R residue, reducing the direct cation-cation clash (Figure 3C). This is consistent with the observed poor activation through the dimeric IL2RB / IL2RG complex, but unimpeded activation through the trimeric IL2RA / IL2RB / IL2G complex. Finally, the Q50W mutation was modelled as the analogue to the human H36W, based on the human IL2 / IL2R structure. While the H36W mutation was predicted to disrupt the interaction, based on the crystal structure of the complex, the Q50W was initially only checked for the effect on the stability of the monomer. Subsequent modelling of the mouse complex revealed the Q50W side-chain falls within a hydrophobic groove of IL2RB with potential TT-stacking interactions with F101 and F135 residues (Figure 3D). The larger hydrophobic side-chain may therefore enhance binding, providing preferred signalling through IL2RB.

[0207] Example 4 In vivo functional testing of V126R IL2 mutein

[0208] As proof-of-principle for the utility of the developed IL2 muteins for in vivo use, we sought to test the Treg-promoting V126R IL2 mutein in mice. We used an AAV- encoding system that delivers cargo to the lung epithelium, using an AAV6.2 capsid and a club cell-specific promoter (Figure 4A). Transduced cells, expressing the appropriate transcription factors to activate the delivered Scgblal promoter, are able to drive the production of IL2 in the lung tissue, without giving systemic IL2 production (manuscript in preparation). The system results in rapid production of IL2, with free IL2 levels dropping as IL2-consuming cells build up (Figure 4B). The V126R IL2 mutein built up at higher initial levels than the wildtype IL2 (Figure 4B), consistent with a cellular restriction in the consumption sources. The expression of wildtype IL2 in the lung increased the representation of lung Tregs, while Tregs in other tissues remained normal (Figure 4C). The V126R IL2 mutein demonstrated a similar tissue-restriction, with a further elevation of Treg numbers compared to wildtype IL2 (Figure 4C). This was achieved without expansion of the resident CD8 T cell population (Figure 4D), demonstrating improved in vivo properties of the V126R IL2 mutant in Treg expansion.

[0209] Example 5 Combinatorial generation of a CD8 T cell superkine

[0210] The muteins Q50W and Y79K both demonstrated different desirable properties as a CD8 T cell-promoting IL2 mutant. Q50W increased CD8 T cell signalling, while Y79K boosted production and impeded Treg signalling (Figure 2C,D). To determine if we could engineer a CD8 T cell superkine with all three properties, we sought to combine these mutations. Combination of Q50W Y79K rescued the mild production defect of Q50W, giving a similar production boost achieved by Y79K alone (Figure 1A. Upon titration, the Q50W Y79K mutant showed decreased Treg responses by ~30 fold compared to both WT and Q50W alone (Figure 5A), giving a defect intermediate to that observed by Y79K. In CD8 T cells, responses to Q50W Y79K, mimicked that of Q50W (Figure 5B). The combination of Q50WY79K therefore creates a cytokine with the ideal properties of the two parental mutants, with improved production, enhanced CD8 T cell responsiveness and impeded Treg responsiveness.

[0211] Example 6 SolubiS human IL2 muteins have altered cellular specificity

[0212] Similar to the murine muteins, to determine the impact of the human IL2 mutations on receptor specificity, we first used a functional assay based on detection of signalling within human Tregs or CD8 T cells in mixed PBMCs. Of the mutations designed to prevent IL2 binding to the IL2RB (H36W, D40R, N108H, V111 R), two of the mutations (D40R, N108H) performed poorly, with detrimental impacts on the pSTAT5 response in Tregs (Figure 6A). This suggests these muteins have a net defect in bioactivity due to poor recruitment of IL2RB to the IL2 / IL2RA complex, potentially exacerbated by poor production (D40R=70.83 ng / ml, N108H 7.65 ng / ml). Three muteins, H36W, V111 R and N108H T151 R, by contrast, achieved the desired properties of maintained near-normal pSTAT5 responsiveness in Tregs, with also having a sharply reduced signal in CD8 T cells (Figure 6B). Notably, the V111 R mutein is the human homolog of V126R, where the structural modelling suggests IL2 / IL2RA complex binding may be rescued by a conformational shift in the mutein residue (Figure 3C). For the residues designed to boost production, N110R, T143N and T151 R were neutral to Treg signalling, although T143N was detrimental when combined with mutations designed to impede IL2RB binding (Figure 6A). The H36W, V111 R and N108H T151 R muteins were therefore candidates for Treg-promoting muteins, with poor IL2RB signalling, with V111 R having marginally better production of the three. This production was improved above wildtype values through ad hoc combination with N110R T151 R, the SolubiS mutation with the best initial production values (Figure 1B).

[0213] Of the muteins designed to prevent binding to IL2RA (E82K, Y65K, Y65K E82K) all three modestly reduced Treg responses as designed (Figure 6A). However, E82K and Y65K also did not produce pSTAT5 responses in CD8 T cells, potentially due to production issues lowering the assay concentration (E82K = 10.9 ng / ml, Y65K = 9.5 ng / ml) (Figure 6B), making them less useful as candidate cytokines. Interestingly, the combined Y65K E82K mutein stimulated marked responses in CD8 T cells despite production issues (used at a concentration 6-fold less than WT, 30.4 ng / ml) (Figure 6B). The Y65K E82K mutation therefore met the biological requirements for a CD8 T cell-promoting mutein, although the coupling with poorer production would impede therapeutic use. Fortunately, we found that while N110R T151 R in isolation demonstrated no increase in production, when in combination with Y65K E82K it rescued the marked defect in production caused by the Y65K E82K mutation and so it was decided to take forward Y65K E82K N110R T151 R (Figure 1 B).

[0214] Example 6 Combination mutants result in human IL2 muteins with enhanced cellular selectivity

[0215] Based on this biological characterisation, four human muteins were of interest for further testing: V111 R of particular interest for potential Treg specificity, and the V111 R N110R T151 R combination with boosted production; Y65K E82K, of particular interest for potential CD8 T cell specificity, and the Y65K E82K N110R T151 R production-boosted version. We therefore characterised these four cytokines in the PBMC pSTAT assay for titrated responses to Treg and CD8 T cell activation.

[0216] For the potential Treg-boosting V111 R and V111 R N110R T151 R muteins, a titration of the muteins on human PBMCs had intact pSTAT responses from Tregs down to 10ng / ml, with a drop-off compared to wildtype IL2 at lower concentrations (Figure 7A). By contrast, CD8 T cell responses were muted ~100-fold, even at the highest doses tested (Figure 7B). These results confirm that the V111 R mutein, like the analogous V126R mouse mutein, has enhanced specificity for Tregs while maintaining near normal bioactivity.

[0217] For the potential CD8 T cell-selective muteins, Y65K E82K and Y65K E82K N110R T151 R, production runs were tested in the same PBMC pSTAT assay. In titrations both the Y65K E82K and Y65K E82K N110R T151 R muteins showed sharply diminished Treg responses (-3000 fold) (Figure 7C). By contrast, CD8 T cell responses were akin to WT (Figure 7D), demonstrating strong CD8 T cell selectivity. The response curves for Treg and CD8 T cells were highly similar (Figure 7C,D), suggesting near-complete elimination of IL2RA binding and activation of Tregs only through the IL2RB-IL2RG complex, as in CD8 T cells. With the enhanced production values of the mutein Y65K E82K N110R T151 R (Figure 1B), the combination mutein had overall superior properties as a CD8 T cell-stimulating muteins.

[0218] Discussion Protein solubility is adapted to endogenous protein abundance in the cell, where protein folding occurs within the context of specific chaperones and potential interaction partners. For use in biotechnology or therapeutics, proteins require production in recombinant incubators, typically in distinct cell types to the natural producer and frequently at concentrations that are several orders of magnitude above their physiological concentration. This often results in protein aggregation, reducing the total protein production yield and increasing costs, creating barriers in affordability and therefore availability of these drugs for patients. Aggregation is a complex process, influenced by physicochemical parameters such as protein and ion concentrations, pH, and temperature contributes, making it challenging to identify sequences that will aggregate under native conditions. The solution to this challenge lies in the distinction between aggregation-prone regions that are thermodynamically protected by folding and those that occur in aggregation-competent conformations that can form without major unfolding transitions.

[0219] Using the SolubiS method, human IL2 was identified to have two aggregation-prone regions, while the mouse IL2 only has one. The first aggregation-prone region in human IL2 can be broken by the N110R or N110E, mutations. Interestingly, the N110R solution identified by SolubiS is the naturally-occurrent residue present in mouse IL2 (R125), indicating that the mouse has found the same gatekeeper solution of an arginine sidechain in this location. The second aggregation-prone region was predicted to be improved by T143N or T151 R (human) or A158N or T166R (mouse). T166R showed production improvements in the mouse as a solo mutation. A158N improved production when in combination with V126R, while the T166R was required to restore the production cost of N123H. In the human muteins, the N110E residue was detrimental to both production and functional activity, either in isolation or in combination with specificity-enhancing mutations. While the N110R, T151 R and T143N mutations did not boost production alone, they helped boost production of the impaired Y65K, E82K and V111 R proteins. In addition to the cost savings advantages that the aggregationprotecting mutations could deliver for IL2 production, a therapeutic protein with annual sales at US$60 million, the results here demonstrate the extension of the utility of the SolubiS method for improving production yields to the cytokine family of proteins, with growing therapeutic importance. Beyond the biotechnology value of increasing protein production, here the SolubiS method was used to simultaneously improve both the production level and the functional properties of the native IL2 protein, through the introduction of single amino acid changes. This functional improvement is theoretically easier for the mutations designed for enhanced specificity for CD8 T cells. As CD8 T cells generally do not express high levels of IL2RA, blocking IL2RA binding can substantially reduce Treg responses while having negligible impact on CD8 T cell responses. Indeed, we have identified Y65K E82K (human) and Y79K (mouse) muteins which have simple cationcation repulsion against the IL2RA, while maintaining signalling through IL2RB / IL2RG complexes. In addition we have identified a murine CD8 T cell superkine Q50W with enhanced IL2RB binding, which, when combined with Y79K becomes a CD8 T cell superkine with greater cellular selectivity. By contrast, muteins to enhance specificity for Tregs face a potential “functionality tax”. While IL2 first binds IL2RA, the recruitment of IL2RB into the complex enhances signalling through the trimeric receptor. Thus any mutations which completely block IL2RB binding will kill CD8 T cell responses, but also dampen down bioactivity on Tregs. This “functionality tax” was observed on all Treg muteins, with the exception of V111 R (human) I V126R (mouse). The success of these muteins likely lies in the structure of IL2 being nearly identical whether free or bound to the receptor, with the exception of the beginning of helix C. Following binding to IL2RA, the beginning of helix C is slightly unwound to move forward by 1.0 A, a confirmation change thought to prime superior binding to IL2RB via a hydrogen bond of N88 of IL2 to R42 of IL2RB. The muteins V126R (mouse) and V111 R (human), when in the unbound state of IL2, create a cation-cation repulsion to R41-R42, blocking IL2RB binding. However the binding of IL2 to IL2RA shifts the V126R / V111 R residues back, enabling recruitment of IL2RB. This conformational-dependency of the V126R / V111 R mutein effect may explain why interaction with CD8 T cells, via IL2RB, results in -1000- fold reduction in signalling activity, while Tregs, first interacting with IL2RA, are still able to recruit IL2RB for full signalling capacity. This advantage of specificity without loss of bioactivity is unlike that observed in some other Treg-specific muteins, where the loss of IL2RB binding is accompanied by an aggregate defect in signalling capacity even in Tregs. The uniqueness of this solution also allows the potential combination with other mutations that directly enhance IL2RA binding, potentially creating a Treg superkine. Notably, V111 R (when annotated as V91 R) has been previously described as an IL2 antagonist. We did not investigate competitive binding of V111 R and wildtype IL2, but based on lack of signal in CD8 T cells, if V111 R still bound IL2RB / IL2RG in a nonsignalling conformation, it could potentially act as an antagonist in CD8 T cells, while as acting as a bioactive signaller in Tregs.

[0220] IL2 was first approved for use in humans in in 1992 for the treatment renal carcinoma in the form of Aldesleukin in high doses, to trigger CD8 T cell responses to aid tumour clearance. The short half-life of IL2, and low affinity for the IL2RB-IL2RG receptor, means larges bolus doses were needed to reach the therapeutic concentration needed to stimulate CD8 T cells. This resulted in severe side effects such as capillary leak syndrome being common in the initial clinical trials, and an efficacy in tumour clearance which did not reach the levels observed with the later advent of immune checkpoint blockade. Since this early clinical use, the conceptual shift in the understanding of IL2 biology warranted re-orientation of the drug to anti-inflammatory utility. As such, clinical trials have been run for a multiple low-dose IL2 treatment regime, successful at elevating Treg frequency, in the context of inflammatory diseases such as Graft Versus Host Disease and Systemic Lupus Erythematosus amongst others. Although trials have shown success in reducing disease symptoms, dosing remains a challenge due to the small therapeutic window. The muteins discussed here may help fix many of the caveats of IL2 therapy. Firstly, increased production efficiency of IL2 will help reduce costs making therapies more accessible to patients. Secondly increased specificity of the muteins will help ameliorate some of the side effects. It can be hypothesised that due to the selectivity of muteins Y65K E82K and Y65K E82K N110R T151 R to CD8 T cells, smaller doses may stimulate better CD8 T cell responses, likely decreasing occurrence of severe side effects, whilst also increasing half-life due to less consumption by off- target Tregs. Likewise, but with an inverted hierarchy of specificity, the V111 R or V111 R N110R T151 R muteins, with enhanced selectivity for Tregs, may further decrease the dose required to reach therapeutic Treg stimulation. By combining selective IL2 muteins like those discussed here with novel delivery systems such as AAVs we can address many of the limiting characteristics of IL2 therapy. The added selectivity of the mutein, given alongside the tissue specificity and stable-production capacity of the AAV, address the issues of half-life, therapeutic window and off-target responses. Together these approaches have the potential to further enhance the efficacy of IL2 in the clinic for both anti- and pro- inflammatory uses.

[0221] Example 7 In vivo expansion of CD8+ T cells or Tregs using an AAV delivery system Mouse muteins V126R or Q50W Y79K are encoded in an AAV-PHP.b based vector delivery system and administered to mice. Mice are tested for brain expansion of Tregs or CD8+T cells.

[0222] Human muteins Y65K E82K N110R T151 R or V111 R N110R T151 R are encoded in an AAV9 based vector delivery system and administered to mice. Mice are tested for brain expansion of Tregs or CD8+T cells.

[0223] Example 8

[0224] Methods:

[0225] Figures 8A-D: Mice were injected with wild type human IL2 (AAV9.GFAP.hulL2), the Treg mutein IL2 (AAV9.GFAP.mutein hulL2), or control (AAV9.GFAP.GFP) intravenously via a tail vein injection at a dose of 1-5x10A10vg per mouse. Animals were administered minocycline (50mg / kg) via oral gavage every day for 14 days prior to being culled and tissues collected. Levels of IL2 were measured in the brain via ELLA assay (Biotechne). The proportion of Tregs (% of CD4+ T cells) in the brain and liver were quantified via flow cytometry.

[0226] Figures 8E-g: Mice were injected with the Treg mutein IL2 (AAV9.GFAP.mutein hulL2) intravenously via a tail vein injection (1X10A1° vg per mouse) or via intrathecal route via the cisterna magna (5x10A9or 1X10A1° vg per mouse). Animals were administered minocycline (50mg / kg) via oral gavage every day for 14 days prior to being culled and tissues collected. A control group received no vector and minocycline only. The proportion of Tregs (% of CD4+ T cells) in the brain and liver were quantified via flow cytometry.

[0227] Figure 9a, b and c: An Experimental Autoimmune Encephalomyelitis (EAE) model of multiple sclerosis was induced in mice. Mice were injected with wild type human IL2 (AAV9.GFAP.hulL2) or the Treg mutein IL2 (AAV9.GFAP.mutein hul L2) intravenously via a tail vein injection at a dose of 5x10A9vg per mouse 5 days after induction of EAE and prior to symptom onset. Clinical scores were assessed daily for 3 weeks.

[0228] Results: The Treg mutein showed enhanced production of IL2 in the brain relative to wild type human IL2 (Fig. 8A). This was associated with an increase in Treg expansion in the brain (Fig. 8B) and liver (Fig. 8C) in mutein treated animals. The ratio of Tregs to CD8 T cells was higher in the mutein treated animals, confirming the mutein IL2 more efficiently targets expansion of immunosuppressive Tregs than wild type IL2 (Fig. 8D).

[0229] Intrathecal administration was tested to assess if direct administration to the cerebrospinal fluid would prevent off-target Treg expansion in the liver. Intrathecal administration successfully induced Treg expansion in the brain (Fig. 8E) and abrogated Treg expansion in the liver as seen with intravenous (systemic) delivery of the vector (Fig. 8F).

[0230] In a mouse model of MS (EAE), the Treg mutein delayed symptom onset and disease severity. Wild type IL2 also reduced disease severity from approximately 2 weeks onwards relative to GFP controls (Fig. 9, Table 2). When looking at the cumulative scores over the total 3 weeks, the Treg mutein group demonstrated the lowest clinical score, and therefore, reduced disease severity compared to other groups (Fig. 9a, b, c).

[0231] Table 2. EAE data showing delayed onset of disease and reduced disease severity in the AAV9. mutein IL2 treated mice at 5e9.

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[0288] The invention is further described in the following clauses:

[0289] 1. A modified interleukin-2 (IL2) polypeptide i. wherein the modified IL2 polypeptide is a modified human IL2 polypeptide and comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. an amino acid substitution at position 151 , ii. a K at position at position 65 and a K at position 82, iii. a W at position 36, iv. an R at position 110, and / or v. an N at position 143 or ii. wherein the modified IL2 polypeptide is a modified murine IL2 polypeptide and comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 2: i. an amino acid substitution at position 50, ii. an amino acid substitution at position 79, iii. an amino acid substitution at position 126, iv. an amino acid substitution at position 158, v. an amino acid substitution at position 166, vi. an amino acid substitution at position 54, vii. an amino acid substitution at position 123, viii. an amino acid substitution at position 96, and / or ix. an amino acid substitution at position 79. The modified IL2 polypeptide of clause 1 , wherein the modified human IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. an R at position 110 and / or an N at position 143; and / or ii. an R at position 110 and / or an amino acid substitution at position 151 ; and / or iii. an amino acid substitution at position 151 and further comprises an amino acid substitution at position 108; and / or iv. a K at position 65 and a K at position 82 and further comprises an amino acid substitution at position 108; or wherein the modified murine IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 2: i. an amino acid substitution at one or more of position 126 and / or 158; and / or ii. an amino acid substitution at one or more of position 50 and / or 79; iii. an amino acid substitution at one or more of position 123 and / or 166; and / or iv. an amino acid substitution at one or more of position 79 and / or 96; and / or v. an amino acid substitution at one or more of position 79, 96 and / or 158; and / or vi. an amino acid substitution at one or more of position 79, 96 and / or 166. The modified IL2 polypeptide of clause 1 or 2, wherein the modified human IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. an amino acid substitution at positions 110 and 143, wherein the amino acid substitution at position 110 comprises N110R and the amino acid substitution at position 143 comprises T143N; and / or ii. an amino acid substitution at positions 110 and 151 , wherein the amino acid substitution at position 110 comprises N110R; or wherein the modified murine IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 2: i. an amino acid substitution at positions 126 and 158; and / or ii. an amino acid substitution at positions 50 and 79; and / or iii. an amino acid substitution at positions 123 and 166; and / or iv. an amino acid substitution at positions 79 and 96; and / or v. an amino acid substitution at positions 79, 96 and 158; and / or vi. an amino acid substitution at positions 79, 96 and 166. The modified IL2 polypeptide of any one of the preceding clauses, wherein the modified human IL2 polypeptide further comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. an amino acid substitution at one or more of position 65 and / or 82; and / or. ii. an amino acid substitution at position 111. 5. The modified IL2 polypeptide of clause 4, wherein the modified human IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. an amino acid substitution at positions 65, 82, 110 and 151 and wherein the amino acid substitution at position 110 comprises N110R; and / or ii. an amino acid substitution at positions 111 , 110, and 151 , and wherein the amino acid substitution at position 110 comprises N110R.

[0290] 6. The modified IL2 polypeptide of any one of the preceding clauses, wherein the modified human IL2 polypeptide comprises an amino acid substitution selected from H36W, Y65K, E82K, N110R, T143N and / or T151 R or wherein the modified murine IL2 polypeptide comprises an amino acid substitution selected from Q50W, D54R, Y79K, E96K, V126R, A158N, T166R, and / or N123H.

[0291] 7. The modified IL2 polypeptide of any one of clauses 2 to 6, wherein the amino acid substitution at position 108 comprises N108H.

[0292] 8. The modified IL2 polypeptide of any one of clauses 4 to 7, wherein the amino acid substitution at position 111 comprises V111 R.

[0293] 9. The modified I L2 polypeptide of clauses 7 or 8, wherein the modified human I L2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. an R at position 110 and an R position 151 ; and / or ii. an R at position 110 and an N at position 143; and / or iii. an R at position 111 and an N at position 143; and / or iv. an H at position 108 and an R at position 151 ; and / or v. a K at position 65, a K at position 82, an R at position 110, an R at position 151 ; and / or vi. an R at position 111 , an R at position 110 and an R at position 151 ; or wherein the modified murine IL2 polypeptide with comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 2: i. an R at position 126 and an N at position 158; and / or ii. an H at position 123 and an R at position 166; and / or iii. a K at position 79 and a K at position 96; and / or iv. a K at position 79, a K at position 96 and an N at position 158; and / or v. a K at position 79, a K at position 96 and an R at position 166; and / or vi. a W at position 50 and a K at position 79.

[0294] 10. The modified IL2 human polypeptide according to clause 1 , wherein the modified human polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. V111 R, N110R and T151 R; ii. H36W or iii. N108H and T151 R.

[0295] 11. The modified IL2 human polypeptide according to clause 1 , wherein the modified human polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. Y65K, E82K, N110R, and T151 R or ii. Y65K and E82K.

[0296] 12. The modified IL2 human polypeptide according to clause 1 , wherein the modified human polypeptide comprises the following amino acid substitutions with reference to SEQ ID NO: 1 : N110R and T151 R.

[0297] 13. The modified IL2 murine polypeptide according to clause 1 , wherein the modified murine polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 2: i. V126R or ii. V126R and A158N.

[0298] 14. The modified IL2 murine polypeptide according to clause 1 , wherein the modified murine polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 2: i. Q50W and Y79K; ii. Q50W or iii. Y79K. 15. The modified IL2 murine polypeptide according to clause 1 , wherein the modified murine polypeptide comprises an amino acid substitution at T166R with reference to SEQ ID NO: 2:

[0299] 16. The modified IL2 polypeptide according to any one of the proceeding clauses, wherein the modified polypeptide further comprises an amino acid substitution at position 40, wherein said substitution is optionally D40R.

[0300] 17. The modified IL2 polypeptide according to any one of the proceeding clauses, wherein the substitution alters binding of IL2 to IL2RA or IL2RB, and / or reduces or prevents IL2 aggregation.

[0301] 18. The modified IL2 polypeptide according to clause 17, wherein the substitution that alters binding of IL2 to IL2RA or IL2RB reduces or abolishes binding of IL2 to IL2RA or IL2RB.

[0302] 19. The modified IL2 polypeptide according to any one of the preceding clauses, wherein the modified IL2 polypeptide comprises SEQ ID NO: 7 to 11 or a sequence with at least 90% sequence identity thereto; or SEQ ID NO: 12 to 17 or a sequence with at least 90% sequence identity thereto.

[0303] 20. The modified IL2 polypeptide according to any one of the proceeding clauses, wherein said modified IL2 polypeptide is conjugated to one or more moiety selected from an enzyme, radioisotope, half-life extending moiety, label, therapeutic molecule or other chemical moiety.

[0304] 21. The modified IL2 polypeptide according to clause 20, wherein said half-life extending moiety is selected from the group consisting of an albumin binding moiety, a transferrin binding moiety, a polyethylene glycol molecule, a recombinant polyethylene glycol molecule, human or murine serum albumin, a fragment of human or murine serum albumin, and an albumin binding peptide, an antibody or fragment thereof that binds to human or murine serum albumin or an antibody Fc domain.

[0305] 22. A nucleic acid construct comprising a nucleic acid encoding a modified IL2 polypeptide of any one of the preceding clauses. 23. A viral vector comprising the construct according to clause 22.

[0306] 24. The viral vector according to clause 23, wherein the viral vector is an adeno- associated viral virus vector and comprises one or more of the following vectors: AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV- rhS, AAV-rh10, AAV-rh39, AAV-rh43, AAVAnc80, AAV 2 / ShH10, AAV-S vector, AAV6.2, Adv5, oncolytic vectors.

[0307] 25. A pharmaceutical composition comprising the modified IL2 polypeptide, viral vector or construct of any of the preceding clauses.

[0308] 26. The modified IL2 polypeptide according to any one of clauses 1 to 21 , a construct according to clause 22, a viral vector according to clauses 23 to 24, or a pharmaceutical composition according to clause 25 for use in the treatment of disease.

[0309] 27. The modified IL2 polypeptide according to any one of clauses 1 to 21 , a construct according to clause 22, a viral vector according to clauses 23 to 24, or a pharmaceutical composition according to clause 25 for use according to clause 26, wherein said disease is selected from cancer, an immune disorder, neurological disease, inflammatory disorder, allergy, transplant rejection, viral infection, immune deficiency or other immune system-related disorder.

[0310] 28. A method for treating a cancer, an immune disorder, neurological disease, inflammatory disorder, allergy, transplant rejection, viral infection, immune deficiency or other immune system-related disorder comprising administering a therapeutically effective amount of a modified IL2 polypeptide according to any one of clauses 1 to 21 , a construct according to clause 22, a viral vector according to clauses 23 to 24, or a pharmaceutical composition according to clause 25.

[0311] 29. The modified IL2 polypeptide according to any one of clauses 1 to 21 , a construct according to clause 22, a viral vector according to clauses 23 to 24, or a pharmaceutical composition according to clause 25 , for a use according to 26 or 27, or a method according to clause 28, wherein said cancer is selected from bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, breast cancer, brain cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, kidney cancer, sarcoma of soft tissue, cancer of the urethra, cancer of the bladder, renal cancer, lung cancer, non-small cell lung cancer, thymoma, urothelial carcinoma leukemia, prostate cancer, mesothelioma, adrenocortical carcinoma, lymphomas, such as such as Hodgkin's disease, non-Hodgkin's, gastric cancer, and multiple myelomas.

[0312] 30. The modified IL2 polypeptide according to any one of clauses 1 to 21 , a construct according to clause 22, a viral vector according to clauses 23 to 24, or a pharmaceutical composition according to clause 25, for a use according to 26 or 27, or a method according to clause 28, wherein said immune disorder is an autoimmune disorder.

[0313] 31. The modified IL2 polypeptide, construct, pharmaceutical composition, use or method according to clause 30, wherein said autoimmune disorder is autoimmune disease is alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison’s disease, autoimmune diseases of the adrenal glands, and autoimmune hemolytic anemia, Autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Bechet syndrome, bullous pemphigoid, cardiomyopathy, stomatitis diarrhea dermatitis, chronic fatigue immune dysfunction syndrome, Chronic inflammatory demyelinating polyneuropathy, Chusch's syndrome, scar pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, idiopathic mixed cold bulb Proteinemia, diabetes, eosinophilic fasciitis, fibromyalgia- fibromyositis, glomerulonephritis, Graves' disease, Guerrilla-Barr syndrome, Hashimoto's thyroiditis, Hen-Sher's disease Purpura, idiopathic pulmonary fibrosis, idiopathic / autoimmune thrombocytopenic purpura, IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere syndrome, mixed connective tissue disease, multiple sclerosis, 1 Type or immune-mediated diabetes, myasthenia gravis, pemphigus-related diseases, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis, dermatomuscularis Inflammation, primary aglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Reye's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Jaglen syndrome, systemic myotonia syndrome, systemic lupus erythematosus, Svet's syndrome, Still's disease, lupus erythematosus, Gaoan's arteritis, transient arteritis / giant cell arteritis, ulcerative colitis, grapevine Meningitis, vasculitis, vitiligo, Wegener's granulomatosis.

[0314] 32. A kit comprising a modified IL2 polypeptide according to any one of clauses 1 to 21 , a construct according to clause 22, a viral vector according to clauses 23 to 24, or a pharmaceutical composition according to clause 25 optionally together with a reagent and / or instructions for use.

[0315] 33. A nucleic acid encoding the modified IL2 polypeptide according to any one of clauses 1 to 21.

[0316] 34. A vector comprising a nucleic acid according to clause 33.

[0317] 35. A host cell comprising a nucleic acid according to clause 33 or a vector according to clause 34.

[0318] 36. The host cell according to clause 35 wherein said host cell is a bacterial, yeast, viral or mammalian cell.

[0319] 37. A method for producing a modified IL2 polypeptide according to any one of clauses 1 to 21 comprising expressing a nucleic acid encoding the modified IL2 polypeptide in a host cell and isolating the modified IL2 polypeptide from the host cell.

[0320] 38. A method for making an IL2 modified polypeptide with reduced aggregation comprising the steps of

[0321] 1) predicting aggregation-prone regions from an IL2 polypeptide, 2) identifying amino acid mutations that abolish the aggregation propensity of these segments without affecting the thermodynamic stability of the polypeptide and

[0322] 3) expressing a modified IL2 polypeptide with reduced aggregation.

[0323] 39. The method of clause 38, wherein said polypeptide comprises SEQ ID NO: 1 or SEQ ID NO: 2.

[0324] 40. A vector delivery system comprising a nucleic acid encoding an IL2 mutein according to any one of clauses 1 to 21 or a construct according to clause 17.

[0325] 41 . The vector delivery system according to clause 40, wherein said vector comprises a viral vector.

[0326] 42. The vector delivery system according to clause 41 , wherein said viral vector is an adeno-associated viral virus vector and comprises one or more of the following vectors: AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-rh8, AAV-rh10, AAV-rh39, AAV-rh43, AAVAnc80, AAV 2 / ShH10, AAV-S vector, AAV6.2, Adv5, oncolytic vectors.

[0327] 43. An in vivo, in vitro or ex vivo method of promoting Treg responses, comprising contacting a population of T cells with an effective amount of a modified IL2 polypeptide according to any one of clauses 1 to 16.

[0328] 44. A method of promoting Treg responses, comprising introducing an amino acid substitution as defined in of any one of clauses 1 to 21 into an IL2 polypeptide.

[0329] 45. The method of clause 43 or 44, wherein the modified human IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. an H at position 108 and an R at position 151 ; or ii. a W at position 36; or iii. an R at position 111 , an R at position 110 and an R at position 151. 46. An in vivo, in vitro or ex vivo method of promoting CD8+ T cell responses, comprising contacting a population of T cells with an effective amount of a modified IL2 polypeptide according to any one of clauses 1 to 21.

[0330] 47. A method of promoting CD8+ T cell responses, comprising introducing an amino acid substitution as defined in any one of clauses 1 to 16 into an IL2 polypeptide.

[0331] 48. The method of clause 46 or 47, wherein the modified human IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. a K at position 65 and a K at position 82; or ii. a K at position 65, a K at position 82, an R at position 110 and an R at position 151.

[0332] 49. A method of decreasing the aggregation of IL2 polypeptide, comprising introducing an amino acid substitution as defined in any one of clauses 1 to 16 into an IL2 polypeptide.

[0333] 50. The method of clause 49, wherein the modified IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. an R at position 111 and an N at position 143; ii. an R at position 110 and an R at position 151 ; iii. an R at position 110 and an N at position 143; iv. a K at position 65, a K at position 82, an R at position 110, and an R at position 151 ; or v. an R at position 111 , an R at position 110 and an R at position 151.

[0334] Sequences

[0335] SEQ ID NO: 1 Full length Human IL2

[0336] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK GSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0337] SEQ ID NO: 2 Full length murine IL2 MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDL

[0338] QELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSF

[0339] QLEDAENFISNIRVTWKLKGSDNTFECQFDDESATWDFLRRWIAFCQSIISTSPQ

[0340] SEQ ID NO: 3 Human IL2 signal sequence

[0341] MYRMQLLSCIALSLALVTNS

[0342] SEQ ID NO: 4 Human mature IL2 sequence

[0343] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQC

[0344] LEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFL

[0345] NRWITFCQSIISTLT

[0346] SEQ ID NO: 5 Mouse IL2 signal sequence

[0347] MYSMQLASCVTLTLVLLVNS

[0348] SEQ ID NO: 6 Mouse mature IL2 sequence

[0349] APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLT

[0350] FKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTWKLKG

[0351] SDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ

[0352] SEQ ID NO: 7 Y65K E82K N110R T151R (human modified IL2 polypeptide; CD8 specificity)

[0353] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR

[0354] MLTFKFKMPKKATELKHLQCLEEKLKPLEEVLNLAQSKNFHLRPRDLISNIRVIVLELK

[0355] GSETTFMCEYADETATIVEFLNRWITFCQSIISRLT

[0356] SEQ ID NO: 8 Y65K E82K (human modified IL2 polypeptide; CD8 specificity)

[0357] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR

[0358] MLTFKFKMPKKATELKHLQCLEEKLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK

[0359] GSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0360] SEQ ID NO: 9 V111R N110R T151R (human modified IL2 polypeptide; Treg specificity)

[0361] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR

[0362] MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIRRIVLELK

[0363] GSETTFMCEYADETATIVEFLNRWITFCQSIISRLT SEQ ID NO: 10 H36W (human modified IL2 polypeptide; Treg specificity)

[0364] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEWLLLDLQMILNGINNYKNPKLTR

[0365] MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK

[0366] GSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0367] SEQ ID NO: 11 N108H T151 R (human modified IL2 polypeptide; Treg specificity)

[0368] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR

[0369] MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISHINVIVLELK

[0370] GSETTFMCEYADETATIVEFLNRWITFCQSIISRLT

[0371] SEQ ID NO: 12 V126R (murine modified IL2 polypeptide; Treg specificity)

[0372] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDL

[0373] QELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSF

[0374] QLEDAENFISNIRRTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ

[0375] SEQ ID NO: 13 V126R A158N (murine modified IL2 polypeptide; Treg specificity)

[0376] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDL

[0377] QELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSF

[0378] QLEDAENFISNIRRTVVKLKGSDNTFECQFDDESATVVDFLRRWINFCQSIISTSPQ

[0379] SEQ ID NO: 14 Q50WY79K (murine modified IL2 polypeptide; CD8 specificity)

[0380] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEWLLMDL

[0381] QELLSRMENYRNLKLPRMLTFKFKLPKQATELKDLQCLEDELGPLRHVLDLTQSKSF

[0382] QLEDAENFISNIRVTWKLKGSDNTFECQFDDESATWDFLRRWIAFCQSIISTSPQ

[0383] SEQ ID NO: 15 Q50W (murine modified IL2 polypeptide; CD8 specificity)

[0384] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEWLLMDL

[0385] QELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSF

[0386] QLEDAENFISNIRVTWKLKGSDNTFECQFDDESATWDFLRRWIAFCQSIISTSPQ

[0387] SEQ ID NO: 16 Y79K (murine modified IL2 polypeptide; CD8 specificity)

[0388] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDL

[0389] QELLSRMENYRNLKLPRMLTFKFKLPKQATELKDLQCLEDELGPLRHVLDLTQSKSF

[0390] QLEDAENFISNIRVTWKLKGSDNTFECQFDDESATWDFLRRWIAFCQSIISTSPQ

[0391] SEQ ID NO: 17 T166R (murine modified IL2 polypeptide; production enhancing) MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDL

[0392] QELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSF

[0393] QLEDAENFISNIRVTWKLKGSDNTFECQFDDESATWDFLRRWIAFCQSIISRSPQ

[0394] SEQ ID NO: 18 >hlL2_1_N110R

[0395] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR

[0396] MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIRVIVLELK

[0397] GSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0398] SEQ ID NO: 19 >hlL2_2_N110E

[0399] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR

[0400] MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIEVIVLELK

[0401] GSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0402] SEQ ID NO: 20 >hlL2_3_T143N

[0403] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR

[0404] MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK

[0405] GSETTFMCEYADETATIVEFLNRWINFCQSIISTLT

[0406] SEQ ID NO: 21 >hlL2_4_T151 R

[0407] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR

[0408] MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK

[0409] GSETTFMCEYADETATIVEFLNRWITFCQSIISRLT

[0410] SEQ ID NO: 22 >hlL2_6_D40R

[0411] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLRLQMILNGINNYKNPKLTR

[0412] MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK

[0413] GSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0414] SEQ ID NO: 23 >hlL2_7_N108H

[0415] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR

[0416] MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISHINVIVLELK

[0417] GSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0418] SEQ ID NO: 24 >hlL2_8_V111R MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR

[0419] MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINRIVLELK

[0420] GSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0421] SEQ ID NO: 25 >hlL2_9_N110R_T143N

[0422] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR

[0423] MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIRVIVLELK

[0424] GSETTFMCEYADETATIVEFLNRWINFCQSIISTLT

[0425] SEQ ID NO: 26 >hlL2_10_N110R_T151R

[0426] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR

[0427] MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIRVIVLELK

[0428] GSETTFMCEYADETATIVEFLNRWITFCQSIISRLT

[0429] SEQ ID NO: 27 >hlL2_11_V111 R_T143N

[0430] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR

[0431] MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINRIVLELK

[0432] GSETTFMCEYADETATIVEFLNRWINFCQSIISTLT

[0433] SEQ ID NO: 28 >mlL2_13_A158N

[0434] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDL

[0435] QELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSF

[0436] QLEDAENFISNIRVTWKLKGSDNTFECQFDDESATWDFLRRWINFCQSIISTSPQ

[0437] SEQ ID NO: 29 >mlL2_14_T166R

[0438] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDL

[0439] QELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSF

[0440] QLEDAENFISNIRVTWKLKGSDNTFECQFDDESATWDFLRRWIAFCQSIISRSPQ

[0441] SEQ ID NO: 30 >mlL2_16_D54R

[0442] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMRL

[0443] QELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSF

[0444] QLEDAENFISNIRVTWKLKGSDNTFECQFDDESATWDFLRRWIAFCQSIISTSPQ

[0445] SEQ ID NO: 31 >mlL2_17_N123H MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDL

[0446] QELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSF

[0447] QLEDAENFISHIRVTWKLKGSDNTFECQFDDESATWDFLRRWIAFCQSIISTSPQ

[0448] SEQ ID NO: 32 >mlL2_19_V126R_A158N

[0449] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDL

[0450] QELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSF

[0451] QLEDAENFISNIRRTVVKLKGSDNTFECQFDDESATVVDFLRRWINFCQSIISTSPQ

[0452] SEQ ID NO: 33 >mlL2_10_N123H_T166R

[0453] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDL

[0454] QELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSF

[0455] QLEDAENFISHIRVTWKLKGSDNTFECQFDDESATWDFLRRWIAFCQSIISRSPQ

[0456] Wild type and a modified IL2 nucleic acid sequence are shown below.

[0457] For experimental purposes, a His tag was included immediately upstream of the stop codon.

[0458] SEQ ID NO: 34 Mouse WT

[0459] IL2ATGTACAGCATGCAGCTCGCATCCTGTGTCACATTGACACTTGTGCTCCTTGT

[0460] CAACAGCGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCA

[0461] GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGG

[0462] ACCTACAGGAGCTCCTGAGCAGGATGGAGAATTACAGGAACCTGAAACTCCCCA

[0463] GGATGCTCACCTTCAAATTTTACTTGCCCAAGCAGGCCACAGAATTGAAAGATCTT

[0464] CAGTGCCTAGAAGATGAACTTGGACCTCTGCGGCATGTTCTGGATTTGACTCAAA

[0465] GCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACT

[0466] GTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTC

[0467] AGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATC

[0468] TCAACAAGCCCTCAATAA

[0469] SEQ ID NO: 35 Human WT

[0470] IL2ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACA

[0471] AACAGTGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTT

[0472] ACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACT

[0473] CACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAA

[0474] CATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGC TCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAA

[0475] TAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTGAATATGCTGATGAG

[0476] ACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATC TCAACACTGACTTGA SEQ ID NO: 36 hlL2 (V111R, N110R, T151 R)

[0477] ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAA

[0478] CAGTGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTAC

[0479] TGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCA

[0480] CCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACAT CTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTC

[0481] AAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAGAAGAATA

[0482] GTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTGAATATGCTGATGAGAC

[0483] AGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTC AAGACTGACTTGA

Claims

Claims1. A modified interleukin-2 (IL2) polypeptide i. wherein the modified IL2 polypeptide is a modified human IL2 polypeptide and comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. an amino acid substitution at position 111 and one or more of: an R at position 110, an N at position 143 and / or an amino acid substitution at position 151 , ii. an amino acid substitution at position 151 , iii. a W at position 36 and / or iv. an R at position 110 and an N at position 143, or ii. wherein the modified IL2 polypeptide is a modified murine IL2 polypeptide and comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 2: i. an amino acid substitution at position 126, ii. an amino acid substitution at position 158, iii. an amino acid substitution at position 166, iv. an amino acid substitution at position 54, and / or v. an amino acid substitution at position 123.

2. The modified IL2 polypeptide of claim 1 , wherein the modified human IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. amino acid substitution at position 151 and further comprises an R at position 110; and / or ii. an amino acid substitution at position 151 and further comprises an amino acid substitution at position 108; and / or wherein the modified murine IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 2: vii. an amino acid substitution at one or more of position 126 and / or 158; and / or viii. an amino acid substitution at one or more of position 123 and / or 166; and / or3. The modified IL2 polypeptide of claim 1 or 2, wherein the modified human IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : iii. an amino acid substitution at positions 110 and 143, wherein the amino acid substitution at position 110 comprises N110R and the amino acid substitution at position 143 comprises T143N; and / or iv. an amino acid substitution at positions 110 and 151 , wherein the amino acid substitution at position 110 comprises N110R; or wherein the modified murine IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 2: vii. an amino acid substitution at positions 126 and 158; and / or viii. an amino acid substitution at positions 123 and 166; and / or4. The modified IL2 polypeptide of any one of the preceding claims, wherein the modified human IL2 polypeptide with an amino acid substitution at position 151 further comprises an amino acid substitution at position 111 with reference to SEQ ID NO: 1.

5. The modified IL2 polypeptide of claim 1 or 4, wherein the modified human IL2 polypeptide comprises an amino acid substitution at positions 111 , 110 and 151 with reference to SEQ ID NO: 1 , and wherein the amino acid substitution at position 110 comprises N110R.

6. The modified IL2 polypeptide of claim 5 wherein the modified human IL2 polypeptide comprises the following amino acid substitutions V111 R, N110R and T151 R.

7. The modified IL2 polypeptide of any one of the preceding claims, wherein the modified human IL2 polypeptide comprises an amino acid substitution selected from H36W, N110R, T143N and / or T151 R or wherein the modified murine IL2 polypeptide comprises an amino acid substitution selected from D54R, V126R, A158N, T166R, and / or N123H.

8. The modified IL2 polypeptide of any one of claims 2 to 7, wherein the amino acid substitution at position 108 comprises N108H.

9. The modified IL2 polypeptide of any one of claims 1 to 7, wherein the amino acid substitution at position 111 comprises V111 R.

10. The modified IL2 polypeptide of claims 7 or 9, wherein the modified human IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. an R at position 110 and an R position 151 ; and / or ii. an R at position 110 and an N at position 143; and / or iii. an R at position 111 and an N at position 143; and / or iv. an H at position 108 and an R at position 151 ; and / or v. an R at position 111 , an R at position 110 and an R at position151 ; or wherein the modified murine IL2 polypeptide with comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 2: vii. an R at position 126 and an N at position 158; and / or viii. an H at position 123 and an R at position 166.11 . The modified IL2 human polypeptide according to claim 1 , wherein the modified human polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. V111 R, N110R and T151 R; ii. H36W or iii. N108H and T151 R.

12. The modified IL2 human polypeptide according to claim 1 , wherein the modified human polypeptide comprises the following amino acid substitutions with reference to SEQ ID NO: 1 : N11 OR and T151 R.

13. The modified IL2 murine polypeptide according to claim 1 , wherein the modified murine polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 2: i. V126R or ii. V126R and A158N.

14. The modified IL2 murine polypeptide according to claim 1 , wherein the modified murine polypeptide comprises an amino acid substitution at T166R with reference to SEQ ID NO: 2:

15. The modified IL2 polypeptide according to any one of the proceeding claims, wherein the modified polypeptide further comprises an amino acid substitution at position 40, wherein said substitution is optionally D40R.

16. The modified IL2 polypeptide according to any one of the proceeding claims, wherein the substitution alters binding of IL2 to IL2RB, and / or reduces or prevents IL2 aggregation.

17. The modified IL2 polypeptide according to claim 16, wherein the substitution that alters binding of IL2 to IL2RB reduces or abolishes binding of IL2 to IL2RB.

18. The modified IL2 polypeptide according to any one of the preceding claims, wherein the modified IL2 polypeptide comprises SEQ ID NO: 9 to 11 or a sequence with at least 90% sequence identity thereto; or SEQ ID NO: 12 to 13 or 17 or a sequence with at least 90% sequence identity thereto.

19. The modified IL2 polypeptide according to any one of the proceeding claims, wherein said modified IL2 polypeptide is conjugated to one or more moiety selected from an enzyme, radioisotope, half-life extending moiety, label, therapeutic molecule or other chemical moiety.

20. The modified IL2 polypeptide according to claim 19, wherein said half-life extending moiety is selected from the group consisting of an albumin binding moiety, a transferrin binding moiety, a polyethylene glycol molecule, a recombinant polyethylene glycol molecule, human or murine serum albumin, a fragment of human or murine serum albumin, and an albumin binding peptide, an antibody or fragment thereof that binds to human or murine serum albumin or an antibody Fc domain.

21. An isolated nucleic acid or nucleic acid construct comprising a nucleic acid encoding a modified IL2 polypeptide of any one of the preceding claims.

22. A viral vector comprising the nucleic acid or nucleic acid construct according to claim 21.

23. The viral vector according to claim 21 , wherein the viral vector is an adeno- associated viral virus vector and comprises one or more of the following vectors: AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV- rhS, AAV-rh10, AAV-rh39, AAV-rh43, AAVAnc80, AAV 2 / ShH10, AAV-S vector, AAV6.2, Adv5, lentiviral vector or oncolytic vector.

24. A pharmaceutical composition comprising the modified IL2 polypeptide according to any one of claims 1 to 20, a nucleic acid or nucleic acid construct according to claim 21 or a viral vector according to claims 22 to 23.

25. The modified IL2 polypeptide according to any one of claims 1 to 20, a nucleic acid or nucleic acid construct according to claim 21 , a viral vector according to claims 22 to 23 or a pharmaceutical composition according to claim 24 for use in the treatment of disease.

26. The modified IL2 polypeptide according to any one of claims 1 to 20, a nucleic acid or nucleic acid construct according to claim 21 , a viral vector according to claims 22 to 23 or a pharmaceutical composition according to claim 24 for use according to claim 25, wherein said disease is selected from cancer, an immune disorder, neurological disease, inflammatory disorder, allergy, transplant rejection, viral infection, immune deficiency or other immune system-related disorder.

27. A method for treating a cancer, an immune disorder, neurological disease, inflammatory disorder, allergy, transplant rejection, viral infection, immune deficiency or other immune system-related disorder comprising administering a therapeutically effective amount of a modified IL2 polypeptide according to any one of claims 1 to 20, a nucleic acid or nucleic acid construct according to claim 22, a viral vector according to claims 22 to 23 or a pharmaceutical composition according to claim 24.

28. The modified IL2 polypeptide according to any one of claims 1 to 20, a nucleic acid or nucleic acid construct according to claim 21 , a viral vector according toclaims 23 to 24 or a pharmaceutical composition according to claim 24 for a use according to 25 or 26, or a method according to claim 27, wherein said cancer is selected from bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, breast cancer, brain cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, kidney cancer, sarcoma of soft tissue, cancer of the urethra, cancer of the bladder, renal cancer, lung cancer, non-small cell lung cancer, thymoma, urothelial carcinoma leukemia, prostate cancer, mesothelioma, adrenocortical carcinoma, lymphomas, such as such as Hodgkin's disease, non-Hodgkin's, gastric cancer, and multiple myelomas.

29. The modified IL2 polypeptide according to any one of claims 1 to 20, a nucleic acid or nucleic acid construct according to claim 21 , a viral vector according to claims 22 to 22 or a pharmaceutical composition according to claim 24 for a use according to 25 or 26, or a method according to claim 27, wherein said immune disorder is an autoimmune disorder.

30. The modified IL2 polypeptide, nucleic acid or nucleic acid construct, viral vector, pharmaceutical composition, use or method according to claim 29, wherein said autoimmune disorder is autoimmune disease is alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison’s disease, autoimmune diseases of the adrenal glands, and autoimmune hemolytic anemia, Autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Bechet syndrome, bullous pemphigoid, cardiomyopathy, stomatitis diarrhea dermatitis, chronic fatigue immune dysfunction syndrome, Chronic inflammatory demyelinating polyneuropathy, Chusch's syndrome, scar pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, idiopathic mixed cold bulb Proteinemia, diabetes, eosinophilic fasciitis, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guerrilla-Barr syndrome, Hashimoto's thyroiditis, Hen-Sher's disease Purpura, idiopathic pulmonary fibrosis, idiopathic / autoimmune thrombocytopenic purpura, IgA neuropathy, juvenile arthritis, lichen planus,lupus erythematosus, Meniere syndrome, mixed connective tissue disease, multiple sclerosis, 1 Type or immune-mediated diabetes, myasthenia gravis, pemphigus-related diseases, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis, dermatomuscularis Inflammation, primary aglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Reye's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Jaglen syndrome, systemic myotonia syndrome, systemic lupus erythematosus, Svet's syndrome, Still's disease, lupus erythematosus, Gaoan's arteritis, transient arteritis / giant cell arteritis, ulcerative colitis, grapevine Meningitis, vasculitis, vitiligo, Wegener's granulomatosis.

31. A kit comprising a modified IL2 polypeptide according to any one of claims 1 to 20, a nucleic acid or nucleic acid construct according to claim 21 , a viral vector according to claims 22 to 23 or a pharmaceutical composition according to claim 24, optionally together with a reagent and / or instructions for use.

32. A host cell comprising a nucleic acid or nucleic acid construct according to claim 21 or a vector according to claim 22 or 23.

33. The host cell according to claim 32 wherein said host cell is a bacterial, yeast, viral or mammalian cell.

34. A method for producing a modified IL2 polypeptide according to any one of claims 1 to 20 comprising expressing a nucleic acid encoding the modified IL2 polypeptide in a host cell and isolating the modified IL2 polypeptide from the host cell.

35. A method for making an IL2 modified polypeptide with reduced aggregation comprising the steps of1) predicting aggregation-prone regions from an IL2 polypeptide,2) identifying amino acid mutations that abolish the aggregation propensity of these segments without affecting the thermodynamic stability of the polypeptide and3) expressing a modified IL2 polypeptide with reduced aggregation.

36. The method of claim 35, wherein said polypeptide comprises SEQ ID NO: 1 or SEQ ID NO: 2.

37. A vector delivery system comprising a nucleic acid encoding an IL2 mutein according to any one of claims 1 to 20 or a construct according to claim 21 .

38. The vector delivery system according to claim 37, wherein said vector comprises a viral vector.

39. The vector delivery system according to claim 38, wherein said viral vector is an adeno-associated viral virus vector and comprises one or more of the following vectors: AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-rh8, AAV-rh10, AAV-rh39, AAV-rh43, AAVAnc80, AAV 2 / ShH10, AAV-S vector, AAV6.2, Adv5, lentiviral vector or oncolytic vector.

40. An in vivo, in vitro or ex vivo method of promoting Treg responses, comprising contacting a population of T cells with an effective amount of a modified IL2 polypeptide according to any one of claims 1 to 20 or a construct according to claim 21.

41. A method of promoting Treg responses, comprising introducing an amino acid substitution as defined in of any one of claims 1 to 20 into an IL2 polypeptide.

42. The method of claim 40 or 41 , wherein the modified human IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. an H at position 108 and an R at position 151 ; or ii. a W at position 36; or iii. an R at position 111 , an R at position 110 and an R at position 151.

43. A method of decreasing the aggregation of IL2 polypeptide, comprising introducing an amino acid substitution as defined in any one of claims 1 to 20 into an IL2 polypeptide.

44. The method of claim 43, wherein the modified IL2 polypeptide comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 1 : i. an R at position 111 and an N at position 143; ii. an R at position 110 and an R at position 151 ; iii. an R at position 110 and an N at position 143; iv. an R at position 111 , an R at position 110 and an R at position 151.

Images