Interleukine-2 muteins with enhanced specificity towards CD8+ t cells and improved production capacity

Abstract

The present invention relates to modified IL polypeptides for enhanced specificity of IL2, in particular for CD8 T 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 half-life 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 / fn 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 IL2 / IL2R orthogonal pairs that do not cross-react with the native system.

[0005] While IL2 muteins provide enhanced specificity for either the Treg 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, NK cells or CD8 T cell lineage responses (thus promoting Treg, NK cells or CD8 T cell respectively), in particular CD8 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. Moreover, mutations generated by the inventors also work by disrupting CD25-binding and therefore reducing Treg responses while keeping CD8 responses intact.

[0011] These muteins, designed with both cellular specificity for either Treg 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 or by disrupt CD25-binding and / or reduces aggregation of the IL2 polypeptide. In particular, the invention therefore relates to isolated modified IL2 polypeptides comprising one or more amino acid substitution that promotes and 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 the following amino acid substitutions with reference to SEQ ID NO: 1 : a K at position at position 65 and a K at position 82, 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 96, and / or iii. an amino acid substitution at position 79.

[0014] 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 : iii. an amino acid substitution at position 151 , iv. an R position 110 and / or v. an N at position 143. or wherein the modified murine IL2 polypeptide and further comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 2: i. an amino acid substitution at position 166 and / or ii. an amino acid substitution at position 158.

[0015] In one embodiment, the amino acid substitution at position 151 is T151 R.

[0016] In one embodiment, the modified human IL2 polypeptide further comprises an R at position 151 and an R at position 110.

[0017] The invention also relates to a nucleic acid construct comprising a nucleic acid encoding a modified IL2 polypeptide as described above.

[0018] The invention also relates to a viral vector comprising the construct as described above.

[0019] The invention also relates to an isolated immune cell, wherein immune cell comprises a chimeric antigen receptor, wherein said cell expresses the modified IL2 polypeptide or the nucleic acid construct as described above.

[0020] The invention also relates to a pharmaceutical composition comprising the modified IL2 polypeptide, the nucleic acid construct, the viral vector or the isolated immune cell as described above.

[0021] In one embodiment, the pharmaceutical composition further comprises an immune cell expressing a chimeric antigen receptor, optionally where the immune cell is a T cell or a NK cell.

[0022] The invention also relates to the modified IL2 polypeptide, the nucleic acid construct, the viral vector or the isolated immune cell as described above for use in the treatment of disease, for example 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. The invention also 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 the modified IL2 polypeptide, the nucleic acid construct, the viral vector or the isolated immune cell as described above.

[0023] The invention also relates to a kit comprising the modified IL2 polypeptide, the nucleic acid construct, the viral vector or the isolated immune cell as described above optionally together with a reagent and / or instructions for use.

[0024] The invention also relates to an isolated nucleic acid encoding the modified IL2 polypeptide as described above. The invention also relates to a nucleic acid construct comprising said nucleic acid.

[0025] The invention also relates to a vector comprising a nucleic acid as described above.

[0026] The invention also relates to a host cell comprising a nucleic acid according as described above or a vector as described above.

[0027] The invention also 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.

[0028] The invention also relates to a vector delivery system comprising a nucleic acid encoding an IL2 mutein or a construct as described above.

[0029] The invention also relates to 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 as described above.

[0030] The invention also relates to a method of promoting CD8+ T cell responses, comprising introducing an amino acid substitution as described above into an IL2 polypeptide.

[0031] The invention also relates to a combination comprising the modified IL2 polypeptide, viral vector or construct as described above and an immune cell expressing a chimeric antigen receptor, e.g. a CAR-T cell. The invention also relates to a combination therapy comprising administering the modified IL2 polypeptide, a construct, a viral vector, or a pharmaceutical composition as described above and an immune cell expressing a chimeric antigen receptor, for example for treating cancer, e.g. wherein the 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, and wherein the immune cell is administered before, at the same time, or after the modified IL2 polypeptide, viral vector or construct.

[0032] The invention also relates to method of increasing the expansion and persistence of T cell therapy, comprising the step of modifying an immune cell to express the modified IL2 polypeptide viral vector or construct as described above.

[0033] Figures

[0034] The invention is illustrated in the following non-limiting figures.

[0035] 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 Q50WY79K relate to ad hoc combination.

[0036] 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.

[0037] 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 I L2 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).

[0038] Figure 3. Structural modelling of murine IL2 muteins with the trimeric IL2R complex. A) Structure of IL2 in complex with 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.

[0039] 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.

[0040] 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 I L2 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).

[0041] 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 T reg 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). N110R, N110E, T143N, T151R, 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.

[0042] Figure 7. Cellular selectivity of Human IL2 muteins is maintained with addition of production boosting mutations N110R T151 R. 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.

[0043] Figure 8. In vitro pSTAT5 Assay (mouse). Frequency of phospho-STAT5+ cells in Tregs and CD8+ T cells following exposure to titrated doses various fusion constructs. Each point represents four independent replicates, with data presented as the mean ± s.e.m.

[0044] Figure 9. In vitro pSTAT5 Assay (human). Frequency of phospho-STAT5+ cells in Tregs and CD8+ T cells following exposure to titrated doses various fusion constructs. Each point represents four independent replicates, with data presented as the mean ± s.e.m. Figure 10. In vivo experimental design. C57BL / 6J mice receive intracranial surgery via stereotactic injection to implant CT-2A tumour cells. CT-2A cells are transformed to express reporter genes: Firefly-luciferase and tdTomato, and GD2-GD3 synthase enzymes to produce the glycolipid antigen GD2. Live imaging is performed weekly to track tumour growth (isoflurane anaesthetic, IP administration of 100uL 30mg / mL D-luciferin, 8-minute induction and 1 minute acquisition on the Perkin Elmer I VIS system).

[0045] Figure 11. Tumour volume assessment by bioluminescence imaging. Bioluminescence measured as total flux (photons / second) 7 days after intracranial surgery, and again immediately prior to humane killing. Due to concerns about mouse wellbeing for groups 2-3 and 5-6 weekly imaging was not performed. Samples as listed on the right are shown in graph from left to right.

[0046] Figure 12. Survival curves. Mouse survival (days post- surgery) was assessed by Kaplan Meier statistical analysis, with Log-rank (Mantel-Cox) test for two-way comparison of selected curves. (GraphPad Prism 10.6). Humane killing (HK) for any cause, left. Humane Killing (HK) for only neurological causes, right).

[0047] Figure 13. Clinical scoring. Scoring from clinical record sheets at humane endpoint. Endpoints requiring humane killing were pre-defined as: Neurological signs of tumour (head-tilt, circling); Cumulative clinical score of 4 (reluctance to move, hunching, ruffled coat, body condition); weight loss of >15%.

[0048] Detailed Description

[0049] 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.

[0050] 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).

[0051] Modified IL2 polypeptides (muteins)

[0052] The invention relates to modified human IL2 polypeptides and to modified murine IL2 polypeptides.

[0053] 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.

[0054] In particular, in one aspect, the invention relates to a modified interleukin-2 (IL2) polypeptide wherein the modified IL2 polypeptide is a modified human IL2 polypeptide and comprises the following amino acid substitutions with reference to SEQ ID NO: 1 : a K at position at position 65 and a K at position 82, or 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 96, and / or iii. an amino acid substitution at position 79.

[0055] 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:

[0056] 1. an amino acid substitution at position 151 ,

[0057] 2. an R position 110 and / or

[0058] 3. an N at position 143.

[0059] In one embodiment, the amino acid substitution at position 151 is T151R.

[0060] In one embodiment, the modified human IL2 polypeptide comprises an R at position 151 and an R at position 110.

[0061] In one embodiment, the modified murine IL2 polypeptide comprises the following amino acid substitutions with reference to SEQ ID NO: 2: i. an amino acid substitution at position 50 and ii. an amino acid substitution at position 79.

[0062] In one embodiment, the mutations are Q50W and Y79K.

[0063] In one embodiment, the modified murine IL2 polypeptide further comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 2: iii. an amino acid substitution at position 158 iv. an amino acid substitution at position 126 v. an amino acid substitution at position 54 vi. an amino acid substitution at position 123 and / or vii. an amino acid substitution at position 166.

[0064] In one embodiment, the modified murine IL2 polypeptide further comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 2: i. an amino acid substitution at position 158 and / or ii. an amino acid substitution at position 166. 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 I L2 polypeptide” or “human I L2 mutein” means a polypeptide variant of a human wild type IL2.

[0065] 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.

[0066] 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) according to the invention. 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.

[0067] 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.

[0068] SEQ ID NO: 34 refers to the murine wild type IL2 nucleotide sequence. SEQ ID NO: 38 refers to the human wild type IL2 nucleotide sequence. A modified nucleic acid sequence encoding modified polypeptides is also provided herein and part of the invention, i.e. SEQ ID NO: 39.

[0069] 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.

[0070] In one embodiment, the murine IL2 polypeptide is a mouse IL2 polypeptide.

[0071] 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. 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[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. 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. 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.

[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. 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.

[0081] 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.

[0082] 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.

[0083] In one embodiment, the modified human IL2 polypeptide comprises H36W, Y65K, E82K, N110R, T143N and / or T151R. In one embodiment, the modified human IL2 polypeptide comprises Y65K, E82K, N110R and T151 R. In one embodiment, the modified murine IL2 polypeptide comprises Q50W, D54R, Y79K, E96K, V126R, A158N, T166R, and / or N123H. In one embodiment, the modified murine IL2 polypeptide comprises Q50W and Y79K.

[0084] In one embodiment, the amino acid substitution at position 108 comprises N108H.

[0085] In one embodiment, the amino acid substitution at position 111 comprises V111 R.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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. In one embodiment, the modified human polypeptide comprises the following amino acid substitutions with reference to SEQ ID NO: 1 : N110R and T151 R.

[0090] 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.

[0091] 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.

[0092] In one embodiment, the modified murine polypeptide comprises an amino acid substitution at T166R with reference to SEQ ID NO: 2:

[0093] 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.

[0094] 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.

[0095] In one embodiment, the modified polypeptides of the invention further comprise 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.

[0096] 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.

[0097] 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.

[0098] Table A. Amino Acid Residues and Examples of Conservative Amino Acid Substitutions

[0099] 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.

[0100] 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. 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.

[0101] A problem in the art is that enhancing specificity for either the Treg 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.

[0102] 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.

[0103] 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.

[0104] In one embodiment, the amino substitution does not alter or does not substantially alter the bioactivity of CD8+ T cells and / or Tregs.

[0105] 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. V111R, N110R and T151 R; ii. H36W or

[0106] Hi. N108H and T151 R.

[0107] 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.

[0108] 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: N110R and T151 R.

[0109] 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: i. V126R or ii. V126R and A158N.

[0110] 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

[0111] Hi. Y79K. 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.

[0112] 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 Tregs. 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.

[0113] 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.

[0114] 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 T regs 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.

[0115] In one embodiment, the amino acid substitution increases the bioactivity / responses of CD8 T cells and / or Tregs. 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.

[0116] 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 T regs” 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. In one embodiment, the modified IL2 polypeptide is conjugated to one or more moieties 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. In one embodiment, the therapeutic molecule is an antibody or antigen fragment thereof that specifically binds to CD8, wherein the fragment is selected from a F(ab')2, Fab, Fv, scFv, heavy chain, light chain, variable heavy (VH), variable light (VL) chain, CDR region, single VH or VL domain, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, and bis-scFv. In one embodiment, the fragment is an scFv.

[0117] For example, the modified IL2 polypeptide may be fused or conjugated to an scFv, for example the modified IL2 polypeptide comprises the following amino acid modifications: Y65K, E82K, N110R and T151 R.

[0118] The antibody may be an immune checkpoint inhibitor, for example a PD-1 , PDL1 , PDL2, LAG3, TIM3 or CTLA4 inhibitor. The PD-1 inhibitor may be Cemiplimab (Libtayo), Nivolumab (Opdivo) or Pembrolizumab (Keytruda).

[0119] In one embodiment, the modified IL2 polypeptide may be fused or conjugated to a costimulatory molecule that [provides a “signal 2”, for example CD80. In one embodiment, the modified IL2 polypeptide may be fused or conjugated to an antibody or antigen fragment that binds a tumor specific antigen, e.g. co-stimulatory molecule, BCMA, CD19, B7H3, mesothelin, for example in a CAR as described further below.

[0120] 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.

[0121] 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 half-life 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).

[0122] Constructs and vector delivery systems

[0123] According to another aspect, the invention relates to a construct comprising a nucleic acid encoding a modified IL2 polypeptide of the invention.

[0124] 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.

[0125] 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.

[0126] 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. In other embodiments, the promoter is CAG 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.

[0127] 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.

[0128] 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.

[0129] Pharmaceutical composition 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.

[0130] Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. Preferably, the compositions are administered parenterally. Parenteral administration may include intratumoral, intracavitary, intrapleural, or intracerebroventricular administration.

[0131] 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. 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] Compositions can take the form of one or more dosage units.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] Exemplary therapeutic applications

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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. 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.

[0149] 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.

[0150] In one embodiment, the tumor is a non-solid tumor. Examples of non-solid tumors include leukemia, multiple myeloma and lymphoma.

[0151] In one embodiment, the cancer is locally advanced unresectable, metastatic, or recurrent cancer.

[0152] 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.

[0153] 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 one embodiment, the fragment is selected from a F(ab')2, Fab, Fv, scFv, heavy chain, light chain, variable heavy (VH), variable light (VL) chain, CDR region, single VH or VL domain, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, and bis-scFv. In one embodiment, the fragment is an scFv. As described elsewhere herein, the modified polypeptides, constructs, vectors or pharmaceutical composition of the invention may be administered together with an immune cell that expresses a chimeric antigen receptor.

[0154] 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.

[0155] Exemplary kits

[0156] 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.

[0157] 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.

[0158] 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.

[0159] Exemplary nucleic acids, vectors and host cells

[0160] 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. 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.

[0161] Furthermore, the invention relates to a vector comprising at least one nucleic acid as defined above.

[0162] 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.

[0163] 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.

[0164] Methods for identifying modified polypeptide (muteins)

[0165] According to one aspect, the invention further comprises a method for identifying a modified IL2 polypeptide comprising the steps of

[0166] 1) predicting aggregation-prone regions from an IL2 polypeptide,

[0167] 2) identifying amino acid mutations that abolish the aggregation propensity of these segments without affecting the thermodynamic stability of the polypeptide and

[0168] 3) expressing a modified IL2 polypeptide with reduced aggregation.

[0169] In one embodiment, the method for identifying a modified polypeptide comprises the SolubiS method (https: / / solubis.switchlab.org / about).

[0170] 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 / ).

[0171] 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 : / / f o I dxs u i te . erg . e u / ) .

[0172] 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.

[0173] Exemplary methods for altering T cell responses, alter the ratio of non aggregated: aggregated IL2 production

[0174] 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.

[0175] 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.

[0176] 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. In one embodiment, the modified IL2 polypeptide is a modified human IL2 polypeptide as described herein.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] In one embodiment, the modified IL2 polypeptide is a modified human IL2 polypeptide as described herein.

[0181] 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.

[0182] 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.

[0183] 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 151 ; or v. an R at position 111 , an R at position 110 and an R at position 151.

[0184] 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.

[0185] 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.

[0186] The above methods may be an in vitro, ex vivo or in vivo method for application in a subject, cell, tissue or cell culture.

[0187] Chimeric antigen receptor and immune cells

[0188] In one embodiment, the invention relates to an isolated immune cell, wherein the immune cell comprises a chimeric antigen receptor, wherein said immune cell expresses the modified human or murine I L2 polypeptide or the nucleic acid construct as described herein.

[0189] In one aspect the invention also relates to a combination comprising the modified human or murine IL2 polypeptide, viral vector or construct as described herein and an immune cell, wherein immune cell comprises a chimeric antigen receptor. In one embodiment, the isolated immune cell is a T cell or NK cell.

[0190] In one aspect, the invention also relates to a combination therapy comprising administering the modified human or murine IL2 polypeptide, a construct, a viral vector or a pharmaceutical composition described herein and an immune cell expressing a chimeric antigen receptor.

[0191] The combination therapy may be for treatment of cancer, e.g. wherein in the 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.

[0192] The immune cell expressing the chimeric antigen receptor cell is administered before, at the same time, or after the modified human or murine IL2 polypeptide, viral vector or nucleic acid construct.

[0193] The invention also relates to a method of increasing the expansion and persistence of immune cell, e.g. T cell, therapy, comprising the step of modifying an immune cell to express the modified human or murine IL2 polypeptide, the nucleic acid construct or the viral vector as described herein. In a further step, the immune cell is administered to a subject in need thereof. The invention also relates to a method of increasing the expansion and persistence of immune cell, e.g. T cell, therapy, comprising administering an immune cell expressing a CAR and administering a modified human or murine IL2 polypeptide, the nucleic acid construct or the viral vector as described herein.

[0194] The invention also relates to a pharmaceutical composition comprising an immune cell as described above or comprising a modified human or murine IL2 polypeptide, the nucleic acid construct or the viral vector as described herein and an immune cell expressing a CAR. The terms "Chimeric antigen receptor" or "CAR" or "CARs" as used herein refer to engineered receptors, which graft an antigen specificity onto cells (for example T cells such as naive T cells, central memory T cells, effector memory T cells or combination thereof) thus combining the antigen binding properties of the antigen binding domain with the lytic capacity and self renewal of T cells. CARs are also known as artificial T cell receptors, chimeric T cell receptors or chimeric immunoreceptors. The term “antigen binding domain or “antigen-specific targeting domain" as used herein refers to the region of the CAR which targets and binds to specific antigens as explained above. When a CAR is expressed in a host cell, this domain forms the extracellular domain (ectodomain).

[0195] A skilled person would know that a CAR comprises additional elements.

[0196] A skilled person would also know that such elements of a CAR (other than antigen-specific targeting domain described herein) are well known in the art. Thus, the invention is not limited to specific domains of the CAR in addition to the antigen-specific targeting domain described herein.

[0197] First generation CARs have been tested in various phase I clinical studies in patients with cancer. Second generation CARs and third generation CARs have also been described are more complex with three or more signalling domains (reviewed in Sadelain et al., Curr Opin Immunol, 21 (2): 215-223, 2009, Sterner, R.C., Sterner, R.M. CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J. 11 , 69, 2021). CARs are also described in US2004043401 , W02019200007 and WO2021108613, all incorporated herein by reference.

[0198] Exemplary domains of a CAR are listed below. As will also be apparent, the CAR may comprise additional domains as explained below.

[0199] Antigen binding domain

[0200] The antigen binding domain used in a CAR of the invention may be a tumor antigen. The tumor may be a solid or non-solid tumor. Tumor antigens are known to the skilled person and include CD19 and BCMA for B-cell malignancies like leukemia, lymphoma, and multiple myeloma, and CD38, CD33, CD123, and CLL-1 for acute myeloid leukemia (AML). Targets for solid tumors include HER2 and PSMA. Further, non-limiting targets may include epidermal growth factor receptor (EGFR) (nonsmall cell lung cancer, epithelial carcinoma, and glioma), variant III of the epidermal growth factor receptor (EGFRvlll) (glioblastoma), human epidermal growth factor receptor 2(HER2) (ovarian cancer, breast cancer, glioblastoma, colon cancer, osteosarcoma, and medulloblastoma), mesothelin (mesothelioma, ovarian cancer, and pancreatic adenocarcinoma), arcinoembryonic antigen (CEA) (pancreatic adenocarcinoma, breast cancer, and colorectal carcinoma), ganglioside 2(GD2) (neuroblastoma and melanoma), lnterleukin-13Ra2 (glioma), Glypican-3 (hepatocellular carcinoma), Carbonic anhydrase IX (CAIX) (renal cell carcinoma), L1 cell adhesion molecule(LI-CAM) (neuroblastoma, melanoma, and ovarian adenocarcinoma), Cancer antigen 125 (CA 125) (epithelial ovarian cancer), Cluster of differentiation 133 (CD 133) (glioblastoma and cholangiocarcinoma) Fibroblast activation protein(FAP) (malignant pleural mesothelioma) Cancer / testis antigen 1 B(CTAG1 B) (melanoma and ovarian cancer), Mucin 1 (seminal vesicle cancer), mesothelin (ung, ovarian, colon, pancreatic and mesothelioma cancer), B7H3 (non-small- cell lung cancer (NSCLC) and prostate cancer) or Folate receptor- a (FR-a) (ovarian cancer).

[0201] The antigen binding domain may be an antibody or antigen binding fragment thereof as defined herein. In one embodiment, the antigen binding domain is an scFV.

[0202] The Intracellular (Cytoplasmic) Domain

[0203] The intracellular (cytoplasmic) domain of the CAR can provide activation of at least one of the normal effector functions of the immune cell. The CAR of the invention may thus further comprise an intracellular signaling domain. An "intracellular signaling domain", "cytoplasmic domain" or “endodomain” is the domain that transmits activation signals to T cells and directs the cell to perform its specialized function.

[0204] An “intracellular signaling domain,” as the term is used herein, refers to an intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes an immune effector function of the CAR containing cell, e.g., a CAR-T cell or CAR- expressing NK cell. Examples of immune effector function, e.g., in a CAR-T cell or CAR- expressing NK cell, include cytolytic activity and helper activity, including the secretion of cytokines. In an embodiment, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation. In an embodiment, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation. For example, in the case of a CART, a primary intracellular signaling domain can comprise a cytoplasmic sequence of a T cell receptor, and a costimulatory intracellular signaling domain can comprise cytoplasmic sequence from co-receptor or costimulatory molecule.

[0205] The intracellular domain may comprise at least in part an activating domain, preferably comprised of a CD3 family member such as CD3 zeta, CD3 epsilon, CD3 gamma, or portions thereof.

[0206] Examples of domains that transduce the effector function signal and can be used according to the invention include but are not limited to the chain of the T-cell receptor complex or any of its homologs (e.g., q chain, FcsRIy and chains, MB1 (Igalpha) chain, B29 (Igbeta) chain, human CD3zeta chain, CD3 gamma or other CD3 polypeptides (A, 5 and E), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lek, Fyn, Lyn, etc.) and other molecules involved in T-cell transduction, such as CD2, CD5, 0X40 and CD28.

[0207] It will be appreciated that suitable intracellular molecules may also include but are not limited to, 4-1 BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function- associated antigen-1 (LFA-I, CDI-la / CDI8), CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-I, B7-H3, CDS, ICAM-I, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, IT GAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-I, ITGAM, CD1 lb, ITGAX, CD1 Ic, ITGB1 , CD29, ITGB2, CD 18, LFA-I, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1 , CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylOS), SLAM (SLAMF1 , CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CDI9a, a ligand that specifically binds with CD83, or any combination thereof. Other intracellular signaling domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.

[0208] In some embodiment, the cytoplasmic domain of the CAR can be designed to comprise the CD3 zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the invention. For example, the cytoplasmic domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region.

[0209] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the invention may be linked to each other in a random or specified order.

[0210] The term "zeta" or alternatively "zeta chain", "CD3-zeta" or "TCR-zeta" is defined as the protein provided as GenBan Acc. No. BAG36664.1 , or the equivalent residues from a nonhuman species, e.g., mouse, rodent, monkey, ape and the like, and a "zeta stimulatory domain" or alternatively a "CD3-zeta stimulatory domain" or a "TCR-zeta stimulatory domain" is defined as the amino acid residues from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit an initial signal necessary for T cell activation. In one aspect the cytoplasmic domain of zeta comprises residues 52 through 164 of GenBank Acc. No. BAG36664.1.

[0211] The extracellular signaling domain

[0212] The CAR may also comprise an extracellular signaling domain. The extracellular domain is beneficial for signaling and for an efficient response of lymphocytes to an antigen. Extracellular domains may be derived from (i.e., comprise) CD28, CD28T, OX-40, 4- 1 BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1 , CDI-la / CDI8), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1 , B7-H3, CDS, ICAM-I, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL- 2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, IT GAL, CD1 la, LFA-I, ITGAM, CD1 lb, ITGAX, CD1 Ic, ITGB1 , CD29, ITGB2, CD 18, LFA-I, ITGB7, NKG2D, TNFR2, TRAN CE / R ANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1 , CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylOS), SLAM (SLAMF1 , CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76 or PAG / Cbp, a ligand that specifically binds to CD83, or any combination thereof. The extracellular domain may be derived either from a natural or from a synthetic source.

[0213] Hinge region

[0214] In one embodiment, the CAR of the invention further comprises a hinge or spacer region which connects the extracellular antigen binding domain and the transmembrane domain. In particular, extracellular domains often comprise a hinge portion. This hinge or spacer region can be used to achieve different lengths and flexibility of the resulting CAR. Examples of the hinge or spacer region that can be used according to the invention include, but are not limited to, Fc fragments of antibodies or fragments or derivatives thereof, hinge regions of antibodies, or fragments or derivatives thereof, CH2 regions of antibodies, CH3 regions of antibodies, artificial spacer sequences, for example peptide sequences, or combinations thereof. Other hinge or spacer region will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. In one embodiment, the hinge is an lgG4 hinge or a CD8A hinge, an immunoglobulin (Ig) sequence or other suitable molecule to achieve the desired special distance from the target cell. In some embodiments, the entire extracellular region comprises a hinge region. In some embodiments, the hinge region comprises CD28T, or the EC domain of CD28.

[0215] The transmembrane domain

[0216] The CAR can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the CAR.

[0217] A "transmembrane domain" (TMD) as used herein refers to the region of the CAR which crosses the plasma membrane and is connected to the endoplasmic signaling domain and the antigen binding domain, in case of the latter optionally via a hinge. In one embodiment, the transmembrane domain of the CAR of the invention is the transmembrane region of a transmembrane protein (for example Type I transmembrane proteins), an artificial hydrophobic sequence or a combination thereof. In one embodiment, the transmembrane domain comprises the CD3zeta domain or CD28 transmembrane domain.

[0218] In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0219] Transmembrane regions of particular use in this invention may be derived from (i.e. comprise) CD28, CD28T, OX-40, 4-1 BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function- associated antigen-1 (LFA-I, CDI-la / CDI8), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP- 10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM- 1 , B7-H3, CDS, ICAM-I, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-I, ITGAM, CD1 lb, ITGAX, CD1 Ic, ITGB1 , CD29, ITGB2, CD 18, LFA-I, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1 , CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylOS), SLAM (SLAMF1 , CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP- 76, PAG / Cbp, a ligand that specifically binds to CD83, or any combination thereof. Other transmembrane domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.

[0220] Optionally, short linkers may form linkages between any or some of the extracellular, transmembrane, and intracellular domains of the CAR.

[0221] In one embodiment, the CAR of the invention further comprises one or more co- stimulatory domains to enhance CAR-T cell activity after antigen specific engagement. Inclusion of this domain in the CAR of the invention enhances the proliferation, survival and / or development of memory cells. The term “costimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. The co-stimulatory domain is located intracellularly.

[0222] The co-stimulatory domain is a functional signaling domain obtained from a protein selected from the following group: CD28, CD137 (4-IBB), CD134 (0X40), DapIO, CD27, CD2, CD5, ICAM-1 , LFA-1 (CD1 la / CD18), Lek, TNFR-I, TNFR-II, Fas, CD30, CD40 or combinations thereof. Other co-stimulatory domains (e.g., from other proteins) will be apparent to those of skill in the art. Multiple co- stimulatory domains can be included in a single CAR to recruit multiple signaling pathways. In one embodiment, the co-stimulatory domain is obtained from 4-1 BB. The term "4-1 BB" refers to a member of the TNFR superfamily with an amino acid sequence provided as GenBank Acc. No. AAA62478.2. In one embodiment, the term "4- 1 BB costimulatory domain" refers to amino acid residues 214-255 of GenBank Acc. No. AAA62478.2.

[0223] In one embodiment, the CAR of the invention further comprises a "linker domain" or "linker region" that connects different domains of the CAR. This domain includes an oligo- or polypeptide region from about 1 to 100 amino acids in length. Suitable linkers will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.

[0224] In one aspect ,the CAR comprises an optional leader sequence at the amino-terminus (N- ter) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain during cellular processing and localization of the CAR to the cellular membrane. In one embodiment, the leader sequence is a CD8A domain.

[0225] In one embodiment, in the aspects relating to immune cells expressing a CAR cells and aspects that relate to co-administration of the modified IL2 polypeptide with a chimeric antigen receptor, the modified IL2 polypeptide is a modified human IL2 polypeptide and comprises the following amino acid substitutions with reference to SEQ ID NO: 1 : a K at position at position 65 and a K at position 82, or 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: iv. an amino acid substitution at position 50, v. an amino acid substitution at position 96, and / or vi. an amino acid substitution at position 79.

[0226] 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 :

[0227] 1. an amino acid substitution at position 151 ,

[0228] 2. an R position 110 and / or

[0229] 3. an N at position 143.

[0230] In one embodiment, the amino acid substitution at position 151 is T151 R.

[0231] In one embodiment, the modified human IL2 polypeptide further comprises an R at position 151 and an R at position 110.

[0232] 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.

[0233] 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.

[0234] "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.

[0235] The invention is now further described in the non-limiting examples.

[0236] Examples

[0237] 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.

[0238] Methods iL2 mutation design

[0239] Mutations were designed based on four structures of human IL2: 1m47 (IL2 alone, resolution of 1.99 A), 1z92 (IL2 with IL2RA, resolution of 2.8 A), 2b5i (IL2 with IL2RA / I L2RB / I L2RB, 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-IL2 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 hl 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.

[0240] Structural models of IL2 in complex with the trimeric receptor was performed based off the 2erj structure of the human IL2 1 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).

[0241] Cloning and molecular biology

[0242] 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 Pfl23ll (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).

[0243] IL2 production and measurement

[0244] 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.

[0245] 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.

[0246] Phospho- STAT Flow cytometry

[0247] 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.

[0248] 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 (400-0.02ng / 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.

[0249] In vivo gene delivery and assessment For gene therapy studies using adeno-associated vectors male and female C57BL / 6 wildtype 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.

[0250] 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.

[0251] 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).

[0252] 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).

[0253] Results Example 1 Directed disruption of IL2 aggregation sites designed by SolubiS

[0254] A key limitation in the production capacity of proteins is the aggregation of nascent polypeptides, mediated through aggregation-prone hydrophobic stretches. As protein- protein 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).

[0255] Table 1 IL2 muteins design strategy

[0256] Example 2 Production screening of IL2 muteins identifies point mutations with improved production capacity

[0257] 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 T 116R, 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, V111 R T143N trended towards increased production but no mutants were significantly elevated and several had production defects (Figure 1 B). The combination of N110R T151 R 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).

[0258] Example 3 SolubiS murine IL2 muteins have altered cellular specificity

[0259] 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).

[0260] 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 ( ~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.

[0261] 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 I 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 sidechain falls within a hydrophobic groove of IL2RB with potential n-stacking interactions with F101 and F135 residues (Figure 3D). The larger hydrophobic side-chain may therefore enhance binding, providing preferred signalling through IL2RB.

[0262] Example 4 In vivo functional testing of V126R IL2 mutein

[0263] 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.

[0264] Example 5 Combinatorial generation of a CD8 T cell superkine

[0265] 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 Q50W Y79K 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.

[0266] Example 6 SolubiS human IL2 muteins have altered cellular specificity

[0267] 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 1 B).

[0268] 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 1B).

[0269] Example 6 Combination mutants result in human IL2 muteins with enhanced cellular selectivity

[0270] 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.

[0271] 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.

[0272] 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.

[0273] Discussion

[0274] 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.

[0275] 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 side-chain 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 N 110R, 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 aggregation-protecting 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 cation-cation 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) / 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 non-signalling conformation, it could potentially act as an antagonist in CD8 T cells, while as acting as a bioactive signaller in Tregs.

[0276] 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 T regs, 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 halflife, 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.

[0277] Example 7 In vivo expansion of CD8+ T cells or Tregs using an AA V delivery system

[0278] 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. Human muteins Y65K E82K N110R T151R or V111 R N110R T151 R are encoded in an

[0279] AAV9 based vector delivery system and administered to mice. Mice are tested for brain expansion of Tregs or CD8+T cells.

[0280] Example 8 In vitro pSTAT5 Assay

[0281] Methods

[0282] Cloning

[0283] Fragments for the various expression constructs were either PCR amplified or purchased as geneBlocks from Integrated DNA Technologies (IDT). PCRs were performed using the Phusion High-Fidelity DNA Polymerase (Thermo Scientific, cat. no. F-530XL), as recommended by the manufacturer. Fragments were assembled using the NEBuilder HiFi DNA Assembly Master mix (New England Biolabs, cat. no. E2621S), as recommended by the manufacturer. Briefly, gene fragments and the linearized CMV-expression vector harbouring an mCherry reporter were pooled and combined with 5 ul of the NEBuilder HiFi DNA Assembly Master mix. The mixture was incubated at 50 degrees Celsius for 1 hour, with 5 ul of the assembly reaction transformed into 100 ul of MAX Efficiency Stbl2 Competent Cells (Invitrogen, cat. no. 10268019). Transformants were selected in Luria Bertani-ampicillin coated plates, and positive clones confirmed by Sanger sequencing.

[0284] HEK293T cell culture

[0285] HEK293T cells (American Type Culture Collection, cat. no. CRL-3216) were cultured in HEK maintenance media [DM EM (Gibco ThermoFisher, cat. no. 21980-032) supplemented with 10% heat inactivated fetal calf serum (Sigma-aldrich, cat. no. F7524), and 2 mM L- glutamine (Gibco ThermoFisher, cat. no 35050-038)]. Cells were confirmed to be mycoplasma negative using the MycoStrip® mycoplasma detection kit (InvivoGen, cat. no. rep-mys-10), as recommended by the manufacturer.

[0286] Transfection

[0287] HEK293T cells were seeded in 24-well plates at a density of 125,000 cells per well. 24- hours post-seeding, cells were transfected with 1 microgram of expression plasmid using the FuGeneHD transfection reagent (Promega, cat. no. E2311), as recommended by the manufacturer. 48-hours post- transfection, supernatants were harvested, centrifuged at 500xg for 5 minutes to pellet debris, and stored at -80° Celsius until further use. T ransfection efficiencies were determined by assessing the frequency of mCherry positive cells by flow cytometry on the Cytek Aurora Spectral Analyser. Flow cytometric plots were generated using FlowJo version 10.8.1.

[0288] Quantification of Mouse IL2 from HEK293T Supernatants using ELISA

[0289] F96 maxisorp nunc-immunoplates (ThermoFisher, cat. no. 442404) were coated with monoclonal anti-mouse IL2 antibody (BioLegend, cat. no. 503704) at 1 ug / ml (50 ul / well) in PBS overnight. The following day, the plate was flicked to discard the supernatant, and then washed twice with PBS with 0.07% Tween 20 (PBS-T; 150 ul / well). The plate was blocked for 1 hour with PBS with 2% BSA (150 ul / well), and the supernatant discarded by flicking the plate. The cell culture supernatants were then added on the plate (50 ul / well), and incubated at room temperature for 1 hour. His-tagged mouse IL2 (GeneTex, cat. no. GTX00285-pro) protein standards were also concurrently added unto plate and left for 1 hour. The plate was washed four times with PBS-T, and then incubated with horse radish peroxidase- conjugated anti-6xHis tag antibody (BioLegend, cat. no. 652503, 1 :2000 dilution) at 50 ul / well. The supernatant was flicked-off, plate washed four times with PBS-T and finally 50 ul / well of tetramethylbenzidine (TMB) solution (FisherScientific, cat. no. 12750000) was added unto the plate. After 10 minutes, the colorimetric reaction was stopped by the addition of 50 ul / well of 1 M HCI. The absorbance at 450 nm was then read using a multiplate reader. Protein concentrations were extrapolated from the standard curve using the GraphPad prism 9 software (version 9.5.1).

[0290] Quantification of Human IL2 from HEK293T Supernatants using His-taq ELISA

[0291] The concentration of IL2 from cell culture supernatants of HEK293T cells transfected with the human IL2 expression constructs were quantified using the GenScript His Tag ELISA Detection Kit (Cat. no. L00436), as recommended by the manufacturer.

[0292] Spleens were smashed between two glass slides, and filtered through a 100 urn nitex filter into a 15 ml conical tube. The filter was rinsed once with 10 ml of FACS buffer, and the tube inverted to mix its contents. The tube was centrifuged for 5 minutes at 4° Celsius and 600xg (acceleration 9, deceleration 9, radius 17.3), and the supernatant discarded. The pellet was counted, and 2 x 106cells seeded unto a Il-bottom 96-well plate for IL2 stimulation and antibody staining.

[0293] Mouse IL2 stimulation experiments

[0294] Splenocytes were isolated from spleens of C57BL / 6 as described earlier. Two million splenocytes were seeded unto a V-bottom 96-well plate (ThermoFisher, cat. no. 611V96) and blocked with 150 ul of 2.4G2 for 30 minutes at room temperature. The plate was spun down at 600xg for 5 minutes at 4° Celsius, and then flicked to discard the supernatant. The pellet was resuspended in 100 ul of eFlour780 viability dye (1 :4000, ThermoFisher, cat. no. 65-0865-18) and incubated for 15 minutes at room temperature. The plate was topped with 150 ul of FACS buffer and spun down at 600xg for 5 minutes at 4° Celsius. It was then surface stained for 1 hour at 4° Celsius with mixture composed of the following antibodies: anti-CD4-BUV395 (1 :500, BD Biosciences, cat. no. 563790), anti-CD25-BV421 (1 :200, BioLegend, cat. no. 102043), and eFlour780 viability dye (1 :4000, ThermoFisher, cat. no. 65-0865-18). The plate was topped with 150 ul of FACS buffer and spun down at 600xg for 5 minutes at 4° Celsius. The plate was washed two more times with FACS buffer after which the pellet was resuspended in 100 ul of the IL2 supernatant media at the desired concentration of choice. The plate was stimulated for 25 minutes at 37° Celsius, after which, 100 ul of Formalin (4% Formaldehdye) was added unto the plate to stop the reaction. The plate was incubated in a dark room for 30 minutes at room temperature, and then centrifuged at 600xg for 5 minutes. The plate was flicked to discard the supernatant, and then the pellet was resuspended in 100 ul of ice-cold 100% methanol and incubated for 30 minutes at 4° Celsius. The cells was washed with 100 ul of Phospho-staining buffer (TBS, 2.5% FCS, and 2.5 mM EDTA), and then centrifuged at 600xg for 5 minutes at room temperature. The cells were then stained overnight in the dark at room temperature with the following antibodies: anti-FoxP3-eFluor450 (1 :100, ThermoFisher, cat. no. 48-5773-82), anti-CD3-Spark Blue 550 (1 :5000, BioLegend, cat. no 100260), anti-CD8-PerCP-eFluor710 (1 :5000, ThermoFisher, cat. no. 46-0081-80), anti-phospho-STAT5-eFluor610 (1 :200, ThermoFisher, cat. no. 61-9010-42). The next day, the cells were washed with Phospho- staining buffer, centrifuged at 600xg at room temperature for 5 minutes, and finally resuspended in 150 ul of Phospho-staining buffer and run on the Cytek Aurora Spectral Analyser. Flow cytometric plots were generated using FlowJo version 10.8.1.

[0295] Human Ficoll Paque Peripheral Blood Mononuclear Cells (PBMCs) Isolation Human leukocyte cones were purchased from the Cambridge University Hospitals Blood service. PBMCs were isolated using Ficoll-Paque PLUS density gradient media (Cytiva, cat. no. 17144002), as recommended by the manufacturer. Briefly, 15 ml of Ficoll-Paque PLUS was added unto a 50-ml conical tube. Afterwards, 10 ml of blood was mixed with 10 ml of PBS supplemented with 2% FCS. This Blood-PBS mixture was then carefully layered on top of the Ficoll-Paque, trying to avoid mixing between the two interfaces as much as possible. The sample was centrifuged in swing-bucket centrifuge at 400xg, for 30 minutes at 20° Celsius with the acceleration / deceleration rates at 9 / 0. After centrifugation, the top layer was carefully aspirated and discarded, while the lymphocyte (middle) layer transferred unto a new 50-ml conical tube. The PBMCs were washed with 15 ml of PBS with 2% FCS, and then centrifuged at 100xg for 10 minutes at 20° Celsius. The supernatant was discarded and the lymphocyte layer was washed two more times with PBS to ensure that all Ficoll- Paque was removed. Finally, the PBMCs were resuspended in Roswell Park Memorial Institute (RPMI-1640) medium (Gibco ThermoFisher, cat. no. 21875-034) supplemented with 10% DMSO at a density of 50 million cells per ml, and then stored at -80° Celsius until use.

[0296] Human IL2 stimulation experiments

[0297] Frozen PBMCs were thawed and resuspended in 15 ml of human T cell media [RPMI-1640 (ThermoFisher, cat. no 11875093), 0.01 M HEPES buffer (ThermoFisher, cat. no. 15630080), 1 % volume / volume Penicilin-Streptomycin (ThermoFisher, cat. no. 15140148), 1 mM Sodium Pyruvate (ThermoFisher, cat. no. 11360-039), 2 mM Glutamine (ThermoFisher, cat. no. 35050-038), 1 % volume / volume Non-essential amino acids (ThermoFisher, cat. no. 11140-035)]. It was centrifuged at 500xg for 5 minutes at room temperature. The supernatant was discarded and the cells were resuspended in 5 ml human T cell media and then counted. Two million PBMCs per well were then seeded unto a V-bottom (ThermoFisher, cat. no. 611V96) 96-well plate. The plate was spun at 500xg for 5 minutes at 4° Celsius and flicked to discard the supernatant. The cell pellets were resuspended in 50 ul of Hanks’ Balanced Salt solution (Sigma-Aldrich, cat. no. H9269) supplemented with 0.01 M HEPES buffer, 2 mM EDTA, 2.5% FCS, 10% human antibody serum (insert brand), and FcX truStain (1 :100, BioLegend, cat. no. 422301). 50 ul of eFlour780 viability dye (1 :2000, ThermoFisher, cat. no. 65-0865-18) supplemented with 20 ug / ml of DNAsel (Sigma, cat. no. DN25) was added to every well, and the plate was incubated at 37° Celsius for 10 minutes. Afterwards, the plate was centrifuged at 500xg for 5 minutes at 4° Celsius and flicked to discard the supernatant. The cell pellets were then surface stained for 1 hour at 37° Celsius with the following antibodies: anti-CD4-SB436 (1 :100, ThermoFisher, cat. no. 62-00047-42), and anti-CD3-SB550 (1 :100, BioLegend, cat. no. 344852). The plate was then centrifuged at 500xg for 5 min at 4° Celsius and flicked to discard the supernatant. The cells were then stimulated with 100 ul HEK293T supernatant containing IL2 at the desired concentration for 25 minutes at 37° Celsius. At the end of the stimulation period, 100 ul of Formalin (4% Formaldehyde) was added unto plate, and left to incubate for 30 minutes in the dark at room temperature. The plate was centrifuged at 500xg for 5 minutes at 4° Celsius and then the supernatant was discarded. 100 ul of ice-cold methanol was added unto the plate and the plate was incubated at 4° Celsius for 30 minutes. After the incubation, 100 ul of Phospho-staining buffer was added to every well, and the plate was similarly centrifuged at 500xg for 5 minutes at room temperature. The plate was then stained overnight in Phospho-staining buffer containing the following antibodies: anti-CD8-SV538 (1 :200, BioLegend, cat. no. 303805), anti-FoxP3-eFluor660 (1 :500, ThermoFisher, cat. no. 50-4776-42), anti-CD25-PE-Fire700 (1 :200, BioLegend, cat. no. 356146), anti-phospho-STAT5-eFluor610 (1 :200, ThermoFisher, cat. no. 61-9010-42). The following morning, the plate was centrifuged at 500xg for 5 minutes at room temperature. It was washed with Phospho-staining buffer, and similarly centrifuged. Finally, the cells were resuspended in 150 ul of Phospho-staining buffer and run on the Cytek Aurora Spectral Analyser. Flow cytometric plots were generated using FlowJo version 10.8.1.

[0298] Results: Mouse

[0299] Mouse splenocytes were stimulated with titrated doses of various synthetic IL2 constructs and then pSTAT5 levels in CD8+ T cells and Tregs were examined (Figure 8). The assay confirmed that the Q50W Y79K mutein exhibited enhanced CD8 specificity compared to wild type IL2, showing approximately a 50-fold higher preference for CD8+ T cells and a 10- fold reduction in Treg activation.

[0300] Fusing the YTS anti-CD8 scFv clone to wild type IL2 produced a similar effect, enhancing CD8 response by 100-fold while reducing Treg activation by 10-fold. Meanwhile, fusing Q50W Y79K to YTS resulted in a 1 ,000-fold increase in CD8+ response and a more than 1 ,000-fold reduction in Treg activation, yielding an overall 106-fold improvement over wild type IL2. Results: Human

[0301] Human PBMCS were stimulated with titrated doses of various synthetic IL2 constructs and then pSTAT5 levels in CD8+ T cells and Tregs were examined (Figure 9). The assay confirmed that the CD8-boosting effect was primarily mediated by the crefmirlimabV2 scFv, whereas the Treg-dampening effect was attributable to the Y465 E82K N1100R T151 R mutein.

[0302] The Y65K E82K N110R T151 R mutein led to a 1 ,000-fold reduction in Treg responses compared to wild type IL2. Crefmirlimabv2 scFv displayed a 1 ,000-fold enhancement of CD8+ T cell responses. Overall, Crefmirlimabv2-(G4S)2- Y65K E82K N110R T151 R exhibited the best CD8 / Treg response profile.

[0303] Example 9 AAV-IL2 and CAR-T cell combination

[0304] Summary.

[0305] AAV-G FAP- IL-2 vectors can be combined with GD2-targeting CAR-T cells to control growth of GFAP+ GD2+ brain tumours.

[0306] Experimental design and methodology.

[0307] An intracranial syngeneic brain tumour models was employed. 30,000 CT-2A (GD2+GFAP+Luc+tdTomato+) cells were implanted via stereotactic injection to the right brain hemisphere of C57BL / 6J mice (female, 10 weeks old). Seven days after surgery, implantation was confirmed by bioluminescence imaging (BLI). Live imaging was repeated at 7-day intervals unless mice were deemed to unwell to undergo the procedure (Figure 10).

[0308] Mice were divided into 6 groups of equivalent average BLI signal and assigned to treatment groups:

[0309] 1. AAV-GFAP-eGFP

[0310] 2. AAV-GFAP-wildtype IL2

[0311] 3. AAV-GFAP-mutein IL2 (Q50W, Y79K)

[0312] 4. AAV-GFAP-eGFP +murine GD2-CAR-T cells

[0313] 5. AAV-GFAP-wildtype IL2 +murine GD2-CAR-T cells

[0314] 6. AAV-GFAP-mutein IL2 (Q50W, Y79K) +murine GD2-CAR-T cells On Day 9 mice received a single IV injection of 10A10 viral particles in 100uL PBS+0.1% Mouse Serum Albumin. On Day 12 mice in groups 4-6 received an IV injection of 3 million GD2-CAR-T cells in 100uL PBS+0.1% Mouse Serum Albumin. Mice were monitored daily, weighed twice weekly, until they reached humane endpoint.

[0315] Experimental results.

[0316] Tumour volume as measure by non-invasive live imaging.

[0317] Over the course of the experiment bioluminescence (Total flux: photons / sec) increased 100- fold for all treatment groups except those receiving the combination of AAV-GFAP-mutein IL-2 and GD2-CAR-T cells (Figure 11). Group 6 mice (AAV-GFAP-mutein IL-2 and GD2- CAR-T cells) had an average 10-fold reduction in bioluminescence from their Day 7 starting point, indicating a reduction in tumour volume.

[0318] Survival curves.

[0319] Any-cause mortality was assessed by Kaplan-Meier survival analysis. Group 6 mice (AAV- GFAP-mutein IL-2 and GD2-CAR-T cells) had significantly longer survival compared to Group 5 mice (AAV-GFAP-wildtype IL-2 and GD2-CAR-T cells) [p=0.013] (Figure 12). When non-neurological endpoints were censored and only tumour cause of death was considered Group 6 had significantly longer survival compared to Group 1 (AAV-GFAP- GFP), 4 (AAV-GFAP-GFP + GD2-CAR-T cells) and 5 (AAV-GFAP-wildtype IL-2 + GD2- CAR-T cells) [p=0.011 ; p=0.015; p=0.017], Group 3 (AAV-GFAP-mutein IL2 (Q50W, Y79K)) also had significantly longer survival than Group 1 or Group 4 [p=0.047; p=0.044]. One mouse in Group 6 was alive with a clinical score of 0 at end of experiment.

[0320] Clinical scoring.

[0321] All mice (5 / 5) in Group 1 (AAV-GFAP-GFP) and Group 4 (AAV-GFAP-GFP and GD2-CAR- T cells) were humanely killed due to Neurological Score, indicating tumour-related morality (Figure 13). No mice from Group 6 (AAV-GFAP-mutein IL-2 and GD2-CAR-T cells) were humanely killed for Neurological Score. 4 of 5 mice from Group 6 were humanly killed due to a combination of Clinical Score and weight-loss, and one was alive at the end of experiment (Day 35). 3 of 5 mice from Group 3 (AAV-GFAP-mutein IL2 (Q50W, Y79K)) and 1 mouse each from Group 2 (AAV-GFAP-wildtype IL-2) and Group 5 ((AAV-GFAP-mutein IL2 (Q50W, Y79K)) were also humanly killed for Clinical Score rather than Neurological Score. Piloerection was an indicator of treatment toxicity and was observed in all mice that had a non-neurological cause of death: 4 of 5 mice from Group 6, 3 of 5 mice from Group 3 and 1 mouse each in Groups 2 and 5.

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[0379] The invention is further described in the following clauses:

[0380] 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. 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.

[0381] 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.

[0382] 7. The modified IL2 polypeptide of any one of clauses 2 to 6, wherein the amino acid substitution at position 108 comprises N108H.

[0383] 8. The modified IL2 polypeptide of any one of clauses 4 to 7, wherein the amino acid substitution at position 111 comprises V111 R.

[0384] 9. The modified IL2 polypeptide of clauses 7 or 8, 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. 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. 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. V111R, N110R and T151 R; ii. H36W or

[0385] Hi. N108H and T151 R.

[0386] 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.

[0387] 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.

[0388] 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.

[0389] 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 Hi. Y79K.

[0390] 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: 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.

[0391] 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.

[0392] 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.

[0393] 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.

[0394] 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.

[0395] 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.

[0396] 22. A nucleic acid construct comprising a nucleic acid encoding a modified IL2 polypeptide of any one of the preceding clauses.

[0397] 23. A viral vector comprising the construct according to clause 22. 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-rh8, AAV-rh10, AAV-rh39, AAV-rh43, AAVAnc80, AAV 2 / ShH10, AAV-S vector, AAV6.2, Adv5, oncolytic vectors.

[0398] 25. A pharmaceutical composition comprising the modified IL2 polypeptide, viral vector or construct of any of the preceding clauses.

[0399] 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.

[0400] 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.

[0401] 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 .

[0402] 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. 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. 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.

[0403] 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.

[0404] 33. A nucleic acid encoding the modified IL2 polypeptide according to any one of clauses 1 to 21.

[0405] 34. A vector comprising a nucleic acid according to clause 33.

[0406] 35. A host cell comprising a nucleic acid according to clause 33 or a vector according to clause 34.

[0407] 36. The host cell according to clause 35 wherein said host cell is a bacterial, yeast, viral or mammalian cell.

[0408] 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.

[0409] 38. A method for making an IL2 modified polypeptide with reduced aggregation comprising the steps of

[0410] 1) predicting aggregation-prone regions from an IL2 polypeptide,

[0411] 2) identifying amino acid mutations that abolish the aggregation propensity of these segments without affecting the thermodynamic stability of the polypeptide and

[0412] 3) expressing a modified IL2 polypeptide with reduced aggregation.

[0413] 39. The method of clause 38, wherein said polypeptide comprises SEQ ID NO: 1 or SEQ ID NO: 2. 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.

[0414] 41. The vector delivery system according to clause 40, wherein said vector comprises a viral vector.

[0415] 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.

[0416] 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.

[0417] 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.

[0418] 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.

[0419] 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 .

[0420] 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.

[0421] 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.

[0422] 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.

[0423] 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.

[0424] Sequences

[0425] SEQ ID NO: 1 Full length Human IL2 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMP KKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV EFLNRWITFCQSIISTLT

[0426] SEQ ID NO: 2 Full length murine IL2 MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMEN YRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKL KGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ

[0427] SEQ ID NO: 3 Human IL2 signal sequence MYRMQLLSCIALSLALVTNS

[0428] SEQ ID NO: 4 Human mature IL2 sequence

[0429] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE

[0430] EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0431] SEQ ID NO: 5 Mouse IL2 signal sequence MYSMQLASCVTLTLVLLVNS

[0432] SEQ ID NO: 6 Mouse mature IL2 sequence

[0433] APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQA

[0434] TELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATW DFLRRWIAFCQSIISTSPQ

[0435] SEQ ID NO: 7 Y65K E82K N110R T151 R (human modified IL2 polypeptide; CD8 specificity)

[0436] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFKMP

[0437] KKATELKHLQCLEEKLKPLEEVLNLAQSKNFHLRPRDLISNIRVIVLELKGSETTFMCEYADETATIV EFLNRWITFCQSIISRLT

[0438] SEQ ID NO: 8 Y65K E82K (human modified IL2 polypeptide; CD8 specificity)

[0439] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFKMP

[0440] KKATELKHLQCLEEKLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV EFLNRWITFCQSIISTLT

[0441] SEQ ID NO: 9 V111 R N110R T151 R (human modified IL2 polypeptide; Treg specificity)

[0442] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMP

[0443] KKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIRRIVLELKGSETTFMCEYADETATIV EFLNRWITFCQSIISRLT

[0444] SEQ ID NO: 10 H36W (human modified IL2 polypeptide; Treg specificity)

[0445] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEWLLLDLQMILNGINNYKNPKLTRMLTFKFYMP

[0446] KKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV EFLNRWITFCQSIISTLT

[0447] SEQ ID NO: 11 N108H T151 R (human modified IL2 polypeptide; Treg specificity)

[0448] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMP

[0449] KKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISHINVIVLELKGSETTFMCEYADETATIV EFLNRWITFCQSIISRLT

[0450] SEQ ID NO: 12 V126R (murine modified IL2 polypeptide; Treg specificity)

[0451] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMEN

[0452] YRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRRTVVKL

[0453] KGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ

[0454] SEQ ID NO: 13 V126R A158N (murine modified IL2 polypeptide; Treg specificity) MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMEN

[0455] YRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRRTVVKL

[0456] KGSDNTFECQFDDESATVVDFLRRWINFCQSIISTSPQ

[0457] SEQ ID NO: 14 Q50WY79K (murine modified IL2 polypeptide; CD8 specificity)

[0458] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEWLLMDLQELLSRME

[0459] NYRNLKLPRMLTFKFKLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVK

[0460] LKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ

[0461] SEQ ID NO: 15 Q50W (murine modified IL2 polypeptide; CD8 specificity)

[0462] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEWLLMDLQELLSRME

[0463] NYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVK

[0464] LKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ

[0465] SEQ ID NO: 16 Y79K (murine modified IL2 polypeptide; CD8 specificity)

[0466] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMEN

[0467] YRNLKLPRMLTFKFKLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKL

[0468] KGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ

[0469] SEQ ID NO: 17 T166R (murine modified IL2 polypeptide; production enhancing)

[0470] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMEN

[0471] YRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKL

[0472] KGSDNTFECQFDDESATVVDFLRRWIAFCQSIISRSPQ

[0473] SEQ ID NO: 18 >hlL2_1_N110R

[0474] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMP

[0475] KKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIRVIVLELKGSETTFMCEYADETATIV

[0476] EFLNRWITFCQSIISTLT

[0477] SEQ ID NO: 19 >hlL2_2_N110E

[0478] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMP

[0479] KKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIEVIVLELKGSETTFMCEYADETATIV

[0480] EFLNRWITFCQSIISTLT

[0481] SEQ ID NO: 20 >hlL2_3_T143N

[0482] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMP

[0483] KKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV

[0484] EFLNRWINFCQSIISTLT SEQ ID NO: 21 >hlL2_4_T151 R

[0485] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMP

[0486] KKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV EFLNRWITFCQSIISRLT

[0487] SEQ ID NO: 22 >hlL2_6_D40R

[0488] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLRLQMILNGINNYKNPKLTRMLTFKFYMP

[0489] KKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV

[0490] EFLNRWITFCQSIISTLT

[0491] SEQ ID NO: 23 >hlL2_7_N108H

[0492] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMP

[0493] KKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISHINVIVLELKGSETTFMCEYADETATIV EFLNRWITFCQSIISTLT

[0494] SEQ ID NO: 24 >hlL2_8_V111 R

[0495] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMP

[0496] KKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINRIVLELKGSETTFMCEYADETATIV EFLNRWITFCQSIISTLT

[0497] SEQ ID NO: 25 >hlL2_9_N110R_T143N

[0498] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMP

[0499] KKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIRVIVLELKGSETTFMCEYADETATIV EFLNRWINFCQSIISTLT

[0500] SEQ ID NO: 26 >hlL2_10_N110R_T151 R

[0501] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMP

[0502] KKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIRVIVLELKGSETTFMCEYADETATIV EFLNRWITFCQSIISRLT

[0503] SEQ ID NO: 27 > h I L2_11_V111 R_T143N

[0504] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMP

[0505] KKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINRIVLELKGSETTFMCEYADETATIV EFLNRWINFCQSIISTLT

[0506] SEQ ID NO: 28 >mlL2_13_A158N

[0507] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMEN

[0508] YRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKL

[0509] KGSDNTFECQFDDESATVVDFLRRWINFCQSIISTSPQ SEQ ID NO: 29 >mlL2_14_T166R

[0510] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMEN

[0511] YRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKL

[0512] KGSDNTFECQFDDESATVVDFLRRWIAFCQSIISRSPQ

[0513] SEQ ID NO: 30 >mlL2_16_D54R

[0514] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMRLQELLSRMEN

[0515] YRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKL

[0516] KGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ

[0517] SEQ ID NO: 31 >mlL2_17_N123H

[0518] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMEN

[0519] YRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISHIRVTVVKL

[0520] KGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ

[0521] SEQ ID NO: 32 >mlL2_19_V126R_A158N

[0522] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMEN

[0523] YRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRRTVVKL

[0524] KGSDNTFECQFDDESATVVDFLRRWINFCQSIISTSPQ

[0525] SEQ ID NO: 33 >mlL2_10_N123H_T166R

[0526] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMEN

[0527] YRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISHIRVTVVKL

[0528] KGSDNTFECQFDDESATVVDFLRRWIAFCQSIISRSPQ

[0529] For experimental purposes, a His Tag was included upstream, of the stop codon in the following nucleotide sequences:

[0530] SEQ ID NO: 34 Mouse WT IL2

[0531] ATGTACAGCATGCAGCTCGCATCCTGTGTCACATTGACACTTGTGCTCCTTGTCAACAGCGCA

[0532] CCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAGCAGCAGCAGCA

[0533] GCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAGCTCCTGAGCAGGATG

[0534] GAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAAGCAG

[0535] GCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGAACTTGGACCTCTGCGGCATGTTCTG

[0536] GATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAG

[0537] TAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGC

[0538] AACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCC

[0539] TCAATAA SEQ ID NO: 35 Mouse Q50WY79K

[0540] ATGTACAGCATGCAGCTCGCATCCTGTGTCACATTGACACTTGTGCTCCTTGTCAACAGCGCA

[0541] CCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAGCAGCAGCAGCA

[0542] GCAGCAGCAGCAGCACCTGGAGTGGCTGTTGATGGACCTACAGGAGCTCCTGAGCAGGATG

[0543] GAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTAAGTTGCCCAAGCAG

[0544] GCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGAACTTGGACCTCTGCGGCATGTTCTG

[0545] GATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAG

[0546] TAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGC

[0547] AACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCC TCAATAA

[0548] SEQ ID NO: 36 mWTIL2-(G4S)4-YTS

[0549] ATGTACAGCATGCAGCTCGCATCCTGTGTCACATTGACACTTGTGCTCCTTGTCAACAGCGCA

[0550] CCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAGCAGCAGCAGCA

[0551] GCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAGCTCCTGAGCAGGATG

[0552] GAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAAGCAG

[0553] GCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGAACTTGGACCTCTGCGGCATGTTCTG

[0554] GATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAG

[0555] TAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGC

[0556] AACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCC

[0557] TCAAGGCGGTGGTGGATCGGGCGGCGGGGGCAGCGGGGGTGGTGGTTCTGGTGGAGGGG

[0558] GCTCAGAAGTGAAGCTGCAGGAAAGCGGCGGAGGCCTGGTGCAGCCCGGCAGAAGCCTGAA

[0559] GCTGAGCTGTGCCGCCAGCGGCTTCAACTTCAACGACTACTGGATGGGCTGGGTCCGACAG

[0560] GCTCCTGGCAAGGGCCTGGAATGGATCGGCGAGATCAACAAGGACAGCAGCACCATCAACTA

[0561] CACCCCCAGCCTGAAGGACAAGTTCACCATCAGCAGAGACAACGCCCAGAACACCCTGTACC

[0562] TGCAGATGAGCAAGCTGGGCAGCGAGGACACCGCCATCTACTACTGCGCCAGAGCCAGAGG

[0563] CATGATGGTGCTGATCATCCCCCACTACTTCGACTACTGGGGCCAGGGCGTGATGGTCACCG

[0564] TGTCCAGCGGCGGTGGTGGATCGGGCGGCGGGGGCAGCGGGGGTGGTGGTTCTGGTGGAG

[0565] GGGGCTCAGACATCGTGCTGACCCAGAGCCCCGCTATGGCCATGAGCCCTGGCGAGAGAAT

[0566] CACAATCAGCTGCAGAGCCAGCGAGAGCGTGTCCACCAGAATGCACTGGTATCAGCAGAAGC

[0567] CCGGCCAGCAGCCCAAGCTGCTGATCTACGGCGCCAGCAACCTGGAATCCGGCGTGCCAGC

[0568] CAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCGACCCCGTGGAAGCC

[0569] AACGACACCGCCACCTATTTCTGCCAGCAGTCTTGGTACGACCCCTGGACCTTCGGTGGAGG CACCAAGCTGGAACTGAAGTAA

[0570] SEQ ID NO: 37 Q50W Y79K-(G4S)4-YTS ATGTACAGCATGCAGCTCGCATCCTGTGTCACATTGACACTTGTGCTCCTTGTCAACAGCGCA

[0571] CCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAGCAGCAGCAGCA

[0572] GCAGCAGCAGCAGCACCTGGAGTGGCTGTTGATGGACCTACAGGAGCTCCTGAGCAGGATG

[0573] GAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTAAGTTGCCCAAGCAG

[0574] GCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGAACTTGGACCTCTGCGGCATGTTCTG

[0575] GATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAG

[0576] TAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGC

[0577] AACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCC

[0578] TCAAGGCGGTGGTGGATCGGGCGGCGGGGGCAGCGGGGGTGGTGGTTCTGGTGGAGGGG

[0579] GCTCAGAAGTGAAGCTGCAGGAAAGCGGCGGAGGCCTGGTGCAGCCCGGCAGAAGCCTGAA

[0580] GCTGAGCTGTGCCGCCAGCGGCTTCAACTTCAACGACTACTGGATGGGCTGGGTCCGACAG

[0581] GCTCCTGGCAAGGGCCTGGAATGGATCGGCGAGATCAACAAGGACAGCAGCACCATCAACTA

[0582] CACCCCCAGCCTGAAGGACAAGTTCACCATCAGCAGAGACAACGCCCAGAACACCCTGTACC

[0583] TGCAGATGAGCAAGCTGGGCAGCGAGGACACCGCCATCTACTACTGCGCCAGAGCCAGAGG

[0584] CATGATGGTGCTGATCATCCCCCACTACTTCGACTACTGGGGCCAGGGCGTGATGGTCACCG

[0585] TGTCCAGCGGCGGTGGTGGATCGGGCGGCGGGGGCAGCGGGGGTGGTGGTTCTGGTGGAG

[0586] GGGGCTCAGACATCGTGCTGACCCAGAGCCCCGCTATGGCCATGAGCCCTGGCGAGAGAAT

[0587] CACAATCAGCTGCAGAGCCAGCGAGAGCGTGTCCACCAGAATGCACTGGTATCAGCAGAAGC

[0588] CCGGCCAGCAGCCCAAGCTGCTGATCTACGGCGCCAGCAACCTGGAATCCGGCGTGCCAGC

[0589] CAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCGACCCCGTGGAAGCC

[0590] AACGACACCGCCACCTATTTCTGCCAGCAGTCTTGGTACGACCCCTGGACCTTCGGTGGAGG CACCAAGCTGGAACTGAAGTAA

[0591] SEQ ID NO: 38 Human WT IL2

[0592] ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGTGCAC

[0593] CTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGAT

[0594] GATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTT

[0595] ACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTC

[0596] TGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAG

[0597] CAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTGAATATGCT

[0598] GATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCT CAACACTGACTTGA

[0599] SEQ ID NO: 39 Y65K E82K N110R T151 R

[0600] ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGTGCAC

[0601] CTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGAT

[0602] GATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTA AGATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAAAGCTCAAACCTC

[0603] TGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAG

[0604] CAATATCAGAGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTGAATATGCT

[0605] GATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCT CAAGACTGACTTGA

[0606] SEQ ID NO: 40 CrefmirlimabV2-(G4S)2-hWTIL2

[0607] ATGGAAACCGACACCCTGCTGCTGTGGGTGCTGCTGCTCTGGGTCCCAGGCTCCACCGGTGA

[0608] CGTCCAGATAACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAC

[0609] TTGCAGGACAAGTAGGAGTATTAGTCAATATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGT

[0610] TCCTAAGCTCCTGATCTATTCTGGATCCACTCTGCAATCTGGAGTCCCATCTCGGTTCAGTGG

[0611] CAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAAC

[0612] TTATTACTGTCAACAGCATAATGAAAACCCGCTCACGTTCGGCGGAGGGACCAAGGTGGAGAT

[0613] CAAAGGCTCCACATCCGGCGGAGGCTCTGGCGGTGGATCTGGCGGAGGCGGCTCATCCGAA

[0614] GTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGC

[0615] TGCGCGGCGAGCGGCTTTAACATTAAAGATACCTATATTCATTTTGTGCGCCAGGCGCCGGG

[0616] CAAAGGCCTGGAATGGATTGGCCGCATTGATCCGGCGAACGATAACACCCTGTATGCGAGCA

[0617] AATTTCAGGGCAAAGCGACCATTAGCGCGGATACCAGCAAAAACACCGCGTATCTGCAGATG

[0618] AACAGCCTGCGCGCGGGAGATACCGCGGTGTATTATTGCGGCCGCGGCTATGGCTATTATGT

[0619] GTTTGATCATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGGGGTGGTGGTTCTGGT

[0620] GGAGGGGGCTCAGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTA

[0621] CTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGA

[0622] TGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGA

[0623] AGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGA

[0624] CCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACAT

[0625] TCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTT

[0626] TTGTCAAAGCATCATCTCAACACTGACTTGA

[0627] SEQ ID NO: 41 CrefmirlimabV2-(G4S)2- Y65K E82K N1100R T151 R

[0628] ATGGAAACCGACACCCTGCTGCTGTGGGTGCTGCTGCTCTGGGTCCCAGGCTCCACCGGTGA

[0629] CGTCCAGATAACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAC

[0630] TTGCAGGACAAGTAGGAGTATTAGTCAATATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGT

[0631] TCCTAAGCTCCTGATCTATTCTGGATCCACTCTGCAATCTGGAGTCCCATCTCGGTTCAGTGG

[0632] CAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAAC

[0633] TTATTACTGTCAACAGCATAATGAAAACCCGCTCACGTTCGGCGGAGGGACCAAGGTGGAGAT

[0634] CAAAGGCTCCACATCCGGCGGAGGCTCTGGCGGTGGATCTGGCGGAGGCGGCTCATCCGAA

[0635] GTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGC

[0636] TGCGCGGCGAGCGGCTTTAACATTAAAGATACCTATATTCATTTTGTGCGCCAGGCGCCGGG CAAAGGCCTGGAATGGATTGGCCGCATTGATCCGGCGAACGATAACACCCTGTATGCGAGCA

[0637] AATTTCAGGGCAAAGCGACCATTAGCGCGGATACCAGCAAAAACACCGCGTATCTGCAGATG

[0638] AACAGCCTGCGCGCGGGAGATACCGCGGTGTATTATTGCGGCCGCGGCTATGGCTATTATGT

[0639] GTTTGATCATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGGGGTGGTGGTTCTGGT GGAGGGGGCTCAGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTA

[0640] CTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGA

[0641] TGCTCACATTTAAGTTTAAGATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGA

[0642] AGAAAAGCTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGA

[0643] CCCAGGGACTTAATCAGCAATATCAGAGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACA TTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCT

[0644] TTTGTCAAAGCATCATCTCAAGACTGACTTGA

Claims

Claims1. A modified interleukin-2 (IL2) polypeptide i. wherein the modified IL2 polypeptide is a modified human IL2 polypeptide and comprises the following amino acid substitutions with reference to SEQ ID NO: 1: a K at position at position 65 and a K at position 82, 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 96, and / or iii. an amino acid substitution at position 79.

2. The modified IL2 polypeptide of claim 1, 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 position 151, ii. an R position 110 and / or iii. an N at position 143 or wherein the modified murine IL2 polypeptide further comprises one or more of the following amino acid substitutions with reference to SEQ ID NO: 2: i. an amino acid substitution at position 166 and / or ii. an amino acid substitution at position 158.

3. The modified IL2 polypeptide of claim 2, wherein the amino acid substitution at position 151 is T151 R.

4. The modified IL2 polypeptide of claim 2 or 3, wherein the modified human IL2 polypeptide comprises an R at position 151 and an R at position 110.

5. The modified IL2 polypeptide of a preceding claim, 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 50 and / or 79; and / or ix. an amino acid substitution at one or more of position 79 and / or 96; and / or x. an amino acid substitution at one or more of position 79, 96 and / or 158; and / or xi. an amino acid substitution at one or more of position 79, 96 and / or 166.

6. The modified IL2 polypeptide of any one of claims 1 or 5, 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 50 and 79; and / or viii. an amino acid substitution at positions 79 and 96; and / or ix. an amino acid substitution at positions 79, 96 and 158; and / or x. an amino acid substitution at positions 79, 96 and 166.

7. The modified IL2 polypeptide of any one preceding claims 1 to 4, wherein the modified human IL2 polypeptide comprises an amino acid substitution selected from Y65K, E82K, N110R, T143N and / or T151 R or wherein the modified murine IL2 polypeptide comprises an amino acid substitution selected from Q50W, Y79K, E96K, A158N, and / or T166R.

8. The modified IL2 polypeptide of any of the preceding claims, 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, a K at position 82, an R at position 110, 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: vii. a K at position 79 and a K at position 96; and / or viii. a K at position 79, a K at position 96 and an N at position 158; and / or ix. a K at position 79, a K at position 96 and an R at position 166; and / or x. a W at position 50 and a K at position 79.

9. 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. Q50W and Y79K; ii. Q50W or iii. Y79K.

10. 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:

11. The modified IL2 polypeptide according to any one of the proceeding claims, wherein the substitution alters binding of IL2 to IL2RA, and / or reduces or prevents IL2 aggregation.

12. The modified IL2 polypeptide according to claim 11 , wherein the substitution that alters binding of IL2 to IL2RA reduces or abolishes binding of IL2 to IL2RA.

13. The modified IL2 polypeptide according to any one of the preceding claims, wherein the modified IL2 polypeptide comprises SEQ ID NO: 7 to 8 or a sequence with at least 90% sequence identity thereto; or SEQ ID NO: 14 to 17 or a sequence with at least 90% sequence identity thereto.

14. 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.

15. The modified IL2 polypeptide according to claim 14, 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.

16. The modified IL2 polypeptide according to claim 14, wherein the therapeutic molecule is an antibody or antigen fragment thereof that specifically binds to CD8, wherein the fragment is selected from a F(ab')2, Fab, Fv, scFv, heavy chain, light chain, variable heavy (VH), variable light (VL) chain, CDR region, single VH or VL domain, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, and bis-scFv.

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

18. A viral vector comprising the nucleic acid or nucleic acid construct according to claim 17.

19. The viral vector according to claim 18, 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-rh8, AAV-rh10, AAV-rh39, AAV-rh43, AAVAnc80, AAV 2 / ShH10, AAV-S vector, AAV6.2, Adv5, oncolytic vector or lentiviral vector.

20. An isolated immune cell, wherein the immune cell comprises a chimeric antigen receptor, wherein said cell expresses the modified IL2 polypeptide of any of claims 1 to 16 or the nucleic acid or nucleic acid construct of claim 17.

21. The isolated immune cell of claim 20 wherein the immune cell is a T cell or NK cell.

22. The isolated immune cell of claim 20 or 21 wherein the chimeric antigen receptor targets a tumor antigen.

23. A pharmaceutical composition comprising the modified IL2 polypeptide of any of claims 1 to 16, the nucleic acid nucleic acid or nucleic acid construct of claim 17, the viral vector of claim 18 or claim 19 or the isolated immune cell of claims 20 to 22.

24. The pharmaceutical composition according to claim 23 comprising the modified IL2 polypeptide of any of claims 1 to 16, the nucleic acid or nucleic acid construct of claim 17, the viral vector of claim 18 or claim 19, further comprising an immune cellexpressing a chimeric antigen receptor, optionally where the immune cell is a T cell of NK cell.

25. The modified IL2 polypeptide according to any one of claims 1 to 16, a nucleic acid or nucleic acid construct according to claim 17, a viral vector according to claims 17 to 19 or a pharmaceutical composition according to claim 23 or claim 24 for use in the treatment of disease.

26. The modified IL2 polypeptide according to any one of claims 1 to 16, the nucleic acid or nucleic acid construct according to claim 17, a viral vector according to claims 18 to 19 or a pharmaceutical composition according to claim 23 or 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 16, a nucleic acid or nucleic acid construct according to claim 17, a viral vector according to claims 18 to 19, or a pharmaceutical composition according to claim 25 or claim 26.

28. The modified IL2 polypeptide according to any one of claims 1 to 16, a nucleic acid or nucleic acid construct according to claim 17, a viral vector according to claims 18 to 19 or a pharmaceutical composition according to claim 23 or 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, glioblastoma, 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, nonHodgkin's, gastric cancer, and multiple myelomas.

29. The pharmaceutical composition according to 22 or 24 for a use according to claim 25 or 26 or a method according to claim 28, wherein said cancer is glioblastoma.

30. A kit comprising a modified IL2 polypeptide according to any one of claims 1 to 16, a nucleic acid or nucleic acid construct according to claim 17, a viral vector according to claims 18 to 19 or a pharmaceutical composition according to claim 23 or 24 optionally together with a reagent and / or instructions for use.

31. A host cell comprising a nucleic acid or nucleic acid construct according to claim 17 or a vector according to claim 18.

32. The host cell according to claim 31 wherein said host cell is a bacterial, yeast, viral or mammalian cell.

33. A method for producing a modified IL2 polypeptide according to any one of claims 1 to 16 comprising expressing a nucleic acid encoding the modified IL2 polypeptide in a host cell and isolating the modified IL2 polypeptide from the host cell.

34. A method for making an IL2 modified polypeptide that promotes CD8+ T cell responses comprising the step of introducing an amino acid substitution as defined in any one of claims 1 to 16 into an IL2 polypeptide.

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

36. A vector delivery system comprising a nucleic acid encoding an IL2 mutein according to any one of claims 1 to 16 or a nucleic acid or nucleic acid construct according to claim 17.

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

38. The vector delivery system according to claim 36, 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 vector or lentiviral vector.

39. 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 claims 1 to 16.

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

41. The method of claim 41 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. 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.

42. A combination comprising the modified IL2 polypeptide, viral vector or construct of any of claims 1 to 16 and an immune cell expressing a chimeric antigen receptor.

43. A combination therapy comprising administering the modified IL2 polypeptide according to any one of claims 1 to 16, a nucleic acid or nucleic acid construct according to claim 17, a viral vector according to claims 18 to 19 or a pharmaceutical composition according to claim 23 or 24 and an immune cell expressing a chimeric antigen receptor, wherein the immune cell expressing the chimeric antigen receptor cell is administered before, at the same time, or after the modified IL2 polypeptide, viral vector, nucleic acid or nucleic acid construct or pharmaceutical composition.

44. A method of increasing the expansion and persistence of T cell therapy, comprising the step of modifying a T cell to express the modified I L2 polypeptide of any of claims 1 to 16, the nucleic acid or nucleic acid construct of claim 17 or the viral vector of claim 18 or claim 19.

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