Treg targeted il-2 fusion proteins and therapeutic use thereof

Abstract

The present disclosure provides human IL-2 fusion proteins that selectively activate and / or expand regulatory T cells which comprise (i) an IL-2 polypeptide, which IL-2 polypeptide optionally is modified to attenuate IL-2R13 / yc binding, (ii) an antibody or antibody fragment which binds to an antigen other than CD25 expressed on Tregs, e.g., one of CTLA-4, TIGIT, GITR, ICOS, TGF beta receptor, TNFR-2, CD39, or CD73, and (iii) a half-life extending moiety, e.g., a serum albumin, an Fc or immunoglobulin constant region, or polyethylene glycol; wherein (i), (ii) and (iii) are comprised in any order from amino terminal to carboxy terminal in the human IL-2 fusion protein, and these moieties optionally are directly linked to each other and / or are indirectly linked to each other, further optionally via a linker peptide. The present disclosure also provides methods of therapy or prophylaxis using these human IL- 2 fusion proteins for treatment of a neuroinflammatory, autoimmune, or inflammatory condition or a pathologic symptom associated with any of said conditions.

Description

[0001] ATTORNEY DOCKET NO.1143252.008013 Treg targeted IL-2 Fusion Proteins and Therapeutic Use Thereof GRANTS

[0001] The instant application was made with government support under grant number 1R21AI174202 awarded by the National Institute of Allergy and Infectious Diseases. The government has certain rights in the invention. RELATED APPLICATIONS

[0002] The instant application claims priority to U.S. Prov. Appl. No.63 / 669,462, filed on July 10, 2024, the contents of which are incorporated by reference in their entirety. SEQUENCE LISTING DISCLOSURE

[0003] The contents of the electronic sequence listing (1143252o008013.xml; Size: 65,673 bytes; and Date of Creation: July 7, 2025) is herein incorporated by reference in its entirety. BACKGROUND

[0004] Interleukin-2 (IL2) was first identified as a T cell growth factor in 1976 (Malek. Annu Rev Immunol.2008;26:453-479. IL2 exerts its biological effects by binding and signaling through IL2 receptors (IL2Rs), which may be composed of different combinations of IL2RA (also known as IL2Rα or CD25), IL2RB (also known as IL2Rβ or common beta chain (βc), or CD122), and IL2RG (also known as common gamma chain (γc) or CD132) (Smith. Annu Rev Cell Biol.1989;5:397-425). IL2RA has a short intracellular tail and does not participate in the signaling process. Functional IL-2Rs may take a trimeric form IL2RA / B / G (also known as IL2Rα / β / γc) or a dimeric form IL2RB / G (also known as IL2Rβ / γc). Compared to the affinity of IL2RA to IL2 (about 10 nM), the dimeric IL2RB / G has higher affinity (about 1 nM), and the trimeric IL2RA / B / G has even higher affinity (about 10 pM) (Ross SH, Cantrell DA. Annu Rev Immunol.2018;36:411-433).

[0005] Because the trimeric IL2RA / B / G is constitutively and predominantly expressed on regulatory T (Treg) cells, native IL2 in general preferentially expands and activates Treg cells over other T cell subsets (Malek. Annu Rev Immunol.2008;26:453-479). Regulatory T (Treg) cells are a subset of CD4+ T cells that is involved in maintaining immune homeostasis and self-tolerance by inhibiting the pro- inflammatory activities of CD4+ and CD8+ effector T cells, natural killer cells, and antigen-presenting cells. Tregs hold great promise for the treatment of autoimmune and inflammatory diseases that affect between 5-10% of the population. Tregs constitutively express high level of CD25 (IL-2Ra) and thus have higher sensitivity to IL-2 than effector T cells (Teff). Low dose IL-2 and re-engineered CD25 dependent IL-2 that preferentially activate Treg while do not act on Teff have been widely employed to treat a variety of autoimmune and inflammatory diseases, but with limited clinical success thus ATTORNEY DOCKET NO.1143252.008013 far. One limitation of current CD25 (IL-2Ra) based targeting of Tregs is that CD25 (IL-2Ra) is also upregulated during Teff activation.

[0006] Also, the clinical use of IL2 is further challenged by its short half-life (about 7 minutes), which makes it difficult to circumvent toxicities (Lotze et al., J Immunol.1985 Oct;135(4):2865-75;Yang et al., J Clin Oncol.2003 Aug 15;21(16):3127-32.).

[0007] Therefore there is a need for improved IL2-based therapeutics and therapies using same. The present invention addresses this need. SUMMARY

[0008] Immune response is essential for protection from infection and malignancy, however unwanted self-antigen targeted or excessive uncontrolled immune response can lead to autoimmune and inflammatory diseases. These two kinds of diseases consist of more than 50 diseases that include rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Type 1 diabetes mellitus (T1DM), multiple sclerosis (MS), inflammatory bowel disease (IBD), dermatitis (eczema) and affect more than 5% of human population(1, 2).

[0009] Regulatory T cell (Treg) is a subpopulation of CD4 T cells that possess potent immuno- suppressive activity. By acting directly on effector T cells (Teff) or indirectly on antigen presenting cells (APC), Treg shuts down the excess immune response and thus holds great promise for the treatment of autoimmune and inflammatory diseases(3, 4).

[0010] As noted above, Tregs hold great promise for the treatment of autoimmune and inflammatory diseases that affect between 5-10% of the population. However, a limitation of current CD25 (IL-2Ra) based targeting of Tregs is that CD25 (IL-2Ra) is also upregulated during Teff activation. To better discriminate between Treg and activated pathogenic Teff, additional cell surface targets are needed.

[0011] Interleukin-2 (IL-2) was identified as T cell growth factor in 1976 and later it was found that IL-2 is essential for the genesis, survival, expansion, and activation of Treg(5). More importantly, since Tregs express a high affinity IL-2 receptor composed of IL-2Rα, IL-2Rβ and common gamma (γc), it has higher IL-2 sensitivity than resting Teff that only expresses medium affinity IL-2 receptor composed of IL-2Rβ and γc(6, 7).

[0012] Interleukin-2 (IL-2), either in low dose or re-engineered forms are being actively exploited to selectively expand regulatory T cells (Treg) for the treatment of autoimmune or inflammatory diseases. However, current IL-2Rα based Treg selectivity does not consider the well-established fact that IL-2Rα is also upregulated during effector T cell (Teff) activation. The shared expression of IL- ATTORNEY DOCKET NO.1143252.008013 2Rα on both Treg and activated pathogenic Teff makes IL-2Rα based discrimination unreliable and unpredictable, which helps to explain the underwhelming performance of current IL-2 in clinical trials.

[0013] The difference in intrinsic IL-2 sensitivity between Treg and Teff has led to the assumption that low dose IL-2 regimen will preferentially act on Treg(8). Although some promising signs was shown in early small clinical trials, more recent results showed that even low dose IL-2 can activate pathogenic T cells(9). To improve Treg selectivity, recent years have witnessed a trend to enhance IL- 2Rα dependence by attenuating its IL-2Rβ binding. Different protein engineering technologies such as mutagenesis, site-specific conjugation and antibody competing has been employed to block IL- 2Rβ binding. Different Treg selective IL-2 formulations have been tested in tens of clinical trials but without conclusive therapeutic benefits. Recently, Eli Lilly, Celgene, Amgen, Roche and Moderna have terminated the clinical development of their candidates, questioning the validity of current IL- 2Rα based Treg selectivity(10).

[0014] It has been well recognized that IL-2Rα is also upregulated during Teff activation(11). Such shared expression of IL-2Rα on both Teff and Treg diminishes its utility in discriminating these two populations and points out to the necessity of other more specific Treg targets.

[0015] To identify Treg surface targets potentially suitable for IL-2 delivery, five markers (CLTA-4, CD39, TNFR2, TIGIT, GITR) were selected based on their density, specificity, and functionality(12). IL- 2 was fused to targeting antibodies that bind to these five markers and the therapeutic efficacy of the putative Treg targeted IL-2 fusion protein was assessed in a humanized xenogeneic GvHD model. It was surprisingly found that the incorporation of a Treg targeting antibody fusion had profound impact on the activity of IL-2 and also demonstrated that CD39 targeting elicited significant delayed disease onset and survival extension as compared to other targeted IL-2’s.

[0016] In addition to CD25 (IL-2Ra), Tregs also highly express cell surface proteins including immune checkpoint inhibitors (CTLA-4, TIGIT), co-stimulatory receptors (GITR, ICOS), cytokine receptors (TGF beta receptor, TNFR-2) and enzymes (CD39, CD73). However, their actual performance in Treg targeted IL-2 delivery was far from certain absent experimental evidence in support of efficacy.

[0017] Therefore, the present invention is in part based on the inventors’ prediction that fusion of IL-2 to particular surface markers expressed on Treg will result in an IL-2 fusion which can discriminate it from Teff more effectively than IL-2Rα. Based on the density, specificity, and functionality of Treg surface markers, five candidates (CTLA-4, CD39, TNFR2, GITR, TIGIT) were chosen for targeted IL-2 delivery to Treg.

[0018] As disclosed infra, the inventors fused IL-2 to antibodies which target specific antigens expressed on Tregs including anti-CTLA-4, TIGIT, GITR, TNFR2 and CD39 antibodies and the in vivo ATTORNEY DOCKET NO.1143252.008013 efficacy of the resultant fusion proteins was assessed in a xenogeneic GVHD model, in which PBMCs were engrafted into NSG mice. Surprisingly it was discovered that these IL-2 fusion proteins much more effectively ameliorated GvHD disease as compared to untargeted IL-2, apparently because they more effectively promote the activation and / or expansion of regulatory T cells compared to wild-type IL-2 and other known IL-2 fusion proteins.

[0019] Accordingly, based on the foregoing, the present disclosure relates to IL-2 fusion polypeptides that more effectively activate and / or expand regulatory T cells (compared to wild-type IL-2) which comprise (i) an IL-2 polypeptide, which IL-2 polypeptide optionally is modified to attenuate IL-2Rβ / γc binding; (ii) an antibody or antibody fragment which binds to an antigen other than CD25 expressed on Tregs; and (iii) at least one half-life extending moiety;

[0020] wherein (i), (ii) and (iii) are comprised in any order from amino terminal to carboxy terminal in the human IL-2 fusion protein, and these moieties optionally are directly linked to each other and / or are indirectly linked to each other, further optionally via a linker peptide.

[0021] In exemplary embodiments the moieties (i), (ii) and (iii) in the IL-2 fusion polypeptide are oriented from N to C terminal as follows: (i) IL-2 polypeptide—(ii) antibody or antibody fragment which binds to an antigen other than CD25 expressed on Tregs—(iii) half-life extending moiety, optionally human serum albumin (HSA) or a human Ig Fc region.

[0022] In exemplary embodiments the (ii) the antibody or antibody fragment in the human IL-2 fusion protein binds to an immune checkpoint inhibitor, co-stimulatory receptor, cytokine receptor or an enzyme expressed on human Tregs, optionally selected from CTLA-4, TIGIT, GITR, ICOS, TGF beta receptor, TNFR-2, CD39, or CD73, and further optionally (ii) the antibody or antibody fragment binds to CD39.

[0023] In some exemplary embodiments the (iii) half-life extending moiety in the human IL-2 fusion protein is selected from a serum albumin, an Fc or immunoglobulin constant region, an unstructured polypeptide, an adnectin, transferrin, CTP (28 amino acid C-terminal peptide (CTP) of hCG with its 4 O-glycans), or a fragment of any of the foregoing; a lipid, O- and N-glycosyl residues, a carbohydrate polymer, cholesterol, poly(ethylene glycol) (PEG), monomethoxy PEG (mPEG), a branched or unbranched acyl group, a branched or unbranched C8-C30 acyl group, a branched or unbranched alkyl group, and a branched or unbranched C8-C30 alkyl group. ATTORNEY DOCKET NO.1143252.008013

[0024] In some exemplary embodiments the (iii) half-life extending moiety in the human IL-2 fusion protein is a serum albumin, optionally human serum albumin, or a human Fc region, which optionally contains a mutation which ablates or reduces at least one effector function such as glycosylation, FcR binding or complement binding and / or a mutation which enhances FcRN binding.

[0025] In some exemplary embodiments the (ii) antibody or antibody fragment in the human IL-2 fusion protein is a scFv, a Fab, F(ab')2or VHH antibody.

[0026] In some exemplary embodiments the (iii) half-life extending moiety in the human IL-2 fusion protein comprises one or more polyethylene glycol(s).

[0027] In some exemplary embodiments the (iii) half-life extending moiety in the human IL-2 fusion protein comprises one or more polyethylene glycols which optionally are attached to one or more residues in the human IL-2 fusion further optionally by the incorporation and attachment to a non- encoded amino acid.

[0028] In some exemplary embodiments the human IL-2 fusion protein comprises a human IL-2 mutein that comprises at least one mutation which reduces IL-2Rβ / γc binding.

[0029] In some exemplary embodiments the human IL-2 fusion protein comprises a human IL-2 mutein that comprises at least one mutation at a residue selected from residue 16, 20, 88, 91, 126 optionally selected from N88D, N88R, H16L, D20T, V91R, and Q126T.

[0030] In some exemplary embodiments the human IL-2 fusion protein comprises a human IL-2 mutein that comprises a from N88D mutation.

[0031] In some exemplary embodiments the invention provides pharmaceutical compositions comprising a human IL-2 fusion protein according to any of the foregoing, and a pharmaceutically acceptable carrier.

[0032] In some exemplary embodiments the invention provides a nucleic acid which encodes a human IL-2 fusion protein according to any of the foregoing or an expression vector containing said nucleic acid.

[0033] In some exemplary embodiments the invention provides a recombinant cell which expresses a nucleic acid which encodes a human IL-2 fusion protein according to any of the foregoing or an expression vector containing said nucleic acid.

[0034] In some exemplary embodiments the invention provides a recombinant cell which expresses a nucleic acid which encodes a human IL-2 fusion protein according to any of the foregoing or an expression vector containing said nucleic acid, which optionally is an immune cell further optionally selected from PMBCs, a T cell, a T cell progenitor cell, a CD4+ T cell, a helper T cell, a regulatory T cell, a CD8+ T cell, a naïve T cell, an effector T cell, a memory T cell, a stem cell memory T (TSCM) cell, a central memory T (TCM) cell, an effector memory T (TEM) cell, a terminally differentiated ATTORNEY DOCKET NO.1143252.008013 effector memory T cell, a tumor-infiltrating lymphocyte (TIL), an immature T cell, a mature T cell, a cytotoxic T cell, a mucosa-associated invariant T (MAIT) cell, a TH1 cell, a TH2 cell, a TH3 cell, a TH17 cell, a TH9 cell, a TH22 cell, a follicular helper T cells, and a / b T cell, a g / d T cell, a Natural Killer T (NKT) cell, a cytokine-induced killer (CIK) cell, a lymphokine-activated killer (LAK) cell, a perforin- deficient cell, a granzyme-deficient cell, a B cell, a myeloid cell, a monocyte, a macrophage, an eosinophil, a neutrophil, and a dendritic cell, further optionally a Treg.

[0035] In some exemplary embodiments the invention provides a lipid nanoparticle comprising a human IL-2 fusion protein according to any of the foregoing or a nucleic acid encoding.

[0036] In some exemplary embodiments the invention provides a pharmaceutical composition comprising a lipid nanoparticle comprising a human IL-2 fusion protein according to any of the foregoing or a nucleic acid encoding, and a pharmaceutically acceptable carrier or excipient.

[0037] In some exemplary embodiments the invention provides a method of therapy or prophylaxis, which comprises the administration of a human IL-2 fusion protein according to any of the foregoing, or a composition containing, to a subject in need thereof.

[0038] In some exemplary embodiments the invention provides a method of therapy or prophylaxis, which comprises the administration of a cell which expresses a human IL-2 fusion protein according to any of the foregoing, or a composition containing, to a subject in need thereof.

[0039] In some exemplary embodiments the invention provides a method of therapy or prophylaxis, which comprises the administration of a nanoparticle which comprises a human IL-2 fusion protein according to any of the foregoing, or a nucleic acid encoding, or a lipid nanoparticle containing to a subject in need thereof.

[0040] In some exemplary embodiments the method of therapy or prophylaxis, is for treatment of a neuroinflammatory, autoimmune, or inflammatory condition or a pathologic symptom associated with any of said conditions.

[0041] In some exemplary embodiments the method of therapy or prophylaxis, is for the treatment or prevention of an autoimmune disease selected from one or more of Acromegaly, Acquired aplastic anemia, Acquired hemophilia, Agammaglobulinemia, primary, Alopecia areata, Ankylosing spondylitis (AS), Anti-NMDA receptor encephalitis, Antiphospholipid syndrome (APS) | catastrophic antiphospholipid syndrome (CAPS) / Asherson's syndrome, Arteriosclerosis, Autoimmune Addison’s disease (AAD), Autoimmune autonomic ganglionopathy (AAG) / autoimmune dysautonomia | autoimmune gastrointestinal dysmotility (AGID), Autoimmune encephalitis | acute disseminated encephalomyelitis (ADEM), Autoimmune gastritis, Autoimmune hemolytic anemia (AIHA), Autoimmune hepatitis (AIH), Autoimmune hyperlipidemia, Autoimmune hypophysitis, Autoimmune inner ear disease (AIED), Autoimmune lymphoproliferative syndrome (ALPS), Autoimmune ATTORNEY DOCKET NO.1143252.008013 myelofibrosis, Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune pancreatitis (AIP), Autoimmune polyglandular syndromes, types I, II, & III (APS type 1, APS type 2, APS type 3, APECED), Autoimmune progesterone dermatitis, Autoimmune retinopathy (AIR), Autoimmune sudden sensorineural hearing loss (SNHL), Balo disease, Behçet’s disease, Birdshot chorioretinopathy / birdshot uveitis, Bullous pemphigoid, Castleman disease, Celiac disease, Chagas disease, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic urticaria (CU), Churg-Strauss syndrome / eosinophilic granulomatosis with polyangiitis (EGPA), Cogan’s syndrome, Cold agglutinin disease, CREST syndrome | limited cutaneous systemic sclerosis, Crohn’s disease (CD), Cronkhite-Canada syndrome (CSS), Cryptogenic organizing pneumonia (COP), Dermatitis herpetiformis, Dermatomyositis, Diabetes, type 1, Discoid lupus, Dressler’s syndrome / postmyocardial infarction / postpericardiotomy syndrome, Eczema / Atopic Dermatitis, Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum, Essential mixed cryoglobulinemia, Evans syndrome, Fibrosing alveolitis / Idiopathic pulmonary fibrosis (IPF), Giant cell arteritis / temporal arteritis / Horton’s disease, Giant Cell Myocarditis, Glomerulonephritis, Goodpasture’s syndrome / anti- GBM / anti-TBM disease, Granulomatosis with polyangiitis (GPA) / Wegener’s granulomatosis, Grave’s disease / thyroid eye disease, Guillain-Barré syndrome (GBS), Hashimoto’s thyroiditis / chronic lymphocytic thyroiditis / autoimmune thyroiditis, Henoch-Schönlein purpura / IgA vasculitis, Hidradenitis suppurativa, Hurst’s disease / acute hemorrhagic leukoencephalitis (AHLE), Hypogammaglobulinemia, IgA nephropathy / Berger's disease, Immune-mediated necrotizing myopathy (IMNM), Immune thrombocytopenia (ITP) / autoimmune thrombocytopenic purpura / autoimmune thrombocytopenia, Inclusion body myositis, IgG4-related sclerosing disease (ISD), Interstitial cystitis, Juvenile idiopathic arthritis / Adult-onset Still's disease. Juvenile polymyositis | Juvenile dermatomyositis | juvenile myositis, Kawasaki disease, Lambert-Eaton myasthenic syndrome (LEMS), Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD) | linear IgA bullous dermatosis (LABD), Lupus nephritis, Lyme disease / chronic Lyme disease / post-treatment Lyme disease syndrome (PTLDS), Lymphocytic colitis / microscopic colitis, Lymphocytic hypophystitis / autoimmune hypophystitis, Ménière’s disease, Microscopic polyangiitis (MPA) / ANCA-associated vasculitis, Mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease, Multifocal motor neuropathy, Multiple sclerosis (MS), Myalgic encephalomyelitis (ME) / Chronic fatigue syndrome (CFS), Myasthenia gravis (MG), Narcolepsy, Neuromyelitis Optica / Devic's disease, Ocular cicatricial pemphigoid, Opsoclonus- myoclonus syndrome (OMS), Palindromic rheumatism, Paraneoplastic cerebellar degeneration, Paraneoplastic pemphigus, Parry-Romberg syndrome (PRS) / Hemifacial atrophy (HFA) / Progressive facial hemiatrophy, Paroxysmal nocturnal hemoglobinuria (PNH), Peripheral uveitis / pars planitis, ATTORNEY DOCKET NO.1143252.008013 PANS / PANDAS, Parsonage-Turner syndrome, Pemphigus gestationis / herpes gestationis, Pemphigus foliaceus, Pemphigus vulgaris, Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Polymyalgia rheumatica, Polymyositis, Postural orthostatic tachycardia syndrome (POTS), Primary biliary cirrhosis (PBC) / primary biliary cholangitis, Primary sclerosing cholangitis (PSC), Psoriasis, Palmoplantar Pustulosis, Psoriatic arthritis, Pulmonary fibrosis, idiopathic (IPF), Pure red cell aplasia (PRCA), Pyoderma gangrenosum, Rasmussen's encephalitis, Raynaud’s syndrome / phenomenon, Reactive arthritis / Reiter’s syndrome, Reflex sympathetic dystrophy syndrome (RSD) / Complex regional pain syndrome (CRPS), Relapsing polychondritis, Restless leg syndrome (RLS) / Willis-Ekbom disease, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidt syndrome / autoimmune polyendocrine syndrome type II, Scleritis, Scleroderma, Sclerosing Mesenteritis / Mesenteric Panniculitis, Serpiginous choroidopathy, Sjögren’s syndrome, Stiff person syndrome (SPS), Small fiber sensory neuropathy, Systemic lupus erythematosus (SLE), Subacute bacterial endocarditis (SBE), Subacute cutaneous lupus, Susac syndrome, Sydenham's chorea, Sympathetic ophthalmia, Takayasu’s arteritis (vasculitis), Testicular autoimmunity (vasculitis, orchitis), Tolosa-Hunt syndrome, Transverse myelitis (TM), Tubulointerstitial nephritis uveitis syndrome (TINU), Ulcerative colitis (UC), Undifferentiated connective tissue disease (UCTD), Uveitis | anterior / intermediate / posterior, Vasculitis, VEXAS Syndrome, Vitiligo, and Vogt-Koyanagi-Harada syndrome (VKH), and / or to prevent or inhibit at least one pathologic symptom associated therewith.

[0042] In some exemplary embodiments the method of therapy or prophylaxis, is for the treatment or prevention of Addison disease, arthritis, celiac disease, lupus, Grave’s disease, myasthenia gravis, multiple sclerosis, ITP, rheumatoid arthritis, colitis, inflammatory bowel disease, pernicious anemia, Hashimoto’s thyroiditis Sjogren’s disease, asthma, type 2 diabetes, and autoimmune type I diabetes and / or to prevent or inhibit at least one pathologic symptom associated therewith.

[0043] In some exemplary embodiments the method of therapy or prophylaxis, is for the treatment or prevention of an inflammatory disease selected from the group consisting of Fatty liver disease, Endometriosis, Type 2 diabetes, mellitus, Type 1 diabetes mellitus, Inflammatory bowel disease (IBD), Asthma, Rheumatoid arthritis, asthma, Obesity, Fibromyalgia, Lupus SLE, osteoarthritis, Rheumatoid Arthritis, Shingles Herpes Zoster, and Vasculitis and / or to prevent or inhibit at least one pathologic symptom associated therewith.

[0044] In some exemplary embodiments the method of therapy or prophylaxis, is for the treatment or prevention of a neurodegenerative or neuroinflammatory disease, e.g., Alzheimer's disease, Amyotrophic lateral sclerosis, Friedreich ataxia, Huntington's disease, Lewy body disease, aphasia, Parkinson's disease or Spinal muscular atrophy and / or to prevent or inhibit at least one pathologic symptom associated therewith. ATTORNEY DOCKET NO.1143252.008013

[0045] In some exemplary embodiments the method of therapy or prophylaxis of any one of the foregoing further comprises the administration of an anti-inflammatory agent, optionally a small molecule, fusion protein or an antibody and / or an agent used to treat autoimmunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIG.1 contains a schematic structure of HSA fused wild type and variant IL-2 and SDS-PAGE analysis of the purified proteins.

[0047] FIG.2 contains ELISA receptor binding analysis results of experiments comparing the binding affinity of WT IL-2 and IL-2(N88D) to IL-2Rα and IL-2R. As is shown in the figure these two proteins showed identical IL-2Rα binding, however the IL-2(N88D) showed diminished IL-2Rβ binding.

[0048] FIG.3 provides contains the results of cellular signaling assays showing that WT IL-2 can induce pSTAT5 formation in both Treg and CD8 T cells (as IL-2 signaling leads to STAT5 phosphorylation). In these experiments pSTAT5 content in different population of immune cells were detected by flow cytometry. As can be seen from the data in the figure IL-2(N88D) retained the ability to act on Tregs but was unable to trigger signaling in CD8 T cells.

[0049] FIG.4 contains the results of experiments evaluating the effects of WT IL-2 and IL-2(N88D) in a GVHD model. After the engrafting of human PBMC into immune-deficient NSG mice, human CD8 T will recognize and attack murine cells, leading to autoimmune-like and highly inflammatory GvHD. As can be seen from the results in the figure the disease progressed rapidly for both the group administered WT IL-2 and the group administered the IL-2 mutein (IL-2(N88D)). The medium survival time (MST) of WT IL-2 and IL-2(N88D) are 10.5d and 11.25d, respectively, indicating that neither the WT IL-2 and IL-2(N88D) was efficacious in this autoimmune / inflammatory disease model.

[0050] FIG.5 shows the schematic structure of an exemplary CTLA-4 targeted IL-2(N88D) and its SDS-PAGE analysis. Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) is a protein receptor preferentially expressed on the surface of Tregs, that acts as an immune checkpoint and which inhibits the activation of T cells by competing with the stimulatory receptor CD28 for binding to CD80 / CD86 on antigen-presenting cells (APCs)(17, 18). CTLA-4-targeted antibodies, such as ipilimumab selectively depletes Treg by the ADCC effector function of Fc and has been approved for the treatment of melanoma(19). As is shown in the figure, anti-CTLA-4 scFv from ipilimumab was fused in tandem with IL-2(N88D) to produce an IL-2 fusion (anti-CTLA-4-IL-2 (N88D)) that potentially may be used to deliver IL-2 to CTLA-4 expressing Tregs.

[0051] FIG.6 contains the results of receptor and target binding assays which compared Protein- protein interactions of IL-2(N88D) by ELISA. As can be seen from the results anti-CTLA-4 scFv confers ATTORNEY DOCKET NO.1143252.008013 high CTLA-4 binding to the anti-CTLA-4-IL-2 (N88D) fusion, while only slightly decreasing IL-2Rα binding.

[0052] FIG.7 contains the results of cellular signaling assays comparing the effects of the anti-CTLA- 4-IL-2 (N88D) fusion and IL-2(N88D) on Treg activity. AS can be seen from the figure the anti-CTLA-4- IL-2 (N88D) fusion much more potently enhanced Treg activity in relation to IL-2(N88D).

[0053] FIG.8 compares the efficacy of the anti-CTLA-4-IL-2 (N88D) fusion and IL-2(N88D) in xenogeneic GvHD model. As can be seen from the figure, the anti-CTLA-4-IL-2 (N88D) fusion significantly delayed the onset of GvHD and extended the survival of NSG mice compared to the untargeted IL-2 IL-2(N88D) (12d vs 22.5d).

[0054] FIG.9 shows the schematic structure of TNFR2, CD39, GITR and TIGIT targeted IL-2 and SDS- PAGE analysis of purified proteins. Essentially, to facilitate targeted delivery of IL-2 to CD39(25), TNFR2(26), GITR(27) and TIGIT(28) expressing Tregs, VHH antibodies were fused in tandem with IL- 2(N88D).

[0055] FIG.10 compares the efficacy of different Treg targeted IL-2 fusions in a humanized xenogeneic GvHD model. As can be seen from the figure, the efficacy of these different targeted IL-2 fusions differed greatly in this humanized xenogeneic GvHD model. As is shown the MST for GITR, TIGIT, TNFR2 and CD39 targeted IL-2 are 16d, 41.75d, 37.5d and 51d, respectively. Accordingly, CD39 targeting showed the most promising delayed disease onset and survival extension. DETAILED DESCRIPTION Definitions

[0056] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0057] Although various embodiments and examples of the present invention have been described referring to certain molecules, compositions, methods, or protocols, it is to be understood that the present invention is not limited to the particular molecules, compositions, methods, or protocols described herein, as theses may vary. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0058] It should be understood that, unless clearly indicated otherwise, in any methods disclosed or claimed herein that comprise more than one step, the order of the steps to be performed is not restricted by the order of the steps cited. ATTORNEY DOCKET NO.1143252.008013

[0059] Throughout this disclosure, numerical features are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiments. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range to the tenth of the unit of the lower limit unless the context clearly dictates otherwise. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual values within that range, for example, 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, unless the context clearly dictates otherwise.

[0060] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0061] It must also be noted that, unless the context clearly dictates otherwise, the singular forms “a,” “an,” and “the” as used herein and in the appended claims include plural refence. Thus, the reference to “a cell” refers to one or more cells and equivalents thereof known to those skilled in the art, and so forth. Similarly, the reference to “a nucleic acid” refers to one or more nucleic acid molecules and equivalents thereof known to those skilled in the art, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by a person of skilled in the art.

[0062] As used herein, the term “about” or “approximately” when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 10%. For example, as used herein, the expression “about 100” includes 90 and 110 and all values in between (e.g., 91, 92, 93, 99, 99.1, 99.2, 99.3, 99.4, 100, 100.8, 100.9, 101, 106, 107, 108, 109, etc.).

[0063] It is understood that aspects and embodiments of the disclosure described herein include “comprising,” “consisting,” and “consisting essentially of” aspects and embodiments. Transitional phrases such as “comprising,” “including,” “having,” “containing,” “involving,” “composed of,” and the like are to be understood to be open-ended, namely, to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively. ATTORNEY DOCKET NO.1143252.008013

[0064] It will be further understood that all transitional terms such as “comprises,” “comprising,” “including,” “having,” “containing,” “involving,” “composed of,” and the like, when used in this specification, are to be understood to be open-ended, namely, to specify the presence of stated features, integers, steps, operations, elements, and / or components, but not to preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Only the transitional phrases “consists of” or “consisting of” shall be closed transitional phrases. The semi-closed transitional phrase “consists essentially of” or “consisting essentially of” shall be understood to specify the presence of stated features, integers, steps, operations, elements, and / or components and to allow the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof as long as they do not materially affect the basic characteristics of stated features, integers, steps, operations, elements, and / or components. For example, in some embodiments, “cells consisting essentially of T cells” may encompass a population of cells about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of which are T cells.

[0065] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0066] A standard form of “antibody” or “immunoglobulin (Ig)” molecules may comprise two heavy chains and two immunoglobulin light chains or multiple units each comprising two pairs of heavy and light chains interconnected by disulfide bonds. Antibodies may be of one of the five major classes, IgA, IgD, IgE, IgG, and IgM and may be further classified based on the subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Each heavy chain is comprised of: a heavy chain variable domain (VH); and a heavy chain constant region (CH), which is typically comprised of a CH1 domain, a hinge, a CH2 domain and a CH3 domain.

[0067] The numbering of amino acid residues in the antibody constant region may be performed by the EU-index or EU numbering system, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). The EU numbering system is used in the present specification unless otherwise specified.

[0068] According to IMGT (the international ImMunoGeneTics information system for immunoglobulins or antibodies, T cell receptors, MH, immunoglobulin superfamily IgSF and MhSF), the CH1 domain is the amino acid positions (or simply referred to as “positions” herein) 118-215 (EU numbering) and the hinge region is the amino acid positions 216-230 (EU numbering). The term “CH1 domain” is used in a broad sense herein to encompass any naturally occurring, corresponding heavy chain constant domain and / or region allotypes and variants thereof, which may comprise ATTORNEY DOCKET NO.1143252.008013 fewer or more amino acids and / or amino acid modification(s) and to refer to a heavy chain region comprising at least 80% of the heavy chain positions 118-215 (EU numbering)) and in some instances also comprising a portion of the hinge region (a portion of heavy chain positions 216-230 (EU numbering)) is included (e.g., up to position 218 or 220). Human IgG1 CH1 domain reference sequences are provided herein as SEQ ID NOS: 61 and 62. A human IgG4 CH1 domain reference sequence is provided herein as SEQ ID NO: 71. These CH1 domain reference sequences are intended to be exemplary as Applicant intends for “CH1 domain” sequences to include any naturally occurring CH1 domain allotype or allelic variant.

[0069] According to IMGT, the hinge is the amino acid positions 216-230 (EU numbering). The term “hinge” is used in a broad sense herein to refer to a heavy chain region comprising at least at least 60% of the hinge positions 216-230 (EU numbering). Human IgG1 and IgG4 hinge reference sequences, corresponding to the amino acid positions 231-340 according to EU numbering, are provided herein as SEQ ID NOS: 63 and 73, respectively, which are exemplary amino acid sequence of a wildtype (WT) hinge. These hinge reference sequences are intended to be exemplary as Applicant intends for “hinge” sequences to include any naturally occurring hinge allotype or allelic variant.

[0070] According to IMGT, the CH2 domain is the amino acid positions (or simply referred to as “positions” herein) 231-340 (EU numbering). The term “CH2 domain” is used in a broad sense herein to refer to a heavy chain region comprising at least at least 80% of the heavy chain positions 231-340 (EU numbering)). Human IgG1 and IgG4 CH2 domain reference sequences, corresponding to the amino acid positions 231-340 according to EU numbering, are provided herein as SEQ ID NOS: 64 and 74, respectively, which are exemplary amino acid sequences of a wild-type (WT) CH2 domain. These CH2 domain reference sequences are intended to be exemplary as Applicant intends for “CH2 domain” sequences to include any naturally occurring CH2 domain allotype or allelic variant.

[0071] According to IMGT, the CH3 domain is the amino acid positions (or simply referred to as “positions” herein) 341-446 (EU numbering). The term “CH3 domain” is used in a broad sense herein to refer to a heavy chain region comprising at least seven consecutive amino acid positions of the heavy chain positions 341-446 (EU numbering)). Human IgG1 CH3 domain reference sequences are provided herein as SEQ ID NOS: 65-68. A human IgG4 CH1 domain reference sequence is provided herein as SEQ ID NO: 75. These CH3 domain reference sequences are intended to be exemplary as Applicant intends for “CH3 domain” sequences to include any naturally occurring CH3 domain allotype or allelic variant. In some embodiments, a CH3 domain may further comprise lysine at the C terminus (i.e., K447, according to EU numbering). ATTORNEY DOCKET NO.1143252.008013

[0072] A “Fc region” is a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region, including native sequence Fc regions and variant Fc regions. A human IgG heavy chain Fc region can extend from Asp221, Thr223, or Thr225 (in case of IgG1) or Tyr (one residue after K218, in case of IgG4), to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0073] The phrase “effector function” of an antibody refers to biological activities attributable to the Fc region of an antibody, which varies by antibody class or isotype. Exemplary effector functions include: complement (e.g., C1q) binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0074] A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. (See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307- 331). Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic- hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine. In some embodiments, conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, in some embodiments, a conservative replacement comprises any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 144345. In some embodiments, a “moderately conservative” replacement comprises any change having a nonnegative value in a PAM250 log- likelihood matrix.

[0075] The term “cytokines” as used herein refers to a broad category of small proteins that are involved in cell signaling. “Chemokines” are a family of cytokines generally involved in mediating ATTORNEY DOCKET NO.1143252.008013 chemotaxis. Generally, their release has some effect on the behavior of cells around them. Cytokines may be involved in autocrine signaling, paracrine signaling, and / or endocrine signaling as immunomodulating agents. Cytokines may include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Cytokines are produced by a broad range of cells, including but not limited to immune cells like macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts, epithelial cells, and various stromal cells and in particular some cytokines are expressed by Tregs.

[0076] The phrase “targeted IL-2 fusion” or “Il-2 fusion polypeptide” as used herein generally refers to a fusion protein comprising (i) an IL-2 polypeptide, typically a human IL-2 polypeptide, which IL-2 polypeptide preferably comprises at least on mutation which attenuates IL-2Rβ binding and / or enhances IL-2Rα binding, (ii) an antibody or antibody fragment which binds to an antigen other than CD25 expressed on Tregs, and optionally (iii) a half-life extending moiety, further optionally a serum albumin, an Fc or immunoglobulin constant region, an unstructured polypeptide, an adnectin, transferrin, CTP (28 amino acid C-terminal peptide (CTP) of hCG with its 4 O-glycans), or a fragment of any of the foregoing; a lipid, O- and N-glycosyl residues, a carbohydrate polymer, cholesterol, poly(ethylene glycol) (PEG), monomethoxy PEG (mPEG), a branched or unbranched acyl group, a branched or unbranched C8-C30 acyl group, a branched or unbranched alkyl group, and a branched or unbranched C8-C30 alkyl group.

[0077] The term "EC50" refers to the "half maximal effective concentration", which value measures the effectiveness of compound (e.g. an IL-2 fusion protein) towards a biological or biochemical utility. This quantitative measure indicates the quantity or concentration required for a particular compound to elicit a given biological process to half of the maximal response.

[0078] An “effective amount” of a cell disclosed herein or a composition (e.g., pharmaceutical composition) described herein, is at least the minimum amount required to achieve the desired therapeutic or prophylactic result, e.g., a measurable improvement (e.g., in a symptom, severity, grade, or progression) of or prevention of a particular disease, disorder, or condition, e.g., an acute or chronic autoimmune or inflammatory disorder. An effective amount may vary according to inter alia disease state, age, sex, and weight of the patient, and the ability of the active ingredient (e.g. cell) to elicit a desired response in the individual and, in some instances, by co-administering one or more additional therapeutic agents. For therapeutic use, beneficial or desired results include clinical results such as decreasing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication such as via targeting, delaying the progression of the disease, and / or prolonging survival. In the case of an autoimmune or ATTORNEY DOCKET NO.1143252.008013 inflammatory disorder, an effective amount of the drug may have the effect of reducing autoantibodies; reducing CD4+ and / or CD8+ T cell activation; reducing CD4+ and / or CD8+ T cell proliferation; reducing inflammation; reducing the levels of proinflammatory cytokines; and / or relieving to some extent one or more of the symptoms associated with the disorder. An effective amount can be administered in one or more administrations. As is understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “effective amount” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved. An “effective amount” may be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0079] The terms "enteral," "enterally," “oral,” "orally," "non-parenteral," "non- parenterally," and the like, refer to administration of a compound or composition to an individual by a route or mode along the alimentary canal. Examples of "oral" routes of administration of a composition include, without limitation, swallowing liquid or solid forms of a composition from the mouth, administration of a composition through a nasojejunal or gastrostomy tube, intraduodenal administration of a composition, and rectal administration, e.g., using suppositories for the lower intestinal tract of the alimentary canal.

[0080] The term “format” as used herein in relation to a particular “targeted IL-2 fusion” or “IL-2 fusion polypeptide” refers to the structure and orientation of the different polypeptides in the targeted IL-2 fusion, i.e., the (i) IL-2 polypeptide, (ii) an antibody or antibody fragment which binds to an antigen other than CD25 expressed on Tregs, and (iii) optionally a half-life extending moiety, compared to a referenced targeted IL-2 fusion structure containing the same moieties, i.e., the domains of the targeted IL-2 fusion, i.e., the (i) IL-2 polypeptide, (ii) an antibody or antibody fragment which binds to an antigen other than CD25 expressed on Tregs, and (iii) optionally a half- life extending moiety, are placed in essentially the same orientation as in the referenced targeted IL- 2 fusion protein structure. This targeted IL-2 fusion polypeptide may additionally comprise other moieties such as one or more linkers, signal peptides, tags (e.g., 6His tag, FLAG tag, etc) may be added or removed as appropriate. For example, in an exemplary “Format” a targeted IL-2 fusion may comprise a first polypeptide comprising an IL2 domain, an anti-CD39 antibody or scFv, and a half-life extending moiety such as a serum albumin in the direction from the N-terminus to the C-terminus optionally directly linked or attached via linker peptides. Each domain may or may not contain ATTORNEY DOCKET NO.1143252.008013 modifications (e.g., amino acid sequence changes, glycosylation, etc.) relative to the corresponding domain or part of a wild-type sequence.

[0081] “Human albumin”, also known as “HSA”, the most abundant protein in human blood. HSA regulates blood plasma colloid osmotic pressure and acts as a carrier protein for a wide range of endogenous molecules including hormones, fatty acids, and metabolites, as well as exogenous drugs. HSA is often used in protein- or polypeptide-based therapeutics for its role in increasing in vivo half-life. In humans, albumin is encoded by the albumin (gene symbol ALB) gene on chromosome 4, with gene location 4q13.3 (NCBI). In some embodiments, HSA may for example have the canonical amino acid sequence of SEQ ID NO: XX (according to NCBI Reference Sequence: NP_000468.1 (without the signal peptide and propeptide)). In some embodiments, HSA may encompass another HSA sequence or a fragment thereof, another HSA isoform, and / or and their variants comprising the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like.

[0082] “IL2”, also known as “interleukin 2”, “IL-2”, “TCGF”, or “lymphokine”, is a member of the IL2 cytokine subfamily which includes IL4, IL7, IL9, IL15, IL21, erythropoietin, and thrombopoietin. IL2 may be produced by various immune cells including activated CD4+ and CD8+ T cells and is important for the proliferation of T and B cells. In humans, IL2 is encoded by the interleukin 2 (gene symbol IL2) gene on chromosome 4, with gene location 4q27 (NCBI). In some embodiments, human IL2 may for example have the canonical amino acid sequence of SEQ ID NO: 1 (according to NCBI Reference Sequence: NP_000577.2 (without the signal peptide)). In some embodiments, human IL2 may encompass such a human IL2 sequence, another IL2 isoform, and / or and their variants comprising the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like. In preferred embodiments the IL-2 contains at least one modification which attenuates IL-2Rβ binding and / or enhances IL-2Rα binding. In some embodiments the Il-2 may comprise a human IL-2 mutein that comprises at least one mutation at a residue selected from residue 16, 69, 74, 88, 103, and 106, optionally selected from N88D, N103R, V106D, V69A, Q74P, and H16L; and further optionally comprises a N88D mutation.

[0083] “IL2RA”, also known as “interleukin 2 receptor subunit alpha”, “CD25”, “IL2Rα”, “IL-2RA”, “IL-2Rα”, “p55”, “IMD41”, “TCGFR”, or “IDDM10”, binds to IL2 with low affinity (with KDabout 10 nM) but, together with the common beta (βc) receptor subunit (also known as “IL2RB”, “IL2Rβ”, or CD122) and the common gamma chain (γc) receptor subunit (also known as “IL2RG”, “IL2Rγ” or CD132), constitutes the high-affinity (with KDof about 10 pM), trimeric IL2A / B / G receptor. In humans, IL2RA is encoded by the interleukin 2 receptor subunit alpha (gene symbol IL2RA) gene on chromosome 10, with gene location 10p15.1 (NCBI). In some embodiments, human IL2RA may for ATTORNEY DOCKET NO.1143252.008013 example have the canonical amino acid sequence according to NCBI Reference Sequence: NP_000408.1 (without the signal peptide)), in which positions 1-219 correspond to the extracellular domain, positions 220-238 correspond to the transmembrane domain, and positions 239-251 correspond to the intracellular domain. In some embodiments, human IL2RA may encompass such a human IL2RA sequence, another IL2RA isoform, and / or and their variants comprising the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like.

[0084] An “isolated” biological component (such as an isolated protein, nucleic acid, vector, or cell) refers to a component that has been substantially separated or purified away from its environment or other biological components in the cell of the organism in which the component naturally occurs, for instance, other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles. Nucleic acids and proteins may be “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant technology as well as chemical synthesis. An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment such as, for example, in a host cell.

[0085] The term “linker” refers to a construct of variable length connecting two or more domains or portions of a polypeptide or connecting two or more polypeptides. In some cases, a linker is used to confer flexibility, improved spatial organization, proximity, etc and in such a case may be referred to as a flexible linker. Exemplary linkers may comprise one or more amino acids, optionally between 1- 50 amino acids, such as one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty amino acids. In some embodiments, the linker may predominantly or entirely consist of G, S, and / or A amino acid residues. In some embodiments, the linker may comprise an amino acid sequence which comprises or consists of the amino acid sequence selected from the group consisting of GGGGS, which may also be called “G4S”), GGGS, which may also be called “G3S”), GGGGGS, which may also be called “G5S”), G, GG, GGG, GS, SG, GGS (which may also be called “G2S”), GSG, SGG, GSS, SGS, and SSG. In some embodiments, the linker may comprise an amino acid sequence which comprises or consists of multiple repeats (e.g., two, three, four, five, or more repeats) of the amino acid sequence selected from the group consisting of G, GS, SG, GGS, GSG, SGG, GSS, SGS, SSG, and any combinations thereof. When the linker comprises or consists of multiple repeats of G5S, G4S, G3S, G2S, GS, or G, the linker may optionally called a (G5S)n linker, a (G4S)n linker, a (G3S)n linker, a (G2S)n linker, a (GS)n linker, or a (G)n linker, respectively (n is a natural number, optionally selected from 1-20, e.g., 2, 3, 4, 5, etc). In particular embodiments, the linker may comprise two or three repeats of SEQ ID ATTORNEY DOCKET NO.1143252.008013 NO: 51, i.e., have the sequence of GGGGSGGGGS or GGGGSGGGGSGGGGS, respectively, and may optionally be called a (G4S)2 linker or a (G4S)3 linker, respectively.

[0086] The term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice, rats, and hamsters, and mammals of the order Logomorpha, such as rabbits. The mammals may be from the order Carnivora, including Felines (cats) and Canines (dogs). The mammals may be from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). The mammals may be of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes).

[0087] The term "nucleic acid" or "polynucleotide" refers to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA / DNA hybrids. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A nucleic acid may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracil, other sugars and linking groups such as fluororibose and thiolate, and nucleotide branches. The sequence of nucleotides may be further modified after polymerization, such as by conjugation, with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides or solid support. The polynucleotides can be obtained by chemical synthesis, recombinantly, or derived from a microorganism.

[0088] The term "parenteral" or “parenterally” as used herein includes any route of administration of a compound or composition, characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intrasynovial injection or infusions; and kidney dialytic infusion techniques. In a preferred embodiment, parenteral administration of the compositions of the present invention comprises subcutaneous or intraperitoneal administration. ATTORNEY DOCKET NO.1143252.008013

[0089] A “pharmaceutical composition” refers to a preparation in such form as to permit the biological activity of an active ingredient contained therein, such as a cell described herein, to be effective and which preferably contains no additional components which are unacceptably toxic to a subject to which the composition would be administered.

[0090] A “pharmaceutical carrier”, as used herein, includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, and absorption delaying agents that are physiologically compatible. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In one embodiment, the carrier is suitable for parenteral, intravenous, intraperitoneal, intramuscular, or sublingual administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the present disclosure is contemplated. Supplementary active compounds can also be incorporated into the compositions. In some embodiments, the carrier may be a liquid, in which an active therapeutic agent is formulated. The excipient generally does not provide any pharmacological activity to the formulation, though it may provide chemical and / or biological stability, and release characteristics. Exemplary formulations can be found, for example, in Remington’s Pharmaceutical Sciences, Gennaro, A. editor, 19th edition, Philadelphia, PA: Williams and Wilkins (1995), which is incorporated by reference.

[0091] As used herein, the term "polypeptide" refers to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, to include disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.

[0092] As used herein, the term "protein" refers to a compound comprised of one or more polypeptides. When a protein comprises two or more polypeptides, the polypeptides may covalently joined (e.g., disulfide bond) to or noncovalently paired (e.g., hydrogen bond) with each other to form a complex.

[0093] The term “recombinant” generally refers to any protein, polypeptide, or cell expressing a gene of interest that is produced by genetic engineering methods. Therefore, the term “recombinant” as used with respect to a protein or polypeptide, means a protein or polypeptide produced by expression of a recombinant polynucleotide. "Recombinant," as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, ATTORNEY DOCKET NO.1143252.008013 restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature.

[0094] The term “host cell” refers to cells into which an exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. In some embodiments, a cell according to the present disclosure may be host cell.

[0095] The term “recombinant cell” or “host cell” refers to cells into which an exogenous nucleic acid sequence has been introduced, including the progeny of such cells. Such cells include transformants and transformed cells, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages.

[0096] A polynucleotide or polypeptide has a certain percent “sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. The terms “identical” or “identity” when used in the context of two or more nucleic acids or polypeptide sequences, refer to the number or percentage of residues that are the same in a sequence of interest and a reference sequence. The percentage can be calculated by optimally aligning the sequence of interest to the reference sequence; comparing the two sequences over the entire length of the reference sequence; determining the number of positions at which the identical amino acid residue or nucleic acid base occurs in both sequences to yield the number of matched positions; dividing the number of matched positions by the total number of positions in the reference sequence adjusted by adding the number of gap positions introduced into the reference sequence in generating the alignment; and multiplying the result by 100 to yield the percentage of sequence identity. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Sequence identity may be determined by using the stand-alone executable BLAST engine program for blasting two sequences (bl2seq), which can be retrieved from the National Center for Biotechnology Information (NCBI) ftp site or over the worldwide web at ncbi.nlm.nih.gov / BLAST / , using the default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; which is incorporated herein by reference in its entirety).

[0097] The term "subject" as used herein may be any living organisms, preferably a mammal. In some embodiments, the subject is a primate such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some examples, the patient or subject is a ATTORNEY DOCKET NO.1143252.008013 validated animal model for disease and / or for assessing toxic outcomes. The subject may also be referred to as “patient” in the art. The subject may have a disease or may be healthy.

[0098] The term "transfected," "transformed," or "transduced" refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A "transfected" or "transformed" or "transduced" cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0099] As used herein, the term "treat," "treatment," or "treating" generally refers to the clinical procedure for reducing or ameliorating the progression, severity, and / or duration of a disease or of a condition, or for ameliorating one or more conditions or symptoms (preferably, one or more discernible ones) of a disease. The type of disease or condition to be treated may be, for example, but are not limited to, autoimmune and inflammatory disorders, diseases and conditions. In specific embodiments, the effect of the “treatment” may be evaluated by the amelioration of at least one measurable physical parameter of a disease, resulting from the administration of a targeted IL-2 fusion protein according to the present disclosure. The parameter may be, for example, gene expression profiles, the mass of disease-affected tissues, inflammation-associated markers, the number or frequency of disease-associated cells, the presence or absence of certain cytokines or chemokines or other disease-associated molecules, the presence or absence of a certain cell type e.g., a certain immune cells such as T cells or a specific subset thereof, and may not necessarily discernible by the patient. In other embodiments "treat", "treatment," or "treating" may result in the inhibition of the progression of a disease, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. In other embodiments the terms "treat", "treatment" and "treating" refer to the reduction or stabilization of cancerous tissue or cells. Additionally, the terms “treat,” and “prevent” as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete cure or prevention. Rather, there are varying degrees of treatment effects or prevention effects of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment or prevention effects of a disease in a mammal. Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the disease being treated or prevented. Also, for purposes herein, “prevention” can encompass delaying the onset of the disease, or a symptom or condition thereof, especially an acute, intermittent or chronic autoimmune or inflammatory disease.

[0100] A "vector" is a compound or a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous ATTORNEY DOCKET NO.1143252.008013 vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, viruses, and virus-like particles (VLPs). Thus, the term "vector" includes an autonomously replicating plasmid, a self-replicating RNA, or a viral particle. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0101] An "expression vector" is a vector, e.g., plasmid, minicircle, viral vector, liposome, and the like as discussed herein or as known in the art, comprising a region which encodes a gene product of interest, and is used for effecting the expression of the gene product in an intended target cell. An expression vector also comprises control elements, e.g., promoters, enhancers, UTRs, miRNA targeting sequences, etc., operatively linked to the encoding region to facilitate expression of the gene product in the target. The combination of control elements and a gene or genes to which they are operably linked for expression is sometimes referred to as an "expression cassette," a large number of which are known and available in the art or can be readily constructed from components that are available in the art.

[0102] A "promoter" as used herein encompasses a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis, i.e., a minimal sequence sufficient to direct transcription. Promoters and corresponding protein or polypeptide expression may be ubiquitous, meaning strongly active in a wide range of cells, tissues and species or cell-type specific, tissue- specific, or species specific. Promoters may be “constitutive,” meaning continually active, or “inducible,” meaning the promoter can be activated or deactivated by the presence or absence of biotic or abiotic factors. Also included in the nucleic acid constructs or vectors of the present disclosure are enhancer sequences that may or may not be contiguous with the promoter sequence. Enhancer sequences influence promoter-dependent gene expression and may be located in the 5' or 3' regions of the native gene.

[0103] "Operatively linked" or "operably linked" refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.

[0104] The term “Protein drug conjugate” or “PDC” refers to a compound wherein one or more copies of a drug, herein an IL-2 fusion protein, are directly or indirectly linked to a polymeric carrier. PDC conjugates often are divided into three different units: i.e., a solubilizing zone, drug–polymer ATTORNEY DOCKET NO.1143252.008013 chemical linkers, and a transport system. The first unit comprises a polymer which is generally used to solubilize all of the macromolecules without toxicity. The second unit is the drug linked to the polymer via a chemical linker. The last unit is the area of the targeting ligand located in the hydrophilic region of the polymer which can enhance the ability to carry the entire macromolecule to biological target sites. PDCs may provide intrinsic benefits over the “naked” or “free” drug such as enhanced drug solubility and activity, modified pharmacokinetic profile, reduced toxicity, polymer- specific effects, and drug combination along the polymer chain. The selection of polymeric macromolecular carriers, the desired target (intracellular, lymphatic system, etc.), the type of conjugation (direct or indirect), the chemistry of the linker, and the molecular weight (MW) are considered key parameters in PDC design. Also, the design of an appropriate polymeric carrier is often influenced by its proposed route of administration and frequency of dosing. PDCs and linkers and polymers finding use in the synthesis thereof are known in the art (see e.g., “Innovative Design of Targeted Nanoparticles: Polymer–Drug Conjugates for Enhanced Cancer Therapy”, by Varaporn Buraphacheep Junyaprasert and Parichart Thummarati, Pharmaceutics 2023, 15(9), 2216; https: / / doi.org / 10.3390 / pharmaceutics15092216; and “Polymer–drug and polymer–protein conjugated nanocarriers: Design, drug delivery, imaging, therapy, and clinical applications”, Haochen Guo and Peng Mi, WIRES Nanomedicine and Nanotechnology, Vol 16(4): July / Aug 2024).

[0105] The term “wildtype” or “native” as used herein refers to a nucleotide sequence, e.g., gene, or gene product, e.g., RNA or protein, that is present in a wild-type cell, tissue, organ or organism. The term “variant” as used herein in relation to a polynucleotide or polypeptide refers to a mutant of a reference polynucleotide or polypeptide sequence, for example a native or wild-type polynucleotide or polypeptide sequence, i.e., having less than 100% sequence identity with the reference polynucleotide or polypeptide sequence. Put another way, a variant polypeptide or polynucleotide comprises at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, and amino acid deletion) or at least one nucleic acid different (e.g., base substitution, base insertion, and base deletion), respectively, relative to a reference polypeptide or polynucleotide sequence, e.g., a native or wild-type, polypeptide or polynucleotide sequence. For example, a variant polypeptide or polynucleotide may be a polypeptide or polynucleotide having a sequence identity of 50% or more, 60% or more, or 70% or more to a full-length native or wild-type, polypeptide or polynucleotide sequence, e.g., an identity of 75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99% identity with the full-length native or wild-type, polypeptide or polynucleotide sequence. Variants may also include variant fragments of a reference, e.g., native, sequence sharing a sequence identity of 70% or more with a fragment of the reference, ATTORNEY DOCKET NO.1143252.008013 e.g., native, sequence, e.g., an identity of 75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99% identity to the native or wild-type sequence. Targeted IL-2 Fusion proteins

[0106] In one aspect the present disclosure provides targeted IL-2 fusion proteins that preferentially deliver IL-2 to Tregs, and which promote the activation and / or expansion of regulatory T cells, which comprise: (i) an IL-2 polypeptide, optionally a human IL-2 polypeptide, which IL-2 polypeptide further optionally is modified to attenuate IL-2Rβ / γc binding; (ii) an antibody or antibody fragment which binds to an antigen other than CD25 expressed on Tregs; and (iii) preferably a half-life extending moiety;

[0107] wherein (i), (ii) and (iii) if present may be comprised in any order from amino terminal to carboxy terminal in the human IL-2 fusion protein, and wherein these moieties may be directly linked to each other or may be indirectly linked to each other, e.g., via one or more linker peptide.

[0108] In some instances the moieties in the targeted IL-2 fusion protein are oriented from N to C terminal as follows:

[0109] (i) IL-2 polypeptide—(ii) antibody or antibody fragment which binds to an antigen other than CD25 expressed on Tregs—(iii) half-life extending moiety, optionally human serum albumin (HSA) or a human Ig Fc region.

[0110] In some instances the (i) IL-2 polypeptide or IL-2 domain comprised in the subject targeted IL-2 fusion proteins comprises at least one mutation which attenuates IL-2Rβ / γc binding.

[0111] In certain embodiments, the IL2 domain may comprise one or more amino acid substitutions, insertions, and / or deletions relative to a wildtype IL2 (e.g., SEQ ID NO: 1) and / or one or more amino acid modifications (e.g., phosphorylation, methylation, acetylation, amidation, formation of pyrrolidone carboxylic acid, isomerization, hydroxylation, sulfation, flavin-binding, cysteine oxidation, and / or nitrosylation, and / or conjugation to another molecule such as a PEG), which reduce or abrogate binding to IL2Rβ / γc. The one or more amino acid substitutions, insertions, and / or deletions may be at any appropriate amino acid positions, and such positions may for example be determined or selected structurally, such as based on crystal structure, and / or using mutagenesis. In some cases, one or more amino acid positions in the IL2-IL2Rβ / γc binding surface may be altered. In some cases, a position of interest or a combination of positions of interest in the ATTORNEY DOCKET NO.1143252.008013 IL2 domain may be substituted and binding to IL2Rβ / γc may be tested to determine which position(s) to be altered.

[0112] In certain embodiments, the IL2 domain may comprise at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO: 1. In some instances, the substitution(s) may be or may comprise a non-conservative amino acid substitution(s). In some instances, the substitution(s) may be or may comprise a substitution to an aliphatic amino acid. In certain instances, the substitution(s) may be or may comprise a substitution to alanine, glycine, isoleucine, leucine, proline, or valine. In particular instances, the substitution(s) may be or may comprise a substitution to alanine or glycine.

[0113] In some exemplary embodiments, the IL2 domain may comprise at least one mutation at a residue selected from residue 16, 20, 88, 91 and 126, and further optionally may comprise a mutation selected from N88D, N88R, H6L, D20T, Q126T and further optionally is N88D (wherein the residue numbering is the same as the residue numbering in wild-type human IL-2).

[0114] In any of the embodiments above, in some cases, the IL2 domain may comprise one or more amino acid substitutions, insertions, and / or deletions, for example, to prevent aggregation. In certain cases, the cysteine residue at position 125 may be substituted to another amino acid. In particular cases, the cysteine residue at position 125 may be substituted to alanine or serine.

[0115] In any of the embodiments above, in some cases, the IL2 domain may comprise one or more amino acid substitutions, insertions, and / or deletions, for example, to alter (e.g., remove or add) potential glycosylation. In certain cases, threonine or serine may be substituted to an amino acid other than threonine or serine to remove a potential O-glycosylation site. In certain cases, the threonine residue at position 3 may be substituted to another amino acid (e.g., an amino acid other than threonine or serine). In particular cases, the threonine residue at position 3 may be substituted to alanine.

[0116] In some instances the (ii) antibody or antibody fragment, e.g., an scFv, Fab or VHH antibody, which antibody or antibody fragment targets the IL-2 fusion proteins and the IL-2 therein preferentially to Tregs binds to an immune checkpoint inhibitor, co-stimulatory receptor, cytokine receptor or an enzyme expressed on human Tregs. Optionally the (ii) antibody or antibody fragment which targets binds to any of CTLA-4, TIGIT, GITR, ICOS, TGF beta receptor, TNFR-2, CD39, or CD73.

[0117] In some preferred embodiments the (ii) antibody or antibody fragment which targets the IL- 2 fusion to Tregs binds to CD39, e.g., an anti-CD39 scFv or anti-CD39 Fab.

[0118] In some instances the (iii) half-life extending moiety is selected from a serum albumin, an Fc or immunoglobulin constant region, an unstructured polypeptide, an adnectin, transferrin, CTP (28 ATTORNEY DOCKET NO.1143252.008013 amino acid C-terminal peptide (CTP) of hCG with its 4 O-glycans), or a fragment of any of the foregoing; a lipid, O- and N-glycosyl residues, a carbohydrate polymer, cholesterol, poly(ethylene glycol) (PEG), monomethoxy PEG (mPEG), a branched or unbranched acyl group, a branched or unbranched C8-C30 acyl group, a branched or unbranched alkyl group, and a branched or unbranched C8-C30 alkyl group.

[0119] In some instances the (iii) half-life extending moiety is a serum albumin, optionally human serum albumin, or a human Fc region, which optionally contains a mutation which ablates or reduces at least one effector function such as glycosylation, FcR binding or complement binding and / or a mutation which enhances FcRN binding.

[0120] In some instances the (ii) antibody or antibody fragment which targets the IL-2 fusion proteins and the IL-2 therein preferentially to Tregs is a scFv, a Fab, F(ab')2 or VHH antibody.

[0121] In some instances either or both of the (i) IL-2 polypeptide and the (ii) antibody or antibody fragment which targets the IL-2 fusion to Tregs binds to CD39, are PEGylated, optionally via an inserted non-encoded amino acid. In some instances the targeted IL-2 fusion polypeptide may comprise a linker to which is attached to one or more poly ethylene glycol residues. Scaffold domain

[0122] In some embodiments, the IL-2 fusion protein may comprise a scaffold domain or half-life extender. Without wishing to be bound by theory, the scaffold domain may bring the IL-2 fusion protein to a desired size (which may for example slow in vivo clearance), and / or allow the cytokine protein to have a prolonged in vivo half-life, an increased stability, an improved production yield and / or purification feasibility, a desired polarity and / or net charge, and / or desired conformation.

[0123] In certain embodiments, the scaffold domain may comprise an albumin domain, such as a domain derived from human albumin (also known as human serum albumin or HSA) or a HSA domain. Wildtype human albumin is known to have an in vivo half-life of about 3 weeks, which is much longer than that of typical peptides, which is often as short as about 2-30 minutes due to the clearance by the kidneys.

[0124] Therefore, in some cases, the scaffold domain may comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%, or at least 99.5% identity to wild-type serum albumin, preferably human serum albumin.

[0125] In certain cases, a threonine or serine in the HSA domain may be substituted to an amino acid other than threonine or serine to remove a potential O-glycosylation site. In certain cases, arginine in the HSA domain may be substituted to an amino acid other than arginine to remove a potential N-glycosylation site. ATTORNEY DOCKET NO.1143252.008013

[0126] In certain cases, a cysteine residue in the HSA domain may be substituted to another amino acid to prevent an intermolecular or intramolecular disulfide bond formation and / or to prevent aggregation. In some instances, the cysteine residue at position 34 may be substituted to another amino acid (e.g., serine, alanine, glycine). In certain instances, the HSA domain may comprise C34S.

[0127] In certain embodiments, the scaffold domain may comprise an immunoglobulin constant region sequence or part thereof (e.g., a CH1 domain, a hinge, a CH2 domain, a CH3 domain, a Fc region, etc). In case of human IgG1, the Fc region corresponds to positions Asp221 (or Thr223, or Thr225) to Lys447 (or Gly446), according to EU numbering. In case of IgG4, the Fc region corresponds to Tyr (one residue after K218) to Lys447 (or Gly446), according to the EU numbering. In mammalian bodies, antibodies are recycled and maintained in circulation by binding to neonatal Fc receptors (FcRn).

[0128] Therefore, in some cases, the scaffold domain or half-life extender may comprise an Fc domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%, or at least 99.5% identity to the Fc domain of a human IgG, e.g., IgG1, IgG4, IgG2, and / or IgG2.

[0129] In certain cases, the Fc domain may comprise one or more amino acid substitutions which increases in vivo half-life (e.g., increased binding to FcRn) and / or which reduces effector functions (e.g., by reduced binding to Fc receptors such as FcγR or complements such as C1q). Without wishing to be bound by theory, reduced effector functions in some instances may provide reduced side effects. Any appropriate substitution(s) at any appropriate position(s) (e.g., Wang et al., Protein Cell. 2018 Jan;9(1):63-73.; Liu et al., Antibodies (Basel).2020 Nov 17;9(4):64.) may be incorporated to the Fc domain of a IL-2 fusion protein according to the present disclosure. Formats

[0130] The IL-2 fusion proteins according to the present disclosure comprising at least an IL2 domain, an antibody or antibody fragment which binds to an antigen expressed on Tregs (other than CD25) and optionally a scaffold protein or half-life extender as described herein may take and may comprise different formats.

[0131] In some embodiments, the domains contained in the IL-2 fusion protein may be joined directly or indirectly via a linker. In certain embodiments, the linker may be a peptide linker, such as a flexible peptide linker. In certain embodiments, the linker may comprise about 1-50 amino acids and may comprise an amino acid sequence comprising or consisting of one or more small amino acids, such as glycine (G), serine (S), and / or alanine (A). In certain embodiments, the linker may comprise a linker unit amino acid sequence selected from the group consisting of G, GG, GGG, GS, ATTORNEY DOCKET NO.1143252.008013 SG, GGS, GSG, SGG, GSS, SGS, and SSG or multiple repeats of any of such linker unit amino acid sequences. In particular embodiments, the linker may comprise a (G4S)2linker or a (G4S)3linker.

[0132] In certain embodiments, the IL-2 fusion protein may comprise (i) an IL-2 polypeptide, (ii) an antibody or antibody fragment that targets Tregs and (iii) a scaffold or half-life extending moiety such as human serum albumin in the direction from the N-terminus to the C-terminus.

[0133] In certain embodiments, the IL-2 fusion protein may comprise (i) an antibody or antibody fragment that targets Tregs (ii) an IL-2 polypeptide, and (iii) a scaffold or half-life extending moiety such as human serum albumin in the direction from the N-terminus to the C-terminus.

[0134] In certain embodiments, the IL-2 fusion protein may comprise (i) a scaffold or half-life extending moiety such as human serum albumin (ii) an antibody or antibody fragment that targets Tregs and (iii) an IL-2 polypeptide from the N-terminus to the C-terminus.

[0135] In certain embodiments, the IL-2 fusion protein may comprise (i) an IL-2 polypeptide, (ii) a scaffold or half-life extending moiety such as human serum albumin and (iii) an antibody or antibody fragment that targets Tregs in the direction from the N-terminus to the C-terminus.

[0136] In any of the above formats, the moieties may be directly linked or linked by inclusion of one or more linker peptides.

[0137] In any of the formats, in some cases, the scaffold domain or half-life extender may be or may comprise HSA or a variant thereof. In any of the formats, in some cases, the scaffold domain may be or may comprise a Fc domain, e.g., any of the Fc domains described herein.

[0138] In certain embodiments, the IL-2 fusion protein may comprise two or more IL2 domains, two or more antibody or antibody fragments that target Tregs and / or two or more scaffold domains or half-life extender moieties.

[0139] In certain embodiments, the IL-2 fusion protein may comprise two Fc domains (e.g., as two scaffold domains). In some cases, the two Fc domains may be paired with each other, e.g., via one or more disulfide bonds.

[0140] In any of the formats described herein, when the IL-2 fusion protein comprises two or more polypeptides, the polypeptides may be covalently joined (e.g., via a disulfide bond) with each other and / or noncovalently paired with each other. Nucleic acids and vectors

[0141] In one aspect the present disclosure provides nucleic acids (or polynucleotides) and vectors encoding any of the IL-2 fusion proteins described above.

[0142] A nucleic acid according to the present disclosure may comprise one nucleic acid (or polynucleotides) molecule or two or more nucleic acid (or polynucleotides) molecules. A vector according to the present disclosure may comprise one vector or two or more vectors. ATTORNEY DOCKET NO.1143252.008013

[0143] In some embodiments, the IL-2 fusion protein encoded by a nucleic acid or a vector according to the present disclosure may comprise (i) an IL-2 polypeptide, (ii) an antibody or antibody fragment that targets Tregs and (iii) a scaffold or half-life extending moiety such as human serum albumin in the direction from the N-terminus to the C-terminus.

[0144] In some embodiments, the IL-2 fusion protein encoded by a nucleic acid or a vector according to the present disclosure may comprise (i) an antibody or antibody fragment that targets Tregs (ii) an IL-2 polypeptide, and (iii) a scaffold or half-life extending moiety such as human serum albumin in the direction from the N-terminus to the C-terminus.

[0145] In some embodiments, the IL-2 fusion protein encoded by a nucleic acid or a vector according to the present disclosure may comprise a (i) a scaffold or half-life extending moiety such as human serum albumin (ii) an antibody or antibody fragment that targets Tregs and (iii) an IL-2 polypeptide from the N-terminus to the C-terminus.

[0146] In some embodiments, the IL-2 fusion protein encoded by a nucleic acid or a vector according to the present disclosure may comprise a (i) an IL-2 polypeptide, (ii) a scaffold or half-life extending moiety such as human serum albumin and (iii) an antibody or antibody fragment that targets Tregs in the direction from the N-terminus to the C-terminus.

[0147] In any of the formats, the moieties in the encoded IL-2 fusion protein may be directly linked or linked by inclusion of one or more nucleic acids which encode one or more linker peptides.

[0148] In any of the formats, the scaffold domain or half-life extender may be or may comprise a HSA polypeptide; or may comprise a Fc domain, e.g., any of the Fc domains described herein.

[0149] In certain embodiments, the IL-2 fusion protein encoded by a nucleic acid or a vector according to the present disclosure may comprise two or more IL2 domains, two or more antibody or antibody fragments that target Tregs and / or two or more scaffold domains or half-life extender moieties.

[0150] In certain embodiments, IL-2 fusion protein encoded by a nucleic acid or a vector according to the present disclosure may comprise two Fc domains (e.g., as two scaffold domains). In some cases, the two Fc domains may be paired with each other, e.g., via one or more disulfide bonds.

[0151] In any of the formats described herein in instances wherein a nucleic acid or a vector encoding a IL-2 fusion protein comprises two or more polypeptides, the polypeptides may be covalently joined (e.g., via a disulfide bond) with each other and / or noncovalently paired with each other. Polymer-Drug Conjugates ATTORNEY DOCKET NO.1143252.008013

[0152] In some embodiments one or more IL-2 fusion proteins according to the present disclosure comprising at least an IL2 domain, an antibody or antibody fragment which binds to an antigen expressed on Tregs (other than CD25) and optionally a scaffold protein or half-life extender as described herein may be directly or indirectly conjugated to a polymer producing a polymer–drug conjugate (PDCs). PDCs have shown great promise in enhancing the efficacy and safety of biologics e.g., during cancer therapy. These conjugates combine the advantageous properties of both polymers and drugs, leading to improved pharmacokinetics, controlled drug release, and targeted delivery, e.g., to tumor and inflammatory or autoimmune tissues.

[0153] Different types of polymers may be used in such PDCs including synthetic polymers, such as poly(€-caprolactone) (PCL), D-α-tocopheryl polyethylene glycol (TPGS), and polyethylene glycol (PEG), as well as natural polymers such as hyaluronic acid (HA). The polymer is chosen to optimize desired properties, such as stability, biocompatibility, and controlled drug release.

[0154] Different strategies for conjugating drugs to polymers are known in the art including covalent bonding, which enables a stable linkage between the polymer and the drug, ensuring controlled release and minimizing premature drug release. The use of polymers can extend the circulation time of the drug, facilitating enhanced accumulation within tumor tissues through the enhanced permeability and retention (EPR) effect. This, in turn, results in improved drug efficacy and reduced systemic toxicity.

[0155] Several IL-2 fusion proteins according to the invention may be covalently bound to polymeric carriers, linear or branched, e.g., through bioresponsive linkers which e.g., result in improved stability with the diversity, specificity, and functionality of biomolecules. PDCs may also improve drug solubility and loading capacity, improve pharmacokinetic profiles by controlling and maintaining drug release, and increase drug half-life by decreasing immune system recognition. In addition, they can increase drug accumulation specificity at the target site through passive and active transport.

[0156] The design and synthesis of PDCs that can interact effectively with biological systems generally involves attachment to free functional groups that can be conjugated directly to polymer backbones through chemical linkers, e.g., R-OH and R-C=O-R. These functional groups can be linked to R-C=O-OH and R-HN2of the polymer to form ester and hydrazone linkers, respectively. PDCs also enable the codelivery of drugs and / or bioactive molecules with different properties in one nanoparticle, making them multifunctional.

[0157] Different types of polymers may be employed for producing PDCs including by way of example pH-sensitive polymers, light-sensitive polymers, enzyme-(cleavable) sensitive polymers, redox-sensitive polymers, hypoxia-sensitive polymers, among others. Specific polymers finding ATTORNEY DOCKET NO.1143252.008013 known usage in PDCs include e.g., PEG, PVP, HPMA, poly(€-caprolactone) (PCL), poly(lactic-co- glycolic acid) (PLGA), D-α-tocopheryl polyethylene glycol (TPGS), hyaluronic acid (HA), dextran, alginate, pectin, and starch.

[0158] In some embodiments the PDC may comprise a pH-responsive polymer which acts as a trigger for drug release related to physiological conditions. A pH-sensitive bond may be used to covalently link the IL-2 fusion proteins to a polymeric carrier. For instance compared to normal tissues, the pH of the tumor tissue environment and intracellularly in the lysosomes is slightly acidic (pHnormal cells= 7.20–7.45, pHcancer environments= 6.50–6.90, and pHlysosomes= 4.5–6.5). Therefore, pH- tumor tissues and lysosomes, while they are stable at physiological pH promote selective release at tumor sites. Many acid-labile linkers have been reported to improve drug release profiles, such as hydrazone, amide, imine, cis-acotinyl, oxime, ketal, and acetal. An exemplary pH-responsive chemical bond is the hydrazone bond, because of its acute responsiveness in drug delivery behavior.

[0159] In some embodiments the PDC may comprise a temperature-responsive polymer which undergoes a reversible phase transition from a hydrophilic to hydrophobic state in response to an external temperature stimulus, allowing the controlled release of drugs at elevated temperatures e.g., those associated with cancer tissues as a trigger to cleave the linker between the polymer and the drug. Because of its temperature sensitivity, poly(N-isopropylacrylamide) (PNIPAM) is a commonly used temperature-responsive polymer for drug conjugation in cancer therapy as the lower critical solution temperature (LCST) of PNIPAM is approximately 32 °C.

[0160] In some embodiments the PDC may comprise an enzyme-responsive polymer which triggers drug release at the target site. In such embodiments one or more IL-2 fusion proteins according to the invention are chemically linked to polymer carriers through enzyme-responsive bonding that can be cleaved by specific enzymes at the target site. Examples of enzyme-responsive polymers and linkers and their therapeutic applications are generally known in the art. Cells

[0161] In one aspect the present disclosure provides cells which express any of the Il-2 fusion proteins described herein, and / or comprise or transformed / transfected / transduced with any of the nucleic acids and / or any of the vectors described herein.

[0162] For expressing a IL-2 fusion protein, any appropriate cells may be used. For example, cells may be: (i) prokaryotic cells, such as gram-negative bacteria and gram-positive bacteria; or (ii) eukaryotic cells, such as yeast, filamentous fungi, protozoa, insect cells, plant cells, and mammalian ATTORNEY DOCKET NO.1143252.008013 cells (reviewed in Frenzel A. et al. Front Immunol.2013; 4: 217. Published online 2013 Jul 29. doi: 10.3389 / fimmu.2013.00217).

[0163] Specific examples of gram-negative bacteria that are suited for production of IL-2 fusion proteins include, but are not limited to, E. coli, Proteus mirabilis, and Pseudomonas putida. Specific examples of gram-positive bacteria include, but are not limited to, Bacillus brevis, Bacillus subtilis, Bacillus megaterium, Lacto-bacilluszeae / casei, and Lactobacillus paracasei. Specific examples of yeast bacteria that are suited for production of IL-2 fusion proteins include, but are not limited to, Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Kluyveromyces lactis, and Yarrowia lipolytica. Specific examples of filamentous fungi that are suited for production of IL-2 fusion proteins include, but are not limited to, the genera Trichoderma and Aspergillus, A. niger (subgenus A. awamori), Aspergillus oryzae, and Chrysosporium lucknowense. Specific examples of protozoa that are suited for production of IL-2 fusion proteins include, but are not limited to, Leishmania tarentolae. Specific examples of insect cells that are suited for production of IL-2 fusion proteins include, but are not limited to, insect cell lines like Sf-9 and Sf-21 or SfSWT-1 "Mimic™" cells of Spodoptera frugiperda, DS2 cells of Drosophila melanogaster, High Five cells (BTI-TN-5B1-4) of Trichopulsia ni, or Schneider2 (S2) cells of D. melanogaster. They can be efficiently transfected with insect-specific viruses from the family of Baculoviridae, particularly the Autographa californica nuclear polyhedrosis virus (AcNPV). Specific examples of mammalian cells that are suited for production of IL-2 fusion proteins include, but are not limited to, Chinese hamster ovary (CHO) cells, the human embryonic retinal cell line Per.C6 [Crucell, Leiden, Netherlands], CHO-derived cell lines such as K1-, DukXB11-, Lec13, and DG44- cell lines, mouse myeloma cells such as SP 2 / 0, YB 2 / 0, and NS0 cells, GS-NSO, hybridoma cells, baby hamster kidney (BHK) cells, and the human embryonic kidney cell line HEK293, HEK293T, HEK293E, and human neuronal precursor cell line AGE1.HN (Probiogen, Berlin, Germany). Specific examples of plant cells that are suited for production of IL-2 fusion proteins include but are not limited to BY2 or NT1 cells of N. tabacum, Bengal, Donjin, or Taipie cells of Oryza sativa, and cells of Hordeum vulgare.

[0164] Alternatively, genetically modified organisms such as transgenic plants and transgenic animals may be used. Exemplary plants that may be used include, but are not limited to, tobacco, maize, duckweed, Chlamydomonas reinhardtii, Nicotiana tabacum, Nicotianaben thamiana, and Nicotiana benthamiana. Exemplary animals that may be used include, but are not limited to mouse, rat, and chicken. Compositions ATTORNEY DOCKET NO.1143252.008013

[0165] In one aspect the present disclosure provides compositions comprising: (I) at least one of: (a) any of the IL-2 fusion proteins described herein; (b) any of the nucleic acids described herein; (c) any of the vectors described herein; and / or (d) any of the isolated, recombinant, and / or host cells or a population of cells comprising any of the isolated, recombinant, and / or host cells; and (II) a pharmaceutically acceptable carrier. Such a composition may also be referred to as a pharmaceutical composition. Carrier

[0166] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn- protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos.2005 / 0260186 and 2006 / 0104968.

[0167] Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0168] In some embodiments, a pharmaceutical composition according to the present disclosure may be used for any of the in vivo and / or treatment methods described herein. Additional agents ATTORNEY DOCKET NO.1143252.008013

[0169] In some embodiments, the pharmaceutical composition may be used alone without an additional agent.

[0170] In certain embodiments, an additional agent(s) may be included therein such as an anti- inflammatory agent or immunosuppressant. In some instances the anti-inflammatory or immunosuppressant may comprise a small molecule or a biologic.

[0171] Examples of such anti-inflammatory agents include nonsteroidal anti-inflammatory drugs (NSAIDs) e.g., aspirin, ibuprofen, and naproxen; paracetamol, Antileukotrienes or leukotriene- related enzyme inhibitors (arachidonate 5-lipoxygenase) or leukotriene receptor antagonists (cysteinyl leukotriene receptors), and the like.

[0172] Other examples of anti-inflammatory agents include glucocorticoids or steroids. Specific examples include Group A – Hydrocortisone type steroids such as Hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, prednisolone, methylprednisolone, and prednisone; a Group B – Acetonide type steroids such as (Amcinonide, budesonide, desonide, fluocinolone cetonide, fluocinonide, halcinonide, triamcinolone acetonide, and Deflazacort (O-isopropylidene derivative); a Group C – Betamethasone type steroids such as declometasone, betamethasone, dexamethasone, fluocortolone, halometasone, and mometasone; Group D1 and D2 esters–such as Group D1 and D2 esters Alclometasone dipropionate, betamethasone dipropionate, betamethasone valerate, clobetasol propionate, clobetasone butyrate, fluprednidene acetate, and mometasone furoate; and Group D2 – Labile prodrug esters such as Ciclesonide, cortisone acetate, hydrocortisone aceponate, hydrocortisone acetate, hydrocortisone buteprate, hydrocortisone butyrate, hydrocortisone valerate, prednicarbate, and tixocortol pivalate.

[0173] Examples of biologic anti-inflammatory agents include TNF inhibitors (TNF blockers or anti- TNFs), B cell inhibitors, interleukin inhibitors, (e.g., IL-1, IL-6 and / or IL-17 inhibitors). Specific examples of TNF inhibitors include adalimumab (Humira), certolizumab pegol (Cimzia), etanercept (Enbrel), golimumab (Simponi, Simponi Aria), infliximab (Remicade), and biosimilars thereof such as Amjevita, Avsola, Inflectra, and Renflexis; specific examples of B cell inhibitors include belimumab (Benlysta) and rituximab (Rituxan), and rituximab biosimilars such as Riabni, Ruxience and Truxima; specific examples of interleukin inhibitors include anakinra (Kineret), canakinumab (Ilaris), guselkumab (Tremfya), ixekizumab (Taltz), risankizumab (Skyrizi), sarilumab (Kevzara), secukinumab (Cosentyx), tocilizumab (Actemra) and ustekinumab (Stelara) and Selective Co-stimulation Modulators include Abatacept (Orencia). Preferred examples of the foregoing include Remicade® (infliximab), Humira® (adalimumab), Simponi® (golimumab) and Cimzia® (certolizumab pegol). ATTORNEY DOCKET NO.1143252.008013

[0174] Such other anti-inflammatory agents may be administered by various means e.g., topically on the skin, eye, and mucous membranes; via inhalation for delivery to the nasal mucosa, sinuses, bronchi, and lungs; orally and / or systemically.

[0175] Other agents used to treat autoimmunity which may be used in combination with the subject IL-2 fusion proteins such as methotrexate, Cytostatics, Alkylating agents such as nitrogen mustards (cyclophosphamide), nitrosoureas, platinum compounds, and others; Cyclophosphamide; Antimetabolites such as folic acid analogues, methotrexate; purine analogues, such as azathioprine and mercaptopurine; pyrimidine analogues, such as fluorouracil and protein synthesis inhibitors; Azathioprine and mercaptopurine; Azathioprine; Cytotoxic antibiotics such as dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin. Treatment methods and uses

[0176] In another aspect the present disclosure provides therapeutic methods, such as methods of treating a disease, disorder, or condition in a subject and methods of stimulating an immune response such as T cell response in a subject.

[0177] In some embodiments, such a method may comprise administering to the subject an effective amount of: at least one of: (a) any of the IL-2 fusion proteins described herein; (b) any of the nucleic acids described herein; (c) any of the vectors described herein; (d) any of the isolated, recombinant, and / or host cells or a population of cells comprising any of the isolated, recombinant, and / or host cells; and / or (e) any of the compositions described herein.

[0178] In any of the aspects and embodiments herein including methods and uses, in some cases, the subject may be a mammal and, in particular, a human.

[0179] In any of the aspects and embodiments herein including methods and uses, in some cases, the subject may have or may have a risk of developing a disease, a disorder, or a condition, particularly an acute or chronic autoimmune or inflammatory disorder.

[0180] In any of the aspects and embodiments herein including methods and uses, in some cases, the disease, disorder, or condition may be a neuroinflammatory, autoimmune, or inflammatory condition or a pathologic symptom associated with any of said conditions.

[0181] In any of the aspects and embodiments herein including methods and uses, in some cases, the therapy or prophylaxis is for the treatment or prevention of an autoimmune disease selected from one or more of Acromegaly, Acquired aplastic anemia, Acquired hemophilia, Agammaglobulinemia, primary, Alopecia areata, Ankylosing spondylitis (AS), Anti-NMDA receptor encephalitis, Antiphospholipid syndrome (APS) | catastrophic antiphospholipid syndrome (CAPS) / Asherson's syndrome, Arteriosclerosis, Autoimmune Addison’s disease (AAD), Autoimmune ATTORNEY DOCKET NO.1143252.008013 autonomic ganglionopathy (AAG) / autoimmune dysautonomia | autoimmune gastrointestinal dysmotility (AGID), Autoimmune encephalitis | acute disseminated encephalomyelitis (ADEM), Autoimmune gastritis, Autoimmune hemolytic anemia (AIHA), Autoimmune hepatitis (AIH), Autoimmune hyperlipidemia, Autoimmune hypophysitis, Autoimmune inner ear disease (AIED), Autoimmune lymphoproliferative syndrome (ALPS), Autoimmune myelofibrosis, Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune pancreatitis (AIP), Autoimmune polyglandular syndromes, types I, II, & III (APS type 1, APS type 2, APS type 3, APECED), Autoimmune progesterone dermatitis, Autoimmune retinopathy (AIR), Autoimmune sudden sensorineural hearing loss (SNHL), Balo disease, Behçet’s disease, Birdshot chorioretinopathy / birdshot uveitis, Bullous pemphigoid, Castleman disease, Celiac disease, Chagas disease, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic urticaria (CU), Churg-Strauss syndrome / eosinophilic granulomatosis with polyangiitis (EGPA), Cogan’s syndrome, Cold agglutinin disease, CREST syndrome | limited cutaneous systemic sclerosis, Crohn’s disease (CD), Cronkhite-Canada syndrome (CSS), Cryptogenic organizing pneumonia (COP), Dermatitis herpetiformis, Dermatomyositis, Diabetes, type 1, Discoid lupus, Dressler’s syndrome / postmyocardial infarction / postpericardiotomy syndrome, Eczema / Atopic Dermatitis, Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum, Essential mixed cryoglobulinemia, Evans syndrome, Fibrosing alveolitis / Idiopathic pulmonary fibrosis (IPF), Giant cell arteritis / temporal arteritis / Horton’s disease, Giant Cell Myocarditis, Glomerulonephritis, Goodpasture’s syndrome / anti- GBM / anti-TBM disease, Granulomatosis with polyangiitis (GPA) / Wegener’s granulomatosis, Grave’s disease / thyroid eye disease, Guillain-Barré syndrome (GBS), Hashimoto’s thyroiditis / chronic lymphocytic thyroiditis / autoimmune thyroiditis, Henoch-Schönlein purpura / IgA vasculitis, Hidradenitis suppurativa, Hurst’s disease / acute hemorrhagic leukoencephalitis (AHLE), Hypogammaglobulinemia, IgA nephropathy / Berger's disease, Immune-mediated necrotizing myopathy (IMNM), Immune thrombocytopenia (ITP) / autoimmune thrombocytopenic purpura / autoimmune thrombocytopenia, Inclusion body myositis, IgG4-related sclerosing disease (ISD), Interstitial cystitis, Juvenile idiopathic arthritis / Adult-onset Still's disease. Juvenile polymyositis | Juvenile dermatomyositis | juvenile myositis, Kawasaki disease, Lambert-Eaton myasthenic syndrome (LEMS), Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD) | linear IgA bullous dermatosis (LABD), Lupus nephritis, Lyme disease / chronic Lyme disease / post-treatment Lyme disease syndrome (PTLDS), Lymphocytic colitis / microscopic colitis, Lymphocytic hypophystitis / autoimmune hypophystitis, Ménière’s disease, Microscopic polyangiitis (MPA) / ANCA-associated vasculitis, Mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease, Multifocal motor neuropathy, Multiple sclerosis (MS), ATTORNEY DOCKET NO.1143252.008013 Myalgic encephalomyelitis (ME) / Chronic fatigue syndrome (CFS), Myasthenia gravis (MG), Narcolepsy, Neuromyelitis Optica / Devic's disease, Ocular cicatricial pemphigoid, Opsoclonus- myoclonus syndrome (OMS), Palindromic rheumatism, Paraneoplastic cerebellar degeneration, Paraneoplastic pemphigus, Parry-Romberg syndrome (PRS) / Hemifacial atrophy (HFA) / Progressive facial hemiatrophy, Paroxysmal nocturnal hemoglobinuria (PNH), Peripheral uveitis / pars planitis, PANS / PANDAS, Parsonage-Turner syndrome, Pemphigus gestationis / herpes gestationis, Pemphigus foliaceus, Pemphigus vulgaris, Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Polymyalgia rheumatica, Polymyositis, Postural orthostatic tachycardia syndrome (POTS), Primary biliary cirrhosis (PBC) / primary biliary cholangitis, Primary sclerosing cholangitis (PSC), Psoriasis, Palmoplantar Pustulosis, Psoriatic arthritis, Pulmonary fibrosis, idiopathic (IPF), Pure red cell aplasia (PRCA), Pyoderma gangrenosum, Rasmussen's encephalitis, Raynaud’s syndrome / phenomenon, Reactive arthritis / Reiter’s syndrome, Reflex sympathetic dystrophy syndrome (RSD) / Complex regional pain syndrome (CRPS), Relapsing polychondritis, Restless leg syndrome (RLS) / Willis-Ekbom disease, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidt syndrome / autoimmune polyendocrine syndrome type II, Scleritis, Scleroderma, Sclerosing Mesenteritis / Mesenteric Panniculitis, Serpiginous choroidopathy, Sjögren’s syndrome, Stiff person syndrome (SPS), Small fiber sensory neuropathy, Systemic lupus erythematosus (SLE), Subacute bacterial endocarditis (SBE), Subacute cutaneous lupus, Susac syndrome, Sydenham's chorea, Sympathetic ophthalmia, Takayasu’s arteritis (vasculitis), Testicular autoimmunity (vasculitis, orchitis), Tolosa-Hunt syndrome, Transverse myelitis (TM), Tubulointerstitial nephritis uveitis syndrome (TINU), Ulcerative colitis (UC), Undifferentiated connective tissue disease (UCTD), Uveitis | anterior / intermediate / posterior, Vasculitis, VEXAS Syndrome, Vitiligo, and Vogt-Koyanagi-Harada syndrome (VKH), and / or to prevent or inhibit at least one pathologic symptom associated therewith.

[0182] In any of the aspects and embodiments herein including methods and uses, in some cases, the therapy or prophylaxis is for the treatment or prevention of Addison disease, arthritis, celiac disease, lupus, Grave’s disease, myasthenia gravis, multiple sclerosis, ITP, rheumatoid arthritis, colitis, inflammatory bowel disease, pernicious anemia, Hashimoto’s thyroiditis Sjogren’s disease, asthma, type 2 diabetes, and autoimmune type I diabetes and / or to prevent or inhibit at least one pathologic symptom associated therewith.

[0183] In any of the aspects and embodiments herein including methods and uses, in some cases, the therapy or prophylaxis is for the treatment or prevention of an inflammatory disease selected from the group consisting of Fatty liver disease, Endometriosis, Type 2 diabetes, mellitus, Type 1 diabetes mellitus, Inflammatory bowel disease (IBD), Asthma, Rheumatoid arthritis, asthma, Obesity, ATTORNEY DOCKET NO.1143252.008013 Fibromyalgia, Lupus SLE, osteoarthritis, Rheumatoid Arthritis, Shingles Herpes Zoster, and Vasculitis and / or to prevent or inhibit at least one pathologic symptom associated therewith.

[0184] In any of the aspects and embodiments herein including methods and uses, in some cases, the therapy or prophylaxis is for the treatment or prevention of a neurodegenerative or neuroinflammatory disease, e.g., Alzheimer's disease, Amyotrophic lateral sclerosis, Friedreich ataxia, Huntington's disease, Lewy body disease, aphasia, Parkinson's disease or Spinal muscular atrophy and / or to prevent or inhibit at least one pathologic symptom associated therewith.

[0185] In any of the aspects and embodiments herein including methods and uses, in some cases, the therapy or prophylaxis may further include the administration of an additional agent(s) such as an anti-inflammatory agent or immunosuppressant. In some instances the anti-inflammatory or immunosuppressant may comprise a small molecule or a biologic.

[0186] Examples of such anti-inflammatory agents which may be administered in association with the subject IL-2 fusion polypeptides, nucleic acids, cells which express said IL-2 fusion polypeptides and compositions containing include nonsteroidal anti-inflammatory drugs (NSAIDs) e.g., aspirin, ibuprofen, and naproxen; paracetamol, Antileukotrienes or leukotriene-related enzyme inhibitors (arachidonate 5-lipoxygenase) or leukotriene receptor antagonists (cysteinyl leukotriene receptors), and the like.

[0187] Other examples of anti-inflammatory agents include glucocorticoids or steroids. Specific examples include Group A – Hydrocortisone type steroids such as Hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, prednisolone, methylprednisolone, and prednisone; a Group B – Acetonide type steroids such as (Amcinonide, budesonide, desonide, fluocinolone cetonide, fluocinonide, halcinonide, triamcinolone acetonide, and Deflazacort (O-isopropylidene derivative); a Group C – Betamethasone type steroids such as declometasone, betamethasone, dexamethasone, fluocortolone, halometasone, and mometasone; Group D1 and D2 esters–such as Group D1 and D2 esters Alclometasone dipropionate, betamethasone dipropionate, betamethasone valerate, clobetasol propionate, clobetasone butyrate, fluprednidene acetate, and mometasone furoate; and Group D2 – Labile prodrug esters such as Ciclesonide, cortisone acetate, hydrocortisone aceponate, hydrocortisone acetate, hydrocortisone buteprate, hydrocortisone butyrate, hydrocortisone valerate, prednicarbate, and tixocortol pivalate.

[0188] Examples of biologic anti-inflammatory agents which may be administered in association with the subject IL-2 fusion polypeptides, nucleic acids, cells which express said IL-2 fusion polypeptides and compositions containing include TNF inhibitors (TNF blockers or anti-TNFs), B cell inhibitors, interleukin inhibitors, (e.g., IL-1, IL-6 and / or IL-17 inhibitors). Specific examples of TNF inhibitors include adalimumab (Humira), certolizumab pegol (Cimzia), etanercept (Enbrel), ATTORNEY DOCKET NO.1143252.008013 golimumab (Simponi, Simponi Aria), infliximab (Remicade), and biosimilars thereof such as Amjevita, Avsola, Inflectra, and Renflexis; specific examples of B cell inhibitors include belimumab (Benlysta) and rituximab (Rituxan), and rituximab biosimilars such as Riabni, Ruxience and Truxima; specific examples of interleukin inhibitors include anakinra (Kineret), canakinumab (Ilaris), guselkumab (Tremfya), ixekizumab (Taltz), risankizumab (Skyrizi), sarilumab (Kevzara), secukinumab (Cosentyx), tocilizumab (Actemra) and ustekinumab (Stelara) and Selective Co-stimulation Modulators include Abatacept (Orencia). Preferred examples of the foregoing include Remicade® (infliximab), Humira® (adalimumab), Simponi® (golimumab) and Cimzia® (certolizumab pegol).

[0189] Such other anti-inflammatory agents may be administered by various means e.g., topically on the skin, eye, and mucous membranes; via inhalation for delivery to the nasal mucosa, sinuses, bronchi, and lungs; orally and / or systemically.

[0190] Other agents used to treat autoimmunity which may be administered in association with the subject IL-2 fusion polypeptides, nucleic acids, cells which express said IL-2 fusion polypeptides and compositions containing include methotrexate, Cytostatics, Alkylating agents such as nitrogen mustards (cyclophosphamide), nitrosoureas, platinum compounds, and others; Cyclophosphamide; Antimetabolites such as folic acid analogues, methotrexate; purine analogues, such as azathioprine and mercaptopurine; pyrimidine analogues, such as fluorouracil and protein synthesis inhibitors; Azathioprine and mercaptopurine; Azathioprine; Cytotoxic antibiotics such as dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin. Administration route and dosing

[0191] In any of the aspects and embodiments of methods described herein, an effective amount of such an inventive IL-2 fusion protein (and optionally any additional agent), a nucleic acid or vector encoding, or a cell comprising such or a pharmaceutical composition comprising such may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In certain embodiments, the administration is subcutaneous administration, which may exhibit a less toxic response in the subject compared to intravenous injection. Dosing can be by any suitable route (e.g., injections, such as intravenous or subcutaneous injections) and may depend in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein. ATTORNEY DOCKET NO.1143252.008013

[0192] IL-2 fusion proteins of the disclosure would be formulated (e.g., as a pharmaceutical composition), dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The IL-2 fusion protein need not, but may optionally be, formulated (e.g., as a pharmaceutical composition) with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents may depend on the amount of the IL-2 fusion protein present in the composition, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically / clinically determined to be appropriate.

[0193] For the prevention or treatment of a disease, a disorder, or a condition, the appropriate dosage of a IL-2 fusion protein of the disclosure (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the severity and course of the disease, whether the IL-2 fusion protein is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the IL-2 fusion protein, and the discretion of the attending physician. In certain embodiments, the IL-2 fusion protein may be suitably administered to the patient at one time or over a series of treatments.

[0194] As a general proposition, an effective amount (e.g., therapeutically effective amount) of the IL-2 fusion protein administered to humans may be in the range of about 0.01 to about 100 mg / kg of patient body weight whether by one or more administrations. In some embodiments, a IL-2 fusion protein may be administered at about 0.01 to about 45 mg / kg, about 0.01 to about 40 mg / kg, about 0.01 to about 35 mg / kg, about 0.01 to about 30 mg / kg, about 0.01 to about 25 mg / kg, about 0.01 to about 20 mg / kg, about 0.01 to about 15 mg / kg, about 0.01 to about 10 mg / kg, about 0.01 to about 5 mg / kg, or about 0.01 to about 1 mg / kg daily, for example. In one embodiment, a IL-2 fusion protein described herein is administered to a human at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg or about 1400 mg on day 1 of 21-day cycles. The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the IL-2 fusion protein would be in the range from about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or ATTORNEY DOCKET NO.1143252.008013 any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, every week or every three weeks (e.g., such that the patient receives from about two to about twenty, or, for example, about six doses). An initial higher loading dose, followed by one or more lower doses, may be administered. The progress of this therapy is easily monitored by conventional techniques and assays. Monotherapy and combination therapy

[0195] In any of the aspects and embodiments of methods and uses described herein, an effective amount of such a IL-2 fusion protein may be administered alone to the subject.

[0196] In some embodiments, an effective amount of such a IL-2 fusion protein may be administered in combination with at least one additional agent to the subject. The additional agent(s) (e.g., therapeutic agents and / or adjuvants) may be or may comprise any appropriate agents including but not limited to those described herein. In certain embodiments, the additional agent(s) may be contained in a pharmaceutical composition together with a IL-2 fusion protein according to the present disclosure. In certain embodiments, the additional agent(s) may not be contained in the pharmaceutical composition comprising a IL-2 fusion protein according to the present disclosure but may be administered together (e.g., simultaneously) with the IL-2 fusion protein. In certain embodiments, the additional agent(s) may be administered separately from (e.g., prior to or following the administration of) the IL-2 fusion protein. In some cases, administration of the IL-2 fusion protein and administration of the additional agent(s) may occur within about one month, or within about one, two or three weeks, or within about one, two, three, four, five, or six days, of each other.

[0197] In some embodiments, an effective amount of such a IL-2 fusion protein may be administered in combination with at least one additional therapy to the subject. The additional therapy(ies) may be or may comprise radiation therapy (e.g., gamma irradiation), surgery, bone marrow transplantation, chemotherapy, or any combination of the foregoing. In certain embodiments, the additional therapy(ies) may be administered together (e.g., simultaneously) with the IL-2 fusion protein. In certain embodiments, the additional therapy(ies) may be administered separately from (e.g., prior to or following the administration of) the IL-2 fusion protein. In some cases, administration of the IL-2 fusion protein and administration of the additional therapy(ies) may occur within about one month, or within about one, two or three weeks, or within about one, two, three, four, five, or six days, of each other. ATTORNEY DOCKET NO.1143252.008013

[0198] Any IL-2 fusion proteins of the disclosure may be used in combination therapies described herein, such as in combination with other agents used to treat inflammatory and / or autoimmune dosorders. Manufacturing methods

[0199] In a further aspect, the present disclosure provides manufacturing methods, such as methods of manufacturing any of the IL-2 fusion proteins described herein and methods of manufacturing any of the isolated, recombinant, and / or host cells or any of the populations of cells comprising isolated, recombinant, and / or host cells according to the present disclosure.

[0200] Any of the IL-2 fusion proteins described herein may be produced or manufactured using any appropriate methods, including recombinant methods, e.g., in vitro, ex vivo, or in vivo.

[0201] In some embodiments, a method of manufacturing a IL-2 fusion protein according to the present disclosure may comprise culturing a cell according to the present disclosure in a condition that allows for expression of the IL-2 fusion protein and harvesting and purifying the IL-2 fusion protein from the cell culture.

[0202] In some embodiments, a method of manufacturing a cell or a population of cells according to the present disclosure may introducing a nucleic acid and / or a vector according to the present disclosure into one or more cells. Such a method may be performed in vitro, ex vivo, or in vivo.

[0203] Physical methods for introducing a nucleic acid into a cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.

[0204] Biological methods for introducing a nucleic acid of interest into a cell include the use of DNA and RNA vectors. In certain embodiments, nucleic acid encoding the IL-2 fusion protein may be isolated and inserted into one or more vectors (e.g., viral vectors, plasmids, and the like) for further cloning and / or expression in an isolated, recombinant, and / or cell. Such nucleic acids may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the IL-2 fusion protein). Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes ATTORNEY DOCKET NO.1143252.008013 simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos.5,350,674 and 5,585,362.

[0205] Chemical means for introducing a nucleic acid into a cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0206] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In some cases, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a "collapsed" structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0207] The example provided below illustrates exemplary embodiments of the present disclosure. These examples are not meant to constrain the present invention to any particular application or theory of operation. EXAMPLE Materials and Methods i. Protein expression and purification

[0208] The genes encoding IL-2, IL-2(N88D), human serum albumin (HSA), and antibody fragments were designed according to preferred Pichia pastoris codon usage and synthesized by Genewiz. Hisx6 tag was appended to the N-terminus of IL-2 to facilitate purification. The proteins were expressed from Pichia pastoris with pPIC9 as vector and GS115 as host, both from Invitrogen. For pilot-scale protein production, recombinant Pichia pastoris was cultured in a 2.5L bioreactor ATTORNEY DOCKET NO.1143252.008013 (Applikon Biotechnology) and the secreted protein was purified to homogeneity by sequential chromatographic purification composed of immobilized metal affinity chromatography (IMAC) (Ni Sepharose FF, GE Healthcare), hydrophobic interaction chromatography (HIC) (Phenyl HP, GE Healthcare), size exclusion chromatography (SEC) (G-25 Fine, GE Healthcare) and ion exchange chromatography (IEC) (Q Sepharose FF, GE Healthcare)(13). ii. Protein-protein interaction assay

[0209] The binding between IL-2 and its receptor or antibody and its target were determined by ELISA. Flat Nunc-Immuno 96-well plates (Thermo Fisher Scientific) were coated overnight at 4 °C with 1 µg / well of purified proteins in PBS. The plates were then washed with 0.1% Tween 20 (RPI) and blocked for 1 hour at room temperature (RT) with 1% BSA (RPI) in 0.1% Tween 20. After washing, serial diluted IL-2Rα, Fc tag, IL-2Rβ, Fc tag and CTLA-4, Fc tag (Acro Biosystems) were added to plates in blocking solution and incubated for 1 h at RT. Plates were then washed and incubated with horseradish peroxidase (HRP) conjugated goat anti-human IgG Fc secondary antibody (Thermo Fisher Scientific) for 1 h at RT in the dark. Following a final wash, plates were developed using 1- Step™ Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) for 5–10 minutes until the reaction was stopped by the addition of 1.8 M H2SO4. The plates were read at 450 nm absorbance using a microplate reader. iii. Cellular signaling assay

[0210] The immune cell signaling capacity of IL-2 was determined by Ex vivo human peripheral blood mononuclear cell (PBMC) STAT5 phosphorylation assay. Human peripheral blood mononuclear cells (PBMCs) were freshly isolated from platelet cones by centrifugation over Histopaque. Washed cells were either untreated or stimulated by dilutions of recombinant proteins in BD™ Staining Buffer, for 30 mins at 37oC, concurrently staining with anti-human CD127-AF647 (Cat. No.558598). The cells were fixed using BD™ Cytofix Fixation Buffer, and permeabilized using BD™ Phosflow Perm Buffer III, as per BD protocols. The fixed cells were further stained with anti-human STAT5-AF488 (Cat No.612598), anti-human CD4-PerCP Cy5.5 (Cat. No.560650), anti-human CD8-APC Cy7 (Cat. No. 557760), anti-human CD25-BV421 (Cat. No.562442) for 30 minutes on ice. Samples were analyzed by flow cytometry on a BioRad Cell analyzer ZE5 (five laser, 27 color capability; made available by the Dartmouth Shared Resources and NCI Cancer Center Support Grant 5P30CA023108-37). Fluorescence compensation settings were established using single-color BD™ CompBead control samples and the software compensation procedure. FlowJo Software was utilized for cell sub- population definition, as described in Protocol III of the BD Phosflow™ Protocols for Human PBMCs). iv. Efficacy in xenogeneic GvHD model

[0211] The efficacy of IL-2 was tested in auto-immune like and highly inflammatory humanized xenogeneic GvHD model. Human PBMC were obtained from DartLab and purified by Ficoll-Hypaque ATTORNEY DOCKET NO.1143252.008013 density centrifugation and suspended in RPMI-1640. PBMC were activated overnight with 12.5 ng ml−1 anƟ-CD3 and 25 ng ml−1 anƟ-CD28. NOD.Cg-Prkdcscid IL2rγtm1Wjl (NSG) mice were purchased from the Dartmouth Mouse Modeling Shared Resource. NSG recipient mice received an intravenous injection of 30 × 106 PBMCs. The next day of PBMC injection, the mice were treated with 10 ug / mouse dose of IL-2. The mice received an intraperitoneal injection of IL-2 every other day for 6 doses. Mice were weighed two to three times weekly, and the appearance of GVHD-like symptoms including weight loss (>20%), hunched posture, ruffled fur, reduced mobility, and tachypnoea was used to determine time of euthanasia and is indicated as time of survival. RESULTS i. IL-2(N88D) fails to ameliorate xenogeneic GvHD

[0212] Asn at position 88 of IL-2 (N88) plays an important role in IL-2Rβ binding. IL-2 muteins such as N88D showed dramatically compromised IL-2Rβ binding. IL-2(N88D) demonstrated improved Treg selectivity in ex vivo cellular signaling assay and in vivo cellular expansion test in normal animal(14). To improve their in vivo half-life, both WT IL-2 and IL-2(N88D) were fused to human serum albumin (HSA)(15). Fusion proteins were secreted from Pichia pastoris and purified to homogeneity (See Fig. 1 which contains an exemplary schematic structure of HSA fused wild type and variant IL-2 and SDS- PAGE analysis of the purified proteins). ii. Comparison of IL-2Rα and IL-2Rβ binding affinity of WT IL-2 and IL-2(N88D)

[0213] The IL-2Rα and IL-2Rβ binding affinity of WT IL-2 and IL-2(N88D) were compared by ELISA. As illustrated by the receptor binding analysis in Figure 2, these two proteins showed identical IL-2Rα binding, however the IL-2(N88D) showed diminished IL-2Rβ binding (See Fig.2). iii. IL-2 signaling leads to STAT5 phosphorylation.

[0214] As shown in by the Cellular signaling assay results in Figure 3, the content of pSTAT5 in different population of immune cells were detected by flow cytometry. WT IL-2 can induce pSTAT5 formation in both Treg and CD8 T cells. IL-2(N88D) retained the ability to act on Treg while was unable to trigger signaling in CD8 T cells (See Fig.3). iv. Comparison of Efficacy of WT IL-2 and IL-2 Mutein (N88D) ATTORNEY DOCKET NO.1143252.008013

[0215] After the engrafting of human PBMC into immune-deficient NSG mice, human CD8 T will recognize and attack murine cells, leading to autoimmune-like and highly inflammatory GvHD(16). As is shown in Figure 4, the efficacy in xenogeneic GvHD model Disease progressed rapidly for both groups, and the medium survival time (MST) of WT IL-2 and IL-2(N88D) are 10.5d and 11.25d, respectively, indicating lack of efficacy (See Fig.4). v. CTLA-4 targeting improves Treg selectivity of IL-2

[0216] Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) is a protein receptor preferentially expressed on the surface of Tregs, that acts as an immune checkpoint. It inhibits the activation of T cells by competing with the stimulatory receptor CD28 for binding to CD80 / CD86 on antigen-presenting cells (APCs)(17, 18). CTLA-4-targeted antibodies, such as ipilimumab selectively depletes Treg by the ADCC effector function of Fc and has been approved for the treatment of melanoma(19). To deliver IL-2 to CTLA-4 expressing Treg, as is shown in the schematic structure of CTLA-4 targeted IL-2(N88D) and its SDS-PAGE analysis, Anti-CTLA-4 scFv from ipilimumab was fused in tandem with IL-2(N88D) (20)(See Fig.5). vi. Protein-protein interaction

[0217] As is shown in Figure 6, a protein-protein interaction assay by ELISA showed that anti-CTLA-4 scFv confers high CTLA-4 binding, while only slightly decreasing IL-2Rα binding (See Fig.6). vii. Cellular signaling assay

[0218] As is shown in Figure 7, a cellular signaling assay revealed that anti-CTLA-4 scFv selectively enhanced Treg activity (See Fig.7). viii. Efficacy in xenogeneic GvHD model

[0219] When compared to untargeted IL-2, CTLA-4 targeting significantly delayed the onset of GvHD and extended the survival of NSG mice (12d vs 22.5d) (See Fig.8). ix. Fusion of IL-2 to Other Targets (CD39, TNFR2, GITR and TIGIT)

[0220] The promising results of CTLA-4 targeted IL-2 encouraged us to explore additional Treg specific targets, i.e., CD39, TNFR2, GITR and TIGIT. ATTORNEY DOCKET NO.1143252.008013

[0221] CD39, also known as ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1), is expressed on the surface of Tregs. It hydrolyzes extracellular ATP and ADP into AMP, which is further converted into adenosine by CD73, another ectonucleotidase. Adenosine acts through its receptors to exert anti-inflammatory effects, such as inhibiting T cell activation and promoting tissue protection(21).

[0222] Tumor Necrosis Factor Receptor 2 (TNFR2) is a receptor for tumor necrosis factor-alpha (TNF-α) and is distinctively expressed on Tregs, particularly those with strong suppressive capabilities. Engagement of TNFR2 by TNF-α or agonistic antibodies leads to enhanced survival, proliferation, and suppressive function of Tregs(22).

[0223] Glucocorticoid-Induced TNFR-Related Protein (GITR) is a receptor belonging to the tumor necrosis factor receptor (TNFR) superfamily and is expressed on various immune cells, including Tregs and effector T cells. The engagement of GITR by its ligand GITRL, typically expressed on antigen-presenting cells (APCs), can lead to diverse effects depending on the cellular context. In Tregs, GITR signaling can disrupt their suppressive activity, leading to reduced inhibition of effector T cells and enhanced immune responses(23).

[0224] T cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory receptor expressed on T cells, including Tregs, and its role is pivotal in dampening immune activation. TIGIT competes with the stimulatory receptor CD226 (DNAM-1) for binding to shared ligands such as CD155 and CD112 on antigen-presenting cells (APCs) and other cell types. When TIGIT engages its ligands, it transduces inhibitory signals that suppress T cell proliferation and cytokine production, contributing to immune homeostasis(24).

[0225] Figure 9 contains the schematic structure of TNFR2, CD39, GITR and TIGIT targeted IL-2 and SDS-PAGE analysis of purified proteins. As shown therein to facilitate targeted delivery of IL-2 to CD39(25), TNFR2(26), GITR(27) and TIGIT(28) expressing Tregs, VHH antibodies were fused in tandem with IL-2(N88D) (See Fig.9). x. Efficacy of IL-2 Fused to Other Targets (CD39, TNFR2, GITR and TIGIT) in GVHD Model

[0226] As noted above, the promising results of CTLA-4 targeted IL-2 encouraged the inventors to explore additional Treg specific targets, i.e., CD39, TNFR2, GITR and TIGIT. As is shown in Figure 10, the efficacy of these differently targeted IL-2 differed greatly in humanized xenogeneic GvHD model and the MST for GITR, TIGIT, TNFR2 and CD39 targeted IL-2 are 16d, 41.75d, 37.5d and 51d, respectively. CD39 targeting showed the most promising delayed disease onset and survival extension (See Fig.10). ATTORNEY DOCKET NO.1143252.008013 References Cited in Specification 1. Wang L, Wang FS, Gershwin ME.2015. Human autoimmune diseases: a comprehensive update. J Intern Med 278:369-395. 2. Surace AEA, Hedrich CM.2019. The Role of EpigeneƟcs in Autoimmune / Inflammatory Disease. Front Immunol 10:1525. 3. Sakaguchi S, Mikami N, Wing JB, Tanaka A, Ichiyama K, Ohkura N.2020. Regulatory T Cells and Human Disease. Annu Rev Immunol 38:541-566. 4. Raffin C, Vo LT, Bluestone JA.2020. 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[0002] ATTORNEY DOCKET NO.1143252.008013 SEQUENCE APPENDIX IL-2 Protein Sequence: SEQ ID NO: 1 HHHHHHAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTggggsgsggggsDAHKSEVA HRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYG EMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRY KAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTD LTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDV CKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNC ELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPV SDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL IL-2 DNA sequence: SEQ ID NO: 2 CATCATCACCATCACCATGCCCCAACCTCCTCCTCCACCAAGAAGACCCAATTGCAACTGGAGCACTTGCTGTT GGACTTGCAAATGATCCTGAACGGTATCAACAACTACAAGAACCCAAAGCTGACCAGAATGCTGACCTTCAAG TTCTACATGCCAAAGAAGGCTACCGAGCTGAAGCACTTGCAATGTTTGGAGGAGGAGTTGAAGCCATTGGAG GAGGTCCTGAACTTGGCTCAATCCAAGAACTTCCACCTGAGACCAAGAGACTTGATCTCCAACATCAACGTCA TCGTCTTGGAGCTGAAGGGTTCCGAGACCACCTTCATGTGTGAGTACGCTGACGAGACCGCTACCATCGTCGA GTTCTTGAACAGATGGATCACCTTCTCCCAATCCATCATCTCCACCCTGACCggtggtggaggttctggatctggtggagg tggttctGACGCTCACAAGTCTGAAGTTGCTCACAGATTCAAGGACTTGGGTGAAGAAAACTTCAAGGCTTTGGT TTTGATTGCTTTCGCTCAATACTTGCAACAATCTCCATTCGAAGACCACGTTAAGTTGGTTAACGAAGTTACTG AATTTGCTAAGACTTGTGTTGCTGACGAATCTGCTGAAAACTGTGACAAGTCTTTGCACACTTTGTTCGGTGAC AAGTTGTGTACTGTTGCTACTTTGAGAGAAACTTACGGTGAAATGGCTGACTGTTGTGCTAAGCAAGAACCAG AAAGAAACGAATGTTTCTTGCAACACAAGGACGACAACCCAAACTTGCCAAGATTGGTTAGACCAGAAGTCG ACGTTATGTGTACTGCTTTCCACGACAACGAAGAAACTTTCTTGAAGAAGTACTTGTACGAAATTGCTAGAAG ACACCCATACTTCTACGCTCCAGAATTGTTGTTCTTCGCTAAGAGATACAAGGCTGCTTTCACTGAATGTTGTCA AGCTGCTGACAAGGCTGCTTGTTTGTTGCCAAAGTTGGACGAATTGAGAGACGAAGGTAAGGCTTCTTCTGCT AAGCAAAGATTGAAGTGTGCTTCTTTGCAAAAGTTCGGTGAAAGAGCTTTCAAAGCTTGGGCTGTTGCTAGAT TGTCTCAAAGATTCCCAAAGGCTGAATTTGCTGAAGTTTCTAAGTTGGTTACTGACTTGACTAAGGTTCACACT GAATGTTGTCACGGTGACTTGTTGGAATGTGCTGACGACAGAGCTGACTTGGCTAAGTACATTTGTGAAAACC AAGACTCTATTTCTTCTAAGTTGAAGGAATGTTGTGAAAAGCCATTGTTGGAAAAGTCTCACTGTATTGCTGAA GTTGAAAACGACGAAATGCCAGCTGACTTGCCATCTTTGGCTGCTGACTTCGTTGAATCTAAGGACGTTTGTA AGAACTACGCTGAAGCTAAGGACGTTTTCTTGGGTATGTTCTTGTACGAATACGCTAGAAGACACCCAGACTA CTCTGTTGTTTTGTTGTTGAGATTGGCTAAGACTTACGAAACTACTTTGGAAAAGTGTTGTGCGGCCGCTGACC CACACGAATGTTACGCTAAGGTTTTCGACGAATTTAAGCCATTGGTTGAAGAACCACAAAACTTGATTAAGCA AAACTGTGAATTGTTCGAACAATTGGGTGAATACAAGTTCCAAAACGCTTTGTTGGTTAGATACACTAAGAAG GTTCCACAAGTTTCTACTCCAACTTTGGTTGAAGTTTCTAGAAACTTGGGTAAGGTTGGTTCTAAGTGTTGTAA GCACCCAGAAGCTAAGAGAATGCCATGTGCTGAAGACTACTTGTCTGTTGTTTTGAACCAATTGTGTGTTTTGC ACGAAAAGACTCCAGTTTCTGACAGAGTTACTAAGTGTTGTACTGAATCTTTGGTTAACAGAAGACCATGTTTC TCTGCTTTGGAAGTTGACGAAACTTACGTTCCAAAGGAATTTAACGCTGAAACTTTCACTTTCCACGCTGACAT TTGTACTTTGTCTGAAAAGGAAAGACAAATTAAGAAGCAAACTGCTTTGGTTGAATTGGTTAAGCACAAGCCA AAGGCTACTAAGGAACAATTGAAGGCTGTTATGGACGACTTCGCTGCTTTCGTTGAAAAGTGTTGTAAGGCTG ATTORNEY DOCKET NO.1143252.008013 ACGACAAGGAAACTTGTTTCGCTGAAGAAGGTAAGAAGTTGGTTGCTGCTTCTCAAGCTGCTTTGGGTTTGTA A IL-2(N88D) Protein Sequence: SEQ ID NO: 3 HHHHHHAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTggggsgsggggsDAHKSEVA HRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYG EMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRY KAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTD LTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDV CKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNC ELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPV SDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAV MDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL IL-2(N88D) DNA Sequence: SEQ ID NO: 4 CATCATCACCATCACCATGCCCCAACCTCCTCCTCCACCAAGAAGACCCAATTGCAACTGGAGCACTTGCTGTT GGACTTGCAAATGATCCTGAACGGTATCAACAACTACAAGAACCCAAAGCTGACCAGAATGCTGACCTTCAAG TTCTACATGCCAAAGAAGGCTACCGAGCTGAAGCACTTGCAATGTTTGGAGGAGGAGTTGAAGCCATTGGAG GAGGTCCTGAACTTGGCTCAATCCAAGAACTTCCACCTGAGACCAAGAGACTTGATCTCCGACATCAACGTCA TCGTCTTGGAGCTGAAGGGTTCCGAGACCACCTTCATGTGTGAGTACGCTGACGAGACCGCTACCATCGTCGA GTTCTTGAACAGATGGATCACCTTCTCCCAATCCATCATCTCCACCCTGACCggtggtggaggttctggatctggtggagg tggttctGACGCTCACAAGTCTGAAGTTGCTCACAGATTCAAGGACTTGGGTGAAGAAAACTTCAAGGCTTTGGT TTTGATTGCTTTCGCTCAATACTTGCAACAATCTCCATTCGAAGACCACGTTAAGTTGGTTAACGAAGTTACTG AATTTGCTAAGACTTGTGTTGCTGACGAATCTGCTGAAAACTGTGACAAGTCTTTGCACACTTTGTTCGGTGAC AAGTTGTGTACTGTTGCTACTTTGAGAGAAACTTACGGTGAAATGGCTGACTGTTGTGCTAAGCAAGAACCAG AAAGAAACGAATGTTTCTTGCAACACAAGGACGACAACCCAAACTTGCCAAGATTGGTTAGACCAGAAGTCG ACGTTATGTGTACTGCTTTCCACGACAACGAAGAAACTTTCTTGAAGAAGTACTTGTACGAAATTGCTAGAAG ACACCCATACTTCTACGCTCCAGAATTGTTGTTCTTCGCTAAGAGATACAAGGCTGCTTTCACTGAATGTTGTCA AGCTGCTGACAAGGCTGCTTGTTTGTTGCCAAAGTTGGACGAATTGAGAGACGAAGGTAAGGCTTCTTCTGCT AAGCAAAGATTGAAGTGTGCTTCTTTGCAAAAGTTCGGTGAAAGAGCTTTCAAAGCTTGGGCTGTTGCTAGAT TGTCTCAAAGATTCCCAAAGGCTGAATTTGCTGAAGTTTCTAAGTTGGTTACTGACTTGACTAAGGTTCACACT GAATGTTGTCACGGTGACTTGTTGGAATGTGCTGACGACAGAGCTGACTTGGCTAAGTACATTTGTGAAAACC AAGACTCTATTTCTTCTAAGTTGAAGGAATGTTGTGAAAAGCCATTGTTGGAAAAGTCTCACTGTATTGCTGAA GTTGAAAACGACGAAATGCCAGCTGACTTGCCATCTTTGGCTGCTGACTTCGTTGAATCTAAGGACGTTTGTA AGAACTACGCTGAAGCTAAGGACGTTTTCTTGGGTATGTTCTTGTACGAATACGCTAGAAGACACCCAGACTA CTCTGTTGTTTTGTTGTTGAGATTGGCTAAGACTTACGAAACTACTTTGGAAAAGTGTTGTGCGGCCGCTGACC CACACGAATGTTACGCTAAGGTTTTCGACGAATTTAAGCCATTGGTTGAAGAACCACAAAACTTGATTAAGCA AAACTGTGAATTGTTCGAACAATTGGGTGAATACAAGTTCCAAAACGCTTTGTTGGTTAGATACACTAAGAAG GTTCCACAAGTTTCTACTCCAACTTTGGTTGAAGTTTCTAGAAACTTGGGTAAGGTTGGTTCTAAGTGTTGTAA GCACCCAGAAGCTAAGAGAATGCCATGTGCTGAAGACTACTTGTCTGTTGTTTTGAACCAATTGTGTGTTTTGC ACGAAAAGACTCCAGTTTCTGACAGAGTTACTAAGTGTTGTACTGAATCTTTGGTTAACAGAAGACCATGTTTC TCTGCTTTGGAAGTTGACGAAACTTACGTTCCAAAGGAATTTAACGCTGAAACTTTCACTTTCCACGCTGACAT ATTORNEY DOCKET NO.1143252.008013 TTGTACTTTGTCTGAAAAGGAAAGACAAATTAAGAAGCAAACTGCTTTGGTTGAATTGGTTAAGCACAAGCCA AAGGCTACTAAGGAACAATTGAAGGCTGTTATGGACGACTTCGCTGCTTTCGTTGAAAAGTGTTGTAAGGCTG ACGACAAGGAAACTTGTTTCGCTGAAGAAGGTAAGAAGTTGGTTGCTGCTTCTCAAGCTGCTTTGGGTTTGTA A CTLA4-IL2(N88D) Protein Sequence SEQ ID NO: 5 HHHHHHAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTggggsgsggggsEIVLTQSPG TLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYY CQQYGSSPWTFGQGTKVEIKGGGGGGSGGGGSGGGGSGGQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTM HWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYW GQGTLVTVSSggggsgsggggsDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVAD ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNE ETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERA FKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAAD PHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPE AKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKE RQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL CTLA4-IL2(N88D) DNA Sequence SEQ ID NO: 6 CATCATCACCATCACCATGCCCCAACCTCCTCCTCCACCAAGAAGACCCAATTGCAACTGGAGCACTTGCTGTT GGACTTGCAAATGATCCTGAACGGTATCAACAACTACAAGAACCCAAAGCTGACCAGAATGCTGACCTTCAAG TTCTACATGCCAAAGAAGGCTACCGAGCTGAAGCACTTGCAATGTTTGGAGGAGGAGTTGAAGCCATTGGAG GAGGTCCTGAACTTGGCTCAATCCAAGAACTTCCACCTGAGACCAAGAGACTTGATCTCCGACATCAACGTCA TCGTCTTGGAGCTGAAGGGTTCCGAGACCACCTTCATGTGTGAGTACGCTGACGAGACCGCTACCATCGTCGA GTTCTTGAACAGATGGATCACCTTCTCCCAATCCATCATCTCCACCCTGACCggtggtggaggttctggatctggtggagg tggttctGAGATCGTCTTGACCCAATCTCCAGGTACCTTGTCCTTGTCTCCAGGTGAGAGAGCTACCTTGTCCTGT AGAGCTTCCCAATCCGTCGGTTCCTCCTACTTGGCTTGGTACCAACAAAAGCCAGGTCAAGCTCCAAGATTGTT GATCTACGGTGCTTTCTCCAGAGCTACTGGTATCCCAGACAGATTCTCTGGTTCTGGTTCTGGTACCGACTTCA CCTTGACCATCTCCAGATTGGAGCCAGAGGACTTCGCTGTCTACTACTGTCAACAATACGGTTCCTCTCCATGG ACCTTCGGTCAAGGTACCAAGGTCGAGATCAAGGGAGGTGGTGGTGGAGGTTCTGGAGGTGGTGGATCTGG TGGAGGTGGTTCTGGTGGACAAGTCCAATTGGTCGAGTCTGGTGGTGGTGTCGTCCAACCAGGTAGATCCTT GAGATTGTCCTGTGCTGCTTCTGGTTTCACCTTCTCCTCCTACACCATGCACTGGGTCAGACAAGCTCCAGGTA AGGGTTTGGAGTGGGTCACCTTCATCTCCTACGACGGTAACAACAAGTACTACGCTGACTCCGTCAAGGGTAG ATTCACCATCTCCAGAGACAACTCCAAGAACACCTTGTACTTGCAAATGAACTCCTTGAGAGCTGAGGACACT GCTATCTACTACTGTGCTAGAACTGGTTGGTTGGGTCCATTCGACTACTGGGGTCAAGGTACCTTGGTCACCG TCTCCTCCGggtggtggaggttctggatctggtggaggtggttctGACGCTCACAAGTCTGAAGTTGCTCACAGATTCAAGG ACTTGGGTGAAGAAAACTTCAAGGCTTTGGTTTTGATTGCTTTCGCTCAATACTTGCAACAATCTCCATTCGAA GACCACGTTAAGTTGGTTAACGAAGTTACTGAATTTGCTAAGACTTGTGTTGCTGACGAATCTGCTGAAAACT GTGACAAGTCTTTGCACACTTTGTTCGGTGACAAGTTGTGTACTGTTGCTACTTTGAGAGAAACTTACGGTGAA ATGGCTGACTGTTGTGCTAAGCAAGAACCAGAAAGAAACGAATGTTTCTTGCAACACAAGGACGACAACCCA AACTTGCCAAGATTGGTTAGACCAGAAGTCGACGTTATGTGTACTGCTTTCCACGACAACGAAGAAACTTTCTT ATTORNEY DOCKET NO.1143252.008013 GAAGAAGTACTTGTACGAAATTGCTAGAAGACACCCATACTTCTACGCTCCAGAATTGTTGTTCTTCGCTAAGA GATACAAGGCTGCTTTCACTGAATGTTGTCAAGCTGCTGACAAGGCTGCTTGTTTGTTGCCAAAGTTGGACGA ATTGAGAGACGAAGGTAAGGCTTCTTCTGCTAAGCAAAGATTGAAGTGTGCTTCTTTGCAAAAGTTCGGTGAA AGAGCTTTCAAAGCTTGGGCTGTTGCTAGATTGTCTCAAAGATTCCCAAAGGCTGAATTTGCTGAAGTTTCTAA GTTGGTTACTGACTTGACTAAGGTTCACACTGAATGTTGTCACGGTGACTTGTTGGAATGTGCTGACGACAGA GCTGACTTGGCTAAGTACATTTGTGAAAACCAAGACTCTATTTCTTCTAAGTTGAAGGAATGTTGTGAAAAGCC ATTGTTGGAAAAGTCTCACTGTATTGCTGAAGTTGAAAACGACGAAATGCCAGCTGACTTGCCATCTTTGGCT GCTGACTTCGTTGAATCTAAGGACGTTTGTAAGAACTACGCTGAAGCTAAGGACGTTTTCTTGGGTATGTTCTT GTACGAATACGCTAGAAGACACCCAGACTACTCTGTTGTTTTGTTGTTGAGATTGGCTAAGACTTACGAAACTA CTTTGGAAAAGTGTTGTGCGGCCGCTGACCCACACGAATGTTACGCTAAGGTTTTCGACGAATTTAAGCCATT GGTTGAAGAACCACAAAACTTGATTAAGCAAAACTGTGAATTGTTCGAACAATTGGGTGAATACAAGTTCCAA AACGCTTTGTTGGTTAGATACACTAAGAAGGTTCCACAAGTTTCTACTCCAACTTTGGTTGAAGTTTCTAGAAA CTTGGGTAAGGTTGGTTCTAAGTGTTGTAAGCACCCAGAAGCTAAGAGAATGCCATGTGCTGAAGACTACTTG TCTGTTGTTTTGAACCAATTGTGTGTTTTGCACGAAAAGACTCCAGTTTCTGACAGAGTTACTAAGTGTTGTAC TGAATCTTTGGTTAACAGAAGACCATGTTTCTCTGCTTTGGAAGTTGACGAAACTTACGTTCCAAAGGAATTTA ACGCTGAAACTTTCACTTTCCACGCTGACATTTGTACTTTGTCTGAAAAGGAAAGACAAATTAAGAAGCAAACT GCTTTGGTTGAATTGGTTAAGCACAAGCCAAAGGCTACTAAGGAACAATTGAAGGCTGTTATGGACGACTTCG CTGCTTTCGTTGAAAAGTGTTGTAAGGCTGACGACAAGGAAACTTGTTTCGCTGAAGAAGGTAAGAAGTTGG TTGCTGCTTCTCAAGCTGCTTTGGGTTTGTAA CD39-IL2(N88D) Protein Sequence SEQ ID NO: 7 HHHHHHAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTggggsgsggggsQVQLQESG GGLVQAGGSLRLACTVSRGTFSEYSMGWFRQAPGQERDFVTAISGFGHITHYADSVKGRFTISRDNAANTVYLQM NTLKPEDTAVYYCAAAWQTSPRRMMQMAGSITWGQGTQVTVSSggggsgsggggsDAHKSEVAHRFKDLGEENF KALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQE PERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAA DKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVF LGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQ NALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESL VNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKC CKADDKETCFAEEGKKLVAASQAALGL CD39-IL2(N88D) DNA Sequence SEQ ID NO: 8 CATCATCACCATCACCATGCCCCAACCTCCTCCTCCACCAAGAAGACCCAATTGCAACTGGAGCACTTGCTGTT GGACTTGCAAATGATCCTGAACGGTATCAACAACTACAAGAACCCAAAGCTGACCAGAATGCTGACCTTCAAG TTCTACATGCCAAAGAAGGCTACCGAGCTGAAGCACTTGCAATGTTTGGAGGAGGAGTTGAAGCCATTGGAG GAGGTCCTGAACTTGGCTCAATCCAAGAACTTCCACCTGAGACCAAGAGACTTGATCTCCGACATCAACGTCA TCGTCTTGGAGCTGAAGGGTTCCGAGACCACCTTCATGTGTGAGTACGCTGACGAGACCGCTACCATCGTCGA GTTCTTGAACAGATGGATCACCTTCTCCCAATCCATCATCTCCACCCTGACCggtggtggaggttctggatctggtggagg tggttctCAAGTCCAATTGCAAGAGTCTGGTGGTGGTTTGGTCCAAGCTGGTGGTTCCTTGAGATTGGCTTGTAC CGTCTCCAGAGGTACCTTCTCCGAGTACTCCATGGGTTGGTTCAGACAAGCTCCAGGTCAAGAGAGAGACTTC GTCACTGCTATCTCTGGTTTCGGTCACATCACCCACTACGCTGACTCCGTCAAGGGTAGATTCACCATCTCCAG ATTORNEY DOCKET NO.1143252.008013 AGACAACGCTGCTAACACCGTCTACTTGCAAATGAACACCTTGAAGCCAGAGGACACTGCTGTCTACTACTGT GCTGCTGCTTGGCAAACCTCTCCAAGAAGAATGATGCAAATGGCTGGTTCCATCACCTGGGGTCAAGGTACCC AAGTCACCGTCTCCTCCggtggtggaggttctggatctggtggaggtggttctGACGCTCACAAGTCTGAAGTTGCTCACAG ATTCAAGGACTTGGGTGAAGAAAACTTCAAGGCTTTGGTTTTGATTGCTTTCGCTCAATACTTGCAACAATCTC CATTCGAAGACCACGTTAAGTTGGTTAACGAAGTTACTGAATTTGCTAAGACTTGTGTTGCTGACGAATCTGCT GAAAACTGTGACAAGTCTTTGCACACTTTGTTCGGTGACAAGTTGTGTACTGTTGCTACTTTGAGAGAAACTTA CGGTGAAATGGCTGACTGTTGTGCTAAGCAAGAACCAGAAAGAAACGAATGTTTCTTGCAACACAAGGACGA CAACCCAAACTTGCCAAGATTGGTTAGACCAGAAGTCGACGTTATGTGTACTGCTTTCCACGACAACGAAGAA ACTTTCTTGAAGAAGTACTTGTACGAAATTGCTAGAAGACACCCATACTTCTACGCTCCAGAATTGTTGTTCTTC GCTAAGAGATACAAGGCTGCTTTCACTGAATGTTGTCAAGCTGCTGACAAGGCTGCTTGTTTGTTGCCAAAGT TGGACGAATTGAGAGACGAAGGTAAGGCTTCTTCTGCTAAGCAAAGATTGAAGTGTGCTTCTTTGCAAAAGTT CGGTGAAAGAGCTTTCAAAGCTTGGGCTGTTGCTAGATTGTCTCAAAGATTCCCAAAGGCTGAATTTGCTGAA GTTTCTAAGTTGGTTACTGACTTGACTAAGGTTCACACTGAATGTTGTCACGGTGACTTGTTGGAATGTGCTGA CGACAGAGCTGACTTGGCTAAGTACATTTGTGAAAACCAAGACTCTATTTCTTCTAAGTTGAAGGAATGTTGT GAAAAGCCATTGTTGGAAAAGTCTCACTGTATTGCTGAAGTTGAAAACGACGAAATGCCAGCTGACTTGCCAT CTTTGGCTGCTGACTTCGTTGAATCTAAGGACGTTTGTAAGAACTACGCTGAAGCTAAGGACGTTTTCTTGGGT ATGTTCTTGTACGAATACGCTAGAAGACACCCAGACTACTCTGTTGTTTTGTTGTTGAGATTGGCTAAGACTTA CGAAACTACTTTGGAAAAGTGTTGTGCGGCCGCTGACCCACACGAATGTTACGCTAAGGTTTTCGACGAATTT AAGCCATTGGTTGAAGAACCACAAAACTTGATTAAGCAAAACTGTGAATTGTTCGAACAATTGGGTGAATACA AGTTCCAAAACGCTTTGTTGGTTAGATACACTAAGAAGGTTCCACAAGTTTCTACTCCAACTTTGGTTGAAGTT TCTAGAAACTTGGGTAAGGTTGGTTCTAAGTGTTGTAAGCACCCAGAAGCTAAGAGAATGCCATGTGCTGAA GACTACTTGTCTGTTGTTTTGAACCAATTGTGTGTTTTGCACGAAAAGACTCCAGTTTCTGACAGAGTTACTAA GTGTTGTACTGAATCTTTGGTTAACAGAAGACCATGTTTCTCTGCTTTGGAAGTTGACGAAACTTACGTTCCAA AGGAATTTAACGCTGAAACTTTCACTTTCCACGCTGACATTTGTACTTTGTCTGAAAAGGAAAGACAAATTAAG AAGCAAACTGCTTTGGTTGAATTGGTTAAGCACAAGCCAAAGGCTACTAAGGAACAATTGAAGGCTGTTATG GACGACTTCGCTGCTTTCGTTGAAAAGTGTTGTAAGGCTGACGACAAGGAAACTTGTTTCGCTGAAGAAGGTA AGAAGTTGGTTGCTGCTTCTCAAGCTGCTTTGGGTTTGTAA TNFR2-IL2(N88D) Protein Sequence SEQ ID NO: 9 HHHHHHAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTggggsgsggggsQVQLVESG GGLVQPGGSLRLSCAASGSTASIYAMGWYLQVPEKQRELVAEITEGVNTPYAYADSVKGRFAVSRDNAKNMVFLR MDSLKPEDTAVYYCHALLAGPGGGGYDYWGQGTLVTVSSggggsgsggggsDAHKSEVAHRFKDLGEENFKALVLIA FAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECF LQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLL PKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECAD DRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYE YARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRY TKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCF SALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKE TCFAEEGKKLVAASQAALGL TNFR2-IL2(N88D) DNA Sequence SEQ ID NO: 10 ATTORNEY DOCKET NO.1143252.008013 CATCATCACCATCACCATGCCCCAACCTCCTCCTCCACCAAGAAGACCCAATTGCAACTGGAGCACTTGCTGTT GGACTTGCAAATGATCCTGAACGGTATCAACAACTACAAGAACCCAAAGCTGACCAGAATGCTGACCTTCAAG TTCTACATGCCAAAGAAGGCTACCGAGCTGAAGCACTTGCAATGTTTGGAGGAGGAGTTGAAGCCATTGGAG GAGGTCCTGAACTTGGCTCAATCCAAGAACTTCCACCTGAGACCAAGAGACTTGATCTCCGACATCAACGTCA TCGTCTTGGAGCTGAAGGGTTCCGAGACCACCTTCATGTGTGAGTACGCTGACGAGACCGCTACCATCGTCGA GTTCTTGAACAGATGGATCACCTTCTCCCAATCCATCATCTCCACCCTGACCggtggtggaggttctggatctggtggagg tggttctCAAGTTCAATTGGTCGAGTCTGGTGGTGGTTTGGTCCAACCAGGTGGTTCCTTGAGATTGTCCTGTGCT GCTTCTGGTTCCACTGCTTCCATCTACGCTATGGGTTGGTACTTGCAAGTCCCAGAGAAGCAAAGAGAGTTGG TCGCTGAGATCACCGAGGGTGTCAACACTCCATACGCTTACGCTGACTCCGTCAAGGGTAGATTCGCTGTCTC CAGAGACAACGCTAAGAACATGGTCTTCTTGAGAATGGACTCCTTGAAGCCAGAGGACACTGCTGTCTACTAC TGTCACGCTTTGTTGGCTGGTCCAGGTGGTGGTGGTTACGACTACTGGGGTCAAGGTACCTTGGTTACCGTTT CCTCCggtggtggaggttctggatctggtggaggtggttctGACGCTCACAAGTCTGAAGTTGCTCACAGATTCAAGGACTT GGGTGAAGAAAACTTCAAGGCTTTGGTTTTGATTGCTTTCGCTCAATACTTGCAACAATCTCCATTCGAAGACC ACGTTAAGTTGGTTAACGAAGTTACTGAATTTGCTAAGACTTGTGTTGCTGACGAATCTGCTGAAAACTGTGA CAAGTCTTTGCACACTTTGTTCGGTGACAAGTTGTGTACTGTTGCTACTTTGAGAGAAACTTACGGTGAAATGG CTGACTGTTGTGCTAAGCAAGAACCAGAAAGAAACGAATGTTTCTTGCAACACAAGGACGACAACCCAAACTT GCCAAGATTGGTTAGACCAGAAGTCGACGTTATGTGTACTGCTTTCCACGACAACGAAGAAACTTTCTTGAAG AAGTACTTGTACGAAATTGCTAGAAGACACCCATACTTCTACGCTCCAGAATTGTTGTTCTTCGCTAAGAGATA CAAGGCTGCTTTCACTGAATGTTGTCAAGCTGCTGACAAGGCTGCTTGTTTGTTGCCAAAGTTGGACGAATTG AGAGACGAAGGTAAGGCTTCTTCTGCTAAGCAAAGATTGAAGTGTGCTTCTTTGCAAAAGTTCGGTGAAAGA GCTTTCAAAGCTTGGGCTGTTGCTAGATTGTCTCAAAGATTCCCAAAGGCTGAATTTGCTGAAGTTTCTAAGTT GGTTACTGACTTGACTAAGGTTCACACTGAATGTTGTCACGGTGACTTGTTGGAATGTGCTGACGACAGAGCT GACTTGGCTAAGTACATTTGTGAAAACCAAGACTCTATTTCTTCTAAGTTGAAGGAATGTTGTGAAAAGCCATT GTTGGAAAAGTCTCACTGTATTGCTGAAGTTGAAAACGACGAAATGCCAGCTGACTTGCCATCTTTGGCTGCT GACTTCGTTGAATCTAAGGACGTTTGTAAGAACTACGCTGAAGCTAAGGACGTTTTCTTGGGTATGTTCTTGTA CGAATACGCTAGAAGACACCCAGACTACTCTGTTGTTTTGTTGTTGAGATTGGCTAAGACTTACGAAACTACTT TGGAAAAGTGTTGTGCGGCCGCTGACCCACACGAATGTTACGCTAAGGTTTTCGACGAATTTAAGCCATTGGT TGAAGAACCACAAAACTTGATTAAGCAAAACTGTGAATTGTTCGAACAATTGGGTGAATACAAGTTCCAAAAC GCTTTGTTGGTTAGATACACTAAGAAGGTTCCACAAGTTTCTACTCCAACTTTGGTTGAAGTTTCTAGAAACTT GGGTAAGGTTGGTTCTAAGTGTTGTAAGCACCCAGAAGCTAAGAGAATGCCATGTGCTGAAGACTACTTGTCT GTTGTTTTGAACCAATTGTGTGTTTTGCACGAAAAGACTCCAGTTTCTGACAGAGTTACTAAGTGTTGTACTGA ATCTTTGGTTAACAGAAGACCATGTTTCTCTGCTTTGGAAGTTGACGAAACTTACGTTCCAAAGGAATTTAACG CTGAAACTTTCACTTTCCACGCTGACATTTGTACTTTGTCTGAAAAGGAAAGACAAATTAAGAAGCAAACTGCT TTGGTTGAATTGGTTAAGCACAAGCCAAAGGCTACTAAGGAACAATTGAAGGCTGTTATGGACGACTTCGCTG CTTTCGTTGAAAAGTGTTGTAAGGCTGACGACAAGGAAACTTGTTTCGCTGAAGAAGGTAAGAAGTTGGTTG CTGCTTCTCAAGCTGCTTTGGGTTTGTAA GITR-IL2(N88D) Protein Sequence SEQ ID NO: 11 HHHHHHAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTggggsgsggggsQVQLQESG GGLVQAGGSLRLSCAASGSVFSIDAMGWYRLAPGQQRELVAVLNGISSAKYADSVKGRFTISGDSAKNAVYLQMD GLKPEDTAVYYCYADVSTGWGRDAHGYWGQGTQVTVSSggggsgsggggsDAHKSEVAHRFKDLGEENFKALVLIA FAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECF LQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLL PKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECAD DRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYE ATTORNEY DOCKET NO.1143252.008013 YARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRY TKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCF SALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKE TCFAEEGKKLVAASQAALGL GITR-IL2(N88D) DNA Sequence SEQ ID NO: 12 CATCATCACCATCACCATGCCCCAACCTCCTCCTCCACCAAGAAGACCCAATTGCAACTGGAGCACTTGCTGTT GGACTTGCAAATGATCCTGAACGGTATCAACAACTACAAGAACCCAAAGCTGACCAGAATGCTGACCTTCAAG TTCTACATGCCAAAGAAGGCTACCGAGCTGAAGCACTTGCAATGTTTGGAGGAGGAGTTGAAGCCATTGGAG GAGGTCCTGAACTTGGCTCAATCCAAGAACTTCCACCTGAGACCAAGAGACTTGATCTCCGACATCAACGTCA TCGTCTTGGAGCTGAAGGGTTCCGAGACCACCTTCATGTGTGAGTACGCTGACGAGACCGCTACCATCGTCGA GTTCTTGAACAGATGGATCACCTTCTCCCAATCCATCATCTCCACCCTGACCggtggtggaggttctggatctggtggagg tggttctCAAGTTCAATTGCAAGAGTCTGGTGGTGGTTTGGTCCAAGCTGGTGGTTCCTTGAGATTGTCCTGTGC TGCTTCTGGTTCCGTCTTCTCCATCGACGCTATGGGTTGGTACAGATTGGCTCCAGGTCAACAAAGAGAGTTG GTCGCTGTCTTGAACGGTATCTCCTCTGCTAAGTACGCTGACTCCGTCAAGGGTAGATTCACCATCTCTGGTGA CTCTGCTAAGAACGCTGTCTACTTGCAAATGGACGGTTTGAAGCCAGAGGACACTGCTGTCTACTACTGTTAC GCTGACGTCTCCACTGGTTGGGGTAGAGACGCTCACGGTTACTGGGGTCAAGGTACCCAAGTTACCGTTTCCT CCggtggtggaggttctggatctggtggaggtggttctGACGCTCACAAGTCTGAAGTTGCTCACAGATTCAAGGACTTGGG TGAAGAAAACTTCAAGGCTTTGGTTTTGATTGCTTTCGCTCAATACTTGCAACAATCTCCATTCGAAGACCACG TTAAGTTGGTTAACGAAGTTACTGAATTTGCTAAGACTTGTGTTGCTGACGAATCTGCTGAAAACTGTGACAA GTCTTTGCACACTTTGTTCGGTGACAAGTTGTGTACTGTTGCTACTTTGAGAGAAACTTACGGTGAAATGGCTG ACTGTTGTGCTAAGCAAGAACCAGAAAGAAACGAATGTTTCTTGCAACACAAGGACGACAACCCAAACTTGCC AAGATTGGTTAGACCAGAAGTCGACGTTATGTGTACTGCTTTCCACGACAACGAAGAAACTTTCTTGAAGAAG TACTTGTACGAAATTGCTAGAAGACACCCATACTTCTACGCTCCAGAATTGTTGTTCTTCGCTAAGAGATACAA GGCTGCTTTCACTGAATGTTGTCAAGCTGCTGACAAGGCTGCTTGTTTGTTGCCAAAGTTGGACGAATTGAGA GACGAAGGTAAGGCTTCTTCTGCTAAGCAAAGATTGAAGTGTGCTTCTTTGCAAAAGTTCGGTGAAAGAGCTT TCAAAGCTTGGGCTGTTGCTAGATTGTCTCAAAGATTCCCAAAGGCTGAATTTGCTGAAGTTTCTAAGTTGGTT ACTGACTTGACTAAGGTTCACACTGAATGTTGTCACGGTGACTTGTTGGAATGTGCTGACGACAGAGCTGACT TGGCTAAGTACATTTGTGAAAACCAAGACTCTATTTCTTCTAAGTTGAAGGAATGTTGTGAAAAGCCATTGTTG GAAAAGTCTCACTGTATTGCTGAAGTTGAAAACGACGAAATGCCAGCTGACTTGCCATCTTTGGCTGCTGACT TCGTTGAATCTAAGGACGTTTGTAAGAACTACGCTGAAGCTAAGGACGTTTTCTTGGGTATGTTCTTGTACGA ATACGCTAGAAGACACCCAGACTACTCTGTTGTTTTGTTGTTGAGATTGGCTAAGACTTACGAAACTACTTTGG AAAAGTGTTGTGCGGCCGCTGACCCACACGAATGTTACGCTAAGGTTTTCGACGAATTTAAGCCATTGGTTGA AGAACCACAAAACTTGATTAAGCAAAACTGTGAATTGTTCGAACAATTGGGTGAATACAAGTTCCAAAACGCT TTGTTGGTTAGATACACTAAGAAGGTTCCACAAGTTTCTACTCCAACTTTGGTTGAAGTTTCTAGAAACTTGGG TAAGGTTGGTTCTAAGTGTTGTAAGCACCCAGAAGCTAAGAGAATGCCATGTGCTGAAGACTACTTGTCTGTT GTTTTGAACCAATTGTGTGTTTTGCACGAAAAGACTCCAGTTTCTGACAGAGTTACTAAGTGTTGTACTGAATC TTTGGTTAACAGAAGACCATGTTTCTCTGCTTTGGAAGTTGACGAAACTTACGTTCCAAAGGAATTTAACGCTG AAACTTTCACTTTCCACGCTGACATTTGTACTTTGTCTGAAAAGGAAAGACAAATTAAGAAGCAAACTGCTTTG GTTGAATTGGTTAAGCACAAGCCAAAGGCTACTAAGGAACAATTGAAGGCTGTTATGGACGACTTCGCTGCTT TCGTTGAAAAGTGTTGTAAGGCTGACGACAAGGAAACTTGTTTCGCTGAAGAAGGTAAGAAGTTGGTTGCTG CTTCTCAAGCTGCTTTGGGTTTGTAA TIGIT-IL2(N88D) Protein Sequence SEQ ID NO: 13 ATTORNEY DOCKET NO.1143252.008013 HHHHHHAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTggggsgsggggsQVQLQESG GGSVQPGGSLRLSCAASGFSFSMSVMYWVRQAPGKELEWVSFINSGGRTYYADSVKGRFTISRDDAKNTLYLQLN SLTTEDTALYYCARDSRGTFRGQGTQVTVSSggggsgsggggsDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPN LPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEG KASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICE NQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSV VLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTP TLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYV PKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKL VAASQAALGL TIGIT-IL2(N88D) DNA Sequence SEQ ID NO: 14 CATCATCACCATCACCATGCCCCAACCTCCTCCTCCACCAAGAAGACCCAATTGCAACTGGAGCACTTGCTGTT GGACTTGCAAATGATCCTGAACGGTATCAACAACTACAAGAACCCAAAGCTGACCAGAATGCTGACCTTCAAG TTCTACATGCCAAAGAAGGCTACCGAGCTGAAGCACTTGCAATGTTTGGAGGAGGAGTTGAAGCCATTGGAG GAGGTCCTGAACTTGGCTCAATCCAAGAACTTCCACCTGAGACCAAGAGACTTGATCTCCGACATCAACGTCA TCGTCTTGGAGCTGAAGGGTTCCGAGACCACCTTCATGTGTGAGTACGCTGACGAGACCGCTACCATCGTCGA GTTCTTGAACAGATGGATCACCTTCTCCCAATCCATCATCTCCACCCTGACCggtggtggaggttctggatctggtggagg tggttctCAAGTTCAATTGCAAGAGTCTGGTGGTGGTTCCGTCCAACCAGGTGGTTCCTTGAGATTGTCCTGTGCT GCTTCTGGTTTCTCCTTCTCCATGTCCGTCATGTACTGGGTCAGACAAGCTCCAGGTAAGGAGTTGGAGTGGG TCTCCTTCATCAACTCTGGTGGTAGAACCTACTACGCTGACTCCGTCAAGGGTAGATTCACCATCTCCAGAGAC GACGCTAAGAACACCTTGTACTTGCAATTGAACTCCTTGACCACCGAGGACACTGCTTTGTACTACTGTGCTAG AGACTCCAGAGGTACCTTCAGAGGTCAAGGTACCCAAGTTACCGTTTCCTCCggtggtggaggttctggatctggtggag gtggttctGACGCTCACAAGTCTGAAGTTGCTCACAGATTCAAGGACTTGGGTGAAGAAAACTTCAAGGCTTTGG TTTTGATTGCTTTCGCTCAATACTTGCAACAATCTCCATTCGAAGACCACGTTAAGTTGGTTAACGAAGTTACTG AATTTGCTAAGACTTGTGTTGCTGACGAATCTGCTGAAAACTGTGACAAGTCTTTGCACACTTTGTTCGGTGAC AAGTTGTGTACTGTTGCTACTTTGAGAGAAACTTACGGTGAAATGGCTGACTGTTGTGCTAAGCAAGAACCAG AAAGAAACGAATGTTTCTTGCAACACAAGGACGACAACCCAAACTTGCCAAGATTGGTTAGACCAGAAGTCG ACGTTATGTGTACTGCTTTCCACGACAACGAAGAAACTTTCTTGAAGAAGTACTTGTACGAAATTGCTAGAAG ACACCCATACTTCTACGCTCCAGAATTGTTGTTCTTCGCTAAGAGATACAAGGCTGCTTTCACTGAATGTTGTCA AGCTGCTGACAAGGCTGCTTGTTTGTTGCCAAAGTTGGACGAATTGAGAGACGAAGGTAAGGCTTCTTCTGCT AAGCAAAGATTGAAGTGTGCTTCTTTGCAAAAGTTCGGTGAAAGAGCTTTCAAAGCTTGGGCTGTTGCTAGAT TGTCTCAAAGATTCCCAAAGGCTGAATTTGCTGAAGTTTCTAAGTTGGTTACTGACTTGACTAAGGTTCACACT GAATGTTGTCACGGTGACTTGTTGGAATGTGCTGACGACAGAGCTGACTTGGCTAAGTACATTTGTGAAAACC AAGACTCTATTTCTTCTAAGTTGAAGGAATGTTGTGAAAAGCCATTGTTGGAAAAGTCTCACTGTATTGCTGAA GTTGAAAACGACGAAATGCCAGCTGACTTGCCATCTTTGGCTGCTGACTTCGTTGAATCTAAGGACGTTTGTA AGAACTACGCTGAAGCTAAGGACGTTTTCTTGGGTATGTTCTTGTACGAATACGCTAGAAGACACCCAGACTA CTCTGTTGTTTTGTTGTTGAGATTGGCTAAGACTTACGAAACTACTTTGGAAAAGTGTTGTGCGGCCGCTGACC CACACGAATGTTACGCTAAGGTTTTCGACGAATTTAAGCCATTGGTTGAAGAACCACAAAACTTGATTAAGCA AAACTGTGAATTGTTCGAACAATTGGGTGAATACAAGTTCCAAAACGCTTTGTTGGTTAGATACACTAAGAAG GTTCCACAAGTTTCTACTCCAACTTTGGTTGAAGTTTCTAGAAACTTGGGTAAGGTTGGTTCTAAGTGTTGTAA GCACCCAGAAGCTAAGAGAATGCCATGTGCTGAAGACTACTTGTCTGTTGTTTTGAACCAATTGTGTGTTTTGC ACGAAAAGACTCCAGTTTCTGACAGAGTTACTAAGTGTTGTACTGAATCTTTGGTTAACAGAAGACCATGTTTC TCTGCTTTGGAAGTTGACGAAACTTACGTTCCAAAGGAATTTAACGCTGAAACTTTCACTTTCCACGCTGACAT ATTORNEY DOCKET NO.1143252.008013 TTGTACTTTGTCTGAAAAGGAAAGACAAATTAAGAAGCAAACTGCTTTGGTTGAATTGGTTAAGCACAAGCCA AAGGCTACTAAGGAACAATTGAAGGCTGTTATGGACGACTTCGCTGCTTTCGTTGAAAAGTGTTGTAAGGCTG ACGACAAGGAAACTTGTTTCGCTGAAGAAGGTAAGAAGTTGGTTGCTGCTTCTCAAGCTGCTTTGGGTTTGTA A Wildtype human albumin (HSA) Sequence SEQ ID NO: 15 DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTV ATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPE LLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAE VSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAAD FVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQ NLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCV LHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATK EQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL Variant HSA (C34S) Albumin Sequence SEQ ID NO: 16 DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTV ATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPE LLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAE VSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAAD FVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQ NLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCV LHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATK EQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL G4S linker unit: GGGGS SEQ ID NO: 17 G3S linker unit: GGGS SEQ ID NO: 18 G5S linker unit: GGGGGS SEQ ID NO: 19 GSGGGS linker unit: GSGGGS SEQ ID NO: 20 GGSGS linker unit: GGSGS SEQ ID NO: 21 GSGSG linker unit: GSGSG SEQ ID NO: 22 ATTORNEY DOCKET NO.1143252.008013 G4S)2 linker: GGGGSGGGGS SEQ ID NO: 23 (G4S)3 linker: GGGGSGGGGSGGGGS SEQ ID NO: 24 Exemplary Immunoglobulin constant region sequences SEQ

Claims

ATTORNEY DOCKET NO.1143252.008013 CLAIMS What is claimed is: CLAIMS 1. A targeted human IL-2 fusion protein that promotes the activation and / or expansion of regulatory T cells which comprises (i) an IL-2 polypeptide, optionally a human IL-2 polypeptide or a variant possessing at least 90, 95, 96, 97, 98 or 99 percent identity to the IL-2 polypeptide of SEQ ID NO: 1, which IL-2 polypeptide optionally is modified to attenuate IL-2Rβ / γc binding; (ii) an antibody or antibody fragment which binds to an antigen other than CD25 expressed on Tregs; and (iii) optionally a half-life extending moiety; wherein (i), (ii) and (iii) if present are comprised in any order from amino terminal to carboxy terminal in the human IL-2 fusion protein, and these moieties are directly linked to each other and / or are indirectly linked to each other, further optionally via one or more linker peptides.

2. The human IL-2 fusion protein of claim 1, wherein the moieties are oriented from N to C terminal as follows: (i) IL-2 polypeptide—(ii) antibody or antibody fragment which binds to an antigen other than CD25 expressed on Tregs—(iii) half-life extending moiety, optionally human serum albumin (HSA) or a human Ig Fc region.

3. The human IL-2 fusion protein of claim 1 or 2, wherein (ii) the antibody or antibody fragment binds to an immune checkpoint inhibitor, co-stimulatory receptor, cytokine receptor or an enzyme expressed on human Tregs.

4. The human IL-2 fusion protein of claim 1 or 2, wherein (ii) the antibody or antibody fragment binds to an antigen expressed on human Tregs selected from CTLA-4, TIGIT, GITR, ICOS, TGF beta receptor, TNFR-2, CD39, or CD73.

5. The human IL-2 fusion protein of claim 1 or 2, wherein (ii) the antibody or antibody fragment binds to CD39.

6. The human IL-2 fusion protein of any of the previous claims, wherein the half-life extending moiety is selected from a serum albumin, an Fc or immunoglobulin constant region, an unstructured polypeptide, an adnectin, transferrin, CTP (28 amino acid C-terminal peptide (CTP) of hCG with its 4ATTORNEY DOCKET NO.1143252.008013 O-glycans), or a fragment of any of the foregoing; a lipid, O- and N-glycosyl residues, a carbohydrate polymer, cholesterol, poly(ethylene glycol) (PEG), monomethoxy PEG (mPEG), a branched or unbranched acyl group, a branched or unbranched C8-C30 acyl group, a branched or unbranched alkyl group, and a branched or unbranched C8-C30 alkyl group.

7. The human IL-2 fusion protein of any of the previous claims, wherein the half-life extending moiety is a serum albumin, optionally human serum albumin, further optionally a variant possessing at least 90, 95, 96, 97, 98 or 99 percent identity to the HSA polypeptide of SEQ ID NO: 15 or 16, or a human Fc region, optionally an Fc region of any of SEQ ID NO.25-SEQ ID NO: 36, which Fc region optionally contains a mutation which ablates or reduces at least one effector function such as glycosylation, FcR binding or complement binding and / or a mutation which enhances FcRN binding.

8. The human IL-2 fusion protein of any of the previous claims, wherein the (ii) antibody or antibody fragment is a scFv, a Fab, F(ab')2or VHH antibody.

9. The human IL-2 fusion protein of any of the previous claims, which is PEGylated, optionally via a non-encoded amino acid.

10. The human IL-2 fusion protein of any of the previous claims, wherein the human IL-2 is a mutein that comprises at least one mutation which reduces IL-2Rβ / γc binding.

11. The human IL-2 fusion protein of any of the previous claims, wherein the human IL-2 is a mutein that comprises at least one mutation at a residue selected from residue 16, 20, 88, 91, 126, optionally selected from N88D, N88R, V91R, H16L, D20T, and Q126T.

12. A polymer–drug conjugate (PDC) comprising one or more human IL-2 fusion proteins of any of the previous claims directly or indirectly conjugated to a polymer which optionally is chosen to optimize desired properties, such as stability, biocompatibility, and controlled drug release.

13. The polymer–drug conjugate (PDC) of claim 12, wherein said one or more human IL-2 fusion proteins are directly or indirectly to a synthetic or natural occurring polymer, optionally poly(€- caprolactone) (PCL), D-α-tocopheryl polyethylene glycol (TPGS), polyethylene glycol (PEG), hyaluronic acid (HA), PVP, HPMA, poly(lactic-co-glycolic acid) (PLGA), dextran, alginate, pectin, or starch.

14. The polymer–drug conjugate (PDC) of claim 12 or 13, wherein said one or more human IL-2 fusion proteins are directly or indirectly to the polymer through a bioresponsive linker.ATTORNEY DOCKET NO.1143252.008013 15. The polymer–drug conjugate (PDC) of claim 12, 13 or 14, wherein said one or more human IL-2 fusion proteins are directly or indirectly to a insensitive polymer e.g., a pH-sensitive polymer, light- sensitive polymer, enzyme-(cleavable) sensitive polymer, redox-sensitive polymer, or hypoxia- sensitive polymer.

16. A pharmaceutical composition comprising a human IL-2 fusion protein or PDC comprising according to any of the previous claims, and a pharmaceutically acceptable carrier.

17. A nucleic acid which encodes a human IL-2 fusion protein or PDC comprising according to any of the previous claims or an expression vector containing said nucleic acid.

18. A recombinant cell which expresses a nucleic acid according to claim 17.

19. The recombinant cell of claim 18, which optionally is an immune cell further optionally selected from PMBCs, a T cell, a T cell progenitor cell, a CD4+ T cell, a helper T cell, a regulatory T cell, a CD8+ T cell, a naïve T cell, an effector T cell, a memory T cell, a stem cell memory T (TSCM) cell, a central memory T (TCM) cell, an effector memory T (TEM) cell, a terminally differentiated effector memory T cell, a tumor-infiltrating lymphocyte (TIL), an immature T cell, a mature T cell, a cytotoxic T cell, a mucosa-associated invariant T (MAIT) cell, a TH1 cell, a TH2 cell, a TH3 cell, a TH17 cell, a TH9 cell, a TH22 cell, a follicular helper T cells, and a / b T cell, a g / d T cell, a Natural Killer T (NKT) cell, a cytokine-induced killer (CIK) cell, a lymphokine-activated killer (LAK) cell, a perforin-deficient cell, a granzyme-deficient cell, a B cell, a myeloid cell, a monocyte, a macrophage, an eosinophil, a neutrophil, and a dendritic cell.

20. The recombinant cell of claim 20, which is a Treg.

21. A lipid nanoparticle comprising a human IL-2 fusion protein or a PDC comprising according to any of the previous claims or a nucleic acid encoding, and a pharmaceutically acceptable carrier or excipient.

22. A method of therapy or prophylaxis, which comprises the administration of a human IL-2 fusion protein or PDC comprising or a recombinant cell which expresses a human IL-2 fusion protein or PDC according to any of the previous claims, or a composition containing, to a subject in need thereof.

23. A method of therapy or prophylaxis, which comprises the administration of a nanoparticle which comprises a human IL-2 fusion protein or a PDC comprising according to any of the previous claims, or a nucleic acid encoding, to a subject in need thereof.ATTORNEY DOCKET NO.1143252.008013 24. A method of therapy or prophylaxis, which comprises the administration of a human IL-2 fusion protein according to any of the previous claims, or a nucleic acid encoding, to a subject in need thereof, wherein said human IL-2 fusion protein is conjugated to a polymer which acts as a nanocarrier that provides for targeted delivery, controlled release, and / or enhanced stability of said human IL-2 fusion protein.

25. The method of therapy or prophylaxis of any one of claims 22-24, wherein the therapy or prophylaxis is for treatment of a neuroinflammatory, autoimmune, or inflammatory condition or a pathologic symptom associated with any of said conditions.

26. The method of therapy or prophylaxis of claim 25 wherein the therapy or prophylaxis is for the treatment or prevention of an autoimmune disease selected from one or more of Acromegaly, Acquired aplastic anemia, Acquired hemophilia, Agammaglobulinemia, primary, Alopecia areata, Ankylosing spondylitis (AS), Anti-NMDA receptor encephalitis, Antiphospholipid syndrome (APS) | catastrophic antiphospholipid syndrome (CAPS) / Asherson's syndrome, Arteriosclerosis, Autoimmune Addison’s disease (AAD), Autoimmune autonomic ganglionopathy (AAG) / autoimmune dysautonomia | autoimmune gastrointestinal dysmotility (AGID), Autoimmune encephalitis | acute disseminated encephalomyelitis (ADEM), Autoimmune gastritis, Autoimmune hemolytic anemia (AIHA), Autoimmune hepatitis (AIH), Autoimmune hyperlipidemia, Autoimmune hypophysitis, Autoimmune inner ear disease (AIED), Autoimmune lymphoproliferative syndrome (ALPS), Autoimmune myelofibrosis, Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune pancreatitis (AIP), Autoimmune polyglandular syndromes, types I, II, & III (APS type 1, APS type 2, APS type 3, APECED), Autoimmune progesterone dermatitis, Autoimmune retinopathy (AIR), Autoimmune sudden sensorineural hearing loss (SNHL), Balo disease, Behçet’s disease, Birdshot chorioretinopathy / birdshot uveitis, Bullous pemphigoid, Castleman disease, Celiac disease, Chagas disease, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic urticaria (CU), Churg- Strauss syndrome / eosinophilic granulomatosis with polyangiitis (EGPA), Cogan’s syndrome, Cold agglutinin disease, CREST syndrome | limited cutaneous systemic sclerosis, Crohn’s disease (CD), Cronkhite-Canada syndrome (CSS), Cryptogenic organizing pneumonia (COP), Dermatitis herpetiformis, Dermatomyositis, Diabetes, type 1, Discoid lupus, Dressler’s syndrome / postmyocardial infarction / postpericardiotomy syndrome, Eczema / Atopic Dermatitis, Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum, Essential mixed cryoglobulinemia, Evans syndrome, Fibrosing alveolitis / Idiopathic pulmonary fibrosis (IPF), Giant cell arteritis / temporal arteritis / Horton’s disease, Giant Cell Myocarditis, Glomerulonephritis, Goodpasture’s syndrome / anti-GBM / anti-TBM disease, Granulomatosis with polyangiitis (GPA) / Wegener’s granulomatosis, Grave’s disease / thyroid eye disease, Guillain-Barré syndrome (GBS), Hashimoto’s thyroiditis / chronic lymphocytic thyroiditis / autoimmune thyroiditis, Henoch- Schönlein purpura / IgA vasculitis, Hidradenitis suppurativa, Hurst’s disease / acute hemorrhagic leukoencephalitis (AHLE), Hypogammaglobulinemia, IgA nephropathy / Berger's disease, Immune- mediated necrotizing myopathy (IMNM), Immune thrombocytopenia (ITP) / autoimmune thrombocytopenic purpura / autoimmune thrombocytopenia, Inclusion body myositis, IgG4-related sclerosing disease (ISD), Interstitial cystitis, Juvenile idiopathic arthritis / Adult-onset Still's disease. Juvenile polymyositis | Juvenile dermatomyositis | juvenile myositis, Kawasaki disease, Lambert- Eaton myasthenic syndrome (LEMS), Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD) | linear IgA bullous dermatosis (LABD), LupusATTORNEY DOCKET NO.1143252.008013 nephritis, Lyme disease / chronic Lyme disease / post-treatment Lyme disease syndrome (PTLDS), Lymphocytic colitis / microscopic colitis, Lymphocytic hypophystitis / autoimmune hypophystitis, Ménière’s disease, Microscopic polyangiitis (MPA) / ANCA-associated vasculitis, Mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease, Multifocal motor neuropathy, Multiple sclerosis (MS), Myalgic encephalomyelitis (ME) / Chronic fatigue syndrome (CFS), Myasthenia gravis (MG), Narcolepsy, Neuromyelitis Optica / Devic's disease, Ocular cicatricial pemphigoid, Opsoclonus-myoclonus syndrome (OMS), Palindromic rheumatism, Paraneoplastic cerebellar degeneration, Paraneoplastic pemphigus, Parry-Romberg syndrome (PRS) / Hemifacial atrophy (HFA) / Progressive facial hemiatrophy, Paroxysmal nocturnal hemoglobinuria (PNH), Peripheral uveitis / pars planitis, PANS / PANDAS, Parsonage-Turner syndrome, Pemphigus gestationis / herpes gestationis, Pemphigus foliaceus, Pemphigus vulgaris, Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Polymyalgia rheumatica, Polymyositis, Postural orthostatic tachycardia syndrome (POTS), Primary biliary cirrhosis (PBC) / primary biliary cholangitis, Primary sclerosing cholangitis (PSC), Psoriasis, Palmoplantar Pustulosis, Psoriatic arthritis, Pulmonary fibrosis, idiopathic (IPF), Pure red cell aplasia (PRCA), Pyoderma gangrenosum, Rasmussen's encephalitis, Raynaud’s syndrome / phenomenon, Reactive arthritis / Reiter’s syndrome, Reflex sympathetic dystrophy syndrome (RSD) / Complex regional pain syndrome (CRPS), Relapsing polychondritis, Restless leg syndrome (RLS) / Willis-Ekbom disease, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidt syndrome / autoimmune polyendocrine syndrome type II, Scleritis, Scleroderma, Sclerosing Mesenteritis / Mesenteric Panniculitis, Serpiginous choroidopathy, Sjögren’s syndrome, Stiff person syndrome (SPS), Small fiber sensory neuropathy, Systemic lupus erythematosus (SLE), Subacute bacterial endocarditis (SBE), Subacute cutaneous lupus, Susac syndrome, Sydenham's chorea, Sympathetic ophthalmia, Takayasu’s arteritis (vasculitis), Testicular autoimmunity (vasculitis, orchitis), Tolosa-Hunt syndrome, Transverse myelitis (TM), Tubulointerstitial nephritis uveitis syndrome (TINU), Ulcerative colitis (UC), Undifferentiated connective tissue disease (UCTD), Uveitis | anterior / intermediate / posterior, Vasculitis, VEXAS Syndrome, Vitiligo, and Vogt-Koyanagi-Harada syndrome (VKH), and / or to prevent or inhibit at least one pathologic symptom associated therewith.

27. The method of therapy or prophylaxis of claim 25 wherein the therapy or prophylaxis is for the treatment or prevention of Addison disease, arthritis, celiac disease, lupus, Grave’s disease, myasthenia gravis, multiple sclerosis, ITP, rheumatoid arthritis, colitis, inflammatory bowel disease, pernicious anemia, Hashimoto’s thyroiditis Sjogren’s disease, asthma, type 2 diabetes, and autoimmune type I diabetes and / or to prevent or inhibit at least one pathologic symptom associated therewith.

28. The method of therapy or prophylaxis of claim 25 wherein the therapy or prophylaxis is for the treatment or prevention of an inflammatory disease selected from the group consisting of Fatty liver disease, Endometriosis, Type 2 diabetes, mellitus, Type 1 diabetes mellitus, Inflammatory bowel disease (IBD), Asthma, Rheumatoid arthritis, asthma, Obesity, Fibromyalgia, Lupus SLE, osteoarthritis, Rheumatoid Arthritis, Shingles Herpes Zoster, and Vasculitis and / or to prevent or inhibit at least one pathologic symptom associated therewith.

29. The method of therapy or prophylaxis of claim 25 wherein the therapy or prophylaxis is for the treatment or prevention of a neurodegenerative or neuroinflammatory disease, e.g., Alzheimer'sATTORNEY DOCKET NO.1143252.008013 disease, Amyotrophic lateral sclerosis, Friedreich ataxia, Huntington's disease, Lewy body disease, aphasia, Parkinson's disease or Spinal muscular atrophy and / or to prevent or inhibit at least one pathologic symptom associated therewith.

30. The method of therapy or prophylaxis of any one of claims 22-29, which further comprises the administration of an anti-inflammatory agent, optionally a small molecule or an antibody.

31. The method of therapy or prophylaxis of any one of claims 22-30, which further comprises the administration of an additional agent(s) such as an anti-inflammatory agent or immunosuppressant, optionally a small molecule or a biologic and / or at least one agent selected from nonsteroidal anti- inflammatory drugs (NSAIDs) e.g., aspirin, ibuprofen, and naproxen; paracetamol, Antileukotrienes or leukotriene-related enzyme inhibitors (arachidonate 5-lipoxygenase) or leukotriene receptor antagonists (cysteinyl leukotriene receptors), and the like and / or a glucocorticoid or steroid, optionally Group A – Hydrocortisone type steroids such as Hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, prednisolone, methylprednisolone, and prednisone; Group B – Acetonide type steroids such as Amcinonide, budesonide, desonide, fluocinolone cetonide, fluocinonide, halcinonide, triamcinolone acetonide, and Deflazacort (O-isopropylidene derivative); Group C – Betamethasone type steroids such as declometasone, betamethasone, dexamethasone, fluocortolone, halometasone, and mometasone; Group D1 and D2 esters–such as Group D1 and D2 esters Alclometasone dipropionate, betamethasone dipropionate, betamethasone valerate, clobetasol propionate, clobetasone butyrate, fluprednidene acetate, and mometasone furoate; and Group D2 – Labile prodrug esters such as Ciclesonide, cortisone acetate, hydrocortisone aceponate, hydrocortisone acetate, hydrocortisone buteprate, hydrocortisone butyrate, hydrocortisone valerate, prednicarbate, and tixocortol pivalate; TNF inhibitors (TNF blockers or anti-TNFs), B cell inhibitors, interleukin inhibitors, (e.g., IL-1, IL-6 and / or IL-17 inhibitors such as adalimumab (Humira), certolizumab pegol (Cimzia), etanercept (Enbrel), golimumab (Simponi, Simponi Aria), infliximab (Remicade); B cell inhibitors such as belimumab (Benlysta) and rituximab (Rituxan), and rituximab; interleukin inhibitors such as anakinra (Kineret), canakinumab (Ilaris), guselkumab (Tremfya), ixekizumab (Taltz), risankizumab (Skyrizi), sarilumab (Kevzara), secukinumab (Cosentyx), tocilizumab (Actemra) and ustekinumab (Stelara); Abatacept (Orencia), Remicade® (infliximab), Humira® (adalimumab), Simponi® (golimumab) and Cimzia® (certolizumab pegol) and / or at least one agent selected from methotrexate, Cytostatics, Alkylating agents such as nitrogen mustards, (cyclophosphamide), nitrosoureas, platinum compounds; Cyclophosphamide; Antimetabolites such as folic acid analogues, methotrexate; purine analogues, such as azathioprine and mercaptopurine; pyrimidine analogues, such as fluorouracil and protein synthesis inhibitors; Azathioprine and mercaptopurine; Azathioprine; Cytotoxic antibiotics such as dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin.

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