Therapeutic muteins

Abstract

Modified cytokines are disclosed that contain one or more amino acid modifications relative to wild type. These modified cytokines exhibit improved activity at acidic pH and often exhibit reduced activity at neutral pH compared to the activity of wild type cytokines. The modified cytokines of the present disclosure are for use as medicines and / or for treating and / or preventing immunological conditions or cancer.

Description

[Technical field]

[0001] The present invention provides modified molecules with improved activity at acidic pH for use in the treatment of various diseases and / or conditions. More particularly, the present disclosure provides modified cytokines, including IL-2, for use in the treatment of cancer. [Background technology]

[0002] The tumor microenvironment (TME) plays an important role in tumor differentiation and immune evasion, thus antagonizing the antitumor response induced by cytokines (Non-Patent Document 1). The cellular and molecular basis that defines the immunosuppressive properties of the TME has been extensively studied (Non-Patent Documents 2, 3). However, how its unique physiochemical properties affect cytokine responses remains largely unknown. Acidosis is a characteristic of the TME. Tumor cells have high glycolytic activity, which leads to excessive production of lactic acid, resulting in an acidic environment with a pH of about 6.2 to 6.5. This is in contrast to the pH of normal tissues, which is 7.4 (Non-Patent Documents 1, 4). It is currently unknown how the acidic TME affects cytokine-receptor binding and cytokine signaling. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Hanahan, D. & Weinberg, RA Hallmarks of Cancer: The Next Generation. Cell 144, 646-674 (2011). [Non-Patent Document 2] Brand, A. et al. LDHA-Associated Lactic Acid Production Blunts Tumor Immunosurveillance by T and NK Cells. Cell Metabolism 24, 657-671 (2016). [Non-Patent Document 3] Huber, V. et al. Cancer acidity: An ultimate frontier of tumor immune escape and a novel target of immunomodulation. Seminars in Cancer Biology 43, 74-89 (2017). [Non-Patent Document 4] Renner, K. et al. Restricting Glycolysis Preserves T Cell Effector Functions and Augments Checkpoint Therapy. Cell Reports 29, 135-150.e9 (2019). [Non-Patent Document 5] Mitra, S. & Leonard, WJ Biology of IL-2 and its therapeutic modulation: Mechanisms and strategies. J. Leukoc. Biol. 103, 643-655 (2018). [Non-Patent Document 6] Rosenberg, SA IL-2: The First Effective Immunotherapy for Human Cancer. The Journal of Immunology 192, 5451-5458 (2014). [Non-Patent Document 7] Smith, KA & Cantrell, DA Interleukin 2 regulates its own receptors.PNAS 82, 864-868 (1985). [Non-Patent Document 8] Pipkin, ME et al. Interleukin-2 and Inflammation Induce Distinct Transcriptional Programs that Promote the Differentiation of Effector Cytolytic T Cells. Immunity 32, 79-90 (2010). [Non-Patent Document 9] Calcinotto, A. et al. Modulation of microenvironment acidity reverses anergy in human and murine tumor-infiltrating T lymphocytes.Cancer Res. 72, 2746-2756 (2012). [Non-Patent Document 10] Mitra, S. et al. Interleukin-2 Activity Can Be Fine Tuned with Engineered Receptor Signaling Clamps. Immunity 42, 826-838 (2015). [Non-Patent Document 11] Krieg, C., Letourneau, S., Pantaleo, G. & Boyman, O. Improved IL-2 immunotherapy by selective stimulation of IL-2 receptors on lymphocytes and endothelial cells. Proc Natl Acad Sci USA 107, 11906- 11911 (2010). [Non-Patent Document 12] Wu, H. et al. T-cells produce acidic niches in lymph nodes to suppress their own effector functions. Nature Communications 11, 4113 (2020). [Non-Patent Document 13] Caudana, P. et al. IL2 / Anti-IL2 Complex Combined with CTLA-4, But Not PD-1, Blockade Rescues Antitumor NK Cell Function by Regulatory T-cell Modulation. Cancer Immunol Res 7, 443-457 (2019). Non-Patent Document 14 Wang, X., Rickert, M. & Garcia, K. C. Structure of the Quaternary Complex of Interleukin-2 with Its α, β, and γc Receptors. Science 310, 1159-1163 (2005). Non-Patent Document 15 Krutzik, P. O. & Nolan, G. P. Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nature Methods 3, 361-368 (2006). Non-Patent Document 16 Boder, E. T. & Wittrup, K. D. Yeast surface display for screening combinatorial polypeptide libraries. Nature Biotechnology 15, 553 (1997). Non-Patent Document 17 You, C., Richter, C. P., Loechte, S., Wilmes, S. & Piehler, J. Dynamic submicroscopic signaling zones revealed by pair correlation tracking and localization microscopy. Anal Chem 86, 8593-8602 (2014). [Non-Patent Document 18] Moraga, I. et al. Tuning cytokine receptor signaling by re-orienting dimer geometry with surrogate ligands. Cell 160, 1196-1208 (2015). [Non-Patent Document 19] Wilmes, S. et al. Receptor dimerization dynamics as a regulatory valve for plasticity of type I interferon signaling. J Cell Biol 209, 579-593 (2015). [Non-Patent Document 20] Vogelsang, J. et al. A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes. Angew Chem Int Ed Engl 47, 5465-5469 (2008). [Non-Patent Document 21] Serge, A., Bertaux, N., Rigneault, H. & Marguet, D. Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes. Nat Methods 5, 687-694 (2008). [Non-Patent Document 22] Chen LQ, Pagel MD. Evaluating pH in the Extracellular Tumor Microenvironment Using CEST MRI and Other Imaging Methods. Adv Radiol. 2015;2015:206405 Summary of the Invention [Problem to be solved by the invention]

[0004] Interleukin-2 (IL-2) cytokines function as potent master regulators of immune activity, making IL-2 a powerful intermediary for coordinating immune responses to more effectively combat disease. In resting lymphocytes, IL-2 transmits signals through the IL-2 receptor (Kd: about 10-9 M) with moderate affinity, consisting of IL-2Rβ and IL-2Rγ. In contrast, activated lymphocytes additionally express IL-2Rα, which binds to IL-2Rβ and IL-2Rγ to form a high affinity receptor (Kd: about 10-11 M), which responds strongly to IL-2 in vivo and effectively regresses tumors (Non-Patent Document 5). For this reason, IL-2 has been used clinically for 30 years as part of the immunotherapy of malignant tumors (Non-Patent Document 6). However, the broad pleiotropy of IL-2, including its role in simultaneously promoting both effector and regulatory T (Treg) cells, has resulted in limited efficacy and high toxicity, preventing its widespread use (Non-Patent Document 6). For this reason, numerous efforts have been made to manipulate IL-2 activity to selectively promote the proliferation of effector cells for the treatment of cancer. However, it is not fully understood how the extracellular chemical environment (such as the acidic pH found in the tumor cavity) affects IL-2 activity. Previous studies using acidic pH to disrupt IL-2 binding from the surface of T lymphocytes suggested that this cytokine is responsive to changes in pH. However, it is not known which of the IL-2-binding receptors are pH-responsive and how acidic pH affects the IL-2 response. [Means for solving the problem]

[0005] The present disclosure provides modified cytokines that contain one or more amino acid modifications (e.g., one or more amino acid substitutions) relative to the wild type. The inventors have found that these modified cytokines have increased activity at acidic pH and often decreased activity at neutral pH compared to the activity of the wild type cytokine. In the present disclosure, modified cytokines are also referred to as cytokine muteins.

[0006] The tumor microenvironment (TME) plays an important role in tumor differentiation and immune evasion, and can antagonize certain antitumor responses induced by cytokines. A characteristic feature of the TME is acidosis. Tumor cells have high glycolytic activity, which leads to excessive production of lactic acid, resulting in an acidic environment with a pH of approximately 6.2 to 6.5. This is in contrast to the neutral pH (e.g., pH 7.4) found in normal tissues.

[0007] The activity of certain cytokines, such as interleukin 2 (IL-2), is important in the development and maintenance of various aspects of the host immune response to disease, such as T cell immunity. Cytokines promote the proliferation and induction of immune cells and immune processes. However, cytokine function can be responsive to changes in pH. For example, the binding of a cytokine to its receptor can be a pH-responsive or pH-dependent process. As discussed above, certain diseases, including cancer, are characterized by the generation of an acidic microenvironment that can adversely affect cytokine receptor binding and ultimately reduce the efficacy of cytokine-based therapies.

[0008] As discussed above, pH-responsive cytokines (i.e., cytokines that exhibit reduced activity / receptor binding under acidic conditions) may be modified by alteration (e.g., substitution) of one or more amino acids in the wild-type primary sequence. Modified cytokines according to the present disclosure may exhibit increased activity at acidic pH and / or reduced activity at neutral pH. This feature makes them useful in medicine, particularly in the treatment and / or prevention of immunological diseases and / or cancer.

[0009] Modified cytokines with therapeutic potential, e.g., for use in medicine, include: modifying the wild-type cytokine sequence to produce a modified cytokine; contacting the modified cytokine with a ligand or a cell; determining whether the modified cytokine binds to a ligand and / or activates a cell, and modified cytokines that bind to a ligand and / or activate a cell may be used in medicine or for the treatment and / or prevention of an immunological condition or cancer.

[0010] The step of contacting the modified cytokine with a ligand may include contacting the modified cytokine with a ligand fragment, the ligand fragment being a cytokine-binding fragment. Similarly, the cell may express the ligand and / or its cytokine-binding fragment.

[0011] The method for identifying a cytokine mutein, preferably a pH-resistant cytokine mutein, may further comprise the step of generating a library comprising nucleic acids encoding a cytokine mutein or fragments thereof, the cytokine mutein comprising one or more amino acid substitutions (e.g., including conservative substitutions); (ii) one or more amino acid deletions; (iii) one or more amino acid additions; and (iv) one or more sequence inversions (all of which are described / defined hereinafter).

[0012] Mutations may be made in at least one residue involved in the binding of cytokines to their corresponding ligands or receptors. The residues involved in the binding profiles of different cytokines can be determined by analyzing the structural data of the functional interactions of those cytokines with their receptors. Mutations may be random or predefined.

[0013] The method may further comprise expressing the nucleic acid library to obtain a cytokine mutein library. The cytokine muteins contained in the library may be expressed on the surface of an expression vehicle, such as a cell, a phage virus, e.g., a yeast cell.

[0014] The cytokine to be modified (by any of the methods or procedures described herein) may be selected from granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), IL-6, IL-11, IL-12, growth hormone (GF1), erythropoietin (EPO), prolactin (PRL), leukemia inhibitory factor (LIF), oncostatin (OSM), thrombopoietin (TPO), or a functional fragment / variant of any of these cytokines.

[0015] In one teaching, the cytokine to be modified may be CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CCLle, CCL2, CCL3, CCL3L1, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CX3CL1, XCL1, XCL2, or a functional fragment / variant of any of these cytokines. The cytokine to which modification is made may be selected from the group consisting of IFN-α (alpha), IFN-β (beta), IFN-γ (gamma), IFN-ε (epsilon), IFN-κ (kappa), IFN-ω (omega), IFN-τ (tau), IFN-ζ (zeta), IFN-δ (delta), IFN-λ (lambda), or a functional fragment / variant of any of these cytokines.

[0016] The cytokines to be modified may include functional fragments or variants of IFN-α (alpha), IFN-β (beta), IFN-γ (gamma), IFN-ε (epsilon), IFN-κ (kappa), IFN-ω (omega), IFN-τ (tau), IFN-ζ (zeta), or IFN-δ (delta), IFN-λ (lambda).

[0017] In another teaching, the cytokines that are modified include IL-1, IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, I L-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-17L , IL-17A / L, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28A, IL-28B, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, or a functional fragment / variant of any of these cytokines.

[0018] The cytokine to be modified may be granulocyte-macrophage colony-stimulating factor (GM-CSL), macrophage colony-stimulating factor (M-CSL), tumor necrosis factor alpha (TNL-α), transforming growth factor beta (TGL-β), ILN-γ (gamma), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-12, or a functional fragment / variant of any of these cytokines.

[0019] In one teaching, the cytokine may be selected from the group consisting of TNF-α (alpha), TNF-β (beta), TNF-γ (gamma), CD252, CD154, CD178, CD70, CD153, 4-1BB-L, LTa, iΤβ, LIGHT, TWEAK, APRIL, BAFF, TL1A, GITRL, OX40L, CD40L, FASL, CD27L, CD30L, 4-1BBL, TRAIL, FLT3 ligand, G-CSF, GM-CSF, IFNα / β / ω, IFNy, LIF, M-CSF, MIF, OSM, stem cell factor, TGFpi, TGFp2, TGF33, TSLP ligand, TRAIL, RANKL, AP03L, CD256, CD257, CD258, TL1, AITRL, EDA1, or functional fragments / variants of these cytokines. The cytokine may be TNF-α (alpha), TNF-β (beta), TNF-γ (gamma), CD252, CD154, CD178, CD70, CD153, 4-1BB-L, TRAIL, RANKL, AP03L, CD256, CD257, CD258, TL1, AITRL, EDAL, or a functional fragment / variant of any of these cytokines. In a preferred embodiment, the cytokine is an interleukin, more preferably IL-2 or IL-10.

[0020] The step of modifying the wild-type cytokine comprises: (i) replacing an amino acid in the wild-type primary sequence with another amino acid. This type of substitution may be a conservative substitution; and / or (ii) deleting an amino acid from the wild-type primary sequence by another amino acid; and / or (iii) adding amino acids to the wild-type primary sequence; and / or (iv) inverting a portion of the wild-type primary amino acid sequence.

[0021] The step of contacting the modified cytokine with the ligand or cells may be carried out under acidic conditions, e.g., at a pH of about 7.5 to less than about 7.2, e.g., less than about pH 7.4 or pH 7.3. Alternatively, the step of contacting the modified cytokine with the ligand or cells may be carried out at a pH of about 4.0 to about 7.0. The step of contacting the modified cytokine with the ligand or cells may be carried out at a pH of about 4.5 or about 4.8 to about pH 5.5 or about pH 6.5, or about pH 5.0 to about pH 6.9, e.g., about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3 or about 6.4. The optional step of determining whether the modified cytokine activates a cell may include contacting the cell with the modified cytokine and detecting, for example, proliferation and / or enlargement of the cell, increased expression of a cell surface marker, or / and expression of other cytokines or molecules from the cell.

[0022] Useful cytokine muteins may be identified using directed evolution / iterative selection cycles in which the cytokine muteins are contacted with decreasing concentrations of the cytokine receptor / ligand, thereby identifying cytokine muteins with the best binding affinity to the receptor.

[0023] The repeated selection cycles involve first (one or several cycles) binding to cytokine receptor multimers, preferably receptor tetramers, followed by (one or several cycles) binding to cytokine receptor monomers. Receptor multimers can be obtained, for example, by binding biotinylated receptors to streptavidin or via other ligand / binder interactions.

[0024] Iterative selection rounds may involve binding decreasing concentrations of receptor (e.g., 100 nM tetramer, 1 μM tetramer, 100 nM monomer; see also FIG. 2b). Without wishing to be bound by theory, decreasing receptor concentration may identify muteins with high receptor affinity.

[0025] The cytokine muteins that have bound to the receptor in the various selection rounds may then be expressed using an expression vector / vehicle that contains a nucleic acid encoding the relevant mutein. Thus, the method may further comprise the step of isolating and / or sequencing the nucleic acid contained in the expression vehicle / vector that bound to the receptor via the expressed mutein.

[0026] The method may further comprise contacting the cytokine mutein with the corresponding receptor or a binding fragment thereof at a pH of at least 7.2, preferably about pH 7.4. Additionally, the method may comprise contacting the corresponding wild-type cytokine with the corresponding receptor or a binding fragment thereof. Using either or both of these method steps, a user can determine the binding affinity of the cytokine mutein and the wild-type cytokine under each condition. The method may further comprise selecting muteins that bind with lower affinity to the corresponding receptor compared to the wild-type cytokine at each pH.

[0027] The method may further comprise the step of selecting a cytokine mutein which binds to a corresponding receptor or a binding fragment thereof with higher affinity at a pH of about 4.0 to about 7.0 than at least pH 7.2, preferably about pH 7.4. Preferably, this step selects a mutein which is characterized by binding to a corresponding receptor with lower affinity compared to the wild-type cytokine at least at pH 7.2, preferably about pH 7.4.

[0028] The present invention further relates to libraries comprising nucleic acids encoding the above-described cytokine muteins, and to cytokine mutein libraries.

[0029] The technology described herein may be applied to interleukin-2 (IL-2), which promotes T cell proliferation and controls various effector functions. IL-2 induces cytotoxic functions, including, for example, the production of IFNγ. Critical to the function of IL-2 is its binding activity. IL-2 receptors include, for example, IL2Rα, IL2Rβ, and IL2Rγ. For convenience, these receptors are collectively referred to as "IL-2 receptors."

[0030] IL-2 has been used as an immunotherapy for malignant tumors. However, some of the key functions of IL-2 are responsive to changes in pH, and in particular, the binding of IL-2 to its receptor is a pH-responsive process. Without wishing to be bound by theory, the acidic pH found in the TME inhibits IL-2 responses, for example, by inhibiting binding to IL-2Rα. The acidic tumor microenvironment (TME) adversely affects IL-2 receptor binding and affects IL-2 signaling. As a result, tumors experience reduced activation of STAT5 by IL-2 and reduced secretion of IFNγ / TNFα by CD8+ T cells. This combination may reduce the efficacy of IL-2-based therapies, especially when used to treat cancer.

[0031] The present disclosure provides IL-2 muteins that are pH tolerant and retain important therapeutic functions at acidic extracellular pH. Furthermore, the particular therapeutic functions imparted to these IL-2 muteins are more potent or effective at acidic pH than at neutral or other pH. Without wishing to be bound by theory, this has the advantage that the IL-2 muteins described herein are selective for the treatment of diseased cells / tissues, particularly those that induce or generate an acidic microenvironment.

[0032] The IL-2 muteins of the present disclosure include binds to any one of the disclosed IL-2 receptors; and / or binds to IL-2Rα; and / or binds to IL-2 receptor or IL-2Rα with higher affinity at a pH selected from about 4.0 to about 7.0, preferably from about 5 to about 6.5, than at a pH selected from about 7.2 to about 7.5; and / or binds to the IL-2 receptor or IL-2Rα with lower affinity at a pH of about 7.2 to about 7.5 compared to the wild-type IL-2 molecule; and / or binds to the IL-2 receptor or IL-2Rα with higher affinity than the wild-type IL-2 molecule at a pH selected from about pH 4.0 to about pH 7.0, preferably about pH 5 to about pH 6.5; and / or Causes activation of STAT5; and / or It induces stronger activation of STAT5 at pH 6.5 than at pH 7.2.

[0033] The IL-2 muteins of the present disclosure bind to an IL-2 receptor (including, for example, IL-2Rα) with higher affinity at a pH selected from about pH 4.0 to about 7.0, and the higher affinity binding at about pH 4.0 to about 7.0 is characterized by a binding constant Kd that is about 0.3; about 0.5; about 0.8; about 1; about 1.5; about 2; about 2.5, or about 3 orders of magnitude lower than the binding constant Kd for binding at a pH of about 7.2 to about 7.5.

[0034] Furthermore, binding of the IL-2 mutein to an IL-2 receptor (e.g., IL-2Rα) with lower affinity compared to the wild-type IL-2 molecule at a pH of about 7.2 to about 7.5 may be characterized by a higher binding constant Kd for the IL-2 mutein, on the order of about 0.3; about 0.5; about 0.8; about 1; about 1.5; about 2; about 2.5 or about 3, compared to the wild-type IL-2 molecule.

[0035] The binding of the IL-2 mutein to an IL-2 receptor (e.g., IL-2Rα) with higher affinity compared to the wild-type IL-2 molecule at a pH selected from about pH 4.0 to about 7.0 may be characterized by a binding constant Kd that is on the order of about 0.3; about 0.5; about 0.8; about 1; about 1.5; about 2; about 2.5 or about 3 lower for the IL-2 mutein compared to the wild-type IL-2 molecule.

[0036] Without wishing to be bound by theory, binding of IL-2 muteins to their high affinity receptor complexes may lead to stronger phosphorylation of STAT5 at pH 6.5 than at pH 7.2 by stabilizing the cytokine-cytokine receptor complex. Furthermore, (again without wishing to be bound by theory), IL-2 muteins may potently induce proliferation of activated T cells expressing high affinity receptor complexes in acidic microenvironments such as those found in the tumor microenvironment (TME) and tertiary lymphoid structures (TLS).

[0037] Due to the high activity of the IL-2 muteins of the present invention in the tumor microenvironment (TME) and tertiary lymphoid structures (TLS), and the relatively low activity in the periphery, such as in blood, the IL-2 muteins of the present invention can overcome the dose-limiting toxicity problems associated with prior art IL-2 therapies. Furthermore, when combined with other therapeutic molecules, such as antibodies against checkpoint inhibitors, the action of the IL-2 molecules of the prior art limits the dosage of such other molecules due to combined toxicity in the periphery. Thus, the selective activity of the IL-2 muteins can reduce toxicity in combination therapies and allow for higher doses of other therapeutic molecules, such as antibodies against checkpoint inhibitors, thereby increasing the therapeutic efficacy of such therapies. Combination therapies (including the cytokine muteins of the present disclosure, the IL-2 muteins, and some other therapeutic / active agents) are described elsewhere herein.

[0038] The present invention will now be described with reference to the following figures. [Brief description of the drawings]

[0039] [Figure 1A] [Figure 1a], [Figure 1b], [Figure 1c], [Figure 1d], [Figure 1e], [Figure 1f], [Figure 1g], [Figure 1h], [Figure 1i], [Figure 1j], [Figure 1k] Low pH reduces the activity of IL-2. (Figure 1A) Dose response of STAT5 phosphorylation in preactivated CD8+ T cells stimulated with IL-2 at pH 7.5 or 6.5 (top panel) and signaling kinetics at suboptimal (10 pM) and saturating doses (10 nM) (bottom panel). Graphs represent the mean ± standard deviation of three independent duplicate experiments. [Figure 1B] (FIG. 1B) Microscale thermophoresis (MST) analysis of the interaction of IL-2 with IL-2Rα at different pH. Kd values ​​are shown for each condition. Data are means ± standard deviation. [Figure 1C] (FIG. 1C) Flowchart of the in vivo experiment. [Figure 1D] (Fig. 1D) Tumor growth in B16.SIY WT or LDHA / B DKO-bearing mice treated with 100 μl PBS or 20 μg Fc4-IL-2 (equivalent to 7 μg IL-2) by ip injection for 5 days. Treatment was initiated when tumors reached 50–100 mm3 in size. Graph shows one representative experiment out of three independent experiments (n=6 per group). *p=0.0238 (B16.SIY WT+Fc4-IL-2 vs. B16.SIY DKO+Fc4-IL-2); *p=0.0242 (B16.SIY DKO+PBS vs. B16.SIY DKO+Fc4-IL-2). Data are means ± standard deviation (sem) and significance was determined by one-way ANOVA with Tukey's correction. ns=not significant. [Figure 1E] (Figure 1E-K) Analysis of tumors from mice sacrificed 15 days after tumor inoculation. (Figure 1E) Percentage of CD8+ T cells shown. **p=0.001 (B16.SIY WT+PBS vs. B16.SIY WT+Fc4-IL-2); **p=0.0018 (B16.SIY DKO+PBS vs. B16.SIY DKO+Fc4-IL-2). [Figure 1F](FIG. 1F) The percentages of CD8+ T cells and Treg cells are shown. [Figure 1G] (Figures 1G-1I) Cells stimulated with PMA / ionomycin were analyzed for cytokine production. Percentages of IFN-γ+ (Figure 1G), TNF+ (Figure 1H), IFN-γ+TNF+ (Figure 1I) and CD8+ T cells are shown. (Figure 1G) **p=0.0013; ****p<0.0001. [Figure 1H] (Figure 1H)****p<0.0001. [Figure 1I] (Figure 1I)****p<0.0001. [Figure 1J] Percentage of PD1+CD8+ T cells is shown. *p=0.0191 (B16.SIY WT+PBS vs. B16.SIY WT+Fc4-IL-2); *p=0.0102 (B16.SIY WT+Fc4-IL-2 vs. B16.SIY DKO+Fc4-IL-2); **p=0.0074. [Figure 1K] (Fig. 1K) Percentage of TIM3+CD8+ T cells is shown. **p=0.0067. (Fig. 1E-K) Graphs are pooled results from three independent experiments. Significance was determined by one-way ANOVA with Tukey's correction. ns=not significant. Data are means ± SD, and each symbol represents one tumor type (Fig. 1E,F,J,K) or two tumor types pooled (Fig. 1G-I). [Figure 2A] [Figure 2a], [Figure 2b], [Figure 2c], [Figure 2d], [Figure 2e], [Figure 2f], [Figure 2g] show the selection of pH-tolerant IL-2 variants. (Figure 2A) Shows the IL-2 protein library (blue) expressed on the yeast surface and interacting with biotinylated IL-2Rα tetramer (yellow). Amino acids mutated during the construction of the IL-2 library are shown in red. [Figure 2B] (FIG. 2B) Mean fluorescence intensity (MFI) of yeast representing the IL-2 library after each selection round at pH 5 is shown. [Figure 2C] (Figure 2C) Structure of the IL-2-IL-2Rα receptor complex. IL-2Rα is shown in yellow and IL-2 in blue. Mutations identified within Switch-2 are highlighted in red and shown on the right. [Figure 2D] (FIG. 2D) Dose-dependent binding of serial dilutions of IL-2Rα to the surface of yeast expressing IL-2WT or Switch-2 at different pH levels. Graphs represent the mean ± standard deviation of two independent experiments. [Figure 2E] (Figure 2E) Quantification of IL-2 / IL-2Rα interaction at the plasma membrane of live cells by two-color TIRF microscopy using labeled IL-2Rα and IL-2 (left panel) and a graph of IL-2 binding normalized to IL-2Rα cell surface expression. Data are means ± standard deviation, and each data point represents the results of one cell. Significance was calculated by the Kolmogorov-Smirnov test. ****p<0.0001. [Figure 2F] (Fig. 2F, G) Dose-response curves of phosphorylated STAT5 (pSTAT5) induced by IL-2 WT and Switch-2 at pH 7.5 and pH 6.5 in freshly isolated (Fig. 2F) and preactivated (Fig. 2G) CD8 T cells. Graphs represent the mean ± standard deviation of three independent duplicate experiments. [Figure 2G] (Fig. 2F, G) Dose-response curves of phosphorylated STAT5 (pSTAT5) induced by IL-2 WT and Switch-2 at pH 7.5 and pH 6.5 in freshly isolated (Fig. 2F) and preactivated (Fig. 2G) CD8 T cells. Graphs represent the mean ± standard deviation of three independent duplicate experiments. [Figure 3A] [Fig. 3A], [Fig. 3B], [Fig. 3C] Functional in vitro activity of IL-2 C1 at acidic pH. (Fig. 3A-C) Analysis of cytokines expressed by preactivated CD8+ T cells after 3 days of culture at pH 7.5 or 6.5 in the presence of 10 nM IL-2 WT or Switch-2. Cells were stimulated with PMA / ionomycin. (Fig. 3A) Supernatants of stimulated cells were analyzed by Luminex assay. ○ indicates amount of released cytokine normalized to control condition (IL-2 WT pH 7.5=100). Data are averages of 3 different donors. [Figure 3B](Fig. 3B,C) Graphs show percentage of IFN-γ+ cells (Fig. 3B) and TNF+ cells (Fig. 3C). Data are mean ± SD, each symbol represents one donor. (Fig. 3B) *p=0.0414 (IL-2 WT pH7.5 vs. IL-2 WT pH6.5); *p=0.0233 (IL-2 WT pH6.5 vs. Switch-2 pH6.5); *p=0.0195 (Switch-2 pH7.5 vs. Switch-2 pH6.5), one-way ANOVA with Tukey's correction. [Figure 3C] (Figure 3C) **p=0.0027 (IL-2 WT pH7.5 vs. IL-2 WT pH6.5); **p=0.0015 (IL-2 WT pH6.5 vs. Switch-2 pH6.5), one-way ANOVA with Tukey's correction. [Figure 3D] (FIG. 3D) Principal component analysis (PCA) of RNA-seq data. Pre-activated CD8+ T cells from 3 different donors were stimulated for 4 h after O / N resting. [Figure 3E] (FIG. 3E) GSEA analysis of Switch-2- and IL-2 WT-stimulated CD8+ T cells at pH 7.5 (upper panel) and pH 6.5 (lower panel). [Figure 3F] (FIG. 3F, G) Heatmaps of the top 476 variable and significant genes (FIG. 3F) and the set of T cell-specific genes (FIG. 3G), respectively. Gene expression is shown as z-score. [Figure 3G] (FIG. 3F, G) Heatmaps of the top 476 variable and significant genes (FIG. 3F) and the set of T cell-specific genes (FIG. 3G), respectively. Gene expression is shown as z-score. [Figure 4A][Figure 4a], [Figure 4bc], [Figure 4d], [Figure 4e], [Figure 4f], [Figure 4g], [Figure 4i], [Figure 4j] Switch-2 improves survival and stimulates antitumor immune responses. (Figure 4A) Pulmonary edema (lung wet weight) was assessed by measuring lung weight before and after drying in mice treated with PBS, 20 μg or 50 μg of Fc4-IL-2 WT or Switch-2. Data are the mean ± standard deviation of two independent experiments, and each symbol represents a mouse. ***p=0.0002 (Fc4-IL-2 WT 20 μg vs. Fc4-Switch-2 20 μg); ****p<0.0001. [Figure 4B] (Figure 4B, C) The percentage of NK in the blood (Figure 4B) and lymph nodes (Figure 4C) of mice treated with PBS, 20 μg or 50 μg of Fc4-IL-2 WT or Switch-2. (Figure 4B) *p=0.0338 (comparison of Fc4-IL-2 WT 20 μg to Fc4-Switch-2 20 μg); ***p=0.0006 (comparison of Fc4-IL-2 WT 50 μg to Fc4-Switch-2 50 μg); ****p<0.0001. [Figure 4C] (Figure 4C) **p=0.0031 (Fc4-IL-2 WT 20 μg vs. Fc4-Switch-2 20 μg); ***p=0.0014 (PBS vs. Fc4-IL-2 WT 20 μg); ***p<0.0001. [Figure 4D] (FIG. 4D) Flow chart of the in vivo experiment. [Figure 4E] (Figure 4E) Tumor growth in B16.SIY WT-bearing mice treated with 100 μl PBS or 20 μg Fc4-IL-2 WT or Fc4-Switch-2 by ip injection for 5 days. Treatment was initiated when tumors reached 50–100 mm3 in size (n=6 per group). Graph shows representative results of three independent experiments. ***p=0.0005 (PBS vs. Fc4-Switch-2), ***p=0.0007 (Fc4-IL-2 WT vs. Fc4-Switch-2). Data are means ± standard deviations, and significance was determined by one-way ANOVA with Tukey's correction. [Figure 4F] (FIG. 4F-L) Analysis of tumors obtained from mice sacrificed 15 days after tumor inoculation. (FIG. 4F) Percentage of Ki67+CD8+ T cells is shown. ***p=0.0006. [Figure 4G] (Figure 4G) The percentage of NK1.1+CD122+ cells is shown. *p=0.0359 (PBS vs. Fc4-IL-2 WT); *p=0.0334 (Fc4-IL-2 WT vs. Fc4-Switch-2); ****p<0.0001 (Figure 4I, Figure 4J). [Figure 4I] The percentages of CD8+IFN-γ+ (Fig. 4i) and CD8+TNF+ (Fig. 4j) T cells after stimulation with PMA / ionomycin are shown. (Fig. 4i) *p=0.0465; **p=0.0035; ****p<0.0001. [Figure 4J] (Figure 4J) ***p=0.0001;****p<0.0001. [Figure 4K] (FIG. 4K) The percentage of PD1+CD8+ T cells is shown. *p=0.0158. [Figure 4L] (Figure 4L) The percentage of TIM3+CD8+ T cells is shown. (Figure 4A-C,E-L) Data are the mean ± standard deviation, and each symbol represents one mouse (Figure 4A-C,F,G,K,L) or two mice pooled (Figure 4I,J) from two (Figure 4A-C) and three (Figure 4F-L) independent experiments. Significant differences were determined by one-way ANOVA with Tukey's correction. [Diagram 5][Figure 5ab], [Figure 5cd] Flow cytometric analysis of pH-dependent binding of yeast that exhibited IL-2 WT at pH 7 (Figure 5a) and pH 5 (Figure 5c) or IL-2 MUT1 at pH 7 (Figure 5b) and pH 5 (Figure 5d). Figure 5B shows flow cytometric analysis of pH-dependent binding of yeast that exhibited IL-2 WT at pH 7 (Figure 5a) and pH 5 (Figure 5c) or IL-2 MUT1 at pH 7 (Figure 5b) and pH 5 (Figure 5d). Figure 5C shows flow cytometric analysis of pH-dependent binding of yeast that exhibited IL-2 WT at pH 7 (Figure 5a) and pH 5 (Figure 5c) or IL-2 MUT1 at pH 7 (Figure 5b) and pH 5 (Figure 5d). FIG. 5D shows analysis of pH-dependent binding of yeast expressing IL-2 WT at pH 7 (FIG. 5a) and pH 5 (FIG. 5c) or IL-2 MUT1 at pH 7 (FIG. 5b) and pH 5 (FIG. 5d) by flow cytometry. [Figure 6] Alignment of sequence IDs 1, 2-8 is shown. SEQ ID NO: 1: mature human IL-2, accession number P60568; SEQ ID NO: 3: mature mouse IL-2, accession number P04351; SEQ ID NO: 4: mature rat IL-2, accession number P17108; SEQ ID NO: 5: mature porcine IL-2, accession number P26891; SEQ ID NO: 6: mature fox IL-2, accession number Q25BC3; SEQ ID NO: 7: mature canine IL-2, accession number NP_001003305; and SEQ ID NO: 8: mature macaque IL-2, accession number P68291. Residues 37, 38, 41, 42, 43, 64 of SEQ ID NO: 1 and the respective residues of the other SEQ ID NOs are shown in bold. [Figure 7] 1 shows additional IL-2 pH resistance mutants. FIG. 1 is a dot plot of IL-2 mutants bound to IL-2Rα at pH 5 and pH 7. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] It should be noted that the terms "comprising," "comprising," and / or "having" are used to indicate that aspects and embodiments of the invention "comprise" one or more particular features. It should be understood that the above terms may also encompass aspects and / or embodiments that "consist essentially of" or "consist of" the relevant feature or features.

[0041] The IL-2 muteins of the present disclosure are modified relative to a wild-type or reference IL-2 sequence. For example, the IL-2 muteins of the present disclosure contain one or more amino acid modifications relative to a wild-type or reference sequence. The amino acid modification includes the substitution of a wild-type or reference amino acid with another amino acid. Such substitutions may be conservative in that a wild-type redidue is replaced by another residue having the same or similar structural, chemical, and / or physiochemical properties. "Conservative" amino acid substitutions are made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0042] Substitutions may also be "non-conservative" in that wild-type residues are replaced with amino acids from a different class, e.g., structurally dissimilar, chemically dissimilar, and / or physiochemically heterologous or dissimilar amino acids.

[0043] The amino acid modification may include the deletion of an amino acid residue from the wild type or reference sequence. Other amino acid modifications include the insertion of one or more amino acids into the wild type / reference sequence. The amino acid modification may also include the inversion of a particular part or part of the wild type / reference sequence.

[0044] The IL-2 muteins of the disclosure may include (relative to a wild-type or reference sequence) one or more of these modifications, such as one or more (e.g., 2, 3, 4, 5, 6 or more) amino acid substitutions, one or more (e.g., 2, 3, 4, 5, 6 or more) deletions of amino acid residues, and / or one or more (e.g., 2, 3, 4, 5, 6 or more) additions of amino acid residues. The modified sequence may further include an inversion of one or more (e.g., 2, 3, 4, 5, 6 or more) portions of the wild-type or reference sequence.

[0045] The reference or wild-type IL-2 sequence may include the human mature IL-2 sequence represented herein by SEQ ID NO:1.

[0046] The sequence of IL-2 is highly conserved across various mammalian species, as evidenced by the sequence alignment shown in Figure 6. Thus, in alternative embodiments, the wild-type IL-2 sequence may comprise the mature IL-2 sequence from mouse (SEQ ID NO:3), rat (SEQ ID NO:4), pig (SEQ ID NO:5), fox (SEQ ID NO:6), dog (SEQ ID NO:7), or macaque (SEQ ID NO:8) as disclosed in Table 1.

[0047] [Table 1-1] [Table 1-2]

[0048] In view of the above, modified IL-2 molecules or IL-2 muteins according to the present disclosure may contain one or more amino acid modifications relative to the sequences of SEQ ID NOs: 1, 2-8.

[0049] In one teaching, the one or more amino acid modifications include: (i) one or more amino acid substitutions (e.g., including conservative substitutions); (ii) a deletion of one or more amino acids; (iii) the addition of one or more amino acids; and (iv) one or more sequence inversions are selected.

[0050] The modified IL-2 molecule or IL-2 mutein may contain a mutation at any one or more residues selected from residue 35 to residue 45, residue 58 to residue 71, and / or residue 107 to residue 112 of SEQ ID NO:1, 4, 5, 8, or each of SEQ ID NO:3, 6, or 7. In one teaching, the modified IL-2 molecule or IL-2 mutein may contain a mutation at any one or more residues from residue 37 to residue 43, residue 60 to residue 69, residue 109 to residue 110 of SEQ ID NO:1, 4, 5, or 8, or each of SEQ ID NO:3, 6, or 7. The modified IL-2 molecule or IL-2 mutein may comprise a mutation in any one or more of residues 37, 38, 41, 42, 43, 60, 61, 63, 64, 66, 68, 69, 109, and 110 of SEQ ID NO: 1, 4, 5, or 8, or the respective residues of SEQ ID NO: 3, 6, or 7. The modified IL-2 molecule or IL-2 mutein may comprise a mutation in any one or more of residues 37, 38, 41, 42, 43, and 64 of SEQ ID NO: 1, 4, 5, or 8, or the respective residues of SEQ ID NO: 3, 6, or 7. The term "respective residue" defines the corresponding residue of SEQ ID NO: 1, 4, 5, 8 in SEQ ID NO: 3, 6, or 7, as follows:

[0051] [Table 2]

[0052] The present disclosure provides IL-2 muteins comprising an amino acid substitution at residue 37 (relative to residues of SEQ ID NO: 1, 4, 5, or 8, or SEQ ID NO: 3, 6, or 7, respectively, or to the wild-type / reference sequence). By way of example, an IL-2 mutein of the present disclosure may comprise a threonine to histidine, arginine, or serine substitution at residue 37. In one teaching, and in addition to the amino acid modification at position 37, an IL-2 mutein may further comprise one or more modifications at one or more other residues. For example, an IL-2 mutein may comprise an amino acid modification at residue 37 and one or more additional amino acid modifications at any of positions 38, 41, 42, 43, and / or 64.

[0053] The present disclosure provides IL-2 muteins comprising an amino acid substitution at residue 38 (relative to SEQ ID NO: 1, 4, 5, or 8 or wild type / reference sequence). By way of example, the IL-2 muteins of the present disclosure may comprise an arginine to leucine, valine, isoleucine, or alanine substitution at residue 38. In one teaching, and in addition to the amino acid modification at position 38, the IL-2 muteins may further comprise one or more modifications at one or more other residues. For example, the IL-2 muteins may comprise an amino acid modification at residue 38 and one or more additional amino acid modifications at any of positions 37, 42, 41, 43, and / or 64. Similarly, the same embodiments are provided based on SEQ ID NO: 3, 6, or 7, and the respective residues of SEQ ID NO: 3, 6, or 7.

[0054] The present disclosure provides IL-2 muteins comprising an amino acid substitution at residue 41 (relative to SEQ ID NO: 1, 4, 5, or 8 or wild type / reference sequence). By way of example, the IL-2 muteins of the present disclosure may comprise a threonine to serine, glycine, or aspartic acid substitution at residue 41. In one teaching, and in addition to the amino acid modification at position 41, the IL-2 muteins may further comprise one or more modifications at one or more other residues. For example, the IL-2 muteins may comprise an amino acid modification at residue 41 and one or more additional amino acid modifications at any of positions 37, 38, 42, 43, and / or 64. Similarly, the same embodiments are provided based on SEQ ID NO: 3, 6, or 7, and the respective residues of SEQ ID NO: 3, 6, or 7.

[0055] The present disclosure provides IL-2 muteins comprising an amino acid substitution at residue 42 (relative to SEQ ID NO: 1, 4, 5, or 8 or wild type / reference sequence). By way of example, an IL-2 mutein of the present disclosure may comprise a phenylalanine to tyrosine substitution at residue 42. In one teaching, and in addition to the amino acid modification at position 42, an IL-2 mutein may further comprise one or more modifications at one or more other residues. For example, an IL-2 mutein may comprise an amino acid modification at residue 42 and one or more additional amino acid modifications at any of positions 37, 38, 41, 43, and / or 64. Similarly, the same embodiments are provided based on SEQ ID NO: 3, 6, or 7, and the respective residues of SEQ ID NO: 3, 6, or 7.

[0056] The present disclosure provides IL-2 muteins comprising an amino acid substitution at residue 43 (relative to SEQ ID NO: 1, 4, 5, or 8 or the wild type / reference sequence). By way of example, an IL-2 mutein of the present disclosure may comprise a lysine to glycine substitution at residue 43. In one teaching, and in addition to the amino acid modification at position 43, an IL-2 mutein may further comprise one or more modifications at one or more other residues. For example, an IL-2 mutein may comprise an amino acid modification at residue 43 and one or more additional amino acid modifications at any of positions 37, 38, 41, 42, and / or 64.

[0057] The present disclosure provides IL-2 muteins comprising an amino acid substitution at residue 64 (relative to SEQ ID NO: 1, 4, 5, or 8 or the wild type / reference sequence). By way of example, the IL-2 muteins of the present disclosure may comprise a non-conservative amino acid substitution of lysine, preferably with an acidic amino acid, most preferably a glutamic acid substitution at residue 64. In one teaching, and in addition to the amino acid modification at position 64, the IL-2 muteins may further comprise one or more modifications at one or more other residues. For example, the IL-2 muteins may comprise an amino acid modification at residue 64 and one or more additional amino acid modifications at any of positions 37, 38, 41, 42, and / or 43.

[0058] In summary, the present disclosure includes the following IL-2 muteins:

[0059] [Table 3]

[0060] The present disclosure provides IL-2 muteins comprising at least modifications at positions 37, 38, 41, 43 of SEQ ID NOs:1, 4, 5, 8, and at least one additional modification at positions 42 or 64, or at each of SEQ ID NOs:3, 6, or 7.

[0061] The present disclosure provides IL-2 muteins comprising amino acid modifications at residues 37, 38, 41, 43, and 64 of SEQ ID NOs:1, 4, 5, 8, or at each of residues 3, 6, or 7.

[0062] IL-2 muteins of the disclosure may comprise a sequence characterized by one or more of the following amino acid mutations (relative to SEQ ID NO: 1 or 8 or the wild-type / reference sequence): (i) T37H; and / or (ii) R38L; and / or (iii) T41S; and / or (iv) F42Y; and / or (v)K43G.

[0063] Thus, an IL-2 mutein according to the present disclosure may comprise SEQ ID NO:2.

[0064] SEQ ID NO:2

[0065] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLHLML SYGFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS

[0066] IL-2 muteins of the disclosure may comprise a sequence characterized by one or more of the following amino acid mutations (relative to SEQ ID NO: 1 or 8 or the wild-type / reference sequence): (i) T37S; and / or (ii) R38A; and / or (iii) T41D; and / or (iv) -K43G; and / or (v)-K64E. Thus, an IL-2 mutein according to the present disclosure may comprise SEQ ID NO:9. SEQ ID NO:9 APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLSAML DFGFYMPKKA TELKHLQCLE EELEPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS

[0067] IL-2 muteins of the disclosure may comprise a sequence characterized by one or more of the following amino acid mutations (relative to SEQ ID NO: 1 or 8 or the wild-type / reference sequence): (i) T37S; and / or (ii) R38L; and / or (iii) T41G; and / or (iv)-F42Y; and / or (v)-K43G.

[0068] Thus, an IL-2 mutein according to the present disclosure may comprise SEQ ID NO:10.

[0069] SEQ ID NO:10

[0070] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLSLML GYGFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS

[0071] IL-2 muteins of the disclosure may comprise a sequence characterized by one or more of the following amino acid mutations (relative to SEQ ID NO: 1 or 8 or the wild-type / reference sequence): (i) T37S; and / or (ii) R38V; and / or (iii) T41G; and / or (iv) K43G.

[0072] Thus, an IL-2 mutein according to the present disclosure may comprise SEQ ID NO:11.

[0073] SEQ ID NO:11

[0074] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLSVML GFGFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS

[0075] IL-2 muteins of the disclosure may comprise a sequence characterized by one or more of the following amino acid mutations (relative to SEQ ID NO: 1 or 8 or the wild-type / reference sequence): (i) T37R; and / or (ii) R38V; and / or (iii) T41G; and / or (iv) K43G.

[0076] Thus, an IL-2 mutein according to the present disclosure may comprise SEQ ID NO:12.

[0077] SEQ ID NO:12

[0078] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLRVML GFGFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS

[0079] IL-2 muteins of the disclosure may comprise a sequence characterized by one or more of the following amino acid mutations (relative to SEQ ID NO: 1 or 8 or the wild-type / reference sequence): (i) T37S; and / or (ii) R38I; and / or (iii) T41G; and / or (iv) K43G.

[0080] Thus, an IL-2 mutein according to the present disclosure may comprise SEQ ID NO:13.

[0081] SEQ ID NO:13

[0082] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLSIML GFGFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS

[0083] An IL-2 mutein may include a functional fragment of any of the modified molecules or muteins described herein. In one teaching, the IL-2 muteins of the present disclosure may comprise, consist essentially of, or consist of a functional fragment of the sequence provided by SEQ ID NO: 2, 9, 10, 11, 12, or 13. A "functional" fragment may include a fragment that retains one or more functions imparted to a longer / full or complete IL-2 mutein. For example, a fragment of the present disclosure may retain one or more functions of a mutein that includes the entire sequence of SEQ ID NO: 2, 9, 10, 11, 12, or 13. Such functions may include, for example, the ability to bind to IL-2Rα; and / or the ability to bind to IL-2Rα with a higher affinity at pH 6.5 than at pH 7.2; and / or the ability to induce a stronger activation of STAT5 at pH 6.5 than at pH 7.2.

[0084] The mutein fragments may be tested for any function (e.g., binding, T cell activation, and / or immune effector function) using any number of different assays. By way of example, a binding assay may involve contacting the IL-2 mutein fragment to be tested with IL-2Rα, and detection of binding of the fragment to IL-2Rα in such an assay indicates that the fragment retains the required binding function. Additionally, the fragments may be tested for their ability to promote T cell proliferation and / or immune effector function using a contacting with CD8+ T cells assay. Functional fragments stimulate CD8+ T cells to proliferate and / or produce effector cytokines. Any of the binding, T cell activation, and / or effector function assays may be performed at an acidic pH, e.g., a pH below pH 7.4, e.g., about 6.1, about 6.2, about 6.3, about 6.4, about 6.5 (or any other pH described herein). This not only tests the functional capabilities of the fragments, but also determines whether the fragment retains the characteristic of acid resistance. The results of these assays may be compared to the results of positive and / or control assays using, for example, wild-type IL-2, IL-2 muteins and / or IL-2 (mutein) fragments with known or predetermined function. Control assays may be performed at a different pH, for example neutral pH, to test for fragments that exhibit stronger activity / function at acidic pH than at neutral pH.

[0085] A fragment of SEQ ID NO:2, 9, 10, 11, 12 or 13 may have any number of residues between about 10 and about n-1 (where n=130; i.e., the total number of residues in SEQ ID NO:2, 9, 10, 11, 12 or 13). By way of example, a fragment may contain 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125 or 129 amino acid residues of SEQ ID NO:2, 9, 10, 11, 12 or 13.

[0086] A fragment of SEQ ID NO:2 may include at least residue numbers 57, 58, 61, 62, and 63. In one teaching, a fragment of SEQ ID NO:2, 9, 10, 11, 12, or 13 may include residues 57-63. A fragment containing any of these selected residues may further include fragments of the sequence immediately upstream and / or downstream thereof.

[0087] Additionally, IL-2 muteins of the present disclosure may exhibit a degree of sequence identity or homology to the sequences of SEQ ID NOs: 2, 9, 10, 11, 12 or 13. For example, useful fragments may include sequences that are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical or homologous to the sequences of SEQ ID NOs: 2, 9, 10, 11, 12 or 13.

[0088] In view of the above, the term IL-2 mutein encompasses not only the specific examples provided by SEQ ID NO:2, 9, 10, 11, 12 or 13, but also functional fragments thereof, molecules having a degree of sequence identity / homology to SEQ ID NO:2, 9, 10, 11, 12, 13, and / or other IL-2-derived molecules containing one or more of the amino acid modifications described.

[0089] The present disclosure further provides nucleic acids encoding any of the modified IL-2 muteins described herein. For example, the present disclosure provides nucleic acids encoding IL-2 muteins having one or more amino acid modifications relative to the reference or wild-type sequence. The nucleic acid may be DNA or RNA, and is preferably mRNA.

[0090] The disclosure provides nucleic acids encoding SEQ ID NOs: 2, 9, 10, 11, 12, 13, or any functional fragment thereof.

[0091] The nucleic acids of the disclosure may be codon optimized for expression in a host cell, such as a microbial host cell.

[0092] Disclosed herein is a vector comprising the nucleic acid of the present disclosure. For example, the present disclosure provides a vector comprising the nucleic acid encoding the IL-2 mutein, SEQ ID NO: 2, 9, 10, 11, 12, 13, or a functional fragment thereof.

[0093] Also disclosed is a host cell transformed with the nucleic acid or vector of the present disclosure. The host cell can be a eukaryotic or prokaryotic cell. The host cell can be a mammalian cell, an insect cell, or a plant cell. The host cell can be a microbial cell, such as a bacterium (such as E. coli). The host cell can be a T cell, preferably a T cell that comprises a chimeric antigen receptor (CAR).

[0094] Also disclosed herein are viruses that contain the nucleic acids of the present disclosure.

[0095] Methods for producing a mutein of the disclosure may include transforming a host cell with a nucleic acid or vector encoding an IL-2 mutein of the disclosure and inducing expression of the nucleic acid encoding the IL-2 mutein. In such methods, the expressed IL-2 mutein may be harvested, extracted or purified from the host cell or the medium in which the host cell is cultured.

[0096] The disclosure also provides a method for identifying a pH-resistant IL-2 mutein, the method comprising: mutating or modifying the IL-2 molecule to produce an IL-2 mutein; contacting the IL-2 mutein with IL-2Rα under acidic conditions to identify muteins that bind to IL-2Rα and have pH resistance.

[0097] The mutated or modified IL-2 molecule may comprise a wild-type IL-2 sequence or other IL-2 reference sequence. For example, the modified or modified IL-2 molecule may comprise SEQ ID NO: 1 or a functional fragment thereof.

[0098] Mutating or modifying an IL-2 molecule to generate an IL-2 mutein may include introducing one or more amino acid modifications into a wild-type or reference IL-2 sequence. A variety of techniques may be used for this purpose, including, for example, PCR-based methods that utilize the use of primers containing degenerate codons (e.g., NDT) to randomly mutate specific residues in the wild-type IL-2 sequence. Any mutein may be tested for its ability to bind to IL-2Rα.

[0099] The step of contacting the modified IL-2 (IL-2 mutein) with IL-2Rα may include contacting the modified IL-2 with an IL-2Rα fragment, where the IL-2Rα fragment is an IL-2 binding fragment. The IL-2 binding fragment of IL-2Rα may include an ectodomain. The IL-2Rα or any binding fragment thereof may be bound to a binding site, such as, for example, biotin. The IL-2Rα may include a detectable label, for example, a fluorescent label, for identifying the mutein bound to the receptor.

[0100] The method for identifying a pH-tolerant IL-2 mutein may further comprise the step of generating a library comprising nucleic acids encoding IL-2 muteins or fragments thereof, the IL-2 muteins comprising one or more amino acid substitutions (e.g., including conservative substitutions); (ii) one or more amino acid deletions; (iii) one or more amino acid additions; and (iv) one or more sequence inversions (all of which are described / defined later in this specification).

[0101] Mutating or modifying an IL-2 molecule to produce an IL-2 mutein may include introducing a mutation (e.g., a substitution, addition, deletion, or inversion) into one or more of residues 35 to 45, 58 to 71, and / or 107 to 112 of SEQ ID NO:1. In one teaching, mutating or modifying an IL-2 molecule to produce an IL-2 mutein may include introducing a mutation into one or more of residues 37 to 43, 60 to 69, and 109 to 110 of SEQ ID NO:1. Mutating or modifying an IL-2 molecule to produce an IL-2 mutein may include introducing a mutation into one or more of residues 37, 38, 41, 42, 43, 60, 61, 63, 64, 66, 68, 69, 109, and 110 of SEQ ID NO:1. The step of mutating or modifying the IL-2 molecule to generate an IL-2 mutein may include introducing a mutation at any one or more of residues 37, 38, 41, 42 and 43 of SEQ ID NO:1.

[0102] The nucleic acids of the generated library may encode muteins that include substitutions of naturally occurring or wild-type amino acids with other amino acids; for example, substitutions of naturally occurring or wild-type amino acids with other amino acids selected from the amino acids G, V, L, I, C, A, E, S, R, H, D, N, F, and Y. The nucleic acid library can be generated by nested PCR using primers with degenerate codons for any one or more of the above amino acids.

[0103] The method may further comprise expressing the nucleic acid library to obtain an IL-2 mutein library. The IL-2 muteins contained in the library may be expressed on the surface of an expression vehicle, such as a cell, a phage virus, e.g., a yeast cell.

[0104] The step of contacting the IL-2 mutein with IL-2Rα may be carried out at a range of different pH. For example, the step of contacting the IL-2 mutein with IL-2Rα may be carried out at a pH below about pH 7.4. For example, the contacting step may be carried out at a pH between about pH 4.0 or pH 5.0 and about pH 7.0 or pH 7.3. For example, the contacting step may be carried out at a pH between about pH 4.5 or pH 4.8 and about pH 6.0 or pH 6.5. The contacting step may be carried out at pH 5.5, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1 or pH 7.2. In this type of method, the mutein bound to IL-2Rα at an acidic pH may be identified as an IL-2 mutein that may have acid resistance. Additionally or alternatively, IL-2 muteins that exhibit stronger receptor (IL-2Rα) binding at acidic pH than their respective wild-type counterparts may be identified as IL-2 muteins that may be acid resistant.

[0105] Useful IL-2 muteins may be identified using directed evolution / iterative selection cycles in which the IL-2 muteins are contacted with decreasing concentrations of IL-2Rα (e.g., the IL-2Rα ectodomain), allowing the identification of IL-2 muteins with the best binding affinity to the receptor.

[0106] The repeated selection cycles involve first (one or several cycles) binding to cytokine receptor multimers, preferably receptor tetramers, followed by (one or several cycles) binding to cytokine receptor monomers. Receptor multimers can be obtained, for example, by binding biotinylated receptors to streptavidin or via other ligand / binder interactions.

[0107] Iterative selection rounds may involve binding decreasing concentrations of receptor (e.g., 100 nM tetramer, 1 μM tetramer, 100 nM monomer; see also FIG. 2b). Without wishing to be bound by theory, decreasing receptor concentration can identify muteins with high receptor affinity.

[0108] The IL-2 muteins identified that bind to the receptor in the selection round may then be expressed using an expression vehicle / vector that contains a nucleic acid encoding the relevant mutein.Thus, the method may further comprise the step of isolating and / or sequencing the nucleic acid contained in the expression vehicle that binds to the receptor via the expressed mutein.

[0109] The method may further comprise contacting the identified IL-2 muteins with the corresponding receptor or binding fragment thereof at a pH of at least 7.2, preferably about 7.4, to identify muteins that bind to the corresponding receptor with lower affinity at each pH compared to the wild-type cytokine.

[0110] The present invention further relates to libraries comprising nucleic acids encoding the above-described cytokine muteins, and to cytokine mutein libraries.

[0111] The acid-resistant IL-2 muteins may find application in the treatment and / or prevention of a variety of diseases and / or conditions, including cancer.

[0112] Thus, the disclosure provides a method for identifying an IL-2 mutein for use in the treatment of cancer, the method comprising: mutating or modifying the IL-2 molecule to produce an IL-2 mutein; contacting an IL-2 mutein with IL-2Rα and identifying a mutein that binds to IL-2Rα.

[0113] Again, the step of mutating or modifying the IL-2 molecule to generate an IL-2 mutein may include introducing one or more amino acid modifications into the wild-type or reference IL-2 sequence. Additionally, the step of contacting the IL-2 mutein with IL-2Rα may be performed at an acidic pH. The objective is to identify IL-2 muteins capable of binding to IL-2Rα at an acidic pH. As the tumor microenvironment may be acidic, IL-2 muteins that are pH resistant will be most useful in the treatment and / or prevention of cancer.

[0114] The present disclosure also provides pH-tolerant IL-2 muteins obtainable by a method comprising mutating or modifying an IL-2 molecule to produce an IL-2 mutein, and contacting the IL-2 mutein with IL-2Rα under acidic conditions to identify muteins that bind to IL-2Rα and are therefore pH-resistant. The IL-2 molecule to be mutated or modified may comprise a wild-type IL-2 sequence or other IL-2 reference sequence. For example, the IL-2 molecule to be modified may comprise SEQ ID NO: 1 or a functional fragment thereof. The acidic conditions may be adjusted as described above. Muteins that retain the ability to bind to IL-2Rα under acidic conditions would be highly useful as agents for use in the treatment of cancers in which the TME is acidic and inhibits the function of standard IL-2-based therapeutics. Preferably, the disclosed IL-2 muteins and fusion proteins are for use in the treatment of said cancers in which the extracellular pH (pHe) of the TME in the cancer tumor is less than about pH 7.4, less than 7.2, preferably less than 7.0, preferably less than about 6.8, and most preferably less than about 6.6. Such cancer types may be, for example, lymphoma cancer or solid cancer. Treating cancer may include determining the extracellular pH of the patient's TME prior to administering the IL-2 muteins, fusion proteins or related compositions disclosed herein. The pH of the TME may be determined by fluorescence imaging, PET, 1 H magnetic resonance spectroscopy (MRS), 31 P-MRS, 19 F MRS, hyperpolarization 13It can be determined according to various methods known in the art, including C MRS, magnetic resonance imaging (MRI), in particular CEST MRI as disclosed in Chen (Non-Patent Document 22), the entire contents of which are incorporated herein by reference. Preferably, the pHe of the TME is determined by MRI.

[0115] The present disclosure further provides modified IL-2 molecules for use in methods, compositions and medicaments for the treatment and / or prevention of various diseases and / or conditions.

[0116] Thus, the present disclosure provides IL-2 muteins for use in medicine.

[0117] In one teaching, the disclosure provides a protein comprising SEQ ID NO: 2, 9, 10, 11, 12, 13, or a functional fragment thereof, for use in medicine. Definitions of functional fragments are provided elsewhere herein.

[0118] The disclosure further provides nucleic acids encoding any of the disclosed IL-2 muteins for use in medicine. In one teaching, the disclosure provides nucleic acids encoding proteins comprising SEQ ID NO:2, 9, 10, 11, 12, 13, or fragments thereof, for use in medicine.

[0119] The present disclosure relates to For use as a medicine, any of the disclosed IL-2 muteins; and / or A protein comprising SEQ ID NO: 2, 9, 10, 11, 12, 13 or a fragment thereof; and / or a nucleic acid encoding any of the disclosed IL-2 muteins; and / or Nucleic acids encoding proteins comprising SEQ ID NO: 2, 9, 10, 11, 12, 13, or fragments thereof are provided.

[0120] In this regard, the IL-2 muteins described herein may be utilized or used as immunotherapeutics.

[0121] The present disclosure provides modified IL-2 molecules for use in the treatment or prevention of an immunological condition.

[0122] The present disclosure provides modified IL-2 molecules for use in the treatment or prevention of cancer.

[0123] In one teaching, the term "cancer" includes cancers whose (tumor) cells are characterized by the overproduction of lactic acid. The term "cancer" also includes cancers that result in tumors forming an acidic microenvironment.

[0124] Also disclosed is the use of a modified IL-2 molecule of the present disclosure in the manufacture of a medicament for the treatment or prevention of (i) cancer, or (ii) an immunological condition.

[0125] The present disclosure further provides a method for treating or preventing cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of any of the modified IL-2 molecules described herein.

[0126] Subjects to which the modified molecules of the present disclosure are administered include human or animal subjects suffering from an immunological condition and / or cancer. The subject may also be any human or animal subject predisposed and / or susceptible to an immunological condition or cancer that can be treated and / or prevented by the use of IL-2.

[0127] Without wishing to be bound by theory, a further advantage associated with the muteins of the present disclosure is that they are less toxic than wild-type or unmodified IL-2 molecules. The muteins of the present disclosure bind to IL-2Rα with higher affinity at acidic pH, induce stronger STAT5 activation at pH 6.5 than at pH 7.2, and are better at inducing cytotoxic T cell proliferation compared to wild-type IL-2 molecules. Thus, while high levels of systemic toxicity have hindered the therapeutic use of IL-2, the muteins provided by the present disclosure are selectively active in acidic tumor microenvironments due to reduced activity at neutral pH. In summary, again without wishing to be bound by theory, the IL-2 muteins of the present disclosure induce a strong response in acidic tumor microenvironments, but are less likely to cause systemic toxicity (than wild-type (or unmodified) IL-2 molecules) due to reduced activity at neutral pH.

[0128] The disclosure may further provide a fusion protein comprising a cytokine mutein or an IL-2 mutein described herein.

[0129] The fusion protein may further comprise one or more other molecules bound, linked or fused to the cytokine mutein or IL-2 mutein. In one teaching, the other molecule may be bound, linked or fused to the C-terminus, N-terminus or N-terminus and C-terminus of the cytokine mutein or IL-2 mutein. In another teaching, the fusion may comprise another molecule directed to the cytokine mutein or IL-2 mutein.

[0130] Thus, the disclosure provides a fusion protein comprising: (i) a cytokine mutein; or (ii) any of the disclosed IL-2 muteins; or (iii) A protein comprising SEQ ID NO: 2, 9, 10, 11, 12, 13, or a fragment thereof.

[0131] Other molecules of the fusion protein of the present disclosure include: a cytokine, cytokine mutein, or fragment thereof; Interleukin molecules or fragments thereof Polypeptide Binding Domains Antibodies or fragments thereof; single chain antibody; It may comprise VHH.

[0132] A fusion protein of the present disclosure may comprise an IL-2 mutein and at least one or more additional distinct cytokines, as described herein.

[0133] The polypeptide binding domains contained in the fusion proteins of the present disclosure may bind to or exhibit specificity / affinity for tumor antigens or checkpoint molecules. Checkpoint molecules are negative regulators of immune responses, such as costimulatory receptors present on the surface of some immune cells and ligands for said receptors. The checkpoint molecules (which the polypeptide binding molecules bind to or exhibit specificity / affinity for) may be selected from CD27, CD137, 2B4, TIGIT, CD155, CD160, ICOS, HVEM, CD40L, LIGHT, LAIR1, OX40, DNAM-1, PD-L1, PD1, PD-L2, CTLA-4, CD8, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDOL, ID02, TDO, KIR, LAG-3, TIM-3, or VISTA.

[0134] As mentioned above, the polypeptide binding domain comprised in the fusion of the invention may bind to or exhibit specificity / affinity for a tumor antigen, and the term "tumor antigen" includes any of the following: EpCAM, EGFR, HER-2, HER-3, c-Met, FoIR, PSMA, CD38, BCMA, CEA, 5T4, AFP, B7-H3, cadherin-6, CAIX, CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD40, CD352, CD37, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, CLDN18.2, DLL3, EphA2, ED-B fibronectin, FAP, FGFR2, FGFR3, GPC3, gpA33, FLT-3, gpNMB, HPV-16, and the like. E6, HPV-16 E7, ITGA2, ITGA3, SLC39A6, MAGE, mesothelin, Muc1, Muc16, NaPi2b, Nectin-4, P-cadherin, NY-ESO-1, PRLR, PSCA, PTK7, ROR1, SLC44A4, SLTRK5, SLTRK6, STEAP1, TIM1, Trop2, or WT1. The polypeptide binding domain may bind to a hematological tumor antigen, and the hematological tumor antigen may be expressed by lymphocytes. Examples of such tumor antigens include ADIR, AURKA, BCR-ABL, BMI1, CML28, CML66, cyclin A1, DDX3Y, DKK1, FMOD, FRAME, G250 / CAIX, HAGE, HM1.24, hTERT, LPP, MAG EA3, MAGEA3, MEF2D, MLL, MPP1, MUC1, myeloperoxidase, NEWREN60, NY-ESO-1, PANE1, PRAME, proteinase 3, PTPN20A / B, RHAMM, ROR1, SLAMF7, survivin, TEX14, WT1, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD52, CD123, CD269, CD138, HM1.24, and SLAMF7. The term (blood) tumor antigen includes surface antigens such as, for example, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD52, CD123, CD269, CD138, HM1.24, SLAMF7.

[0135] The polypeptide domain for use in the fusion of the present disclosure binds to or exhibits affinity / specificity for an antigen expressed by regulatory T cells. The antigen expressed by regulatory T cells may be included in the cell surface markers of regulatory T cells. The antigen expressed by regulatory T cells may be selected from CTLA4, CD25, OX40, GITR, TNFRII, NRP1, TIGIT, CCR8, LAYN, MAGEH1, CD27, ICOS, LAG-3, TIM-3, CD30, IL-1R2, IL-21R, 4-1BB, PDL-1, and PDL-2.

[0136] The fusion protein of the present disclosure may comprise an anti-Ox40 antibody or a fragment thereof. Useful examples include the antibodies disclosed in WO 2015 / 132580 or US 2019 / 275084 (the entire disclosures of which are incorporated herein by reference).

[0137] The antibody for use in the fusion protein of the present disclosure may include an antagonist antibody or an agonist fragment thereof. Any fragment of these antibodies may be used, and this fragment also shows the required antagonist / agonist activity. Useful agonist antibodies may be, for example, those disclosed in WO2020 / 006509, WO2018 / 045110, WO2017 / 214092, WO2019 / 072868 (the relevant contents of all these documents are incorporated herein by reference).

[0138] The antibody for use in the fusion protein of the present disclosure may comprise a pH-responsive antibody or a fragment thereof, which may retain the characteristic of being pH-responsive. A pH-responsive antibody exhibits different antigen-binding kinetics at different pHs. For example, without wishing to be bound by theory, a pH-responsive antibody may bind to an antigen with higher or lower affinity at an acidic pH than it binds to the same antigen at a different (e.g., neutral or alkaline) pH. In one teaching, a pH-responsive antibody may exhibit high affinity for an antigen at an acidic pH (the increase being an increase compared to the affinity of the antibody for the same antigen at a different (e.g., neutral or alkaline) pH). The pH-responsive antibody (or fragment thereof) may bind (or have affinity / specificity for) CTLA-4 (disclosed in WO 2019 / 152413, the entire disclosure of which is incorporated herein by reference), PD-L1 (disclosed in WO 2017 / 161976, the entire disclosure of which is incorporated herein by reference), VISTA (disclosed in U.S. Patent Application Publication Nos. 2020 / 20055936 and WO 2019 / 183040, the entire disclosures of which are incorporated herein by reference). Fusion proteins of the present disclosure may include an anti-CD3 antibody disclosed in WO 2020 / 247932, an anti-EPCAM antibody disclosed in WO 2020 / 252095, or a pH-responsive antibody disclosed in WO 2018 / 218076.

[0139] Under conditions present at the tumor site, certain cytokines, such as IL-2, are conditionally active. Thus, the fusion proteins of the present disclosure may include a polypeptide domain that conditionally inactivates the cytokine muteins / IL-2 muteins of the present disclosure. The polypeptide domain that conditionally inactivates IL-2 may be a polypeptide that blocks or reduces the binding of IL-2 to its receptor. The polypeptide domain that conditionally inactivates an interleukin is known in the art as a masking moiety or domain. Activation is promoted by a conformational change in the fusion protein or by the release of the polypeptide domain from the fusion protein. The masking domain may be fused to IL-2 via a polypeptide linker. The polypeptide linker is preferably cleaved under conditions of the tumor microenvironment. Interleukin fusion proteins containing releasable masking moieties are disclosed, for example, in WO 2020 / 069398 by Xilio.

[0140] A fusion protein of the present disclosure (which fusion protein comprises a cytokine mutein or IL-2 mutein of the present disclosure) may comprise a half-life extending molecule. For example, a fusion of the present disclosure may comprise a cytokine mutein or IL-2 mutein (as defined herein) fused or conjugated to a half-life extending molecule. The half-life extending molecule comprises an immunoglobulin fragment, preferably an Fc molecule, a polypeptide binding domain that binds to a blood serum protein, preferably a polypeptide binding domain that binds to albumin or a polymer.

[0141] In this respect, the term "Fc molecule" may include human IgG1 Fc. In one teaching, a useful IgG1 Fc molecule may include one or more mutations that alter the effector function of the Fc. As an example, a human IgG1 may include a substitution of N297, such as a substitution of N297G. In another teaching, a useful human IgG Fc molecule may include a substitution or deletion of the C-terminal lysine.

[0142] The fusions of the present disclosure may include a linker moiety that links the mutein component to the other components of the fusion. Suitable linkers may include those disclosed in WO2021 / 030602, the relevant contents of which are incorporated herein by reference. In one teaching, the fusions of the present disclosure may include a cytokine / IL-2 mutein linked (via any short peptide linker) to a linker that links the Fc and human IL-2 mutein portions of the protein.

[0143] The polymer (included in the fusions of the present disclosure) may include a polyethylene glycol molecule.

[0144] The present disclosure further provides fusion proteins of the present disclosure for use in methods, compositions and medicaments for the treatment and / or prevention of various diseases and / or conditions.

[0145] By way of example, the present disclosure provides any of the disclosed fusion proteins for use in medicine. In one teaching, the present disclosure provides a fusion protein comprising a cytokine mutein or an IL-2 mutein, a protein comprising SEQ ID NO: 2, 9, 10, 11, 12, 13, or a fragment of any of these, for use in medicine.

[0146] The disclosed fusion proteins can be utilized or used as immunotherapeutics.

[0147] The present disclosure provides any one of the disclosed fusion proteins for use in treating an immunological condition.

[0148] The present disclosure provides the fusion protein of the present disclosure for use in the treatment of cancer. In one teaching, the term "cancer" includes cancers whose (tumor) cells are characterized by excessive production of lactic acid. The term "cancer" includes any cancer that results in tumors forming an acidic microenvironment.

[0149] Also disclosed is the use of a fusion protein of the present disclosure in the manufacture of a medicament for the treatment of (i) cancer, or (ii) an immunological condition.

[0150] The present disclosure further provides a method of treating cancer, the method comprising administering a therapeutically effective amount of any of the fusion proteins disclosed herein to a subject in need thereof.

[0151] Subjects to which the fusion proteins of the present disclosure are administered include human or animal subjects suffering from an immunological condition and / or cancer, and may be any human or animal subject predisposed to and / or susceptible to an immunological condition or cancer that can be treated and / or prevented using the fusion proteins of the present disclosure.

[0152] It should be noted that any of the disclosed cytokine muteins, IL-2 muteins or fusion proteins may be provided in the form of a composition. Such compositions can be used as pharmaceuticals. The composition of the present disclosure may be, for example, a pharmaceutical composition that includes one or more pharma- ceutically acceptable excipients.

[0153] Alternatively, compositions for use as pharmaceuticals according to the present disclosure may comprise a polynucleotide, preferably RNA, most preferably mRNA, encoding any of the disclosed cytokine muteins, IL-2 muteins or fusion proteins.

[0154] The disclosed compositions comprising proteins or polynucleotides, preferably mRNA, may be administered, for example, systemically or locally by intratumoral or extratumoral administration.

[0155] Compositions comprising any of the disclosed cytokine muteins, IL-2 muteins, or fusion proteins may further comprise one or more additional active or therapeutic agents. For example, the compositions may comprise an anti-tumor antigen antibody, a checkpoint molecule, an antibody against a checkpoint molecule, a tumor antigen, a steroid, and / or a CAR T cell.

[0156] Any of the therapeutic treatments described herein may further include the use of one or more additional active or therapeutic moieties, such as anti-tumor antigen antibodies, checkpoint molecules, antibodies against checkpoint molecules, tumor antigens, steroids and / or CAR T cells, which may be administered separately, before, during (simultaneously or together), or after administration of the cytokine muteins, IL-2 muteins or fusion proteins of the disclosure.

[0157] In one teaching, the additional active or therapeutic moiety is selected from CD27, CD137, 2B4, TIGIT, CD155, CD160, ICOS, HVEM, CD40L, LIGHT, LAIR1, OX40, DNAM-1, PD-L1, PD1, PD-L2, CTLA-4, CD8, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDOL, ID02, TDO, KIR, LAG-3, TIM-3, or VISTA, and is most preferably PD-L1, PD1, or PD-L2.

[0158] For example, the disclosure provides the cytokine muteins, IL-2 muteins and / or fusion proteins of the disclosure, together with one or more additional therapeutically or pharma- ceutical active moieties, for use in methods, compositions and medicaments for the treatment and prevention of cancer (as defined herein) and / or immunological conditions.

[0159] Any of the disclosed cytokine muteins, IL-2 muteins or fusion proteins may be used as an adjuvant. An "adjuvant" is a compound that enhances, modulates or strengthens the host immune response to one or more antigens co-administered with the adjuvant. Thus, the disclosed cytokine muteins, IL-2 muteins or fusion proteins may be used in combination with one or more antigens to enhance, modulate or strengthen the host immune response to one or more antigens. The one or more antigens may include, for example, microbial, bacterial and / or viral antigens.

[0160] Thus, the present disclosure further provides a method of improving an immune response to an antigen in a host, the method comprising administering to the host an antigen and any of the disclosed cytokine muteins, IL-2 muteins or fusion proteins. Without wishing to be bound by theory, the cytokine muteins, IL-2 muteins or fusion proteins act as adjuvants that enhance, modulate or strengthen the immune response to the antigen in the host.

[0161] The present disclosure further provides a vaccine composition comprising an antigen and any of the disclosed cytokine muteins, IL-2 muteins, or fusion proteins. In such vaccine compositions, the cytokine mutein, IL-2 mutein, or fusion protein components act or function as an adjuvant. In one teaching, the vaccine is a tumor vaccine. EXAMPLES

[0162] Materials and Methods Cell culture and media B16.SIY WT and B16.SIY LDHA / B DKO (kindly provided by Marina Kreutz, University of Regensburg) were cultured in RPMI 1640 with GlutaMAX supplemented with 10% fetal bovine serum (FBS) and penicillin / streptomycin. HeLa cells stably transfected with SNAPf-IL-2Rα were cultured at 37°C and 5% CO2 in MEM medium supplemented with Earle's balanced salts, glutamine, 10% FBS, non-essential amino acids, and HEPES buffer. For baculovirus preparation and protein production, Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five) cells were cultured in SF900 III SFM medium (Invitrogen; 12658027) and Insect Xpress medium (Lonza; BELN12-730Q), respectively. Human T cells were cultured in RPMI 1640 with GlutaMAX (Gibco, 61870036) supplemented with 10% FBS, minimal non-essential amino acids, 1 mM sodium pyruvate, and penicillin / streptomycin. When adjusting the pH of the medium for short-term or long-term experiments, the medium was acidified using HCl and 20 mM HEPES pH 6.5 was added to stabilize the pH at 6.5. An equal volume of HEPES pH 7.5 was added to the pH 7.5 medium. For mouse T cells, the medium was additionally supplemented with 50 μM β-mercaptoethanol.

[0163] Protein production Human IL-2 wild type (WT; residues 1-133) and Switch-2 were cloned in frame with an N-terminal gp67 and a C-terminal histidine tag into the pFB-CT10HF vector, human IL-2Rα ectodomain (residues 1-217) was cloned into the same vector with a C-terminal biotin acceptor peptide (BAP)-LNDIFEAQKIEWHW followed by a histidine tag, and for in vivo experiments, the Fc portion of human IgG4 was cloned into the N-terminus of IL-2 WT and Switch-2. Proteins were produced using the baculovirus expression system. Briefly, the vectors were recombined in DH10Bac bacteria (Gibco), and the resulting bacmids were used to produce baculovirus. Baculoviruses were produced and amplified in Spodoptera frugiperda (Sf9) cells and used for protein expression by infecting Trichoplusia ni (High Five) cells. Two days after infection, proteins released into the cell culture supernatant were captured using His-Pur Ni-NTA resin (Invitrogen; 88222). Proteins were purified by size exclusion on a Superdex 75 Increase column (GE Healthcare; 29-1487-21). Proteins were stored in 10 mM HEPES (pH 7.2) and 150 mM NaCl (HBS buffer). For IL-2Rα, proteins were reduced with 10 mM cysteine, alkylated with 20 mM iodoacetamide 14, and biotinylated using BirA ligase in the presence of 100 μM biotin. For microscopy experiments, IL-2 WT and Switch-2 were cloned in frame with N-terminal mannose-binding protein (MBP) and YbbR tags (DSLEFIASKLA peptide), as well as a C-terminal histidine tag into the pMAL vector. Proteins were expressed in BL21 E. coli cells by O / N induction with 1 mM IPTG at 20°C. The periplasmic fraction was isolated by osmotic shock and the recombinant protein was captured on His-Pur Ni-NTA resin. Proteins were purified by size exclusion on a Superdex 75 Increase column.

[0164] Microscale Thermophoresis (MST) MST was performed using an NT.115 Pico MST instrument (Nano Temper Technologies GmbH) equipped with red and blue filter sets. IL2 WT and Switch-2 were diluted to 200 nM in PBS buffer containing 0.05% Tween (PBS-T) and labeled with Monolith His-Tag Labeling Kit RED-tris-NTA (Nano Temper; MO-L018). RED-tris-NTA dye was diluted to 100 nM in PBS-T. The mixture was incubated for 30 min at room temperature (RT) in the dark. IL-2Rα ectodomain (25 μM) was diluted in a 1:1 ratio with a 16 gradient. The labeled proteins were then mixed with IL-2Rα ectodomain in a 1:1 ratio and incubated for 15 min in the dark. The capillaries were then individually filled and loaded into the instrument. Data were acquired using medium MST power and 20% LED. Data were analyzed using MO Control Software (Nano Temper). MST diagrams were generated using MO Affinity Analysis (Nano Temper) and GraphPad Prism 7.

[0165] Isolation and culture of human T cells Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated from buffy coats (Etablissement Francais du Sang) by density gradient centrifugation using Pancoll human (Pan Biotech, P04-60500). 200x106 PBMCs were stained with 15μl of anti-CD8 FITC antibody (Clone HIT8a; Biolegend, 300906) for 15 min at 4°C, washed and then incubated with 70μl of anti-FITC microbeads (Miltenyi, 130-048-701). CD8+ T cells were isolated by magnetic separation using LS columns (Miltenyi, 130-042-401) and activated with coated anti-CD3 antibody (clone OKT3; Biolegend, 317326) and 2 μg / ml soluble anti-CD28 antibody (clone CD28.2; Biolegend, 302934) in complete medium for 3 days. Activation was always performed at neutral pH 7.5 unless otherwise specified. For proliferation assays, CD8+ T cells were labeled with CellTrace Violet (Thermo Scientific, C34557) before T cell activation. To purify mRNA, activated CD8+ T cells were rested O / N, transferred to complete medium pH 7.5 or 6.5, and stimulated with 10 nM IL-2 WT or Switch-2 for 4 h. For CD8+ T cells used for proteome analysis, activated cells were cultured in the presence of 10 nM IL-2 WT or Switch-2 in medium at pH 7.5 or 6.5 for 48 h, washed twice with PBS, and the dried cell pellets were frozen. Activated CD8+ T cells used for cytokine expression analysis and secretome analysis were cultured in the presence of 10 nM IL-2 WT or Switch-2 in medium at pH 7.5 or 6.5 for 3 days and then stimulated for 4 h. For cytokine expression analysis by flow cytometry, a cell stimulation cocktail containing transport inhibitors (eBioscience; 00-4975-93) was used. Supernatants for Luminex analysis were harvested after stimulation with a cell stimulation cocktail (eBioscience; 00-4970-93).CD4+ cells were isolated using 40 μl of anti-CD4 FITC antibody (clone A161A1; Biolegend; 357406) following the same protocol as for CD8+ T cell isolation.

[0166] Signal transduction experiments For signaling experiments, activated CD8+ T cells were rested O / N and then stimulated with the indicated amounts of IL-2 WT or Switch-2 in medium at pH 7.5 or 6.5 for 15 min. For time course experiments, cells were stimulated with 10 nM or 10 pM IL-2 for 6 h, 3 h, 2 h, 1 h, 30 min, and 15 min. IL-2 signaling in Treg cells was assessed after stimulation of freshly isolated total CD4 cells for 15 min.

[0167] Flow cytometry analysis Human CD8 cells were incubated with Zombie aqua Fixable viability kit (Biolegend; 423101) for 20 min at 4 °C, and then stained for surface markers with anti-human CD8 FITC, anti-human CD3 BV711 (clone UCHT1; Biolegend; 300463), anti-human CD25 APC (clone M-A251; Biolegend, 356110), anti-human CD122 PE-Cy7 (clone TU27; Biolegend; 339013), anti-human CD132 PE (clone TUGh4; Biolegend; 338605), and anti-human CD69 BV650 (clone FN50; Biolegend; 310933) in MACS buffer (Miltenyi; 130-091-221) for 30 min. For analysis of cytokine expression, cells stained with surface markers were fixed and permeabilized using the BD Cytofix / Cytoperm kit (BD Biosciences; 554714). Anti-human IL-2 BV421 (clone MQ1-17H12; Biolegend, 500328), anti-human TNFα PE / Dazzle 594 (clone Mab11; Biolegend, 502946), and anti-human IFNγ APC (clone B27; Biolegend, 506510) were used. All antibodies were used at 1:100. For dose-response and kinetic experiments, stimulated cells were immediately fixed with 2% PFA for 15 min at room temperature. Cells were then washed with PBS and permeabilized with ice-cold methanol for 30 min on ice, followed by fluorescent bar encoding as previously described (Non-Patent Document 15). Briefly, individual wells were stained with different concentrations of a combination of PacificBlue (Thermo Scientific; 10163) and DyLight800 NHS dye (Thermo Scientific; 46421).Sixteen bar-encoded samples were pooled and stained with 1:100 anti-STAT5 PE (clone 47 / Stat5; BD Biosciences; 612567), 1:100 anti-ERK1 / 2 AF647 (clone 4B11B69; Biolegend, 677504), 1:50 anti-Akt AF647 (clone 193H2; Cell Signaling Technologies, 2337S), and 1:100 anti-S6R PE (clone D57.2.2E; Cell Signaling Technologies; 5316S) in MACS buffer for 1 h at room temperature. For Treg cell signaling experiments, samples were washed and stained with 1:10 anti-human FoxP3 AF647 (clone 259D / C7; BD Biosciences; 560045) using the FoxP3 / transcription factor staining buffer set (eBioscience; 00-5523-00). Single cell suspensions of mouse spleens and lymph nodes were obtained by mechanical disruption. Tumors were digested with collagenase (Sigma, C6885) and DNase I (StemCell, 07470). After treatment with TruStain FcX (anti-mouse CD16 / 32) Antibody (Biolegend; 101320), samples were stained using the same procedure as described above.The antibodies used were anti-mouse CD3 PerCP-Cy5.5 (clone 17A2; Biolegend; 100218), anti-mouse CD4 BV605 (clone RM4-5; Biolegend; 100548), anti-mouse CD4 AF700 (clone GK1.5; Biolegend; 100430), anti-mouse CD8 AF488 (clone 53-6.7; Biolegend; 100723), anti-mouse CD45 BV711 (clone 30-F11; Biolegend; 103147), anti-mouse CD122 PE / Dazzle 594 (clone TM-β1; Biolegend; 123217), anti-mouse PD-1 BV785 (clone 29F-1A12; Biolegend; 135225), and anti-mouse TIM3. Antibodies against the following antibodies were used: BV421 (clone RMT3-23; Biolegend; 119723), anti-mouse FoxP3 PE (clone FJK-16s; eBioscience; 12-5773-82), anti-mouse Ki67 PE-Cy5 (clone SolA15; eBioscience; 15-5698-82), anti-mouse NK1.1 BV605 (clone PK136; Biolegend; 108739), anti-mouse TNFα BV605 (clone MP6-XT22; Biolegend; 506329), and anti-mouse IFNγ APC (clone XMG1.2; Biolegend; 505809). Flow cytometry was performed using an LSR Fortessa X20 (BD) and data were analyzed using FlowJo software (TreeStar Inc, version 10).

[0168] Animal models Six-week-old female C57Bl / 6JRj mice (Janvier) were subcutaneously injected in the right flank with 30.000 B16.SIY WT or B16.SIY LDHA / B DKO dissolved in PBS and Matrigel (1:1) (Corning; 356232). 20 μg of Fc4-IL-2 WT or Switch-2 were administered intraperitoneally (ip) from day 7 to day 11. Tumors were measured using a caliper and tumor volumes were calculated using the formula length × width 2 / 2. For the analysis of TILs, mice were sacrificed on day 15 after tumor injection. For toxicity studies, 20 μg or 50 μg of Fc4-IL-2 WT or Switch-2 were administered ip for 5 consecutive days and mice were sacrificed the day after the last injection. The wet weight of the lungs was measured after collection, and the lung edema (wet lung weight) was evaluated by subtracting the dry weight after drying overnight at 80°C.

[0169] IL-2 library construction and selection IL-2 cDNA was cloned into pCT302 vector for expression in yeast, adapting the previously described yeast protocol (Non-Patent Document 16). Eight overlapping primers were assembled to generate an IL-2 library, with two primers containing the homology region required for combination with the pCT302 vector (see Table 1). Three of the primers had NDT codons (encoding amino acids G, V, L, IC, S, R, H, D, N, F, and Y) to randomly mutate residues T37, R38, T41, F42, F43, E60, E61, E63, L66, E68, V69, D109, and E110. The PCR product was further amplified with Lib Fw and Lib Rv primers (Table 1) at a final concentration of 10 μM to obtain at least 25 μg of DNA. S. cerevisiae EBY100 strain was transformed by electroporation with 25 μg of insert DNA and 5 μg of linearized purified plasmid. Transfected yeast were grown in SDCAA medium at 30°C for 1 day and in SGCAA medium at 20°C for 2 days for each selection round. 5x10 7The library, with a size of 10, was screened by magnetic activated cell sorting (MACS) using LS columns (Miltenyi; 130-042-401). 10 cells were used, and in subsequent rounds 10 8 100 nM of cells were used to ensure at least 10-fold coverage in each round. Different concentrations of biotinylated IL-2Rα ectodomain were used to select pH-tolerant IL-2 variants. More specifically, the first two rounds used 100 nM IL-2Rα tetramer at pH 5, the third round used 1 μM IL-2Rα monomer, and the fourth round used 100 nM IL-2Rα monomer. IL-2Rα tetramer was generated by incubating IL-2Rα and streptavidin (SA)-Alexa647 in a 4:1 ratio.

[0170] [Table 4-1] [Table 4-2]

[0171] Nano Differential Scanning Fluorometry (NanoDSF) IL2 WT and Switch-2 (10 μM) were analyzed using a Tycho NT.6 (NanoTemper, Munich, Germany) applying a standard capillary (10 μl) for each HBS buffer condition (pH 7-4). Thermal unfolding profiles were recorded within a temperature gradient between 35 °C and 95 °C. Inflection temperature (Ti) values ​​were determined automatically using the integrated software.

[0172] Single-molecule fluorescence imaging For microscopy experiments, HeLa cells stably transfected with SNAPf-IL-2Rα were transferred to 25 mm glass coverslips coated with poly-L-lysine-grafted (polyethylene glycol) copolymer functionalized with RGD to minimize nonspecific binding (Non-Patent Document 17). Single-molecule imaging experiments were performed by total internal reflection fluorescence (TIRF) microscopy using an inverted microscope (Olympus IX71) equipped with a triple-line total internal reflection (TIR) ​​illumination condenser (Olympus) and a back-7 illumination electron multiplying (EM) CCD camera (iXon DU897D, Andor Technology) as described in previous publications (Non-Patent Documents 18, 19). For TIR illumination of the samples, a 150x magnification, 1.45 numerical aperture objective (UAPO 150x / 1.45 TIRFM, Olympus) was used. All experiments were performed at room temperature in phenol red-free medium supplemented with oxygen scavengers and photoprotectants with redox activity to minimize photodegradation (20).

[0173] For stoichiometric cell surface labeling of SNAPf-tagged IL-2Rα, cells were incubated with 80 nM premixed BG-dye solution (95% BG-488 and 5% BG-Dy547) at 37 °C for 15 min and washed five times with prewarmed PBS to remove unreacted dye. Dy547P1 / Dy647P1-conjugated ybbR-IL-2 WT and Switch-2 were added at a concentration of 1 nM 5 min before imaging experiments. For single-molecule experiments, orange (Dy547 / Dy547P1) and red (DY647P1) emitting fluorescent dyes were excited simultaneously with a 561 nm fiber laser (2RU-VFL-P-500-560-B1R, MPB Communications) and a 642 nm fiber laser (2RU-VFL-P-500-642-B1R, MPB Communications). Fluorescence was filtered with a pentaband polychromic mirror (zt405 / 488 / 561 / 640 / 730rpc, Semrock), and excitation light was blocked with a pentaband bandpass emission filter (BrightLine HC 440 / 521 / 607 / 694 / 809, Semrock). A four-color image splitter (QuadView, QV2, Photometrics) with three dichroic beam splitters at 565 nm, 630 nm, and 735 nm (480dcxr, 565dcxr, 640dcxr, Chroma) and four single bandpass emission filters (BrightLine HC 438 / 24, BrightLine HC 520 / 35, BrightLine HC 600 / 37, BrightLine HC 685 / 40, Chroma) was used to simultaneously acquire both channels with one back-illuminated EMCCD camera (iXon Ultra 897, Andor Technologies). For each cell, image stacks of 150 frames were recorded with a time resolution of 32 ms / frame.

[0174] Single molecule localization was performed using the multiple target tracing (MTT) algorithm (21). To ratiometrically quantify ligand binding, the localization of Dy647P1 (IL-2 WT or Switch-2) over 30 frames was normalized to that of Dy547 (IL-2Rα) or Dy547P1 (IL-2 WT), respectively.

[0175] Single-molecule fluorescence imaging For microscopy experiments, HeLa cells stably transfected with SNAPf-IL-2Rα were transferred to 25 mm glass coverslips coated with poly-L-lysine-grafted (polyethylene glycol) copolymer functionalized with RGD to minimize nonspecific binding (Non-Patent Document 17). Single-molecule imaging experiments were performed by total internal reflection fluorescence (TIRF) microscopy using an inverted microscope (Olympus IX71) equipped with a triple-line total internal reflection (TIR) ​​illumination condenser (Olympus) and a back-7 illumination electron multiplying (EM) CCD camera (iXon DU897D, Andor Technology) as described in previous publications (Non-Patent Documents 18, 19). For TIR illumination of the samples, a 150x magnification, 1.45 numerical aperture objective (UAPO 150x / 1.45 TIRFM, Olympus) was used. All experiments were performed at room temperature in phenol red-free medium supplemented with oxygen scavengers and photoprotectants with redox activity to minimize photodegradation (20).

[0176] For stoichiometric cell surface labeling of SNAPf-tagged IL-2Rα, cells were incubated with 80 nM premixed BG-dye solution (95% BG-488 and 5% BG-Dy547) at 37 °C for 15 min and washed five times with prewarmed PBS to remove unreacted dye. Dy547P1 / Dy647P1-conjugated ybbR-IL-2 WT and Switch-2 were added at a concentration of 1 nM 5 min before imaging experiments. For single-molecule experiments, orange (Dy547 / Dy547P1) and red (DY647P1) emitting fluorescent dyes were excited simultaneously with a 561 nm fiber laser (2RU-VFL-P-500-560-B1R, MPB Communications) and a 642 nm fiber laser (2RU-VFL-P-500-642-B1R, MPB Communications). Fluorescence was filtered with a pentaband polychromic mirror (zt405 / 488 / 561 / 640 / 730rpc, Semrock), and excitation light was blocked with a pentaband bandpass emission filter (BrightLine HC 440 / 521 / 607 / 694 / 809, Semrock). A four-color image splitter (QuadView, QV2, Photometrics) with three dichroic beam splitters at 565 nm, 630 nm, and 735 nm (480dcxr, 565dcxr, 640dcxr, Chroma) and four single bandpass emission filters (BrightLine HC 438 / 24, BrightLine HC 520 / 35, BrightLine HC 600 / 37, BrightLine HC 685 / 40, Chroma) was used to simultaneously acquire both channels with one back-illuminated EMCCD camera (iXon Ultra 897, Andor Technologies). For each cell, an image stack of 150 frames was recorded with a time resolution of 32 ms / frame. Single molecule localization was performed using the Multiple Target Tracing (MTT) algorithm (Non-Patent Document 21).To ratiometrically quantitate ligand binding, the localization of Dy647P1 (IL-2 WT or Switch-2) over 30 frames was normalized to the localization of Dy547 (IL-2Rα) or Dy547P1 (IL-2 WT), respectively.

[0177] Luminex analysis Cell supernatants were measured on a custom 36-multiplex R&D Systems Luminex panel (R&D Systems) at the University of Dundee Immunoassay Biomarker Core Laboratory. Samples were diluted 2-fold as instructed in the assay instructions. A wash step was performed using a Bio-Plex Pro wash station. Multiplex assay plates were measured on a Bio-plex 200 analyzer using Bio-plex Manager software v6.1. Thirty-six analyte-specific antibodies are precoated onto microparticles.

[0178] Standards, samples, and a cocktail of all microparticles were added to each well. The plate was covered with a foil plate sealer and incubated at room temperature for 2 hours with shaking at 800 ± 50 rpm. At this stage, the immobilized antibodies bind to the analytes of interest. The plate was washed three times using the Bio-Plex Pro wash station as per the assay instructions.

[0179] A diluted biotinylated antibody cocktail specific for the analyte of interest was added to each well. The plate was covered with a foil plate sealer and incubated at room temperature for 1 hour with shaking at 800±50 rpm.

[0180] After this, the plate was washed as described above. Diluted streptavidin-phycoerythrin conjugate (Streptavidin-PE) was added to each well. The plate was covered with a foil plate sealer and incubated for 30 minutes with shaking at 800±50 rpm. After this, the plate was washed as described above. The microparticles were resuspended in wash buffer. The plate was placed on a plate shaker set at 800±50 rpm for 2 minutes. The plate was read immediately using a Bio-plex 200 analyzer. The blank mean MFI was subtracted from the mean duplicate fluorescence intensity (MFI) measurements for each standard and sample. Five-parameter logistic (5-PL) curve fit standard curves were generated for each analyte using Bio-plex Manager v6.1 software. The software was also used to calculate results that accounted for sample dilution.

[0181] RNA sequence analysis RNA from human CD8+ T cells was purified using the Quick-RNA Microprep kit (Zymo Research; R1051). Library preparation and sequencing were performed by Novogene.

[0182] Isolation and activation of mouse CD8+ T cells Mouse CD8+ T cells were isolated from mouse spleens using the MagniSort Mouse CD8+ T Cell Enrichment Kit (eBioscience; 8804-6822-74). Cells were activated with coated anti-CD3 antibody (clone 145-2C11; Biolegend; 100340) and 2 μg / ml soluble anti-CD28 antibody (clone 37.51; Biolegend; 102116) in complete medium for 3 days.

[0183] statistical analysis Comparisons between multiple groups were performed using one-way analysis of variance with Tukey's correction. Survival curves were presented as Kaplan-Meier curves, and statistical significance was determined by the Log-rank test. All analyses were performed using Prism 9 software (GraphPad).

[0184] result Acidic pH of the TME inhibited IL-2 immunotherapy Previous studies have suggested that IL-2 binding to its receptor is a pH-responsive process (Non-Patent Document 7). We therefore investigated whether acidic pH, as found in the TME, affects IL-2 signaling and activity. IL-2 induced significantly lower levels of STAT5 phosphorylation in preactivated CD8+ T cells cultured at pH 6.5 than in cells cultured at pH 7.5 (Fig. 1a and Extended Data Fig. 1a). Similar results were seen when the medium was acidified with lactate (Extended Data Fig. 1b). When HCl or lactate was replaced by NaCl, the effect of acidic pH on IL-2 signaling was found to be tonicity independent, as equimolar NaCl failed to reproduce the effect of pH on signaling. However, in IL-2Rα-negative cells, which react weakly with IL-2, IL-2 activated STAT5 to a similar extent at pH 6.5 and pH 7.5. This suggests that the binding of IL-2 to IL-2Rα is pH-responsive (Extended Data Fig. 1c, d). Indeed, yeast-displayed IL-2 consistently bound worse to IL-2Rα at pH 6 and pH 5 than at pH 7 (Fig. 1b). We confirmed these results by measuring the dissociation constant (Kd) of the recombinant protein (Extended Data Fig. 1e). We next investigated whether the acidic pH found in the TME adversely affects IL-2 treatment. Since the release of lactate by tumors is one of the main causes of acidity in the TME, we used B16 melanoma cells, which do not express lactate dehydrogenases A and B (LDHA / B), as described in the prior art, to evaluate the effect of pH on IL-2 treatment (Non-Patent Document 4). Mice were injected with B16 wild-type (WT) or B16 LDHA / B double knockout (DKO) and treated with IL-2 conjugated to the Fc portion of human IgG4 (Fc4-IL-2) (Fig. 1c).Fc4-IL-2 therapy minimized tumor growth or increased survival in mice bearing B16 WT tumors (Fig. 1d, e and Extended Data Fig. 2a). However, in B16 DKO tumor-bearing mice, Fc4-IL-2 therapy significantly inhibited tumor growth and prolonged survival (Fig. 1d, e and Extended Data Fig. 2a). Mice treated with Fc4-IL-2 had a higher percentage of infiltrating CD8+ T cells and a slight increase in CD8+ / Treg cells in both B16 WT and DKO tumors compared with untreated mice (Fig. 1f, g and Extended Data Fig. 2b). Comparing B16 WT and DKO tumors treated with Fc4-IL-2, no significant differences were observed (Fig. 1f, g). In contrast, CD8+ tumor-infiltrating lymphocytes (TILs) from Fc4-IL-2-treated B16 DKO tumors produced significantly more IFNγ and TNFα (Fig. 1h,i and Extended Data Fig. 2c,d) and had a less compromised CD8+ T cell phenotype, as indicated by lower expression of PD1 and TIM3, compared with Fc4-IL-2-treated B16 WT tumors (Fig. 1j,k). Our data indicate that the acidic pH found in the TME profoundly inhibits IL-2 responses by inhibiting binding to IL-2Rα, ultimately impairing IL-2 immunotherapy.

[0185] Engineering pH-tolerant IL-2 variants Next, given IL-2's limited ability to function at acidic extracellular pH, we generated a variant library of IL-2 bound to Aga2p for yeast surface display with the goal of improving IL-2 binding to IL-2Rα at acidic pH (Fig. 2a). In vitro directed evolution was performed at pH 5 with decreasing concentrations of the IL-2Rα ectodomain in each round (Fig. 2b), and we identified a single IL-2 variant, termed Switch-2, featuring the mutations T37H, R38L, T41S, F42Y, and K43G (Fig. 2c and Extended Data Fig. 3a). Switch-2 not only exhibited stronger binding to IL-2Rα at low pH, but also displayed pH-switchable behavior, characterized by lower binding at neutral pH compared with IL-2 WT (Fig. 2d and Extended Data Fig. 3b). We next used single-molecule total internal reflection fluorescence microscopy (TIRF) to assess the ability of IL-2 to interact with IL-2Rα on the membrane of live cells at neutral (7) and acidic pH (6) (Fig. 2e). To this end, IL-2Rα fused to an N-terminal SNAPf tag was stably expressed in HeLa cells at a physiologically relevant density (copies / cell) and labeled with a mixed fluorescent dye, DY547 and BG-DY647. We then added Dy547P1 / Dy647P1-conjugated ybbR-IL-2 WT and Switch-2, and dynamically monitored the interaction of IL-2 and IL-2Rα at the single-cell level by dual-color co-tracking of individual cytokine receptor dimers (Fig. 2e, Extended Data Fig. 2c, and Movie S1). These experiments showed that recruitment of IL-2 to the IL-2Rα receptor was increased at neutral pH, but only weakly interacted at pH 6.5. In contrast, Switch-2 showed the opposite relationship, exhibiting a strong ligand-receptor interaction at acidic pH and minimal interaction at neutral pH, again confirming pH-switchable IL-2Rα binding properties.

[0186] We next examined whether acidic pH affected IL-2 stability. Analysis of thermal unfolding profiles showed that IL-2 WT and Switch-2 had comparable stabilities that were not affected by low pH (Extended Data Fig. 3d). This indicates that low pH specifically inhibits IL-2 signaling not by decreasing protein stability but specifically by disrupting cytokine-receptor interaction. We next examined the function of Switch-2 at acidic pH. First, we examined the phosphorylation levels of STAT5 induced by IL-2 WT and Switch-2 at pH 7.5 and pH 6.5 in freshly isolated and preactivated CD8+ T cells. In freshly isolated CD8+ T cells that do not express IL-2Rα, both IL-2 WT and Switch-2 induced comparable STAT5 activation at pH 6.5 and pH 7.5 (Fig. 2f, g). However, in preactivated CD8+ T cells, IL-2 WT induced stronger STAT5 activation at pH 7.5 than at pH 6.5 (Fig. 2f, g). On the other hand, Switch-2 showed the opposite behavior, inducing stronger STAT5 activation at pH 6.5 than at pH 7.5 (Fig. 2f, g). Kinetic studies yielded similar results (Extended Data Fig. 4a). Interestingly, IL-2 WT-mediated phosphorylation of ERK1 / 2 was also affected by acidic pH, whereas in contrast, no obvious differences were observed in IL-2-mediated phosphorylation of Akt and S6R (Extended Data Fig. 4b–d). Moreover, Switch-2 induced stronger ERK1 / 2 and Akt phosphorylation in preactivated CD8+ T cells stimulated at pH 6.5 (Extended Data Fig. 4b, d). Moreover, stimulation of Treg cells with IL-2 WT and Switch-2 at pH 6.5 and pH 7.5 produced results comparable to those obtained with CD8+ T cells (Extended Data Fig. 4e).

[0187] Switch-2 induced functional T cells at acidic pH IL-2 promotes T cell proliferation and effector function by inducing cytotoxic functions, including the production of IFNγ8. However, acidic pH has been reported to inhibit T cell proliferation during activation (Extended Data Fig. 5a) (Non-Patent Document 9) and T cell effector function (Non-Patent Document 2). Therefore, we investigated the ability of CD8+ T cells stimulated with either IL-2 or Switch-2 to proliferate and produce effector cytokines under neutral and acidic pH conditions. CD8+ T cells were first activated with anti-CD3 and anti-CD28 activation beads at pH 7.5, and then switched to either pH 7.5 or 6.5 medium in the presence of IL-2 WT or Switch-2. As expected, IL-2 WT induced CD8+ T cell proliferation at pH 7.5, but not at pH 6.5 (Extended Data Fig. 5b). On the other hand, Switch-2 induced CD8+ T cell proliferation at both pH 7.5 and pH 6.5 (Extended Data Fig. 5b). We next examined the cytokine secretion profile by activated CD8+ T cells stimulated with IL-2 WT or Switch-2 at pH 7.5 or 6.5. Again, IL-2 WT strongly induced cytokine secretion by CD8+ T cells at pH 7.5, but nearly lost activity when cells were cultured at pH 6.5 (Fig. 3a). On the other hand, Switch-2, almost in mirror image of IL-2 WT, strongly induced cytokine release by CD8+ T cells at pH 6.5 than at pH 7.5 (Fig. 3a). Furthermore, at acidic pH, cells grown in Switch-2 secreted cytokines associated with effector functions, such as IFNγ8, GMCSF, and TNFα, whereas cells grown in IL-2 expressed little of these cytokines. We then confirmed these results for IFNγ and TNFα using intracellular staining and flow cytometry (Fig. 3b–d).

[0188] Switch-2 exhibits potent antitumor effects with low toxicity Given the striking effects of increased activity at acidic pH and decreased activity at neutral pH in a series of in vitro assays, we hypothesized that Switch-2 might increase activity in acidic tissue niches but induce less systemic toxicity. To validate its in vivo efficacy in mouse models, we first examined the activity of Switch-2 in mouse CD8+ T cells. Importantly, Switch-2 caused stronger STAT5 activation at pH 6.5 than at pH 7.5 in mouse CD8+ T cells (Extended Data Fig. 6a). Given the short half-life of free IL-2, we conjugated IL-2 or "Switch-2" to non-lytic Fc to extend its half-life and improve its function (Non-Patent Document 10). To compare the effects of IL-2 variants in mediating systemic toxicity in addition to immune cell activation in peripheral blood and tissue niches, WT C57Bl / 6 mice were treated with high doses of IL-2 variants. One of the major side effects of high-dose IL-2 therapy is vascular leak syndrome (Non-Patent Document 11). As expected, high doses of IL-2 WT induced significant pulmonary edema (Fig. 4a). Surprisingly, high doses of Switch-2 significantly reduced pulmonary edema (Fig. 4a). This is consistent with lower proportions of natural killer (NK) and Treg cells in the periphery in mice injected with Switch-2 (Fig. 4b and Extended Data Fig. 6b-c), and consistent with Switch-2 being less active at pH 7.5. Lymph nodes (LNs) are characterized by an acidic pH (Non-Patent Document 12). Consistent with this, we found that Switch-2 increased the number of NK and Treg cells in LNs to a greater or similar extent than IL-2 WT (Fig. 4c and Extended Data Fig. 6d). These data indicate that Switch-2 also exhibits activity biased toward the acidic pH niche in vivo.

[0189] High doses of IL-2 administration can potently activate cytotoxic T-cell and NK cell-mediated tumor killing. However, its therapeutic efficacy is limited by insufficient activation of TILs within the TME. Our data show that intratumoral pH strongly limits IL-2 activity within the TME (Fig. 1d-k and Extended Data Fig. 2). Indeed, many TILs within tumors are dysfunctional (Res: Rosenberg SA). Importantly, TILs isolated from tumors can be reactivated and expanded in vitro in the presence of IL-2. These data suggest that inappropriate targeting and functioning of IL-2 within the TME may limit its efficacy in vivo. We hypothesized that reduced binding of Switch-2 to circulating cells at neutral pH would improve its targeting to activated immune cells expressing CD25 within the TME and lymphoid niche. We next investigated the antitumor activity of Switch-2 in a B16 melanoma tumor model (Non-Patent Document 13), which is sensitive to NK- and cytotoxic T lymphocyte (CTL)-mediated killing. B16-bearing mice were injected with Fc4-IL-2 WT or Fc4-Switch-2, and tumor growth and survival were measured (Fig. 4d). Fc4-IL-2 WT therapy only slightly inhibited tumor growth and extended survival (Fig. 4e, f). On the other hand, Fc4-Switch-2 therapy strongly delayed tumor growth and extended survival (Fig. 4e, f). To examine the difference in immune responses between IL-2 and Switch-2 therapy, B16-infiltrating T cells were analyzed by flow cytometry 4 days after the end of treatment (Fig. 4g and Extended Data Fig. 6e-g). Switch-2 and IL-2 WT slightly increased the CD8-Treg ratio compared with PBS-treated mice, but the difference was not significant (Extended Data Fig. 6e). However, Switch-2 induced stronger proliferation of CD8+ TILs and increased numbers of infiltrating NK cells (Fig. 4g, h).Moreover, Switch-2 induced stronger IFNγ and TNFα production by CD8+ T cells than IL-2 WT (Fig. 4i, j).Notably, CD8+ TILs in the Switch-2 treatment group had significantly reduced levels of exhaustion markers measured by PD1 expression and TIM3 expression compared with the IL-2 WT group (Extended Data Fig. 6f, g). Taken together, these data indicate that Switch-2 induces a more potent response in the acidic TME while causing less systemic toxicity than IL-2 WT. Importantly, these results challenge the dogma that selective binding of IL-2 to intermediate IL-2Rα affinity receptor complexes is required for its antitumor effect, and show that acidic pH in tumors is a negative barrier to optimal IL-2 responses via high affinity receptor complexes additionally expressing CD25. Furthermore, we have demonstrated a rational protein engineering approach to design and develop "Switch-2" that can potently activate TILs to mediate effective immune responses as a single agent in poorly immunogenic B16 melanoma tumors. [Industrial Applicability]

[0190] A practical possibility lies in the therapeutic exploitation of the pH-responsive nature of IL-2: Switch-2 potently activates cytotoxic CD8+ T cells and NK cells, resulting in a robust antitumor immune response with minimal toxicity, potentially revolutionizing current IL-2 therapy.

Claims

1. An acid-resistant pH IL-2 mutein, wherein the amino acid sequence of the mutein contains one or more amino acid modifications relative to the wild-type IL-2 sequence, and the mutein contains a substitution of threonine with histidine, arginine, or serine at residue 37 of SEQ ID NOs: 1, 4, 5, 8, an IL-2 mutein.

2. The IL-2 mutein according to claim 1, wherein the IL-2 mutein binds to IL-2Rα with higher affinity at an acidic pH such as a pH selected from about 4.0 to about 7.

3. The IL-2 mutein according to claim 2, wherein the IL-2 mutein binds to IL-2Rα with higher affinity at an acidic pH of about 5 to about 6.5 than at a pH selected from about 7.2 to about 7.

5.

4. The IL-2 mutein according to claim 1 or 2, wherein the mutein further contains one or more modifications at any of positions 38, 41, 42, 43, and / or 64 of SEQ ID NOs: 1, 4, 5, 8.

5. Relative to the sequence of SEQ ID NO: 1 or 8, the IL-2 mutein has the following combination of amino acid substitutions: (A) (i) T37H; and optionally (ii) R38L; and / or (iii) T41S; and / or (iv) F42Y; and / or (v) K43G, (B) (i) T37S; and optionally (ii) R38A; and / or (iii) T41D; and / or (iv) K43G; and / or (v) K64E, (C) (i) T37S; and optionally (ii) R38L; and / or (iii) T41G; and / or (iv) F42Y; and / or (v) K43G, (D) (i) T37S; and optionally (ii) R38V; and / or (iii) T41G; and / or (iv) K43G, (E) (i) T37R; and optionally (ii) R38V; and / or (iii) T41G; and / or (iv) K43G, or (F) (i) T37S; and optionally (ii) R38I; and / or (iii) T41G; and / or (iv) K43G The IL-2 mutein according to claim 1, comprising any of the above.

6. Relative to the sequence of SEQ ID NO: 1 or 8, the IL-2 mutein has the following amino acid substitutions: (i) T37H; (ii) R38L; (iii) T41S; (iv) F42Y; and (v) K43G all five of which are included. The IL-2 mutein according to claim 1.

7. The IL-2 mutein according to claim 1, wherein the mutein comprises SEQ ID NO: 2, 9, 10, 11, 12 or 13 or a functional fragment thereof.

8. The IL-2 mutein according to claim 1, wherein the mutein comprises SEQ ID NO: 2 or a functional fragment thereof.

9. A fusion protein comprising the IL-2 mutein according to claim 1 or a functional fragment thereof, wherein the fusion protein further comprises an additional polypeptide molecule or polypeptide fragment, and optionally, the additional polypeptide molecule or peptide fragment is a cytokine or a fragment thereof; an interleukin molecule or a fragment thereof; a polypeptide binding domain; an antibody or a fragment thereof; a single-chain antibody; or a VHH, or the IL-2 mutein is fused to a half-life extended molecule, and optionally, the half-life extended molecule comprises an immunoglobulin fragment, an Fc molecule, a polypeptide binding domain that binds to a blood serum protein, a polypeptide binding domain that binds to albumin, or a polymer.

10. The polypeptide binding domain binds to a tumor antigen or a checkpoint molecule, and optionally, the polypeptide binding domain binds to at least one checkpoint molecule selected from CD27, CD137, 2B4, TIGIT, CD155, CD160, ICOS, HVEM, CD40L, LIGHT, LAIR1, OX40, DNAM-1, PD-L1, PD1, PD-L2, CTLA-4, CD8, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDOl, ID02, TDO, KIR, LAG-3, TIM-3, or VISTA, or the polypeptide binding domain binds to an antigen expressed by regulatory T cells. The fusion protein according to claim 9.

11. The fusion protein according to claim 9, wherein the polymer comprises a polyethylene glycol molecule.

12. A nucleic acid encoding the IL-2 mutein according to claim 1 or the fusion protein according to claim 9.

13. A vector comprising the nucleic acid according to claim 12.

14. A host cell comprising the nucleic acid according to claim 12 or the vector according to claim 13, optionally wherein the host cell is a T cell.

15. The host cell according to claim 14, wherein the T cell comprises a chimeric antigen receptor (CAR).

16. The IL-2 mutein according to claim 1, the fusion protein according to claim 9, the nucleic acid according to claim 12, the vector according to claim 13, or the host cell according to claim 14, for use in medicine, optionally for use in the treatment or prevention of an immunological condition, cancer, or an infectious disease, or for use as an adjuvant.

17. A composition comprising the IL-2 mutein according to claim 1, the fusion protein according to claim 9, the nucleic acid according to claim 12, the vector according to claim 13, or the host cell according to claim 14, optionally wherein the composition is a pharmaceutical composition, optionally comprising one or more pharmaceutically acceptable excipients, and further optionally wherein the composition or pharmaceutical composition comprises another therapeutic moiety or pharmaceutically active agent.

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