De novo design of potent and selective interleukin mimetics

Abstract

To solve the problem in which considerable potential of central immune cytokine interleukins such as IL-2 and IL-4 for cancer treatment has sparked numerous efforts to improve their therapeutic properties by mutation and / or chemical modification.SOLUTION: De novo designed polypeptides that bind to IL-2 receptor βγc heterodimer (IL-2Rβγc), IL-4 receptor αγc heterodimer (IL-4Rαγc), or IL-13 receptor α subunit (IL-13Rα) are disclosed, as are methods for using and designing the polypeptides.SELECTED DRAWING: Figure 3E

Description

[Technical Field]

[0001] cross reference This application claims priority to U.S. Provisional Patent Application Nos. 62 / 689,769, filed June 25, 2018, and 62 / 768,733, filed November 16, 2018, each of which is incorporated herein by reference in its entirety. [Background technology]

[0002] The great potential of interleukins, the central immune cytokines such as IL-2 and IL-4, for cancer therapy has triggered numerous efforts to improve their therapeutic properties by mutation and / or chemical modification. However, these approaches have been limited to the IL-2 receptor α subunit (IL-2Rα), IL-4 receptor αγ subunit (IL-4Rα), and IL-4 receptor αγ subunit (IL-4Rα). c Heterodimer (IL-4Rαγ c ), and undesirable properties such as poor stability and binding to the IL-13 receptor α subunit (IL-13Rα). Summary of the Invention

[0003] In one aspect, a method is provided in which a computational device determines a structure of a plurality of residues of a protein, the structure of the plurality of residues providing a specific receptor binding interface, and the computational device determines a plurality of designed residues using a mimetic design protocol provided by the computational device, the plurality of designed residues providing the specific receptor binding interface, the plurality of designed residues being different from the plurality of residues.

[0004] The computing device determines one or more connected helix structures that connect the plurality of designed residues. The computing device determines a first protein backbone of the protein by assembling the one or more connected helix structures and the plurality of designed residues across a plurality of combinations. The computing device designs a second protein backbone of the protein based on the first protein backbone for flexibility and a low-energy structure. The computing device generates an output related to at least the second protein backbone.

[0005] Also included are non-naturally occurring proteins prepared by the methods described herein. The non-naturally occurring proteins can be cytokines, such as non-naturally occurring IL-2 or IL-4 (also referred to herein as IL-2, IL-2 / 15 mimics, or IL-4 mimics).

[0006] In another aspect, a computing device is provided, the computing device including one or more processors and data storage configured to store at least computer-readable instructions that, when executed by the one or more processors, cause the computing device to perform functions including: determining a structure of a plurality of residues of a protein that provides a specific receptor-binding interface; determining a plurality of designed residues using a mimetic design protocol, the plurality of designed residues providing the specific receptor-binding interface, the plurality of designed residues being different from the plurality of residues; determining one or more connected helix structures connecting the plurality of designed residues; determining a first protein backbone of the protein by assembling the one or more connected helix structures and the plurality of designed residues across a plurality of combinations; designing a second protein backbone of the protein for flexibility and a low-energy structure based on the first protein backbone; and generating an output related to at least the second protein backbone of the protein.

[0007] In another aspect, a non-transitory computer-readable medium is provided. The non-transitory computer-readable medium is configured to store at least computer-readable instructions that, when executed by one or more processors of a computing device, cause the computing device to perform functions, including determining a structure of a plurality of residues of a protein that provides a specific receptor-binding interface; determining a plurality of designed residues using a mimetic design protocol, where the plurality of designed residues provide the specific receptor-binding interface and the plurality of designed residues are different from the plurality of residues; determining one or more interlocking helix structures connecting the plurality of designed residues; determining a first protein backbone of the protein by assembling the one or more interlocking helix structures and the plurality of designed residues across a plurality of combinations; designing a second protein backbone of the protein for flexibility and a low-energy structure based on the first protein backbone; and generating an output related to at least the second protein backbone of the protein.

[0008] In another aspect, an apparatus is provided, comprising: means for determining a structure of a plurality of residues of a protein that provides a specific receptor binding interface; means for determining a plurality of designed residues using a mimetic design protocol, where the plurality of designed residues provides the specific receptor binding interface and the plurality of designed residues are different from the plurality of residues; means for determining one or more connected helix structures connecting the plurality of designed residues; means for determining a first protein backbone of the protein by assembling the one or more connected helix structures and the plurality of designed residues across a plurality of combinations; means for designing a second protein backbone of the protein for flexibility and a low-energy structure based on the first protein backbone; and means for generating an output related to at least the second protein backbone of the protein.

[0009] In another aspect, a non-naturally occurring polypeptide is provided, comprising domains X1, X2, X3, and X4, (a) X1 is [ka] a peptide comprising an amino acid sequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to (SEQ ID NO: 1); (b) X2 is a helical peptide at least 8 amino acids in length; (a) X3 is [ka] (SEQ ID NO: 2), (a) X4 is [ka] (SEQ ID NO: 3), X1, X2, X3, and X4 can be in any order within the polypeptide; An amino acid linker may be present between any of the domains, The polypeptide is an IL-2 receptor βγ c Heterodimer (IL-2Rβγ c ), IL-4 receptor αγ c Heterodimer (IL-4Rαγ c ), or binds to the IL-13 receptor alpha subunit (IL-13Rα).

[0010] In other aspects, pharmaceutical compositions are provided, comprising one or more polypeptides disclosed herein and a pharmaceutically acceptable carrier, a recombinant nucleic acid encoding a polypeptide disclosed herein, an expression vector comprising the recombinant nucleic acid disclosed herein, and a recombinant host cell comprising one or more expression vectors disclosed herein. In a further aspect, methods of treating cancer are provided, comprising administering to a subject having cancer one or more polypeptides, recombinant nucleic acids, expression vectors comprising the recombinant nucleic acids, and / or recombinant host cells disclosed herein, or pharmaceutical compositions thereof, in an amount effective to treat the tumor. [Brief explanation of the drawings]

[0011] The following figures are according to an exemplary embodiment.

[0012] [Figure 1]Computational design of de novo cytokine mimetics. Figure 1A) The designed mimic has four helices, three of which mimic IL-2 and interact with hIL-2Rβγc, and the fourth holds the first three in place. Top: In the first-generation design, each of the IL-2 core elements (helices H1–H4) was independently idealized using fragment assembly from a clustered ideal fragment database (size: 4 aa). Bottom: In the second-generation design, the core elements were instead constructed using parametric equations that reproduced the shape of each unrealized helix, allowing the length of each helix to be varied by + / - 8 aa. Figure 1B) Pairs of helices were reconnected using ideal loop fragments (size: 4 aa or 7 aa, gen-1 and gen-2, respectively, see "Methods"). Representative examples are shown with newly constructed elements connecting each pair of helices. Figure 1C) The helical hairpins generated in Figure 1B were assembled in all possible combinations to generate a fully connected protein scaffold. (Figure 1D) Designs and experimentally matured versions were tested for binding by yeast display, and those showing high-affinity binding were recombinantly expressed (E. coli) and tested for binding using surface plasmon resonance and IL-2-like phospho-STAT5 (pSTAT5). Results for three first-generation designs and ten second-generation designs are shown as filled symbols in the 2D plot. The open star represents Neoleukin-2 / 15, and the arrowhead is from its parent (unoptimized) design. [Figure 2]Characterization of Neoleukin-2 / 15. Figure 2A) From top to bottom: In surface plasmon resonance experiments, Neoleukin-2 / 15 does not bind to human or mouse IL-2Rα but binds to both human and mouse IL-2Rβ with similar affinity (Kds of approximately 11.2 nM and 16.1 nM for the human and mouse receptors, respectively). Like natural IL-2, Neoleukin-2 / 15 binds poorly to the γc receptor but exhibits cooperative binding to both human and mouse IL-2Rβγc (Kds of approximately 18.8 nM and 38.4 nM for the human and mouse heterodimeric receptors, compared with approximately 193.6 nM and 300.9 nM for native hIL-2 and Super-2; see Table E1). Figure 2B) Top: In vitro pSTAT5 signaling studies demonstrate that Neoleukin-2 / 15 induces IL-2-like signaling in human cells (EC50) and activates human YT-1 NK cells with nearly identical potency, regardless of IL-2Rα expression (CD25+ and CD25- cells, respectively, with EC50s of approximately 73.0 pM and 49.2 pM). Bottom: Similarly, ex vivo experiments with mouse CD4+ primary cells demonstrate that Neoleukin-2 / 15 can also induce potent IL-2-like signaling in mouse cells, independent of IL-2Rα expression (EC50s of approximately 24 pM and 129 pM for CD25+ and CD25- cells, respectively). Figure 2C) Top: Binding experiments (OCTET) show that Neoleukin-2 / 15 can be incubated at 80°C for 2 hours without significant loss of binding, whereas human and mouse IL-2 rapidly lose activity. Bottom: Ex vivo experiments with cultured mouse splenocytes, which require IL-2 for survival, demonstrate that Neoleukin-2 / 15 still effectively drives cell survival (approximately 70% relative luminescence at 10 ng / ml) even after 1 hour of incubation at 95°C, whereas mIL2 and Super-2 are virtually inactive (approximately 10% and 0.1%, respectively, at 10 ng / ml). [Figure 3]Structure of Neoleukin-2 / 15 (Neo-2 / 15) and its ternary complex with mIL-2Rβγc. Figure 3A) Top: Structural alignment of Neoleukin-2 / 15 (Neo-2 / 15) A chain with the designed model (rmsd 1.11 Å, for 100 Cα atoms). Bottom: Detail of interface helices H1, H3, and H4 (numbered according to hIL-2; see Figure 1). Interface side chains are shown as sticks. Figure 3B) Crystal structure of the ternary complex of Neo-2 / 15 with mIL-2Rβ and γc (rmsd 1.27 Å, for 93 modeled Cα atoms of Neo-2 / 15 in the ternary complex). Figure 3C) Structural alignment of monomeric Neo-2 / 15 (A chain) with Neo-2 / 15 in the ternary complex (rmsd 1.71 Å for 93 modeled Cα atoms in the ternary complex). Helix H4 shifts by approximately 4.0 Å in the ternary complex compared to the monomeric crystal structure. Figure 3D): Crystal structure of hIL-2 (schematic representation). The regions that interact with IL-2Rβ and γc are labeled. The loop-rich region of hIL-2 that interacts with IL-2Rα is absent in the de novo mimic Neo-2 / 15. Figure 3E): Crystal structure of Neoleukin-2 / 15 from the ternary complex in "b") (schematic representation). The regions that interact with IL-2Rβ and γc are labeled. The loop-rich region of hIL-2 that interacts with IL-2Rα is absent in the de novo mimic Neo-2 / 15. [Figure 4]Immunogenicity, immunostimulatory activity, and therapeutic activity of Neoleukin-2 / 15. Figure 4A) Dose-escalation effects of Neoleukin-2 / 15 (Neo-2 / 15) on naive mouse T cells. Naive C57BL / 6 mice were treated daily with the indicated concentrations of Neoleukin-2 / 15 or mIL-2 (n = 2–3 per group). 14 days later, spleens were harvested and analyzed by flow cytometry using the indicated markers. The bar graph shows that mIL-2 dose-dependently enhanced CD4+ Treg proliferation, whereas Neo-2 / 15 had little or no effect on Treg proliferation. Neoleukin-2 / 15 resulted in a higher CD8+:Treg ratio compared to mIL-2. Figure 4B) Effect of Neo-2 / 15 in a mouse airway inflammation model (20 μg / day / mouse, 7 days). Similar to naive mice, Neo-2 / 15 did not increase the frequency of antigen-specific CD4+Foxp3+ Tregs in lymphoid organs and was equally effective as mIL-2 in increasing the frequency of lung-resident CD8+ T cells (Thy1.2-, by intravascular labeling). (Figure 4C) Neoleukin-2 / 15 has no detectable immunogenicity. C57BL / 6 mice were inoculated with 5 × 105 B16F10 cells by subcutaneous injection. Starting on day 1, mice were treated daily with Neoleukin-2 / 15 (10 μg) or equimolar mIL-2 by intraperitoneal (ip) injection (n = 10 for each group). After 14 days, serum (antiserum) was collected and IgG was detected by ELISA on plates coated with fetal bovine serum (FBS 10%, negative control), Neoleukin-2 / 15, mIL-2, hIL-2, or ovalbumin (OVA) as a negative control (dotted line indicates the mean of the negative control). Anti-Neo-2 / 15 polyclonal antibody was used as a positive control (black, n = 2) and did not cross-react with mIL-2 or hIL-2. (Figure 4D) C57BL / 6 mice were immunized with 500 μg of KO Neo-2 / 15 in complete Freund's adjuvant and boosted with 500 μg of KO Neo-2 / 15 in incomplete Freund's adjuvant on days 7 and 15.Reactivity to KO Neo-2 / 15 and native Neo-2 / 15, as well as cross-reactivity with mouse IL-2, was determined by incubating serum (diluted 1:1,000 in PBS) with plate-bound KO Neo-2 / 15, Neo-2 / 15, or mouse IL-2, as indicated. Serum binding was detected using an anti-mouse secondary antibody conjugated to HRP, followed by incubation with TMB. Data are reported as optical density at 450 nm. Top: naive mouse serum; bottom: immunized mouse serum. Figures 4E-4G) Therapeutic effect of Neoleukin-2 / 15. Figure 4E) BALB / C mice were inoculated with CT26 tumors. Starting on day 6, mice were treated daily with intraperitoneal injections of mIL-2 or Neoleukin-2 / 15 (10 μg) or left untreated (n = 5 per group). Tumor growth curves (top, only data from surviving mice are shown). Survival curves (bottom). Mice were euthanized when body weight loss exceeded 10% of initial body weight or when tumor size reached 1,300 mm3. Figure 4F) C57BL / 6 mice were inoculated with B16 tumors as in "a" (Figure 4F). Starting on day 1, mice were treated daily with intraperitoneal injections of Neoleukin-2 / 15 (10 μg) or equimolar mIL-2 (n = 10 per group). Twice-weekly treatment with TA99 was added on day 3. Mice were euthanized when body weight loss exceeded 10% of initial body weight or when tumor size reached 2,000 mm3. Tumor growth curves (top and bottom left). Survival curves; inset shows mean body weight change (top right). Quantification of cause of death (bottom right). Figure 4G) Neo-2 / 15 induces a higher CD8+:Treg ratio than murine IL-2. C57BL / 6 mice were inoculated with B16 tumors and treated daily by intraperitoneal injection, as indicated. Treatment with TA99 (bottom plot) began on day 5 and continued twice weekly. When tumors reached 2,000 mm3, tumors were harvested from mice and analyzed by flow cytometry. The CD8:Treg cell ratio was calculated by dividing the percentage of CD45+CD3+ cells that were CD8+ by the percentage that were CD4+CD25+FoxP3+. [Figure 5]Therapeutic effect of Neoleukin-2 / 15 on colon cancer. Figure 5A) BALB / C mice were inoculated with CT26 tumors. Starting on day 9 and ending on day 14, mice were treated daily with intraperitoneal injections of the indicated concentrations of mIL-2 or Neoleukin-2 / 15, or left untreated (n = 5 per group). Tumor growth curve (top, only data from surviving mice are shown). Survival curve (bottom). Mice were euthanized when body weight loss exceeded 10% of their initial weight or when tumor size reached 1,300 mm3. Figures 5B-5D) Bar graphs compare T cell populations in BALB / C mice (n = 3 per group) inoculated with CT26 tumors and treated with daily intraperitoneal injections of 10 μg of Neoleukin-2 / 15 or 10 μg of mIL-2 starting on day 6, or left untreated (No Tx). On day 14, the percentage of Treg cells (CD4+ CD45+ FoxP3+, upper graph) and the CD8:Treg cell ratio ((CD45+ CD3+ CD8+) / Treg, lower graph) were assessed in the tumor (Figure 5B), adjacent inguinal lymph nodes (LN) (Figure 5C), and spleen (Figure 5D). [Figure 6]Therapeutic effect of Neoleukin-2 / 15 on melanoma. Figures 6A-6E) Tumor growth curves (bottom) and survival curves (top) of C57BL / 6 mice inoculated with B16 tumors and treated with low doses (1 μg / mouse / day, a-b) or high doses (10 μg / mouse / day, c-d) of Neoleukin-2 / 15. Starting on day 1, mice (n = 5 per group) were treated daily with intraperitoneal injections of 1 μg / mouse of single-agent Neoleukin-2 / 15 or equimolar mIL-2 (n = 5 per group) (Figure 6A), or in combination with twice-weekly treatment with TA99 (starting on day 5) (Figure 6B). Mice were euthanized when tumor size reached 2,000 mm3. As indicated, C57BL / 6 mice were inoculated with B16 tumors and treated daily by intraperitoneal injection. As in Figures 6C-6D (a-b), starting on day 4, mice were treated intraperitoneally daily with 10 μg / mouse Neoleukin-2 / 15 or equimolar mIL-2, either alone (Figure 6C) or in combination with TA99 twice weekly starting on day 4 (Figure 6D). Mice were euthanized when tumor size reached 2,000 mm3. The therapeutic effect of Neoleukin-2 / 15 was dose-dependent (higher doses were better) and enhanced in the presence of the antibody TA99. Experiments were performed once. For all growth curves, data are means ± standard error (SEM). Results were analyzed by one-way analysis of variance (95% CI), except for survival curves, which were assessed using the Mantel-Cox test (95% CI). [Figure 7]We reengineered Neoleukin-2 / 15 into a human interleukin-4 (hIL-4) mimic (Neoleukin-4). Figure 7A) Neo-2 / 15 structurally aligned to the structure of IL-4 complexed with IL-4Rα and γc (PDB code 3BPL). The 14 IL-4 residues that contact IL-4Rα and were grafted into Neo-2 / 15 are labeled. Figure 7B) The new protein Neoleukin-4 (Neo-4) has 16 amino acid mutations compared to Neo-2 / 15. These mutations are labeled; 13 of these are derived from IL-4 residues that mediate contact with IL-4Rα (shown in panel "a"), three of which (H8M, K68I, and I98F, underlined in the figure) were introduced by directed evolution using random mutagenesis and screening for high-binding affinity variants. Figure 7C) Biolayer interferometry data show that Neo-4, like IL-4, binds to IL-4Rα alone and has no affinity for γc alone, but binds to γc in the presence of IL-4Rα in solution. [Figure 8] Stimulatory effect of Neoleukin-2 / 15 on human CAR-T cells. Human primary CD4 (top) or CD8 (bottom) T cells, stimulated with anti-CD3 / CD28 (Figure 8A) or unstimulated (Figure 8B), were cultured with the indicated concentrations of human IL2 or Neoleukin-2 / 15. T cell proliferation is measured as the fold change relative to T cells cultured without IL2 supplementation. Neoleukin-2 / 15 is as effective as native IL-2 in inducing proliferation of stimulated CAR-T cells and more effective than native IL-2 in inducing proliferation of unstimulated CAR-T cells, particularly unstimulated CD8 CAR-T cells. [Figure 9]Overall sequence conservation at binding residues in each of the four common helices, combining information from three different de novo designed IL-2 mimetics. Sequence logos were generated using combined data from binding experiments (using heterodimeric murine IL-2Rβγc) from three independent SSM mutagenesis libraries: G2_neo2_40_1F_seq27, G2_neo2_40_1F_seq29, and G2_neo2_40_1F_seq36 (Figures 11-13). All of these proteins are functional high-affinity de novo mimics of mouse and human IL-2, and although some have different topologies from Neo-2 / 15, all share four helices: H1 (Figure 9A, 1-22 of Neo-2 / 15 are SEQ ID NO: 248, 6-27 of IL-2 are SEQ ID NO: 249, 1-15 of IL-15 are SEQ ID NO: 250), H3 (Figure 9B, 34-55 of Neo-2 / 15 are SEQ ID NO: 251, 8 of IL-2 are SEQ ID NO: 252), H4 (Figure 9C, 35-56 of Neo-2 / 15 are SEQ ID NO: 253, 8 of IL-2 are SEQ ID NO: 254), H5 (Figure 9D, 36-57 of Neo-2 / 15 are SEQ ID NO: 255), H6 (Figure 9E, 37-59 of Neo-2 / 15 are SEQ ID NO: 256), H7 (Figure 9F, 38-60 of Neo-2 / 15 are SEQ ID NO: 257), H8 (Figure 9H, 39-61 of Neo-2 / 15 are SEQ ID NO: 258), H9 (Figure 9H, 40-41 of Neo-2 / 15 are SEQ ID NO: 259), H10 (Figure 9H, 42-43 of Neo-2 / 15 are SEQ ID NO: 260), H11 (Figure 9H, 44-58 of Neo-2 / 15 are SEQ ID NO: 261), H12 (Figure 9H, 45-60 of Neo-2 / 15 are SEQ ID NO: 262), H13 (Figure 9H, 46-59 of Neo- Neo-2 / 15 contains amino acids 2-103 (SEQ ID NO: 252), IL-15 contains amino acids 59-80 (SEQ ID NO: 253), H2' (Figure 9C, Neo-2 / 15 contains amino acids 58-76 (SEQ ID NO: 254), IL-2 contains amino acids 50-68 (SEQ ID NO: 255), IL-15 contains amino acids 34-52 (SEQ ID NO: 256), and H4 (Figure 9D, Neo-2 / 15 contains amino acids 80-100 (SEQ ID NO: 257), IL-2 contains amino acids 111-131 (SEQ ID NO: 258), IL-15 contains amino acids 93-113 (SEQ ID NO: 259). Each logo independently represents the helix combination information. The line graph below each logo indicates the probability score of each amino acid in the Neo-2 / 15 sequence (higher values ​​indicate higher conservation). Horizontal solid lines highlight positions where Neo-2 / 15 amino acids have a probability score of 30% or higher (i.e., these amino acids are more commonly involved in receptor binding because they are more enriched overall in the binding population across all de novo IL-2 mimetics tested). The topology of each Neo-2 / 15 helix is ​​shown to the left of each logo. The sequences of the Neo-2 / 15 helices and the corresponding helical sequences (structurally aligned) in human IL-2 and IL-15 are shown below the graphs, highlighting features of the Neo-2 / 15 helices and binding interface. [Figure 10]Experimental optimization of G1_neo2_40. Figures 10A-10C) Heat maps of the G1_neo2_40 single-site mutagenesis library showing enrichment at specific positions after successive rounds of increasing selection using 50 nM (Figure 10A), 2 nM (Figure 10B), and 0.1 nM (Figure 10C) of the IL-2Rβγc heterodimer. Based on these enrichment data, a combinatorial library was designed with a nucleotide diversity of 1.5 x 10. Figure 10D) The amino acid residues available in the initial combinatorial library are depicted to indicate residues predicted to be advantageous (shown above the original sequence) and deleterious (shown below the original sequence). In the depiction of the original sequence, black indicates residues represented in the combinatorial library, and gray indicates residues not represented in the combinatorial library. [Figure 11] Experimental optimization of G2_neo2_40_1F_seq27. Heat maps of the single-site mutagenesis library of G2_neo2_40_1F_seq27 showing enrichment at specific positions after successive rounds of selection using increasing concentrations of IL-2Rβγc heterodimer at 10 nM (Figure 11A), 1 nM (Figure 11B), 0.1 nM (Figure 11C), and 0.1 nM (Figure 11D). Based on these enrichment data, a combinatorial library was designed with a nucleotide diversity of 5.3 x 10. (Figure 11E) The available amino acid residues in the initial combinatorial library are depicted, indicating residues predicted to be advantageous; black indicates residues in the starting sequence represented in the combinatorial library. [Figure 12]Experimental optimization of G2_neo2_40_1F_seq29. Heat maps of the single-site mutagenesis library of G2_neo2_40_1F_seq29 showing enrichment at specific positions after successive rounds of increasing selection with 10 nM (Figure 12A), 1 nM (Figure 12B), 0.1 nM (Figure 12C), and 0.1 nM (Figure 12D) of the IL-2Rβγc heterodimer. Based on these enrichment data, a combinatorial library was designed with a nucleotide diversity of 2.9 x 10. (Figure 12E) The available amino acid residues in the initial combinatorial library are depicted, indicating residues predicted to be advantageous; black indicates residues in the starting sequence represented in the combinatorial library. [Figure 13] Experimental optimization of G2_neo2_40_1F_seq36. Heat maps of the single-site mutagenesis library of G2_neo2_40_1F_seq36 showing enrichment at specific positions after successive rounds of increasing selection with 10 nM (Figure 13A), 1 nM (Figure 13B), 0.1 nM (Figure 13C), and 0.1 nM (Figure 13D) of the IL-2Rβγc heterodimer. Based on these enrichment data, a combinatorial library was designed with a nucleotide diversity of 2.7 x 10. (Figure 13E) The available amino acid residues in the initial combinatorial library are depicted, indicating residues predicted to be advantageous; black indicates residues in the starting sequence represented in the combinatorial library. [Figure 14] Circular dichroism (CD) thermal denaturation experiments of several IL-2 / IL-15 de novo designed mimics (first generation). Figure 14A) Thermal denaturation curves and Figure 14B) wavelength scans. [Figure 15A-B] Experimentally optimized circular dichroism (CD) thermal denaturation experiments of several IL-2 / IL-15 de novo designed mimics (first generation). Figure 15A) Thermal denaturation curves and Figure 15B) wavelength scans. [Figure 16] Circular dichroism thermal melting of IL-2 / IL-15 mimetic designs (2nd generation). Figures 16A and 16C) Thermal denaturation curves and Figures 16B and 16D) Wavelength scans. [Figure 17]Characterization of Neoleukin-2 / 15 expression, purification, and thermal denaturation. Figure 17A) SDS Tris-Tricine gel electrophoresis showing expression and affinity column purification. Figure 17B) Circular dichroism at 222 nm shows robust thermal stability during thermal melting from 25°C to 95°C. Figure 17C) Circular dichroism wavelength scans at 25°C, 95°C, and then again at 25°C show that Neoleukin-2 / 15 does not completely melt even at 95°C and fully refolds after cooling to 25°C. [Figure 18]Single-disulfide-stapled variants of Neoleukin-2 / 15 with higher thermal stability. Structural models of disulfide-stabilized variants of Neoleukin-2 / 15 are shown with the positions of the mutated residues labeled and the disulfide bonds indicated. Two strategies were used to generate the disulfide variants: (18A) internal placement and terminal attachment at residues 38 and 75. (18B) For the terminal variants, three residues were added to each end to limit any distortion to the starting structure that would otherwise be required to form disulfide bonds. CD spectra of the internal and terminal disulfide variants at 25°C, 95°C, and after cooling to 25°C are shown below their structural models. Both variants show negligible signal loss at 95°C and complete refolding upon cooling. (18C) Thermal melting of each variant was performed by monitoring the CD signal at 222.0 nm over a range of temperatures. Each disulfide variant exhibits improved stability compared to the native variant. (Figure 18D) The binding strength of each variant to IL-2Rβγc was measured by biolayer interferometry. These data demonstrate that, as opposed to disrupting the binding interaction, the introduction of a disulfide bond improves binding of the mimetic to IL-2Rβγc. Both disulfide-bonded variants showed improved binding to IL-2Rβγc (Kds of approximately 1.3±0.49 and 1.8±0.26 nM for the internal and external disulfide staples, respectively, compared to 6.9±0.61 nM for Neo-2 / 15 under the same experimental conditions), consistent with the expected effect of disulfide-induced stabilization of the protein's binding site. [Figure 19]Robustness of Neoleukin-2 / 15 to single-point cysteine ​​mutants at non-binding interface positions. Figure 19A) Schematic showing the locations of point mutants in Neolukin-2 / 15 that can be individually mutated to cysteines without interfering with protein expression or binding to IL-2Rβγc. The positions were chosen to avoid interference with receptor binding. Figure 19B) Association kinetics of Neolukin-2 / 15 cysteine ​​mutants with IL-2Rβγc measured using biolayer interferometry. All variants associate with the receptor nearly identically to Neo-2 / 15. [Figure 20] Characterization of Neoleukin-4 expression, purification, and thermal denaturation. Figure 20A) SDS Tris-Tricine gel electrophoresis showing expression and affinity column purification. Figure 20B) Circular dichroism at 222 nm shows robust temperature stability during thermal melting from 25°C to 95°C. Figure 20C) Circular dichroism wavelength scans at 25°C, 95°C, and then again at 25°C show that Neoleukin-4 does not completely melt even at 95°C and fully refolds after cooling to 25°C. [Figure 21]Cytokine levels in nonhuman primates in response to Neo-2 / 15 or Neo-2 / 15-PEG. Two nonhuman primates (NHPs) per group, one male and one female per group, were assigned to treatment with either vehicle, Neo-2 / 15, or Neo-2 / 15-PEG (containing Neo-2 / 15 with the E62C single cysteine ​​mutation linked to PEG40K). Animals received either 0 (vehicle), 0.1, 0.2, or 0.3 mg / kg Neo-2 / 15, or 0.05, 0.10, or 0.15 mg / kg Neo-2 / 15-PEG by intravenous bolus. Animals treated with Neo-2 / 15 PEG received 0 mg / kg Neo-2 / 15 PEG by intravenous bolus. Cytokine samples were collected at 0, 4, 8, and 24 hours post-dose. Cytokine serum samples were prepared, frozen below -70°C, and shipped for analysis. Samples were analyzed on a Luminex multiplex immunoassay system. Several cytokines, including IL-10 (Figures 21A-21B) and IL-15 (Figures 21C-21D), demonstrated significant differences in the time course of cytokine production, consistent with a more sustained pharmacodynamic effect of the PEGylated molecules. [Figure 22] 1 is a block diagram of an exemplary computing network. [Figure 23A] 1 is a block diagram of an exemplary computing device. [Figure 23B] FIG. 1 is a block diagram of an exemplary network of computing devices arranged as a cloud-based server system. [Figure 24] 1 is a flowchart of the method. DETAILED DESCRIPTION OF THE INVENTION

[0013] As used herein, unless otherwise indicated, the terms "a" and "an" shall be construed to mean "one," "at least one," or "one or more." Unless otherwise required by context, as used herein, the singular shall include the plural and the plural shall include the singular.

[0014] Unless the context clearly requires otherwise, throughout the detailed description and the claims, words like "comprise," "comprising," and the like are to be construed in the inclusive or exhaustive sense, as opposed to the exclusive sense, i.e., "including, but not limited to." Words using the singular or plural also include the plural and singular, respectively. Furthermore, the words "herein," "above," and "below," and words of similar import, when used in this application, refer to this application as a whole and not to any particular portions of this application.

[0015] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0016] All embodiments of any aspect of the invention can be used in combination unless the context clearly indicates otherwise.

[0017] In one aspect, the invention provides a non-naturally occurring polypeptide comprising domains X1, X2, X3, and X4, (a) X1 is [ka] (SEQ ID NO: 1), (b) X2 is a helical peptide at least 8 amino acids in length; (c) X3 is [ka] (SEQ ID NO: 2), (d) X4 is [ka] (SEQ ID NO: 3), X1, X2, X3, and X4 can be in any order within the polypeptide; An amino acid linker may be present between any of the domains, The polypeptide is an IL-2 receptor βγ c Heterodimer (IL-2Rβγ c ), IL-4 receptor αγ c Heterodimer (IL-4Rαγ c ), or the IL-13 receptor alpha subunit (IL-13Rα). In various embodiments, the polypeptide binds to IL-2Rβγ with a binding affinity of 200 nM or less, 100 nM or less, 50 nM or less, or 25 nM or less. c or IL-4Rαγ c Combine with.

[0018] In one aspect, the invention provides a non-naturally occurring polypeptide comprising domains X1, X2, X3, and X4, (a) X1 is [ka] (SEQ ID NO: 1), (b) X2 is a helical peptide at least 8 amino acids in length; (c) X3 is [ka] (SEQ ID NO: 2), (d) X4 is [ka] (SEQ ID NO: 3), X1, X2, X3, and X4 can be in any order within the polypeptide; An amino acid linker may be present between any of the domains, The polypeptide is an IL-2 receptor βγ c Heterodimer (IL-2Rβγ c In various embodiments, the polypeptide binds to IL-2Rβγ with a binding affinity of 200 nM or less, 100 nM or less, 50 nM or less, or 25 nM or less. c Combine with.

[0019] In one aspect, the invention provides a non-naturally occurring polypeptide comprising domains X1, X2, X3, and X4, (a) X1 is the amino acid sequence [ka] (SEQ ID NO: 1), (b) X2 is a helical peptide at least 8 amino acids in length; (c) X3 is the amino acid sequence [ka] (SEQ ID NO: 2), (d) X4 is the amino acid sequence [ka] (SEQ ID NO: 3), X1, X2, X3, and X4 can be in any order within the polypeptide; An amino acid linker may be present between any of the domains, The polypeptide is an IL-2 receptor βγc Heterodimer (IL-2Rβγ c In various embodiments, the polypeptide binds to IL-2Rβγ with a binding affinity of 200 nM or less, 100 nM or less, 50 nM or less, or 25 nM or less. c Combine with.

[0020] As shown in the Examples below, the polypeptides of the present disclosure are capable of binding to (a) IL-2 receptor βγ c Heterodimer (IL-2Rβγ c IL-2 and interleukin-15 (IL-15) mimetics that bind to the IL-2Rα or IL-15Rα receptor but do not have the binding sites for IL-2Rα or IL-15Rα; or (b) IL-4 receptor αγ c Heterodimer (IL-4Rαγ c ) or IL-13 receptor α subunit (IL-13Rα) (Native IL-4 and the IL-4 mimics described herein cross-react with the IL-13 receptor, forming IL-4Rα / IL13Rα heterodimers). This design is ultrastable and binds to human and mouse IL-2Rβγ with higher affinity than the native cytokine. c or IL-4Rαγ c and induces downstream cell signaling independently of IL-2Rα and IL-15Rα, or independently of IL-13Rα. This polypeptide can be used, for example, to treat cancer.

[0021] The term protein mimetic, as used herein, refers to a protein that mimics a specific aspect of the function of another protein. The two proteins typically have different amino acid sequences and / or different structures. Specifically, de novo mimetics of IL-2 and IL-15 are provided herein. The aspect of IL-2 and IL-15 function that these mimetics mimic is the induction of IL-2Rβγc heterodimerization, which leads to STAT5 phosphorylation. Because both IL-2 and IL-15 signal through IL-2Rβγc heterodimerization, these mimetics mimic this biological function of both IL-2 and IL-15. These mimetics may be referred to herein as IL-2 mimetics, IL-15 mimetics, or both IL-2 and IL-15 mimetics.

[0022] De novo mimetics of IL-4 are also provided. These mimetics can mimic specific functions of IL-4. The functions of IL-4 that these mimics mimic include IL-4Rαγ c The primary objective of this study is to induce IL-4Rα / IL-13Rα heterodimerization (and / or IL-4Rα / IL-13Rα heterodimerization).

[0023] Native hIL-2 is composed of four helices connected by a long irregular loop. The N-terminal helix (H1) interacts with both the β and γ subunits, the third helix (H3) interacts with the β subunit, and the C-terminal helix (H4) interacts with the γ subunit. The interaction surface of the α subunit is formed by the irregular second helix (H2) and two long loops, one connecting H1 to H2 and the other connecting H3 to H4. Idealized proteins were designed and constructed in which H1, H3, and H4 were replaced with idealized structural domains, including but not limited to, helices and β strands (referred to as domains X1, X3, and X4, respectively), inspired by H1, H3, and H4, to form the IL-2Rβγ protein. c or IL-4Rαγ cThe peptide domains exhibit an interface, and H2 is replaced with an idealized helix (designated domain X2) that provides better packing. As shown in the Examples, extensive mutational studies have been performed, demonstrating that the amino acid sequence of each peptide domain can be extensively modified without losing binding to the IL-2 or IL-4 receptor, respectively, and that the domains can be arranged in any order while retaining binding to the IL-2 or IL-4 receptor. The polypeptides may contain L-amino acids and glycine, D-amino acids and glycine, or combinations thereof.

[0024] Thus, X1, X2, X3, and X4 can be in any order within the polypeptide, and in non-limiting embodiments, the order can be X1-X2-X3-X4, X1-X3-X2-X4, X1-X4-X2-X3, X3-X2-X1-X4, X4-X3-X2-X1, X2-X3-X4-X1, X2-X1-X4-X3, etc.

[0025] The domains can be separated by an amino acid linker of any length and amino acid composition. There is no need for a linker, and in one embodiment, there is no linker between any of the domains. In other embodiments, amino acid linkers can be present between one, two, or all three junctions between domains X1, X2, X3, and X4. The linker can be any length that is deemed appropriate for the intended use.

[0026] In various embodiments, X1 is a peptide comprising an amino acid sequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1. In other embodiments, X3 is a peptide comprising an amino acid sequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:2. In a further embodiment, X4 is a peptide comprising an amino acid sequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:3.

[0027] In one embodiment, the polypeptide is an IL-2 / 15 mimetic, wherein (i) X1 includes one or both of the following: H at residue 2 and Y at residue 5, and / or (ii) X3 includes one, two, three, four, or all five of the following: Y at residue 1, F at residue 3, N at residue 4, L at residue 7, and I at residue 8. In a further embodiment, (iii) X4 includes I at residue 8.

[0028] In another embodiment, the polypeptide is an IL-4 mimetic, wherein (i) X1 comprises an E at residue 2 and a K at residue 5, and (ii) X3 comprises an F at residue 1, a K at residue 3, an R at residue 4, an R at residue 7, and an N at residue 8. In a further embodiment, (iii) X4 comprises an F at residue 8.

[0029] In all of these embodiments, X1, X3, and X4 can be of any suitable length, meaning that each domain can contain any suitable number of additional amino acids other than the peptides of SEQ ID NOs: 1, 2, and 3, respectively. [ka] (SEQ ID NO: 4), and X3 is a peptide comprising an amino acid sequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical along its length to (SEQ ID NO: 4), and X3 is a peptide [ka] (SEQ ID NO: 5), and X4 is a peptide comprising an amino acid sequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical along its length to (SEQ ID NO: 5), and X4 is a peptide [ka] (SEQ ID NO: 6) along its length.

[0030] In one embodiment, X1 is a peptide [ka] (SEQ ID NO: 4), and X3 is a peptide comprising an amino acid sequence at least 80% identical along its length to the peptide [ka] (SEQ ID NO: 5), and X4 is a peptide comprising an amino acid sequence at least 80% identical along its length to the peptide [ka] (SEQ ID NO: 6) along its length.

[0031] In one embodiment, X1 is a peptide [ka] (SEQ ID NO: 4), and X3 is a peptide comprising an amino acid sequence at least 85% identical along its length to the peptide [ka] (SEQ ID NO: 5), and X4 is a peptide comprising an amino acid sequence at least 85% identical along its length to the peptide [ka] (SEQ ID NO: 6) along its length.

[0032] In one embodiment, X1 is a peptide [ka] (SEQ ID NO: 4), and X3 is a peptide comprising an amino acid sequence at least 90% identical along its length to the peptide [ka] (SEQ ID NO: 5), and X4 is a peptide comprising an amino acid sequence at least 90% identical along its length to the peptide [ka] (SEQ ID NO: 6) along its length.

[0033] In one embodiment, X1 is a peptide [ka] (SEQ ID NO: 4), and X3 is a peptide comprising an amino acid sequence at least 95% identical along its length to the peptide [ka] (SEQ ID NO: 5), and X4 is a peptide comprising an amino acid sequence at least 95% identical along its length to the peptide [ka] (SEQ ID NO: 6) along its length.

[0034] In one embodiment, X1 is a peptide [ka] (SEQ ID NO: 4), and X3 is a peptide containing an amino acid sequence 100% identical along its length to the peptide [ka] (SEQ ID NO: 5), and X4 is a peptide containing an amino acid sequence 100% identical along its length to the peptide [ka] (SEQ ID NO: 6) along its length.

[0035] In one embodiment, the polypeptide is an IL-2 / 15 mimetic, wherein (i) X1 includes one, two, three, four, or all five of the following: L at residue 7, H at residue 8, H at residue 11, Y at residue 14, and M at residue 18; and / or (ii) X3 includes one, two, three, four, five, six, seven, or all eight of the following: D at residue 3, Y at residue 4, F at residue 6, N at residue 7, L at residue 10, I at residue 11, E at residue 13, or E at residue 14. In a further embodiment, (iii) X4 includes I at residue 19.

[0036] In one embodiment of the IL-2 mimetic, the amino acid substitution relative to the reference peptide domain (ie, SEQ ID NO: 1, 2, 3, 4, 5, or 6) does not occur at the amino acid (AA) residues marked in bold.

[0037] In another embodiment, the polypeptide is an IL-4 / IL-13 mimetic, X1 is a peptide [ka] (SEQ ID NO: 8), X3 is a peptide [ka] (SEQ ID NO: 9), X4 is a peptide [ka] (SEQ ID NO: 10),

[0038] (i) X1 contains I at residue 7, T or M at residue 8, E at residue 11, K at residue 14, and S at residue 18; (ii) X3 contains R at residue 3, F at residue 4, K at residue 6, R at residue 7, R at residue 10, N at residue 11, W at residue 13, and G at residue 14.

[0039] In a further embodiment, (iii) X4 comprises an F at residue 19.

[0040] In one embodiment, the amino acid substitution relative to the reference peptide domain is a conservative amino acid substitution. As used herein, a "conservative amino acid substitution" refers to the substitution of a given amino acid with a residue having similar physiochemical properties, such as the substitution of one aliphatic residue for another (such as Ile, Val, Leu, or Ala for another), or the substitution of one polar residue for another (such as between Lys and Arg, between Glu and Asp, or between Gln and Asn). Other such conservative substitutions, such as the substitution of entire regions with similar hydrophobic properties, are known. Polypeptides containing conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that the desired activity, such as antigen-binding activity and specificity of the native or reference polypeptide, is retained. Amino acids can be grouped according to the similarity of their side chain properties (A.L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile, (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln, (3) acidic: Asp, Glu, (4) basic: His, Lys, Arg, (5) residues that influence chain orientation: Gly, Pro, (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will require exchanging a member of one of these classes for another class.Particular conservative substitutions include, for example, substituting Ala for Gly or Ser, Arg for Lys, Asn for Gln or His, Asp for Glu, Cys for Ser, Gln for Asn, Glu for Asp, Gly for Ala or Pro, His for Asn or Gln, Ile for Leu or Val, Leu for Ile or Val, Lys for Arg, Gln, or Glu, Met for Leu, Tyr, or Ile, Phe for Met, Leu, or Tyr, Ser for Thr, Thr for Ser, Trp for Tyr, Tyr for Trp, and / or Phe for Val, Ile, or Leu.

[0041] In one embodiment, the amino acid residue of X1 for SEQ ID NO:4 is selected from the group consisting of: [Table 1]

[0042] In one embodiment, the polypeptide is an IL-4 mimetic, where position 7 is I, position 8 is M or T, position 11 is E, position 14 is K, and position 18 is S.

[0043] In another embodiment, the polypeptide is an IL-2 mimetic and one, two, three, four, or five of the following are not true: position 7 is I; position 8 is M or T; position 11 is E; position 14 is K; and position 18 is S.

[0044] In another embodiment, the amino acid residue of X3 for SEQ ID NO: 5 is selected from the group consisting of: [Table 2]

[0045] In another embodiment, the polypeptide is an IL-4 / IL-13 mimetic, where position 3 is R, position 4 is F, position 6 is K, position 7 is R, position 10 is R, position 11 is N, position 13 is W, and position 14 is G.

[0046] In another embodiment, the polypeptide is an IL-2 mimetic wherein one, two, three, four, five, six, seven, or all eight of the following are not true: position 3 is R, position 4 is F, position 6 is K, position 7 is R, position 10 is R, position 11 is N, position 13 is W, and position 14 is G.

[0047] In any such embodiment, the polypeptide further allows for a cysteine ​​at position 17 relative to SEQ ID NO: 5, in addition to amino acid residues H, K, L, N, and R. Thus, the amino acid residue at X3 relative to SEQ ID NO: 5 can be selected from the group consisting of: [Table 3]

[0048] In another embodiment, the amino acid residue of X4 for SEQ ID NO: 6 is selected from the group consisting of: [Table 4]

[0049] In another embodiment, the polypeptide is an IL-4 / IL-13 mimetic and position 19 is I. In another embodiment, the polypeptide is an IL-2 mimetic and position 19 is not I.

[0050] In any such embodiment, the polypeptide further allows for a cysteine ​​at position 3 relative to SEQ ID NO:6, in addition to the E, G, H, and K amino acid residues.

[0051] Thus, the amino acid residue of X4 for SEQ ID NO: 6 can be selected from the group consisting of: [Table 5]

[0052] As described herein, domain X2 is a structural domain and therefore can use any amino acid sequence that allows other related domains to be linked (depending on the order of the domains) and to fold. The required length depends on the structure of the protein to be produced and can be 8 amino acids or more. In one exemplary and non-limiting embodiment, X2 is [ka] (SEQ ID NO: 7) along its length at least 20%, 27%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to (SEQ ID NO: 7). In a further embodiment, the amino acid residues of X2 relative to SEQ ID NO: 7 are selected from the group consisting of: [Table 6]

[0053] In another embodiment, the polypeptide is an IL-4 / IL-13 mimetic and position 11 is I. In another embodiment, the polypeptide is an IL-2 mimetic and position 11 is not I.

[0054] In any of such embodiments, the polypeptide further allows for a cysteine ​​at position 5 or 16 relative to SEQ ID NO:7.

[0055] Alternatively, in any of such embodiments, the polypeptide further allows for a cysteine ​​at position 1, 2, 5, 9 or 16 relative to SEQ ID NO:7.

[0056] Thus, the amino acid residue of X2 for SEQ ID NO: 7 can be selected from the group consisting of: [Table 7]

[0057] In another embodiment, the polypeptide comprises a polypeptide that is at least at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to an amino acid sequence selected from the group consisting of the following polypeptides (i.e., SEQ ID NOs: 11-94, 103-184, 190-243, and 245-247). The underlined residues are linkers and are optional, and each residue of the linker, when present, can comprise any amino acid. For each variant below, two SEQ ID NOs: a first SEQ ID NO containing the optional and variable linker positions and a second SEQ ID NO recites the sequence shown below. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 8-7] [Table 8-8]

[0058] For each variant below, two SEQ ID NOs are provided: a first SEQ ID NO that lists the sequence shown below and a second SEQ ID NO that includes optional and variable linker positions. >Neoleukin-2 / 15_R50C (SEQ ID NO: 190) [ka] >Neoleukin-2 / 15_R50C (SEQ ID NO: 217) [ka] >Neoleukin-2 / 15_E53C (SEQ ID NO: 191) [ka] >Neoleukin-2 / 15_E53C (SEQ ID NO: 218) [ka] >Neoleukin-2 / 15_D56C (SEQ ID NO: 192) [ka] >Neoleukin-2 / 15_D56C (SEQ ID NO: 219) [ka] >Neoleukin-2 / 15_K58C (SEQ ID NO: 193) [ka] >Neoleukin-2 / 15_K58C (SEQ ID NO: 220) [ka] >Neoleukin-2 / 15_D59C (SEQ ID NO: 194) [ka] >Neoleukin-2 / 15_D59C (SEQ ID NO: 221) [ka] >Neoleukin-2 / 15_E62C (SEQ ID NO: 195) [ka] >Neoleukin-2 / 15_E62C (SEQ ID NO: 222) [ka] >Neoleukin-2 / 15_R66C (SEQ ID NO: 196) [ka] >Neoleukin-2 / 15_R66C (SEQ ID NO: 223) [ka] >Neoleukin-2 / 15_E69C (SEQ ID NO: 197) [ka] >Neoleukin-2 / 15_E69C (SEQ ID NO: 224) [ka] >Neoleukin-2 / 15_R73C (SEQ ID NO: 198) [ka] >Neoleukin-2 / 15_R73C (SEQ ID NO: 225) [ka] >Neoleukin-2 / 15_T77C (SEQ ID NO: 199) [ka] >Neoleukin-2 / 15_T77C (SEQ ID NO: 226) [ka] >Neoleukin-2 / 15_E82C (sequence number 200) [ka] >Neoleukin-2 / 15_E82C (SEQ ID NO: 227) [ka] >Neoleukin-2 / 15_E85C (SEQ ID NO: 201) [ka] >Neoleukin-2 / 15_E85C (SEQ ID NO: 228) [ka] >Neoleukin-2 / 15_R50C_R73C (SEQ ID NO: 202) [ka] >Neoleukin-2 / 15_R50C_R73C (SEQ ID NO: 229) [ka] >Neoleukin-2 / 15_E53C_R73C (SEQ ID NO: 203) [ka] >Neoleukin-2 / 15_E53C_R73C (SEQ ID NO: 230) [ka] >Neoleukin-2 / 15_D56C_R73C (SEQ ID NO: 204) [ka] >Neoleukin-2 / 15_D56C_R73C (SEQ ID NO: 231) [ka] >Neoleukin-2 / 15_K58C_R73C (SEQ ID NO: 205) [ka] >Neoleukin-2 / 15_K58C_R73C (SEQ ID NO: 232) [ka] >Neoleukin-2 / 15_D59C_R73C (SEQ ID NO: 206) [ka] >Neoleukin-2 / 15_D59C_R73C (SEQ ID NO: 233) [ka] >Neoleukin-2 / 15_E62C_R73C (SEQ ID NO: 207) [ka] >Neoleukin-2 / 15_E62C_R73C (SEQ ID NO: 234) [ka] >Neoleukin-2 / 15_R66C_R73C (SEQ ID NO: 208) [ka] >Neoleukin-2 / 15_R66C_R73C (SEQ ID NO: 235) [ka] >Neoleukin-2 / 15_R50C_E82C (SEQ ID NO: 209) [ka] >Neoleukin-2 / 15_R50C_E82C (SEQ ID NO: 236) [ka] >Neoleukin-2 / 15_E53C_E82C (SEQ ID NO: 210) [ka] >Neoleukin-2 / 15_E53C_E82C (SEQ ID NO: 237) [ka] >Neoleukin-2 / 15_D56C_E82C (SEQ ID NO: 211) [ka] >Neoleukin-2 / 15_D56C_E82C (SEQ ID NO: 238) [ka] >Neoleukin-2 / 15_K58C_E82C (SEQ ID NO: 212) [ka] >Neoleukin-2 / 15_K58C_E82C (SEQ ID NO: 239) [ka] >Neoleukin-2 / 15_D59C_E82C (SEQ ID NO: 213) [ka] >Neoleukin-2 / 15_D59C_E82C (SEQ ID NO: 240) [ka] >Neoleukin-2 / 15_E62C_E82C (SEQ ID NO: 214) [ka] >Neoleukin-2 / 15_E62C_E82C (SEQ ID NO: 241) [ka] >Neoleukin-2 / 15_R66C_E82C (SEQ ID NO: 215) [ka] >Neoleukin-2 / 15_R66C_E82C (SEQ ID NO: 242) [ka] >Neoleukin-2 / 15_E69C_E82C (SEQ ID NO: 216) [ka] >Neoleukin-2 / 15_E69C_E82C (SEQ ID NO: 243) [ka]

[0059] In one embodiment, the polypeptide comprises a polypeptide that is at least at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to an amino acid sequence selected from the group consisting of SEQ ID NOs: 90, 181, and 247.

[0060] In another embodiment, the polypeptide comprises a polypeptide identical to the amino acid sequence of SEQ ID NO: 90, 181, or 247, wherein the polypeptide (i) does not bind to human or mouse IL-2Rα, (ii) binds to human IL2RB with an affinity of about 11.2 nM, (iii) binds to mouse IL2RB with an affinity of about 16.1 nM, and (iv) binds to human IL-2Rβγ with an affinity of about 18.8 nM. c and (v) binds to murine IL-2Rβγ with an affinity of about 3.4 nM. c Combine with.

[0061] In any of these embodiments of a full-length polypeptide, the polypeptide can be an IL-4 / IL-13 mimetic, where position 7 is I, position 8 is T or M, position 11 is E, position 14 is K, position 18 is S, position 33 is Q, position 36 is R, position 37 is F, position 39 is K, position 40 is R, position 43 is R, position 44 is N, position 46 is W, and position 47 is G. In a further embodiment, position 68 is I and position 98 is F.

[0062] In any of these embodiments of a full-length polypeptide, the polypeptide may be an IL-2 mimetic wherein one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or all fourteen of the following are not true: position 7 is I, position 8 is T or M, position 11 is E, position 14 is K, position 18 is S, position 33 is Q, position 36 is R, position 37 is F, position 39 is K, position 40 is R, position 43 is R, position 44 is N, position 46 is W, and position 47 is G. In further embodiments, one or both of the following are not true: position 68 is I, and position 98 is F.

[0063] In one embodiment, the IL-2 mimetic polypeptide of any embodiment or combination of embodiments disclosed herein has a three-dimensional structure with structural coordinates that have a root mean square deviation of the backbone or alpha carbon atoms of common residues of less than 2.5 Å, less than 1.5 Å, or less than 1 Å when superimposed onto the backbone or alpha carbon atoms of the three-dimensional structure of native IL-2.

[0064] In another embodiment, the IL-2 mimetic polypeptide of any embodiment or combination of embodiments disclosed herein has a three-dimensional structure with structural coordinates that have a root mean square deviation of the backbone atoms or alpha carbon atoms of common residues of less than 2.5 Å, less than 1.5 Å, or less than 1 Å when superimposed onto the backbone atoms or alpha carbon atoms of a three-dimensional structure having the structural coordinates of Table E2.

[0065] In a further embodiment, the IL-2 mimetic polypeptide of any embodiment or combination of embodiments disclosed herein is a polypeptide that binds to the murine IL-2 receptor βγ cMouse IL-2 receptor βγ in a ternary complex with c When in a ternary complex with IL-2, it has a three-dimensional structure with structural coordinates that have a root mean square deviation of the backbone atoms or alpha carbon atoms of common residues of less than 2.5 Å, less than 1.5 Å, or less than 1 Å when superimposed onto the backbone atoms or alpha carbon atoms of the three-dimensional structure of native IL-2.

[0066] In another embodiment, the IL-4 mimetic polypeptide of any embodiment or combination of embodiments disclosed herein has a three-dimensional structure with structural coordinates that, when superimposed onto the backbone or alpha carbon atoms of the three-dimensional structure of native IL-4, contain root mean square deviations of the backbone or alpha carbon atoms of common residues of less than 2.5 Å, less than 1.5 Å, or less than 1 Å.

[0067] In each of these embodiments, the three-dimensional structure of the polypeptide may be determined using computational modeling, or alternatively, the three-dimensional structure of the polypeptide may be determined using crystallographically determined structural data.

[0068] In one embodiment of any embodiment or combination of embodiments disclosed herein, X1, X2, X3, and X4 are alpha-helical domains. In another embodiment, the amino acid length of each of X1, X2, X3, and X4 is independently at least about 8, 10, 12, 14, 16, 19, or more amino acids. In other embodiments, the amino acid length of each of X1, X2, X3, and X4 is independently no more than 1000, 500, 400, 300, 200, 100, or 50 amino acids. In various further embodiments, the amino acid length of each of X1, X2, X3, and X4 is independently about 8 to 1000, 8 to 500, 8 to 400, 8 to 300, 8 to 200, 8 to 100, 8 to 50, 10 to 1000, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 50, 12 to 1000, 12 to 500, 12 to 400, 12 to 300, 12 to 200, The amino acid sequence may be 12-100, 12-50, 14-1000, 14-500, 14-400, 14-300, 14-200, 14-100, 14-50, 16-1000, 16-500, 16-400, 16-300, 16-200, 16-100, 16-50, 19-1000, 19-500, 19-400, 19-300, 19-200, 19-100, or about 19-50 amino acids in length.

[0069] In another embodiment, the IL-2 mimetic polypeptide of any embodiment or combination of embodiments disclosed herein, X1 binds to the β and γ subunits of the human IL-2 receptor. In another embodiment of the IL-2 mimetic polypeptide of any embodiment or combination of embodiments disclosed herein, X2 does not bind to the human IL-2 receptor. In another embodiment of the IL-2 mimetic polypeptide of any embodiment or combination of embodiments disclosed herein, X3 binds to the β subunit of the human IL-2 receptor. In a further embodiment of the IL-2 mimetic polypeptide of any embodiment or combination of embodiments disclosed herein, X4 binds to the γ subunit of the human IL-2 receptor. In another embodiment or any embodiment or combination of embodiments disclosed herein, the IL-2 mimetic polypeptide does not bind to the α subunit of the human or mouse IL-2 receptor. In one embodiment, the binding to the receptor is specific binding as determined by surface plasmon resonance at biologically relevant concentrations. In another embodiment, the IL-2 mimetic peptide of any embodiment or combination of embodiments disclosed herein binds to the IL-2 receptor βγ subunit. c Heterodimer (IL-2Rβγ c ) and binds with a binding affinity of 200 nM or less, 100 nM or less, 50 nM or less, or 25 nM or less. In further embodiments of the IL-2 mimetic polypeptide of any embodiment or combination of embodiments disclosed herein, the affinity of the polypeptide for the human and mouse IL-2 receptors is about equal to or exceeds the affinity of native IL-2.

[0070] In one embodiment of the IL-4 mimetic polypeptide of any embodiment or combination disclosed herein, the IL-4 receptor αγ c Heterodimer (IL-4Rαγ c) and binds with a binding affinity of 200 nM or less, 100 nM or less, 50 nM or less, or 25 nM or less. In another embodiment of the IL-4 mimetic polypeptide of any embodiment or combination of embodiments disclosed herein, the affinity of the polypeptide for the human and mouse IL-4 receptor is about equal to or exceeds the affinity of native IL-4.

[0071] In one embodiment of the IL-2 mimetic polypeptide of any embodiment or combination of embodiments disclosed herein, the polypeptide stimulates phosphorylation of STAT5 in cells expressing the IL-2 receptor with about the same or greater potency as native IL-2. In another embodiment of the IL-2 mimetic polypeptide of any embodiment or combination of embodiments disclosed herein, the polypeptide stimulates phosphorylation of IL-2 receptor βγ in cells expressing the IL-2 receptor with about the same or greater potency as native IL-2. c It stimulates STAT5 phosphorylation in cells that express the heterodimer but lack IL-2 receptor alpha.

[0072] In another embodiment, the IL-2 mimetic polypeptide of any embodiment or combination of embodiments disclosed herein demonstrates a thermostability that is about equal to or exceeds that of native IL-2.

[0073] In further embodiments, the polypeptides of any embodiment or combination of embodiments disclosed herein maintain or recover at least 70%, 80%, or 90% of their folded structure after a thermal stability test, and / or maintain or recover at least 80% of their ellipticity spectrum after a thermal stability test, and / or maintain or recover at least 70% or 80% of their activity after a thermal stability test. In one embodiment, such activity is determined by a STAT5 phosphorylation assay. In another embodiment, thermal stability is measured by circular dichroism (CD) spectroscopy at 222 nm. In a further embodiment, the thermal stability test comprises heating the polypeptide from 25°C to 95°C over a 1-hour time frame, cooling the polypeptide to 25°C over a 5-minute time frame, and monitoring ellipticity at 222 nm.

[0074] The polypeptides described herein can be chemically synthesized or recombinantly expressed (if the polypeptide is genetically encodable). The polypeptides can be linked to other compounds, such as stabilizing compounds to promote increased in vivo half-life, including, but not limited to, albumin, PEGylation (attachment of one or more polyethylene glycol chains), HESylation, PASylation, glycosylation, or can be produced as Fc-fused or deimmunized variants. Such linkages can be covalent or non-covalent. For example, the addition of a polyethylene glycol ("PEG") containing moiety can include attachment of a PEG group linked to a maleimide group ("PEG-MAL") to a cysteine ​​residue of the polypeptide. Suitable examples of PEG-MAL are methoxyPEG-MAL 5 kD, methoxyPEG-MAL 20 kD, methoxy(PEG)2-MAL 40 kD, methoxyPEG(MAL)2 5 kD, methoxyPEG(MAL)2 20 kD, methoxyPEG(MAL)2 40 kD, or any combination thereof. See also U.S. Patent No. 8,148,109. In other embodiments, the PEG may comprise branched PEG and / or multiple PEG chains.

[0075] In one embodiment, the stabilizing compound, including but not limited to a PEG-containing moiety, is linked at a cysteine ​​residue of the polypeptide. In another embodiment, the cysteine ​​residue is present in the X2 domain. In some embodiments, the cysteine ​​residue is present, for example, at any one of several positions in the X2 domain. In some such embodiments, the X2 domain is at least 19 amino acids in length, and the cysteine ​​residue is at position 1, 2, 5, 9, or 16 relative to those 19 amino acids. In a further embodiment, the stabilizing compound, including but not limited to a PEG-containing moiety, is linked to a cysteine ​​residue via a maleimide group, including but not limited to, linked to the cysteine ​​residue at amino acid residue 62 relative to SEQ ID NO:90.

[0076] In some embodiments, the polypeptide is a Neo-2 / 15 polypeptide, and amino acids of Neo-2 / 15 have been mutated to cysteine ​​residues for attachment of stabilizing moieties (e.g., PEG-containing moieties) thereto. In some embodiments, the polypeptide is a Neo-2 / 15 polypeptide, and amino acids at positions 50, 53, 62, 69, 73, 82, 56, 58, 59, 66, 77, or 85 relative to SEQ ID NO: 90, 181, or 247, or combinations thereof, have been mutated to cysteine ​​residues for attachment of stabilizing moieties (e.g., PEG-containing moieties) thereto. Thus, in further embodiments, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the full length amino acid sequence of SEQ ID NO: 90, 181, or 247 [Neo-2 / 15], and in which one, two, three, four, five, or all six of the following mutations are present: R50C, E53C, E62C, E69C, R73C and / or E82C.

[0077] In further embodiments, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the full length amino acid sequence of SEQ ID NO: 90, 181, or 247, and in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all 12 of the following mutations are present: D56C, K58C, D59C, R66C, T77C, E85C, R50C, E53C, E62C, E69C, R73C and / or E82C.

[0078] In further embodiments, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the full length of an amino acid sequence selected from the group consisting of SEQ ID NOs: 190-243.

[0079] In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to an amino acid sequence selected from the group consisting of SEQ ID NOs: 190 and 217. In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to the amino acid sequence of SEQ ID NO:190.

[0080] In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to an amino acid sequence selected from the group consisting of SEQ ID NOs: 191 and 218. In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to the amino acid sequence of SEQ ID NO:191.

[0081] In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to an amino acid sequence selected from the group consisting of SEQ ID NOs: 192 and 219. In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to the amino acid sequence of SEQ ID NO:192.

[0082] In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to an amino acid sequence selected from the group consisting of SEQ ID NOs: 193 and 220. In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to the amino acid sequence of SEQ ID NO:193.

[0083] In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to an amino acid sequence selected from the group consisting of SEQ ID NOs: 194 and 221. In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to the amino acid sequence of SEQ ID NO:194.

[0084] In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to an amino acid sequence selected from the group consisting of SEQ ID NOs: 195 and 222. In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to the amino acid sequence of SEQ ID NO:195.

[0085] In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to an amino acid sequence selected from the group consisting of SEQ ID NOs: 196 and 223. In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to the amino acid sequence of SEQ ID NO:196.

[0086] In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to an amino acid sequence selected from the group consisting of SEQ ID NOs: 197 and 224. In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to the amino acid sequence of SEQ ID NO:197.

[0087] In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to an amino acid sequence selected from the group consisting of SEQ ID NOs: 198 and 225. In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to the amino acid sequence of SEQ ID NO:198.

[0088] In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to an amino acid sequence selected from the group consisting of SEQ ID NOs: 199 and 226. In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to the amino acid sequence of SEQ ID NO:199.

[0089] In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to an amino acid sequence selected from the group consisting of SEQ ID NOs: 200 and 227. In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to the amino acid sequence of SEQ ID NO:200.

[0090] In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to an amino acid sequence selected from the group consisting of SEQ ID NOs: 201 and 228. In one embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along its length to the amino acid sequence of SEQ ID NO:201.

[0091] In another embodiment, the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical to the full length of an amino acid sequence selected from the group consisting of SEQ ID NOs: 195, 207, 214, 222, 234, and 241; or the polypeptide comprises a polypeptide that is at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical to the full length of an amino acid sequence selected from the group consisting of SEQ ID NOs: 195, 207, and 214.

[0092] In a further embodiment, the polypeptide further comprises a targeting domain. In this embodiment, the polypeptide can target a desired target. The targeting domain can be covalently or non-covalently bound to the polypeptide. In the embodiment in which the targeting domain is non-covalently bound to the polypeptide, any suitable means for such non-covalent binding can be used, including, but not limited to, a streptavidin-biotin linker.

[0093] In another embodiment, the targeting domain, if present, is a translational fusion with the polypeptide. In this embodiment, the polypeptide and targeting domain may be directly adjacent to each other in the translational fusion, or may be linked by a polypeptide linker suitable for the intended purpose. Exemplary such linkers include, but are not limited to, those disclosed in WO2016 / 178905, WO2018 / 153865 (especially page 13), and WO2018 / 170179 (especially paragraphs

[0316] -

[0317] ). In other embodiments, suitable linkers include, but are not limited to, GGGGG (SEQ ID NO: 95), GSGGG (SEQ ID NO: 96), GGGGGG (SEQ ID NO: 97), GGSGGG (SEQ ID NO: 98), GGSGGSGGGSGGSGSG (SEQ ID NO: 99), GSGGSGGGSGGSGSG (SEQ ID NO: 100), GGSGGSGGGSGGSGGGSGGGSGGGGS (SEQ ID NO: 101), and [GGGGX] n (SEQ ID NO: 102), where X is Q, E or S, and n is 2 to 5.

[0094] The targeting domain is a polypeptide domain or small molecule that binds to a target of interest. In one non-limiting embodiment, the targeting domain binds to a cell surface protein. In this embodiment, the cell can be any cell type of interest that contains a surface protein that can be bound by a suitable targeting domain. In one embodiment, the cell surface protein is present on the surface of a cell selected from the group consisting of tumor cells, tumor vasculature cells, tumor microenvironment cells (e.g., fibroblasts, infiltrating immune cells, or stromal elements), other cancer cells, and immune cells (including, but not limited to, CD8+ T cells, regulatory T cells, dendritic cells, NK cells, or macrophages). When the cell surface protein is on the surface of a tumor cell, a vascular cell, or a tumor microenvironment cell (e.g., a fibroblast, an infiltrating immune cell, or a stromal element), any suitable tumor cell, vascular cell, or tumor microenvironment cell surface marker includes, but is not limited to, EGFR, EGFRvIII, Her2, HER3, EpCAM, MSLN, MUC16, PSMA, TROP2, ROR1, RON, PD-L1, CD47, CTLA-4, CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD40, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC, HLD-DR, carcinoembryonic antigen (CEA), TAG-72, MUC1, folate binding protein, A33, G250, prostate-specific membrane antigen (PSMA), ferritin, GD2, GD3, GM2, Le y, CA-125, CA19-9, epidermal growth factor, p185HER2, IL-2 receptor, EGFRvIII (de2-7 EGFR), fibroblast activation protein, tenascin, metalloproteinase, endosialin, vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6, HPV E7, Her-2 / neu, MAGE A3, non-mutated p53, NY-ESO-1, MelanA / MART1, Ras mutant, gp100, p53 mutant, PR1, bcr-abl, tyronsinase, survivin, PSA, hTERT, sarcoma translocation breakpoint protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, fucosyl-GM1, mesothelin (MSLN), PSCA, MAGE Al, sLe (animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TESL sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, legumain, Tie3, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, TRAIL1, MUC16, MAGE A4, MAGE C2, GAGE, EGFR, CMET, HER3, MUC15, CA6, NAPI2B, TROP2, CLDN6, CLDN16, CLDN18.2, CLorf186, RON, LY6E, FRA, DLL3, PTK7, STRA6, TMPRSS3, TMPRSS4, TMEM238, UPK1B, VTCN1, LIV1, ROR1, and Fos-related antigen 1.

[0095] In other embodiments, if the cell surface protein is on the surface of a tumor cell, vasculature cell, or tumor microenvironment cell (e.g., fibroblasts, infiltrating immune cells, or stromal elements), any suitable tumor cell, vasculature cell, or tumor microenvironment cell surface marker can be targeted, including but not limited to, targets from the following list: (1) BMPR1B (bone morphogenetic protein receptor type IB, Genbank accession number NM.sub.--001203), (2) E16 (LAT1, SLC7A5, Genbank accession number NM.sub.--003486); (3) STEAP1 (six-transmembrane epithelial antigen of the prostate, Genbank accession number NM.sub.--012449); (4) 0772P (CA125, MUC16, Genbank accession number AF361486), (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession number NM.sub.--005823), (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute transporter family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession number NM.sub.--006424), (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession number AB040878); (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession number AY358628), (9) ETBR (endothelin type B receptor, Genbank accession number AY275463), (10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession number NM.sub.--017763), (11) STEAP2 (HGNC-8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, prostate six-transmembrane epithelial antigen 2, six-transmembrane prostate protein, Genbank accession number AF455138), (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession number NM.sub.--017636), (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratoma-derived growth factor, Genbank accession number NP.sub.--003203 or NM.sub.--003212), (14) CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor) or Hs.73792, Genbank accession number M26004), (15) CD79b (IGb (immunoglobulin-related beta), B29, Genbank accession number NM.sub.--000626), (16) FcRH2 (IFGP4, IRTA4, SPAP1A (phosphatase anchor protein 1a SH2 domain containing), SPAP1B, SPAP1C, Genbank accession number NM_--030764), (17) HER2 (Genbank accession number M11730), (18) NCA (Genbank accession number M18728), (19) MDP (Genbank accession number BC017023), (20)IL20R.α. (Genbank accession number AF184971), (21) Brevican (Genbank accession number AF229053), (22) Ephb2R (Genbank accession number NM_--004442), (23) ASLG659 (Genbank accession number AX092328), (24) PSCA (Genbank accession number AJ297436), (25) GEDA (Genbank accession number AY260763), (26) BAFF-R (Genbank accession number NP_--443177.1), (27) CD22 (Genbank accession number NP-001762.1), (28) CD79a (CD79A, CD79α, immunoglobulin-related α, B cell-specific protein that covalently interacts with Igβ (CD79B) and forms a complex with IgM molecules on the surface, transducing signals involved in B-cell differentiation. Genbank accession number NP_--001774.1), (29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense and is involved in the development of HIV-2 infection, possibly AIDS, lymphoma, myeloma, and leukemia. Genbank accession number NP_--001707.1), (30) HLA-DOB (β subunit of MHC class II molecule (Ia antigen) that binds peptides and presents them to CD4+ T lymphocytes, Genbank accession number NP_--002111.1), (31) P2X5 (P2X ligand-gated ion channel 5, a purinergic receptor that is an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, and its deficiency may contribute to the pathophysiology of idiopathic detrusor instability. Genbank accession number NP_--002552.2) (32) CD72 (B cell differentiation antigen CD72, Lyb-2, Genbank accession number NP_--001773.1), (33) LY64 (lymphocyte antigen 64 (RP105), a type I membrane protein of the leucine-rich repeat (LRR) family, regulates B cell activation and apoptosis, and loss of function is associated with increased disease activity in patients with systemic lupus erythematosus. Genbank accession number NP_--005573.1). (34) FCRH1 (Fc receptor-like protein 1, a putative receptor for immunoglobulin Fc domains containing C2-type Ig-like and ITAM domains, may have a role in B lymphocyte differentiation; Genbank accession number NP_--443170.1), or (35) IRTA2 (immunoglobulin superfamily receptor translocation-associated 2, an immune receptor with a possible role in B-cell development and lymphomagenesis; gene deregulation by translocation occurs in some B-cell malignancies. Genbank accession number NP_--112571.1).

[0096] In another embodiment, the targeting domain binds to an immune cell surface marker. In this embodiment, the target can be a cell surface protein on any suitable immune cell, including but not limited to, CD8+ T cells, regulatory T cells, dendritic cells, NK cells, or macrophages. The targeting domain can be any suitable immune cell surface marker (either endogenous or engineered immune cells, including but not limited to, engineered CAR-T cells), including but not limited to, CD3, CD4, CD8, CD19, CD20, CD21, CD25, CD37, CD30, CD33, CD40, CD68, CD123, CD254, PD-1, B7-H3, and CTLA-4. In another embodiment, the targeting domain binds to PD-1, PDL-1, CTLA-4, TROP2, B7-H3, CD33, CD22, carbonic anhydrase IX, CD123, nectin-4, tissue factor antigen, CD154, B7-H3, B7-H4, FAP (fibroblast activation protein), or MUC16, and / or the targeting domain binds to PD-1, PDL-1, CTLA-4, TROP2, B7-H3, CD33, CD22, carbonic anhydrase IX, CD123, nectin-4, tissue factor antigen, CD154, B7-H3, B7-H4, FAP (fibroblast activation protein), or MUC16.

[0097] In all of these embodiments, the targeting domain can be any suitable polypeptide that binds to a target of interest and can be incorporated into the polypeptides of the present disclosure. In non-limiting embodiments, the targeting domain can include, but is not limited to, scFv, F(ab), F(ab'), B cell receptor (BCR), DARPin, affibody, monobody, nanobody, diabody, antibody (including monospecific or bispecific antibody), cell-targeting oligopeptide including, but not limited to, RGD, integrin-binding peptide, de novo designed binder, aptamer, bicyclic peptide, conotoxin, small molecule such as folate, and virus that binds to the cell surface.

[0098] In another embodiment, the polypeptide comprises at least one disulfide bond (i.e., 1, 2, 3, 4, or more disulfide bonds). Any suitable disulfide bond can be used, such as a disulfide bond connecting two different helices. In one embodiment, the disulfide bond comprises a disulfide bond connecting helix 1 (X1) and helix 4 (X4). The disulfide bond may improve the thermal stability of the polypeptide, for example, compared to a substantially similar polypeptide that does not have a disulfide bond connecting the two domains together.

[0099] The polypeptides and peptide domains of the present invention may contain additional residues at the N-terminus, C-terminus, or both that are not present in the polypeptides or peptide domains of the present disclosure. These additional residues are not included in determining the percent identity of the polypeptides or peptide domains of the present disclosure relative to the reference polypeptide. Such residues may be any residues suitable for the intended use, including, but not limited to, detection tags (i.e., fluorescent proteins, antibody epitope tags, etc.), adapters, ligands suitable for purification purposes (e.g., His tags), other peptide domains that add functionality to the polypeptide, etc. Residues suitable for attachment of such groups may include cysteine, lysine, or p-acetylphenylalanine residues, or may be tags such as amino acid tags suitable for reaction with transglutaminase as disclosed in U.S. Patent Nos. 9,676,871 and 9,777,070.

[0100] In a further aspect, the present invention provides nucleic acids, including isolated nucleic acids, encoding the polypeptides of the present invention, which can be genetically encoded.The isolated nucleic acid sequences can include RNA or DNA.Such isolated nucleic acid sequences can include, but are not limited to, polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, transport signals, secretion signals, nuclear localization signals, and cell membrane localization signals, and can include additional sequences useful for facilitating purification and / or expressing the encoded proteins.Based on the teachings herein, it will be clear to those skilled in the art which nucleic acid sequences encode the polypeptides of the present disclosure.

[0101] In another aspect, the present invention provides a recombinant expression vector comprising an isolated nucleic acid of any aspect of the present invention operably linked to a suitable control sequence. A "recombinant expression vector" includes a vector in which a nucleic acid coding region or gene is operably linked to any control sequence capable of effecting expression of the gene product. A "control sequence" operably linked to a nucleic acid sequence of the present invention is a nucleic acid sequence capable of effecting expression of a nucleic acid molecule. Control sequences need not be contiguous with a nucleic acid sequence, provided that they function to direct its expression. Thus, for example, intervening untranslated but transcribed sequences can be present between the promoter sequence and the nucleic acid sequence, and the promoter sequence would still be considered "operably linked" to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors include, but are not limited to, plasmid and viral-based expression vectors. The promoter may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of several inducible promoters, including but not limited to, tetracycline, ecdysone, steroid-responsive). The expression vector must be replicable in the host organism, either episomally or by integration into host chromosomal DNA. In various embodiments, the expression vector may comprise a plasmid, a viral-based vector (including but not limited to, a retroviral vector or an oncolytic virus), or any other suitable expression vector. In some embodiments, the expression vector may be administered in the methods of the present disclosure to express a polypeptide in vivo for therapeutic benefit. In non-limiting embodiments, the expression vector may be used to transfect or transduce a cell therapy target (including but not limited to, CAR-T cells or tumor cells) to carry out the therapeutic methods disclosed herein.

[0102] In a further aspect, the present disclosure provides a host cell comprising the recombinant expression vector disclosed herein, wherein the host cell can be either prokaryotic or eukaryotic. Cells can be transiently or stably engineered to incorporate the expression vector of the present invention using techniques including, but not limited to, bacterial transformation, calcium phosphate co-precipitation, electroporation, or liposome-, DEAE-dextran-, polycation-, or viral-mediated transfection (see, e.g., Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press); Culture of Animal Cells: A Manual of Basic Technique, 2000; and others). nd Ed. (R.I. Freshney, 1987. Liss, Inc. New York, NY). A method for producing a polypeptide according to the present invention is an additional part of the present invention. The method comprises (a) culturing a host under conditions conducive to expression of the polypeptide according to this aspect of the disclosure, and (b) optionally recovering the expressed fusion protein. Expressed polypeptides can be recovered from cell-free extracts, but preferably they are recovered from the culture medium.

[0103] In a further aspect, the present disclosure provides an antibody that selectively binds to a polypeptide of the present disclosure. The antibody may be a polyclonal antibody, a monoclonal antibody, a humanized antibody, or a fragment thereof, and may be produced using techniques known to those skilled in the art. As used herein, "selectively binds" refers to the preferential binding of an antibody to a polypeptide of the present disclosure as opposed to one or more other biomolecules, structures, cells, tissues, etc., as is well understood by those skilled in the art.

[0104] In another aspect, the present disclosure provides a pharmaceutical composition comprising one or more polypeptides, nucleic acids, expression vectors, and / or host cells of the present disclosure and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present disclosure can be used, for example, in the methods of the present disclosure described below. In addition to the polypeptide of the present disclosure, the pharmaceutical composition can include (a) a lyoprotectant, (b) a surfactant, (c) a bulking agent, (d) a tonicity adjusting agent, (e) a stabilizer, (f) a preservative, and / or (g) a buffering agent.

[0105] In some embodiments, the buffer in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer, or an acetate buffer. The pharmaceutical composition may also contain a lyoprotectant, such as sucrose, sorbitol, or trehalose. In certain embodiments, the pharmaceutical composition contains a preservative, such as benzalkonium chloride, benzethonium, chlorhexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the pharmaceutical composition contains a bulking agent, such as glycine. In yet other embodiments, the pharmaceutical composition includes a surfactant, such as polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleate, or a combination thereof. The pharmaceutical composition may also include a tonicity adjuster, such as a compound that makes the formulation substantially isotonic or isosmotic with human blood. Exemplary tonicity adjusters include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine, and arginine hydrochloride. In other embodiments, the pharmaceutical composition further comprises a stabilizer, e.g., a molecule that, when combined with the protein of interest, substantially prevents or reduces chemical and / or physical instability of the protein of interest in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0106] The polypeptide, nucleic acid, expression vector, and / or host cell may be the only active agent in the pharmaceutical composition, or the composition may further comprise one or more other active agents suitable for the intended use.

[0107] In a further aspect, the present disclosure provides a method for treating and / or limiting cancer, comprising administering to a subject in need thereof a therapeutically effective amount of one or more polypeptides, nucleic acids, expression vectors, and / or host cells, salts thereof, conjugates thereof, or pharmaceutical compositions thereof of the present disclosure to treat and / or limit cancer. Where the method comprises treating cancer, one or more polypeptides, nucleic acids, expression vectors, and / or host cells are administered to a subject already diagnosed with cancer. As used herein, "treat" or "treating" means achieving one or more of the following: (a) reducing the size or volume of a tumor and / or metastasis in a subject; (b) limiting the increase in size or volume of a tumor and / or metastasis in a subject; (c) improving survival; (d) reducing the severity of symptoms associated with cancer; (e) limiting or preventing the occurrence of symptoms associated with cancer; or (f) inhibiting the worsening of symptoms associated with cancer.

[0108] When the method includes limiting the onset of cancer, one or more polypeptides, nucleic acids, expression vectors, and / or host cells are administered prophylactically to a subject who is not known to have cancer but may be at risk of cancer. As used herein, "limiting" means limiting the onset of cancer in a subject at risk of cancer, including, but not limited to, a subject with a family history of cancer, a subject who is genetically predisposed to cancer, a subject who exhibits symptoms of cancer, etc.

[0109] The method can be used to treat or limit the occurrence of any suitable cancer, including, but not limited to, colon cancer, melanoma, renal cell carcinoma, squamous cell carcinoma of the head and neck, gastric cancer, urothelial carcinoma, Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, pancreatic cancer, Merkel cell carcinoma, colorectal cancer, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, multiple myeloma, ovarian cancer, cervical cancer, and any tumor type selected by a diagnostic test such as microsatellite instability, tumor mutational burden, PD-L1 expression level, or Immunoscore assay (developed by the Society for Immunotherapy of Cancer).

[0110] The subject can be any subject having or at risk of developing cancer, hi one embodiment, the subject is a mammal, including but not limited to, a human, dog, cat, horse, or cow.

[0111] In a further aspect, the present disclosure provides a method for modulating an immune response in a subject by administering to the subject a polypeptide, recombinant nucleic acid, expression vector, recombinant host cell, or pharmaceutical composition of the present disclosure.

[0112] As used herein, a regulated "immune response" refers to a response by a cell of the immune system, such as a B cell, a T cell (CD4 or CD8), a regulatory T cell, an antigen-presenting cell, a dendritic cell, a monocyte, a macrophage, a NKT cell, a NK cell, a basophil, an eosinophil, or a neutrophil, to a stimulus. In some embodiments, the response is specific to a particular antigen (an "antigen-specific response") and refers to a response by CD4 T cells, CD8 T cells, or B cells via their antigen-specific receptors. In some embodiments, the immune response is a T cell response, such as a CD4+ response or a CD8+ response. Such responses by these cells may include, for example, cytotoxicity, proliferation, cytokine or chemokine production, trafficking, or phagocytosis, and may depend on the nature of the immune cell receiving the response. In some embodiments of the compositions and methods described herein, the regulated immune response is T cell-mediated.

[0113] In some embodiments, the immune response is an anti-cancer immune response. In some such embodiments, an IL-2 mimetic described herein is administered to a subject with cancer to modulate an anti-cancer immune response in the subject.

[0114] In some embodiments, the immune response is a tissue repair immune response. In some such embodiments, an IL-4 mimetic described herein is administered to a subject in need thereof to modulate a tissue repair immune response in the subject.

[0115] In some embodiments, the immune response is a wound healing immune response. In some such embodiments, an IL-4 mimetic described herein is administered to a subject in need thereof to modulate a wound healing immune response in the subject.

[0116] In some embodiments, a method is provided for regulating an immune response to a second therapeutic agent in a subject. In some such embodiments, the method comprises administering to the subject a polypeptide of the present disclosure in combination with an effective amount of the second therapeutic agent. The second therapeutic agent can be, for example, a chemotherapeutic agent or an antigen-specific immunotherapeutic agent. In some embodiments, the antigen-specific immunotherapeutic agent comprises a chimeric antigen receptor T cell (CAR-T cell). In some embodiments, the polypeptide of the present disclosure enhances the subject's immune response to the therapeutic agent. The immune response can be enhanced, for example, by improving T cell responses (including CAR-T cell responses), enhancing innate T cell immune responses, reducing inflammation, inhibiting regulatory T cell activity, or a combination thereof.

[0117] In some embodiments, the cytokine mimics of the present invention, e.g., the IL-4 mimics described herein, are impregnated or otherwise associated with a biomaterial, and the biomaterial is introduced into a subject. In some embodiments, the biomaterial is a component of an implantable medical device, and the device will be coated with the biomaterial, for example. Such medical devices include, for example, vascular and arterial grafts. IL-4 and / or IL-4-associated biomaterials can be used, for example, to promote wound healing and / or tissue repair and regeneration.

[0118] As used herein, a "therapeutically effective amount" refers to an amount of a polypeptide, nucleic acid, expression vector, and / or host cell effective to treat and / or limit cancer. The polypeptide, nucleic acid, expression vector, and / or host cell are typically formulated as a pharmaceutical composition, such as those disclosed above, in a unit-dose formulation containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles, and can be administered via any suitable route, including but not limited to, orally, by inhalation spray, intravenously, subcutaneously, intraperitoneally, and intravesically. In a specific embodiment, the polypeptide, nucleic acid, expression vector, and / or host cell are administered mucosally, including but not limited to, intraocularly, by inhalation, or intranasally. In another specific embodiment, the polypeptide, nucleic acid, expression vector, and / or host cell are administered orally. Such specific embodiments may be administered via drops, a nebulizer, a spray, or other suitable formulation.

[0119] Any suitable dosage range can be used, as determined by the attending medical professional. Dosage regimens can be adjusted to provide the optimum desired response (e.g., therapeutic or prophylactic response). A suitable dose range can be, for example, 0.1 μg / kg to 100 mg / kg body weight, or it can be 0.5 μg / kg to 50 mg / kg, 1 μg / kg to 25 mg / kg, or 5 μg / kg to 10 mg / kg body weight. In some embodiments, the recommended dose can be less than 0.1 mcg / kg, particularly when administered topically. In other embodiments, the recommended dose is less than 0.1 mcg / kg body weight / m 2 (i.e., body surface area) and / or it can be administered in fixed doses (e.g., 0.5-100 mg). The polypeptide, nucleic acid, expression vector, and / or host cell can be delivered in a single bolus or can be administered two or more times (e.g., 2, 3, 4, 5 or more times) as determined by the attending healthcare professional.

[0120] The engineered polypeptides, nucleic acids, expression vectors, and / or host cells can be administered as the sole prophylactic or therapeutic agent, or can be administered in conjunction with (i.e., combined or separately) one or more other prophylactic or therapeutic agents, including, but not limited to, tumor resection, chemotherapy, radiation therapy, immunotherapy, etc.

[0121] Example of a computing environment Figure 22 is a block diagram of an example computational network. Some or all of the techniques disclosed herein, including but not limited to those disclosed as part of and / or implemented by the software Rosetta software suite, Rosetta Script, PyRosetta, the Rosetta application, and / or other computer software and hardware described herein, may be part of and / or executed by a computing device. For example, Figure 1 illustrates a protein design system 102 configured to communicate with client devices 104a, 104b, and 104c and a protein database 108 over a network 106. In some embodiments, protein design system 102 and / or protein database 108 may be computing devices configured to execute some or all of the methods and techniques described herein, including but not limited to method 300 and functionality described as part of or associated with Rosetta. Protein database 108, in some embodiments, may store information associated with and / or used by Rosetta.

[0122] Network 106 may correspond to a LAN, a wide area network (WAN), a corporate intranet, the public Internet, or any other type of network configured to provide a communication path between networked computing devices. Network 106 may also correspond to a combination of one or more LANs, WANs, corporate intranets, and / or the public Internet.

[0123] While FIG. 22 shows only three client devices 104a, 104b, and 104c, the distributed application architecture may serve tens, hundreds, or thousands of client devices. Furthermore, client devices 104a, 104b, and 104c (or any additional client devices) may be any type of computing device, such as a typical laptop computer, a desktop computer, a network terminal, a wireless communication device (e.g., a cell phone or smartphone), etc. In some embodiments, client devices 104a, 104b, and 104c may be dedicated to use of the problem-solving / Rosetta software suite. In other embodiments, client devices 104a, 104b, and 104c may be used as general-purpose computers configured to perform several tasks and that do not need to be dedicated to use of the problem-solving / Rosetta software suite. In still other embodiments, some or all of the functionality of protein design system 102 and / or protein database 108 may be incorporated into client devices, such as client devices 104a, 104b, and / or 104c.

[0124] Computing Environment Architecture 23A is a block diagram of an exemplary computing device (e.g., system). In particular, computing device 200 shown in FIG. 23A may be configured to include components and / or perform one or more functions of some or all of the methods and techniques described herein, such as method 300 and functionality described as being part of or associated with Rosetta. Computing device 200 may include a user interface module 201, a network communication interface module 202, one or more processors 203, data storage 204, and a protein synthesis device 220, all of which may be coupled together via a system bus, network, or other connection mechanism 205.

[0125] The user interface module 201 may be operable to transmit data to and / or receive data from external user input / output devices. For example, the user interface module 201 may be configured to transmit data to and / or receive data from user input devices such as a keyboard, keypad, touchscreen, computer mouse, trackball, joystick, camera, voice recognition module, and / or other similar devices. The user interface module 201 may also be configured to provide output to a user display device, such as one or more cathode ray tubes (CRTs), liquid crystal displays (LCDs), light emitting diodes (LEDs), displays using digital light processing (DLP) technology, printers, light bulbs, and / or other similar devices now known or later developed. The user interface module 201 may also be configured to generate audible output, such as a speaker, speaker jack, audio output port, audio output device, earphones, and / or other similar devices.

[0126] The network communication interface module 202 may include one or more wireless interfaces 207 and / or one or more wired interfaces 208 configurable to communicate over a network, such as the network 106 shown in Figure 22. The wireless interface 207 may include one or more wireless transmitters, receivers, and / or transceivers, such as a Bluetooth transceiver, a Zigbee transceiver, a Wi-Fi transceiver, a WiMAX transceiver, and / or other similar types of wireless transceivers configurable to communicate over a wireless network. The wired interface 208 may include one or more wired transmitters, receivers, and / or transceivers, such as an Ethernet transceiver, a Universal Serial Bus (USB) transceiver, or similar transceiver configurable to communicate over twisted pair, one or more wires, coaxial cable, optical fiber link, or similar physical link to a wired network.

[0127] In some embodiments, the network communication interface module 202 can be configured to provide reliable, secure, and / or authenticated communications. For each communication described herein, information to ensure reliable communications (i.e., guaranteed message delivery) can be provided, possibly as part of the message header and / or footer (e.g., packet / message sequencing information, encapsulation header(s) and / or footer(s), size / time information, and transmission verification information such as CRC and / or parity check values). Communications can be secured (e.g., coded or encrypted) and / or decrypted / decrypted using one or more cryptographic protocols and / or algorithms, such as, but not limited to, DES, AES, RSA, Diffie-Hellman, and / or DSA. Similarly, other cryptographic protocols and / or algorithms can be used, or can be used to secure (and then decrypt / decrypt) communications in addition to those enumerated herein.

[0128] The processor 203 may include one or more general-purpose processors and / or one or more special-purpose processors (e.g., digital signal processors, application-specific integrated circuits, etc.). The processor 203 may be configured to execute computer-readable program instructions 206 contained in data storage 204 and / or other instructions described herein. The data storage 204 may include one or more computer-readable storage media that may be read and / or accessed by at least one of the processors 203. The one or more computer-readable storage media may be integrated, in whole or in part, with at least one processor 203 and may include volatile and / or non-volatile storage components, such as optical, magnetic, organic, or other memory or disk storage. In some embodiments, the data storage 204 may be implemented using a single physical device (e.g., one optical, magnetic, organic, or other memory or disk storage unit), while in other embodiments, the data storage 204 may be implemented using two or more physical devices.

[0129] Data storage 204 can include computer-readable program instructions 206 and possibly additional data. For example, in some embodiments, data storage 204 can store some or all of the data utilized by the protein design system and / or protein database (e.g., protein design system 102, protein database 108). In some embodiments, data storage 204 can further include storage necessary to execute at least some of the methods and techniques described herein and / or at least some of the functionality of the devices and networks described herein.

[0130] In some embodiments, computing device 200 includes protein synthesis device 220. Protein synthesis device can use commands and / or data provided by processor 203 and / or data storage 204 to synthesize (or generate) polypeptides based on input data provided to protein synthesis device 220. For example, some or all of the functionality of protein synthesis device 220 can be performed by a semi-automated or automated peptide synthesizer.

[0131] 23B illustrates a network 106 of computing clusters 209a, 209b, 209c arranged as a cloud-based server system, according to an exemplary embodiment. The data and / or software for protein design system 102 can be stored on one or more cloud-based devices that store program logic and / or data for cloud-based applications and / or services. In some examples, protein design system 102 can be a single computing device resident in a single computing center. In other examples, protein design system 102 can include multiple computing devices within a single computing center, or even multiple computing devices located in multiple computing centers in different geographic locations.

[0132] In some examples, the data and / or software of protein design system 102 may be encoded as computer-readable information stored on a tangible computer-readable medium (or computer-readable storage medium) and accessible by client devices 104a, 104b, and 104c and / or other computing devices. In some examples, the data and / or software of protein design system 102 may be stored on a single disk drive or other tangible storage medium, or may be implemented on multiple disk drives or other tangible storage media located in one or more different geographic locations.

[0133] Figure 23B shows a cloud-based server system according to an exemplary embodiment. In Figure 23B, the functionality of protein design system 102 may be distributed among three computing clusters 209a, 209b, and 209c. Computing cluster 209a may include one or more computing devices 200a, cluster storage array 210a, and cluster router 211a coupled by a local cluster network 212a. Similarly, computing cluster 209b may include one or more computing devices 200b, cluster storage array 210b, and cluster router 211b coupled by a local cluster network 212b. Similarly, computing cluster 209c may include one or more computing devices 200c, cluster storage array 210c, and cluster router 211c coupled by a local cluster network 212c.

[0134] In some embodiments, each of the computing clusters 209a, 209b, and 209c may have the same number of computing devices, the same number of cluster storage arrays, and the same number of cluster routers. However, in other embodiments, each computing cluster may have a different number of computing devices, a different number of cluster storage arrays, and a different number of cluster routers. The number of computing devices, cluster storage arrays, and cluster routers in each computing cluster varies depending on the computing task(s) assigned to each computing cluster.

[0135] In computational cluster 209a, for example, computing device 200a can be configured to perform various computational tasks for protein design system 102. In one embodiment, various functionality of protein design system 102 can be distributed among one or more computing devices 200a, 200b, and 200c. Computing devices 200b and 200c in computational clusters 209b and 209c can be configured similarly to computing device 200a in computational cluster 209a. On the other hand, in some embodiments, computing devices 200a, 200b, and 200c can be configured to perform different functions.

[0136] In some embodiments, computational tasks and stored data associated with protein design system 102 may be distributed among computing devices 200a, 200b, and 200c based at least in part on the processing requirements of protein design system 102, the processing power of computing devices 200a, 200b, and 200c, the latency of network connections between computing devices within each computing cluster and between the computing clusters themselves, and / or other factors that may contribute to cost, speed, fault tolerance, resilience, efficiency, and / or other design goals of the overall system architecture.

[0137] Cluster storage arrays 210a, 210b, and 210c of computing clusters 209a, 209b, and 209c may be data storage arrays and include disk array controllers configured to manage read and write access to groups of hard disk drives. The disk array controllers, alone or in combination with their respective computing devices, may also be configured to manage backup or redundant copies of data stored in the cluster storage arrays to protect against disk drive or other cluster storage array failures and / or network failures, preventing one or more computing devices from accessing one or more cluster storage arrays.

[0138] Similar to the manner in which the functionality of protein design system 102 may be distributed among computing devices 200a, 200b, and 200c of computing clusters 209a, 209b, and 209c, various active and / or backup portions of these components may be distributed among cluster storage arrays 210a, 210b, and 210c. For example, some cluster storage arrays may be configured to store portions of the data and / or software of protein design system 102, while other cluster storage arrays may store separate portions of the data and / or software of protein design system 102. Additionally, some cluster storage arrays may be configured to store backup versions of data stored in other cluster storage arrays.

[0139] Cluster routers 211a, 211b, and 211c in computing clusters 209a, 209b, and 209c may include network equipment configured to provide internal and external communications for the computing clusters. For example, cluster router 211a of computing cluster 209a may include one or more Internet switching and routing devices configured to provide (i) local area network communications between computing device 200a and cluster storage array 201a via local cluster network 212a, and (ii) wide area network communications between computing cluster 209a and computing clusters 209b and 209c via wide area network connection 213a to network 106. Cluster routers 211b and 211c may include network equipment similar to cluster router 211a, and cluster routers 211b and 211c may perform similar network functions with respect to computing clusters 209b and 209b as cluster router 211a performs with respect to computing cluster 209a.

[0140] In some embodiments, the configuration of cluster routers 211a, 211b, and 211c may be based at least in part on the data communication requirements of the computing devices and cluster storage arrays, the data communication capabilities of the network equipment of cluster routers 211a, 211b, and 211c, the latency and throughput of local networks 212a, 212b, 212c, the latency, throughput, and cost of wide area network connections 213a, 213b, and 213c, and / or other factors that may contribute to cost, speed, fault tolerance, resilience, efficiency, and / or other design goals of the moderation system architecture.

[0141] Example of operation method 24 is a flowchart of an example method 300. Method 300 may be performed by a computing device such as computing device 200 described in the context of at least FIG. 2A. At least examples of method 300 described below are discussed above.

[0142] Method 300 can begin at block 310, where a computational device can determine a structure of a plurality of residues of a protein using the computational device, where the structure of the plurality of residues provides a particular receptor binding interface. As will be appreciated by a skilled practitioner, determining a structure of a plurality of residues of a protein, where the structure of the plurality of residues provides a particular receptor binding interface, is typically the identification of original residues of a native protein that bind to a particular receptor binding interface, and the plurality of designed residues are identified residues that can bind to the same receptor binding interface.

[0143] In block 320, the computational device can determine a plurality of designed residues using the mimetic design protocol, the plurality of designed residues providing a particular receptor binding interface, and the plurality of designed residues differing from the plurality of residues.

[0144] In some examples, determining a plurality of designed residues using a mimetic design protocol can include determining an idealized residue using a database of idealized residues, where the idealized residue is associated with a designed residue of the plurality of designed residues. In some of these examples, determining an idealized residue using the database of idealized residues can include searching the database of idealized residues for one or more idealized fragments associated with the idealized residue, and determining the idealized residue by reconstructing the associated designed residue using the one or more idealized fragments. In some of these examples, reconstructing the associated designed residue using the one or more idealized fragments can include reconnecting the one or more pairs of idealized fragments by using combinatorial fragment assembly of the one or more pairs of idealized fragments, and determining whether the one or more pairs of idealized fragments connect two or more of the plurality of designed residues using Cartesian constrained backbone minimization. In some of these examples, reconstructing associated designed residues using one or more idealized fragments may include using a database of idealized residues to verify that overlapping fragments of idealized residues are idealized fragments, verifying whether the idealized residues do not clash with a target receptor associated with a particular receptor binding interface, and after verifying that the idealized residues do not clash with a target receptor associated with a particular receptor binding interface, using the database of idealized residues to determine the most likely amino acid at each position of the idealized residues. In some of these examples, determining a first protein backbone of the protein by assembling one or more connected helix structures and a plurality of designed residues across a plurality of combinations may include combinatorially recombining one or more pairs of idealized fragments, recombining one or more pairs of idealized fragments, and determining a first protein backbone of the protein using the recombined pair of idealized fragments.In some of these embodiments, combining and recombining one or more pairs of idealized fragments may include ranking the one or more pairs of idealized fragments based on interconnection lengths between the idealized fragments of the one or more pairs of idealized fragments.

[0145] In other examples, determining a plurality of designed residues using a mimetic design protocol may include determining idealized residues using one or more parametric equations that represent the shapes of the designed residues of the plurality of designed residues, and determining a single fragment that closes the idealized residue with at least one designed residue of the plurality of designed residues. In some of these examples, the designed residues may include a helical structure, and the one or more parametric equations may include equations related to the phi (φ) and psi (ψ) angles of the helical structure. In some of these examples, the equations related to the φ and ψ angles of the helical structure may include one or more terms related to the angular pitch of the φ and ψ angles of the helical structure.

[0146] In block 330, the computational device can determine one or more connecting helix structures connecting the plurality of designed residues.

[0147] In block 340, the computational device may determine a first protein backbone for the protein by assembling one or more connected helical structures and a plurality of designed residues across a plurality of combinations.

[0148] In block 350, the computational device may design a second protein backbone of the protein based on the first protein backbone for flexibility and low-energy structure.

[0149] At block 360, the computing device may generate output related to at least the second protein backbone of the protein. In some examples, generating output related to the second protein backbone of the protein may include designing one or more molecules based on the second protein backbone of the protein.

[0150] In other examples, generating an output related to the second protein backbone of the protein may include generating a synthetic gene for the protein based on the second protein backbone of the protein, expressing the specific protein in vivo using the synthetic gene, and purifying the specific protein. In some of these examples, expressing the specific protein sequence in vivo using the synthetic gene may include expressing the specific protein sequence in one or more E. coli bacteria containing the synthetic gene.

[0151] In other examples, generating output related to the second protein backbone of the protein may include generating one or more images including at least a portion of the second protein backbone of the protein.

[0152] In other examples, the computing device may include a protein synthesis device. Then, generating an output related to at least a second protein backbone of the protein may include synthesizing the at least a second protein backbone of the protein using the protein synthesis device.

[0153] In one embodiment, the method is for designing a protein mimetic, as exemplified herein.

[0154] Also included are non-naturally occurring proteins prepared by the computational methods described herein. The non-naturally occurring protein can be a cytokine, for example, a non-naturally occurring IL-2 or IL-4 mimetic.

[0155] The items set forth herein are exemplary and are presented solely for the purpose of exemplary discussion of embodiments of the present invention, to provide what is believed to be the most useful and understandable explanation of the principles and conceptual aspects of various embodiments of the present invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, but rather the detailed description is presented by way of figures and / or examples that will make clear to those skilled in the art how some forms of the invention may be embodied in practice.

[0156] The above definitions and explanations are intended and intended to control in any future constructions unless clearly and unambiguously changed in the examples below, or unless the construction becomes meaningless or essentially meaningless upon application of the meaning. If the construction of a term becomes meaningless or essentially meaningless, the definition shall remain in accordance with Webster's Dictionary, 3 rd The term "biological information" should be obtained from dictionaries known to those skilled in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004), or the Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

[0157] The above description provides specific details for a thorough understanding and enabling of embodiments of the present disclosure. However, those skilled in the art will understand that the disclosure may be practiced without these details. In other instances, well-known structures and functions have not been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments of the present disclosure. The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments and examples of the present disclosure have been described herein for illustrative purposes, those skilled in the art will recognize that various equivalent modifications are possible within the scope of the present disclosure.

[0158] All references cited herein are incorporated by reference. Aspects of the disclosure can be modified, if necessary, to employ the systems, functions, and concepts of the above references and applications to provide still further embodiments of the disclosure. These and other modifications can be made to the disclosure in light of the detailed description.

[0159] Specific elements of any of the foregoing embodiments can be combined with or substituted for elements of other embodiments. Additionally, although advantages associated with certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily need to exhibit such advantages to fall within the scope of the present disclosure.

[0160] The above detailed description, with reference to the accompanying drawings, describes various features and functions of the disclosed systems, devices, and methods. In the drawings, like symbols generally identify like components unless otherwise clear from context. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the drawings, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0161] With respect to any or all of the ladder diagrams, scenarios, and flowcharts in the figures as described herein, each block and / or communication may represent the processing of information and / or the transmission of information according to an exemplary embodiment. Alternative embodiments are included within the scope of these exemplary embodiments. In these alternative embodiments, for example, functions described as blocks, transmissions, communications, requests, responses, and / or messages may be executed in an order different from that shown or discussed, including substantially concurrently or in reverse order, depending on the functionality involved. Furthermore, more or fewer blocks and / or functions may be used with any of the ladder diagrams, scenarios, and flowcharts described herein, and these ladder diagrams, scenarios, and flowcharts may be combined with each other, in part or in whole.

[0162] The blocks representing processing of information may correspond to circuitry that can be configured to perform specific logical functions of the methods or techniques described herein. Alternatively or additionally, the blocks representing processing of information may correspond to modules, segments, or portions of program code (including associated data). The program code may include one or more instructions executable by a processor to implement specific logical functions or operations in the method or technique. The program code and / or associated data may be stored in any type of computer-readable medium, such as a storage device, including a disk drive or hard drive, or other storage medium.

[0163] Computer-readable media may also include non-transitory computer-readable media, such as computer-readable media that store short-term data, such as register memory, processor cache, and / or random access memory (RAM). Computer-readable media may also include non-transitory computer-readable media that store program code and / or data for longer periods, such as secondary or permanent long-term storage, such as read-only memory (ROM), optical or magnetic disks, and / or compact disc read-only memory (CD-ROM). Computer-readable media may also be any other volatile or non-volatile storage system. Computer-readable media may be considered, for example, as computer-readable storage media or tangible storage devices. Furthermore, blocks representing one or more information transmissions may correspond to information transmissions between software and / or hardware modules in the same physical device. However, other information transmissions may occur between software and / or hardware modules in different physical devices.

[0164] Many modifications and variations of the present disclosure are possible in light of the above teachings. [Example]

[0165] We describe a computational approach to design de novo cytokine mimetics that repeat functional sites of natural cytokines but are otherwise unrelated in topology or amino acid sequence. This strategy is applicable to IL-2 and interleukin-15 (IL-15). 15 was used to design a de novo mimetic of the IL-2 receptor βγ c Heterodimer (IL-2Rβγ c ) 16,17 The design is ultrastable and binds to human and mouse IL-2Rβγ with higher affinity than the native cytokine. cIt binds to mouse IL-2Rβγ and induces downstream cell signaling independently of IL-2Rα and IL-15Rα. The crystal structure of the experimentally optimized mimetic, Neoleukin-2 / 15, closely matches the design model and is related to the murine IL-2Rβγ. c We provide the first structural information on the complex. Neoleukin-2 / 15 has highly effective therapeutic activity compared with IL-2 in mouse models of melanoma and colon cancer, with reduced toxicity and no signs of immunogenicity. This strategy for constructing ultrastable de novo mimetics can be readily applied to numerous natural cytokines and other signaling proteins, enabling the generation of superior therapeutic candidates with enhanced clinical profiles.

[0166] Due to the potent biological activity of natural protein hormones and cytokines, extensive efforts have been made to improve their potential therapeutic efficacy through protein engineering. Such efforts have sought to simplify manufacturing, extend half-life, and modulate receptor interactions. 18-20 However, there are inherent challenges in developing new therapeutic agents when starting from naturally occurring, bioactive proteins. First, most natural proteins are only marginally stable. 21-25 However, amino acid substitutions aimed at increasing efficacy can reduce expression or cause aggregation, making manufacturing and storage difficult. More drastic modifications, such as deletion or fusion of functional or targeting domains, are often infeasible and can dramatically alter pharmacokinetic properties and tissue penetration. 19 Second, the immune response to the engineered variants may be directed against endogenous molecules. 26-35 Computational design approaches have been developed to generate analogs of natural proteins with improved therapeutic properties that circumvent these challenges, focusing on engineering de novo cytokine mimetics that exhibit specific subsets of receptor binding interfaces optimal for treating disease.

[0167] Many cytokines bind to multiple different receptor subunits.15 , 16, 36-39 It interacts with the receptor subunit and, like most naturally occurring proteins, contains non-ideal structural features that compromise stability but are important for function. A computational protocol was developed in which structural elements that interact with the desired receptor subunit are spatially fixed and an idealized globular protein structure is constructed to support these elements. We extended previous efforts by using combinatorial fragment assembly to support short linear epitopes with the parametric construction of disembodied helices combined with knowledge-based loop closure (Figure 1a-b). This approach was applied to the human IL-2Rβγ subunit. c (hIL-2Rβγ c The potential for mutations was tested by de novo designing stable idealized proteins with interaction surfaces that mimic the interaction surfaces of human IL-2 (hIL-2) and human IL-15 (hIL-15) for IL-2 receptor alpha (IL-2Rα), but completely lacking the interaction surface of IL-2 receptor alpha (IL-2Rα). 9,44,45 (e.g., F42A mutation in Super-2, H9 9 also known as ) or PEGylated (e.g., NKTR-214 9,13 Previous efforts to remove the α-interacting region of hIL-2 by either β- or γ-interacting with hIL-2R have resulted in the formation of hIL-2Rβγ. c significantly reduced the stability, binding and / or potency of the cytokine to the receptor, while failing to completely eliminate the α interaction.

[0168] IL-2Rβγ cComputational design of an IL-2 / IL-15 mimetic that binds and activates IL-2 receptor: Native hIL-2 is composed of four helices connected by a long irregular loop. The N-terminal helix (H1) interacts with both the β and γ subunits of the IL-2 receptor, the third helix (H3) interacts with the β subunit, and the C-terminal helix (H4) interacts with the γ subunit. The interaction surface of the α subunit is formed by the irregular second helix (H2) and two long loops, one connecting H1 to H2 and the other connecting H3 and H4. An ideal protein was designed that reproduced the interface formed by H1, H3, and H4 with β and γ subunits and replaced H2 with a regular helix that provided better packing. Helices H1, H3, and H4 (see Figure 1a) were used as templates for the binding site, while helix H2 (H2') was reconstructed using a database of highly represented cluster fragments (see Methods). Pairs of helices are linked with loops extracted from the same database (see Figure 1b), and the resulting helix hairpins are joined to the fully linked scaffold (see Figure 1c), which is then run using Rosetta. 46-48 Combinatorial flexible scaffold design calculations were performed on hIL-2Rβγ c The top four computational designs, and eight single-disulfide stapled variations (see Table S1), were selected for experimental characterization by yeast display (see Methods). Eight designs were found to bind to a fluorescently tagged β-γ chimeric IL-2 receptor at low nanomolar concentrations. The best non-disulfide design (G1_neo2_40) was subjected to site-saturation mutagenesis followed by murine IL-2Rβγ. c (mIL-2Rβγ cSubstitutions that increased affinity for the STAT5 protein (see Figure 10) were selected and combined. The optimized design (recombinantly expressed in E. coli) was found to induce pSTAT5 signaling in vitro in IL-2-responsive mouse cells at low nanomolar or even picomolar concentrations (see Table E1), but exhibited relatively low thermal stability (Tm below approximately 45°C, see Figures 14 and 15). To improve stability, we repeated the computational design protocol, starting with the scaffold of the highest-affinity first-round design (G1_neo2_40_1F, topology: H1→H4→H2'→H3) and combining a loop-building process with parametric variation of helix length (+ / - 8 amino acids, see the bottom panel of Figure 1a). This second approach improved the quality of the model by allowing the exploration of substantially more combinations of loops connecting each pair of helices. The 14 best second-generation designs, along with 27 Rosetta sequence redesigns of G1_neo2_40_1F (see Table S3), were experimentally characterized, and all but one was found to bind the IL-2 receptor at low nanomolar concentrations (Figure 1d, Table E1, and Figure S16). The three highest affinity and stability designs (one sequence redesign and two new mimetics) were selected as mIL-2Rβγ. c The binding of human and mouse IL-2Rβγ was subjected to site-saturation mutagenesis (Figures 11-13). c Substitutions that increased affinity for both IL-2 and mIL-2 were selected and combined. Mature designs (see Table S4) showed enhanced binding while maintaining hyperstability (see Table E1). The top design, Neoleukin-2 / 15 (also referred to herein as Neo-2 / 15), is a 100-residue protein with a novel topology and sequence that is completely distinct from human or mouse IL-2 (29% sequence identity over 89 residues with hIL-2 and 16% sequence identity over 76 residues aligned with mIL-2 in a structure-topology-independent alignment, see Table E1).

[0169] Functional characterization of Neoleukin-2 / 15: Neoleukin-2 / 15 binds to human and mouse IL-2Rβγ with high affinity. c(Kd of approximately 38 nM and approximately 19 nM, respectively), but does not interact with IL-2Rα (Fig. 2a). c The affinity of Neoleukin-2 / 15 for hIL-2Rβγ is significantly higher than that of the corresponding native IL-2 cytokine. In contrast to native IL-2, Neoleukin-2 / 15 induces IL-2Rα-independent signaling in both human and mouse IL-2-responsive cells (Figure 2b, top) and in primary mouse T cells (Figure 2b, bottom). Due to its high binding affinity, Neoleukin-2 / 15 activates IL-2Rα cells more potently than native human or mouse IL-2. In primary cells, Neoleukin-2 / 15 is more active against IL-2Rα- cells and less active against IL-2Rα+ cells compared to Super-2, likely due to its complete lack of IL-2Rα binding. Neoleukin-2 / 15 is ultrastable (see Figure 17), retaining hIL-2Rβγ even after 2 hours of incubation at 80°C. c While hIL-2 and Super-2 do not lose their binding affinity to IL-2, they are completely inactivated within 10 minutes (inactivation half-lives of approximately 4.2 and 2.6 minutes, respectively, Figure 2c). Similarly, in ex vivo primary cell cultures, Neoleukin-2 / 15 effectively promoted T cell survival after boiling at 95°C for 60 minutes, although these conditions inactivated both IL-2 and Super-2 (Figure 2c, bottom). Thermal denaturation studies have been performed on many other engineered mimetics, demonstrating their thermal stability as well (see Figures 14-16). This unprecedented stability of cytokine-like molecules not only eliminates the need for cold-chain storage, but also suggests robustness against mutations (see Figures 13 and 18-19), gene fusions, and chemical modifications, far exceeding those of native IL-2, potentially contributing to the development of improved or novel therapeutic properties (see Figure 7).

[0170] Monomeric Neoleukin-2 / 15 and mIL-2Rβγ cThe X-ray crystal structure of Neoleukin-2 / 15 was determined and found to be very similar to the computationally designed model (rmsd C α = 1.1–1.3 Å, 6 copies of the asymmetric unit (Fig. 3a). Mouse IL-2Rβγ c The crystal structure of Neoleukin-2 / 15 in a ternary complex with the mouse receptor (Figure 3B, Table E2) has been elucidated. This may be the first example of a de novo designed protein enabling the structure determination of a previously unsolved native receptor complex. The crystal structure alignment of the Neoleukin-2 / 15 designed model and the mouse ternary complex structure, rmsd C The α and γ are 1.27 and 1.29 Å, respectively (Fig. 3c). The helix order of Neoleukin-2 / 15 (IL-2 numbering) is H1 → H3 → H2' → H4 (see Fig. 1a and Fig. 3a, d). The H1-H3 loop is disordered in the ternary complex, but helix H3 is in close agreement with the predicted structure. Compared to the monomeric structure, there is also outward movement of helix H4 and the H2'-H4 loop (Fig. 3c). Neoleukin-2 / 15 interacts with mIL-2Rβ through helices H1 and H3 and with γ through helices H1 and H4. c These regions align closely with both the computational model (Figure 3a) and the monomer crystal structure (Figure 3c). 49 Structural alignment to the crystal structure of Neoleukin-2 / 15 reveals a close correspondence between the helical backbone of Neoleukin-2 / 15 and hIL-2 in the binding site, despite the different topologies of the two proteins (Fig. 3d-e). c Although there are several side chain interactions between L and Y, others, e.g., L19Y, arose during the computational design process.

[0171] Therapeutic use of Neoleukin-2 / 15: IL-2 has had limited clinical use primarily due to toxicity 50-52The interactions responsible for IL-2 toxicity in humans are not fully understood, but in mouse models, toxicity is T cell independent and is attenuated in animals lacking the IL-2Rα chain (CD25+). Thus, much effort has been directed at re-engineering IL-2 to weaken its interaction with IL-2Rα, but mutations in the CD25 binding site can be highly destabilizing. 6 The inherent low stability of IL-2 and its tightly evolved dependency on CD25 present barriers to the translation of re-engineered IL-2 compounds. 53,54 The focus has been on IL-15 because it does not have affinity for CD25 but binds to IL-2Rβγ. c However, IL-15 relies on trans-presentation by the IL-15α (CD215) receptor, which is primarily present on antigen-presenting cells and natural killer cells. The low stability of native IL-15 and its dependence on trans-presentation also pose substantial barriers to reengineering efforts. 53-55 .

[0172] In a dose-escalation study in naive mice, mIL-2 inhibited CD25+ cells, consistent with its preferential binding to CD25+ cells. 41,56,57 , preferentially expand regulatory T cells, whereas Neoleukin-2 / 15 predominantly expands CD8 + Although Neoleukin-2 / 15 stimulates T cell proliferation (Figure 4a), even the highest dose tested does not or only minimally induces regulatory T cell proliferation. Similarly, in a mouse model of airway inflammation, Neoleukin-2 / 15 induces a small proportion of tissue-resident CD8+ T cells, but not CD4+ T cells in lymphoid organs. + Foxp3 + Thy1.2 without increasing antigen-specific Tregs - CD44 + CD8 + resulting in an increase in T cells (Fig. 4b).

[0173] De novo protein design avoids the structural limitations of natural cytokines, but can potentially induce anti-drug antibodies. To test whether Neoleukin-2 / 15 induces an anti-drug response, tumor-bearing mice were treated with Neoleukin-2 / 15 daily for 2 weeks, and no evidence of anti-drug antibodies was observed in any of the treated animals (Figure 4c, left panel). A similar lack of immune response was observed for other de novo designed therapeutic candidates. 41 Polyclonal antibodies against Neoleukin-2 / 15 were generated by vaccinating mice with an inactive Neoleukin-2 / 15 mutant (KONeoleukin) in complete Freund's adjuvant. These polyclonal anti-Neoleukin-2 / 15 antibodies did not cross-react with human or mouse IL-2 (Figure 4c). The lack of binding to native IL-2 suggests that even if there is an immune response to Neoleukin-2 / 15, this response is unlikely to cross-react with endogenous IL-2. Furthermore, due to the low sequence identity between Neoleukin-2 / 15 and hIL-2 (<30%, see Table E1), an autoimmune response to host IL-2 is much more likely with previously engineered hIL-2 variants (e.g., Super-2, see Table E1), which differ from endogenous IL-2 by only a few mutations.

[0174] The therapeutic efficacy of Neoleukin-2 / 15 was tested in mouse models of the less immunogenic B16F10 melanoma and the more immunogenic CT26 colon cancer. Single-agent treatment with Neoleukin-2 / 15 resulted in a dose-dependent delay in tumor growth in both cancer models. In CT26 colon cancer, single-agent treatment demonstrated improved efficacy over that observed with recombinant mIL-2 (Figures 4d and 5). In B16F10 melanoma, combined treatment with the anti-melanoma antibody TA99 (anti-TRP1) significantly delayed tumor growth, whereas TA99 treatment alone had little effect (Figures 4e and 6). In long-term survival experiments (8 weeks), Neoleukin-2 / 15 in combination with TA99 demonstrated significantly reduced toxicity and an overall superior therapeutic effect compared to mIL-2 (Figure 4e). Mice treated with the combination of mIL-2 and TA99 steadily lost weight and overall health declined to the point of euthanasia, whereas little decline was observed with the combination of Neoleukin-2 / 15 and TA99 (Figure 4e). Consistent with a therapeutic benefit, Neoleukin-2 / 15 treatment significantly reduced intratumoral CD8:T cell proliferation. reg This resulted in a significant increase in the ratio of β-glucan to β-glucan (Figure 4f and Figure 5), which has previously been correlated with an effective anti-tumor immune response. 58 CD8:T by Neoleukin-2 / 15 reg The increase in the ratio was dose and antigen dependent (Figure 4f). Optimal therapeutic effects were obtained at high doses and in combination with other immunotherapies (see Figure 6). Collectively, these data suggest that Neoleukin-2 / 15 exerts the expected homeostatic benefits derived from IL-2, such as immune-enhancing activity, but not CD25. + These enhanced properties and low toxicity may allow for the routine use of Neoleukin-2 / 15 for other immunotherapies where recombinant IL-2 is not widely used. As an example of such use, we investigated the potential application of Neoleukin-2 / 15 to enhance CAR-T cell therapy (see Figure 8). 6 NSG mice inoculated with 0.8 × 10 RAJI tumor cells were either left untreated or administered 0.8 × 106 Mice were treated with either anti-CD19 CAR-T cells (infused 7 days after tumor cell inoculation) or similarly treated with anti-CD19 CAR-T cells in addition to either 20 μg / day of human IL-2 or Neoleukin-2 / 15 on days 8–14 after tumor inoculation. As expected, Neoleukin-2 / 15 significantly enhanced the antitumor effect of CAR-T cell therapy in this model, slowing tumor growth and prolonging mouse survival (data not shown).

[0175] De novo design of protein mimetics has the potential to transform the field of protein-based therapeutics, enabling the development of biosuperior molecules with enhanced therapeutic properties and reduced side effects, not only for cytokines but for virtually any biologically active molecule with a known or precisely predictable structure. Due to the incremental nature of current conventional engineering approaches (e.g., one- to three-amino acid substitutions, chemical modifications at single sites), most of the shortcomings of the parent molecule are inevitably inherited, often in an exacerbated form, in the resulting engineered variant. By constructing mimetics de novo, these shortcomings can be completely avoided. Unlike engineered variants of recombinant IL-2 and hIL-2, Neoleukin-2 / 15 can be solubly expressed in E. coli (see Figure 17), retains activity at high temperatures, does not interact with IL-2Rα, and is robust to substantial sequence changes, allowing for the engineering of new functions (Figure 7). Due to their small size and high stability, de novo designed proteins are likely to be less immunogenic, and in contrast to incremental variants of hIL-2, any antibody response to the mimetics is unlikely to cross-react with the native parent cytokine. Due to their high stability and robustness, as well as their tailored interaction surfaces, designed mimetics are likely to be particularly potent in next-generation therapeutics that combine the functionalities of different proteins, e.g., targeted versions of Neoleukin-2 / 15.

[0176] Robust modularity of Neoleukin-2 / 15. Disulfide stapling and re-engineering into an IL-4 mimetic: Neoleukin-2 / 15 is highly modular, allowing its properties to be easily tuned, such as increasing its stability or altering its binding preference. This modularity and robustness were demonstrated through computational design of Neoleukin-2 / 15. 59 We took advantage of the introduction of a single disulfide staple, which enhances stability while preserving the function of Neoleukin-2 / 15. To this end, two orthogonal strategies were used. First, disulfide bridges were introduced by searching for pairs of positions with favorable geometric configurations, followed by minimizing backbone flexibility. In the final design, a single disulfide was introduced between residues 38 and 75 to stabilize helices H3 and H2. In the second approach, the N- and C-termini of Neoleukin-2 / 15 were remodeled to allow the introduction of a single disulfide staple encompassing the entire protein (after removal of the terminal P and S residues, the sequences CNSN (SEQ ID NO: 260) and NFQC (SEQ ID NO: 261) were added to the N- and C-termini, respectively; see Figure 18). Both disulfide stapling strategies enhanced the stability of Neoleukin-2 / 15 (melting temperature Tm > 95°C) while leaving its sequence and function largely unaffected (see Figure 18). The modular nature of Neoleukin-2 / 15 was used to alter its binding preference. All cytokines in the interleukin-2 family are γ- c They interact with IL-2Rβ and share a common architecture. Therefore, it was hypothesized that Neoleukin-2 / 15 could be converted into another cytokine mimic of the IL-2 family by changing only half of the amino acids in the binding site that interacts with IL-2Rβ (helices H1 and H3). As a proof of concept, human interleukin-4 (hIL-4) was chosen as the target because it shares extensive structural homology with IL-2 and has potential applications in regenerative medicine. 60,61By aligning the Neo-2 / 15 model to the structure of human IL-4 bound to its IL-4 receptor, and by mutating 14 residues of Neo-2 / 15 to match the amino acids of IL-4 at those structural positions that mediate the interaction between IL-4 and IL-4r, we found that Neo-2 / 15 binds to the human IL-4 receptor (IL-4Rα and γ). c It does not bind to the human IL-2 receptor (IL-2Rβ and γ c The resulting protein, Neoleukin-4, was engineered to contain a total of 16 mutations (Figure 7). Binding was further optimized by directed evolution using random mutagenesis and screening for high-binding affinity variants. Neoleukin-2 / 15 was derived from Neoleukin-2 / 15 by introducing two additional amino acid substitutions and modifying one of the 14 original residues transplanted from the IL-4 protein. The resulting optimized design, Neoleukin-4 (see Table S5), was recombinantly expressed and purified from E. coli and tested for binding. Neoleukin-4 binds with high affinity to the IL-4Rα receptor and IL-4Rαγ. c Neoleukin-4 binds cooperatively to the IL-13 receptor (see Figure 7), but does not bind to or has no affinity for the IL-2 receptor (data not shown). Neoleukin-4 retains the superior thermostability of Neoleukin-2 / 15 (see Figures 20b, c) and, as expected, binds to the IL-13 receptor (data not shown), given the natural cross-reactivity of IL-4 to the IL-13 receptor. Overall, this indicates that Neoleukin-2 / 15 is robust enough to function as a modular scaffold, and that substantial rational sequence changes can be introduced to alter its function or physical properties in highly predictable ways.

[0177] method Computational design of de novo cytokine mimetics: IL-2Rβγ as a template for design. cWe began by defining the structure of hIL-2 in quaternary complex with its receptor. After inspection, residues comprising the binding site were defined as hotspots using Rosetta metadata (PDBInfoLabels). The structure was fed into a novel mimetic design protocol programmed in PyRosetta to automatically detect core-secondary structure elements comprising the target template and generate a resulting de novo mimetic scaffold containing complete RosettaScripts-compatible information for design. Briefly, the mimetic construction algorithm works as follows: In the first generation of designs, each core element was idealized by reconstructing it using loops from a clustered database of highly idealized fragments (fragment size 4 amino acids). After idealization, the mimetic construction protocol aims to reconnect the idealized elements through all possible pairwise combinations. This is done using combinatorial fragment assembly of sequence-independent fragments from the database, followed by Cartesian constrained scaffold minimization of potential solutions (i.e., when the N- and C-termini of the constructed fragments are close enough to connect two secondary structures). After minimization, solutions were verified to contain highly ideal fragments (i.e., all overlapping fragments that make up two connected elements are also included in the database) and to ensure that the backbone does not clash with the target (context) receptor. Next, the passing backbone solutions were characterized using the same database of fragments to determine the most likely amino acid at each position (this information was coded into the design metadata). The connected secondary structure pair solutions were then combined and recombined to produce a fully connected backbone by using the connected building blocks of graph theory. Because the number of solutions grows exponentially with each element pair, at each step of fragment combination, designs were ranked to prioritize those with shorter interconnections between pairs of core elements, and only the top solutions were retained to proceed to the next step. The fully connected solutions were then characterized by layer (interface, core, non-core surface, surface) to restrict the possible amino acid identities to those compatible with the layer.Finally, all information regarding hotspots, amino acids, and layers of compatible building fragments was combined (hotspots take precedence over amino acid probability, which in turn takes precedence over layers). These fully characterized scaffolds were then passed to RosettaScripts for flexible scaffold design and filtering (see rosetta-script in Appendix A). For the second-generation design, two approaches were taken. In the first approach, a sequence redesign of the best first-generation optimized design was performed (G1_neo2_40_1F, see Appendix B). In the second approach, new mimetics were engineered using G1_neo2_40_1F as the target template. This second-generation mimetic design protocol is similar to that described for the first generation, with two key differences. First, the core fragment was no longer constructed from fragments. Instead, parametric equations for the repeating φ and ψ angles (ω fixed at 180°) were found to yield a repeating secondary structure that closely replicated each of the target helices, allowing for "pitch" in the φ and ψ angles for every X-amino acid (final parameters: H1, H2, H3, H4). The use of these parametric equations, combined with the loop-building process (maximum / minimum 8 amino acids), allowed for the size of each core element in the target structure to be arbitrarily altered (expanded or contracted), ensuring that a reduction in core element size would not remove hot spots from the binding site. A second difference in the second-generation design was that instead of reconnecting core elements of the secondary structure, a fragment size of 7 amino acids was used, and combinatorial assembly of more than two fragments was not permitted (i.e., a single fragment must be able to close a pair of secondary structures). The remaining design algorithms were essentially similar to those employed in the first generation (see Appendix C). The Rosetta energy functions used were "talaris2013" and "talaris2014" for the first and second generation designs, respectively.

[0178] The database of highly idealized fragments used to design the de novo mimetic scaffolds was constructed using a novel Rosetta application, “kcenters_clustering_of_fragments,” using an extensive database of non-redundant publicly available protein structures from the RCSB Protein Data Bank (consisting of 16767 PDBs for the 4-mer database used for the first generation design and 7062 PDBs for the 7-mer database used for the second generation design).

[0179] Yeast display: Yeast was transformed with the gene encoding the protein to be displayed along with the linearized pETcon3 vector. The vector was linearized by 100x overdigestion with NdeI and XhoI (New England Biolabs) and then purified by gel extraction (Qiagen). The gene contained 50-base overlaps with the vector at both the 5' and 3' ends, allowing homologous recombination to place the gene in frame between the AGA2 gene and the myc tag on the vector. As previously described, yeast were grown in C-Trp-Ura medium and then induced in SGCAA medium. After 12–18 h of induction, cells were washed with cold display buffer (50 mM NaPO4 pH 8, 20 mM NaCl, 0.5% BSA) and incubated with various concentrations of biotinylated receptors (either human or mouse IL-2Rα, IL-2Rβ, IL-2Rγ, or human IL-4Rα) at ​​4°C with agitation. After approximately 30 min, the cells were washed again in chilled buffer and then incubated with FITC-conjugated anti-c-Myc antibody (3 × 10 6 The yeast were incubated with biotinylated IL-2Rγ (1 μL per cell) and streptavidin-phycoerythrin (1 μL per 100 μL of yeast volume) for 5 minutes on ice. Yeast were then washed and counted by flow cytometry (Accuri C6) or sorted by FACS (Sony SH800). In experiments where the initial receptor incubation was performed with biotinylated IL-2Rγ in combination with non-biotinylated IL-4Rα, the non-biotinylated receptor was provided in molar excess.

[0180] Mutagenesis and affinity maturation: For error-prone PCR-based mutagenesis, the design to be mutated was cloned into the pETcon3 vector and amplified using the MutaGene II mutagenesis kit (Invitrogen) according to the manufacturer's instructions, resulting in a mutation frequency of approximately 1% per nucleotide. 1 μg of this mutated gene was amplified with 1 μg of linearized pETcon3 vector in 10 mL of 1000 ribosomal DNA. 8 The vector was electroporated into EBY100 yeast with a transformation efficiency of approximately 1. The yeast was successively induced and selected multiple times with gradually decreasing concentrations of receptor until the population converged. Between selections, the yeast was regrowthed in C-Trp-Ura medium.

[0181] Site-saturation mutagenesis (SSM) libraries were constructed from synthetic DNA in Genscript. For each amino acid in each design template, forward and reverse primers were designed to generate 5' and 3' PCR products containing a degenerate NNK codon. Amplification of the "left" and "right" products with COF and COR primers yielded a series of template products, each consisting of a degenerate NNK codon at a different residue position. For each design, these products were pooled to generate the SSM library. The SSM library was transformed into Saccharomyces cerevisiae strain EBY100 cells by electroporation with the linearized pETCON3 vector using the protocol previously described by Benatuil et al.

[0182] A combinatorial library was constructed from synthetic DNA from Genscript containing ambiguous nucleotides and similarly transformed into the linearized pETCON3 vector.

[0183] Protein expression: The gene encoding the designed protein sequence was synthesized and cloned into the pET-28b(+) E. coli plasmid expression vector (GenScript, N-terminal 6xHis tag and thrombin cleavage site). The plasmid was then transformed into chemically competent E. coli Lemo21 cells (NEB). Protein expression was performed using Terrific Broth and M salts, and OD 600 Cultures were grown at 37°C until an RI of approximately 0.8 was reached, after which expression was induced with 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) and the temperature was reduced to 18°C. After approximately 18 hours of expression, cells were harvested, lysed at 18,000 psi using a Microfluidics M110P microfluidizer, and centrifuged at 24,000 g for 20 minutes to clarify the soluble fraction. The soluble fraction was purified by Immobilized Metal Affinity Chromatography (Qiagen), followed by FPLC size exclusion chromatography (Superdex 75 10 / 300 GL, GE Healthcare). Purified Neoleukin-2 / 15 was characterized by mass spectrometry (MS) verification of molecular weight species in solution (Thermo Scientific), size exclusion-multiangle laser light scattering (SEC-MALLS) (Agilent, Wyatt) to verify monomeric state and molecular weight, SDS-PAGE, and endotoxin levels (Charles River).

[0184] hIL-2 (1-133 amino acids), hIL-2Rα (1-217 amino acids), hIL-2Rβ (1-214 amino acids), hIL-2Rγ (1-232 amino acids), mIL-2 (1-149 amino acids), mIL-2Rα ectodomain (1-213 amino acids), mIL-2Rβ ectodomain (1-215 amino acids), and mγ c Human and mouse IL-2 complex components, including the ectodomain (amino acids 1–233), were synthesized as previously described. 17,49, secreted and purified using a baculovirus expression system. All proteins were purified to >98% homogeneity using a Superdex 200 sizing column (GE Healthcare) equilibrated with HBS. Purity was confirmed by SDS-PAGE analysis. For expression of biotinylated human IL-2 and mouse IL-2 receptor subunits, proteins containing the C-terminal biotin acceptor peptide (BAP)-LNDIFEAQKIEWHE (SEQ ID NO: 262) were expressed and purified as described via Ni-NTA affinity chromatography, then biotinylated using soluble BirA ligase enzyme in 0.5 mM Bicine pH 8.3, 100 mM ATP, 100 mM magnesium acetate, and 500 mM biotin (Sigma). Excess biotin was removed by size-exclusion chromatography on a Superdex 200 column equilibrated with HBS.

[0185] Neoleukin-2 crystal and co-crystal structures: C-terminally 6xHis-tagged endoglycosidase H (endoH) with murine IL-2Rβ and IL-2Rγ were separately expressed in Hi-five cells using the baculovirus system as previously described. IL-2Rγ was grown in the presence of 5 μM kifunensine. After approximately 72 h, the secreted proteins were purified from the medium by passage through a Ni-NTA agarose column and eluted with 200 mM imidazole in HBS buffer (150 mM NaCl, 10 mM HEPES pH 7.3). EndoH was exchanged into HBS buffer by diafiltration. mIL-2Rγ was deglycosylated by overnight incubation with endoH at 1:75 (w / w). mIL-2Rβ and mIL-2Rγ were further purified and buffer-exchanged by FPLC using an S200 column (GE Life Sciences).

[0186] Monomeric Neoleukin-2 / 15 was concentrated to 12 mg / ml and crystallized by vapor diffusion from 2.4 M sodium malonate, pH 7.0. Crystals were collected and flash-frozen without further cryoprotection. Crystals diffracted at 2.0 Å resolution at beamline 12-2 at the Stanford Synchrotron Radiation Laboratory and indexed and integrated using XDS (Kabsch, 2010). Space groups were assigned with Pointless (Evans, 2006), and scaling was performed with Aimless (Evans and Murshudov, 2013) in the CCP4 suite (Winn et al., 2013). The structure was solved by molecular replacement in Phaser (McCoy et al., 2007) using our predicted model as the search ensemble, with six protomers arranged in the asymmetric unit. After initial reconstruction with Autobuild ( Terwilliger et al., 2008 ), iterative cycles of manual reconstruction and refinement were performed using Coot ( Emsley et al., 2010 ) and Phenix ( Adams et al., 2010 ).

[0187] To crystallize the ternary Neoleukin:mIL-2Rβ:mIL-2Rγ complex, the three proteins were combined in an equimolar ratio, digested overnight with 1:100 (w / w) carboxypeptidases A and B to remove the purification tag, and purified by FPLC using an S200 column. Fractions containing all three proteins were pooled and concentrated to 20 mg / ml. Initial needle-shaped crystals were formed by vapor diffusion from 0.1 M imidazole pH 8.0, 1 M sodium citrate, and used to prepare microseed stocks for subsequent use in microseed matrix screening (MMS, (D'Arcy et al., 2014)). After one MMS cycle, crystals grown in the same precipitant were cryoprotected in 30% ethylene glycol, harvested, and anisotropically diffracted at 3.4 Å x 3.8 Å x 4.1 Å resolution at beamline 23ID-B at the Advanced Photon Source. The structure was solved by molecular replacement in Phaser using the human IL-2Rβ and IL-2Rγ structures (pdb ID 2B5I) as the search ensemble. This resulted in an electron density map from which two polyalanine alpha helices could be manually constructed. Following rigid-body refinement in Phenix, the electron density for the two unmodeled alpha helices, along with the BC loop and several aromatic side chains, was visualized, allowing docking of monomeric Neoleukin. Two further rounds of MMS and an additive screen (Hampton Research) were used to produce crystals grown by vapor diffusion using 150 nL of protein, 125 nL of well solution containing 0.1 M Tris pH 7.5, 5% dextran sulfate, 2.1 M ammonium sulfate, and 25 nL of a microseed stock containing 1.3 M ammonium sulfate, 50 mM Tris pH 7.5, 50 mM imidazole pH 8.0, and 300 mM sodium citrate. Crystals cryoprotected in 3 M sodium malonate were flash frozen and diffracted anisotropically to 2.5 Å x 3.7 Å x 3.8 Å on beamline 5.0.1 at the Advanced Light Source.After processing the data in XDS, an elliptical resolution limit was applied using the STARANISO server (Bruhn et al., 2017). Rapid convergence of the model was obtained by refinement against these reflections using TLS and targeted restraints to the high-resolution human receptor (PDB id 2B5I) and Neoleukin-2 / 15 structures in Buster (Smart et al., 2012; Bricogne et al., 2016), followed by manual rebuilding in Coot, followed by a final round of refinement without targeted restraints in Phenix. Structural diagrams were created in PyMol (Schrodinger, LLC. 2010. The PyMOL Molecular Graphics System, Version 2.1.0). The software used in this project was installed and configured by SBGrid (Morin et al., 2013).

[0188] Cell line: unmodified YT-1 64 and IL-2Rα + YT-1 human natural killer cells 65 were cultured in RPMI complete medium (RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, minimal non-essential amino acids, sodium pyruvate, 25 mM HEPES, and penicillin-streptomycin [Gibco]). CTLL-2 cells purchased from ATCC were cultured in RPMI supplemented with 10% T-STIM culture supplement containing ConA (Corning). All cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. A subpopulation of YT-1 cells expressing IL-2Rα was purified via magnetic selection as previously described. 17 The enrichment and persistence of IL-2Rα expression was monitored by analyzing the binding of PE-conjugated anti-human IL-2Rα (Biolegend) antibody on an Accuri C6 flow cytometer (BD Biosciences).

[0189] Circular dichroism (CD): Far-UV CD measurements were performed using an AVIV Model 420 spectrometer in 1 mm pathlength cuvettes in PBS buffer (pH 7.4) at a protein concentration of approximately 0.20 mg / ml (unless otherwise specified in the text). The temperature was melted from 25 to 95°C and the absorbance signal at 222 nm was monitored (2°C / min steps with 30 s equilibration between each step). Wavelength scans (195–260 nm) were collected at 25°C and 95°C, and again at 25°C after rapid refolding (~5 min).

[0190] Binding Studies: Surface Plasmon Resonance (SPR): For IL-2 receptor affinity titration studies, biotinylated human or mouse IL-2Rα, IL-2Rβ, and IL-2Rγ receptors were immobilized on streptavidin-coated chips and analyzed on a Biacore T100 instrument (GE Healthcare). To subtract nonspecific binding, an irrelevant biotinylated protein was immobilized in the reference channel. To minimize mass transfer effects, less than 100 response units (RU) of each ligand were immobilized. Three-fold serial dilutions of hIL-2, mIL-2, Super-2, or engineered IL-2 mimics were flowed over the immobilized ligands for 60 seconds, and dissociation was measured for 240 seconds. IL-2Rβγ cFor binding studies, saturating concentrations of hIL-2Rβ (3 μM) or mIL-2Rβ (5 μM) were added to the indicated concentrations of hIL-2 or mIL-2, respectively. Surface regeneration was performed for all interactions using a 15-second exposure to 1 M MgCl2 in 10 mM sodium acetate, pH 5.5. SPR experiments were performed at 25 °C in HBS-P+ buffer (GE Healthcare) supplemented with 0.2% bovine serum albumin (BSA), and all binding studies were performed at a flow rate of 50 L / min to prevent analyte rebinding. Data were visualized and processed using Biacore T100 Evaluation Software Version 2.0 (GE Healthcare). Fitting of equilibrium titration curves and determination of equilibrium binding / dissociation (KD) values ​​were performed using GraphPad Prism, assuming all binding interactions were first-order. Biolayer interferometry: Binding data were collected on an Octet RED96 (ForteBio, Menlo Park, CA) and processed using the instrument's integrated software using a 1:1 binding model. Biotinylated target receptors, either human or mouse IL-2Rα, IL-2Rβ, IL-2Rγ, or human IL-4Rα, were functionalized onto streptavidin-coated biosensors (SA ForteBio) at 1 μg / ml for 300 seconds in binding buffer (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, 0.5% nonfat dry milk). Analyte proteins were diluted from concentrated stocks into the binding buffer. After measuring a baseline with binding buffer alone, the biosensor was immersed in a well containing 100 nM of the design protein (binding), and then the sensor was reimmersed in the baseline well (dissociation) to monitor the binding kinetics. In binding experiments, while IL-2Rγ was bound to the sensor, either IL-2Rβ or IL-4Rα was supplemented into the solution, with the supplemented proteins provided in a 2.5-fold molar excess.

[0191] STAT5 phosphorylation studies: In vitro studies: approximately 2x10 5 YT-1, IL-2Rα +YT-1 or CTLL-2 cells were plated in each well of a 96-well plate and resuspended in RPMI complete medium containing serial dilutions of hIL-2, mIL-2, Super-2, or engineered IL-2 mimic. Cells were stimulated for 15 minutes at 37°C, immediately fixed by adding formaldehyde to 1.5% and incubated at room temperature for 10 minutes. Cells were permeabilized by resuspending in ice-cold 100% methanol for 30 minutes at 4°C. Fixed and permeabilized cells were washed twice with FACS buffer (phosphate-buffered saline [PBS] pH 7.2 containing 0.1% bovine serum albumin) and incubated for 2 hours with AlexaFluor® 647-conjugated anti-STAT5pY694 (BD Biosciences) diluted in FACS buffer. Cells were then washed twice with FACS buffer and MFI was measured using a CytoFLEX flow cytometer (Beckman-Coulter). Dose-response curves were fitted to a logistic model, and the mean fluorescence intensity (MFI) of unstimulated cells was subtracted and normalized to the maximum signal intensity, after which the half-maximal effective concentration (EC) was calculated using GraphPad Prism data analysis software. 50 The values ​​were calculated. Experiments were performed in triplicate and repeated three times with similar results. Ex vivo studies: Spleens and lymph nodes were obtained from wild-type C57BL / 6J mice or B6;129S4-Il2ra mice purchased from Jackson Laboratory. tm1Dw CD4+ T cells were collected from (CD25KO) mice and prepared into a single cell suspension in sorting buffer (2% fetal bovine serum in phosphate-buffered saline, pH 7.2). CD4+ T cells were enriched through negative selection by staining the cell suspension with biotin-conjugated anti-B220, CD8, NK1.1, CD11b, CD11c, Ter119, and CD19 antibodies (1:100) on ice for 30 minutes. After washing with sorting buffer, anti-biotin microbeads (Miltenyi Biotec) were added to the cells at 100 μg / ml. 7 20 μL of PBS was added to the cell suspension per total cell count and incubated on ice for 20 minutes. The cells were washed, resuspended, and negatively selected using an EasySep Magnet (STEMCELL Technologies). Approximately 1 x 10 cells were cultured. 5Enriched cells were added to each well of a 96-well plate in RPMI complete medium containing 5% FCS and 10-fold serial dilutions of mIL-2, Super-2, or Neoleukin-2 / 15. Cells were stimulated for 20 minutes at 37°C in 5% CO2, fixed with 4% PFA, and incubated at 4°C for 30 minutes. After fixation, cells were harvested, washed twice with sorting buffer, and permeabilized again with 500 μL of 90% ice-cold methanol in dH2O for 30 minutes on ice. Cells were washed twice with Perm / Wash buffer (BD Biosciences) and stained with anti-CD4-PerCP (1:300), anti-CD44-Alexa Fluor 700 (1:200), anti-CD25-PE-Cy7 (1:200), and 5 μL of anti-pSTAT5-PE pY694 per sample in Perm / Wash buffer for 45 minutes at room temperature in the dark. Cells were washed with Perm / Wash and resuspended in sorting buffer for analysis on a BD LSR II flow cytometer (BD Biosciences).

[0192] In vivo mouse airway inflammation experiments: C57BL / 6J mice were purchased from Jackson Laboratory. Mice were inoculated intranasally with 20 μL of whole house dust mite antigen (Greer) resuspended in PBS, for a total of 23 μg of Derp1 per mouse. From days 1 to 7, mice received daily intraperitoneal injections of 20 μg of mIL-2 in sterile PBS (pH 7.2), a molar equivalent of Neoleukin-2 / 15 in sterile PBS, or no injection. On day 8, circulating T cells were intravascularly labeled and tetramer-positive cells were enriched from lymph nodes and spleens or lungs as previously described (Hondowicz, Immunity, 2016). Both the flow-through and bound fractions from the column were saved for flow cytometry analysis. Cells were surface stained with antibodies and analyzed on a BD LSR II flow cytometer (BD Biosciences). Animal model: C57BL / 6 mice were purchased from Jackson Laboratory or bred in-house. BALB / c mice were purchased from Charles River. Animals were maintained according to protocols approved by the Institutional Animal Care and Use Committee of the Dana-Farber Cancer Institute (DFCI), the Direcao Geral de Veterinaria, and the iMM Lisboa ethical committee.

[0193] In vivo mouse experiments of colorectal cancer: CT26 cells were provided by Jocelyne Demengeot's research group at the Instituto Gulbenkian de Ciencia (IGC), Portugal. On day 0, 5x10^5 cells were injected subcutaneously (sc) into the flanks of BALB / c mice with 50 μL of a 1:1 mixture of Dulbecco's modified Eagle's medium (Gibco) and Matrigel (Corning). From day 6, when tumor volumes reached approximately 100 mm3, Neoleukin-2 / 15 and mIL-2 (Peprotech) in 50 μL of PBS (Gibco) were administered daily via intraperitoneal (ip) injection. Treatment with anti-PD-1 antibody (Bio X Cell) was performed via intraperitoneal injection of 200 μg (in PBS) per mouse twice weekly. Mice were sacrificed when tumor volumes reached 1,300 mm3.

[0194] In vivo melanoma experiments: B16F10 cells were purchased from ATCC. On day 0, 5 × 10 cells were cultured in 500 μL of Hank's balanced salt solution (Gibco). 5 Cells were inoculated by subcutaneous injection. Starting on day 1, Neoleukin-2 / 15 and mIL-2 (Peprotech) in 200 μL of LPS-free PBS (Teknova) were administered daily by intraperitoneal (ip) injection. Additional treatments with 150 μg / mouse of TA99 (a gift from Noor Momin and Dane Wittrup, Massachusetts Institute of Technology) were added several days later, as indicated. Mice were sacrificed when tumor volumes reached 2,000 mm3.

[0195] Flow cytometry: Resected tumors were minced, enzymatically digested (Miltenyi Biotec), and passed through a 40 μm filter. Cells from the spleen and tumor-draining lymph nodes were dispersed into PBS through a 40 μm cell strainer using the back of a 1 mL syringe plunger. All cell suspensions were washed once with PBS, and the cell pellets were resuspended in 2% inactivated fetal bovine serum containing fluorophore-conjugated antibodies. Cells were incubated at 4°C for 15 minutes before being fixed, permeabilized, and stained using the BioLegend FoxP3 staining kit. Samples were analyzed on a BD Fortessa flow cytometer. The antibodies used in melanoma experiments (BioLegend) were CD45-BV711 (clone 30-F11), CD8-BV650 (53-6.7), CD4-BV421 (GK1.5), TCRβ-BV510 (H57-597), CD25-AF488 (PC61), and FoxP3-PE (MF-14). The antibodies used in colon cancer experiments (eBioscience) were CD45-BV510 (30-F11), CD3-BV711 (17A2), CD49b-FITC (DX5), CD4-BV605 (GK1.5), CD8-PECy7 (53-6.7), and Foxp3-APC (FJK-16s). Dead cells were excluded using Fixable Viability Dye eFluor 780 (eBioscience).

[0196] Generation of anti-Neoleukin-2 / 15 polyclonal antibodies: Mice were intraperitoneally injected with 500 μg of K-ONeoleukin in 200 μL of a 1:1 emulsion of PBS and complete Freund's adjuvant. On days 7 and 15, mice were boosted with 500 μg of K-ONeoleukin in 200 μL of a 1:1 emulsion of PBS and incomplete Freund's adjuvant. On day 20, serum was collected and Neoleukin-2 / 15 recognition was confirmed by ELISA.

[0197] Enzyme-linked immunosorbent assay (ELISA): High-binding 96-well plates (Corning) were coated overnight at 4°C with 100 ng / mL Neoleukin-2 / 15, mIL-2 (Peprotech), hIL-2 (Peprotech), or ovalbumin (Sigma-Aldrich) in carbonate buffer. Antibody binding to the target proteins was detected using 75 ng / mL HRP-conjugated sheep anti-mouse IgG (GE Healthcare). Plates were developed with tetramethylbenzidine and HCl. Absorbance was measured at 450 nm using an EnVision multimode plate reader (PerkinElmer).

[0198] T cell proliferation assay: Cells were isolated from mouse spleens using the EasySep T Cell Isolation Kit (Stemcell Technologies). They were plated at a density of 10,000 cells / well in RPMI medium in 96-well culture plates. The medium was supplemented with normal or heat-treated Neoleukin-2 / 15, rmIL-2, or Super-2. After 5 days of culture at 37°C, cell viability and proliferation were measured using the CellTiter-Glo Luminescent Cell Viability Assay (Promega).

[0199] Statistical and power analysis: In vivo mouse airway inflammation experiment; MIKEL in vivo mouse colon cancer experiment; CARLOS in vivo mouse melanoma experiment. Comparison of survival rates of tumor-bearing mice was performed using the log-rank (Mantel-Cox) test. Comparison of weight loss in tumor-bearing mice was performed using a two-tailed t-test. P values ​​of less than 0.05 were considered significant. The minimum group size was determined using G*Power for an expected large effect size (Cohen's d = 1.75).

[0200] Biolayer interferometry analysis of mouse serum albumin (MSA) fusion with Neoleukin-2 / 15. When Neoleukin-2 / 15 is genetically fused to MSA for extended half-life, the cytokine mimic maintains its original binding affinity (33.5 ± 0.2 nM) for mouse IL-2RBeta and IL-2RGamma (data not shown). The constructs utilized in this study were as follows: Optional: (HisTag TEV cleavage site in brackets) Mouse serum albumin (italics) Linker Neo2 / 15(bold) [ka] (SEQ ID NO: 244)

[0201] Biotin-mIL2γ was immobilized on a streptavidin biosensor, and the concentration of MSA-Neo2 was titrated from 729 to 1 nM in the presence of a saturating concentration of mIL2Beta. Biolayer interferometry was performed as described above. Binding data were collected with an Octet RED96 (ForteBio, Menlo Park, CA) and processed using the instrument's integrated software using a 1:1 binding model. Biotinylated target receptors, either human or mouse IL-2Rα, IL-2Rβ, IL-2Rγ, or human IL-4Rα, were functionalized onto streptavidin-coated biosensors (SA ForteBio) at 1 μg / ml for 300 s in binding buffer (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, 0.5% nonfat dry milk). Analyte proteins were diluted from concentrated stocks into binding buffer. After a baseline measurement in binding buffer alone, biosensors were immersed in wells containing 100 nM of design protein (binding), and then the sensors were immersed again in baseline wells (dissociation) to monitor binding kinetics.

[0202] In vivo experiments with CAR-T cells: in vitro T cell proliferation assay. Primary human T cells were obtained from healthy donors. Peripheral blood mononuclear cells (PBMCs) were separated by centrifugation on Ficoll-Hypaque (Sigma). T cells were isolated using the EasySep™ CD8 or CD4 negative isolation kit (STEMCELL Technologies). To stimulate T cells, T cells were thawed and incubated with anti-CD3 / CD28 Dynabeads (Gibco) at a 1:1 ratio in medium supplemented with 50 IU / ml (3.1 ng / ml) IL2. After 4 days of incubation, the beads were removed. Stimulated or freshly thawed unstimulated T cells were plated at 30,000 or 50,000 cells / well in a 96-well format and cultured in triplicate with the indicated concentrations of IL2 or Neoleukin-2 / 15. After 3 days, proliferation was measured using CellTiter-Glo 2.0 (Promega).

[0203] In vivo RAJI experiments: 6-8 week-old NSG mice were obtained from the Jackson Laboratory. 0.5*10^6 RAJI tumor cells transduced with ffluc / eGFP were injected into NSG mice via the tail vein. Seven days after tumor injection, mice were intravenously injected with lentiviral-transduced anti-CD19 CAR T cells (0.4*10^6 CD4 and 0.4*10^6 CD8) prepared as described (Liu et al., 2016). 20 μg / mouse of hIL2 or Neoleukin-2 / 15 was administered intraperitoneally 8-16 days after tumor injection.

[0204] Preparation of PEGylated polypeptides: Neo-2 / 15 stocks containing single or double cysteine ​​mutations were dialyzed into phosphate buffer (pH 7.0) and adjusted to 1.0–2.0 mg / ml. TCEP was added at a 10:1 molar ratio to protein and incubated at RT for 10 min to reduce disulfides. Maleimide-modified PEG40k (PEG40k-MA) or PEG30k (PEG30k-MA) powder was added directly to the reduced protein solution at a 10:1 PEG:cysteine ​​molar ratio and incubated for 2 h with stirring. Aliquots were taken directly from the reaction mixture for SDS-PAGE. These data demonstrate the rapid, spontaneous, and nearly quantitative formation of covalent bonds between PEG40k-MA or PEG30k-MA and Neo-2 / 15 cysteine ​​mutants at the expected stoichiometry.

[0205] Treatment with Neo-2 / 15 and PEGylated Neo-2 / 15-E62C (Neo-2 / 15-PEG) demonstrated changes in the levels of multiple inflammatory markers: Two nonhuman primates (NHPs), one male and one female per group, were assigned to treatment with either vehicle (Group 1), Neo-2 / 15 (no PEG) (Groups 2-4), or Neo-2 / 15 PEG (Groups 5-7, single cysteine ​​mutation of E62C and PEG40K). Animals treated with vehicle or Neo-2 / 15 (no PEG) were administered by intravenous (IV) bolus on study days 1, 2, 3, 4, 5, 6, and 7 (once daily for 1 week) at dose levels of either 0 (vehicle) or 0.07, 0.21, or 0.14 mg / kg / day of Neo-2 / 15 (no PEG) (Groups 2, 3, and 4, respectively). Animals treated with Neo-2 / 15 PEG were administered by intravenous bolus on study days 1 and 7 at dose levels of 0.05, 0.15, or 0.10 mg / kg / day of Neo-2 / 15 PEG (Groups 5, 6, and 7, respectively). Cytokine samples were collected on days 1 and 7 at 0, 4, 8, and 24 hours post-dose. Serum samples for cytokines were prepared, frozen at <-70°C, and shipped for analysis. Samples were analyzed on a Luminex multiplex immunoassay system. Several cytokines, including IL-15 and IL-10, demonstrated significant differences in the time course of cytokine production, consistent with a more sustained pharmacodynamic effect of the PEGylated molecules.

[0206] Targeted Neo-2 / 15 fusions retained their IL-2R binding affinity and demonstrated antitumor efficacy. Selected targeting domains were fused to the N- or C-terminus of Neo-2 / 15 via peptide linkers and tested in vitro to characterize their binding affinity to human and mouse IL-2R by biolayer interferometry. Results confirmed that fusion of Neo-2 / 15 to either the N- or C-terminus did not interfere with its ability to bind to IL-2R. Subsequent in vitro flow cytometry studies confirmed that these fusion proteins were able to bind to target receptors on the surface of cells. The efficacy of the targeting constructs was evaluated in in vivo mouse experiments, demonstrating that targeting Neo-2 / 15 moieties to tumor cells or immune cells had beneficial antitumor effects compared with non-targeted controls (data not shown).

[0207] Fusions tested include, but are not limited to, the following: (i) fusion of an anti-CD47 nanobody to the C-terminus of Neo2 / 15 via a linker of SEQ ID NO: 100; (b) fusion of an anti-CD47 nanobody to the N-terminus of Neo2 / 15 via a linker of SEQ ID NO: 100; (c) fusion of an anti-CTLA4 nanobody to the C-terminus of Neo2 / 15 via a linker of SEQ ID NO: 100; (d) fusion of an anti-CTLA4 nanobody to the N-terminus of Neo2 / 15 via a linker of SEQ ID NO: 100; (e) fusion of an anti-PDL-1 nanobody to the C-terminus of Neo2 / 15 via a linker of SEQ ID NO: 100; and (f) fusion of an anti-PDL-1 nanobody to the N-terminus of Neo2 / 15 via a linker of SEQ ID NO: 100.

[0208] Fusion of albumin to Neo-2 / 15 maintained IL-2R binding affinity. Mouse serum albumin (MSA) was fused to the N-terminus of Neo-2 / 15 via a peptide linker and tested in vitro by biolayer interferometry to characterize its binding affinity to mouse IL-2R. Biotin-mIL2γ was immobilized on a streptavidin biosensor, and the concentration of MSA-Neo-2 was titrated from 729 to 1 nM in the presence of saturating concentrations of mIL2Beta. These fusions maintained IL-2R binding ability (data not shown).

[0209] PEGylated and non-PEGylated Neo-2 / 15 do not induce significant anti-drug antibody (ADA) responses in non-human primates (NHPs). The potential for ADA induction of PEGylated and non-PEGylated Neo-2 / 15 (PEGylated Neo-2 / 15: E62C single cysteine ​​mutation and PEG40K) was tested in non-human primates. Animals were intravenously administered one compound for one week: PEGylated Neo-2 / 15 on days 1 and 7, or wild-type Neo-2 / 15 on days 1–7. Blood was then collected at various time points and analyzed for the presence of antibodies specific to the administered compound. Each dose group consisted of one male and one female macaque. Non-PEGylated Neo-2 / 15 was administered via daily intravenous bolus injections at 0.1 mg / kg, 0.2 mg / kg, or 0.3 mg / kg for seven consecutive days. PEGylated Neo-2 / 15 was administered via intravenous bolus injection at 0.015 mg / kg, 0.050 mg / kg, or 0.10 mg / kg on days 1 and 7. The vehicle control group received an equal volume of saline daily for seven consecutive days. Approximately 750 μl of blood was collected from each animal via the cephalic or saphenous vein for ADA analysis on study days 1 (pre-dose), 22, 29, and 43. Serum was extracted from the blood using serum separator tubes on wet ice and then stored at -80°C until analysis. All cynomolgus monkeys administered either vehicle or PEGylated Neo-2 / 15 tested negative for ADA on days 22, 29, and 43, demonstrating that PEGylated Neo-2 / 15, a computationally designed protein completely foreign to the macaque immune system, did not elicit a detectable immune response, even after repeated administration. Both macaques (1 male, 1 female) that received the vehicle control tested negative for ADA to wild-type Neo-2 / 15 on days 1, 15, 22, and 28. All animals (3 males, 2 females) in the group that received non-PEGylated Neo-2 / 15 tested negative for ADA on day 1 (pre-dose). Of these, 3 of 5 (60%) remained negative for ADA on days 22, 29, and 43. The remaining 2 animals subsequently tested positive for ADA on days 22, 29, or 43.One subject tested positive on days 22 and 29 but returned negative on day 43. In that subject, the ADA response was low and transient, suggesting minimal clinical significance. Another subject tested positive on days 22, 29, and 43. In that subject, the measured ADA concentration was well below 100 ng / ml, so clinical relevance was unclear.

[0210] Data Table Table E1. Characterization of several de novo designed mimetics of IL-2 / IL-15. This table shows the Kd, EC of de novo IL-2 / IL-15 mimetics and reference cytokines: mIL-2Rβ, mIL-2Rβγc. 50 , structural alignment (MICAN) against hIL-2 (PDB:2B5I) and mIL-2 (PDB: 63 ), the parent of each molecule, its amino acid length, and the sequence of the de novo IL-2 mimetic are shown. "N / S" means non-significant, and "N / A" means non-available. [Table 9-1] [Table 9-2] [Table 9-3] [Table 10] [Table 11] [Table 12] [Table 13-1] [Table 13-2] [Table 13-3] [Table 14] Neoleukin-2 / 15-H8Y-K33E:H1->H3->H2'->H4 [ka] (SEQ ID NO: 94)

[0211] The binding of Neoleukin-2 / 15-H8Y-K33E to the IL2 receptor, measured by biolayer interferometry, was found to have higher binding affinity for IL2-Rbeta than Neoleukin-2, both when tested against IL2Rbeta alone and when tested against the IL2Rbeta-gamma complex, and this increased affinity was primarily due to an improved off-rate from IL2-Rbeta. [Table 15] Appendix A. RosettaScripts XML protocol for mimic first generation sequence design. <rosettascripts> <scorefxns> <sfxn6 weights="talaris2013.wts" / > <sfxn6da weights="talaris2013_downAla.wts" / > < / scorefxns> <filters> <ssprediction name=""sspred”" cmd="" / work / dadriano / PROGRAMS / psipred / runpsipred_single”" use_probability=""0”" use_svm=""0”" threshold="0.80" confidence=""1” / "> <scoretype name=""rama”" scorefxn=""SFXN6”" score_type=""rama”" threshold="0.0" confidence=""0”" / > <packstat name="pack" threshold="0.63" confidence="1 / "> <holes name="holes" threshold="1.2" confidence="0 / "> <scoretype name=""score”" scorefxn=""SFXN6”" score_type=""total_score”" threshold="0.0" confidence=""0”" / > <residuecount name=""nres”" confidence=""0”" / > <calculatorfilter name=""score_res”" equation=""SCORE / NRES”" threshold=""-1.7”" confidence=""1”"> <score name=""SCORE”" filter_name=""score”" / > <nres name=""NRES”" filter_name="”nres”" / > < / calculatorfilter> <compoundstatement name="filt"> <and filter_name="sspred" / > <and filter_name="rama" / > <and filter_name="score_res" / > <and filter_name="pack" / > < / compoundstatement> < / holes> < / packstat> < / ssprediction> < / filters> <taskoperations> <initializefromcommandline name=""init” / "> <includecurrent name=""inclcur” / "> <limitaromachi2 name="limitchi2" / > DisallowIfNonnative name="not_aa_H” disallow_aas="H” / > <readresfile name=""resfile”" filename="". / input.resfile”" / > < / includecurrent> < / initializefromcommandline> < / taskoperations> <movers> <dssp name="dssp / "> <FastDesign name="fdesign” task_operations="init,resfile,limitchi2” scorefxn="SFXN6dA” allow_design="1” only_design_worst_region="0” design_by_psipred="0” design_by_frag_qual="0” repeats="2” clear_designable_residues="0” max_redesigns=”2000” / > <fastrelax name="relax" / > <parsedprotocol name=""complexDesign”"> <add mover_name=""fdesign”" / > <add mover_name=""relax”" / > <add mover_name=""dssp”" / > < / parsedprotocol> <loopover name=""fastDesignProtein”" mover_name=""complexDesign”" filter_name="filt" drift="0" iterations=""10”" ms_whenfail="FAIL_DO_NOT_RETRY / "> < / loopover> < / dssp> < / movers> <APPLY_TO_POSE> < / APPLY_TO_POSE> <protocols> <add mover_name="fastDesignProtein" / > <add filter_name="sspred" / > <add filter_name="pack" / > <add filter_name="score" / > <add filter_name="score_res" / > <add filter_name="holes" / > <add filter_name="rama" / > < / protocols> < / rosettascripts> Appendix B. RosettaScripts XML protocol for the sequence redesign of G1_Neo2_40_1F (i.e., second generation). <rosettascripts> <scorefxns> <SFXN6_vanilla weights=". / talaris2014_cart.wts” symmetric=0 / > <SFXN6dA_vanilla weights=". / talaris2014_cart_downAla.wts” symmetric=0 / > <SFXN6dA_norep_elect weights=". / talaris2014_cart_downAla.wts” symmetric=0 > <reweight scoretype="fa_rep" weight="0.05" / > <reweight scoretype="fa_elec" weight="1.0" / > < / SFXN6dA_norep_elect > <SFXN6dA_elect weights=". / talaris2014_cart_downAla.wts” symmetric=0 > <reweight scoretype="fa_elec" weight="2.0" / > < / SFXN6dA_elect > < / scorefxns> <movers> <switchchainorder name=""keep_only_chain_A”" chain_order=""1” / "> < / switchchainorder> < / movers> <filters> <!--Not Enabled--> <holes name=""holes_disabled”" threshold=""1.2”" confidence=""0” / "> <scoretype name=""score_disabled”" scorefxn=""SFXN6_vanilla”" score_type=""total_score”" threshold="0.0" confidence=""0”" / > <residuecount name=""nres_disabled”" confidence=""0”" / > <packstat name="packstat_disabled" threshold=""0.65”" repeats=""3”" confidence=""0”" / > <ssprediction name=""sspred_disabled”" cmd="" / work / dadriano / PROGRAMS / psipred / runpsipred_single”" use_probability=""0”" use_svm=""0”" threshold="0.85" confidence=""0” / "> <buriedunsathbonds name=""unsat_core_disabled”" cutoff="0" task_operations=""only_core_residues”" jump_number="0" confidence=""0” / "> <rmsdsimple name=""rmsd1_chainA_disabled”" reference_name=""reference_conformation”" chain=""1”" align=""1”" threshold=""1.0”" confidence=""0” / "> <rmsdsimple name=""rmsd2_chainA_disabled"reference_name="reference_conformation”" chain=""1”" align=""1”" threshold=""1.0”" confidence=""0” / "> <rmsdsimple name=""rmsd3_chainA_disabled”" reference_name=""reference_conformation”" chain=""1”" align=""1”" threshold=""1.0”" confidence=""0” / "> <calculatorfilter name=""score_res_disabled”" equation=""SCORE / NRES”" threshold=""-1.8”" confidence=""0”"> <score name=""SCORE”" filter_name=""score_disabled”" / > <nres name=""NRES”" filter_name=""nres_disabled”" / > < / calculatorfilter> <!--Enabled--> <packstat name=""packstat_enabled”" threshold=""0.65”" repeats=""3”" confidence=""1”" / > <ssprediction name=""sspred_enabled”" cmd="" / work / dadriano / PROGRAMS / psipred / runpsipred_single”" use_probability=""0”" use_svm=""0”" threshold="0.85" confidence=""1” / "> <calculatorfilter name=""score_res_enabled”" equation=""SCORE / NRES”" threshold=""-1.8”" confidence=""1”"> <score name=""SCORE”" filter_name=""score_disabled”" / > <nres name=""NRES”" filter_name=""nres_disabled”" / > < / calculatorfilter> <cavityvolume name=""cav_vol_disabled”" / > <buriedunsathbonds name=""unsat_core_enabled”" cutoff="0" task_operations=""only_core_residues”" jump_number="0" confidence=""1” / "> <rmsdsimple name=""rmsd1_chainA_enabled”" reference_name=""reference_conformation”" chain=""1”" align=""1”" threshold=""1.0”" confidence=""1” / "> <rmsdsimple name=""rmsd2_chainA_enabled”" reference_name=""reference_conformation”" chain=""1”" align=""1”" threshold=""1.0”" confidence=""1” / "> <rmsdsimple name=""rmsd3_chainA_enabled”" reference_name=""reference_conformation”" chain=""1”" align=""1”" threshold=""1.0”" confidence=""1” / "> <compoundstatement name="all_enabled_filters"> <and filter_name="sspred_enabled" / > <and filter_name="score_res_enabled" / > <and filter_name="packstat_enabled" / > <and filter_name="unsat_core_enabled" / > < / compoundstatement> < / rmsdsimple> < / rmsdsimple> < / rmsdsimple> < / buriedunsathbonds> < / ssprediction> < / rmsdsimple> < / rmsdsimple> < / rmsdsimple> < / buriedunsathbonds> < / ssprediction> < / holes> < / filters> <filters> <!--Chain A Filters--> <!--Not Enabled--> <movebeforefilter name=""packstat_chainA_disabled”" mover=""keep_only_chain_A”" filter="packstat_disabled" confidence=""0” / "> <movebeforefilter name=""sspred_chainA_disabled”" mover=""keep_only_chain_A”" filter="sspred_disabled" confidence=""0” / "> <movebeforefilter name=""score_res_chainA_disabled”" mover=""keep_only_chain_A”" filter="score_res_disabled" confidence=""0” / "> <movebeforefilter name=""cav_vol_chainA_disabled”" mover=""keep_only_chain_A”" filter="cav_vol_disabled" confidence=""0” / "> <movebeforefilter name=""unsat_core_chainA_disabled”" mover=""keep_only_chain_A”" filter="unsat_core_disabled" confidence=""0” / "> <!--Enabled--> <movebeforefilter name=""packstat_chainA_enabled”" mover=""keep_only_chain_A”" filter="packstat_enabled" confidence=""1” / "> <movebeforefilter name=""sspred_chainA_enabled”" mover=""keep_only_chain_A”" filter="sspred_enabled" confidence=""1” / "> <movebeforefilter name=""score_res_chainA_enabled”" mover=""keep_only_chain_A”" filter="score_res_enabled" confidence=""1” / "> <movebeforefilter name=""all_enabled_filters_chainA”" mover=""keep_only_chain_A”" filter="all_enabled_filters" confidence=""1” / "> <movebeforefilter name=""unsat_core_chainA_enabled”" mover=""keep_only_chain_A”" filter="unsat_core_enabled" confidence=""1” / "> < / movebeforefilter> < / movebeforefilter> < / movebeforefilter> < / movebeforefilter> < / movebeforefilter> < / movebeforefilter> < / movebeforefilter> < / movebeforefilter> < / movebeforefilter> < / movebeforefilter> < / filters> <RESIDUE_SELECTORS> <residuepdbinfohaslabel name=""hotspots”" property=""HOTSPOTB”" / > < / RESIDUE_SELECTORS> <taskoperations> <initializefromcommandline name=""init” / "> <includecurrent name=""inclcur” / "> <limitaromachi2 name="limitchi2" / > <disallowifnonnative name=""only_native_H”" disallow_aas=""H” / "> <readresfile name=""resfile”" filename="". / input.resfile”" / > <preventchainfromrepacking name=""not_chain_B”" chain=""2”" / > <preventchainfromrepacking name=""not_chain_C”" chain=""3”" / > <preventchainfromrepacking name=""not_chain_D”" chain=""4”" / > <!--Select designable residues by sasa and packable by flag--> <selectbysasa name=""only_core_residues”" mode=""mc”" probe_radius="2.0" core_asa="20.0" surface_asa="30.0" core="1" boundary="0" surface="0" verbose="1" / > <!--Restrict Hotspots to Repacking--> <operateonresiduesubset name=""hotspot_onlyrepack”" selector=""hotspots”"> <restricttorepackingrlt / > < / operateonresiduesubset> <!--Layer Design as Tom Helped to set omit operations.Thanks Tom L.:)--> <layerdesign name=""layer_all”" layer=""all”" use_sidechain_neighbors=""True”" pore_radius=""2.0”" verbose=""true”"> <norepackdisulfides name=""disulf”"> <all aa=""c”" specification=""fixed”" operation=""omit”" / > < / norepackdisulfides> <operateonresiduesubset name=""hotspot_onlyrepack_layerdesignOmit”" selector=""hotspots”"> <preventrepackingrlt / > <all specification=""fixed”" operation=""omit”" / > < / operateonresiduesubset> <readresfile name=""resfile_layerdesignOmit”" filename="". / input_fix.resfile”"> <all specification=""fixed”" operation=""omit”" / > < / readresfile> <core> <all append=""M”" / > < / core> <boundary> <all append=""M”" / > < / boundary> < / layerdesign> <layerdesign name=""layer_boundary_surface”" layer=""boundary_surface”" use_sidechain_neighbors=""True”" pore_radius=""2.0”" verbose=""true”"> <norepackdisulfides name=""disulf”"> <all aa=""c”" specification=""fixed”" operation=""omit”" / > < / norepackdisulfides> <operateonresiduesubset name=""hotspot_onlyrepack_layerdesignOmit”" selector=""hotspots”"> <preventrepackingrlt / > <all specification=""fixed”" operation=""omit”" / > < / operateonresiduesubset> <readresfile name=""resfile_layerdesignOmit”" filename="". / input_fix.resfile”"> <all specification=""fixed”" operation=""omit”" / > < / readresfile> <core> <all append=""M”" / > < / core> <boundary> <all append=""M”" / > < / boundary> < / layerdesign> < / disallowifnonnative> < / includecurrent> < / initializefromcommandline> < / taskoperations> <movers> <saveposemover name=""save_RMSDreference_conformation”" reference_name=""reference_conformation” / "> <addconstraintstocurrentconformationmover name="constrainCA" task_operations=""init,resfile,inclcur,limitchi2,only_native_H,layer_all,hotspot_onlyrepack,not_chain_B,not_chain_C,not_chain_D”" CA_only="1" / > <clearconstraintsmover name="clearAllConstraints" / > <PackRotamersMover name="design_all_norep” scorefxn="SFXN6dA_norep_elect” task_operations="init,resfile,inclcur,limitchi2,only_native_H,layer_all,hotspot_onlyrepack,not_chain_B,not_chain_C,not_chain_D” / > <PackRotamersMover name="design_onlyCore_norep” scorefxn="SFXN6dA_norep_elect” task_operations="init,resfile,inclcur,limitchi2,only_native_H,layer_all,hotspot_onlyrepack,only_core_residues,not_chain_B,not_chain_C,not_chain_D” / > <TaskAwareMinMover name="min_vanilla_SC” scorefxn="SFXN6_vanilla” bb="0” chi="1” jump="1” task_operations="init,resfile,inclcur,limitchi2,only_native_H,layer_all,hotspot_onlyrepack,not_chain_B,not_chain_C,not_chain_D” / > <TaskAwareMinMover name="min_vanilla_BBSC” scorefxn="SFXN6_vanilla” bb="1” chi="1” jump="1” task_operations="init,resfile,inclcur,limitchi2,only_native_H,layer_all,hotspot_onlyrepack,not_chain_B,not_chain_C,not_chain_D” / > <FastDesign name="fdesign_all_elec” scorefxn="SFXN6dA_vanilla” task_operations="init,resfile,inclcur,limitchi2,only_native_H,layer_all,hotspot_onlyrepack,not_chain_B,not_chain_C,not_chain_D” only_design_worst_region="0” design_by_psipred="0” design_by_frag_qual="0” repeats="3” clear_designable_residues="0” max_redesigns="2000” > <fastrelax name="fast_relax_vanilla" scorefxn=""SFXN6dA_vanilla”"> <movemap name=""mappyfr”"> <chain number="1" chi="1" bb="1 / "> <chain number="2" chi="0" bb="0 / "> <chain number="3" chi="0" bb="0 / "> <chain number="4" chi="0" bb="0 / "> <jump number="1" setting="0 / "> < / jump> < / chain> < / chain> < / chain> < / chain> < / movemap> < / fastrelax> <parsedprotocol name=""design_all_w_minimize_vanilla”"> <add mover_name=""constrainCA”" / > <!-- START CA-contraints --> <add mover_name=""design_all_norep”" / > <add mover_name=""min_vanilla_SC”" / > <add mover_name=""min_vanilla_BBSC”" / > Add filter_name="rmsd_chainA_enabled” / > <!-- Check RMSD --> <add mover_name=""clearAllConstraints”" / > <!-- END CA-contraints --> < / parsedprotocol> <GenericSimulatedAnnealer name="SA_DesignProtein” mover_name="design_onlyCore_norep” trials="100” periodic_mover="design_all_w_minimize_vanilla” eval_period="20” history="10” bolz_rank="1” recover_low="1” preapply="0” drift="1” checkpoint_file="mc” keep_checkpoint_file="0” filter_name="cav_vol_chainA_disabled” temperature="1.5” sample_type="low” stopping_condition="all_enabled_filters_chainA” > <filters> <and filter_name="unsat_core_chainA_disabled" sample_type=""low”" temperature="0.05" / > <and filter_name=""score_res_chainA_disabled”" sample_type=""low”" temperature="0.05" / > < / filters> <GenericMonteCarlo name="MC_FastDesignProtein” mover_name="fdesign_all_elec” filter_name="cav_vol_chainA_disabled” sample_type="low” trials="3” preapply="0” stopping_condition="all_enabled_filters_chainA” > <filters> <and filter_name=""unsat_core_chainA_disabled”" sample_type=""low”" / > <and filter_name=""score_res_chainA_disabled”" sample_type=""low”" / > < / filters> < / saveposemover> < / movers> <APPLY_TO_POSE> < / APPLY_TO_POSE> <protocols> <add mover_name="save_RMSDreference_conformation" / > <add mover_name="SA_DesignProtein" / > <add filter_name="rmsd1_chainA_enabled" / > <add mover_name="MC_FastDesignProtein" / > <add filter_name="rmsd2_chainA_enabled" / > <add mover_name="fast_relax_vanilla" / > <add filter_name="rmsd3_chainA_enabled" / > <add filter_name="unsat_core_chainA_enabled" / > <add filter_name="score_res_chainA_enabled" / > <add filter_name="sspred_chainA_enabled" / > <add filter_name="packstat_chainA_disabled" / > <add filter_name="cav_vol_chainA_disabled" / > < / protocols> < / rosettascripts> Appendix C. RosettaScripts XML protocol for the sequence design of new mimetics generation 2 (i.e., mimetics based on G1_Neo2_40_1F as template). <rosettascripts> <scorefxns> <SFXN6_vanilla weights=". / talaris2014_cart.wts” symmetric=0 / > <SFXN6dA_vanilla weights=". / talaris2014_cart_downAla.wts” symmetric=0 / > <SFXN6dA_norep_elect weights=". / talaris2014_cart_downAla.wts” symmetric=0 > <reweight scoretype="fa_rep" weight="0.05" / > <reweight scoretype="fa_elec" weight="1.0" / > < / SFXN6dA_norep_elect > <SFXN6dA_elect weights=". / talaris2014_cart_downAla.wts” symmetric=0 > <reweight scoretype="fa_elec" weight="2.0" / > < / SFXN6dA_elect > < / scorefxns> <movers> <switchchainorder name=""keep_only_chain_A”" chain_order=""1” / "> < / switchchainorder> < / movers> <filters> <!--Not Enabled--> <holes name=""holes_disabled”" threshold=""1.2”" confidence=""0” / "> <scoretype name=""score_disabled”" scorefxn=""SFXN6_vanilla”" score_type=""total_score”" threshold="0.0" confidence=""0”" / > <residuecount name=""nres_disabled”" confidence=""0”" / > <packstat name="packstat_disabled" threshold=""0.65”" repeats=""3”" confidence=""0”" / > <ssprediction name=""sspred_disabled”" cmd="" / work / dadriano / PROGRAMS / psipred / runpsipred_single”" use_probability=""0”" use_svm=""0”" threshold="0.85" confidence=""0” / "> <buriedunsathbonds name=""unsat_core_disabled”" cutoff="0" task_operations=""only_core_residues”" jump_number="0" confidence=""0” / "> <rmsdsimple name=""rmsd1_chainA_disabled”" reference_name=""reference_conformation”" chain=""1”" align=""1”" threshold=""1.0”" confidence=""0” / "> <rmsdsimple name=""rmsd2_chainA_disabled”" reference_name=""reference_conformation”" chain=""1”" align=""1”" threshold=""1.0”" confidence=""0” / "> <rmsdsimple name=""rmsd3_chainA_disabled”" reference_name=""reference_conformation”" chain=""1”" align=""1”" threshold=""1.0”" confidence=""0” / "> <calculatorfilter name=""score_res_disabled”" equation=""SCORE / NRES”" threshold=""-1.8”" confidence=""0”"> <score name=""SCORE”" filter_name=""score_disabled”" / > <nres name=""NRES”" filter_name=""nres_disabled”" / > < / calculatorfilter> <!--Enabled--> <packstat name=""packstat_enabled”" threshold=""0.65”" repeats=""3”" confidence=""1”" / > <ssprediction name=""sspred_enabled”" cmd="" / work / dadriano / PROGRAMS / psipred / runpsipred_single”" use_probability=""0”" use_svm=""0”" threshold="0.85" confidence=""1” / "> <calculatorfilter name=""score_res_enabled”" equation=""SCORE / NRES”" threshold=""-1.8”" confidence=""1”"> <score name=""SCORE”" filter_name=""score_disabled”" / > <nres name=""NRES”" filter_name=""nres_disabled”" / > < / calculatorfilter> <cavityvolume name=""cav_vol_disabled”" / > <buriedunsathbonds name=""unsat_core_enabled”" cutoff="0" task_operations=""only_core_residues”" jump_number="0" confidence=""1” / "> <rmsdsimple name=""rmsd1_chainA_enabled”" reference_name=""reference_conformation”" chain=""1”" align=""1”" threshold=""1.0”" confidence=""1” / "> <rmsdsimple name=""rmsd2_chainA_enabled”" reference_name=""reference_conformation”" chain=""1”" align=""1”" threshold=""1.0”" confidence=""1” / "> <rmsdsimple name=""rmsd3_chainA_enabled”" reference_name=""reference_conformation”" chain=""1”" align=""1”" threshold=""1.0”" confidence=""1” / "> <compoundstatement name="all_enabled_filters"> <and filter_name="sspred_enabled" / > <and filter_name="score_res_enabled" / > <and filter_name="packstat_enabled" / > <and filter_name="unsat_core_enabled" / > < / compoundstatement> < / rmsdsimple> < / rmsdsimple> < / rmsdsimple> < / buriedunsathbonds> < / ssprediction> < / rmsdsimple> < / rmsdsimple> < / rmsdsimple> < / buriedunsathbonds> < / ssprediction> < / holes> < / filters> <filters> <!--Chain A Filters--> <!--Not Enabled--> <movebeforefilter name=""packstat_chainA_disabled”" mover=""keep_only_chain_A”" filter="packstat_disabled" confidence=""0” / "> <movebeforefilter name=""sspred_chainA_disabled”" mover=""keep_only_chain_A”" filter="sspred_disabled" confidence=""0” / "> <movebeforefilter name=""score_res_chainA_disabled”" mover=""keep_only_chain_A”" filter="score_res_disabled" confidence=""0” / "> <movebeforefilter name=""cav_vol_chainA_disabled”" mover=""keep_only_chain_A”" filter="cav_vol_disabled" confidence=""0” / "> <movebeforefilter name=""unsat_core_chainA_disabled”" mover=""keep_only_chain_A”" filter="unsat_core_disabled" confidence=""0” / "> <!--Enabled--> <movebeforefilter name=""packstat_chainA_enabled”" mover=""keep_only_chain_A”" filter="packstat_enabled" confidence=""1” / "> <movebeforefilter name=""sspred_chainA_enabled”" mover=""keep_only_chain_A”" filter="sspred_enabled" confidence=""1” / "> <movebeforefilter name=""score_res_chainA_enabled”" mover=""keep_only_chain_A”" filter="score_res_enabled" confidence=""1” / "> <movebeforefilter name=""all_enabled_filters_chainA”" mover=""keep_only_chain_A”" filter="all_enabled_filters" confidence=""1” / "> <movebeforefilter name=""unsat_core_chainA_enabled”" mover=""keep_only_chain_A”" filter="unsat_core_enabled" confidence=""1” / "> < / movebeforefilter> < / movebeforefilter> < / movebeforefilter> < / movebeforefilter> < / movebeforefilter> < / movebeforefilter> < / movebeforefilter> < / movebeforefilter> < / movebeforefilter> < / movebeforefilter> < / filters> <RESIDUE_SELECTORS> <residuepdbinfohaslabel name=""hotspots”" property=""HOTSPOTB”" / > < / RESIDUE_SELECTORS> <taskoperations> <initializefromcommandline name=""init” / "> <includecurrent name=""inclcur” / "> <limitaromachi2 name="limitchi2" / > <disallowifnonnative name=""only_native_H”" disallow_aas=""H” / "> <readresfile name=""resfile”" filename="". / input.resfile”" / > <preventchainfromrepacking name=""not_chain_B”" chain=""2”" / > <preventchainfromrepacking name=""not_chain_C”" chain=""3”" / > <preventchainfromrepacking name=""not_chain_D”" chain=""4”" / > <!--Select designable residues by sasa and packable by flag--> <selectbysasa name=""only_core_residues”" mode=""mc”" probe_radius="2.0" core_asa="20.0" surface_asa="30.0" core="1" boundary="0" surface="0" verbose="1" / > <!--Restrict Hotspots to Repacking--> <operateonresiduesubset name=""hotspot_onlyrepack”" selector=""hotspots”"> <restricttorepackingrlt / > < / operateonresiduesubset> <!--Layer Design as Tom Helped to set omit operations.Thanks Tom L.:)--> <layerdesign name=""layer_all”" layer=""all”" use_sidechain_neighbors=""True”" pore_radius=""2.0”" verbose=""true”"> <norepackdisulfides name=""disulf”"> <all aa=""c”" specification=""fixed”" operation=""omit”" / > < / norepackdisulfides> <operateonresiduesubset name=""hotspot_onlyrepack_layerdesignOmit”" selector=""hotspots”"> <preventrepackingrlt / > <all specification=""fixed”" operation=""omit”" / > < / operateonresiduesubset> <readresfile name=""resfile_layerdesignOmit”" filename="". / input_fix.resfile”"> <all specification=""fixed”" operation=""omit”" / > < / readresfile> <core> <all append=""M”" / > < / core> <boundary> <all append=""M”" / > < / boundary> < / layerdesign> <layerdesign name=""layer_boundary_surface”" layer=""boundary_surface”" use_sidechain_neighbors=""True”" pore_radius=""2.0”" verbose=""true”"> <norepackdisulfides name=""disulf”"> <all aa=""c”" specification=""fixed”" operation=""omit”" / > < / norepackdisulfides> <operateonresiduesubset name=""hotspot_onlyrepack_layerdesignOmit”" selector=""hotspots”"> <preventrepackingrlt / > <all specification=""fixed”" operation=""omit”" / > < / operateonresiduesubset> <readresfile name=""resfile_layerdesignOmit”" filename="". / input_fix.resfile”"> <all specification=""fixed”" operation=""omit”" / > < / readresfile> <core> <all append=""M”" / > < / core> <boundary> <all append=""M”" / > < / boundary> < / layerdesign> < / disallowifnonnative> < / includecurrent> < / initializefromcommandline> < / taskoperations> <movers> <saveposemover name=""save_RMSDreference_conformation”" reference_name=""reference_conformation” / "> <addconstraintstocurrentconformationmover name="constrainCA" task_operations=""init,resfile,inclcur,limitchi2,only_native_H,layer_all,hotspot_onlyrepack,not_chain_B,not_chain_C,not_chain_D”" CA_only="1" / > <clearconstraintsmover name="clearAllConstraints" / > <PackRotamersMover name="design_all_norep” scorefxn="SFXN6dA_norep_elect” task_operations="init,resfile,inclcur,limitchi2,only_native_H,layer_all,hotspot_onlyrepack,not_chain_B,not_chain_C,not_chain_D” / > <PackRotamersMover name="design_onlyCore_norep” scorefxn="SFXN6dA_norep_elect” task_operations="init,resfile,inclcur,limitchi2,only_native_H,layer_all,hotspot_onlyrepack,only_core_residues,not_chain_B,not_chain_C,not_chain_D” / > <TaskAwareMinMover name="min_vanilla_SC” scorefxn="SFXN6_vanilla” bb="0” chi="1” jump="1” task_operations="init,resfile,inclcur,limitchi2,only_native_H,layer_all,hotspot_onlyrepack,not_chain_B,not_chain_C,not_chain_D” / > <TaskAwareMinMover name="min_vanilla_BBSC” scorefxn="SFXN6_vanilla” bb="1” chi="1” jump="1” task_operations="init,resfile,inclcur,limitchi2,only_native_H,layer_all,hotspot_onlyrepack,not_chain_B,not_chain_C,not_chain_D” / > <FastDesign name="fdesign_all_elec” scorefxn="SFXN6dA_vanilla” task_operations="init,resfile,inclcur,limitchi2,only_native_H,layer_all,hotspot_onlyrepack,not_chain_B,not_chain_C,not_chain_D” only_design_worst_region="0” design_by_psipred="0” design_by_frag_qual="0” repeats="3” clear_designable_residues="0” max_redesigns="2000” > <fastrelax name="fast_relax_vanilla" scorefxn=""SFXN6dA_vanilla”"> <movemap name=""mappyfr”"> <chain number="1" chi="1" bb="1 / "> <chain number="2" chi="0" bb="0 / "> <chain number="3" chi="0" bb="0 / "> <chain number="4" chi="0" bb="0 / "> <jump number="1" setting="0 / "> < / jump> < / chain> < / chain> < / chain> < / chain> < / movemap> < / fastrelax> <parsedprotocol name=""design_all_w_minimize_vanilla”"> <add mover_name=""constrainCA”" / > <!-- START CA-contraints --> <add mover_name=""design_all_norep”" / > <add mover_name=""min_vanilla_SC”" / > <add mover_name=""min_vanilla_BBSC”" / > Add filter_name="rmsd_chainA_enabled” / > <!-- Check RMSD --> <add mover_name=""clearAllConstraints”" / > <!-- END CA-contraints --> < / parsedprotocol> <GenericSimulatedAnnealer name="SA_DesignProtein” mover_name="design_onlyCore_norep” trials="100” periodic_mover="design_all_w_minimize_vanilla” eval_period="20” history="10” bolz_rank="1” recover_low="1” preapply="0” drift="1” checkpoint_file="mc” keep_checkpoint_file="0” filter_name="cav_vol_chainA_disabled” temperature="1.5” sample_type="low” stopping_condition="all_enabled_filters_chainA” > <filters> <and filter_name="unsat_core_chainA_disabled" sample_type=""low”" temperature="0.05" / > <and filter_name=""score_res_chainA_disabled”" sample_type=""low”" temperature="0.05" / > < / filters> <GenericMonteCarlo name="MC_FastDesignProtein” mover_name="fdesign_all_elec” filter_name="cav_vol_chainA_disabled” sample_type="low” trials="3” preapply="0” stopping_condition="all_enabled_filters_chainA” > <filters> <and filter_name=""unsat_core_chainA_disabled”" sample_type=""low”" / > <and filter_name=""score_res_chainA_disabled”" sample_type=""low”" / > < / filters> < / saveposemover> < / movers> <APPLY_TO_POSE> < / APPLY_TO_POSE> <protocols> <add mover_name="save_RMSDreference_conformation" / > <add mover_name="SA_DesignProtein" / > <add filter_name="rmsd1_chainA_enabled" / > <add mover_name="MC_FastDesignProtein" / > <add filter_name="rmsd2_chainA_enabled" / > <add mover_name="fast_relax_vanilla" / > <add filter_name="rmsd3_chainA_enabled" / > <add filter_name="unsat_core_chainA_enabled" / > <add filter_name="score_res_chainA_enabled" / > <add filter_name="sspred_chainA_enabled" / > <add filter_name="packstat_chainA_disabled" / > <add filter_name="cav_vol_chainA_disabled" / > < / protocols> < / rosettascripts>

Claims

1. A non-naturally occurring polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 181, wherein the polypeptide binds to the IL-2 receptor βγ c heterodimer (IL-2Rβγ c ).

2. A polypeptide described in claim 1, comprising an amino acid sequence that is at least 95%, at least 98%, or 100% identical to the amino acid sequence of SEQ ID NO:

181.

3. A polypeptide described in claim 2, comprising the amino acid sequence of SEQ ID NO:

181.

4. A polypeptide described in any one of claims 1 to 3, linked to a stabilizing compound.

5. A polypeptide described in claim 1 or 2, comprising a cysteine ​​residue and one or more polyethylene glycol chains linked to the cysteine ​​residue via a maleimide group.

6. A polypeptide described in claim 1 or 2, wherein an amino acid residue of the polypeptide is mutated to a cysteine ​​residue for attachment of a stabilizing moiety.

7. The polypeptide according to claim 1, wherein the affinity of the polypeptide for the human IL-2 receptor βγ c heterodimer (IL-2Rβγ c ) is higher than that of natural IL-2.

8. A polypeptide described in any one of claims 1 to 3, wherein the polypeptide stimulates phosphorylation of STAT5 in cells expressing the IL-2 receptor with greater potency than natural IL-2.

9. A polypeptide described in any one of claims 1 to 3, comprising at least one disulfide bond.

10. A polypeptide described in any one of claims 1 to 3, further comprising a targeting domain.

11. The polypeptide described in claim 10, wherein the targeting domain is a translational fusion with the polypeptide.

12. The polypeptide described in claim 10, wherein the targeting domain binds to a cell surface protein.

13. The polypeptide of claim 12, wherein the cell surface protein is present on the surface of a cell selected from tumor cells, vascular cells, tumor microenvironment cells, and immune cells, and is selected from the group consisting of CD3, CD4, CD8, CD19, CD20, CD21, CD25, CD37, CD30, CD33, CD40, CD68, CD123, CD254, PD-1, B7-H3, and CTLA-4.

14. The polypeptide described in claim 10, wherein the targeting domain binds to a surface marker of tumor cells, tumor vascular cells, or tumor microenvironment cells.

15. The surface marker, EGFR, EGFRvIII, Her2, HER3, EpCAM, MSLN, MUC16, PSMA, TROP2, ROR1, RON, PD -L1, CD47, CTLA-4, CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD40, CD45, CAM PATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC, HLD-DR, carcinoembryonic antigen (CEA), TAG-72, EpCAM, MUC1, folate-binding protein, A33, G250, prostate-specific membrane antigen (PSMA), ferritin, GD2, GD3, GM2, Le y , CA-125, CA19-9, epidermal growth factor, p185HER2, IL-2 receptor, EGFRvIII (de2-7 EGFR), fibroblast activation protein, tenascin, metalloproteinase, endosialin, vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6, HPV E7, Her-2 / neu, MAGE A3, non-mutated p53, NY-ESO-1, MelanA / MART1, Ras mutant, gp100, p53 mutant, PR1, bcr-abl, tyronsinase, survivin, PSA, hTERT, sarcoma translocation breakpoint protein, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, mesothelin (MSLN), PSCA, MAGE Al, sLe (animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TESL sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, legumain, Tie3, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, TRAIL1, MUC16, MAGE A4, MAGE C2, GAGE, EGFR, CMET, HER3, MUC15, CA6, NAPI2B, TROP2, CLDN6, CLDN16, CLDN18.2, CLorf186, RON, LY6E, FRA, DLL3, PTK7, STRA6, TMPRSS3, TMPRSS4, TMEM238, UPK1B, VTCN1, LIV1, ROR1, or Fos-related antigen 1, Or a marker from the list below: (1) BMPR1B (bone morphogenetic protein receptor type IB); (2) E16 (LAT1, SLC7A5); (3) STEAP1 (six-transmembrane epithelial antigen of the prostate); (4) 0772P (CA125, MUC16); (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin); (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b); (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B); (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene); (9) ETBR (endothelin type B receptor); (10) MSG783 (RNF124, hypothetical protein FLJ20315); (11) STEAP2 (HGNC-8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, prostate six-transmembrane epithelial antigen 2, six-transmembrane prostate protein); (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4); (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratoma-derived growth factor); (14) CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor) or Hs.73792); (15) CD79b (IGb (immunoglobulin-related beta), B29); (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C); (17) HER2; (18) NCA; (19) MDP; (20) IL20R. alpha; (21) Brevican; (22) Ephb2R; (23) ASLG659; (24) PSCA; (25) GEDA; (26) BAFF-R; (27) CD22; (28) CD79a (CD79A, CD79α, immunoglobulin-related α, a B cell-specific protein that covalently interacts with Igβ (CD79B), forms a complex with IgM molecules on the surface, and transmits signals involved in B cell differentiation); (29) CXCR5 (Burkitt lymphoma receptor 1, a G protein-coupled receptor activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, and is involved in HIV-2 infection and the development of AIDS, lymphoma, myeloma, and leukemia); (30) HLA-DOB (β subunit of MHC class II molecules (Ia antigens) that bind peptides and present them to CD4+ T lymphocytes); (31) P2X5 (purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, involved in synaptic transmission and neurogenesis, whose deficiency contributes to the pathophysiology of idiopathic detrusor instability); (32) CD72 (B cell differentiation antigen CD72, Lyb-2); (33) LY64 (lymphocyte antigen 64 (RP105), a type I membrane protein of the leucine-rich repeat (LRR) family, which regulates B cell activation and apoptosis and whose loss of function is associated with increased disease activity in patients with systemic lupus erythematosus); (34) FCRH1 (Fc receptor-like protein 1, a receptor for immunoglobulin Fc domains containing C2-type Ig-like and ITAM domains and playing a role in B lymphocyte differentiation); or (35) IRTA2 (Immunoglobulin superfamily receptor translocation-associated 2, an immune receptor with a role in B cell development and lymphomagenesis; gene deregulation due to translocation occurs in some B cell malignancies) 15. The polypeptide of claim 14, 16. The polypeptide of claim 10, wherein the targeting domain binds to an immune cell surface marker selected from the group consisting of CD3, CD4, CD8, CD19, CD20, CD21, CD25, CD37, CD30, CD33, CD40, CD68, CD123, CD254, PD-1, B7-H3, and CTLA-4.

17. The polypeptide of claim 10, wherein the targeting domain is an scFv, F(ab), F(ab')2, B-cell receptor (BCR), DARPin, affibody, monobody, nanobody, diabody, antibody, cell-targeting oligopeptide, RGD, integrin-binding peptide, aptamer, bicyclic peptide, conotoxin, or viral protein.

18. A recombinant nucleic acid encoding a polypeptide described in any one of claims 1 to 3.

19. An expression vector comprising the recombinant nucleic acid of claim 18 operably linked to a promoter.

20. A recombinant host cell comprising the recombinant nucleic acid described in claim 18.

21. A pharmaceutical composition for the treatment of cancer, comprising a polypeptide according to any one of claims 1 to 3.

22. Use of a polypeptide according to any one of claims 1 to 3 in the manufacture of a pharmaceutical composition for the treatment of cancer.

23. Use of the recombinant nucleic acid of claim 18 in the manufacture of a pharmaceutical composition for the treatment of cancer.

24. The cancer is colon cancer, melanoma, renal cell carcinoma, squamous cell carcinoma of the head and neck, gastric cancer, urothelial carcinoma, Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, pancreatic cancer, Merkel cell carcinoma, colorectal cancer, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma 22. The pharmaceutical composition of claim 21, wherein the cancer is pancreatic cancer, multiple myeloma, ovarian cancer, or cervical cancer.

25. The cancer is colon cancer, melanoma, renal cell carcinoma, head and neck squamous cell carcinoma, gastric cancer, urothelial carcinoma, Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, pancreatic cancer, Merkel cell carcinoma, colorectal cancer, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma 23. The use of the polypeptide of claim 22 in the treatment of pancreatic cancer, multiple myeloma, ovarian cancer, or cervical cancer.

26. The cancer is colon cancer, melanoma, renal cell carcinoma, head and neck squamous cell carcinoma, gastric cancer, urothelial carcinoma, Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, pancreatic cancer, Merkel cell carcinoma, colorectal cancer, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma 24. The use of the recombinant nucleic acid of claim 23, wherein the cancer is human pancreatic cancer, multiple myeloma, ovarian cancer, or cervical cancer.

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