Split interleukin mimetics and their use

Abstract

To provide split interleukin mimetics and their use.SOLUTION: Conditionally active receptor agonists that, when activated, bind to IL-2 receptor βγC heterodimer (IL-2RβγC), IL-4 receptor αγC heterodimer (IL-4RαγC), or IL-13 receptor a subunit (IL-13Rα) are disclosed, as are components of the conditionally active receptor agonists and methods for using the conditionally active receptor agonists.SELECTED DRAWING: None

Description

[Technical field]

[0001] cross reference This application claims priority to U.S. Provisional Patent Application No. 62 / 770,152, filed November 20, 2018, which is incorporated by reference in its entirety as a part of this specification. [Background technology]

[0002] The great potential of the major immune cytokines interleukins, such as IL-2 and IL-4 for cancer therapy, has led to numerous attempts to improve their therapeutic properties by mutation and / or chemical modification. However, these approaches are limited by the low stability and limited expression of the IL-2 receptor α subunit (IL-2Rα), IL-4 receptor αγ subunit, and IL-2 receptor αγ subunit, due to their close association with native IL-2 or IL-4. c Heterodimer (IL-4Rαγ c ), or undesirable properties such as binding to the IL-13 receptor α subunit (IL-13Rα). Summary of the Invention

[0003] In one aspect, the disclosure provides a non-naturally occurring conditionally active receptor agonist comprising a first polypeptide component and a second polypeptide component, wherein the first polypeptide component and the second polypeptide component are not present in a fusion protein, and the first polypeptide component and the second polypeptide component collectively comprise domains X1, X2, X3, and X4; (a) X1 is a peptide

[0004] [ka]

[0005] a peptide comprising an amino acid sequence 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 (b) X2 is a helical peptide domain at least 8 amino acids in length; (c) X3 is a peptide

[0006] [ka]

[0007] a peptide comprising an amino acid sequence 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 (d) X4 is a peptide

[0008] [ka]

[0009] a peptide comprising an amino acid sequence 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 (i) the amino acid residue in brackets may be present or absent; (ii) the first polypeptide component comprises at least one of X1, X2, X3, and X4, but does not comprise each of X1, X2, X3, and X4; (iii) the second polypeptide component comprises X1, which is not present in the first polypeptide component; Each of X2, X3, and X4 includes The first polypeptide component and the second polypeptide component are not individually active receptor agonists, and the first polypeptide component and the second polypeptide component interact to activate the IL-2 receptor βγ cHeterodimer (IL-2Rβγ c ), IL-4 receptor αγ c Heterodimer (IL-4Rαγ c ), IL-13 alpha, or forms an active agonist of the IL-4R alpha / IL13R alpha heterodimer. Numerous embodiments of the first and second polypeptides are provided herein. In an exemplary embodiment, (i) the first polypeptide component comprises one of X1, X2, X3, and X4, and the second polypeptide component comprises three of X1, X2, X3, and X4 that are not present in the first polypeptide component; or (ii) the first polypeptide component comprises two of X1, X2, X3, and X4, and the second polypeptide component comprises two of X1, X2, X3, and X4 that are not present in the first polypeptide component. (i) the first polypeptide comprises X1, and the second polypeptide comprises X2, X3, and X4; (ii) the first polypeptide comprises X2 and the second polypeptide comprises X1, X3, and X4; (iii) the first polypeptide comprises X3 and the second polypeptide comprises X1, X2, and X4; (iv) the first polypeptide comprises X4 and the second polypeptide comprises X1, X2, and X3; (v) the first polypeptide comprises X1 and X2, and the second polypeptide comprises X3 and X4; (vi) the first polypeptide comprises X1 and X3, and the second polypeptide comprises X2 and X4; (vii) the first polypeptide comprises X1 and X4, and the second polypeptide comprises X2 and X3; (viii) the first polypeptide comprises X2 and X3, and the second polypeptide comprises X1 and X4; (ix) the first polypeptide comprises X2 and X4, and the second polypeptide comprises X1 and X3; (x) the first polypeptide comprises X3 and X4, and the second polypeptide comprises X1 and X2; (xi) the first polypeptide comprises X1, X2, and X3, and the second polypeptide comprises X4; (xii) the first polypeptide comprises X1, X2, and X4, and the second polypeptide comprises X3; (xiii) the first polypeptide comprises X1, X3, and X4, and the second polypeptide comprises X2; or (xiv) the first polypeptide comprises X2, X3, and X4, and the second polypeptide comprises X1.

[0010] In other exemplary embodiments, the first and second polypeptide components may be non-covalently bound, and / or the first and second polypeptide components may be indirectly bound to each other via a receptor. In further exemplary embodiments, the first polypeptide component further comprises a first targeting domain, and / or the second polypeptide component further comprises a second targeting domain, and in some embodiments, the first targeting domain, if present, is a translational fusion with the first polypeptide, and the second targeting domain, if present, is a translational fusion with the second polypeptide. In some embodiments, the targeting domain may bind to a cell surface protein.

[0011] In another embodiment, the present disclosure provides a polypeptide comprising one, two or three, but not all four, of domains X1, X2, X3 and X4, (a) X1, if present, is a peptide

[0012] [ka]

[0013] a peptide comprising an amino acid sequence 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 (b) X2, if present, is any helical peptide domain; (c) X3, if present, is a peptide

[0014] [ka]

[0015] a peptide comprising an amino acid sequence 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 (d) X4, if present, is a peptide

[0016] [ka]

[0017] a peptide comprising an amino acid sequence 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 The amino acid residues in brackets may or may not be present. Numerous embodiments of polypeptides are provided. In some exemplary embodiments, the polypeptide comprises: (i) a polypeptide that includes X1 and excludes X2, X3, and X4; (ii) a polypeptide that includes X2 and excludes X1, X3, and X4; (iii) a polypeptide that includes X3 and excludes X1, X2, and X4; (iv) a polypeptide that includes X4 and excludes X1, X2, and X3; (v) a polypeptide comprising X1 and X2 and excluding X3 and X4; (vi) a polypeptide comprising X1 and X3 and excluding X2 and X4; (vii) a polypeptide comprising X1 and X4 and excluding X2 and X3; (viii) a polypeptide comprising X2 and X3 and excluding X1 and X4; (ix) a polypeptide comprising X2 and X4 and excluding X1 and X3; (x) a polypeptide comprising X3 and X4 and excluding X1 and X2; (xi) a polypeptide comprising X1, X2, and X3, but excluding X4; (xii) a polypeptide comprising X1, X2, and X4, but excluding X3; (xiii) a polypeptide comprising X1, X3, and X4, and excluding X2; and (xiv) A polypeptide including X2, X3, and X4, but excluding X1.

[0018] In another exemplary embodiment, the polypeptide further comprises a targeting domain, including but not limited to a targeting domain that is a translational fusion with the polypeptide. In some embodiments, the targeting domain is capable of binding to a cell surface protein.

[0019] In other aspects, the disclosure provides an expression vector comprising a nucleic acid encoding a polypeptide, a first polypeptide, or a second polypeptide of any of the disclosed embodiments, a nucleic acid operably linked to a promoter, a nucleic acid and / or an expression vector disclosed herein. and a pharmaceutical composition comprising a conditionally active receptor agonist, polypeptide, nucleic acid, expression vector, or host cell of any of the disclosed embodiments and a pharma- ceutical acceptable carrier.

[0020] The present disclosure also provides a method of treating cancer, the method comprising administering a conditionally active receptor agonist of any embodiment disclosed herein to a subject in need thereof under conditions where the first polypeptide component and the second polypeptide component interact in cells of the tumor to treat the cancer.

[0021] In another aspect, the present disclosure provides a conditionally active receptor agonist, polypeptide, nucleic acid, expression vector, host cell, or pharmaceutical composition of any of the disclosed embodiments for use as an medicament for treating cancer and / or for modulating an immune response in a subject.

[0022] In a further aspect, the disclosure provides a method of stimulating an IL-2 receptor or an IL-4 receptor, the method comprising administering to a subject a conditionally active receptor agonist of any embodiment disclosed herein under conditions in which the first polypeptide component and the second polypeptide component interact at the receptor.

[0023] The following figures conform to exemplary embodiments. [Brief description of the drawings]

[0024] [Figure 1]Figures 1A-1C. Computational design of de novo cytokine mimetics. Figure 1A) The designed non-split mimic has four helices, three mimicking IL-2 interacting with hIL-2Rβγc, while the fourth holds the first three in place. Top: In the first generation design, each of the core elements of IL-2 (helices H1-H4) were individually idealized using fragment assemblies from a clustered ideal fragment database (size: 4 amino acids). Bottom: In the second generation design, the core elements were instead constructed using parametric equations reproducing the shape of each separated helix, allowing the length of each helix to be altered by + / - 8 amino acids. Figure 1B) The helix pairs were relinked using idealized loop fragments (size: 4 or 7 amino acids for gen-1 and gen-2, respectively, see Methods), and a representative example is shown with the newly constructed elements linking each pair of helices. Figure 1C) The helical hairpins created in Figure 1B were assembled in all possible combinations to create a fully linked protein scaffold. [Diagram 2] Properties of neoleukin-2 / 15. Binding experiments (biolayer interferometry) show that incubation of neoleukin-2 / 15 at 80°C for 2 hours does not result in significant loss of binding, whereas human and mouse IL-2 are rapidly inactivated. [Diagram 3]Figures 3A-3C. Reengineering of neoleukin-2 / 15 into a human interleukin-4 (hIL-4) mimic (neoleukin-4). Figure 3A) Neo-2 / 15 structurally aligned to the structure of IL-4 in complex with IL-4Rα and γC (from PDB code 3BPL). The 14 IL-4 residues that contact IL-4Rα and were grafted to Neo-2 / 15 are labeled. Figure 3B) Neo-4, a new protein, with 16 amino acid mutations compared to Neo-2 / 15. These mutations are labeled, 13 of which are derived from IL-4 residues that mediate contact with IL-4Rα (indicated in panel "a"), and three of these (H8M, K68I, and I98F, underlined in the figure) were introduced by directed evolution using random mutagenesis and screening for high binding affinity variants. FIG. 3C) Biolayer interferometry data showing that Neo-4, like IL-4, binds to IL-4Rα alone and has no affinity for γc alone, but binds to γc when IL-4Rα is present in solution. [Figure 4]Figures 4A-4D. 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 created using combined data obtained 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 mimetics of mouse and human IL-2, and although some have different topologies than Neo-2 / 15, all share the four helices H1 (Figure 4A, Neo-2 / 15 1-22 is SEQ ID NO: 04, IL-2 6-27 is SEQ ID NO: 248, and IL-15 1-15 is SEQ ID NO: 249), H3 (Figure 4B, Neo-2 / 15 34-55 is SEQ ID NO: 05, IL-2 82-103 is SEQ ID NO: 250, and IL-15 59-80 is SEQ ID NO: 251), H2' (Figure 4C, Neo-2 / 15 58-76 is SEQ ID NO: 07, IL-2 50-68 is SEQ ID NO: 252, and IL-15 34-52 is SEQ ID NO: 253), and H4 (Figure 4D, Neo-2 / 15 IL-2 80-100 is SEQ ID NO: 06, IL-2 111-131 is SEQ ID NO: 254, and IL-15 93-113 is SEQ ID NO: 255). The logos show the combinatorial information of each helix independently. The line graph below each logo shows the probability score of each amino acid in the Neo-2 / 15 sequence (higher means more conserved). The solid horizontal lines highlight positions where Neo-2 / 15 amino acids have a probability score ≥ 30% (i.e., these amino acids contribute more commonly to receptor binding because they are overall enriched in the binding population of 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 helices in human IL-2 and IL-15 (structurally aligned) are shown below the graphs, highlighting the uniqueness of the Neo-2 / 15 helices and binding interface. [Diagram 5]Figures 5A-5D. Experimental optimization of G1_neo2_40. Figures 5A-5C) Heat maps of the G1_neo2_40 single-site mutagenesis library showing enrichment at specific positions after successive rounds of increasing selection with IL-2Rβγc heterodimer at 50 nM, 2 nM, and 0.1 nM. Based on these enrichment data, a combinatorial library was designed with a nucleotide diversity of 1.5 x 106. Figure 5D) 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 that appear in the combinatorial library and grey indicates residues that do not appear in the combinatorial library. [Figure 6] Figures 6A-6E. Experimental optimization of G2_neo2_40_1F_seq27. Heat maps of the G2_neo2_40_1F_seq27 single-site mutagenesis library showing enrichment at specific positions after successive rounds of increasing selection with Figure 6A) 10 nM, Figure 6B) 1 nM, Figure 6C) 0.1 nM, and Figure 6D) 0.1 nM IL-2RβγC heterodimer. Based on these enrichment data, a combinatorial library was designed with a nucleotide diversity of 5.3 x 106. Figure 6E) The available amino acid residues in the initial combinatorial library are depicted to indicate residues predicted to be advantageous, with black indicating residues in the starting sequence that will appear in the combinatorial library. [Figure 7]Figures 7A-7E. Experimental optimization of G2_neo2_40_1F_seq29. Heat maps of the G2_neo2_40_1F_seq29 single-site mutagenesis library showing enrichment at specific positions after successive rounds of increasing selection with Figure 7A) 10 nM, Figure 7B) 1 nM, Figure 7C) 0.1 nM, and Figure 7D) 0.1 nM IL-2RβγC heterodimer. Based on these enrichment data, a combinatorial library was designed with a nucleotide diversity of 2.9 x 106. Figure 7E) The available amino acid residues in the initial combinatorial library are depicted to indicate residues predicted to be advantageous, with black indicating residues in the starting sequence that will appear in the combinatorial library. [Figure 8] Figures 8A-8E. Experimental optimization of G2_neo2_40_1F_seq36. Heat maps of the G2_neo2_40_1F_seq36 single-site mutagenesis library showing enrichment at specific positions after successive rounds of increasing selection with Figure 8A) 10 nM, Figure 8B) 1 nM, Figure 8C) 0.1 nM, and Figure 8D) 0.1 nM IL-2RβγC heterodimer. Based on these enrichment data, a combinatorial library was designed with a nucleotide diversity of 2.7 x 106. Figure 8E) The available amino acid residues in the initial combinatorial library are depicted to indicate residues predicted to be advantageous, with black indicating residues in the starting sequence that will appear in the combinatorial library. [Figure 9] Figures 9A-9B. Circular dichroism (CD) thermal denaturation experiments of multiple IL-2 / IL-15 de novo designed mimetics, first generation. Figure 9A) Thermal denaturation curves and Figure 9B) wavelength scans. [Figure 10] Figures 10A-10B. Circular dichroism (CD) thermal denaturation experiments of multiple experimentally optimized IL-2 / IL-15 de novo designed mimetics, first generation. Figure 10A) Thermal denaturation curves and Figure 10B) wavelength scans. [Figure 11] Figures 11A-11D. Circular dichroism thermal melting for IL-2 / IL-15 mimetic design, second generation. Figures 11A and 11C) Thermal denaturation curves and Figures 11B and 11D) wavelength scans. [Figure 12]Figures 12A-12C. Expression, purification, and thermal denaturation characteristics of neoleukin-2 / 15. Figure 12A) SDS Tris-Tricine gel electrophoresis showing expression and purification on an affinity column. Figure 12B) Circular dichroism at 222 nm during thermal melting from 25°C to 95°C shows robust temperature stability. Figure 12C) Circular dichroism wavelength scans at 25°C, 95°C, and then again at 25°C, showing that neoleukin-2 / 15 does not completely melt at 95°C and fully refolds after cooling to 25°C. [Figure 13] Figures 13A-13B. Robustness of neolukin-2 / 15 to single point cysteine ​​mutants at non-binding interface positions. Figure 13A) 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. Positions were chosen that avoided interference with receptor binding. Figure 13B) Association kinetics of Neolukin-2 / 15 cysteine ​​mutants with IL-2Rβγc measured using biolayer interferometry. All variants associate with the receptor nearly as well as Neo-2 / 15. [Figure 14] Figures 14A-14C. Expression, purification, and thermal denaturation characteristics of neoleukin-4. Figure 14A) SDS Tris-Tricine gel electrophoresis showing expression and purification on an affinity column. Figure 14B) Circular dichroism at 222 nm during thermal melting from 25°C to 95°C shows robust temperature stability. Figure 14C) Circular dichroism wavelength scans at 25°C, 95°C, and then again at 25°C, showing that neoleukin-4 does not completely melt at 95°C and fully refolds after cooling to 25°C. [Figure 15]Figure 15A-C. Split Neo-2 / 15 variants can reconstitute Neoleukin-2 / 15 activity by binding to the human IL-2 receptor. a. Biolayer interference binding assay of Neo-2 / 15 split between helices H1+H3-H2'-H4 (Neo2A1 and Neo2B1, respectively) to the human IL-2 receptor. Binding data were collected on an Octet RED96 (ForteBio) and processed using ForteBio™ data analysis software version 9.0.0.10. Biotinylated target receptor human γc was immobilized on a streptavidin-coated biosensor (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). After loading the γc target receptor onto the biosensor, baseline measurements were performed by immersing the biosensor in binding buffer only, and then binding kinetics were observed by immersing the biosensor in wells containing the target analyte protein (association step) and then immersing the sensor back into baseline / buffer (dissociation). In the association step, the analyte proteins (i.e., Neoleukin-2 / 15, Neo2A1, Neo2B1, and an equimolar ratio of Neo2A1+Neo2B1) were diluted from concentrated stocks into binding buffer to a final concentration of 100 nM. Human IL-2Rβ was also added to the solution at a saturating concentration (250 nM). b. Biolayer interference binding assay of Neo-2 / 15 split into helices H1-H3+H2'-H4 (Neo2A2 and Neo2B2, respectively) according to the experimental protocol previously described. c. Biolayer interference binding assay of Neo-2 / 15 split into helices H1-H3-H2'+H4 (Neo2A3 and Neo2B3, respectively) according to the experimental protocol described above. [Figure 16]Figure 16A-C. Split-Neo-2 / 15 variants stimulate cell signaling by STAT5 phosphorylation in mouse CTLL-2 cell line. a.CTLL-2 cells were starved for 2 hours prior to assay in cytokine-free medium (RPMI, 10% FBS, +1 mM sodium pyruvate, +2 mM L-glutamine, 1% P / S). Cells were seeded in 96-well plates and resuspended in RPMI medium containing serial dilutions of protein samples: Neoleukin-2 / 15, Neo2A1, Neo2B1, and an equimolar ratio of Neo2A1+Neo2B1. Cells were stimulated for 15 minutes at 37°C and immediately fixed by adding formaldehyde to 1.5% and incubating at room temperature for 10 minutes. Cells were permeabilized by resuspending in cold 100% methanol at 4°C for 30 minutes. Fixed and permeabilized cells were washed twice with FACS buffer (PBS pH 7.2 with 0.1% bovine serum albumin) and incubated with anti-STAT5 pY694 conjugated with Alexa™ Fluor647 (BD Biosciences) diluted 1:50 in FACS buffer for 2 h at room temperature. Cells were then washed twice with FACS buffer and mean fluorescence intensity (MFI) was measured on a Guava easyCyte™ flow cytometer (Millipore). Dose-response curves were fitted to a logistic model and half-maximal effective concentrations (EC50 values) were calculated using GraphPad Prism data analysis software after subtracting the MFI of unstimulated cells and normalizing to the maximum signal intensity. b. Biolayer interference binding assay of Neo-2 / 15 split into helices H1-H3+H2'-H4 (Neo2A3 and Neo2B3, respectively) according to the experimental protocol described above. c. Biolayer interference binding assay of Neo-2 / 15 split into helices H1-H3-H2'+H4 (Neo2A2 and Neo2B2, respectively) according to the experimental protocol described above. [Figure 17]Figure 17A-B. Fusions of split Neo2 / 15 variants and targeting domains bind to the IL-2 receptor. a. Neo2A and Neo2B split protein variants fused to anti-EGFR and anti-Her2 DARPin targeting domains. b. Biolayer interference binding assay of split-Neo-2 / 15 protein fusions to mouse IL-2 receptor. Binding data were collected on an Octet RED96 (ForteBio) and processed using ForteBio™ data analysis software version 9.0.0.10. Biotinylated target receptor human γc was immobilized on a streptavidin-coated biosensor (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). After loading the target receptor onto the biosensor, baseline measurements were performed by immersing the biosensor in binding buffer only, and then binding kinetics were observed by immersing the biosensor in wells containing the target protein (association step) and then immersing the sensor back into baseline / buffer (dissociation). For the association step, the analyte proteins (i.e., aHer2Neo2A1+Neo2B1-aEGFR, aHer2Neo2A2+Neo2B2-aEGFR, aHer2Neo2A3+Neo2B3-aEGFR) were diluted in equimolar amounts from concentrated stocks into binding buffer to a final concentration of 100 nM. Mouse IL-2Rβ was also added to the solution at a saturating concentration (250 nM). [Figure 18]Figure 18A-C. Targeting reconstitution of split Neoleukin-2 / 15 on the surface of K562 cells expressing Her2 and EGFR. a. Neo2A and Neo2B split protein variants fused to anti-EGFR and anti-Her2 DARPin targeting domains. b. Modified K562 tumor cell lines transduced for expression of Her2+ / eGFP+, EGFR+ / iRFP+ or Her2+ / eGFP+ and EGFR+ / iRFP+ surface markers. c. Functional reconstitution assay of Her2-targeted Neo2A variants (aHer2Neo2A1, aHer2Neo2A2 and aHer2Neo2A3) and EGFR-targeted Neo2B mutants (Neo2B1-aEGFR, Neo2B2-aEGFR and Neo2B3-aEGFR) on the surface of the four aforementioned K562 cell lines. All cell lines were mixed in equal ratios (50,000 of each cell type per well) and transferred to V-bottom plates at 200.000 cells / well. Cells were incubated with an equimolar ratio of anti-Her2-Neo2, anti-Her2-Neo2A + anti-EGFR-Neo2B variants at a final concentration of 10 nM in FACS buffer (PBS (Gibco), 0.5% BSA) for 30 min at room temperature. Cells were then washed twice (PBS (Gibco), 0.5% BSA) followed by a 5 min incubation with the following: 50 nM biotinylated soluble human common gamma receptor, 50 nM soluble human IL-2Rβ, and 15 nM streptavidin-phycoerythrin complex (SAPE). Cells were washed again and analyzed by flow cytometry on an LSRII instrument. Her2+ cells were sorted by eGFP fluorescence (Ex / Em=488 / 508), EGFR+ cells were sorted by iRFP fluorescence (Ex / Em=637 / 670), and Her2+ / EGFR+ cells were identified by co-expression of eGFP and iRFP. Reconstitution of Neoleukin-2 / 15 activity and binding to the IL-2 receptor were identified by analyzing PE (Ex / Em=561 / 582) fluorescence at the surface of each subset of cells. [Figure 19]Figure 19A-C. Both fragments of the split-Neo-2 / 15 variant are necessary to allow functional reconstitution on the surface of K562 cells expressing Her2 and EGFR. a. Functional reconstitution assay of the Her2-targeted Neo2A variant (aHer2-Neo2A1) and the EGFR-targeted Neo2B variant (Neo2B1-aEGFR) on the surface of four K562 cell lines expressing the following markers: None, Her2+ / eGFP+, EGFR+ / iRFP+, Her2+ / eGFP+ / EGFR+ / iRFP+. All cell lines were mixed in equal ratios (50,000 of each cell type per well) and transferred to V-bottom plates at 200.000 cells / well. Cells were incubated with anti-Her2-Neo2A1, Neo2B1-anti-EGFR or anti-Her2-Neo2A1+Neo2B1-anti-EGFR at a final concentration of 810 nM in FACS buffer (PBS (Gibco), 0.5% BSA) for 30 min at room temperature. Cells were then washed twice (PBS (Gibco), 0.5% BSA) followed by incubation for 5 min with the following: 50 nM biotinylated soluble human common gamma receptor, 50 nM soluble human IL-2Rβ, and 15 nM streptavidin-phycoerythrin complex (SAPE). Cells were washed again and analyzed by flow cytometry on an LSRII instrument. Her2+ cells were sorted by eGFP fluorescence (Ex / Em=488 / 508), EGFR+ cells were sorted by iRFP fluorescence (Ex / Em=637 / 670), and Her2+ / EGFR+ cells were identified by co-expression of eGFP and iRFP. Reconstitution of Neoleukin-2 / 15 activity and binding to IL-2 receptor was identified by analyzing PE (Ex / Em=561 / 582) fluorescence at the surface of each subset of cells. As observed, both fragments are necessary to reconstitute functional Neoleukin-2 / 15 activity at the surface of double positive cells. b. Same assay as described in (a) using aHer2-Neo2A2 and Neo2B2-aEGFR. c. Same assay as described in (a) using aHer2-Neo2A3 and Neo2B3-aEGFR. [Figure 20]Figure 20A-B. Illustrated alternative mechanism of action of the split Neo-2 platform for highly specific activation of immune cell subtypes. a. Selective targeting of split Neo-2 to two surface markers to stimulate proliferation of specific immune cell subtypes (e.g., CD8+, CAR T cells or regulatory T cells). b. Simultaneous targeting of tumor and T cells with the split Neoleukin-2 / 15 system induces specific proliferation of immune cell subsets in the environment of the targeted tumor cells. [Figure 21] FIG. 21A. Neoleukin-4 split fragments can reconstitute full activity when combined. Biolayer interference binding assay of Neo-4 split into helices H1 (Neo4A1) + H3-H2'-H4 (Neo4B1) to human IL-4 receptor. Binding data were collected on an Octet RED96 (ForteBio) and processed using ForteBio™ data analysis software version 9.0.0.10. Biotinylated target receptor human γc was immobilized on a streptavidin-coated biosensor (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). After loading the target receptor onto the biosensor, a baseline measurement was performed in binding buffer alone, and then binding kinetics were observed by immersing the biosensor in wells containing the indicated concentrations of the target protein (association step) and then immersing the sensor back into baseline / buffer solution (dissociation). In the association step, the analyte proteins (i.e., Neoleukin-4, Neo4A1, Neo4B1 and an equimolar ratio of Neo4A1+Neo4B1) were diluted from concentrated stocks into binding buffer to a final concentration of 100 nM, and human IL-4Rα was also added to the solution at a saturating concentration (250 nM). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] As used herein, unless otherwise indicated, the terms "a" and "an" are intended to mean "one," "at least one," or "one or more." Unless otherwise required by context, as used herein, singular terms shall include the plural and plural terms shall include the singular.

[0026] Unless the context clearly requires otherwise, throughout this description and the claims, words like "comprise," "comprising," and the like are to be construed in an inclusive or exhaustive sense, i.e., "including but not limited to," as opposed to an exclusive sense. Words using the singular or plural also include the plural or singular, respectively. Additionally, 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.

[0027] 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).

[0028] All embodiments of any aspect of the invention can be used in combination unless the context clearly dictates otherwise.

[0029] In a first aspect, the present disclosure provides a non-naturally occurring conditionally active receptor agonist comprising a first polypeptide component and a second polypeptide component, wherein the first polypeptide component and the second polypeptide component are not present in a fusion protein, and the first polypeptide component and the second polypeptide component collectively comprise domains X1, X2, X3, and X4; (a) X1 is a peptide

[0030] [ka]

[0031] a peptide comprising an amino acid sequence 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 (b) X2 is any helical peptide domain; (c) X3 is a peptide

[0032] [ka]

[0033] a peptide comprising an amino acid sequence 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 (d) X4 is a peptide

[0034] [ka]

[0035] a peptide comprising an amino acid sequence 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 (i) the amino acid residue in brackets may be present or absent; (ii) the first polypeptide component comprises at least one of X1, X2, X3, and X4, but does not comprise each of X1, X2, X3, and X4; (iii) the second polypeptide component comprises each of X1, X2, X3, and X4 that is not present in the first polypeptide component; The first polypeptide component and the second polypeptide component are not individually active receptor agonists, and the first polypeptide component and the second polypeptide component interact to activate the IL-2 receptor βγ c Heterodimer (IL-2Rβγ c ), IL-4 receptor αγ c Heterodimer (IL-4Rαγ c ), IL-13alpha, or form active agonists of the IL-4Ralpha / IL13Ralpha heterodimer.

[0036] As shown in the Examples below, as described in detail in PCT Application No. PCT / US2019 / 038703, filed June 29, 2019, and in Silva et al. As described in et al., Nature 565, pg. 186, January 10, 2019, a polypeptide containing all of X1 to X4 was previously synthesized by (a) IL-2 receptor βγ c Heterodimer (IL-2Rβγ c ), but does not have the binding sites for IL-2Rα or IL-15Rα; or (b) an IL-4 receptor αγ c Heterodimer (IL-4Rαγ c ) or IL-13 receptor α subunit (IL-13Rα) (natural IL-4 and the IL-4 mimics described herein cross-react with the IL-13 receptor, forming IL-4Rα / IL13Rα heterodimers). The full-length polypeptide is ultrastable and binds to human and mouse IL-2Rβγ with higher affinity than the natural cytokine. Cor IL-4Rαγ C It has been shown that the IL-1Rα-binding domain binds to IL-2Rα and IL-15Rα and induces downstream cell signaling independent of IL-13Rα. The full-length polypeptide can be used, for example, to treat cancer.

[0037] In contrast, the present disclosure surprisingly demonstrates conditionally active receptor agonists that include distinct first and second polypeptides as recited, which are not receptor agonists individually, but which are both The polypeptide non-covalently interacts with the IL-2 receptor βγ C Heterodimer (IL-2Rβγ c ), IL-4 receptor αγ C Heterodimer (IL-4Rαγ c ), IL-13 alpha, or IL-4R alpha / IL13R alpha heterodimers. The affinity of this non-covalent interaction between the "split components" (i.e., the first and second polypeptides) is such that the interaction occurs only in the presence of the appropriate receptor and only when both split components are co-present. Thus, the conditionally active receptor agonists of the current disclosure can be used in any application in which a polypeptide comprising all of X1-X4 can be used. Furthermore, the conditionally active receptor agonists allow for co-presentation-dependent reconstitution of the agonist and thus conditional activation of the receptor.

[0038] The term protein mimic as used herein refers to a protein that mimics a certain aspect of the function of another protein. The two proteins usually have different amino acid sequences and / or different structures. Provided herein are, among others, conditionally active mimics of IL-2 and IL-15. The aspect of the function of IL-2 and IL-15 that these conditionally active mimics mimic is the induction of heterodimerization of IL-2Rβγc, which leads to phosphorylation of STAT5. Since both IL-2 and IL-15 signal through heterodimerization of IL-2Rβγc, these conditionally active mimics mimic both biological functions of IL-2 and IL-15. These conditionally active mimics may be referred to herein as mimics of IL-2, mimics of IL-15, or mimics of both IL-2 and IL-15.

[0039] Conditionally active mimetics of IL-4 are also provided. These conditionally active mimetics are capable of mimicking a particular function of IL-4. The function of IL-4 that these mimetics mimic is the activation of IL-4Rαγ c The primary objective of this study is to induce IL-4Rα / IL-13Rα heterodimerization (and / or IL-4Rα / IL-13Rα heterodimerization).

[0040] In one embodiment, the first polypeptide component and the second polypeptide interact to bind to the IL-2 receptor βγ C Heterodimer (IL-2Rβγ c In another embodiment, the first polypeptide component and the second polypeptide interact to form an agonist for the IL-4 receptor αγ C Heterodimer (IL-4Rαγ c ), IL-13 alpha, or IL-4R alpha / IL13R alpha heterodimer agonists.

[0041] Native hIL-2 contains four helices connected by long irregular loops. The N-terminal helix (H1) interacts with both the beta and gamma subunits, the third helix (H3) interacts with the beta subunit, the C-terminal helix (H4) interacts with the gamma subunit, and the surface that interacts with the alpha subunit is formed by the irregular second helix (H2) and two long loops, one linking H1 to H2 and the other linking H3 to H4. Design and produce an ideal protein in which H1, H3 and H4 are replaced by ideal structural domains, including but not limited to, IL-2Rβγ stimulated by H1, H3 and H4. c or IL-4Rαγ c The peptide domains comprise a helix and a beta strand (referred to as domains X1, X3, and X4, respectively) that exhibit an interface between H1 and H2, and H3 is replaced with an idealized helix (referred to as 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 maintaining conditional 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. As described herein, the idealized protein can be divided into two polypeptides that, alone, barely bind to the relevant receptor. can be mixed together to reconstitute receptor activity. The protein is typically split at a site that does not interfere with the function of the protein (e.g., the linker section in embodiments that use linkers). In addition, the split protein can contain any combination of domains, such that the X1, X2, X3, and X4 domains of the unsplit protein can be looped together in any order.

[0042] Thus, X1, X2, X3, and X4 can be in any order in the first and second polypeptides, and in a non-limiting embodiment, (i) the first polypeptide comprises X1, and the second polypeptide comprises X2, X3, and X4; (ii) the first polypeptide comprises X2 and the second polypeptide comprises X1, X3, and X4; (iii) the first polypeptide comprises X3 and the second polypeptide comprises X1, X2, and X4; (iv) the first polypeptide comprises X4 and the second polypeptide comprises X1, X2, and X3; (v) the first polypeptide comprises X1 and X2, and the second polypeptide comprises X3 and X4; (vi) the first polypeptide comprises X1 and X3, and the second polypeptide comprises X2 and X4; (vii) the first polypeptide comprises X1 and X4, and the second polypeptide comprises X2 and X3; (viii) the first polypeptide comprises X2 and X3, and the second polypeptide comprises X1 and X4; (ix) the first polypeptide comprises X2 and X4, and the second polypeptide comprises X1 and X3; (x) the first polypeptide comprises X3 and X4, and the second polypeptide comprises X1 and X2; (xi) the first polypeptide comprises X1, X2, and X3, and the second polypeptide comprises X4; (xii) the first polypeptide comprises X1, X2, and X4, and the second polypeptide comprises X3; (xiii) the first polypeptide comprises X1, X3, and X4, and the second polypeptide comprises X2; or (xiv) the first polypeptide comprises X2, X3, and X4, and the second polypeptide comprises X1.

[0043] When the first and / or second polypeptide comprises two or more domains of X1, X2, X3, and X4, the domains may be separated by an amino acid linker of any suitable length or amino acid composition in some embodiments. There is no need for a linker, and in one embodiment, there is no linker between any of the domains. In other embodiments, an amino acid linker may be present between 0, 1, or 2 junctions between the domains of X1, X2, X3, and X4 in the first and / or second polypeptide. The amino acid linker may be of any length that is deemed appropriate for the application. In some embodiments, the linker is referred to as a linker even though it is at the N-terminus or C-terminus and does not link two domains. In some embodiments, the linker is referred to as a linker because it was present in a non-split protein that links two domains.

[0044] In all of these embodiments, X1, X3, and X4 can be any suitable length. This means that each domain may contain any suitable number of additional amino acids other than the peptides of SEQ ID NOs: 4, 5, and 6, respectively. The residues in brackets are optional and may or may not be present. As will be appreciated by those of skill in the art, this may include, for example,

[0045] [ka]

[0046] This means that the N-terminal 6 amino acids and the C-terminal 5 amino acid residues are optional. As will be further understood by those skilled in the art, (i) if one N-terminal amino acid is absent, it is the most N-terminal amino acid (i.e., the N-terminal P residue in SEQ ID NO: 4), (ii) if two N-terminal amino acids are absent, it is the two most N-terminal amino acids (i.e., the N-terminal PK dipeptide in SEQ ID NO: 4), (iii) if one C-terminal amino acid residue is absent, it is the most C-terminal amino acid (i.e., the C-terminal I residue in SEQ ID NO: 4), (iv) if two C-terminal amino acid residues are absent, it is the two most C-terminal amino acids (i.e., the C-terminal NI dipeptide in SEQ ID NO: 4), etc. Thus, as exemplified above, it will be clear to those skilled in the art that one or more optional amino acid residues may be absent, and that the amino acids absent at any residue are adjacent from the relevant peptide terminus.

[0047] In one embodiment, X1 is a peptide

[0048] [ka]

[0049] and X3 is a peptide having identity to the full length of peptide

[0050] [ka]

[0051] and X4 is a peptide having the same identity as the full length of

[0052] [ka]

[0053] and a peptide having identity to the entire length of at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%, respectively.

[0054] In various embodiments, X1 is a peptide

[0055] [ka]

[0056] X3 is a peptide having an amino acid sequence identical to the full length of the peptide

[0057] [ka]

[0058] X4 is a peptide having an amino acid sequence identical to the full length of the peptide

[0059] [ka]

[0060] and a peptide comprising an amino acid sequence having identity to the full length of, respectively, at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%.

[0061] In certain embodiments, (i) X1 is a peptide

[0062] [ka]

[0063] X3 is a peptide having an amino acid sequence having at least 70% identity to the full length of

[0064] [ka]

[0065] X4 is a peptide having an amino acid sequence having at least 70% identity to the full length of

[0066] [ka]

[0067] A peptide comprising an amino acid sequence having at least 70% identity to the full length of (ii) X1 is a peptide

[0068] [ka]

[0069] X3 is a peptide having an amino acid sequence having at least 70% identity to the full length of

[0070] [ka]

[0071] X4 is a peptide having an amino acid sequence having at least 70% identity to the full length of

[0072] [ka]

[0073] A peptide comprising an amino acid sequence having at least 70% identity to the full length of (iii) X1 is a peptide

[0074] [ka]

[0075] X3 is a peptide having an amino acid sequence having at least 85% identity to the entire length of

[0076] [ka]

[0077] X4 is a peptide having an amino acid sequence having at least 85% identity to the entire length of the peptide

[0078] [ka]

[0079] A peptide comprising an amino acid sequence having at least 85% identity to the full length of (iv) X1 is a peptide

[0080] [ka]

[0081] X3 is a peptide having an amino acid sequence having 100% identity with the full length of

[0082] [ka]

[0083] X4 is a peptide having an amino acid sequence having 100% identity with the full length of the peptide

[0084] [ka]

[0085] A peptide having an amino acid sequence having 100% identity to the full length of (v) X1 is a peptide

[0086] [ka]

[0087] X3 is a peptide having an amino acid sequence having at least 85% identity to the entire length of

[0088] [ka]

[0089] X4 is a peptide having an amino acid sequence having at least 85% identity to the entire length of the peptide

[0090] [ka]

[0091] or a peptide comprising an amino acid sequence having at least 85% identity to the full length of (vi) X1 is a peptide

[0092] [ka]

[0093] X3 is a peptide having an amino acid sequence having 100% identity with the full length of

[0094] [ka]

[0095] X4 is a peptide having an amino acid sequence having 100% identity with the full length of the peptide

[0096] [ka]

[0097] It is a peptide containing an amino acid sequence that has 100% identity with the full-length sequence of

[0098] In these embodiments, different versions of SEQ ID NOs: 4, 5, and 6 are shown which have the same primary amino acid sequence but differ at any residue position as indicated in brackets.

[0099] In a further embodiment, X1 is a peptide

[0100] [ka]

[0101] a peptide comprising an amino acid sequence 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 X3 is a peptide

[0102] [ka]

[0103] and / or a peptide comprising an amino acid sequence 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 X4 is a peptide

[0104] [ka]

[0105] is a peptide comprising an amino acid sequence 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

[0106] In another embodiment, any of the residues of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 are present: (a) X1 is a peptide

[0107] [ka]

[0108] a peptide comprising an amino acid sequence 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 (c) X3 is a peptide

[0109] [ka]

[0110] a peptide comprising an amino acid sequence 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 (d) X4 is a peptide

[0111] [ka]

[0112] is a peptide comprising an amino acid sequence 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

[0113] In another embodiment, selected residues of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 are present. (a) X1 is a peptide

[0114] [ka]

[0115] a peptide comprising an amino acid sequence 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 (c) X3 is a peptide

[0116] [ka]

[0117] a peptide comprising an amino acid sequence 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 X4 is a peptide

[0118] [ka]

[0119] is a peptide comprising an amino acid sequence 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

[0120] In various embodiments, any residue of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 is present. In the formula, X1 is a peptide

[0121] [ka]

[0122] X3 is a peptide having an amino acid sequence identical to the full length of the peptide

[0123] [ka]

[0124] X4 is a peptide having an amino acid sequence identical to the full length of the peptide

[0125] [ka]

[0126] and a peptide comprising an amino acid sequence having identity to the full length of, respectively, at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%.

[0127] In certain embodiments, (i) X1 is a peptide

[0128] [ka]

[0129] X3 is a peptide having an amino acid sequence having at least 65% identity to the full length of

[0130] [ka]

[0131] X4 is a peptide having an amino acid sequence having at least 65% identity to the full length of

[0132] [ka]

[0133] A peptide comprising an amino acid sequence having at least 65% identity to the full length of (ii) X1 is a peptide

[0134] [ka]

[0135] X3 is a peptide having an amino acid sequence having at least 75% identity to the entire length of

[0136] [ka]

[0137] A peptide having an amino acid sequence having at least 75% identity to the full length of X4 is a peptide

[0138] [ka]

[0139] A peptide comprising an amino acid sequence having at least 75% identity to the full length of (iii) X1 is a peptide

[0140] [ka]

[0141] X3 is a peptide having an amino acid sequence having at least 80% identity to the full length of

[0142] [ka]

[0143] X4 is a peptide having an amino acid sequence having at least 80% identity to the full length of

[0144] [ka]

[0145] A peptide comprising an amino acid sequence having at least 80% identity to the full length of (iv) X1 is a peptide

[0146] [ka]

[0147] X3 is a peptide having an amino acid sequence having at least 90% identity to the entire length of

[0148] [ka]

[0149] X4 is a peptide having an amino acid sequence having at least 90% identity to the entire length of the peptide

[0150] [ka]

[0151] A peptide comprising an amino acid sequence having at least 90% identity to the full length of (v) X1 is a peptide

[0152] [ka]

[0153] X3 is a peptide having an amino acid sequence having 100% identity with the full length of

[0154] [ka]

[0155] X4 is a peptide having an amino acid sequence having 100% identity with the full length of the peptide

[0156] [ka]

[0157] A peptide having an amino acid sequence having 100% identity to the full length of (vi) X1 is a peptide

[0158] [ka]

[0159] X3 is a peptide having an amino acid sequence having at least 80% identity to the full length of

[0160] [ka]

[0161] X4 is a peptide having an amino acid sequence having at least 80% identity to the full length of

[0162] [ka]

[0163] A peptide comprising an amino acid sequence having at least 80% identity to the full length of (vii) X1 is a peptide

[0164] [ka]

[0165] X3 is a peptide having an amino acid sequence having at least 90% identity to the entire length of

[0166] [ka]

[0167] X4 is a peptide having an amino acid sequence having at least 90% identity to the entire length of the peptide

[0168] [ka]

[0169] A peptide comprising an amino acid sequence having at least 90% identity to the full length of (viii) X1 is a peptide

[0170] [ka]

[0171] X3 is a peptide having an amino acid sequence having 100% identity with the full length of

[0172] [ka]

[0173] X4 is a peptide having an amino acid sequence having 100% identity with the full length of the peptide

[0174] [ka]

[0175] A peptide having an amino acid sequence having 100% identity to the full length of (ix) X1 is a peptide

[0176] [ka]

[0177] X3 is a peptide having an amino acid sequence having at least 80% identity to the full length of

[0178] [ka]

[0179] X4 is a peptide having an amino acid sequence having at least 80% identity to the full length of

[0180] [ka]

[0181] A peptide comprising an amino acid sequence having at least 80% identity to the full length of (x) X1 is a peptide

[0182] [ka]

[0183] X3 is a peptide having an amino acid sequence having at least 90% identity to the entire length of

[0184] [ka]

[0185] X4 is a peptide having an amino acid sequence having at least 90% identity to the full length of the peptide

[0186] [ka]

[0187] or a peptide comprising an amino acid sequence having at least 90% identity to the full length of (xi) X1 is a peptide

[0188] [ka]

[0189] X3 is a peptide having an amino acid sequence having 100% identity with the full length of

[0190] [ka]

[0191] X4 is a peptide having an amino acid sequence having 100% identity with the full length of the peptide

[0192] [ka]

[0193] It is a peptide containing an amino acid sequence that has 100% identity with the full-length sequence of

[0194] In one embodiment, the conditionally active receptor agonist is an IL-2 / 15 mimetic that is a conditionally active receptor agonist: (i) X1 comprises one, two, three, four or all five of L at residue 7, H at residue 8, H at residue 11, Y at residue 14, and M at residue 18, with numbering based on the presence of any residue relative to SEQ ID NO: 4; and / or (ii) X3 comprises one, two, three, four, five, six, seven or all eight of 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, and the numbering is made relative to SEQ ID NO:5 based on which any residue is present. In a further embodiment, (iii) X4 comprises I at residue 19, and the numbering is made relative to SEQ ID NO:6 based on which any residue is present.

[0195] In SEQ ID NOs: 4, 5, and 6, some amino acid residues are marked in bold.

[0196] [ka]

[0197] In, amino acid residues E10, L13, Y14, D15, and L17 (numbered based on the arbitrary residues present) are marked in bold,

[0198] [ka]

[0199] In, amino acid residues L1, Y4, N7, L10, I11, and I15 (numbered based on the arbitrary residues present) are marked in bold,

[0200] [ka]

[0201] In the figure, amino acid residues I12, Q16, and W18 (numbered based on the arbitrary residues present) are marked in bold.

[0202] In one embodiment, (a) the amino acid substitutions of X1 relative to the amino acid (AA) sequence of SEQ ID NO:4 occur at no more than three amino acid residues marked in bold, or at no more than two amino acid residues marked in bold, or at no more than one amino acid residue marked in bold, or at no amino acid residues marked in bold; (b) the amino acid substitutions of X3 relative to the amino acid sequence of SEQ ID NO:5 occur at no more than three amino acid residues marked in bold, or at no more than two amino acid residues marked in bold, or at no more than one amino acid residue marked in bold, or at no amino acid residues marked in bold, and / or (c) the amino acid substitutions of X4 relative to the amino acid sequence of SEQ ID NO:6 occur at no more than two amino acid residues marked in bold, or at no more than one amino acid residue marked in bold, or at no amino acid residues marked in bold.

[0203] In another embodiment, the amino acid substitution at X2 relative to the amino acid sequence of SEQ ID NO:7 does not occur at an amino acid residue marked in bold.

[0204] In another embodiment of an IL-2 mimetic that is a conditionally active receptor agonist, no amino acid substitutions relative to the reference peptide domain (i.e., SEQ ID NO: 4, 5, or 6) occur at the amino acid residues marked in bold. As shown below, SEQ ID NOs: 4, 5, and 6 each contain the bolded residues involved in binding to the receptor.

[0205] [ka]

[0206] Amino acid residues E10, L13, Y14, D15, and L17 (numbered based on any residues present) are invariant in this embodiment;

[0207] [ka]

[0208] Amino acid residues L1, Y4, N7, L10, I11, and I15 (numbered based on any residues present) are invariant in this embodiment;

[0209] [ka]

[0210] Amino acid residues I12, Q16, and W18 (numbered based on any residues present) are invariant in this embodiment.

[0211] In a further embodiment, X2 is a peptide

[0212] [ka]

[0213] In the case of a peptide comprising an amino acid sequence 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 SEQ ID NO: 1, amino acid residue W13 is invariant and the residues in brackets are optional. In one embodiment, the optional residue is present, and in another embodiment, the optional residue is not present.

[0214] In another embodiment of the IL-2 mimetic that is a conditionally active receptor agonist, amino acid substitutions relative to the reference peptide domain (i.e., SEQ ID NO: 4, 5, or 6) do not occur at 4, 3, or 2 or more amino acid residues marked in bold.

[0215] In another embodiment, the conditionally active receptor agonist is an IL-4 / IL-13 mimetic that is a conditionally active receptor agonist; X1 is a peptide

[0216] [ka]

[0217] 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 X3 is a peptide

[0218] [ka]

[0219] a peptide comprising an amino acid sequence at least 37%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical along its length to X4 is a peptide

[0220] [ka]

[0221] 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 (i) X1 contains an I at residue 7, a T or M at residue 8, an E at residue 11, a K at residue 14, and an S at residue 18; (ii) X3 contains an R at residue 3, an F at residue 4, a K at residue 6, an R at residue 7, an R at residue 10, an N at residue 11, a W at residue 13, and a G at residue 14.

[0222] In a further embodiment, (iii) X4 comprises F at residue 19.

[0223] In various embodiments, X1 is

[0224] [ka]

[0225] and X3 is a peptide comprising an amino acid sequence having identity to the full length of

[0226] [ka]

[0227] and X4 is a peptide comprising an amino acid sequence having identity to the full length of

[0228] [ka]

[0229] and wherein the amino acid sequence has at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identity to the full length of (i) X1 is a peptide

[0230] [ka]

[0231] and X3 is a peptide comprising an amino acid sequence at least 65% identical along its length to

[0232] [ka]

[0233] and X4 is a peptide comprising an amino acid sequence at least 65% identical along its length to

[0234] [ka]

[0235] and a peptide comprising an amino acid sequence at least 65% identical along its length to (ii) X1 is a peptide

[0236] [ka]

[0237] and X3 is a peptide comprising an amino acid sequence at least 75% identical along its length to

[0238] [ka]

[0239] and X4 is a peptide comprising an amino acid sequence at least 75% identical along its length to

[0240] [ka]

[0241] and a peptide comprising an amino acid sequence at least 75% identical along its length to (iii) X1 is a peptide

[0242] [ka]

[0243] and X3 is a peptide comprising an amino acid sequence at least 80% identical along its length to

[0244] [ka]

[0245] and X4 is a peptide comprising an amino acid sequence at least 80% identical along its length to

[0246] [ka]

[0247] or a peptide comprising an amino acid sequence that is at least 80% identical along its length to (iv) X1 is a peptide

[0248] [ka]

[0249] and X3 is a peptide comprising an amino acid sequence at least 90% identical along its length to

[0250] [ka]

[0251] and X4 is a peptide comprising an amino acid sequence at least 90% identical along its length to

[0252] [ka]

[0253] and a peptide comprising an amino acid sequence at least 90% identical along its length to In either case, (A) X1 contains an I at residue 7, a T or M at residue 8, an E at residue 11, a K at residue 14, and an S at residue 18; (B) X3 contains an R at residue 3, an F at residue 4, a K at residue 6, an R at residue 7, an R at residue 10, an N at residue 11, a W at residue 13, and a G at residue 14.

[0254] In another embodiment of the conditionally active receptor agonist IL-4 / IL-13 mimetic, no amino acid substitutions relative to the reference peptide domain (i.e., SEQ ID NO: 8, 9, or 10) occur at the amino acid residues marked in bold. As shown below, SEQ ID NOs: 8, 9, and 10 each contain the bold residues.

[0255] [ka]

[0256] Amino acid residues E10, E11, A12, L13, K14, D15, A16 and L17 are unchanged in this embodiment.

[0257] [ka]

[0258] Amino acid residues F4, A5, K6, R7, F8, E9, R10, and N11 are unchanged in this embodiment.

[0259] [ka]

[0260] Amino acid residues I11, I12, T13, I14, L15, Q16, S17, W18, F19, and F20 are unchanged in this embodiment. In a further embodiment, X2 is a peptide

[0261] [ka]

[0262] and at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or or a peptide containing 100% identical amino acid sequence, amino acid residue W13 is invariant.

[0263] In another embodiment, the amino acid substitutions relative to the reference peptide domain are conservative amino acid substitutions. As used herein, "conservative amino acid substitution" means that a given amino acid can be replaced with a residue that has similar physiochemical properties, such as replacing one aliphatic residue with another (such as Ile, Val, Leu, or Ala with each other), or replacing one polar residue with another (such as between Lys and Arg, between Glu and Asp, or between Gln and Asn). Other such conservative substitutions are known, such as replacement of entire regions with similar hydrophobic properties. Polypeptides that contain conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that the desired activity, such as the antigen binding activity and specificity of the native or reference polypeptide, is retained. Amino acids can be grouped according to the similarity of the properties of their side chains (ALLehninger, in Biochemistry, second ed., pp.73-75, Worth Publishers, New York (1975)). (1) non-polar: 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 affect chain orientation: Gly, Pro, (6) aromatic: Trp, Tyr, Phe. Nonconservative substitutions involve exchanging a member of one of these classes for another class.Particular conservative substitutions include, for example, Ala to Gly or Ser, Arg to Lys, Asn to Gln or His, Asp to Glu, Cys to Ser, Gln to Asn, Glu to Asp, Gly to Ala or Pro, His to Asn or Gln, Ile to Leu or Val, Leu to Ile or Val, Lys to Arg, Gln, or Glu, Met to Leu, Tyr, or Ile, Phe to Met, Leu, or Tyr, Ser to Thr, Thr to Ser, Trp to Tyr, Tyr to Trp, and / or Phe to Val, Ile, or Leu.

[0264] In one embodiment, the amino acid residue of X1 for SEQ ID NO:4 is selected from the group consisting of:

[0265] [Table 1]

[0266] In one embodiment, the conditionally active receptor agonist is a conditionally active 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.

[0267] In another embodiment, the conditionally active receptor agonist is a conditionally active IL-2 mimetic wherein 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.

[0268] In another embodiment, the amino acid residue of X3 for SEQ ID NO:5 is selected from the group consisting of:

[0269] [Table 2]

[0270] In another embodiment, the conditionally active receptor agonist is a conditionally active 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.

[0271] In another embodiment, the conditionally active receptor agonist is a conditionally active 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.

[0272] In any such embodiment, the conditionally active receptor agonist further comprises a cysteine ​​at position 17 relative to SEQ ID NO:5 in addition to amino acid residues H, K, L, N and R, or a cysteine ​​at position 20 relative to SEQ ID NO:5 in addition to amino acid residues A, C, E, F, G, M, S and Y. Thus, in this embodiment, the amino acid residue at X3 relative to SEQ ID NO:5 may be selected from the group consisting of:

[0273] [Table 3]

[0274] In another embodiment, the amino acid residue of X4 for SEQ ID NO:6 is selected from the group consisting of:

[0275] [Table 4]

[0276] In another embodiment, the conditionally active receptor agonist is a conditionally active IL-4 / IL-13 mimetic and position 19 is not I. In another embodiment, the conditionally active receptor agonist is a conditionally active IL-2 mimetic and position 19 is not I.

[0277] In any of such embodiments, the conditionally active receptor agonist further comprises a cysteine ​​at position 3 relative to SEQ ID NO: 6, in addition to the amino acid residues E, G, H and K. Thus, in this embodiment, the amino acid residue at X4 relative to SEQ ID NO: 6 may be selected from the group consisting of:

[0278] [Table 5]

[0279] As described herein, domain X2 is a structural domain, and therefore any amino acid sequence that allows it to link with other related domains (i.e., within the same polypeptide or upon non-covalent interaction of the first and second polypeptides) and fold them can be used. The required length depends on the specifics of the first and second polypeptides used and can be 8 amino acids or more. In one exemplary and non-limiting embodiment, X2 is

[0280] [ka]

[0281] 7. A peptide comprising an amino acid sequence 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 along its length to:

[0282] [Table 6]

[0283] 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.

[0284] In any such embodiment, the polypeptide further comprises a cysteine ​​at position 5 or 16 relative to SEQ ID NO:7.

[0285] Alternatively, in any such embodiment, the polypeptide further comprises a cysteine ​​at position 1, 2, 5, 9 or 16 relative to SEQ ID NO:7.

[0286] Thus, the amino acid residue of X2 for SEQ ID NO:7 may be selected from the group consisting of:

[0287] [Table 7]

[0288] In various specific embodiments, (i) X2 is

[0289] [ka]

[0290] and a peptide comprising an amino acid sequence at least 70% identical along its length to (ii) X2 is

[0291] [ka]

[0292] and a peptide comprising an amino acid sequence that is at least 80% identical along its length to (iii) X2 is

[0293] [ka]

[0294] and a peptide comprising an amino acid sequence at least 90% identical along its length to (iv) X2 is a peptide

[0295] [ka]

[0296] A peptide having an amino acid sequence having at least 65% identity to the full length of X1 is a peptide

[0297] [ka]

[0298] X3 is a peptide having an amino acid sequence having at least 65% identity to the full length of

[0299] [ka]

[0300] X4 is a peptide having an amino acid sequence having at least 65% identity to the full length of

[0301] [ka]

[0302] A peptide comprising an amino acid sequence having at least 65% identity to the full length of (v) X2 is a peptide

[0303] [ka]

[0304] X1 is a peptide having an amino acid sequence having at least 75% identity to the entire length of the peptide

[0305] [ka]

[0306] X3 is a peptide having an amino acid sequence having at least 75% identity to the entire length of

[0307] [ka]

[0308] X4 is a peptide having an amino acid sequence having at least 75% identity to the full length of the peptide

[0309] [ka]

[0310] A peptide comprising an amino acid sequence having at least 75% identity to the full length of (vi) X2 is a peptide

[0311] [ka]

[0312] X1 is a peptide having an amino acid sequence having at least 80% identity to the full length of

[0313] [ka]

[0314] X3 is a peptide having an amino acid sequence having at least 80% identity to the full length of

[0315] [ka]

[0316] X4 is a peptide having an amino acid sequence having at least 80% identity to the full length of

[0317] [ka]

[0318] A peptide comprising an amino acid sequence having at least 80% identity to the full length of (vii) X2 is a peptide

[0319] [ka]

[0320] X1 is a peptide having an amino acid sequence having at least 90% identity to the full length of

[0321] [ka]

[0322] X3 is a peptide having an amino acid sequence having at least 90% identity to the entire length of

[0323] [ka]

[0324] X4 is a peptide having an amino acid sequence having at least 90% identity to the full length of the peptide

[0325] [ka]

[0326] A peptide comprising an amino acid sequence having at least 90% identity to the full length of (viii) X2 is a peptide

[0327] [ka]

[0328] and X1 is a peptide having an amino acid sequence having at least 100% identity to the entire length of the peptide

[0329] [ka]

[0330] X3 is a peptide having an amino acid sequence having at least 100% identity to the full length of

[0331] [ka]

[0332] X4 is a peptide having an amino acid sequence having at least 100% identity to the full length of the peptide

[0333] [ka]

[0334] It is a peptide comprising an amino acid sequence having at least 100% identity to the full length of

[0335] In exemplary embodiments (i) through (viii) above, any amino acid residue set forth in SEQ ID NO: 7 is present. In exemplary embodiments (i) through (viii) above, the peptides X1, X3, and X4 are set forth in SEQ ID NOs: 4, 5, and 6. In exemplary embodiments (i) through (viii) above, the peptides X1, X3, and X4 are set forth in SEQ ID NOs: 320, 321, and 322.

[0336] In various embodiments, (i) the first polypeptide component comprises one of X1, X2, X3, and X4, and the second polypeptide component comprises three of X1, X2, X3, and X4 that are not present in the first polypeptide component; or (ii) the first polypeptide component comprises two of X1, X2, X3, and X4, and the second polypeptide component comprises two of X1, X2, X3, and X4 that are not present in the first polypeptide component.

[0337] In a further embodiment, (a) the first polypeptide is a fusion protein that includes X1 and excludes X2, X3, and X4, and the second polypeptide includes X3-Z1-X2-Z2-X4 and excludes X1; (b) the first polypeptide is a fusion protein that includes X4 and excludes X1, X2, and X3, and the second polypeptide includes X1-Z1-X3-Z2-X2 and excludes X4; (c) the first polypeptide is a fusion protein comprising X1-Z1-X3 and excluding X2 and X4, and the second polypeptide is a fusion protein comprising X2-Z1-X4 and excluding X1 and X3; Each of Z1 and Z2 is independently any amino acid linker. Z1 and / or Z2 may contain any number of amino acid residues to separate domains in the first and / or second polypeptide as deemed appropriate for the application. 1 and / or Z2 linker may be of any suitable length and amino acid composition. In one embodiment, Z1 and Z2 are both absent, in another embodiment, 1 and 2 are both present, and in a further embodiment, one of Z1 and Z2 is present and the other is absent.

[0338] In another embodiment, X1, X2, X3, and X4 are peptides that are 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 the X1, X2, X3, and X4 domains shown below (SEQ ID NOs: 4-7), respectively. The residues in brackets include tides and may or may not be present.

[0339] [ka]

[0340] In other embodiments, X1, X2, X3, and X4 are peptides comprising amino acid sequences at least 80% identical to the full length of the X1, X2, X3, and X4 domains shown below (SEQ ID NOs: 4-7), respectively, and the residues in brackets may or may not be present.

[0341] [ka]

[0342] In other embodiments, X1, X2, X3, and X4 are peptides comprising amino acid sequences at least 90% identical to the full length of the X1, X2, X3, and X4 domains shown below (SEQ ID NOs: 4-7), respectively, and the residues in brackets may or may not be present.

[0343] [ka]

[0344] In other embodiments, X1, X2, X3, and X4 are 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 the X1, X2, X3, and X4 domains shown below, respectively, and the residues in brackets may be present or absent.

[0345] [ka]

[0346] In other embodiments, X1, X2, X3, and X4 are peptides comprising amino acid sequences at least 80% identical to the full length of the X1, X2, X3, and X4 domains, respectively, shown below, where the residues in brackets may or may not be present.

[0347] [ka]

[0348] In other embodiments, X1, X2, X3, and X4 are peptides comprising amino acid sequences at least 90% identical to the full length of the X1, X2, X3, and X4 domains, respectively, shown below, where the residues in brackets may or may not be present.

[0349] [ka]

[0350] In one embodiment, one or more or all of any of the amino acids are present, and in another embodiment, one or more or all of any of the amino acids are absent. (i) X1 is a peptide comprising an amino acid sequence having at least 55% identity to the full length of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 320, X2 is a peptide comprising an amino acid sequence having at least 55% identity to the full length of the amino acid sequence of SEQ ID NO: 7, X3 is a peptide comprising an amino acid sequence having at least 55% identity to the full length of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 321, and X4 is a peptide comprising an amino acid sequence having at least 55% identity to the full length of the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 322; (ii) X1 has at least the full length of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 320. X2 is a peptide comprising an amino acid sequence having at least 75% identity to the full length of the amino acid sequence of SEQ ID NO:7; X3 is a peptide comprising an amino acid sequence having at least 75% identity to the full length of the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:321; X4 is a peptide comprising an amino acid sequence having at least 75% identity to the full length of the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:322; (iii) X1 is a peptide comprising an amino acid sequence having at least 80% identity to the full length of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 320, X2 is a peptide comprising an amino acid sequence having at least 80% identity to the full length of the amino acid sequence of SEQ ID NO: 7, X3 is a peptide comprising an amino acid sequence having at least 80% identity to the full length of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 321, and X4 is a peptide comprising an amino acid sequence having at least 80% identity to the full length of the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 322; (iv) X1 is a peptide comprising an amino acid sequence having at least 90% identity to the full length of the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:320, X2 is a peptide comprising an amino acid sequence having at least 90% identity to the full length of the amino acid sequence of SEQ ID NO:7, X3 is a peptide comprising an amino acid sequence having at least 90% identity to the full length of the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:321, and X4 is a peptide comprising an amino acid sequence having at least 90% identity to the full length of the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:322, or (vi) X1 is a peptide comprising an amino acid sequence having 100% identity to the entire length of the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:320, X2 is a peptide comprising an amino acid sequence having 100% identity to the entire length of the amino acid sequence of SEQ ID NO:7, X3 is a peptide comprising an amino acid sequence having 100% identity to the entire length of the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:321, and X4 is a peptide comprising an amino acid sequence having 100% identity to the entire length of the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:322.

[0351] In the above exemplary embodiments (i) to (vi), the X1, X3, and X4 peptides are set forth in SEQ ID NOs: 4, 5, and 6. In the above exemplary embodiments (i) to (vi), the X1, X3, and X4 peptides are set forth in SEQ ID NOs: 320, 321, and 322.

[0352] In another embodiment, the first and second polypeptides are at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to a pair of first and second polypeptides set forth below (the underlined or "X" residues are optional and, when present, each residue in any domain may comprise any amino acid). (i) First Polypeptide X1 (Neo2A)

[0353] [ka]

[0354] and A second polypeptide X3-X2'-X4 (Neo2B)

[0355] [ka]

[0356] (ii) First Polypeptide X1-X3-X2'

[0357] [ka]

[0358] and Second Polypeptide X4

[0359] [ka]

[0360] (iii) First Polypeptide X1-X3

[0361] [ka]

[0362] and A second polypeptide X2-X4

[0363] [ka]

[0364] (iv) First Polypeptide X1 (Neo4A)

[0365] [ka]

[0366] A second polypeptide X3-X2'-X4 (Neo4B)

[0367] [ka]

[0368] (v) First Polypeptide X1 (Neo2A)

[0369] [ka]

[0370] and A second polypeptide X3-X2'-X4 (Neo2B)

[0371] [ka]

[0372] (vi) First Polypeptide X1-X3-X2'

[0373] [ka]

[0374] and Second Polypeptide X4

[0375] [ka]

[0376] (vii) First Polypeptide X1-X3

[0377] [ka]

[0378] and A second polypeptide X2-X4

[0379] [ka]

[0380] (viii) First Polypeptide X1 (Neo4A)

[0381] [ka]

[0382] A second polypeptide X3-X2'-X4 (Neo4B)

[0383] [ka]

[0384] (ix) First polypeptide>Neo4_H1-H3'

[0385] [ka]

[0386] Second Polypeptide >Neo4_H2-H4

[0387] [ka]

[0388] (x) First polypeptide>Neo4_H1-H3'

[0389] [ka]

[0390] Second Polypeptide >Neo4_H2-H4

[0391] [ka]

[0392] (xi) First Polypeptide Neo4_H1-H3'-H2_

[0393] [ka]

[0394] The second polypeptide Neo4_H4

[0395] [ka]

[0396] (xii) First Polypeptide Neo4_H1-H3'-H2_

[0397] [ka]

[0398] The second polypeptide Neo4_H4

[0399] [ka]

[0400] (xiii) First Polypeptide (X1)

[0401] [ka]

[0402] Second Polypeptide (X3-X2'-X4)

[0403] [ka]

[0404] In an exemplary embodiment, the first and second polypeptides are peptides that comprise amino acid sequences at least 80% identical to a pair of first and second polypeptides set forth in embodiments (i)-(viii) above (wherein the underlined or "X" residues are optional, and where present, each residue in any domain may comprise any amino acid).

[0405] In an exemplary embodiment, the first and second polypeptides are peptides that comprise amino acid sequences at least 90% identical to a pair of first and second polypeptides set forth in embodiments (i)-(viii) above (wherein the underlined or "X" residues are optional, and where present, each residue in any domain may comprise any amino acid).

[0406] In an exemplary embodiment, the first and second polypeptides are A peptide that comprises an amino acid sequence that is 100% identical to a pair of first and second polypeptides set forth in embodiments (i)-(viii) (where the underlined or "X" residues are optional, and where present, each residue of any domain may comprise any amino acid).

[0407] In various further embodiments, X1, X2, X3, and X4 comprise peptides that are at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the X1, X2, X3, and X4 domains (as defined in Table 1, but listed as H1, H2, H3, and H4 domains) present within the amino acid sequences of SEQ ID NOs: 11-94, 190-216, 247, and SEQ ID NOs: 275-300, respectively.

[0408] Table 1 provides two SEQ ID NOs for many variants. The first SEQ ID NO describes the position of the linker as optional and variable (indicated underlined in the table), and the second SEQ ID NO includes the position of the linker, if necessary. Table 1 shows the domain arrangement of the polypeptides of SEQ ID NOs: 11-94, 190-216, 247, and SEQ ID NOs: 275-300 (see column 2), while the sequences show the underlined amino acid linkers that separate the domains. See, for example, SEQ ID NO: 11, which has the domain arrangement H1->H4->H2'->H3 (corresponding to the X1-X4-X2-X3 arrangement).

[0409] [ka]

[0410] Based on this arrangement, it will be apparent to one of skill in the art that in SEQ ID NO: 11, the X1 domain is STKKWQLQAEHALLDWQMALNK (SEQ ID NO: 271), the X4 domain is ENLNRAITAAQSWIS (SEQ ID NO: 272), the X2 domain is LDKAEDIRRNSDQARREAEK (SEQ ID NO: 273), and the X3 domain is RDLISNAQVILLEAR (SEQ ID NO: 274). Similarly, the amino acid sequences of SEQ ID NOs: 11-94, 190-216, 247, and SEQ ID NOs: 275-300 for each of the X1, X2, X3, and X4 domains will be apparent to one of skill in the art based on the teachings of this specification. As will be appreciated by one of skill in the art, the X1, X2, X3, and / or X4 amino acids can include additional (1, 2, 3, 4, or 5 or more) amino acids at the N-terminus and / or C-terminus to the X1, X2, X3, and X4 domains set forth in SEQ ID NOs: 11-94, 190-216, 247, and SEQ ID NOs: 275-300.

[0411] [Table 8-1]

[0412] [Table 8-2]

[0413] [Table 8-3]

[0414] [Table 8-4]

[0415] [Table 8-5]

[0416] [Table 8-6]

[0417] [Table 8-7]

[0418] [Table 8-8]

[0419] [Table 8-9]

[0420] In certain embodiments, X1, X2, X3, and X4 are 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the X1, X2, X3, and X4 domains (as defined in Table 1, but listed as H1, H2, H3, and H4 domains) present in the amino acid sequence of type 1 or type 2 of SEQ ID NO:90, which have the same primary amino acid sequence but slightly differ in optional / variable linker residues. In various embodiments, this embodiment includes variants of the X1, X2, X3, and / or X4 domains present in type 1 or type 2 of SEQ ID NO:90, which incorporate mutations relative to the primary amino acid sequence of SEQ ID NO:90 as set forth in SEQ ID NOs:275-300. Immerse.

[0421] 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, or 19 amino acids in length. 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 length. In further various embodiments, the length of each of the amino acids 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 20 The length may be 0, 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.

[0422] In one embodiment, the first polypeptide component and / or the second polypeptide component comprises at least one disulfide bond.

[0423] In another embodiment, the first and second polypeptide components are non-covalently linked. As described herein, the first and second polypeptide components are not individually active receptor agonists, but rather the first and second polypeptide components interact to activate the IL-2 receptor βγ receptor. c Heterodimer (IL-2Rβγ c ), IL-4 receptor αγ c Heterodimer (IL-4Rαγ c), IL-13 alpha, or IL-4R alpha / IL13R alpha heterodimer. Thus, in this embodiment, the first and second polypeptides may interact to form an active agonist. The interaction may be any suitable interaction, such as a non-covalent interaction. The interaction may include a direct non-covalent bond of the first and second polypeptides, or an indirect interaction. In one embodiment, the first and second polypeptide components interact with the IL-2 receptor βγ C Heterodimer (IL-2Rβγ c ), IL-4 receptor αγ C Heterodimer (IL-4Rαγ c ), IL-13alpha, or the IL-4Ralpha / IL13Ralpha heterodimer.

[0424] Methods for determining receptor binding are known in the art and described herein, for example, biolayer interference binding assay. In some embodiments, when the first polypeptide component and the second polypeptide interact with a receptor of interest, they coexist and bind to the receptor with a binding affinity of 1000nm or less, 200nm or less, 100nm or less, 50nM or less, or 25nM or less. For example, the split IL-2 mimics of the present invention coexist and bind to the IL-2 receptor βγ with a binding affinity of 1000nm or less, 200nm or less, 100nm or less, 50nM or less, or 25nM or less. c Heterodimer (IL-2Rβγ c Similarly, by way of example, the split IL-4 mimetics of the invention can colocalize with the IL-4 receptor αγ with a binding affinity of 1000 nm or less, 200 nm or less, 100 nm or less, 50 nM or less, or 25 nM or less. c Heterodimer (IL-4Rαγ c In some embodiments, receptor agonism of a receptor to which the split mimetic co-localizes and binds is measured by STAT5 phosphorylation.

[0425] In another embodiment, the present disclosure provides a method for the preparation of a polypeptide comprising one or more of the domains X1, X2, X3, and X4. or three, but not all four, (a) X1, if present, is a peptide

[0426] [ka]

[0427] a peptide comprising an amino acid sequence 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 (b) X2, if present, is any helical peptide domain; (c) X3, if present, is a peptide

[0428] [ka]

[0429] a peptide comprising an amino acid sequence 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 (d) X4, if present, is a peptide

[0430] [ka]

[0431] a peptide comprising an amino acid sequence 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 The amino acid residues in brackets may or may not be present.

[0432] The polypeptides of this aspect can be used, for example, to generate a conditionally active receptor agonist of any embodiment or combination of embodiments disclosed herein (i.e., the polypeptides of this aspect are either the first or second polypeptides of the conditionally active receptor agonist of the present disclosure). Thus, as will be apparent to one of skill in the art, all embodiments and combinations of the first and second polypeptides disclosed above, and all embodiments and combinations of the X1, X2, X3, and X4 domains described above, are equally applicable to the polypeptides of this aspect of the present disclosure. In one embodiment, (a) the amino acid substitutions of X1 relative to the amino acid (AA) sequence of SEQ ID NO:4 occur at no more than three amino acid residues marked in bold, or at no more than two amino acid residues marked in bold, or at no more than one amino acid residue marked in bold, or at no amino acid residues marked in bold; (b) the amino acid substitutions of X3 relative to the amino acid sequence of SEQ ID NO:5 occur at no more than three amino acid residues marked in bold, or at no more than two amino acid residues marked in bold, or at no more than one amino acid residue marked in bold, or at no amino acid residues marked in bold, and / or (c) the amino acid substitutions of X4 relative to the amino acid sequence of SEQ ID NO:6 occur at no more than two amino acid residues marked in bold, or at no more than one amino acid residue marked in bold, or at no amino acid residues marked in bold.

[0433] In another embodiment, the amino acid substitution at X2 relative to the amino acid sequence of SEQ ID NO:7 does not occur at an amino acid residue marked in bold.

[0434] In various embodiments, the polypeptide may be selected from the group consisting of:

[0435] (i) a polypeptide that includes X1 and excludes X2, X3, and X4; (ii) a polypeptide that includes X2 and excludes X1, X3, and X4; (iii) a polypeptide that includes X3 and excludes X1, X2, and X4; (iv) a polypeptide that includes X4 and excludes X1, X2, and X3; (v) a polypeptide comprising X1 and X2 and excluding X3 and X4; (vi) a polypeptide comprising X1 and X3 and excluding X2 and X4; (vii) a polypeptide comprising X1 and X4 and excluding X2 and X3; (viii) a polypeptide comprising X2 and X3 and excluding X1 and X4; (ix) a polypeptide comprising X2 and X4 and excluding X1 and X3; (x) a polypeptide comprising X3 and X4 and excluding X1 and X2; (xi) a polypeptide comprising X1, X2, and X3, but excluding X4; (xii) a polypeptide comprising X1, X2, and X4, but excluding X3; (xiii) a polypeptide comprising X1, X3, and X4, and excluding X2; and (xiv) A polypeptide comprising X2, X3, and X4, but excluding X1.

[0436] In one embodiment, the polypeptide comprises 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 a first polypeptide or a second polypeptide described below (underlined residues are optional, and when present, each optional residue may comprise any amino acid). (i) First Polypeptide X1 (Neo2A)

[0437] [ka]

[0438] and A second polypeptide X3-X2'-X4 (Neo2B)

[0439] [ka]

[0440] (ii) First Polypeptide X1-X3-X2'

[0441] [ka]

[0442] and Second Polypeptide X4

[0443] [ka]

[0444] (iii) First Polypeptide X1-X3

[0445] [ka]

[0446] and A second polypeptide X2-X4

[0447] [ka]

[0448] (iv) First Polypeptide X1 (Neo4A)

[0449] [ka]

[0450] A second polypeptide X3-X2'-X4 (Neo4B)

[0451] [ka]

[0452] (v) First Polypeptide X1 (Neo2A)

[0453] [ka]

[0454] and A second polypeptide X3-X2'-X4 (Neo2B)

[0455] [ka]

[0456] (vi) First Polypeptide X1-X3-X2'

[0457] [ka]

[0458] and Second Polypeptide X4

[0459] [ka]

[0460] (vii) First Polypeptide X1-X3

[0461] [ka]

[0462] and A second polypeptide X2-X4

[0463] [ka]

[0464] (viii) First Polypeptide X1 (Neo4A)

[0465] [ka]

[0466] A second polypeptide X3-X2'-X4 (Neo4B)

[0467] [ka]

[0468] (ix) First polypeptide>Neo4_H1-H3'

[0469] [ka]

[0470] Second Polypeptide >Neo4_H2-H4

[0471] [ka]

[0472] (x) First polypeptide>Neo4_H1-H3'

[0473] [ka]

[0474] Second Polypeptide >Neo4_H2-H4

[0475] [ka]

[0476] (xi) First Polypeptide Neo4_H1-H3'-H2_

[0477] [ka]

[0478] The second polypeptide Neo4_H4

[0479] [ka]

[0480] (xii) First Polypeptide Neo4_H1-H3'-H2_

[0481] [ka]

[0482] The second polypeptide Neo4_H4

[0483] [ka]

[0484] (xiii) First Polypeptide (X1)

[0485] [ka]

[0486] Second Polypeptide (X3-X2'-X4)

[0487] [ka]

[0488] In exemplary embodiments, the polypeptide comprises an amino acid sequence at least 80% identical to a first or second polypeptide set forth in embodiments (i)-(viii) above (wherein the underlined or "X" residues are optional, and where present, each residue in any domain may comprise any amino acid).

[0489] In exemplary embodiments, the polypeptide comprises an amino acid sequence at least 90% identical to a first or second polypeptide set forth in embodiments (i)-(viii) above (wherein the underlined or "X" residues are optional, and where present, each residue in any domain may comprise any amino acid).

[0490] In exemplary embodiments, the polypeptide comprises an amino acid sequence at least 100% identical to a first or second polypeptide set forth in embodiments (i)-(viii) above (wherein the underlined or "X" residues are optional, and where present, each residue in any domain may comprise any amino acid).

[0491] In another embodiment, X1, X2, X3, and X4, if present, comprise an amino acid sequence 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 X1, X2, X3, and X4 domains (as defined in Table 1) present within an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-94, 190-216, 247, and SEQ ID NOs: 275-300, respectively. In certain embodiments, X1, X2, X3, and X4, if present, comprise amino acid sequences that are at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the X1, X2, X3, and X4 domains (as defined in Table 1, but listed as H1, H2, H3, and H4 domains) present in the Type 1 or Type 2 amino acid sequence of SEQ ID NO:90, respectively. In various embodiments, the forms have the same primary amino acid sequence but differ slightly in optional / variable linker residues. In various embodiments, the embodiments include variants of the X1, X2, X3, and / or X4 domains present in Form 1 or Form 2 of SEQ ID NO:90, which incorporate mutations relative to the primary amino acid sequence of SEQ ID NO:90 as set forth in SEQ ID NOs:275-300.

[0492] The first polypeptide, second polypeptide, and polypeptide described herein can be chemically synthesized or recombinantly expressed (if the polypeptide is genetically encodable). The polypeptide can be linked to other compounds, such as stabilizing compounds that promote increased half-life in vivo, including but not limited to albumin, PEGylation (attachment of one or more polyethylene glycol chains), HESylation, PASylation, glycosylation, or 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 the 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 methoxy PEG-MAL 5kD, methoxy PEG-MAL 20kD, methoxy (PEG)2-MAL 40kD, methoxy PEG (MAL)2 5kD, methoxy PEG (MAL)2 20kD, methoxy PEG (MAL)2 40kD, or any combination thereof. See also U.S. Patent No. 8,148,109. In other embodiments, PEG may include branched PEG and / or multiple PEG chains.

[0493] In one embodiment, the stabilizing compound, including but not limited to a PEG-containing moiety, is linked to a cysteine ​​residue of a 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 long, and the cysteine ​​residue is at the 1st, 2nd, 5th, 9th, or 16th position relative to the 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.

[0494] In further embodiments, the first polypeptide, the second polypeptide, and the polypeptide may further comprise a targeting domain.In this embodiment, the conditional receptor agonist can be directed to a target of interest.The targeting domain may be covalently or non-covalently bound to the first polypeptide, the second polypeptide, and / or the polypeptide.In the embodiment in which the targeting domain is non-covalently bound, any suitable means for such non-covalent binding may be used, including but not limited to streptavidin-biotin linker.

[0495] In another embodiment, the target domain, if present, is a translational fusion with the polypeptide. In this embodiment, the polypeptide and the target 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. Such exemplary linkers include, but are not limited to, those disclosed in WO2016 / 178905, WO2018 / 153865 (particularly page 13), and WO2018 / 170179 (particularly paragraphs

[0316] to

[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), GGSGGSGGGSGGSGSGSG (SEQ ID NO: 99), GSGGSGGGSGGSGSG (SEQ ID NO: 100), GGSGGSGGGSGGSGGGSGGGSGGGSG (SEQ ID NO: 101), and [GGGGX] n (SEQ ID NO: 102), in which X is Q, E or S, and n is 2 to 5.

[0496] The targeting domain is a polypeptide domain or a small molecule that binds to a target of interest. In one non-limiting embodiment, the targeting domain binds to a cell surface protein, and 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 vascular component 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). Any suitable tumor cell, vascular cell, or tumor microenvironment cell surface marker may be targeted, where the cell surface protein is present on the surface of a tumor cell, vascular cell, or tumor microenvironment cell (e.g., fibroblasts, infiltrating immune cells, or stromal elements), including 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, 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, p53 non-mutant, 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, Fos-related antigen 1, BMPR1B (bone morphogenetic protein receptor-type IB, Genbank accession number NM.001203), E16 (LAT1, SLC7A5, Genbank accession number NM-003486), STEAP1 (six-transmembrane epithelial antigen of the prostate, Genbank accession number NM-003486), bank accession number NM012449), 0772P (CA125, MUC16, Genbank accession number AF361486), MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession number NM005823), Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession number NM006424), Sema5b (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), PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA2700050C12, RIKEN cDNA2700050C12 gene, Genbank accession number AY3 58628), ETBR (endothelin type B receptor, Genbank accession no. AY275463), MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession no. NM017763), STEAP2 (HGNC.sub.--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 protein of the prostate, Genbank accession no. AF4551). 38), TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession number NM017636), CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratoma-derived growth factor, Genbank accession numbers NP003203 or NM003212), CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor) or Hs.73792, Ge Genbank accession number M26004), CD79b (IGb (immunoglobulin-related beta), B29, Genbank accession number NM000626), FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank accession number NM030764), HER2 (Genbank accession number M11730), NCA (Genbank accession number M18728), MDP (Genbank accession number BC017023 ), IL20Ralpha, (Genbank accession no. AF184971), brevican (Genbank accession no. AF229053), Ephb2R (Genbank accession no. NM004442), ASLG659 (Genbank accession no. AX092328), PSCA (Genbank accession no. AJ297436), GEDA (Genbank accession no. AY260763), BAFF-R (Genbank accession no. NP443177.1), and CD22 (Genbank accession no. NP-001762).1), CD79a (CD79A, CD79 alpha, immunoglobulin-related alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with IgM molecules, transduces signals involved in B cell differentiation, Genbank accession number NP001774.1), CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, and may play a role in the prevention of HIV-2 infection, and possibly A. Involved in the development of IDS, lymphoma, myeloma, and leukemia, Genbank accession number NP001707.1), HLA-DOB (beta subunit of MHC class II molecule (Ia antigen) that binds peptides and presents them to CD4+ T lymphocytes, Genbank accession number NP002111.1), P2X5 (purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, and deficiency may be involved in the pathogenesis of idiopathic detrusor instability. These may contribute to the pathology of leukine-rich repeat (LRR) family of type I membrane proteins, CD72 (B cell differentiation antigen CD72, Lyb-2, Genbank accession number NP001773.1), LY64 (lymphocyte antigen 64 (RP105), and loss of function is associated with increased disease activity in patients with systemic lupus erythematosus, Genbank accession number NP005573.1). These may include, but are not limited to, IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative receptor for immunoglobulin Fc domains containing C2-type Ig-like and ITAM domains that may be involved in B-lymphocyte differentiation, Genbank Accession No. NP443170.1), or IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, an immune receptor putatively involved in B-cell development and lymphomagenesis, deregulation of the gene by translocation occurs in some B-cell malignancies, Genbank Accession No. NP112571.1).

[0497] In another embodiment, the targeting domain binds to an immune cell surface marker. In this embodiment, the target is a cell surface protein of any suitable immune cell, including, but not limited to, a CD8+ T cell, a regulatory T cell, a dendritic cell, a NK cell, or a macrophage. The targeting domain may target any suitable immune cell surface marker (whether 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.

[0498] In all these embodiments, the targeting domain is 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')2, B cell receptor (BCR), DARPin, affibody, monobody, nanobody, diabody, antibody (including monospecific or bispecific antibodies), and cell targeting oligopeptides include, but are not limited to, RGD integrin binding peptides, de novo designed binders, aptamers, bicyclic peptides, conotoxins, small molecules such as folate, and viruses that bind to cell surfaces.

[0499] The first polypeptide, second polypeptide, and polypeptide of the present disclosure may contain additional residues at the N-terminus, C-terminus, or both ends that are not present in the first polypeptide, second polypeptide, and polypeptide of the present disclosure, and these additional residues are not included in determining the percent identity of the polypeptide or peptide domain of the present disclosure to the reference polypeptide. Such residues can be any residue suitable for the application, including, but not limited to, detection tags (i.e., fluorescent proteins, antibody epitope tags, etc.), adapters, ligands suitable for purification purposes (such as His tags), other peptide domains that add functionality to the polypeptide, etc. Residues suitable for attachment of such groups can include cysteine, lysine, or p-acetylphenylalanine residues, or can be tags, such as amino acid tags suitable for reaction with transglutaminase as disclosed in U.S. Pat. Nos. 9,676,871 and 9,777,070.

[0500] In a further aspect, the present invention provides nucleic acid, including isolated nucleic acid, encoding the first polypeptide, second polypeptide, and polypeptide of the present disclosure, which can be genetically encoded.The isolated nucleic acid sequence can include RNA or DNA.Such isolated nucleic acid sequence includes additional sequences useful for facilitating the expression and / or purification of the encoded protein, including, but not limited to, polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, transport signals, and secretion signals, nuclear localization signals, and plasma membrane localization signals.Based on the teachings of this specification, it will be clear to one skilled in the art which nucleic acid sequence encodes the polypeptide of the present invention.

[0501] In another aspect, the disclosure provides an expression vector comprising a nucleic acid of any aspect of the invention operably linked to a suitable control sequence. An "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 a gene product. A "control sequence" operably linked to a nucleic acid sequence of the invention is a nucleic acid sequence capable of effecting expression of a nucleic acid molecule. Control sequences need not be contiguous, so long as they function to direct expression of the nucleic acid sequence. Thus, for example, an intervening non-translated but transcribed sequence may be present between the promoter sequence and the nucleic acid sequence, and the promoter sequence may be a non-translated but transcribed sequence. may still be considered "operably linked" to the coding sequence. Such other 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 expression vectors. In mammalian systems, the control sequences used to drive expression of the disclosed nucleic acid sequences 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 a number of inducible promoters, including but not limited to, tetracycline, ecdysone, steroid responsive). The expression vector must be replicable in the host organism, either as an episome or by integration into the host chromosomal DNA. In various embodiments, the expression vector may comprise a plasmid, a viral 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 disclosed methods to express the polypeptide in vivo for therapeutic benefit. In a non-limiting embodiment, the expression vectors can be used to transfect or transduce cell therapy targets (including but not limited to, CAR-T cells or tumor cells) to achieve the therapeutic methods disclosed herein.

[0502] In a further aspect, the disclosure provides a host cell comprising the expression vector and / or nucleic acid disclosed herein, the host cell can be either prokaryotic or eukaryotic. The cell can be transiently or stably modified 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, The Journal of Clinical Chemistry, 1999, 144:1311-1321, 2000). nd Ed. (R.I. Freshney. 1987. Liss, Inc. New York, NY)). A method for producing a polypeptide according to the invention is an additional part of the present invention. The method comprises the steps of (a) culturing a host under conditions conducive to expression of a polypeptide according to this aspect of the invention, and (b) optionally recovering the expressed polypeptide. The expressed polypeptide can be recovered from a cell-free extract, but is preferably recovered from the culture medium.

[0503] In another aspect, the present disclosure provides a pharmaceutical composition comprising one or more of the conditionally active receptor agonists, polypeptides, nucleic acids, expression vectors, and / or host cells of any aspect or embodiment of the present disclosure, and a pharma- ceutical acceptable carrier.The pharmaceutical composition of the present disclosure can be used, for example, in the methods of the present disclosure described below.The pharmaceutical composition can comprise, in addition to the polypeptide of the present disclosure, (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.

[0504] 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 include a lyoprotectant, such as sucrose, sorbitol, or trehalose. In certain embodiments, the pharmaceutical composition may include 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 the like. In other embodiments, the pharmaceutical composition includes 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 combinations thereof. The pharmaceutical composition may also include a tonicity adjuster, such as a compound that renders 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 the 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.

[0505] The conditionally active receptor agonist, 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 appropriate for the application.

[0506] In a further aspect, the present disclosure provides a method for treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a conditionally active receptor agonist, polypeptide, nucleic acid, expression vector, and / or host cell of any embodiment or combination of embodiments disclosed herein under conditions in which the first and second polypeptide components interact in cells of the tumor to treat the cancer. In embodiments for administering a conditionally active receptor agonist, the first and second polypeptides may be administered together or in separate pharmaceutical formulations.

[0507] 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 metastases in a subject, (b) limiting any increase in size or volume of a tumor and / or metastases in a subject, (c) improving survival rates, (d) reducing the severity of symptoms associated with cancer, (e) limiting or preventing the progression of symptoms associated with cancer, and (f) inhibiting the worsening of symptoms associated with cancer.

[0508] The methods can be used to treat 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, the number of genetic mutations in the tumor, PD-L1 expression levels, or an immunoscore assay (developed by the Society for Immunotherapy of Cancer).

[0509] The subject can be any subject with cancer, hi one embodiment, the subject is a mammal, including but not limited to humans, dogs, cats, horses, cows, etc.

[0510] In one embodiment, a first targeting domain binds to a cell marker and a second targeting domain binds to a second, distinct cell marker, and these two markers are targeted to the same or nearby cells. Co-expression of the markers occurs more commonly in tumors than in other tissues, and the first and second polypeptides interact only after the first targeting domain binds to a first cell marker and the second targeting domain binds to a second cell marker. This embodiment uses, for example, two targeting domains that bind to separate markers that are not themselves enriched in tumor cells, but whose co-expression is enriched in tumors.

[0511] In another embodiment, the first polypeptide component comprises a first targeting domain and the second polypeptide component comprises a second targeting domain, the first targeting domain binds to a first tumor cell marker and the second targeting domain binds to a second tumor cell marker, which may be the same as or different from the first tumor cell marker, and the first and second polypeptides interact only after the first targeting domain binds to the first tumor cell marker and the second targeting domain binds to the second tumor cell marker.

[0512] In another embodiment, the first polypeptide component comprises a first targeting domain and the second polypeptide component comprises a second targeting domain, wherein the first targeting domain binds to a tumor cell marker and the second targeting domain binds to an immune cell marker (including, but not limited to, a CD8+ T cell, a T regulatory cell, a dendritic cell, or a macrophage), and the first and second polypeptides interact only after the first targeting domain binds to the tumor cell marker and the second targeting domain binds to the immune cell marker.

[0513] In one embodiment, the first targeting domain binds to a cell marker and the second targeting domain binds to a second distinct cell marker, co-expression of these two markers occurs more commonly in tumor cells than in some other cell type, and the first and second polypeptides interact only after the first targeting domain binds to the first cell marker and the second targeting domain binds to the second cell marker on the same cell. This embodiment uses, for example, two targeting domains that bind to distinct markers that are not themselves enriched in tumor cells, but whose co-expression is enriched in tumors.

[0514] In a further embodiment, the first polypeptide component comprises a first targeting domain and the second polypeptide component comprises a second targeting domain, wherein the first targeting domain binds to a first immune cell marker (including but not limited to, CD8+ T cells, T regulatory cells, dendritic cells, or macrophages) and the second targeting domain binds to a second immune cell marker (including but not limited to, CD8+ T cells, T regulatory cells, dendritic cells, or macrophages), which can be the same or different from the first immune cell marker, and wherein the first and second polypeptides interact only after the first targeting domain binds to the first immune cell marker and the second targeting domain binds to the second immune cell marker.

[0515] In a further aspect, the present disclosure provides a method for modulating an immune response in a subject by administering to the subject a conditionally active receptor agonist, polypeptide, nucleic acid, expression vector, host cell, or pharmaceutical composition of the present disclosure. In one embodiment, the method comprises administering to the subject a conditionally active receptor agonist under conditions in which the first polypeptide component and the second polypeptide component interact in an immune cell to modulate the immune response.

[0516] 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 for a particular antigen (an "antigen-specific response") and is mediated by CD4 T cells, CD8 T cells, or CD4 T cells through their antigen-specific receptors. refers to a response by B cells. 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, depending on the nature of the immune cell that receives the response. In some embodiments of the compositions and methods described herein, the immune response that is regulated is T cell mediated.

[0517] In some embodiments, the immune response is an anti-cancer immune response. In some such embodiments, the conditionally active IL-2 mimetics described herein are administered to a subject having cancer to regulate an anti-cancer immune response in the subject.

[0518] In some embodiments, the immune response is a tissue repair immune response. In some such embodiments, the conditionally active IL-4 mimetics described herein are administered to a subject in need thereof to regulate the subject's tissue repair immune response.

[0519] In some embodiments, the immune response is a wound healing immune response. In some such embodiments, the conditionally active IL-4 mimetics described herein are administered to a subject in need thereof to regulate the subject's wound healing immune response.

[0520] In some embodiments, a method is provided for regulating 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 a 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 response (including CAR-T cell response), enhancing innate T cell immune response, reducing inflammation, inhibiting the activity of regulatory T cells, or a combination thereof.

[0521] In some embodiments, the conditionally active cytokine mimic of the present invention, for example, the conditionally active IL-4 mimic described herein, is 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, for example, coated with the biomaterial. Such medical devices include, for example, vascular and arterial grafts. Conditionally active IL-4 and / or IL-4-associated biomaterials can be used, for example, to promote wound healing and / or tissue repair and regeneration.

[0522] In another aspect, the disclosure provides a method of stimulating an IL-2 receptor or an IL-4 receptor, the method comprising administering to a subject a conditionally active receptor agonist of any embodiment or combination of embodiments disclosed herein under conditions in which the first polypeptide component and the second polypeptide component interact at the receptor.

[0523] As used herein, a "therapeutically effective amount" refers to an amount of a conditionally active receptor agonist, polypeptide, nucleic acid, expression vector, and / or host cell effective to treat and / or limit the disease (e.g., cancer) being treated. The conditionally active receptor agonist, polypeptide, nucleic acid, expression vector, and / or host cell are typically formulated as a pharmaceutical composition, such as those disclosed above, and can be administered via any suitable route, including, but not limited to, oral, ocular, intravenous, subcutaneous, intraperitoneal, and intravesicular, via inhalation spray, in a dosage unit containing conventional pharma- ceutically acceptable carriers, adjuvants, and vehicles. In a particular embodiment, the polypeptide, nucleic acid, expression vector, and / or host cell are administered mucosally, intraocularly, by inhalation, In another specific embodiment, the polypeptide, nucleic acid, expression vector, and / or host cell is administered orally, including, but not limited to, intranasal or intranasal administration. Such specific embodiments may be administered via drops, nebulizer, spray, or other suitable formulations.

[0524] Any suitable dosage range can be used, as determined by the attending medical practitioner. Dosage regimens can be adjusted to provide the optimum response desired (e.g., therapeutic or prophylactic response). A suitable dosage range for the conditionally active receptor agonist or polypeptide can be, for example, 0.1 ug / kg to 100 mg / kg body weight, or alternatively, 0.5 μg / kg to 50 mg / kg, 1 ug / kg to 25 mg / kg, or 5 ug / kg to 10 mg / kg body weight. In some embodiments, the recommended dosage can be less than 0.1 mcg / kg, especially when administered topically. In other embodiments, the recommended dosage is less than 0.1 mcg / kg body weight / m 2 (i.e., based on body surface area) and / or can be administered in a fixed dose (e.g., 0.05-100 mg). The conditionally active receptor agonist, 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, or 5 or more times) as determined by the attending physician.

[0525] The conditionally active receptor agonists, polypeptides, nucleic acids, expression vectors, and / or host cells may be administered as the sole prophylactic or therapeutic agent or may be administered together (i.e., in combination or separately) with one or more other prophylactic or therapeutic agents, including, but not limited to, tumor resection, chemotherapy, radiation therapy, immunotherapy, and the like.

[0526] Numerous modifications and variations of the present disclosure are possible in light of the above teachings. EXAMPLES

[0527] Example 1. We describe a computational approach to design de novo cytokine mimetics that recapitulate functional sites of native cytokines but are otherwise unrelated in topology or amino acid sequence. This strategy is directed at the IL-2 receptor βγ C Heterodimer (IL-2Rβγ c The design was used to design de novo non-cleavable mimetics of IL-2 and interleukin 15 (IL-15) that bind to human and mouse IL-2Rβγ with higher affinity than the native cytokines, but do not possess the binding sites for IL-2Rα or IL-15Rα. C and induce downstream cell signaling independent of IL-2Rα and IL-15Rα. The crystal structure of the experimentally optimized mimetic, neoleukin-2 / 15, is very close to the designed model and binds to mouse IL-2Rβγ c We provide the first structural information for the complex. Neoleukin-2 / 15 has highly effective therapeutic activity, reduced toxicity, and no signs of immunogenicity compared to IL-2 in mouse models of melanoma and colon cancer.

[0528] Many cytokines interact with multiple different receptor subunits and, like most natural proteins, contain 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(s) are fixed in space and an idealized globular protein structure is constructed to support these elements. Combinatorial fragment assembly was used to support short linear epitopes with parametric construction of isolated helices combined with knowledge-based loop closure (Figure 1a-b). This approach was applied to the construction of human IL-2Rβγ c (hIL-2Rβγ c The present invention provides a stable idealized protein having an interaction surface that mimics the interaction surface of human IL-2 (hIL-2) and human IL-15 (hIL-15) for IL-2 receptor alpha (IL-2Rα), but completely lacks the interaction surface of IL-2 receptor alpha (IL-2Rα). This was tested by attempting to design proteins de novo.

[0529] IL-2Rβγ c Computational design of a non-split IL-2 / IL-15 mimic that binds and activates: Native hIL-2 contains four helices connected by long irregular loops. The N-terminal helix (H1) interacts with both the beta and gamma subunits of the IL-2 receptor, the third helix (H3) interacts with the beta subunit, the C-terminal helix (H4) interacts with the gamma subunit, and the surface that interacts with the alpha subunit is formed by an irregular second helix (H2) and two long loops, one linking H1 to H2 and the other linking H3 and H4. An ideal protein was designed that reproduces the interface formed by H1, H3, and H4 with beta and gamma, and replaces H2 with a regular helix that provides better packing. Helices H1, H3, and H4 (see Fig. 1a) were used as binding site templates, while helix H2 was reconstructed (H2') using a database of highly represented clustered fragments (see Methods). The helix pairs were linked with loops extracted from the same database (see Fig. 1b), and the resulting helical hairpins were joined to a fully linked scaffold (see Fig. 1c), and Rosetta™ combinatorial flexible scaffold sequence design calculations were used to design the binding site for hIL-2Rβγ. C (see Methods). The top four computational designs and eight single disulfide bracketed mutations (see Table 2) were selected for experimental characterization by yeast display (see Methods). The eight designs were found to bind fluorescently tagged beta-gamma chimeric IL-2 receptor at low nanomolar concentrations. The best non-disulfide design (G1_neo2_40) was identified by site-saturation mutagenesis followed by murine IL-2Rβγ c The mIL-2Rβγ c, see Figure 5). The optimized design, recombinantly expressed in E. coli, was found to induce pSTAT5 signaling in IL-2-responsive mouse cells in vitro at low nanomolar or even picomolar concentrations (Table 3, Silva et al., Nature 565, pg. 186, January 2013). 10,2019), however, the thermal stability was relatively low (Tm~<45 °C, see Figures 9 and 10). To improve stability, the computational design protocol was repeated, starting from 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 bottom panel of Figure 1a). This second approach improved the quality of the models by allowing the exploration of substantially more combinations of loops connecting each pair of helices. Along with 27 Rosetta™ sequence redesigns of G1_neo2_40_1F (see Table 4), the 14 best designs of the second generation were experimentally characterized and all but one were found to bind the IL-2 receptor at low nanomolar concentrations. The three highest affinity and stability designs (one sequence redesign and two novel mimetics) were characterized for MIL-2Rβγ C Site-saturation mutagenesis of the binding sites (Figures 6-8) followed by analysis of both human and mouse IL-2Rβγ C The mature designs (see Table 5) showed enhanced binding while maintaining hyperstability (see Silva et al., Nature 565, pg. 186, January 10, 2019). The best 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 different from human or mouse IL-2 (29% sequence identity with hIL-2 over 89 residues and 16% sequence identity with mIL-2 over 76 aligned residues in a structural topology-independent alignment).

[0530] Functional characterization of neoleukin-2 / 15: Neoleukin-2 / 15 inhibits human and mouse IL-2Rβγ c It binds with high affinity to IL-2Rα and does not interact with IL-2Rα (Silva et al., Nature 565, pg. 186, January 10, 2019). Human and mouse IL-2 receptors (IL-2Rβ and IL -2Rβγ c The affinity of Neoleukin-2 / 15 for IL-2Rα 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, and in primary mouse T cells (see Silva et al., Nature 565, pg. 186, January 10, 2019). 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+ compared to Super-2, likely due to a complete lack of IL-2Rα binding. Neoleukin-2 / 15 was ultrastable (see FIG. 12 ) and maintained hIL-2Rβγ expression even after 2 hours of incubation at 80° C. CWhile neither of these lose their binding affinity for IL-2 or Super-2, hIL-2 and Super-2 are fully inactivated after 10 min (half inactivation time = ~4.2 min and ~2.6 min, respectively, Figure 2). Similarly, in ex vivo primary cell cultures, neoleukin-2 / 15 effectively promoted T cell survival after boiling at 95 °C for 60 min, while these conditions inactivated both IL-2 and Super-2 (see Silva et al., Nature 565, pg. 186, January 10, 2019). Thermal denaturation studies were performed on many other engineered mimetics, and their thermal stability was demonstrated as well (see Figure 11). This unprecedented stability of a cytokine-like molecule not only eliminates the need for cold chain storage, but also suggests robustness against mutations (see Figures 8 and 13), gene fusions, and chemical modifications that significantly exceed those of native IL-2 (see Figure 3).

[0531] Therapeutic Applications of Neoleukin-2 / 15: Clinical use of IL-2 has been limited primarily by toxicity. The interactions responsible for IL-2 toxicity in humans are not fully understood, but in mouse models, toxicity is T cell independent and is mitigated 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. The inherent low stability of IL-2, and the tightly evolved CD25 dependency, have been barriers to the translation of re-engineered IL-2 compounds. IL-2Rβγ c Other efforts have focused on IL-15, which dimerizes to induce signaling similar to IL-2 but has no affinity for CD25. However, IL-15 is dependent on trans-presentation, primarily by the IL-15α (CD215) receptor, which is displayed by antigen-presenting cells and natural killer cells. The low stability of native IL-15 and its dependence on trans-presentation also pose major barriers to reengineering efforts.

[0532] Dose-escalation studies in naive mice show that mIL-2 preferentially expands regulatory T cells, consistent with preferential binding to CD25+ cells, whereas neoleukin-2 / 15 promotes proliferation primarily of CD8+ T cells, with no or minimal induction of regulatory T cell proliferation at only the highest dose tested. Similarly, in a mouse model of airway inflammation, neoleukin-2 / 15 induces only a small proportion of tissue-resident CD8+ T cells, whereas mIL-2 preferentially expands regulatory T cells in lymphoid organs. + Foxp3 + Thy1.2 without increasing antigen-specific Tregs - CD44 + CD8 + resulting in an increase in T cells (data not shown, see Silva et al., Nature 565, pg. 186, January 10, 2019).

[0533] The therapeutic efficacy of neoleukin-2 / 15 was tested in the poorly immunogenic B16F10 melanoma and the more immunogenic CT26 colon cancer mouse models. 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 showed improved efficacy over that observed with recombinant mIL-2 (Silva et al., Nature 565, pg. In B16F10 melanoma, combined treatment with the anti-melanoma antibody TA99 (anti-TRP1) resulted in significant delay in tumor growth, while TA99 treatment alone had little effect (Silva et al., Nature 565, pg. 186, January 10, 2019). In long-term survival experiments (8 weeks), neoleukin-2 / 15 in combination with TA99 showed significantly reduced toxicity and overall superior therapeutic efficacy compared to mIL-2 (Silva et al., Nature 565, pg. 186, January 10, 2019). 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 (see Silva et al., Nature 565, pg. 186, January 10, 2019). Consistent with the therapeutic effect, neoleukin-2 / 15 treatment significantly reduced intratumoral CD8:T reg (see Silva et al., Nature 565, pg. 186, January 10, 2019), which is already an effective antitumor immune response. 58 Correlates with neoleukin-2 / 15-induced CD8:T reg The increase in the ratio was dose- and antigen-dependent (see Silva et al., Nature 565, pg. 186, January 10, 2019), with optimal therapeutic effects obtained at higher doses and in combination with other immunotherapies (see Silva et al., Nature 565, pg. 186, January 10, 2019). Collectively, these data suggest that neoleukin-2 / 15 exhibits the expected homeostatic benefits derived from IL-2, such as its immunostimulatory activity, but not CD25. + The therapeutic effect of neoleukin-2 / 15 was tested in a CAR-T model. 6 NSG mice inoculated with 0.8 × 10 RAJI tumor cells were left untreated or 6Mice were treated with either 1000 μg / day of anti-CD19 CAR-T cells (infused for 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 was shown to significantly enhance the antitumor effect of CAR-T cell therapy, slowing tumor growth and prolonging mouse survival (see Silva et al., Nature 565, pg. 186, January 10, 2019).

[0534] 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; 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).

[0535] Robust modularity of neoleukin-2 / 15. Disulfide stapling and reengineering into an IL-4 mimetic: Because neoleukin-2 / 15 is highly modular, its properties can be easily tuned, such as increasing its stability or altering its binding preference. This modularity and robustness was exploited by introducing, through computational design, a single disulfide staple that enhances the stability of neoleukin-2 / 15 while preserving its function. In one example, a disulfide bridge was introduced by searching for pairs of positions with favorable geometric configurations, followed by minimizing the flexible backbone. In the final design, a single disulfide was introduced between residues 38 and 75 to stabilize helices H3 and H2. This strategy increased the stability of neoleukin-2 / 15 (Tm>95°C) while its sequence and function were largely unaffected (see Silva et al., Nature 565, pg.186, January 10, 2019). Using the modular nature of neoleukin-2 / 15, we have identified its binding preferences. All cytokines in the interleukin-2 family are γ c and share a common architecture. It was therefore hypothesized that neoleukin-2 / 15 could be converted into another cytokine mimic of the IL-2 family by changing only half the amino acids in the binding site that interacts with IL-2Rβ (helices H1 and H3). As proof of concept, human interleukin-4 (hIL-4) was chosen as a target since it shares extensive structural homology with IL-2 and has potential applications in regenerative medicine. By aligning the Neo-2 / 15 model to the structure of human IL-4 bound to the IL-4 receptor and mutating 14 residues of Neo-2 / 15 to match the amino acids of IL-4 at the structural positions that mediate the interaction of IL-4 with IL-4r, Neo-2 / 15 was shown to bind to the human IL-4 receptors (IL-4Rα and γ). C It binds to the human IL-2 receptor (IL-2Rβ and γ CThe binding was further optimized by directed evolution using random mutagenesis and screening for high binding affinity variants, creating a new protein, Neoleukin-4, with a total of 16 mutations from Neoleukin-2 / 15 by introducing two additional amino acid substitutions and modifying one of the 14 original residues grafted from the IL-4 protein. The resulting optimally designed neoleukin-4 (see Table 6) was recombinantly expressed and purified from E. coli and tested for binding. Neoleukin-4 binds with high affinity to the IL-4Rα receptor and cooperates with IL-4Rαγ C (see Fig. 3) and does not bind with any affinity to the IL-2 receptor (data not shown), Neoleukin-4 retains the superior thermostability of neoleukin-2 / 15 (see Fig. 14b, c) and binds to the IL-13 receptor, as expected given the natural cross-reactivity of IL-4 with the IL-13 receptor (data not shown).

[0536] method Computational design of de novo cytokine mimetics: Design of de novo cytokine mimetics using IL-2Rβγ as a template cWe started by defining the structure of hIL-2 in quaternary complex with the receptor. After inspection, the residues that make up the binding site were defined as hotspots using Rosetta™ metadata (PDBInfoLabels). The structure was fed into a new mimetic design protocol programmed in PyRosetta™, which automatically detects the core-secondary structure elements that make up the target template and generates a de novo mimetic scaffold that is obtained equipped with full RosettaScripts™ compatible information for design. Briefly, the mimetic building 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 building protocol aims to reconnect the idealized elements with all possible pairwise combinations. To do this, we use combinatorial fragment assembly of sequence-independent fragments from the database, followed by backbone minimization of Cartesian constraints of potential solutions (i.e., when the N- and C-termini of the building fragments are close enough to connect two secondary structures). After minimization, solutions are verified to contain highly ideal fragments (i.e., any overlapping fragments that make up the two connected elements are also included in the database) and that the scaffold does not clash with the target (context) receptor. The passing scaffold solutions were then 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 generate a fully connected scaffold by using the connected building blocks of graph theory. Since the number of solutions grows exponentially for each pair of elements, at each step of fragment combination, we ranked the designs to favor those with shorter interconnections between pairs of core elements, and kept only the top solutions to proceed to the next step. The fully connected solutions were then ranked by layer (interface, core, non-core surface, surface) to restrict the possible amino acid identities to those that are layer compatible. Each was characterized. Finally, all information regarding hotspots, amino acids and layers of compatible building fragments were combined (hotspots are prioritized over amino acid probability, which in turn is prioritized over layers). These fully characterized scaffolds were then passed to RosettaScripts™ for flexible scaffold design and filtering. 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, ). In the second approach, new mimetics were engineered using G1_neo2_40_1F as the target template. This second generation mimetic design protocol was similar to that described for the first generation, with two important differences. First, the core fragment was no longer built from fragments, but instead allowed a "pitch" in the φ and ψ angles for every X-amino acid (final parameters: H1, H2, H3, H4) to give the helix the possibility of having curvature, by finding parametric equations for repetitive φ and ψ angles (ω fixed at 180°) that resulted in a repetitive secondary structure that reproduced each of the target helices as closely as possible, and the use of these parametric equations, in combination with the loop building process (max / min 8 amino acids), allowed the size of each of the core elements in the target structure to be changed at will (increasing or decreasing the size), so that a reduction in the size of the core elements would not remove hot spots from the binding site. The second difference in the second generation design was that instead of relinking the 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 allowed (i.e., a single fragment should be able to close a pair of secondary structures). The rest of the design algorithm was essentially similar to that employed in the first generation. The Rosetta™ energy functions used were "talaris2013" and "talaris2014" for the first and second generation designs, respectively.

[0537] A database of highly idealized fragments used for de novo mimetic scaffold design was constructed with the new Rosetta™ application “kcenters_clustering_of_fragments” using an extensive database of publicly available non-redundant protein structures from the RCSB Protein Data Bank, which consisted 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.

[0538] Yeast display: Yeast was transformed with genes encoding proteins for display together with a linearized pETcon3 vector. The vector was linearized by 100-fold overdigestion with NdeI and XhoI (New England Biolabs) and then purified by gel extraction (Qiagen). The genes contained 50-base overlaps with the vector at both the 5' and 3' ends, such that by homologous recombination the genes were placed in frame between the AGA2 gene and the myc tag on the vector. Yeast were grown in C-Trp-Ura medium as described above and then induced in SGCAA medium. After 12-18 hours of induction, cells were washed with chilled display buffer (50 mM NaPO4 pH 8, 20 mM NaCl, 0.5% BSA) and incubated with various concentrations of biotinylated receptors (human or mouse IL-2Rα, IL-2Rβ, IL-2Rγ, or human IL-4Rα) with agitation at 4°C. After approximately 30 min, the cells were washed again in cold buffer and then stained with FITC-conjugated anti-c-Myc antibody (3 × 10 6 The yeast were then washed and counted by flow cytometry (Accuri C6) or analyzed by FACS (Sony). In experiments where the initial receptor incubation was performed in combination with biotinylated IL-2Rγ and non-biotinylated IL-4Rα, the non-biotinylated receptor was provided in molar excess.

[0539] 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 at 10 8 The receptor was electroporated into EBY100 yeast with a transformation efficiency of about 1. Yeast were successively induced and selected multiple times with gradually decreasing concentrations of receptor until the population converged. Between each selection, yeast were regrown in C-Trp-Ura medium.

[0540] Site-saturation mutagenesis (SSM) libraries were constructed from synthetic DNA in Genscript. For each amino acid of each design template, forward and reverse primers were designed such that PCR amplification resulted in a 5' PCR product containing a degenerate NNK codon, and a 3' PCR product, respectively. 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 obtain the SSM library. The SSM library was transformed by electroporation into tailored Saccharomyces cerevisiae strain EBY100 cells with the linearized pETCON3 vector using the protocol previously described by Benatuil et al.

[0541] A combinatorial library was constructed from Genscript synthetic DNA containing ambiguous nucleotides and similarly transformed into the linearized pETCON3 vector.

[0542] Protein expression: Genes encoding the designed protein sequences were synthesized and cloned into pET-28b(+) E. coli plasmid expression vector (GenScript, N-terminal 6xHis tag and thrombin cleavage site). The plasmids were 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 A of about 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 and lysed at 18,000 psi using a Microfluidics M110P microfluidizer, followed by centrifugation 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 / 300GL, GE Healthcare). Purified neoleukin-2 / 15 was characterized by mass spectrometry (MS) verification of molecular weight of species in solution (Thermo Scientific), size exclusion-multi-angle laser light scattering (SEC-MALLS) (Agilent, Wyatt) to verify monomeric state and molecular weight, SDS-PAGE, and endotoxin levels (Charles River).

[0543] hIL-2 (amino acids 1-133), hIL-2Rα (amino acids 1-217), hIL-2Rβ (amino acids 1-214), hIL-2Rγ (amino acids 1-232), mIL-2 (amino acids 1-149), MIL-2Rα extracellular domain (amino acids 1-213), mIL-2Rβ extracellular domain (amino acids 1-215), and mγ C The components of the human and mouse IL-2 complex containing the extracellular domain (amino acids 1 to 233) have been previously 17,49The proteins were secreted and purified using the baculovirus expression system as described. All proteins were purified to >98% homogeneity using a Superdex™ 200 sizing column (GE Healthcare) equilibrated with HBS. Purity was confirmed by SDS-P Confirmed by AGE analysis. For expression of biotinylated human IL-2 and mouse IL-2 receptor subunits, proteins containing C-terminal biotin acceptor peptide (BAP)-LNDIFEAQKIEWHE (SEQ ID NO: 303) were expressed and purified by Ni-NTA affinity chromatography as described, and then biotinylated with 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 Superdex200 column equilibrated with HBS.

[0544] Circular dichroism (CD): Far-UV CD measurements were performed with an AVIV spectrometer model 420 in PBS buffer (pH 7.4) in 1 mm path length cuvettes at a protein concentration of approximately 0.20 mg / ml (unless otherwise stated in the text). Melting was performed at temperatures from 25 to 95 °C and the absorbance signal was monitored at 222 nm (steps of 2 °C / min, 30 s equilibration per step). Wavelength scans (195-260 nm) were collected at 25 °C and 95 °C and again after rapid refolding (approximately 5 min) at 25 °C.

[0545] STAT5 phosphorylation studies: In vitro studies: Approximately 2x10 5 YT-1, IL-2Rα +YT-1 or CTLL-2 cells were seeded into each well of a 96-well plate and resuspended in RPMI complete medium containing serial dilutions of hIL-2, mIL-2, Super-2, or modified IL-2 mimics. Cells were stimulated for 15 min at 37°C and immediately fixed by adding formaldehyde to 1.5% and incubating at room temperature for 10 min. Permeabilization of cells was achieved by resuspending in ice-cold 100% methanol for 30 min 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 with anti-STAT5 pY694 conjugated with Alexa Fluor® 647 (BD Biosciences) diluted in FACS buffer for 2 h at room temperature. Cells were then washed twice with FACS buffer and MFI was measured on a CytoFLEX™ flow cytometer (Beckman-Coulter). Dose-response curves were fitted with a logistic model to estimate the half-maximal effective concentration (EC 50 Values ​​(%) are calculated using GraphPad Prism data analysis software after subtracting the mean fluorescence intensity (MFI) of unstimulated cells and normalizing to the maximum signal intensity. Experiments were performed in triplicate and in triplicate with similar results. Ex vivo studies: spleens and lymph nodes were cultured from wild-type C57BL / 6J or B6, 129S4-Il2ra tm1Dw CD4+ T cells are harvested from (CD25KO) mice and brought to a single cell suspension in sorting buffer (phosphate-buffered saline pH 7.2 containing 2% fetal bovine serum). CD4+ T cells are enriched by negative selection by staining the cell suspension with biotin-conjugated anti-B220, CD8, NK1.1, CD11b, CD11c, Ter119, and CD19 antibodies at 1:100 for 30 min on ice. After washing with sorting buffer, 100 µg of anti-Biotin MicroBeads™ (Miltenyi Biotec) are added to the cells. 7 Add 20 μL per total cells to the cell suspension and incubate on ice for 20 minutes. After washing and resuspending the cells, negative selection is performed using an EasySep™ magnet (STEMCELL Technologies). Approximately 1x10 5Add 100 μL of enriched cells to each well of a 96-well plate in RPMI complete medium containing 10-fold serial dilutions of mIL-2, Super-2, or Neoleukin-2 / 15 and 5% FCS. Stimulate cells for 20 min at 37°C in 5% CO2, fix with 4% PFA, and incubate at 4°C for 30 min. After fixation, harvest cells, wash twice with sorting buffer, and fix again with 500 μL of 90% ice-cold methanol in dH2O for 30 min on ice for permeabilization. Perm / Wash cells. Wash twice with Perm / Wash Buffer (BD Biosciences) and stain with 5 μL per sample of Perm / Wash buffer containing anti-CD4-PerCP (1:300), anti-CD44-Alexa Fluor700 (1:200), anti-CD25-PE-Cy7 (1:200) and anti-pSTAT5-PE pY694 for 45 min at room temperature in the dark. Wash with m / Wash and resuspend in sorting buffer for analysis on a BD LSRII flow cytometer (BD Biosciences). Data Table

[0546] [Table 9]

[0547] [Table 10-1]

[0548] [Table 10-2]

[0549] [Table 11-1]

[0550] [Table 11-2]

[0551] [Table 11-3]

[0552] [Table 12]

[0553] [ka]

[0554] The binding of Neoleukin-2 / 15-H8Y-K33E to the IL2 receptor, measured by biolayer interferometry, was found to have a higher binding affinity to IL2-R beta than Neoleukin-2 when tested against both IL2R beta alone and the IL2R beta-gamma complex, and this increased affinity was primarily due to an improved off-rate from IL2-R beta.

[0555] [Table 13]

[0556] Example 2: Split cytokine mimetics for targeted immunotherapy De novo proteins are designed according to the rules of ideal protein structure, offering rare biochemical properties such as extreme thermostability and mutational robustness. Thus, de novo designed proteins are ideal candidates for use in the development of conditionally active protein therapeutics. Here, we report the development of a split cytokine mimetic for highly targeted immunotherapy based on Neoleukin-2 / 15, a recently developed de novo designed IL-2 mimetic protein. This system enables the delivery of conditionally active therapeutic proteins that reconstitute their activity by colocalizing on the surface of target cells. We identified potential split sites and demonstrated successful reconstitution of Neoleukin-2 / 15 activity by binding to the IL-2 receptor, cell signaling, and colocalization-dependent activation on the surface of target tumor cells. We also demonstrate this application to Neoleukin-4, another de novo designed cytokine mimetic.

[0557] To identify potential split sites for Neoleukin-2 / 15 (Neo2), the protein structure was evaluated to find split positions that would minimize detrimental effects on protein function. As a result, three exemplary split positions were defined: (i) between helical elements H1 (Neo2A1) and H3'-H2-H4 (Neo2B1), (ii) between helical elements H1-H3' (Neo2A2) and H2-H4 (Neo2B2), and (iii) between helical elements H1-H3'-H2 (Neo2A3) and H4 (Neo2B3) (Figure 15). Split Neo2 fragments were characterized by analyzing their binding ability to the human IL-2 receptor (Figure 15) and signaling in IL-2-responsive CTTL-2 cells (Figure 16). While individual split fragments in most cases had negligible binding and signaling ability to IL-2Rβγ, equimolar combinations of complementary split fragments showed potent activity.

[0558] The development of split-Neo2 allows for coexistence-dependent reconstitution of the proteins, thus allowing for conditional activation of split-Neo2. To allow for the coexistence of the Neo2A and Neo2B fragments, we first performed gene fusions to the targeting domains (Figure 17). Neo2A1, Neo2A2, and Neo2A3 were fused to the C-terminus of an ankyrin repeat protein (DARPin) designed to target Her2 by a 15-residue flexible linker (GSGGSGGGSGGSGSG, SEQ ID NO: 100). Neo2B1, Neo2B2, and Neo2B3 were fused to the N-terminus of an ankyrin repeat protein (DARPin) designed to target EGFR by a 15-residue flexible linker (GSGGSGGGSGGSGSG, SEQ ID NO: 100). We then assessed the ability of the split fragments to reconstitute binding to IL-2Rβγ after combination, demonstrating that the fusions did not interfere with the reconstitution ability of split-Neo-2 (Figure 17).

[0559] To evaluate the colocalization-dependent activation and target selectivity of the split-Neo2 system, an in vitro assay was performed with split Neo-2 / 15 targeting EGFR and Her2 on the surface of a modified K562 cell line (Figure 18a). Four modified cell lines were used: (i) no surface receptor, (ii) Her2+ / eGFP+, (iii) EGFR+ / iRFP+, and (iv) Her2+ / eGFP+EGFR+ / iRFP+ (Figure 18b). The four cell lines were mixed in equal ratios and incubated with the targeted split-Neo2 fragments for 15 min. Subsequently, the cells were washed and incubated with a mixture of biotinylated IL-2 receptor and fluorescent streptavidin-phycoerythrin complex (SAPE). Finally, the cells were analyzed by fluorescence-activated cell sorting (FACS). Successful reconstitution of Neo-2 activity resulted in fluorescent labeling with PE on the surface of the target cells (Figure 18c). When full Neo2 was targeted with a single anti-Her2 targeting domain (aHer2-Neo2), strong IL-2 receptor binding was observed on the surface of Her2+ and Her2+ / EGFR+. Similar binding was observed when split fragments of Neo2A1 and Neo2B1 were both targeted to Her2 (aHer2-Neo2A1+aHer2-Neo2B1), demonstrating that both fragments could be delivered to the same cell surface markers, as needed. Furthermore, highly selective rearrangements were observed on the surface of double-positive Her2+ / EGFR+ when each split fragment was targeted to either Her2 or EGFR (aHer2-Neo2A1+Neo2B1-aEGFR, aHer2-Neo2A2+Neo2B2-aEGFR, aHer2-Neo2A3+Neo2B3-aEGFR). Further studies demonstrated that for each combination of split Neo2 fragments, both fragments had to be present simultaneously to be able to recruit IL-2 receptors on the surface of K562 cells (Figure 19). These results demonstrate the high selectivity of split cytokine mimetics to reconstitute desired activities specifically on the surface of target cells.

[0560] The application of split cytokine mimicry technology is not limited to targeting tumor-associated antigens. In a given application, targeting a specific subset of immune cells to be selectively stimulated would be beneficial to direct the immune response to treat the disease. For example, this application would help to specifically increase the proliferation of CD8+ cytotoxic T cells, natural killer cells, or engineered CAR T cells to enhance anti-tumor responses, as well as target regulatory T cells to suppress potent immunity (Figure 20a). Furthermore, in some scenarios, a mixed targeting approach could be deployed, i.e., one split fragment targets tumor cells or tumor microenvironment, while the other fragment is delivered to immune cells (Figure 20b).

[0561] Finally, to demonstrate that the methodology described here can be transferred to other de novo designed interleukins, we created a new conditionally active IL-4 mimetic (Figure 21). To achieve this, we successfully introduced specific mutations into split Neo-2 that alter the binding interface to the hIL-2 beta receptor in order to bind to the hIL-4 alpha receptor. Sequences of exemplary split Neoleukin-2 / 15 variants used in Example 2 H1 (Neo2A1) PKKKIQLHAEHALYDALMILNIVKTNS (SEQ ID NO: 256) H3-H2'-H4 Neo2B1) TNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO: 257) H1-H3-H2'Neo2A3 PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTAS (SEQ ID NO: 258) H4 (Neo2B3) TTASEDEQEEMANAIITILQSWIFS (SEQ ID NO: 259) H1-H3 (Neo2A2) PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGD (SEQ ID NO: 260) H2'-H4 (Neo2B2) DQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO: 261) Exemplary split Neoleukin-4 sequences used in Example 2 H1 Neo4A1)

[0562] [ka]

[0563] H3-H2'-H4 (also known as Neo4B1)

[0564] [ka]

[0565] Exemplary constructs used in Example 2 X2-Z1-X3-Z2-X4-E01_EGFR_DARPin

[0566] [ka]

[0567] G3_Her2_DARPin_X1

[0568] [ka]

[0569] G3_Her2_DARPin_X2-Z1-X3-Z3-X4

[0570] [ka]

[0571] X2-Z1-X3-Z2-X4-E01_EGFR_DARPin

[0572]

change

[0573] X2-Z2-X4-E01_EGFR_DARPin

[0574]

change

[0575] X4-E01_EGFR_DARPin

[0576]

change

[0577] G3_Her2_DARPin_X1-Z1-X3

[0578]

change

[0579] G3_Her2_DARPin_X1-Z1-X3-Z2-X2

[0580]

change

Claims

1. A non-naturally occurring conditionally active receptor agonist comprising a first polypeptide component and a second polypeptide component, said first polypeptide component and said second polypeptide component being absent from a fusion protein, said first polypeptide component and said second polypeptide component collectively comprising domains X1, X2, X3, and X4; (a) X1 is a peptide 【Chemistry 1】 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 the full length of (b) X2 is any helical peptide domain; (c) X3 is a peptide 【Chemistry 2】 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 the full length of (d) X4 is a peptide 【Chemistry 3】 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 the full length of (i) the amino acid residue within brackets may be present or absent; (ii) the first polypeptide component comprises at least one of X1, X2, X3, and X4, but does not comprise each of X1, X2, X3, and X4; (iii) the second polypeptide component comprises each of X1, X2, X3, and X4 that is not present in the first polypeptide component; The first polypeptide component and the second polypeptide component are not individually active receptor agonists, and the first polypeptide component and the second polypeptide component interact to activate the IL-2 receptor βγ ｃ Heterodimer (IL-2Rβγ ｃ ), IL-4 receptor αγ ｃ Heterodimer (IL-4Rαγ ｃ ), IL-13alpha, or a conditionally active receptor agonist that forms an active agonist of the IL-4Ralpha / IL13Ralpha heterodimer.

2. (a) X1 is a peptide 【Chemistry 4】 A peptide comprising an amino acid sequence that is at least 70% identical to the full length of (b) X2 is any helical peptide domain; (c) X3 is a peptide 【Chemistry 5】 A peptide comprising an amino acid sequence that is at least 70% identical to the full length of (d) X4 is a peptide 【Chemistry 6】 2. The conditionally active receptor agonist of claim 1, which is a peptide comprising an amino acid sequence that is at least 70% identical to the full length of

3. (a) X1 is a peptide 【Chemistry 7】 A peptide comprising an amino acid sequence that is at least 85% identical to the full length of (b) X2 is any helical peptide domain; (c) X3 is a peptide 【Chemistry 8】 A peptide comprising an amino acid sequence that is at least 85% identical to the full length of (d) X4 is a peptide 【Chemistry 9】 2. The conditionally active receptor agonist of claim 1, which is a peptide comprising an amino acid sequence that is at least 85% identical to the full length of

4. (a) X1 is a peptide 【Chemistry 10】 a peptide comprising the same amino acid sequence as the full length of (b) X2 is any helical peptide domain; (c) X3 is a peptide 【Chemistry 11】 a peptide comprising the same amino acid sequence as the full length of (d) X4 is a peptide 【Chemistry 12】 2. The conditionally active receptor agonist of claim 1, which is a peptide comprising an amino acid sequence identical to the full length of

5. (a) X1 is a peptide 【Chemistry 13】 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 the full length of (b) X3 is a peptide 【Chemistry 14】 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 the full length of (c) X4 is a peptide 【Chemistry 15】 2. The conditionally active receptor agonist of claim 1, which 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 to the full length of SEQ ID NO:

6.

6. (a) X1 is a peptide 【Chemistry 16】 A peptide comprising an amino acid sequence at least 65% identical to the full length of (b) X3 is a peptide 【Chemistry 17】 A peptide comprising an amino acid sequence at least 65% identical to the full length of (c) X4 is a peptide 【Chemistry 18】 2. The conditionally active receptor agonist of claim 1, which is a peptide comprising an amino acid sequence that is at least 65% identical to the full length of

7. (a) X1 is a peptide 【Chemistry 19】 A peptide comprising an amino acid sequence that is at least 75% identical to the full length of (b) X3 is a peptide 【Chemistry 20】 A peptide comprising an amino acid sequence that is at least 75% identical to the full length of (c) X4 is a peptide 【Chemistry 21】 2. The conditionally active receptor agonist of claim 1, which is a peptide comprising an amino acid sequence that is at least 75% identical to the full length of

8. (a) X1 is a peptide 【Chemical 22】 A peptide comprising an amino acid sequence that is at least 80% identical to the full length of (b) X3 is a peptide 【Chemistry 23】 A peptide comprising an amino acid sequence that is at least 80% identical to the full length of (c) X4 is a peptide 【Chemistry 24】 2. The conditionally active receptor agonist of claim 1, which is a peptide comprising an amino acid sequence that is at least 80% identical to the full length of

9. (a) X1 is a peptide 【Chemistry 25】 A peptide comprising an amino acid sequence that is at least 90% identical to the full length of (b) X3 is a peptide 【Chemistry 26】 A peptide comprising an amino acid sequence that is at least 90% identical to the full length of (c) X4 is a peptide 【Chemical 27】 2. The conditionally active receptor agonist of claim 1, which is a peptide comprising an amino acid sequence that is at least 90% identical to the full length of

10. The conditionally active receptor agonist of any one of claims 1 to 9, wherein X2 is at least 8 amino acids in length.

11. X2 is a peptide 【Chemistry 28】 11. The conditionally active receptor agonist of any one of claims 1 to 10, which is a peptide comprising an amino acid sequence 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

12. (i) X1 comprises one, two, three, four, or all five of L at residue 7, H at residue 8, H at residue 11, Y at residue 14, and M at residue 18, and numbering is made relative to SEQ ID NO: 4 based on the presence of any residue; and / or (ii) X3 comprises one, two, three, four, five, six, seven or all eight of 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, and numbering is based on the presence of any residue with respect to SEQ ID NO:

5.

13. (iii) X4 comprises an I at residue 19, and numbering is based on the presence of any residue relative to SEQ ID NO:

6.

14. X1 is a peptide 【Chemical 29】 and 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 X3 is a peptide 【Chemistry 30】 and a peptide comprising an amino acid sequence at least 37%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical along its length to X4 is a peptide 【Chemistry 31】 and 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 (i) X1 comprises an I at residue 7, a T or M at residue 8, an E at residue 11, a K at residue 14, and an S at residue 18; (ii) The conditionally active receptor agonist of any one of claims 1 to 11, wherein X3 comprises an R at residue 3, an F at residue 4, a K at residue 6, an R at residue 7, an R at residue 10, an N at residue 11, a W at residue 13, and a G at residue 14.

15. X1 is a peptide 【Chemistry 32】 and a peptide comprising an amino acid sequence at least 65% identical along its length to X3 is a peptide 【Chemical 33】 and a peptide comprising an amino acid sequence at least 65% identical along its length to X4 is a peptide 【Chemical 34】 and a peptide comprising an amino acid sequence at least 65% identical along its length to (i) X1 comprises an I at residue 7, a T or M at residue 8, an E at residue 11, a K at residue 14, and an S at residue 18; (ii) The conditionally active receptor agonist of any one of claims 1 to 11, wherein X3 comprises an R at residue 3, an F at residue 4, a K at residue 6, an R at residue 7, an R at residue 10, an N at residue 11, a W at residue 13, and a G at residue 14.

16. X1 is a peptide 【Chemistry 35】 and a peptide comprising an amino acid sequence at least 75% identical along its length to X3 is a peptide 【Chemical 36】 and a peptide comprising an amino acid sequence at least 75% identical along its length to X4 is a peptide 【Chemical 37】 and a peptide comprising an amino acid sequence at least 75% identical along its length to (i) X1 comprises an I at residue 7, a T or M at residue 8, an E at residue 11, a K at residue 14, and an S at residue 18; (ii) The conditionally active receptor agonist of any one of claims 1 to 11, wherein X3 comprises an R at residue 3, an F at residue 4, a K at residue 6, an R at residue 7, an R at residue 10, an N at residue 11, a W at residue 13, and a G at residue 14.

17. X1 is a peptide 【Chemical 38】 and a peptide comprising an amino acid sequence at least 80% identical along its length to X3 is a peptide 【Chemical Formula 39】 and a peptide comprising an amino acid sequence at least 80% identical along its length to X4 is a peptide 【Chemistry 40】 and a peptide comprising an amino acid sequence at least 80% identical along its length to (i) X1 comprises an I at residue 7, a T or M at residue 8, an E at residue 11, a K at residue 14, and an S at residue 18; (ii) The conditionally active receptor agonist of any one of claims 1 to 11, wherein X3 comprises an R at residue 3, an F at residue 4, a K at residue 6, an R at residue 7, an R at residue 10, an N at residue 11, a W at residue 13, and a G at residue 14.

18. X1 is a peptide 【Chemistry 41】 and a peptide comprising an amino acid sequence at least 90% identical along its length to X3 is a peptide 【Chemistry 42】 and a peptide comprising an amino acid sequence at least 90% identical along its length to X4 is a peptide 【Chemistry 43】 and a peptide comprising an amino acid sequence at least 90% identical along its length to (i) X1 comprises an I at residue 7, a T or M at residue 8, an E at residue 11, a K at residue 14, and an S at residue 18; (ii) X3 is R at residue 3, F at residue 4, K at residue 6, R at residue 7, R at residue 10; 12. The conditionally active receptor agonist of any one of claims 1 to 11, comprising an N at residue 11, a W at residue 13, and a G at residue 14.

19. (iii) X4 comprises F at residue 19. The conditionally active receptor agonist of any one of claims 14 to 18.

20. 20. The conditionally active receptor agonist of any one of claims 1 to 19, wherein amino acid (AA) substitutions relative to the reference peptide domain occur at no more than three amino acid residues marked in bold, or at no more than two amino acid residues marked in bold, or at no more than one amino acid residue marked in bold, or at no amino acid residues marked in bold.

21. The conditionally active receptor agonist of any one of claims 1 to 20, wherein the amino acid substitutions relative to the reference peptide domain are conservative amino acid substitutions.

22. 22. The conditionally active receptor agonist of any one of claims 1 to 21, wherein the amino acid residues related to SEQ ID NO: 4 are selected from the group consisting of: 【Table 1】

23. 23. The conditionally active receptor agonist of claim 22, wherein position 7 is I, position 8 is M or T, position 11 is E, position 14 is K, and position 18 is S.

24. 23. The conditionally active receptor agonist of claim 22, wherein one, two, three, four, or five of the following are not true: 7th is I; 8th is M or T; 11th is E; 14th is K; and 18th is S.

25. 25. The conditionally active receptor agonist of any one of claims 1 to 24, wherein the amino acid residues relative to SEQ ID NO:5 are selected from the group consisting of: 【Table 2】

26. 26. The conditionally active receptor agonist of any one of claims 1 to 25, comprising a cysteine ​​substitution at position 17 or 20 relative to SEQ ID NO:

5.

27. 27. The conditionally active receptor agonist of claim 25 or 26, wherein the third position is R, the fourth position is F, the sixth position is K, the seventh position is R, the tenth position is R, the eleventh position is N, the thirteenth position is W, and the fourteenth position is G.

28. 27. The conditionally active receptor agonist of claim 25 or 26, wherein one, two, three, four, five, six, seven or all eight of the following are not true: the third is R, the fourth is F, the sixth is K, the seventh is R, the tenth is R, the eleventh is N, the thirteenth is W, and the fourteenth is G.

29. 29. The conditionally active receptor agonist of any one of claims 1 to 28, wherein the amino acid residues relative to SEQ ID NO:6 are selected from the group consisting of: 【Table 3】

30. 30. The conditionally active receptor agonist of any one of claims 1 to 29, comprising a cysteine ​​substitution at position 3 relative to SEQ ID NO:

6.

31. 32. The conditionally active receptor agonist of claim 30 or 31, wherein position 19 is I.

32. 32. The conditionally active receptor agonist of claim 30 or 31, wherein position 19 is not I.

33. X2, 【Chemistry 44】 33. The conditionally active receptor agonist of any one of claims 1 to 32, which is a peptide comprising an amino acid sequence that is 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

34. 34. The conditionally active receptor agonist of claim 11 or 33, wherein the amino acid residues relative to SEQ ID NO: 7 are selected from the group consisting of: 【Table 4】

35. 35. The conditionally active receptor agonist of claim 11, 33, or 34, comprising a cysteine ​​substitution at position 1, 2, 5, 9, 12, or 16 relative to SEQ ID NO:

7.

36. 36. The conditionally active receptor agonist of claim 34 or 35, wherein position 11 is I.

37. 36. The conditionally active receptor agonist of claim 34 or 35, wherein position 11 is not I.

38. (i) the first polypeptide component comprises one of X1, X2, X3, and X4, and the second polypeptide component comprises three of X1, X2, X3, and X4 that are not present in the first polypeptide component; or (ii) the first polypeptide component comprises two of X1, X2, X3, and X4, and the second polypeptide component comprises two of X1, X2, X3, and X4 that are not present in the first polypeptide component.

39. (i) the first polypeptide comprises X1, and the second polypeptide comprises X2, X3, and X4; (ii) the first polypeptide comprises X2 and the second polypeptide comprises X1, X3, and X4; (iii) the first polypeptide comprises X3 and the second polypeptide comprises X1, X2, and X4; (iv) the first polypeptide comprises X4 and the second polypeptide comprises X1, X2, and X3; (v) the first polypeptide comprises X1 and X2, and the second polypeptide comprises X3 and X4; (vi) the first polypeptide comprises X1 and X3, and the second polypeptide comprises X2 and X4; (vii) the first polypeptide comprises X1 and X4, and the second polypeptide comprises X2 and X3; (viii) the first polypeptide comprises X2 and X3, and the second polypeptide comprises X1 and X4; (ix) the first polypeptide comprises X2 and X4, and the second polypeptide comprises X1 and X3; (x) the first polypeptide comprises X3 and X4, and the second polypeptide comprises X1 and X2; (xi) the first polypeptide comprises X1, X2, and X3, and the second polypeptide comprises X4; (xii) the first polypeptide comprises X1, X2, and X4, and the second polypeptide comprises X3; (xiii) the first polypeptide comprises X1, X3, and X4, and the second polypeptide comprises X2; or (xiv) The conditionally active receptor agonist of any one of claims 1-37, wherein the first polypeptide comprises X2, X3, and X4, and the second polypeptide comprises X1.

40. (a) the first polypeptide is a fusion protein that includes X1 and excludes X2, X3, and X4, and the second polypeptide includes X3-Z1-X2-Z2-X4 and excludes X1; (b) the first polypeptide is a fusion protein comprising X4 and excluding X1, X2, and X3, and the second polypeptide comprises X1-Z1-X3-Z2-X2 and excluding X4; or (c) the first polypeptide is a fusion protein comprising X1-Z1-X3 and excluding X2 and X4, and the second polypeptide is a fusion protein comprising X2-Z1-X4 and excluding X1 and X3; 40. The conditionally active receptor agonist of any one of claims 1 to 39, wherein each of Z1 and Z2 is independently any amino acid linker.

41. 41. The conditionally active receptor agonist of any one of claims 1-40, wherein X1, X2, X3, and X4 comprise amino acid sequences 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 the X1, X2, X3, and X4 domains shown below, respectively, and the residues in brackets may be present or absent. 【Chemistry 45】

42. 42. The conditionally active receptor agonist of any one of claims 1-41, wherein the first and second polypeptides comprise amino acid sequences at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical to a pair of first and second polypeptides set forth below (wherein the underlined or "X" residues are optional and, if present, each residue of any domain may comprise any amino acid): (i) First Polypeptide X1 (Neo2A) 【Chemistry 46】 and Second Polypeptide X3-X2'-X4 (Neo2B) 【Chemistry 47】 (ii) First Polypeptide X1-X3-X2' 【Chemistry 48】 and Second Polypeptide X4 【Chemistry 49】 (iii) First Polypeptide X1-X3 【Chemistry 50】 and Second Polypeptide X2-X4 【Chemistry 51】 (iv) First Polypeptide X1 (Neo4A) 【Chemistry 52】 Second Polypeptide X3-X2'-X4 (Neo4B) 【Chemistry 53】 (v) First Polypeptide X1 【Chemical 54】 and Second Polypeptide X3-X2'-X4 【Chemistry 55】 (vi) First Polypeptide X1-X3-X2' 【Chemistry 56】 and Second Polypeptide X4 【Chemistry 57】 (vii) First Polypeptide X1-X3 【Chemistry 58】 and Second Polypeptide X2-X4 【Chemistry 59】 (viii) First Polypeptide X1 【Chemistry 60】 Second Polypeptide X3-X2'-X4 【Chemistry 61】 (ix) First Polypeptide>Neo4_X1-X3' 【Chemistry 62】 Second Polypeptide>Neo4_X2-X4 【Chemistry 63】 (x) First Polypeptide>Neo4_X1-X3' 【Chemistry 64】 Second Polypeptide>Neo4_X2-X4 【Chemistry 65】 (xi) First Polypeptide Neo4_X1-X3'-X2 【Chemistry 66】 Second Polypeptide Neo4X4 【Chemistry 67】 (xii) First Polypeptide Neo4_X1-X3'-X2 【Chemistry 68】 Second Polypeptide Neo4_X4 【Chemistry 69】 (xiii) First Polypeptide (X1) 【Chemistry 70】 Second Polypeptide (X3-X2'-X4) 【Chemistry 71】

43. 42. The conditionally active receptor agonist of any one of claims 1-41, wherein X1, X2, X3, and X4 comprise amino acid sequences that are at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical, respectively, to the X1, X2, X3, and X4 domains (as defined in Table 1) present within the amino acid sequences of SEQ ID NOs: 11-94, 190-216, 247, and SEQ ID NOs: 275-300, respectively.

44. 44. The conditionally active receptor agonist of any one of claims 1 to 43, wherein the first polypeptide component and / or the second polypeptide component comprises at least one disulfide bond.

45. 45. The conditionally active receptor agonist of any one of claims 1 to 44, wherein said first polypeptide component and said second polypeptide component are non-covalently linked.

46. 45. The conditionally active receptor agonist of any one of claims 1 to 44, wherein the first polypeptide component and the second polypeptide component are indirectly bound to each other via a receptor.

47. 47. The conditionally active receptor agonist of any one of claims 1 to 46, wherein the first polypeptide component further comprises a first targeting domain or the second polypeptide component further comprises a second targeting domain.

48. 47. The conditionally active receptor agonist of any one of claims 1 to 46, wherein the first polypeptide component further comprises a first targeting domain and the second polypeptide component further comprises a second targeting domain.

49. 49. The conditionally active receptor agonist of claim 47 or 48, wherein the first targeting domain, if present, is a translational fusion with the first polypeptide, and the second targeting domain, if present, is a translational fusion with the second polypeptide.

50. 50. The conditionally active receptor agonist of any one of claims 47 to 49, wherein the first targeting domain and the second targeting domain are both present, and the first targeting domain and the second targeting domain are identical.

51. 50. The conditionally active receptor agonist of any one of claims 47 to 49, wherein the first targeting domain and the second targeting domain are both present, and the first targeting domain and the second targeting domain are different.

52. The first targeting domain and / or the second targeting domain are targeted to a cell surface protein.

52. The conditionally active receptor agonist of any one of claims 47 to 51, which binds to a substrate, respectively.

53. 53. The conditionally active receptor agonist of claim 52, wherein the cell surface protein is present on the surface of a cell selected from the group consisting of tumor cells, vascular components, the tumor microenvironment (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), e.g., immune cell surface markers 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.

54. 54. The conditionally active receptor agonist of claim 52 or 53, wherein the first targeting domain and the second targeting domain bind to a surface marker of a tumor cell, a tumor vascular component cell, or a tumor microenvironment cell, respectively.

55. The surface markers of the tumor cells, vascular component cells, or tumor microenvironment cells are 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, 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, p53 non-mutant, 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 (animals), 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, XAGE 1, 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, Fos-related antigen 1, BMPR1B (bone morphogenetic protein receptor-type IB, Genbank accession number NM.001203), E16 (LAT1, SLC7A5, Genbank accession number NM-003486), STE AP1 (six-transmembrane epithelial antigen of the prostate, Genbank accession number NM012449), 0772P (CA125, MUC16, Genbank accession number AF361486), MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession number NM005823), Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession number NM006424), Sema5b (FLJ1037 2, 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 no. AB040878), PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA2700050C12, RIKEN cDNA2700050C12 gene, Genbank accession number AY358628), ETBR (endothelin type B receptor, Genbank accession number AY275463), MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession number NM017763), STEAP2 (HGNC.sub.--8639, IPCA-1, PCA NAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six-transmembrane epithelial antigen 2 of the prostate, six-transmembrane protein, Genbank accession no. AF455138), TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM017636), CRIPTO (CR, CR1, CRGF, CRIP TO, TDGF1, teratoma-derived growth factor, Genbank accession no. NP003203 or NM003212), CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor) or Hs.73792, Genbank accession no. M26004), CD79b (IGb (immunoglobulin-related beta), B29, Genbank accession no. NM000626), FcRH2 (IFGP4, IRTA4, SPAP1A (phosphatase anchor SH2 domain containing protein 1a), SPAP1B, SPAP1C, Genbank Accession No. NM030764), HER2 (Genbank Accession No. M11730), NCA (Genbank Accession No. M18728), MDP (Genbank Accession No. BC017023), IL20Ralpha, (Genbank Accession No. AF184971), brevican (Genbank Accession No. AF229053), Ephb2R (Genbank Accession No. NM004442),ASLG659 (Genbank Accession No. AX092328), PSCA (Genbank Accession No. AJ297436), GEDA (Genbank Accession No. AY260763), BAFF-R (Genbank Accession No. NP443177.1), CD22 (Genbank Accession No. NP-001762.1), and CD79a (CD79A, CD79 alpha, immunoglobulin-related alpha, Ig beta (CD79B) are B cell-specific proteins that covalently interact with IgM molecules and form complexes with IgM molecules on the surface, and are involved in B cell differentiation signaling. , Genbank Accession No. NP001774.1), CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor activated by the CXCL13 chemokine that functions in lymphocyte migration and humoral defense and is involved in HIV-2 infection and possibly the development of AIDS, lymphoma, myeloma, and leukemia, Genbank Accession No. NP001707.1), HLA-DOB (beta subunit of MHC class II molecule (Ia antigen) that binds and presents peptides to CD4+ T lymphocytes, Genbank Accession No. NP00211 1.1), P2X5 (P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, and deficiency may contribute to the pathophysiology of idiopathic detrusor instability, Genbank Accession No. NP002552.2), CD72 (B cell differentiation antigen CD72, Lyb-2, Genbank Accession No. NP001773.1), LY64 (Lymphocyte antigen 64 (RP105), a type I membrane protein of the leucine-rich repeat (LRR) family, is involved in the activation of B cells. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 108, 109, 109, 109, 109, 109, 110, 111, 112, 113, 114,Translocation-induced gene deregulation occurs in some B-cell malignancies, Genb, 55. The conditionally active receptor agonist of claim 54, wherein the conditionally active receptor agonist is selected from the group that may include, but is not limited to, the agonist of claim 54 (ank registration number NP112571.1).

56. The first targeting domain and / or the second targeting domain bind to an immune cell surface marker, including, but not limited to, a CD8+ T cell, a regulatory T cell, a dendritic cell, a NK cell, or a macrophage, and the immune cell surface marker 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 56. The conditionally active receptor agonist of any one of claims 47-55, which may include, but is not limited 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.

57. 54. The conditionally active receptor agonist of claim 52 or 53, wherein the first targeting domain and the second targeting domain each bind to a T cell surface marker, including but not limited to, a CD8+ T cell, a regulatory T cell, a dendritic cell, or a macrophage.

58. 54. The conditionally active receptor agonist of claim 52 or 53, wherein either the first targeting domain or the second targeting domain binds to a surface marker of a tumor cell, a vascular component cell, or a tumor microenvironment cell (e.g., fibroblasts, infiltrating immune cells, or stromal elements), and the other targeting domain binds to an immune cell surface marker (including, but not limited to, CD8+ T cells, regulatory T cells, dendritic cells, or macrophages), which may include, but are not limited to, CD3, CD4, CD8, CD19, CD20, CD21, CD25, CD37, CD30, CD33, CD40, CD68, CD123, CD254, PD-1, B7-H3, and CTLA-4.

59. The first targeting domain and / or the second targeting domain is an scFv, F(ab), F(ab') ２ 59. The conditionally active receptor agonist of any one of claims 47-58, which may include, but is not limited to, B cell receptors (BCRs), DARPins, affibodies, monobodies, nanobodies, diabodies, antibodies (including monospecific or bispecific antibodies), and cell targeting oligopeptides include, but are not limited to, RGD integrin binding peptides, de novo designed binders, aptamers, bicyclic peptides, conotoxins, small molecules such as folate, and viruses that bind to the cell surface.

60. The first targeting domain and / or the second targeting domain is an scFv, F(ab), F(ab') ２ 59. The conditionally active receptor agonist of any one of claims 47-58, which may include, but is not limited to, B cell receptors (BCRs), DARPins, affibodies, monobodies, nanobodies, diabodies, and antibodies (including monospecific or bispecific antibodies).

61. X2, 【Chemical 72】 61. The conditionally active receptor agonist of any one of claims 1 to 60, which is a peptide comprising an amino acid sequence that is at least 70% identical along its length to: and wherein the residues in brackets are optional.

62. X2, 【Chemical Formula 73】 61. The conditionally active receptor agonist of any one of claims 1 to 60, which is a peptide comprising an amino acid sequence that is at least 80% identical along its length to: and wherein the residues in brackets are optional.

63. X2, 【Chemical 74】 61. The conditionally active receptor agonist of any one of claims 1 to 60, which is a peptide comprising an amino acid sequence that is at least 90% identical along its length to: and wherein the residues in brackets are optional.

64. A polypeptide comprising one, two or three, but not all four, of the domains X1, X2, X3 and X4, (a) X1, when present, is a peptide 【Chemistry 75】 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 the full length of (b) X2, if present, is any helical peptide domain; (c) X3, when present, is a peptide 【Chemical Formula 76】 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 the full length of (d) X4, when present, is a peptide 【Chemical 77】 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 the full length of A polypeptide in which the amino acid residues in brackets may or may not be present.

65. (a) X1, when present, is a peptide 【Chemical Formula 78】 A peptide comprising an amino acid sequence that is at least 70% identical to the full length of (b) X2, if present, is any helical peptide domain; (c) X3, when present, is a peptide 【Chemical 79】 A peptide comprising an amino acid sequence that is at least 70% identical to the full length of (d) X4, when present, is a peptide 【Chemistry 80】 The polypeptide of claim 64, which is a peptide comprising an amino acid sequence that is at least 70% identical to the entire length of the polypeptide.

66. (a) X1, when present, is a peptide 【Chemistry 81】 A peptide comprising an amino acid sequence that is at least 85% identical to the full length of (b) X2, if present, is any helical peptide domain; (c) X3, when present, is a peptide 【Chemistry 82】 A peptide comprising an amino acid sequence that is at least 85% identical to the full length of (d) X4, when present, is a peptide 【Chemistry 83】 The polypeptide of claim 64, which is a peptide comprising an amino acid sequence that is at least 85% identical to the full length of the polypeptide of claim 64.

67. (a) X1, when present, is a peptide 【Chemistry 84】 a peptide comprising the same amino acid sequence as the full length of (b) X2, if present, is any helical peptide domain; (c) X3, when present, is a peptide 【Chemistry 85】 a peptide comprising the same amino acid sequence as the full length of (d) X4, when present, is a peptide 【Chemistry 86】 65. The polypeptide of claim 64, which is a peptide comprising the same amino acid sequence as the full length of said polypeptide.

68. (a) X1, when present, is a peptide 【Chemistry 87】 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 the full length of (c) X3, when present, is a peptide 【Chemistry 88】 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 the full length of (d) X4, when present, is a peptide 【Chemistry 89】 65. The polypeptide of claim 64, which is a peptide comprising an amino acid sequence 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

69. (a) X1, when present, is a peptide 【Chemistry 90】 A peptide comprising an amino acid sequence at least 65% identical to the full length of (c) X3, when present, is a peptide 【Chemistry 91】 A peptide comprising an amino acid sequence at least 65% identical to the full length of (d) X4 is a peptide 【Chemistry 92】 65. The polypeptide of claim 64, which is a peptide comprising an amino acid sequence that is at least 65% identical to the full length of

70. (a) X1, when present, is a peptide 【Chemistry 93】 A peptide comprising an amino acid sequence that is at least 75% identical to the full length of (c) X3, when present, is a peptide 【Chemistry 94】 A peptide comprising an amino acid sequence that is at least 75% identical to the full length of (d) X4, when present, is a peptide 【Chemistry 95】 69. The polypeptide of claim 68, which is a peptide comprising an amino acid sequence that is at least 75% identical to the full length of

71. (a) X1, when present, is a peptide 【Chemistry 96】 A peptide comprising an amino acid sequence that is at least 80% identical to the full length of (c) X3, when present, is a peptide 【Chemistry 97】 A peptide comprising an amino acid sequence that is at least 80% identical to the full length of (d) X4, when present, is a peptide 【Chemistry 98】 69. The polypeptide of claim 68, which is a peptide comprising an amino acid sequence that is at least 80% identical to the full length of

72. (a) X1, when present, is a peptide 【Chemistry 99】 A peptide comprising an amino acid sequence that is at least 90% identical to the full length of (c) X3, when present, is a peptide 【Chemistry 100】 A peptide comprising an amino acid sequence that is at least 90% identical to the full length of (d) X4, when present, is a peptide 【Chemistry 101】 69. The polypeptide of claim 68, which is a peptide comprising an amino acid sequence that is at least 90% identical to the full length of

73. X2, when present, is a peptide 【Chemistry 102】 73. The polypeptide of any one of claims 64-72, 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 the full length of

74. X2, when present, is a peptide 【Chemistry 103】 73. The polypeptide of any one of claims 64 to 72, comprising an amino acid sequence that is at least 75% identical to the full length of:

75. X2, when present, is a peptide 【Chemistry 104】 73. The polypeptide of any one of claims 64 to 72, comprising an amino acid sequence that is at least 85% identical to the full length of:

76. X2, when present, is a peptide 【Chemistry 105】 73. The polypeptide of any one of claims 64 to 72, comprising an amino acid sequence that is at least 90% identical to the full length of:

77. X2, when present, is a peptide 【Chemistry 106】 73. The polypeptide of any one of claims 64 to 72, comprising an amino acid sequence that is at least 80% identical to the full length of:

78. X2, when present, is a peptide 【Chemistry 107】 73. The polypeptide of any one of claims 64 to 72, comprising an amino acid sequence that is at least 90% identical to the full length of:

79. (i) a polypeptide that includes X1 and excludes X2, X3, and X4; (ii) a polypeptide that includes X2 and excludes X1, X3, and X4; (iii) a polypeptide that includes X3 and excludes X1, X2, and X4; (iv) a polypeptide that includes X4 and excludes X1, X2, and X3; (v) a polypeptide comprising X1 and X2 and excluding X3 and X4; (vi) a polypeptide comprising X1 and X3 and excluding X2 and X4; (vii) a polypeptide comprising X1 and X4 and excluding X2 and X3; (viii) a polypeptide comprising X2 and X3 and excluding X1 and X4; (ix) a polypeptide comprising X2 and X4 and excluding X1 and X3; (x) a polypeptide comprising X3 and X4 and excluding X1 and X2; (xi) a polypeptide comprising X1, X2, and X3, and excluding X4; (xii) a polypeptide comprising X1, X2, and X4, but excluding X3; (xiii) a polypeptide comprising X1, X3, and X4, but excluding X2; and (xiv) a polypeptide comprising X2, X3, and X4, and excluding X1.

80. 80. The polypeptide of any one of claims 64-79, wherein the polypeptide comprises X1, wherein X1 comprises one, two, three, four, or all five of: L at residue 7, H at residue 8, H at residue 11, Y at residue 14, and M at residue 18, and numbering is based on the presence of any residue relative to SEQ ID NO:

4.

81. 81. The polypeptide of any one of claims 64-80, wherein the polypeptide comprises X3, wherein X3 comprises one, two, three, four, five, six, seven or all eight of: 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, and numbering is made relative to SEQ ID NO:5 based on the presence of any residue.

82. 82. The polypeptide of any one of claims 64-81, wherein the polypeptide comprises X4, wherein X4 comprises an I at residue 19, and numbering is relative to SEQ ID NO:6 based on the presence of any residue.

83. X1 is present, and X1 is a peptide 【Chemistry 108】 80. The polypeptide of any one of claims 64-79, wherein the polypeptide comprises 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:

84. X3 is present, and X3 is a peptide 【Chemistry 109】 84. The polypeptide of any one of claims 64-79 or 83, wherein the polypeptide comprises an amino acid sequence at least 37%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical along its length to:

85. X4 is present, and X4 is a peptide 【Chemistry 110】 85. The polypeptide of any one of claims 64-79, 84, or 84, which is a peptide comprising an amino acid sequence that is 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

86. 86. The polypeptide of claim 85, wherein X4 comprises F at residue 19.

87. 87. The polypeptide of any one of claims 64 to 86, wherein amino acid (AA) substitutions relative to the reference peptide domain occur at no more than three amino acid residues marked in bold, or at no more than two amino acid residues marked in bold, or at no more than one amino acid residue marked in bold, or at no amino acid residues marked in bold.

88. A polypeptide according to any one of claims 64 to 87, wherein the amino acid substitutions relative to the reference peptide domain are conservative amino acid substitutions.

89. 89. The polypeptide of any one of claims 64 to 88, wherein X1 is present and the amino acid residues relative to SEQ ID NO:4 are selected from the group consisting of: 【Table 5】

90. 90. The polypeptide of claim 89, wherein the 7th position is I, the 8th position is M or T, the 11th position is E, the 14th position is K, and the 18th position is S.

91. 90. The polypeptide of claim 89, wherein one, two, three, four, or five of the following are not true: 7th is I, 8th is M or T, 11th is E, 14th is K, and 18th is S.

92. 92. The polypeptide of any one of claims 64 to 91, wherein X3 is present and the amino acid residues relative to SEQ ID NO:5 are selected from the group consisting of: 【Table 6】

93. 93. The polypeptide of claim 92, comprising a cysteine ​​substitution at position 17 or 20 relative to SEQ ID NO:

5.

94. The polypeptide of claim 92 or 93, wherein the third position is R, the fourth position is F, the sixth position is K, the seventh position is R, the tenth position is R, the eleventh position is N, the thirteenth position is W, and the fourteenth position is G.

95. The polypeptide of claim 92 or 93, wherein one, two, three, four, five, six, seven or all eight of the following are not true: the third is R, the fourth is F, the sixth is K, the seventh is R, the tenth is R, the eleventh is N, the thirteenth is W, and the fourteenth is G.

96. 96. The polypeptide of any one of claims 64 to 95, wherein X4 is present and the amino acid residue relative to SEQ ID NO:6 is selected from the group consisting of: 【Table 7】

97. 97. The polypeptide of claim 96, comprising a cysteine ​​substitution at position 3 relative to SEQ ID NO:

6.

98. 97. The polypeptide of claim 95 or 96, wherein the 19th residue is I.

99. 97. The polypeptide of claim 95 or 96, wherein position 19 is not I.

100. 100. The polypeptide of any one of claims 64 to 99, wherein X4 is present and the amino acid residue relative to SEQ ID NO:7 is selected from the group consisting of: 【Table 8】

101. 101. The polypeptide of claim 100, comprising a cysteine ​​substitution at position 1, 2, 5, 9, 12, or 16 relative to SEQ ID NO:

7.

102. 102. The polypeptide of claim 101 or 101, wherein the 11th position is I.

103. 102. The polypeptide of claim 100 or 101, wherein the 11th residue is not I.

104. 104. The polypeptide of any one of claims 64-103, wherein the polypeptide comprises 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 a first polypeptide or a second polypeptide listed below (underlined residues are optional, and when present, any each residue may comprise any amino acid). (i) First Polypeptide X1 (Neo2A) 【Chemistry 111】 and Second Polypeptide X3-X2'-X4 (Neo2B) 【Chemistry 112】 (ii) First Polypeptide X1-X3-X2' 【Chemistry 113】 Second Polypeptide X4 【Chemistry 114】 (iii) First Polypeptide X1-X3 【Chemistry 115】 and Second Polypeptide X2-X4 【Chemistry 116】 (iv) First Polypeptide X1 (Neo4A) 【Chemistry 117】 Second Polypeptide X3-X2'-X4 (Neo4B) 【Chemistry 118】 (v) First Polypeptide X1 (Neo2A) 【Chemistry 119】 and Second Polypeptide X3-X2'-X4 (Neo2B) 【Chemistry 120】 (vi) First Polypeptide X1-X3-X2' 【Chemistry 121】 Second Polypeptide X4 【Chemistry 122】 (vii) First Polypeptide X1-X3 【Chemistry 123】 and Second Polypeptide X2-X4 【Chemical 124】 (viii) First Polypeptide X1 (Neo4A) 【Chemistry 125】 Second Polypeptide X3-X2'-X4 (Neo4B) 【Chemistry 126】 (ix) First Polypeptide>Neo4_X1-X3' 【Chemistry 127】 Second Polypeptide>Neo4_X2-X4 【Chemistry 128】 (x) First Polypeptide>Neo4_X1-X3' 【Chemistry 129】 Second Polypeptide>Neo4_X2-X4 【Chemistry 130】 (xi) First Polypeptide Neo4_X1-X3'-X2 【Chemistry 131】 Second Polypeptide Neo4_X4 【Chemistry 132】 (xii) First Polypeptide Neo4_X1-X3'-X2 【Chemistry 133】 Second Polypeptide Neo4_X4 【Chemistry 134】 (xiii) First Polypeptide (X1) 【Chemistry 135】 Second Polypeptide (X3-X2'-X4) 【Chemistry 136】

105. 104. The polypeptide of any one of claims 64-103, wherein X1, X2, X3, and X4, if present, comprise an amino acid sequence 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 X1, X2, X3, and X4 domains (as defined in Table 1) present within the amino acid sequences of SEQ ID NOs: 11-94, 190-216, 247, and SEQ ID NOs: 275-300, respectively.

106. The polypeptide of any one of claims 64 to 105, wherein the polypeptide comprises at least one disulfide bond.

107. The polypeptide of any one of claims 64 to 106, wherein the polypeptide further comprises a targeting domain.

108. 108. The polypeptide of claim 107, wherein the targeting domain is a translational fusion with the polypeptide.

109. The polypeptide of claim 108, wherein the targeting domain binds to a cell surface protein.

110. The polypeptide of claim 109, wherein the cell surface protein is present on the surface of a cell selected from the group consisting of tumor cells, vascular components, the tumor microenvironment (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), e.g., an immune cell surface marker, 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.

111. The polypeptide of any one of claims 107-110, wherein the targeting domain binds to a surface marker of a tumor cell, a vascular component cell, or a tumor microenvironment cell (e.g., a fibroblast, an infiltrating immune cell, or a stromal element).

112. The surface markers of the tumor cells, vascular component cells, or tumor microenvironment cells (e.g., fibroblasts, infiltrating immune cells, or stromal elements) are EGFR, EGFRvIII, Her2, HER3, EpCAM, MSLN, MUC16, PSMA, TROP2, ROR1, RON, PD-L1, CD47, CTLA-4, CD5, CD19, CD20, CD2 5, CD37, CD30, CD33, CD40, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC, HDL-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, p53 non-mutant, 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 (animals), 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, Fos-related antigen 1, BMPR1B (bone morphogenetic protein Protein receptor-type IB, Genbank accession number NM.001203), E16 (LAT1, SLC7A5, Genbank accession number NM-003486), STEAP1 (six-transmembrane epithelial antigen of the prostate, Genbank accession number NM012449), 0772P (CA125, MUC16, Genbank accession number AF361486), MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor , mesothelin, Genbank accession number NM005823), Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession number NM006424), Sema5b (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), PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA2700050C12, RIKEN cDNA2700050C12 gene, Genbank accession number AY358628), ETBR (endothelin type B receptor, Genbank accession number AY275463), MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession number NM0 17763), STEAP2 (HGNC.sub.--8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six-transmembrane epithelial antigen 2 of the prostate, six-transmembrane prostate protein, Genbank accession no. AF455138), TrpM4 (BR22450, FLJ20041, TRPM4, TRP M4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession number NM017636), CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratoma-derived growth factor, Genbank accession numbers NP003203 or NM003212), CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor ) or Hs. 73792, Genbank Accession No. M26004), CD79b (IGb (immunoglobulin-related beta), B29, Genbank Accession No. NM000626), FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank Accession No. NM030764),HER2 (Genbank Accession No. M11730), NCA (Genbank Accession No. M18728), MDP (Genbank Accession No. BC017023), IL20Ralpha, (Genbank Accession No. AF184971), brevican (Genbank Accession No. AF229053), Ephb2R (Genbank Accession No. NM004442), ASLG659 (Genbank Accession No. AX092328), PSCA (Genbank Accession No. AJ297436), GEDA (Genbank Accession No. AY260763), BAFF- R (Genbank accession number NP443177.1), CD22 (Genbank accession number NP-001762.1), CD79a (CD79A, CD79 alpha, immunoglobulin-related alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex with IgM molecules on the surface, transduces signals involved in B cell differentiation, Genbank accession number NP001774.1), CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor activated by the CXCL13 chemokine, is a B cell-specific protein that mediates B cell differentiation, Genbank accession number NP001774.1), and CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor activated by the CXCL13 chemokine, is a B cell-specific protein that mediates B cell differentiation, Genbank accession number NP001774.1). HLA-DOB (beta subunit of MHC class II molecule (Ia antigen) that binds peptides and presents them to CD4+ T lymphocytes; Genbank Accession No. NP002111.1); P2X5 (purinergic receptor P2X ligand-gated ion channel 5; an ion channel gated by extracellular ATP that may be involved in synaptic transmission and neurogenesis; deficiency is particularly may contribute to the pathophysiology of chronic detrusor instability, Genbank Accession No. NP002552.2), CD72 (B cell differentiation antigen CD72, Lyb-2, Genbank Accession No. NP001773.1), LY64 (lymphocyte antigen 64 (RP105), a type I membrane protein of the leucine-rich repeat (LRR) family that regulates B cell activation and apoptosis, and loss of function is associated with increased disease activity in patients with systemic lupus erythematosus, Genbank Accession No. NP005573.1), FCRH1 (Fc receptor-like protein 1,A putative receptor for immunoglobulin Fc domains containing C2-type Ig-like and ITAM domains; 112. The polypeptide of claim 111, which may be involved in B-lymphocyte differentiation, Genbank Accession No. NP443170.1), or IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, an immune receptor putatively involved in B-cell development and lymphomagenesis, gene deregulation by translocation occurs in some B-cell malignancies, Genbank Accession No. NP112571.1).

113. 101. The polypeptide of any one of claims 107-100, wherein the targeting domain binds to an immune cell surface marker, including but not limited to, a CD8+ T cell, a regulatory T cell, a dendritic cell, or a macrophage, which may include, but is not limited to, CD3, CD4, CD8, CD19, CD20, CD21, CD25, CD37, CD30, CD33, CD40, CD68, CD123, CD254, PD-1, B7-H3, and CTLA-4.

114. The targeting domain is an scFv, F(ab), F(ab') ２ , B cell receptors (BCR), DARPins, affibodies, monobodies, nanobodies, diabodies, antibodies (including monospecific or bispecific antibodies), and cell-targeting oligopeptides include, but are not limited to, RGD integrin binding peptides, de novo designed binders, aptamers, bicyclic peptides, conotoxins, small molecules such as folate, and viruses that bind to the cell surface.

115. A nucleic acid encoding a polypeptide according to any one of claims 64 to 114, or the first or second polypeptide component according to any one of claims 1 to 63.

116. 116. An expression vector comprising the nucleic acid of claim 115 operably linked to a promoter.

117. A host cell comprising the nucleic acid of claim 115 and / or the expression vector of claim 116.

118. A pharmaceutical composition comprising a conditionally active receptor agonist according to any one of claims 1 to 63, a polypeptide according to any one of claims 64 to 114, a nucleic acid according to claim 115, an expression vector according to claim 116, or a host cell according to claim 117, and a pharma- ceutically acceptable carrier.

119. 64. A method of treating cancer comprising administering to a subject in need thereof the conditionally active receptor agonist of any one of claims 1-63 under conditions wherein said first and second polypeptide components interact in cells of said tumor to treat said cancer.

120. The method of claim 119, wherein the first polypeptide component comprises a first targeting domain, the second polypeptide component comprises a second targeting domain, the first targeting domain binds to a first tumor cell marker, and the second targeting domain binds to a second tumor cell marker, which may be the same as or different from the first tumor cell marker, and the first and second polypeptides interact only after the first targeting domain binds to the first tumor cell marker and the second targeting domain binds to the second tumor cell marker.

121. The first polypeptide component comprises a first targeting domain, and the second polypeptide 121. The method of claim 120, wherein the moiety comprises a second targeting domain, wherein the first targeting domain binds to a tumor cell marker and the second targeting domain binds to an immune cell marker (including, but not limited to, a CD8+ T cell, a regulatory T cell, a dendritic cell, or a macrophage), and wherein the first and second polypeptides interact only after the first targeting domain binds to the tumor cell marker and the second targeting domain binds to the immune cell marker.

122. 121. The method of claim 120, wherein the first polypeptide component comprises a first targeting domain and the second polypeptide component comprises a second targeting domain, wherein the first targeting domain binds to a first immune cell marker (including but not limited to, CD8+ T cells, regulatory T cells, dendritic cells, or macrophages), and the second targeting domain binds to a second immune cell marker (including but not limited to, CD8+ T cells, regulatory T cells, dendritic cells, or macrophages), which may be the same as or different from the first immune cell marker, and wherein the first and second polypeptides interact only after the first targeting domain binds to the first immune cell marker and the second targeting domain binds to the second immune cell marker.

123. The method of claims 119-122, wherein the cancer is selected from the group consisting of 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 mutation count, PD-L1 expression level, or Immunoscore assay (developed by the Society for Immunotherapy of Cancer).

124. A conditionally active receptor agonist according to any one of claims 1 to 63, a polypeptide according to any one of claims 64 to 114, a nucleic acid according to claim 115, an expression vector according to claim 116, or a host cell according to claim 117, or a pharmaceutical composition according to claim 118, for use as a medicament for treating cancer and / or modulating an immune response in a subject.

125. 130. A method of modulating an immune response in a subject, comprising administering to the subject a conditionally active receptor agonist according to any one of claims 1 to 63, a polypeptide according to any one of claims 64 to 114, a nucleic acid according to claim 115, an expression vector according to claim 116, or a host cell according to claim 117, or a pharmaceutical composition according to claim 118.

126. The method of claim 125, wherein the immune response is an anti-cancer immune response, a tissue repair immune response, or a wound healing immune response.

127. 127. The method of claim 125 or 126, wherein the conditionally active receptor agonist, nucleic acid, expression vector, host cell, or pharmaceutical composition is administered as a component of a biomaterial.

128. 64. A method of stimulating the IL-2 receptor or the IL-4 receptor, comprising administering to a subject the conditionally active receptor agonist of any one of claims 1-63 under conditions in which the first and second polypeptide components interact at the receptor.

129. The first polypeptide component and the second polypeptide interact with each other to bind to an IL-2 receptor. Condition βγ Ｃ Heterodimer (IL-2Rβγ Ｃ 64. The conditionally active receptor agonist of any one of claims 1 to 63, which forms an active agonist of

130. The first polypeptide component and the second polypeptide interact to bind to the IL-4 receptor αγ Ｃ Heterodimer (IL-4Rαγ Ｃ ), IL-13 alpha, or an active agonist of an IL-4R alpha / IL13R alpha heterodimer.

131. 64. The conditionally active receptor agonist of any one of claims 1 to 63, wherein the amino acid residues in brackets are present.

132. 64. The conditionally active receptor agonist of any one of claims 1 to 63, wherein the amino acid residue within the brackets is absent.

133. 115. The polypeptide of any one of claims 64 to 114, wherein the amino acid residues in brackets are present.

134. A polypeptide according to any one of claims 64 to 114, wherein the amino acid residues in brackets are absent.

135. 64. The conditionally active receptor agonist of any one of claims 1-63, wherein X1, X3, and X4 comprise amino acid sequences at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the full length of the X1, X2, X3, and X4 domains, respectively, as shown below, and the residues in brackets may be present or absent. 【Chemistry 137】

136. 64. The conditionally active receptor agonist of any one of claims 1-63, wherein X1, X3, and X4 comprise amino acid sequences at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the full length of the X1, X2, X3, and X4 domains, respectively, as shown below, and the residues in brackets may be present or absent. 【Chemistry 138】

137. X2 is an amino acid sequence 【Chemistry 139】 137. The conditionally active receptor agonist of any one of claims 1-63, 135, and 136, comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the full length of

138. 64. The conditionally active receptor agonist of any one of claims 1 to 63, wherein X1, X2, X3, and X4 each comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the X1, X2, X3, and X4 domains, respectively, present within the type 1 or type 2 amino acid sequence of SEQ ID NO:90, as defined in Table 1.

138. 135. The polypeptide of any one of claims 64-114, 133 or 134, wherein X1, X3 and X4, if present, each comprise an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98% or 100% identical to the full length of the X1, X2, X3 and X4 domains shown below, respectively, and the residues in brackets may be present or absent. 【Chemistry 140】

139. X2, if present, is the amino acid sequence 【Chemistry 141】 139. The polypeptide of any one of claims 64-114, 133, 134 or 138, comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the full length of

140. 135. The polypeptide of any one of claims 64-114, 133, 134, wherein X1, X2, X3, and X4, if present, are at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical, respectively, to the X1, X2, X3, and X4 domains (as defined in Table 1) present within the type 1 or type 2 amino acid sequence of SEQ ID NO:90, as defined in Table 1.

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