Separated interleukin mimetics and their uses

Abstract

The present invention relates to the resolution of interleukin mimetics and their uses. Disclosed are conditionally active receptor agonists that bind to an IL-2 receptor [beta] [gamma] c heterodimer (IL-2R [beta] [gamma] c), an IL-4 receptor [alpha] [gamma] c heterodimer (IL-4R [alpha] [gamma] c), or an IL-13 receptor [alpha] subunit (IL-13R [alpha]) upon activation, as well as components of the conditionally active receptor agonists and methods for using the conditionally active receptor agonists.

Description

[0001] This application is a divisional application of the invention application with the application date of November 19, 2019, the Chinese application number of 201980070569.6, and the invention name of "Split interleukin mimetics and their uses".

[0002] Cross-reference

[0003] This application claims the priority of U.S. Provisional Patent Application Serial No. 62 / 770,152, filed on November 20, 2018, which is incorporated herein by reference in its entirety. Technical Field

[0004] The present invention relates to split interleukin mimetics and their uses. Background Art

[0005] The promising potential of central immunocytokines interleukin (such as IL-2 and IL-4) for cancer treatment has inspired numerous efforts to improve their therapeutic properties by mutation and / or chemical modification. However, because these approaches are closely tied to native IL-2 or IL-4, they cannot eliminate unwanted properties such as low stability and binding to the α subunit of the IL-2 receptor (IL-2Rα), the IL-4 receptor αγ c heterodimer (IL-4Rαγ c ), or the α subunit of the IL-13 receptor (IL-13Rα). Summary of the Invention

[0006] In one 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 do not exist as a fusion protein, and wherein the first polypeptide component and the second polypeptide component together comprise domains X1, X2, X3, and X4, wherein:

[0007] (a) X1 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 the peptide ;

[0008] (b) X2 is any helical peptide domain that is at least 8 amino acids in length;

[0009] (c) X3 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 the peptide ; and

[0010] (d) X4 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 the peptide wherein:

[0011] (i) the amino acid residues in parentheses may be present or absent;

[0012] (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; and

[0013] (iii) the second polypeptide component comprises each of X1, X2, X3, and X4 that is not present in the first polypeptide component; wherein the first polypeptide component and the second polypeptide component alone are not active receptor agonists, and wherein the first polypeptide component and the second polypeptide component interact to form an active agonist of an IL-2 receptor βγ

[0014] heterodimer (IL-2Rβγ c ), an IL-4 receptor αγ c heterodimer (IL-4Rαγ c ), an IL-13α, or an IL-4Rα / IL13Rα heterodimer. Numerous embodiments of the first and second polypeptides are provided herein. In an exemplary embodiment, c

[0015] (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

[0016] (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. In other exemplary embodiments,

[0017] (i) the first polypeptide comprises X1 and the second polypeptide comprises X2, X3, and X4;

[0018] (ii) the first polypeptide comprises X2 and the second polypeptide comprises X1, X3, and X4;

[0019] (iii) the first polypeptide comprises X3 and the second polypeptide comprises X1, X2, and X4;

[0020] ​(iv) The first polypeptide comprises X4 and the second polypeptide comprises X1, X2, and X3;

[0021] (v) The first polypeptide comprises X1 and X2 and the second polypeptide comprises X3 and X4;

[0022] (vi) The first polypeptide comprises X1 and X3 and the second polypeptide comprises X2 and X4;

[0023] (vii) The first polypeptide comprises X1 and X4 and the second polypeptide comprises X2 and X3;

[0024] (viii) The first polypeptide comprises X2 and X3 and the second polypeptide comprises X1 and X4;

[0025] (ix) The first polypeptide comprises X2 and X4 and the second polypeptide comprises X1 and X3;

[0026] (x) The first polypeptide comprises X3 and X4 and the second polypeptide comprises X1 and X2;

[0027] (xi) The first polypeptide comprises X1, X2, and X3 and the second polypeptide comprises X4;

[0028] (xii) The first polypeptide comprises X1, X2, and X4 and the second polypeptide comprises X3;

[0029] (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.

[0030] In other exemplary embodiments, the first polypeptide component and the second polypeptide component can be non-covalently associated, and / or the first polypeptide component and the second polypeptide component can bind to each other indirectly via a receptor. In yet another exemplary embodiment, the first polypeptide component further comprises a first targeting domain and / or the second polypeptide component further comprises a second targeting domain; in some embodiments, the first targeting domain, when present, is a translational fusion with the first polypeptide, and wherein the second targeting domain, when present, is a translational fusion with the second polypeptide. In some embodiments, the targeting domain can bind to a cell surface protein.

[0031] In another aspect, the present disclosure provides polypeptides comprising 1, 2, or 3 but not all 4 of the domains X1, X2, X3, and X4, wherein:

[0032] (a) X1, when present, is a peptide comprising a peptide having 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

[0033] (b) X2 is any helical peptide domain when present;

[0034] (c) X3 is a peptide containing when present a peptide having 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

[0035] (d) X4 is a peptide containing when present a peptide having 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

[0036] The amino acid residues in parentheses may be present or absent. Numerous embodiments of the polypeptide are provided. In some exemplary embodiments, the polypeptide comprises:

[0037] (i) a polypeptide comprising X1 and excluding X2, X3, and X4;

[0038] (ii) a polypeptide comprising X2 and excluding X1, X3, and X4;

[0039] (iii) a polypeptide comprising X3 and excluding X1, X2, and X4;

[0040] (iv) a polypeptide comprising X4 and excluding X1, X2, and X3;

[0041] (v) a polypeptide comprising X1 and X2 and excluding X3 and X4;

[0042] (vi) a polypeptide comprising X1 and X3 and excluding X2 and X4;

[0043] (vii) a polypeptide comprising X1 and X4 and excluding X2 and X3;

[0044] (viii) a polypeptide comprising X2 and X3 and excluding X1 and X4;

[0045] (ix) a polypeptide comprising X2 and X4 and excluding X1 and X3;

[0046] (x) a polypeptide comprising X3 and X4 and excluding X1 and X2;

[0047] (xi) A polypeptide comprising X1, X2, and X3 and excluding X4;

[0048] (xii) A polypeptide comprising X1, X2, and X4 and excluding X3;

[0049] (xiii) A polypeptide comprising X1, X3, and X4 and excluding X2; and

[0050] (xiv) A polypeptide comprising X2, X3, and X4 and excluding X1.

[0051] In another exemplary embodiment, the polypeptide further comprises a targeting domain, including but not limited to a targeting domain as a translational fusion with the polypeptide. In some embodiments, the targeting domain can bind to a cell surface protein.

[0052] In other aspects, the present disclosure provides a nucleic acid encoding a polypeptide, a first polypeptide, or a second polypeptide of any of the disclosed embodiments; an expression vector comprising the nucleic acid operably linked to a promoter; a host cell comprising the nucleic acid and / or expression vector disclosed herein; and a pharmaceutical composition comprising a conditional active receptor agonist, polypeptide, nucleic acid, expression vector, or host cell of any of the disclosed embodiments and a pharmaceutically acceptable carrier.

[0053] The present disclosure also provides a method for treating cancer, which comprises administering to a subject in need thereof a conditional active receptor agonist of any of the disclosed embodiments herein under conditions where the first polypeptide component and the second polypeptide component interact at the cells of the tumor to treat the cancer.

[0054] In another aspect, the present disclosure provides a conditional active receptor agonist, polypeptide, nucleic acid, expression vector, host cell, or pharmaceutical composition of any of the disclosed embodiments for use as a medicament in a subject for treating cancer and / or for modulating an immune response.

[0055] In yet another aspect, the present disclosure provides a method for activating an IL-2 receptor or an IL-4 receptor, which comprises administering to a subject a conditional active receptor agonist of any of the disclosed embodiments herein under conditions where the first polypeptide component and the second polypeptide component interact at the receptor. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The following figures are in accordance with exemplary embodiments:

[0057] Figures 1A - 1C . Computational design of de novo cytokine mimics. Figure 1A ) Design of non-split mimics with four helices; three mimic IL-2 binding to hIL-2Rβγ cThe interaction of the fourth one holds the first three in place. Top: In the first-generation design, each core element (helices H1-H4) of IL-2 was independently idealized using fragment-assembly from a clustered library of ideal fragments (size: 4 a.a.); Bottom: In the second-generation design, core elements were instead constructed using parametric equations that recapitulate the shape of each non-physical helix, allowing the length of each helix to vary by + / - 8 a.a. Figure 1B ) Reconnection of paired helices using ideal loop fragments (sizes of 4 a.a. or 7 a.a. for generations 1 and 2, respectively, see Methods) shows representative examples of the newly constructed elements connecting each pair of helices; Figure 1C ) Assemble in all possible combinations Figure 1B the helical hairpins generated in to generate a fully connected protein backbone.

[0058] Figure 2 . Characterization of Neoleukin-2 / 15. Binding experiments (Biolayer Interferometry) show that Neoleukin-2 / 15 can be incubated at 80 °C for 2 hours without any detectable loss of binding, while human and murine IL-2 rapidly lose activity.

[0059] Figures 3A - 3C . Reprogramming of Neoleukin-2 / 15 into a human interleukin-4 (hIL-4) mimic (Neoleukin-4). Figure 3A ) Neo-2 / 15 is structurally aligned with the complex of IL-4Rα and γ c in the structure of IL-4 from the PDB code 3BPL. Fourteen IL-4 residues that contact IL-4Rα are marked and transplanted into Neo-2 / 15. Figure 3B ) Neoleukin-4 (Neo-4), a new protein with 16 amino acid mutations compared to Neo-2 / 15. Mark these mutations; 13 of these are derived from Figure 3A the IL-4 residues depicted in that mediate contact with IL-4Rα, and 3 of them (H8M, K68I, and I98F, underlined in the figure) were introduced by directed evolution using random mutagenesis and screening for high-binding affinity variants. Figure 3C ) Biolayer interferometry data show that Neo-4, which binds to IL-4Rα alone like IL-4, has no affinity for γ alone c but binds γ when IL-4Rα is present in solution c .

[0060] Figures 4A - 4D。The overall sequence conservation of the binding residues of each of the four common helices, combining information from three different de novo designed IL-2 mimetics. Three independent SSM mutagenesis libraries from G2_neo2_40_1F_seq27, G2_neo2_40_1F_seq29, and G2_neo2_40_1F_seq36 were used to generate sequence logos ([ Figures 11A - 13B ) using combined data from binding experiments (using the heterodimeric murine IL-2Rβγc). All of these proteins are functional high-affinity de novo mimetics of murine and human IL-2, some with different topologies from Neo-2 / 15, but all contain four helices, H1([ Figure 4A ; Neo-2 / 15 1-22 is SEQ ID NO:04, IL-2 6-27 is SEQ ID NO:248, 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, IL-15 59-80 is SEQ IDNO:251), H2′([ Figure 4C ; Neo-2 / 15 58-76 is SEQ ID NO:07, IL-2 50-68 is SEQ ID NO:252, IL-15 34-52 is SEQ ID NO:253) and H4([ Figure 4D ; Neo-2 / 15 80-100 is SEQ ID NO:06, IL-2 111-131 is SEQ ID NO:254, IL-15 93-113 is SEQ ID NO:255). The logos independently display the combined information for each helix. Below each logo, a line graph shows the probability score for each amino acid in the Neo-2 / 15 sequence (higher mean is more conserved). The solid horizontal line highlights positions where Neo-2 / 15 amino acids have a probability score of ≥30% (i.e., these amino acids more generally contribute to receptor binding as they are overall enriched in the binding population across all de novo IL-2 mimetics tested). The topology of each helix in Neo-2 / 15 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 figure, highlighting the uniqueness of the Neo-2 / 15 helices and the binding interface.

[0061] Figures 5A - 5D 。Experimental optimization of G1_neo2_40. Figures 5A - 5C ) Heatmap of the G1_neo2_40 single-site mutagenesis library, showing with Figure 5A ) 50 nM, Figure 5B ) 2 nM, andFigure 5C ) 0.1 nM IL-2Rβγ c Enrichment at specific positions after successive rounds of incremental selection with the heterodimer. Based on these enrichment data, a combinatorial library with a nucleotide diversity of 1.5 x 10 6 was designed. Figure 5D ) Depicts the amino acid residues available in the initial combinatorial library, indicating predicted favorable (shown above the original sequence) and deleterious (shown below the original sequence) residues; in the depiction of the original sequence, black indicates residues represented in the combinatorial library and gray indicates residues not represented in the combinatorial library.

[0062] Figures 6A - 6E . Experimental optimization of G2_neo2_40_1F_seq27. Heatmap of the G2_neo2_40_1F_seq27 single-site mutagenesis library, showing treatment with Figure 6A ) 10 nM, Figure 6B ) 1 nM, Figure 6C ) 0.1 nM, and Figure 6D ) 0.1 nM IL-2Rβγ c Enrichment at specific positions after successive rounds of incremental selection with the heterodimer. Based on these enrichment data, a combinatorial library with a nucleotide diversity of 5.3 x 10 6 was designed. Figure 6E ) Depicts the amino acid residues available in the initial combinatorial library, indicating predicted favorable residues; black indicates residues in the starting sequence represented in the combinatorial library.

[0063] Figures 7A - 7E . Experimental optimization of G2_neo2_40_1F_seq29. Heatmap of the G2_neo2_40_1F_seq29 single-site mutagenesis library, showing treatment with Figure 7A ) 10 nM, Figure 7B ) 1 nM, Figure 7C ) 0.1 nM, and Figure 7D ) 0.1 nM IL-2Rβγ c Enrichment at specific positions after successive rounds of incremental selection with the heterodimer. Based on these enrichment data, a combinatorial library with a nucleotide diversity of 2.9 x 10 6 was designed. Figure 7E ) Depicts the amino acid residues available in the initial combinatorial library, indicating predicted favorable residues; black indicates residues in the starting sequence represented in the combinatorial library.

[0064] Figures 8A - 8E . Experimental optimization of G2_neo2_40_1F_seq36. Heatmap of the G2_neo2_40_1F_seq36 single-site mutagenesis library, showing treatment with Figure 8A ) 10 nM, Figure 8B) 1 nM, Figure 8C ) 0.1 nM, and Figure 8D ) 0.1 nM IL-2Rβγ c Enrichment at specific positions after successive rounds of incremental selection with the heterodimer. Based on these enrichment data, a combinatorial library with a nucleotide diversity of 2.7x10 6 was designed. Figure 8E ) Depicts the amino acid residues available in the initial combinatorial library, indicating predicted favorable residues; black indicates residues in the starting sequence represented in the combinatorial library.

[0065] Figures 9A - 9B . Circular dichroism (CD) thermal denaturation experiments on multiple 1st-generation IL-2 / IL-15 de novo-designed mimetics. Figure 9A ) Thermal denaturation curves and Figure 9B ) Wavelength scans.

[0066] Figures 10A - 10B . Circular dichroism (CD) thermal denaturation experiments on multiple 1st-generation experimentally optimized IL-2 / IL-15 de novo-designed mimetics. Figure 10A ) Thermal denaturation curves and Figure 10B ) Wavelength scans.

[0067] Figures 11A - 11D . Circular dichroism thermal melting of 2nd-generation IL-2 / IL-15 mimetic design. Figure 11A and Figure 11C ) Thermal denaturation curves and Figure 11B and Figure 11D ) Wavelength scans.

[0068] Figures 12A - 12C . Expression, purification, and thermal denaturation characterization of Neoleukin-2 / 15. Figure 12A ) SDS Tris-Tricine gel electrophoresis showing expression and purification on the affinity column. Figure 12B ) Circular dichroism at 222 nm during thermal melting from 25 °C to 95 °C, showing 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 fully unfold at 95 °C and fully refolds after cooling back to 25 °C.

[0069] Figures 13A - 13B . Robustness of Neoleukin-2 / 15 to single-point cysteine mutants at non-binding interface positions. Figure 13A ) Schematic showing the positions of point mutants in Neoleukin-2 / 15 that can be individually mutated to cysteine without interfering with protein expression or binding to IL-2Rβγ c while avoiding interference with receptor binding. Figure 13B) Binding kinetics of Neoleukin-2 / 15 cysteine mutants to IL-2Rβγ measured by biolayer interferometry. All variants associated with the receptor in a manner roughly similar to Neo-2 / 15. c

[0070] Figures 14A - 14C Expression, purification, and thermal denaturation characterization 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, showing 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 fully unfold at 95 °C and fully refolds upon cooling back to 25 °C.

[0071] Figures 15A - 15C Split Neo-2 / 15 variants can reconstruct Neoleukin-2 / 15 activity by binding to the human IL-2 receptor. 15A. Biolayer interferometry binding assay of split Neo-2 / 15 between helices H1+H3-H2’-H4 (Neo2A1 and Neo2B1, respectively) to the human IL-2 receptor. Binding data were collected in an Octet RED96 (ForteBio) and analyzed using ForteBio TMProcessed with data analysis software version 9.0.0.10. Biotinylated target receptor human γc was immobilized at 1 μg / ml on streptavidin-coated biosensors (SA ForteBio) in binding buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, 0.5% skim milk powder) for 300 seconds. After loading the γc target receptor onto the biosensor, baseline measurements were performed by dipping the biosensor into separate binding buffer, and then binding kinetics were monitored by dipping the biosensor into wells containing the target analyte protein (binding step), and then redipping the sensor into baseline / buffer (dissociation). For the binding step, analyte proteins (i.e., Neoleukin-2 / 15, Neo2A1, Neo2B1, and Neo2A1+Neo2B1 at equimolar ratio) were diluted from concentrated stock solutions in binding buffer to a final concentration of 100 nM. Human IL-2Rβ in solution was also added at a saturating concentration (250 nM). 15B. Biolayer interferometry binding assay of Neo-2 / 15 split into helices H1-H3+H2’-H4 (Neo2A2 and Neo2B2 respectively) followed the above experimental protocol. 15C. Biolayer interferometry binding assay of Neo-2 / 15 split into helices H1-H3-H2’+H4 (Neo2A3 and Neo2B3 respectively) followed the above experimental protocol.

[0072] Figures 16A - 16C . The Neo-2 / 15 variants were split to stimulate cell signaling through STAT5 phosphorylation in murine CTLL-2 cell line. 16A. CTLL-2 cells were starved for 2 hours in cytokine-free medium (RPMI, 10% FBS, +1 mM sodium pyruvate, +2 mM L-glutamine, 1% P / S) before the assay. The cells were dispensed into 96-well plates and resuspended in RPMI medium containing serial dilutions of protein samples: Neoleukin-2 / 15, Neo2A1, Neo2B1, and Neo2A1+Neo2B1 at equimolar ratio. The cells were stimulated at 37 °C for 15 minutes and immediately fixed by adding formaldehyde to 1.5% and incubating at room temperature for 10 minutes. The cells were permeabilized by resuspending in cold 100% methanol at 4 °C for 30 minutes. The fixed and permeabilized cells were washed twice with FACS buffer (PBS pH 7.2 containing 0.1% bovine serum albumin) and incubated with Alexa TM Fluor 647-conjugated anti-STAT5 pY694 (BD Biosciences) diluted 1:50 in FACS buffer for 2 hours at room temperature. Then the cells were washed twice in FACS buffer and analyzed on a Guava easyCyte TMThe mean fluorescence intensity (MFI) was measured on a flow cytometer (Millipore). The dose-response curve was fitted to a logistic model and the half-maximal effective concentration (EC50 value) was calculated using GraphPad Prism data analysis software after subtracting the MFI of unstimulated cells and normalizing against the maximum signal intensity. 16B. Biolayer interferometry binding assays of Neo-2 / 15 split into helices H1-H3+H2’-H4 (Neo2A3 and Neo2B3, respectively), following the above experimental protocol. 16C. Biolayer interferometry binding assays of Neo-2 / 15 split into helices H1-H3-H2’+H4 (Neo2A2 and Neo2B2, respectively), following the above experimental protocol.

[0073] Figures 17A - 17B . Split Neo2 / 15 variants fused to targeting domains bind to the IL-2 receptor. 17A. Split Neo2A and Neo2B protein variants fused to anti-EGFR and anti-Her2 DARPin targeting domains. 17B. Biolayer interferometry binding assays of split Neo-2 / 15 protein fusions to the murine IL-2 receptor. Binding data were collected on an Octet RED96 (ForteBio) and processed using ForteBio TM data analysis software version 9.0.0.10. Biotinylated target receptor human γc was immobilized at 1 μg / ml in binding buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, 0.5% skim milk) on streptavidin-coated biosensors (SA ForteBio) for 300 s. After loading the target receptor onto the biosensors, baseline measurements were performed by dipping the biosensors into separate binding buffer, followed by monitoring the binding kinetics by dipping the biosensors into wells containing the target protein (binding step), and then re-dipping the sensors into baseline / buffer (dissociation). For the binding step, analyte proteins (i.e., aHer2Neo2A1+Neo2B1-aEGFR, aHer2Neo2A2+Neo2B2-aEGFR, aHer2Neo2A3+Neo2B3-aEGFR) were diluted from concentrated stock solutions in binding buffer to a final concentration of 100 nM in equimolar amounts. Mouse IL-2Rβ in solution was also added at a saturating concentration (250 nM).

[0074] Figures 18A - 18CTargeted reconstitution of Neoleukin-2 / 15 on the surface of K562 cells expressing Her2 and EGFR. 18A. Neo2A and Neo2B split protein variants fused to anti-EGFR and anti-Her2 DARPin targeting domains. 18B. Engineered K562 tumor cell lines transduced to express Her2+ / eGFP+, EGFR+ / iRFP+ or Her2+ / eGFP+ and EGFR+ / iRFP+ surface markers. 18C. Functional reconstitution assay of Her2-targeted Neo2A variants (aHer2Neo2A1, aHer2Neo2A2 and aHer2Neo2A3) and EGFR-targeted Neo2B variants (Neo2B1-aEGFR, Neo2B2-aEGFR and Neo2B3-aEGFR) on the surface of four of the above K562 cell lines. All cell lines were mixed at equal ratios (50,000 cells of each cell type per well) and transferred to V-bottom plates at 200,000 cells / well. Cells were incubated with equimolar ratios 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 minutes at room temperature. Cells were then washed twice (PBS (Gibco), 0.5% BSA) and subsequently incubated for 5 minutes with the following components: 50 nM biotinylated soluble human common γ receptor, 50 nM soluble human IL-2Rβ and 15 nM streptavidin-phycoerythrin conjugate (SAPE). Cells were washed again and analyzed by flow cytometry in an LSRII instrument. eGFP fluorescence (Ex / Em = 488 / 508) was sorted for Her2+ cells, iRFP fluorescence (Ex / Em = 637 / 670) was sorted for EGFR+ cells, 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 was identified by analyzing PE (Ex / Em = 561 / 582) fluorescence on the surface of cells in each subset.

[0075] Figures 19A - 19C。Both fragments of the split Neo-2 / 15 variant are required for functional reconstitution on the surface of K562 cells expressing Her2 and EGFR. 19A. Functional reconstitution assays of the Her2-targeting Neo2A variant (aHer2-Neo2A1) and the EGFR-targeting 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+. Mix all cell lines at equal ratios (50,000 cells of each cell type per well) and transfer to a V-bottom plate at 200,000 cells / well. Incubate the cells 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 minutes at room temperature. Then wash the cells twice (PBS (Gibco), 0.5% BSA), and subsequently incubate for 5 minutes with the following components: 50 nM biotinylated soluble human common γ receptor, 50 nM soluble human IL-2Rβ, and 15 nM streptavidin-phycoerythrin conjugate (SAPE). Wash the cells again and analyze by flow cytometry in an LSRII instrument. Sort eGFP fluorescence (Ex / Em = 488 / 508) for Her2+ cells, sort iRFP fluorescence (Ex / Em = 637 / 670) for EGFR+ cells, and identify Her2+ / EGFR+ cells by co-expression of eGFP and iRFP. Identify reconstitution of Neoleukin-2 / 15 activity and binding to the IL-2 receptor by analyzing PE (Ex / Em = 561 / 582) fluorescence on the surface of cells in each subset. As observed, both fragments are required for reconstitution of functional Neoleukin-2 / 15 activity on the surface of double-positive cells. 19B. The same assay described in 19A, using aHer2-Neo2A2 and Neo2B2-aEGFR. 19C. The same assay described in 19A, using aHer2-Neo2A3 and Neo2B3-aEGFR.

[0076] Figures 20A - 20B 。Illustration of alternative modes of action of the split Neo-2 platform for highly specific activation of immune cell subtypes. 20A. Selective targeting of two surface markers by split Neo-2 to stimulate expansion of specific immune cell subtypes (e.g., CD8+, CAR-T cells, or regulatory T cells). 20B. Simultaneous tumor and T cell targeting of the split Neoleukin-2 / 15 system to induce subset-specific proliferation of immune cells in the environment of target tumor cells.

[0077] Figure 21 。The split fragments of Neoleukin-4 can reconstruct full activity upon combination. Biolayer interferometry binding assay of Neo-4, split into helix H1 (Neo4A1)+H3-H2’-H4 (Neo4B1), to the human IL-4 receptor. Binding data were collected in an Octet RED96 (ForteBio) and processed using ForteBio TM data analysis software version 9.0.0.10. The biotinylated target receptor human γc was immobilized at 1 μg / ml in binding buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, 0.5% non-fat dry milk) on streptavidin-coated biosensors (SA ForteBio) for 300 seconds. After loading the target receptor onto the biosensors, baseline measurements were performed in separate binding buffer, and then binding kinetics were monitored by dipping the biosensors into wells containing the indicated concentration of the target protein (binding step), and then re-dipping the sensors into baseline / buffer (dissociation). For the binding step, the analyte proteins (i.e., Neoleukin-4, Neo4A1, Neo4B1, and an equimolar ratio of Neo4A1+Neo4B1) were diluted from concentrated stock solutions in binding buffer to a final concentration of 100 nM. Human IL-4Rα in solution was also added at a saturating concentration (250 nM). Detailed Description

[0078] As used herein and unless otherwise specified, the terms “a” and “an” are used to mean “one,” “at least one,” or “one or more.” Unless the context otherwise requires, singular terms used herein shall include the plural and plural terms shall include the singular.

[0079] Unless the context clearly requires otherwise, throughout the specification and claims, the words “comprise,” etc., are to be construed in an inclusive sense, rather than an exclusive or exhaustive sense; that is, the meaning of “including, but not limited to.” The use of the singular or plural words also includes the plural or singular respectively. Additionally, as used in this application, the words “herein,” “above,” and “below,” and words of similar import, shall refer to this application as a whole and not to any particular part of this application.

[0080] As used herein, the amino acid residue abbreviations are 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).

[0081] All embodiments of any aspect of the invention can be used in combination, unless the context clearly dictates otherwise.

[0082] In a first aspect, the present disclosure provides a non-naturally occurring conditional active receptor agonist comprising a first polypeptide component and a second polypeptide component, wherein the first polypeptide component and the second polypeptide component do not exist as a fusion protein, and wherein the first polypeptide component and the second polypeptide component together comprise domains X1, X2, X3, and X4, wherein:

[0083] (a) X1 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 the peptide ;

[0084] (b) X2 is any helical peptide domain;

[0085] (c) X3 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 the peptide ; and

[0086] (d) X4 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 the peptide ;

[0087] wherein:

[0088] (i) The amino acid residues in parentheses may be present or absent;

[0089] (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; and

[0090] (iii) The second polypeptide component comprises each of X1, X2, X3, and X4 that is not present in the first polypeptide component; wherein the first polypeptide component and the second polypeptide component alone are not active receptor agonists, and wherein the first polypeptide component and the second polypeptide component interact with each other to form an IL-2 receptor βγ c heterodimer (IL-2Rβγ c ), an IL-4 receptor αγ c heterodimer (IL-4Rαγ c ), an IL-13α, or an active agonist of an IL-4Rα / IL13Rα heterodimer.

[0091] As shown in the following examples, as detailed in the PCT application with serial number PCT / US2019 / 038703 filed on June 29, 2019, and as described in Silva et al., Nature 565, pg. 186, January 10, 2019, polypeptides comprising all of X1-X4 have previously been shown to be (a) mimetics of IL-2 and interleukin-15 (IL-15) that bind to the IL-2 receptor βγ c heterodimer (IL-2Rβγ c ), but do not have binding sites for IL-2Rα or IL-15Rα, or (b) mimetics of IL-4 that bind to the IL-4 receptor αγ c heterodimer (IL-4Rαγ c ) or the IL-13 receptor α subunit (IL-13Rα) (native IL-4 and the IL-4 mimetics described herein cross-react with the IL-13 receptor to form an IL-4Rα / IL13Rα heterodimer). The full-length polypeptides have been shown to be highly stable, bind to human and murine IL-2Rβγ c or IL-4Rαγ c with higher affinity than the native cytokines, and initiate downstream cell signaling independently of IL-2Rα and IL-15Rα, or independently of IL-13Rα. The full-length polypeptides can be used, for example, in the treatment of cancer.

[0092] In contrast, the present disclosure surprisingly demonstrates a conditional active receptor agonist that comprises the separate first and second polypeptides, which alone are not receptor agonists but can interact non-covalently to form an IL-2 receptor βγ c heterodimer (IL-2Rβγ c ), an IL-4 receptor αγ cHeterodimer (IL-4Rαγ c ), an active agonist of IL-13α, or an IL-4Rα / IL13Rα heterodimer. The affinity of this non-covalent interaction between the "split components" (i.e., the first polypeptide and the second polypeptide) causes the interaction to occur only in the presence of a suitable receptor and also only when the two split components are co-localized. Thus, the conditional active receptor agonists of the present disclosure can be used for any use in which polypeptides including all of X1-X4 can be used. Moreover, the conditional active receptor agonists are capable of achieving co-localization-dependent reconstitution of the agonist and thus conditional activation of the receptor.

[0093] As used herein, the term protein mimetic refers to a protein that mimics certain aspects of the function of another protein. The two proteins typically have different amino acid sequences and / or different structures. Exemplarily, provided herein are conditional active mimetics of IL-2 and IL-15. The aspects of the functions of IL-2 and IL-15 that these conditional active mimetics mimic are induction of IL-2Rβγc heterodimerization, which results in STAT5 phosphorylation. Since both IL-2 and IL-15 signal via heterodimerization of IL-2Rβγc, these conditional active mimetics mimic this biological function of both IL-2 and IL-15. These conditional active mimetics can be referred to herein as mimetics of IL-2, IL-15, or both IL-2 and IL-15.

[0094] Also provided are conditional active mimetics of IL-4. These conditional active mimetics are capable of mimicking certain functions of IL-4. The functions of IL-4 that these mimetics mimic are induction of IL-4Rαγ c heterodimerization (and / or IL-4Rα / IL-13Rα heterodimerization).

[0095] In one embodiment, the first polypeptide component and the second polypeptide component interact to form an agonist of the IL-2 receptor βγ c heterodimer (IL-2Rβγ c ). In another embodiment, the first polypeptide component and the second polypeptide component interact to form an agonist of the IL-4 receptor αγ c heterodimer (IL-4Rαγ c ), IL-13α, or an IL-4Rα / IL13Rα heterodimer.

[0096] Native hIL-2 contains four helices connected by irregular long loops. The N-terminal helix (H1) interacts with both the β and γ subunits, the third helix (H3) interacts with the β subunit, and the C-terminal helix (H4) interacts with the γ subunit; the α subunit interaction surface is formed by the irregular second helix (H2) and two long loops, one connecting H1 and H2 and the other connecting H3 and H4. An idealized protein was designed and generated in which H1, H3, and H4 were replaced with idealized domains, including but not limited to helices and β-strands that exhibit the IL-2Rβγ c or IL-4Rαγ c interface (designated domains X1, X3, and X4, respectively), and in which H2 was replaced with an idealized helix (designated domain X2) that provides better packing. As shown in the examples, extensive mutagenesis studies have been carried out, demonstrating that the amino acid sequences of each peptide domain can be extensively modified without loss of binding to the IL-2 or IL-4 receptor, and that the domains can be placed in any order while retaining conditional binding to the IL-2 or IL-4 receptor. The polypeptide can comprise L-amino acids and glycine, D-amino acids and glycine, or a combination thereof. As described herein, the idealized protein can be split into two polypeptides that separately have negligible binding to the relevant receptor but can reconstitute receptor activity when mixed together. Typically, the protein is split at a site that does not interfere with its function (e.g., the linker portion in linker-containing embodiments). Additionally, just as the X1, X2, X3, and X4 domains in the non-split protein can be looped together in any order, the split proteins can comprise any combination of domains.

[0097] Thus, X1, X2, X3, and X4 can be in any order in the first and second polypeptides; in non-limiting embodiments:

[0098] (i) The first polypeptide comprises X1 and the second polypeptide comprises X2, X3, and X4;

[0099] (ii) The first polypeptide comprises X2 and the second polypeptide comprises X1, X3, and X4;

[0100] (iii) The first polypeptide comprises X3 and the second polypeptide comprises X1, X2, and X4;

[0101] (iv) The first polypeptide comprises X4 and the second polypeptide comprises X1, X2, and X3;

[0102] (v) The first polypeptide comprises X1 and X2 and the second polypeptide comprises X3 and X4;

[0103] (vi) The first polypeptide comprises X1 and X3 and the second polypeptide comprises X2 and X4;

[0104] (vii) The first polypeptide comprises X1 and X4 and the second polypeptide comprises X2 and X3;

[0105] (viii) The first polypeptide comprises X2 and X3 and the second polypeptide comprises X1 and X4;

[0106] (ix) The first polypeptide comprises X2 and X4 and the second polypeptide comprises X1 and X3;

[0107] (x) The first polypeptide comprises X3 and X4 and the second polypeptide comprises X1 and X2;

[0108] (xi) The first polypeptide comprises X1, X2, and X3 and the second polypeptide comprises X4;

[0109] (xii) The first polypeptide comprises X1, X2, and X4 and the second polypeptide comprises X3;

[0110] (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.

[0111] When the first polypeptide and / or the second polypeptide comprises more than one domain of X1, X2, X3, and X4, the domains may in some embodiments be separated by an amino acid linker of any suitable length or composition of amino acids. There is no requirement for a linker; in one embodiment, there is no linker between any domains. In other embodiments, an amino acid linker may be present between 0, 1, or 2 junctions between the domains X1, X2, X3, and X4 in the first polypeptide and / or the second polypeptide. The amino acid linker may be of any length, such as is considered appropriate for the intended use. In some aspects, the linker is at the N-terminus or C-terminus and is called a linker even though it does not join two domains together. In some aspects, the linker is called a linker because it is present in a non-split protein and joins two domains together.

[0112] In all of these embodiments, X1, X3, and X4 can be of any suitable length, meaning that each domain can separately contain any suitable number of additional amino acids other than the peptides of SEQ ID NO: 4, 5, and 6. The residues in parentheses are optional and thus may be present or absent. As would be appreciated by one of ordinary skill in the art, this means for example The 6 N-terminal amino acids and the 5 C-terminal amino acid residues are optional. As will be further understood by those skilled in the art: (i) if one N-terminal amino acid is missing, then it will be 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 missing, then it will be 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 missing, then it will be 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 missing, then it will be the two most C-terminal amino acids (i.e., the C-terminal NI dipeptide in SEQ ID NO: 4), and so on. Thus, those skilled in the art will appreciate that one or more optional amino acid residues can be missing, and the missing amino acids from the optional residues are contiguous with the relevant peptide end, as illustrated above.

[0113] In one embodiment, respectively, X1 comprises a peptide having 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 the peptide In one embodiment, respectively, X1 comprises a peptide having 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 the peptide In one embodiment, respectively, X3 comprises a peptide having 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 the peptide and X4 comprises a peptide having 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 the peptide

[0114] In various embodiments, respectively, X1 is a peptide comprising an amino acid sequence having 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 the peptide or ; X3 is a peptide comprising an amino acid sequence having 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 the peptide or a peptide having an amino acid sequence with 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; and X4 is a peptide comprising or a peptide having an amino acid sequence with 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.

[0115] In a specific embodiment,

[0116] (i) X1 is a peptide comprising an amino acid sequence with at least 70% identity to the full length of the peptide or ; X3 is a peptide comprising an amino acid sequence with at least 70% identity to the full length of the peptide or ; and X4 is a peptide comprising an amino acid sequence with at least 70% identity to the full length of the peptide or ;

[0117] (ii) X1 is a peptide comprising an amino acid sequence with at least 70% identity to the full length of the peptide or ; X3 is a peptide comprising an amino acid sequence with at least 70% identity to the full length of the peptide or ; and X4 is a peptide comprising an amino acid sequence with at least 70% identity to the full length of the peptide or ;

[0118] (iii) X1 is a peptide comprising an amino acid sequence with at least 85% identity to the full length of the peptide or ; X3 is a peptide comprising an amino acid sequence with at least 85% identity to the full length of the peptide or ; and X4 is a peptide comprising an amino acid sequence with at least 85% identity to the full length of the peptide or ;

[0119] (iv) X1 is a peptide having an amino acid sequence with 100% identity to the full length of the peptide or ; X3 is a peptide comprising an amino acid sequence with at least 85% identity to the full length of the peptide or a peptide having an amino acid sequence that is 100% identical to the full length; and X4 is a peptide comprising or a peptide having an amino acid sequence that is 100% identical to the full length;

[0120] (v) X1 is a peptide comprising an amino acid sequence that is at least 85% identical to the full length of the peptide ; X3 is a peptide comprising an amino acid sequence that is at least 85% identical to the full length of the peptide ; and X4 is a peptide comprising an amino acid sequence that is at least 85% identical to the full length of the peptide ; or (vi) X1 is a peptide having an amino acid sequence that is 100% identical to the full length of the peptide ; X3 is a peptide having an amino acid sequence that is 100% identical to the full length of the peptide ; and X4 is a peptide having an amino acid sequence that is 100% identical to the full length of the peptide .

[0121] In these embodiments, different forms of SEQ ID NO:4, 5, and 6 are shown to have the same primary amino acid sequence but differ in the positions of the optional residues indicated by parentheses.

[0122] In still other embodiments,

[0123] X1 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 the peptide ;

[0124] X3 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 the peptide ; and / or

[0125] X4 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 the peptide .

[0126] In another embodiment in which the optional residues of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 are present,

[0127] (a) X1 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 the peptide ;

[0128] (c) X3 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 the peptide ; and

[0129] (d) X4 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 the peptide .

[0130] In another embodiment in which the selected optional residues of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 are present,

[0131] (a) X1 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 the peptide ;

[0132] (c) X3 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 the peptide ; and

[0133] (d) X4 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 the peptide .

[0134] In various embodiments in which the optional residues of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 are present, respectively, X1 is a peptide comprising an amino acid sequence having 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 the peptide ; X3 is a peptide comprising an amino acid sequence having 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 the peptide or ; and X4 is a peptide comprising an amino acid sequence having 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 the peptide or .

[0135] In a specific embodiment,

[0136] (i) X1 is a peptide comprising an amino acid sequence having at least 65% identity to the full length of the peptide or ; X3 is a peptide comprising an amino acid sequence having at least 65% identity to the full length of the peptide or ; and X4 is a peptide comprising an amino acid sequence having at least 65% identity to the full length of the peptide or ;

[0137] (ii) X1 is a peptide comprising an amino acid sequence having at least 75% identity to the full length of the peptide or ; X3 is a peptide comprising an amino acid sequence having at least 75% identity to the full length of the peptide or ; and X4 is a peptide comprising an amino acid sequence having at least 75% identity to the full length of the peptide or ;

[0138] (iii) X1 is a peptide comprising an amino acid sequence having at least 80% identity to the full length of the peptide or ; X3 is a peptide comprising an amino acid sequence having at least 80% identity to the full length of the peptide or a peptide having an amino acid sequence with at least 80% identity to the full length; and X4 is a peptide comprising or a peptide having an amino acid sequence with at least 80% identity to the full length;

[0139] (iv) X1 is a peptide comprising an amino acid sequence with at least 90% identity to the full length of the peptide or ; X3 is a peptide comprising an amino acid sequence with at least 90% identity to the full length of the peptide or ; and X4 is a peptide comprising an amino acid sequence with at least 90% identity to the full length of the peptide or ;

[0140] (v) X1 is a peptide comprising an amino acid sequence with 100% identity to the full length of the peptide or ; X3 is a peptide comprising an amino acid sequence with 100% identity to the full length of the peptide or ; and X4 is a peptide comprising an amino acid sequence with 100% identity to the full length of the peptide or ;

[0141] (vi) X1 is a peptide comprising an amino acid sequence with at least 80% identity to the full length of the peptide ; X3 is a peptide comprising an amino acid sequence with at least 80% identity to the full length of the peptide ; and X4 is a peptide comprising an amino acid sequence with at least 80% identity to the full length of the peptide ;

[0142] (vii) X1 is a peptide comprising an amino acid sequence with at least 90% identity to the full length of the peptide ; X3 is a peptide comprising an amino acid sequence with at least 90% identity to the full length of the peptide ; and X4 is a peptide comprising an amino acid sequence with at least 90% identity to the full length of the peptide ;

[0143] (viii) X1 is a peptide comprising an amino acid sequence with 100% identity to the full length of the peptide ; X3 is a peptide comprising an amino acid sequence with 100% identity to the full length of the peptide ; and X4 is a peptide comprising an amino acid sequence with 100% identity to the full length of the peptide ;

[0144] (ix) X1 is a peptide comprising an amino acid sequence having at least 80% identity to the full length of the peptide ; X3 is a peptide comprising an amino acid sequence having at least 80% identity to the full length of the peptide ; and X4 is a peptide comprising an amino acid sequence having at least 80% identity to the full length of the peptide ;

[0145] (x) X1 is a peptide comprising an amino acid sequence having at least 90% identity to the full length of the peptide ; X3 is a peptide comprising an amino acid sequence having at least 90% identity to the full length of the peptide ; and X4 is a peptide comprising an amino acid sequence having at least 90% identity to the full length of the peptide ; or

[0146] (xi) X1 is a peptide comprising an amino acid sequence having 100% identity to the full length of the peptide ; X3 is a peptide comprising an amino acid sequence having 100% identity to the full length of the peptide ; and X4 is a peptide comprising an amino acid sequence having 100% identity to the full length of the peptide .

[0147] In one embodiment, the conditionally active receptor agonist is a conditionally active receptor agonist IL-2 / 15 mimic and (i) X1 comprises 1, 2, 3, 4, or all 5 of the following: L at residue 7, H at residue 8, H at residue 11, Y at residue 14, or M at residue 18, wherein the numbering is relative to SEQ ID NO:4 based on the presence of optional residues; and / or (ii) X3 comprises 1, 2, 3, 4, 5, 6, 7, or all 8 of the following: D at residue 3, Y at residue 4, F at residue 6, N at residue 7, L at residue 10, I at residue 11, E at residue 13, or E at residue 14, wherein the numbering is relative to SEQ ID NO:5 based on the presence of optional residues. In yet another embodiment, (iii) X4 comprises I at residue 19, wherein the numbering is relative to SEQ ID NO:6 based on the presence of optional residues.

[0148] In SEQ ID NO:4, 5, and 6, some amino acid residues are shown in bold. In , the amino acid residues E10, L13, Y14, D15, and L17 (numbered based on the presence of optional residues) are shown in bold; in , the amino acid residues L1, Y4, N7, L10, I11, and I15 (numbered based on the presence of optional residues) are shown in bold; and in In which, amino acid residues I12, Q16, and W18 (numbered based on the presence of optional residues) are shown in bold.

[0149] In one embodiment:

[0150] (a) AA substitutions in X1 relative to the AA sequence of SEQ ID NO: 4 occur at no more than 3 AA residues shown in bold, or at no more than 2 AA residues shown in bold, or at no more than 1 AA residue shown in bold, or do not occur at AA residues shown in bold;

[0151] (b) AA substitutions in X3 relative to the AA sequence of SEQ ID NO: 5 occur at no more than 3 AA residues shown in bold, or at no more than 2 AA residues shown in bold, or at no more than 1 AA residue shown in bold, or do not occur at AA residues shown in bold; and / or

[0152] (c) AA substitutions in X4 relative to the AA sequence of SEQ ID NO: 6 occur at no more than 2 AA residues shown in bold, or at no more than 1 AA residue shown in bold, or do not occur at AA residues shown in bold.

[0153] In another embodiment, AA substitutions in X2 relative to the AA sequence of SEQ ID NO: 7 do not occur at AA residues shown in bold.

[0154] In another embodiment of the conditional active receptor agonist IL-2 mimetic, amino acid substitutions relative to the reference peptide domains (i.e., SEQ ID NO: 4, 5, or 6) do not occur at AA residues shown in bold. As shown below, each of SEQ ID NO: 4, 5, and 6 includes bold residues involved in binding to the receptor:

[0155] ● Amino acid residues E10, L13, Y14, D15, and L17 (numbered based on the presence of optional residues) are unchanged in this embodiment;

[0156] ● Amino acid residues L1, Y4, N7, L10, I11, and I15 (numbered based on the presence of optional residues) are unchanged in this embodiment; and

[0157] ● Amino acid residues I12, Q16, and W18 (numbered based on the presence of optional residues) are unchanged in this embodiment.

[0158] In yet another embodiment, when X2 is a peptide containing When the peptide has 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 thereof, wherein the residues in parentheses are optional and the amino acid residue W13 remains unchanged. In one embodiment, the optional residue is present; in another embodiment, the optional residue is absent.

[0159] In another embodiment of the conditional active receptor agonist IL-2 mimetic, amino acid substitutions relative to the reference peptide domain (i.e., SEQ ID NO: 4, 5, or 6) do not occur at more than 3, 2, or 1 of the AA residues marked in bold.

[0160] In another embodiment, the conditional active receptor agonist is a conditional active receptor agonist IL-4 / IL-13 mimetic, and

[0161] X1 is a peptide comprising along its length 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 peptide;

[0162] X3 is a peptide comprising along its length an amino acid sequence that is at least 37%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the peptide; and

[0163] X4 is a peptide comprising along its length 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 peptide,

[0164] wherein

[0165] (i) X1 includes I at residue 7, T or M at residue 8, E at residue 11, K at residue 14, and S at residue 18; and

[0166] (ii) X3 includes R at residue 3, F at residue 4, K at residue 6, R at residue 7, R at residue 10, N at residue 11, W at residue 13, and G at residue 14.

[0167] In yet another embodiment, (iii) X4 comprises F at residue 19.

[0168] In various embodiments, X1 is a peptide comprising an amino acid sequence having 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 , X3 is a peptide comprising an amino acid sequence having 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 , and X4 is a peptide comprising an amino acid sequence having 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 . In a specific embodiment,

[0169] (i) X1 is a peptide comprising an amino acid sequence that is at least 65% identical along its length to the peptide ; X3 is a peptide comprising an amino acid sequence that is at least 65% identical along its length to the peptide ; and X4 is a peptide comprising an amino acid sequence that is at least 65% identical along its length to the peptide ;

[0170] (ii) X1 is a peptide comprising an amino acid sequence that is at least 75% identical along its length to the peptide ; X3 is a peptide comprising an amino acid sequence that is at least 75% identical along its length to the peptide ; and X4 is a peptide comprising an amino acid sequence that is at least 75% identical along its length to the peptide ;

[0171] (iii) X1 is a peptide comprising an amino acid sequence that is at least 80% identical along its length to the peptide ; X3 is a peptide comprising an amino acid sequence that is at least 80% identical along its length to the peptide ; and X4 is a peptide comprising an amino acid sequence that is at least 80% identical along its length to the peptide ; or (iv) X1 is a peptide comprising an amino acid sequence that is at least 90% identical along its length to the peptide ; X3 is a peptide comprising an amino acid sequence that is at least 90% identical along its length to the peptide ; and X4 is a peptide comprising an amino acid sequence that is at least 90% identical along its length to the peptide A peptide having an amino acid sequence that is at least 90% identical;

[0172] In each case, where

[0173] (A) X1 comprises I at residue 7, T or M at residue 8, E at residue 11, K at residue 14, and S at residue 18; and

[0174] (B) X3 comprises R at residue 3, F at residue 4, K at residue 6, R at residue 7, R at residue 10, N at residue 11, W at residue 13, and G at residue 14.

[0175] In another embodiment of the conditional active receptor agonist IL-4 / IL-13 mimetic, amino acid substitutions relative to the reference peptide domains (i.e., SEQ ID NO: 8, 9, or 10) do not occur at the AA residues marked in bold. As shown below, each of SEQ ID NO: 8, 9, and 10 includes bold residues:

[0176] ● The amino acid residues E10, E11, A12, L13, K14, D15, A16, and L17 are unchanged in this embodiment;

[0177] ● The amino acid residues F4, A5, K6, R7, F8, E9, R10, and N11 are unchanged in this embodiment;

[0178] ● The amino acid residues I11, I12, T13, I14, L15, Q16, S17, W18, F19, and F20 are unchanged in this embodiment.

[0179] In yet another embodiment, when X2 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 the peptide the amino acid residue W13 is unchanged.

[0180] 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 having similar physicochemical characteristics, e.g., 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; Glu and Asp; or Gln and Asn). Other such conservative substitutions are known, e.g., substitutions of entire regions having similar hydrophobicity characteristics. Polypeptides containing conservative amino acid substitutions can be tested in any of the assays described herein to confirm retention of the desired activity of the native or reference polypeptide, e.g., antigen-binding activity and specificity. Amino acids can be grouped according to the similarity of their side-chain properties (A.L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be grouped 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 affecting chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions would entail exchanging a member of one of these classes for a member of another. For example, specific conservative substitutions include: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to Ile or to Leu.

[0181] In one embodiment, the amino acid residue in X1 relative to SEQ ID NO:4 is selected from the group consisting of:

[0182]

[0183]

[0184] In one embodiment, the conditionally active receptor agonist is a conditionally active IL-4 mimetic, and position 7 is I, position 8 is M or T, position 11 is E, position 14 is K, and position 18 is S.

[0185] In another embodiment, the conditionally active receptor agonist is a conditionally active IL-2 mimetic, and one, two, three, four, or five of the following are not true: position 7 is I, position 8 is M or T, position 11 is E, position 14 is K, and position 18 is S.

[0186] In another embodiment, the amino acid residue in X3 relative to SEQ ID NO:5 is selected from the group consisting of:

[0187]

[0188]

[0189] In another embodiment, the conditionally active receptor agonist is a conditionally active IL-4 / IL-13 mimetic and 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.

[0190] In another embodiment, the conditionally active receptor agonist is a conditionally active IL-2 mimetic and 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.

[0191] In any such embodiment, the conditionally active receptor agonist further permits a cysteine at position 17 relative to SEQ ID NO:5 other than amino acid residues of H, K, L, N, and R, or a cysteine at position 20 relative to SEQ ID NO:5 other than amino acid residues of A, C, E, F, G, M, S, and Y. Thus, in this embodiment, the amino acid residue in X3 relative to SEQ ID NO:5 can be selected from the group consisting of:

[0192]

[0193] In another embodiment, the amino acid residue in X4 relative to SEQ ID NO:6 is selected from the group consisting of:

[0194]

[0195] In another embodiment, the conditionally active receptor agonist is a conditionally active IL-4 / IL-13 mimetic and position 19 is I. In another embodiment, the conditionally active receptor agonist is a conditionally active IL-2 mimetic and position 19 is not I.

[0196] In any such embodiment, outside of the amino acid residues of E, G, H, and K, the conditionally active receptor agonist further permits a cysteine at position 3 relative to SEQ ID NO:6. Thus, in this embodiment, the amino acid residue in X4 relative to SEQ ID NO:6 can be selected from the group consisting of:

[0197]

[0198]

[0199] As documented herein, domain X2 is a domain and thus can be any amino acid sequence that links (i.e., in the same polypeptide or after non-covalent interaction of the first and second polypeptides) related other domains and permits their folding. The required length will depend on the details of the first and second polypeptides used and can be 8 amino acids or longer. In an illustrative and non-limiting embodiment, X2 is a peptide comprising along its length an amino acid sequence that is or at least 20%, 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In one embodiment, the amino acid change relative to the amino acid sequence of SEQ ID NO:7 is a conservative amino acid substitution. In another embodiment, the W13 amino acid residue is unchanged. In yet another embodiment, the amino acid residue in X2 relative to SEQ ID NO:7 is selected from the group consisting of:

[0200]

[0201] 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.

[0202] In any such embodiment, the polypeptide further permits a cysteine at position 5 or 16 relative to SEQ ID NO:7.

[0203] Alternatively, in any such embodiment, the polypeptide further permits a cysteine at position 1, 2, 5, 9 or 16 relative to SEQ ID NO:7.

[0204] Thus, the amino acid residue in X2 relative to SEQ ID NO:7 can be selected from the group consisting of:

[0205]

[0206] In various specific embodiments,

[0207] (i) X2 is a peptide comprising an amino acid sequence that is at least 70% identical along its length to ;

[0208] (ii) X2 is a peptide comprising an amino acid sequence that is at least 80% identical along its length to ;

[0209] (iii) X2 is a peptide comprising an amino acid sequence that is at least 90% identical along its length to ;

[0210] (iv) X2 is a peptide comprising an amino acid sequence that has at least 65% identity to the full length of the peptide ; X1 is a peptide comprising an amino acid sequence that has at least 65% identity to the full length of the peptide or ; X3 is a peptide comprising an amino acid sequence that has at least 65% identity to the full length of the peptide or ; and X4 is a peptide comprising an amino acid sequence that has at least 65% identity to the full length of the peptide or ;

[0211] (v) X2 is a peptide comprising an amino acid sequence that has at least 75% identity to the full length of the peptide ; X1 is a peptide comprising an amino acid sequence that has at least 75% identity to the full length of the peptide or ; X3 is a peptide comprising an amino acid sequence that has at least 75% identity to the full length of the peptide or a peptide having an amino acid sequence with at least 75% identity to the full length; and X4 is a peptide containing or a peptide having an amino acid sequence with at least 75% identity to the full length;

[0212] (vi) X2 is a peptide containing an amino acid sequence with at least 80% identity to the full length of the peptide ; X1 is a peptide containing an amino acid sequence with at least 80% identity to the full length of the peptide or ; X3 is a peptide containing an amino acid sequence with at least 80% identity to the full length of the peptide or ; and X4 is a peptide containing an amino acid sequence with at least 80% identity to the full length of the peptide or ;

[0213] (vii) X2 is a peptide containing an amino acid sequence with at least 90% identity to the full length of the peptide ; X1 is a peptide containing an amino acid sequence with at least 90% identity to the full length of the peptide or ; X3 is a peptide containing an amino acid sequence with at least 90% identity to the full length of the peptide or ; and X4 is a peptide containing an amino acid sequence with at least 90% identity to the full length of the peptide or ; or

[0214] (viii) X2 is a peptide having an amino acid sequence with 100% identity to the full length of the peptide ; X1 is a peptide having an amino acid sequence with 100% identity to the full length of the peptide or ; X3 is a peptide having an amino acid sequence with 100% identity to the full length of the peptide or ; and X4 is a peptide having an amino acid sequence with 100% identity to the full length of the peptide or .

[0215] In the illustrative embodiments of (i) to (viii) above, the optional amino acid residues in SEQ ID NO:7 are present. In the illustrative embodiments of (i) to (viii) above, the peptides of X1, X3, and X4 are shown in SEQ ID NO.4, 5, and 6. In the illustrative embodiments of (i) to (viii) above, the peptides of X1, X3, and X4 are shown in SEQ ID NO.320, 321, and 322.

[0216] In various embodiments,

[0217] (i) The first polypeptide component comprises one of X1, X2, X3, and X4, and the second polypeptide component comprises the three of X1, X2, X3, and X4 that are not present in the first polypeptide component; or

[0218] (ii) The first polypeptide component comprises two of X1, X2, X3, and X4, and the second polypeptide component comprises the two of X1, X2, X3, and X4 that are not present in the first polypeptide component.

[0219] In still other embodiments,

[0220] (a) The first polypeptide comprises X1 and excludes X2, X3, and X4; and the second polypeptide is a fusion protein comprising X3-Z1-X2-Z2-X4 and excluding X1;

[0221] (b) The first polypeptide comprises X4 and excludes X1, X2, and X3; and the second polypeptide is a fusion protein comprising X1-Z1-X3-Z2-X2 and excluding X4; or

[0222] (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;

[0223] wherein each of Z1 and Z2 is independently an optional amino acid linker. When considered appropriate for the intended use, Z1 and / or Z2 may comprise any number of amino acid residues to separate the domains within the first and / or second polypeptides. The Z1 and / or Z2 linker can be of any suitable length and amino acid composition. In one embodiment, both Z1 and Z2 are absent; in another embodiment, both Z1 and Z2 are present; in still another embodiment, one of Z1 and Z2 is present and the other is absent.

[0224] In other embodiments, X1, X2, X3, and X4 each comprise a peptide 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 X1, X2, X3, and X4 domains (SEQ ID NO: 4 - 7) shown below, where the residues in parentheses may be present or absent:

[0225] X1:

[0226] X2:

[0227] X3:

[0228] X4:

[0229] In other embodiments, X1, X2, X3, and X4 are peptides comprising amino acid sequences that are at least 80% identical to the full length of the X1, X2, X3, and X4 domains (SEQ ID NO: 4 - 7) shown below, where the residues in parentheses may be present or absent:

[0230] X1:

[0231] X2:

[0232] X3:

[0233] X4:

[0234] In other embodiments, X1, X2, X3, and X4 are peptides comprising amino acid sequences that are at least 90% identical to the full length of the X1, X2, X3, and X4 domains (SEQ ID NO: 4 - 7) shown below, where the residues in parentheses may be present or absent:

[0235] X1:

[0236] X2:

[0237] X3:

[0238] X4:

[0239] In other embodiments, respectively, X1, X2, X3, and X4 are respectively 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, where the residues in parentheses may be present or absent:

[0240] X1:

[0241] X2:

[0242] X3:

[0243] X4:

[0244] In other embodiments, X1, X2, X3, and X4 are respectively peptides comprising amino acid sequences that are at least 80% identical to the full length of the X1, X2, X3, and X4 domains shown below, where the residues in parentheses may be present or absent:

[0245] X1:

[0246] X2:

[0247] X3:

[0248] X4:

[0249] In other embodiments, X1, X2, X3, and X4 are respectively peptides comprising amino acid sequences that are at least 90% identical to the full length of the X1, X2, X3, and X4 domains shown below, where the residues in parentheses may be present or absent:

[0250] X1:

[0251] X2:

[0252] X3:

[0253] X4:

[0254] In one embodiment, one or more or all of the optional amino acids are present; in another embodiment, one or more or all of the optional amino acids are absent. In other embodiments,

[0255] (i) X1 is a peptide comprising an amino acid sequence having at least 55% identity to the full-length 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 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 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 amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 322;

[0256] (ii) X1 is a peptide comprising an amino acid sequence having at least 75% identity to the full-length 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 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 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 75% identity to the full-length amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 322;

[0257] (iii) X1 is a peptide comprising an amino acid sequence having at least 80% identity to the full-length 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 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 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 amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 322;

[0258] (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 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 100% 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 100% 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 100% identity to the full length of the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:322.

[0259] In the exemplary embodiments of (i) to (vi) above, the peptides of X1, X3, and X4 are shown in SEQ ID NO.4, 5, and 6. In the exemplary embodiments of (i) to (vi) above, the peptides of X1, X3, and X4 are shown in SEQ ID NO.320, 321, and 322.

[0260] In another embodiment, the first polypeptide and the second polypeptide 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 shown below (underlined residues or "X" residues are optional and each residue in the optional region may contain any amino acid):

[0261] (i)

[0262] First polypeptide X1 (Neo2A)

[0263] PKKKIQLHAEHALYDALMILNI VKTNS (SEQ ID NO:256) and

[0264] Second polypeptide X3-X2'-X4 (Neo2B)

[0265] TNSPPAEEKLEDYAFNFELILEEIARLFESG DQ KDEAEKAKRMKEWMKRIKT TAS EDEQEEMANAIITILQSWIFS(SEQ ID NO:257);

[0266] (ii)

[0267] The first polypeptide X1-X3-X2’

[0268] PKKKIQLHAEHALYDALMILNI VKTNSPPAEEK LEDYAFNFELILEEIARLFESG DQ KDEAEKAKRMKEWMKRIKTTAS(SEQ ID NO:258) and

[0269] The second polypeptide X4

[0270] TTASE DEQEEMANAIITILQSWIFS(SEQ ID NO:259);

[0271] (iii)

[0272] The first polypeptide X1-X3

[0273] PKKKIQLHAEHALYDALMILNI VKTNSPPAEEK LEDYAFNFELILEEIARLFES GD (SEQ ID NO:260) and

[0274] The second polypeptide X2-X4

[0275] DQKDEAEKAKRMKEWMKRIKT TAS EDEQEEMANAIITILQSWIFS(SEQ ID NO:261);

[0276] (iv)

[0277] The first polypeptide X1(Neo4A)

[0278] PKKKIQIMAEEALKDALSILNI VKTNS (SEQ ID NO:262) and

[0279] The second polypeptide X3-X2’-X4(Neo4B)

[0280] TNSPPAEE QLERFAKRFERNLWGIARLFESG DQKDEAEKAKRMIEWMKRIKT TAS EDEQEEMANAIITILQSWFFS (SEQ ID NO:263);

[0281] (v)

[0282] First polypeptide X1 (Neo2A)

[0283] PKKKIQLHAEHALYDALMILNI XXXXX (SEQ ID NO:311) and

[0284] Second polypeptide X3-X2’-X4 (Neo2B)

[0285] XXXXXXXXX LEDYAFNFELILEEIARLFESG XX KDEAEKAKRMKEWMKRIKT XXX EDEQEEMANAIITILQSWIFS (SEQ ID NO:264);

[0286] (vi)

[0287] First polypeptide X1-X3-X2’

[0288] PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESG XX KDEAEKAKRMKEWMKRIKTTAS (SEQ ID NO:265) and

[0289] Second polypeptide X4

[0290] XXXXX DEQEEMANAIITILQSWIFS (SEQ ID NO:266);

[0291] (vii)

[0292] First polypeptide X1-X3

[0293] PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESXX GD (SEQ ID NO:267) and

[0294] Second polypeptide X2-X4

[0295] DQKDEAEKAKRMKEWMKRIKT XXXEDEQEEMANAIITILQSWIFS (SEQ ID NO:268);

[0296] (viii)

[0297] The first polypeptide X1 (Neo4A)

[0298] PKKKIQIMAEEALKDALSILNI XXXXX (SEQ ID NO:269) and

[0299] The second polypeptide X3 - X2’ - X4 (Neo4B)

[0300] XXXXXXXX QLERFAKRFERNLWGIARLFESG XX KDEAEKAKRMIEWMKRIKT XXX EDEQEEMANAIITILQSWFFS (SEQ ID NO:270);

[0301] (ix)

[0302] The first polypeptide >Neo4_H1 - H3’

[0303] PKKKIQIMAEEALKDALSILNI VKTNSPPAEE QLERFAKRFERNLWGIARLFES GD (SEQ ID NO:312) and

[0304] The second polypeptide >Neo4_H2 - H4

[0305] DQ KDEAEKAKRMIEWMKRIK TTAS EDEQEEMANAIITILQSWFFS (SEQ ID NO:313);

[0306] (x)

[0307] The first polypeptide >Neo4_H1 - H3’

[0308] PKKKIQIMAEEALKDALSILNIXXXXXXXXXXQLERFAKRFERNLWGIARLFESXX (SEQ ID NO:314) and

[0309] The second polypeptide >Neo4_H2 - H4

[0310] XXKDEAEKAKRMIEWMKRIKXXXXEDEQEEMANAIITILQSWFFS (SEQ ID NO:315);

[0311] (xi)

[0312] The first polypeptide Neo4_H1-H3’-H2

[0313] PKKKIQIMAEEALKDALSILNI VKTNSPPAEEQ LERFAKRFERNLWGIARLFES GDQ KDEAEKAKRMIEWMKRIK TTA (SEQ ID NO:316) and

[0314] The second polypeptide Neo4_H4

[0315] TTASE DEQEEMANAIITILQSWFFS (SEQ ID NO:317);

[0316] (xii)

[0317] The first polypeptide Neo4_H1-H3’-H2

[0318] PKKKIQIMAEEALKDALSILNIXXXXXXXXXXXLERFAKRFERNLWGIARLFESXXXKDEAEKAKRMIEWMKRIKXXX (SEQ ID NO:318) and

[0319] The second polypeptide Neo4_H4

[0320] XXXXX DEQEEMANAIITILQSWFFS (SEQ ID NO:319);

[0321] (xiii)

[0322] The first polypeptide (X1)

[0323] PKKKIQLHAEHALYDALMILNI VGGSS (SEQ ID NO:323), or

[0324] SKEA IQLHAEHALYDALMILNIVKTNS (SEQ ID NO:324), or

[0325] P IQLHAEHALYDALMILNIV (SEQ ID NO:325) and

[0326] Second polypeptide (X3-X2'-X4)

[0327] PK LEDYAFNFELILEEIARLFESG DQ KDEAEKAKRMKEWMKRIKT TAS EDEQEEMANAIITILQSWIFS (SEQ ID NO:326), or

[0328] GGSSGG LEDYAFNFELILEEIARLFESG GSSGG KDEAEKAKRMKEWMKRIT GGSSGG DEQEEMANAIITILQSWIFS (SEQ ID NO:327), or

[0329] GGSSGG LEDYAFNFELILEEIARLFES GGSSGGGG EAEKAKRMKEWMKRI GGSSGG DEQEEMANAIITILQSWIFS (SEQ ID NO:328).

[0330] In an exemplary embodiment, the first polypeptide and the second polypeptide are peptides comprising an amino acid sequence that is at least 80% identical to a pair of first and second polypeptides shown in embodiments (i)-(viii) above (underlined residues or "X" residues are optional and each residue in the optional domain may comprise any amino acid when present).

[0331] In an exemplary embodiment, the first polypeptide and the second polypeptide are peptides comprising an amino acid sequence that is at least 90% identical to a pair of first and second polypeptides shown in embodiments (i)-(viii) above (underlined residues or "X" residues are optional and each residue in the optional domain may comprise any amino acid when present).

[0332] In an exemplary embodiment, the first polypeptide and the second polypeptide are peptides comprising an amino acid sequence that is 100% identical to a pair of first and second polypeptides shown in embodiments (i)-(viii) above (underlined residues or "X" residues are optional and each residue in the optional domain may comprise any amino acid when present).

[0333] In various further embodiments, X1, X2, X3, and X4 each comprise a peptide 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 present within the amino acid sequences of SEQ ID NOs: 11-94, 190-216, 247, and 275-300, respectively (as defined in Table 1, although listed as the H1, H2, H3, and H4 domains).

[0334] Table 1 provides two SEQ ID NOs for many variants, listing the first SEQ ID NO (underlined in the table) of the linker position as optional and variable, and the second SEQ ID NO that requires the inclusion of the linker position. Table 1 shows the domain arrangement of the polypeptides of SEQ ID NOs: 11-94, 190-216, 247, and 275-300 (see the second column), while the sequences show the underlined amino acid linkers separating 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):

[0335] STKKWQLQAEHALLDWQMALNK SPEPN ENLNRAITAAQSWIS TGKID LDKAEDIRRNSDQARREAEK R GIDV RDLISNAQVILLEAR (SEQ ID NO: 11).

[0336] As would be apparent to one of ordinary skill in the art based on this alignment, 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 each of the X1, X2, X3, and X4 domains SEQ ID NO:11 - 94, 190 - 216, 247, and 275 - 300 would be clear to one of ordinary skill in the art based on the teachings herein. As would be appreciated by one of ordinary skill in the art, the X1, X2, X3, and / or X4 amino acids may include additional (1, 2, 3, 4, 5, or more) amino acids at the N - terminus and / or C - terminus relative to the X1, X2, X3, and X4 domains shown in SEQ ID NO:11 - 94, 190 - 216, 247, and 275 - 300.

[0337] Table 1

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355] In one specific embodiment, respectively, 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, although listed as the H1, H2, H3, and H4 domains) present within the amino acid sequence of SEQ ID NO: 90 form 1 or 2. SEQ ID NO: 90 form 1 or 2 has the same primary amino acid sequence but varies slightly in optional / variable linker residues. In various embodiments, this embodiment may include variants of the X1, X2, X3, and / or X4 domains present in SEQ ID NO: 90 form 1 or 2, incorporating the mutations shown in SEQ ID NO: 275 - 300 relative to the SEQ ID NO: 90 primary amino acid sequence.

[0356] In one embodiment of any of the embodiments or combinations of embodiments disclosed herein, X1, X2, X3, and X4 are α - helix domains. In another embodiment, the amino acid length of each of X1, X2, X3, and X4 is independently at least about 8, 10, 12, 14, 16, 19, or more amino acids in 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 various further embodiments, the amino acid length of each of X1, X2, X3, and X4 is independently between about 8 - 1000, 8 - 500, 8 - 400, 8 - 300, 8 - 200, 8 - 100, 8 - 50, 10 - 1000, 10 - 500, 10 - 400, 10 - 300, 10 - 200, 10 - 100, 10 - 50, 12 - 1000, 12 - 500, 12 - 400, 12 - 300, 12 - 200, 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.

[0357] In one embodiment, the first polypeptide component and / or the second polypeptide component comprises at least one disulfide bond.

[0358] In another embodiment, the first polypeptide component and the second polypeptide component are non-covalently associated. As documented herein, the first polypeptide component and the second polypeptide component alone are not active receptor agonists, and wherein the first polypeptide component and the second polypeptide component interact to form an IL-2 receptor βγ c heterodimer (IL-2Rβγ c ), an IL-4 receptor αγ c heterodimer (IL-4Rαγ c ), an IL-13α, or an active agonist of an IL-4Rα / IL13Rα heterodimer. Thus, in this embodiment, the first polypeptide and the second polypeptide can interact to form an active agonist. Such interaction can be any suitable interaction, such as a non-covalent interaction. The interaction can involve direct non-covalent binding of the first and second polypeptides, or indirect interaction. In one embodiment, the first polypeptide component and the second polypeptide component bind indirectly to each other via a receptor, such as an IL-2 receptor βγ c heterodimer (IL-2Rβγ c ), an IL-4 receptor αγ c heterodimer (IL-4Rαγ c ), an IL-13α, or an IL-4Rα / IL13Rα heterodimer.

[0359] Methods for determining binding to a receptor are known in the art and are described herein, such as biolayer interferometry binding assays. In some embodiments, when the first polypeptide component and the second polypeptide component interact at their respective receptors, they co-localize to bind the receptor 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. For example, the split IL-2 mimetic of the present invention co-localizes to bind the IL-2 receptor βγ c heterodimer (IL-2Rβγ c ) 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. Similarly, as an example, the split IL-4 mimetic of the present invention co-localizes to bind the IL-4 receptor αγ c heterodimer (IL-4Rαγ c)。In some aspects, agonism of the receptor to which the split mimetic co-localizes and binds is measured by STAT5 phosphorylation.

[0360] In another aspect, the present disclosure provides polypeptides comprising 1, 2, or 3 but not all 4 of the domains X1, X2, X3, and X4, wherein:

[0361] (a) When present, X1 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 the peptide ;

[0362] (b) When present, X2 is any helical peptide domain;

[0363] (c) When present, X3 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 the peptide ; and

[0364] (d) When present, X4 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 the peptide ; and

[0365] the amino acid residues in parentheses may be present or absent.

[0366] The polypeptides of this aspect can be used, for example, to generate conditional active receptor agonists of any of the embodiments or combinations of embodiments disclosed herein (i.e., the polypeptides of this aspect are either the first polypeptide or the second polypeptide of the conditional active receptor agonists of the present disclosure). Thus, as will be clear to those skilled in the art, all embodiments and combinations of embodiments of the first and second polypeptides disclosed above, and all embodiments and combinations of embodiments of the X1, X2, X3, and X4 domains described above, equally apply to the polypeptides of this aspect of the present disclosure. In one embodiment,

[0367] (a) AA substitutions relative to the AA sequence of SEQ ID NO: 4 in X1 occur at no more than 3 bolded AA residues, or at no more than 2 bolded AA residues, or at no more than 1 bolded AA residue, or do not occur at bolded AA residues;

[0368] (b) The AA substitutions in X3 relative to the AA sequence of SEQ ID NO:5 occur at no more than 3 AA residues marked in bold, or at no more than 2 AA residues marked in bold, or at no more than 1 AA residue marked in bold, or do not occur at the AA residues marked in bold; and / or

[0369] (c) The AA substitutions in X4 relative to the AA sequence of SEQ ID NO:6 occur at no more than 2 AA residues marked in bold, or at no more than 1 AA residue marked in bold, or do not occur at the AA residues marked in bold.

[0370] In another embodiment, the AA substitutions in X2 relative to the AA sequence of SEQ ID NO:7 do not occur at the AA residues marked in bold.

[0371] In various embodiments, the polypeptide may be selected from the group consisting of:

[0372] (i) a polypeptide comprising X1 and excluding X2, X3, and X4;

[0373] (ii) a polypeptide comprising X2 and excluding X1, X3, and X4;

[0374] (iii) a polypeptide comprising X3 and excluding X1, X2, and X4;

[0375] (iv) a polypeptide comprising X4 and excluding X1, X2, and X3;

[0376] (v) a polypeptide comprising X1 and X2 and excluding X3 and X4;

[0377] (vi) a polypeptide comprising X1 and X3 and excluding X2 and X4;

[0378] (vii) a polypeptide comprising X1 and X4 and excluding X2 and X3;

[0379] (viii) a polypeptide comprising X2 and X3 and excluding X1 and X4;

[0380] (ix) a polypeptide comprising X2 and X4 and excluding X1 and X3;

[0381] (x) a polypeptide comprising X3 and X4 and excluding X1 and X2;

[0382] (xi) a polypeptide comprising X1, X2, and X3 and excluding X4;

[0383] (xii) a polypeptide comprising X1, X2, and X4 and excluding X3;

[0384] (xiii) A polypeptide comprising X1, X3, and X4 and excluding X2; and

[0385] (xiv) A polypeptide comprising X2, X3, and X4 and excluding X1.

[0386] In one embodiment, the polypeptide comprises 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 first polypeptide or the second polypeptide listed below (the underlined residues are optional and each optional residue, when present, can comprise any amino acid):

[0387] (i)

[0388] First polypeptide X1 (Neo2A)

[0389] PKKKIQLHAEHALYDALMILNI VKTNS (SEQ ID NO:256) and

[0390] Second polypeptide X3-X2’-X4 (Neo2B)

[0391] TNSPPAEEK LEDYAFNFELILEEIARLFESG DQ KDEAEKAKRMKEWMKRIKT TAS EDEQEEMANAIITILQSWIFS (SEQ ID NO:257);

[0392] (ii)

[0393] First polypeptide X1-X3-X2’

[0394] PKKKIQLHAEHALYDALMILNI VKTNSPPAEEK LEDYAFNFELILEEIARLFESG DQ KDEAEKAKRMKEWMKRIKTTAS (SEQ ID NO:258) and

[0395] Second polypeptide X4

[0396] TTASE DEQEEMANAIITILQSWIFS (SEQ ID NO:259);

[0397] (iii)

[0398] First polypeptide X1-X3

[0399] PKKKIQLHAEHALYDALMILNI VKTNSPPAEEK LEDYAFNFELILEEIARLFES GD (SEQ ID NO:260) and

[0400] The second polypeptide X2-X4

[0401] DQKDEAEKAKRMKEWMKRIKT TAS EDEQEEMANAIITILQSWIFS (SEQ ID NO:261);

[0402] (iv)

[0403] The first polypeptide X1 (Neo4A)

[0404] PKKKIQIMAEEALKDALSILNI VKTNS (SEQ ID NO:262) and

[0405] The second polypeptide X3-X2’-X4 (Neo4B)

[0406] TNSPPAEE QLERFAKRFERNLWGIARLFESG DQ KDEAEKAKRMIEWMKRIKT TAS EDEQEEMANAIITILQSWFFS (SEQ ID NO:263);

[0407] (v)

[0408] The first polypeptide X1 (Neo2A)

[0409] PKKKIQLHAEHALYDALMILNI XXXXX (SEQ ID NO:311) and

[0410] The second polypeptide X3-X2’-X4 (Neo2B)

[0411] XXXXXXXXX LEDYAFNFELILEEIARLFESG XX KDEAEKAKRMKEWMKRIKT XXX EDEQEEMANAIITILQSWIFS (SEQ ID NO:264);

[0412] (vi)

[0413] The first polypeptide X1-X3-X2’

[0414] PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESG XX KDEAEKAKRMKEWMKRIKTTAS (SEQ ID NO:265) and

[0415] The second polypeptide X4

[0416] XXXXX DEQEEMANAIITILQSWIFS (SEQ ID NO:266);

[0417] (vii)

[0418] The first polypeptides X1 - X3

[0419] PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESXX GD (SEQ ID NO:267) and

[0420] The second polypeptides X2 - X4

[0421] DQKDEAEKAKRMKEWMKRIKT XXX EDEQEEMANAIITILQSWIFS (SEQ ID NO:268);

[0422] (viii)

[0423] The first polypeptide X1 (Neo4A)

[0424] PKKKIQIMAEEALKDALSILNI XXXXX (SEQ ID NO:269) and

[0425] The second polypeptide X3 - X2’ - X4 (Neo4B)

[0426] XXXXXXXX QLERFAKRFERNLWGIARLFESG XX KDEAEKAKRMIEWMKRIKT XXX EDEQEEMANAIITILQSWFFS (SEQ ID NO:270);

[0427] (ix)

[0428] The first polypeptide >Neo4_H1 - H3’

[0429] PKKKIQIMAEEALKDALSILNI VKTNSPPAEEQLERFAKRFERNLWGIARLFES GD (SEQ ID NO:312) and

[0430] Second polypeptide > Neo4_H2-H4

[0431] DQ KDEAEKAKRMIEWMKRIK TTAS EDEQEEMANAIITILQSWFFS(SEQ ID NO:313);

[0432] (x)

[0433] First polypeptide > Neo4_H1-H3’

[0434] PKKKIQIMAEEALKDALSILNIXXXXXXXXXXQLERFAKRFERNLWGIARLFESXX(SEQ ID NO:314) and

[0435] Second polypeptide > Neo4_H2-H4

[0436] XX KDEAEKAKRMIEWMKRIKXXXXEDEQEEMANAIITILQSWFFS(SEQ ID NO:315);

[0437] (xi)

[0438] First polypeptide Neo4_H1-H3’-H2

[0439] PKKKIQIMAEEALKDALSILNI VKTNSPPAEEQ LERFAKRFERNLWGIARLFES GDQ KDEAEKAKRMIEWMKRIK TTA (SEQ ID NO:316) and

[0440] Second polypeptide Neo4_H4

[0441] TTASE DEQEEMANAIITILQSWFFS(SEQ ID NO:317);

[0442] (xii)

[0443] First polypeptide Neo4_H1-H3’-H2

[0444] PKKKIQIMAEEALKDALSILNIXXXXXXXXXXXLERFAKRFERNLWGIARLFESXXXKDEAEKAKRMIEWMKRIKXXX (SEQ ID NO:318) and

[0445] The second polypeptide Neo4_H4

[0446] XXXXX DEQEEMANAIITILQSWFFS (SEQ ID NO:319);

[0447] (xiii)

[0448] The first polypeptide (X1)

[0449] PKKKIQLHAEHALYDALMILNI VGGSS (SEQ ID NO:323), or

[0450] SKEA IQLHAEHALYDALMILNIVKTNS (SEQ ID NO:324), or

[0451] P IQLHAEHALYDALMILNIV (SEQ ID NO:325) and

[0452] The second polypeptide (X3 - X2’ - X4)

[0453] PK LEDYAFNFELILEEIARLFESG DQ KDEAEKAKRMKEWMKRIKT TAS EDEQEEMANAIITILQSWIFS (SEQ ID NO:326), or

[0454] GGSSGG LEDYAFNFELILEEIARLFESG GSSGG KDEAEKAKRMKEWMKRIT GGSSGG DEQEEMANAIITILQSWIFS (SEQ ID NO:327), or

[0455] GGSSGG LEDYAFNFELILEEIARLFES GGSSGGGG EAEKAKRMKEWMKRI GGSSGG DEQEEMANAIITILQSWIFS (SEQ ID NO:328).

[0456] In an illustrative embodiment, the polypeptide comprises an amino acid sequence that is at least 80% identical to the first or second polypeptide shown in embodiments (i)-(viii) above (underlined residues or "X" residues are optional and each residue of the optional domain can comprise any amino acid when present).

[0457] In an illustrative embodiment, the polypeptide comprises an amino acid sequence that is at least 90% identical to the first or second polypeptide shown in embodiments (i)-(viii) above (underlined residues or "X" residues are optional and each residue of the optional domain can comprise any amino acid when present).

[0458] In an illustrative embodiment, the polypeptide comprises an amino acid sequence that is 100% identical to the first or second polypeptide shown in embodiments (i)-(viii) above (underlined residues or "X" residues are optional and each residue of the optional domain can comprise any amino acid when present).

[0459] In another embodiment, when present, 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 selected from the group consisting of SEQ ID NOs: 11-94, 190-216, 247, and 275-300. In a specific embodiment, when present, 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, although listed as H1, H2, H3, and H4 domains) present within the amino acid sequence of SEQ ID NO: 90 form 1 or 2, SEQ ID NO: 90 form 1 or 2 having the same primary amino acid sequence but differing slightly in optional / variable linker residues. In various embodiments, this embodiment can include variants of the X1, X2, X3, and / or X4 domains present in SEQ ID NO: 90 form 1 or 2, incorporating the mutations shown in SEQ ID NOs: 275-300 relative to the SEQ ID NO: 90 primary amino acid sequence.

[0460] The first polypeptide, second polypeptide, and polypeptide (when the polypeptide is genetically encodable) described herein can be chemically synthesized or recombinantly expressed. The polypeptide can be linked to other compounds, such as stabilizing compounds to promote an extended in vivo half-life, including but not limited to albumin, PEGylation (attachment of one or more polyethylene glycol chains), HESylation, PASylation, glycosylation, or the polypeptide can be generated as an Fc fusion or as a deimmunized variant. Such linkages can be covalent or non-covalent. For example, adding a module containing polyethylene glycol (“PEG”) can involve attaching a PEG group (“PEG-MAL”) linked to a maleimide group 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, the PEG can comprise branched PEG and / or multiple PEG chains.

[0461] In one embodiment, a stabilizing compound (including but not limited to a module containing PEG) is linked to a cysteine residue in the polypeptide. In another embodiment, the cysteine residue is present in the X2 domain. In some embodiments, the cysteine residue is present in any one of a plurality of positions in, for example, the X2 domain. In some such embodiments, the X2 domain is at least 19 amino acids in length and the cysteine residue is at position 1, 2, 5, 9, or 16 relative to those 19 amino acids. In yet another embodiment, the stabilizing compound (including but not limited to a module containing PEG) is linked to the cysteine residue via a maleimide group.

[0462] In yet another embodiment, the first polypeptide, second polypeptide, and polypeptide can further comprise a targeting domain. In this embodiment, the conditional active receptor agonist can be targeted to a target of interest. The targeting domain can be covalently or non-covalently bound to the first polypeptide, second polypeptide, and / or polypeptide. In embodiments in which the targeting domain is non-covalently bound, any means suitable for such non-covalent binding can be used, including but not limited to a streptavidin-biotin linker.

[0463] In another embodiment, when present, the targeting domain is a translational fusion with the polypeptide. In this embodiment, the polypeptide and the targeting domain may be directly contiguous to each other in the translational fusion or may be linked by a polypeptide linker suitable for the intended purpose. Exemplary such linkers include, but are not limited to, those disclosed in WO2016178905, WO2018153865 (especially on page 13), and WO2018170179 (especially in paragraphs

[0316] -

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

[0464] A targeting domain is a polypeptide domain or small molecule that binds to a target of interest. In one non-limiting embodiment, the targeting domain binds to a cell surface protein; in this embodiment, the cell can be any cell type of interest that includes a surface protein capable of being 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 (such as fibroblasts, infiltrating immune cells, or stromal elements), other cancer cells, and immune cells (including but not limited to CD8+ T cells, T regulatory cells, dendritic cells, NK cells, or macrophages). When the cell surface protein is on the surface of a tumor cell, vascular component cell, or tumor microenvironment cell (such as fibroblasts, infiltrating immune cells, or stromal elements), any suitable tumor cell, vascular component cell, or tumor microenvironment cell surface marker can be targeted, including but not limited to EGFR, EGFRvIII, Her2, HER3, EpCAM, MSLN, MUC16, PSMA, TROP2, ROR1, RON, PD-L1, CD47, CTLA-4, CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD40, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC, HLD-DR, carcinoembryonic antigen (CEA), TAG-72, 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 (de2-7EGFR), fibroblast activation protein, tenascin, metalloproteinase, endosialin, vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6, HPV E7, Her-2 / neu, MAGE A3, non-mutant p53, NY-ESO-1, MelanA / MART1, Ras mutant, gp100, mutant p53, PR1, bcr-abl, tyrosinase, 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, MAGEA1, 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, XAGE 1, podoplanin, Tie 3, 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 NM001203); E16 (LAT1, SLC7A5, Genbank accession number NM003486); STEAP1 (prostate transmembrane protein, androgen-induced 1, 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 cotransporter 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 cDNA 2700050C12, RIKEN cDNA2700050C12 gene, Genbank accession number AY358628); ETBR (endothelin B receptor, Genbank accession number AY275463); MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession number NM017763); STEAP2 (HGNC.sub.--8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, prostate transmembrane epithelial antigen 2 with six transmembrane domains, six transmembrane prostate protein, Genbank accession number AF455138); TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession number NM017636); CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession number NP003203 or NM003212); CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein Barr virus receptor) or Hs.73792, Genbank accession number M26004); CD79b (IGb (immunoglobulin related β), 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); IL20Rα (Genbank accession number AF184971); Brevican (Genbank accession number AF229053); Ephb2R (Genbank accession number NM004442); ASLG659 (Genbank accession number AX092328); PSCA (Genbank accession number AJ297436); GEDA (Genbank accession number AY260763); BAFF-R (Genbank accession number NP443177.1); CD22 (Genbank accession number NP001762.1); CD79a (CD79A, CD79α, immunoglobulin related α, B cell-specific protein, covalently interacts with Igβ (CD79B) and forms a complex with Ig M molecules on the surface, transduces signals involved in B cell differentiation, Genbank accession number NP001774.1); CXCR5 (Burkitt lymphoma receptor 1, G protein-coupled receptor, activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and possibly the development of AIDS, lymphoma, myeloma, and leukemia, Genbank accession number NP001707.1); HLA-DOB (beta subunit of MHC class II molecule (Ia antigen), 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, defects may contribute to the pathophysiology of idiopathic detrusor instability, Genbank accession number NP002552.2); CD72 (B cell differentiation antigen CD72, Lyb-2, Genbank accession number NP001773.1); LY64 (lymphocyte antigen 64 (RP105), a type I membrane protein of the leucine-rich repeat (LRR) family, regulates B cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosus, Genbank accession number NP005573.1); FCRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain containing C2-type Ig-like and ITAM domains, may have a role in B lymphocyte differentiation, Genbank accession number NP443170.1); or IRTA2 (immunoglobulin superfamily receptor translocation-associated 2, a putative immunoreceptor that may have a role in B cell development and lymphoma genesis; gene dysregulation caused by translocation occurs in some B cell malignancies, Genbank accession number NP112571.1).

[0465] In another embodiment, the targeting domain binds to an immune cell surface marker. In this embodiment, the target can be a cell surface protein of any suitable immune cell, including but not limited to CD8+ T cells, T regulatory cells, dendritic cells, NK cells, or macrophages. The targeting domain can 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 wherein 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.

[0466] In all such embodiments, the targeting domain can be any suitable polypeptide that binds to a target of interest and can be incorporated into the polypeptides of the present disclosure. In non-limiting embodiments, the targeting domain can include, but is not limited to, scFv, F(ab), F(ab’) that bind to the cell surface 2 , B cell receptor (BCR), DARPin, affibody, monobody, nanobody, diabody, antibodies (including monospecific or bispecific antibodies), cell-targeting oligopeptides, including but not limited to RGD integrin-binding peptides, de novo designed binders, aptamers, bicyclic peptides, conotoxins, small molecules such as folic acid, and viruses.

[0467] The first polypeptide, second polypeptide, and polypeptides of the present disclosure can have additional residues at the N-terminus, C-terminus, or both that are not present in the first polypeptide, second polypeptide, and polypeptides of the present disclosure; these additional residues are not included when determining the percent identity of the polypeptides or peptide domains of the present disclosure relative to a reference polypeptide. Such residues can be any residues suitable for the intended use, including but not limited to detection tags (i.e., fluorescent proteins, antibody epitope tags, etc.), linkers, ligands suitable for purification purposes (His tags, etc.), other peptide domains that add functionality to the polypeptide, etc. Residues suitable for attaching 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. Patent Nos. 9,676,871 and 9,777,070.

[0468] In yet another aspect, the invention provides nucleic acids encoding the first polypeptide, second polypeptide, and polypeptides of the present disclosure that can be genetically encoded, including isolated nucleic acids. The isolated nucleic acid sequence can comprise RNA or DNA. Such isolated nucleic acid sequences can comprise additional sequences useful for promoting the expression and / or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretion signals, nuclear localization signals, and plasma membrane localization signals. Based on the teachings herein, which nucleic acid sequences would encode the polypeptides of the invention will be apparent to those skilled in the art.

[0469] In another aspect, the present disclosure provides expression vectors that comprise a nucleic acid of any aspect of the present invention operably linked to a suitable control sequence. An "expression vector" includes a vector that operably links a nucleic acid coding region or gene to any control sequence capable of effecting the expression of the gene product. A "control sequence" operably linked to a nucleic acid sequence of the present invention is a nucleic acid sequence capable of effecting the expression of the nucleic acid molecule. The control sequence need not be contiguous with the nucleic acid sequence so long as it functions to direct its expression. Thus, for example, there may be intervening untranslated but transcribed sequences between the promoter sequence and the nucleic acid sequence, and the promoter sequence can still be considered to be "operably linked" to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors include, but are not limited to, plasmid and viral-based expression vectors. Control sequences for driving the expression of the disclosed nucleic acid sequences in mammalian systems can 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 variety 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 can comprise a plasmid, a viral-based vector (including but not limited to retroviral vectors or oncolytic viruses), or any other suitable expression vector. In some embodiments, the expression vector can be administered in the methods of the present disclosure for therapeutic benefit to express a polypeptide in vivo. In non-limiting embodiments, the expression vector can be used to transfect or transduce a cell target (including but not limited to CAR-T cells or tumor cells) to effect the therapeutic methods disclosed herein.

[0470] In yet another aspect, the present disclosure provides host cells that comprise the expression vectors and / or nucleic acids disclosed herein, wherein the host cells can be prokaryotic or eukaryotic. Techniques including but not limited to bacterial transformation, calcium phosphate co-precipitation, electroporation, or liposome, DEAE-dextran, polycation, or virus-mediated transfection can be used to transiently or stably modify the cells to incorporate the expression vectors of the present invention (see, for example, Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press); Culture of Animal Cells: A Manual of Basic Technique, 2 ndEd. (R. I. Freshney, 1987, Liss, Inc. New York, NY)). The method of generating a polypeptide according to the present invention is another part of the present invention. The method comprises the following steps: (a) culturing a host according to this aspect of the present invention under conditions conducive to polypeptide expression, and (b) optionally recovering the expressed polypeptide. The expressed polypeptide can be recovered from a cell-free extract, but it is preferably recovered from the culture medium.

[0471] In another aspect, the present disclosure provides a pharmaceutical composition comprising one or more conditionally active receptor agonists, polypeptides, nucleic acids, expression vectors, and / or host cells of any aspect or embodiment of the present disclosure and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present disclosure can be used, for example, in the disclosed methods described below. In addition to the polypeptides of the present disclosure, the pharmaceutical composition can comprise (a) a cryoprotectant; (b) a surfactant; (c) a filler; (d) an osmotic pressure regulator; (e) a stabilizer; (f) a preservative; and / or (g) a buffer.

[0472] 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 can also include a cryoprotectant, such as sucrose, sorbitol, or trehalose. In certain embodiments, the pharmaceutical composition includes a preservative, such as benzalkonium chloride, benzethonium chloride, chlorhexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chloromethanol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the pharmaceutical composition includes a filler, such as glycine. In still other embodiments, the pharmaceutical composition includes a surfactant, such as polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleate, or a combination thereof. The pharmaceutical composition can also include an osmotic pressure regulator, such as a compound that renders the formulation substantially isotonic or iso-osmotic with human blood. Exemplary osmotic pressure regulators include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine, and arginine hydrochloride. In other embodiments, the pharmaceutical composition additionally includes a stabilizer, such as a molecule that substantially prevents or alleviates the chemical and / or physical instability of the protein of interest in lyophilized or liquid form when combined with the protein of interest. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0473] A conditional active receptor agonist, polypeptide, nucleic acid, expression vector, and / or host cell can be the sole active agent in a pharmaceutical composition, or the composition can further comprise one or more other active agents suitable for the intended use.

[0474] In yet another aspect, the present disclosure provides methods for treating cancer, which comprise administering to a subject in need thereof a therapeutically effective amount of a conditional 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 a first polypeptide component and a second polypeptide component interact at the cells of a tumor to treat cancer. In embodiments in which a conditional active receptor agonist is administered, the first and second polypeptides can be administered together or can be administered in separate pharmaceutical formulations.

[0475] As used herein, "treating" or "treatment" means achieving one or more of the following: (a) reducing the size or volume of a tumor and / or metastasis in a subject; (b) limiting any increase in the size or volume of a tumor and / or metastasis in a subject; (c) prolonging survival; (d) reducing the severity of symptoms associated with cancer; (e) limiting or preventing the occurrence of symptoms associated with cancer; and (f) inhibiting the worsening of symptoms associated with cancer.

[0476] Any suitable cancer can be treated using the methods, including but not limited to colon cancer, melanoma, renal cell carcinoma, head and neck squamous cell carcinoma, gastric cancer, urothelial carcinoma of the bladder, Hodgkin 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 lymphoma, multiple myeloma, ovarian cancer, cervical cancer, and any tumor type selected by diagnostic tests such as microsatellite instability, tumor mutational burden, PD-L1 expression level, or immune scoring assays (such as those developed by The Society for Immunotherapy of Cancer).

[0477] The subject can be any subject having cancer. In one embodiment, the subject is a mammal, including but not limited to a human, dog, cat, horse, cow, etc.

[0478] In one embodiment, the first targeting domain binds to a cell marker and the second targeting domain binds to a second and different cell marker, and wherein the co-expression of these two markers on the same or nearby cells is more commonly found in tumors than in other tissues, and wherein the first polypeptide and the second polypeptide interact only after the first targeting domain binds to the first cell marker and the second targeting domain binds to the second cell marker. For example, this embodiment employs two targeting domains that bind to different markers that are not enriched on tumor cells by themselves but are enriched in tumors by their co-expression.

[0479] 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 first tumor cell marker and the second targeting domain binds to a second tumor cell marker that may be the same as or different from the first tumor cell marker, and wherein the first polypeptide and the second polypeptide 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.

[0480] 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 CD8+ T cells, T regulatory cells, dendritic cells, or macrophages), and wherein the first polypeptide and the second polypeptide interact only after the first targeting domain binds to the tumor cell marker and the second targeting domain binds to the immune cell marker.

[0481] In one embodiment, the first targeting domain binds to a cell marker and the second targeting domain binds to a second and different cell marker, and wherein the co-expression of these two markers is more commonly found on tumor cells than on some other types of cells, and wherein the first polypeptide and the second polypeptide 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. For example, this embodiment employs two targeting domains that bind to different markers that are not enriched on tumor cells by themselves but are enriched in tumors by their co-expression.

[0482] In yet 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 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) that may be the same as or different from the first immune cell marker, and wherein the first polypeptide and the second polypeptide 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.

[0483] In yet another aspect, the present disclosure provides methods for modulating an immune response in a subject by administering to the subject a conditional active receptor agonist, polypeptide, nucleic acid, expression vector, host cell, or pharmaceutical composition of the present disclosure. In one embodiment, the method comprises administering a conditional active receptor agonist to a subject under conditions in which a first polypeptide component and a second polypeptide component interact at an immune cell to modulate an immune response.

[0484] As used herein, a “modulated immune response” refers to the response of cells of the immune system, such as B cells, T cells (CD4 or CD8), regulatory T cells, antigen presenting cells, dendritic cells, monocytes, macrophages, NKT cells, NK cells, basophils, eosinophils, or neutrophils, to a stimulus. In some embodiments, the response is specific to a particular antigen (“antigen-specific response”) and refers to the response of CD4 T cells, CD8 T cells, or B cells via their antigen-specific receptors. In some embodiments, the immune response is a T cell response, such as a CD4+ response or a CD8+ response. Such responses of these cells can include, for example, cytotoxicity, proliferation, cytokine or chemokine production, trafficking, or phagocytosis, and can depend on the nature of the immune cell undergoing the response. In some embodiments of the compositions and methods described herein, the modulated immune response is T cell-mediated.

[0485] In some aspects, the immune response is an anti-cancer immune response. In some such aspects, the conditional active IL-2 mimetic described herein is administered to a subject having cancer to modulate the anti-cancer immune response in the subject.

[0486] In some aspects, the immune response is a tissue repair immune response. In some such aspects, the conditional active IL-4 mimetic described herein is administered to a subject in need thereof to modulate the tissue repair immune response in the subject.

[0487] In some aspects, the immune response is a wound healing immune response. In some such aspects, the conditional active IL-4 mimetic described herein is administered to a subject in need thereof to modulate the wound healing immune response in the subject.

[0488] In some aspects, methods are provided for modulating an immune response to a second therapeutic agent in a subject. In some such aspects, the method comprises administering to the subject a combination of a polypeptide of the present disclosure and 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 aspects, the antigen-specific immunotherapeutic agent comprises chimeric antigen receptor T cells (CAR-T cells). In some aspects, the polypeptides of the present disclosure enhance the immune response of the subject to the therapeutic agent. The immune response can be enhanced, for example, by improving T cell responses (including CAR-T cell responses), boosting innate T cell immune responses, reducing inflammation, inhibiting T regulatory cell activity, or combinations thereof.

[0489] In some aspects, a conditional active cytokine mimetic of the invention, such as a conditional active IL-4 mimetic as described herein, is infiltrated or otherwise combined with a biomaterial, and the biomaterial is introduced into a subject. In some aspects, the biomaterial is a component of an implantable medical device, and the device is, for example, coated with the biomaterial. Such medical devices include, for example, vascular and arterial grafts. Conditional active IL-4 and / or IL-4-related biomaterials can be used, for example, to promote wound healing and / or tissue repair and regeneration.

[0490] In another aspect, the present disclosure provides methods for agonizing an IL-2 receptor or an IL-4 receptor, which comprise administering to a subject a conditional active receptor agonist of any embodiment or combination of embodiments disclosed herein under conditions in which a first polypeptide component and a second polypeptide component interact at the receptor.

[0491] As used herein, "therapeutically effective amount" refers to an amount of a conditional active receptor agonist, polypeptide, nucleic acid, expression vector, and / or host cell effective to treat and / or limit a disease to be treated (such as cancer). Conditional active receptor agonists, polypeptides, nucleic acids, expression vectors, and / or host cells are typically formulated into pharmaceutical compositions, such as those disclosed above, and can be administered via any suitable route in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles, including but not limited to orally, by inhalation spray, ophthalmically, intravenously, subcutaneously, intraperitoneally, and intracapsularly. In one particular embodiment, the polypeptide, nucleic acid, expression vector, and / or host cell is administered mucosally, including but not limited to intravitreally, by inhalation, or intranasally. In another particular embodiment, the polypeptide, nucleic acid, expression vector, and / or host cell is administered orally. Such particular embodiments can be administered via drops, nebulizers, sprays, or other suitable formulations.

[0492] Any suitable dosage range determined by the attending medical staff can be used. The dosage regimen can be adjusted to provide the optimal desired response (e.g., therapeutic or prophylactic response). A suitable dosage range for a conditional active receptor agonist or polypeptide can be, for example, 0.1 μg / kg to 100 mg / kg body weight; alternatively, it can be 0.5 μg / kg to 50 mg / kg; 1 μg / kg to 25 mg / kg, or 5 μg / kg to 10 mg / kg body weight. In some embodiments, the recommended dosage can be less than 0.1 mcg / kg, especially if administered locally. In other embodiments, the recommended dosage can be based on weight / m 2 (i.e., body surface area), and / or it can be administered at a fixed dose (e.g., 0.05 - 100 mg). The conditional active receptor agonist, polypeptide, nucleic acid, expression vector, and / or host cell can be delivered as a single bolus, or it can be administered more than once (e.g., 2, 3, 4, 5, or more times), as determined by the attending physician.

[0493] The conditional active receptor agonist, polypeptide, nucleic acid, expression vector, and / or host cell can be administered as the sole prophylactic or therapeutic agent, or it can be administered in combination with one or more other prophylactic or therapeutic agents (i.e., combined or separately), including but not limited to tumor resection, chemotherapy, radiotherapy, immunotherapy, etc.

[0494] Numerous modifications and variations of the present disclosure are possible in light of the above teachings.

[0495] Examples

[0496] Example 1

[0497] A computational approach for designing de novo cytokine mimics that recapitulate the functional sites of native cytokines but are otherwise unrelated in topology or amino acid sequence is described. This strategy is used to design de novo non-split mimics of interleukin-2 (IL-2) and interleukin-15 (IL-15) that bind the IL-2 receptor βγ c heterodimer (IL-2Rβγ c ) but lack binding sites for IL-2Rα or IL-15Rα. The designs are highly stable, bind human and murine IL-2Rβγ c with higher affinity than native cytokines, and initiate downstream cell signaling independent of IL-2Rα and IL-15Rα. The crystal structure of the experimentally optimized mimic Neoleukin-2 / 15 is very close to the design model and provides the first structural information on the murine IL-2Rβγ c complex. Neoleukin-2 / 15 has highly potent therapeutic activity compared to IL-2 in murine models of melanoma and colon cancer, with reduced toxicity and no signs of immunogenicity.

[0498] Many cytokines interact with multiple different receptor subunits and, like most naturally occurring proteins, contain non-ideal structural features that compromise stability but are important for function. A computational scheme was developed in which structural elements that interact with a desired receptor subunit are fixed in space and an idealized globular protein structure is constructed to support these elements. Combinatorial fragment assembly was used to support linear short epitopes, and a parameterized construction of entity-free helices coupled with knowledge-based loop closure ( Figures 1A - 1B ) was performed. The approach was tested by attempting to de novo design a stable idealized protein with an interaction surface that mimics the interaction surfaces of human IL-2 (hIL-2) and human IL-15 (hIL-15) with human IL-2Rβγ c (hIL-2Rβγ c ) but completely lacks the interaction surface of the IL-2 receptor α (IL-2Rα).

[0499] Computational design of non-split IL-2 / IL-15 mimics that bind and activate IL-2Rβγ c : Native hIL-2 contains four helices connected by irregular long loops. The N-terminal helix (H1) interacts with both the β and γ subunits of the IL-2 receptor, the third helix (H3) interacts with the β subunit, and the C-terminal helix (H4) interacts with the γ subunit; the α subunit interaction surface is formed by the irregular second helix (H2) and two long loops, one connecting H1 and H2 and the other connecting H3 and H4. An idealized protein that recapitulates the interface with β and γ formed by H1, H3, and H4 was designed, and H2 was replaced with regular helices that provide better packing. Helices H1, H3, and H4 (see Figure 1A ) were used as templates for the binding sites, and a database of highly representative clustered fragments was used to reconstruct helix H2 (H2’) (see Methods). Loops extracted from the same database were used to connect pairs of helices (see Figure 1B ), the resulting helical hairpins were assembled into a fully connected backbone (see Figure 1C ), and RosettaTM combinatorial flexible backbone sequence design calculations were performed in the presence of hIL-2Rβγ c (see Methods). The top four computational designs and eight single disulfide stapled variants (see Table 2) were selected for experimental characterization by yeast display (see Methods). Eight designs were found to bind the fluorescently tagged β-γ chimeric IL-2 receptor at low nanomolar concentrations. The best non-disulfide design (G1_neo2_40) was subjected to site-saturation mutagenesis, followed by selection and combination of affinity-enhancing substitutions for murine IL-2Rβγ c (mIL-2Rβγ c ) (see Figures 5A - 5D)。Upon recombinant expression in E. coli with optimized designs, they were found to elicit pSTAT5 signaling on IL-2-responsive murine cells at low nanomolar or even picomolar concentrations (Table 3, see Silva et al., Nature 565, pg. 186, January 10, 2019), but with relatively low thermal stability (Tm ~ <45 °C, see Figures 9A - 9B and 10A-10B). To improve stability, design schemes were recalculated, starting with the backbone of the highest-affinity first-round design (G1_neo2_40_1F, topology: H1->H4->H2’->H3), coupling loop construction with parametric variation in helix length (+ / -8 amino acids, see Figure 1A bottom inset). This second approach improved the quality of the model by exploring substantially more combinations of loops connecting each pair of helices. Fourteen of the best designs of the second generation were experimentally characterized, along with 27 Rosetta TM sequence redesigned of G1_neo2_40_1F (see Table 4), and all designs except one were found to bind the IL-2 receptor at low nanomolar concentrations. Three of the highest-affinity and stability designs (one sequence redesigned and two new mimetics) were submitted to site-saturation mutagenesis for binding to mIL-2Rβγ c ( Figures 6A - 6E and 8A-8E), followed by selection and combination of affinity-enhancing substitutions for both human and murine IL-2Rβγ c . The mature designs (see Table 5) showed enhanced binding while retaining high stability (see Silva et al., Nature 565, pg. 186, January 10, 2019). The top design, Neoleukin-2 / 15 (also referred to herein as Neo-2 / 15), is a 100-residue protein with a new topology and sequence quite different from human or murine IL-2 (in a structure-topology-agnostic pairwise alignment, 29% sequence identity with hIL-2 over 89 residues, and 16% sequence identity with mIL-2 over 76 aligned residues).

[0500] Functional characterization of Neoleukin-2 / 15: Neoleukin-2 / 15 binds to human and murine IL-2Rβγ with high affinity c , but does not interact with IL-2Rα (see Silva et al., Nature 565, pg. 186, January 10, 2019). Neoleukin-2 / 15 binds to human and murine IL-2 receptors (IL-2Rβ and IL-2Rβγ c) with an affinity significantly higher than that of the corresponding native IL-2 cytokine. In contrast to native IL-2, Neoleukin-2 / 15 triggers IL-2Rα-independent signaling in both human and mouse IL-2-responsive cells, and in mouse primary T cells (see Silva et al., Nature 565, pg.186, January 10, 2019). Neoleukin-2 / 15 activates IL-2Rα- cells more potently than native human or mouse IL-2, consistent with its higher binding affinity. In primary cells, Neoleukin-2 / 15 is more active against IL-2Rα- cells and less active against IL-2Rα+ than Super-2, presumably due to its complete lack of IL-2Rα binding. Neoleukin-2 / 15 is highly stable (see Figures 12A - 12C ) and did not lose the ability to bind hIL-2Rβγ after incubation at 80°C for 2 hours c The binding affinity of hIL-2 and Super-2 was completely inactivated after 10 minutes (half-inactivation time was 4.2 minutes and 2.6 minutes, respectively). Figure 2 ). Similarly, in ex vivo primary cell cultures, Neoleukin-2 / 15 effectively drives T cell survival after boiling at 95°C for 60 minutes, while these conditions inactivate both IL-2 and Super-2 (see Silva et al., Nature 565, pg.186, January 10, 2019). Many other designed mimetics have been subjected to thermal denaturation studies, which also demonstrate their thermal stability (see Figures 11A - 11D In addition to eliminating the requirement for cold chain storage, this unprecedented stability of cytokine-like molecules suggests increased resistance to mutations (see Figures 8A - 8E and 13A-13B), genetically fused and chemically modified, far exceeding the robustness of natural IL-2 (see Figures 3A - 3C ).

[0501] Therapeutic Applications of Neoleukin-2 / 15: The clinical use of IL-2 is primarily limited by toxicity. Although the interactions responsible for IL-2 toxicity in humans are not fully understood, toxicity is T cell independent in murine models and ameliorated in animals deficient in the IL-2Rα chain (CD25+). Thus, many efforts have been directed toward reengineering IL-2 to attenuate interaction with IL-2Rα, but mutations in the CD25 binding site can be highly destabilizing. The intrinsic low stability of IL-2 and its tight evolutionary dependence on CD25 have been obstacles to the translation of reengineered IL-2 compounds. Other efforts have focused on IL-15 because it binds to IL-2Rβγ by dimerizing IL-2Rβγ. cElicits signaling similar to IL-2 but has no affinity for CD25. However, IL-15 relies on trans-presentation by the IL-15α (CD215) receptor, which is mainly displayed on antigen-presenting cells and natural killer cells. The low stability of native IL-15 and its dependence on trans-presentation also pose substantial obstacles to re-engineering efforts.

[0502] Dose escalation studies in naive mice showed that mIL-2 preferentially expanded regulatory T cells, consistent with preferential binding to CD25+ cells, while Neoleukin-2 / 15 mainly drove CD8 + T cell expansion and did not induce or only minimally induced regulatory T cell expansion at the highest dose tested. Similarly, in a murine model of airway inflammation that typically induces a small percentage of tissue-resident CD8+ T cells, Neoleukin-2 / 15 generated Thy1.2 - CD44 + CD8 + T cell increases without increasing CD4 in lymphoid organs + Foxp3 + antigen-specific Tregs (data not shown; see Silva et al., Nature 565, pg. 186, January 10, 2019).

[0503] The therapeutic efficacy of Neoleukin-2 / 15 was tested in murine models of poorly immunogenic B16F10 melanoma and highly immunogenic CT26 colon carcinoma. Single-agent treatment with Neoleukin-2 / 15 resulted in a dose-dependent delay in tumor growth in both cancer models. In CT26 colon carcinoma, single-agent treatment showed improved efficacy compared to that observed with recombinant mIL-2 (see Silva et al., Nature 565, pg. 186, January 10, 2019). In B16F10 melanoma, co-treatment with the anti-melanoma antibody TA99 (anti-TRP1) resulted in a significant delay in tumor growth, while treatment with TA99 alone had little effect (see Silva et al., Nature 565, pg. 186, January 10, 2019). In a long-term survival experiment (8 weeks), the combination of Neoleukin-2 / 15 and TA99 showed substantially reduced toxicity and overall superior therapeutic efficacy compared to mIL-2 (see Silva et al., Nature 565, pg. 186, January 10, 2019). Mice treated with the combination of mIL-2 and TA99 steadily lost weight, and their overall health declined to the point where euthanasia was required, while little decline was observed for the combination of Neoleukin-2 / 15 and TA99 (see Silva et al., Nature 565, pg. 186, January 10, 2019). Consistent with the therapeutic benefit, treatment with Neoleukin-2 / 15 led to a significant increase in the intratumoral CD8:T reg ratio (see Silva et al., Nature 565, pg. 186, January 10, 2019), which has previously been associated with an effective anti-tumor immune response 58 . The increase in the CD8:T reg ratio by Neoleukin-2 / 15 was dose- and antigen-dependent (see Silva et al., Nature 565, pg. 186, January 10, 2019); optimal therapeutic efficacy was obtained at higher doses and in combination with other immunotherapies (see Silva et al., Nature 565, pg. 186, January 10, 2019). Collectively, these data show that Neoleukin-2 / 15 exhibits predicted homeostatic benefits derived from its IL-2-like immunostimulatory activity but without the adverse effects associated with preferential binding to CD25 + . The therapeutic efficacy of Neoleukin-2 / 15 was tested in a CAR-T model. Mice were inoculated with 0.5x10 6NSG mice with Raji tumor cells were left untreated, treated with 0.8x10 6 anti-CD19 CAR-T cells (infused 7 days after tumor cell inoculation), or treated with anti-CD19 CAR-T cells plus 20 μg / day human IL-2 or Neoleukin-2 / 15 from days 8 - 14 after tumor inoculation. As expected, Neoleukin-2 / 15 showed a significant enhancement of the anti-tumor effect of CAR-T cell therapy, slowing tumor growth and prolonging mouse survival (see Silva et al., Nature 565, pg. 186, January 10, 2019).

[0504] De novo design of protein mimics has the potential to transform the field of protein-based therapies, enabling the development of bio-excellent molecules with enhanced therapeutic properties and reduced side effects. Different from engineered variants of recombinant IL-2 and hIL-2, Neoleukin-2 / 15 can be expressed solubly in Escherichia coli (see Figures 17A - 17B ), retains activity at higher temperatures, does not interact with IL-2Rα and is robust to substantial sequence changes that allow the engineering of new functions ( Figures 7A - 7E ).

[0505] Robust modularity of Neoleukin-2 / 15. Disulfide elbow pins and re-engineering into an IL-4 mimic: Neoleukin-2 / 15 is highly modular, allowing easy modulation of its properties, such as enhancing its stability or modifying its binding preferences. Through computational design, this modularity and robustness were exploited by introducing a stability-enhancing single disulfide elbow pin that retains the function of Neoleukin-2 / 15. In one example, by searching for pairs of positions with favorable geometric arrangements, followed by flexible backbone minimization to introduce the disulfide bridge. The final design introduced a single disulfide between residues 38 and 75, which stabilizes helices H3 and H2. This strategy increased the stability of Neoleukin-2 / 15 (Tm > 95 °C), while leaving most of its sequence and function unaffected (see Silva et al., Nature 565, pg. 186, January 10, 2019). Using the modularity properties of Neoleukin-2 / 15 to modify its binding preferences. All cytokines in the interleukin-2 family bind to γ cInteract and share a common architecture. Thus, it was hypothesized that Neoleukin-2 / 15 could be converted into another cytokine mimic of the IL-2 family by simply changing the amino acids in only half of the binding sites (helices H1 and H3) that interact with IL-2Rβ. As proof of concept, human interleukin-4 (hIL-4) was chosen as the target because it shares extensive structural homology with IL-2 and has potential applications in regenerative medicine. Neo-2 / 15 was modified as follows to bind to the human IL-4 receptor (comprising IL-4Rα and γ c ) and not to the human IL-2 receptor (comprising IL-2Rβ and γ c ), namely, by aligning the Neo-2 / 15 model with the structure of human IL-4 bound to its IL-4 receptor and mutating 14 residues in Neo-2 / 15 to match the amino acids in IL-4 that mediate the interaction between IL-4 and IL4R at those structural positions ( Figures 3A - 3C ). Binding was further optimized by directed evolution, namely, using random mutagenesis and screening for high-binding affinity variants, which introduced two additional amino acid substitutions and modified one of the 14 original residues transplanted from the IL-4 protein, thereby creating the new protein Neoleukin-4, which has a total of 16 mutations relative to Neoleukin-2 / 15. The resulting optimized design, Neoleukin-4 (see Table 6), was recombinantly expressed, purified, and tested for binding in Escherichia coli. Neoleukin-4 binds to the IL-4Rα receptor with high affinity and co-binds IL-4Rαγ c (see Figures 3A - 3C ), and does not bind to the IL-2 receptor with any affinity (data not shown). Neoleukin-4 retains the excellent thermal stability properties of Neoleukin-2 / 15 (see Figure 14B , 14C), 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).

[0506] Methods

[0507] Computational design of de novo cytokine mimics: The design of de novo cytokine mimics began with defining the structure of hIL-2 in the quaternary complex with the IL-2Rβγ c receptor as a template for design. After inspection, the residues constituting the binding site were defined as hotspots using Rosetta TM metadata (PDBInfoLabels). The structure was input into a new mimic design protocol, which was programmed in PyRosetta TM and was able to automatically detect the core-secondary structure elements constituting the target-template and generate a complete RosettaScripts TMThe obtained compatibility information is used to simulate the backbone from scratch for design purposes. Briefly, the mimic construction algorithm works as follows. For the first-generation design, each core-element is idealized by reconstructing loops using a clustered database of highly ideal fragments (fragment size 4 amino acids). After idealization, the mimic construction scheme aims to reconnect the idealized elements pairwise in all possible combinations. To do so, it uses combinatorial fragment assembly of sequence-agnostic fragments from the database, followed by Cartesian-constrained backbone minimization to obtain potential solutions (i.e., where the N and C termini of the constructed fragments are close enough to connect two secondary structures). After minimization, the solutions are verified to contain highly ideal fragments (i.e., each overlapping fragment that makes up the two connecting elements is also included in the database) and that the backbone has no clashes with the target (background) receptor. Then, the passed backbone solutions are profiled using the same fragment database to determine the most likely amino acid at each position (this information is encoded in the metadata of the design). Next, the pairwise-connected secondary structure solutions are recombined using graph-theoretic connected components to generate a fully connected backbone. Since the number of solutions grows exponentially with each pair of elements, at each fragment combination step, we rank the designs, favoring those with shorter interconnects between the pairwise core elements, and only retain the top solutions to proceed to the next step. Then, the fully connected solutions are profiled by layer (interface, core, non-core-surface, surface) to constrain the identity of the possible amino acids to be layer-compatible. Finally, all information on hotspots, compatible building-fragment amino acids, and layers is combined (hotspots take precedence over amino acid probabilities, and amino acid probabilities take precedence over layers). These fully profiled backbones are then passed to RosettaScripts TM For flexible backbone design and filtering. For the second-generation design, two approaches are followed. In the first approach, sequence redesign of the best first-generation optimized design (G1_neo2_40_1F) is performed. In the second approach, G1_neo2_40_1F is used as a target template to engineer new mimics. The mimic design scheme in this second generation is similar to that described for the first generation, but there are two key differences. First, instead of constructing core-fragments from fragments, we instead discover repeats that result in repeating secondary structures that most closely recapitulate each target helix and the parametric equations for ψ angles (ω fixed at 180°), with each X-amino acid allowed The "pitch" at the θ and ψ angles allows the helix to potentially have curvature (ultimate parameters: H1, H2, H3, H4). The use of these parametric equations allows for arbitrary changes in the size of each core - element in the target structure (either increasing or decreasing the size), which couples (max / min 8.a.a.) the loop construction process and does not allow for the reduction of the core - element size to remove hotspots from the binding site. The second difference in the second - generation design is that instead of reconnecting the secondary - structure core - elements, we use a 7 - amino - acid fragment size and do not allow the combinatorial assembly of more than one fragment (i.e., a single fragment must be able to close a pair of secondary structures). The rest of the design algorithm is essentially similar to that followed in the first generation. The Rosetta TM energy functions used in the first and second generations are "talaris2013" and "talaris2014", respectively.

[0508] The database of highly ideal fragments used for de novo backbone design was constructed with the new Rosetta TM application "kcenters_clustering_of_fragments" using a broad database of non - redundant publicly available protein structures from the RCSB Protein Data Bank. The 4 - mer database for the first - generation design contains 16767 PDBs, while the 7 - mer database for the second - generation design contains 7062 PDBs.

[0509] Yeast display: Yeast is transformed with the gene encoding the protein to be displayed together with the linearized pETcon3 vector. The vector is linearized by 100 - fold over - digestion with NdeI and XhoI (New England Biolabs) and then purified by gel extraction (Qiagen). The gene includes 50 - base overlaps with the vector at both the 5' and 3' ends such that homologous recombination places the gene in - frame between the AGA2 gene and the myc - tag on the vector. Yeast is grown in C - Trp - Ura medium and then induced in SGCAA medium as previously described. 12 - 18 hours after induction, the cells are washed in chilled display buffer (50 mM NaPO 4 pH 8, 20 mM NaCl, 0.5% BSA) and incubated with different concentrations of biotinylated receptors (human or murine IL - 2Rα, IL - 2Rβ, IL - 2Rγ, or human IL - 4Rα) with shaking at 4°C. After approximately 30 minutes, the cells are washed again in chilled buffer and then incubated with FITC - conjugated anti - c - Myc antibody (1 μL per 3x10 6The cells were incubated with streptavidin-phycoerythrin (1 μL per 100 μL volume of yeast) on ice for 5 minutes. The yeast was then washed and counted by flow cytometry (Accuri C6) or sorted by FACS (Sony SH800). For experiments in which the initial receptor incubation was performed with a combination of biotinylated IL-2Rγ and non-biotinylated IL-4Rα, the non-biotinylated receptor was provided in molar excess.

[0510] Mutagenesis and affinity maturation: For error-prone PCR-based mutagenesis, the design to be mutated was cloned into the pETcon3 vector using the MutaGene TM II mutagenesis kit (Invitrogen) and amplified according to the manufacturer's instructions to generate a mutation frequency of approximately 1% per nucleotide. 1 μg of this mutated gene was electroporated into EBY100 yeast together with 1 μg of linearized pETcon3 vector, and the transformation efficiency was on the order of 10 8 . Yeast induction and sorting were performed continuously for multiple times, and the receptor concentration was gradually decreased until the population converged. Yeast regrew in C-Trp-Ura medium between each sorting.

[0511] A site-saturation mutagenesis (SSM) library was constructed from synthetic DNA from Genscript. For each amino acid on each design template, forward and reverse primers were designed such that PCR amplification would generate 5' PCR products and 3' PCR products with degenerate NNK codons, respectively. Amplification of the "left" and "right" products by COF and COR primers generated a series of template products, each consisting of a degenerate NNK codon at a different residue position. For each design, these products were combined to generate the SSM library. The SSM library was transformed by electroporation into conditioned Saccharomyces cerevisiae strain EBY100 cells together with the linearized pETcon3 vector using the protocol previously described by Benatuil et al.

[0512] A combinatorial library was constructed from synthetic DNA containing divergent nucleotides from Genscript and similarly transformed into the linearized pETcon3 vector.

[0513] Protein expression: Genes encoding the designed protein sequences were synthesized and cloned into the pET-28b(+) Escherichia coli plasmid expression vector (GenScript, N-terminal 6xHis tag and thrombin cleavage site). The plasmid was then transformed into chemically competent Escherichia coli Lemo21 cells (NEB). Protein expression was carried out using Terrific Broth TM and M salts, and the cultures were grown at 37 °C until OD 600Reached approximately 0.8, then induced expression with 1 mM isopropyl β-D-thiogalactopyranoside (IPTG), and lowered the temperature to 18 °C. After expressing for approximately 18 hours, harvested the cells and lysed them with a Microfluidics M110P microfluidizer at 18,000 psi, then clarified the soluble fraction by centrifugation at 24,000 g for 20 minutes. Purified the soluble fraction by immobilized metal affinity chromatography (Qiagen), followed by FPLC size exclusion chromatography (Superdex TM 75 10 / 300GL, GE Healthcare). Characterized the purified Neoleukin-2 / 15 by mass spectrometry (MS) (to verify the molecular weight of the species in solution (Thermo Scientific)), size exclusion - multi-angle laser scattering (SEC-MALLS) (to verify the monomeric state and molecular weight (Agilent, Wyatt)), SDS-PAGE, and endotoxin level (Charles River).

[0514] As previously described 17,49 , secreted and purified the human and mouse IL-2 complex components using a baculovirus expression system, including hIL-2 (a.a. 1-133), hIL-2Rα (a.a. 1-217), hIL-2Rβ (a.a. 1-214), hIL-2Rγ (a.a. 1-232), mIL-2 (a.a. 1-149), mIL-2Rα extracellular domain (a.a. 1-213), mIL-2Rβ extracellular domain (a.a. 1-215), and mγ c extracellular domain (a.a. 1-233). Purified all proteins to >98% homogeneity using a Superdex TM 200 size column (GE Healthcare) equilibrated in HBS. Verified the purity by SDS-PAGE analysis. For the expression of biotinylated human IL-2 and mouse IL-2 receptor subunits, expressed the proteins containing the C-terminal biotin acceptor peptide (BAP)-LNDIFEAQKIEWHE (SEQ ID NO:303) and purified them via Ni-NTA affinity chromatography as described, then biotinylated them with soluble BirA ligase in 0.5 mM Bicine pH 8.3, 100 mM ATP, 100 mM magnesium acetate, and 500 mM biotin (Sigma). Removed the excess biotin by size exclusion chromatography on a Superdex 200 column equilibrated in HBS.

[0515] Circular dichroism (CD): Far-UV CD measurements were performed using an AVIV Model 420 spectrometer in PBS buffer (pH 7.4) in a 1-mm pathlength cuvette at a protein concentration of ~0.20 mg / ml (unless otherwise stated in the text). Thermal unfolding was from 25 to 95 °C and the absorbance signal at 222 nm was monitored (step size of 2 °C / min with a 30-s equilibration step). Wavelength scans (195 - 260 nm) were collected at 25 °C and 95 °C and again at 25 °C after rapid refolding (~5 minutes).

[0516] STAT5 phosphorylation studies: In vitro studies: Approximately 2 x 10 5 YT-1, IL-2Rα + YT-1, or CTLL-2 cells were dispensed 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 engineered IL-2 mimetics. Cells were stimulated at 37 °C for 15 minutes and immediately fixed by adding formaldehyde to 1.5% and incubating at room temperature for 10 minutes. Permeabilization of the cells was achieved by resuspending in ice-cold 100% methanol for 30 minutes at 4 °C. The 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 Alexa 647-conjugated anti-STAT5 pY694 (BD Biosciences) diluted in FACS buffer for 2 hours at room temperature. The cells were then washed twice in FACS buffer and the mean fluorescence intensity (MFI) was determined on a CytoFLEX TM flow cytometer (Beckman-Coulter). After subtracting the mean fluorescence intensity (MFI) of unstimulated cells and normalizing against the maximum signal intensity, the dose-response curve was fitted to a logarithmic model and the half-maximal effective concentration (EC 50 value) was calculated using GraphPad Prism data analysis software. Experiments were performed in triplicate and carried out three times with similar results. In vivo studies: Spleen and lymph nodes were harvested from wild-type C57BL / 6J or B6; 129S4-Il2ra tm1Dw (CD25KO) mice and made into single-cell suspensions in sorting buffer (phosphate-buffered saline pH 7.2 containing 2% fetal bovine serum). CD4+ T cells were 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 on ice for 30 minutes. After washing with sorting buffer, anti-biotin MicroBeads were added at 20 μL per 10 7 total cells TM(Miltenyi Biotec) was added to the cell suspension and incubated on ice for 20 minutes. The cells were washed, resuspended, and then negative selection was performed using EasySep TM Magnets (STEMCELL Technologies). Approximately 1 x 10 5 enriched cells were added to each well of a 96-well plate together with 10-fold serial dilutions of mIL-2, Super-2, or Neoleukin-2 / 15 in RPMI complete medium with 5% FCS. The cells were stimulated at 37 °C in 5% CO 2 for 20 minutes, fixed with 4% PFA and incubated at 4 °C for 30 minutes. After fixation, the cells were harvested, washed twice with sorting buffer, and fixed again on ice for 30 minutes in 500 μL of dH 2 O containing 90% ice-cold methanol for permeabilization. The cells were washed twice with permeabilization / wash buffer (BD Biosciences) and stained in the dark at room temperature for 45 minutes with permeabilization / wash buffer containing anti-CD4-PerCP (1:300), anti-CD44-AlexaFluor 700 (1:200), anti-CD25-PE-Cy7 (1:200), and 5 μL of anti-pSTAT5-PE pY694 per sample. The cells were washed with permeabilization / wash buffer and resuspended in sorting buffer for analysis on a BD LSRII flow cytometer (BD Biosciences).

[0517] Data Table

[0518] Table 2: Amino acid sequences of the top 12 first-round designs. Ten designs (G1_neo2_35-44) were characterized by yeast display experiments, and all but two (G1_neo2_35 and G1_neo2_44) were found to bind fluorescently labeled chimeric ILRβγ at low nanomolar concentrations via flow cytometry screening of the first-round protein conjugates designed. c The indicated designs were expressed on yeast and incubated with 2 nM hIL-2Rβγ c or 0 nM IL-2Rβγ c (data not shown).

[0519]

[0520] Table 3: Amino acid sequences of the experimentally optimized first-round designs.

[0521]

[0522]

[0523] Table 4: Amino acid sequences of the second-round design. G2_neo2_40_1F_seq02 to G2_neo2_40_1F_seq28 correspond to the 27 Rosetta TM resequences of G1_neo2_40_1F; G2_neo2_40_1F_seq29 to G2_neo2_40_1F_seq42 represent 14 de novo mimetic designs.

[0524]

[0525]

[0526]

[0527] Table 5: Amino acid sequences of the second-round design optimized experimentally.

[0528]

[0529]

[0530] Neoleukin-2 / 15-H8Y-K33E: H1->H3->H2’->H4

[0531] PKKKIQLYAEHALYDALMILNI VKTNSPPAEE ELEDYAFNFELILEEIARLFES GD QKDE AEKAKRMKEWMKRIKT TAS EDEQEEMANAIITILQSWIFS (SEQ ID NO:94)

[0532] Binding of Neoleukin-2 / 15-H8Y-K33E to the IL2 receptor was measured by biolayer interferometry and was found to have a higher binding affinity for IL2-Rβ than Neoleukin-2, both when tested against isolated IL2Rβ and against the IL2Rβ-γ complex. This increased affinity is mostly attributable to an improved dissociation rate from IL2-Rβ.

[0533] Table 6: Amino acid sequences of interleukin-4 mimetic designs based on Neoleukin-2 / 15 re-engineering.

[0534]

[0535] Example 2: Split cytokine mimetics for targeted immunotherapy

[0536] De novo proteins are designed to follow the rules of ideal protein structures, endowing them with unusual biochemical properties such as extreme thermal stability and mutational robustness. Thus, de novo designed proteins are ideal candidates for developing conditional active protein therapeutics. Here, we report the development of split cytokine mimics for highly targeted immunotherapy, based on the recently developed de novo designed IL-2 mimic protein, Neoleukin-2 / 15. This system is capable of delivering conditional active therapeutic proteins that reconstitute their activity through colocalization on the surface of target cells. We identified potential split sites and demonstrated successful reconstitution of Neoleukin-2 / 15 activity through binding to the IL-2 receptor, cell signaling, and colocalization-dependent activation on the surface of target tumor cells. We also demonstrated the application of this to another de novo designed cytokine mimic, Neoleukin-4.

[0537] To identify potential split sites of Neoleukin-2 / 15 (Neo2), we evaluated the protein structure to find split positions that would minimize the deleterious effects on the function of the protein. As a result, we defined three illustrative split positions: (i) between the helical elements H1 (Neo2A1) and H3’-H2-H4 (Neo2B1), (ii) between the helical elements H1-H3’ (Neo2A2) and H2-H4 (Neo2B2), (iii) between the helical elements H1-H3’-H2 (Neo2A3) and H4 (Neo2B3) ( Figures 15A - 15C ). The split Neo2 fragments were characterized by analyzing their binding ability to the human IL-2 receptor ( Figures 15A - 15C ) and signaling on IL-2-responsive CTTL-2 cells ( Figures 16A - 16C ). Each split fragment showed negligible binding and signaling ability to IL-2Rβγ in most cases, but robust activity after equimolar combination of complementary split fragments.

[0538] The development of split Neo2 enables colocalization-dependent reconstitution of the protein and thus conditional activation of split Neo2. To enable colocalization of the Neo2A and Neo2B fragments, we first implemented genetic fusions with targeting domains ( Figures 17A - 17B)。Neo2A1, Neo2A2, and Neo2A3 were fused to the C-terminus of a Her2-targeted designed ankyrin repeat protein (DARPin) via a 15-residue flexible linker (GSGGSGGGSGGSGSG; SEQ ID NO: 100). Neo2B1, Neo2B2, and Neo2B3 were fused to the N-terminus of an EGFR-targeted DARPin via a 15-residue flexible linker (GSGGSGGGSGGSGSG; SEQ ID NO: 100). Then, we evaluated their ability to reconstitute binding to IL-2Rβγ upon split fragment recombination, demonstrating that the fusions did not interfere with their split Neo-2 reconstitution ability ( Figures 17A - 17B )。

[0539] To evaluate the co-localization-dependent activation and targeting selectivity of the split Neo2 system, we performed in vitro assays on the surface of an engineered K562 cell line with split Neo-2 / 15 targeting EGFR and Her2 ( Figure 18A )。We used four engineered cell lines: (i) no surface receptors, (ii) Her2+ / eGFP+, (iii) EGFR+ / iRFP+, (iv) Her2+ / eGFP+EGFR+ / iRFP+ ( Figure 18B )。The four cell lines were mixed at equal ratios and incubated with the targeted split Neo2 fragments for 15 minutes. Subsequently, the cells were washed and incubated with a mixture of biotinylated IL-2 receptor and fluoresceinated streptavidin-phycoerythrin conjugate (SAPE). Finally, the cells were analyzed by fluorescence-activated cell sorting (FACS). Successful reconstitution of Neo-2 activity led to fluorescent labeling of PE on the surface of the target cells ( Figure 18C )。When targeting intact Neo2 with a single anti-Her2 targeting domain (aHer2-Neo2), we observed strong IL-2 receptor binding on the surface of Her2+ and Her2+ / EGFR+. Similar binding was observed when the split fragments Neo2A1 and Neo2B1 were both targeted to Her2 (aHer2-Neo2A1 + aHer2-Neo2B1), demonstrating that these two fragments can be delivered to the same cell surface marker when needed. Additionally, when each split fragment targeted either Her2 or EGFR (aHer2-Neo2A1 + Neo2B1-aEGFR, aHer2-Neo2A2 + Neo2B2-aEGFR, aHer2-Neo2A3 + Neo2B3-aEGFR), we observed highly selective reconstitution on the surface of double-positive Her2+ / EGFR+. Other studies have demonstrated that for each combination of split Neo2 fragments, the simultaneous presence of the two fragments is necessary to achieve IL-2 receptor recruitment on the surface of K562 cells ( Figures 19A - 19C) These results demonstrate the high selectivity of split cytokine mimics to specifically reconstitute the desired activity on the surface of target cells.

[0540] The application of split cytokine mimic technology is not limited solely to targeting tumor-associated antigens. For a given application, targeting a specific subset of immune cells to be selectively stimulated can be beneficial for guiding the immune response to treat diseases. For example, such applications can be useful for specifically priming the expansion of CD8+ cytotoxic T cells, natural killer cells, or engineered CAR-T cells to enhance their anti-tumor responses, and targeting regulatory T cells to dampen strong immune responses ( Figure 20A ). Moreover, in some scenarios, a hybrid targeting approach can be deployed, where one split fragment targets tumor cells or the tumor microenvironment while delivering another fragment to immune cells ( Figure 20B ).

[0541] Finally, to demonstrate the transferability of the methods described herein to other de novo designed interleukins, we created a new conditionally active IL-4 mimic ( Figure 21 ). To achieve this, we successfully introduced specific mutations in the split Neo-2 that modify the binding interface to the hIL-2β receptor to bind to the hIL-4α receptor.

[0542] Exemplary split Neoleukin-2 / 15 variant sequences used in Example 2

[0543] H1(Neo2A1)

[0544] PKKKIQLHAEHALYDALMILNIVKTNS(SEQ ID NO:256)

[0545] H3-H2’-H4(Neo2B1)

[0546] TNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS(SEQ ID NO:257)

[0547] H1-H3-H2’(Neo2A3)

[0548] PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTAS(SEQ ID NO:258)

[0549] H4(Neo2B3)

[0550] TTASEDEQEEMANAIITILQSWIFS (SEQ ID NO:259)

[0551] H1-H3 (Neo2A2)

[0552] PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGD (SEQ ID NO:260)

[0553] H2’-H4 (Neo2B2)

[0554] DQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO:261) Exemplary split Neoleukin-4 sequence used in Example 2

[0555] H1 (Neo4A1)

[0556] PKKKIQIMAEEALKDALSILNIVKTNS (SEQ ID NO:262)

[0557] H3-H2’-H4 (also referred to as Neo4B1)

[0558] TNSPPAEEQLERFAKRFERNLWGIARLFESGDQKDEAEKAKRMIEWMKRIKTTASEDEQEEMANAIITILQSWFFS (SEQ ID NO:263)

[0559] Exemplary constructs used in Example 2

[0560] X2-Z1-X3-Z2-X4-E01_EGFR_DARPin

[0561]

[0562] G3_Her2_DARPin_X1

[0563]

[0564] G3_Her2_DARPin_X2-Z1-X3-Z3-X4

[0565]

[0566] X2-Z1-X3-Z2-X4-E01_EGFR_DARPin

[0567]

[0568] ID NO: 307)

[0569] X2-Z2-X4-E01_EGFR_DARPin

[0570]

[0571]

[0572] X4-E01_EGFR_DARPin

[0573]

[0574] G3_Her2_DARPin_X1-Z1-X3

[0575]

[0576] G3_Her2_DARPin_X1-Z1-X3-Z2-X2

[0577]

[0578] The present invention relates to the following embodiments.

[0579] 1. 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 do not exist as a fusion protein, and wherein the first polypeptide component and the second polypeptide component together comprise domains X1, X2, X3, and X4, wherein:

[0580] (a) X1 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 the peptide ;

[0581] (b) X2 is any helical peptide domain;

[0582] (c) X3 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 the peptide ; and

[0583] (d) X4 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 the peptide a peptide having 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

[0584] wherein:

[0585] (i) the amino acid residues in parentheses may be present or absent;

[0586] (ii) the first polypeptide component comprises at least one but not every one of X1, X2, X3, and X4; and

[0587] (iii) the second polypeptide component comprises every one of X1, X2, X3, and X4 that is not present in the first polypeptide component;

[0588] wherein the first polypeptide component and the second polypeptide component alone are not active receptor agonists, and wherein the first polypeptide component and the second polypeptide component interact to form an active agonist of an IL-2 receptor βγ c heterodimer (IL-2Rβγ c ), an IL-4 receptor αγ c heterodimer (IL-4Rαγ c ), an IL-13α, or an IL-4Rα / IL13Rα heterodimer.

[0589] 2. The conditional active receptor agonist of claim 1, wherein:

[0590] (a) X1 is a peptide having an amino acid sequence that is at least 70% identical to the full length of the peptide or ;

[0591] (b) X2 is any helical peptide domain;

[0592] (c) X3 is a peptide having an amino acid sequence that is at least 70% identical to the full length of the peptide or ; and

[0593] (d) X4 is a peptide having an amino acid sequence that is at least 70% identical to the full length of the peptide or ;

[0594] 3. The conditional active receptor agonist of claim 1, wherein:

[0595] (a) X1 is a peptide having an amino acid sequence that is identical to the peptide or a peptide having an amino acid sequence that is at least 85% identical to the full length of

[0596] (b) X2 is any helical peptide domain;

[0597] (c) X3 is a peptide comprising an amino acid sequence that is at least 85% identical to the full length of the peptide or ; and

[0598] (d) X4 is a peptide comprising an amino acid sequence that is at least 85% identical to the full length of the peptide or .

[0599] 4. The conditional active receptor agonist of claim 1, wherein:

[0600] (a) X1 is a peptide comprising an amino acid sequence that is identical to the full length of the peptide or ;

[0601] (b) X2 is any helical peptide domain;

[0602] (c) X3 is a peptide comprising an amino acid sequence that is identical to the full length of the peptide or ; and

[0603] (d) X4 is a peptide comprising an amino acid sequence that is identical to the full length of the peptide or .

[0604] 5. The conditional active receptor agonist of claim 1, wherein:

[0605] (a) X1 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 the peptide ;

[0606] (b) X3 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 the peptide ; and

[0607] (c) X4 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 the peptide a peptide having 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

[0608] 6. The conditional active receptor agonist of claim 1, wherein:

[0609] (a) X1 is a peptide comprising an amino acid sequence that is at least 65% identical to the full length of the peptide or ;

[0610] (b) X3 is a peptide comprising an amino acid sequence that is at least 65% identical to the full length of the peptide or ; and

[0611] (c) X4 is a peptide comprising an amino acid sequence that is at least 65% identical to the full length of the peptide or .

[0612] 7. The conditional active receptor agonist of claim 1, wherein:

[0613] (a) X1 is a peptide comprising an amino acid sequence that is at least 75% identical to the full length of the peptide or ;

[0614] (b) X3 is a peptide comprising an amino acid sequence that is at least 75% identical to the full length of the peptide or ; and

[0615] (c) X4 is a peptide comprising an amino acid sequence that is at least 75% identical to the full length of the peptide or .

[0616] 8. The conditional active receptor agonist of claim 1, wherein:

[0617] (a) X1 is a peptide comprising an amino acid sequence that is at least 80% identical to the full length of the peptide or ;

[0618] (b) X3 is a peptide comprising an amino acid sequence that is at least 80% identical to the full length of the peptide or ; and

[0619] (c) X4 is a peptide comprising an amino acid sequence that is at least 80% identical to the full length of the peptide or .

[0620] 9. The conditional active receptor agonist of claim 1, wherein:

[0621] (a) X1 is a peptide comprising an amino acid sequence that is at least 90% identical to the full length of the peptide or ;

[0622] (b) X3 is a peptide comprising an amino acid sequence that is at least 90% identical to the full length of the peptide or ; and

[0623] (c) X4 is a peptide comprising an amino acid sequence that is at least 90% identical to the full length of the peptide or .

[0624] 10. The conditional active receptor agonist of any one of claims 1-9, wherein X2 has a length of at least 8 amino acids.

[0625] 11. The conditional active receptor agonist of any one of claims 1-10, wherein:

[0626] X2 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 the peptide , where the amino acid residues in parentheses may be present or absent.

[0627] 12. The conditional active receptor agonist of any one of claims 1-11, wherein:

[0628] (i) X1 comprises 1, 2, 3, 4, or all 5 of the following: L at residue 7, H at residue 8, H at residue 11, Y at residue 14, or M at residue 18, where the numbering is relative to SEQ ID NO:4 based on the presence of optional residues; and / or

[0629] (ii) X3 comprises 1, 2, 3, 4, 5, 6, 7, or all 8 of the following: D at residue 3, Y at residue 4, F at residue 6, N at residue 7, L at residue 10, I at residue 11, E at residue 13, or E at residue 14, where the numbering is relative to SEQ ID NO:5 based on the presence of optional residues.

[0630] 13. The conditional active receptor agonist of any one of claims 1-12, wherein:

[0631] (iii) X4 includes I at residue 19, wherein the numbering is relative to SEQ ID NO:6 in the presence of optional residues.

[0632] 14. A conditional active receptor agonist according to any one of claims 1-11, wherein:

[0633] X1 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 the peptide ;

[0634] X3 is a peptide comprising an amino acid sequence that is at least 37%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical along its length to the peptide ; and

[0635] X4 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 the peptide ;

[0636] wherein

[0637] (i) X1 includes I at residue 7, T or M at residue 8, E at residue 11, K at residue 14, and S at residue 18; and

[0638] (ii) X3 includes R at residue 3, F at residue 4, K at residue 6, R at residue 7, R at residue 10, N at residue 11, W at residue 13, and G at residue 14.

[0639] 15. A conditional active receptor agonist according to any one of claims 1-11, wherein:

[0640] X1 is a peptide comprising an amino acid sequence that is at least 65% identical along its length to the peptide ;

[0641] X3 is a peptide comprising an amino acid sequence that is at least 65% identical along its length to the peptide ; and

[0642] X4 is a peptide comprising an amino acid sequence that is at least 65% identical along its length to the peptide ;

[0643] wherein

[0644] (i) X1 comprises I at residue 7, T or M at residue 8, E at residue 11, K at residue 14, and S at residue 18; and

[0645] (ii) X3 comprises R at residue 3, F at residue 4, K at residue 6, R at residue 7, R at residue 10, N at residue 11, W at residue 13, and G at residue 14.

[0646] 16. A conditional active receptor agonist according to any one of claims 1 - 11, wherein:

[0647] X1 is a peptide comprising an amino acid sequence that is at least 75% identical along its length to the peptide at least 75% identical amino acid sequence;

[0648] X3 is a peptide comprising an amino acid sequence that is at least 75% identical along its length to the peptide at least 75% identical amino acid sequence; and

[0649] X4 is a peptide comprising an amino acid sequence that is at least 75% identical along its length to the peptide at least 75% identical amino acid sequence,

[0650] wherein

[0651] (i) X1 comprises I at residue 7, T or M at residue 8, E at residue 11, K at residue 14, and S at residue 18; and

[0652] (ii) X3 comprises R at residue 3, F at residue 4, K at residue 6, R at residue 7, R at residue 10, N at residue 11, W at residue 13, and G at residue 14.

[0653] 17. A conditional active receptor agonist according to any one of claims 1 - 11, wherein:

[0654] X1 is a peptide comprising an amino acid sequence that is at least 80% identical along its length to the peptide at least 80% identical amino acid sequence;

[0655] X3 is a peptide comprising an amino acid sequence that is at least 80% identical along its length to the peptide at least 80% identical amino acid sequence; and

[0656] X4 is a peptide comprising an amino acid sequence that is at least 80% identical along its length to the peptide at least 80% identical amino acid sequence,

[0657] wherein

[0658] (i) X1 comprises I at residue 7, T or M at residue 8, E at residue 11, K at residue 14, and S at residue 18; and

[0659] (ii) X3 comprises R at residue 3, F at residue 4, K at residue 6, R at residue 7, R at residue 10, N at residue 11, W at residue 13, and G at residue 14.

[0660] 18. A conditional active receptor agonist according to any one of claims 1 - 11, wherein:

[0661] X1 is a peptide comprising an amino acid sequence that is at least 90% identical along its length to the peptide having at least 90% identity;

[0662] X3 is a peptide comprising an amino acid sequence that is at least 90% identical along its length to the peptide having at least 90% identity; and

[0663] X4 is a peptide comprising an amino acid sequence that is at least 90% identical along its length to the peptide having at least 90% identity,

[0664] wherein

[0665] (i) X1 comprises I at residue 7, T or M at residue 8, E at residue 11, K at residue 14, and S at residue 18; and

[0666] (ii) X3 comprises R at residue 3, F at residue 4, K at residue 6, R at residue 7, R at residue 10, N at residue 11, W at residue 13, and G at residue 14.

[0667] 19. A conditional active receptor agonist according to any one of claims 14 - 18, wherein

[0668] (iii) X4 comprises F at residue 19.

[0669] 20. A conditional active receptor agonist according to any one of claims 1 - 19, wherein the amino acid (AA) substitutions relative to the reference peptide domain occur at no more than 3 bold - marked AA residues, or at no more than 2 bold - marked AA residues, or at no more than 1 bold - marked AA residue, or at no bold - marked AA residues.

[0670] 21. A conditional active receptor agonist according to any one of claims 1 - 20, wherein the amino acid substitutions relative to the reference peptide domain are conservative amino acid substitutions.

[0671] 22. A conditional active receptor agonist according to any one of claims 1 - 21, wherein the amino acid residues relative to SEQ ID NO:4 are selected from the group consisting of:

[0672]

[0673] 23. The conditional 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.

[0674] 24. The conditional active receptor agonist of claim 22, 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.

[0675] 25. The conditional active receptor agonist of any one of claims 1-24, wherein the amino acid residues relative to SEQ ID NO:5 are selected from the group consisting of:

[0676]

[0677]

[0678] 26. The conditional active receptor agonist of any one of claims 1-25, which comprises a cysteine substitution at position 17 or 20 relative to SEQ ID NO:5.

[0679] 27. The conditional active receptor agonist of claim 25 or 26, wherein 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.

[0680] 28. The conditional 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: 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.

[0681] 29. The conditional active receptor agonist of any one of claims 1-28, wherein the amino acid residues relative to SEQ ID NO:6 are selected from the group consisting of:

[0682]

[0683]

[0684] 30. The conditional active receptor agonist of any one of claims 1-29, which comprises a cysteine substitution at position 3 relative to SEQ NO:6.

[0685] 31. The conditional active receptor agonist of claim 30 or 31, wherein position 19 is I.

[0686] 32. A conditional active receptor agonist of claim 30 or 31, wherein position 19 is not I.

[0687] 33. A conditional active receptor agonist of any one of claims 1-32, wherein X2 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 at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% the same amino acid sequence.

[0688] 34. A conditional 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:

[0689]

[0690] 35. A conditional active receptor agonist of claim 11, 33 or 34, which comprises a cysteine substitution at position 1, 2, 5, 9, 12, or 16 relative to SEQ NO:7.

[0691] 36. A conditional active receptor agonist of claim 34 or 35, wherein position 11 is I.

[0692] 37. A conditional active receptor agonist of claim 34 or 35, wherein position 11 is not I.

[0693] 38. A conditional active receptor agonist of any one of claims 1-37, wherein:

[0694] (i) the first polypeptide component comprises one of X1, X2, X3, and X4, and the second polypeptide component comprises the three that are not present in the first polypeptide component among X1, X2, X3, and X4; or

[0695] (ii) the first polypeptide component comprises two of X1, X2, X3, and X4, and the second polypeptide component comprises the two that are not present in the first polypeptide component among X1, X2, X3, and X4.

[0696] 39. A conditional active receptor agonist of any one of claims 1-37, wherein (i) the first polypeptide comprises X1 and the second polypeptide comprises X2, X3, and X4;

[0697] (ii) the first polypeptide comprises X2 and the second polypeptide comprises X1, X3, and X4;

[0698] (iii) the first polypeptide comprises X3 and the second polypeptide comprises X1, X2, and X4;

[0699] (iv) The first polypeptide comprises X4 and the second polypeptide comprises X1, X2, and X3;

[0700] (v) The first polypeptide comprises X1 and X2, and the second polypeptide comprises X3 and X4;

[0701] (vi) The first polypeptide comprises X1 and X3, and the second polypeptide comprises X2 and X4;

[0702] (vii) The first polypeptide comprises X1 and X4, and the second polypeptide comprises X2 and X3;

[0703] (viii) The first polypeptide comprises X2 and X3, and the second polypeptide comprises X1 and X4;

[0704] (ix) The first polypeptide comprises X2 and X4, and the second polypeptide comprises X1 and X3;

[0705] (x) The first polypeptide comprises X3 and X4, and the second polypeptide comprises X1 and X2;

[0706] (xi) The first polypeptide comprises X1, X2, and X3 and the second polypeptide comprises X4;

[0707] (xii) The first polypeptide comprises X1, X2, and X4 and the second polypeptide comprises X3;

[0708] (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.

[0709] 40. A conditional active receptor agonist according to any one of claims 1-39, wherein:

[0710] (a) The first polypeptide comprises X1 and excludes X2, X3, and X4; and the second polypeptide is a fusion protein comprising X3-Z1-X2-Z2-X4 and excluding X1;

[0711] (b) The first polypeptide comprises X4 and excludes X1, X2, and X3; and the second polypeptide is a fusion protein comprising X1-Z1-X3-Z2-X2 and excluding X4; or

[0712] (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,

[0713] wherein each of Z1 and Z2 is independently an optional amino acid linker.

[0714] 41. A conditional active receptor agonist according to any one of claims 1 - 40, wherein X1, X2, X3, and X4 each comprise an amino acid sequence having 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 the X1, X2, X3, and X4 domains shown below, where the residues in parentheses may be present or absent:

[0715] X1:

[0716] X2:

[0717] X3:

[0718] X4:

[0719] 42. A conditional active receptor agonist according to any one of claims 1 - 41, wherein the first polypeptide and the second polypeptide comprise an amino acid sequence having at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identity to a pair of first and second polypeptides shown below (the underlined residues or "X" residues are optional and each residue in the optional domain may comprise any amino acid when present):

[0720] (i)

[0721] First polypeptide X1 (Neo2A)

[0722] PKKKIQLHAEHALYDALMILNI VKTNS (SEQ ID NO:256) and

[0723] Second polypeptide: X3 - X2’ - X4 (Neo2B)

[0724] TNSPPAEEK LEDYAFNFELILEEIARLFESG DQ KDEAEKAKRMKEWMKRIKT TAS EDEQEEMANAIITILQSWIFS (SEQ ID NO:257);

[0725] (ii)

[0726] First polypeptide X1 - X3 - X2’

[0727] PKKKIQLHAEHALYDALMILNIVKTNSPPAEEK LEDYAFNFELILEEIARLFESG DQ KDEAEKAKRMKEWMKRIKTTAS (SEQ ID NO:258) and

[0728] The second polypeptide X4

[0729] TTASE DEQEEMANAIITILQSWIFS (SEQ ID NO:259);

[0730] (iii)

[0731] The first polypeptides X1 - X3

[0732] PKKKIQLHAEHALYDALMILNI VKTNSPPAEEK LEDYAFNFELILEEIARLFES GD (SEQ ID NO:260) and

[0733] The second polypeptides X2 - X4

[0734] DQKDEAEKAKRMKEWMKRIKT TAS EDEQEEMANAIITILQSWIFS (SEQ ID NO:261); (iv)

[0735] The first polypeptide X1 (Neo4A)

[0736] PKKKIQIMAEEALKDALSILNI VKTNS (SEQ ID NO:262) and

[0737] The second polypeptide X3 - X2’ - X4 (Neo4B)

[0738] TNSPPAEE QLERFAKRFERNLWGIARLFESG DQ KDEAEKAKRMIEWMKRIKT TAS EDEQEEMANAIITILQSWFFS (SEQ ID NO:263);

[0739] (v)

[0740] The first polypeptide X1

[0741] PKKKIQLHAEHALYDALMILNI XXXXX (SEQ ID NO:311) and

[0742] The second polypeptide: X3 - X2’ - X4

[0743] XXXXXXXXX LEDYAFNFELILEEIARLFESG XX KDEAEKAKRMKEWMKRIKT XXX EDEQEEMANAIITILQSWIFS(SEQ ID NO:264);

[0744] (vi)

[0745] The first polypeptide X1-X3-X2’

[0746] PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESG XX KDEAEKAKRMKEWMKRIKTTAS(SEQ ID NO:265) and

[0747] The second polypeptide X4

[0748] XXXXX DEQEEMANAIITILQSWIFS(SEQ ID NO:266);

[0749] (vii)

[0750] The first polypeptide X1-X3

[0751] PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESXX GD (SEQ ID NO:267) and

[0752] The second polypeptide X2-X4

[0753] DQKDEAEKAKRMKEWMKRIKT XXX EDEQEEMANAIITILQSWIFS(SEQ ID NO:268);(viii)

[0754] The first polypeptide X1

[0755] PKKKIQIMAEEALKDALSILNI XXXXX (SEQ ID NO:269) and

[0756] The second polypeptide X3-X2’-X4

[0757] XXXXXXXX QLERFAKRFERNLWGIARLFESG XX KDEAEKAKRMIEWMKRIKTXXX EDEQEEMANAIITILQSWFFS (SEQ ID NO:270);

[0758] (ix)

[0759] First polypeptide > Neo4_X1-X3’

[0760] PKKKIQIMAEEALKDALSILNI VKTNSPPAEE QLERFAKRFERNLWGIARLFES GD (SEQ ID NO:312) and

[0761] Second polypeptide > Neo4_X2-X4

[0762] DQ KDEAEKAKRMIEWMKRIK TTAS EDEQEEMANAIITILQSWFFS (SEQ ID NO:313);

[0763] (x)

[0764] First polypeptide > Neo4_X1-X3’

[0765] PKKKIQIMAEEALKDALSILNIXXXXXXXXXXQLERFAKRFERNLWGIARLFESXX (SEQ ID NO:314) and

[0766] Second polypeptide > Neo4_X2-X4

[0767] XX KDEAEKAKRMIEWMKRIKXXXXEDEQEEMANAIITILQSWFFS (SEQ ID NO:315);

[0768] (xi)

[0769] First polypeptide Neo4_X1-X3’-X2

[0770] PKKKIQIMAEEALKDALSILNI VKTNSPPAEEQ LERFAKRFERNLWGIARLFES GDQ KDEAEKAKRMIEWMKRIK TTA (SEQ ID NO:316) and

[0771] Second polypeptide Neo4_X4

[0772] TTASEDEQEEMANAIITILQSWFFS (SEQ ID NO:317);

[0773] (xii)

[0774] The first polypeptide Neo4_X1-X3'-X2

[0775] PKKKIQIMAEEALKDALSILNIXXXXXXXXXXXLERFAKRFERNLWGIARLFESXXXKDEAEKAKRMIEWMKRIKXXX (SEQ ID NO:318) and

[0776] The second polypeptide Neo4_X4

[0777] XXXXX DEQEEMANAIITILQSWFFS (SEQ ID NO:319); or (xiii)

[0778] The first polypeptide (X1)

[0779] PKKKIQLHAEHALYDALMILNI VGGSS (SEQ ID NO:323), or

[0780] SKEA IQLHAEHALYDALMILNIVKTNS (SEQ ID NO:324), or

[0781] P IQLHAEHALYDALMILNIV (SEQ ID NO:325) and

[0782] The second polypeptide (X3-X2'-X4)

[0783] PK LEDYAFNFELILEEIARLFESG DQ KDEAEKAKRMKEWMKRIKT TAS EDEQEEMANAIITILQSWIFS (SEQ ID NO:326), or

[0784] GGSSGG LEDYAFNFELILEEIARLFESG GSSGG KDEAEKAKRMKEWMKRIT GGSSGG DEQEEMANAIITILQSWIFS (SEQ ID NO:327), or

[0785] GGSSGGLEDYAFNFELILEEIARLFES GGSSGGGG EAEKAKRMKEWMKRI GGSSGG DEQEEMANAIITILQSWIFS (SEQ ID NO:328).

[0786] 43. A conditional active receptor agonist according to any one of claims 1 - 41, wherein X1, X2, X3, and X4 respectively 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) present within the amino acid sequences of SEQ ID NOs: 11 - 94, 190 - 216, 247, and 275 - 300, respectively.

[0787] 44. A conditional active receptor agonist according to any one of claims 1 - 43, wherein the first polypeptide component and / or the second polypeptide component comprises at least one disulfide bond.

[0788] 45. A conditional active receptor agonist according to any one of claims 1 - 44, wherein the first polypeptide component and the second polypeptide component are non-covalently associated.

[0789] 46. A conditional active receptor agonist according to any one of claims 1 - 44, wherein the first polypeptide component and the second polypeptide component are indirectly bound to each other via a receptor.

[0790] 47. A conditional active receptor agonist according to any one of claims 1 - 46, wherein the first polypeptide component further comprises a first targeting domain or the second polypeptide component further comprises a second targeting domain.

[0791] 48. A conditional active receptor agonist according to any one of claims 1 - 46, wherein the first polypeptide component further comprises a first targeting domain and the second polypeptide component further comprises a second targeting domain.

[0792] 49. A conditional active receptor agonist according to claim 47 or 48, wherein the first targeting domain, when present, is a translational fusion with the first polypeptide, and wherein the second targeting domain, when present, is a translational fusion with the second polypeptide.

[0793] 50. A conditional active receptor agonist according to any one of claims 47 - 49, wherein both the first targeting domain and the second targeting domain are present, and wherein the first targeting domain and the second targeting domain are the same.

[0794] 51. A conditional active receptor agonist according to any one of claims 47 - 49, wherein both the first targeting domain and the second targeting domain are present, and wherein the first targeting domain and the second targeting domain are different.

[0795] 52. A conditional active receptor agonist according to any one of claims 47 - 51, wherein the first targeting domain and / or the second targeting domain each bind to a cell surface protein.

[0796] 53. The conditional 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, 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, T regulatory cells, dendritic cells, NK cells, or macrophages), such 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.

[0797] 54. The conditional active receptor agonist of claim 52 or 53, wherein the first targeting domain and the second targeting domain each bind to a tumor cell, tumor vascular component cell, or tumor microenvironment cell surface marker.

[0798] 55. The conditional active receptor agonist of claim 54, wherein the tumor cell, vascular component cell, or tumor microenvironment cell surface marker is selected from including but not limited to EGFR, EGFRvIII, Her2, HER3, EpCAM, MSLN, MUC16, PSMA, TROP2, ROR1, RON, PD - L1, CD47, CTLA - 4, CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD40, CD45, CAMPATH - 1, BCMA, CS - 1, PD - L1, B7 - H3, B7 - DC, HLD - DR, carcinoembryonic antigen (CEA), TAG - 72, 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 (de2-7EGFR), 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, Tyrosinase, 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 A1, 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, XAGE 1, Legumain, Tie 3, 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 NM001203); E16 (LAT1, SLC7A5, Genbank accession number NM003486); STEAP1 (prostate transmembrane protein, androgen-induced 1, 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); Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession number AB040878); PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession number AY358628); ETBR (endothelin B receptor, Genbank accession number AY275463); MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession number 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 Prostate 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, CRIPTO, TDGF1, Teratocarcinoma-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-Associated β), B29, Genbank Accession No. NM000626); FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 Domain-Containing Phosphatase-Anchoring Protein 1a), SPAP1B, SPAP1C, Genbank Accession No. NM030764); HER2 (Genbank Accession No. M11730); NCA (Genbank Accession No. M18728); MDP (Genbank Accession No. BC017023); IL20Rα (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. NP001762.1); CD79a (CD79A, CD79α, Immunoglobulin-Associated α, B-Cell-Specific Protein, Covalently Interacts with Igβ (CD79B) and Forms a Complex with Ig M Molecules on the Surface, Transduces Signals Involved in B-Cell Differentiation, Genbank Accession No. NP001774.1); CXCR5 (Burkitt's Lymphoma Receptor 1, G-Protein-Coupled Receptor, Activated by the CXCL13 Chemokine, Functions in Lymphocyte Migration and Humoral Defense, Plays a Role 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), 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, deficiency may contribute to the pathophysiology of idiopathic detrusor instability, Genbank accession number NP002552.2); CD72 (B cell differentiation antigen CD72, Lyb-2, Genbank accession number NP001773.1); LY64 (lymphocyte antigen 64 (RP105), a type I membrane protein of the leucine-rich repeat (LRR) family, regulates B cell activation and apoptosis, loss of function is associated with elevated disease activity in patients with systemic lupus erythematosus, Genbank accession number NP005573.1); FCRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain containing C2-type Ig-like and ITAM domains, may have a role in B lymphocyte differentiation, Genbank accession number NP443170.1); or IRTA2 (immunoglobulin superfamily receptor translocation-associated 2, a putative immunoreceptor that may have a role in B cell development and lymphoma genesis; gene dysregulation caused by translocation occurs in some B cell malignancies, Genbank accession number NP112571.1).

[0799] 56. The conditional active receptor agonist of any one of claims 47 - 55, wherein the first targeting domain and / or the second targeting domain binds to an immune cell surface marker (including but not limited to CD8+ T cells, T regulatory cells, dendritic cells, NK cells, or macrophages), and wherein the immune cell surface marker may include 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, and / or, wherein 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.

[0800] 57. The conditional 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 CD8+ T cells, T regulatory cells, dendritic cells, or macrophages).

[0801] 58. The conditional 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 (such as a fibroblast, an infiltrating immune cell, or a stromal element), and wherein the other targeting domain binds to a surface marker of an immune cell (including but not limited to a CD8+ T cell, a T regulatory cell, a dendritic cell, or a macrophage), and wherein the surface marker of the immune cell can include 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.

[0802] 59. The conditional active receptor agonist of any one of claims 47-58, wherein the first targeting domain and / or the second targeting domain can include but not limited to an scFv, F(ab), F(ab’) 2 , B cell receptor (BCR), DARPin, affibody, monobody, nanobody, diabody, antibody (including a monospecific or bispecific antibody), a cell-targeting oligopeptide, including but not limited to an RGD integrin-binding peptide, a de novo designed conjugate, an aptamer, a cyclic peptide, a conotoxin, a small molecule, such as folic acid, and a virus.

[0803] 60. The conditional active receptor agonist of any one of claims 47-58, wherein the first targeting domain and / or the second targeting domain can include but not limited to an scFv, F(ab), F(ab’) 2 , B cell receptor (BCR), DARPin, affibody, monobody, nanobody, diabody, and antibody (including a monospecific or bispecific antibody).

[0804] 61. The conditional active receptor agonist of any one of claims 1-60, wherein X2 is a peptide comprising an amino acid sequence that is at least 70% identical along its length, wherein the residues in parentheses are optional.

[0805] 62. The conditional active receptor agonist of any one of claims 1-60, wherein X2 is a peptide comprising an amino acid sequence that is at least 80% identical along its length, wherein the residues in parentheses are optional.

[0806] 63. The conditional active receptor agonist of any one of claims 1-60, wherein X2 is a peptide comprising an amino acid sequence that is at least A peptide having an amino acid sequence that is at least 90% identical, wherein the residues in parentheses are optional.

[0807] 64. A polypeptide that comprises 1, 2, or 3, but not all 4, of domains X1, X2, X3, and X4, wherein:

[0808] (a) When present, X1 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 the peptide ;

[0809] (b) When present, X2 is any helical peptide domain;

[0810] (c) When present, X3 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 the peptide ; and

[0811] (d) When present, X4 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 the peptide ;

[0812] The amino acid residues in parentheses may be present or absent.

[0813] 65. The polypeptide of claim 64, wherein:

[0814] (a) When present, X1 is a peptide comprising an amino acid sequence that is at least 70% identical to the full length of the peptide or ;

[0815] (b) When present, X2 is any helical peptide domain;

[0816] (c) When present, X3 is a peptide comprising an amino acid sequence that is at least 70% identical to the full length of the peptide or ; and

[0817] (d) When present, X4 is a peptide comprising an amino acid sequence that is at least 70% identical to the full length of the peptide or ;

[0818] 66. The polypeptide of claim 64, wherein:

[0819] (a) X1, when present, is a peptide comprising an amino acid sequence that is at least 85% identical to the full length of the peptide or ;

[0820] (b) X2, when present, is any helical peptide domain;

[0821] (c) X3, when present, is a peptide comprising an amino acid sequence that is at least 85% identical to the full length of the peptide or ; and

[0822] (d) X4, when present, is a peptide comprising an amino acid sequence that is at least 85% identical to the full length of the peptide or .

[0823] 67. The polypeptide of claim 64, wherein:

[0824] (a) X1, when present, is a peptide comprising an amino acid sequence that is identical to the full length of the peptide or ;

[0825] (b) X2, when present, is any helical peptide domain;

[0826] (c) X3, when present, is a peptide comprising an amino acid sequence that is identical to the full length of the peptide or ; and

[0827] (d) X4, when present, is a peptide comprising an amino acid sequence that is identical to the full length of the peptide or .

[0828] 68. The polypeptide of claim 64, wherein (a) X1, when present, 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 the peptide ;

[0829] (c) X3, when present, 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 the peptide ; and

[0830] (d) X4, when present, 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 the peptide a peptide having 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

[0831] 69. The polypeptide of claim 64, wherein (a) when X1 is present, it is a peptide comprising an amino acid sequence that is at least 65% identical to the full length of the peptide or ;

[0832] (c) when X3 is present, it is a peptide comprising an amino acid sequence that is at least 65% identical to the full length of the peptide or ; and

[0833] (d) X4 is a peptide comprising an amino acid sequence that is at least 65% identical to the full length of the peptide or .

[0834] 70. The polypeptide of claim 68, wherein (a) when X1 is present, it is a peptide comprising an amino acid sequence that is at least 75% identical to the full length of the peptide or ;

[0835] (c) when X3 is present, it is a peptide comprising an amino acid sequence that is at least 75% identical to the full length of the peptide or ; and

[0836] (d) when X4 is present, it is a peptide comprising an amino acid sequence that is at least 75% identical to the full length of the peptide or .

[0837] 71. The polypeptide of claim 68, wherein (a) when X1 is present, it is a peptide comprising an amino acid sequence that is at least 80% identical to the full length of the peptide or ;

[0838] (c) when X3 is present, it is a peptide comprising an amino acid sequence that is at least 80% identical to the full length of the peptide or ; and

[0839] (d) when X4 is present, it is a peptide comprising an amino acid sequence that is at least 80% identical to the full length of the peptide or .

[0840] 72. The polypeptide of claim 68, wherein (a) when X1 is present, it is a peptide comprising an amino acid sequence that is at least 80% identical to the full length of the peptide or a peptide having an amino acid sequence that is at least 90% identical to the full length of

[0841] (c) X3, when present, is a peptide comprising an amino acid sequence that is at least 90% identical to the full length of the peptide or ; and

[0842] (d) X4, when present, is a peptide comprising an amino acid sequence that is at least 90% identical to the full length of the peptide or .

[0843] 73. The polypeptide of any one of claims 64 - 72, wherein X2, when present, comprises 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 peptide , wherein the residues in parentheses are optional.

[0844] 74. The polypeptide of any one of claims 64 - 72, wherein X2, when present, comprises an amino acid sequence that is at least 75% identical to the full length of the peptide , wherein the residues in parentheses are optional.

[0845] 75. The polypeptide of any one of claims 64 - 72, wherein X2, when present, comprises an amino acid sequence that is at least 85% identical to the full length of the peptide , wherein the residues in parentheses are optional.

[0846] 76. The polypeptide of any one of claims 64 - 72, wherein X2, when present, comprises an amino acid sequence that is at least 90% identical to the full length of the peptide , wherein the residues in parentheses are optional.

[0847] 77. The polypeptide of any one of claims 64 - 72, wherein X2, when present, comprises an amino acid sequence that is at least 80% identical to the full length of the peptide , wherein the residues in parentheses are optional.

[0848] 78. The polypeptide of any one of claims 64 - 72, wherein X2, when present, comprises an amino acid sequence that is at least 90% identical to the full length of the peptide , wherein the residues in parentheses are optional.

[0849] 79. The polypeptide of any one of claims 64 - 78, consisting of the group consisting of:

[0850] (i) a polypeptide comprising X1 and excluding X2, X3, and X4;

[0851] (ii) A polypeptide comprising X2 and excluding X1, X3, and X4;

[0852] (iii) A polypeptide comprising X3 and excluding X1, X2, and X4;

[0853] (iv) A polypeptide comprising X4 and excluding X1, X2, and X3;

[0854] (v) A polypeptide comprising X1 and X2 and excluding X3 and X4;

[0855] (vi) A polypeptide comprising X1 and X3 and excluding X2 and X4;

[0856] (vii) A polypeptide comprising X1 and X4 and excluding X2 and X3;

[0857] (viii) A polypeptide comprising X2 and X3 and excluding X1 and X4;

[0858] (ix) A polypeptide comprising X2 and X4 and excluding X1 and X3;

[0859] (x) A polypeptide comprising X3 and X4 and excluding X1 and X2;

[0860] (xi) A polypeptide comprising X1, X2, and X3 and excluding X4;

[0861] (xii) A polypeptide comprising X1, X2, and X4 and excluding X3;

[0862] (xiii) A polypeptide comprising X1, X3, and X4 and excluding X2; and

[0863] (xiv) A polypeptide comprising X2, X3, and X4 and excluding X1.

[0864] 80. The polypeptide of any one of claims 64 - 79, wherein the polypeptide comprises X1, and X1 comprises 1, 2, 3, 4, or all 5 of the following: L at residue 7, H at residue 8, H at residue 11, Y at residue 14, or M at residue 18, wherein the numbering is relative to SEQ ID NO:4 based on the presence of optional residues.

[0865] 81. The polypeptide of any one of claims 64 - 80, wherein the polypeptide comprises X3, and X3 comprises 1, 2, 3, 4, 5, 6, 7, or all 8 of the following: D at residue 3, Y at residue 4, F at residue 6, N at residue 7, L at residue 10, I at residue 11, E at residue 13, or E at residue 14, wherein the numbering is relative to SEQ ID NO:5 based on the presence of optional residues.

[0866] 82. A polypeptide according to any one of claims 64 - 81, wherein the polypeptide comprises X4, wherein X4 comprises I at residue 19, and wherein the numbering is relative to SEQ ID NO:6 in the presence of optional residues.

[0867] 83. A polypeptide according to any one of claims 64 - 79, wherein X1 is present, wherein X1 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 the peptide, and wherein X1 comprises I at residue 7, T or M at residue 8, E at residue 11, K at residue 14, and S at residue 18. 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 the peptide, and wherein X1 comprises I at residue 7, T or M at residue 8, E at residue 11, K at residue 14, and S at residue 18.

[0868] 84. A polypeptide according to any one of claims 64 - 79 or 83, wherein X3 is present, wherein X3 is a peptide comprising an amino acid sequence that is at least 37%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical along its length to the peptide, and wherein X3 comprises R at residue 3, F at residue 4, K at residue 6, R at residue 7, R at residue 10, N at residue 11, W at residue 13, and G at residue 14. at least 37%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical along its length to the peptide, and wherein X3 comprises R at residue 3, F at residue 4, K at residue 6, R at residue 7, R at residue 10, N at residue 11, W at residue 13, and G at residue 14.

[0869] 85. A polypeptide according to any one of claims 64 - 79, 84, and 84, wherein X4 is present, wherein X4 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 the peptide. 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 the peptide.

[0870] 86. The polypeptide of claim 85, wherein X4 comprises F at residue 19.

[0871] 87. A polypeptide according to any one of claims 64 - 86, wherein the amino acid (AA) substitutions relative to the reference peptide domain occur at no more than 3 bold - marked AA residues, or at no more than 2 bold - marked AA residues, or at no more than 1 bold - marked AA residue, or do not occur at bold - marked AA residues.

[0872] 88. A polypeptide according to any one of claims 64 - 87, wherein the amino acid substitutions relative to the reference peptide domain are conservative amino acid substitutions.

[0873] A polypeptide according to any one of claims 64 - 88, wherein X1 is present, and wherein the amino acid residue relative to SEQ ID NO:4 is selected from the group consisting of:

[0874]

[0875]

[0876] 90. The polypeptide of claim 89, wherein position 7 is I, position 8 is M or T, position 11 is E, position 14 is K, and position 18 is S.

[0877] 91. The polypeptide of claim 89, 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.

[0878] 92. A polypeptide according to any one of claims 64 - 91, wherein X3 is present, and wherein the amino acid residue relative to SEQ ID NO:5 is selected from the group consisting of:

[0879]

[0880] 93. The polypeptide of claim 92, which comprises a cysteine substitution at position 17 or 20 relative to SEQ ID NO:5.

[0881] 94. The polypeptide of claim 92 or 93, wherein 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.

[0882] 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: 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.

[0883] 96. A polypeptide according to any one of claims 64 - 95, wherein X4 is present, and wherein the amino acid residue relative to SEQ ID NO:6 is selected from the group consisting of:

[0884]

[0885] 97. The polypeptide of claim 96, which comprises a cysteine substitution at position 3 relative to SEQ NO:6.

[0886] 98. The polypeptide of claim 95 or 96, wherein position 19 is I.

[0887] The polypeptide of claim 95 or 96, wherein position 19 is not I.

[0888] 100. The polypeptide of any one of claims 64 - 99, wherein X4 is present, and wherein the amino acid residue relative to SEQ ID NO:7 is selected from the group consisting of:

[0889]

[0890]

[0891] 101. The polypeptide of claim 100, which comprises a cysteine substitution at position 1, 2, 5, 9, 12, or 16 relative to SEQ NO:7.

[0892] 102. The polypeptide of claim 101 or 101, wherein position 11 is I.

[0893] 103. The polypeptide of claim 100 or 101, wherein position 11 is not I.

[0894] 104. The polypeptide of any one of claims 64 - 103, wherein the polypeptide comprises 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 first polypeptide or the second polypeptide listed below (the underlined residues are optional and each optional residue, when present, can comprise any amino acid):

[0895] (i)

[0896] First polypeptide X1 (Neo2A)

[0897] PKKKIQLHAEHALYDALMILNI VKTNS (SEQ ID NO:256) or

[0898] Second polypeptide X3 - X2’ - X4 (Neo2B)

[0899] TNSPPAEEK LEDYAFNFELILEEIARLFESG DQ KDEAEKAKRMKEWMKRIKT TAS EDEQEEMANAIITILQSWIFS (SEQ ID NO:257);

[0900] (ii)

[0901] First polypeptide X1 - X3 - X2’

[0902] PKKKIQLHAEHALYDALMILNI VKTNSPPAEEK LEDYAFNFELILEEIARLFESG DQ KDEAEKAKRMKEWMKRIKTTAS (SEQ ID NO:258) or

[0903] The second polypeptide X4

[0904] TTASE DEQEEMANAIITILQSWIFS (SEQ ID NO:259);

[0905] (iii)

[0906] The first polypeptides X1 - X3

[0907] PKKKIQLHAEHALYDALMILNI VKTNSPPAEEK LEDYAFNFELILEEIARLFES GD (SEQ ID NO:260) or

[0908] The second polypeptides X2 - X4

[0909] DQKDEAEKAKRMKEWMKRIKT TAS EDEQEEMANAIITILQSWIFS (SEQ ID NO:261); (iv)

[0910] The first polypeptide X1 (Neo4A)

[0911] PKKKIQIMAEEALKDALSILNI VKTNS (SEQ ID NO:262) or

[0912] The second polypeptides X3 - X2’ - X4 (Neo4B)

[0913] TNSPPAEE QLERFAKRFERNLWGIARLFESG DQ KDEAEKAKRMIEWMKRIKT TAS EDEQEEMANAIITILQSWFFS (SEQ ID NO:263);

[0914] (v)

[0915] The first polypeptide X1 (Neo2A)

[0916] PKKKIQLHAEHALYDALMILNI XXXXX (SEQ ID NO:311) or

[0917] Second polypeptide X3-X2'-X4 (Neo2B)

[0918] XXXXXXXXX LEDYAFNFELILEEIARLFESG XX KDEAEKAKRMKEWMKRIKT XXX EDEQEEMANAIITILQSWIFS (SEQ ID NO:264);

[0919] (vi)

[0920] First polypeptide X1-X3-X2'

[0921] PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESG XX KDEAEKAKRMKEWMKRIKTTAS (SEQ ID NO:265) or

[0922] Second polypeptide X4

[0923] XXXXX DEQEEMANAIITILQSWIFS (SEQ ID NO:266);

[0924] (vii)

[0925] First polypeptide X1-X3

[0926] PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESXX GD (SEQ ID NO:267) or

[0927] Second polypeptide X2-X4

[0928] DQKDEAEKAKRMKEWMKRIKT XXX EDEQEEMANAIITILQSWIFS (SEQ ID NO:268);

[0929] (viii)

[0930] First polypeptide X1 (Neo4A)

[0931] PKKKIQIMAEEALKDALSILNI XXXXX (SEQ ID NO:269) or

[0932] Second polypeptide X3-X2'-X4 (Neo4B)

[0933] XXXXXXXX QLERFAKRFERNLWGIARLFESG XX KDEAEKAKRMIEWMKRIKT XXX EDEQEEMANAIITILQSWFFS(SEQ ID NO:270);

[0934] (ix)

[0935] First polypeptide > Neo4_X1-X3’

[0936] PKKKIQIMAEEALKDALSILNI VKTNSPPAEE QLERFAKRFERNLWGIARLFES GD (SEQ ID NO:312) or

[0937] Second polypeptide > Neo4_X2-X4

[0938] DQ KDEAEKAKRMIEWMKRIK TTAS EDEQEEMANAIITILQSWFFS(SEQ ID NO:313);

[0939] (x)

[0940] First polypeptide > Neo4_X1-X3’

[0941] PKKKIQIMAEEALKDALSILNIXXXXXXXXXXQLERFAKRFERNLWGIARLFESXX(SEQ ID NO:314) or

[0942] Second polypeptide > Neo4_X2-X4

[0943] XX KDEAEKAKRMIEWMKRIKXXXXEDEQEEMANAIITILQSWFFS(SEQ ID NO:315);

[0944] (xi)

[0945] First polypeptide Neo4_X1-X3’-X2

[0946] PKKKIQIMAEEALKDALSILNI VKTNSPPAEEQ LERFAKRFERNLWGIARLFES GDQ KDEAEKAKRMIEWMKRIK TTA (SEQ ID NO:316) or

[0947] Second polypeptide Neo4_X4

[0948] TTASE DEQEEMANAIITILQSWFFS (SEQ ID NO:317);

[0949] (xii)

[0950] First polypeptide Neo4_X1-X3’-X2

[0951] PKKKIQIMAEEALKDALSILNIXXXXXXXXXXXLERFAKRFERNLWGIARLFESXXXKDEAEKAKRMIEWMKRIKXXX (SEQ ID NO:318) or

[0952] Second polypeptide Neo4_X4

[0953] XXXXX DEQEEMANAIITILQSWFFS (SEQ ID NO:319);

[0954] (xiii)

[0955] First polypeptide (X1)

[0956] PKKKIQLHAEHALYDALMILNI VGGSS (SEQ ID NO:323), or

[0957] SKEA IQLHAEHALYDALMILNIVKTNS (SEQ ID NO:324), or

[0958] P IQLHAEHALYDALMILNIV (SEQ ID NO:325) or

[0959] Second polypeptide (X3-X2’-X4)

[0960] PK LEDYAFNFELILEEIARLFESG DQ KDEAEKAKRMKEWMKRIKT TAS EDEQEEMANAIITILQSWIFS (SEQ ID NO:326), or

[0961] GGSSGG LEDYAFNFELILEEIARLFESG GSSGG KDEAEKAKRMKEWMKRIT GGSSGGDEQEEMANAIITILQSWIFS (SEQ ID NO:327), or

[0962] GGSSGG LEDYAFNFELILEEIARLFES GGSSGGGG EAEKAKRMKEWMKRI GGSSGG DEQEEMANAIITILQSWIFS (SEQ ID NO:328).

[0963] 105. A polypeptide according to any one of claims 64 - 103, wherein when X1, X2, X3, and X4 are present, they 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) present within the amino acid sequences of SEQ ID NOs: 11 - 94, 190 - 216, 247, and 275 - 300, respectively.

[0964] 106. A polypeptide according to any one of claims 64 - 105, wherein the polypeptide comprises at least one disulfide bond.

[0965] 107. A polypeptide according to any one of claims 64 - 106, wherein the polypeptide further comprises a targeting domain.

[0966] 108. The polypeptide of claim 107, wherein the targeting domain is a translational fusion with the polypeptide.

[0967] 109. The polypeptide of claim 108, wherein the targeting domain binds to a cell surface protein.

[0968] 110. The polypeptide of claim 109, wherein the cell surface protein is present on the surface of cells selected from the group consisting of tumor cells, vascular components, tumor microenvironment (such as fibroblasts, infiltrating immune cells, or stromal elements), other cancer cells, and immune cells (including but not limited to CD8+ T cells, T regulatory cells, dendritic cells, NK cells, or macrophages), and such immune cell surface markers 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.

[0969] 111. The polypeptide according to 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 (such as a fibroblast, an infiltrating immune cell, or a stromal element).

[0970] The polypeptide of claim 111, wherein the surface marker of the tumor cell, vascular component cell, or tumor microenvironment cell (such as fibroblast, infiltrating immune cell, or stromal element) is selected from including but not limited to EGFR, EGFRvIII, Her2, HER3, EpCAM, MSLN, MUC16, PSMA, TROP2, ROR1, RON, PD-L1, CD47, CTLA-4, CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD40, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC, HLD-DR, carcinoembryonic antigen (CEA), TAG-72, 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, tyrosinase, 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, MAGEAl, 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, XAGE 1, legumain, Tie 3, 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 NM001203); E16 (LAT1, SLC7A5, Genbank accession number NM003486); STEAP1 (prostate transmembrane protein, androgen-induced 1, 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); Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession number AB040878); PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession number AY358628); ETBR (endothelin B receptor, Genbank accession number AY275463); MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession number NM017763); STEAP2 (HGNC.sub.--8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, Prostate cancer-associated gene 1, Prostate cancer-associated protein 1, Prostate transmembrane epithelial antigen 2 with six transmembrane domains, Six-transmembrane prostate protein, Genbank accession number AF455138); TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, Transient receptor potential cation channel, subfamily M, member 4, Genbank accession number NM017636); CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, Teratocarcinoma-derived growth factor, Genbank accession number NP003203 or NM003212); CD21 (CR2 (Complement receptor 2) or C3DR (C3d / Epstein Barr virus receptor) or Hs.73792, Genbank accession number M26004); CD79b (IGb (Immunoglobulin-related β), B29, Genbank accession number NM000626); FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain-containing phosphatase-anchoring protein 1a), SPAP1B, SPAP1C, Genbank accession number NM030764); HER2 (Genbank accession number M11730); NCA (Genbank accession number M18728); MDP (Genbank accession number BC017023); IL20Rα (Genbank accession number AF184971); Brevican (Genbank accession number AF229053); Ephb2R (Genbank accession number NM004442); ASLG659 (Genbank accession number AX092328); PSCA (Genbank accession number AJ297436); GEDA (Genbank accession number AY260763); BAFF-R (Genbank accession number NP443177.1); CD22 (Genbank accession number NP001762.1); CD79a (CD79A, CD79α, Immunoglobulin-related α, B cell-specific protein, covalently interacts with Igβ (CD79B) and forms a complex with Ig M molecules on the surface, transduces signals involved in B cell differentiation, Genbank accession number NP001774.1); CXCR5 (Burkitt lymphoma receptor 1, G protein-coupled receptor, activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and possibly the development of AIDS, lymphoma, myeloma, and leukemia, Genbank accession number NP001707.1); HLA-DOB (beta subunit of MHC class II molecule (Ia antigen), 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, defects may contribute to the pathophysiology of idiopathic detrusor instability, Genbank accession number NP002552.2); CD72 (B cell differentiation antigen CD72, Lyb-2, Genbank accession number NP001773.1); LY64 (lymphocyte antigen 64 (RP105), a type I membrane protein of the leucine-rich repeat (LRR) family, regulates B cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosus, Genbank accession number NP005573.1); FCRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain containing C2-type Ig-like and ITAM domains, may have a role in B lymphocyte differentiation, Genbank accession number NP443170.1); or IRTA2 (immunoglobulin superfamily receptor translocation-associated 2, a putative immunoreceptor that may have a role in B cell development and lymphoma genesis; gene dysregulation caused by translocation occurs in some B cell malignancies, Genbank accession number NP112571.1).

[0971] 113. 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 CD8+ T cells, T regulatory cells, dendritic cells, or macrophages), and wherein the immune cell surface marker may include 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.

[0972] 114. The polypeptide of any one of claims 107 - 113, wherein the targeting domain may include but not limited to scFv, F(ab), F(ab’) that bind to the cell surface 2 , B cell receptor (BCR), DARPin, affibody, monobody, nanobody, diabody, antibody (including monospecific or bispecific antibodies), cell-targeting oligopeptides, including but not limited to RGD integrin-binding peptides, de novo designed conjugates, aptamers, bicyclic peptides, conotoxins, small molecules such as folic acid, and viruses.

[0973] 115. A nucleic acid encoding the polypeptide of any one of claims 64 - 114, or the first polypeptide component or the second polypeptide component of any one of claims 1 - 63.

[0974] 116. An expression vector comprising the nucleic acid of claim 115 operably linked to a promoter.

[0975] 117. A host cell comprising the nucleic acid of claim 115 and / or the expression vector of claim 116.

[0976] 118. A pharmaceutical composition comprising a conditional active receptor agonist of any one of claims 1 - 63, a polypeptide of any one of claims 64 - 114, the nucleic acid of claim 115, the expression vector of claim 116, or the host cell of claim 117, and a pharmaceutically acceptable carrier.

[0977] 119. A method for treating cancer comprising administering to a subject in need thereof a conditional active receptor agonist of any one of claims 1 - 63 to treat the cancer under conditions in which the first polypeptide component and the second polypeptide component interact at the cells of the tumor.

[0978] 120. The method of claim 119, 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 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 wherein the first polypeptide and the second polypeptide 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.

[0979] 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 tumor cell marker and the second targeting domain binds to an immune cell marker (including but not limited to CD8+ T cells, T regulatory cells, dendritic cells, or macrophages), and wherein the first polypeptide and the second polypeptide interact only after the first targeting domain binds to the tumor cell marker and the second targeting domain binds to the immune cell marker.

[0980] 122. 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, T regulatory cells, dendritic cells, or macrophages) and the second targeting domain binds to a second immune cell marker that can be the same as or different from the first immune cell marker (including but not limited to CD8+ T cells, T regulatory cells, dendritic cells, or macrophages), and wherein the first polypeptide and the second polypeptide 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.

[0981] 123. The method of any one of claims 119-122, wherein the cancer is selected from the group consisting of colon cancer, melanoma, renal cell carcinoma, head and neck squamous cell carcinoma, gastric cancer, bladder urothelial carcinoma, Hodgkin 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 lymphoma, multiple myeloma, ovarian cancer, cervical cancer, and any tumor type selected by diagnostic tests such as microsatellite instability, tumor mutational burden, PD-L1 expression level, or immune scoring assays (such as those developed by the Society for Immunotherapy of Cancer).

[0982] 124. A conditionally active receptor agonist of any one of claims 1-63, a polypeptide of any one of claims 64-114, a nucleic acid of claim 115, an expression vector of claim 116, a host cell of claim 117, or a pharmaceutical composition of claim 118, for use as a medicament in a subject for treating cancer and / or for modulating an immune response.

[0983] 125. A method for modulating an immune response in a subject, comprising administering to the subject a conditionally active receptor agonist of any one of claims 1-63, a polypeptide of any one of claims 64-114, a nucleic acid of claim 115, an expression vector of claim 116, a host cell of claim 117, or a pharmaceutical composition of claim 118.

[0984] 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.

[0985] 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.

[0986] 128. A method for activating an IL-2 receptor or an IL-4 receptor, comprising administering to a subject a conditionally active receptor agonist of any one of claims 1-63 under conditions in which the first polypeptide component and the second polypeptide component interact at the receptor.

[0987] 129. A conditionally active receptor agonist of any one of claims 1-63, wherein the first polypeptide component and the second polypeptide component interact to form an active agonist of IL-2 receptor βγ c heterodimer (IL-2Rβγ c ).

[0988] 130. A conditionally active receptor agonist of any one of claims 1-63, wherein the first polypeptide component and the second polypeptide component interact to form an active agonist of IL-4 receptor αγ c heterodimer (IL-4Rαγ c ), IL-13α, or an IL-4Rα / IL13Rα heterodimer.

[0989] 131. A conditionally active receptor agonist of any one of claims 1-63, wherein the amino acid residues in parentheses are present.

[0990] 132. A conditionally active receptor agonist of any one of claims 1-63, wherein the amino acid residues in parentheses are absent.

[0991] 133. A polypeptide of any one of claims 64-114, wherein the amino acid residues in parentheses are present.

[0992] 134. A polypeptide of any one of claims 64-114, wherein the amino acid residues in parentheses are absent.

[0993] 135. A conditionally active receptor agonist of any one of claims 1-63, wherein X1, X3, and X4 each comprise an amino acid sequence that is 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, wherein the residues in parentheses may be present or absent:

[0994] X1:

[0995] X3:

[0996] X4:

[0997] A conditional active receptor agonist according to any one of claims 1 - 63, wherein X1, X3, and X4 each comprise an amino acid sequence that is 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, where the residues in parentheses may be present or absent:

[0998] X1:

[0999] X3:

[1000] X4:

[1001] 137. A conditional active receptor agonist according to any one of claims 1 - 63, 135, and 136, wherein X2 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the full length of the amino acid sequence wherein the residues in parentheses are optional.

[1002] 138. A conditional active receptor agonist according to any one of claims 1 - 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 present within the amino acid sequence of SEQ ID NO:90 form 1 or form 2 as defined in Table 1.

[1003] 138. A polypeptide according to any one of claims 64 - 114, 133, and 134, wherein when present, X1, X3, and X4 each comprise an amino acid sequence that is 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, where the residues in parentheses may be present or absent:

[1004] X1:

[1005] X3:

[1006] X4:

[1007] 139. A polypeptide according to any one of claims 64 - 114, 133, 134, and 138, wherein when present, X2 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the full length of the amino acid sequence wherein the residues in parentheses are optional.

[1008] A polypeptide according to any one of claims 64 - 114, 133, and 134, wherein when present, X1, X2, X3, and X4 are at least 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 sequence of SEQ ID NO: 90, Form 1 or Form 2, as defined in Table 1.

Claims

1. A conditionally active receptor agonist comprising a first polypeptide component and a second polypeptide component, wherein the first polypeptide component and the second polypeptide component do not exist as a fusion protein, and wherein the first polypeptide and the second polypeptide each have an amino acid sequence of a pair of first and second polypeptide components selected from options (i)-(iii) or (xiii): (i) First polypeptide X1 (Neo2A) PKKKIQLHAEHALYDALMILNIVKTNS (SEQ ID NO:256) and Second polypeptide: X3-X2'-X4 (Neo2B) TNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO:257); (ii) First polypeptide X1-X3-X2' PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTAS (SEQ ID NO:258) and Second polypeptide X4 TTASEDEQEEMANAIITILQSWIFS (SEQ ID NO:259); (iii) First polypeptide X1-X3 PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLF ESGD (SEQ ID NO:260) and Second polypeptide X2-X4 DQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO:261); or (xiii) First polypeptide (X1) PKKKIQLHAEHALYDALMILNIVGGSS (SEQ ID NO:323), or SKEAIQLHAEHALYDALMILNIVKTNS (SEQ ID NO:324), or PIQLHAEHALYDALMILNIV (SEQ ID NO:325) and Second polypeptide (X3-X2'-X4) PKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASED EQEEMANAIITILQSWIFS (SEQ ID NO: 326), or GGSSGGLEDYAFNFELILEEIARLFESGGSSGGKDEAEKAKRMKEWMKRITGGSSGGDEQEEMANAIITILQSWIFS (SEQ ID NO: 327), or GGSSGGLEDYAFNFELILEEIARLFESGGSSGGGGEAEKAKRMKEWMKRI GGSSGGDEQEEMANAIITILQSWIFS (SEQ ID NO: 328), wherein the first polypeptide component and the second polypeptide component are not individually active receptor agonists, and wherein the first polypeptide component and the second polypeptide component interact to form an active agonist of the IL-2 receptor βγ c heterodimer.

2. The conditional active receptor agonist of claim 1, wherein the first polypeptide component and the second polypeptide component are non-covalently associated.

3. The conditional active receptor agonist of claim 1, wherein the first polypeptide component and the second polypeptide component are indirectly bound to each other via a receptor.

4. The conditional active receptor agonist of claim 1, wherein the first polypeptide component further comprises a first targeting domain and the second polypeptide component further comprises a second targeting domain.

5. The conditional active receptor agonist of claim 4, wherein the first targeting domain is a translational fusion with the first polypeptide component, and wherein the second targeting domain is a translational fusion with the second polypeptide component.

6. The conditional active receptor agonist of claim 4, which further comprises a first polypeptide linker connecting the first polypeptide component to the first targeting domain and / or a second polypeptide linker connecting the second polypeptide component to the second targeting domain.

7. The conditional active receptor agonist of claim 4, wherein the first targeting domain and the second targeting domain are the same.

8. The conditional active receptor agonist of claim 4, wherein the first targeting domain and / or the second targeting domain each bind to a cell surface protein.

9. The conditional active receptor agonist of claim 1, wherein the first targeting domain and / or the second targeting domain is selected from the group consisting of: scFv, F(ab), F(ab’) 2 , B cell receptor (BCR), DARPin, affibody, monobody, nanobody, diabody, and antibody.

10. A pharmaceutical composition comprising the conditional active receptor agonist of claim 1 and a pharmaceutically acceptable carrier.

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