Operated CD25 polypeptide and its use

Abstract

Provided herein are engineered polypeptides that comprise a combination of spatially related topological constraints, at least one of which is derived from a CD25 reference target, and methods for selecting the engineered polypeptides. Further provided are methods for using the engineered polypeptides, including positive and / or negative selection molecules, in methods for screening libraries of binding molecules, such as antibodies. Further provided herein are CD25 antibodies selected using these engineered polypeptides.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 902,334, filed Sep. 18, 2019; and U.S. Provisional Patent Application No. 62 / 767,431, filed Nov. 14, 2018, the disclosures of which are incorporated herein by reference in their entirety.

Background Art

[0002] The CD25 protein is the alpha chain of the interleukin - 2 (IL - 2) receptor and is a transmembrane protein present on regulatory T cells and activated T cells. In a normal state, regulatory T cells constitutively express CD25 and act to suppress the expansion and proliferation of effector T cells. Regulatory T cells maintain a healthy state and inhibit effector T cells from reacting to self - antigens or over - reacting to foreign antigens. In a normal protective immune response, effector T cells increase after contact with a foreign antigen and overcome the inhibition by regulatory T cells. However, in the case of a proliferative disease, cancer cells may increase the amount of regulatory T cells, thereby disabling the healthy immune response by limiting the production of effector T cells against cancer cells. Therefore, there is interest in therapeutic agents that alter the proliferation of CD25 - expressing regulatory T cells to suppress the immune system, for example, for use in cancer treatment. These therapeutic agents may include CD25 - targeting antibodies.

[0003] CD25 - targeting antibodies can be produced by immunizing animals with a CD25 immunogen, but current methods of developing CD25 immunogens often result in unpredictable and undesirable characteristics, such as antibody confusion or low cross - reactivity across species.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Accordingly, there is a need in the art for new engineered polypeptides having structural and / or kinetic similarity to CD25 or portions thereof, such as engineered polypeptides designed to mimic epitopes outside the IL-2 binding site.

Means for Solving the Problems

[0005] In one aspect, the present disclosure provides an engineered polypeptide that shares at least 46% structural and / or kinetic identity with a CD25 reference target, wherein the CD25 reference target is a portion of CD25 selected from CD25 residues 55-63, 13-20:127-132, 5-17, 5-11:156-163, 77-89, 147-157, 11-14 or 44-56.

[0006] In embodiments, the engineered polypeptide shares at least 60% structural and / or kinetic identity with the CD25 reference target. In embodiments, the engineered polypeptide shares at least 80% structural and / or kinetic identity with the CD25 reference target. In embodiments, the engineered polypeptide shares at least 80% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1-16. In embodiments, the engineered polypeptide shares at least 46% structural and / or kinetic identity with the CD25 reference target, and the CD25 reference target is a portion of CD25 selected from CD25 residues 55-63, 13-20:127-132, 5-17, 5-11:156-163, 77-89, 147-157, 11-14 or 44-56. In embodiments, the engineered polypeptide shares at least 80% structural and / or kinetic identity with the CD25 reference target. In embodiments, the structural and / or kinetic identity to the CD25 reference target is determined using the structure of CD25 deposited in PDB ID number 2ERJ, chain A. In embodiments, the engineered polypeptide includes an N-terminal modification or a C-terminal modification and optionally includes N-terminal biotin-PEG2- or C-terminal -GSGSGK-biotin.

[0007] In embodiments, between 10% and 98% of the amino acids of the engineered polypeptide satisfy one or more CD25 reference target-derived constraints. In embodiments, the amino acids that satisfy one or more CD25 reference target-derived constraints have a structural homology with the CD25 reference target with a backbone root mean square deviation (RSMD) of less than 8.0 Å. In embodiments, the amino acids that satisfy one or more CD25 reference target-derived constraints have a van der Waals surface area overlap with the reference between 30 Å 2 and 3000 Å 2 . In embodiments, the CD25 reference target-derived constraints are independently selected from the group consisting of interatomic distance; atomic fluctuation; atomic energy; chemical descriptor; solvent exposure; amino acid sequence similarity; bioinformatics descriptor; non-covalent binding propensity; phi angle; psi angle; van der Waals radius; secondary structure propensity; amino acid adjacency; and amino acid contact. In embodiments, the engineered polypeptide shares a structural similarity of 46% to 96% or higher RMSIP with the reference target across the amino acids of the polypeptide that satisfy one or more reference target-derived constraints.

[0008] In another aspect, the present disclosure provides a CD25-specific antibody comprising an antigen-binding domain that specifically binds to a CD25 epitope selected from CD25 residues 55-63, 13-20:127-132, 5-17, 5-11:156-163, 77-89, 147-157, 11-14 or 44-56. In embodiments, the antibody competes with an epitope-specific reference binder for binding to CD25, and the epitope-specific binder is IL-2, daclizumab, basiliximab and / or 7G7B6. In embodiments, the antibody does not compete with an off-target reference binder, and the off-target binder is IL-2, daclizumab, basiliximab and / or 7G7B6. In embodiments, the antibody has a k -2 of less than 10 -3 / s, less than 10 -4 / s or less than 10 off / s, where k off is measured using biolayer interferometry with soluble human CD25. In embodiments, the antibody is 10-2 between / s and 10 -5 k between / s and off having k, where k off is measured using biolayer interferometry with soluble human CD25. In embodiments, the antibody has a K of less than 100 nM, less than 25 nM or less than 5 nM, where K D is measured using biolayer interferometry with soluble human CD25. In embodiments, the antibody has a K D between 100 nM and 1 nM, where K D is measured using biolayer interferometry with soluble human CD25. In embodiments, the antibody has a K D is measured using biolayer interferometry with soluble human CD25.

[0009] In embodiments, the antibody specifically binds to cells expressing CD25. In embodiments, the antibody binds to cells expressing CD25 with an average fluorescence intensity (MFI) of at least 10 4 or at least 10 5 In embodiments, the antibody binds to cells expressing CD25 with an average fluorescence intensity (MFI) between 10 4 and 10 6 In embodiments, the antibody does not bind to CD25(−) cells. In embodiments, the antibody binds to CD25(−) cells with an average fluorescence intensity (MFI) of less than 10 3 In embodiments, the antibody comprises any one of the six CDRs of combinations 1 - 126 of Table 7D.

[0010] In an embodiment, the antibody comprises six complementarity determining regions (CDRs) for any one of YU390-B12, YU397-F01, YU397-D01, YU398-A11, YU404-H01, YU400-B07, YU400-D09, YU401-B01, YU401-G07, YU404-C02, YU403-G07, YU403-G05, YU391-B12, YU400-A03, YU400-D02, YU392-A09, YU392-B11, YU392-B12, YU392-E05, YU392-E06, YU392-G08, YU389-A03, YU392-G09, YU392-G12, YU392-H02, YU392-H04, YU402-F01, YU389-B11, YU394-D08, or YU390-A11 provided in Tables 3A and 3B.

[0011] In embodiments, the antibody comprises a heavy chain variable region and a light chain variable region that share at least 90%, 95%, 99% or 100% sequence identity with the heavy chain variable region and the light chain variable region of YU390-B12, YU397-F01, YU397-D01, YU398-A11, YU404-H01, YU400-B07, YU400-D09, YU401-B01, YU401-G07, YU404-C02, YU403-G07, YU403-G05, YU391-B12, YU400-A03, YU400-D02, YU392-A09, YU392-B11, YU392-B12, YU392-E05, YU392-E06, YU392-G08, YU389-A03, YU392-G09, YU392-G12, YU392-H02, YU392-H04, YU402-F01, YU389-B11, YU394-D08, or YU390-A11 provided in Table 5. In embodiments, the antibody is a full-length immunoglobulin G monoclonal antibody. In embodiments, the antibody comprises a single-chain variable fragment (scFv) that shares at least 90%, 95%, 99% or 100% sequence identity with the scFv sequence of YU390-B12, YU397-F01, YU397-D01, YU398-A11, YU404-H01, YU400-B07, YU400-D09, YU401-B01, YU401-G07, YU404-C02, YU403-G07, YU403-G05, YU391-B12, YU400-A03, YU400-D02, YU392-A09, YU392-B11, YU392-B12, YU392-E05, YU392-E06, YU392-G08, YU389-A03, YU392-G09, YU392-G12, YU392-H02, YU392-H04, YU402-F01, YU389-B11, YU394-D08, or YU390-A11 provided in Table 5.

[0012] In embodiments, the antibody is a human antibody. In embodiments, the antibody is a humanized antibody. In embodiments, the antibody is a chimeric antibody. In embodiments, the antibody comprises a murine variable domain and a human constant domain. In embodiments, the antibody also binds to cynomologous monkey CD25.

[0013] In another aspect, the disclosure provides a pharmaceutical composition comprising any antibody of the disclosure and, optionally, a pharmaceutically acceptable excipient. In another aspect, the disclosure provides a method of treating a subject in need of treatment, the method comprising administering to the subject a therapeutically effective amount of any antibody or pharmaceutical composition of the disclosure. In embodiments, the subject has cancer. In embodiments, the subject has an autoimmune disease or disorder. In another aspect, the disclosure provides a method of depleting the number of regulatory T cells in a subject, the method comprising administering to the subject a therapeutically effective amount of any antibody or pharmaceutical composition of the disclosure. In embodiments, the subject has cancer. In embodiments, the subject has an autoimmune disease or disorder.

[0014] In another aspect, the disclosure provides a kit comprising any antibody of the disclosure or an antibody of a pharmaceutical composition of the disclosure.

[0015] In some aspects, engineered immunogens having at least 60% sequence similarity to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11 are provided herein. In some embodiments, the engineered immunogen has at least 80% similarity to its sequence. In other embodiments, the engineered immunogen has at least 90% similarity to its sequence. In certain embodiments, the engineered immunogen shares at least one feature with CD25. In still further embodiments, the engineered immunogen binds to an antibody of CD25. In some embodiments, the engineered immunogen has a higher binding affinity for an antibody of CD25 at a pH below 7.0 as compared to its binding affinity at a pH between about 7.3 and about 7.5. In some embodiments, the engineered immunogen has a higher binding affinity for an antibody of CD25 at a pH between about 6.4 and about 6.6 as compared to its binding affinity at a pH between about 7.3 and about 7.5.

[0016] In still other embodiments, provided herein is a method of producing an antibody, the method comprising immunizing an animal with an engineered immunogen having at least 60% sequence similarity to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11; and producing an antibody. In some embodiments of the method, the antibody is an antibody against CD25. In certain embodiments, the antibody exhibits a higher binding affinity for CD25 at a pH below 7.0 as compared to its binding affinity at a pH between about 7.3 and about 7.5. In still further embodiments, the antibody exhibits a higher binding affinity for CD25 at a pH between about 6.4 and about 6.6 as compared to its binding affinity at a pH between about 7.3 and about 7.5. In some embodiments, the antibody does not block the binding of CD25 to IL-2. In other embodiments, the antibody blocks the binding of CD25 to IL-2. The method according to any one of claims 8 to 11, wherein the antibody does not block the binding of CD25 to IL-2. In some embodiments, the antibody prevents the heterotrimerization of IL-2R-alpha, IL-2R-beta, and IL-2R-gamma. In certain embodiments, the antibody is capable of binding to both the cis and trans orientations of CD25.

[0017] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent Office upon request and payment of the necessary fee. This application can be understood by reference to the following description, taken in conjunction with the accompanying drawings.

Brief Description of the Drawings

[0018]

Figure 1

[0019]

Figure 2

[0020]

Figure 3A

Figure 3B

Figure 3C

[0021]

Figure 4A

Figure 4B

[0022]

Figure 5

[0023]

Figure 6

[0024]

Figure 7

[0025]

Figure 8

[0026]

Figure 9

[0027]

Figure 10

[0028]

Figure 11

[0029]

Figure 12

[0030]

Figure 13

[0031]

Figure 14A

Figure 14B

[0032]

Figure 15

[0033]

Figure 16

[0034]

Figure 17

[0035]

Figure 18

[0036]

Figure 19

[0037]

Figure 20

[0038]

Figure 21

[0039]

Figure 22

[0040]

Figure 23

[0041]

Figure 24

[0042]

Figure 25

[0043]

Figure 26

[0044]

Figure 27

[0045]

Figure 28

[0046]

Figure 29

[0047]

Figure 30

[0048]

Figure 31

[0049]

Figure 32

[0050]

Figure 33

[0051]

Figure 34

DETAILED DESCRIPTION OF THE INVENTION

[0052] Engineered polypeptides that share structural and / or kinetic identity with a portion of the CD25 target are provided herein. The epitopes of interest include, but are not limited to, the 8 epitopes shown in FIG. 6. In some embodiments, the selected epitopes do not overlap with the binding sites (epitopes) for IL-2, daclizumab, and / or basiliximab. In some embodiments, the epitope overlaps with the epitope for 7G7B6. In some embodiments, the selected epitope is selected from 55-63, 12-20:127-132 (discontinuous epitope), 5-17, 5-11:156-163 (discontinuous epitope), 77-89, 147-157, 11-14, or 44-56. In some embodiments, the engineered polypeptide is conformationally stable and presents a CD25 epitope involved in the interaction with an antibody that specifically binds to CD25. In some embodiments, the engineered polypeptide presents a surface portion of CD25 that is known not to interact with an antibody that specifically binds to CD25. Such engineered polypeptides can be used, for example, to select and / or produce an antibody that specifically binds to CD25. I. Engineered Polypeptides.

[0053] In some embodiments, the engineered polypeptides provided herein share at least 46% structural and / or kinetic identity with a CD25 reference target, which is a portion of CD25 selected from those listed in the following table. As generally provided herein, % structural / kinetic identity is the root mean square inner product (RMSIP) identity (provided above in this specification) × 100%. In some embodiments, structural identity refers to sequence identity.

[0054] [Table 1]

[0055] In some embodiments, the engineered polypeptides provided herein are

[0056]

Table 2

[0057] In some embodiments, the polypeptide shares at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% structural and / or kinetic identity with the CD25 reference target. In some embodiments, the polypeptide shares at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity with the CD25 reference target.

[0058] In some embodiments, the engineered polypeptide is designed to mimic a selected CD25 epitope. For example, in some embodiments, the polypeptide comprises a mesoscale engineered molecule, such as a mesoscale engineered polypeptide. Methods of selecting a mesoscale engineered polypeptide, as well as compositions comprising such engineered polypeptides and methods of using the same are provided herein. For example, methods of using an engineered polypeptide in in vitro selection of antibodies are provided herein.

[0059] The engineered polypeptides of the present disclosure are between 1 kDa and 10 kDa and are referred to herein as “mesoscale.” Engineered polypeptides of this size may have certain advantages in some embodiments, such as protein-like functionality, a large theoretical space for selecting candidates, cell permeability, and / or structural and kinetic variability. The terms mesoscale peptide and mesoscale polypeptide are used interchangeably herein, and the term mesoscale molecule (MEM) is intended to cover these.

[0060] The methods provided herein include identifying a plurality of spatially related topological constraints that may in part be derived from a CD25 reference target, constructing a combination of these constraints, comparing candidate peptides to this combination, and selecting candidates having constraints that overlap with this combination. By using spatially related topological constraints, different aspects of the engineered polypeptide can be included in the combination depending on the intended use, or desired function, or another desired characteristic. Further, in some embodiments, not all of the constraints need to be derived from the CD25 reference target. Through such methods, in some embodiments, the selected engineered polypeptide is not simply a variation of the CD25 reference target (e.g., obtainable via single reference peptide mutagenesis or progressive modification), but can have an overall structure different from the reference peptide while still retaining the desired functional characteristics and / or important substructures.

[0061] Methods of using such engineered polypeptides are further provided herein, including methods of programmable in vitro selection using one or more engineered polypeptides. Such selection can be used, for example, in the identification of antibodies.

[0062] These methods and engineered polypeptides are described in more detail below. II. Methods of Selecting Engineered Polypeptides

[0063] In some aspects, a method of selecting an engineered polypeptide, comprising identifying one or more topological features of a CD25 reference target; designing spatially related constraints for each topological feature to produce a combination of CD25 reference target-derived constraints; comparing the spatially related topological features of a candidate peptide to the combination derived from the CD25 reference target; and Selecting candidate peptides having spatially related topological features that overlap with a combination of constraints derived from a CD25 reference target A method including the same is provided herein.

[0064] In some embodiments, one or more additional spatially related topological constraints not derived from the CD25 reference target are included in the combination. a. Spatially related topological constraints

[0065] The engineered polypeptides described herein are selected based on how closely they match a combination of spatially related topological constraints. This combination can also be described using the mathematical concept of a "tensor". In such a combination (or tensor), each constraint is described independently in three-dimensional space (e.g., spatially related), and the combination of these constraints in three-dimensional space provides a "map" of the representation of different desired features and their desired levels (if applicable) with respect to position. This map, in some embodiments, is not based on a linear or otherwise predetermined amino acid backbone, and thus can allow flexibility in the structure that can satisfy the desired combination being described. For example, in some embodiments, the "map" includes spatial regions where defined constraint limits can be appropriately satisfied by two adjacent amino acids - in some embodiments, these amino acids can be directly bonded (e.g., two consecutive amino acids), while in other embodiments, the amino acids are not directly bonded to each other but can be brought together spatially by peptide folding (e.g., not consecutive amino acids). The individual constraints themselves also do not necessarily have to be based on structure, and can include, for example, chemical descriptors and / or functional descriptors. In some embodiments, the constraints include structural descriptors, such as a desired secondary structure or amino acid residues. In certain embodiments, each constraint is selected independently.

[0066] For example, FIG. 1 is a schematic diagram showing the construction of a representative combination of spatially related topological constraints. The three constraints in FIG. 1 are arrangement, nearest neighbor distance, and atomic motion, and the nearest neighbor distance and atomic motion are combined in one picture. As shown, some constraints are mapped independently of the backbone location (e.g., atomic motion of a particular side chain), thus allowing a much more diverse structural conformation to be attempted compared to simply varying one or more positions on the reference scaffold. The three different constraints and their spatial descriptions are combined into a matrix (e.g., a tensor), and then a series of candidate peptides can be compared to this combination to identify new engineered polypeptides that meet the desired criteria. In some embodiments, one or more additional non-reference-derived constraints are also included in the combination. The comparison of candidate peptides to the defined combination can be performed, for example, using in silico methods for evaluating the constraints of each candidate peptide against the desired combination and a ratio of how well the candidates match. These candidates with the desired level of overlap with the defined combination can then be synthesized and evaluated using standard peptide synthesis methods known to those of skill in the art.

[0067] In some embodiments, the combination of constraints includes at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, between 3 and 12, between 3 and 10, between 3 and 8, between 3 and 6, or 3, or 4, or 5, or 6, independently selected spatially related topological constraints. One or more of the constraints are derived from a CD25 reference target. In some embodiments, each of the constraints is derived from a CD25 reference target. In other embodiments, at least one constraint is derived from a CD25 reference target and the remaining constraints are not derived from a reference target. For example, in some embodiments, a constraint between 1 and 9, between 1 and 7, between 1 and 5, or between 1 and 3 is derived from a CD25 reference target and a constraint between 1 and 9, between 1 and 7, between 1 and 5, or between 1 and 3 is not derived from a CD25 reference target.

[0068] Once a combination of constraints is constructed, a series of candidate peptides are compared to this combination to identify one or more new engineered polypeptides that meet the desired criteria. In some embodiments, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200 or at least 250 or more candidate peptides are compared to this combination to identify one or more new engineered polypeptides that meet the desired criteria. In some embodiments, for example, more than 250 candidate peptides, more than 300 candidate peptides, more than 400 candidate peptides, more than 500 candidate peptides, more than 600 candidate peptides, or more than 750 candidate peptides are compared. In some embodiments, a topology feature simulation is used to evaluate the overlap of the topology features of the candidate peptides compared to the combination of constraints, if present. In some embodiments, one or more candidate peptides are also compared to a CD25 reference target, and if present, the overlap of the topology features of the candidate peptides with those of the CD25 reference target is evaluated. In some embodiments, the engineered polypeptide is identified from computer samples of more than 5, more than 10, more than 20, more than 30, more than 40, more than 50, more than 60, more than 70, more than 80, more than 90, or more than 100 distinct peptides, and a topology feature simulation, and the engineered polypeptide is selected, and the selected engineered polypeptide has the highest overlap of topology features compared to the CD25 reference target among the sampled population.

[0069] Spatially related topological constraints used to construct a desired combination (e.g., a desired tensor) can be selected independently from each of a wide group of possible features. These can include, for example, constraints that describe structural, dynamical, chemical, or functional features, or any combination thereof.

[0070] Structural constraints can include, for example, interatomic distances, amino acid sequence similarity, solvent exposure, phi angles, psi angles, secondary structure or amino acid contacts, or any combination thereof.

[0071] Dynamical constraints can include, for example, atomic fluctuations, atomic energies, van der Waals radii, amino acid adjacencies, or non-covalent binding tendencies. Atomic energies can include, for example, pairwise attractive energy between two atoms, pairwise repulsive energy between two atoms, atomic-level solvation energy, pairwise charge attractive energy between two atoms, pairwise hydrogen bond attractive energy between two atoms, or non-covalent binding energy, or any combination thereof.

[0072] Chemical features can include, for example, chemical descriptors. Such chemical descriptors can include, for example, hydrophobicity, polarity, atomic volume, atomic radius, net charge, logP, HPLC retention time, van der Waals radius, charge pattern or H-bond pattern, or any combination thereof.

[0073] Functional features can include, for example, bioinformatics descriptors, biological responses, or biological functions. Bioinformatics descriptors can include, for example, BLOSUM similarity, pKa, zScale, Cruciani Properties, Kidera Factors, VHSE-scale, ProtFP, MS-WHIM score, T-scale, ST-scale, transmembrane tendency, protein buried region, helix tendency, sheet tendency, coil tendency, turn tendency, immunogenic tendency, presence of antibody epitope, and / or presence of protein interface, or any combination thereof.

[0074] In some embodiments, designing the constraints incorporates information about per-residue energy, per-residue interactions, per-residue fluctuations, per-residue interatomic distances, per-residue chemical descriptors, per-residue solvent exposure, per-residue amino acid sequence similarity, per-residue bioinformatics descriptors, per-residue non-covalent propensity, per-residue phi / psi angles, per-residue van der Waals radii, per-residue secondary structure propensity, per-residue amino acid adjacency, or per-residue amino acid contacts. In some embodiments, these features are used for a subset of the total residues in the CD25 reference target, or a subset of the total residues of the total combination of constraints, or a combination thereof. In some embodiments, one or more different features are used for one or more different residues. That is, in some embodiments, one or more features are used for a certain subset of residues and at least one different feature is used for a different subset of residues. In some embodiments, one or more of these features used to design one or more constraints are determined by computer simulation. Suitable computer simulation methods can include, for example, molecular dynamics simulation, Monte Carlo simulation, coarse-grained simulation, Gaussian network model, machine learning, or any combination thereof.

[0075] In some embodiments, multiple constraints are selected from one category. For example, in some embodiments, the combination includes two or more constraints that are independently a certain type of biological response. In some embodiments, two or more constraints are independently a certain type of secondary structure. In certain embodiments, two or more constraints are independently a certain type of chemical descriptor. In other embodiments, the combination does not include overlapping categories of constraints.

[0076] In some embodiments, one or more constraints are independently associated with a biological response or biological function. In some embodiments, this constraint is a spatially defined atomic level constraint, or a spatially defined shape / area / volume level constraint (e.g., a characteristic shape / area / volume that can be satisfied by several different atomic compositions), or a spatially defined kinetic level constraint (e.g., a characteristic kinetics or set of kinetics that can be satisfied by several different atomic compositions).

[0077] In some embodiments, one or more constraints are derived from a protein structure or peptide structure associated with a biological function or biological response. For example, in some embodiments, one or more constraints are derived from an extracellular domain, such as a G protein-coupled receptor (GPCR) extracellular domain or an ion channel extracellular domain. In some embodiments, one or more constraints are derived from a protein-protein interface junction. In some embodiments, one or more constraints are derived from a protein-peptide interface junction, such as an MHC-peptide or GPCR-peptide interface. In certain embodiments, the atoms or amino acids constrained by such a protein or peptide structure are atoms or amino acids associated with a biological function or biological response. In some embodiments, the atoms or amino acids in the engineered polypeptide constrained by such a protein or peptide structure are atoms or amino acids derived from a CD25 reference target. In some embodiments, one or more constraints are derived from a polymorphic region of the CD25 reference target (e.g., a region subject to allelic variation between individuals).

[0078] In some embodiments, one or more atoms associated with a biological function or biological response are selected from the group consisting of carbon, oxygen, nitrogen, hydrogen, sulfur, phosphorus, sodium, potassium, zinc, manganese, magnesium, copper, iron, molybdenum, and nickel. In certain embodiments, the atom is selected from the group consisting of oxygen, nitrogen, sulfur, and hydrogen.

[0079] In some embodiments where one of the constraints is one or more amino acids related to a biological function or biological response and / or the engineered polypeptide comprises one or more amino acids related to a biological function or biological response, the one or more amino acids are independently selected from the group consisting of the 20 proteinogenic naturally occurring amino acids, non-proteinogenic naturally occurring amino acids, and non-natural amino acids. In some embodiments, the non-natural amino acids are chemically synthesized. In certain embodiments, the one or more amino acids are selected from the 20 proteinogenic naturally occurring amino acids. In other embodiments, the one or more amino acids are selected from non-proteinogenic naturally occurring amino acids. In still further embodiments, the one or more amino acids are selected from non-natural amino acids. In still further embodiments, the one or more amino acids are selected from a combination of the 20 proteinogenic naturally occurring amino acids, non-proteinogenic naturally occurring amino acids, and non-natural amino acids.

[0080] The combination of constraints used to select the engineered polypeptides described herein includes at least one constraint derived from the CD25 reference target, although in some embodiments, one or more of the constraints of the combination are not derived from the CD25 reference target. Thus, in certain embodiments, the selected engineered polypeptide comprises one or more features not shared with the CD25 reference target.

[0081] In some embodiments, one or more constraints derived from a CD25 reference target and used in a combination describe the opposite of a feature observed in the CD25 reference target. Thus, for example, the CD25 reference target may have a particular pattern of positive charge, and the constraint related to charge is derived from this CD25 reference target, and the derived constraint describes a neutral or negative charge in a similar pattern. Thus, in some embodiments, one or more reverse constraints are derived from the CD25 reference target and included in the combination. Such reverse constraints may be useful, for example, in selecting engineered polypeptides as control molecules for a particular assay or panning method or as negative selection molecules in the programmable in vitro selection methods described herein.

[0082] In some embodiments, the combination of spatially defined topological constraints includes one or more non-reference-derived topological constraints. In some embodiments, one or more non-reference-derived topological constraints force or stabilize one or more secondary structure elements, force atomic fluctuations, change the total hydrophobicity of the peptide, change the solubility of the peptide, change the total charge of the peptide, enable detection in labeled or unlabeled assays, enable detection in in vitro assays, enable detection in in vivo assays, enable capture from complex mixtures, enable enzymatic processing, enable cell membrane permeability, enable binding to secondary targets, or change immunogenicity. In certain embodiments, one or more non-reference-derived topological constraints constrain one or more atoms or amino acids in a combination of constraints (or subsequently selected peptide) derived from a CD25 reference target. For example, in some embodiments, the combination of constraints includes a secondary structure derived from a CD25 reference target, and the combination of constraints also includes constraints that stabilize the secondary structure element (e.g., via additional hydrogen bonds, or hydrophobic interactions, or side chain stacking, or salt bridges, or disulfide bonds), and this stabilizing constraint does not exist in the CD25 reference target. In another example, in some embodiments, the combination of constraints (or subsequently selected peptide) includes one or more atoms or amino acids derived from a CD25 reference target, and the combination of constraints also includes constraints that force atomic fluctuations in at least a portion of the atoms or amino acids derived from the target reference, and this constraint does not exist in the target reference. In some embodiments, one or more non-reference-derived constraints are inverse constraints. For example, in some embodiments, two combinations of constraints are constructed to select engineered polypeptides with inverse characteristics. In some such embodiments, the first combination of constraints will include one or more constraints derived from a CD25 reference target, and one or more constraints not derived from a CD25 reference target; the second combination of constraints will include the same one or more constraints derived from a CD25 reference target, and one or more inverses of the non-CD25 reference target constraints of the first combination. b. CD25 reference target

[0083] Any suitable CD25 reference target can be used to induce one or more spatially related topological constraints for use in the methods provided herein. In some embodiments, the CD25 reference target is the full-length native protein. In other embodiments, the CD25 reference target is a portion of the full-length native protein. In still further embodiments, the CD25 reference target is a non-native protein, or a portion thereof.

[0084] In some embodiments, the CD25 reference target is

[0085] [Table 3] selected from.

[0086] For example, in some embodiments, the CD25 reference target is a portion of CD25, such as an epitope or predicted epitope. In some embodiments, the methods provided herein can be used to select one or more engineered polypeptides that are immunogenic and can be used to generate one or more antibodies that specifically bind to the protein from which the target reference is derived. In still further embodiments, the methods provided herein can then be used to select one or more engineered polypeptides that can then be used to select one or more binding partners of a protein of interest, such as an antibody, a phage displaying a Fab, or a phage displaying a scFv. c. Comparison of Constraints

[0087] In some embodiments, one or more constraints (e.g., reference-derived or non-reference-derived) are determined by molecular simulations (e.g., molecular dynamics) or laboratory measurements (e.g., NMR), or a combination thereof. When the constraints are induced and combined, the engineered polypeptide candidates are generated, in some embodiments, using computational protein design (e.g., Rosetta). In some embodiments, other methods of sampling the peptide space are used. Then, a dynamics simulation can be performed on the candidate engineered polypeptides to obtain the parameters of the selected constraints. A covariance matrix of atomic fluctuations is generated for the CD25 reference target, and a covariance matrix is generated for each residue in each of the candidate engineered polypeptides, and these covariance matrices are compared to determine overlap. Principal component analysis is performed to calculate the eigenvectors and eigenvalues for each covariance matrix - one covariance matrix for the CD25 reference target and one covariance for each of the candidate engineered polypeptides - and the eigenvector with the largest eigenvalue is retained.

[0088] The eigenvectors describe the first, second, third, Nth dominant motions observed in a set of simulated molecular structures. Without wishing to be bound by any theory, if the candidate engineered polypeptide moves like the CD25 reference target, its eigenvectors will be similar to the eigenvectors of the CD25 reference target. The similarity of the eigenvectors corresponds to those components (3D vectors centered on each CA atom) that are aligned and point in the same direction.

[0089] In some embodiments, this similarity between the eigenvector of the candidate engineered polypeptide and the eigenvector of the CD25 reference target is calculated using the inner product of the two eigenvectors. The inner product value is 0 if the two eigenvectors are 90 degrees relative to each other, or 1 if the two eigenvectors point exactly in the same direction. Without wishing to be bound by theory, the ordering of the eigenvectors is based on their eigenvalues, which may not necessarily be the same between different molecules due to the probabilistic nature by which molecular dynamics (MD) simulations sample the underlying energy landscapes of two different molecules. Thus, in some embodiments, inner products between multiple hierarchically ranked eigenvectors are required (e.g., eigenvector 1 of the engineered polypeptide × eigenvectors 2, 3, 4, etc. of the CD25 reference target). Further, molecular motion is complex and more than one (or more than a few) dominant / principal modes of motion may be involved. Accordingly, in some embodiments, inner products for all pairs of eigenvectors in the candidate engineered polypeptide and the CD25 reference target are calculated. This results in a matrix of inner products, the dimension of which is determined by the number of eigenvectors analyzed. For example, for 10 eigenvectors, the matrix of inner products is 10×10. This matrix of inner products can be converted to a single value by calculating the root mean square of the squares of the 100 (in the case of 10×10) inner products. This is the root mean square inner product (RMSIP). From this comparison, one or more candidate engineered polypeptides having similarity to a defined combination of constraints are selected. d. Further steps

[0090] In some embodiments, the selection of one or more engineered polypeptides involves one or more additional steps. For example, in some embodiments, engineered polypeptide candidates are selected based on similarity to a defined combination of spatially related topological constraints as described herein, and then undergo one or more analyses to determine one or more additional features and one or more structural modifications to impart or enforce a desired feature. For example, in some embodiments, selected candidates are analyzed, e.g., via molecular dynamics simulations, to determine the overall stability of the molecule and / or the propensity for a particular folded structure. In some embodiments, one or more modifications are made to the engineered polypeptide to impart or enhance a desired level of stability or a desired propensity for a desired folded structure. Such modifications can include, for example, the introduction of one or more crosslinks (e.g., disulfide bonds), salt bridges, hydrogen bond interactions or hydrophobic interactions, or any combination thereof.

[0091] The methods provided herein can further include assaying one or more selected engineered polypeptides for one or more desired features, such as a desired binding interaction or activity. Any suitable assay can be used as needed to measure the desired feature.

[0092] In other aspects, engineered polypeptides, such as engineered polypeptides selected via the methods described herein, are provided herein. In some embodiments, the engineered polypeptide has a molecular mass between 1 kDa and 10 kDa and contains up to 50 amino acids. In certain embodiments, the engineered polypeptide has a molecular mass between 2 kDa and 10 kDa, between 2 kDa and 10 kDa, between 3 kDa and 10 kDa, between 4 kDa and 10 kDa, between 5 kDa and 10 kDa, between 6 kDa and 10 kDa, between 7 kDa and 10 kDa, between 8 kDa and 10 kDa, between 9 kDa and 10 kDa, between 1 kDa and 9 kDa, between 1 kDa and 8 kDa, between 1 kDa and 7 kDa, between 1 kDa and 6 kDa, between 1 kDa and 5 kDa, between 1 kDa and 4 kDa, between 1 kDa and 3 kDa, or between 1 kDa and 2 kDa. In certain embodiments, the engineered polypeptide contains up to 45 amino acids, up to 40 amino acids, up to 35 amino acids, up to 30 amino acids, up to 25 amino acids, up to 20 amino acids, at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, or at least 40 amino acids.

[0093] In certain embodiments, the engineered polypeptide comprises a combination of spatially related topological constraints, one or more of which are constraints derived from the CD25 reference target. Any of the constraints described herein may, in some embodiments, be used in combination. In still further embodiments, between 10% and 98% of the amino acids of the engineered polypeptide satisfy one or more constraints derived from the CD25 reference target (e.g., if the engineered polypeptide comprises 50 amino acids, between 5 and 49 amino acids satisfy one or more constraints derived from the CD25 reference target). In some embodiments, between 20% and 98%, between 30% and 98%, between 40% and 98%, between 50% and 98%, between 60% and 98%, between 70% and 98%, between 80% and 98%, between 90% and 98%, between 10% and 90%, between 10% and 80%, between 10% and 70%, between 10% and 60%, between 10% and 50%, between 10% and 40%, between 10% and 30% or between 10% and 20% of the amino acids of the engineered polypeptide satisfy one or more constraints derived from the CD25 reference target. In still further embodiments, one or more amino acids that satisfy one or more constraints derived from the CD25 reference target have a structural homology with the CD25 reference target with a root mean square deviation (RSMD) of less than 8.0 Å, less than 7.5 Å, less than 7.0 Å, less than 6.5 Å, less than 6.0 Å, less than 5.5 Å or less than 5.0 Å. In some embodiments, the engineered polypeptide has a molecular mass between 1 kDa and 10 kDa; contains at most 50 amino acids; a combination of spatially related topological constraints, one or more of which are constraints derived from the CD25 reference target; between 10% and 98% of the amino acids of the engineered polypeptide satisfy one or more constraints derived from the CD25 reference target; and the amino acids that satisfy one or more constraints derived from the CD25 reference target have a structural homology with the CD25 reference target with a root mean square deviation (RSMD) of less than 8.0 Å.

[0094] In some embodiments, the amino acids of the engineered polypeptide that satisfy one or more CD25 reference target-derived constraints have a sequence homology between 10% and 90%, between 20% and 90%, between 30% and 90%, between 40% and 90%, between 50% and 90%, between 60% and 90%, between 70% and 90%, or between 80% and 90% with respect to the CD25 reference target. In some embodiments, the amino acids that satisfy one or more CD25 reference target-derived constraints are between 30 Å 2 and 3000 Å 2 or between 100 Å 2 and 3000 Å 2 or between 250 Å 2 and 3000 Å 2 or between 500 Å 2 and 3000 Å 2 or between 750 Å 2 and 3000 Å 2 or between 1000 Å 2 and 3000 Å 2 or between 1250 Å 2 and 3000 Å 2 or between 1500 Å 2 and 3000 Å 2 or between 1750 Å 2 and 3000 Å 2 or between 2000 Å 2 and 3000 Å 2 or between 2250 Å 2 and 3000 Å 2 or between 2500 Å 2 and 3000 Å 2 or between 2750 Å 2 and 3000 Å 2 and have a van der Waals surface area overlap with the reference between.

[0095] The combination of constraints satisfied by the engineered polypeptide can include two or more, three or more, four or more, five or more, six or more, or seven or more CD25 reference target-derived constraints. The combination can include one or more constraints not derived from the CD25 reference target, as described elsewhere in this disclosure. These reference-derived constraints, and, if present, non-reference-derived constraints, can each independently be any of the constraints described herein, for example, any of the structural, kinetic, chemical, or functional features described herein, or any combination thereof.

[0096] In some embodiments, the engineered polypeptide comprises at least one structural difference when compared to the CD25 reference target. Such structural differences can include, for example, differences in sequence, number of amino acid residues, total number of atoms, total hydrophilicity, total hydrophobicity, total positive charge, total negative charge, one or more secondary structures, shape factor, Zernike descriptors, van der Waals surface, nodes and edges of a structural graph, volumetric surface, electrostatic potential surface, hydrophobic potential surface, local diameter, local surface features, skeleton model, charge density, hydrophilic density, surface-to-volume ratio, amphiphilic density or surface roughness, or any combination thereof. In some embodiments, the difference in one or more features (e.g., one or more features described herein) is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or higher than 100% when compared to the feature in the CD25 reference target, where applicable for the type of feature. For example, in some embodiments, the difference is the total number of atoms, and the engineered polypeptide has at least 10%, at least 20% or at least 30% more atoms than the CD25 reference target, or at least 10%, at least 20% or at least 30% fewer atoms than the CD25 reference target. In some embodiments, the difference is in the total positive charge, and the total positive charge of the engineered polypeptide is at least 10%, at least 20%, at least 30%, at least 40% or at least 50% greater (e.g., more positive) than the CD25 reference target, while in other embodiments, the total positive charge of the engineered polypeptide is at least 10%, at least 20%, at least 30%, at least 40% or at least 50% less (e.g., less positive) than the CD25 reference target.

[0097] In some embodiments, the combination of spatially defined topological constraints includes one or more secondary structure elements that are not present in the CD25 reference target. Thus, in some embodiments, the engineered polypeptide includes one or more secondary structure elements that are not present in the CD25 reference target. In some embodiments, the combination and / or the engineered polypeptide includes one secondary structure element, two secondary structure elements, three secondary structure elements, four secondary structure elements, or more than four secondary structure elements that are not found in the CD25 reference target. In some embodiments, each secondary structure element is independently selected from the group consisting of helices, sheets, loops, turns, and coils. In some embodiments, each secondary structure element that is not present in the CD25 reference target is independently an α-helix, β-bridge, β-strand, 3 10 helix, π-helix, turn, loop, or coil.

[0098] In certain embodiments, the CD25 reference target includes one or more atoms associated with a biological response or biological function (e.g., as described herein); the engineered polypeptide includes one or more atoms associated with a biological response or biological function (e.g., as described herein); and the atomic fluctuations of these atoms in the engineered polypeptide overlap the atomic fluctuations of these atoms in the CD25 reference target. Thus, for example, in some embodiments, the atoms themselves are different atoms, but their atomic fluctuations overlap. In other embodiments, the atoms are the same atoms and their atomic fluctuations overlap. In still further embodiments, the atoms are independently the same or different. In some embodiments, the overlap is a root mean square inner product (RMSIP) greater than 0.25. In some embodiments, the overlap is an RMSIP greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, greater than 0.5, greater than 0.55, greater than 0.6, greater than 0.65, greater than 0.7, greater than 0.75, greater than 0.8, greater than 0.85, greater than 0.9, or greater than 0.95. In certain embodiments, the RMSIP is

[0099]

Number

[0100] In some embodiments, the engineered polypeptide comprises atoms or amino acids (or combinations thereof) associated with a biological response or biological function, at least some of these atoms or amino acids or combinations are derived from the CD25 reference target, and the specific constraints of the set of atoms or amino acids in the engineered polypeptide and the set in the CD25 reference target can be described by a matrix. In some embodiments, the matrix is an L×L matrix. In other embodiments, the matrix is an S×S×M matrix. In still further embodiments, the matrix is an L×2 phi / psi angle matrix.

[0101] For example, in some embodiments, the atomic fluctuations of atoms or amino acids in an engineered polypeptide associated with a biological response or biological function are described by an L×L matrix; a portion of these atoms or amino acids is derived from a CD25 reference target; and the atomic fluctuations in this portion of the CD25 reference target are described by an L×L matrix. In some embodiments, the adjacency (with respect to amino acid position) of each set is described by a corresponding L×L matrix. In certain embodiments, the mean percentage error (MPE) across all matrix elements (i,j) of the L×L atomic fluctuation or adjacency matrix of the engineered polypeptide is 75% or less when compared to the corresponding (i,j) element in the atomic fluctuation or adjacency matrix of the CD25 reference target for a portion of the engineered polypeptide derived from the CD25 reference target. In some embodiments, the MPE is less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, or less than 40% when compared to the corresponding element in the CD25 reference matrix for a portion of the engineered polypeptide derived from the CD25 reference target. In some embodiments where the matrix represents atomic fluctuations, L is the number of amino acid positions, and the (i,j) value in the atomic fluctuation matrix element is the sum of the intramolecular atomic fluctuations for the i-th and j-th amino acids if the (i,j) interatomic distance is 7 Å or less, or zero if the (i,j) interatomic distance is greater than 7 Å or if (i,j) is on the diagonal. Alternatively, in some embodiments, the interatomic distance can serve as a weighting factor for the atomic fluctuation matrix element (i,j) instead of a multiplier of 0 or 1. In certain embodiments, the i-th and j-th atomic fluctuations and interatomic distances can be determined by molecular simulations (e.g., molecular dynamics) and / or laboratory measurements (e.g., NMR). In other embodiments where the matrix represents adjacency, L is the number of amino acid positions, and the value in the adjacency matrix element (i,j) is the intramolecular interatomic distance between the i-th amino acid and the j-th amino acid if the interatomic distance is 7 Å or less, or zero if the interatomic distance is greater than 7 Å or if (i,j) is on the diagonal.Alternatively, in some embodiments, the interatomic distance can act as a weighting factor for the adjacency matrix element (i,j) instead of a multiplier of 0 or 1. In certain embodiments, the interatomic distance between the i-th and j-th atoms can be determined by molecular simulation (e.g., molecular dynamics) and / or laboratory measurement (e.g., NMR).

[0102] In certain embodiments, an atom or amino acid in the engineered polypeptide that is associated with a response or function has an average percentage error (MPE) of less than 75% compared to a reference described by a chemical descriptor vector of topological constraints and the same chemical descriptor for a portion of the engineered polypeptide derived from the CD25 reference target, and each i-th element in the chemical descriptor vector corresponds to an amino acid position index. In some embodiments, the MPE is less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45% or less than 40% compared to a reference described by the same chemical descriptor for a portion of the engineered polypeptide derived from the CD25 reference target.

[0103] In yet further embodiments, the matrix is an L×2 phi / psi angle matrix, and an atom or amino acid in the engineered polypeptide that is associated with a response or function has an MPE of less than 75% with respect to the reference phi / psi angle matrix in a portion of the engineered polypeptide derived from the reference target, where L is the number of amino acid positions, and the phi, psi values are of dimensions (L,1) and (L,2), respectively. In some embodiments, the MPE is less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45% or less than 40% with respect to the reference phi / psi angle matrix in a portion of the engineered polypeptide derived from the reference target. In some embodiments, the phi / psi values are determined by molecular simulation (e.g., molecular dynamics), knowledge-based structure prediction or laboratory measurement (e.g., NMR).

[0104] In some embodiments, the matrix is an S×S×M secondary structure element interaction matrix, and the atoms or amino acids in the engineered polypeptide that are associated with a response or function have an average percentage error (MPE) of less than 75% compared to a reference secondary structure element relationship matrix in a portion of the engineered polypeptide derived from a reference target, where S is the number of secondary structure elements and M is the number of interaction descriptors. In some embodiments, the MPE is less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, or less than 40% compared to a reference secondary structure element relationship matrix in a portion of the engineered polypeptide derived from a reference target. Interaction descriptors can include, for example, hydrogen bonds, hydrophobic packing, van der Waals interactions, ionic interactions, covalent bridges, chirality, orientation or distance, or any combination thereof. In the secondary structure element interaction matrix subscript, (i,j,m) is the value of the mth interaction descriptor between the ith and jth secondary structure elements.

[0105] The average percentage error (MPE) for different matrices described herein is

[0106]

Number

[0107] In some embodiments, the engineered polypeptide has less than 75% MPE as compared to the CD25 reference target. In certain embodiments, the engineered polypeptide has less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45% or less than 40% MPE as compared to the CD25 reference target. In some embodiments, the MPE is determined by total topological constraint distance (TCD), topological clustering coefficient (TCC), Euclidean distance, power distance, Soergel distance, Canberra distance, Sørensen distance, Jaccard distance, Mahalanobis distance, Hamming distance, Quantitative Estimate of Likeness (QEL) or chain topological parameter (CTP). e. Secondary structure elements

[0108] In some embodiments, at least a portion of the engineered polypeptide is topologically constrained to one or more secondary structure elements. In some embodiments, the atoms or amino acids in the engineered polypeptide that are associated with a biological response or biological function are topologically constrained to one or more secondary structure elements. In some embodiments, the secondary structure elements are independently a sheet, helix, turn, loop or coil. In some embodiments, the secondary structure elements are independently an α-helix, β-bridge, β-strand, 3 10 helix, π-helix, turn, loop or coil. In certain embodiments, one or more of the secondary structure elements to which at least a portion of the engineered polypeptide is topologically constrained are present in the CD25 reference target. In some embodiments, at least a portion of the engineered polypeptide is topologically constrained to a combination of secondary structure elements, each element being independently selected from the group consisting of a sheet, helix, turn, loop and coil. In still further embodiments, each element is independently selected from the group consisting of an α-helix, β-bridge, β-strand, 3 10 helix, π-helix, turn, loop and coil.

[0109] In some embodiments, the secondary structure element is a parallel or antiparallel sheet. In some embodiments, the sheet secondary structure comprises two or more residues. In some embodiments, the sheet secondary structure comprises 50 or fewer residues. In still further embodiments, the sheet secondary structure comprises between two and 50 residues. The sheet can be parallel or antiparallel. In some embodiments, the parallel sheet secondary structure can be described as having two parallel strands i, j (the N-termini of the i strand and the j strand are in opposite orientations), and a hydrogen bonding pattern of residues i:j. In some embodiments, the antiparallel sheet secondary structure can also be described as having two antiparallel strands i, j (the N-termini of the i strand and the j strand are in the same orientation), and a hydrogen bonding pattern of residues i:j-1, i:j+1. In certain embodiments, the strand orientation and hydrogen bonding can be determined by knowledge-based or molecular dynamics simulations and / or laboratory measurements.

[0110] In some embodiments, the secondary structure element is a helix. The helix can be clockwise or counterclockwise. In some embodiments, the helix has a residues per turn value between 2.5 and 6.0, and a pitch between 3.0 Å and 9.0 Å. In some embodiments, the residues per turn and the pitch are determined by knowledge-based or molecular dynamics simulations and / or laboratory measurements.

[0111] In some embodiments, the secondary structure element is a turn. In some embodiments, the turn comprises between two and seven residues, and one or more inter-residue hydrogen bonds. In some embodiments, the turn comprises two, three, or four inter-residue hydrogen bonds. In certain embodiments, the turn is determined by knowledge-based or molecular dynamics simulations and / or laboratory measurements.

[0112] In yet further embodiments, the secondary structure element is a coil. In certain embodiments, the coil comprises between 2 and 20 residues and zero predicted inter-residue hydrogen bonds. In some embodiments, these coil parameters are determined by knowledge-based or molecular dynamics simulations and / or laboratory measurements.

[0113] In yet further embodiments, the engineered polypeptide comprises one or more atoms or amino acids derived from a CD25 reference target, and these atoms or amino acids have a secondary structure. In some embodiments, these atoms or amino acids are associated with a biological response or biological function. In some embodiments, the secondary structure motif vector of the atoms or amino acids in the engineered polypeptide, for a portion of the engineered polypeptide derived from the CD25 reference target, has a cosine similarity greater than 0.25 compared to the secondary structure motif vector of the CD25 reference target, where the length of the vector is the number of secondary structure motifs, and the value at the i-th vector position defines the identity of the secondary structure motif (e.g., helix, sheet) derived from a lookup table. In some embodiments, each motif comprises two or more amino acids. In certain embodiments, the motifs include, for example, α-helix, β-bridge, β-strand, 3 10 helix, π-helix, turn and loop. In some embodiments, the cosine similarity is greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45 or greater than 0.5 compared to the secondary structure motif vector of the CD25 reference target, for a portion of the engineered polypeptide derived from the CD25 reference target. The cosine similarity can be calculated by

[0114]

Number

[0115] In some embodiments, one or more atoms or amino acids of engineered polypeptides derived from a CD25 reference target may be compared to the corresponding atoms or amino acids of the CD25 reference target using a total topological constraint distance (TCD). In some embodiments, the total TCD of the atoms or amino acids of these engineered polypeptides derived from a CD25 reference target is + / - 75% compared to the TCD distance of the corresponding atoms in the CD25 reference target, and two intramolecular topological constraints interact if their pairwise distance is 7 Å or less. In some embodiments, the atoms or amino acids in the engineered polypeptides being compared are associated with a biological function or biological response. The i-th and j-th pairwise distances of two atoms or amino acids may be determined in some embodiments by molecular simulation (e.g., molecular dynamics) and / or laboratory measurements (e.g., NMR). An exemplary equation for calculating the total topological constraint distance (TCD) is:

[0116]

number

[0117] In some embodiments, one or more atoms or amino acids of an engineered polypeptide derived from a CD25 reference target can be compared to the corresponding atoms or amino acids of the CD25 reference target using a chain topology parameter (CTP). In some embodiments, the CTP of these atoms or amino acids of the engineered polypeptides is + / - 50% compared to the CTP of the corresponding atoms or amino acids in the CD25 reference target, and the intramolecular topological interactions are at a pairwise distance of 7 Å or less. In some embodiments, the atoms or amino acids in the engineered polypeptides being compared are related to a biological function or biological response. In some embodiments, the pairwise distance of the i-th and j-th can be determined by molecular simulation (e.g., molecular dynamics) and / or laboratory measurement (e.g., NMR). Exemplary equations for evaluating CTP are

[0118] [Number] where i and j are position indices for the amino acids (i,j), and S ij is the difference between the topological constraints S(i) and S(j), Δ(i,j) = 1 when the amino acid (i,j) is within the 7 Å chain topological interaction threshold, L is the number of amino acid positions in the peptide or corresponding CD25 reference target, and N is the total number of intramolecular contacts in the engineered polypeptide or CD25 reference target that satisfy the 7 Å topological interaction threshold. Alternatively, in some embodiments, Δ(i,j) can act as a weighting factor for the difference instead of a multiplier of 0 or 1. ij

[0119] ​In some embodiments, one or more atoms or amino acids of the engineered polypeptide derived from the CD25 reference target can be compared to the atoms or amino acids of the corresponding CD25 reference target using a quantitative estimate of likeness (QEL). In some embodiments, the QEL of these atoms or amino acids of the engineered polypeptide is + / - 50% compared to the QEL of the corresponding atoms or amino acids in the CD25 reference target. In some embodiments, the atoms or amino acids in the engineered polypeptide being compared are associated with a biological function or biological response. Exemplary equations for determining the QEL are

[0120] [Number] where di is a topological constraint for the i-th amino acid or atomic position, or a composition function (e.g., a linear regression function) that combines multiple topological constraints for the i-th amino acid or atomic position, and n is the number of amino acid or atomic positions in the peptide or CD25 reference target.

[0121] In some embodiments, one or more atoms or amino acids of the engineered polypeptide derived from the CD25 reference target can be compared to the corresponding atoms or amino acids of the CD25 reference target using the topological clustering coefficient (TCC) vector and the mean percentage error (MPE). In some embodiments, the TCC vector and MPE are less than 75% compared to the TCC of the corresponding atoms or amino acids in the CD25 reference target, and each element (i) of the vector is the topological clustering coefficient for the i-th amino acid position, where the intramolecular cluster is defined by an interaction edge distance of 7 Å or less from the i-th amino acid position and two edges: i-j, j-l. In some embodiments, the atoms or amino acids in the engineered polypeptide being compared are associated with a biological function or biological response. In some embodiments, the i-th, j-th, and l-th edge distances can be determined by molecular simulations (e.g., molecular dynamics) and / or laboratory measurements (e.g., NMR). An exemplary equation for evaluating the topological clustering coefficient for the i-th position is

[0122] [Number] where Δ(i,j) = 1, Δ(i,l) = 1, Δ(j,l) = 1 when the intramolecular amino acid positions: (i,j), (i,l), (j,l) are within the interaction edge threshold of 7 Å, respectively, and S ijl is the combination (e.g., sum) of the topological constraints for the i-th, j-th, and l-th amino acids, L is the number of amino acid positions in the peptide vector or the corresponding CD25 reference target vector, and N c is the number of intramolecular interacting amino acid positions for the i-th amino acid that satisfy the 7 Å edge threshold from the i-th amino acid and the two edges: i-j, j-l. Alternatively, in some embodiments, Δ(i,j), Δ(i,l), and Δ(j,l) can act as weighting factors for the clustering coefficient vector element (i) instead of being multipliers of 0 or 1.

[0123] In yet further embodiments, one or more atoms or amino acids of the engineered polypeptide derived from the CD25 reference target can be compared to the corresponding atoms or amino acids of the CD25 reference target using an L×M topological constraint matrix and the mean percentage error (MPE) of the Euclidean distance, power distance, Soergel distance, Canberra distance, Sørensen distance, Jaccard distance, Mahalanobis distance, or Hamming distance across all M dimensions. The L×M matrix element (l,m) contains the mth constraint value for the lth amino acid position, where L is the number of amino acid positions and M is the number of distinct topological constraints. In some embodiments, the MPE of the L×M matrix of the engineered polypeptide is less than 75% compared to the matrix of the corresponding atoms or amino acids of the CD25 reference target. In some embodiments, the MPE is less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, or less than 45%. In some embodiments, the atoms or amino acids in the engineered polypeptide being compared are associated with a biological function or biological response. III. Programmable In Vitro Selection

[0124] In other aspects, methods are further provided herein for using the engineered polypeptides described herein when selecting binding partners using a series of programmed selection steps, where at least one selection step comprises evaluating the interaction of a pool of potential binding partners with the engineered polypeptide.

[0125] In some embodiments, methods are provided herein for manipulating the selection of binding molecules using two or more selection molecules. In some embodiments, the method comprises subjecting a pool of candidate binding molecules to at least one round of selection, each round comprising at least one negative selection step in which at least a portion of the pool is screened against a negative selection molecule, and at least one positive selection step in which at least a portion of the pool is screened against a positive selection molecule. In some embodiments, the method comprises at least 2 rounds, at least 3 rounds, at least 4 rounds, at least 5 rounds, at least 6 rounds, at least 7 rounds, at least 8 rounds, at least 9 rounds, at least 10 rounds, or more rounds, each round independently comprising at least one negative selection step and at least one positive selection step. In some embodiments, each round independently comprises more than one negative selection step or more than one positive selection step, or a combination thereof. FIG. 5 provides an exemplary schematic diagram showing in detail three rounds of selection, wherein the first and third rounds comprise more than one negative selection step, and the first round further comprises more than one positive selection round. As shown in the scheme, two negative selection molecules (“baits”) are used in the first round, and three negative selection molecules are used in the third round. Further, two positive selection molecules are used in the first round.

[0126] In some embodiments where the method includes more than one round, each negative and positive selection molecule is independently selected. In other embodiments, the same negative selection molecule or the same positive selection molecule, or combinations thereof, can be used in more than one round. For example, in FIG. 5, the same negative selection molecule used in round 1 is used again in round 3, and an additional third negative selection molecule is also included in round 3. The order of the negative selection step and the positive selection step can, in certain embodiments, be independently selected within each round of selection. Thus, for example, in some embodiments, the method includes one or more rounds of selection, and each round first includes a negative selection step and then a positive selection step. In other embodiments, the method includes one or more rounds of selection, and each round first includes a positive selection step and then a negative selection step. In still further embodiments, the method includes one or more rounds of selection, and each round independently includes a negative selection step and a positive selection step, and in each round, the negative selection step is independently before or after the positive selection step.

[0127] Such methods of selection use positive (+) and negative (−) steps to manipulate a library of candidate binding molecules towards or away from a particular desired characteristic, such as binding specificity or binding affinity. By using multiple steps with both positive and negative selection molecules, the pool of candidates can be directed in a stepwise fashion to select for desired characteristics and against undesired characteristics. Further, in some embodiments, the order of each step within each round, and the order of the rounds relative to each other, can direct the selection in different directions. Thus, for example, in some embodiments, a method that includes one round using a (+) selection followed by a (−) selection will result in a different final pool of candidates than if the (−) selection is first and then the (+) selection. From this, it can be inferred that for methods that include multiple rounds, the order of the selection steps can result in different final pools of selected candidates even when the same positive and negative selection molecules are used overall.

[0128] In some embodiments, a selection molecule having the reverse characteristics of another selection molecule is used. This can be useful, for example, to ensure that candidate binding partners identified using a positive selection molecule (or excluded due to a negative selection molecule) are identified (or excluded) due to the desired trait (or undesired trait), rather than due to separate non - relevant binding interactions. To remove binding partners that are bound through non - relevant interactions, a reverse selection molecule having a structure and characteristics similar or identical to the selection molecule, except for the residue / structure that confers the desired trait (or undesired trait), can be used. For example, if interaction with a specific charge pattern in a positive selection molecule is desired, a reverse negative selection molecule in which the residues providing that charge pattern are replaced with uncharged residues and / or residues of the opposite charge can be used. Thus, for a particular selection molecule, multiple different corresponding reverse selection molecules may be possible.

[0129] In the selection methods provided herein, at least one of the selection molecules is an engineered polypeptide as described herein. In some embodiments, more than one engineered polypeptide is used. In some embodiments, each engineered polypeptide is independently a positive or negative selection molecule. In certain embodiments, each selection molecule used in one or more rounds of selection is independently an engineered polypeptide. In other embodiments, at least one molecule that is not an engineered polypeptide is used as a selection molecule. Such selection molecules that are not engineered polypeptides can include, for example, naturally occurring polypeptides or portions thereof. In other embodiments, one or more selection molecules that are not engineered polypeptides can include, for example, non-naturally occurring polypeptides or portions thereof. For example, in some embodiments, one or more selection molecules (e.g., positive or negative selection molecules) are an immunogen, an antibody, a cell surface receptor, or a transmembrane protein, or a signaling protein, or a multi-protein complex, or a peptide-protein complex, or any portion thereof, or any combination thereof. In some embodiments, one or more selection molecules are CD25, or any portion of CD25.

[0130] The positive and negative features selected for or against it in each step can be selected from various traits and can be adjusted depending on the desired characteristics of the one or more final binding molecules obtained. Such desired characteristics can depend, for example, on the intended use of the one or more binding molecules. For example, in some embodiments, the methods provided herein are used to screen antibody candidates for one or more positive features, such as high specificity, and one or more negative features, such as cross-reactivity. It should be understood that what is considered a positive feature in one context can be a negative feature in another context, and vice versa. Thus, the positive selection molecules in one series of selection rounds can be, in some embodiments, negative selection molecules in a different series of selection rounds, or when selecting different types of binding molecules, or when selecting the same type of binding molecules for different purposes.

[0131] In some embodiments, each selection feature is selected independently from the group consisting of amino acid sequence, polypeptide secondary structure, molecular dynamics, chemical characteristics, biological function, immunogenicity, CD25 reference target(s) multispecificity, cross-species CD25 reference target reactivity, selectivity for desired CD25 reference target(s) over undesired reference target(s), selectivity for reference target(s) within a sequence and / or structurally homologous family, selectivity for reference target(s) having similar protein function, selectivity for distinct desired reference target(s) from a larger family of undesired targets having high sequence and / or structural homology, selectivity for distinct reference target alleles or mutations, selectivity for chemical modifications at the residue level of distinct reference targets, selectivity for cell type, selectivity for tissue type, selectivity for tissue environment, tolerance for structural diversity of reference target(s), tolerance for sequence diversity of reference target(s), and tolerance for kinetic diversity of reference target(s). In some embodiments, each selection feature is a different type of selection feature. In other embodiments, two or more selection features are of the same type but are different features. For example, in some embodiments, two or more selection features are polypeptide secondary structure, one is a positive selection for a desired polypeptide secondary structure, and one is a negative selection for an undesired polypeptide secondary structure. In some embodiments, two or more selection features are selectivity for cell type, the positive selection feature is selectivity for a particular desired cell type, and the negative selection feature is selectivity for a particular undesired cell type. In some embodiments, two or more, three or more, four or more, five or more or six or more selection features are of the same type.

[0132] In some embodiments, the selection feature is binding to an engineered polypeptide of the present disclosure. For example, the engineered polypeptides shown in FIGS. 7, 1, 8 and 9 can be used to select for antibodies (or other binding agents) that specifically bind to the epitopes shown in FIGS. 6 and 7. Exemplary selection strategies are provided in Table 10.

[0133] In yet another aspect, provided herein is a composition comprising two or more selection steering polypeptides, wherein each polypeptide is independently a positive selection molecule comprising one or more positive steering features or a negative selection molecule comprising one or more negative steering features. Such features, in some embodiments, are amino acid sequence, polypeptide secondary structure, molecular dynamics, chemical characteristics, biological function, immunogenicity, multi-specificity for reference target(s), cross-species reference target reactivity, selectivity for desired reference target(s) over undesired reference target(s), selectivity for reference target(s) within a sequence- and / or structurally homologous family, selectivity for reference target(s) having similar protein functions, selectivity for distinct desired reference target(s) from a larger family of undesired targets having high sequence and / or structural homology, selectivity for distinct reference target alleles or mutations, selectivity for chemical modifications at the residue level of distinct reference targets, selectivity for cell type, selectivity for tissue type, selectivity for tissue environment, tolerance for structural diversity of reference target(s), tolerance for sequence diversity of reference target(s), and tolerance for kinetic diversity of reference target(s), and may be selected from the group consisting of.

[0134] Accordingly, in a further aspect, provided herein is a method of screening a library of binding molecules using a selection steering composition described herein, wherein each round of selection comprises a negative selection step of screening at least a portion of the pool against a negative selection molecule; and a positive selection step of screening at least a portion of the pool for a positive selection molecule; and the order of the selection steps within each round, and the order of the rounds, results in a selection of a subset of the pool that is different from the alternative order.

[0135] In some embodiments, the binding partner being evaluated using a composition of selection steering polypeptides described herein or a screening method described herein is a phage library, such as a Fab-containing phage library; or a cell library, such as a B cell library or a T cell library.

[0136] In some embodiments of the screening methods provided herein, the method includes the selection of two or more, three or more, four or more, five or more, six or more, or seven or more rounds. In some embodiments where there are more than one round, each round includes a different set of selected molecules. In other embodiments where there are more than one round, at least two rounds include the same negative selected molecule, the same positive selected molecule, or both.

[0137] In some embodiments of the screening method, the method includes analyzing a subset of the pool before proceeding to the next round of selection. In certain embodiments, each subset pool analysis is independently selected from the group consisting of peptide / protein biosensor binding, peptide / protein ELISA, peptide library binding, cell extract binding, cell surface binding, cell activity assay, cell proliferation assay, cell death assay, enzyme activity assay, gene expression profile, protein modification assay, Western blot, and immunohistochemistry. In some embodiments, the gene expression profile includes whole sequence repertoire analysis of the subset pool, such as next-generation sequencing. In some embodiments, statistical and / or information science scoring, or machine learning training, is used to evaluate one or more subsets of the pool in one or more rounds of selection.

[0138] In some embodiments, the identity and / or order of the positive and / or negative selected molecules for subsequent rounds is determined by analyzing a subset pool from one round of selection. In some embodiments, statistical and / or information science scoring, or machine learning training, is used to evaluate one or more subsets of the pool in one or more rounds of selection to determine the identity and / or order of the positive and / or negative selected molecules for subsequent rounds (e.g., the next round, or a further round in the program).

[0139] In yet further embodiments, the method of selection includes modifying the subset pool obtained from a selection round before proceeding to the next selection round. Such modifications can include, for example, genetic mutations of the subset pool, genetic depletion of the subset pool (e.g., selecting a subset of the subset pool to proceed to selection), genetic enrichment of the subset pool (e.g., increasing the size of the pool), chemical modification of at least a portion of the subset pool, or enzymatic modification of at least a portion of the subset pool, or any combination thereof. In some embodiments, statistical and / or information science scoring, or machine learning training, is used to evaluate the subset pool and determine one or more modifications to be made before proceeding with the modified subset pool to selection. In certain embodiments, such statistical and / or information science scoring, or machine learning training, is also used to determine the identity and / or order of positive and / or negative selection molecules for subsequent rounds of selection.

[0140] Any suitable assay can be used to evaluate the binding of the pool of binding partners to the selection molecules at each step. In some embodiments, the binding is evaluated directly, for example, by directly detecting a label on the binding partner. Such labels can include, for example, fluorescent labels such as fluorophores or fluorescent proteins. In other embodiments, the binding is evaluated indirectly, for example, using a sandwich assay. In a sandwich assay, the binding partner binds to the selection molecule, and then a labeled secondary reagent is added to label the bound binding partner. The labeled secondary reagent is then detected. Examples of sandwich assay components include His-tagged binding partners detected using an anti-His tag antibody or a His-tag specific fluorescent probe; biotinylated binding partners detected using labeled streptavidin or labeled avidin; or unlabeled binding partners detected using an anti-binding partner antibody.

[0141] In some embodiments, the binding partners selected in each step are identified based on binding signals or dose responses using a number of available detection methods. These detection methods can include, for example, imaging, fluorescence-activated cell sorting (FACS), mass spectrometry, or biosensors. In some embodiments, a hit threshold is defined (e.g., median signal), and anything having a signal above that signal is flagged as a putative hit motif. IV. Use of Engineered Polypeptides for Producing Antibodies

[0142] The engineered polypeptides provided herein and identified by the methods provided herein can be used, for example, to produce one or more antibodies. In some embodiments, the antibodies are monoclonal or polyclonal antibodies. Thus, in some embodiments, antibodies produced by immunizing an animal with an immunogen, where the immunogen is an engineered polypeptide provided herein, are provided herein. In some embodiments, the animal is a human, rabbit, mouse, hamster, monkey, etc. In certain embodiments, the monkey is a cynomolgus monkey, macaque monkey, or rhesus monkey. Immunizing an animal with an engineered polypeptide can include, for example, administering to the animal at least one dose of a composition comprising the peptide and optionally an adjuvant. In some embodiments, generating an antibody from an animal includes isolating B cells that express the antibody. Some embodiments further include fusing the B cells with myeloma cells to create hybridomas that express the antibody. In some embodiments, antibodies generated using an engineered polypeptide can cross-react with humans and monkeys, e.g., cynomolgus monkeys. a. Characteristics of Engineered Polypeptides

[0143] The engineered polypeptides provided herein have one or more features in common with CD25. In some embodiments, these exhibit at least one feature of the surface of CD25, such as a functional interfacial surface that binds to a binding partner of CD25. In some embodiments, the binding partner is an antibody that specifically binds to CD25. In some embodiments, the engineered polypeptide exhibits at least one feature of a portion of the surface of CD25 that is not known to interact with an antibody to CD25.

[0144] In some embodiments of certain types of features, the engineered polypeptide presents a mimic of the functional interface (e.g., binding surface) of CD25, but the features shared by the engineered polypeptide are best described as being shared overall with CD25. For example, one shared feature can be the binding between a binding partner of CD25 and CD25, which occurs between the functional binding interface of CD25, but the structure and orientation of the functional binding interface are supported by the remainder of the CD25 protein.

[0145] Such shared features can include, for example, structural or functional metrics, or combinations thereof. At least one shared feature can include, for example, one or more structural similarities, conformational entropy similarities, one or more chemical descriptor similarities, one or more functional binding similarities, or one or more phenotypic similarities, or any combination thereof. In certain embodiments, the engineered polypeptide shares one or more of these features with at least a portion of the surface of CD25, such as a functional interface, such as a binding surface.

[0146] In some embodiments, the engineered polypeptide has a structural similarity to CD25 (or a portion of the surface of CD25, such as a binding surface), and this structural similarity is evaluated by the backbone root mean square deviation (RMSD) or the side chain RMSD. RMSD assesses the average distance between atoms and can be applied to three-dimensional structures to compare how two separate similar structures exist in three-dimensional space. In some embodiments, the RMSD of the backbone or amino acid side chains or both between the engineered polypeptide and CD25 (or the functional interface of CD25) is lower than the RMSD between CD25 (or the functional interface of CD25) and a different molecule. In some embodiments, it is a portion of CD25 (or a portion of the functional interface of CD25) that is being compared to the engineered polypeptide. RMSD can be evaluated, for example, using the experimentally measured or simulated structure of the engineered polypeptide; and the experimentally measured or simulated structure of CD25 (or its functional interface). In some embodiments, the engineered polypeptide is considered to be structurally similar to CD25 if the backbone of the engineered polypeptide has an average RMSD of 6.0 Å or less compared to the backbone of the x-ray structure of CD25.

[0147] In some embodiments, the engineered polypeptide has a conformational entropy similar to CD25 (or a portion of the surface of CD25, such as the binding surface), and this conformational entropy is evaluated using, for example, the experimentally measured or simulated structure of the engineered polypeptide and the experimentally measured structure or molecular dynamics simulated motion of CD25 (or a portion thereof). In such simulations, in some embodiments, the experimentally measured structure or molecular dynamics simulated motion of CD25 (or a portion thereof, such as a portion of the binding surface) is used. In certain embodiments, the conformational entropy of the engineered polypeptide is considered similar to that of CD25 (or a portion thereof) if the engineered polypeptide molecular dynamics ensemble run under standard physiological conditions has all states where all non-hydrogen atom positions have an RMSD ≤ 6.0 Å when compared to the known x-ray crystal structure of CD25 (or a portion thereof).

[0148] In still other embodiments, the engineered polypeptide has one or more chemical descriptors similar to CD25 (or a portion thereof, such as the binding surface). In other embodiments, the engineered polypeptide has one or more chemical descriptors complementary to the binding partner of CD25 (e.g., an antibody to CD25). Such chemical descriptors (which can be similar or complementary) can include, for example, hydrophobic patterns, H-bonding patterns, atomic volume / radius, charge patterns or atomic occupancy patterns, or any combination thereof. These chemical descriptors can, in some embodiments, be evaluated using the experimentally measured or simulated structure of the engineered polypeptide and the experimentally measured or simulated structure of CD25 (or a portion thereof, such as the binding surface).

[0149] In other embodiments, the engineered polypeptide has a functional binding similar to CD25. For example, in some embodiments, the engineered polypeptide has a binding to a CD25 binding partner or a fragment thereof. In some embodiments, the binding partner is a fragment of the native binding partner or a modified native binding partner. Such modifications can include, for example, a fusion protein comprising at least a fragment of the native binding partner; labeling with a chromophore; labeling with a fluorophore; labeling with biotin; or labeling with a His tag. In some embodiments, the engineered polypeptide has a binding to a CD25 binding partner that is within about two orders of magnitude or within about one order of magnitude of the binding of CD25 to the binding partner. In some embodiments, the similarity of the binding is evaluated by comparing the binding constant (Kd), or the inhibition constant (Ki), or the on-rate of the binding, or the off-rate of the binding, or the binding affinity of the binding pair, or the Gibbs free energy of the binding (ΔG). In some embodiments, the binding partner is an antibody against CD25.

[0150] In some embodiments, the binding constant (K d ) of the engineered polypeptide to the CD25 binding partner is within 1000-fold, 800-fold, 600-fold, 400-fold, 200-fold, 100-fold, 90-fold, 80-fold, 70-fold, 60-fold, 50-fold, 40-fold, 30-fold, 20-fold, 10-fold, 8-fold, 6-fold, 4-fold, 2-fold, 1.5-fold, 1.2-fold or about the same as the K d of CD25 to the binding partner. In other embodiments, the inhibition constant (K i ) of the engineered polypeptide to the CD25 binding partner is the K iwithin 1000-fold, 800-fold, 600-fold, 400-fold, 200-fold, 100-fold, 90-fold, 80-fold, 70-fold, 60-fold, 50-fold, 40-fold, 30-fold, 20-fold, 10-fold, 8-fold, 6-fold, 4-fold, 2-fold, 1.5-fold, 1.2-fold or approximately the same as it. In still further embodiments, the on-rate of binding of the engineered polypeptide to its CD25 binding partner is similar to the on-rate of binding of CD25 to the binding partner. In some embodiments, the on-rate of binding of the engineered polypeptide to its CD25 binding partner is within 1000-fold, 800-fold, 600-fold, 400-fold, 200-fold, 100-fold, 90-fold, 80-fold, 70-fold, 60-fold, 50-fold, 40-fold, 30-fold, 20-fold, 10-fold, 8-fold, 6-fold, 4-fold, 2-fold, 1.5-fold, 1.2-fold or approximately the same as the on-rate of binding of CD25 to the binding partner. In other embodiments, the off-rate of binding of the engineered polypeptide to its CD25 binding partner is similar to the off-rate of binding of CD25 to the binding partner. In some embodiments, the off-rate of binding of the engineered polypeptide to its CD25 binding partner is within 1000-fold, 800-fold, 600-fold, 400-fold, 200-fold, 100-fold, 90-fold, 80-fold, 70-fold, 60-fold, 50-fold, 40-fold, 30-fold, 20-fold, 10-fold, 8-fold, 6-fold, 4-fold, 2-fold, 1.5-fold, 1.2-fold or approximately the same as the off-rate of binding of CD25 to the binding partner. In still further embodiments, the binding affinity of the engineered polypeptide for the CD25 binding partner is similar to the binding affinity of CD25 for the binding partner.In some embodiments, the binding affinity of the engineered polypeptide for the CD25 binding partner is within 1000-fold, 800-fold, 600-fold, 400-fold, 200-fold, 100-fold, 90-fold, 80-fold, 70-fold, 60-fold, 50-fold, 40-fold, 30-fold, 20-fold, 10-fold, 8-fold, 6-fold, 4-fold, 2-fold, 1.5-fold, 1.2-fold or about the same as the binding affinity of CD25 for the binding partner. In some embodiments, the Gibbs free energy of binding of the engineered polypeptide to the CD25 binding partner is within 1000-fold, 800-fold, 600-fold, 400-fold, 200-fold, 100-fold, 90-fold, 80-fold, 70-fold, 60-fold, 50-fold, 40-fold, 30-fold, 20-fold, 10-fold, 8-fold, 6-fold, 4-fold, 2-fold, 1.5-fold, 1.2-fold or about the same as the Gibbs free energy of binding of CD25 to the binding partner. In some embodiments, the CD25 binding partner is an antibody to CD25.

[0151] In still other embodiments, the engineered polypeptide shares sequence similarity with CD25 or a portion thereof (e.g., the binding surface of CD25). The similarity can be compared to the contiguous amino acid sequence of CD25 (or a portion thereof) or the non-contiguous sequence of CD25 (or a portion thereof). For example, in certain embodiments, the binding surface of CD25 is formed by a non-contiguous amino acid sequence and the engineered polypeptide has sequence similarity with at least a portion of the non-contiguous sequence forming the surface. In other embodiments, the engineered polypeptide has sequence similarity with at least a portion of the contiguous amino acid sequence forming the binding surface of CD25. In some embodiments, the binding surface of CD25 includes an epitope that binds to an antibody to CD25.

[0152] In some embodiments, the engineered polypeptide has a sequence that is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical to a contiguous sequence of CD25, such as a portion of the contiguous sequence that forms the binding surface of CD25. In certain embodiments, the engineered polypeptide has a sequence that is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical to a non - contiguous sequence of CD25, such as a portion of the non - contiguous sequence that forms the binding surface of CD25. In certain embodiments, the engineered polypeptide has a sequence that is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical to a contiguous portion of the binding surface of CD25. In still further embodiments, the engineered polypeptide has a sequence that is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical to two or more non - contiguous portions of the binding surface of CD25. In some embodiments, the engineered polypeptide has a sequence that is at least partially identical (as described herein) to the binding surface of CD25, and the binding surface comprises an epitope that binds to one or more antibodies against CD25.

[0153] In certain embodiments, the sequence similarity between the engineered polypeptide and CD25 (or a portion thereof), if present, is evaluated using the peptide portion(s) of the engineered polypeptide without a linker. In certain embodiments, one or more linking portions are considered to be the same as, for example, when the engineered polypeptide comprises one or more linkers containing amino acids. b. Engineered polypeptide

[0154] In some embodiments, the engineered polypeptide comprises more than one peptide, for example, at least two peptides, or at least three peptides, or more peptides. In some embodiments, the engineered polypeptide comprises between 1 and 10 peptides, between 1 and 8 peptides, between 1 and 6 peptides, between 1 and 4 peptides, between 2 and 10 peptides, between 2 and 8 peptides, between 2 and 6 peptides, or between 2 and 4 peptides.

[0155] In some embodiments, the engineered polypeptide comprises between 2 and 100 amino acids, for example, between 2 and 80 amino acids, between 2 and 70 amino acids, between 2 and 60 amino acids, between 2 and 50 amino acids, between 2 and 40 amino acids, between 2 and 30 amino acids, between 2 and 25 amino acids, between 2 and 20 amino acids, between 2 and 15 amino acids, between 5 and 30 amino acids, between 5 and 25 amino acids, between 5 and 20 amino acids, between 5 and 15 amino acids, or between 9 and 15 amino acids.

[0156] In certain embodiments, the engineered polypeptide comprises more than one peptide, e.g., at least two peptides, or at least three peptides, or at least four peptides, or more peptides, and each peptide independently comprises between 1 and 100 amino acids, or between 2 and 100 amino acids, e.g., between 2 and 80 amino acids, between 2 and 70 amino acids, between 2 and 60 amino acids, between 2 and 50 amino acids, between 2 and 40 amino acids, between 2 and 30 amino acids, between 2 and 25 amino acids, between 2 and 20 amino acids, between 2 and 15 amino acids, between 5 and 30 amino acids, between 5 and 25 amino acids, between 5 and 20 amino acids, between 5 and 15 amino acids or between 9 and 15 amino acids.

[0157] In some embodiments, the engineered polypeptide comprises only naturally occurring amino acids. In other embodiments, the engineered polypeptide comprises non-natural amino acids, e.g., a combination of naturally occurring and non-natural amino acids.

[0158] In some embodiments where the engineered polypeptide comprises two or more peptides, each peptide independently exhibits at least one characteristic of CD25 or a portion thereof (e.g., a binding surface). In some embodiments, each peptide independently exhibits 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 characteristics of CD25 or a portion thereof. In some embodiments, the characteristic is shared with a portion of CD25 that interacts with an antibody to CD25.

[0159] In some embodiments, the engineered polypeptide has at least one characteristic that is complementary to a binding partner of CD25, e.g., an antibody to CD25.

[0160] In some embodiments, the peptide of the engineered polypeptide shares one or more structural similarities with CD25 or a portion thereof. The structural similarity can be evaluated, in some embodiments, by backbone RMSD or side chain RMSD. For example, in certain embodiments, the RMSD of the backbone or amino acid side chains or both between the peptide of the engineered polypeptide and CD25 (or a portion thereof) is lower than the RMSD between CD25 (or a portion thereof) and a different molecule (e.g., a different peptide). In some embodiments, a portion of CD25 is compared to a peptide, e.g., a portion of the surface of CD25, e.g., the surface that interacts with an antibody to CD25. The RMSD of the structural similarity can be evaluated, for example, using the experimentally measured or simulated structure of the peptide and the experimentally measured or simulated structure of CD25 or a portion thereof. In some embodiments, the peptide of the engineered polypeptide is considered to be structurally similar to CD25 (or a portion thereof) if the backbone of the peptide has an average RMSD of 6.0 Å or less compared to the backbone of the known x-ray structure of CD25 or a portion thereof.

[0161] In some embodiments, the engineered polypeptide has a conformational entropy similar to CD25 or a portion thereof. In some embodiments, the experimentally measured structure of the peptide or the motion simulated by molecular dynamics is used to compare the conformational entropy to the experimentally measured structure or the simulated structure of CD25 or a portion thereof. The conformational entropy is considered to be similar, in some embodiments, if the peptide molecular dynamics ensemble run under standard physiological conditions has all states where all non-hydrogen atom moieties have an RMSD ≦ 6.0 Å compared to the known x-ray crystal structure of CD25 or a portion thereof. In some embodiments, a portion of CD25 is compared to a peptide, e.g., the surface portion of CD25 that interacts with an antibody to CD25.

[0162] In further embodiments, the similarity between the peptide of the engineered polypeptide and CD25 (or a portion thereof) can be one or more chemical descriptors. In some embodiments, the peptide has one or more chemical descriptors common to CD25 (or a portion thereof) or one or more chemical descriptors complementary to a binding partner of CD25 (e.g., an antibody to CD25). Chemical descriptors can include, for example, hydrophobicity patterns, H-bonding patterns, atomic volume / radius, charge patterns or atomic occupancy patterns, or any combination thereof. In some embodiments, the peptide of the engineered polypeptide has one or more hydrophobicity patterns, H-bonding patterns, atomic volume / radius, charge patterns or atomic occupancy patterns that are similar to CD25 or a portion thereof or complementary to a binding partner of CD25 (e.g., an antibody to CD25), or any combination thereof. In some embodiments, the similarity comprises having in common one or more of the same chemical descriptors, e.g., the same hydrophobicity pattern, H-bonding pattern, atomic volume / radius, charge pattern or atomic occupancy pattern. Complementary chemical descriptors include, for example, a peptide having a positive charge pattern that complements the negative charge pattern of a binding partner of CD25, e.g., an antibody to CD25. These chemical descriptors can be evaluated in some embodiments using the experimentally measured or simulated structure of the peptide and the experimentally measured or simulated structure of CD25 or the CD25 binding partner (e.g., for complementary assessment).

[0163] For example, in some embodiments, the engineered polypeptide binds to a binding partner of CD25, similar to the binding of CD25 to a binding partner (e.g., IL-2). In some embodiments, the binding partner is a native binding partner, a fragment of a native binding partner, or a modified native binding partner or fragment thereof, or an antibody that specifically binds to CD25. In some embodiments, the binding partner binds under certain circumstances but not under other circumstances. In some embodiments, the binding partner binds under pathological conditions or under non-pathological conditions. The binding partner may, for example, be constitutively expressed or be the product of an accidental gene, or may comprise a protein or fragment thereof. In certain embodiments, the binding partner is a fragment of a native binding partner or a modified native binding partner. In some embodiments, the modification includes a fusion protein comprising at least a fragment of a native binding partner; labeling with a chromophore; labeling with a fluorophore; labeling with biotin; or labeling with a His tag.

[0164] In some embodiments, the engineered polypeptide has binding to a binding partner of CD25 that is within about two orders of magnitude or within about one order of magnitude of the binding of CD25 to its binding partner. In some embodiments, the similarity of the binding is evaluated by comparing the binding constant (Kd), or inhibition constant (Ki), or on-rate of binding, or off-rate of binding, or binding affinity of the binding pair, or Gibbs free energy of binding (ΔG). In some embodiments, the binding partner is an antibody to CD25.

[0165] In some embodiments, the binding constant (K d ) of the engineered polypeptide to its CD25 binding partner is the K dWithin 1000 times, 800 times, 600 times, 400 times, 200 times, 100 times, 90 times, 80 times, 70 times, 60 times, 50 times, 40 times, 30 times, 20 times, 10 times, 8 times, 6 times, 4 times, 2 times, 1.5 times, 1.2 times or approximately the same as that. In other embodiments, the inhibition constant (K i ) of the engineered polypeptide and the CD25 binding partner is the K iwithin 1000-fold, 800-fold, 600-fold, 400-fold, 200-fold, 100-fold, 90-fold, 80-fold, 70-fold, 60-fold, 50-fold, 40-fold, 30-fold, 20-fold, 10-fold, 8-fold, 6-fold, 4-fold, 2-fold, 1.5-fold, 1.2-fold or approximately the same as it. In still further embodiments, the on-rate of binding of the engineered polypeptide to its CD25 binding partner is similar to the on-rate of binding of CD25 to its binding partner. In some embodiments, the on-rate of binding of the engineered polypeptide to its CD25 binding partner is within 1000-fold, 800-fold, 600-fold, 400-fold, 200-fold, 100-fold, 90-fold, 80-fold, 70-fold, 60-fold, 50-fold, 40-fold, 30-fold, 20-fold, 10-fold, 8-fold, 6-fold, 4-fold, 2-fold, 1.5-fold, 1.2-fold or approximately the same as the on-rate of binding of CD25 to its binding partner. In other embodiments, the off-rate of binding of the engineered polypeptide to its CD25 binding partner is similar to the off-rate of binding of CD25 to its binding partner. In some embodiments, the off-rate of binding of the engineered polypeptide to its CD25 binding partner is within 1000-fold, 800-fold, 600-fold, 400-fold, 200-fold, 100-fold, 90-fold, 80-fold, 70-fold, 60-fold, 50-fold, 40-fold, 30-fold, 20-fold, 10-fold, 8-fold, 6-fold, 4-fold, 2-fold, 1.5-fold, 1.2-fold or approximately the same as the off-rate of binding of CD25 to its binding partner. In still further embodiments, the binding affinity of the engineered polypeptide for its CD25 binding partner is similar to the binding affinity of CD25 for its binding partner.In some embodiments, the binding affinity of the engineered polypeptide to the CD25 binding partner is within 1000-fold, 800-fold, 600-fold, 400-fold, 200-fold, 100-fold, 90-fold, 80-fold, 70-fold, 60-fold, 50-fold, 40-fold, 30-fold, 20-fold, 10-fold, 8-fold, 6-fold, 4-fold, 2-fold, 1.5-fold, 1.2-fold or about the same as the binding affinity of CD25 to the binding partner. In some embodiments, the Gibbs free energy of binding of the engineered polypeptide to the CD25 binding partner is within 1000-fold, 800-fold, 600-fold, 400-fold, 200-fold, 100-fold, 90-fold, 80-fold, 70-fold, 60-fold, 50-fold, 40-fold, 30-fold, 20-fold, 10-fold, 8-fold, 6-fold, 4-fold, 2-fold, 1.5-fold, 1.2-fold or about the same as the Gibbs free energy of binding of CD25 to the binding partner. In some embodiments, the CD25 binding partner is an antibody to CD25.

[0166] In some embodiments, the engineered polypeptide has sequence similarity to CD25 or a portion thereof. In some embodiments, the engineered polypeptide has sequence similarity to a portion of the surface of CD25 that binds to an antibody of CD25. In certain embodiments, the sequence similarity is compared to a contiguous amino acid sequence of CD25. In other embodiments, the sequence similarity is compared to a non-contiguous sequence of CD25. For example, in certain embodiments, the binding surface of folded CD25 is formed by non-contiguous amino acid sequences, and the engineered polypeptide has sequence similarity to at least a portion of the non-contiguous sequences that form the surface. In some embodiments, the engineered polypeptide has sequence similarity to at least a portion of a contiguous amino acid sequence that forms the binding surface of CD25. In some embodiments, the engineered polypeptide has a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to at least a portion of a contiguous sequence of CD25, such as a contiguous sequence that forms the binding surface. In certain embodiments, the engineered polypeptide has a sequence that is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical to at least a portion of a non-contiguous sequence of CD25, such as a non-contiguous sequence that forms the binding surface. In certain embodiments, the engineered polypeptide has a sequence that is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical to a contiguous portion of CD25.In yet further embodiments, the engineered polypeptide has a sequence that is at least 40% identical, at least 45% identical, at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical or at least 90% identical to two or more non - contiguous portions of CD25. In some embodiments, for an engineered polypeptide comprising at least two peptides, two or more peptides of the engineered immunogen share sequence similarity independently with CD25, e.g., the binding surface of CD25. In some embodiments, the portion of CD25 that shares sequence similarity with the engineered polypeptide is the surface that binds to an antibody against CD25. c. Linking moiety

[0167] The engineered polypeptides provided herein may include a linking moiety. When present, the linking moiety can be, for example, independently, a cross - linker or a linker.

[0168] In some embodiments, the engineered polypeptide comprises N peptides and N - 1 linking moieties; or N peptides and N - 1 linking moieties; or N peptides and N linking moieties; or N peptides and N + 1 linking moieties; or N peptides and N + 2 linking moieties; or N peptides and N - 2 linking moieties, where N is 3 or more.

[0169] In some embodiments, the engineered polypeptide comprises at least 1 linker moiety, at least 2 linker moieties, at least 3 linker moieties, at least 4 linker moieties, at least 5 linker moieties, at least 6 linker moieties, from 1 to 6 linker moieties, from 1 to 5 linker moieties, from 1 to 4 linker moieties, from 1 to 3 linker moieties, 1 linker moiety, or 2 linker moieties. In some embodiments, each linker moiety is independently a crosslink or a linker. In certain embodiments, each linker moiety is a crosslink. In other embodiments, each linker moiety is a linker. In still further embodiments, at least 1 linker moiety is a crosslink and the remaining linker moieties are independently a crosslink or a linker. In other embodiments, at least 1 linker moiety is a linker and the remaining linker moieties are independently a crosslink or a linker.

[0170] Crosslinking includes, for example, covalent bonds between the side chain of one amino acid and a portion of another amino acid. The amino acids can independently be natural or non-natural amino acids. In some embodiments, crosslinking includes covalent bonds between the side chains of two amino acids, or between the side chain of one amino acid and the amine or carboxyl group of another amino acid. Crosslinks can form within one peptide or between two separate peptides. In some embodiments, the engineered polypeptides provided herein include a mixture of both intra-peptide and inter-peptide crosslinks. In some embodiments, the crosslink is a disulfide bond between two thiol groups of an amino acid side chain, for example, a disulfide bond between two cysteines. In some embodiments, the crosslink is an amide bond between the amine group and carboxylic acid group of two amino acids, wherein at least one of the amine group and carboxylic acid group is positioned on the side chain of the amino acid (e.g., the amide bond is not a backbone amide bond). In some embodiments, the crosslink is an amide bond formed between diaminopimelic acid and aspartic acid. In some embodiments, the amide crosslink is a lactam. In some embodiments, the crosslink is an oxime. In some embodiments, the crosslink is a hydrazone. In some embodiments, crosslinking includes a covalent bond between the side chain of one amino acid and a portion of another amino acid, wherein one or both of the side chain and the portion are modified to form the covalent bond. Such modifications can include, for example, oxidation, reduction, reaction with a catalyst that forms an intermediate, or other modifications known to those of skill in the art.

[0171] Linkers include, for example, molecules covalently attached to at least two sites of a peptide or molecules covalently attached between at least two peptides. A linker can bind to two sites within one peptide or between two separate peptides or can be a combination of both. For example, a linker containing more than two peptide binding sites can form both intra-peptide and inter-peptide bonds. In an engineered polypeptide comprising at least two peptides and at least one linker, the peptides and the linker can be connected in a variety of different conformations. For example, the engineered polypeptide can have a pattern such as peptide-linker-peptide that ends with a peptide. In some embodiments, the engineered polypeptide includes a linker that forms a branching point, for example, a linker that binds independently to three peptides. In some embodiments, the engineered polypeptide includes a linker having three peptide binding sites, and the linker binds to only two peptides.

[0172] In some embodiments, the linker comprises one or more amino acids. The amino acids forming part of the linker can, in some embodiments, be separately identified from the engineered polypeptide. In certain embodiments, the linker is a region that separates and presents the peptides of the engineered polypeptide in a structural, chemical, and / or kinetic manner that reflects the structure and / or function of the functional interface of the surface protein. In yet further embodiments, the linker has no function by itself when not connected to the peptide of the engineered polypeptide, e.g., does not exhibit binding to the binding partner of CD25. In some embodiments, each linker independently comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6 or more amino acids. In some embodiments, each linker independently comprises 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids or 6 amino acids. The amino acids forming part of the linker can, in some embodiments, be naturally occurring amino acids or non-naturally occurring amino acids. Each linker can, in some embodiments, independently comprise one or more alpha-amino acids, one or more beta-amino acids or one or more gamma-amino acids, or any combination thereof. In certain embodiments, the linker independently comprises cyclic beta residues. Cyclic beta residues can include, for example, APC or ACPC. In yet further embodiments, the linker can comprise one or more glycine residues, one or more serine residues or one or more proline residues. In some embodiments, the linker has an amino acid sequence selected from the group consisting of AP, GP, GSG, (GGGGS)n, (GSG)n, GGGSGGGGS, GGGGSGGGS, (PGSG)n and PGSGSG, wherein n is an integer between 1 and 10. In some embodiments, the engineered polypeptide comprises at least one linker, each linker comprising no amino acids, or each linker comprising no natural amino acids, or each linker comprising at least one non-natural amino acid.

[0173] In some embodiments, the linker comprises a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). A linker comprising PEG can comprise, for example, at least 3 PEG monomer units, at least 4 PEG monomer units, at least 5 PEG monomer units, at least 6 PEG monomer units, at least 7 PEG monomer units, at least 8 PEG monomer units, at least 9 PEG monomer units, at least 10 PEG monomer units, at least 11 PEG monomer units, at least 12 PEG monomer units or more than 12 PEG monomer units. In some embodiments of the linker comprising PEG, the PEG comprises monomer units between 3 and 12, between 3 and 6, between 6 and 12 or between 4 and 8. In some embodiments, the engineered polypeptide comprises at least one linker comprising PEG3 (comprising 3 monomer units), PEG6 or PEG12. In some embodiments, at least one linker is independently PEG3, PEG6 or PEG12. In a further embodiment, the linker comprises multi-arm PEG. For example, in certain embodiments, at least one linker independently comprises 4-arm PEG or 8-arm PEG. In certain embodiments, each arm independently comprises monomer units between 3 and 12, or between 3 and 6, or between 6 and 12, or between 4 and 8. In certain embodiments, each arm of the multi-arm PEG comprises the same number of monomer units, such as 4-arm or 8-arm PEG, and each arm comprises 3 monomer units, 6 monomer units or 12 monomer units.

[0174] In other embodiments, the linker comprises a dendrimer. Dendrimers include, for example, molecules having a tree-like branched architecture that includes a symmetric core from which molecular moieties radiate, where the branch points form new layers within the molecule. Each new branch point introduces a new and larger layer, and these radial extensions often terminate in functional groups at the outer terminal surface of the dendrimer. Thus, increasing the number of branch points in turn amplifies the possible number of terminal functional groups at the surface.

[0175] In some embodiments, at least one linker comprises a small molecule that is neither an amino acid nor a polymer. In some embodiments, at least one linker comprises a benzodiazepine. In some embodiments, the linker comprises a portion that is a product of a sulfhydryl-maleimide reaction and can be a pyrrolidinedione moiety (e.g., a pyrrolidine-2,5-dione moiety). In some embodiments, the linker comprises an amidine moiety. In some embodiments, the linker comprises a thioether moiety.

[0176] In some embodiments, at least one linker comprises trans-pyrrolidine-3,4-dicarboxamide.

[0177] In some embodiments where the engineered polypeptide comprises at least two linkers (e.g., embodiments where the engineered polypeptide comprises at least two linking moieties and each linking moiety is independently a linker or a crosslink, or each linking moiety is independently a linker), each linker is independently any of the linkers described herein. For example, in some embodiments, each linker is independently a linker comprising one or more amino acids, a linker comprising a polymer, a linker comprising a dendrimer, or a linker comprising a small molecule that is neither an amino acid nor a polymer.

[0178] One or more linking moieties of the engineered polypeptide can confer specific structural or functional features of interest, or combinations thereof. For example, in some embodiments, the linking moiety is present in the engineered polypeptide to confer a structural or functional feature, or combination thereof. Such structural features can include, for example, increased structural flexibility, decreased structural flexibility, a directional feature, increased length or decreased length. Directional features that can be targeted can include, for example, a structural turn, or maintenance of a linear structure. Functional features can include, for example, enhanced solubility, one or more protonation sites, one or more proteolytic sites, one or more sites for enzymatic modification, one or more oxidation sites, a label, or a capture handle. In some embodiments, the linker includes one or more functional features or one or more structural features, or combinations thereof.

[0179] In some embodiments, one or more linkers independently introduce a structural "turn" into the engineered polypeptide. Examples of such linkers include Gly-Pro, Ala-Pro, and trans-pyrrolidine-3,4-dicarboxamide. In some embodiments, one or more linkers present in the engineered polypeptide increase the structural flexibility of the engineered polypeptide compared to the absence of a linker or the selection of a different linker. For example, a linker that is longer and / or less sterically hindered than another linker can, in some embodiments, result in a molecule with higher structural flexibility than if no linker were present or if another linker were used instead. In other embodiments, one or more linking moieties, such as a linker that is shorter and / or more sterically hindered than another linker, or a cross-link at a position or of a type that reduces the flexibility of one or more peptides, independently decrease the structural flexibility in the engineered polypeptide. The presence of a cross-link at a particular position between specific peptides or between specific amino acid side chains can result in a molecule with lower structural flexibility than if the cross-link were at a different position or between different side chains (e.g., a disulfide or amide cross-link) or if no cross-link were present. d. Additional Components

[0180] In some embodiments, the engineered polypeptides provided herein include one or more additional components. For example, in some embodiments, the engineered polypeptide includes one or more moieties that bind the engineered polypeptide to a solid surface, such as beads or a flat surface. In some embodiments, the binding moiety includes a polymer (e.g., PEG) or biotin, or a combination thereof. In some embodiments, binding the engineered polypeptide to a solid surface can enable, for example, the evaluation of one or more characteristics of the engineered polypeptide, such as the evaluation of binding to a binding partner of CD25 (e.g., an antibody to CD25). e. Sequence Similarity

[0181] In some embodiments, the engineered polypeptides provided herein are those having one of the sequences listed in Table 1:

[0182] [Table 4] and having one of the sequences listed in it.

[0183] In some embodiments, the engineered polypeptide has at least 60% sequence similarity to any one of SEQ ID NOs: 1-21. In some embodiments, the engineered polypeptide has at least 70% sequence similarity to any one of SEQ ID NOs: 1-21. In some embodiments, the engineered polypeptide has at least 80% sequence similarity to any one of SEQ ID NOs: 1-21. In some embodiments, the engineered polypeptide has at least 90% sequence similarity to any one of SEQ ID NOs: 1-21. In some embodiments, the engineered polypeptide has at least 95% sequence similarity to any one of SEQ ID NOs: 1-21. In some embodiments, the engineered polypeptide comprises any one of SEQ ID NOs: 1-21. In certain embodiments, the engineered polypeptide has any one of SEQ ID NOs: 1-21.

[0184] In some embodiments, the engineered polypeptide comprises any one of SEQ ID NOs: 1-21 and is modified at the N-terminus and / or C-terminus. For example, in some embodiments, the C-terminus or N-terminus is covalently attached to another molecule. In still further embodiments, the engineered polypeptide comprises any one of SEQ ID NOs: 1-21 and one or more amino acids at the N-terminus and / or C-terminus.

[0185] In some embodiments, the N-terminal molecule is biotin-PEG2:

[0186] [Chemical formula] as follows.

[0187] In some embodiments, the C-terminal molecule is a linker followed by biotin (e.g., -GSGSGK-biotin). Other linkers suitable for attaching biotin to the C-terminus of the engineered polypeptide include GSG, GSS, GGS, GGSGGS, GSSGSS, GSGK, GSSK, GGSK, GGSGGSK, GSSGSSK, and the like. V. Method for Selecting an Engineered Polypeptide

[0188] Also provided herein are methods for selecting an engineered polypeptide described herein. Such methods can include, for example, using iterative optimization of the structural features of the engineered polypeptide.

[0189] In some embodiments, one or more sections of CD25 are identified as target interfaces. In some embodiments, at least a portion of the identified section(s) binds to an antibody to CD25. Thus, for example, in some embodiments, a portion of CD25 that is an epitope for one or more antibodies is identified as a target interface. In other embodiments, a section of CD25 is identified as a target interface where it is unknown whether it does not bind to an antibody or whether antibody binding occurs. In certain embodiments, the crystal structure for at least a portion of CD25 is unknown and the initial selection of the target interface includes molecular dynamics simulations of CD25 and CD25 binding. In some embodiments, one or more initial input sequences are obtained from the identified section(s), and each sequence, independently, is contiguous or non-contiguous. When developing engineered polypeptide candidates, at least a portion of the interfacial residues of each sequence are retained and one or more linker segments are incorporated into the sequence to provide desired structural and dynamical characteristics. In some embodiments, one or more non-interfacial residues are added to the sequence or one or more residues in the input sequence are replaced with one or more non-interfacial residues to achieve desired structural and dynamical characteristics compared to the structure and dynamics of the cognate target. In some embodiments, these non-interfacial residues are not derived from the target interface of CD25, or do not share one or more characteristics with the target interface of CD25, or share fewer characteristics than the retained interfacial residues and / or do not share characteristics as strongly with the target interface of CD25. These intervening non-interfacial residues can, in some embodiments, form part or all of an amino acid linker.

[0190] Next, in some embodiments, an initial design (or designs) is generated and molecular dynamics simulated to determine the flexibility and overall stability of the design. If this initial design does not meet the RMSD requirements, in some embodiments, this can undergo iterative optimization of one or more linker moieties (e.g., one or more crosslinks or intervening linker residues) using computational mutagenesis. During this optimization, in some embodiments, the interface residues are fixed, while one or more of the linker moieties are altered or removed or added. The iterative optimization can be repeated until the RMSD of the engineered polypeptide compared to the target interface and structural order metrics meets certain requirements (e.g., ≤6.0 Å and ≥0.25, respectively. Here, the structural order is a normalized scale from 0 to 1, where 1 = complete structural stability).

[0191] In some embodiments, the intervening structurally stable residue regions can range from 1 to 50 amino acids in length. In certain embodiments, these intervening structurally stable residue regions are linkers, e.g., amino acid linkers. In some embodiments, the relatively small size of the engineered polypeptides produced by certain embodiments of the methods provided herein (e.g., as compared to approaches that graft an interface onto a larger structurally stabilizing scaffold) can enable chemical synthesis of the molecule, as opposed to larger molecules that may require in vitro expression systems. Further, in some embodiments, the methods provided herein enable incorporation of non-natural amino acids into intervening or interface positions, which can enable fine control of interface manipulation by novel moieties and properties, e.g., post-translational modifications, solubility, cell permeability, enzyme reactivity, pH sensitivity, oxidative sensitivity, etc. In yet further embodiments, the engineered polypeptides can be selected by virtue of a higher likelihood of species cross-reactivity or disease-related mutant reactivity in selected antibodies when the engineered polypeptide is used as an immunogen or epitope-bait.

[0192] In some embodiments, the optimized molecule is an engineered polypeptide provided herein. In other embodiments, the optimized molecule is a candidate engineered polypeptide that can undergo further evaluation, further adjustment, or be used to generate a peptide library or a library of candidate engineered polypeptides, or any combination thereof. In certain embodiments, the method further comprises generating a peptide library or a library of candidate engineered polypeptides using the candidate engineered polypeptides, and then contacting the library with a binding partner of CD25 (e.g., an antibody to CD25). The peptide library can include, for example, peptides that are smaller than the candidate engineered polypeptides, share at least some sequence similarity therewith, and may have certain residues replaced with other residues. The library of candidate engineered polypeptides can include, for example, variations of the candidate engineered polypeptides.

[0193] In some embodiments, the peptides of the peptide library contain between 2 and 15 amino acids, between 5 and 15 amino acids, between 10 and 15 amino acids, between 2 and 10 amino acids, or between 5 and 10 amino acids. In some embodiments, the total number of amino acids in each peptide of the library includes both surface amino acids and structural amino acids, which may include, for example, linker amino acids. The engineered polypeptide candidate library can be prepared, for example, by varying one or more amino acids or linker moieties in the candidates to create new library members. The engineered polypeptide candidates in the engineered polypeptide candidate library, in some embodiments, independently contain between 5 and 40 amino acids, between 10 and 35 amino acids, between 15 and 35 amino acids, or between 20 and 30 amino acids. In some embodiments, the total number of amino acids in each engineered polypeptide candidate of the candidate library can include both surface amino acids and structural amino acids, which may include, for example, linker amino acids in some embodiments. The peptide library and the engineered polypeptide candidate library, in some embodiments, independently contain between 5,000 and 100,000 members, between 5,000 and 80,000 members, between 5,000 and 60,000 members, between 5,000 and 40,000 members, between 5,000 and 30,000 members, between 10,000 and 25,000 members, between 15,000 and 20,000 members, or about 17,000 members (e.g., distinct peptides or distinct engineered polypeptide candidates). In some embodiments, multiple separate libraries are generated and evaluated. In certain embodiments, the library members do not contain specific crosslinks. For example, in some embodiments, libraries are evaluated in which the library members do not have disulfide crosslinks.

[0194] In some embodiments for generating candidates for a candidate library, one or more linking moieties are added or removed or repositioned in the design of the original engineered polypeptide candidate. For example, in some embodiments, disulfide bridges are removed or added or their positions are shifted. In other embodiments, lactam bridges are removed or added or their positions are shifted. In some embodiments, one or more amino acid residues are replaced. Binding of a CD25 binding partner to a peptide library or an engineered polypeptide candidate library or both (if present) can provide additional information for further refining the design of the engineered polypeptide or for selecting the engineered polypeptide. Additional information from screening these libraries can be used, for example, to vary the engineered polypeptide, for example, to increase its binding affinity to a binding partner of CD25. An engineered polypeptide candidate library can, in some embodiments, provide additional information regarding the effect of a particular linker moiety on binding interactions (including the presence or position of such moieties), such as disulfide bonds and crosslinks including lactams. A peptide or an engineered polypeptide candidate library or both can, in some embodiments, be used to identify common motifs (e.g., amino acid patterns or linking moieties, or combinations thereof) that can increase the binding affinity or binding specificity to a binding partner of CD25 or provide other desired characteristics. Evaluating the binding of cognate binding partners to members of a peptide or an engineered polypeptide candidate library or both can provide additional structural and functional information, which can be used for further refining the design of the engineered polypeptide or for selecting an engineered polypeptide candidate. a. Selection by binding under varying pH

[0195] In some embodiments, the engineered polypeptide is selected based at least in part on its structural flexibility at physiological pH as compared to its structural flexibility at a lower pH. For example, CD25 can be overexpressed on tumor cells, and thus, in some embodiments, binding of an antibody to CD25 with a higher affinity in the tumor microenvironment may be desired. Thus, in some embodiments, an engineered polypeptide that is less flexible at a lower pH as compared to the same engineered polypeptide at physiological pH, or an engineered polypeptide in which one or more amino acids have a specific orientation at a lower pH, or an engineered polypeptide that has a higher binding affinity or binding selectivity at a lower pH may be desirably selected. In many cancerous tumors, the growth rate of cancerous cells can outstrip the available oxygen supply in parts of the tumor, creating a hypoxic microenvironment within the tumor. The level of oxygen in a tissue can affect the pH of the tissue environment, and low oxygen levels can result in a decreased pH (including, for example, due to the accumulation of acidic metabolites from anaerobic glycolysis). Thus, in some embodiments, selecting an engineered polypeptide that has a higher binding at a lower pH (e.g., has a desirable structural feature that results in a binding interaction), but a reduced binding at physiological pH (e.g., has fewer, or no, desirable structural features that result in a binding interaction) can result in an engineered polypeptide that can produce an antibody that has a higher binding to a desired target in the tumor as compared to binding when not in the tumor. Physiological pH is typically between about 7.35 and about 7.45, for example, about 7.4. The pH of the tumor microenvironment can be, for example, less than about 7.45, less than about 7.45, between about 7.45 and about 6.0, between about 7.0 and about 6.0, between about 6.8 and about 6.2, between about 6.7 and about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9 or about 7.0. In some embodiments, the engineered polypeptide can be evaluated at different pHs using computational methods, such as molecular dynamics simulations. In other embodiments, the engineered polypeptide is selected based on differential pH characteristics using in vitro methods.Suitable in vitro methods can include, for example, phage panning at different pHs. For example, an antibody phage display library can be used to pan for one or more engineered polypeptides at physiological pH, and phages that bind at that pH can be discarded. Then, a second round of panning can be performed at a lower pH, and phages that bind to one or more engineered polypeptides at the lower pH can be selected. In some embodiments, engineered polypeptides that do not bind phages at a lower pH or bind phages with a similar affinity at both the lower pH and physiological pH may not be as desirable for use in generating antibodies that target tumor cells. b. Reverse peptide evaluation

[0196] In still further embodiments, selecting an engineered polypeptide can include comparing the binding of the engineered polypeptide to the binding of a reverse-engineered polypeptide. The reverse-engineered polypeptide can be based on the engineered polypeptide, but one or more of the interfacial-interacting amino acid residues (e.g., based on the surface of CD25) are replaced with amino acids that exhibit reverse characteristics. For example, an amino acid having a large sterically bulky hydrophobic side chain can be replaced with an amino acid having a smaller side chain, or a hydrophilic side chain, or a smaller and hydrophilic side chain. In some embodiments, an amino acid having a hydrogen-bond donating side chain can be replaced with an amino acid having a hydrogen-bond accepting side chain or an amino acid having a side chain that does not hydrogen bond. Binding characteristics that can be compared using the engineered polypeptide and the reverse-engineered polypeptide can include, in some embodiments, specificity and / or affinity. Comparing the binding characteristics of the engineered polypeptide to the binding characteristics of the reverse-engineered polypeptide can, in some embodiments, help select an engineered polypeptide in which the interfacial-interacting amino acids drive the binding interaction rather than being characteristics of a linking moiety such as a linker. In some embodiments, engineered polypeptides in which the binding is driven by a linking moiety such as a linker may not be as desirable because they can exhibit off-target binding or other undesirable binding characteristics.

[0197] In further embodiments, the method further comprises modifying the selected engineered polypeptide. c. Binding evaluation

[0198] As described herein, in some embodiments, the method of selecting an engineered polypeptide provided herein comprises evaluating the binding of an engineered polypeptide candidate to a protein or fragment thereof, such as a binding partner of CD25 (e.g., an antibody to CD25). For example, in some embodiments, a library of engineered polypeptide candidates or a peptide library is screened for binding to a binding partner of CD25.

[0199] The binding of a protein or fragment thereof (e.g., a binding partner of CD25) to one or more peptides or engineered polypeptide candidates (e.g., members of a library) can be evaluated in various ways. In some embodiments, the binding is evaluated directly, for example, by directly detecting a label on the protein or fragment thereof. Such labels can include, for example, fluorescent labels such as fluorophores or fluorescent proteins. In other embodiments, the binding is evaluated indirectly, for example, using a sandwich assay. In a sandwich assay, a peptide or engineered polypeptide candidate (e.g., a member of a library) binds to the binding partner, and then a labeled secondary reagent is added to label the bound binding partner. The labeled secondary reagent is then detected. Examples of sandwich assay components include His-tagged binding partners detected using an anti-His tag antibody or a His tag-specific fluorescent probe; biotin-labeled binding partners detected using labeled streptavidin or labeled avidin; or unlabeled binding partners detected using an anti-binding partner antibody.

[0200] In some embodiments, the peptide of interest or engineered polypeptide candidate is identified based on binding signals or dose responses using any of a number of available detection methods. These detection methods can include, for example, imaging, fluorescence-activated cell sorting (FACS), mass spectrometry, or biosensors. In some embodiments, a hit threshold is defined (e.g., median signal), and anything having a signal above that signal is flagged as a putative hit motif.

[0201] For the development of combinatorial libraries, peptides identified from peptide libraries based on binding to a protein or fragment thereof can, in some embodiments, be further clustered into distinct groups using sequence or structural information, or combinations thereof. This grouping can be performed, for example, using commonly available sequence alignment, chemical descriptors, structural prediction, and entropy prediction information science tools (e.g., MUSCLE, CLUSTALW, PSIPRED, AMBER, Hydropathy Calculator, and Isoelectric Point Calculator) and clustering algorithms (e.g., K-Means, Gibbs, and Hierarchical). Clusters of motifs (e.g., structural or functional motifs) present in peptide hits can be identified from this analysis. Individual peptide motif hits can also be identified. Using these motif clusters and individual motifs, in some embodiments, design rules can be devised that define one or more of the sequence, structural, and chemical features of motifs that appear to drive protein interactions at the target interface. In some embodiments, the structure of the target interface is not necessary for the identification of these interface motif design rules. Rather, the design rules can, in some embodiments, be derived from the analysis of peptides identified from screening a peptide library.

[0202] In some embodiments, the binding assay is about 10 5has a sensitive kinetic range. Thus, in some embodiments, the engineered polypeptide candidate has a binding event with a CD25 binding partner within 10 5 signal brackets of the native CD25:binding partner signal and is identified as being of interest. The type of signal can vary depending on what type of assay is being used or how the signal is being evaluated. For example, in some embodiments, the signal is a response unit in a sensorgram, a fluorescent signal in an image-based readout, or an enzymatic readout in an enzyme-based assay. The signal for the binding event can be measured relative to the CD25:binding partner signal.

[0203] In some embodiments, the engineered polypeptide candidate is modified prior to evaluating binding. For example, in some embodiments, biotin, PEG or another binding moiety, or combinations thereof, are attached to the C-terminus or N-terminus of the peptide to enable it to be used in a binding evaluation system. For example, in some embodiments, biotin-PEG12- is covalently attached to the N-terminus of the engineered polypeptide. In other embodiments, the engineered polypeptide candidate is modified with -GSGSGK-PEG4-biotin at the C-terminus. In certain embodiments, such biotin-modified engineered polypeptide candidates are then attached to streptavidin beads via the biotin moiety and the bead-supported immunogen is evaluated for binding to the binding partner of CD25. VI. Use of Engineered Polypeptides and CD25 Antibodies

[0204] The engineered polypeptides provided herein and identified by the methods provided herein can be used, for example, to produce one or more antibodies that specifically bind to CD25. In some embodiments, the antibodies are monoclonal or polyclonal antibodies.

[0205] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an intact immunoglobulin of polyclonal or monoclonal origin, or a fragment thereof, and can be derived from a natural or recombinant source.

[0206] The term "antibody fragment" or "antibody binding domain" refers to at least one portion of an antibody or a recombinant variant thereof that contains an antigen-binding domain, i.e., the antigenic determinant variable region of an intact antibody, which is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen and its defined epitope. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2 and Fv fragments, single-chain (sc)Fv ("scFv") antibody fragments, linear antibodies, single domain antibodies (abbreviated as "sdAb") (either VL or VH), camelid VHH domains, and multispecific antibodies formed from antibody fragments.

[0207] The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing the variable region of the light chain and at least one antibody fragment containing the variable region of the heavy chain, wherein the light chain and heavy chain variable regions are continuously linked via a short flexible polypeptide linker and can be expressed as a single polypeptide chain, and the scFv retains the specificity of the intact antibody from which it is derived.

[0208] With respect to an antibody, "heavy chain variable region" or "VH" (or, in the case of a single domain antibody, such as a nanobody, "VHH") refers to a fragment of the heavy chain that contains three CDRs inserted between adjacent stretches known as framework regions, which framework regions are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.

[0209] Unless otherwise specified, as used herein, an scFv can have the VL and VH variable regions in either order with respect to the N- and C-termini of the polypeptide, and the scFv can contain VL-linker-VH or VH-linker-VL.

[0210] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in an antibody molecule in its naturally occurring conformation. Kappa ("Κ").

[0211] Accordingly, in some embodiments, antibodies produced by immunizing an animal with an immunogen, where the immunogen is an engineered polypeptide provided herein, are provided herein. In some embodiments, the animal is a human, rabbit, mouse, hamster, monkey, etc. In certain embodiments, the monkey is a cynomolgus monkey, rhesus monkey or macaque monkey. Immunizing an animal with an engineered polypeptide can include, for example, administering to the animal at least one dose of a composition comprising the immunogen and optionally an adjuvant. In some embodiments, generating an antibody from an animal includes isolating B cells that express the antibody. Some embodiments further include fusing the B cells with myeloma cells to create hybridomas that express the antibody. In some embodiments, antibodies generated using an engineered polypeptide can cross-react with humans and monkeys, such as cynomolgus monkeys.

[0212] In certain embodiments, a method of generating an antibody further comprises determining one or more epitopes for the antibody. In some embodiments, the method comprises screening an antibody for binding to two or more epitopes, for example, by contacting an epitope library with the antibody and evaluating binding of the antibody to the epitopes of the library. In certain embodiments, an antibody that binds to two or more epitopes is discarded. In some embodiments, the engineered polypeptide mimics one epitope of CD25. In other embodiments, the engineered polypeptide mimics two or more epitopes of CD25. In certain embodiments, screening an antibody for binding to two or more epitopes, where the engineered polypeptide mimics two or more epitopes of CD25, comprises contacting an epitope library with the antibody, evaluating binding of the antibody to the epitopes of the library, and discarding one or more antibodies that bind to two or more epitopes that are not the epitopes mimicked by the engineered polypeptide.

[0213] In some embodiments, an antibody produced using the engineered polypeptide provided herein specifically binds to CD25. In certain embodiments, the antibody does not block binding of IL-2 to CD25 when the antibody is bound to CD25.

[0214] In some embodiments, the antibody is a non-IL-2 blocking antibody (non-IL-2 blocker) - that is, the binding of the antibody to CD25 does not disrupt or prevent the binding of the IL-2 ligand to CD25 (IL-2 alpha chain), and does not affect IL-2 mediated signaling, for example, signaling via the IL-2 / JAK3 / STAT-5 signaling pathway. In some embodiments, the antibody binds to an epitope different from the epitope to which the 7G7B6 antibody binds without disrupting the binding of the IL-2 ligand to CD25 (IL-2 alpha chain). In some embodiments, the antibody does not disrupt the binding of the IL-2 ligand to CD25, but disrupts the trimerization of the beta, gamma, and alpha (CD25) chains of the IL-2 receptor.

[0215] In some embodiments, the antibody is an IL-2 blocking antibody. For example, the antibody disrupts or prevents the binding of the IL-2 ligand to the alpha, beta, and / or gamma chains of the receptor, and reduces or inhibits IL-2 mediated signaling. In certain embodiments, the antibody disrupts or prevents the binding of the IL-2 ligand to CD25. In some embodiments, the antibody disrupts or prevents the binding of the IL-2 ligand to CD25 and binds to an epitope different from the epitope to which either daclizumab or basiliximab binds.

[0216] In some embodiments, the CD25 antibody is a partially blocking antibody that, not completely but partially, disrupts the binding of the IL-2 ligand to the alpha, beta, and / or gamma chains of the IL-2 receptor (CD25), and / or, not completely but partially, reduces IL-2 mediated signaling.

[0217] In some embodiments, the antibody disrupts or prevents the heterotrimerization of the alpha, beta, and gamma IL-2 chains. In some embodiments, the antibody does not block the binding of the IL-2 ligand to CD25, but disrupts or prevents the heterotrimerization of the alpha, beta, and gamma IL-2R chains. In certain embodiments, the antibody selectively binds to Treg cells. In other embodiments, the antibody selectively binds to Teff cells.

[0218] In still further embodiments, it is evaluated whether an antibody produced using the engineered polypeptides provided herein blocks the binding of CD25 to IL-2. In some embodiments, an antibody that does not block CD25 binding to IL-2 is selected. In other embodiments, an antibody that blocks CD25 binding to IL-2 is selected. Such blocking or non-blocking can be evaluated, for example, by coupling CD25 to a biosensor tip and assessing binding by the antibody in the presence and absence of IL-2. In some embodiments, the antibody is expressed with a 6×His tag that can be used with Ni-NTA in flow cytometry to evaluate antibody binding and the blocking or non-blocking of IL-2 binding to CD25. In certain embodiments, antibody binding is evaluated at physiological pH (e.g., between about pH 7.3 and about pH 7.5, or about pH 7.4), and also at the pH of the tumor microenvironment (e.g., between about pH 6.4 and about pH 6.6, or about pH 6.5). In certain embodiments, the blocking / non-blocking activity is compared to the binding of an IL-2 blocking antibody (e.g., daclizumab or basiliximab). In certain embodiments, the blocking / non-blocking activity is compared to the binding of an IL-2 non-blocking antibody (e.g., antibody 7G7B6). In certain embodiments, the blocking / non-blocking activity is compared to both an IL-2 blocking antibody and an IL-2 non-blocking antibody.

[0219] In some embodiments, the antibody is an agonist antibody to CD25. In other embodiments, the antibody is an antagonist antibody to CD25.

[0220] In some embodiments, the antibody binds to CD25 in a trans orientation. In other embodiments, the antibody binds to CD25 in a cis orientation. In still further embodiments, the antibody is capable of binding to CD25 in either a cis or trans conformation.

[0221] The originating antibody clone can be identified by the indicated ID, e.g., the clone ID in Table 2. For example, the antibody can include the antibody clone "YU389-A01" heavy chain complementary determining region shown in row 1 of Table 2.

[0222] In some embodiments, the antibody has CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each independently selected from those disclosed in Table 2.

[0223] [Table 5] TIFF0007695881000014.tif219140 TIFF0007695881000015.tif218139 TIFF0007695881000016.tif115140

[0224] In some embodiments, CDR-H1 is selected from: GGTFSSYA, GGSISSGGYY, GFTFSSYG, GYTFTSYY, GYTFTSYG, GYTFTDYY, GGSISSGGYS, GGSISSSNW, GYSFTSYW, GFTFSNYG, GFTFSSSA, GFTFSSYW, GFIFSRHA, GYTFNNYG, GFTFSSYA, GYTFTTYA, GFTFNNAW, GFTFSSYE, GYSFTTYW, GYSFNTYW, GFTFRRYW, GYSFSTYW, GFAFSSYG, GYKFANYW, GYTFKNFG, GFTFSSYS, GDSISSSSYY, and GGSISRSNW;

[0225] In some embodiments, CDR-H2 is selected from IIPIFGTA, IIPIFGTA, IYYSGST, ISYDGSNK, INPSGGST, ISAYNGNT, IMPIFDTA, VDPEDGET, IYHSGST, IYPGDSDT, ISHDGHVK, IKQDGSEK, ISVYNGDI, INTNTGDP, IKSKTDGGTT, ISSSGSTI, ISSRGSTI, IYPSDSDT, ISGRKGNT, ISSSSSYI, INHSGST, IYHTGST, and ISYDGNNK;

[0226] In some embodiments, CDR-H3 is selected from AREMYYYYGMDV, AREMYYYYGMDV, ARGNLWSGYYF, AKELLEGAFDI, ARDRVTMVRGALAY, ARERSYYGMDV, ASWSERIGYQYGLDV, ARDILGLDY, ATEDTAMGGIDY, ATEGRYGMDV, AVEGGRAPGTYYYDSSGLAY, ARAGYYYGMDV, ARDLGTMVRGVIEPYYFDY, ARGVRGTGFDP, ARDRNGYFQH, AKDLLGELSFFDY, ARLENNWDYGGWFDP, ARDRSYYGMDV, ARDKGYYGMDV, AKEISPRSSVGWPLDY, ARDFWSGYNELGGMDV, ARTWFGEFFDY, ARVIGGWFDP, ARGRLAYGDTEGFDY, ARDILRGESSILDH, ARDRYYYGMDV, ARDLLGSGYDIIDY, ARVWGKNGDFDY, ARDRFHYGMDV, ARDRGDY, TTEGVELLSFGGAPFDY, ARRRGGGFDY, AREKGSWFDP, ARDRGDRVGGLVFDY, ARQVAGGLDY, ARDRGYYGMDV, FRFGEGFDY, ARDGGYYFDD, ARDFRMDV, ARDAYAYGLDV, ARDLMNYGMDV, AREYDYGDYVFDY, ARLENNWDYGGWFDP, ARDYYYYGMDV, ARDIGYYYGMDV, ARVGDGYSLDY, AKAITSIEPY, AKGQGDGMDV, ARLGWGMDV, ARVWGDTTLGYGMDV, AIPWDAELGNYGMDV, ARGRWSGLGDY, ARARGGRYFDY, ARDQLAARRGYYYGMDV, AKGDVNYGMDV, ARDFYYGSGSYPNGYYYGMDV, ARDFNPFSITIFEMDV, ANLAMGQYFDY, ARDLGEAKSSSPHEPDY, ARDQEMYYFDY, ARGKGSYAFDI, and AKGYSSSPGDY;

[0227] In some embodiments, CDR-L1 is selected from QSISSY, QSISSY, SSNIGNNF, QSISNY, NIETKS, KLGDKY, QSVSNY, QTISQW, SSNIGSNY, NFNIGNNL, RNIWSY, QSISSW, QSVSSR, QTISGL, DIESEM, NIGSKS, QSIGNY, QGISSW, QSVSSTY, QDISNY, NIESES, SSDVGAYNY, QDINNY, QGISNS, SSNIGNNY, EGIRTS, QGTSSW, SSDVGGYNY, QSVSNNY, QGINSY, QAVRID, QSISRY, QSIGYW, SSNVGSNY, QSIKNY, QDIKRR, SGSIASSY, NSNVGNNY, SLRSYY, KLGERF, SGSVSTSYY, SSNIGRNY, EDIRMY, QGISTY, SSNVGSRT, NIGTKS, NIGSKT, QSINSY, SSNIGSNT, QSIITY, QSLLHSDGKTY, and GGNIARNY.

[0228] In some embodiments, CDR-L2 is selected from AAS, AAS, DST, DDD, KDN, GAS, KAS, RNN, SNN, AND, DAF, DDS, AAT, AVS, DAS, GVS, DNN, DVS, RAS, GTS, EDN, DND, GKN, QYI, NTD, RNH, EGS, DGR, TAS, DDT, EVS, and EDD.

[0229] In some embodiments, CDR-L3 is selected from QQSYSTPPT, QQSYSTPPT, GSWDTNLSGYV, QVWDSSSGHREV, QAWDSSTYV, QQYNHWPPL, QQYSGDSMYT, AAWDDSLSGVV, AAWDDSLNGVV, ATWDDSLSGVV, QQSHSTPIT, QQYNSYSRT, QQYTNWPQT, LQYDRYSGA, QVWHTTNDHVL, QVWDSSSDHWV, QQSKQIPYT, QQSYSLPLT, QQFDISGGLI, QQYDNLPLT, QVWDSSSDHTVA, SSYTTTDTFV, QQYDNLPYT, QQYYSTPPH, QQSYSTPLT, QVWDSSSDHVV, GTWDSSLSAYV, QQTHTWPWT, QQANSFPLT, QQSYSTPYT, SSYTSSSTYV, QRYGSSPR, QQVHSFPFT, LQHNTFPYT, QQSHSTPLT, QQYNSYPFT, QQYNSSPLMYT, QQTYSTPLT, QQANTFPQT, QSYDGSSVV, GSWEARESVFV, QQTYNDPPT, NSRDSSGNHVV, QTWDGSIVV, VLYMGSGIWV, ATWDDALSGWV, SSYTSSSTLVV, QQSYSTPWT, SSYTSSSTWV, LQDYNYPPA, QQYYDDPQ, QQLNGYPTT, AAWDDSLIGHV, QVWDTSGDLHWA, QQSYTTPLT, QVWDSSSDLLWV, GTWDSSLSALV, AAWDDSLNGPV, MQTKQLPLT, QQANSFPPT, QSYDGNNHMV, and SSYTSSSTLWV.

[0230] In some embodiments, the antibody has CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each independently selected from those disclosed in Table 3A and Table 3B. It is possible to generate new antibodies by combining CDRs from different antibodies in any combination. Gene synthesis and high-throughput screening technologies enable those skilled in the art to test all combinations of the six CDRs without undue experimentation.

[0231]

Table 6

[0232]

Table 7

[0233] In some embodiments, the antibody has any one of the six CDRs of any of the combinations provided in Table 4.

[0234]

Table 8

[0235] In some embodiments, the antibody is an scFv selected from Table 5, or any antibody having an antigen-binding domain derived from an scFv in Table 5. In embodiments, the full-length heavy and light chain variable regions are extracted from the scFv sequences in Table 5 and used to generate soluble Fab fragments, monoclonal antibodies, bispecific antibodies, or any other type of antibody known in the art. If the scFv in Table 5 is a VH:VL scFv, it is possible to reverse the order of the heavy and light chains to generate a VL:VH scFv. If the scFv in Table 5 is a VL:VH scFv, it is possible to reverse the order of the heavy and light chains to generate a VH:VL scFv.

[0236]

Table 9

[0237] In some embodiments, the antibody has CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each independently selected from those disclosed in Tables 14A and 14B. In some embodiments, the antibody has CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each independently selected from any one clone listed in Tables 14A and 14B. In some embodiments, the antibody has CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each independently selected from those disclosed in groups in Tables 15A and 15B. The present disclosure provides antibodies having CDRs from individual clones, or antibodies having CDRs from any one CDR that matches with any other five CDRs. The antibodies identified in Tables 14A and 14B are derived from a murine phage display library. Known methods can be used to convert these CDRs into humanized or chimeric antibodies. VII. Use of CD25 Antibodies

[0238] In some embodiments, the CD25 antibodies provided herein are useful for use in a therapeutic agent, for example, in the treatment of proliferative diseases or disorders such as cancer, or in the treatment of autoimmune diseases.

[0239] Accordingly, provided herein is a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a therapeutic CD25 antibody. In some embodiments, the cancer is a primary cancer. In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer involves solid tumors; in other embodiments, the cancer involves liquid tumors, such as blood-based cancers. In an exemplary embodiment, the CD25 antibody is a non-IL-2 blocking antibody.

[0240] Accordingly, provided herein is a method of treating an autoimmune-related disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a therapeutic CD25 antibody. In an exemplary embodiment, the CD25 antibody is a non-IL-2 blocking antibody.

[0241] As used herein, a subject refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports or pet animals such as dogs, horses, rabbits, cows, pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc. The subject can be male or female.

[0242] Administration of any of the therapeutic CD25 antibodies provided herein can be administered in combination with other known drugs / treatments (e.g., small molecule drugs or biologics). The administration can be sequential or concurrent.

[0243] In vivo administration of the therapeutic CD25 antibodies described herein can be carried out intravenously, intratumorally, intracranially, intralesionally (e.g., intralesional injection, direct contact diffusion), intracavitary (intraperitoneal, intrathoracic, intrauterine, intrarectal), intraperitoneally, intramuscularly, subcutaneously, topically, orally, transdermally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In an exemplary embodiment, the route of administration is by intravenous injection.

[0244] A therapeutically effective amount of a therapeutic antibody will generally be administered. The appropriate dosage of the therapeutic antibody can be determined based on the severity of the disease, the clinical condition of the subject, the clinical history of the subject and response to treatment, as well as the discretion of the attending physician. VIII. Diagnostic Uses

[0245] The CD25 antibodies provided herein can be used for diagnostic and detection purposes. Depending on the application, the CD25 antibodies can be detected and quantified in vivo or in vitro.

[0246] The CD25 antibodies provided herein can be modified for use in various immunoassays. These immunoassays include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), Western blot, radioimmunoassay (RIA), flow cytometry, radioimmunoassay, immunofluorescence assay, spectrophotometry, radiography, electrophoresis, high performance liquid chromatography (HPLC) or thin layer chromatography (TLC).

[0247] The CD25 antibodies provided herein can include a detectable label that is detectable, for example, by spectroscopic, photochemical, biochemical, immunochemical, fluorescent, electrical, optical or chemical means. Labels useful in the present invention include, but are not limited to, fluorescent dyes, radiolabels, enzymes, colorimetric labels, avidin or biotin.

[0248] In some embodiments, the CD25 antibody is radiolabeled with an isotope useful for imaging by a nuclear medicine device (SPECT, PET or scintigraphy). VIII. Pharmaceutical Compositions

[0249] The present disclosure provides compositions comprising a therapeutic CD25 antibody, and in some embodiments, the compositions are sterile. Pharmaceutical compositions generally comprise an effective amount of the therapeutic antibody in a pharmaceutically acceptable excipient. IX. Kits and Manufactured Articles

[0250] The present disclosure also provides a kit comprising any of the CD25 antibodies described herein for either therapeutic use or diagnostic use. In some embodiments, the kit further comprises a component selected from a secondary antibody, a reagent for immunohistochemical analysis, a pharmaceutically acceptable excipient, and instructions for use, and any combination thereof. In some embodiments, the kit comprises one or more of the therapeutic compositions described herein together with one or more pharmaceutically acceptable excipients.

[0251] The present application also provides a manufactured article comprising any one of the therapeutic or diagnostic compositions or kits described herein. Examples of the manufactured article include vials (e.g., sealed vials).

[0252] The description provided herein shows a number of exemplary configurations, methods, parameters, etc. However, it should be recognized that such description is not intended to be a limitation on the scope of the present disclosure, but rather is provided as an explanation of exemplary embodiments.

Examples

[0253] The following examples are illustrative only and in no way limit any aspect of the present disclosure.

Examples

[0254] Development of Engineered Immunogens Sharing Features of CD25 The crystal structure of CD25 was obtained. Some of the available crystal structures for CD25 lack the loop sections of protein mobility. Molecular dynamics simulations were performed to gain a better understanding of this mobility loop and the binding interaction of CD25 with IL-2.

[0255] Different sections of CD25 were selected as inputs for developing engineered immunogens. Some of these regions are shown in FIGS. 34B and 34C. These inputs were used in the ROSETTA program to improve the overall desirable structural and dynamical properties of the interface residues. This process involved varying the structural (non-interface) parts of the segments to stabilize and reproduce the structure, conformation, dynamics, and other properties of the interface residues according to the context of the native CD25 from which they were derived. The stability and flexibility of the segments under development were also analyzed, and the sequences were adjusted as necessary to change these parameters. For example, the N-terminus or C-terminus can be extended by the addition of one or more amino acids to add desired properties. The effect of cross-linking on the engineered immunogen candidates was also evaluated using disulfide bonds formed between the side chains of different amino acid residues. At each stage of the design operations - amino acid addition, cross-linking, and structural residue optimization - each of the many design candidates was quantitatively evaluated using the native scoring and energy functions of the ROSETTA program. Candidates with the best ROSETTA energies were advanced to subsequent stages of the design and ultimately to evaluation and validation by molecular dynamics simulations. In addition to evaluating these parameters at physiological pH (e.g., approximately pH 7.4), the parameters were also evaluated at the tumor microenvironment pH (e.g., approximately pH 6.5) in some examples.

[0256] Quantitative metrics for ranking different designs using molecular dynamics (MD) simulations included similarity to CD25, evaluated via RMSD; and the structural flexibility of the candidates. FIGS. 33A and 33B show exemplary comparisons of stability vs. RMSD at physiological pH for exemplary engineered immunogens developed using the input sections shown in FIG. 32 (left arrow for FIG. 33A, right arrow for FIG. 33B). FIG. 33C is an exemplary comparison of stability vs. RMSD at tumor microenvironment pH for the exemplary immunogen of FIG. 33B. Representative scoring algorithms are presented below.

[0257] [Number]

[0258] Structural similarity was calculated using the root mean square deviation (RMSD) between the atomic coordinates of each peptide conformation in the MD ensemble and the reference structure after RMS alignment to the reference structure. The RMSD was calculated by using a computationally designed engineered immunogen candidate structure as the reference structure or by using an experimentally characterized (e.g., X-ray crystal structure) structure as the reference. In these simulations, all residues including candidate functional interface residues (in some simulations) and candidate structural residues (in other simulations) were compared to the reference.

[0259] An ensemble of conformations sampled by MD was clustered into groups (clusters) that are structurally similar to each other based on RMSD. Disorder was evaluated as the proportion of conformations in the MD ensemble that could not be grouped into clusters of similar conformations due to their structural differences (e.g., high RMSD) from all other conformations in the ensemble. Thus, engineered immunogen candidates with more disorder than alternative candidates were more flexible. Order was evaluated as the proportion of conformations in the MD ensemble that were grouped into clusters of similar conformations (low RMSD). Engineered immunogen candidates with higher order than alternative candidates had lower flexibility, where a higher proportion of that ensemble of conformations went into fewer clusters than the alternative candidates.

[0260] The clusters with the engineered immunogen candidates added were compared to the reference structure using RMSD. If the RMSD of a cluster was below a threshold value of 4 angstroms, this cluster was considered to be ordered (e.g., low flexibility) and similar to the reference (structural similarity). Engineered immunogen candidates having a high proportion of their ensembles meeting this criterion of low flexibility and high structural similarity were predicted to be more active than alternative candidates having a low proportion of their ensembles meeting this criterion of low flexibility and high structural similarity.

[0261] This quantitative analysis was combined with a qualitative analysis of the MD trajectories regarding biophysical, biological and physicochemical interactions and used to select a given immunogen candidate for in vitro evaluation. Table 6 below lists the 11 engineered immunogens prepared as described above.

[0262]

Table 10

Example

[0263] In Vitro Evaluation of Engineered Immunogens The binding of the engineered immunogens prepared in Example 1 was evaluated using an antibody against CD25. The engineered immunogens were modified with a -GSGSGK-biotin group at the C-terminus and then separately bound to streptavidin-coated biosensor tips. A buffer containing the CD25 antibody was flowed over the tips during a 300-second association phase and then the flowed solution was switched to a buffer without the CD25 antibody to measure dissociation from the biosensor tips. A control with no protein or engineered immunogen initially bound to the tip was also run to evaluate any background binding of the CD25 antibody to the tip. A second control with full-length CD25 biotinylated and bound to the biosensor tip was performed to demonstrate the binding level of the CD25 antibody to full-length CD25. The data obtained from these biosensor experiments were used to qualitatively rank the binding of the engineered immunogens.

Example

[0264] Evaluation of Engineered Immunogens by Phage Panning The engineered immunogens provided herein are evaluated using phage panning technology.

[0265] A mouse HuCD25 immunized phage library is transformed by electroporation in TG1 using standard phage display protocols and phage are grown by addition of CM13. The TG1 culture secreting phage is PEG precipitated with PEG / NaCl after incubation on ice for 1 hour. Exemplary libraries that can be used include 7807, 7808, 7809 and 7810.

[0266] Tumor Microenvironment (TME) pH Subtractive Selection: Phage panning is performed at physiological pH and TME pH. Subtractive panning is performed by absorption for 1 hour on ELISA plates coated with 10 ug / ml full-length CD25 (400 nM) in PBST pH 7.4 to deplete antibodies that bind with high affinity to full-length CD25 at physiological pH. 3 x 10 11 pfu phage (1000-fold representation of 3 x 10 8 is first performed by counterselection. The resulting phage supernatant is collected and the pH is adjusted to pH 6.5 with PBST. Subsequent phage panning selections are performed at pH 6.5.

[0267] Pre-clear the panning selection after 1 hour of incubation with 25 microliters of streptavidin Dynabeads without antigen. Then, add the phage to a new pre-blocked Eppendorf LoBind tube. Add the biotinylated engineered immunogen (e.g., as described in Example 1) at a concentration of 100 nM (in some cases, with an additional 500 mM NaCl added to reduce non-specific binding of the immunogen to the phage) for 40 minutes to 1 hour. Next, incubate the sample with 25 microliters of streptavidin beads or streptavidin-coated plate at RT for 1 hour. Pellet the sample and wash using a magnet / magnetic beads, or if using a plate, wash 7 - 10 times with PBST. Replace the tube twice to remove residual phage.

[0268] To elute the phage, add 50 - 800 μL of glycine pH 2.2 to the beads and plate respectively, incubate for 10 minutes or less, and then neutralize with high pH Tris 9.0. Add the eluted phage to 1 - 5 ml of freshly grown TG1 (OD600 ~ 0.5) and incubate for 20 - 30 minutes.

[0269] Plate a fractional log dilution series and transfer the rest to 25 ml of 2×YT. Reserve 1 ml of glycerol stock for subsequent panning rounds and add helper phage / IPTG at OD600 ~ 0.5.

[0270] Perform the selection against the engineered immunogen at pH 6.5 once more, along with the counter-selection at pH 7.4. Subsequently evaluate the periplasmic extract using phage ELISA and octet screening.

[0271] To ensure that the Fab phage binds to full-length CD25 in addition to the engineered immunogen, the final selection using full-length CD25 can be appropriately performed using full-length CD25 instead of the engineered immunogen (two rounds of selection against the engineered immunogen, followed by one round of selection against full-length CD25).

[0272] To perform the selection using full-length CD25, the panning selection was pre-cleared with 25 microliters of streptavidin Dynabeads. After adding the phage to a new pre-blocked Eppendorf LoBind tube, biotinylated full-length CD25 was added at a concentration of 100 nM for 1 hour. The sample was then incubated with 25 microliters of streptavidin beads at RT for 1 hour. The pelleting, washing, and elution steps follow those described above.

Example

[0273] Phage ELISA protocol and biosensor / Octet screening ELISA / Extract preparation: Perform phage ELISA and periplasm extract preparation for Fab Octet screening.

[0274] Dilute the CD25 antigen, add it to the ELISA plate wells and incubate. After incubation, wash the wells twice with PBS, then block by adding BSA and then incubating at 25 °C for 2 hours. Dilute the phage two-fold in 1×PBST 1.0% BSA, pH 6.5, add 50 microliters per well and incubate at room temperature for 5 minutes. Flick the blocking solution from the wells, add 50 μL of the diluted phage preparation to each well and incubate at room temperature for 1 hour. Wash the ELISA plate wells 3 - 5 times with 200 microliters of PBST pH 6.5. Dilute the HRP-conjugated anti-M13 antibody (Abcam, ab50370) 1:5000 in 1×PBST 1.0% BSA pH 6.5. Add 50 microliters of the diluted secondary antibody conjugate to each well and incubate at room temperature for 1 hour. Wash the ELISA plate wells 3 - 5 times with 200 microliters of PBST pH 6.5. Prepare the ECL Lumo substrate (e.g., Supersignal ELISA Pico Chemiluminescent Substrate) as a 1:1 mixture as described. Add 50 microliters of the substrate solution to each well, incubate at room temperature for 5 - 60 minutes and then read.

[0275] Inoculate colonies in 0.03 - 4 ml of 2×YT 0.2% glucose with 0.1 ml of an overnight culture (1 ml of culture in a 96-well plate or 4 ml of culture in a 14 ml falcon tube). Incubate these at 37 °C at 250 - 700 rpm until the OD600 reaches approximately 0.5 - 1.0. Induce the cultures with 50 - 400 μL of 0.025 - 0.1 M IPTG. In some cases, reduce the temperature to 30 °C while shaking at 250 rpm. Then incubate these overnight. Recover 1 - 4 ml of the culture by pelleting at 3400 rcf for 10 - 15 minutes. Discard the supernatant. Resuspend the culture in 50 - 75 μL of PPB buffer (30 mM Tris-HCl, pH 8.0, 1 mM EDTA, 20% sucrose) with 1× Halt protease inhibitor and incubate on a rocking platform at room temperature for 5 minutes or at 4 °C for 10 minutes. Then resuspend the culture in 150 - 225 μL of cold ddH2O with 1× Halt protease inhibitor and incubate on a rocking platform at room temperature for 1 hour or at 4 °C for 1 - 2 hours. Spin the lysate suspension at 15000 rcf at 4 °C for 10 - 15 minutes. Collect and dilute the supernatant.

[0276] Fab Expression and Purification Protocol: Inoculate cell cultures, grow overnight, then induce with 50 μL of 25 mM - 1 M IPTG. Reduce the temperature to 30 °C and the rpm to 150. Incubate overnight. Recover 50 ml of the culture or plates by pelleting at 3400 rcf for 15 minutes. Discard the supernatant. Place the cell pellet from 50 mL of the culture in a -80 °C freezer for 1 hour, while for the culture grown on plates, add 75 μL of PPB with 1×Halt Protease Inhibitor, without EDTA (Thermo Fisher Scientific) and vortex. Shake the plate at 1000 rpm at 4 °C for 10 minutes. Add 225 uL volume of cold water containing 1×Halt Protease Inhibitor, without EDTA (Thermo Fisher Scientific) to each well. Mix the samples and shake at maximum speed, i.e., 1000 rpm at 4 °C for 1 - 2 hours. Spin the plate at 3500 rpm at 4 °C for 10 minutes. Transfer the supernatant (PPE) to a new plate and store at -20 °C. Take the cell pellet from 50 mL of the culture out of the freezer and add 5 ml of PBS, 10 mM imidazole, along with 2.5 mg / ml lysozyme and 1×Halt Protease Inhibitor, without EDTA (Thermo Fisher Scientific). After thawing the pellet at room temperature for 30 minutes, centrifuge the lysate at 3400 rcf for 15 minutes. Take out the supernatant and discard the pellet. For Fab purification, add 500 μL of Ni-NTA resin (pre-washed and pelleted) or use a Ni-NTA spin column. Incubate with the clarified lysate for 30 minutes - 1 hour. Spin this at 1500 rcf. Wash these 5 times with 1 ml of PBS, 10 mM imidazole. Discard the buffer after each spin. Add and mix 1 ml of PBS, 200 mM imidazole, incubate for 30 minutes, and spin at 1500 rcf for 15 minutes. After determining the protein concentration, store the eluted protein at 4 °C or 20 °C. Use a Zeba column for desalting / buffer exchange.

[0277] Octet / Biosensor Screening: For Octet Koff rate screening in cell supernatants, 50 μL of lysate is used in a 384-well Pall ForteBio Octet plate. Data is collected on an Octet RED 384 (MD ForteBio). Briefly, human CD25 is coupled to the AR2G tip (1 μg / ml). For data collection, the baseline is evaluated in PBST 1% BSA buffer for 60 seconds. The tip is then transferred to 50 μL of lysate and association is measured for 300 seconds. Finally, the tip is transferred to PBST 1% BSA buffer. The tip is then regenerated with 200 mM Tris-glycine, pH 2.5 and neutralized with PBST, 1% BSA. For data analysis, double references (no CD25 on the tip as well as blank reference wells) are performed with Octet HT 11.0 software for reference subtraction.

Example

[0278] Evaluation of Antibodies Generated from Immunogens Antibodies are produced by immunizing mice with the engineered immunogens described herein. These antibodies are evaluated for cross-reactivity, cross-blocking, affinity, and dissociation rate (off-rate).

[0279] Protocol for determining cross-reactivity by a biosensor (Octet Red 384, Pall Forte Bio): Using this protocol, the ability of individual test clones (anti-human CD25 mouse monoclonal) to bind to targets (antigens) from human, cynomolgus monkey, and mouse species is determined. The target protein is covalently coupled via a primary amine to a dextran-coated sensor tip or the 6×His-tagged target protein is affinity captured on an anti-6×His monoclonal antibody-coated sensor tip. The monoclonal supernatant in solution is bound to the antigen on the biosensor tip. The net binding signal is the binding signal subtracted from the corresponding signal of the binding of blank or antigen-coated tip to blank medium or buffer. A signal > 3× background binding is considered an actual binding event.

[0280] Protocol for cross-blocking by a biosensor: This method is for determining whether individual test clones (anti-human CD25 mouse monoclonal) can cross-block a control antibody. Cross-blocking may indicate that the test clone recognizes an epitope overlapping with the corresponding epitope of the control antibody. Further, this may imply that the test antibody may have functional characteristics similar to those of the control antibody. For this protocol, the control antibody is covalently coupled via a primary amine to a dextran-coated sensor tip. The target antigen in solution is bound to the control antibody. After this step, the test antibody in solution is bound to the antigen in a sandwich format. If the test antibody can bind to the antigen, it is shown that it does not cross-block the control antibody, but non-binding can be interpreted as the ability to cross-block the control antibody.

[0281] Protocol for Affinity Determination by Biosensor: This method is used to determine the affinity of individual test clones for an antigen when the concentration of the test antibody is known. A capture molecule, such as Protein G or an anti-mouse IgG monoclonal or anti-human IgG monoclonal, is coated onto the biosensor tip. The test clones are captured on the capture molecule-coated surface. The antigen in solution is allowed to associate and dissociate with these test clones for a period in the range of 60 to 600 seconds for the association phase and 60 to 1800 seconds for the dissociation phase. The resulting data (or "sensorgram") is then fitted using either a 1:1 Langmuir model or a 2:1 heterogeneous model. The former assumes that the interaction pairs are homogeneous when the 2:1 model for fitting the data gives a better fit, which indicates that the clone requires further subcloning due to inherent heterogeneity. The data curve fit provides the dissociation constant as the ratio of the on-rate constant and the off-rate constant.

[0282] Protocol for dissociation rate (off-rate) estimation by biosensor: This method is used to estimate the dissociation rate constant of a test clone when the antibody concentration is unknown or when the test clone requires further subcloning. A capture molecule, such as Protein G or an anti-mouse IgG monoclonal or anti-human IgG monoclonal, is coated on the biosensor tip. The test clone is captured on the capture molecule-coated surface. The test clones are allowed to associate and dissociate with the antigen in solution for a period ranging from 60 to 600 seconds for the association phase and from 60 to 1800 seconds for the dissociation phase. The resulting data (or "sensorgram") is then fit using either a 1:1 Langmuir model or a 2:1 heterogeneous model. The former assumes that the interaction pairs are homogeneous when the 2:1 model provides a better fit for fitting the data, which indicates that the clone requires further subcloning due to inherent heterogeneity. The data is fit only for the dissociation rate (off-rate) constant and not for the binding rate (on-rate) (or association) rate constant. This provides an estimate of the dissociation rate (off-rate) constant that can be used to rank the test clones.

Example

[0283] Selection of Engineered Polypeptides Using the CD25 Moiety as a Reference Target The sequence and three-dimensional (3D) structure of CD25 were retrieved from the Protein Data Bank (PDB) (PDB ID number 2ERJ, chain A): ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGSSSHSSWDNQCQCTSSATRSTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTG

[0284] As shown in Figure 6, the putative therapeutic epitopes of CD25 were identified as reference targets for the selection of engineered polypeptides. The residue positions and epitope sequences with respect to SEQ ID NO: 1 are provided in Table 7.

[0285]

Table 11

[0286] Interatomic distances and amino acid descriptor topologies were determined. The interatomic distances and amino acid descriptor topologies of the reference targets were obtained using molecular dynamics simulations, and a covariance matrix of atomic fluctuations was generated for the epitopes in the reference targets. Next, different engineered polypeptide candidates were generated using computational protein design (e.g., Rosetta), molecular dynamics simulations performed on the candidates, and the determined interatomic distances and amino acid descriptor topologies. A covariance matrix of atomic fluctuations was generated for the reference target epitopes and for the residues in the candidates corresponding to the residues in the reference target epitopes.

[0287] Principal component analysis was performed to calculate the eigenvectors and eigenvalues for each covariance matrix - one covariance matrix for each reference target and one covariance for each of the candidates. Only the eigenvectors with the largest eigenvalues were retained. The eigenvectors describe the first, second, third, Nth dominant motions observed in the set of simulated molecular structures. If a candidate moves like the reference epitope, its eigenvector will be similar to the eigenvector of the reference target (epitope). The similarity of the eigenvectors corresponds to those components (3D vectors centered on each CA atom) that are aligned and point in the same direction. This similarity between the eigenvector of the candidate and the eigenvector of the reference target was calculated using the inner product of the two eigenvectors. The inner product value was 0 if the two eigenvectors were 90 degrees with respect to each other, or 1 if the two eigenvectors pointed exactly in the same direction.

[0288] The ordering of the eigenvectors is based on their eigenvalues, which, due to the probabilistic nature by which molecular dynamics simulations sample the energy landscapes underlying two different molecules, need not necessarily be the same between those different molecules, so the inner products between multiple hierarchically ranked eigenvectors were required (e.g., candidate eigenvector 1 × reference target eigenvectors 2, 3, 4, etc.). Further, although not desired to be bound by any theory, molecular motion is complex and more than one (or more than a few) dominant / principal modes of motion can be involved.

[0289] To address these two issues, the inner product between all pairs of eigenvectors in the candidate and reference target was calculated. This results in a matrix of inner products, the dimension of which is determined by the number of eigenvectors analyzed - for 10 eigenvectors, the matrix of inner products is 10×10. This matrix of inner products was converted to a single value by calculating the root mean square value of the squares of the inner products. This is the root mean square inner product (RMSIP).

[0290] Principal component analysis (PCA) makes the 3L×3L dimensional coordinate covariance matrix (where L is the number of atoms) into a set of eigenvectors Φ (reference target) and Ψ (MEM), and eigenvalues Λ. The set Φ contains N eigenvectors φ i for the reference target, and the set Ψ contains N eigenvectors ψ j for MEM, and the eigenvectors are ordered in their respective sets by their associated eigenvalues. The eigenvector with the largest eigenvalue constitutes the largest proportion of the total coordinate covariance. The inner product of each φ i and ψ j eigenvectors is calculated to compare the similarity of motion between the reference target and MEM. The root mean square of the squares of all inner product combinations of φ i and ψ j eigenvectors gives the total similarity of motion of the engineered polypeptide candidate (MEM) to the reference target (RMSIP).

[0291] As shown in Figure 7, in each engineered polypeptide, the epitope residues (gold) and positions in 3D space with added scaffold residues (gray) are selected by this computer-aided design procedure. The residue positions and epitope sequences for PDB ID number 2ERJ, chain A are provided in Tables 8 and 9. The crosslinking positions refer to the expected presence of intracellular disulfide bond formation in each MEM array.

[0292]

Table 12

[0293]

Chemical formula

[0294]

Table 13

Examples

[0295] Selection of antibodies using MEM-programmed in vitro selection Thirty-two different panning strategies (S1 - S32), each including 3 rounds of positive selection, were devised (Table 10). Each program used at least one engineered polypeptide as the selection molecule. Conventional selection using the conventional method (CD25 as the positive target) was also included. Bovine serum albumin (BSA) was used as the negative target for selection against non-specific binding.

[0296] The panning protocol started with a human naive scFv library and the panning was performed in solution using a selection molecule conjugated to biotin (but still in solution). For each round, the starting pool was first combined with a negative selection molecule (BSA) in solution and then streptavidin-coated substrate (e.g., magnetic beads) was applied to the mixture to bind the negative selection molecule. Thus, all phages bound to the negative selection molecule in the pool were bound to the streptavidin-coated support. The remaining solution was removed and then this flow-through was subjected to a positive selection step. The flow-through was combined with and bound to a positive selection molecule (antigen 1) and then streptavidin-coated solid substrate was applied to the mixture. In this step, the bound phages were retained while the remaining unbound phages were removed. Then the bound phages were eluted. Escherichia coli (E. coli) was transfected with the eluted phages using a 30-minute cultivation, the transfected cells were split for next-generation sequencing for analysis and DNA isolation, and then the phages were amplified for use in subsequent panning rounds. For each panning program, negative selection was performed first and positive selection was performed next in each round.

[0297]

Table 14

[0298] For each strategy, 384 clones were selected for ELISA response analysis against full-length CD25 after 3 rounds of panning (Figure 9). Data are shown using the sorted strategies designated by epitope (Figure 6). For each epitope, at least one strategy yielded clones capable of binding to CD25. Different strategies using the same engineered polypeptide were observed to enrich distinct subsets of high-affinity clones (Figure 10, black bars). As shown in Table 11, most of the MEM-programmed selection strategies produced anti-CD25 hits more productively than conventional full-length panning.

[0299]

Table 15

[0300] 1475 of the hits were selected for further characterization because they met one of the following two criteria in ELISA: 1) >10:1 signal-to-noise (s / n) in full-length CD25 ELISA; or 2) >3:1 s / n in MEM ELISA and >5:1 s / n in CD25 ELISA. Confirmation test by biolayer interferometry

[0301] The affinity of different scFv antibodies was evaluated using a single-cycle kinetics assay design on a ForteBio® Octet RED384™ biolayer interferometry instrument. His-tagged scFv was immobilized on an anti-his biosensor (Fortebio® HIS1K). Full-length CD25 analyte was washed away from the sensor tip, and binding of molecules in the analyte to the scFv was recorded. Each assay was run in duplicate. Controls were also run using buffer only (to control for sensor drift) and a separate control of polyclonal IgG isotype antibody purified from human serum (to control for non-specific IgG binding).

[0302] As shown in Figure 11, a bio-layer interferometry of 1475 anti-CD25 scFvs is identified by phage display panning. It is shown that 1433 hits (97%) are confirmed to bind to CD25. The observed K D range of these hits is 10 - 200 nM, and the median K D was 28.5 nM. As shown in Figure 11, most screening strategies generated scFvs with high affinity for CD25. Only scFvs with k -3 less than 10 / s are shown. Appropriate K off values are given on the y-axis. As shown in Figure 12, most of the panning strategies yielded at least one hit with k D less than 10 / s. -3 / s. off Confirmation test by flow cytometry

[0303] The CD25 specificity of different scFv antibodies was evaluated by flow cytometry using cells expressing CD25 [CD25(+)] or cells not expressing CD25 [CD25(-)]. As shown in Figure 13, out of 1248 scFv hits analyzed in this assay, 1160 (93%) specifically bind to CD25(+) cells. Sequence analysis of hits

[0304] Next-generation sequencing was performed on the phage panned in each round. As shown in Figure 15, MEM-directed panning concentrates the CDR diversity of the antibody library in a strategy-dependent manner. Each round of selection decreased the repertoire diversity (Figure 16) and concentrated the CDR lengths to the preferred lengths for each MEM (Figure 17).

[0305] Individual scFvs were sequenced using the Sanger sequencing method. The complete protein sequences for each scFv are provided in Table 5. Immunoglobulin gene usage and complementarity-determining regions are provided in Tables 12 and 2, respectively.

[0306]

Table 16

[0307] Analysis of CDR and germline usage suggests that the 1475 sequenced scFvs correspond to at least 126 distinct clones. This set includes 40 different VH+JH framework selections and 35 VL+JL framework selections. The unique CDR sequences include

[0308]

Table 17

[0309] Sequence analysis applied to the scFvs against individual target epitopes identifies common CDR usage patterns within each set of antibodies:

[0310] For CD25 epitope 1 (55~63), the CDRs used include the following:

[0311]

Table 18

[0312] For CD25 epitope 2 (13~20:127~132), the CDRs used include the following:

[0313]

Table 19

[0314] For CD25 epitope 3 (5 - 17), the CDRs used include the following:

[0315]

Table 20

[0316] For CD25 epitope 4 (5 - 11:156 - 163), the CDRs used include the following:

[0317]

Table 21

[0318] For CD25 epitope 5 (77 - 89), the CDRs used include the following:

[0319]

Table 22

[0320] For CD25 epitope 6 (147 - 157), the CDRs used include the following:

[0321]

Table 23

[0322] For CD25 epitope 7 (11 - 14), the CDRs used include the following:

[0323]

Table 24

[0324] For CD25 epitope 8 (44 - 56), the CDRs used include the following:

[0325]

Table 25

Example

[0326] Confirmation of epitope specificity by competitive binding One hundred and twenty-six anti-CD25 clones were subjected to epitope resolution using the four-target competitive binding assay shown in Figure 18. The binding sites for IL-2, daclizumab, and basiliximab shown in the figure are based on X-ray crystallographic structure determination. The binding site for 7G7B6 is based on peptide mapping.

[0327] The cross-competition assay was performed in a classical sandwich format. This involves immobilizing the first antibody on the biosensor, followed by incubation with the antigen and then the second sandwich antibody. His-tagged scFv was expressed and purified in situ on the biosensor using His-tag capture from the supernatant. Biosensor His-tag capture was standardized across scFv clones by monitoring the tip-loading response to consistent levels for all scFv measurements. Each scFv was individually captured on an anti-His biosensor (Fortebio HIS1K). Baseline measurements were obtained in running buffer. CD25 was then captured by the antibody. Finally, each of various competitive analytes, including IL-2, 7G7B6, basiliximab, or daclizumab, was added. The competitive analyte can bind to the captured CD25 only if the binding epitope of the competitive analyte does not overlap with the binding epitope of the immobilized scFv.

[0328] As shown in Figure 19, the full-length CD25 panining clones are dominated by the IL-2 interface epitope. Most clones are blocked by IL-2, daclizumab, and basiliximab, but not by 7G7B6.

[0329] As shown in Figure 20, the 147-157 epitope MEM engineered clones mainly bind at the intended epitope. Most of the clones are blocked by daclizumab, but not by IL-2, basiliximab, or 7G7B6.

[0330] As shown in Figure 21, the 6-17 epitope MEM engineered clones mainly bind at the intended epitope. Most of the clones are blocked by 7G7B6, but not by IL-2, daclizumab, or basiliximab.

[0331] As shown in Figure 22, the 13-20:127-132 epitope MEM engineered clones mainly bind at the intended epitope. Most of the clones are blocked by 7G7B6, but not by IL-2, daclizumab, or basiliximab.

[0332] As shown in Figure 23, the 44-56 epitope MEM engineered clones mainly bind at the intended epitope. The clones were divided into two profiles. In Profile 1, the clones are blocked by 7G7B6, but not by IL-2, daclizumab, or basiliximab. In Profile 2, the clones are blocked by IL-2, daclizumab, and basiliximab, but not by 7G7B6. These blocking profiles indicate binding to the intended epitope from different approach angles.

[0333] As shown in Fig. 24, the 55 - 63 epitope MEM engineered clones bind predominantly at the intended epitope. The clones were divided into three profiles. In Profile 1, the clones are blocked by 7G7B6, but not by IL - 2, daclizumab, or basiliximab. In Profile 2, the clones are blocked by IL - 2, daclizumab, and basiliximab, but not by 7G7B6. These blocking profiles indicate binding to the intended epitope from different approach angles. In Profile 3, the clones are blocked by IL - 2 and 7G7B6, but not by daclizumab or basiliximab. These blocking profiles indicate binding to the intended epitope from different approach angles.

Example

[0334] Mapping of Functional Epitopes by Alanine Mutagenesis Alanine mutations were designed to confirm or reject the ability of the MEM engineered clones to bin the intended epitope (Fig. 25). Alanine mutagenesis was chosen as an orthogonal method for binning antibodies because it acts on functional epitopes rather than structural epitopes defined by competition assays. Various pairs of surface - accessible residues were selected for mutagenesis. Computer modeling was used to confirm that the alanine mutations selected for use in these assays do not affect either global or local stability. For example, Fig. 26 shows the results for the modeling of alanine mutations within the 145 - 157 epitope. For each mutant and wild - type: RMSD from three independent 100 ns MD simulations in explicit solvent for each of eight different starting apo - CD25 conformations used with reference to the crystal structure. As shown in Figs. 27 - 29, the alanine mutant versions of CD25 have binding responses to basiliximab, daclizumab, and 7G7B6, respectively.

[0335] As intended, binding scFv hits from in vitro selection using the 147 - 157 epitope - targeted engineered polypeptide are consistent with specificity for the intended portion of CD25.

[0336] Each of the 117 scFvs from the screening campaign was tested against four alanine mutation pairs (Figure 31). Diversity of functional epitopes was observed. MEM - engineered hits have distinct in - epitope alanine substitution position sensitivities.

Example

[0337] Confirmation test of antibodies in immunoglobulin G (IgG1) form Thirty antibodies were selected as full - length immunoglobulins for further testing. Heavy and light chain sequences were cloned, expressed, and purified in human immunoglobulin G (IgG1) form. Binding to CD25 was evaluated by Octet® as shown in Table 13.

[0338]

Table 26

Example

[0339] Panning - biased library of mouse antibody sequences A phage display library was generated from the immunoglobulin genes of mice immunized with full - length CD25. This library biased towards CD25 - binding antibodies was panned against the indicated engineered polypeptide to obtain the complementarity - determining region sequences shown in Table 14A and Table 14B.

[0340]

Table 27

[0341]

Table 28

[0342] Array analysis suggests that these antibodies derived from clonal lineages can be grouped as shown in Tables 15A and 15B.

[0343]

Table 29

[0344]

Table 30

Claims

1. A CD25-specific antibody comprising an antigen-binding domain that specifically binds to a polypeptide consisting of the amino acid sequence of SWDNQCQCT, The CD25-specific antibody, comprising the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 and the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3, which are any one of the combinations (1) to (12) presented in the following table. 【Table 1】

2. Whether the CD25-specific antibody competes with an epitope-specific reference binder for binding to CD25, and whether the epitope-specific reference binder is IL-2, daclizumab, basiliximab, and / or 7G7B6; Whether the CD25-specific antibody does not compete with an off-target reference binder, and whether the off-target reference binder is IL-2, daclizumab, basiliximab, and / or 7G7B6; The binding of the CD25-specific antibody to IL-2 is a) D77A and Q79A; b) Q81A and T83A; c) D77A and N78A; d) T35A and Q151A; e) M39A and M147A; f) H33A and T35A; g) K37A and Y149A; h) E30A and H33A; i) D27A and E30A; j) R176A and Q179A; k) Q181A and I183A; l) E100A and R104A; m) Q101A and K105A; n) K102A and K105A; o) K169A and T171A; p) K174A and R176A; q) T175A and R176A; (r) M170A and H172A; (s) N70A and S71A; (t) S72A and H73A; (u) S74A and S75A; and (v) L23A and D25A is disrupted by a mutation to IL-2 selected from; the CD25-specific antibody has a k －２ of less than 10 －３ / s, less than 10 －４ / s or less than 10 ｏｆｆ / s, where the k ｏｆｆ is measured using biolayer interferometry with soluble human CD25; the CD25-specific antibody has a k －２ between 10 －５ / s and 10 ｏｆｆ / s, where the k ｏｆｆ is measured using biolayer interferometry with soluble human CD25; the CD25-specific antibody has a K Ｄ of less than 100 nM, less than 25 nM or less than 5 nM, where the K Ｄ is measured using biolayer interferometry with soluble human CD25; or the CD25-specific antibody has a K Ｄ between 100 nM and 1 nM, where the K Ｄ is measured using biolayer interferometry with soluble human CD25; The CD25-specific antibody according to claim 1.

3. specifically binds to cells expressing CD25; binds to cells expressing CD25 with an average fluorescence intensity (MFI) of at least 10 ４ or at least 10 ５ ; binds to cells expressing CD25 with an average fluorescence intensity (MFI) between 10 ４ and 10 ６ ? Do not bind to CD25(−) cells; or 10 ３ Bind to CD25(−) cells with an average fluorescence intensity (MFI) of less than; The CD25-specific antibody according to claim 1 or 2.

4. A pharmaceutical composition comprising any one of the CD25-specific antibodies according to claims 1 to 3 and optionally a pharmaceutically acceptable excipient.

5. A composition for treating a subject in need of treating or modulating cancer, an autoimmune disease, an autoimmune disorder or depleting regulatory T cells, comprising a therapeutically effective amount of the CD25-specific antibody according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 4.

6. Is a humanized antibody; Is a chimeric antibody; Contains mouse variable domains and human constant domains; or Also binds to cynomolgus monkey CD25; The CD25-specific antibody according to any one of claims 1 to 3.

7. A kit comprising the CD25-specific antibody according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 4.

Images