Manipulated CD25 polypeptide and its use

Abstract

The present invention provides novel engineered polypeptides having structural and / or kinetic similarities to CD25 or a portion thereof, such as engineered polypeptides designed to mimic an epitope outside the IL-2 binding site. [Solution] Manipulated polypeptides comprising a combination of spatially related topological constraints in which at least one constraint originates from the CD25 reference target, and methods for selecting such manipulated polypeptides are provided herein. Further provided are methods for using manipulated polypeptides, including positive and / or negative selection molecules, in a method for screening a library of binding molecules such as antibodies. CD25 antibodies selected using these manipulated polypeptides are further provided herein.

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 on September 18, 2019; and U.S. Provisional Patent Application No. 62 / 767,431, filed on November 14, 2018, the disclosures of which are incorporated herein by reference in their entireties.

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 foreign antigens and overcome the inhibition by regulatory T cells. However, in the case of proliferative diseases, cancer cells may increase the amount of regulatory T cells, thereby disabling the healthy immune response by restricting the generation of effector T cells against cancer cells. Therefore, for example, there is interest in therapeutic agents that modify the proliferation of CD25 - expressing regulatory T cells to suppress the immune system for use in cancer treatment. These therapeutic agents may include CD25 - targeting antibodies.

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

Summary of the Invention

Problems to be Solved by the Invention

[0004] Therefore, there is a need in the field for novel engineered polypeptides having structural and / or kinetic similarities to CD25 or its moieties, such as engineered polypeptides designed to mimic the epitopes outside the IL-2 binding site. [Means for solving the problem]

[0005] In one embodiment, the 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 one embodiment, the engineered polypeptide shares at least 60% structural and / or kinetic identity with the CD25 reference target. In another embodiment, the engineered polypeptide shares at least 80% structural and / or kinetic identity with the CD25 reference target. In yet another embodiment, the engineered polypeptide shares at least 80% sequence identity with an amino acid sequence selected from SEQ ID NOs: 1-16. In yet another embodiment, the engineered polypeptide shares at least 46% structural and / or kinetic identity with the CD25 reference target, where 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 yet another embodiment, the engineered polypeptide shares at least 80% structural and / or kinetic identity with the CD25 reference target. In the embodiment, structural and / or dynamic identity to the CD25 reference target is determined using the structure of CD25 deposited in the A chain, PDB ID number 2ERJ. In the embodiment, the manipulated polypeptide includes an N-terminal modification or a C-terminal modification, and optionally includes an N-terminal biotin-PEG2- or C-terminal-GSGSGK-biotin.

[0007] In the embodiment, 10% to 98% of the amino acids in the manipulated polypeptide satisfy one or more constraints derived from the CD25 reference target. In the embodiment, amino acids that satisfy one or more constraints derived from the CD25 reference target have structural homology with the CD25 reference target in a mean squared skeletal deviation (RSMD) of less than 8.0 Å. In the embodiment, amino acids that satisfy one or more constraints derived from the CD25 reference target have a 30 Å 2 ~3000Å 2 There is a van der Waals surface area overlap with the reference between them. In embodiments, the constraints derived from the CD25 reference target 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 bonding tendency; phi angle; psi angle; van der Waals radius; secondary structure tendency; amino acid adjacency; and amino acid contact. In embodiments, the manipulated polypeptide shares a structural similarity of 46% to 96% or higher with the reference target across amino acids of the polypeptide satisfying one or more reference target-derived constraints.

[0008] In another embodiment, the 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 an embodiment, the antibody competes with an epitope-specific reference conjugate for CD25 binding, the epitope-specific conjugate being IL-2, daclizumab, basioliximab, and / or 7G7B6. In an embodiment, the antibody does not compete with an off-target reference conjugate, the off-target conjugate being IL-2, daclizumab, basioliximab, and / or 7G7B6. In an embodiment, the antibody is 10 -2 Less than / s, 10 -3 / s less than or 10 -4 k less than / s off It has, k offIt is measured using biolayer interferometry with soluble human CD25. In embodiments, the antibody has a k -2 between 10 -5 / s and 10 off / s, and k off is measured using biolayer interferometry with soluble human CD25. In embodiments, the antibody has a K D less than 100 nM, less than 25 nM or less than 5 nM, and 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, and 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 to from Table 7D.

[0010] In the embodiment, the antibodies are provided in Tables 3A and 3B: 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, YU It includes six complementarity determination regions (CDRs) for any one of the following: 389-A03, YU392-G09, YU392-G12, YU392-H02, YU392-H04, YU402-F01, YU389-B11, YU394-D08, or YU390-A11.

[0011] In the embodiment, the antibodies are 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-B 12, comprising heavy chain variable regions and light chain variable regions that each share at least 90%, 95%, 99%, or 100% sequence identity with the heavy chain variable regions and light chain variable regions of 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. In embodiments, the antibody is a full-length immunoglobulin G monoclonal antibody. In the embodiment, the antibodies are 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, provided in Table 5. The scFv contains a single-strand variable fragment (scFv) sequence that shares at least 90%, 95%, 99%, or 100% sequence identity with 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.

[0012] In the embodiments, the antibody is a human antibody. In the embodiments, the antibody is a humanized antibody. In the embodiments, the antibody is a chimeric antibody. In the embodiments, the antibody contains a mouse variable domain and a human constant domain. In the embodiments, the antibody also binds to cynomolgus monkey CD25.

[0013] In another embodiment, the Disclosure provides a pharmaceutical composition comprising any antibody of the Disclosure and, as appropriate, pharmaceutically acceptable excipients. In another embodiment, the Disclosure provides a method for treating a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of any antibody or pharmaceutical composition of the Disclosure. In an embodiment, the subject has cancer. In an embodiment, the subject has an autoimmune disease or disorder. In another embodiment, the Disclosure provides a method for depleting the number of regulatory T cells in a subject, comprising administering to the subject a therapeutically effective amount of any antibody or pharmaceutical composition of the Disclosure. In an embodiment, the subject has cancer. In an embodiment, the subject has an autoimmune disease or disorder.

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

[0015] In some embodiments, manipulated immunogens having at least 60% sequence similarity to sequences selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 are provided herein. In some embodiments, the manipulated immunogen has at least 80% similarity to its sequence. In other embodiments, the manipulated immunogen has at least 90% similarity to its sequence. In certain embodiments, the manipulated immunogen has at least 90% similarity to its sequence. The immunogen shares at least one characteristic with CD25. In further embodiments, the engineered immunogen binds to an antibody of CD25. In some embodiments, the engineered immunogen has a higher binding affinity to the antibody of CD25 at pH below 7.0 compared to a binding affinity at pH between approximately 7.3 and approximately 7.5. In some embodiments, the engineered immunogen has a higher binding affinity to the antibody of CD25 at pH between approximately 6.4 and approximately 6.6 compared to a binding affinity at pH between approximately 7.3 and approximately 7.5.

[0016] In other embodiments, a method for producing an antibody is provided herein, 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 NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 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 to CD25 at pH below 7.0 compared to a binding affinity at pH between about 7.3 and about 7.5. In further embodiments, the antibody exhibits a higher binding affinity to CD25 at pH between about 6.4 and about 6.6 compared to a binding affinity at 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. A 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 heterotrimerization of IL-2R-alpha, IL-2R-beta, and IL-2R-gamma. In certain embodiments, the antibody is capable of binding to both cis and trans orientations of CD25.

[0017] This patent or application file includes at least one drawing in color. A copy of this patent or patent application accompanied by the color drawing(s) will be provided by the Patent Office upon request and payment of the required fees. This application can be understood by reference to the following description, to be interpreted in conjunction with the attached drawings. [Brief explanation of the drawing]

[0018] [Figure 1] Figure 1 provides a schematic diagram illustrating the construction of three exemplary combinations of spatially related topological constraints for use in selecting the manipulated polypeptides described herein.

[0019] [Figure 2] Figure 2 provides a schematic diagram of the steps involved in some exemplary methods for determining spatially related topological constraints derived from a reference, and their use in selecting manipulated polypeptides. Manipulated polypeptides are referred to herein as mesoscale molecules, MEMs, or mesoscale peptides.

[0020] [Figure 3A] Figures 3A–3C provide schematic diagrams illustrating the selection of a group of manipulated polypeptides using the methods described herein. Figure 3A shows the extraction of spatially relevant topological information about the interface of interest under reference and its use in defining topological constraints for use in selecting manipulated polypeptides. Figure 3B provides a schematic diagram detailing the in silico screening process, showing how mismatched candidates are discarded and candidates that match the topology are retained. Figure 3C shows the top 12 selected manipulated polypeptide candidates identified. [Figure 3B] Figures 3A–3C provide schematic diagrams illustrating the selection of a group of manipulated polypeptides using the methods described herein. Figure 3A shows the extraction of spatially relevant topological information about the interface of interest under reference and its use in defining topological constraints for use in selecting manipulated polypeptides. Figure 3B provides a schematic diagram detailing the in silico screening process, showing how mismatched candidates are discarded and candidates that match the topology are retained. Figure 3C shows the top 12 selected manipulated polypeptide candidates identified. [Figure 3C]Figures 3A–3C provide schematic diagrams illustrating the selection of a group of manipulated polypeptides using the methods described herein. Figure 3A shows the extraction of spatially relevant topological information about the interface of interest under reference and its use in defining topological constraints for use in selecting manipulated polypeptides. Figure 3B provides a schematic diagram detailing the in silico screening process, showing how mismatched candidates are discarded and candidates that match the topology are retained. Figure 3C shows the top 12 selected manipulated polypeptide candidates identified.

[0021] [Figure 4A] Figures 4A–4B provide schematic diagrams of a second set illustrating the selection of different groups of manipulated polypeptides based on different sets of reference parameters using the methods described herein. Figure 4A shows the extraction of spatially relevant topological information and the construction of a topological matrix. Figure 4B provides a list of the top eight manipulated polypeptide candidates selected by comparing the candidates with topological constraints and in silico. [Figure 4B] Figures 4A–4B provide schematic diagrams of a second set illustrating the selection of different groups of manipulated polypeptides based on different sets of reference parameters using the methods described herein. Figure 4A shows the extraction of spatially relevant topological information and the construction of a topological matrix. Figure 4B provides a list of the top eight manipulated polypeptide candidates selected by comparing the candidates with topological constraints and in silico.

[0022] [Figure 5] Figure 5 is a schematic diagram providing an overview of an exemplary programmable in vitro selection design using the engineered polypeptides described herein, and also using native proteins as positive (T) or negative (X) selection molecules.

[0023] [Figure 6]Figure 6 shows a diagram of eight epitopes on CD25 outside the IL-2 interface that were targeted for the generation of the manipulated polypeptides of this disclosure.

[0024] [Figure 7] Figure 7 shows 16 engineered polypeptides designed to mimic eight epitopes outside the IL-2 interface on CD25. In each figure, the CD25 target epitope residues are shown in gold. The scaffold residues designed to support these epitope residues are shown in gray.

[0025] [Figure 8] Figure 8 shows the computer-determined deviation of the engineered polypeptide from the target epitope. The engineered polypeptide exhibits structural and dynamic similarities to the target epitope (46%–96% RMSIP).

[0026] [Figure 9] Figure 9 shows ELISA analyses of 384 anti-CD25 scFv clones per in vitro selection strategy. Eight CD25 epitopes were targeted using 32 programmed selection strategies. The numbers indicate the ELISA analysis of individual scFv from each selection strategy. Each scFv was tested by ELISA against full-length CD25. Selection strategies S1-S32 are designated by epitope numbers 1-8, corresponding to the epitopes shown in Figure 6.

[0027] [Figure 10] Figure 10 shows that MEM-programmed selection schemes enrich distinct high-affinity clone subsets. Histograms of two different selection strategies (Scheme A and Scheme B) for each of the three MEM polypeptides are shown. The schemes in the right panel produced a higher number of high-affinity clones. Panning with full-length CD25 produced relatively fewer high-affinity clones.

[0028] [Figure 11]Figure 11 shows data from biolayer interferometry for 1433 anti-CD25 scFv identified by phage display panning. The y-axis plots the koff (1 / s) for each clone. The observed median kon was 1.35 × 10⁵ (1 / Ms). The KD estimate assumes a kon of 4.5 × 10⁴ (1 / Ms). Of the 1475 screen hits tested, 1433 (97%) were confirmed to bind to CD25. The plot shows the dissociation rate (off-rate) distribution for the 1433 confirmed hits.

[0029] [Figure 12] Figure 12 shows data from biolayer interferometry for anti-CD25 scFv identified by phage display panning. Hits are identified by the panning strategy used. Data are shown only for hits with koff less than 10⁻³ / s.

[0030] [Figure 13] Figure 13 shows flow cytometry data for anti-CD25 scFv identified by phage display panning. The CD25 specificity of different scFv antibodies was evaluated by flow cytometry using CD25-expressing cells [CD25(+)] or CD25-non-expressing cells [CD25(-)].

[0031] [Figure 14A] Figures 14A–14B show data from flow cytometry for anti-CD25 scFv identified by phage display panning. Hits are identified by the panning strategy used. Figure 14A shows binding to CD25(+) cells. Figure 14B shows binding to control CD25(-) cells. [Figure 14B]Figures 14A–14B show data from flow cytometry for anti-CD25 scFv identified by phage display panning. Hits are identified by the panning strategy used. Figure 14A shows binding to CD25(+) cells. Figure 14B shows binding to control CD25(-) cells.

[0032] [Figure 15] Figure 15 shows the amino acid residue enrichment at each CDR H3 position in a typical enrichment strategy (S12).

[0033] [Figure 16] Figure 16 shows a graph of sequence diversity between each round of MEM or CD25-steered in vitro selection.

[0034] [Figure 17] Figure 17 shows a graph of CDR length between each round of MEM or CD25-driven in vitro selection.

[0035] [Figure 18] Figure 18 shows a ribbon diagram of CD25 indicating the appropriate binding sites for IL-2 and three antibodies (daclizumab, Tusk 7G7B6, and basiliximab) used in epitope degradation using a four-target competitive binding assay.

[0036] [Figure 19] Figure 19 shows that full-length CD25 panning clones are dominated by the IL-2 interface epitope. Most clones are blocked by IL-2, daclizumab, and basioliximab, but not by 7G7B6.

[0037] [Figure 20]Figure 20 shows that the 147-157 epitope MEM manipulator clones primarily bind to their intended epitopes. Most clones are blocked by daclizumab, but not by IL-2, basioliximab, or 7G7B6.

[0038] [Figure 21] Figure 21 shows that the 6-17 epitope MEM manipulator clones primarily bind to their intended epitopes. Most clones are blocked by 7G7B6, but not by IL-2, daclizumab, or basioliximab.

[0039] [Figure 22] Figure 22 shows that the 13-20:127-132 epitope MEM manipulator clones primarily bind to their intended epitopes. Most clones are blocked by 7G7B6, but not by IL-2, daclizumab, or basioliximab.

[0040] [Figure 23] Figure 23 shows that the 44-56 epitope MEM manipulator clone primarily binds to the intended epitopes. The clone was split into two profiles. In profile 1, the clone is blocked by 7G7B6 but not by IL-2, daclizumab, or basioliximab. In profile 2, the clone is blocked by IL-2, daclizumab, and basioliximab but not by 7G7B6. These blocking profiles demonstrate binding to the intended epitopes from different approach angles.

[0041] [Figure 24]Figure 24 shows that the 55-63 epitope MEM-manipulated clone primarily binds to the intended epitopes. The clone was divided into three profiles. In profile 1, the clone is blocked by 7G7B6 but not by IL-2, daclizumab, or basioliximab. In profile 2, the clone is blocked by IL-2, daclizumab, and basioliximab but not by 7G7B6. These blocking profiles demonstrate binding to the intended epitopes from different approach angles. In profile 3, the clone is blocked by IL-2 and 7G7B6 but not by daclizumab or basioliximab. These blocking profiles demonstrate binding to the intended epitopes from different approach angles.

[0042] [Figure 25] Figure 25 shows alanine mutations designed to confirm or reject binning of intended epitopes by the MEM manipulator clone. Eight epitopes are shown in color. The site of the residue mutated to alanine is indicated by a red bar.

[0043] [Figure 26] Figure 26 shows that alanine mutations in the 147–157 CD25 epitopes do not affect either global or local stability. For each mutant and wild-type: RMSD from three independent 100 ns MD simulations in explicit solvent for each of the eight different starting apo-CD25 configurations, using the crystal structure as a reference.

[0044] [Figure 27] Figure 27 shows the reliability of Ala mutant epitope mapping demonstrated using basiliximab control antibody. The binding response of the Ala mutant supports the crystal structure of the basiliximab epitope. The basiliximab-CD25 epitope, as determined from the X-ray crystal structure, is shown in orange.

[0045] [Figure 28] Figure 28 shows the reliability of the Ala mutant epitope mapping demonstrated using a daclizumab control antibody. The binding response of the Ala mutant supports the crystal structure of the daclizumab epitope. The daclizumab-CD25 epitope, as determined from the X-ray crystal structure, is shown in orange. The inset in the lower left shows an epitope zoom illustrating the effect of T175A on daclizumab binding.

[0046] [Figure 29] Figure 29 shows the reliability of Ala mutant epitope mapping demonstrated using a 7G7B6 control antibody. The binding response of the Ala mutant supports the peptide mapping of the 7G7B6 epitope.

[0047] [Figure 30] Figure 30 shows the epitope mapping of MEM-programmed selected hits for 147–157 epitopes. Most hits exhibit susceptibility to ala mutations in the intended epitopes.

[0048] [Figure 31] Figure 31 shows the sensitivity of various MEM-manipulated antibody hits to alanine substitution. Functional epitope diversity is observed. MEM-manipulated hits have distinct in-epitope alanine substitution site sensitivities.

[0049] [Figure 32] Figure 32 shows a model of CD25 (ribbon) binding with IL-2 ligands (space-filling), IL-2R-gamma, and IL-2R-beta. The left and right arrows indicate selected sections of CD25 used to develop engineered immunogens that mimic CD25.

[0050] [Figure 33]Figure 33A is an exemplary graph of the evaluation of molecular stability versus mean squared deviation (RMSD) at physiological pH for a manipulated immunogen developed using the CD25 section indicated by the left arrow in Figure 32 as the initial input. Figure 33B is an exemplary graph of the evaluation of molecular stability versus mean squared deviation (RMSD) at physiological pH for a manipulated immunogen developed using the CD25 section indicated by the right arrow in Figure 32 as the initial input. Figure 33C is an exemplary graph of the evaluation of molecular stability versus mean squared deviation (RMSD) at tumor microenvironment pH (lower pH) for the manipulated immunogen in Figure 2B (developed using the CD25 section indicated by the right arrow in Figure 32 as the initial input).

[0051] [Figure 34] Figure 34A is a model of IL-2 binding with the IL-2R complex, showing the CD25 section (ribbon), IL-2 (1), IL-2R-gamma (2), and IL-2R-beta (3). Figure 34B is another representation of IL-2 binding with the IL-2R complex, listing the regions of CD25 used as input to develop different selected exemplary engineered immunogens. Figure 34C is another representation of IL-2 binding with the IL-2R complex, listing the regions of CD25 used as input to develop different selected exemplary engineered immunogens. [Modes for carrying out the invention]

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

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

[0054] [Table 1]

[0055] In some embodiments, the operated polypeptide provided herein is

[0056] [Table 2] It shares 80% sequence identity with the amino acid sequence selected from the available options.

[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 dynamic 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, e.g., a mesoscale engineered polypeptide. Methods for selecting a mesoscale engineered polypeptide, as well as compositions comprising this engineered polypeptide and methods for using the same, are provided herein. For example, antibody in vitro A method for using the workspace manipulated in the selection is provided herein.

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

[0060] The methods provided herein include identifying a number of spatially related topological constraints, some of which may be derived from the CD25 reference target; constructing combinations of these constraints; comparing candidate peptides to these combinations; and selecting candidates having constraints that overlap with these combinations. By using spatially related topological constraints, different embodiments of the engineered polypeptide can be included in the combination, depending on the intended use, desired function, or other desired features. Furthermore, in some embodiments, not all constraints need to be derived from the CD25 reference target. Through such methods, in some embodiments, the selected engineered polypeptide may have a different overall structure from the reference peptide, while still retaining desired functional features and / or important substructures, rather than simply being a variation of the CD25 reference target (which may be obtained, e.g., through single-reference peptide mutagenesis or progressive modification).

[0061] Methods using the manipulated polypeptide, including a programmable in vitro selection method using one or more manipulated polypeptides, are further provided herein. Such selection may be used, for example, in the identification of antibodies.

[0062] These methods and manipulated polypeptides are described in more detail below. II. How to select a manipulated polypeptide

[0063] In some embodiments, a method for selecting an manipulated polypeptide, Identifying one or more topological features of the CD25 reference target; Design spatially related constraints for each topological feature to produce combinations of constraints derived from the CD25 reference target; Comparing the spatially related topological features of candidate peptides with those derived from combinations of the CD25 reference target; and Select candidate peptides that have spatially related topological features that overlap with the constraint combinations derived from the CD25 reference target. Methods including the above are 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 manipulated 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 different desired features for a given position and their desired level (if applicable). In some embodiments, this map is not based on a linear or otherwise predetermined amino acid skeleton and can therefore satisfy the desired combination described. This can allow for flexibility in the structure. For example, in some embodiments, the “map” includes a spatial region in which a given constraint can be adequately satisfied by two adjacent amino acids – in some embodiments, these amino acids can be directly bound (e.g., two consecutive amino acids), while in other embodiments, the amino acids are not directly bound to each other but can be spatially brought together by peptide folding (e.g., not consecutive amino acids). The separate constraints themselves do not necessarily have to be structural and may include, for example, chemical descriptors and / or functional descriptors. In some embodiments, constraints include structural descriptors, e.g., a desired secondary structure or amino acid residues. In certain embodiments, each constraint is selected independently.

[0066] For example, Figure 1 is a schematic diagram showing the construction of a typical combination of spatially related topological constraints. The three constraints in Figure 1 are sequence, nearest neighbor distance, and atomic motion, with nearest neighbor distance and atomic motion combined in one picture. As shown, some constraints are mapped independently of the location of the skeleton (e.g., atomic motion of a particular side chain), thus allowing for a much wider variety of structural configurations to be attempted compared to simply varying one or more positions on a reference scaffold. The three different constraints and their spatial descriptions are combined into a matrix (e.g., a tensor), and a set of candidate peptides can then be compared to this combination to identify novel manipulated polypeptides that satisfy desired criteria. In some embodiments, one or more further non-reference-derived constraints are also included in the combination. Comparison of candidate peptides with a defined combination can be performed, for example, using an in silico method to evaluate the constraints of each candidate peptide on the desired combination and a ratio of how well the candidates match. These candidates with a desired level of overlap with the defined combination can then be synthesized and evaluated using standard peptide synthesis methods known to those skilled in the art.

[0067] In some embodiments, the constraint combination 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, 4, 5, or 6 independently selected spatially related topological constraints. One or more of the constraints originate from the CD25 reference target. In some embodiments, each of the constraints originates from the CD25 reference target. In other embodiments, at least one constraint originates from the CD25 reference target, and the remaining constraints do not originate from the reference target. For example, in some embodiments, the constraints between 1 and 9, 1 and 7, 1 and 5, or 1 and 3 originate from the CD25 reference target, while the constraints between 1 and 9, 1 and 7, 1 and 5, or 1 and 3 do not originate from the CD25 reference target.

[0068] Once a constraint combination is constructed, a set of candidate peptides are compared to this combination to identify one or more novel engineered polypeptides that satisfy 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 novel engineered polypeptides that satisfy 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, topological feature simulations are performed. If present, the combination of constraints is used to evaluate the overlap of topological features of candidate peptides compared to the CD25 reference target. In some embodiments, one or more candidate peptides are also compared to the CD25 reference target, and if present, the overlap of topological features of the candidate peptides with the topological features of the CD25 reference target is evaluated. In some embodiments, the manipulated polypeptides are identified from computer samples and topological feature simulations 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 the manipulated polypeptides are selected, and the selected manipulated polypeptides have the highest topological feature overlap with the CD25 reference target among the total sampled population.

[0069] The spatially related topological constraints used to construct a desired combination (e.g., a desired tensor) can be independently selected from a broad set of possible features. These may include, for example, constraints describing structural, dynamic, chemical, or functional features, or any combination thereof.

[0070] Structural constraints may include, for example, interatomic distance, amino acid sequence similarity, solvent exposure, phi angle, psi angle, secondary structure or amino acid contact, or any combination thereof.

[0071] Dynamical constraints may include, for example, atomic fluctuations, atomic energies, van der Waals radii, amino acid proximity, or non-covalent bonding tendencies. Atomic energies may include, for example, pairwise attraction energy between two atoms, pairwise repulsion energy between two atoms, atomic-level solvation energy, pairwise charge attraction energy between two atoms, pairwise hydrogen bond attraction energy between two atoms, or non-covalent bonding energy, or any combination thereof.

[0072] Chemical characteristics may include, for example, chemical descriptors. Such chemical descriptors may 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 may include, for example, bioinformatics descriptors, biological responses, or biological functions. Bioinformatics descriptors may include, for example, BLOSUM similarity, pKa, zScale, Cruciani Properties, Kidera Factors, VHSE-scale, ProtFP, MS-WHIM score, T-scale, ST-scale, transmembrane tendency, protein embedding region, helix tendency, sheet tendency, coil tendency, turn tendency, immunogenicity tendency, presence of antibody epitopes, and / or presence of protein interfaces, or any combination thereof.

[0074] In some embodiments, designing constraints incorporates information about residue-level energy, residue-level interactions, residue-level fluctuations, residue-level interatomic distances, residue-level chemical descriptors, residue-level solvent exposure, residue-level amino acid sequence similarity, residue-level bioinformatics descriptors, residue-level non-covalent bonding tendencies, residue-level phi / pse angle, residue-level van der Waals radius, residue-level secondary structure tendencies, residue-level amino acid adjacency, or residue-level 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 in the total combination of constraints, or combinations 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 subset of residues. At least one different feature is used for different subsets 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 may include, for example, molecular dynamics simulations, Monte Carlo simulations, coarse-grained simulations, Gaussian network models, machine learning, or any combination thereof.

[0075] In some embodiments, multiple constraints are selected from a single category. For example, in some embodiments, the combination includes two or more constraints that are independently a type of biological response. In some embodiments, two or more constraints are independently a type of secondary structure. In certain embodiments, two or more constraints are independently a 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(s) 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 dynamical level constraint (e.g., a characteristic dynamic or set of dynamics that can be satisfied by several different atomic compositions).

[0077] In some embodiments, one or more constraints originate from a protein or peptide structure associated with a biological function or biological response. For example, in some embodiments, one or more constraints originate from an extracellular domain, e.g., a G protein-coupled receptor (GPCR) extracellular domain or an ion channel extracellular domain. In some embodiments, one or more constraints originate from a protein-protein interface junction. In some embodiments, one or more constraints originate from a protein-peptide interface junction, e.g., an MHC-peptide or GPCR-peptide interface. In certain embodiments, the atom or amino acid constrained to such a protein or peptide structure is an atom or amino acid associated with a biological function or biological response. In some embodiments, the atom or amino acid in the manipulated polypeptide constrained to such a protein or peptide structure is an atom or amino acid derived from a CD25 reference target. In some embodiments, one or more constraints originate from a polymorphic region of the CD25 reference target (e.g., a region that contributes to inter-individual allele variation).

[0078] In some embodiments, one or more atoms associated with a biological function or 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 atoms are selected from the group consisting of oxygen, nitrogen, sulfur, and hydrogen.

[0079] In some embodiments, one of the constraints is one or more amino acids associated with a biological function or biological response, and / or the engineered polypeptide contains one or more amino acids associated with a biological function or biological response, the one or more amino acids are independently selected from the group consisting of 20 naturally occurring proteinogenic amino acids, naturally occurring non-proteinogenic amino acids, and unnatural amino acids. In some embodiments, the unnatural amino acids are chemically synthesized. In a particular embodiment, one or more amino acids are selected from 20 naturally occurring proteinogenic amino acids. In other embodiments, one or more amino acids are selected from naturally occurring non-proteinogenic amino acids. In further embodiments, one or more amino acids are selected from unnatural amino acids. In further embodiments, one or more amino acids are a combination of 20 naturally occurring proteinogenic amino acids, naturally occurring non-proteinogenic amino acids, and unnatural amino acids. Selected from.

[0080] The constraint combinations used to select the manipulated polypeptides described herein include at least one constraint derived from the CD25 reference target, however, in some embodiments, one or more constraints in the combination do not originate from the CD25 reference target. Thus, in certain embodiments, the selected manipulated polypeptide includes one or more features not shared with the CD25 reference target.

[0081] In some embodiments, one or more constraints derived from the CD25 reference target and used in the combination describe the inverse of features observed in the CD25 reference target. For example, the CD25 reference target may have a particular pattern of positive charge, and a charge-related constraint derived from this CD25 reference target describes a similar pattern of neutral or negative charge. Therefore, in some embodiments, one or more inverse constraints derived from the CD25 reference target are included in the combination. Such inverse constraints may be useful in selecting engineered polypeptides, for example, as a control molecule for a particular assay or panning method, or as a negative select molecule in a programmable in vitro selection method described herein.

[0082] In some embodiments, a spatially defined combination of 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 structural elements, force atomic fluctuations, alter the total hydrophobicity of a peptide, alter the solubility of a peptide, alter the total charge of a 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 alter immunogenicity. In certain embodiments, one or more non-reference-derived topological constraints restrain one or more atoms or amino acids in a combination of constraints (or subsequently selected peptides) derived from the CD25 reference target. For example, in some embodiments, the constraint combination includes a secondary structure derived from the CD25 reference target, and the constraint combination also includes constraints that stabilize the secondary structure elements (e.g., via further hydrogen bonding, or hydrophobic interactions, or side-chain stacking, or salt bridges, or disulfide bonds), and these stabilizing constraints are not present in the CD25 reference target. In another example, in some embodiments, the constraint combination (or subsequently selected peptide) includes one or more atoms or amino acids derived from the CD25 reference target, and the constraint combination also includes constraints that force atomic fluctuations in at least a portion of the atoms or amino acids derived from the target reference, and these constraints are not present in the target reference. In some embodiments, one or more non-reference-derived constraints are inverse constraints. For example, in some embodiments, two constraint combinations are constructed to select an engineered polypeptide having inverse features. In some such embodiments, a first combination of constraints would include one or more constraints derived from the CD25 reference target and one or more constraints not derived from the CD25 reference target; a second combination of constraints would include the same one or more constraints derived from the CD25 reference target and one or more inverse non-CD25 reference target constraints of the first combination. b.CD25 reference target

[0083] Any suitable CD25 reference target may be used to induce one or more spatially relevant topological constraints for use in the methods provided herein. In some embodiments, the CD25 reference target is a full-length native protein. In other embodiments, the CD25 reference target is a portion of a full-length native protein. In 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, e.g., an epitope or a predicted epitope. In some embodiments, the methods provided herein may be used to select one or more engineered polypeptides that are immunogens and can be used to produce one or more antibodies that specifically bind to the protein from which the target reference originates. In further embodiments, the methods provided herein may be used to select one or more engineered polypeptides that can then be used to select one or more binding partners for a protein of interest, e.g., an antibody, a phage displaying Fab, or a phage displaying 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 simulation (e.g., molecular dynamics) or laboratory measurements (e.g., NMR), or a combination thereof. Once the constraints are induced and combined, engineered polypeptide candidates are generated, in some embodiments, using computational protein design (e.g., Rosetta). In some embodiments, other methods for sampling the peptide space are used. Dynamic simulations may then be performed on the engineered polypeptide candidates to obtain the parameters of the selected constraints. A covariance matrix of atomic fluctuations is generated for the CD25 reference target, and covariance matrices are generated for each residue in each engineered polypeptide candidate, and these covariance matrices are compared to determine overlap. Principal component analysis is performed on each covariance matrix - CD25 reference target A single covariance matrix is ​​created for each of the candidate manipulated polypeptides, and the eigenvectors and eigenvalues ​​for each covariance are calculated, with the eigenvector having the largest eigenvalue being retained.

[0088] Eigenvectors describe the 1st, 2nd, 3rd, and Nth dominant motions observed in a simulated set of molecular structures. While we do not wish to be constrained by any particular theory, if a candidate engineered polypeptide behaves similarly to the CD25 reference target, its eigenvectors will be similar to those of the CD25 reference target. The similarity of eigenvectors corresponds to their aligned components (3D vectors centered on each CA atom) pointing in the same direction.

[0089] In some embodiments, this similarity between the eigenvectors of a candidate engineered polypeptide and the eigenvectors of the CD25 reference target is calculated using the dot product of the two eigenvectors. The dot product value is 0 if the two eigenvectors are 90 degrees to each other, or 1 if the two eigenvectors point in exactly the same direction. Although we do not wish to be constrained by theory, the ordering of eigenvectors is based on their eigenvalues, and since the eigenvalues ​​may not necessarily be the same between those different molecules due to the probabilistic nature of molecular dynamics (MD) simulations sampling the underlying energy landscape of two different molecules, in some embodiments, the dot product between multiple differentially ranked eigenvectors is required (e.g., eigenvector 1 of the engineered polypeptide × eigenvectors 2, 3, 4 of the CD25 reference target, etc.). Furthermore, molecular motion is complex, and the motion of more than one (or more than a few) dominant / major modes may be involved. Therefore, in some embodiments, the dot product between all pairs of eigenvectors in the candidate engineered polypeptide and the CD25 reference target is calculated. This yields an inner product matrix, the dimension of which is determined by the number of eigenvectors analyzed. For example, for 10 eigenvectors, the inner product matrix is ​​10 × 10. This inner product matrix can be reduced to a single value by calculating the root mean square value of the inner product of 100 (in the case of 10 × 10). This is the root mean square inner product (RMSIP). From this comparison, one or more candidate manipulated polypeptides that have similarity to the given combination of constraints are selected. d. Further steps

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

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

[0092] In other embodiments, manipulated polypeptides, such as manipulated polypeptides selected via the methods described herein, are provided herein. In some embodiments, the manipulated polypeptides have a molecular weight between 1 kDa and 10 kDa and contain up to 50 amino acids. In a particular installation, the manipulated polypeptide has molecular masses 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 manipulated polypeptide comprises 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 CD25 reference target-derived constraints. Any constraints described herein may be used in combination in some embodiments. In further embodiments, between 10% and 98% of the amino acids in the engineered polypeptide satisfy one or more CD25 reference target-derived constraints (for example, if the engineered polypeptide contains 50 amino acids, between 5 and 49 amino acids satisfy one or more CD25 reference target-derived constraints). In some embodiments, between 20% and 98%, 30% and 98%, 40% and 98%, 50% and 98%, 60% and 98%, 70% and 98%, 80% and 98%, 90% and 98%, 10% and 90%, 10% and 80%, 10% and 70%, 10% and 60%, 10% and 50%, 10% and 40%, 10% and 30%, or 10% and 20% of the amino acids of the manipulated polypeptide satisfy one or more CD25 reference target-derived constraints. In further embodiments, one or more amino acids satisfying one or more constraints derived from the CD25 reference target have structural homology with the CD25 reference target in skeletal 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 weight between 1 kDa and 10 kDa; contains up to 50 amino acids; is a combination of spatially related topological constraints in which one or more constraints are derived from the CD25 reference target; between 10% and 98% of the amino acids in the engineered polypeptide satisfy one or more constraints derived from the CD25 reference target; and the amino acids satisfying one or more constraints derived from the CD25 reference target have structural homology with the CD25 reference target in skeletal mean square deviation (RSMD) of less than 8.0 Å.

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

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

[0096] In some embodiments, the manipulated polypeptide exhibits at least one structural difference when compared to the CD25 reference target. Such structural differences may 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 factors, Zernike descriptors, van der Waals surfaces, nodes and edges of the structural graph, volumetric surface, electrostatic potential surface, hydrophobic potential surface, local diameter, local surface features, skeleton model, charge density, hydrophilicity 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%, compared to the feature in the CD25 reference target, where applicable to the type of feature. For example, in some embodiments, the difference is the total number of atoms, and the manipulated 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 lies in the total positive charge, where the total positive charge of the manipulated 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 manipulated 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 spatially defined combination of topological constraints includes one or more secondary structural elements not present in the CD25 reference target. Therefore, in some embodiments, the manipulated polypeptide includes one or more secondary structural elements not present in the CD25 reference target. In some embodiments, the combination and / or manipulated polypeptide includes one, two, three, four, or more secondary structural elements not found in the CD25 reference target. In some embodiments, each secondary structural element is independently selected from the group consisting of helices, sheets, loops, turns, and coils. In some embodiments, each secondary structural element not present in the CD25 reference target is independently an α-helix, β-bridge, β-strand, 3 10 It is a helix, π-helix, turn, loop, or coil.

[0098] In certain embodiments, the CD25 reference target comprises one or more atoms associated with a biological response or biological function (e.g., as described herein); the engineered polypeptide comprises 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 with the atomic fluctuations of these atoms in the CD25 reference target. Therefore, for example, in some embodiments, the atoms themselves may be different atoms, but their atomic fluctuations overlap. In one embodiment, the atoms are the same atoms, and their atomic fluctuations overlap. In further embodiments, the atoms are independent, the same or different. In some embodiments, the overlap is an 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 a particular embodiment, the RMSIP is

[0099]

number

[0100] In some embodiments, the engineered polypeptide contains atoms or amino acids (or combinations thereof) associated with a biological response or biological function, and at least a portion of these atoms or amino acids or combinations are derived from the CD25 reference target, and specific constraints on 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 further embodiments, the matrix is ​​an L×2 phi / psi angular matrix.

[0101] For example, in some embodiments, atomic fluctuations of atoms or amino acids in the 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 originate from the 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 positions) of each set is described by a corresponding L×L matrix. In a particular embodiment, 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 for a portion of the engineered polypeptide derived from the CD25 reference target compared to the corresponding (i,j) element in the atomic fluctuation or adjacency matrix 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%, less than 45%, or less than 40% of the manipulated polypeptide derived from the CD25 reference target, compared to the corresponding element in the CD25 reference target matrix. In some embodiments where the matrix exhibits atomic fluctuations, L is the number of amino acid positions, and the (i,j) value in the atomic fluctuation matrix element is either the sum of the intramolecular atomic fluctuations for the i-th and j-th amino acids, respectively, 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 diagonal. Alternatively, in some embodiments, the interatomic distance may act as a weighting coefficient 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 may be determined by molecular simulation (e.g., molecular dynamics) and / or laboratory measurements (e.g., NMR). In other embodiments where the matrix indicates adjacency, L is the number of amino acid positions, and the value in the adjacency matrix element (i,j) is either the intramolecular interatomic distance between the i-th and j-th amino acids, respectively, 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 diagonal. Alternatively, in some embodiments, the interatomic distance is expressed as an adjacency matrix element instead of a multiplier of 0 or 1. It can act as a weighting coefficient for (i,j). In certain embodiments, the i-th and j-th interatomic distances may be determined by molecular simulations (e.g., molecular dynamics) and / or laboratory measurements (e.g., NMR).

[0102] In certain embodiments, the atoms or amino acids in the manipulated polypeptide that are associated with the response or function have a mean percentage error (MPE) of less than 75% compared to the chemical descriptor vector of the topological constraint and the reference described by the same chemical descriptor, for a portion of the manipulated polypeptide derived from the CD25 reference target, where each i-th element in the chemical descriptor vector corresponds to an amino acid position subscript. 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 the reference described by the same chemical descriptor for a portion of the manipulated polypeptide derived from the CD25 reference target.

[0103] In further embodiments, the matrix is ​​an L×2 phi / psi angular matrix, where the atoms or amino acids associated with the response or function in the engineered polypeptide have an MPE of less than 75% with respect to the reference phi / psi angular 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 and psi values ​​are 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 angular 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 structural prediction, or laboratory measurements (e.g., NMR).

[0104] In some embodiments, the matrix is ​​an S×S×M secondary structure element interaction matrix, where the atoms or amino acids associated with the response or function in the engineered polypeptide have a mean percentage error (MPE) of less than 75% compared to the reference secondary structure element relation matrix in a portion of the engineered polypeptide derived from the 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 the reference secondary structure element relation matrix in a portion of the engineered polypeptide derived from the reference target. Interaction descriptors may 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) = the value of the m-th interaction descriptor between the i-th and j-th secondary structure elements.

[0105] The mean percentage error (MPE) for the different matrices described herein is:

[0106]

number

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

[0108] In some embodiments, at least a portion of the manipulated polypeptide is topologically constrained to one or more secondary structural elements. In some embodiments, atoms or amino acids in the manipulated polypeptide that are associated with a biological response or biological function are topologically constrained to one or more secondary structural elements. In some embodiments, the secondary structural elements are independently sheets, helices, turns, loops, or coils. In some embodiments, the secondary structural elements are independently α-helices, β-bridges, β-strands, 3 10 These are helices, π-helices, turns, loops, or coils. In certain embodiments, at least a portion of the manipulated polypeptide is topologically constrained to one or more secondary structural elements present in the CD25 reference target. In some embodiments, at least a portion of the manipulated polypeptide is topologically constrained to a combination of secondary structural elements, each element independently selected from the group consisting of sheets, helices, turns, loops, and coils. In further embodiments, each element is an α-helix, a β-bridge, a β-strand, or 3 10 The shapes are independently selected from the group consisting of helices, π-helices, turns, loops, and coils.

[0109] In some embodiments, the secondary structure elements are parallel or antiparallel sheets. In some embodiments, the sheet secondary structure contains two or more residues. In some embodiments, the sheet secondary structure contains 50 residues or less. In further embodiments, the sheet secondary structure includes residues between two and 50. The sheet can be parallel or antiparallel. In some embodiments, a parallel sheet secondary structure may be described as having two parallel strands i,j (the N-terminuses of the i and j strands are oppositely oriented) and a hydrogen bonding pattern between residues i:j. In some embodiments, an antiparallel sheet secondary structure may also be described as having two antiparallel strands i,j (the N-terminuses of the i and j strands are the same orientation) and a hydrogen bonding pattern between residues i:j-1,i:j+1. In certain embodiments, the orientation of the strands and the hydrogen bonding may be determined by knowledge-based or molecular dynamics simulations and / or laboratory measurements.

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

[0111] In some embodiments, the secondary structural element is a turn. In some embodiments, a turn comprises 2 to 7 residues and one or more inter-residue hydrogen bonds. In some embodiments, a 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 further embodiments, the secondary structural element is a coil. In certain embodiments, the coil includes between 2 and 20 residues and zero predicted interresidial hydrogen bonds. In some embodiments, these coil parameters are determined by knowledge-based or molecular dynamics simulations and / or laboratory measurements.

[0113] In further embodiments, the engineered polypeptide comprises one or more atoms or amino acids derived from the 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 vectors of atoms or amino acids in the engineered polypeptide have a cosine similarity greater than 0.25 compared to the secondary structure motif vectors of the CD25 reference target for a portion of the engineered polypeptide derived from 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 may be, for example, α-helix, β-bridge, β-strand, 3 10 This includes helices, π-helices, turns, and loops. 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 for a portion of the manipulated polypeptide derived from the CD25 reference target, compared to the secondary structure motif vector of the CD25 reference target. The cosine similarity is,

[0114]

number

[0115] In some embodiments, one or more atoms or amino acids of an engineered polypeptide derived from a CD25 reference target may be compared to the corresponding atoms or amino acids of the CD25 reference target using the total topological constraint distance (TCD). In some embodiments, the total TCD of these engineered polypeptide atoms or amino acids derived from the CD25 reference target is + / - 75% compared to the TCD distance of the corresponding atoms in the CD25 reference target, and the two intramolecular topological constraints are interacting if their pairwise distance is 7 Å or less. In some embodiments, the atoms or amino acids in the engineered polypeptide being compared are associated with a biological function or biological response. The i-th and j-th pairwise distances of the two atoms or amino acids may, in some embodiments, be determined 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 may be compared to the corresponding atoms or amino acids of the CD25 reference target using chain topology parameters (CTP). In some embodiments, the CTPs of these engineered polypeptide atoms or amino acids are + / - 50% compared to the CTPs of the corresponding atoms or amino acids in the CD25 reference target, and the intrachain topological interactions are pairwise distances of 7 Å or less. 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 and j-th pairwise distances may be determined by molecular simulations (e.g., molecular dynamics) and / or laboratory measurements (e.g., NMR). An exemplary equation for evaluating CTP is:

[0118]

number

[0119] In some embodiments, one or more atoms or amino acids of an engineered polypeptide derived from a CD25 reference target may be compared to the corresponding atoms or amino acids of the CD25 reference target using a quantitative estimate of likeness (QEL). In some embodiments, the QELs of these engineered polypeptide atoms or amino acids are + / - 50% compared to the QELs 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. An exemplary equation for determining the QEL is:

[0120]

number

[0121] In some embodiments, one or more atoms or amino acids in an engineered polypeptide derived from a CD25 reference target may be compared to the corresponding atoms or amino acids in the CD25 reference target using a topological clustering coefficient (TCC) vector and mean percentage error (MPE). In some embodiments, the TCC vector and MPE are less than 75% of the TCC of the corresponding atoms or amino acids in the CD25 reference target, where each element (i) of the vector is the topological clustering coefficient for the i-th amino acid position, and the intramolecular cluster is defined by an interacting edge distance of 7 Å or less from the i-th amino acid position and two edges: ij,jl. 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 may be determined by molecular simulation (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

[0123] In further embodiments, one or more atoms or amino acids of an engineered polypeptide derived from a CD25 reference target may be compared to the atoms or amino acids of the corresponding 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, Jackard distance, Mahalanobis distance, or Hamming distance across all M dimensions. The L×M matrix element (l,m) contains the m-th constraint value for the l-th 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 atoms or amino acids of the corresponding 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 embodiments, methods using the engineered polypeptide described herein in selecting binding partners using a series of programmed selection steps are further provided herein, wherein at least one selection step includes evaluating the interaction of a pool of potential binding partners with the engineered polypeptide.

[0125] In some embodiments, a method for manipulating the selection of a binding molecule using two or more select molecules is described. , provided herein. 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 for negative selection molecules, and at least one positive selection step in which at least a portion of the pool is screened for positive selection molecules. In some embodiments, the method comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, 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. Figure 5 provides an exemplary schematic diagram illustrating three rounds of selection in detail, in the figure, the first and third rounds comprising more than one negative selection step, and the first round further comprising 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. In addition, two positive selection molecules are used in the first round.

[0126] In some embodiments of the method, which includes more than one round, each negative and positive selection molecule is selected independently. In other embodiments, the same negative selection molecule or the same positive selection molecule, or a combination thereof, may be used in more than one round. For example, in Figure 5, the same negative selection molecule used in round 1 is used again in round 3, and a further third negative selection molecule is also included in round 3. The order of the negative selection steps and positive selection steps may be selected independently within each round of selection in certain embodiments. Thus, for example, in some embodiments, the method includes one or more rounds of selection, each round including a negative selection step first, followed by a positive selection step. In other embodiments, the method includes one or more rounds of selection, each round including a negative selection step and a positive selection step independently, and in each round, the negative selection step independently precedes or follows the positive selection step.

[0127] Selection methods employ positive (+) and negative (-) steps to maneuver a library of candidate binding molecules toward or away from specific desired features, such as binding specificity or binding affinity. By using multiple steps employing both positive and negative selection molecules, the pool of candidates can be directed in a stepwise manner for selection toward desired and undesirable features. Furthermore, 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 toward different directions. Thus, for example, in some embodiments, a method involving one round employing (+) selection followed by (-) selection will result in a different pool of candidates than when (-) selection is first followed by (+) selection. Conjecturing from this, when considering methods involving multiple rounds, the order of the selection steps can result in a different final pool of selected candidates, even when the same positive and negative selection molecules are used as a whole.

[0128] In some embodiments, a selection molecule having the opposite characteristics of another selection molecule is used. This may be useful, for example, to ensure that candidate binding partners identified (or excluded due to a negative selection molecule) using a positive selection molecule are identified (or excluded) due to a desired (or undesired) trait, rather than due to separate, unrelated binding interactions. To remove binding partners that are binding via unrelated interactions, the selection molecule is used, except for residues / structures that convey the desired (or undesired) trait. Reverse select molecules having similar or identical structures and characteristics may be used. For example, if interaction with a specific charge pattern in a positive select molecule is desired, a reverse negative select molecule may be used in which the residue providing that charge pattern is replaced with an uncharged residue and / or a residue with the opposite charge. Thus, for a particular select molecule, multiple different corresponding reverse select 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 a selection molecule that is not an engineered polypeptide may include, for example, a naturally occurring polypeptide or a portion thereof. In other embodiments, one or more selection molecules that are not engineered polypeptides may include, for example, a polypeptide or a portion thereof that does not exist naturally. For example, in some embodiments, one or more selection molecules (e.g., positive or negative selection molecules) are immunogens, antibodies, cell surface receptors, or transmembrane proteins, or signaling proteins, or multiprotein complexes, or peptide-protein complexes, or any portion thereof, or any combination thereof. In some embodiments, one or more selection molecules are CD25, or any portion thereof.

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

[0131] In some embodiments, each selection feature is independently selected from the group consisting of amino acid sequence, polypeptide secondary structure, molecular dynamics, chemical characteristics, biological function, immunogenicity, CD25 reference target(s) polyspecificity, cross-species CD25 reference target reactivity, selectivity for desired CD25 reference targets(s) over undesired reference targets(s), selectivity for reference targets(s) within a family that are sequencely and / or structurally homologous, selectivity for reference targets(s) with similar protein function, selectivity for distinct desired reference targets(s) from a larger family of undesired targets with high sequencely 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 targets(s), tolerance for sequence diversity of reference targets(s), and tolerance for dynamical diversity of reference targets(s). In some embodiments, each selection feature is a different type of selection feature. In other embodiments, the two or more selection features are of the same type but different. For example, in some embodiments, the two or more selection features are polypeptide secondary structures, one being a positive selection for a desired polypeptide secondary structure and the other a negative selection for an undesired polypeptide secondary structure. In some embodiments, the two or more selection features are selection for a cell type. A positive selection feature is selectivity for a specific desired cell type, while a negative selection feature is selectivity for a specific 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 the manipulated polypeptides of the Disclosure. For example, the manipulated polypeptides shown in Figure 7, Table 1, Table 8, and Table 9 may be used to select antibodies (or other binders) that specifically bind to the epitopes shown in Figure 6 and Table 7. An example selection strategy is provided in Table 10.

[0133] In another embodiment, the Specified Reference Reference provides a composition comprising two or more selectively manipulating polypeptides, wherein each polypeptide is independently a positively selective molecule comprising one or more positively manipulating features or a negatively selective molecule comprising one or more negatively manipulating features. Such features may, in some embodiments, be selected from the group consisting of amino acid sequence, polypeptide secondary structure, molecular dynamics, chemical characteristics, biological function, immunogenicity, reference target(s) polyspecificity, cross-species reference target reactivity, selectivity for desired reference targets(s) over undesired reference targets(s), selectivity for reference targets(s) within a family of sequence and / or structural homology, selectivity for reference targets(s) with similar protein function, selectivity for distinct desired reference targets(s) from a larger family of undesired targets with 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 targets(s), tolerance for sequence diversity of reference targets(s), and tolerance for dynamical diversity of reference targets(s).

[0134] Accordingly, in a further embodiment, a method is provided herein for screening a library of binding molecules using the selective manipulator compositions described herein, wherein each round of selection comprises a negative selection step of screening at least a portion of the pool for negative selection molecules; and a positive selection step of screening at least a portion of the pool for positive selection molecules; and the order of the selection steps within each round, and the order of the rounds, results in the selection of a subset of the pool that differs from an alternative order.

[0135] In some embodiments, the binding partners evaluated using the selective manipulation polypeptide compositions described herein or the screening methods described herein are phage libraries, e.g., Fab-containing phage libraries; or cell libraries, e.g., B-cell libraries or T-cell libraries.

[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 selection molecules. In other embodiments where there are more than one round, at least two rounds include the same negative selection molecule, the same positive selection 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 profiling, protein modification assay, Western blotting, and immunohistochemistry. In some embodiments, gene expression profiling includes a complete sequence repertoire analysis of the subset pool, e.g., next-generation sequencing. In some embodiments, statistical and / Alternatively, information science scoring or machine learning training may be used to evaluate one or more subsets of the pool in one or more selection rounds.

[0138] In some embodiments, the identity and / or order of positive and / or negative selection molecules for subsequent rounds is determined by analyzing a subset pool from one selection round. 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 selection rounds to determine the identity and / or order of positive and / or negative selection molecules for subsequent rounds (e.g., the next round, or a later round in the program).

[0139] In further embodiments, the selection method includes modifying the subset pool obtained from a selection round before proceeding to the next selection round. Such modifications may include, for example, genetic mutation 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 the modified subset pool proceeds 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 selection in subsequent rounds.

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

[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 may 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 any signal exceeding that threshold is flagged as a presumed hit motif. IV. Use of engineered polypeptides to produce 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, wherein the immunogen is an engineered polypeptide provided herein, are provided herein. In some embodiments, the animals are humans, rabbits, mice, hamsters, monkeys, etc. In certain embodiments, the monkeys are cynomolgus monkeys, macaques, or rhesus monkeys. Immunizing an animal with a manipulated polypeptide may 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 antibodies from an animal includes isolating B cells that express the antibodies. Some embodiments further include fusing the B cells with myeloma cells to create a hybridoma that expresses the antibodies. In some embodiments, antibodies produced using the manipulated polypeptide can cross-react with humans and monkeys, for example, cynomolgus monkeys. a. Characteristics of the manipulated polypeptide

[0143] The engineered polypeptides provided herein have one or more features in common with CD25. In some embodiments, they represent at least one feature of the surface of CD25, for example, a functional interface 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 represents at least one feature of a portion of the surface of CD25 that is not known to interact with an antibody against CD25.

[0144] In some embodiments of certain types of features, the engineered polypeptide presents a mimicry of the functional interface (e.g., binding surface) of CD25, but the features shared by the engineered polypeptide can be best described as being shared with CD25 as a whole. For example, one shared feature may be the binding between CD25 and its binding partner, which occurs between CD25 and 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 may include, for example, structural metrics or functional metrics, or a combination thereof. At least one shared feature may include, for example, one or more structural similarities, conformational entropy similarities, one or more chemical descriptor similarities, one or more functional bond similarities, or one or more phenotypic similarities, or any combination thereof. In a particular embodiment, the manipulated polypeptide shares one or more of these features with at least a portion of the surface of CD25, for example, a functional interface, for example, a binding surface.

[0146] In some embodiments, the manipulated polypeptide has structural similarity to CD25 (or a portion of the CD25 surface, e.g., the binding surface), and this structural similarity is assessed by the mean squared deviation (RMSD) of the skeleton or side-chain RMSD. RMSD can be applied to three-dimensional structures to assess the average distance between atoms and to compare how two similar, separate structures exist in three-dimensional space. In some embodiments, the RMSD of the skeleton, amino acid side chains, or both between the manipulated 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, this is the portion of CD25 (or the functional interface of CD25) being compared to the manipulated polypeptide. RMSD can be assessed using, for example, the experimentally measured or simulated structure of the manipulated polypeptide; and the experimentally measured or simulated structure of CD25 (or its functional interface). In some embodiments, the manipulated polypeptide is considered structurally similar to CD25 if the backbone of the manipulated 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 manipulated polypeptide has conformational entropy similar to CD25 (or a portion of the surface of CD25, e.g., the binding surface), and this con The conformational entropy is evaluated, for example, using the experimentally measured or simulated structure of the manipulated polypeptide and the experimentally measured structure or molecular dynamics-simulated motion of CD25 (or a portion thereof). In some embodiments, such simulations use the experimentally measured structure or molecular dynamics-simulated motion of CD25 (or a portion thereof, e.g., a portion of the bonding surface). In certain embodiments, the conformational entropy of the manipulated polypeptide is considered analogous to that of CD25 (or a portion thereof) if the manipulated polypeptide molecular dynamics ensemble, performed under standard physiological conditions, has all states where all non-hydrogen atom positions have an RMSD ≤ 6.0 Å, compared to the known X-ray crystal structure of CD25 (or a portion thereof).

[0148] In other embodiments, the manipulated polypeptide has one or more chemical descriptors similar to CD25 (or a portion thereof, e.g., the binding surface). In other embodiments, the manipulated polypeptide has one or more chemical descriptors complementary to the binding partner of CD25 (e.g., an antibody against CD25). Such chemical descriptors (which may be similar or complementary) may include, for example, hydrophobic patterns, H-bonding patterns, atomic volume / radius, charge patterns, or atomic occupation patterns, or any combination thereof. In some embodiments, these chemical descriptors may be evaluated using experimentally measured or simulated structures of the manipulated polypeptide and experimentally measured or simulated structures of CD25 (or a portion thereof, e.g., the binding surface).

[0149] In other embodiments, the engineered polypeptide has a functional binding similar to that of 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 may include, for example, a fusion protein containing 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 assessed by comparing the binding constant (Kd) or inhibition constant (Ki), or the binding rate (on-rate), or the dissociation rate (off-rate), or the binding affinity of the binding pair, or the Gibbs free energy (ΔG) of the binding. In some embodiments, the binding partner is an antibody against CD25.

[0150] In some embodiments, the binding constant (K) between the manipulated polypeptide and its CD25 binding partner is determined. d ) is the K between CD25 and its binding partner d The inhibition constant (K) with the CD25 binding partner of the manipulated polypeptide is within 1000 times, within 800 times, within 600 times, within 400 times, within 200 times, within 100 times, within 90 times, within 80 times, within 70 times, within 60 times, within 50 times, within 40 times, within 30 times, within 20 times, within 10 times, within 8 times, within 6 times, within 4 times, within 2 times, within 1.5 times, within 1.2 times, or approximately the same. In other embodiments, the inhibition constant (K) with the CD25 binding partner of the manipulated polypeptide is used. i ) is the K between CD25 and its binding partner iThe on-rate of the binding of the manipulated polypeptide to the CD25 binding partner is similar to the on-rate of the binding of CD25 to the binding partner. In some embodiments, the on-rate of the binding of the manipulated polypeptide to the CD25 binding partner is similar to the on-rate of the binding of CD25 to the binding partner. In some embodiments, the on-rate of the binding of the manipulated polypeptide to the CD25 binding partner is similar to the on-rate of the binding of CD25 to the binding partner. The ratios are within 0.0, 400, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 8, 6, 4, 2, 1.5, 1.2, or approximately the same. In other embodiments, the off-rate of the binding of the manipulated polypeptide to its CD25 binding partner is similar to the off-rate of the binding of CD25 to its binding partner. In some embodiments, the off-rate of the manipulated polypeptide's binding to its CD25 binding partner is within 1000, 800, 600, 400, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 In some embodiments, the binding affinity between the manipulated polypeptide and the CD25 binding partner is within 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, 10 times, 8 times, 6 times, 4 times, 2 times, 1.5 times, 1.2 times, or approximately the same as the binding affinity between CD25 and the binding partner. In some embodiments, the Gibbs free energy of binding between the manipulated polypeptide and the CD25 binding partner is within 1000, 800, 600, 400, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 1.2, or approximately the same as the Gibbs free energy of binding between CD25 and the binding partner. In some embodiments, the CD25 binding partner is an antibody against CD25.

[0151] In other embodiments, the manipulated polypeptide shares sequence similarity with CD25 or a portion thereof (e.g., the binding surface of CD25). This similarity may be compared to a continuous amino acid sequence or a discontinuous sequence of CD25 (or a portion thereof). For example, in a particular embodiment, the binding surface of CD25 is formed by a discontinuous amino acid sequence, and the manipulated polypeptide has sequence similarity to at least a portion of the discontinuous sequence forming the surface. In other embodiments, the manipulated polypeptide has sequence similarity to at least a portion of the continuous 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 against CD25.

[0152] In some embodiments, the manipulated polypeptide has sequences that are 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 portion of the continuous sequence that forms the binding surface of CD25. In certain embodiments, the manipulated polypeptide has sequences that are 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 portion of the discontinuous sequence that forms the binding surface of CD25. In a particular embodiment, the manipulated polypeptide 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 the contiguous portion of the binding surface of CD25. It has identical sequences. In further embodiments, the manipulated polypeptide has sequences that are 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 discontinuous portions of the CD25 binding surface. In some embodiments, the manipulated polypeptide has sequences that are at least partially identical (as described herein) to the CD25 binding surface, and the binding surface includes an epitope that binds to one or more antibodies against CD25.

[0153] In certain embodiments, the sequence similarity between the manipulated polypeptide and CD25 (or a portion thereof) is evaluated using the peptide portion(s) of the manipulated polypeptide, if present, without linkers. In certain embodiments, one or more linking portions are considered equivalent, for example, if the manipulated polypeptide contains one or more linkers, each containing an amino acid. b. Manipulated polypeptide

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

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

[0156] In a particular embodiment, the manipulated 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, each peptide independently comprising 1 to 100 amino acids, or 2 to 100 amino acids, e.g., 2 to 80 amino acids, 2 to 70 amino acids, 2 to 60 amino acids, 2 to 50 amino acids, 2 to 40 amino acids, 2 to 30 amino acids, 2 to 25 amino acids, 2 to 20 amino acids, 2 to 15 amino acids, 5 to 30 amino acids, 5 to 25 amino acids, 5 to 20 amino acids, 5 to 15 amino acids, or 9 and 15 amino acids.

[0157] In some embodiments, the manipulated polypeptide comprises only naturally occurring amino acids. In other embodiments, the manipulated polypeptide comprises unnatural amino acids, for example, combinations of naturally occurring and unnatural amino acids.

[0158] In some embodiments, the manipulated polypeptide comprises two or more peptides, each peptide independently exhibits at least one feature of CD25 or a portion thereof (e.g., the 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 features of CD25 or a portion thereof. In some embodiments, the features are shared with the portion of CD25 that interacts with the antibody.

[0159] In some embodiments, the manipulated polypeptide has at least one feature that is complementary to a CD25 binding partner, such as an antibody to CD25.

[0160] In some embodiments, the peptide of the manipulated polypeptide shares one or more structural similarities with CD25 or a portion thereof. Structural similarity can be assessed in some embodiments by backbone RMSD or side-chain RMSD. For example, in certain embodiments, the RMSD of the backbone, amino acid side chains, or both between the peptide of the manipulated 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 against CD25. The RMSD of structural similarity can be assessed, for example, using experimentally measured or simulated structures of the peptide and experimentally measured or simulated structures of CD25 or a portion thereof. In some embodiments, the peptide of the manipulated polypeptide is considered structurally similar to CD25 (or a portion thereof) if the peptide backbone has an average RMSD of 6.0 Å or less compared to the backbone of a known X-ray structure of CD25 or a portion thereof.

[0161] In some embodiments, the manipulated polypeptide has conformational entropy similar to that of CD25 or a portion thereof. In some embodiments, the experimentally measured structure or molecular dynamics-simulated motion of the peptide is used to compare the conformational entropy with that of the experimentally measured or simulated structure of CD25 or a portion thereof. In some embodiments, conformational entropy is considered similar if a peptide molecular dynamics ensemble performed under standard physiological conditions has all states where all non-hydrogen atom portions 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, for example, a surface portion of CD25 that interacts with an antibody.

[0162] In further embodiments, the similarity between the peptide of the manipulated polypeptide and CD25 (or a portion thereof) may 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 against CD25). Chemical descriptors may include, for example, hydrophobic patterns, H-bonding patterns, atomic volume / radius, charge patterns, or atomic occupancy patterns, or any combination thereof. In some embodiments, the peptide of the manipulated polypeptide has one or more hydrophobic patterns, H-bonding patterns, atomic volume / radius, charge patterns, or atomic occupancy patterns, or any combination thereof, that are similar to CD25 or a portion thereof or complementary to a binding partner of CD25 (e.g., an antibody against CD25). In some embodiments, the similarity is having the same chemical descriptors in common, e.g., the same hydrophobic patterns, H-bonding patterns, atomic volume / radius, charge patterns, or atomic occupancy patterns. Complementary chemical descriptors include, for example, CD25 binding partners, such as peptides having a positive charge pattern that complements the negative charge pattern of an antibody against CD25. These chemical descriptors may, in some embodiments, be evaluated using experimentally measured or simulated structures of the peptide and experimentally measured or simulated structures of CD25 or its binding partner (for example, for complementary evaluation).

[0163] For example, in some embodiments, the manipulated polypeptide binds to the binding partner of CD25 in a manner similar to the binding of CD25 to its binding partner (e.g., IL-2). In some embodiments, the binding partner is a native binding partner, a fragment of a native binding partner, 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 conditions but not under others. In some embodiments, the binding partner binds under pathological conditions or under non-pathological conditions. The binding partner may be, for example, constitutively expressed, or a product of an incidental gene, or may include 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. Modifications may include, in some embodiments, a fusion protein containing 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 manipulated polypeptide has binding to a CD25 binding partner that is within approximately two orders of magnitude or within approximately one order of magnitude of binding to the CD25 binding partner. In some embodiments, binding similarity is assessed by comparing the binding constant (Kd) or inhibition constant (Ki), or the binding rate (on-rate), or the dissociation rate (off-rate), or the binding affinity of the binding pair, or the Gibbs free energy (ΔG) of the binding. In some embodiments, the binding partner is an antibody against CD25.

[0165] In some embodiments, the binding constant (K) between the manipulated polypeptide and its CD25 binding partner is determined. d ) is K with CD25's binding partner dThe inhibition constant (K) between the manipulated polypeptide and the CD25 binding partner is within 1000 times, within 800 times, within 600 times, within 400 times, within 200 times, within 100 times, within 90 times, within 80 times, within 70 times, within 60 times, within 50 times, within 40 times, within 30 times, within 20 times, within 10 times, within 8 times, within 6 times, within 4 times, within 2 times, within 1.5 times, within 1.2 times, or approximately the same. In other embodiments, the inhibition constant (K) between the manipulated polypeptide and the CD25 binding partner is i ) is the K between CD25 and its binding partner iThe ratio is within 1000 times, within 800 times, within 600 times, within 400 times, within 200 times, within 100 times, within 90 times, within 80 times, within 70 times, within 60 times, within 50 times, within 40 times, within 30 times, within 20 times, within 10 times, within 8 times, within 6 times, within 4 times, within 2 times, within 1.5 times, within 1.2 times, or approximately the same. In further embodiments, the on-rate of binding of the manipulated polypeptide to its CD25 binding partner is similar to the on-rate of binding between CD25 and its binding partner. In some embodiments, the on-rate binding of the manipulated polypeptide to its CD25 binding partner is within 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, 1.2 times, or approximately the same as the on-rate binding of CD25 to its binding partner. In other embodiments, the off-rate dissociation of the manipulated polypeptide to its CD25 binding partner is similar to the off-rate dissociation of CD25 to its binding partner. In some embodiments, the off-rate of the manipulated polypeptide's binding to its CD25 binding partner is within 1000, 800, 600, 400, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 20, 12, or approximately the same as the off-rate of the CD25 binding partner. In further embodiments, the binding affinity of the manipulated polypeptide to its CD25 binding partner is similar to the binding affinity of CD25 to its binding partner. In some embodiments, the binding affinity of the manipulated polypeptide to its CD25 binding partner is within 1000, 800, 600, 400, 2 It is within 00x, within 200x, within 100x, within 90x, within 80x, within 70x, within 60x, within 50x, within 40x, within 30x, within 20x, within 10x, within 8x, within 6x, within 4x, within 2x, within 1.5x, within 1.2x, or approximately the same. In some embodiments, the Gibbs free energy of binding between the manipulated polypeptide and the CD25 binding partner is within 1000, 800, 600, 400, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 1.2, or approximately the same as the Gibbs free energy of binding between CD25 and the binding partner. In some embodiments, the CD25 binding partner is an antibody against CD25.

[0166] In some embodiments, the manipulated polypeptide has sequence similarity to CD25 or a portion thereof. In some embodiments, the manipulated polypeptide has sequence similarity to a portion of the surface of CD25 that binds to an antibody. In certain embodiments, the sequence similarity is compared to a continuous amino acid sequence of CD25. In other embodiments, the sequence similarity is compared to a discontinuous sequence of CD25. For example, in certain embodiments, the binding surface of folded CD25 is formed by a discontinuous amino acid sequence, and the manipulated polypeptide has sequence similarity to at least a portion of the discontinuous sequence forming the surface. In some embodiments, the manipulated polypeptide has sequence similarity to at least a portion of the continuous amino acid sequence forming the binding surface of CD25. In some embodiments, the manipulated polypeptide has a sequence of CD25, for example, 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 the sequence forming the binding surface. In a particular embodiment, the manipulated polypeptide has a discontinuous sequence of CD25, for example, 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 the discontinuous sequence forming the binding surface. In a particular embodiment, the manipulated polypeptide has sequences that are 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 a contiguous portion of CD25.In further embodiments, the manipulated 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 discontinuous portions of CD25. In some embodiments, for a manipulated polypeptide comprising at least two peptides, the two or more peptides of the manipulated immunogen independently share sequence similarity with CD25, for example, the binding surface of CD25. In some embodiments, the portion of CD25 that shares sequence similarity with the manipulated polypeptide is the surface that binds to an antibody against CD25. c. Connecting part

[0167] The manipulated polypeptides provided herein may include linking portions. If present, these linking portions may, for example, independently be crosslinks or linkers.

[0168] In some embodiments, the manipulated polypeptide consists of N peptides and N-1 linkers; or N peptides and N-1 linkers; or N peptides and It contains N linkages; or N peptides and N+1 linkages; or N peptides and N+2 linkages; or N peptides and N-2 linkages, where N is 3 or greater.

[0169] In some embodiments, the manipulated polypeptide includes at least one linkage portion, at least two linkage portions, at least three linkage portions, at least four linkage portions, at least five linkage portions, at least six linkage portions, linkage portions between one and six, linkage portions between one and five, linkage portions between one and four, linkage portions between one and three, one linkage portion, or two linkage portions. In some embodiments, each linkage portion is independently a crosslink or linker. In certain embodiments, each linkage portion is a crosslink. In other embodiments, each linkage portion is a linker. In further embodiments, at least one linkage portion is a crosslink, and the remaining linkage portions are independently crosslinks or linkers. In other embodiments, at least one linkage portion is a linker, and the remaining linkage portions are independently crosslinks or linkers.

[0170] Crosslinking includes, for example, a covalent bond between the side chain of one amino acid and a portion of another amino acid. The amino acids can be native or non-native amino acids independently. In some embodiments, crosslinking includes a covalent bond 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. Crosslinking can be formed within a single peptide or between two separate peptides. In some embodiments, the manipulated polypeptides provided herein include a mixture of both intra-peptide crosslinking and inter-peptide crosslinking. In some embodiments, crosslinking is a disulfide bond between two thiol groups of an amino acid side chain, for example, a disulfide bond between two cysteine ​​groups. In some embodiments, crosslinking is an amide bond between an amine group and a carboxylic acid group of two amino acids, where at least one of the amine group and the carboxylic acid group is positioned on the side chain of the amino acid (e.g., the amide bond is not a skeletal amide bond). In some embodiments, crosslinking is an amide bond formed between diaminopimelic acid and aspartic acid. In some embodiments, the amide crosslinking is a lactam. In some embodiments, the crosslinking is an oxime. In some embodiments, the crosslink is a hydrazone. In some embodiments, the crosslink involves a covalent bond between a side chain of one amino acid and a moiety of another amino acid, and one or both of the side chain and moiety are modified to form a covalent bond. Such modifications may include, for example, oxidation, reduction, reaction with a catalyst to form an intermediate, or other modifications known to those skilled in the art.

[0171] Linkers include, for example, molecules covalently bonded to at least two sites of a peptide or molecules covalently bonded between at least two peptides. A linker may bond to two sites within a single peptide, or to two separate peptides, or a combination of both. For example, a linker containing more than two peptide-binding sites may 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 linker may be linked in a variety of different configurations. For example, an engineered polypeptide may have a pattern such as peptide-linker-peptide, ending in a peptide. In some embodiments, the engineered polypeptide includes a linker that forms a branching point, for example, a linker independently bonded to three peptides. In some embodiments, the engineered polypeptide includes a linker having three peptide-binding sites, and the linker is bonded to only two peptides.

[0172] In some embodiments, the linker comprises one or more amino acids. In some embodiments, the amino acids that form part of the linker may be identified separately from the engineered polypeptide. In certain embodiments, the linker is part of the structure of the functional interface of the interfacial protein and / or These are regions that separate and present the peptides of the engineered polypeptide in a structural, chemical, and / or kinetic manner that reflects their function. In further embodiments, linkers do not have function on their own if they are not attached to the peptides of the engineered polypeptide, for example, they do not show binding to a CD25 binding partner. In some embodiments, each linker independently contains at least one, at least two, at least three, at least four, at least five, at least six or more amino acids. In some embodiments, each linker independently contains one amino acid, two amino acids, three amino acids, four amino acids, five amino acids, or six amino acids. The amino acids that form part of the linker may, in some embodiments, be naturally occurring or non-naturally occurring amino acids. In some embodiments, each linker may independently contain 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 contains a cyclic beta residue. Cyclic beta residues may include, for example, APC or ACPC. In further embodiments, the linker may 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, GGGSGGGS, GGGGSGGGS, (PGSG)n, and PGSGSG, where n is an integer between 1 and 10. In some embodiments, the manipulated polypeptide comprises at least one linker, each linker either contains no amino acids, each linker either contains no native amino acids, or each linker contains at least one non-native amino acid.

[0173] In some embodiments, the linker comprises a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). A PEG-containing linker may, for example, contain 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 PEG-containing linker, the PEG comprises monomer units between 3 and 12, monomer units between 3 and 6, monomer units between 6 and 12, or monomer units between 4 and 8. In some embodiments, the operated polypeptide comprises at least one linker comprising PEG3 (containing 3 monomer units), PEG6, or PEG12. In some embodiments, at least one linker is independently PEG3, PEG6, or PEG12. In further embodiments, the linker comprises multi-armed PEG. For example, in a particular embodiment, at least one linker independently comprises a 4-arm PEG or an 8-arm PEG. In a particular embodiment, each arm independently comprises between 3 and 12 monomer units, or between 3 and 6 monomer units, or between 6 and 12 monomer units, or between 4 and 8 monomer units. In a particular embodiment, each arm of a multi-arm PEG comprises the same number of monomer units, such as a 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 includes a dendrimer. The dendrimer includes a molecule having a tree-like branching architecture, for example, a symmetrical core from which molecular parts extend radially, and the branching points form new layers within the molecule. Each new branching point introduces a new, larger layer, and these radial extensions often terminate with functional groups at the outer terminal surfaces of the dendrimer. Thus, increasing the number of branching points then increases the possible number of terminal functional groups on 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 the product of a sulfhydryl-maleimide reaction, which may 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 in which the manipulated polypeptide comprises at least two linkers (for example, in embodiments in which the manipulated polypeptide comprises at least two linking portions, each linking portion being independently a linker or crosslink, or each linking portion being independently a linker), each linker is independently one 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 linkages in the manipulated polypeptide may confer specific structural or functional features, or a combination thereof, of interest. For example, in some embodiments, linkages are present in the manipulated polypeptide to confer structural or functional features, or a combination thereof. Such structural features may include, for example, increased structural flexibility, decreased structural flexibility, directional features, increased length, or decreased length. Targeted directional features may include, for example, structural turns or maintenance of linear structure. Functional features may include, for example, enhanced solubility, one or more protonation sites, one or more proteolytic sites, one or more enzymatic modification sites, one or more oxidation sites, labels, or capture handles. In some embodiments, the linker includes one or more functional features or one or more structural features, or a combination thereof.

[0179] In some embodiments, one or more linkers independently introduce structural "turns" into the manipulated polypeptide. Examples of such linkers include Gly-Pro, Ala-Pro, and trans-pyrrolidine-3,4-dicarboxamide. In some embodiments, The presence of one or more linkers in an engineered polypeptide increases the structural flexibility of the engineered polypeptide compared to the absence of linkers or the selection of different linkers. For example, a linker that is longer and / or less sterically hindered than another linker may, in some embodiments, result in a molecule with higher structural flexibility than when no linker is present or when a different linker is used instead. In other embodiments, one or more linkages, such as a linker that is shorter and / or more sterically hindered than another linker, or a crosslink in 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 crosslinks at specific positions between particular peptides or between particular amino acid side chains may result in a molecule with lower structural flexibility than when the crosslinks are at different positions or between different side chains (e.g., disulfide or amide crosslinks) or when no crosslinks are present. d. Further ingredients

[0180] In some embodiments, the manipulated polypeptides provided herein comprise one or more further components. For example, in some embodiments, the manipulated polypeptide comprises one or more components that bind the manipulated polypeptide to a solid surface, such as beads or a flat surface. This includes [a specific component]. In some embodiments, the binding portion includes a polymer (e.g., PEG) or biotin, or a combination thereof. In some embodiments, binding the manipulated polypeptide to a solid surface may allow, for example, evaluation of one or more characteristics of the manipulated polypeptide, such as evaluation of binding to a CD25 binding partner (e.g., an antibody against CD25). e. Sequence similarity

[0181] In some embodiments, the manipulated polypeptides provided herein are as shown in Table 1:

[0182] [Table 4] It has one of the arrays listed in [the specified field].

[0183] In some embodiments, the manipulated polypeptide has at least 60% sequence similarity to any one of SEQ ID NOs: 1 to 21. In some embodiments, the manipulated polypeptide has at least 70% sequence similarity to any one of SEQ ID NOs: 1 to 21. In some embodiments, the manipulated polypeptide has at least 80% sequence similarity to any one of SEQ ID NOs: 1 to 21. In some embodiments, the manipulated polypeptide has at least 90% sequence similarity to any one of SEQ ID NOs: 1 to 21. In some embodiments, the manipulated polypeptide has at least 95% sequence similarity to any one of SEQ ID NOs: 1 to 21. In some embodiments, the manipulated polypeptide contains any one of SEQ ID NOs: 1 to 21. In a particular embodiment, the manipulated polypeptide contains any one of SEQ ID NOs: 1 to 21.

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

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

[0186] [ka] That is the case.

[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 manipulated polypeptide include GSG, GSS, GGS, GGSGGS, GSSGSS, GSGK, GSSK, GGSK, GGSGGSK, GSSGSSK, and others. V. How to select a manipulated polypeptide

[0188] Methods for selecting the manipulated polypeptides described herein are further provided herein. Such methods may include, for example, using iterative optimization of the structural features of the manipulated polypeptides.

[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 the antibody of 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 that does not bind to the antibody or where antibody binding is unknown. In certain embodiments, at least a portion of CD25 The crystal structure for the target interface is unknown, and the initial selection of the target interface involves molecular dynamics simulations of CD25 and CD25 binding. In some embodiments, one or more initial input sequences are obtained from identified sections(s), and each sequence is independently continuous or discontinuous. When developing the engineered polypeptide candidate, at least some of the interface residues of each sequence are retained, and one or more linkage portions are incorporated into the sequence to provide desired structural and dynamic features. In some embodiments, one or more non-interface residues are added to the sequence or one or more residues in the input sequence are replaced with one or more non-interface residues to achieve desired structural and dynamic features compared to the structure and dynamics of a congener target. In some embodiments, these non-interface residues are not derived from the target interface of CD25, or do not share one or more features with the target interface of CD25, or share fewer features than the retained interface residues, and / or share features with the target interface of CD25 less strongly. These intermediate non-interface residues may, in some embodiments, form part or all of the amino acid linker.

[0190] Next, in some embodiments, an initial design (or multiple designs) is generated and molecular dynamics simulations are performed to determine the flexibility and overall stability of the design. If this initial design does not meet the RMSD requirements, it may, in some embodiments, undergo iterative optimization of one or more linkages (e.g., one or more crosslinks or intermediate linker residues) using computational mutagenesis. During this optimization, in some embodiments, the interface residues are fixed, but one or more of the linkages are modified, removed, or added. Iterative optimization may be repeated until the RMSD interface residue positions of the manipulated polypeptide, compared to target interface and structural order metrics, meet specific requirements (e.g., ≤6.0 Å and ≥0.25, respectively, where structural order is a normalized scale of 0 to 1, and 1 = perfect structural stability).

[0191] In some embodiments, the intermediate structural stability residue regions may be in the range of 1 to 50 amino acids in length. In certain embodiments, these intermediate structural stability residue regions are linkers, e.g., amino acid linkers. In some embodiments, the relatively small size of the engineered polypeptide produced by certain embodiments of the methods provided herein (e.g., compared to approaches that graft interfaces onto larger structurally stable scaffolds) may enable the chemical synthesis of the molecule, in contrast to larger molecules that may require an in vitro expression system. Furthermore, in some embodiments, the methods provided herein allow for the incorporation of non-native amino acids into intermediate or interfacial positions, which may enable fine-grained control of interfacial manipulation by novel parts and properties, e.g., post-translational modification, solubility, cell permeability, enzyme reactivity, pH sensitivity, oxidation sensitivity, etc. In further embodiments, the engineered polypeptide may be selected for its higher potential for species cross-reactivity or disease-associated mutagenicity 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 may be subject to further evaluation, further adjustment, or 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 engineered polypeptide candidate library using the engineered polypeptide candidate, and then contacting the library with a CD25 binding partner (e.g., an antibody against CD25). The peptide library may contain peptides that are smaller than the engineered polypeptide candidate, share at least some sequence similarity with it, and in which certain residues may be replaced by other residues. The engineered polypeptide candidate library may contain, for example, variations of the engineered polypeptide candidate. It may contain n.

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

[0194] In some embodiments for producing candidates for a candidate library, one or more linking moieties are added to, removed from, or repositioned in the design of the original engineered polypeptide candidate. For example, in some embodiments, disulfide crosslinks are removed, added to, or their position is shifted. In other embodiments, lactam crosslinks are removed, added to, or their position is shifted. In some embodiments, one or more amino acid residues are replaced. Binding of CD25 binding partners to the peptide library or the engineered polypeptide candidate library or both (if present) can provide further information that can be used to further refine the design of the engineered polypeptide or to select the engineered polypeptide. Further information from screening these libraries can be used, for example, to modify the engineered polypeptide, for example, to increase the binding affinity with the CD25 binding partner. In some embodiments, the engineered polypeptide candidate library can provide further information regarding binding interactions (including the presence or position of such moieties), for example, the effect of specific linker moieties on crosslinks, including disulfide bonds and lactams. A library of peptides or engineered polypeptide candidates, or both, may, in some embodiments, be used to identify common motifs (e.g., amino acid patterns or linkages, or combinations thereof) that can increase the binding affinity or specificity to CD25 binding partners or provide other desired features. Evaluating the binding of homologous binding partners to members of the peptide or engineered polypeptide candidate library, or both, can provide further structural and functional information that can be used to further refine the design of engineered polypeptides or to select engineered polypeptide candidates. a. Selection by bonding under changing pH conditions

[0195] In some embodiments, the manipulated polypeptide exhibits structural flexibility and relative flexibility at lower pH levels. The selection is based at least in part on structural flexibility at physiological pH. For example, CD25 may be overexpressed on tumor cells, and therefore, binding of antibodies to CD25 with higher affinity in the tumor microenvironment may be desired in some embodiments. Therefore, in some embodiments, it may be desirable to select an engineered polypeptide that is less flexible at lower pH 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 lower pH, or an engineered polypeptide that has higher binding affinity or binding selectivity at lower pH. In many cancerous tumors, the growth rate of cancer cells may outpace the oxygen supply available in the tumor region, resulting in a hypoxic microenvironment within the tumor. The level of oxygen in tissues can affect the pH of the tissue environment, and hypoxic levels can result in a reduced pH (including, for example, due to the accumulation of acidic metabolites from anaerobic glycolysis). Therefore, in some embodiments, selecting an engineered polypeptide that has higher binding at lower pH (e.g., possesses desirable structural features that result in binding interactions) but reduced binding at physiological pH (e.g., has fewer, less desirable structural features that result in binding interactions) may, in some embodiments, result in an engineered polypeptide that can produce an antibody with higher binding to a desired target in the tumor compared to binding in the absence of 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 may 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 manipulated polypeptides may be evaluated at different pH levels using computer methods, such as molecular dynamics simulations. In other embodiments, the manipulated polypeptides are selected based on differential pH characteristics using in vitro methods. Suitable in vitro methods may include, for example, phage panning at different pH levels.For example, an antibody-phage display library may be used to pan one or more engineered polypeptides at physiological pH, and phages that bind at that pH may be discarded. A second round of panning may then be performed at a lower pH, and phages that bind to one or more engineered polypeptides at the lower pH may be selected. In some embodiments, engineered polypeptides that do not bind to phages at lower pH or that bind to phages with similar affinity at both lower and physiological pH may be less desirable for use in generating antibodies that target tumor cells. b. Reverse peptide evaluation

[0196] In further embodiments, selecting an engineered polypeptide may involve comparing the binding of the engineered polypeptide to the binding of a reverse engineered polypeptide. The reverse engineered polypeptide may 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 exhibiting the opposite characteristics. For example, an amino acid with a large, sterically bulky hydrophobic side chain may be replaced with an amino acid with a smaller side chain, or a hydrophilic side chain, or an amino acid with a smaller and hydrophilic side chain. In some embodiments, an amino acid with a hydrogen bond-donating side chain may be replaced with an amino acid with a hydrogen bond-accepting side chain or an amino acid with a non-hydrogen-bonding side chain. Binding characteristics that can be compared using engineered and reverse engineered polypeptides may include specificity and / or affinity in some embodiments. Comparing the binding characteristics of an engineered polypeptide to those of a reverse engineered polypeptide may, in some embodiments, help in selecting an engineered polypeptide in which the interfacial interacting amino acids drive the binding interaction rather than being a linker or other linking feature. Manipulated polypeptides in which the binding is driven by linkers or other binding components may exhibit off-target binding or other undesirable binding characteristics, and therefore may be less desirable in some embodiments.

[0197] In further embodiments, the method further includes modifying a selected manipulated polypeptide. c. Bonding evaluation

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

[0199] The binding of a protein or fragment of it (e.g., a CD25 binding partner) to one or more peptides or engineered polypeptide candidates (e.g., members of a library) can be evaluated in various ways. In some embodiments, binding is evaluated directly, for example, by directly detecting a label on the protein or fragment. Such labels may include, for example, fluorescent labels, such as fluorophores or fluorescent proteins. In other embodiments, 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 a binding partner, and then a labeled secondary reagent is added to label the bound binding partner. This labeled secondary reagent is then detected. Examples of sandwich assay components include His-tagged binding partners detected using an anti-His-tagged antibody or a His-tagged 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 target peptide or engineered polypeptide candidate is identified based on binding signal or dose-response using one of several available detection methods. These detection methods may 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 any signal exceeding that threshold is flagged as a putative hit motif.

[0201] For combinatorial library development, peptides identified from a peptide library based on their binding to proteins or fragments may, in some embodiments, be further clustered into distinct groups using sequence or structural information, or a combination thereof. This grouping may be performed, for example, using commonly available sequence alignment, chemical descriptors, structural predictions, and entropy prediction information science tools (e.g., MUSCLE, CLUSTALW, PSIPRED, AMBER, Hydropathology 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 may be identified from this analysis. Individual peptide motif hits may also be identified. Using these motif clusters and individual motifs, in some embodiments, design rules may be devised that define one or more of the sequence, structure, and chemical characteristics of a motif that appears to drive protein interactions at a target interface. In some embodiments, the structure of the target interface is not required for the identification of these interface motif design rules. Rather, in some embodiments, the design rules may derive from the analysis of peptides identified by screening a peptide library. ru.

[0202] In some embodiments, the binding assay is performed for approximately 10 minutes. 5It has a sensitivity dynamic range. Therefore, in some embodiments, the engineered polypeptide candidate has a native CD25:binding partner signal of 10 5 A signal is identified as the desired one if it has a binding event with a CD25 binding partner within the signal bracket. The type of signal may vary depending on the type of assay being used or how the signal is evaluated. For example, in some embodiments, the signal may be a response unit in a sensorgram, a fluorescence signal in an image-based readout, or an enzymatic readout in an enzyme-based assay. The signal for a binding event may be measured in comparison to the CD25:binding partner signal.

[0203] In some embodiments, the engineered polypeptide candidate is modified before binding is evaluated. For example, in some embodiments, biotin, PEG, or another binding moiety, or a combination thereof, is attached to the C-terminus or N-terminus of the peptide to enable its use 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 at the C-terminus with -GSGSGK-PEG4-biotin. In certain embodiments, such biotin-modified engineered polypeptide candidate is then bound to streptavidin beads via the biotin moiety, and the bead-supported immunogen is evaluated for binding to its CD25 binding partner. VI. Use of Modified polypeptides and CD25 antibodies

[0204] The manipulated polypeptides provided herein and identified by the methods provided herein may 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 natural or recombinant sources.

[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, e.g., 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 bispecific 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, the "heavy chain variable region" or "VH" (or, in the case of a single-domain antibody, e.g., a nanobody, "VHH") refers to a fragment of the heavy chain containing three CDRs inserted between adjacent stretches known as framework regions, and these framework regions are generally more highly conserved than the CDRs and form a scaffold to support the CDRs. The 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, scFv may have VL and VH variable regions in either order with respect to the N-terminus and C-terminus of the polypeptide, for example, and scFv may contain a VL-linker-VH or a VH-linker-VL.

[0210] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations: kappa ("Κ").

[0211] Accordingly, in some embodiments, antibodies are provided herein that are produced by immunizing an animal with an immunogen, wherein the immunogen is an engineered polypeptide 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 may include, for example, administering to the animal at least one dose of a composition comprising an immunogen and optionally an adjuvant. In some embodiments, generating antibodies from an animal includes isolating B cells that express the antibody. Some embodiments further include fusing the B cells with myeloma cells to create a hybridoma that expresses the antibody. In some embodiments, antibodies produced using an engineered polypeptide can cross-react with humans and monkeys, for example, cynomolgus monkeys.

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

[0213] In some embodiments, antibodies produced using the manipulated polypeptides provided herein bind specifically to CD25. In certain embodiments, the antibody does not block the 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 neither disrupts nor prevents the binding of the IL-2 ligand to CD25 (IL-2 alpha chain) and does not affect IL-2-mediated signaling, such as signaling via the IL-2 / JAK3 / STAT-5 signaling pathway. In some embodiments, the antibody does not disrupt the binding of the IL-2 ligand to CD25 (IL-2 alpha chain) and binds to an epitope different from the epitope to which the 7G7B6 antibody binds. In some embodiments, the antibody does not disrupt the binding of the IL-2 ligand to CD25 (IL-2 alpha chain) but disrupts the trimming 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 IL-2 ligands to the alpha, beta, and / or gamma chains of the receptor, thereby reducing or inhibiting IL-2-mediated signaling. In certain embodiments, the antibody disrupts or prevents the binding of IL-2 ligands to CD25. In some embodiments, the antibody disrupts or prevents the binding of IL-2 ligands to CD25 and binds to an epitope different from the epitope to which either daclizumab or baciliximab binds.

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

[0217] In some embodiments, the antibody disrupts or prevents heterotrimerization of alpha, beta, and gamma IL-2 chains. In some embodiments, the antibody does not block the binding of IL-2 ligand to CD25, but disrupts or prevents heterotrimerization of 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 further embodiments, it is evaluated whether antibodies produced using the manipulated polypeptides provided herein block the binding of CD25 to IL-2. In some embodiments, antibodies that do not block the binding of CD25 to IL-2 are selected. In other embodiments, antibodies that block the binding of CD25 to IL-2 are selected. Such blockade or non-blockade can be evaluated, for example, by coupling CD25 to a biosensor tip and evaluating the binding by the antibody in the presence and absence of IL-2. In some embodiments, the antibody is expressed with a 6×His tag, which can be used with Ni-NTA in flow cytometry to evaluate antibody binding and the blockade or non-blockade of IL-2 binding to CD25. In certain embodiments, antibody binding is evaluated at physiological pH (e.g., between approximately pH 7.3 and approximately pH 7.5, or approximately pH 7.4) and at the pH of the tumor microenvironment (e.g., between approximately pH 6.4 and approximately pH 6.6, or approximately pH 6.5). In certain embodiments, blocking / non-blocking activity is compared to the binding of an IL-2 blocking antibody (e.g., daclizumab or bacliliximab). In certain embodiments, blocking / non-blocking activity is compared to the binding of an IL-2 non-blocking antibody (e.g., antibody 7G7B6). In certain embodiments, 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 against CD25. In other embodiments, the antibody is an antagonist antibody against 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 further embodiments, the antibody can bind to CD25 in either a cis or trans configuration.

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

[0222] In some embodiments, the antibodies are independently selected from those disclosed in Table 2: CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR -Has L3.

[0223] [Table 5] TIFF2026065087000014.tif219140 TIFF2026065087000015.tif218139 TIFF2026065087000016.tif115140

[0224] In some embodiments, CDR-H1 is 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 Selected from 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 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 The following can be selected: 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 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, QQAN SFPLT, QQSYSTPYT, SSYTSSSTYV, QRYGSSPR, QQVHSFPFT, LQHNTFPYT, QQSHSTPLT, QQYNSYPFT, QQYNSSPLMYT, QQTYSTPLT, QQANTFPQT, QSYDGSSVV, GSWEAR Selected from ESVFV, 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 antibodies include CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each independently selected from those disclosed in Tables 3A and 3B. It is possible to generate novel 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 excessive experimentation.

[0231] [Table 6] TIFF2026065087000018.tif217127 TIFF2026065087000019.tif158127

[0232] [Table 7] TIFF2026065087000021.tif21683 TIFF2026065087000022.tif15983

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

[0234] [Table 8] TIFF2026065087000024.tif212141 TIFF2026065087000025.tif210142 TIFF2026065087000026.tif210142 TIFF2026065087000027.tif210142 TIFF2026065087000028.tif211143 TIFF2026065087000029.tif210142 TIFF2026065087000030.tif210142 TIFF2026065087000031.tif92142

[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 sequence in Table 5 and used to generate a soluble Fab fragment, a monoclonal antibody, a bispecific antibody, 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.

[0236] [Table 9] TIFF2026065087000033.tif219134 TIFF2026065087000034.tif219133 TIFF2026065087000035.tif219133 TIFF2026065087000036.tif219133 TIFF2026065087000037.tif214133 TIFF2026065087000038.tif219133 TIFF2026065087000039.tif214134 TIFF2026065087000040.tif214134 TIFF2026065087000041.tif219133 TIFF2026065087000042.tif219133 TIFF2026065087000043.tif219134 TIFF2026065087000044.tif219134 TIFF2026065087000045.tif220134 TIFF2026065087000046.tif219133 TIFF2026065087000047.tif219133 TIFF2026065087000048.tif219134 TIFF2026065087000049.tif215134 TIFF2026065087000050.tif217134 TIFF2026065087000051.tif127133

[0237] In some embodiments, the antibodies include 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 antibodies include CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each independently selected from any one clone enumerated in Tables 14A and 14B. In some embodiments, the antibodies include 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. This disclosure provides antibodies having CDRs from individual clones, or antibodies having CDRs from any one CDR that matches any five other CDRs. The antibodies identified in Tables 14A and 14B are derived from mouse phage display libraries. Known methods can be used to convert these CDRs into humanized or chimeric antibodies. VII. Use of CD25 antibody

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

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

[0240] Accordingly, methods for treating autoimmune-related diseases or disorders are provided herein, comprising administering a therapeutically effective amount of a therapeutic CD25 antibody to a subject in need thereof. In exemplary embodiments, the CD25 antibody is a non-IL-2 blocking antibody.

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

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

[0243] The therapeutic CD25 antibodies described herein may be administered intravenously, intratumorally, intracranially, intralesionally (e.g., intralesion injection, direct contact diffusion), intracavitaryly (intraperitoneal, intrapleural, intrauterine, intrarectal), intraperitoneally, intramuscularly, subcutaneously, topically, orally, perdermally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In exemplary embodiments, the route of administration is intravenous injection.

[0244] A therapeutically effective dose of a therapeutic antibody will generally be administered. The appropriate dosage of the therapeutic antibody may be determined based on the severity of the disease, the patient's clinical condition, the patient's clinical history and response to treatment, and the discretion of the attending physician. VIII.Diagnostic use

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

[0246] The CD25 antibodies provided herein are modifiable for use in various immunoassays. These immunoassays include, but are not limited to, enzyme-linked immunosorbent assays (ELISA), Western blotting, radioimmunoassays (RIA), flow cytometry, immunofluorescence assays, spectrophotometrics, X-ray imaging, electrophoresis, high-performance liquid chromatography (HPLC), or thin-layer chromatography (TLC).

[0247] The CD25 antibodies provided herein may include detectable labels, for example, that are detectable by spectroscopic, photochemical, biochemical, immunochemical, fluorescent, electrical, optical, or chemical methods. Useful labels in the present invention include, but are not limited to, fluorescent dyes, radioactive labels, enzymes, colorimetric labels, avidin, or biotin.

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

[0249] This disclosure provides compositions comprising a therapeutic CD25 antibody, and in some embodiments, the composition is sterile. The pharmaceutical composition generally contains an effective amount of the therapeutic antibody in a pharmaceutically acceptable excipient. IX. Kits and manufactured products

[0250] This disclosure also provides kits comprising any of the CD25 antibodies described herein for either therapeutic or diagnostic use. In some embodiments, the kit further comprises a secondary antibody, reagents for immunohistochemical analysis, pharmaceutically acceptable excipients and instructions for use, and components selected from 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] This application also provides a product comprising any one of the therapeutic or diagnostic compositions or kits described herein. Examples of products include vials (e.g., sealed vials).

[0252] The descriptions provided herein include numerous exemplary configurations, methods, parameters, etc. However, it should be recognized that such descriptions are not intended to limit the scope of this disclosure, but rather are provided as descriptions of exemplary embodiments. [Examples]

[0253] The following embodiments are illustrative examples and are not intended to limit any aspect of this disclosure. [Examples]

[0254] Development of engineered immunogens that share the characteristics of CD25 The crystal structure of CD25 was obtained. Several available crystal structures for CD25 lack the mobile loop section of the protein. Molecular dynamics simulations were performed to gain a better understanding of this mobile 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 Figures 34B and 34C. These inputs were used in the Rosetta program to improve the overall desirable structural and dynamic properties of the interface residues. This process altered the structural (non-interface) portion of the segments to stabilize and reproduce the structural, conformational, dynamical, and other properties of the interface residues in the context of the native CD25 from which they originated. The stability and flexibility of the segments under development were also analyzed, and sequences were adjusted as needed to modify these parameters. For example, the N-terminus or C-terminus could be extended by the addition of one or more amino acids to add desired properties. The effect of crosslinking on 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 operation—amino acid addition, crosslinking, and structural residue optimization—each of the many design candidates was quantitatively evaluated using the Rosetta program's native scoring and energy functions. Candidates with the best Rosetta energy are advanced to the next stage of design, and finally, 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 in some cases at tumor microenvironment pH (e.g., approximately pH 6.5).

[0256] Quantitative metrics for ranking different designs using molecular dynamics (MD) simulations included similarity to CD25, assessed via RMSD, and the structural flexibility of the candidates. Figures 33A and 33B show an exemplary comparison of stability at physiological pH versus RMSD for exemplary manipulated immunogens developed using the input section shown in Figure 32 (left arrow for Figure 33A, right arrow for Figure 33B). This is shown. Figure 33C is an exemplary comparison of stability in tumor microenvironment pH versus RMSD for the exemplary immunogen in Figure 33B. A typical scoring algorithm is presented below.

[0257]

number

[0258] Structural similarity was calculated using the mean squared 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. RMSD was calculated using either a computer-designed manipulated immunogen candidate structure as the reference structure, or an experimentally characterized structure (e.g., X-ray crystal structure) as the reference. In these simulations, all residues, including candidate functional interface residues (in some simulations) and candidate structural residues (in others), were compared to the reference.

[0259] Ensembles of conformations sampled by MD were clustered into groups (clusters) based on structural similarity to one another, according to RMSD. Disorder was assessed as the proportion of conformations in the MD ensemble that could not be grouped into clusters of similar conformations due to structural differences from all other conformations in the ensemble (e.g., high RMSD). Therefore, engineered immunogen candidates with more disorder than alternative candidates were more flexible. Order was assessed 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 were less flexible, and in this case, a higher proportion of their ensemble conformations fell into fewer clusters than the alternative candidates.

[0260] Clusters to which the engineered immunogen candidate was added were compared to the reference structure using RMSD. If the RMSD of a cluster was below a 4-angstrom threshold, the cluster was considered ordered (e.g., low flexibility) and similar to the reference (structural similarity). Engineered immunogen candidates with a high proportion of their ensemble meeting these criteria of low flexibility and high structural similarity are predicted to be more active than alternative candidates with a low proportion of their ensemble meeting these criteria.

[0261] This quantitative analysis was combined with a qualitative analysis of MD trajectories related to 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] [Examples]

[0263] In vitro evaluation of manipulated immunogens The binding of the engineered immunogen prepared in Example 1 is evaluated using an antibody against CD25. The engineered immunogen is modified with a -GSGSGK-biotin group at its C-terminus and then separately bound to a streptavidin-coated biosensor tip. A buffer containing CD25 antibody is passed over the tip during a 300-second association phase, and then the solution is switched to a buffer without CD25 antibody to measure dissociation from the biosensor tip. A control without any of the engineered immunogens or proteins initially bound to the tip is also performed 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 is performed to demonstrate the binding level of the CD25 antibody to full-length CD25. The data obtained from these biosensor experiments are used to qualitatively rank the binding of the engineered immunogen. [Examples]

[0264] Evaluation of manipulated immunogens by phage panning The manipulated immunogens provided herein are evaluated using phage panning techniques.

[0265] A mouse HuCD25 immunized phage library is transformed by electroporation in TG1 using a standard phage display protocol, and phage proliferation is induced by the addition of CM13. The TG1 culture secreting phages is incubated on ice for 1 hour, followed by PEG precipitation with PEG / NaCl. Exemplary libraries that may 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. To deplete antibodies that bind with high affinity to full-length CD25 at physiological pH, subtractive panning is performed by absorption over 1 hour on an ELISA plate coated with 10 ug / ml of full-length CD25 (400 nM) in PBST pH 7.4, resulting in 3 × 10⁶ values ​​at pH 7.4. 11 PFU phage (3 x 10) 8 The first step is to perform a counterselection using a 1000x magnification model. The resulting phage supernatant is collected and its pH is adjusted to 6.5 with PBST. Subsequent phage panning selection is then performed at pH 6.5.

[0267] Panning selections are pre-clarified with 25 microliters of antigen-free streptavidin Dynabeads after 1 hour incubation. The phages are then added to new, pre-blocked Eppendorf LoBind tubes. Biotinylated engineered immunogen (e.g., as described in Example 1) is added at a concentration of 100 nM (in some cases, an additional 500 mM NaCl was added to reduce nonspecific binding of the immunogen to the phages) for 40 minutes to 1 hour. The samples are then incubated at RT for 1 hour with 25 microliters of streptavidin beads or a streptavidin-coated plate. The samples are pelleted and washed using magnets / magnetic beads, or, if using plates, washed 7-10 times with PBST. The tubes are changed twice to remove residual phages.

[0268] To elute the phages, 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 phages to 1-5 ml of newly grown TG1 (OD600 approximately 0.5) and incubate for 20-30 minutes.

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

[0270] Along with counter-selection at pH 7.4, another selection for the manipulated immunogen is performed at pH 6.5. Periplasm extracts are subsequently evaluated using phage ELISA and octet screening.

[0271] To ensure that the fab phage binds to full-length CD25 in addition to the engineered immunogen, a final selection using full-length CD25 may be performed as appropriate, 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 selection using full-length CD25, panning selection was pre-clarified with 25 microliters of streptavidin Dynabeads. After adding phages 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. Pelletization, washing, and elution steps were performed according to the above procedure. [Examples]

[0273] Phage ELISA protocols and biosensors / Octet screening ELISA / Extract Preparation: Perform phage ELISA and periplasmic extract preparation for Fab Octet screening.

[0274] Dilute the CD25 antigen and add it to the ELISA plate wells, then incubate. After incubation, wash the wells twice with PBS, then block by adding BSA and incubating at 25°C for 2 hours. Dilute the phages 2-fold in 1×PBST 1.0% BSA, pH 6.5, add 50 microliters per well, and incubate at room temperature for 5 minutes. Shake off the blocking solution from the wells, add 50 μL of dilute 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. Add the HRP-conjugated anti-M13 antibody (Abcam, ab50370) Dilute 1:5000 with 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) in a 1:1 mixture as described. Add 50 microliters of the substrate solution to each well and incubate at room temperature for 5-60 minutes, then read.

[0275] Inoculate colonies with 0.1 ml of overnight culture (1 ml of culture in a 96-well plate or 4 ml of culture in a 14 ml falcon tube) in 0.03–4 ml of 2 × YT 0.2% glucose. Incubate these at 37°C at 250–700 rpm until the OD600 is 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. Collect 1–4 ml of culture by pelletizing at 3400 rcf for 10–15 minutes. Discard the supernatant. The culture was resuspended with 1×Halt protease inhibitor in 50–75 μL of PPB buffer (30 mM Tris-HCl, pH 8.0, 1 mM EDTA, 20% sucrose) and incubated on a rocking platform at room temperature for 5 minutes or at 4°C for 10 minutes. Then, the culture was resuspended with 1×Halt protease inhibitor in 150–225 μL of cold ddH2O and incubated on a rocking platform at room temperature for 1 hour or at 4°C for 1–2 hours. The lysate suspension was spun at 15000 rcf for 10–15 minutes at 4°C. The supernatant was collected and diluted.

[0276] Fab Expression and Purification Protocol: Cell cultures were inoculated and grown overnight, then induced with 50 μL of 25 mM–1 M IPTG. The temperature was reduced to 30°C and the rpm to 150. Incubation was performed overnight. 50 ml of culture or plate was collected by pelletizing at 3400 rcf for 15 minutes. The supernatant was discarded. Cell pellets from 50 mL of culture were placed in a -80°C freezer for 1 hour, while cultures grown in plates were vortexed with 75 μL of PPB along with 1× Halt protease inhibitor, EDTA-free (Thermo Fisher Scientific). The plates were shaken at 1000 rpm at 4°C for 10 minutes. 225 μL of cold water containing 1× Halt protease inhibitor, EDTA-free (Thermo Fisher Scientific) was added to each well. The samples were mixed and shaken at maximum speed, i.e., 1000 rpm, at 4°C for 1-2 hours. The plate was spun at 3500 rpm at 4°C for 10 minutes. The supernatant (PPE) was transferred to a new plate and stored at -20°C. A cell pellet from 50 mL of culture was removed from the freezer and added with 5 mL of PBS, 10 mM imidazole, 2.5 mg / mL of lysozyme and 1× Halt protease inhibitor, without EDTA (Thermo Fisher Scientific). After thawing the pellet at room temperature for 30 minutes, the lysate was centrifuged at 3400 rcf for 15 minutes. The supernatant was removed and the pellet was discarded. For Fab purification, 500 μL of Ni-NTA resin (pre-washed and pelleted) was added, or a Ni-NTA spin column was used. The clarified lysate was incubated with the added resin for 30 minutes to 1 hour. This was then spun at 1500 rcf. These were washed five times with 1 ml of PBS and 10 mM imidazole. After each spin, the buffer was discarded. 1 ml of PBS and 200 mM imidazole were added and mixed, incubated for 30 minutes, and spun at 1500 rcf for 15 minutes. The eluted proteins were stored at 4°C or 20°C after determining the protein concentration. A Zeba column was used for desalting / buffer exchange.

[0277] Octet / Biosensor Screening: For screening Octet Koff rate in the supernatant, 50 μL of lysate is used in a 384-well Pall For This procedure is used in teBio Octet plates. Data is collected using Octet RED 384 (MD ForteBio). Briefly, human CD25 is coupled to the AR2G tip (1 ug / ml). For data acquisition, baseline is evaluated for 60 seconds in PBST 1% BSA buffer. 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 in 200 mM Tris-glycine, pH 2.5, and neutralized with PBST 1% BSA. For data analysis, dual reference (without CD25 on the tip, and a blank reference well) is performed using Octet HT 11.0 software for reference subtraction. [Examples]

[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-blockage, affinity, and off-rate.

[0279] Protocol for determining cross-reactivity using biosensors (Octet Red 384, Pall Forte Bio): This protocol is used to determine the ability of individual test clones (anti-human CD25 mouse monoclonals) to bind to targets (antigens) derived from human, cynomolgus monkey, and mouse species. The target protein is covalently coupled to a dextran-coated sensor tip via a primary amine, or a 6×His-tagged target protein is affinity-captured onto a sensor tip coated with an anti-6×His monoclonal antibody. 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 for binding of blank medium or buffer to the blank or antigen-coated tip. Signals > 3× background binding are considered actual binding events.

[0280] Protocol for cross-blockage using biosensors: This method is for determining whether individual test clones (anti-human CD25 mouse monoclonals) can cross-block a control antibody. Cross-blockage may indicate that the test clone recognizes an epitope that overlaps with the corresponding epitope of the control antibody. Furthermore, this may imply that the test antibody may have similar functional properties to the control antibody. For this protocol, the control antibody is covalently coupled to a dextran-coated sensor tip via a primary amine. 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 configuration. If the test antibody can bind to the antigen, it is indicated that it does not cross-block the control antibody; however, non-binding can be interpreted as the ability to cross-block the control antibody.

[0281] Protocol for affinity determination using biosensors: This method is used to determine the affinity of individual test clones to the antigen when the concentration of the test antibody is known. A capture molecule, e.g., protein G or anti-mouse IgG monoclonal or anti-human IgG monoclonal, is coated onto the tip of the biosensor. The test clones are captured on the surface coated with the capture molecule. The antigen in solution is allowed to associate and dissociate these test clones over a period ranging from 60 to 600 seconds for the association phase and 60 to 1800 seconds for the dissociation phase. The resulting data (or "sensorgrams") are then processed using a 1:1 Langmuir model or a 2:1 heterogeneous model. Fitting is performed using either of the following models. The former assumes that the interaction pairs are homogeneous, where a 2:1 model yields a better fit for fitting the data, indicating that clones require further subcloning due to inherent heterogeneity. Data curve fitting uses the on-rate constant and The dissociation constant is provided as the ratio of the dissociation rate (off-rate) constant.

[0282] Protocol for Estimating Dissociation Rate (Off-Rate) 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, e.g., protein G or anti-mouse IgG monoclonal or anti-human IgG monoclonal, is coated onto the tip of the biosensor. The test clone is captured on the surface coated with the capture molecule. The antigen in solution is allowed to associate and dissociate these test clones over a period ranging from 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 if the 2:1 model yields a better fit for fitting the data, indicating that the clone requires further subcloning due to its inherent heterogeneity. The data are fitted 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 order test clones. [Examples]

[0283] Selection of manipulated polypeptides using the CD25 portion as a reference target The sequence and three-dimensional (3D) structure of CD25 were searched from the Protein Data Bank (PDB) (PDB ID number 2ERJ, A chain): ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGSSSHSSWDNQCQCTSSATRSTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTG

[0284] As shown in Figure 6, the putative therapeutic epitope of CD25 was identified as a reference target for the selection of engineered polypeptides. The residue positions and epitope sequences for 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 target were obtained using dynamical simulations, and covariance matrices for atomic fluctuations were generated for the epitopes in the reference target. Next, different manipulated polypeptide candidates were generated using computational protein design (e.g., Rosetta), dynamical simulations performed on the candidates, and the determined interatomic distances and amino acid descriptor topologies. Covariance matrices for atomic fluctuations were generated for the reference target epitopes and for residues in the candidates corresponding to residues in the reference target epitopes.

[0287] Principal component analysis was performed to calculate eigenvectors and eigenvalues ​​for each covariance matrix—one covariance matrix for each reference target and one covariance for each candidate. Only the eigenvectors with the largest eigenvalues ​​were retained. Eigenvectors describe the 1st, 2nd, 3rd, and Nth dominant motions observed in the simulated set of molecular structures. If a candidate moves similarly to the reference epitope, its eigenvectors will be similar to those of the reference target (epitope). The similarity of eigenvectors corresponds to their aligned components (3D vectors centered on each CA atom) pointing in the same direction. This similarity between the candidate eigenvectors and the reference target eigenvectors was calculated using the dot product of the two eigenvectors. The dot product value was 0 if the two eigenvectors were 90 degrees to each other, or 1 if the two eigenvectors pointed exactly in the same direction.

[0288] The ordering of eigenvectors is based on their eigenvalues, and since these eigenvalues ​​are not necessarily the same between the different molecules due to the probabilistic nature of molecular dynamics simulations sampling the underlying energy landscapes of two different molecules, an inner product between multiple differentially ranked eigenvectors was required (e.g., candidate eigenvector 1 × reference target eigenvectors 2, 3, 4, etc.). Furthermore, although we do not wish to be constrained by any theory, molecular motion is complex and may involve the motion of more than one (or more than a few) dominant / major modes.

[0289] To solve these two problems, we calculated the dot product between all pairs of eigenvectors in the candidate and reference targets. This yielded a matrix of dot products, the dimension of which was determined by the number of eigenvectors analyzed – for 10 eigenvectors, the dot product matrix is ​​10 × 10. We transformed this matrix of dot products into a single value by calculating the root mean square value of the dot product. This is the root mean square dot product (RMSIP).

[0290] Principal component analysis (PCA) converts a 3L × 3L coordinate covariance matrix (where L is the number of atoms) into a set of eigenvectors Φ (reference target) and Ψ (MEM), as well as eigenvalues ​​Λ. The set Φ consists of N eigenvectors φ about the reference target. i The set Ψ includes N eigenvectors ψ about MEM. j The eigenvectors are ordered within their respective sets by their associated eigenvalues. The eigenvector with the largest eigenvalue constitutes the largest proportion of the total coordinate covariance. i and ψ j The dot product of the eigenvectors is calculated to compare the similarity of motion between the reference target and MEM. φ i and ψ j The root mean square of all dot product combinations of eigenvectors gives the sum of the similarities in motion of the manipulated polypeptide candidate (MEM) to that of the reference target (RMSIP).

[0291] As shown in Figure 7, in each manipulated polypeptide, the scaffold residue (gray), epitope residue (gold), and its position in 3D space are selected by this computer design procedure. PDB ID number 2ERJ, the residue positions and epitope sequences for the A chain are provided in Tables 8 and 9. The crosslinking sites are intracellular dysplasmic dinucleotides in each MEM sequence. This refers to the expected presence of sulfide bond formation.

[0292] [Table 12] TIFF2026065087000056.tif60142

[0293] [ka]

[0294] [Table 13] [Examples]

[0295] Antibody selection using MEM-programmed in vitro selection Each programme devised 32 different panning strategies (S1-S32) including three rounds of positive selection (Table 10). Each program used at least one engineered polypeptide as the selection molecule. Conventional selection using conventional methods (CD25 as the positive target) was also included. Bovine serum albumin (BSA) was used as a negative target for selection against nonspecific binding.

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

[0297] [Table 14] TIFF2026065087000060.tif160161 Primary ELISA screening and hit selection

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

[0299] [Table 15]

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

[0301] The affinity of different scFv antibodies was evaluated using a single-cycle kinetic 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 from the sensor tip, and the binding of molecules in the analyte to scFv was recorded. Each assay was performed in double-chain fashion. Controls were also performed using buffer only (to control sensor drift) and separate controls of polyclonal IgG isotype antibodies purified from human serum (to control nonspecific IgG binding).

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

[0303] The CD25 specificity of different scFv antibodies was evaluated by flow cytometry using CD25-expressing cells [CD25(+)] or CD25-non-expressing cells [CD25(-)]. As shown in Figure 13, of the 1248 scFv hits analyzed in this assay, 1160 (93%) specifically bound to CD25(+) cells. Hit Sequence Analysis

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

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

[0306] [Table 16] TIFF2026065087000063.tif218138 TIFF2026065087000064.tif146138

[0307] Analysis of CDR and germline use 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] TIFF2026065087000066.tif146138 It includes.

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

[0310] For CD25 Epitope 1 (55-63), the CDR used included the following:

[0311] [Table 18]

[0312] Regarding CD25 Epitope 2 (13-20:127-132), the CDR used includes the following:

[0313] [Table 19]

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

[0315] [Table 20]

[0316] Regarding CD25 Epitope 4 (5-11:156-163), the CDR used includes the following:

[0317] [Table 21]

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

[0319] [Table 22]

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

[0321] [Table 23]

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

[0323] [Table 24]

[0324] For CD25 Epitope 8 (44-56), the CDR used included the following:

[0325] [Table 25] [Examples]

[0326] Confirmation of epitope specificity through competitive binding. 126 anti-CD25 clones were subjected to epitope degradation using a four-target competitive binding assay shown in Figure 18. The binding sites for IL-2, daclizumab, and basioliximab shown in the figure are based on X-ray crystallographic structural determination. The binding site for 7G7B6 is based on peptide mapping.

[0327] Cross-competition assays were performed in a classic sandwich format. This involved immobilizing the first antibody onto the biosensor, followed by incubation with the antigen and then the second sandwich antibody. His-tagged scFvs were expressed and purified in situ on the biosensor using His-tag capture from the supernatant. Biosensor His-tag capture was normalized across scFv clones by monitoring the tip loading response to a consistent level across 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 with an antibody. Finally, various competitive analytes, including IL-2, 7G7B6, basiliximab, or daclizumab, were added. A competitive analyte can bind to 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 panning clone is dominated by the IL-2 interface epitope. Most clones are blocked by IL-2, daclizumab, and basioliximab, but not by 7G7B6.

[0329] As shown in Figure 20, the 147-157 epitope MEM manipulator clones primarily bind to their intended epitopes. Most clones are blocked by daclizumab, but not by IL-2, basioliximab, or 7G7B6.

[0330] As shown in Figure 21, the 6-17 epitope MEM manipulator clones primarily bind to their intended epitopes. Most clones are blocked by 7G7B6, but not by IL-2, daclizumab, or basioliximab.

[0331] As shown in Figure 22, the 13-20:127-132 epitope MEM manipulator clones primarily bind to their intended epitopes. Most clones are blocked by 7G7B6, but not by IL-2, daclizumab, or basioliximab.

[0332] As shown in Figure 23, the 44-56 epitope MEM manipulator clone primarily binds to the intended epitopes. The clone was divided into two profiles. In profile 1, the clone is blocked by 7G7B6 but not by IL-2, daclizumab, or basioliximab. In profile 2, the clone is blocked by IL-2, daclizumab, and basioliximab but not by 7G7B6. These blocking profiles demonstrate binding to the intended epitopes from different approach angles.

[0333] As shown in Figure 24, the 55-63 epitope MEM manipulator clone primarily binds to the intended epitopes. The clone was divided into three profiles. In profile 1, the clone is blocked by 7G7B6 but not by IL-2, daclizumab, or basioliximab. In profile 2, the clone is blocked by IL-2, daclizumab, and basioliximab but not by 7G7B6. These blocking profiles demonstrate binding to the intended epitopes from different approach angles. In profile 3, the clone is blocked by IL-2 and 7G7B6 but not by daclizumab or basioliximab. These blocking profiles demonstrate binding to the intended epitopes from different approach angles. [Examples]

[0334] Mapping of functional epitopes by alanine mutagenesis Alanine mutations were designed to confirm or reject binning of the intended epitopes by the MEM manipulator clone (Figure 25). Since alanine mutagenesis acts on functional epitopes rather than structural epitopes defined by the competitive assay, This was chosen as the orthogonal method for binning antibodies. Various pairs of surface-accessible residues were selected for mutagenesis. Computational modeling was used to confirm that the alanine mutations selected for use in these assays did not affect either global or local stability. For example, Figure 26 shows the results for modeling alanine mutations within 145–157 epitopes. For each mutant and wild type: RMSD from three independent 100 ns MD simulations in exposed solvent for each of eight different starting apo-CD25 configurations, with the crystal structure used as a reference. As shown in Figures 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 147–157 epitope-targeted engineered polypeptides are consistent with the intended specificity for the CD25 region.

[0336] Each of the 117 scFvs from the screening campaign was tested against four alanine mutation pairs (Figure 31). Functional epitope diversity was observed. MEM-manipulated hits exhibit distinct in-epitope alanine substitution site sensitivity. [Examples]

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

[0338] [Table 26] [Examples]

[0339] Panning bias library of mouse antibody sequences A phage display library was generated from the immunoglobulin genes of mice immunized with full-length CD25. This CD25-biased library was panned against the manipulated polypeptides shown to obtain the complementarity-determining regions shown in Tables 14A and 14B.

[0340] [Table 27] TIFF2026065087000077.tif97168 TIFF2026065087000078.tif98170 TIFF2026065087000079.tif98170 TIFF2026065087000080.tif97168 TIFF2026065087000081.tif97168 TIFF2026065087000082.tif98170 TIFF2026065087000083.tif99170 TIFF2026065087000084.tif97168 TIFF2026065087000085.tif97168 TIFF2026065087000086.tif98170 TIFF2026065087000087.tif97168 TIFF2026065087000088.tif97168 TIFF2026065087000089.tif98169 TIFF2026065087000090.tif97168 TIFF2026065087000091.tif97168 TIFF2026065087000092.tif7168

[0341]

Table 28

[0342] Sequence 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 that specifically binds to the CD25 epitope; The CD25 epitope is ATFKAMA...MVYYQC, and the CD25-specific antibody contains 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 combination of (1) to (4) shown in Table 1 below; Table 1 The CD25 epitope is DDPPEIPHATFKA, and the CD25-specific antibody contains 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 combination of (1) to (4) shown in Table 2 below; Table 2 The CD25 epitope is DDPPEIP:RWTQPQLI, and the CD25-specific antibody contains 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 combination of (1) to (2) shown in Table 3 below; Table 3 The CD25 epitope is QPEEQKERKTTEM, and the CD25-specific antibody contains 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 combination of (1) to (2) shown in Table 4 below; Table 4 The CD25 epitope is VCKMTHGKTRW, and the CD25-specific antibody contains 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 combination of (1) to (12) shown in Table 5 below; Table 5 The CD25 epitope is IPHA, and the CD25-specific antibody includes 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 combinations of (1) shown in Table 6 below; Table 6 or The CD25 epitope is YMLCTGSSSHSSW, and the CD25-specific antibody comprises 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 combination of (1) to (25) shown in Table 7 below; Table 7 The aforementioned CD25-specific antibody.

2. A CD25-specific antibody competes with an epitope-specific reference conjugate for CD25 binding, wherein the epitope-specific reference conjugate is IL-2, daclizumab, basioliximab, and / or 7G7B6; The CD25-specific antibody does not compete with the off-target reference conjugate, and the off-target reference conjugate is IL-2, daclizumab, basioliximab, 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 It is destroyed by mutations targeting IL-2 selected from; The CD25-specific antibody is 10 －２ / s less than 10 －３ / s less than or 10 －４ k less than / s ｏｆｆ Having the k ｏｆｆ However, is it measured using biolayer interferometry with soluble human CD25? The CD25-specific antibody has a k between 10 －２ / s and 10 －５ / s, and the k ｏｆｆ is measured using biolayer interferometry with soluble human CD25; ｏｆｆ ​ The CD25-specific antibody has a K content of less than 100 nM, less than 25 nM, or less than 5 nM. Ｄ Having the K Ｄ However, this is measured using biolayer interferometry with soluble human CD25; or The CD25-specific antibody has a K concentration between 100 nM and 1 nM. Ｄ Having the K Ｄ However, it is measured using biolayer interferometry with soluble human CD25; The CD25-specific antibody according to claim 1.

3. Does it specifically bind to cells expressing CD25? at least 10 ４ or at least 10 ５ Based on the average fluorescence intensity (MFI), does it bind to cells expressing CD25? 10 ４ and 10 ６ At an average fluorescence intensity (MFI) between [value] and [value], does it bind to cells expressing CD25? It does not bind to CD25(-) cells; or 10 ３ It binds to CD25(-) cells with an average fluorescence intensity (MFI) of less than 1. A CD25-specific antibody according to claim 1 or 2.

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

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

6. Is it a humanized antibody? Is it a chimeric antibody? It includes a mouse variable domain and a human constant domain; or It also binds to CD25 in cynomolgus monkeys; A CD25-specific antibody according to any one of claims 1 to 3.

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

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