Engineered CD25 polypeptides and uses thereof

Abstract

To provide novel engineered polypeptides having structural and / or kinetic similarity to CD25 or portions thereof.SOLUTION: Disclosed are engineered polypeptides that comprise a combination of spatially-associated topological constraints, wherein at least one constraint is derived from a CD25 reference target, and methods of selecting the engineered polypeptides. Further disclosed are methods of using the engineered polypeptides, including positive and / or negative selection molecules, in methods of screening a library of binding molecules, such as antibodies. Further disclosed are CD25 antibodies selected using these engineered polypeptides.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 902,334, filed September 18, 2019; and U.S. Provisional Patent Application No. 62 / 767,431, filed November 14, 2018. No. 6,229,799, the disclosures of which are incorporated herein by reference in their entireties. [Background technology]

[0002] The CD25 protein is the alpha chain of the interleukin-2 (IL-2) receptor. , a transmembrane protein present on regulatory T cells and activated T cells. In this state, regulatory T cells constitutively express CD25 and mediate the expansion and proliferation of effector T cells. Regulatory T cells act to keep the immune system healthy and suppress effector T cells. It prevents the immune system from responding to self-antigens or over-reacting to foreign antigens. In a normal protective immune response, effector T cells expand and regulate after contact with a foreign antigen. However, in proliferative diseases, cancer cells overcome the inhibition of regulatory T cells. Increases the amount of T cells that can fight cancer, thereby limiting the generation of T effector cells against cancer cells. This can disable a healthy immune response, e.g., in cancer treatment. Altering the proliferation of CD25-expressing regulatory T cells to suppress the immune system for use in There is interest in therapeutic agents that target CD25. These therapeutic agents may include antibodies targeting CD25. .

[0003] CD25-targeting antibodies can be produced by immunizing animals with a CD25 immunogen. However, current methods for developing CD25 immunogens have unpredictable and undesirable characteristics, such as anti- This often results in body confusion or low cross-reactivity across species. Summary of the Invention [Problem to be solved by the invention]

[0004] Therefore, structural and / or kinetic similarity to CD25 or portions thereof New engineered polypeptides having epitopes outside the IL-2 binding site, e.g. There is a need in the art for engineered polypeptides that are designed to mimic. [Means for solving the problem]

[0005] In one aspect, the present disclosure provides a method for detecting a CD25 reference target having at least 46% structural and / or functional identity with the CD25 reference target. an engineered polypeptide that shares kinetic identity with the CD25 reference target; 25 residues 55~63, 13~20:127~132, 5~17, 5~11:156~16 CD25 selected from 3, 77-89, 147-157, 11-14, or 44-56 The present invention provides an engineered polypeptide that is a portion of

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

[0007] In embodiments, between 10% and 98% of the amino acids of the engineered polypeptide are one or more In embodiments, one or more CD25 reference target-derived constraints are satisfied. Amino acids that satisfy the constraints have a backbone root mean square deviation (RS) of less than 8.0 Å from the CD25 reference target. In embodiments, one or more CD25 reference target-derived restriction sites are present. The amino acid to be filled is 30Å. 2 ~3000Å 2 Between the reference and van der Waals surface areas In embodiments, the CD25 reference target-derived constraints include interatomic distances; atomic fluctuations; Atomic energy; chemical descriptors; solvent exposure; amino acid sequence similarity; bioinformatic descriptors; Non-covalent bond tendency; Phi angle; Psi angle; Van der Waals radius; Secondary structure tendency; Amino and amino acid contact. The polypeptides identified are those containing amino acids in the polypeptide that satisfy one or more reference target-derived constraints. Across all of the nucleotide sequences, 46% to 96% RMSIP or higher structural similarity to the reference target was observed. share.

[0008] In another aspect, the present disclosure provides a method for the production of CD25 residues 55-63, 13-20:127-132, 5 ~17, 5~11:156~163, 77~89, 147~157, 11~14 or 4 Contains an antigen-binding domain that specifically binds to a CD25 epitope selected from 4 to 56 CD25-specific antibodies are provided. In embodiments, the antibodies are epitopes for binding to CD25. Epitope-specific binders compete with the top-specific reference binders, and the epitope-specific binders are IL-2, daclizumab, and , basioliximab and / or 7G7B6. In embodiments, the antibody does not compete with an off-target reference binder, and the off-target binder is IL-2, daclizumab, basioliximab and / or 7G7B6. So, the antibody is 10 -2 / s, less than 10 -3 / s or less than 10 -4 k less than / s off k off Biolayer Interferometry Using Soluble Human CD25 In an embodiment, the antibody is measured using -2 / s and 10 -5 Between / s k off k off Biolayer interface using soluble human CD25 In embodiments, the antibody has a titer of less than 100 nM, less than 25 nM, or less than 100 nM. K less than or equal to 5 nM D K D Biolayer using soluble human CD25 In embodiments, the antibody is at a concentration between 100 nM and 1 K between nM D K D Biolayer Interferon Using Soluble Human CD25 It is measured using ferrometry.

[0009] In embodiments, the antibody specifically binds to cells expressing CD25. The antibody should be at least 10 4 or at least 10 5 The mean fluorescence intensity (MFI) of CD2 5. In embodiments, the antibody binds to cells expressing 10 4 and 10 6 The average fluorescence between In embodiments, the antibody binds to cells expressing CD25 with high MFI. 5(-) cells. In an embodiment, the antibody does not bind to 10 3 Mean fluorescence intensity (MFI) In an embodiment, the antibody binds to CD25(-) cells at combinations 1 to 7 of Table 7D. 126.

[0010] In embodiments, the antibody is YU390-B12, YU 397-F01, YU397-D01, YU398-A11, YU404-H01, YU 400-B07, YU400-D09, YU401-B01, YU401-G07, YU 404-C02, YU403-G07, YU403-G05, YU391-B12, YU 400-A03, YU400-D02, YU392-A09, YU392-B11, YU 392-B12, YU392-E05, YU392-E06, YU392-G08, YU 389-A03, YU392-G09, YU392-G12, YU392-H02, YU 392-H04, YU402-F01, YU389-B11, YU394-D08, and contains six complementarity determining regions (CDRs) for any one of YU390-A11 .

[0011] In embodiments, the antibody is YU390-B12, YU397-F01, or any of the antibodies provided in Table 5. , YU397-D01, YU398-A11, YU404-H01, YU400-B07 , YU400-D09, YU401-B01, YU401-G07, YU404-C02 , YU403-G07, YU403-G05, YU391-B12, YU400-A03 , YU400-D02, YU392-A09, YU392-B11, YU392-B12 , YU392-E05, YU392-E06, YU392-G08, YU389-A03 , YU392-G09, YU392-G12, YU392-H02, YU392-H04 , YU402-F01, YU389-B11, YU394-D08, or YU390- A11 heavy chain variable region and light chain variable region at least 90%, 95%, 99% or 1 The heavy and light chain variable regions each share 0.000% sequence identity. In embodiments, the antibody is a full-length immunoglobulin G monoclonal antibody. , YU390-B12, YU397-F01, YU397-D01 provided in Table 5, YU398-A11, YU404-H01, YU400-B07, YU400-D09, YU401-B01, YU401-G07, YU404-C02, YU403-G07, YU403-G05, YU391-B12, YU400-A03, YU400-D02, YU392-A09, YU392-B11, YU392-B12, YU392-E05, YU392-E06, YU392-G08, YU389-A03, YU392-G09, YU392-G12, YU392-H02, YU392-H04, YU402-F01, Single-chain variable fragments of YU389-B11, YU394-D08, or YU390-A11 ( scFv) sequence. It contains scFv that

[0012] In embodiments, the antibody is a human antibody. In embodiments, the antibody is a humanized antibody. In embodiments, the antibody is a chimeric antibody. In embodiments, the antibody comprises a murine variable domain. and a human constant domain. In embodiments, the antibody is derived from a cynomolgus monkey (cynomolgus monkey) It also binds to CD25 in the rhesus monkey.

[0013] In another aspect, the disclosure provides a method for the preparation of a pharmaceutical composition comprising administering to a subject an antibody of the present disclosure, optionally in a pharmaceutically acceptable excipient. In another aspect, the present disclosure provides a pharmaceutical composition comprising the agent. A method comprising administering to a subject a therapeutically effective amount of any of the antibodies or pharmaceutical compositions of the present disclosure. In embodiments, the subject has cancer. In another aspect, the present disclosure provides a method for treating a subject suffering from an autoimmune disease or disorder. A method for depleting the number of regulatory T cells in a subject comprising administering to the subject a therapeutically effective amount of any of the compounds of the present disclosure. In embodiments, the subject is provided with a method comprising administering the antibody or pharmaceutical composition to the subject. In embodiments, the subject suffers from an autoimmune disease or disorder.

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

[0015] In some embodiments, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, From sequence number 6, sequence number 7, sequence number 8, sequence number 9, sequence number 10 and sequence number 11 an engineered immune system having at least 60% sequence similarity to a sequence selected from the group consisting of: In some embodiments, the engineered immunogen has a sequence In another embodiment, the engineered immunogen has at least 80% similarity to the In certain embodiments, the engineered The immunogen shares at least one characteristic with CD25. The engineered immunogen binds to an antibody to CD25. In some embodiments, the engineered immunogen The original binding affinity was found to be higher at pH below 7.0 compared to that at pH between about 7.3 and about 7.5. In some embodiments, the engineered antibody has a higher binding affinity for CD25. The immunogens tested had binding affinities at pHs between about 6.4 and about 7.5 compared to binding affinities at pHs between about 7.3 and about 7.5. At a pH between about 6.6 and 7.0, CD25 has a higher binding affinity to the antibody.

[0016] In yet another embodiment, a method of producing an antibody is provided comprising the steps of: No. 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, At least 60 to a sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 11 immunizing an animal with an engineered immunogen having 100% sequence similarity; and producing antibodies. In some embodiments of the method, the antibody is C. In certain embodiments, the antibody has a IgG antibody binding domain between about 7.3 and about 7.5. Higher binding affinity for CD25 at pH below 7.0 compared to pH between In still further embodiments, the antibody exhibits a pM of between about 7.3 and about 7.5. The binding affinity for CD25 was higher at pH between about 6.4 and about 6.6 compared to that at pH 6. In some embodiments, the antibody inhibits the binding of CD25 to IL-2. In other embodiments, the antibody blocks the binding of CD25 to IL-2. Item 12. The method according to any one of Items 8 to 11, wherein the antibody is capable of inhibiting binding of CD25 to IL-2. In some embodiments, the antibody does not block IL-2R-alpha, IL-2R-base. In certain embodiments, the IL-2R-gamma receptor prevents heterotrimerization of IL-2R and IL-2R-gamma. The antibody is capable of binding to both the cis and trans orientations of CD25.

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

[0018] [Figure 1] FIG. 1 provides a schematic diagram showing the construction of an exemplary combination of three spatially related topological constraints for use in selecting engineered polypeptides described herein.

[0019] [Figure 2]Figure 2 provides a schematic representation of the steps involved in some exemplary methods for determining reference-derived, spatially relevant topological constraints and their use in selecting engineered polypeptides, which are referred to herein as meso-scale molecules, MEMs, or meso-scale peptides.

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

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

[0022] [Figure 5] FIG. 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]FIG. 6 shows a diagram of eight epitopes on CD25 outside the IL-2 interface that were targeted for the generation of engineered polypeptides of the present 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 a diagram of the computationally determined deviation of the engineered polypeptide from the target epitope. The engineered polypeptide shows similarity in structure and dynamics to the target epitope (46%-96% RMSIP).

[0026] [Figure 9] Figure 9 shows ELISA analysis of 384 anti-CD25 scFv clones per in vitro selection strategy. Eight CD25 epitopes were targeted using 32 programmed selection strategies. Numbers indicate ELISA analysis of individual scFvs 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, which correspond to the epitopes shown in Figure 6.

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

[0028] [Figure 11]Figure 11 shows data from biolayer interferometry for 1433 anti-CD25 scFvs identified by phage display panning. The y-axis plots koff (1 / s) for each clone. The observed median koff was 1.35 x 105 (1 / Ms). KD estimates assume a koff of 4.5 x 104 (1 / Ms). 1433 of 1475 tested screening hits (97%) are confirmed to bind to CD25. The plot shows the off-rate distribution for the 1433 confirmed hits.

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

[0030] [Figure 13] Figure 13 shows data from flow cytometry for anti-CD25 scFvs identified by phage display panning. The CD25 specificity of different scFv antibodies was assessed by flow cytometry using cells expressing CD25 [CD25(+)] or cells not expressing CD25 [CD25(-)].

[0031] [Figure 14A] Figures 14A-14B show data from flow cytometry for anti-CD25 scFvs 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 scFvs 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] FIG. 15 shows the amino acid residue enrichment at each CDR H3 position in a representative enrichment strategy (S12).

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

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

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

[0036] [Figure 19] Figure 19 shows that full-length CD25 panning clones are dominated by IL-2 interface epitopes. 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-driven clones bind primarily at the intended epitope. 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-driven clones bind primarily at the intended epitope. Most clones are blocked by 7G7B6, but not by IL-2, daclizumab, or basiliximab.

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

[0040] [Figure 23] Figure 23 shows that the 44-56 epitope MEM-driven clones bind primarily at the intended epitope. The clones were divided into two profiles. In profile 1, the clones are blocked by 7G7B6 but not by IL-2, daclizumab, or basioliximab. In profile 2, the clones are blocked by IL-2, daclizumab, and basioliximab but not by 7G7B6. These blocking profiles demonstrate binding to the intended epitope from different angles.

[0041] [Figure 24]Figure 24 shows that the 55-63 epitope MEM-driven clones bind primarily to the intended epitope. The clones were divided into three profiles. In profile 1, the clones are blocked by 7G7B6 but not by IL-2, daclizumab, or basioliximab. In profile 2, the clones are blocked by IL-2, daclizumab, and basioliximab but not by 7G7B6. These blocking profiles demonstrate binding to the intended epitope from different angles. In profile 3, the clones are blocked by IL-2 and 7G7B6 but not by daclizumab or basioliximab. These blocking profiles demonstrate binding to the intended epitope from different angles.

[0042] [Figure 25] Figure 25 shows the alanine mutations designed to confirm or reject MEM-driven clone binning of the intended epitopes. The eight epitopes are indicated by color. The sites of the residues mutated to alanine are indicated by red bars.

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

[0044] [Figure 27] Figure 27 shows the reliability of the Ala mutant epitope mapping demonstrated using a basiliximab control antibody. The binding response of the Ala mutant confirms the crystal structure of the basiliximab epitope. The basiliximab-CD25 epitope revealed by the X-ray crystal structure is shown in orange.

[0045] [Figure 28] Figure 28 shows the reliability of Ala mutant epitope mapping demonstrated using a daclizumab control antibody. The binding response of the Ala mutant confirms the crystal structure of the daclizumab epitope. The daclizumab-CD25 epitope revealed from the X-ray crystal structure is shown in orange. The inset at the bottom left shows an epitope zoom demonstrating the effect of T175A on daclizumab binding.

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

[0047] [Figure 30] Figure 30 shows epitope mapping of MEM-programmed selection hits for epitopes 147-157. Most hits show ala mutation sensitivity in the intended epitope.

[0048] [Figure 31] Figure 31 shows the sensitivity of various MEM-driven antibody hits to alanine substitution. Functional epitope diversity is observed. MEM-driven hits have distinct in-epitope alanine substitution position 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 that were used to develop engineered immunogens that mimic CD25.

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

[0051] [Figure 34] Figure 34A is a model of IL-2 binding to the IL-2R complex, showing CD25 sections (ribbons), IL-2 (1), IL-2R-gamma (2), and IL-2R-beta (3). Figure 34B is another representation of IL-2 binding to the IL-2R complex, listing the regions of CD25 that were used as input to develop different selected exemplary engineered immunogens. Figure 34C is another representation of IL-2 binding to the IL-2R complex, listing the regions of CD25 that were used as input to develop different selected exemplary engineered immunogens. DETAILED DESCRIPTION OF THE INVENTION

[0052] engineered fragments that share structural and / or kinetic identity with a portion of the reference CD25 target Polypeptides of interest include the eight epitopes shown in Figure 6. In some embodiments, the selected epitope The topes identify binding sites (e.g., endothelial cells) for IL-2, daclizumab, and / or basiliximab. In some embodiments, the epitope does not overlap with the epitope for 7G7B6. In some embodiments, the selected epitopes overlap with 55-63, 12- 20:127-132 (non-continuous epitopes), 5-17, 5-11:156-163 (non-continuous epitopes) Consecutive epitopes), 77-89, 147-157, 11-14, or 44-56 In some embodiments, the engineered polypeptide is conformationally stable. and the CD25 epitope involved in the interaction with an antibody that specifically binds to CD25 is In some embodiments, the engineered polypeptide specifically binds to CD25. These engineered CD25 proteins display surface portions of CD25 that are known not to interact with the antibodies. The polypeptides may be used, for example, to select and / or generate antibodies that specifically bind to CD25. It can be used to I. Engineered polypeptides.

[0053] In some embodiments, the engineered polypeptides provided herein are Shares at least 46% structural and / or kinetic identity with target, CD25 reference The target is a portion of CD25 selected from those listed in the table below. As commonly provided in IP) identity (provided hereinabove) × 100%. Structural identity refers to sequence identity.

[0054] [Table 1]

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

[0056] [Table 2] shares 80% sequence identity with an amino acid sequence selected from

[0057] In some embodiments, the polypeptide has a similar affinity to the CD25 reference target by at least 60%, and at least At least 70%, at least 75%, at least 80%, at least 85%, at least 90% % or at least 95% structural and / or kinetic identity. In embodiments, the polypeptide has a similar affinity to the CD25 reference target by at least 60%, at least 70%, or , at least 75%, at least 80%, at least 85%, at least 90% or less They share at least 95% sequence identity.

[0058] In some embodiments, the engineered polypeptide mimics a selected CD25 epitope. For example, in some embodiments, the polypeptide is designed to mimic mesoscale Mesoscale engineered molecules, such as mesoscale engineered polypeptides. A method for selecting an engineered polypeptide of the present invention, as well as a method for producing a polypeptide containing the engineered polypeptide Compositions containing the antibody and methods of using the same are provided herein. Methods of using the engineered polypeptides in selection are provided herein.

[0059] The engineered polypeptides of the present disclosure are between 1 kDa and 10 kDa and are referred to herein as Engineered polypeptides of this size are referred to as "mesoscale" in some embodiments. In this case, certain advantages, such as protein-like functionality, a large theoretical space to select candidates, Mesoscale peptides may have cellular permeability and / or structural and kinetic variability. The terms peptide and mesoscale polypeptide are used interchangeably herein. , the term mesoscale molecules (MEMs) is intended to cover these.

[0060] The methods provided herein involve the generation of multiple spatially distinct CD25 targets, some of which may be derived from a CD25 reference target. Identifying the topological constraints associated with the , comparing the candidate peptide with this combination and determining whether the constraints overlap with this combination. Using spatially related topological constraints By this, different aspects of the engineered polypeptide can be tailored to suit the intended use or desired function, or may be included in a combination depending on other desired characteristics. In this situation, not all of the binding needs to come from the CD25 reference target. In some embodiments, the selected engineered polypeptide is simply a CD25 reference. Variation of a reference target (e.g., peptide mutagenesis or progressive modification of a single reference) (which can be obtained through a mutation) but rather It may have a different overall structure than the reference peptide while still retaining structure.

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

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

[0063] In some aspects, there is provided a method for selecting an engineered polypeptide, comprising: identifying one or more topological features of the CD25 reference target; Spatially relevant constraints were designed for each topological feature to allow for the identification of the CD25 reference target. Producing combinations of constraints; Spatially related topological features of candidate peptides were compared with a set of peptides derived from the CD25 reference target. comparing combinations; and Spatially related topologies overlapping with binding combinations derived from the CD25 reference target and selecting candidate peptides having the desired characteristics. Provided herein is a method comprising:

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

[0065] The engineered polypeptides described herein are designed to have a topology in which they are spatially related. The combination is selected based on how closely it matches the set of constraints. Combinations can also be described using the mathematical concept of a "tensor." (or tensor), each constraint is described independently in three-dimensional space (e.g., spatial The combination of these constraints in 3D space can be used to create different constraints on position, e.g. Provides a representational "map" of desired features and their desired levels (if applicable) This map may, in some embodiments, be a linear or otherwise predetermined It is not based on the amino acid backbone and therefore cannot meet the desired combinations described. For example, in some embodiments, a "map" may be Contains a spatial region in which a defined constraint can be adequately satisfied by two adjacent amino acids - In some embodiments, these amino acids may be directly linked (e.g., two consecutive In other embodiments, the amino acids are not directly bonded to each other but are bonded to peptides. The amino acids can be spaced together by folding of the amino acids (e.g., consecutive amino acids). The separate constraints themselves do not necessarily have to be structure-based, e.g., they can be chemically descriptive. In some embodiments, the constraints may include structural and / or functional descriptors. In certain embodiments, the amino acid sequence includes a desired secondary structure or amino acid residues. Each constraint is selected independently.

[0066] For example, Figure 1 shows the construction of a representative set of spatially related topological constraints. The three constraints in Figure 1 are alignment, nearest neighbor distance, and atomic motion. The parametric distance and atomic motion are combined in one picture. As shown, some constraints is mapped independently of the backbone location (e.g., the atomic motion of a particular side chain), but This allows for much more diverse structures compared to simply varying one or more positions on a reference scaffold. Three different constraints and their spatial description allow for structural configurations to be attempted. are combined into a matrix (e.g., a tensor), and then a set of candidate peptides is generated based on the desired This combination and comparison are performed to identify new engineered polypeptides that meet the criteria of In some embodiments, one or more additional non-reference-derived constraints may also be combined. Comparison of candidate peptides with defined combinations can be used to identify, for example, desired combinations. In silico methods for assessing the constraints of each candidate peptide against the match, and how the candidate This can be done using a ratio of the degree of matching. These candidates with the desired level of overlap can be synthesized using standard peptide synthesis methods known to those skilled in the art. can be synthesized and evaluated using

[0067] In some embodiments, the combination of constraints is at least three, at least four, or at least At least 5, at least 6, at least 7, at least 8, at least 9, at least At least 10, at least 11, at least 12, between 3 and 12, between 3 and 10, Between 3 and 8, between 3 and 6, or 3, or 4, or 5, or 6, unique The constraints include spatially related topological constraints that are selected independently. One or more of the constraints may be In some embodiments, each of the constraints is derived from a CD25 reference target. In other embodiments, at least one constraint is from a CD25 reference target and the remaining The constraints do not come from a reference target. For example, in some embodiments, between 1 and 9 constraints Bundle, constraint between 1 and 7, constraint between 1 and 5, or constraint between 1 and 3 derived from the CD25 reference target, with binding between 1 and 9, binding between 1 and 7, and 1 Restriction between 0 and 5 or between 1 and 3 does not derive from the CD25 reference target .

[0068] Once the set of constraints is constructed, a set of candidate peptides is selected to identify one or more peptides that meet the desired criteria. These combinations are compared to identify new engineered polypeptides. 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 At least 100, at least 125, at least 150, at least 175, at least At least 200 or at least 250 or more candidate peptides are selected to identify the desired This combination is used to identify one or more new engineered polypeptides that meet the criteria. In some embodiments, more than 250 candidate peptides, for example, 3 More than 00 candidate peptides, more than 400 candidate peptides, more than 500 Many candidate peptides, more than 600 candidate peptides, or more than 750 Candidate peptides are compared. In some embodiments, topological feature simulation is performed , if any, of the topological features of the candidate peptide to be compared with the combination of constraints In some embodiments, one or more candidate peptides are used to assess overlap. The CD25 reference target is also compared, and the topological features of the CD25 reference target, if present, are The overlap of the topological features of the candidate peptide with the The polypeptides may be 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 Computational samples of many distinct peptides and topological features simulations The engineered polypeptides identified from the analysis are selected, and the selected engineered polypeptides are The peptides were the highest topoisomers compared to the CD25 reference target in the total sampled population. have overlapping logistical characteristics.

[0069] The spatially related components used to construct the desired combination (e.g., the desired tensor) The associated topological constraints can each be independently selected from a wide range of possible features. These include, for example, constraints that describe structural, dynamic, chemical, or functional characteristics; Any combination thereof may be included.

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

[0071] Dynamical constraints include, for example, atomic fluctuations, atomic energies, van der Waals radii, This may include amino acid proximity or non-covalent bonding propensity. Atomic energy may include, for example, two The pairwise attractive energy between atoms of, the pairwise repulsive energy between two atoms of, atom level solvation energy, pairwise charge attraction energy between two atoms, pairwise hydrogen bond attraction energy, or non-covalent bond energy, or Any combination may be included.

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

[0073] Functional features include, for example, bioinformatic descriptors, biological responses, or biological functions. Bioinformatic descriptors include, for example, BLOSUM similarity, pKa, zScal e, Cruciani Properties, Kidera Factors, VHS E-scale, ProtFP, MS-WHIM score, T-scale, ST-scale, Transmembrane propensity, buried protein region, helix propensity, sheet propensity, coil propensity, turn propensity tropism, immunogenic tendency, presence of antibody epitopes, and / or presence of protein interfaces, or or any combination thereof.

[0074] In some embodiments, designing the constraints involves determining per-residue energies, per-residue interactions, , per-residue fluctuations, per-residue interatomic distances, per-residue chemical descriptors, per-residue solvent exposure, Residue-by-residue amino acid sequence similarity, residue-by-residue bioinformatic descriptors, residue-by-residue non-covalent bonding propensity, residue-by-residue Phi / psi angle per residue, van der Waals radius per residue, secondary structure tendency per residue, It incorporates information about group-by-group amino acid adjacency or residue-by-residue amino acid contacts. In embodiments, these features are a subset of the total residues in the CD25 reference target, or a constraint. used for a subset of the total residues of the total combination of bundles, or their combinations In some embodiments, one or more different features are used for one or more different residues. That is, in some embodiments, one or more features are used for a certain subset of residues. and at least one different feature is used for different subsets of residues. In some embodiments, one of these features is used to design one or more constraints. One or more of these are determined by computer simulation. Simulation methods include, for example, molecular dynamics simulation, Monte Carl Simulation, coarse-grained simulation, Gaussian network model, machine learning, or any combination thereof.

[0075] In some embodiments, multiple constraints are selected from one category. In this embodiment, a combination is defined as 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 this embodiment, the combination does not include overlapping categories of constraints.

[0076] In some embodiments, the one or more constraints independently affect a biological response or a biological function. In some embodiments, this constraint is related to the spatially defined atom(s) level. constraints, or spatially defined shape / area / volume level constraints (e.g., some a characteristic shape / area / volume) or spatially ordered structure that can be satisfied by different atomic compositions of the The dynamic level constraints (e.g., those that can be satisfied by several different atomic compositions) (a characteristic dynamic or set of dynamics that

[0077] In some embodiments, the one or more constraints are associated with a biological function or biological response. derived from a protein or peptide structure. For example, in some embodiments, one or more The binding of the receptor to its extracellular domain, e.g., the extracellular domain of a G protein-coupled receptor (GPCR), In some embodiments, one or more of the binding domains are derived from the extracellular domain of a polypeptide or an ion channel. The bundles originate from protein-protein interfacial junctions. In some embodiments, one or more Constraints can occur at protein-peptide interface junctions, e.g., MHC-peptide or GPCR- In certain embodiments, such protein or peptide structures are derived from peptide interfaces. The structurally constrained atoms or amino acids are atoms associated with biological functions or biological responses. or amino acids. In some embodiments, such protein or peptide structures are confined to The atoms or amino acids in the engineered polypeptide to be bundled are derived from the CD25 reference target. In some embodiments, the one or more constraints are atoms or amino acids that are derived from a polymorphic region of interest (e.g., a region subject to allelic variation between individuals) .

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

[0079] One of the constraints is one or more amino acids associated with a biological function or biological response. and / or engineered polypeptides associated with a biological function or biological response. In some embodiments, the one or more amino acids are selected from the 20 types of amino acids. Proteinogenic naturally occurring amino acids, non-proteinogenic naturally occurring amino acids and and unnatural amino acids. In some embodiments, the unnatural amino acids In certain embodiments, one or more amino acids are chemically synthesized. In another embodiment, one or more of the following amino acids are selected from the group consisting of: The above amino acids are selected from non-proteinogenic naturally occurring amino acids. In further embodiments, one or more amino acids are selected from unnatural amino acids. In another embodiment, the one or more amino acids are selected from the 20 proteinogenic naturally occurring amino acids. amino acids, non-proteinogenic naturally occurring amino acids and unnatural amino acid combinations is selected from.

[0080] The set of constraints used to select the engineered polypeptides described herein The matching includes at least one constraint derived from a CD25 reference target, although in some embodiments In this case, one or more constraints of the combination are not derived from the CD25 reference target. In certain embodiments, the selected engineered polypeptide has a common target sequence with the CD25 reference target. It contains one or more features that are not

[0081] In some embodiments, one of the CD25 reference targets used in the combination is derived from the CD25 reference target. The above constraints describe the inverse of the characteristics observed in the CD25 reference target. For example, a CD25 reference target may have a particular pattern of positive charges, resulting in charge-related binding. The CD25 reference target is derived from this target, and the resulting binding follows a similar pattern but with a neutral charge. Thus, in some embodiments, one or more inverse constraints may be: CD25 reference target and included in the combination. Such counter-constraints can be used, for example, to as a control molecule for the assay or panning method, or for the processes described herein. Engineered polypeptides as negative selection molecules in programmable in vitro selection methods This can be useful in selecting

[0082] In some embodiments, the combination of spatially defined topological constraints includes one or more In some embodiments, one or more non-reference derived topological constraints Logical constraints constrain or stabilize one or more secondary structure elements and enforce atomic fluctuations. , changing the total hydrophobicity of the peptide, changing the solubility of the peptide, changing the total charge of the peptide , allowing detection in labeled or unlabeled assays, and in vitro assays and in vivo assays. , enabling capture from complex mixtures, enabling enzymatic processing, and cell membrane permeability enable binding to a secondary target, or alter immunogenicity. In this state, one or more non-reference derived topological constraints may be added to the CD25 reference target derived constraints. One or more atoms or amino acids in the combination (or subsequently selected peptide) For example, in some embodiments, the binding combination is based on a CD25 reference target. The combination of constraints stabilizes the secondary structure element (e.g., further Hydrogen bonds, hydrophobic interactions, side chain stacking, salt bridges, or dimers The stabilizing constraint also includes a tether (via a sulfide bond) in the CD25 reference target. As another example, in some embodiments, a combination of constraints (or The peptides selected by the nucleotide sequence are selected from one or more atoms or amino acids derived from the CD25 reference target. wherein the combination of constraints comprises at least a portion of an atom or amino acid from the target reference. It also includes constraints that enforce atomic fluctuations in the molecule, which are not present in the target reference. In some embodiments, one or more of the non-reference-derived constraints are inverse constraints. In embodiments, two combinations of constraints select for engineered polypeptides with opposite characteristics. In some such embodiments, the first set of constraints is constructed to select One or more constraints derived from the CD25 reference target and one or more not derived from the CD25 reference target the second combination of constraints would include the same 1 from the CD25 reference target; one or more of the non-CD25 reference target constraints in the first combination, and the reverse of one or more of the non-CD25 reference target constraints in the second combination. It will include: b.CD25 reference target

[0083] Any suitable CD25 reference target may be used in combination with a CD25 antibody for use in the methods provided herein. This can be used to induce one or more spatially related topological constraints. In one embodiment, the CD25 reference target is the full-length native protein. The CD25 reference target is a portion of the full-length native protein. In the present case, the CD25 reference target is a non-native protein, or portion thereof.

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

[0085] [Table 3] is selected from.

[0086] For example, in some embodiments, the CD25 reference target is a portion of CD25, e.g., an epitope. In some embodiments, the present invention provides a method for detecting a target gene, a target protein, or a predicted epitope. The method includes providing one or more antibodies that are immunogens and specifically bind to the protein from which the target reference is derived. used to select one or more engineered polypeptides that can be used to generate antibodies In still further embodiments, the methods provided herein involve detecting a protein of interest. , e.g., antibodies, Fab-displaying phages or scFv-displaying phages One or more procedures that can then be used to select one or more binding partners for the phage. can be used to select a polypeptide that has been c.Compare constraints

[0087] In some embodiments, one or more constraints (e.g., reference-derived or non-reference-derived) are Simulations (e.g., molecular dynamics) or laboratory measurements (e.g., NMR), or is determined by their combination. Once the constraints are induced and combined, the The candidate polypeptides may, in some embodiments, be derived from computational protein design (e.g., In some embodiments, peptide space is sampled using a A dynamic simulation is then performed to determine the geometry of the selected constraints. can be performed on candidate engineered polypeptides to obtain the parameters of A covariance matrix of the child fluctuations is generated for the CD25 reference target, and the covariance matrix is ​​used to The covariance matrices are generated for the residues in each of the constructed polypeptides, and these covariance matrices are used to determine overlap. Principal component analysis is performed to determine the covariance matrices for each of the CD25 reference targets. one covariance matrix for all the candidate engineered polypeptides, and one covariance matrix for each of the candidate engineered polypeptides. Variance - is performed to calculate the eigenvectors and eigenvalues ​​for The eigenvectors with their eigenvalues ​​are retained.

[0088] The eigenvectors are the first, second, and third eigenvectors observed in a set of simulated molecular structures. Describe the 1st, 3rd, and Nth dominant motions. If the candidate engineered polypeptide behaves similarly to the CD25 reference target, then The eigenvectors of will be similar to those of the CD25 reference target. The similarity of the components is determined by their alignment and pointing in the same direction (3D vectors centered on each CA atom). Corresponds to the

[0089] In some embodiments, the eigenvector of the candidate engineered polypeptide and the CD25 reference target are This similarity between the eigenvectors of the target is calculated using the dot product of the two eigenvectors. The dot product is either 0 if the two eigenvectors are at 90 degrees to each other, or or 1 if the two eigenvectors point exactly in the same direction. Although we do not want to be confused, the ordering of eigenvectors is based on their eigenvalues, which are , molecular dynamics (MD) simulations reveal the underlying energy run of two different molecules. Due to the stochastic nature of sampling the landscape, there is not necessarily a difference between these different molecules. Since the numbers may not be the same, in some embodiments, multiple differentially ranked unique The dot product between vectors is required (e.g., the eigenvectors 1× Eigenvectors of the CD25 reference target (e.g., 2, 3, 4). Furthermore, molecular motion is complex, and 1 may involve more than one (or more than a few) dominant / major modes of motion Thus, in some embodiments, the candidate engineered polypeptide and the CD25 reference The dot products between all pairs of eigenvectors at the target are calculated. This results in a matrix of dot products. The dimension is determined by the number of eigenvectors analyzed. For example, if there are 10 eigenvectors, For vectors, the matrix of the dot product is 10x10. This matrix of the dot product is 100(10x 10) to a single value by calculating the root mean square value of the dot product This is the root mean square dot product (RMSIP). From this comparison, the constraint rule One or more candidate engineered polypeptides having similarity to the determined combination are selected. will be done. d. Further processing

[0090] In some embodiments, the selection of one or more engineered polypeptides further comprises one or more further For example, in some embodiments, the engineered candidate polypeptides are As described in and then selecting one or more additional features based on similarity. It is subjected to analysis and one or more structural adjustments to impart or enforce desired characteristics. For example, in some embodiments, the selected candidates are selected based on the overall stability and / or specificity of the molecule. To determine the propensity of the folded structure of a protein, for example, molecular dynamics simulations are used. In some embodiments, one or more modifications are analyzed via a desired level of stability. To impart or enhance the desired propensity for a desired folded structure or Such modifications may include, for example, the addition of one or more cross-linking moieties to the polypeptide. bridges (e.g., disulfide bonds), salt bridges, hydrogen bonding interactions or hydrophobic interactions, or or any combination thereof.

[0091] The methods provided herein can be used to identify one or more desired characteristics, such as a desired binding interaction or assaying one or more selected engineered polypeptides for a specific activity or activity. Any suitable assay may be used as needed to measure the desired characteristic. It can be used.

[0092] In other aspects, engineered polypeptides, e.g., via the methods described herein, Selected engineered polypeptides are provided herein. In some embodiments, the engineered polypeptides The constructed polypeptides have a molecular mass between 1 and 10 kDa and a maximum of 50 amino acids. In certain embodiments, the engineered polypeptide comprises a 2 kDa and a 10 kDa amino acid. Between Da, between 2kDa and 10kDa, between 3kDa and 10kDa, between 4kDa and 1 Between 0kDa, between 5kDa and 10kDa, between 6kDa and 10kDa, 7kDa Between 8kDa and 10kDa, between 9kDa and 10kDa, 1k Between Da and 9kDa, between 1kDa and 8kDa, between 1kDa and 7kDa, 1kD Between a and 6kDa, between 1kDa and 5kDa, between 1kDa and 4kDa, 1kDa It has a molecular mass between 1 and 3 kDa, or between 1 and 2 kDa. In embodiments, the engineered polypeptides may be up to 45 amino acids, up to 40 amino acids, up to Large: 35 amino acids, Max: 30 amino acids, Max: 25 amino acids, Max: 20 amino acids, Small: At least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 2 0 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids It contains at least 40 amino acids.

[0093] In certain embodiments, the engineered polypeptides are spatially related topologically related polypeptides. This includes a combination of constraints, one or more of which are CD25 reference target-derived constraints. Any of the constraints described in this specification may be used in combination in some embodiments. In a further embodiment, between 10% and 98% of the amino acids of the engineered polypeptide are One or more CD25 reference target-derived constraints are met (e.g., the engineered polypeptide is 50 If amino acids are included, between 5 and 49 amino acids are derived from one or more CD25 reference targets. In some embodiments, between 20% and 30% of the amino acids of the engineered polypeptide are Between 98%, between 30% and 98%, between 40% and 98%, between 50% and 98%, between 60% and Between 98%, Between 70% and 98%, Between 80% and 98%, Between 90% and 98%, Between 10% and Between 90%, Between 10% and 80%, Between 10% and 70%, Between 10% and 60%, Between 10% and Between 50%, between 10% and 40%, between 10% and 30%, or between 10% and 20% are In yet a further embodiment, one or more CD25 reference target-derived constraints are satisfied. One or more amino acids that satisfy the constraints from the CD25 reference target are aligned within 8.0 Å of the CD25 reference target. less than, 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 have a structural homology of less than 5.0 Å root mean square deviation (RSMD). In the present case, the engineered polypeptide has a molecular mass between 1 kDa and 10 kDa; and 50 amino acids; at least one of the constraints is a constraint derived from the CD25 reference target. combination of topological constraints related to 10% of the amino acids in the engineered polypeptide ~98% of patients met one or more CD25 reference target-derived restrictions; Amino acids that satisfy the reference target-derived constraints have a backbone mean square distance of less than 8.0 Å from the CD25 reference target. It has structural homology with RSMD.

[0094] In some embodiments, engineered polypeptides that satisfy one or more CD25 reference target-derived constraints. The amino acids of the peptides have between 10% and 90% sequence homology to the CD25 reference target, 2 Between 0% and 90% sequence identity, between 30% and 90%, between 40% and 90% between 50% and 90% sequence identity; between 60% and 90% sequence identity homology, between 70% and 90% sequence homology or between 80% and 90% sequence homology In some embodiments, the amino acids that satisfy one or more CD25 reference target-derived constraints are , 30 Å 2 ~3000Å 2 Between or 100Å 2 ~3000Å 2 Between or 250Å 2 ~3000Å 2 Between or 500Å 2 ~3000Å 2 Between or 750Å 2 ~30 00Å 2 Between or 1000Å 2 ~3000Å 2 Between or 1250Å 2 ~3000 Å 2 Between or 1500Å 2 ~3000Å2 Between or 1750Å 2 ~3000Å 2 Between or 2000Å 2 ~3000Å 2 Between or 2250Å 2 ~3000Å 2 Between , or 2500 Å 2 ~3000Å 2 Between or 2750Å 2 ~3000Å 2 Between has a van der Waals surface area overlap with the reference.

[0095] The engineered polypeptide satisfies two or more, three or more, four or more combinations of constraints. , 5 or more, 6 or more, or 7 or more CD25 reference target-derived restrictions. The target may be one or more antibodies not derived from the CD25 reference target, as described elsewhere in this disclosure. These reference-derived constraints, and non-reference-derived constraints, if present, are independent. In this context, any of the constraints described herein, for example, the structural, Kinetic, chemical or functional characteristics, or any combination thereof. It is possible.

[0096] In some embodiments, the engineered polypeptide has a CD25 affinity when compared to a CD25 reference target. The structural difference may be, for example, a difference in sequence, amino acid residue, or the like. Number of groups, 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, structures Graph nodes and edges, volumetric surfaces, static electrostatic potential surface, hydrophobic potential surface, local diameter, local surface features, skeleton in the model, charge density, hydrophilic density, surface-to-volume ratio, amphiphilic density or surface roughness In some embodiments, one or more The difference in a feature (e.g., one or more features described herein) is relative to the type of feature. At least 10% when compared to the characteristics in the CD25 reference target, if applicable , at least 20%, at least 30%, at least 40%, at least 50%, less At least 60%, at least 70%, at least 80%, at least 90%, at least 10 0% or greater than 100%. For example, in some embodiments, the difference is and the engineered polypeptide has at least 10% less affinity than the CD25 reference target. at least 20% or at least 30% more atoms, or fewer atoms than the CD25 reference target Some examples have at least 10%, at least 20%, or at least 30% fewer atoms. In an embodiment, the difference is in the total positive charge, and the positive charge of the engineered polypeptide is The total charge of the target is at least 10%, at least 20%, less than that of the CD25 reference target. at least 30%, at least 40%, or at least 50% greater (e.g., more positive) However, in other embodiments, the total positive charge of the engineered polypeptide is greater than that of the CD25 reference target. at least 10%, at least 20%, at least 30%, at least 40% or is at least 50% smaller (e.g., less positive).

[0097] In some embodiments, the combination of spatially defined topological constraints is CD25 It contains one or more secondary structure elements that are not present in the reference target. In other words, the engineered polypeptide contains one or more secondary structure elements that are not present in the CD25 reference target. In some embodiments, the combined and / or engineered polypeptide comprises: One secondary structure element not found in the CD25 reference target, two secondary structure elements, three secondary structure elements Contains one primary structure element, four secondary structure elements, or more than four secondary structure elements. In embodiments, each secondary structure element is selected from the group consisting of helices, sheets, loops, turns and coils. In some embodiments, the CD25 reference target is independently selected from the group consisting of: Each secondary structure element can independently be an α-helix, a β-bridge, a β-strand, or 0 helix, π-helix, turn, loop or coil.

[0098] In certain embodiments, the CD25 reference target is a target of a biological response or function (e.g., an engineered polypeptide that includes one or more atoms associated with a polypeptide chain (e.g., those described herein); The peptides are those associated with a biological response or biological function (e.g., those described herein). The atomic fluctuations of these atoms in the engineered polypeptide , overlap with the atomic fluctuations of these atoms in the CD25 reference target. In some embodiments, the atoms themselves are different atoms, but their atomic fluctuations overlap. In an embodiment, the atoms are the same atoms and their atomic fluctuations overlap. In embodiments, the atoms are independently the same or different. In some embodiments, the overlap is 0. Root Mean Square Inner Product (RMSIP) greater than 25. In some embodiments, overlap is 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, 0.8 RMSIP greater than 5, greater than 0.9, or greater than 0.95. In certain embodiments, the RMSIP is

[0099]

number

[0100] In some embodiments, the engineered polypeptides may be used to enhance a biological response or function. Related atoms or amino acids (or combinations thereof), at least a portion of the amino acids or combinations are derived from the CD25 reference target, A set of atoms or amino acids in the engineered polypeptide and a set of atoms or amino acids in the CD25 reference target The specific constraints of the set may be described by a matrix. In some embodiments, the matrix is ​​an L×L In another embodiment, the matrix is ​​an SxSxM matrix. Now the matrix is ​​an L×2 phi / psi angle matrix.

[0101] For example, in some embodiments, engineered polypeptides associated with a biological response or function may be used. The atomic fluctuations of atoms or amino acids in a polypeptide are described by an L × L matrix; A portion of these atoms or amino acids is derived from the CD25 reference target; The atomic fluctuations in the 5 reference target are described by an L×L matrix. , the adjacency (in terms of amino acid positions) of each set is described by the corresponding L × L matrix In certain embodiments, the L×L atomic wobble or adjacency of the engineered polypeptide The mean percentage error (MPE) over all matrix elements (i,j) of a matrix is ​​CD2 5 For the portion of the engineered polypeptide derived from the reference target, the atoms of the CD25 reference target It is 75% or less compared to the corresponding (i,j) element in the fluctuation or adjacency matrix. In some embodiments, the MPE is one of an engineered polypeptide derived from a CD25 reference target. For some elements, the percentage of the target region is less than 70%, less than 65%, compared to the corresponding element in the CD25 reference target matrix. Full, less than 60%, less than 55%, less than 50%, less than 45%, or less than 40%. In some embodiments that represent atomic fluctuations, L is the number of amino acid positions, and the atomic fluctuation matrix The (i, j) value in the element is i if the interatomic distance (i, j) is 7 Å or less. The sum of the intramolecular atomic fluctuations for the jth and jth amino acids, or (i, j) If the interatomic distance is greater than 7 Å or (i,j) is on a diagonal, Alternatively, in some embodiments, the interatomic distance is multiplied by 0 or 1. In addition, it can act as a weighting factor for the atomic fluctuation matrix element (i,j). In this embodiment, the i-th and j-th atomic fluctuations and interatomic distances are calculated by molecular simulation. determined by simulation (e.g., molecular dynamics) and / or laboratory measurements (e.g., NMR) In other embodiments where the matrix represents adjacency, L is the number of amino acid positions and the adjacency The value in the matrix element (i,j) is the i-th value when the interatomic distance is 7 Å or less. The distance between the amino acid and the jth amino acid is 7 is zero if it is greater than Å or if (i,j) is on the diagonal, or In some embodiments, the interatomic distances are multiplied by the adjacency matrix elements, (i,j). In a particular embodiment, the ith and The distance between the jth and jth atoms can be calculated by molecular simulation (e.g., molecular dynamics) and / or It can be determined by laboratory measurements (eg, NMR).

[0102] In certain embodiments, the gene associated with the response or function in the engineered polypeptide is The molecule or amino acid is a portion of the engineered polypeptide derived from the CD25 reference target. The chemical descriptor vector of the topological constraints and the have a mean percentage error (MPE) of less than 75% compared to the reference and have a chemical description Each i-th element in the child vector corresponds to an amino acid position index. MPE is a method for identifying the same polypeptide fragments derived from the CD25 reference target. Compared to the reference described by the biological descriptor, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45% or less than 40%.

[0103] In yet a further embodiment, the matrix is ​​an L×2 phi / psi matrix and is manipulated The atoms or amino acids in the polypeptide that are associated with a response or function are derived from the reference target. In some engineered polypeptides, the α- and β-values ​​are less than 75% with respect to the reference phi / psi angle matrix. where L is the number of amino acid positions, and the phi and psi values ​​are as follows: In some embodiments, the MPE is derived from a reference target. In a portion of the engineered polypeptide, with respect to the reference phi / psi angle matrix, Less than 65%, less than 60%, less than 55%, less than 50%, less than 45% or less than 40% In some embodiments, the phi / psi value is determined by molecular simulations (e.g., molecular dynamics), knowledge-based structure prediction, or laboratory measurements (e.g., NMR) will be done.

[0104] In some embodiments, the matrix is ​​an S×S×M secondary structure element interaction matrix and is manipulated The atoms or amino acids in the polypeptide that are associated with the response or function are derived from the reference target. In the portion of the engineered polypeptide that %, S is the number of secondary structure elements, and M is the number of interaction descriptors. In some embodiments, the MPE is the number of interactions derived from the reference target. In some of the constructed polypeptides, the secondary structure element relationship matrix was less than 70% Full, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, or less than 40% Interaction descriptors include, for example, hydrogen bonding, hydrophobic packing, van der Waals interactions, ionic interactions, covalent bridges, chirality, orientation or distance, or or any combination thereof. The secondary structure element interaction matrix subscripts include (i , j, m) = the mth interaction record between the i-th secondary structure element and the j-th secondary structure element The value of the predicate.

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

[0106]

number

[0107] In some embodiments, the engineered polypeptide has a 75% or higher affinity to a CD25 reference target compared to the CD25 reference target. In certain embodiments, the engineered polypeptide has an MPE of less than 50%. 5 Compared to the reference target: <70%, <65%, <60%, <55%, <50% In some embodiments, the MPE is less than 45% or less than 40% of the total Topological Constraint Distance (TCD), Topological Clustering Coefficient (TCC), Euclidean Power distance, Soergel distance, Cannonball distance Vera distance, Sorensen distance, Jaccard distance, Mahalanobis distance, Hamming distance, La Quantitative Estimate of Likeness ess) (QEL) or chain topology parameters (CTP). e. Secondary structure elements

[0108] In some embodiments, at least a portion of the engineered polypeptide comprises one or more secondary In some embodiments, the engineered polypeptide is topologically constrained to a structural element. The atoms or amino acids associated with a biological response or function of In some embodiments, the secondary structural elements are independently topologically constrained to the structural elements. , a sheet, a helix, a turn, a loop, or a coil. The structural elements can be independently α-helices, β-bridges, β-strands, 3 10 Heli In certain embodiments, the amino acid sequence is a helix, a π-helix, a turn, a loop, or a coil. At least a portion of the engineered polypeptide is topologically constrained by secondary structure elements. One or more of which are present in the CD25 reference target. at least a portion of the peptide is topologically constrained by a combination of secondary structure elements; Each element is independently selected from the group consisting of a sheet, a helix, a turn, a loop, and a coil. In still further embodiments, each element is selected from an α-helix, a β-bridge, a β- Strand, 3 10 Consists of helices, π-helices, turns, loops, and coils are independently selected from the group.

[0109] In some embodiments, the secondary structural elements are parallel or anti-parallel sheets. In some embodiments, the sheet secondary structure comprises two or more residues. In yet a further embodiment, the sheet secondary structure comprises 50 residues or less. The sheets may be parallel or antiparallel. In some embodiments, the parallel sheet secondary structure is formed by two parallel strands i,j (i The N-termini of strand i and strand j are in opposite orientations, and the hydrogen bond pattern of residues i:j In some embodiments, the anti-parallel sheet secondary structure may also be described as having a turn. In addition, two antiparallel strands i, j (i strand and j strand N The ends are in the same orientation), and the hydrogen bonding pattern of residues i:j-1, i:j+1 In certain embodiments, strand orientation and hydrogen bonding can be described using knowledge-based can be determined by molecular dynamics simulations and / or laboratory measurements. do.

[0110] In some embodiments, the secondary structure element is a helix. In some embodiments, the helix may be between 2.5 and 6.0 residues per turn (residues / turn) value of and a pitch between 3.0 Å and 9.0 Å. In some embodiments, the residues / turns and pitches are based on knowledge-based or analytical determined by molecular dynamics simulations and / or laboratory measurements.

[0111] In some embodiments, the secondary structure element is a turn. In some embodiments, the amino acid sequence comprises between 2 and 7 residues, and one or more inter-residue hydrogen bonds. The turn contains two, three, or four inter-residue hydrogen bonds. The model is based on knowledge-based or molecular dynamics simulations and / or laboratory measurements. It is decided that:

[0112] In still further embodiments, the secondary structural element is a coil. , the coil contains between 2 and 20 residues and zero predicted inter-residue hydrogen bonds. In this embodiment, these coil parameters are determined using knowledge-based or molecular dynamics simulations. The ion exchange rate is determined by simulation and / or laboratory measurements.

[0113] In yet a further embodiment, the engineered polypeptide is derived from a CD25 reference target It contains one or more atoms or amino acids, and these atoms or amino acids have a secondary structure. In some embodiments, these atoms or amino acids may be involved in a biological response or In some embodiments, the atom or amino acid in the engineered polypeptide is The secondary structure motif vector is a vector of engineered polypeptides derived from the CD25 reference target. For some, the secondary structure motif vectors were greater than 0.25 compared to the CD25 reference target. The length of the vector is the number of secondary structure motifs, and the i-th The values ​​at the eye vector positions are derived from a lookup table of secondary structure motifs (e.g. In some embodiments, each motif defines the identity of a molecule (e.g., a helix, a sheet). In certain embodiments, the motif includes, for example, an α-helix. cus, β-bridge, β-strand, 3 10 Helix, π-helix, turn and In some embodiments, the cosine similarity is measured using a CD25 reference target. The portion of the engineered polypeptide that corresponds to the secondary structure motif vector of the CD25 reference target is Compared to Torr, greater than 0.3, greater than 0.35, greater than 0.4, 0 Greater than .45 or greater than 0.5. Cosine similarity is

[0114]

number

[0115] In some embodiments, one or more of the engineered polypeptides derived from the CD25 reference target The atoms or amino acids of the corresponding CDs are calculated using the total topological constraint distance (TCD). In some embodiments, the target can be compared to an atom or amino acid of a CD25 reference target. The total TCD of atoms or amino acids of these engineered polypeptides derived from the target is C The TCD distance is + / - 75% compared to the corresponding atom in the D25 reference target, and the two Intramolecular topological constraints are mutually interacting if their pairwise distance is less than 7 Å. In some embodiments, the atoms or amino acids in the engineered polypeptides being compared The amino acid is associated with a biological function or response. The jth pairwise distance may, in some embodiments, be calculated using molecular simulations (e.g., It can be determined by molecular dynamics) and / or laboratory measurements (e.g., NMR). An exemplary equation for calculating the topological constraint distance (TCD) is:

[0116]

number

[0117] In some embodiments, one or more of the engineered polypeptides derived from the CD25 reference target The atoms or amino acids of the corresponding C In some embodiments, these manipulations can be compared to atoms or amino acids of the D25 reference target. The CTP of an atom or amino acid in the engineered polypeptide is the same as the corresponding CTP in the CD25 reference target. + / - 50% of the atomic or amino acid CTP and intrachain topological interactions is a pairwise distance of 7 Å or less. The atoms or amino acids in a polypeptide are associated with a biological function or response. In some embodiments, the i-th and j-th pairwise distances are calculated using a molecular simulation (e.g., It can be determined by molecular dynamics (e.g., molecular dynamics) and / or laboratory measurements (e.g., NMR). An example equation for assessing CTP is:

[0118]

number

[0119] In some embodiments, one or more of the engineered polypeptides derived from the CD25 reference target The atoms or amino acids in the corresponding CD2 5 can be compared to a reference target atom or amino acid. In some embodiments, these engineered The QEL of an atom or amino acid in a selected polypeptide is the QEL of the corresponding atom in the CD25 reference target. or + / - 50% compared to the QEL of the amino acid. The atoms or amino acids in the engineered polypeptides that are An exemplary equation for determining the QEL is:

[0120]

number

[0121] In some embodiments, one or more of the engineered polypeptides derived from the CD25 reference target The atoms or amino acids are represented by a topological clustering coefficient (TCC) vector and a mean The mean percentage error (MPE) was used to determine the atomic or atomic ratio of the corresponding CD25 reference target. In some embodiments, the TCC vector and MPE may be compared to CD25 The TCC of the corresponding atom or amino acid in the reference target is less than 75% and the vector Each element (i) of the formula is the topological clustering coefficient for the i-th amino acid position. and an intramolecular cluster is defined as an interaction edge of 7 Å or less from the i-th amino acid position. The distance is defined by two edges: ij, jl. In some embodiments, the ratio The atoms or amino acids in the engineered polypeptides being compared have biological function or activity. In some embodiments, the i-th, j-th, and l-th edge distances are related to the physical response. are based on molecular simulations (e.g., molecular dynamics) and / or laboratory measurements (e.g., The topological clustering function for the i-th position can be determined by the NMR. An example equation for evaluating the number is:

[0122]

number

[0123] In yet a further embodiment, one of the engineered polypeptides derived from the CD25 reference target The number of atoms or amino acids is determined by the L × M topological constraint matrix and the number of amino acids in all M dimensions. Euclidean distance, power distance, Soergel distance, Canberra distance, Sorensen distance Average percentage error in distance, Jaccard distance, Mahalanobis distance or Hamming distance The difference (MPE) can be used to compare the corresponding atom or amino acid of the CD25 reference target. The L × M matrix element (l, m) contains the mth constraint value for the lth amino acid position. where L is the number of amino acid positions and M is the number of distinct topological constraints. In embodiments, the MPE of an L×M matrix of engineered polypeptides is calculated using the corresponding CD25 reference In some embodiments, M PE 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 are associated with biological functions or biological responses. III. Programmable in vitro selection

[0124] In other embodiments, a series of programmed selection steps are used to select binding partners. Methods of using the engineered polypeptides described herein are also described herein. At least one selection step is provided for the selection of an engineered pool of potential binding partners. and assessing the interaction with the polypeptide.

[0125] In some embodiments, the method involves using two or more selection molecules to steer the selection of binding molecules. In some embodiments, a method comprises: each of the pools includes subjecting the pool to a single round of selection, each round including at least one round of selection in which at least a portion of the pool At least one negative selection step in which the pool is screened against a negative selection molecule; At least one positive selection molecule is screened against at least a portion of the positive selection molecule. In some embodiments, the method includes at least two rounds of sexual selection. rounds, at least 4 rounds, at least 5 rounds, at least 6 rounds, At least 7 rounds, At least 8 rounds, At least 9 rounds, At least 10 rounds It involves 1 or more rounds, each of which includes at least one negative selection. In some embodiments, each lane comprises a selection step and at least one positive selection step. The round may involve more than one negative selection step or more than one positive selection step, or independently include combinations thereof. Figure 5 shows an exemplary selection process detailing three rounds of selection. A schematic diagram is provided, in which the first and third rounds involve more than one negative selection step. The first round further comprises more than one round of positive selection. As shown, two negative selection molecules ("baits") were used in the first round. In the third round, three negative selection molecules are used. In addition, two positive selection molecules are used. The offspring is used in the first round.

[0126] In some embodiments, the method includes more than one round. In other embodiments, the offspring are selected independently using the same negative selection molecule or the same positive selection molecule. The molecules, or combinations thereof, may be used in more than one round. For example, in Figure 5, the same negative selection molecule used in round 1 was used in round 3. A third negative selection molecule is also included in round 3. The order of the selection and positive selection steps may, in certain embodiments, be independent within each round of selection. Thus, for example, in some embodiments, the method may involve one or more rounds of selection. Each round includes a negative selection step first, followed by a positive selection step. In other embodiments, the method comprises one or more rounds of selection, each round consisting of an initially positive In yet a further embodiment, the method comprises a selection step followed by a negative selection step. The selection process involves more than one round of selection, with each round independently comprising a negative selection step and a positive selection step. In each round, the negative selection step may be performed independently before or after the positive selection step. This is after the process.

[0127] Such methods of selection may be directed towards a particular desired characteristic, e.g., binding specificity or binding affinity. To steer the library of candidate binding molecules towards or away from positive ( Use both positive (+) and negative (-) selection molecules. By using multiple steps, the pool of candidates can be enriched for desirable characteristics and for desired In addition, some implementations may be directed in a step-by-step fashion to select against undesirable features. In an embodiment, the order of each step within each round, and the order of the rounds relative to each other, is determined by the selection can be directed in different directions. Thus, for example, in some embodiments, A method involving one round of (-) selection followed by (-) selection is This will result in a different final pool of candidates than if the subsequent (+) selection were performed. From this, it can be inferred that for methods involving multiple rounds, the order of selection steps is Even when sexual selection molecules are used as a whole, different final pools of selected candidates This may result in:

[0128] In some embodiments, a selection molecule is used that has the inverse characteristics of another selection molecule, such as For example, the nucleotide sequence identified using a positive selection molecule (or eliminated due to a negative selection molecule) ) the candidate binding partners are present in the desired form rather than resulting from separate, unrelated binding interactions To ensure that the product has been identified (or eliminated) due to a quality (or undesirable trait). It may be useful to remove binding partners that are bound through unrelated interactions. To do this, the selected molecule is filtered out, excluding residues / structures that convey the desired trait (or undesired traits). Counterselection molecules having similar or identical structures and characteristics to the positive selection molecules can be used. If interaction with a particular charge pattern in a selected molecule is desired, the charge pattern can be provided. Reverse negative selection molecules in which the corresponding residue is replaced by an uncharged residue and / or a residue of the opposite charge. Thus, for a particular selection molecule, multiple different corresponding counterselections can be used. Molecules may be possible.

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

[0130] The positive and negative features selected for or against at each step are A variety of traits can be selected and tailored depending on the desired characteristics of the resulting final binding molecule or molecules. Such desired characteristics may depend, for example, on the intended use of one or more binding molecules. For example, in some embodiments, the methods provided herein include detecting one or more positive features, For example, for high specificity and for one or more negative characteristics, e.g., cross-reactivity. , used to screen antibody candidates. In one context, it is considered a positive feature. It should be understood that a positive trait can be a negative trait in another context, and vice versa. Thus, the positive selection molecules in one series of selection rounds are, in some embodiments, , in different series of selection rounds or when selecting different types of binding molecules, Or as a negative selection molecule to select for the same type of binding molecule but for a different purpose. could be.

[0131] In some embodiments, each selected feature is an amino acid sequence, a polypeptide secondary structure, a molecular dynamics chemistry, chemical features, biological function, immunogenicity, CD25 reference target(s) multispecificity, crossover Seed CD25 reference target reactivity, the desired CD25 reference over the undesired reference target(s) Selectivity of the target(s), sequence and / or structurally homologous reference targets within the family ( Selectivity of the target(s), selectivity of the reference target(s) with similar protein function, high sequence integrity Isolation from a larger family of unwanted targets with sequence and / or structural homology Selectivity of the desired reference target(s), for distinct reference target alleles or mutations Selectivity for residue-level chemical modifications of distinct reference targets, selectivity for cell types, Selectivity for the target, selectivity for the tissue type, selectivity for the tissue environment, structure of the reference target(s) tolerance for sequence diversity of the reference target(s), and tolerance to kinetic variability of the target(s). In some embodiments, each selection feature is a different type of selection feature. The above selection features are different features of the same type. For example, in some embodiments In the present invention, the two or more selection characteristics are polypeptide secondary structure, one of which is a desired polypeptide secondary structure. One is a positive selection for undesired polypeptide secondary structures, and the other is a positive selection for undesired polypeptide secondary structures. In some embodiments, the two or more selection characteristics are negative selections for a cell type. A positive selection characteristic is a preference for a particular desired cell type, and a negative selection characteristic is a preference for a particular desired cell type. , selectivity for a particular undesired cell type. In some embodiments, two or more, three or more At least four, at least five, or at least six selected features are of the same type.

[0132] In some embodiments, the selection characteristic is binding to an engineered polypeptide of the present disclosure. For example, the engineered polypeptides shown in Figure 7, Table 1, Table 8 and Table 9 can be used in conjunction with Figures 6 and Select for antibodies (or other binding agents) that specifically bind to the epitopes shown in Table 7. An example selection strategy is provided in Table 10.

[0133] In yet another embodiment, a composition comprising two or more selection steering polypeptides, wherein each polypeptide The peptides may be independently linked to a positive selection molecule containing one or more positive steering features or one or more negative selection features. Provided herein are compositions that are negative selection molecules that contain steering features. In some embodiments, amino acid sequences, polypeptide secondary structures, molecular dynamics, chemical features, biomolecules, and the like are used. biological function, immunogenicity, reference target(s) multispecificity, cross-species reference target reactivity, undesired Selectivity, sequence and / or Selectivity of reference target(s) within a structurally homologous family, with similar protein function Selective, high sequence and / or structural homology to the reference target(s) desired. Selectivity of distinct desired reference target(s) from a larger family of targets that are not distinct Selectivity for reference target alleles or mutations, at the residue level for distinct reference targets Selectivity to chemical modification, cell type selectivity, tissue type selectivity, tissue environment selectivity selectivity for, tolerance to structural diversity of the reference target(s), reference target(s) Tolerance for sequence diversity of the target(s), as well as tolerance for kinetic diversity of the reference target(s). The degree may be selected from the group consisting of:

[0134] Thus, in a further aspect, the selectivity steering compositions described herein are used to identify binding moieties. A method for screening a library of clones, wherein each round of selection selects a smaller number of clones from the pool. a negative selection step in which at least a portion of the pool is screened against the negative selection molecule; and a positive selection step of screening at least a portion of the The order of selection steps within a round, and the order of rounds, may differ from alternative orders. Methods are provided herein that result in the selection of a pool of candidates.

[0135] In some embodiments, the compositions of the selection steering polypeptides described herein or the The binding partners being evaluated using the screening methods described herein are a phage library, e.g., a Fab-containing phage library; or a cell library - for example, a B cell library or a T cell library.

[0136] In some embodiments of the screening methods provided herein, the method comprises two or more , including the selection of 3 or more, 4 or more, 5 or more, 6 or more or 7 or more rounds. 1 In some embodiments where there are more than two rounds, each round is a different set of In other embodiments where there are more than one round, at least two The two rounds contain the same negative selection molecule, the same positive selection molecule, or both.

[0137] In some embodiments of the screening method, before proceeding to the next round of selection, the method comprises: In certain embodiments, analyzing a subset of the pools. The analysis involves peptide / protein biosensor binding, peptide / protein ELISA , peptide library binding, cell extract binding, cell surface binding, cell activity assay, cells Proliferation assay, cell death assay, enzyme activity assay, gene expression profile, protein Independently selected from the group consisting of quality control assay, Western blot and immunohistochemistry. In some embodiments, the gene expression profile is determined by comparing the complete sequence levels of the subset pool. Partial analysis, for example, next generation sequencing. In some embodiments, statistical and / or or informatics scoring or machine learning training in one or more selection rounds are used to evaluate one or more subsets of the pool.

[0138] In some embodiments, the positive and / or negative selection of molecules for subsequent rounds is performed. The population and / or order can be analyzed by analyzing a subset pool from one selection round. In some embodiments, the method is determined by statistical and / or informatics scoring. , or machine learning training continues in a subsequent round (e.g., the next round, or program The identity and / or number of positively and / or negatively selected molecules for further rounds of selection To determine the order, one or more subsets of the pool are subjected to one or more selection rounds. Used to evaluate.

[0139] In still further embodiments, the method of selection comprises: Such modifications include, for example, modifying the subset pool obtained from the subset. Genetic mutation of the set pool, genetic depletion of the subset pool (e.g., proceed to selection) selecting a subset of a subset pool), genetic enrichment of the subset pool (e.g. (e.g., increasing the size of the pool), chemically modifying at least a portion of the subset pool modification, or enzymatic modification of at least a portion of the subset pool, or any of these. In some embodiments, statistical and / or informatics scores may be included in combination. Machine learning, or machine learning training, evaluates the subset pool and generates modified subset pools. It is used to determine one or more modifications to be made before proceeding with the selection. In embodiments of the present invention, such statistical and / or informatics scoring, or machine learning Training determines the identity and quality of positively and / or negatively selected molecules for subsequent rounds of selection. and / or used to determine the order.

[0140] Any suitable assay may be used to measure the binding of the pool of binding partners to the selected molecule at each step. In some embodiments, binding can be used to assess, for example, the binding partner. The activity of the antibody can be directly assessed by directly detecting the above-mentioned markers. Such markers include, for example, For example, fluorescent labels, such as fluorophores or fluorescent proteins, may be included. In some embodiments, binding is assessed indirectly, for example, using a sandwich assay. In a Deutsch assay, the binding partner binds to a molecule of choice and is then reacted with a labeled secondary reagent. A drug is added to label the bound binding partner. Examples of sandwich assay components include anti-His tag antibodies or H His-tagged binding partners detected using is-tag-specific fluorescent probes; Biotin-labeled proteins detected with streptavidin or labeled avidin or unlabeled binding partners that are detected using anti-binding partner antibodies Includes Na.

[0141] In some embodiments, the binding partners selected at each step may have several benefits. The antibodies are identified based on binding signals or dose responses using available detection methods. These detection methods include, for example, imaging, fluorescence activated cell sorting (FACS), mass spectrometry or In some embodiments, a hit threshold is defined (e.g., a medium median signal), anything with a signal above that signal is a putative hit. flagged as a flop. IV. Use of Engineered Polypeptides to Generate Antibodies

[0142] Engineered strains provided herein and identified by the methods provided herein The resulting polypeptide can be used, for example, to generate one or more antibodies. In some embodiments, the antibody may be a monoclonal or polyclonal antibody. In one embodiment, the antibody is produced by immunizing an animal with an immunogen, Antibodies whose origin is an engineered polypeptide provided herein are provided herein. In some embodiments, the animal is a human, rabbit, mouse, hamster, monkey, or the like. In certain embodiments, the monkey is a cynomolgus monkey, a macaque monkey, or a rhesus monkey. Immunizing an animal with an engineered polypeptide, for example, contains a peptide and is suitably administered as an adjuvant. The method may include administering to the animal at least one dose of a composition comprising the compound. In embodiments, generating antibodies from an animal includes isolating antibody-expressing B cells. Some embodiments include transfecting B cells into a mammalian host to create hybridomas that express antibodies. In some embodiments, the engineered polypeptide further comprises fusing the engineered polypeptide with a myeloma cell. Antibodies generated using the method can cross-react with humans and monkeys, e.g., cynomolgus monkeys. . a. Engineered Polypeptide Characteristics

[0143] The engineered polypeptides provided herein share one or more characteristics with CD25. In some embodiments, these have at least one feature on the surface of CD25, e.g. For example, they exhibit 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. The engineered polypeptides are CD25 polypeptides that are not known to interact with antibodies against CD25. 25 shows at least one feature of a portion of the surface.

[0144] In some embodiments of a particular type of feature, the engineered polypeptide inhibits the functional activity of CD25. presenting a mimic of an interface (e.g., a binding surface) shared by the engineered polypeptides. The characteristics shared by CD25 as a whole can best be described as being shared with CD25. One characteristic to be examined may be the binding between a binding partner of CD25 and CD25, and this Binding occurs with the functional binding interface of CD25, but the structure and orientation of the functional binding interface is supported by the remainder of the CD25 protein.

[0145] Such shared characteristics may include, for example, structural or functional metrics, or Combinations thereof may be included. At least one shared characteristic may include, for example, one The structural similarity, conformational entropy similarity, and one or more chemical Similarity of one or more functional connections, or similarity of one or more phenotypic features, or or any combination thereof. In certain embodiments, engineered poly The peptides may be attached to at least a portion of the surface of CD25, e.g., a functional interface, e.g., a binding surface. , share one or more of these characteristics.

[0146] In some embodiments, the engineered polypeptide is a polypeptide that encodes CD25 (or a polypeptide on the surface of CD25). The structural similarity is determined by the structure of the backbone average. The RMSD is evaluated by the root mean square deviation (RMSD) or side chain RMSD. Evaluate the average distance and compare how two similar, separate structures are located in 3D space In some embodiments, engineered polypeptides may be applied to three-dimensional structures to and CD25 (or the functional interface of CD25) The RMSD of both of them is the difference between CD25 (or the functional interface of CD25) and different molecules. In some embodiments, it is lower than the RMSD between the engineered polypeptide. The RMSD is the portion of CD25 (or a portion of the functional interface of CD25) that is expressed as: For example, experimentally determined or simulated structures of engineered polypeptides. and experimentally determined or simulated structures of CD25 (or its functional interface). In some embodiments, the engineered polypeptide may be evaluated using a modified structure. reported that the backbone of the engineered polypeptide is 6.0 Å larger than that of the CD25 x-ray structure. A molecule is considered structurally similar to CD25 if it has an average RMSD of:

[0147] In some embodiments, the engineered polypeptide is a polypeptide that encodes CD25 (or a polypeptide on the surface of CD25). a conformational entropy similar to that of a portion of the molecule (e.g., a binding surface), and Conformational entropy can be measured experimentally, for example, for engineered polypeptides. The experimentally measured structures of CD25 (or parts thereof) The structure is evaluated using a determined structure or molecular dynamics simulated motion. In some embodiments, the simulation involves the use of CD25 (or a portion thereof, e.g., a binding site) The experimentally measured structure or molecular dynamics simulated motion of a part of the surface is used. In certain embodiments, the conformational endogenous structure of the engineered polypeptide is Tropism is a molecular dynamics analysis of engineered polypeptides performed under standard physiological conditions. The sample was compared to the known x-ray crystal structure of CD25 (or parts thereof) to determine all non-aqueous If the elementary atom positions have all states with RMSD ≦ 6.0 Å, then CD25 (or its (part) is considered similar to that of

[0148] In yet other embodiments, the engineered polypeptide is CD25 (or a portion thereof, e.g., In other embodiments, the engineered surface has one or more similar chemical descriptors (e.g., a binding surface). The polypeptide binds to a binding partner of CD25 (e.g., an antibody against CD25). and one or more chemical descriptors that are similar or complementary to each other. Possible patterns include, for example, hydrophobicity patterns, H-bond patterns, atomic volumes / radii, charge patterns, etc. These may include a morphology or an atom occupation pattern, or any combination thereof. Chemical descriptors, in some embodiments, are experimentally determined structural features of engineered polypeptides. a constructed or simulated structure and CD25 (or a portion thereof, e.g., a binding surface) can be evaluated using experimentally measured or simulated structures.

[0149] In yet other embodiments, the engineered polypeptide has a similar functional binding to CD25. For example, in some embodiments, the engineered polypeptide is a CD25 binding partner. In some embodiments, the binding partner has binding to a native The antibody may be a fragment of the native binding partner or an engineered native binding partner. Such modifications include, for example, fusion proteins containing at least a fragment of the native binding partner. Labeling with chromophores; Labeling with fluorophores; Labeling with biotin; In some embodiments, the engineered polypeptide may be labeled with a His tag. The peptide is within about two orders of magnitude or within about one order of magnitude of binding of CD25 to its binding partner. , and has binding to a binding partner of CD25. In some embodiments, the similarity of binding is Binding constant (Kd), or inhibition constant (Ki), or on-rate of binding or the off-rate of the bond, or the binding affinity of the binding pair, or the binding In some embodiments, the Gibbs free energy (ΔG) of the In this example, the binding partner is an antibody against CD25.

[0150] In some embodiments, the binding constant of the engineered polypeptide with a CD25 binding partner (K d ) is the K between CD25 and its binding partner d Within 1000 times, within 800 times, 60 Within 0x, Within 400x, Within 200x, Within 100x, Within 90x, Within 80x, Within 70 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 In another embodiment, the engineered polypeptide is linked to a CD25 binding partner. Inhibition constant (K i ) is the K between CD25 and its binding partner i Within 1000 times, 800 times or more Within, 600x or less, 400x or less, 200x or less, 100x or less, 90x or less, 80x or more Within 70 times, within 60 times, within 50 times, within 40 times, within 30 times, within 20 times, 10 Within 8 times, within 6 times, within 4 times, within 2 times, within 1.5 times, within 1.2 times or In yet a further embodiment, the CD25 binding of the engineered polypeptide is The on-rate of binding to the binding partner is In some embodiments, the on-rate of binding is similar to that of the engineered polynucleotide. The on-rate of binding of a peptide to its CD25 binding partner is The binding rate of the binding partner is within 1000, 800, and 6 times Within 00 times, within 400 times, within 200 times, within 100 times, within 90 times, within 80 times, Within 0x, within 60x, within 50x, within 40x, within 30x, within 20x, within 10x , within 8 times, within 6 times, within 4 times, within 2 times, within 1.5 times, within 1.2 times or In another embodiment, the CD25 binding partner of the engineered polypeptide is The off-rate of the binding of CD25 to its binding partner is In some embodiments, the off-rate of the engineered polypeptide is similar. The off-rate of CD25 binding to its binding partner is Within 1000 times, 800 times, or 600 times the off-rate with the toner Within, 400 times or less, 200 times or less, 100 times or less, 90 times or less, 80 times or less, 70 times or more Within 60 times, within 50 times, within 40 times, within 30 times, within 20 times, within 10 times, within 8 times Within, within 6 times, within 4 times, within 2 times, within 1.5 times, within 1.2 times or approximately the same In yet a further embodiment, the engineered polypeptide and the CD25 binding partner The binding affinity between CD25 and the binding partner is similar to the binding affinity between CD25 and the binding partner. In this embodiment, the binding affinity of the engineered polypeptide to the CD25 binding partner is The binding affinity of 5 to the binding partner is within 1000-fold, 800-fold, 600-fold, and 4 Within 00 times, within 200 times, within 100 times, within 90 times, within 80 times, within 70 times, 60 Within 50 times, within 40 times, within 30 times, within 20 times, within 10 times, within 8 times, 6 times Within 100 times, 4 times, 2 times, 1.5 times, 1.2 times, or approximately the same. In some embodiments, the binding of the engineered polypeptide to a CD25 binding partner is The Gibbs free energy of binding between CD25 and a binding partner is 10 times the Gibbs free energy. Within 00x, within 800x, within 600x, within 400x, within 200x, within 100x , within 90 times, within 80 times, within 70 times, within 60 times, within 50 times, within 40 times, within 30 times Within, 20 times or less, 10 times or less, 8 times or less, 6 times or less, 4 times or less, 2 times or less, 1.5 times or more In some embodiments, the CD25-binding domain is within 1.2-fold or about the same. The antibody is an antibody against CD25.

[0151] In yet other embodiments, the engineered polypeptide is CD25 or a portion thereof (e.g., For example, it shares sequence similarity with CD25 (or a portion thereof). can be compared to a continuous amino acid sequence of CD25 (or a portion thereof) or a non-contiguous sequence of CD25 (or a portion thereof). For example, in certain embodiments, the binding surface of CD25 is defined by a non-contiguous amino acid sequence. The engineered polypeptides are formed by at least a portion of the non-contiguous sequences that form the surface. In another embodiment, the engineered polypeptide has sequence similarity to the CD25 binding polypeptide. have sequence similarity with at least a portion of the contiguous amino acid sequence forming the binding surface. In embodiments, the binding surface of CD25 contains an epitope that binds to an antibody to CD25. nothing.

[0152] In some embodiments, the engineered polypeptide comprises a continuous sequence of CD25, e.g., At least 40% identical to a portion of the contiguous sequence forming the binding surface of 25, 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% have sequences that are identical, at least 85% identical, or at least 90% identical. In embodiments, the engineered polypeptide comprises a non-contiguous sequence of CD25, e.g., CD25 At least 40% identical to a portion of the non-contiguous sequence that forms the binding surface of 5% identical, at least 50% identical, at least 55% identical, at least 60% identical, at least At least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical 1. Having a sequence that is at least 85% identical or at least 90% identical. In embodiments, the engineered polypeptide is directed against a contiguous portion of the binding surface of CD25. 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% In yet a further embodiment, the engineered polypeptide has a sequence that is identical to that of the CD At least 40% identical to two or more non-contiguous portions of the 25 binding surface, 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% Some embodiments have sequences that are identical, at least 85% identical, or at least 90% identical. In embodiments, the engineered polypeptide has a binding surface at least partially identical to that of CD25. and the binding surface has one or more sequences for CD25. The antibody comprises an epitope that binds to the antibody.

[0153] In certain embodiments, the engineered polypeptide and CD25 (or a portion thereof) The sequence similarity, if any, is determined by the peptide portion of the engineered polypeptide without the linker. In certain embodiments, one or more linking moieties are evaluated using For example, the engineered polypeptide may include one or more linkers comprising amino acids. It is considered to be the same. b. Engineered polypeptide

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

[0155] In some embodiments, the engineered polypeptides are between 2 and 100 amino acids, e.g., For example, between 2 and 80 amino acids, between 2 and 70 amino acids, between 2 and 60 amino acids , between 2 and 50 amino acids, between 2 and 40 amino acids, between 2 and 30 amino acids, Between 2 and 25 amino acids, between 2 and 20 amino acids, between 2 and 15 amino acids, 5 ~30 amino acids, 5-25 amino acids, 5-20 amino acids, 5- It contains between 15 amino acids or between 9 and 15 amino acids.

[0156] In certain embodiments, the engineered polypeptide comprises more than one peptide, e.g. For example, at least two peptides, or at least three peptides, or at least Contains 4 or more peptides, each peptide containing 1 to 100 or between 2 and 100 amino acids, for example, between 2 and 80 amino acids. Acids, between 2 and 70 amino acids, between 2 and 60 amino acids, between 2 and 50 amino acids , between 2 and 40 amino acids, between 2 and 30 amino acids, between 2 and 25 amino acids, Between 2 and 20 amino acids, between 2 and 15 amino acids, between 5 and 30 amino acids, 5 between 25 amino acids, between 5 and 20 amino acids, between 5 and 15 amino acids, or Independently containing between 9 and 15 amino acids.

[0157] In some embodiments, the engineered polypeptide contains only naturally occurring amino acids. In other embodiments, the engineered polypeptides may contain unnatural amino acids, e.g., naturally occurring amino acids. This includes combinations with unnatural amino acids.

[0158] In some embodiments, where the engineered polypeptide comprises two or more peptides, each peptide independently exhibit at least one characteristic of CD25 or a portion thereof (e.g., the binding surface). In some embodiments, each peptide comprises 1 to 10, 100, or 1000 of CD25 or a portion thereof. ~9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1, or two characteristics independently. In some embodiments, the characteristics are correlated with antibodies to CD25. It is shared with the functional portion of CD25.

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

[0160] In some embodiments, the peptide of the engineered polypeptide is CD25 or a portion thereof. In some embodiments, the structural similarity may be a backbone similarity. The RMSD can be evaluated by side chain RMSD or side chain RMSD. For example, in certain embodiments, The scaffolding between the peptide of the engineered polypeptide and CD25 (or a portion thereof) The RMSD of the amino acid side chains or both differs from that of CD25 (or a portion thereof). In some embodiments, the RMSD is lower than the RMSD between the corresponding molecules (e.g., different peptides). A portion of CD25 may be a peptide, e.g., a portion of the surface of CD25, e.g., The RMSD of the structural similarity is compared to the surface that interacts with the corresponding antibody. The experimentally determined or simulated structure of CD25 or its derivatives can be evaluated using experimentally measured or simulated structures of the part. In some embodiments, the peptide of the engineered polypeptide has a peptide backbone that is a CD 25 or a portion thereof, compared to the backbone of the known x-ray structure, with an average RMSD of 6.0 Å or less. If so, it is considered structurally similar to CD25 (or a portion thereof).

[0161] In some embodiments, the engineered polypeptide is a polypeptide similar to CD25 or a portion thereof. In some embodiments, experimentally determined conformational entropy of a peptide The measured structure or molecular dynamics simulated motion is used to determine the conformational ent The tropy was determined by the experimentally determined or simulated structure of CD25 or a portion thereof. Conformational entropy is used to compare the conformational entropy of a molecule to the structure of the molecule. In this state, peptide molecular dynamics ensembles run under standard physiological conditions showed that CD2 Compared with the known x-ray crystal structure of 5 or a portion thereof, all non-hydrogen atom moieties have an RMSD of ≦ 6.0 Å are considered similar. The portion of CD25 that interacts with a peptide, e.g., an antibody to CD25, is a surface portion of CD25. is compared to.

[0162] In a further embodiment, the engineered polypeptide peptide and CD25 (or a portion thereof) The similarity between the peptides (peptides) can be one or more chemical descriptors. The CD25 peptide has one or more chemical descriptors in common with CD25 (or a portion thereof) or CD2 one or more chemicals complementary to the binding partner of 5 (e.g., an antibody to CD25) Chemical descriptors include, for example, hydrophobicity patterns, H-bonding patterns, atom volume / radius, charge pattern or atom occupancy pattern, or any combination thereof In some embodiments, the peptide of the engineered polypeptide may be a peptide of CD25 or or a portion thereof, or a binding partner of CD25 (e.g., an antibody to CD25) one or more hydrophobic patterns, H-bonding patterns, atomic volumes / radii, and electrical potentials that are complementary to the The atom occupancy pattern or atom occupancy pattern, or any combination thereof. In this embodiment, similarity is measured by the same chemical descriptors, e.g., the same hydrophobicity pattern, H-bond pattern, etc. The nuclei have one or more of the following in common: turn, atomic volume / radius, charge pattern, or atomic occupancy pattern. Complementary chemical descriptors include, for example, binding partners of CD25, e.g., Peptides with a positive charge pattern that complements the negative charge pattern of antibodies to CD25 These chemical descriptors, in some embodiments, include experimentally determined The constructed or simulated structures and CD25 or CD25 binding partners Experimentally measured or simulated structures (e.g., for complementary evaluation) can be evaluated using

[0163] For example, in some embodiments, the engineered polypeptide is a binding partner for CD25. (e.g., IL-2) binds to a binding partner of CD25. In this embodiment, the binding partner is a native binding partner, a native binding part a fragment of a native binding partner, or an engineered native binding partner or fragment thereof, or a CD In some embodiments, the binding partner is an antibody that specifically binds to 25. In some embodiments, the binding partner binds to the target protein under certain circumstances but not under other circumstances. , bind under pathological conditions or bind under non-pathological conditions. For example, it may be constitutively expressed or may be the product of a serendipitous gene, or proteins or fragments thereof. The binding partner may be a fragment of the native binding partner or an engineered native binding partner. The modifications may, in some embodiments, involve reducing the fragment size of the native binding partner. Fusion proteins containing chromophores; labeling with fluorophores; biolabeling or labeling with a His tag.

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

[0165] In some embodiments, the binding constant of the engineered polypeptide with a CD25 binding partner (K d ) is the K d Within 1000 times, within 800 times, 60 Within 0x, Within 400x, Within 200x, Within 100x, Within 90x, Within 80x, Within 70 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 In another embodiment, the engineered polypeptide and the CD25 binding partner are the same. Inhibition constant (K i ) is the K between CD25 and its binding partner i Within 1000 times, 800 times or more Within, 600x or less, 400x or less, 200x or less, 100x or less, 90x or less, 80x or more Within 70 times, within 60 times, within 50 times, within 40 times, within 30 times, within 20 times, 10 Within 8 times, within 6 times, within 4 times, within 2 times, within 1.5 times, within 1.2 times or In yet a further embodiment, the CD25 binding of the engineered polypeptide is The on-rate of binding to the binding partner is In some embodiments, the on-rate of binding is similar to that of the engineered polynucleotide. The on-rate of binding of a peptide to its CD25 binding partner is The binding rate of the binding partner is within 1000, 800, and 6 times Within 00 times, within 400 times, within 200 times, within 100 times, within 90 times, within 80 times, Within 0x, within 60x, within 50x, within 40x, within 30x, within 20x, within 10x , within 8 times, within 6 times, within 4 times, within 2 times, within 1.5 times, within 1.2 times or In another embodiment, the CD25 binding partner of the engineered polypeptide is The off-rate of the binding of CD25 to its binding partner is In some embodiments, the off-rate of the engineered polypeptide is similar. The off-rate of CD25 binding to its binding partner is Within 1000 times, 800 times, or 600 times the off-rate with the toner Within, 400x or less, 200x or less, 100x or less, 90x or less, 80x or more, 70x or more Within 60 times, within 50 times, within 40 times, within 30 times, within 20 times, within 10 times, within 8 times Within, within 6 times, within 4 times, within 2 times, within 1.5 times, within 1.2 times or approximately the same In yet a further embodiment, the engineered polypeptide and the CD25 binding partner The binding affinity between CD25 and the binding partner is similar to the binding affinity between CD25 and the binding partner. In this embodiment, the binding affinity of the engineered polypeptide to the CD25 binding partner is The binding affinity of 5 to the binding partner is within 1000-fold, 800-fold, 600-fold, and 4 Within 00 times, within 200 times, within 100 times, within 90 times, within 80 times, within 70 times, 60 Within 50 times, within 40 times, within 30 times, within 20 times, within 10 times, within 8 times, 6 times Within 100 times, 4 times, 2 times, 1.5 times, 1.2 times, or approximately the same. In some embodiments, the binding of the engineered polypeptide to a CD25 binding partner is The Gibbs free energy of binding between CD25 and a binding partner is 10 times the Gibbs free energy. Within 00x, within 800x, within 600x, within 400x, within 200x, within 100x , within 90 times, within 80 times, within 70 times, within 60 times, within 50 times, within 40 times, within 30 times Within, 20 times or less, 10 times or less, 8 times or less, 6 times or less, 4 times or less, 2 times or less, 1.5 times or more In some embodiments, the CD25-binding domain is within 1.2-fold or about the same. The antibody is an antibody against CD25.

[0166] In some embodiments, the engineered polypeptide has a sequence similar to that of CD25 or a portion thereof. In some embodiments, the engineered polypeptide binds to an antibody to CD25. In certain embodiments, the sequence The similarity is compared to the contiguous amino acid sequence of CD25. In other embodiments, the sequence similarity is and compared to non-contiguous sequences of CD25. For example, in certain embodiments, fold-in sequences are The binding surface of engineered CD25 is formed by a non-contiguous amino acid sequence. The polypeptides have sequence similarity to at least a portion of the non-contiguous sequences that form the surface. In some embodiments, the engineered polypeptide comprises a contiguous array of polypeptides that form the binding surface of CD25. In some embodiments, the engineered amino acid sequence has sequence similarity to at least a portion of the amino acid sequence. The polypeptide may comprise at least a contiguous sequence of CD25, e.g., at least a contiguous sequence that forms a binding surface. At least 50% identical, at least 55% identical, at least 60% identical to any part 1. At least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical or have sequences that are at least 99% identical. In certain embodiments, the engineered polynucleotides The peptide may be a non-contiguous sequence of CD25, e.g., at least one of the non-contiguous sequences that form the binding surface. At least 40% identical, at least 45% identical, at least 50% identical to a portion , at least 55% identical, at least 60% identical, at least 65% identical, at least 7 0% identical, at least 75% identical, at least 80% identical, at least 85% identical, or In certain embodiments, the engineered polypeptide has a sequence that is at least 90% identical. The peptide is at least 40% identical to a contiguous portion of CD25, at least 45% identical to a contiguous portion of CD25. 1. 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, In still further embodiments, the sequence of the nucleotide sequence of the present invention is at least 85% identical or at least 90% identical. In some embodiments, the engineered polypeptide may be at least partially engineered to two or more non-contiguous portions of CD25. 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 are also 75% identical, at least 80% identical, at least 85% identical or at least 90% identical In some embodiments, the engineered peptide comprises at least two peptides. For a given polypeptide, two or more peptides of an engineered immunogen The domains independently share sequence similarity with CD25, e.g., the binding surface of CD25. In one embodiment, the portion of CD25 that shares sequence similarity with the engineered polypeptide is C This is a surface that binds antibodies against D25. c. Connecting part

[0167] The engineered polypeptides provided herein may include a linking moiety. In this case, the linking moieties can be, for example, independently, a bridge or a linker.

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

[0169] In some embodiments, the engineered polypeptide comprises at least one linking moiety, at least At least two linking parts, at least three linking parts, at least four linking parts, at least At least 5 connecting parts, at least 6 connecting parts, between 1 and 6 connecting parts, between 1 and 5 connecting parts Connection part between, connection part between 1 and 4, connection part between 1 and 3, 1 connection part In some embodiments, each linking moiety independently comprises a cross-linking moiety. or a linker. In certain embodiments, each linking moiety is a bridge. In yet a further embodiment, at least one linking moiety is a linker. is a bridge, and the remaining linking moieties are, independently, bridges or linkers. In this embodiment, at least one linking moiety is a linker and the remaining linking moieties are independently and a bridge or linker.

[0170] Cross-linking involves, for example, a covalent bond between the side chain of one amino acid and a portion of another amino acid. The amino acids may independently be natural or unnatural amino acids. So, a crosslink involves a bond between the side chains of two amino acids, or between the side chain of one amino acid and another amino acid. The crosslinking involves covalent bonding between the amine or carboxyl groups of one peptide. In some embodiments, the peptides may be formed within a peptide chain or between two separate peptides. The engineered polypeptides provided herein are characterized by intra- and inter-peptide crosslinks. In some embodiments, the crosslinking is between two thiol groups on the amino acid side chains. disulfide bonds between two cysteines, for example. In embodiments, the bridge is an amide bond between the amine and carboxylic acid groups of two amino acids. and at least one of the amine group and the carboxylic acid group is positioned on the side chain of the amino acid. (e.g., amide bonds are not backbone amide bonds). is the amide bond formed between diaminopimelic acid and aspartic acid. In embodiments, the amide bridge is a lactam. In some embodiments, the bridge is an oxime In some embodiments, the bridge is a hydrazone. In some embodiments, the bridge is , which includes a covalent bond between the side chain of one amino acid and a moiety of another amino acid, One or both may be modified to form a covalent bond. Such modifications include, for example, acid These modifications may include oxidation, reduction, reaction with a catalyst to form an intermediate, or other modifications known to those skilled in the art. .

[0171] The linker may be, for example, a molecule or a peptide covalently attached to at least two sites of the peptide. A linker comprises a molecule covalently linked between at least two peptides. It can bind to two sites within a peptide or between two separate peptides. It may be a combination of both, e.g., a peptide containing more than two peptide binding sites. The linker can form both intrapeptide and interpeptide bonds. In an engineered polypeptide comprising one peptide and at least one linker, the peptide The peptide and linker can be connected in a variety of different configurations. For example, engineered polynucleotides The peptides may have a pattern such as peptide-linker-peptide, ending in a peptide. In some embodiments, the engineered polypeptide may include a linker that forms a branch point, e.g., For example, the engineered peptide may comprise a linker that is independently attached to three peptides. The polypeptide comprises a linker having three peptide binding sites, the linker having two Only one peptide is bound.

[0172] In some embodiments, the linker comprises one or more amino acids forming part of the linker. The amino acids that contribute to the synthesis of the polypeptide can, in some embodiments, be identified separately from the engineered polypeptide. In certain embodiments, the linker is a functional interface of the interface protein. engineered polypeptides in structural, chemical and / or kinetic patterns that reflect their function or function. In yet a further embodiment, the linker has no function by itself when not connected to a peptide in an engineered polypeptide. In some embodiments, each linker The anchors are at least one, at least two, at least three, at least four, Each independently contains at least five, six, or more amino acids. In the form, each linker may be one amino acid, two amino acids, three amino acids, four amino acids, or amino acid, five amino acids or six amino acids, independently. The amino acids may, in some embodiments, be naturally occurring or non-naturally occurring amino acids. Each linker may in some embodiments be one or more alpha-amino acids. , one or more beta-amino acids or one or more gamma-amino acids, or any of these. In certain embodiments, the linker may independently comprise any combination of cyclic beta- The cyclic beta residues can include, for example, APC or ACPC. In still further embodiments, the linker comprises one or more glycine residues, one or more serine residues, In some embodiments, the linker may comprise an AP residue or one or more proline residues. ,GP,GSG,(GGGGS)n,(GSG)n,GGGSGGGS,GGGGSG GGS, (PGSG)n and PGSGSG. wherein n is an integer between 1 and 10. In some embodiments, the engineered polypeptide The peptides include at least one linker, and each linker contains either no amino acids or or each linker does not contain a natural amino acid, or each linker contains at least one Contains unnatural amino acids.

[0173] In some embodiments, the linker comprises a polymer. In some embodiments, the polymer , polyethylene glycol (PEG). Linkers containing PEG include, for example, at least 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 monomers units, at least 10 PEG monomer units, at least 11 PEG monomer units , at least 12 PEG monomer units or more than 12 PEG monomer units In some embodiments of the linker comprising PEG, the PEG may comprise between 3 and 12 amino acids. monomer units, between 3 and 6 monomer units, between 6 and 12 monomer units or comprises between 4 and 8 monomer units. In some embodiments, the engineered polypeptide The PEG-12 copolymer contains at least 100% PEG-3 (containing 3 monomer units), PEG6, or PEG12. In some embodiments, at least one linker independently comprises , PEG3, PEG6 or PEG12. In a further embodiment, the linker is For example, in certain embodiments, at least one linker independently comprise a 4-arm PEG or an 8-arm PEG. In certain embodiments, each The arms may be between 3 and 12 monomer units, or between 3 and 6 monomer units, or or between 6 and 12 monomer units, or between 4 and 8 monomer units, independently In certain embodiments, each arm of the multi-arm PEG comprises a 4-arm or an 8-arm. Each arm contains the same number of monomer units, such as PEG, 3 monomer units, 6 or 12 monomer units.

[0174] In other embodiments, the linker comprises a dendrimer. Dendrimers include, for example, Molecules with a tree-like branched architecture containing a symmetric core from which the subunits radiate outward Each new branch point creates a new layer in the molecule. These radial extensions terminate with functional groups at the outer terminal surfaces of the dendrimers. Therefore, increasing the number of branching points will in turn increase the number of terminals on the surface. This amplifies the number of possible end functional groups.

[0175] In some embodiments, at least one linker is a small molecule that is not an amino acid or a polymer. In some embodiments, at least one linker comprises a benzodiazepine. In some embodiments, the linker comprises a pyrrolidinedione moiety (e.g., pyrrolidine-2 a moiety that is the product of a sulfhydryl-maleimide reaction, which may be a 5-dione moiety; In some embodiments, the linker comprises an amidine moiety. The anchor contains a thioether moiety.

[0176] In some embodiments, at least one linker is trans-pyrrolidine-3,4- Contains dicarboxamides.

[0177] In some embodiments, the engineered polypeptide comprises at least two linkers (e.g., For example, the engineered polypeptide comprises at least two linking moieties, each linking moiety independently and each linking moiety is independently a linker. In some embodiments, each linker is independently any of the linkers described herein. For example, in some embodiments, each linker independently comprises a linker comprising one or more amino acids. linkers, polymer-containing linkers, dendrimer-containing linkers, or amino acid-based poly It is a linker that contains a small molecule that is not a mer.

[0178] One or more linking moieties of the engineered polypeptide may be linked to a particular structural or functional group of interest. For example, in some embodiments, a linking moiety may be provided. is engineered to impart structural or functional characteristics, or a combination thereof. Such structural features include, for example, increased structural flexibility. reduced structural flexibility, directional features, increased The directional features that may be targeted include, for example, increased or decreased length. Functional characteristics may include, for example, enhanced structural turn, or maintenance of linear structure. 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 may be included. In this embodiment, the linker may comprise one or more functional features or one or more structural features, or This includes combinations of the above.

[0179] In some embodiments, one or more linkers may be used to provide a structural "tag" in the engineered polypeptide. Examples of such linkers include Gly-Pro, Ala-Pro, and trans-pyrrolidine-3,4-dicarboxamide. In some embodiments, The one or more linkers present in the engineered polypeptide may be Increased structural flexibility of the engineered polypeptide compared to the same or different linker selection. For example, a linker that is longer and / or less sterically hindered than another linker may be added. In some embodiments, the linker may be replaced by a linker that was not present or is replaced by another linker. This can result in molecules with greater structural flexibility than if they were used in combination. , e.g., a linker that is shorter and / or more sterically hindered than another linker; or one or more crosslinks at positions or types that reduce the flexibility of the peptide. The linking moieties independently reduce the structural flexibility in the engineered polypeptide. The presence of crosslinks at specific positions between certain peptides or between certain amino acid side chains indicates that the crosslinks If they are in different positions or between different side chains (e.g., disulfides or amide bridge) or a molecule with less structural flexibility than if the bridge were not present. This can occur. d. Further ingredients

[0180] In some embodiments, the engineered polypeptides provided herein comprise one or more further For example, in some embodiments, the engineered polypeptide comprises an engineered One or more moieties that bind the resulting polypeptide to a solid surface, e.g., a bead or a flat surface. In some embodiments, the binding moiety comprises a polymer (e.g., PEG) or a biomolecule. In some embodiments, the engineered polypeptide comprises a polypeptide sequence selected from the group consisting of a nucleotide sequence, ... Binding the engineered polypeptide to a solid surface can be used, for example, to assess one or more characteristics of the engineered polypeptide. valency, e.g., assessment of binding to a binding partner of CD25 (e.g., an antibody against CD25) This may enable the value to be e. sequence similarity

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

[0182] [Table 4] The sequence has one of the sequences listed in

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

[0184] In some embodiments, the engineered polypeptide comprises any one of SEQ ID NOs: 1-21. modified at the N-terminus or C-terminus or both. In some embodiments, the C-terminus or N-terminus is covalently linked to another molecule. The engineered polypeptide may comprise any one of SEQ ID NOs: 1 to 21; and an N-terminal or includes one or more amino acids at the C-terminus or both.

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

[0186] [ka] is.

[0187] In some embodiments, the C-terminal molecule comprises a linker (e.g., -GSG) followed by biotin. SGK-biotin) by attaching biotin to the C-terminus of the engineered polypeptide. Other suitable linkers include GSG, GSS, GGS, GGSGGS, GSSGSS, G These include SGK, GSSK, GGSK, GGSGGSK, GSSGSSK, etc. V. Methods for Selecting Engineered Polypeptides

[0188] Methods for selecting engineered polypeptides as described herein are further provided herein. Such methods include, for example, iterative optimization of structural features of an engineered polypeptide. It may include using.

[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) is CD2 Thus, for example, in some embodiments, the antibody binds to one or more antibodies. In another embodiment, a portion of CD25 that is an epitope associated with the target interface is identified. The CD25 section either does not bind to antibodies or it is unknown whether antibody binding occurs. In certain embodiments, at least a portion of CD25 is identified as a target interface. The crystal structure of the molecule is unknown, and the initial selection of the target interface is based on CD25 and CD25 bonds. In some embodiments, the method includes performing molecular dynamics simulations of one or more initial inputs. A set of sequences is obtained from the identified section(s), and each sequence is independently In developing engineered polypeptide candidates, the interface residues of each sequence are considered. At least a portion of the groups are retained and one or more linking moieties are provided to provide the desired structural and dynamic characteristics. In some embodiments, the structure and structure of the cognate target are incorporated into the sequence to provide a signature. one or more non-interfaces to achieve the desired structural and dynamic characteristics compared to the dynamics Residues are added to the sequence or one or more residues in the input sequence are added to one or more non-interface regions. In some embodiments, these non-interface residues are replaced with residues that are specific to the target domain of CD25. It is not derived from or does not share one or more characteristics with the target interface of CD25. The residues shared fewer characteristics than the interfacial residues retained by CD25 and / or were not identical to the CD25 target. These intermediate non-interface residues share less strong characteristics with the primary interface. may form part or all of the amino acid linker.

[0190] Next, in some embodiments, an initial design (or designs) is generated, allowing for flexibility in the design. and molecular dynamics simulations to determine overall stability. This initial design is then used in R If the MSD requirements are not met, this may, in some embodiments, be mediated by computational mutagenesis. Derivatization is used to create one or more linking moieties (e.g., one or more bridges or intermediate linker residues). During this optimization, in some embodiments, the interface residues are fixed. However, one or more of the linking moieties may be changed, removed, or added. Iterative optimization evaluates the quality of the engineered polypeptide compared to the target interface and structural order metrics. The RMSD of the interface residue positions meets certain requirements (e.g., ≤ 6.0 Å and ≥ 0.2 Å, respectively). 5. Here, structural order is a normalized scale of 0 to 1, with 1 = perfect structural stability. This can be repeated until the following condition is met:

[0191] In some embodiments, the intermediate structural stability region ranges from 1 to 50 amino acids in length. In certain embodiments, these intermediate structurally stable residue regions may be linked to a linker, For example, an amino acid linker. The relatively small size (e.g., (compared to approaches that graft interfaces onto large, structurally stabilizing scaffolds) The molecule can be chemically synthesized, as opposed to larger molecules that may require in vitro expression systems. Additionally, in some embodiments, the methods provided herein may include the step of or interface positions, which allows for the incorporation of unnatural amino acids into novel moieties and specific properties, such as post-translational modifications, solubility, cell permeability, enzymatic reactivity, pH sensitivity, and oxidation sensitivity. In still further embodiments, the manipulated The polypeptides may be engineered to be used as immunogens or epitope baits. species cross-reactivity or disease-associated mutation reactivity in the selected antibody when can be selected with a higher probability.

[0192] In some embodiments, the optimized molecule is an engineered polypeptide provided herein. In other embodiments, the optimized molecule is further evaluated, further adjusted, or a peptide library or candidate engineered polypeptides. A manipulated version of the candidate may be used to generate a rally, or any combination thereof. In certain embodiments, the method comprises the step of: Use the complement to generate a peptide library or engineered polypeptide candidate library. and then isolating the library from a binding partner of CD25 (e.g., CD2 The peptide library may further comprise contacting the peptide library with, for example, an engineered and share at least some sequence similarity with the constructed polypeptide candidate. However, engineered peptides may include peptides in which certain residues may be replaced with other residues. A polypeptide candidate library may, for example, contain a variety of engineered polypeptide candidates. It may include

[0193] In some embodiments, the peptides in the peptide library are between 2 and 15 amino acids. Acids, 5-15 amino acids, 10-15 amino acids, 2-10 amino acids In some embodiments, the library comprises an amino acid or between 5 and 10 amino acids. The total number of amino acids in each peptide is 0.01, which may include, for example, linker amino acids, and may be 0.01. The engineered polypeptide candidate library contains both amino acids and structural amino acids. For example, one or more amino acids or amino acids in the candidate to generate new library members The engineered polypeptide candidate library can be prepared by varying the linking moiety. The engineered polypeptide candidates in the library may, in some embodiments, contain between 5 and 40 amino acids. amino acids, between 10 and 35 amino acids, between 15 and 35 amino acids or between 20 and 3 In some embodiments, each engineered amino acid in the candidate library independently comprises between 0 and 10 amino acids. In some embodiments, the total number of amino acids in the selected candidate polypeptide is determined by, for example, The peptide may contain amino acids, both interfacial and structural amino acids. The library and engineered polypeptide candidate library may, in some embodiments, comprise 5, Between 000 and 100,000 members, between 5,000 and 80,000 Members, between 5,000 and 60,000 Members, between 5,000 and 40,00 Between 0 and 10,000 members, between 5,000 and 30,000 members, and between 10,000 and 10,000 members Between 15,000 and 25,000 members, Between 15,000 and 20,000 members or about 17,000 members (e.g., distinct peptides or distinct engineered In some embodiments, the libraries may include multiple separate libraries (potential polypeptide candidates). In certain embodiments, library members are generated and evaluated. For example, in some embodiments, library members do not contain disulfide bridges. Libraries without bridges are evaluated.

[0194] In some embodiments for generating candidates for a candidate library, one or more linkages Moieties may be added or removed in the design of the original engineered polypeptide candidate. For example, in some embodiments, disulfide bridges may be removed. Alternatively, the lactam bridge may be added or moved. In some embodiments, a molecule is removed or added or its position is moved. One or more amino acid residues are replaced. CD25 binding partners to the peptide candidate library or both (if present) The binding of the engineered polypeptide may be used to further refine the design of the engineered polypeptide or to further refine the design of the engineered polypeptide. These libraries can provide further information that can be used to select polypeptides. Further information from screening the RI can be used to identify, for example, engineered polypeptides. to alter, for example, to increase the binding affinity of CD25 with its binding partner. The engineered polypeptide candidate library can be used in some embodiments to binding interactions (including the presence or location of such moieties), e.g., disulfide bonds and This provides further information on the influence of specific linker moieties on crosslinking involving lactams. The peptide and / or engineered polypeptide candidate library can be In some embodiments, the binding affinity or specificity of CD25 to a binding partner. Common motifs (e.g., amino acid residues) that may increase the activity or provide other desirable characteristics patterns or linking portions, or a combination thereof) Peptide or engineered polypeptide candidate libraries or cognate binding partners Evaluating binding to members of the IL-11 domain or both provides additional structural and functional information. This can be used to further refine the design of the engineered polypeptide or to The method can be used to select candidate polypeptides. a. Selection by binding under varying pH

[0195] In some embodiments, the engineered polypeptide exhibits relatively low structural flexibility at lower pH. The amino acid sequence is selected at least in part based on its structural flexibility at physiological pH compared to that of the amino acid sequence. CD25 may be overexpressed on tumor cells and therefore contributes to higher expression in the tumor microenvironment. Binding of the antibody to CD25 with affinity may be desirable in some embodiments. In some embodiments, lower than the same engineered polypeptide at physiological pH. engineered polypeptides that are less flexible at low pH, or in which one or more amino acids are Engineered polypeptides with specific orientation at high pH or higher binding at low pH. It is desirable to select engineered polypeptides with binding affinity or selectivity. In many cancerous tumors, the growth rate of cancerous cells is higher than the available This can overwhelm the available oxygen supply, resulting in a hypoxic microenvironment within the tumor. can affect the pH of the tissue environment, and low oxygen levels can result in decreased pH (e.g., (For example, due to the accumulation of acidic metabolites from anaerobic glycolysis). In some cases, the desired binding interaction is at a lower pH, resulting in higher binding. have novel structural features) but have reduced binding at physiological pH (e.g., binding phase Decreased, less, or lacking desirable structural features that result in interactions b) Selecting an engineered polypeptide, in some embodiments, can be performed in cases where the engineered polypeptide is not present in the tumor. and producing antibodies with higher binding to desired targets in tumors compared to the binding of Physiological pH is typically between about 7.35 and about 8.55. The pH of the tumor microenvironment is between about 7.45 and about 7.4, for example, about 7.4. less than about 7.45, between about 7.45 and about 6.0, between about 7.0 and about 6.0, about 6 Between 0.8 and approximately 6.2, between approximately 6.7 and approximately 6.3, approximately 6.4, approximately 6.5, approximately 6.6, approximately In some embodiments, the engineered The polypeptides can be analyzed using computational methods, e.g., molecular dynamics simulations. In other embodiments, the engineered polypeptide may be evaluated at different pHs. Using in vitro methods, the strains are selected based on their differential pH characteristics. This can include, for example, phage panning at different pH levels. The display library pans one or more engineered polypeptides at physiological pH. The second pH can then be used to bind the phage, and the phage that bind can be discarded. Rounds of panning can be performed at lower pH, with one or more engineered nucleotides at lower pH. Phages that bind to the target polypeptide can be selected. or similar binding to phages at both low and physiological pH. The engineered polypeptides bind with affinity to the phage, producing antibodies that target tumor cells. It may not be desirable to use it when creating b. Reverse peptide evaluation

[0196] In yet a further embodiment, selecting the engineered polypeptide comprises selecting the engineered polypeptide. The method may include comparing the binding of the polypeptide to the binding of a de-engineered polypeptide. The engineered polypeptide may be based on an engineered polypeptide, but may contain an interface-interacting amino acid. One or more of the amino acid residues (e.g., based on the surface of CD25) are amino acids that exhibit reverse characteristics. For example, amino acids with large, sterically bulky hydrophobic side chains are more likely to be replaced. amino acids with smaller side chains, or hydrophilic side chains, or smaller and hydrophilic side chains In some embodiments, an amino acid having a hydrogen bond donor side chain may be replaced with a hydrogen It is replaced with an amino acid that has a bond-accepting side chain or an amino acid that does not hydrogen bond. Engineered and reverse engineered polypeptides can be used to compare The binding characteristics may, in some embodiments, include specificity and / or affinity. Comparing the binding characteristics of the engineered polypeptide with the binding characteristics of the de-engineered polypeptide In some embodiments, the interface-interacting amino acids are not characteristic of a linking moiety such as a linker. It may be helpful to select engineered polypeptides that drive binding interactions. Engineered polypeptides driven by linking moieties such as linkers can be used to target off-target molecules. In some embodiments, less desirable binding may be achieved because the antibody may exhibit binding or other undesirable binding characteristics. It may not be appropriate.

[0197] In a further embodiment, the method comprises modifying the selected engineered polypeptide. Further includes: c. Bond evaluation

[0198] As described herein, in some embodiments, the engineered The method for selecting a polypeptide includes selecting a candidate engineered polypeptide from a protein or its fragments, e.g., fragments that bind to a binding partner of CD25 (e.g., an antibody against CD25). For example, in some embodiments, the engineered polypeptide candidate live The library or peptide library is screened for binding to a binding partner of CD25. It will be leaned.

[0199] One or more peptides of a protein or fragment thereof (e.g., a binding partner of CD25) Binding to the peptide or engineered polypeptide candidate (e.g., a member of a library) In some embodiments, binding can be assessed in a variety of ways, e.g., by binding to a protein or The fragments are directly assessed by directly detecting the label on the fragment. can include, for example, a fluorescent label, such as a fluorophore or a fluorescent protein. In this embodiment, binding is assessed indirectly, for example, using a sandwich assay. In sandwich assays, a peptide or engineered polypeptide candidate (e.g., a library member) binds to the binding partner, and then a labeled secondary reagent is A secondary reagent is then added to label the bound binding partner. Examples of sandwich assay components include anti-His tag antibodies or His tag antibodies. His-tagged binding partners detected using tag-specific fluorescent probes; labeled streptavidin Biotin-labeled binding detected using leptavidin or labeled avidin or an unlabeled binding partner that is detected using an anti-binding partner antibody Included.

[0200] In some embodiments, the candidate peptide or engineered polypeptide of interest is a are identified based on binding signal or dose response using available detection methods. These detection methods include, for example, imaging, fluorescence activated cell sorting (FACS), mass spectrometry, or In some embodiments, a hit threshold is defined (e.g., , median signal), anything with a signal above that signal is a putative hit Flagged as a motif.

[0201] For the development of combinatorial libraries, In some embodiments, peptides identified from a peptide library based on the sequence Further clustering into distinct groups using other information, such as structural information, or a combination of these. This grouping can be done, for example, by using publicly available sequence alignments, Chemical descriptors, structural predictions, and entropy prediction informatics tools (e.g., MUS CLE, CLUSTALW, PSIPRED, AMBER, Hydropathy Ca lculator and Isoelectric Point Calculator) and clustering algorithms (e.g., K-Means, Gibbs, and Hie Motifs present in peptide hits can be analyzed using a holomorphic algorithm. Clusters (e.g., structural or functional motifs) can be identified from this analysis. Individual peptide motif hits can also be identified. These motif clusters and individual Each motif is used, in some embodiments, to identify protein interactions at the target interface. Define one or more of the sequence, structure, and chemical features of the motif that appear to be driving Design rules can be devised. In some embodiments, the structure of the target interface is determined by these interface motifs. Identification of the design rules is not necessary for the design of the chip. Rather, the design rules may be used in some embodiments. The results can be derived from the analysis of peptides identified from screening peptide libraries. do.

[0202] In some embodiments, the binding assay is performed using a 5 It has a sensitive kinetic range of Thus, in some embodiments, the engineered polypeptide candidate is a polypeptide that binds native CD25: 10 Partner Signals 5 Signal bracket (signal bracket) If a target antibody has a binding event with a CD25 binding partner, it is identified as being of interest. The type of signal depends on what type of assay is being used or how the signal is generated. For example, in some embodiments, the signal may be , response units in sensorgrams, fluorescent signals in image-based readouts, and is the enzymatic readout in enzyme-based assays. The signal for a binding event is , can be measured relative to the CD25:binding partner signal.

[0203] In some embodiments, the engineered polypeptide candidates are modified prior to assessing binding. For example, in some embodiments, biotin, PEG, or another binding moiety, or The combination of For example, in some embodiments, biotin-P is attached to the C-terminus or N-terminus of the peptide. EG12- is covalently attached to the N-terminus of the engineered polypeptide. The engineered polypeptide candidate was C-terminally modified with -GSGSGK-PEG4-biotin. In certain embodiments, such biotin-modified engineered polypeptides The candidate enzymes are then bound to streptavidin beads via the biotin moiety, and the beads are then Supported immunogens are assessed for binding to binding partners of CD25. VI. Uses of Engineered Polypeptides and CD25 Antibodies

[0204] Engineered strains provided herein and identified by the methods provided herein The resulting polypeptide can be used, for example, to generate one or more antibodies that specifically bind to CD25. In some embodiments, the antibody may be a monoclonal or polyclonal antibody. It is the body.

[0205] The term "antibody," as used herein, refers to an immunoglobulin that specifically binds to an antigen. Antibodies are proteins or polypeptide sequences derived from a molecule. It may be an intact immunoglobulin, or a fragment thereof, of monoclonal or natural origin. It may be derived from natural or recombinant sources.

[0206] The term "antibody fragment" or "antibody binding domain" refers to a fragment that binds to a target, e.g., an antigen and its associated target. antigen binding sufficient to confer recognition and specific binding of the antibody fragment to the desired epitope. At least one of the domains of an antibody, i.e., containing the antigen-determining variable region of an intact antibody Examples of antibody fragments include Fab, Fab', F(ab Fv fragments, single-chain (sc) Fv ("scFv") antibody fragments, linear antibodies, single-chain antibodies, Main antibody (abbreviated as "sdAb") (either VL or VH), camelid These include, but are not limited to, multispecific antibodies formed from VHH domains and antibody fragments. It will not be done.

[0207] The term "scFv" refers to at least one antibody fragment containing the variable region of a light chain and the variable region of a heavy chain. It refers to a fusion protein containing at least one antibody fragment containing a variable region, and the light and heavy chains The variable regions are connected in series via short, flexible polypeptide linkers to form a single polypeptide. The scFv can be expressed as a single chain, and the scFv can be expressed as a single chain from the intact antibody from which it is derived. Preserves the specificity of the body.

[0208] With respect to antibodies, the "heavy chain variable region" or "VH" (or single domain antibodies, e.g., nanoparticles) In the case of a ribozyme, "VHH" refers to a region of the ribozyme that is adjacent to the ribozyme and is known as the framework region. The fragment of the heavy chain containing the three CDRs interleaved between these framework regions , which are generally more highly conserved than the CDRs and form a scaffold that supports the CDRs.

[0209] Unless otherwise specified, as used herein, an scFv refers to a polypeptide that is, for example, located at the N-terminus of the polypeptide. and the VL and VH variable regions may be in either order with respect to the C-terminus, may comprise VL-linker-VH or may comprise VH-linker-VL.

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

[0211] Thus, in some embodiments, an antibody produced by immunizing an animal with an immunogen An antibody, wherein the immunogen is an engineered polypeptide provided herein, is In some embodiments, the animal is a human, a rabbit, a mouse, a hamster, or a mammal. In certain embodiments, the monkey is a cynomolgus monkey, a macaque monkey, or a Immunizing animals with engineered polypeptides can be used to generate immunogens, e.g., rhesus monkeys. administering to the animal at least one dose of a composition comprising In some embodiments, generating antibodies from an animal may include generating antibody-expressing B cells. Some embodiments involve isolating the antibody. In some embodiments, the method further comprises fusing the B cells with myeloma cells to produce the engineered B cells. Antibodies generated using the polypeptides described above can be used in human and monkey, e.g., cynomolgus, monkeys. It can cross-react with

[0212] In certain embodiments, the method for generating antibodies comprises generating one or more epitopes for the antibody. In some embodiments, the method further comprises determining, for example, the epitope activity. by contacting the library with antibodies and assessing binding of the antibodies to epitopes in the library. This involves screening antibodies for binding to more than one epitope. In certain embodiments, antibodies that bind to more than one epitope are discarded. In embodiments, the engineered polypeptide mimics one epitope of CD25. In embodiments, the engineered polypeptide mimics two or more epitopes of CD25. In certain embodiments, the engineered polypeptide binds two or more epitopes of CD25. Screening antibodies for binding to two or more epitopes that mimic contacting the epitope library with antibodies and determining whether the antibodies are directed to epitopes in the library; Evaluating antibody binding and breaking down one or more antibodies that bind to two or more epitopes. These epitopes are mimicked by engineered polypeptides. Not an epitope.

[0213] In some embodiments, the engineered polypeptides provided herein are produced using In certain embodiments, the antibody specifically binds to CD25. When bound to CD25, it does not block the binding of IL-2 to CD25.

[0214] In some embodiments, the antibody is a non-IL-2 blocking antibody (non-IL-2 blocking agent) That is, the binding of the antibody to CD25 is related to the IL-2 ligand CD25 (IL-2 alpha chain) does not disrupt or prevent binding to IL-2, and does not inhibit IL-2-mediated signaling, e.g., IL-2 / JAK It does not affect signaling through the STAT-3 / 5 signaling pathway. In this form, the antibody disrupts the binding of the IL-2 ligand to CD25 (IL-2 alpha chain) It does not bind to the epitope of the 7G7B6 antibody, but binds to an epitope different from that of the 7G7B6 antibody. In one embodiment, the antibody disrupts the binding of IL-2 ligand to CD25 (IL-2 alpha chain). Trimers of the beta, gamma, and alpha (CD25) chains of the IL-2 receptor, but not Destroy the transformation.

[0215] In some embodiments, the antibody is an IL-2 blocking antibody, e.g., the antibody blocks IL-2 receptor activity. disrupts or prevents binding of IL-2 ligand to the IL-1, IL-2 beta and / or gamma chains In certain embodiments, the antibody In some embodiments, the antibody disrupts or prevents the binding of IL-2 ligand to CD25. The body disrupts or prevents the binding of IL-2 ligand to CD25, and The epitope is different from that bound by either baciliximab or ciliximab. Binds to tope.

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

[0217] In some embodiments, the antibody comprises a heterotrimeric IL-2 chain of alpha, beta, and gamma. In some embodiments, the antibody disrupts or prevents merization of IL-2 ligand and CD2 5, but does not block binding to alpha, beta, and gamma IL-2R chain heterotrimers. In certain embodiments, the antibody selectively binds to Treg cells. In other embodiments, the antibody selectively binds to Teff cells.

[0218] In yet further embodiments, the engineered polypeptides provided herein are used to The antibodies produced will be evaluated to determine whether they block the binding of CD25 to IL-2. In embodiments, antibodies are selected that do not block CD25 binding to IL-2. In this case, antibodies that block the binding of CD25 to IL-2 are selected. For example, CD25 is coupled to the tip of a biosensor, and the fragmentation is performed in the presence of IL-2 and In some embodiments, binding by the antibody can be assessed in the presence and absence of the antibody. In this study, antibodies were assessed for binding and blocking or non-blocking of IL-2 binding to CD25. 6xHis tag that can be used with Ni-NTA in flow cytometry to In certain embodiments, antibody binding is observed at physiological pH (e.g., about pH 7.5). between pH 7.3 and about pH 7.5, or about pH 7.4), and in the tumor microenvironment (e.g., between about pH 6.4 and about pH 6.6, or about pH 6.5) In certain embodiments, blocking / non-blocking activity is assessed using an IL-2 blocking antibody (e.g., , daclizumab, or bacliliximab) In certain embodiments, blocking / non-blocking activity is determined by the activity of an IL-2 non-blocking antibody (e.g., In certain embodiments, blocking / non-blocking activity is compared to the binding of the antibody 7G7B6. Both IL-2 blocking and non-IL-2 blocking antibodies are compared.

[0219] In some embodiments, the antibody is an agonistic antibody to CD25. In the present case, the antibody is an antagonist antibody against CD25.

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

[0221] The original antibody clone is identified by the indicated ID, e.g., the clone ID in Table 2. For example, the antibody may be the antibody clone "YU389-A01" shown in row 1 of Table 2. Heavy chain complementary determining region on).

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

[0223] [Table 5] TIFF2025026923000014.tif219140 TIFF2025026923000015.tif218139 TIFF2025026923000016.tif115140

[0224] In some embodiments, CDR-H1 is GGTFSSYA, GGSISSGGYY, GFTFSSYG, GYTFTSYY, GYTFTSYG, GYTFTDYY, GGSISSGGYS, GGSISSSNW, GYSFTSYW, GTFFSNYG, GFTFSSSA, GTFFSSY W, GFIFSRHA, GYTFNNYG, GTFFSSYA, GYTFTTYA, GFTFNNAW, GTFFSSYE, GYSFTTYW, GYSFNTY W, GFTFRRYW, GYSFSTYW, GFAFSSYG, GYKFANYW, GYTFKNFG, GFTFSSYS, GDSISSSSYY, and and GGSISRSNW;

[0225] In some embodiments, CDR-H2 is IIPIFGTA, IIPIFGTA, IYYSGST, ISYDGSNK, INP SGGST, ISAYNGNT, IMPIFDTA, VDPEDGET, IYHSGST, IYPGDSDT, ISHDGHVK, IKQDGSEK, ISVY NGDI, INTNTGDP, IKSKTDGGTT, ISSSGSTI, ISSRGSTI, IYPSDSDT, ISGRKGNT, ISSSSSYI, IN HSGST, IYHTGST, and ISYDGNNK;

[0226] In some embodiments, the CDR-H3 is AREMYYYYGMDV, AREMYYYYGMDV, ARGNLWSGYYF, A KELLEGAFDI, ARDRVTMVRGALAY, ARERSYYGMDV, ASWSERIGYQYGLDV, ARDILGLDY, ATEDTAMGGID Y, ATEGRYGMDV, AVEGGRAPGTYYYDSSGLAY, ARAGYYYGMDV, ARDLGTMVRGVIEPYYFDY, ARGVRGTGF DP, ARDRNGYFQH, AKDLLGELSFFDY, ARLENNWDYGGWFDP, ARDRSYYGMDV, ARDKGYYGMDV, AKEISP RSSVGWPLDY, ARDFWSGYNELGGMDV, ARTWFGEFFDY, ARVIGGWFDP, ARGRLAYGDTEGFDY, ARDILRGE SSILDH, ARDRYYYGMDV, ARDLLGSGYDIIDY, ARVWGKNGDFDY, ARDRFHYGMDV, ARDRGDY, TTEGVEL LSFGGAPFDY, ARRRGGGFDY, AREKGSWFDP, ARDRGDRVGGLVFDY, ARQVAGGLDY, ARDRGYYGMDV, FR FGEGFDY, ARDGGYYFDD, ARDFRMDV, ARDAYAYGLDV, ARDLMNYGMDV, AREYDYGDYVFDY, ARLENNWN YGGWFDP, ARDYYYYGMDV, ARDIGYYYGMDV, ARVGDGYSLDY, AKAITSIEPY, AKGQGDGMDV, ARLGWGM DV, ARVWGDTTLGYGMDV, AIPWDAELGNYGMDV, ARGRWSGLGDY, ARARGGRYFDY, ARDQLAARRGYYYGMD V, AKGDVNYGMDV, ARDFYYGSGSYPNGYYYGMDV, ARDFNPFSITIFEMDV, ANLAMGQYFDY, ARDLGEAKSS SPHEPDY, ARDQEMYYFDY, ARGKGSYAFDI, and AKGYSSSPGDY; selected from.

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

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

[0229] In some embodiments, the CDR-L3 is QQSYSTPPT, QQSYSTPPT, GSWDTNLSGYV, QVWDSSS GHREV, QAWDSSTYV, QQYNHWPPL, QQYSGDSMYT, AAWDDSLSGVV, AAWDDSLNGVV, ATWDDSLSGVV, QQSHSTPIT, QQYNSYSRT, QQYTNWPQT, LQYDRYSGA, QVWHTTNDHVL, QVWDSSSDHWV, QQSKQIPYT , QQSYSLPLT, QQFDISGGLI, QQYDNLPLT, QVWDSSSDHTVA, SSYTTTDTFV, QQYDNLPYT, QQYYSTP PH, QQSYSTPLT, QVWDSSSDHVV, GTWDSSLSAYV, QQTHTWPWT, QQANSFPLT, QQSYSTPYT, SSYTSS STYV, QRYGSSPR, QQVHSFPFT, LQHNTFPYT, QQSHSTPLT, QQYNSYPFT, QQYNSSPLMYT, QQTYSTP LT, QQANTFPQT, QSYDGSSVV, GSWEARESVFV, QQTYNDPPT, NSRDSSGNHVV, QTWDGSIVV, VLYMGS GIWV, ATWDDALSGWV, SSYTSSSTLVV, QQSYSTPWT, SSYTSSSTWV, LQDYNYPPA, QQYYDDPQ, QQLN GYPTT, AAWDDSLIGHV, QVWDTSGDLHWA, QQSYTTPLT, QVWDSSSDLLWV, GTWDSSLSALV, AAWDDSLN Selected from GPV, MQTKQLPLT, QQANSFPPT, QSYDGNNHMV, and SSYTSSSTLWV.

[0230] In some embodiments, the antibody is each independent from those disclosed in Table 3A and Table 3B. CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L CDRs from different antibodies can be combined in any combination. It is possible to generate new antibodies by combining gene synthesis and high-throughput screening. The scanning technology allows one of skill in the art to identify all combinations of the six CDRs without undue experimentation. This allows for testing.

[0231] [Table 6] TIFF2025026923000018.tif217127 TIFF2025026923000019.tif158127

[0232] [Table 7] TIFF2025026923000021.tif21683 TIFF2025026923000022.tif15983

[0233] In some embodiments, the antibody comprises six Cs of any one of the combinations provided in Table 4. Has DR.

[0234] [Table 8] TIFF2025026923000024.tif212141 TIFF2025026923000025.tif210142 TIFF2025026923000026.tif210142 TIFF2025026923000027.tif210142 TIFF2025026923000028.tif211143 TIFF2025026923000029.tif210142 TIFF2025026923000030.tif210142 TIFF2025026923000031.tif92142

[0235] In some embodiments, the antibody is an scFv selected from Table 5, or an scFv in Table 5. In embodiments, the antibody is any antibody having a full-length heavy chain and an antigen-binding domain derived from The light chain variable region was extracted from the scFv sequence in Table 5 and used to generate soluble Fab fragments, monoclonal antibodies, and can be used to generate monospecific antibodies, bispecific antibodies, or any other type of antibody known in the art. When the scFv in Table 5 is a VH:VL scFv, the VL:VH scFv is used. It is possible to reverse the order of the heavy and light chains to generate scF If v is a VL:VH scFv, then the heavy chain and It is possible to reverse the order of the light and dark chains.

[0236] [Table 9] TIFF2025026923000033.tif219134 TIFF2025026923000034.tif219133 TIFF2025026923000035.tif219133 TIFF2025026923000036.tif219133 TIFF2025026923000037.tif214133 TIFF2025026923000038.tif219133 TIFF2025026923000039.tif214134 TIFF2025026923000040.tif214134 TIFF2025026923000041.tif219133 TIFF2025026923000042.tif219133 TIFF2025026923000043.tif219134 TIFF2025026923000044.tif219134 TIFF2025026923000045.tif220134 TIFF2025026923000046.tif219133 TIFF2025026923000047.tif219133 TIFF2025026923000048.tif219134 TIFF2025026923000049.tif215134 TIFF2025026923000050.tif217134 TIFF2025026923000051.tif127133

[0237] In some embodiments, the antibody is unique from those disclosed in Table 14A and Table 14B. independently selected, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR In some embodiments, the antibody has CDR-L2 and CDR-L3. CDR-H1, each independently selected from any one of the clones listed in 4B , CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 In some embodiments, the antibody is selected from those disclosed in groups in Tables 15A and 15B. CDR-H1, CDR-H2, CDR-H3, and CDR-L1, each independently selected The present disclosure provides CDRs from individual clones. or any one CDR matching with any other five CDRs. The antibodies identified in Tables 14A and 14B are derived from mouse CDRs. These CDRs are derived from phage display libraries. or can be used to convert it into a chimeric antibody. VII. Use of CD25 Antibodies

[0238] In some embodiments, the CD25 antibodies provided herein are used in therapeutic agents, e.g., to treat proliferation for use in a genetic disease or disorder, such as cancer, or for use in an autoimmune disease It is useful for

[0239] Thus, administering a therapeutically effective amount of a therapeutic CD25 antibody to a subject in need thereof Provided herein are methods of treating cancer, comprising: In some embodiments, the cancer is a primary cancer. In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer involves a solid tumor; in other embodiments, the cancer involves a liquid tumor, e.g., a blood tumor. In an exemplary embodiment, the CD25 antibody is a non-IL-2 blocking antibody. is.

[0240] Thus, administering a therapeutically effective amount of a therapeutic CD25 antibody to a subject in need thereof Provided herein are methods for treating an autoimmune-related disease or disorder, including: In a typical embodiment, the CD25 antibody is a non-IL-2 blocking antibody.

[0241] As used herein, a subject includes humans, domestic animals, and and farm animals, as well as zoo, sport or pet animals, e.g. For example, dogs, horses, rabbits, cows, pigs, hamsters, gerbils, mice, and ferrets. The term refers to any animal classified as a mammal, including animals, rats, cats, etc. The subject is a male Or it may be female.

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

[0243] In vivo administration of the therapeutic CD25 antibodies described herein can be intravenous, intratumoral, intracranial, or intravenous. Intrathecal, intralesional (e.g., intralesional injection, direct contact diffusion), intracavitary (intraperitoneal, intrapleural (i intravenous, intrauterine, intrarectal, intraperitoneal, intramuscular, subcutaneous, and topical , orally, transdermally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally In an exemplary embodiment, the route of administration is by intravenous injection.

[0244] A therapeutically effective amount of the therapeutic antibody will generally be administered. Suitable dosages of the therapeutic antibody are: severity of the disease, the subject's clinical condition, the subject's clinical history and response to treatment, and the treating physician may be determined at its discretion. VIII.Diagnostic use

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

[0246] The CD25 antibodies provided herein are modified for use in various immunoassays. These immunoassays include enzyme-linked immunosorbent assays (ELISAs). , Western blot, radioimmunoassay (RIA), flow cytometry, radioimmunoassay Immunoassay, immunofluorescence assay, spectrophotometry, radiography, electrophoresis, high performance liquid chromatographic High performance liquid chromatography (HPLC) or thin layer chromatography (TLC) Not limited to these.

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

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

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

[0250] The present disclosure provides, for example, the methods described herein for either therapeutic or diagnostic uses. Kits containing any of the CD25 antibodies described herein are also provided. In some embodiments, the kit The kit includes secondary antibodies, reagents for immunohistochemical analysis, pharmaceutically acceptable excipients and handling materials. and any combination thereof. In some embodiments, the kit comprises one or more pharmaceutically acceptable excipients, together with the The therapeutic compositions may include any one or more of the therapeutic compositions described herein.

[0251] This application relates to any of the therapeutic or diagnostic compositions or kits described herein. Also provided are articles of manufacture containing one or more of the above. Examples of articles of manufacture include vials (e.g., sealed vials). ) is included.

[0252] The description provided herein shows numerous exemplary configurations, methods, parameters, etc. However, such description is not intended to be a limitation on the scope of the present disclosure, but rather It should be appreciated that the present invention is provided as a description of an exemplary embodiment. [Example]

[0253] The following examples are illustrative only and are not meant to limit any aspect of the present disclosure in any way. . [Example]

[0254] Development of engineered immunogens that share CD25 characteristics The crystal structure of CD25 was obtained. The few crystal structures available for CD25 are The protein is missing a flexible loop section. To better understand this flexible loop and the binding interaction of CD25 with IL-2, Got it.

[0255] Different sections of CD25 as input for developing engineered immunogens Some of these areas are shown in Figures 34B and 34C. The set was used in the ROSETTA program to determine the overall desired structural and This process did not alter the structural (non-interface) parts of the segments, improving their dynamic properties. The structure and conformation of the interface residues in the context of the native CD25 from which they were derived are also investigated. Stabilized and reproduced motion, dynamics and other characteristics. Stability of the segment during development. The flexibility and flexibility of the sequences were also analyzed, and the sequences were adjusted as needed to change these parameters. For example, the N-terminus or C-terminus may be modified with one or more The effect of cross-linking on engineered immunogen candidates is also The design was evaluated using disulfide bonds formed between the side chains of different amino acid residues. At each stage of synthesis – amino acid addition, cross-linking and structural residue optimization – ROSE Many settings are implemented using the native scoring and energy functions of the TTA program. Each of the design candidates was quantitatively evaluated. The candidate with the best ROSETTA energy was selected as the design candidate. The next step in the design will be to finally evaluate and validate the results using molecular dynamics simulations. Proceed to the next step. Evaluate these parameters at physiological pH (e.g., approximately pH 7.4). In addition to measuring the pH of the tumor microenvironment, in some cases, the parameters may also be measured. pH 6.5) was also evaluated.

[0256] A formula for ranking different designs using molecular dynamics (MD) simulations Quantitative metrics include similarity to CD25, assessed via RMSD; and the similarity of candidate 33A and 33B show the input section shown in FIG. 33A and 33B). Exemplary Comparison of Stability vs. RMSD at Physiological pH for Engineered Immunogens Figure 33C shows the tumor microenvironment pH for the exemplary immunogens in Figure 33B. An exemplary comparison of stability versus RMSD. Representative scoring algorithms include: Presented below.

[0257]

number

[0258] Structural similarity was calculated for each peptide component in the MD ensemble after RMS alignment to the reference structure. Calculated using the root mean square deviation (RMSD) between the atomic coordinates of the conformation and the reference structure The RMSD was calculated using a computationally designed engineered immunogen candidate structure relative to the reference structure. or experimentally characterized (e.g., X-ray crystal structure) structures as references. In these simulations, the candidate function All residues, including potential interface residues (in some simulations) and candidate structural residues, are included. Groups (in other simulations) were compared to a reference.

[0259] The ensemble of conformations sampled by MD is then analyzed based on RMSD. Based on this, the molecules were clustered into groups (clusters) that were structurally similar to each other. Structural differences from all other conformations in the ensemble (e.g., high RMSD) Due to the lack of conformational similarity, the The ratio of the conformations in the MD ensemble was evaluated. The engineered immunogen candidates, which had more disorder than the standard candidates, were more flexible. , MD grouped into clusters of similar conformations (low RMSD) The proportion of conformations in the ensemble that are more ordered than alternative candidates was evaluated. The engineered immunogen candidate with a conformational A higher proportion of that ensemble fell into fewer clusters than alternative candidates.

[0260] Clusters populated with engineered immunogen candidates are compared to the reference structure using RMSD If the RMSD of a cluster is below a threshold of 4 Å, the cluster is considered to be a cluster. The target is considered to be well-ordered (e.g., low flexibility) and similar to the reference (structural similarity). The ensemble, which fulfills this criterion of low flexibility and high structural similarity, is highly Engineered immunogen candidates with a high degree of flexibility meet this criterion of low flexibility and high structural similarity. are predicted to be more active than alternative candidates that have a lower proportion of their ensemble fulfilling .

[0261] This quantitative analysis is used to analyze biophysical, biological, and physico-chemical interactions. Combined with qualitative analysis of the locus, it is possible to select a given immunogen candidate for in vitro evaluation. Table 6 below lists the 11 engineered immunogens prepared as described above. It is listed.

[0262] [Table 10] [Example]

[0263] In vitro evaluation of engineered immunogens Binding of the engineered immunogens prepared in Example 1 was assessed using antibodies against CD25 The engineered immunogen is modified with a -GSGSGK-biotin group on the C-terminus and then CD2 is bound separately to a streptavidin-coated biosensor tip. 5. A buffer solution containing the antibody was flowed over the tip during a 300 second association phase, and then the flowed solution The buffer is switched to one without the CD25 antibody, and dissociation from the biosensor tip is measured. A control was also run in which the tip did not have any engineered immunogen or protein initially bound. Any background binding of CD25 antibody to the tip is assessed. A second control was performed in which the CD25 IgG was bound to the biosensor tip to measure the ratio of CD25 to full-length CD25. The data obtained from these biosensor experiments demonstrate the level of antibody binding. The binding of the engineered immunogens is then qualitatively ranked. [Example]

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

[0265] The mouse HuCD25-immunized phage library was then purified using standard phage display technology. Transformation by electroporation in TG1 using the following protocol: Phages are propagated by the addition of M13. Phage-secreting TG1 cultures are incubated for 1 hour. After incubation on ice for 1 h, PEG is precipitated with PEG / NaCl. Exemplary libraries that may be used include 7807, 7808, 7809, and 7810. It can be enjoyed.

[0266] Tumor microenvironment (TME) pH subtractive selection: Phage panning was performed at physiological pH and TME pH. Antibodies that bind to full-length CD25 with high affinity at physiological pH To deplete IgG, subtractive panning was performed using 10 μg / mL of IgG in PBST pH 7.4. 1 h on ELISA plates coated with 1 ml of full-length CD25 (400 nM). 3 × 10 at pH 7.4 due to absorption over a period of 11 pfu phage (3 × 10 8 10 of The resulting phage supernatant is collected and diluted to pH 7. The pH is adjusted to 6.5 with PBST. Subsequent phage panning selection is carried out at pH 6.5. To carry out.

[0267] Panning selection was performed after 1 hour of incubation with 25 microliters of no antigen. The phage are then pre-cleared with streptavidin Dynabeads. Add to a new pre-blocked Eppendorf LoBind tube A biotinylated engineered immunogen (such as that described in Example 1) is added to the at a concentration of 100 nM (in some cases, an additional 500 mM NaCl was added to inhibit phage Add PBS for 40 minutes to 1 hour (to reduce non-specific binding of the immunogen to the sample). Add 25 microliters of streptavidin beads or streptavidin-coated Incubate with the coated plate for 1 hour at RT. Pellet the samples. Wash using a magnet / magnetic beads, or if using a plate, with PBST for 7-10 min. Wash 10 times, changing tubes twice to remove residual phage.

[0268] To elute the phage, add 50 to 800 μL of glycine pH 2.2 to each well. Add to the flask and plate, incubate for 10 minutes or less, then add high pH Tris Neutralize with 500 ml of freshly grown TG1 (O Add to a D600 (approximately 0.5) and incubate for 20 to 30 minutes.

[0269] Plate fractional log dilution series and the remainder Transfer 1 ml of the glycerol stock to 25 ml of 2xYT. Set aside for 2 min and add helper phage / IPTG at an OD600 of approximately 0.5.

[0270] Selection against the engineered immunogen at pH 6.5 with counterselection at pH 7.4 Periplasmic extracts are used for phage ELISA and octet sequencing. Cleaning is used and subsequently evaluated.

[0271] Ensure that the Fab phage binds full-length CD25 in addition to the engineered immunogen To achieve this, the final selection was performed using full-length CD25 instead of the engineered immunogen. 25 (two rounds of selection against engineered immunogens, followed by one round of selection against full-length CD25).

[0272] To perform selection with full-length CD25, 25 microliters of streptavidin was added. Pre-cleared panning selection with Dynabeads and new pre-cleared After adding phage to blocked Eppendorf LoBind tubes Biotinylated full-length CD25 was added to the sample at a concentration of 100 nM for 1 hour. The pool was incubated with 25 microliters of streptavidin beads for 1 hour at RT. Pelleting, washing and elution steps follow as above. [Example]

[0273] Phage ELISA Protocol and Biosensor / Octet Screening ELISA / Extract Preparation: Phage ELISA, and Fab Octet Screening Periplasmic extract preparation for cloning is performed.

[0274] The CD25 antigen is diluted and added to the ELISA plate wells and incubated. After incubation, the wells were washed twice with PBS, and then BSA was added and then 25 Block by incubating at 37°C for 2 hours. Dilute 2x in 1.0% BSA, pH 6.5, and use 50 microliters per tube. Add blocking solution and incubate at room temperature for 5 minutes. Shake off the blocking solution from the wells. Add 50 µL of the dilute phage preparation to each well and incubate for 1 h at room temperature. Fill ELISA plate wells with 200 microliters of PBST pH 6.5 for 3-5 minutes. Wash twice. HRP-conjugated anti-M13 antibody (Abcam, ab50370) Dilute 1:5000 in 1x PBST 1.0% BSA pH 6.5. 50 microliters Add 100 ml of diluted secondary antibody conjugate to each well and incubate at room temperature for 1 hour. ELISA plate wells are filled with 200 microliters of PBST pH 6 Wash 3-5 times with 0.5. ECL Lumo substrate (e.g., Supersignal E LISA Pico Chemiluminescent Substrate) A 1:1 mixture of substrate solution was prepared as described in the literature. 50 microliters of substrate solution was added to each well. Add to PBS and incubate at room temperature for 5-60 minutes before reading.

[0275] Colonies were cultured overnight in 0.1 ml of 0.03–4 ml of 2xYT 0.2% glucose. Cultures (1 ml culture in 96-well plates or 14 ml falcon tubes) Inoculate these until the OD600 reaches approximately 0.5-1.0. Incubate at 37 °C at 250-700 rpm. Discard the culture in 50-400 µL of 0 Induce with 0.025 to 0.1 M IPTG. In some cases, increase the temperature and shake at 250 rpm. While shaking, reduce the temperature to 30°C. Then, incubate them overnight. ml of culture is harvested by pelleting at 3400 rcf for 10-15 minutes. Discard the supernatant. Resuspend the culture in 50-75 μL of PBS with 1x Halt protease inhibitor. PPB buffer (30mM Tris-HCl, pH8.0, 1mM EDTA, 20% salt) Resuspend in PBS (cloze) and incubate on a rocking platform for 5 minutes at room temperature or 10 minutes at 4°C. The cultures are then incubated for 150-20 min with 1x Halt protease inhibitor. Resuspend in 225 µL of cold ddH2O and incubate at room temperature on a rocking platform for 1 h or 4 h. Incubate the lysate suspension at 15,000 rcf for 10-2 hours at 4°C. Spin for 15 minutes. Collect supernatant and dilute.

[0276] Fab expression and purification protocol: Cell cultures were inoculated and grown overnight, then 5 min. Induce with 0 μL of 25 mM to 1 M IPTG. Reduce the temperature to 30°C and increase the rpm to 1. The incubation was carried out overnight. The pellet was collected by pelleting at 3400 rcf for 15 minutes. The supernatant was discarded. The cell pellet from the 50 mL culture was placed in a -80°C freezer for 1 hour. On the other hand, cultures grown in plates were supplemented with 1x Halt protease inhibitor, EDTA 75 μL of PPB with none (Thermo Fisher Scientific) Add and vortex. Shake the plate at 1000 rpm for 10 minutes at 4°C. ×Halt protease inhibitor, EDTA-free (Thermo Fisher Scientific Add 225uL of cold water containing ntificial solution to each well. Mix the sample. The plates were shaken at maximum speed, i.e., 1000 rpm, at 4°C for 1-2 hours. Spin at 4 °C for 10 min at RT. Transfer the supernatant (PPE) to a new plate and store at -20 °C. The cell pellet from the 50 mL culture is removed from the freezer and stored in 5 mL of PBS, 10 mM imidazole, 2.5 mg / ml lysozyme and 1x Halt Protease inhibitors, EDTA-free (Thermo Fisher Scientific c) and add the pellet. After thawing the pellet at room temperature for 30 minutes, the lysate was heated at 3400 rcf The supernatant was removed and the pellet was discarded. 500 μL of Ni-NTA resin (pre-washed and pelleted) was added or Alternatively, Ni-NTA spin columns were used. The clarified lysate was incubated for 30 minutes to 1 hour. Incubate. Spin at 1500 rcf. Add 1 ml of PBS, Washed 5 times with 10 mM imidazole. After each spin, the buffer was discarded. Add BS, 200 mM imidazole, mix, and incubate for 30 minutes. The eluted protein was then spun at 1000 rcf for 15 minutes. After determining the protein concentration, , and stored at 4° C. or 20° C. A Zeba column was used for desalting / buffer exchange.

[0277] Octet / biosensor screening: Octet Koff in raw supernatants For rate screening, 50 μL of lysate was added to a 384-well plate containing Pall For Used in teBio Octet plates. Data were analyzed using Octet RED 3 Briefly, human CD25 was isolated from AR2 G tip coupled (1 ug / ml). For data collection, a baseline was Evaluate in PBST 1% BSA buffer for 60 seconds. Then, insert the tip into 50 μL of lysate. Finally, the tip is moved to PBST 1% BSA buffer and the association is measured for 300 seconds. The tip is then regenerated with 200 mM Tris-glycine, pH 2.5. Neutralize with PBST, 1% BSA. For data analysis, double-reference (CD25 on the tip). (none, as well as blank reference wells) were used for reference subtraction. This is done using HT 11.0 software. [Example]

[0278] Evaluation of antibodies generated from immunogens Antibodies were produced by immunizing mice with the engineered immunogens described herein. These antibodies were analyzed for cross-reactivity, cross-blocking, affinity and off-rate (off-ra). te) is evaluated to estimate

[0279] Biosensor (Octet Red 384, Pall Forte Bio) Protocol for determining cross-reactivity of individual test cross-links: This protocol is used to determine cross-reactivity of individual test cross-links. Anti-human CD25 mouse monoclonal antibody was derived from human, cynomolgus monkey, and mouse species. The ability of the target protein to bind to the deoxyribonucleic acid (DEA) is determined by the binding of the target protein to the deoxyribonucleic acid (DEA) via the primary amine. Covalently coupled to a xylan-coated sensor tip or The 6xHis tag is located on the sensor tip coated with anti-6xHis monoclonal antibody. The monoclonal supernatant in solution is then transferred to the biosensor tip to affinity capture the target protein. The net binding signal is measured using a blank or antigen-coated plate. The binding of blank medium or buffer to the tip was subtracted from the corresponding signal. A signal >3× background binding is considered a real binding event.

[0280] Protocol for cross-blocking with biosensors: This method involves cross-blocking individual test clones. The ability of the antibody (anti-human CD25 mouse monoclonal) to cross-block the control antibody was examined. Cross-blocking is the ability of a test clone to cross-block the corresponding epitope of a control antibody. Furthermore, this may indicate that the test antibody recognizes an epitope that overlaps with the target antibody. This suggests that the antibody may have similar functional properties to the control antibody. The reference antibody is covalently linked to a dextran-coated sensor tip via a primary amine. The target antigen in solution is allowed to bind to the control antibody. Then, the test antibody in solution is allowed to bind to the antigen in a sandwich format. If possible, it will be shown that it does not cross-block the control antibody, but non-binding will be the same as the control antibody. This can be interpreted as the ability to cross-block the body.

[0281] Protocol for biosensor affinity determination: This method is used to determine the affinity of test antibodies. The affinity of each test clone to the antigen when the concentration of the antigen is known is determined. Antibodies such as protein G or anti-mouse IgG monoclonal or anti-human IgG monoclonal The test clones are coated onto the biosensor tip. These test clones are then subjected to antigen in solution, which is then captured on a coated surface. Duration ranges from 60 to 600 seconds for the dissociation phase and 60 to 1800 seconds for the dissociation phase The resulting data (or "sensorgram") is then , a 1:1 Langmuir model or a 2:1 heterogeneous model The former fits the data using one of the following two models: If the 2:1 model for This indicates that the clones may require further subcloning due to inherent heterogeneity. The data curve fit indicates that the on-rate constant and The dissociation constant is given as the ratio of the dissociation rate constant and the off-rate constant.

[0282] Protocol for biosensor-based off-rate estimation: The method can be used when the concentration of antibody is unknown or when the test clone is to be further subcloned. Estimate the dissociation rate constant of the test clone if cloning is required. , Protein G, or anti-mouse IgG monoclonal or anti-human IgG monoclonal The test clones are coated onto the capture molecule coating. These test clones are then subjected to the antigen in solution, which is then captured on a surface coated with IgG. for periods ranging from 60 to 600 seconds for the dissociation phase and from 60 to 1800 seconds for the dissociation phase. The resulting data (or "sensorgrams") are then analyzed 1:1 Fit using either the Langmuir model or the 2:1 heterogeneous model. The authors conclude that if a 2:1 model to fit the data yields a better fit, We assume that interaction pairs are homogeneous, which means that clones may be more heterogeneous due to inherent heterogeneity. The data indicate that subcloning consisting of the following is required: The on-rate (or association) constant is fitted only to the No rate constants were fitted; this was used to order the test clones. This provides an estimate of the possible off-rate constants. [Example]

[0283] Selection of engineered polypeptides using the CD25 moiety as a reference target The sequence and three-dimensional (3D) structure of CD25 were obtained from the Protein Data Bank (PDB). Searched (PDB ID number 2ERJ, A chain): ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGSSSHSSWDNQCQCTSSATRSTTKQVTPQPEEQ KERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQ LICTG

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

[0285] [Table 11]

[0286] The interatomic distances and amino acid descriptor topology were determined. The amino acid descriptor topology was obtained using dynamic simulations, and the coherence of atomic fluctuations was analyzed. A covariance matrix was generated for the epitopes in the reference target. The peptide candidates are subjected to computational protein design (e.g., Rosetta), Dynamics simulations were performed using the NMR data, and the interatomic distances and amino acid sequences were determined. The covariance matrix of atomic fluctuations was generated using the descriptor topology. and for residues in the candidate that correspond to residues in the epitope of the reference target. .

[0287] A principal component analysis was performed to obtain covariance matrices - one covariance matrix for each reference target and Calculate the eigenvectors and eigenvalues ​​for each of the covariances - Only the eigenvector with the largest eigenvalue is kept. The first, second, third, Nth observed molecular structure in the set of simulated molecular structures Describes the dominant motion. If a candidate moves similarly to the reference epitope, its eigenvector is , will be similar to the eigenvector of the reference target (epitope). The similarity of the eigenvectors is , with respect to their components (3D vectors centered on each CA atom) aligned and pointing in the same direction. This similarity between the eigenvectors of the candidate and the eigenvectors of the reference target is called the The dot product was calculated using the dot product of the eigenvectors. The dot product value is the sum of the two eigenvectors relative to each other. 0 degrees, or 0 if the two eigenvectors point exactly in the same direction. In this case, the result was 1.

[0288] The ordering of the eigenvectors is based on their eigenvalues, which are then used in the molecular dynamics simulation. tion samples the underlying energy landscape of two different molecules Due to their stochastic nature, the number of molecules is not necessarily the same between them. The dot product between several differentially ranked eigenvectors was required (e.g., the candidate eigenvalues). (eigenvector 1 × eigenvectors of the reference target 2, 3, 4, etc.). Furthermore, However, molecular motions are complex and involve more than one (or several) dominant / major modes of motion (more than one) may be involved.

[0289] To solve these two problems, we first compute all the eigenvectors in the candidate and reference targets. This results in a matrix of dot products whose dimension is determined by the eigenvectors analyzed. The matrix of the inner product is determined by the number of eigenvectors - for 10 eigenvectors, the matrix is ​​10x1 0. This matrix of dot products can be converted into a single matrix 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) is a method for solving a 3L × 3L dimensional coordinate covariance matrix (where L is the number of atoms) as follows: Let us consider the set of eigenvectors Φ (reference target) and Ψ (MEM) and the set of eigenvalues ​​Λ. The set Φ is the N eigenvectors φ for the reference target. i The set Ψ includes N eigenvectors ψ j , and the eigenvectors are expressed by their associated eigenvalues. The eigenvalues ​​with the largest eigenvalues ​​are ordered in their respective sets. The vectors make up the largest proportion of the total coordinate covariation. i and ψ j eigenvectors The dot product of φ is calculated to compare the similarity of the motion between the reference target and the MEM. i and ψ j The root mean square of all dot product combinations of the eigenvectors is the relative position of the manipulated point with the reference target. It gives the sum of the motion similarity of candidate peptides (MEM) (RMSIP).

[0291] As shown in Figure 7, in each engineered polypeptide, scaffold residues (gray) were added. The epitope residues (gold) and their positions in 3D space were selected using this computational design procedure. PDB ID number 2ERJ, residue positions and epitopes relative to A chain. The cross-linking sequences are provided in Tables 8 and 9. The cross-linking positions are the intracellular cross-linking positions in each MEM sequence. Refers to the expected presence of sulfide bond formation.

[0292] [Table 12] TIFF2025026923000056.tif60142

[0293] [ka]

[0294] [Table 13] [Example]

[0295] Selection of antibodies using MEM-programmed in vitro selection We considered 32 different panning strategies (S1–S32), each involving three rounds of positive selection. Each program uses at least one engineered polynucleotide as the selection molecule. The peptide was used for conventional selection using conventional methods (CD25 as a positive target). Bovine serum albumin (BSA) was included as a negative target to select against nonspecific binding. and used it.

[0296] The panning protocol begins with a human naive scFv library and pans: It was performed in solution with the selection molecule bound to biotin (but still in solution). For each round, the starting pool is first combined in solution with the negative selection molecule (BSA), A streptavidin-coated substrate (e.g., magnetic beads) is then added to the mixture. Thus, the total number of negatively selected molecules in the pool bound to the negatively selected molecules was calculated. All phages were allowed to bind to a streptavidin-coated support. The fluid was removed and the flow-through was then subjected to a positive selection step. Combine with positive selection molecule (antigen 1), allow to bind, then streptavidin-coat A solid substrate coated with phages was applied to the mixture. This step retained the bound phages but The remaining unbound phages were removed. The bound phages were then eluted. E. coli was transfected with the eluted phages using a 30 min incubation. Transfect and analyze the transfected cells by next generation sequencing and D The phages are then split for NA isolation and used in subsequent panning rounds. For each panning program, negative selection was performed in each round. The first was performed, and the second was performed positive selection.

[0297] [Table 14] TIFF2025026923000060.tif160161 Primary ELISA screening and hit selection

[0298] For each strategy, 384 clones were selected against full-length CD25 after three rounds of panning. The data were selected for ELISA response analysis (Figure 9). At least one for each epitope is shown using a sorting strategy designated by Two strategies yielded clones capable of binding to CD25. Different peptide-based strategies have been shown to enrich for distinct high-affinity clonal subsets. As shown in Table 11, most MEM programs The optimized selection strategy yields anti-CD25 hits more productively than traditional full-length panning. did.

[0299] [Table 15]

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

[0301] For comparing the affinities of different scFv antibodies using a single-cycle kinetic assay design teBio® Octet RED384™ BioLayer Interface The His-tagged scFv was assayed using an anti-His biosensor (For The full-length CD25 analyte was immobilized on a sensor (tebio® HIS1K). The tip was washed and the binding of molecules in the analyte to the scFv was recorded. Controls were also run: buffer only (to control for sensor drift) and Separate controls of polyclonal IgG isotype antibodies purified from human serum (non-specific (to control for IgG binding) was used.

[0302] As shown in Figure 11, the biolayer interface of 1475 anti-CD25 scFvs The 1433 human phage-display clones were identified by phage display panning. The results show that 97% of the hits were confirmed to bind to CD25. K was noticed D The range is 10-200 nM, with a median K D The value was 28.5 nM. As shown in

[11] , most screening strategies target high affinity CD25 The scFv having the following structure was generated: -3 k less than / s off Only scFvs with Appropriate K D The values ​​are given on the y-axis. Mostly 10 -3 k less than / s off At least one hit was generated with the following sequence: Confirmatory testing by flow cytometry

[0303] The CD25 specificity of different scFv antibodies was assessed using CD25-expressing cells [CD25(+)]. or cells that do not express CD25 [CD25(-)], and As shown in Figure 13, 1248 scFv human antibodies analyzed in this assay Of the total number of cells, 1160 (93%) specifically bound to CD25(+) cells. Sequence analysis of hits

[0304] Next generation sequencing was performed on the panned phages from each round. As shown, MEM-steered panning significantly improved the potency of antibody libraries in a strategy-dependent manner. Concentrating CDR diversity. Each round of selection reduces repertoire diversity (Figure 1 6), CDR lengths were centered around the preferred length for each MEM (Figure 17).

[0305] Individual scFvs were sequenced using Sanger sequencing. The complete protein sequences for the IgG1A, IgG2A, IgG3A, IgG4A, IgG5A, IgG6A, IgG7A, IgG8A, IgG9A, IgG1B, IgG1C, IgG1D, IgG1E, IgG1F, IgG1G, IgG1H, IgG1H, IgG1H, IgG1I, IgG1I, IgG1F, IgG1H, IgG1I, IgG1F, The complementarity determining regions are provided in Tables 12 and 2, respectively.

[0306] [Table 16] TIFF2025026923000063.tif218138 TIFF2025026923000064.tif146138

[0307] Analysis of CDR and germline usage revealed that 1475 sequenced scFvs had at least This suggests that the set represents 126 distinct clones. These include 1 VH+JH framework selection and 35 VL+JL framework selections. The unique CDR sequences include:

[0308] [Table 17] TIFF2025026923000066.tif146138 Includes:

[0309] Sequence analysis applied to scFvs against individual target epitopes revealed common sequences within each set of antibodies. Identify common CDR usage patterns:

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

[0311] [Table 18]

[0312] For CD25 epitope 2 (13-20: 127-132), the CDRs used were: , including the following:

[0313] [Table 19]

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

[0315] [Table 20]

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

[0317] [Table 21]

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

[0319] [Table 22]

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

[0321] [Table 23]

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

[0323] [Table 24]

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

[0325] [Table 25] [Example]

[0326] Confirmation of epitope specificity by competitive binding 126 anti-CD25 clones were tested using a four-target competitive binding assay shown in Figure 18. The epitopes were subjected to cleavage. The binding site for the mab is based on X-ray crystallographic structure determination. is based on peptide mapping.

[0327] Cross-competition assays were performed in a classical sandwich format, in which a first antibody was placed in a buffer. Immobilizing the antigen and then the second sandwich antibody on the biosensor. The His-tagged scFv was expressed and the His-tagged scFv was isolated from the supernatant. Purification was performed in situ on the biosensor using s-tag capture. Advance biosensor His tag capture to consistent levels across all scFv measurements. Normalization across scFv clones was performed by monitoring the end loading response. Each scFv was individually captured on an anti-His biosensor (Fortebio HIS1K). A baseline measurement was taken in running buffer. CD25 was then detected using an antibody. Finally, IL-2, 7G7B6, basiliximab, or daclizumab were administered to the Each of the various competitive analytes was added, including the competitive analyte. The captured epitope was only captured if it did not overlap with the binding epitope of the immobilized scFv. It is capable of binding to CD25.

[0328] As shown in Figure 19, full-length CD25 panning clones target the IL-2 interface epitope. Most clones are dominated by IL-2, daclizumab, and basioxymethane. It is blocked by cimab but not by 7G7B6.

[0329] As shown in Figure 20, the 147-157 epitope MEM-driven clones Most clones bind primarily to epitopes that are blocked by daclizumab. but is blocked by IL-2, basioliximab, and 7G7B6. I can't.

[0330] As shown in Figure 21, the 6-17 epitope MEM-driven clones Most clones bind primarily in the 7G7B6 group. Not blocked by IL-2, daclizumab, or basioliximab .

[0331] As shown in Figure 22, the 13-20:127-132 epitope MEM-driven clones Most clones bind primarily at the intended epitope. It is blocked by IL-2, daclizumab, and basioliximab. It will not be blocked.

[0332] As shown in Figure 23, the 44-56 epitope MEM-driven clones were The clones were divided into two profiles. In group 1, the clone was blocked by 7G7B6 but also by IL-2. It is not blocked by either izumab or basioliximab. The line is blocked by IL-2, daclizumab, and basioliximab, but not by 7G7. These blocking profiles are not blocked by B6. , indicating binding to the intended epitope.

[0333] As shown in Figure 24, the 55-63 epitope MEM-driven clones The clones were divided into three profiles. In group 1, the clone was blocked by 7G7B6 but also by IL-2. It is not blocked by either izumab or basioliximab. The line is blocked by IL-2, daclizumab, and basioliximab, but not by 7G7. These blocking profiles are not blocked by B6. In profile 3, the clones bind to IL-2 and and 7G7B6, but also by daclizumab and basioliximab. These blocking profiles are designed to accommodate the intended impact from different approach angles. Binding to the pitope is shown. [Example]

[0334] Mapping functional epitopes by alanine mutagenesis Alanine mutations were used to bin the intended epitopes in MEM-driven clones. Alanine mutagenesis was performed using a competitive assay. Since it acts on a functional epitope rather than a structural epitope defined by This was chosen as an orthogonal method for binning antibodies. The following pairs were selected for mutagenesis. Using computer modeling, these The alanine mutations selected for use in the assay were shown to improve both global and local stability. For example, Figure 26 shows the structure of the 145-157 epitope. The results of modeling alanine mutations within the group are shown. Regarding: Each of the eight different starting apo-CD25 configurations was analyzed using the crystal structure as a reference. From three independent 100 ns MD simulations in open solvent for each RMSD. As shown in Figures 27-29, the alanine mutant version of CD25 , with binding responses to basiliximab, daclizumab, and 7G7B6, respectively .

[0335] As intended, engineered polypeptides targeting the 147-157 epitope were used. Binding scFv hits from in vitro selection were directed against the intended portion of CD25. Consistent with specificity.

[0336] Each of the 117 scFvs from the screening campaign was cloned into four alanine amyloid nucleotides. Mutation pairs were tested (Fig. 31). Diversity of functional epitopes is observed. The resulting hits have distinct in-epitope alanine substitution position sensitivities. [Example]

[0337] Confirmatory test for immunoglobulin G (IgG1) type antibodies Thirty antibodies were selected as full-length immunoglobulins for further testing. The human and light chain sequences were cloned into a human immunoglobulin G (IgG1) format, expressed, and purified. Binding to CD25 was measured by Octet® as shown in Table 13. and evaluated it.

[0338] [Table 26] [Example]

[0339] Panning-biased library of mouse antibody sequences The phage display library was analyzed by immunoglobulins from mice immunized with full-length CD25. This library, biased towards CD25-binding antibodies, was generated from the phospho-antibody gene. The complementarity determinations shown in Table 14A and Table 14B were obtained by panning against the selected polypeptides. The constant region sequence was obtained.

[0340] [Table 27] TIFF2025026923000077.tif97168 TIFF2025026923000078.tif98170 TIFF2025026923000079.tif98170 TIFF2025026923000080.tif97168 TIFF2025026923000081.tif97168 TIFF2025026923000082.tif98170 TIFF2025026923000083.tif99170 TIFF2025026923000084.tif97168 TIFF2025026923000085.tif97168 TIFF2025026923000086.tif98170 TIFF2025026923000087.tif97168 TIFF2025026923000088.tif97168 TIFF2025026923000089.tif98169 TIFF2025026923000090.tif97168 TIFF2025026923000091.tif97168 TIFF2025026923000092.tif7168

[0341] [Table 28] TIFF2025026923000094.tif97164 TIFF2025026923000095.tif98166 TIFF2025026923000096.tif97163 TIFF2025026923000097.tif98167 TIFF2025026923000098.tif98166 TIFF2025026923000099.tif97163 TIFF2025026923000100.tif98166 TIFF2025026923000101.tif99167 TIFF2025026923000102.tif99166 TIFF2025026923000103.tif97163 TIFF2025026923000104.tif96163 TIFF2025026923000105.tif98166 TIFF2025026923000106.tif100170 TIFF2025026923000107.tif96163 TIFF2025026923000108.tif97163 TIFF2025026923000109.tif10163

[0342] Sequence analysis shows that these antibodies, derived from clonal lineages, are identical to those shown in Tables 15A and 15B. This suggests that the data can be grouped in such a way that

[0343] [Table 29]

[0344] Table 30

Claims

[Claim 1] Shares at least 46% structural and / or kinetic identity with the CD25 reference target. wherein the CD25 reference target is 【Table 1】 An engineered polypeptide which is a portion of CD25 selected from:

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