Novel peptides with affinity for PD-L1

Abstract

This disclosure relates to a class of engineered peptides with binding affinity for programmed death-ligand 1 (PD-L1), and provides a sequence comprising ERNX4AAX7EIL X 11 LPNLX 16 X 17 X 18 QX 20 WAFIWX 26 LX 28 PD-L1-binding peptides of type D. This disclosure also relates to the use of such PD-L1-binding peptides as therapeutic agents, prognostic agents, and / or diagnostic agents.

Description

Invention Field

[0001] This disclosure relates to a class of engineered peptides having binding affinity for programmed death-ligand 1 (hereinafter referred to as PD-L1). This disclosure also relates to the use of such PD-L1 binding peptides as therapeutic agents, prognostic agents, and / or diagnostic agents.

[0002] background

[0003] Under normal physiological conditions, immune checkpoints are crucial for maintaining self-tolerance (i.e., preventing autoimmunity) and regulating immune responses to protect tissues from damage when the immune system responds to pathogenic infections. Sometimes, tumor cells can co-opt certain immune checkpoint pathways to evade immune surveillance mechanisms. Therefore, inhibiting immune checkpoints has become a promising approach in cancer immunotherapy. The two most actively studied immune checkpoint receptors in this regard are cytotoxic T-lymphocyte-associated antigen (CTLA-4; also known as CD152) and programmed cell death protein 1 (PD-1; also known as CD279), which regulate immune responses at different levels. CTLA-4 primarily regulates the immune response in the early stages of T-cell activation, while PD-1 primarily restricts T-cell activity during the effector phase in tissues and tumors (Pardoll, 2012, Nat. Rev. Cancer, 12:252-64).

[0004] PD-1 has two known ligands: programmed death-ligand 1 (PD-L1; also known as human B7 homologue 1, B7-H1, or differentiation cluster 274, CD274) and programmed death-ligand 2 (PD-L2; also known as B7-DC and CD273). Both ligands belong to the B7 immunoglobulin superfamily and are type I transmembrane glycoproteins including IgC- and IgV-type extracellular domains. However, PD-L1 and PD-L2, as well as PD-1, have recently been reported to exist in soluble forms in addition to membrane binding. PD-L1 and PD-L2 share approximately 40% amino acid residue identity. However, PD-L2 expression is primarily limited to antigen-presenting cells, while PD-L1 is expressed in both hematopoietic and non-hematopoietic cells. High tumor expression of PD-L1 is associated with increased invasiveness and poor prognosis (Dai et al., 2014, Cellular Immunology, 290:72-79).

[0005] The clinical significance of targeting the immune checkpoint pathway has been demonstrated by several monoclonal antibodies that inhibit CTLA-4, PD-1, and PD-L1, which work by restoring the protective immune response against tumor cells. Anti-CTLA-4 antibody ipilimumab (ipilimumab) Bristol Myers Squibb was approved by the FDA in 2011 for the treatment of patients with metastatic melanoma, with durable responses observed in 10%–15% of patients. However, ipilimumab is associated with immune-related toxicity, likely due to its role in the initiation phase of the immune response, which also affects normal tissues. A safer approach could be to target the PD-1 / PD-L1 pathway to selectively restore anti-tumor immunity within the tumor microenvironment. Inhibition of the PD-1 / PD-L1 pathway has shown durable responses in 30%–35% of patients with advanced melanoma, resulting in the FDA approval of the anti-PD-1 antibody pembrolizumab (formerly known as lambolizumab) in 2014. Merck and nivolumab (Bristol Myers Squibband Ono Pharmaceutical) (Shin and Ribas, 2015, Curr. Opin. Immunol., 33:23-35; Philips and Atkins, 2015, International Immunology, 27:39-46). The first PD-L1-targeting antibody investigated in clinical trials was MDX-1105, which was evaluated in a phase I study in patients with advanced solid tumors, including melanoma, non-small cell lung cancer (NSCLC), colorectal cancer, renal cell carcinoma, ovarian cancer, pancreatic cancer, gastric cancer, and breast cancer (Momtaz and Postow, 2014, Pharmgenomics Pers Med. 7:357-65). Results demonstrated the potential benefit of PD-L1 blockade. Other antibodies targeting PD-L1 currently in Phase III clinical trials include atezolizumab (MDPL3280A, Genentech), durvalumab (MEDI4736, MedImmune / AstraZeneca, Celgene), and avelumab (MSB0010718C, EMD Serono, Pfizer).

[0006] To improve efficacy and increase the number of patients responding to immunotherapy, targeting the antitumor immune response at multiple levels can be beneficial. This can be achieved through synergistic combinations. For example, preclinical studies of combinations of CTLA-4 and PD-1 blocking antibodies (ipilimumab and nivolumab) have shown excellent antitumor activity, but with similar toxicities to anti-CTLA-4 monotherapy (Shin and Ribas, 2015, ibid.). Furthermore, PD-L1 is considered a potential biomarker due to its abundance in the tumor microenvironment and the strong correlation between PD-L1 tumor expression and response to anti-PD-1 / PD-L1 therapy.

[0007] The high incidence of cancer and infectious diseases, along with significant unmet medical needs, necessitates the development of new treatment modalities. Since tissue penetration is negatively correlated with molecular size, relatively large antibody molecules inherently exhibit poor tissue distribution and penetration capabilities.

[0008] Therefore, the use of monoclonal antibodies is not always optimal for treatment, and there remains a persistent need for agents with high affinity for PD-L1. There is also great interest in providing the use of such molecules in the treatment, diagnosis, and prognosis of PD-L1-related disorders. Invention Overview

[0010] The purpose of this disclosure is to provide novel PD-L1 binders that can be used, for example, for therapeutic, prognostic, and diagnostic applications.

[0011] The purpose of this disclosure is to provide novel multispecific agents, such as bispecific agents, that have affinity for PD-L1 and at least one other antigen.

[0012] The purpose of this disclosure is to provide a molecule that allows for effective treatments for, for example, various forms of cancer and inflammatory diseases, while mitigating the aforementioned and other drawbacks of current therapies.

[0013] The purpose of this disclosure is to provide a molecule suitable for prognostic and diagnostic applications, such as those relating to various forms of cancer and infectious diseases.

[0014] These and other objectives, which will be apparent to those skilled in the art from the content of this disclosure, are satisfied by the various aspects of the invention as claimed in the appended claims and as generally disclosed herein.

[0015] Therefore, in a first aspect of this disclosure, a PD-L1 binding polypeptide is provided, the PD-L1 binding polypeptide comprising a PD-L1 binding motif BM, the motif being composed of an amino acid sequence selected from:

[0016] i)ERNX4AAX7EIL X 11 LPNLX 16 X 17 X 18 QX 20 WAFIWX 26 LX 28 D

[0017] Among them, independently of each other,

[0018] X4 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, and Y;

[0019] X7 is selected from A, E, F, H, N, Q, S, T, V, W, and Y;

[0020] X 11 Selected from A, D, E, F, H, K, L, N, Q, R, S, T, V, W, and Y;

[0021] X 16 Selected from N and T;

[0022] X 17 Selected from A, H, K, N, Q, R, and S;

[0023] X 18 Selected from A, D, E, G, H, K, L, N, Q, R, S, T, V, and Y;

[0024] X 20 Selected from H, I, K, L, N, Q, R, T, V, and Y;

[0025] X 26 Selected from K and S; and

[0026] X 28 Selected from A, D, and E;

[0027] and

[0028] ii) An amino acid sequence that has at least 96% identity with the sequence defined in i).

[0029] The above definition of a class of sequence-related PD-L1-binding peptides is based on a statistical analysis of numerous random peptide variants of a parental scaffold, selected in a selection experiment for their interaction with PD-L1. The identified PD-L1 binding motif, or "BM," corresponds to the target binding region of the parental scaffold, which constitutes two α-helices within a triple-helix bundle protein domain. In the parental scaffold, altered amino acid residues of the two BM helices constitute a binding surface for interaction with the constant Fc portion of the antibody. In this disclosure, random variations of the binding surface residues and subsequent variant selection have replaced Fc interaction capability with the ability to interact with PD-L1.

[0030] As those skilled in the art will recognize, the function of any polypeptide, such as the PD-L1 binding capacity of the polypeptides of this disclosure, depends on the polypeptide's tertiary structure. Therefore, it is possible to make minor alterations to the amino acid sequence of a polypeptide without affecting its function. Consequently, this disclosure includes modified variants of the PD-L1-binding polypeptide that have retained the PD-L1 binding characteristics.

[0031] In this manner, this disclosure also includes PD-L1 binding peptides comprising an amino acid sequence having 96% or greater identity with the peptide defined as in i). For example, it is possible for amino acid residues belonging to a specific functional group of amino acid residues (e.g., hydrophobic, hydrophilic, polar, etc.) to be exchanged for another amino acid residue from the same functional group.

[0032] In some embodiments, such changes can be made at any position in the sequence of the PD-L1 binding peptide as disclosed herein. In other embodiments, such changes can be made only at non-variable positions, also known as scaffold amino acid residues. In such cases, changes at variable positions are not permitted. In other embodiments, such changes can be made only at variable positions. According to one definition of such “variable positions,” these are positions indicated by “X” in sequence i) as defined above. According to another definition, “variable positions” are those positions that are randomized in the selection library of Z variants prior to selection, and thus can be, for example, positions 2, 3, 4, 6, 7, 10, 11, 17, 18, 20, 21, 25, and 28 in sequence i), as illustrated in Example 1.

[0033] The term "% identity," used throughout this specification, can be calculated as follows: The query sequence is aligned with the target sequence using the CLUSTAL W algorithm (Thompson et al., (1994) Nucleic Acids Research, 22:4673-4680). Comparisons are made on a window corresponding to the shortest aligned sequence. In some cases, the shortest aligned sequence may be the target sequence. In other cases, the query sequence may constitute the shortest aligned sequence. Amino acid residues at each position are compared, and the percentage of positions in the query sequence that have the same counterpart in the target sequence is reported as the identity %.

[0034] In another embodiment, a PD-L1 binding peptide is provided, wherein in sequence i)

[0035] X4 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, and Y;

[0036] X7 is selected from E, F, H, N, Q, S, T, V, W, and Y;

[0037] X 11 Selected from A, D, H, L, Q, R, T, V, W, and Y;

[0038] X 16 Selected from N and T;

[0039] X 17 Selected from A, H, K, N, Q, R, and S;

[0040] X 18 Selected from A, D, E, G, H, K, L, N, Q, R, S, T, V, and Y;

[0041] X 20 Selected from H, I, K, L, Q, R, T, V, and Y;

[0042] X 26 Selected from K and S; and

[0043] X 28 Choose from A, D, and E.

[0044] In another embodiment, a PD-L1 binding peptide is provided, wherein in sequence i)

[0045] X4 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, and Y;

[0046] X7 is selected from A, E, F, H, N, Q, S, T, V, W, and Y;

[0047] X 11Selected from A, D, E, F, H, K, L, N, Q, R, S, T, V, W, and Y;

[0048] X 16 Selected from N and T;

[0049] X 17 Selected from A, H, K, N, Q, R, and S;

[0050] X 18 Selected from A, D, E, G, H, K, L, N, Q, R, S, T, V, and Y;

[0051] X 20 Selected from H, I, K, L, N, Q, R, T, V, and Y;

[0052] X 26 Selected from K and S; and

[0053] X 28 Choose from A, D, and E.

[0054] In another embodiment, a PD-L1 binding peptide is provided, wherein in sequence i)

[0055] X4 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, and V;

[0056] X7 is selected from F, H, Q, and Y;

[0057] X 11 Selected from H, Q, W, and Y;

[0058] X 16 Selected from N and T;

[0059] X 17 Selected from A, H, K, N, Q, and S;

[0060] X 18 Selected from A, E, G, H, K, L, N, Q, R, S, T, V, and Y;

[0061] X 20 Selected from H, I, K, Q, R, and V;

[0062] X 26 Selected from K and S; and

[0063] X 28 Choose from A and D.

[0064] As used in this article, “X” n "and "X m"X3" is used to indicate amino acids at positions n and m in sequence i) as defined above, where n and m are integers indicating the positions of amino acids in the sequence when counted from the N-terminus. For example, X3 and X7 indicate amino acids at positions 3 and 7 from the N-terminus of sequence i), respectively.

[0065] In an embodiment according to the first aspect, a polypeptide is provided, wherein X in sequence i) n Independently selected from a set of possible residues according to Table 1. Those skilled in the art will understand that X n It can be selected from any of the listed possible residue groups and the selection is independent of X. m The selection of amino acids in the formula, where n ≠ m. Therefore, the positions X listed in Table 1... n Any of the possible residues can be independently combined with any other possible residues at variable positions listed in Table 1.

[0066] Those skilled in the art will understand that Table 1 should be interpreted as follows: In one embodiment according to the first aspect, a polypeptide is provided, wherein the amino acid residue "X" in sequence i) n "Selected from "possible residues". Therefore, Table 1 discloses several specific and individualized embodiments of the first aspect of this disclosure. For example, in one embodiment according to the first aspect, a polypeptide is provided in which X4 in sequence i) is selected from A, D, E, I, K, L, N, Q, S, and T, and in another embodiment according to the first aspect, a polypeptide is provided in which X4 in sequence i) is selected from A, D, E, I, K, Q, S, and T. For the avoidance of doubt, the listed embodiments can be freely combined in other embodiments. For example, one such combination embodiment is where X4 is selected from A, D, E, I, K, Q, S, and T, and X7 is selected from F, H, Q, and Y, and X... 18 Polypeptides selected from A, L, K, and S.

[0067] Table 1

[0068]

[0069]

[0070] In a particular embodiment of the first aspect, a polypeptide is provided, wherein sequence i) satisfies at least four of the following seven conditions I-VII:

[0071] I.X7 is selected from F, H, Q, and Y;

[0072] II.X 11 Selected from H and Y;

[0073] III.X16 Let T be the value of T.

[0074] IV.X 17 Selected from N, Q, and S;

[0075] VX 20 Selected from H, I, K, and R;

[0076] VI.X 26 For K; and

[0077] VII.X 28 The answer is either A or D.

[0078] In one implementation, sequence i) satisfies at least five of the seven conditions I-VII, such as at least six of the seven conditions I-VII. In a particular implementation, sequence i) satisfies all seven conditions I-VII.

[0079] In some embodiments of the PD-L1 binding peptide according to the first aspect, X7X 11 X 20 Selected from FYK and YYK. In some implementations, X 11 X 17 X 20 Selected from YNK and YQK. In some implementations, X 11 X 18 X 20 For YAK.

[0080] As described in detail in the experimental section below, the selection of PD-L1 binding peptide variants has led to the identification of a large number of individual PD-L1 binding motif (BM) sequences. These sequences constitute individual embodiments according to sequence i). The sequence of the individual PD-L1 binding motif corresponds to Figure 1The amino acid positions 8-36 in SEQ ID NO:1-808 are shown. Therefore, in one embodiment of the PD-L1 binding polypeptide according to this aspect, sequence i) corresponds to the sequence from position 8 to position 36 in the sequence selected from the group consisting of SEQ ID NO:1-808. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 in the sequence selected from the group consisting of SEQ ID NO:1-93 and 774-796, such as the sequence consisting of SEQ ID NO:1-93 and 774-787. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 in the sequence selected from the group consisting of SEQ ID NO:1-93, 775, 776, 779-781 and 784-786, such as the group consisting of SEQ ID NO:1-93, 776, 780, 781, 784 and 786, such as the group consisting of SEQ ID NO:1-93, 776 and 784, or the group consisting of SEQ ID NO:1-93, 776 and 781, for example the group consisting of SEQ ID NO:1-93 and 776 or the group consisting of SEQ ID NO:1-93 and 781 or the group consisting of SEQ ID NO:1-93 and 784. In one embodiment, sequence i) corresponds to the group selected from SEQ ID NO:1-93, 774, 775, and 780-786, such as the sequence from position 8 to position 36 in the sequence of the group composed of SEQ ID NO:1-93, 775, 780, 781, 784, and 786. In one embodiment, sequence i) corresponds to the group selected from SEQ ID NO:1, 2, 17, 776, and 781, such as the sequence from position 8 to position 36 in the sequence of the group composed of SEQ ID NO:1, 2, and 776. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:776. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 in the sequence selected from the group composed of SEQ ID NO:1-93. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 selected from the group consisting of SEQ ID NO: 1, 3-8, 11, 13, 16, 18, 20, 22, 23, 43 and 73. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 selected from the group consisting of SEQ ID NO: 1-24.For example, in one embodiment, sequence i) corresponds to the sequence selected from the group consisting of SEQ ID NO:1-16, such as the sequence from position 8 to position 36 in the group consisting of SEQ ID NO:1, 2, 4, 5, 7, 9, and 10. In another embodiment, sequence i) corresponds to the sequence selected from the group consisting of SEQ ID NO:1, 3-6, 9-10, 12-21, 23, and 24, such as the sequence from position 8 to position 36 in the group consisting of SEQ ID NO:1, 4-6, 9, 14, and 18-21. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 in the group consisting of SEQ ID NO:1-12, 14, and 17-21. In one embodiment, sequence i) corresponds to the sequence selected from the group consisting of SEQ ID NO:1-12 and 17, such as the group consisting of SEQ ID NO:1-5 and 17, such as the sequence from position 8 to position 36 in the group consisting of SEQ ID NO:1, 2, and 17. In one embodiment, sequence i) corresponds to the group selected from SEQ ID NO:1, 4, 5, 6, 9, 14, and 18-21, such as the group selected from SEQ ID NO:4, 5, 18, and 21, such as the sequence from position 8 to position 36 in the sequence of the group selected from SEQ ID NO:4, 5, and 21. In one embodiment, sequence i) corresponds to the group selected from SEQ ID NO:1, 2, 4, 5, and 21, such as the sequence from position 8 to position 36 in the sequence of the group selected from SEQ ID NO:1 and 2. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:21.

[0081] In some embodiments of this disclosure, BM, as defined above, "forms" a portion of a triple-helix bundle protein domain. This is understood to mean that the sequence of BM is "inserted" into or "transplanted" onto the sequence of the original triple-helix bundle domain, such that BM replaces a similar structural motif in the original domain. For example, not wanting to be bound by theory, BM is considered to constitute two of the three helices of the triple-helix bundle and thus can replace any such double-helix motif within the triple-helix bundle. As those skilled in the art will recognize, the replacement of two helices of the triple-helix bundle domain by two BM helices must be done in a manner that does not affect the basic structure of the polypeptide. That is, the overall folding of the Cα backbone of the polypeptide according to this embodiment of the invention is substantially consistent with the overall folding of the triple-helix bundle protein domain to which it forms a portion, for example, having the same secondary structural elements in the same order. Therefore, if the polypeptide according to this embodiment has the same folding as the original domain, then the fact that BM "forms" a portion of the triple-helix bundle domain according to this disclosure implies shared basic structural characteristics, such as those that result in similar CD spectra. Other relevant parameters are known to those skilled in the art.

[0082] In certain embodiments, the PD-L1 binding motif (BM) thus forms part of a triple-helix bundle protein domain. For example, the BM may substantially constitute two α-helices with interconnected loops within the triple-helix bundle protein domain. In certain embodiments, the triple-helix bundle protein domain is selected from domains of bacterial receptor proteins. Non-limiting examples of such domains are the five different triple-helix domains of protein A from Staphylococcus aureus, such as domain B and its derivatives. In some embodiments, the triple-helix bundle protein domain is a variant of protein Z derived from domain B of Staphylococcus aureus protein A (Wahlberg E et al., 2003, PNAS 100(6):3185-3190).

[0083] In some embodiments where the PD-L1 binding peptide forms part of a triple-helix bundle protein domain, as disclosed herein, the PD-L1 binding peptide may include a binding module (BMod) whose amino acid sequence is selected from...

[0084] iii)K-[BM]-DPSQSX a X b LLX c EAKKLX d X e X f Q;

[0085] in,

[0086] [BM] is the PD-L1 binding motif as defined in this paper;

[0087] X a Selected from A and S;

[0088] X b Selected from N and E;

[0089] X c Selected from A, S, and C;

[0090] X d Selected from E, N, and S;

[0091] X e Selected from D, E, and S; and

[0092] X f Selected from A and S; and

[0093] The amino acid sequence that has at least 93% identity with the sequence defined in iv) and iii).

[0094] In some embodiments, the polypeptide may advantageously exhibit high structural stability during production and storage, as well as in vivo, such as tolerance to chemical modifications, changes in physical conditions, and protein hydrolysis.

[0095] As discussed above, polypeptides containing minor alterations to the amino acid sequences described above that do not significantly affect the tertiary structure and function of the polypeptide are also within the scope of this disclosure. Therefore, in some embodiments, sequence iv) has at least 93%, such as at least 95%, such as at least 97%, identity with the sequence defined by iii).

[0096] In one implementation, X in sequence iii) a The answer is A.

[0097] In one implementation, X in sequence iii) a Let it be S.

[0098] In one implementation, X in sequence iii) b Let N be the number of elements in the array.

[0099] In one implementation, X in sequence iii) b The value is E.

[0100] In one implementation, X in sequence iii) c The answer is A.

[0101] In one implementation, X in sequence iii) c Let it be S.

[0102] In one implementation, X in sequence iii) c The answer is C.

[0103] In one implementation, X in sequence iii) d The value is E.

[0104] In one implementation, X in sequence iii) d Let N be the number of elements in the array.

[0105] In one implementation, X in sequence iii) d Let it be S.

[0106] In one implementation, X in sequence iii) e The answer is D.

[0107] In one implementation, X in sequence iii) e The value is E.

[0108] In one implementation, X in sequence iii) e Let it be S.

[0109] In one implementation, X in sequence iii) d X e Selected from EE, ES, SD, SE, and SS.

[0110] In one implementation, X in sequence iii) d X e For ES.

[0111] In one implementation, X in sequence iii) d X e It is SE.

[0112] In one implementation, X in sequence iii) d X e For SD.

[0113] In one implementation, X in sequence iii) f The answer is A.

[0114] In one implementation, X in sequence iii) f Let it be S.

[0115] In one implementation, in sequence iii), X a For A; X b For N; X c For A and X f The answer is A.

[0116] In one implementation, in sequence iii), X a S; Xb For E; X c For A and X f The answer is A.

[0117] In one implementation, in sequence iii), X a For A; X b For N; X c C and X f The answer is A.

[0118] In one implementation, in sequence iii), X a S; X b For E; X c For S and X f Let it be S.

[0119] In one implementation, in sequence iii), X a S; X b For E; X c For C and X f Let it be S.

[0120] In one implementation, in sequence iii), X a For A; X b For N; X c For A; X d X e For ND and X f The answer is A.

[0121] In one implementation, in sequence iii), X a S; X b For E; X c For A; X d X e For ND and X f The answer is A.

[0122] In one implementation, in sequence iii), X a For A; X b For N; X c For C; X d X e For ND and X f The answer is A.

[0123] In one implementation, in sequence iii), X a S; X b For E; X c S; X d X e For ND and X f Let it be S.

[0124] In one implementation, in sequence iii), X a S; X b For E; X c For C; X d X e For ND and X f Let it be S.

[0125] In one implementation, in sequence iii), X a For A; X b For N; X c For A; X d X e For SE and X f The answer is A.

[0126] In one implementation, in sequence iii), X a S; X b For E; X c For A; X d X e For SE and X f The answer is A.

[0127] In one implementation, in sequence iii), X a For A; X b For N; X c For C; X d X e For SE and X f The answer is A.

[0128] In one implementation, in sequence iii), X a S; X b For E; X c S; X d X e For SE and X f Let it be S.

[0129] In one implementation, in sequence iii), X a S; X b For E; X c For C; X d X e For SE and X f Let it be S.

[0130] In one implementation, in sequence iii), X a For A; X b For N; X c For A; X d X e For SD and X f The answer is A.

[0131] In one implementation, in sequence iii), X a S; X b For E; X c For A; X d X e For SD and X f The answer is A.

[0132] In one implementation, in sequence iii), X a For A; X b For N; X c For C; X d X e For SD and X f The answer is A.

[0133] In one implementation, in sequence iii), X a S; X b For E; X c S; X d X e For SD and X f Let it be S.

[0134] In one implementation, in sequence iii), X a S; X b For E; X c For C; X d X e For SD and X f Let it be S.

[0135] In another implementation, sequence iii) corresponds to the selected free... Figure 1The sequence shown is from position 7 to position 55 of the sequence comprising the group consisting of SEQ ID NO:1-808. Therefore, in one embodiment of the PD-L1 binding peptide according to this aspect, sequence iii) corresponds to the sequence from position 7 to position 55 selected from the sequence comprising the group consisting of SEQ ID NO:1-808. In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 selected from the group consisting of SEQ ID NO:1-93 and 774-796, such as the sequence comprising SEQ ID NO:1-93 and 774-787. In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in the sequence selected from the group consisting of SEQ ID NO:1-93, 775, 776, 779-781 and 784-786, such as the group consisting of SEQ ID NO:1-93, 776, 780, 781, 784 and 786, such as the group consisting of SEQ ID NO:1-93, 776 and 784, such as the group consisting of SEQ ID NO:1-93, 776 and 784, or the group consisting of SEQ ID NO:1-93, 776 and 781, for example, the group consisting of SEQ ID NO:1-93 and 776 or the group consisting of SEQ ID NO:1-93 and 781 or the group consisting of SEQ ID NO:1-93 and 784. In one embodiment, sequence iii) corresponds to the sequence selected from the group consisting of SEQ ID NO:1-93, 774, 775, and 780-786, such as the sequence from position 7 to position 55 in the sequence consisting of the group consisting of SEQ ID NO:1-93, 775, 780, 781, 784, and 786. In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in the group consisting of SEQ ID NO:1, 2, 17, 776, and 781, such as the sequence from position 7 to position 55 in the sequence consisting of the group consisting of SEQ ID NO:1, 2, and 776. In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in the sequence selected from the group consisting of SEQ ID NO:1-93. In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 selected from the group consisting of SEQ ID NO: 1, 3-8, 11, 13, 16, 18, 20, 22, 23, 43 and 73. In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 selected from the group consisting of SEQ ID NO: 1-24.For example, in one embodiment, sequence iii) corresponds to the sequence selected from the group consisting of SEQ ID NO:1-16, such as the sequence from position 7 to position 55 in the group consisting of SEQ ID NO:1, 2, 4, 5, 7, 9, and 10. In another embodiment, sequence iii) corresponds to the sequence selected from the group consisting of SEQ ID NO:1, 3-6, 9-10, 12-21, 23, and 24, such as the sequence from position 7 to position 55 in the group consisting of SEQ ID NO:1, 4-6, 9, 14, and 18-21. In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in the group consisting of SEQ ID NO:1-12, 14, and 17-21. In one embodiment, sequence iii) corresponds to the sequence selected from the group consisting of SEQ ID NO:1-12 and 17, such as the sequence consisting of SEQ ID NO:1-5 and 17, such as the sequence from position 7 to position 55 in the group consisting of SEQ ID NO:1, 2, and 17. In one embodiment, sequence iii) corresponds to the group selected from SEQ ID NO:1, 4, 5, 6, 9, 14, and 18-21, such as the group selected from SEQ ID NO:4, 5, 18, and 21, such as the sequence from position 7 to position 55 in the sequence of the group selected from SEQ ID NO:4, 5, and 21. In one embodiment, sequence iii) corresponds to the group selected from SEQ ID NO:1, 2, 4, 5, and 21, such as the sequence from position 8 to position 36 in the sequence of the group selected from SEQ ID NO:1 and 2. In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:21.

[0136] Furthermore, in another embodiment, a PD-L1 binding polypeptide is provided, the PD-L1 binding polypeptide comprising an amino acid sequence selected from the following:

[0137] v)YA-[BMod]-AP;

[0138] Where [BMod] is the PD-L1 binding module as defined in this paper; and

[0139] vi) has an amino acid sequence that is at least 90% identical to the sequence defined in v).

[0140] Optionally, a PD-L1 binding polypeptide is provided, the PD-L1 binding polypeptide comprising an amino acid sequence selected from the following:

[0141] vii)FN-[BMod]-AP;

[0142] Where [BMod] is the PD-L1 binding module as defined in this paper; and

[0143] The sequences defined in viii) and vii) have at least 90% amino acid sequence identity.

[0144] For example, in one embodiment, a PD-L1 binding peptide is provided, the PD-L1 binding peptide being selected from the group consisting of:

[0145] ix)FNK-[BM]-DPSQS ANLLX c EAKKL NDAQA P;

[0146] Where [BM] is the PD-L1 binding motif as defined above, and X c Selected from A and C; and

[0147] The sequences defined in x) and ix) have at least 90% amino acid identity.

[0148] In another embodiment, a PD-L1 binding polypeptide is provided, the PD-L1 binding polypeptide being selected from the group consisting of:

[0149] xi)FAK-[BM]-DPSQS SELLX c EAKKL SESQA P;

[0150] Where [BM] is the PD-L1 binding motif as defined above, and X c Selected from A, S, and C; and

[0151] The sequences defined in xii) and xi) have at least 90% amino acid identity.

[0152] In another embodiment, a PD-L1 binding polypeptide is provided, the PD-L1 binding polypeptide being selected from the group consisting of:

[0153] xiii) FAK-[BM]-DPSQS SELLX c EAKKL NDSQA P;

[0154] Where [BM] is the PD-L1 binding motif as defined above, and X c Selected from A, S, and C;

[0155] The sequences defined in xiv) and xiii) have amino acid sequences that are at least 90% identical.

[0156] In another embodiment, a PD-L1 binding polypeptide is provided, wherein the PD-L1 binding polypeptide is selected from the group consisting of:

[0157] xv)YAK-[BM]-DPSQS SELLX c EAKKL NDSQA P;

[0158] Where [BM] is the PD-L1 binding motif as defined above, and X c Selected from A, S, and C;

[0159] The sequences defined in xvi) and xv) have at least 90% amino acid identity.

[0160] As discussed above, polypeptides containing minor alterations to the amino acid sequences described above that do not significantly affect the tertiary structure and function of the polypeptide also fall within the scope of this disclosure. Therefore, in some embodiments, sequences vi), viii), x), xii), xiv), or xvi) may be, for example, at least 90%, such as at least 92%, such as at least 94%, such as at least 96%, such as at least 98%, respectively identical to the sequences defined by v), vii), ix), xi), xiii), and xv).

[0161] In some embodiments, the PD-L1 binding motif can form part of a polypeptide comprising an amino acid sequence selected from:

[0162] ADNNFNK-[BM]-DPSQSANLLSEAKKLNESQAPK;

[0163] ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK;

[0164] ADNKFNK-[BM]-DPSVSKEILAEAKKLNDAQAPK;

[0165] ADAQQNNFNK-[BM]-DPSQSTNVLGEAKKLNESQAPK;

[0166] AQHDE-[BM]-DPSQSANVLGEAQKLNDSQAPK;

[0167] VDNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK;

[0168] AEAKYAK-[BM]-DPSESSELLSEAKKLNKSQAPK;

[0169] VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK;

[0170] VDAKYAK-[BM]-DPSQSSELLAEAKKLNDSQAPK;

[0171] AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;

[0172] AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAP;

[0173] AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;

[0174] AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAP;

[0175] AEAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK;

[0176] AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK;

[0177] AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAP;

[0178] AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK;

[0179] AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAP;

[0180] AEAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK;

[0181] AEAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK;

[0182] AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK;

[0183] AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAP;

[0184] AEAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK;

[0185] AEAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK;

[0186] AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK;

[0187] AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAP;

[0188] AEAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK;

[0189] AEAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK;

[0190] AEAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK;

[0191] VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;

[0192] VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK;

[0193] VDAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK;

[0194] VDAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK;

[0195] VDAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK;

[0196] VDAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK;

[0197] VDAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK;

[0198] VDAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK;

[0199] VDAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK;

[0200] VDAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK;

[0201] VDAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK;

[0202] VDAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK;

[0203] VDAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK;

[0204] AEAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK; and

[0205] ADAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;

[0206] Where [BM] is the PD-L1 binding motif as defined in this paper.

[0207] In one embodiment, the PD-L1 binding polypeptide comprises an amino acid sequence selected from the following:

[0208] xvii)VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;

[0209] Where [BM] is the PD-L1 binding motif as defined in this paper; and

[0210] The sequences defined in xviii) and xvii) have at least 89% amino acid identity.

[0211] In one embodiment, the PD-L1 binding polypeptide comprises an amino acid sequence selected from the following:

[0212] xix)AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK;

[0213] Where [BM] is the PD-L1 binding motif as defined in this paper; and

[0214] The amino acid sequence that has at least 89% identity with the sequence defined in xix) is xx).

[0215] In one embodiment, the PD-L1 binding polypeptide comprises an amino acid sequence selected from the following:

[0216] xxi)AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;

[0217] Where [BM] is the PD-L1 binding motif as defined in this paper; and

[0218] The amino acid sequences defined in xxii) and xxi) have at least 89% identity.

[0219] In one embodiment, the PD-L1 binding polypeptide comprises an amino acid sequence selected from the following:

[0220] xxiii)AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;

[0221] Where [BM] is the PD-L1 binding motif as defined in this paper; and

[0222] The amino acid sequences defined in xxiv) and xiii) have at least 89% identity.

[0223] Furthermore, polypeptides containing minor alterations to the amino acid sequences described above that do not significantly affect the tertiary structure and function of the polypeptide also fall within the scope of this disclosure. Therefore, in some embodiments, sequences xviii), xx), xxii), or xxiv) may be, for example, at least 89%, such as at least 91%, such as at least 93%, such as at least 94%, such as at least 96%, such as at least 98%, respectively identical to the sequences defined by xvii), xix), xxi), and xxiii).

[0224] The sequence xvii) or xxi) in such polypeptides can be selected freely. Figure 1The group consisting of SEQ ID NO:1-814 is shown in the figure. In one embodiment of the PD-L1 binding polypeptide according to this aspect, sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 selected from the group consisting of SEQ ID NO:1-808. In one embodiment, sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 selected from the group consisting of SEQ ID NO:1-93, 774-796 and 809-814, such as the sequence from position 1 to position 58 of the group consisting of SEQ ID NO:1-93, 774-787 and 809-814. In one embodiment, sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in the sequence selected from the group consisting of SEQ ID NO:1-93, 775, 776, 779-781, 784-786 and 809-814, such as the group consisting of SEQ ID NO:1-93, 776, 780, 781, 784, 786 and 809-814, such as the group consisting of SEQ ID NO:1-93, 776, 781, 784 and 809-814, such as the group consisting of SEQ ID NO:1-93, 776, 784, 809 and 811-814, or the sequence consisting of the group consisting of SEQ ID NO:1-93, 776, 781, 809 and 811-814. In one embodiment, sequence xvii) or xxi) corresponds to a sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO: 1-93, 776, 809 and 811-814, or the group consisting of SEQ ID NO: 1-93, 781, 809 and 811-814, or the group consisting of SEQ ID NO: 1-93, 784 and 811-814. In one embodiment, sequence xvii) or xxi) corresponds to a sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO: 1-93, 774, 775, 780-786 and 810-814, such as the group consisting of SEQ ID NO: 1-93, 775, 780, 781, 784, 786 and 810-814. In one embodiment, sequence xvii) or xxi) corresponds to the group selected from SEQ ID NO: 1-93, 776, 781, and 809-813, such as the sequence from position 1 to position 58 in the sequence of the group composed of SEQ ID NO: 1-93, 781, and 810-813. In one embodiment, sequence xvii) or xxi) corresponds to the group selected from SEQ ID NO: 1, 2, 17, 776, 781, and 809-812, such as the sequence from position 1 to position 58 in the sequence of the group composed of SEQ ID NO: 1, 2, 776, 809, 811, and 812.In one embodiment, sequence xvii) corresponds to the sequence selected from the group consisting of SEQ ID NO:1-93, 776, and 781, such as the sequence from position 1 to position 58 in the sequence consisting of the group consisting of SEQ ID NO:1-93 and 781. In one embodiment, sequence xvii) corresponds to the sequence selected from the group consisting of SEQ ID NO:1, 2, 17, 776, and 781, such as the sequence from position 1 to position 58 in the sequence consisting of the group consisting of SEQ ID NO:1, 2, and 776. In one embodiment, sequence xvii) corresponds to the sequence from position 1 to position 58 in the sequence consisting of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:776. In one embodiment, sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in the sequence selected from the group consisting of SEQ ID NO:1-93 and 811-813. In one embodiment, sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in the sequence selected from the group consisting of SEQ ID NO: 1, 3-8, 11, 13, 16, 18, 22, 23, 43, 73 and 811-813. In one embodiment, sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in the sequence selected from the group consisting of SEQ ID NO: 1-24 and 811-813. For example, in one embodiment, sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in the group consisting of SEQ ID NO: 1-16 and 811-813, such as the sequence consisting of SEQ ID NO: 1, 2, 4, 5, 7, 9, 10, 811 and 812. In another embodiment, sequence xvii) or xxi) corresponds to the group consisting of SEQ ID NO: 1, 3-6, 9-10, 12-21, 23, 24, 811, and 812, such as the sequence from position 1 to position 58 in the group consisting of SEQ ID NO: 1, 4-6, 9, 14, 18-21, 811, and 812. In one embodiment, sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in the group consisting of SEQ ID NO: 1-12, 14, 17-21, and 811-812. In one embodiment, sequence xvii) or xxi) corresponds to the group consisting of SEQ ID NO: 1-12, 17, 811, and 812, such as the group consisting of SEQ ID NO: 1-5, 17, 811, and 812, such as the sequence from position 1 to position 58 in the group consisting of SEQ ID NO: 1, 2, 17, 811, and 812.In one embodiment, sequence xvii) or xxi) corresponds to the group selected from SEQ ID NO: 1, 4, 5, 6, 9, 14, 18, 19, 20, 21, and 811, such as the group selected from SEQ ID NO: 4, 5, 18, and 21, such as the sequence from position 1 to position 58 in the sequence selected from SEQ ID NO: 4, 5, and 21. In one embodiment, sequence xvii)) corresponds to the group selected from SEQ ID NO: 1, 2, 4, 5, and 21, such as the sequence from position 8 to position 36 in the sequence selected from SEQ ID NO: 1 and 2. In one embodiment, sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in SEQ ID NO: 1 or 811. In one embodiment, sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in SEQ ID NO: 2 or 812. In one implementation, the sequence xvii corresponds to the sequence from position 1 to position 58 in SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:21.

[0225] As used in this specification, the terms “PD-L1 binding” and “PD-L1 binding affinity” refer to properties of peptides that can be tested, for example, by ELISA or by using surface plasmon resonance (SPR) technology.

[0226] For example, as described in the following examples, PD-L1 binding affinity can be tested in an experiment in which a peptide sample is captured on an antibody-coated ELISA plate, and biotinylated PD-L1 is added, followed by streptavidin-conjugated HRP. A TMB substrate is added, and a multi-well plate reader, such as Victor, is used. 3 The absorbance at 450 nm is measured using a PerkinElmer. A technician can then interpret the results obtained through such experiments to establish at least a qualitative measurement of the binding affinity of the peptide to PD-L1. If a quantitative measurement is desired, for example, to determine the EC50 value (half-maximum effective concentration) of the interaction, an ELISA can also be used. The response of a dilution series of peptides to biotinylated PD-L1 is measured using an ELISA as described above. The technician can then interpret the results obtained through such experiments, and the EC50 value can be calculated from the results using, for example, GraphPad Prism 5 and nonlinear regression.

[0227] PD-L1 binding affinity can also be tested in experiments in which PD-L1 or a fragment thereof is immobilized on the sensor chip of a surface plasmon resonance (SPR) instrument, and a sample containing the peptide to be tested passes through the chip. Alternatively, the peptide to be tested is immobilized on the instrument's sensor chip, and a sample containing PD-L1 or a fragment thereof passes through the chip. The results obtained by a technician can then be interpreted to at least establish a qualitative measurement of the peptide's binding affinity to PD-L1. If quantitative measurement is desired, for example, to determine the K0 of the interaction... D The binding value can also be determined using surface plasmon resonance (SPR) methods. Binding values ​​can be defined in instruments such as Biacore (GE Healthcare) or ProteOn XPR36 (Bio-Rad). PD-L1 is suitably immobilized on the instrument's sensor chip, and samples of the peptides whose affinity to be determined are prepared by serial dilution and injected in a random sequence. Then, K... D The values ​​can be calculated from the results using, for example, the 1:1 Langmui combined model of BIAevaluation 4.1 software provided by the instrument manufacturer, or other suitable software.

[0228] As used in this disclosure, the terms “albumin binding” and “binding affinity to albumin” refer to the properties of a peptide that can be tested using SPR in a manner similar to the examples described above regarding PD-L1, for example, in a Biacore instrument or a ProteOn XPR36 instrument.

[0229] In one embodiment, the PD-L1 binding peptide is able to bind to PD-L1, such that K interacts with PD-L1. D The value is at most 2×10 -8 M, such as at most 1×10 -8 M, such as at most 1×10 -9 M, such as at most 5×10 -10 M, such as at most 3×10 -10 M.

[0230] In one embodiment, the PD-L1 binding peptide is able to bind to PD-L1, such that k interacts with PD-L1. d The value is at most 1×10 -3 s -1 Such as at most 6×10 -4 s -1 .

[0231] In one embodiment, a PD-L1 binding polypeptide according to any of the foregoing items is provided, the PD-L1 binding polypeptide being capable of binding PD-L1 such that the EC50 value of the interaction is at most 1 × 10⁻⁶. -9 M, such as at most 1×10 -10 M, such as at most 7×10 -11 M.

[0232] The binding of peptides as defined herein to PD-L1 can interfere with in vivo or in vitro signaling via PD-L1. When PD-L1 binds to PD-1, the ligand / receptor interaction inhibits T lymphocyte responses by, for example, inhibiting enzymes involved in T lymphocyte activation. Therefore, blocking the binding of PD-L1 to PD-1 restores T lymphocyte responses. Blocking activity can be quantified, for example, by the half-maximal inhibitory concentration (IC50), a measure of a substance's potency in inhibiting a specific biological or biochemical function. This quantitative measurement indicates how much of a specific substance is required to inhibit a given biological process by half and is frequently used in the art.

[0233] Therefore, in one embodiment, a PD-L1 binding peptide as defined herein is provided, which blocks PD-L1-dependent signaling. In one embodiment, the half-maximum inhibitory concentration (IC50) for blocking is at most 5 × 10⁻⁶. -8 M, such as at most 1×10 -8 M, such as at most 5×10 -9 M, such as at most 3.5 × 10 -9 M, such as at most 1×10 - 9 M, such as at most 5×10 -10 M, such as at most 1×10 -10 M. In one embodiment, the PD-L1 binding peptide can block the interaction between PD-L1 and PD-1.

[0234] In one embodiment, the PD-L1 is human PD-L1. In another embodiment, the PD-L1 is rhesus monkey PD-L1.

[0235] Those skilled in the art will understand that various modifications and / or additions can be made to the PD-L1 binding peptide according to any aspect of the disclosure herein to suit the peptide for a particular application without departing from the scope of this disclosure.

[0236] For example, in one embodiment, a PD-L1 binding polypeptide as described herein is provided, said PD-L1 binding polypeptide having been extended at the C-terminus and / or N-terminus and / or containing additional amino acids. Such polypeptides should be understood as polypeptides having one or more additional amino acid residues at the first and / or last position in the polypeptide chain. Therefore, a PD-L1 binding polypeptide can contain any suitable number of additional amino acid residues, such as at least one additional amino acid residue. Each additional amino acid residue can be added individually or collectively to, for example, improve and / or simplify the production, purification, in vivo or in vitro stability, conjugation, or detection of the polypeptide. Such additional amino acid residues can comprise one or more amino acid residues added for the purpose of chemical conjugation. One such example is the addition of a cysteine ​​residue. Other amino acid residues can also provide "tags" for the purification or detection of peptides, such as His6 tags, (HisGlu)3 tags ("HEHEHE" tags), or "myc" (c-myc) tags or "FLAG" tags, for interaction with tag-specific antibodies or for immobilization metal affinity chromatography (IMAC) in the case of His6 tags.

[0237] In one embodiment, a PD-L1 binding polypeptide as described herein is provided, the PD-L1 binding polypeptide comprising additional amino acids at a C-terminus and / or N-terminus. For example, in one embodiment of a PD-L1 binding polypeptide disclosed herein, the PD-L1 binding polypeptide consists of any of the sequences disclosed herein, having from 0 to 15 additional C-terminal and / or N-terminal residues, such as from 0 to 7 additional C-terminal and / or N-terminal residues. In one embodiment, the PD-L1 binding polypeptide consists of any of the sequences disclosed herein, having from 0 to 15, such as from 0 to 4, such as 3 additional C-terminal residues. In a particular embodiment, the PD-L1 binding polypeptide described herein comprises additional C-terminal residues VDC or VEC.

[0238] Other amino acids, as discussed above, can be coupled to PD-L1 binding peptides by chemical conjugation (using known organic chemical methods) or by any other method, such as expressing PD-L1 binding peptides as fusion proteins or by any other means directly or via linkers, such as amino acid linkers.

[0239] Furthermore, additional polypeptide domains can provide another PD-L1 binding moiety. Therefore, in another embodiment, a PD-L1 binding polypeptide in a multimeric form is provided. The multimer is understood to comprise at least two PD-L1 binding polypeptides as disclosed herein as monomer units, the amino acid sequences of which may be identical or different. The multimeric polypeptide may comprise a suitable number of domains, each domain having a PD-L1 binding motif, and each domain forming a monomer within the multimer. These domains may have the same amino acid sequence, but optionally, they may have different amino acid sequences. In other words, the PD-L1 binding polypeptide of the present invention can form homomeric or heteromeric forms, such as homodimers or heterodimers. In one embodiment, a PD-L1 binding polypeptide is provided wherein the monomer units are covalently coupled together. In another embodiment, the PD-L1 binding polypeptide monomer units are expressed as a fusion protein. In one embodiment, a PD-L1 binding polypeptide in a dimer form is provided. In a particular embodiment, the dimer form is a homodimer form. In another embodiment, the dimer form is a heterodimer form. For clarity, throughout this disclosure, the term "PD-L1 binding polypeptide" is used to include all forms of PD-L1 binding polypeptides, i.e., monomeric and multimeric forms.

[0240] The additional amino acids discussed above can, for example, constitute one or more additional polypeptide domains. These additional polypeptide domains can provide a PD-L1 binding dimer with another function, such as, for example, another binding function, or an enzymatic function, or a toxic function, or a fluorescent signal transduction function, or a combination thereof.

[0241] Furthermore, it may be advantageous for the PD-L1 binding peptide, as defined herein, to be part of a fusion protein or conjugate comprising a second or additional moiety. The second and one or more moiety / moieties of the fusion peptide or conjugate in such proteins may suitably possess the desired biological activity.

[0242] Therefore, in a second aspect of this disclosure, a fusion protein or conjugate is provided, the fusion protein or conjugate comprising a first portion consisting of a PD-L1 binding polypeptide according to the first aspect, and a second portion consisting of a polypeptide having a desired biological activity. In another embodiment, the fusion protein or conjugate may additionally comprise a further portion having a desired biological activity, which may be the same as or different from the biological activity of the second portion.

[0243] Non-limiting examples of desired biological activities include therapeutic activity, binding activity, and enzymatic activity. In one embodiment, the second portion having the desired biological activity is a therapeutically active peptide. In one embodiment, the second portion is an immune response modifier. In another embodiment, the two portions are anticancer agents.

[0244] In one embodiment of the first or second aspect of this disclosure, a PD-L1 binding peptide, fusion protein, or conjugate is provided, said PD-L1 binding peptide, fusion protein, or conjugate comprising an immune response modulator. Other non-limiting examples of immune response modulators include immunomodulators or other anti-inflammatory agents.

[0245] In one embodiment of the first or second aspect of this disclosure, a PD-L1 binding polypeptide, fusion protein, or conjugate is provided, said PD-L1 binding polypeptide, fusion protein, or conjugate comprising an anticancer agent. Non-limiting examples of anticancer agents include agents selected from the group consisting of: auristatin, anthracycline, calicheamycin, compbretastatin, doxorubicin, duocarmycin, CC-1065 antitumor antibiotic, ecteinascidin, geldanamycin, maytansinoid, methotrexate, fungal toxins, taxol, ricin, bouganin, white tree toxin, Pseudomonas exotoxin 38 (PE38), diphtheria toxin (DT), and analogs thereof, derivatives thereof, and combinations thereof. Those skilled in the art will appreciate that non-limiting examples of anticancer agents include all possible variations of the agents, such as the agent olistatin, which is intended to include, for example, olistatin E, olistatin F, olistatin PE, and their derivatives.

[0246] Non-limiting examples of therapeutically active peptides are biomolecules, such as molecules selected from the group consisting of human endogenous enzymes, hormones, growth factors, chemokines, cytokines, and lymphokines.

[0247] Non-limiting examples of binding activity include binding activity that increases the in vivo half-life of the fusion protein or conjugate, and binding activity that exerts a blocking effect on biological activity. One example of such binding activity is binding activity that increases the in vivo half-life of the fusion protein or conjugate. In one embodiment of the fusion protein or conjugate, the in vivo half-life of the fusion protein or conjugate is longer than the in vivo half-life of the polypeptide itself having the desired biological activity. In one embodiment, the in vivo half-life is increased by at least 10-fold, such as at least 25-fold, such as at least 50-fold, such as at least 75-fold, such as at least 100-fold, compared to the in vivo half-life of the fusion protein or conjugate itself.

[0248] The fusion protein or conjugate may contain at least one additional portion. In one particular embodiment, the target is albumin that binds to it, increasing the in vivo half-life of the fusion protein or conjugate. In one embodiment, the albumin-binding activity is provided by the albumin-binding domain (ABD) of a streptococcal G protein or a derivative thereof. Thus, the fusion protein may, for example, comprise a PD-L1 binding polypeptide in monomeric or multimeric form (such as homodimer or heterodimer) and the albumin-binding domain of a streptococcal G protein or a derivative thereof, as defined herein.

[0249] In another embodiment, the provided fusion protein or conjugate wherein the second portion having the desired binding activity is a protein Z based on the B domain of protein A derived from Staphylococcus aureus, which has binding affinity for targets other than PD-L1.

[0250] For example, the fusion protein or conjugate comprising at least one additional portion may comprise [PD-L1 binding polypeptide] - [albumin binding moiety] - [migration moiety having affinity for a selected target]. It should be understood that the three portions in this example can be arranged in any order from the N-terminus to the C-terminus of the polypeptide.

[0251] Those skilled in the art know that the construction of fusion proteins typically involves the use of linkers between functional parts to be fused, and that different types of linkers with varying properties exist, such as flexible amino acid linkers, rigid amino acid linkers, and cleavable amino acid linkers. Linkers have been used, for example, to increase the stability of fusion proteins or improve the folding of fusion proteins to increase fusion protein expression, improve the biological activity of fusion proteins, allow fusion protein targeting, and alter the pharmacokinetics of fusion proteins. Therefore, in one embodiment, the polypeptide according to any aspect disclosed herein further comprises at least one linker, such as at least one selected from: flexible amino acid linkers, rigid amino acid linkers, and cleavable amino acid linkers. In one embodiment, the linker is arranged between the PD-L1 binding polypeptide and another polypeptide domain, such as between a PD-L1 binding domain as disclosed herein and an antibody or its antigen-binding fragment (as further detailed below). Flexible linkers are commonly used in the art when the linked domains require a degree of mobility or interaction, and can be particularly useful in some embodiments of the complex. Such linkers typically comprise small, nonpolar amino acids (e.g., G) or polar amino acids (e.g., S or T). Some flexible linkers are mainly composed of segments of G and S residues, such as (GGGGS). p Adjusting the copy number "p" allows for optimization of the linker to achieve proper separation between functional parts or to maintain necessary inter-part interactions. Besides G and S linkers, other flexible linkers are known in the art, such as G and S linkers containing additional amino acid residues such as T and A to maintain flexibility, and polar amino acid residues to improve solubility. Further non-limiting examples of linkers include GGGGSLVPRGSGGGGS, (GS)3, (GS)4, (GS)8, GGSGGHMGSGG, GGSGGSGGSGG, GGSGG, GGSGGGGG, GGGSEGGGSEGGGSEGGG, AAGAATAA, GGGGG, GGSSG, GGSGGTGGGSG, GGSGGTGGGSG, GGSGSGSGSGSGSGGSG, and GGSGSGSGSGSGSGSGGSG, as well as GT, corresponding to SEQ ID NO:820-836, respectively. Other suitable linkers are known to those skilled in the art.

[0252] In one embodiment, the linker is a flexible linker comprising glycine (G), serine (S), and / or threonine (T) residues. In one embodiment, the linker has a composition selected from (G) n S m ) p and (S) n G m ) pThe general formula is given, wherein independently, n = 1-7, m = 0-7, n+m ≤ 8, and p = 1-7. In one embodiment, n = 1-5. In one embodiment, m = 0-5. In one embodiment, p = 1-5. In a more specific embodiment, n = 4, m = 1, and p = 1-4. In one embodiment, the connector is selected from the group consisting of S4G, (S4G)3, and (S4G)4. In one embodiment, the connector is selected from the group consisting of G4S and (G4S)3. In a particular embodiment, the connector is G4S, and in another embodiment, the connector is (G4S)3.

[0253] Regarding the above description of fusion proteins or conjugates incorporating PD-L1 binding peptides according to this disclosure, it should be noted that the designations of the first, second, and additional portions are for clarity, distinguishing on one hand from one or more PD-L1 binding peptides according to the invention, and on the other hand from portions exhibiting other functions. These designations are not intended to indicate the actual order of different domains in the polypeptide chain of the fusion protein or conjugate. Similarly, for clarity, the first and second monomer units are named to distinguish the units. Thus, for example, the first portion (or monomer unit) may appear without limitation at the N-terminus, middle, or C-terminus of the fusion protein or conjugate.

[0254] Recent advancements have been made in the development of multispecific agents, such as antibodies capable of binding to more than one antigen. These advancements include engineered complementarity-determining regions (CDRs) to satisfy two antigens at a single antibody binding site (Bostrom et al., 2009, Science 323(5921):1610-1614; Schaefer et al., 2011, Cancer Cell 20(4):472-486), the construction of heterodimeric antibodies using engineered Fc units (Carter, 2001, J Immunol Methods 248(1-2):7-15; Schaefer et al., 2011, Proc Natl Acad Sci USA 108(27):11187-11192), and the fusion of helper recognition unit genes into the N-terminus or C-terminus of the light or heavy chain of a full-length antibody (Kanakaraj et al., 2012, MAbs). 4(5):600-613; LaFleur et al., 2013, MAbs 5(2):208-218). Therefore, it may be beneficial to incorporate molecules that have an affinity for PD-L1 as disclosed herein as also having an affinity for another factor, such as a cancer-related factor or an immune response-related factor.

[0255] Therefore, in a third aspect of this disclosure, a complex is provided comprising at least one PD-L1 binding polypeptide and at least one antibody or antigen-binding fragment thereof, wherein the PD-L1 binding polypeptide is as described herein.

[0256] When used herein, the term "complex" is intended to refer to two or more associated polypeptide chains, at least one of which has an affinity for PD-L1, and at least one of which is an antibody or its antigen-binding fragment. These polypeptide chains may each contain different protein domains, and the resulting multi-protein complex may have multiple functions. "Complex" is also intended to refer to two or more polypeptide chains as defined above, covalently linked, for example, by expressing them as a recombinant fusion protein or by chemical conjugation.

[0257] As is well known, an antibody is an immunoglobulin molecule capable of specifically binding to a target (antigen) through at least one antigen recognition site located in the variable region of an immunoglobulin molecule, such as carbohydrates, polynucleotides, lipids, peptides, or others. As used herein, the term "antibody or antigen-binding fragment thereof" includes not only full-length or complete polyclonal or monoclonal antibodies, but also: antigen-binding fragments thereof, such as Fab, Fab′, F(ab′)2, Fab3, Fv, and variants thereof; fusion proteins comprising one or more antibody moieties; humanized antibodies; chimeric antibodies; minibodies; diabodies; triabodies; tetrabodies; linear antibodies; single-chain antibodies; multispecific antibodies (e.g., bispecific antibodies); and any other modifications to the immunoglobulin molecule that include an antigen recognition site of desired specificity, including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Other examples of modified antibodies and their antigen-binding fragments include nanobodies, AlbudAb, DART (dual-affinity retargeting), BiTE (bispecific T-cell engager), TandAb (tandem double-stranded antibody), DAF (dual-action Fab), two-in-one antibodies, SMIP (small modular immunopharmaceuticals), FynomAb (fynomer fused to an antibody), DVD-Ig (double variable domain immunoglobulin), CovX-body (peptide-modified antibody), duobody, and triomAb. This enumeration of variants of antibodies and their antigen-binding fragments is not intended to be limiting, and other suitable variants are known to those skilled in the art.

[0258] Full-length antibodies consist of two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (V0). H ) and the first constant region, the second constant region and the third constant region (C H 1. C H 2 and C H 3). Each light chain contains a light chain variable region (V). L ) and light chain constant region (C L Antibodies are classified into different classes based on the amino acid sequence of the constant structural domains of their heavy chains. There are six major classes of antibodies: IgA, IgD, IgE, IgG, IgM, and IgY, and several of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. As used herein, the term "full-length antibody" refers to any class of antibody, such as IgD, IgE, IgG, IgA, IgM, or IgY (or any of its subclasses). The subunit structures and three-dimensional configurations of the different classes of antibodies are well known.

[0259] An "antigen-binding fragment" is a portion or region of an antibody molecule that retains all or important antigen-binding parts of the corresponding full-length antibody, or a derivative thereof. Antigen-binding fragments may include heavy chain variable regions (V... H ), light chain variable region (V L ), or both. V H and V L Each typically contains three complementary determinant regions, CDR1, CDR2, and CDR3. H or V L The three CDR flanks form the frame regions (FR1, FR2, FR3, and FR4). As briefly listed above, examples of antigen-binding fragments include, but are not limited to: (1) Fab fragments, which are V L -C L Chain and V H -C H (1) a monovalent fragment of chain 1; (2) a Fab' fragment, wherein the Fab' fragment is a Fab fragment having a heavy chain hinge region; (3) an F(ab')2 fragment, wherein the F(ab')2 fragment is a dimer of Fab' fragments connected by heavy chain hinge regions, for example by disulfide bridges in the hinge regions; (4) an Fc fragment; (5) an Fv fragment, wherein the Fv fragment is a V-shaped fragment with an antibody-containing single arm. L and V H The smallest antibody fragment of the structural domain; (6) a single-chain Fv (scFv) fragment, wherein the single-chain Fv (scFv) fragment is the V of scFv therein. H and V LThe domains are linked by peptide linkers to single-chain polypeptides; (7) (scFv)2, wherein the (scFv)2 comprises two V H Structural domain and two V L Structural domains, they are connected via disulfide bridges through two V... H Domain association, and (8) domain antibody, wherein the domain antibody may be a single variable domain (V) of an antibody that specifically binds to an antigen. H or V L Polypeptide.

[0260] Antigen-binding fragments can be prepared using conventional methods. For example, the F(ab′)2 fragment can be produced by pepsin digestion of a full-length antibody molecule, and the Fab fragment can be produced by reducing the disulfide bridge of the F(ab′)2 fragment. Alternatively, fragments can be prepared via recombinant techniques by expressing heavy chain and light chain fragments in suitable host cells (e.g., E. coli cells, yeast cells, mammalian cells, plant cells, or insect cells) and assembling them to form the desired antigen-binding fragment in vivo or in vitro. Single-chain antibodies can be prepared via recombinant techniques by linking a nucleotide sequence encoding a heavy chain variable region and a nucleotide sequence encoding a light chain variable region. For example, a flexible linker can be incorporated between the two variable regions. Methods for preparing both full-length antibodies and their antigen-binding fragments are known to those skilled in the art.

[0261] Therefore, in one embodiment, this aspect of the present disclosure provides a complex as defined herein, wherein the at least one antibody or its antigen-binding fragment is selected from the group consisting of: full-length antibodies, Fab fragments, Fab' fragments, F(ab')2 fragments, Fc fragments, Fv fragments, single-chain Fv fragments, (scFv)2, and domain antibodies. In one embodiment, the at least one antibody or its antigen-binding fragment is selected from full-length antibodies, Fab fragments, and scFv fragments. In a particular embodiment, the at least one antibody or its antigen-binding fragment is a full-length antibody.

[0262] In one embodiment of the complex as defined herein, the antibody or its antigen-binding fragment is selected from the group consisting of monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies and their antigen-binding fragments.

[0263] As used herein, the term "monoclonal antibody" refers to an antibody with monovalent affinity, meaning that each antibody molecule in a sample binds to the same epitope on the antigen. The term "polyclonal antibody," as used herein, refers to a collection of antibodies that react to a specific antigen, but which may contain different antibody molecules, for example, identifying different epitopes on the antigen. Polyclonal antibodies are typically produced by inoculating suitable mammals and purified from mammalian serum. Monoclonal antibodies are produced by the same immune cells of clones of unique parent cells (e.g., hybridoma cell lines). As used herein, the term "human antibody" refers to an antibody having variable and constant regions that substantially correspond to or are derived from antibodies obtained from human subjects. As used herein, the term "chimeric antibody" refers to a recombinant antibody or genetically engineered antibody, such as, for example, a mouse monoclonal antibody containing polypeptides or domains from a different species, such as humans, which are introduced to reduce the immunogenicity of the antibody. The term "humanized antibody" refers to an antibody derived from a non-human species whose protein sequences have been modified to increase their similarity to naturally occurring antibody variants in humans to reduce immunogenicity.

[0264] The complexes described herein can exist, for example, as fusion proteins or conjugates. Therefore, the at least one PD-L1 binding peptide and the at least one antibody or its antigen-binding fragment can be coupled by chemical conjugation (using known organic chemical methods) or by any other method such as expressing the complex as a fusion protein, or otherwise linked directly or via a linker, such as an amino acid linker. Those skilled in the art will appreciate that the above description of the linker sequence of the fusion peptide is equally relevant to the complexes disclosed herein.

[0265] Therefore, in one embodiment, a complex as defined herein is provided, wherein the complex is a fusion protein or a conjugate. In one embodiment, the complex is a fusion protein. In another embodiment, the complex is a conjugate. In one embodiment of the complex, the PD-L1 binding polypeptide is attached to the N-terminus or C-terminus of the heavy chain of the antibody or its antigen-binding fragment. In another embodiment, the PD-L1 binding polypeptide is attached to the N-terminus or C-terminus of the light chain of the antibody or its antigen-binding fragment. In one embodiment, the PD-L1 binding polypeptide is attached to the N-terminus and / or C-terminus of both the light and heavy chains of the antibody or its antigen-binding fragment. For example, the PD-L1 binding peptide may be attached to the N-terminus of the heavy chain only, the N-terminus of the light chain only, the C-terminus of the heavy chain only, the C-terminus of the light chain only, both the N-terminus and C-terminus of the heavy chain, both the N-terminus and C-terminus of the light chain, the C-terminus of the light chain and the N-terminus of the heavy chain only, or both the C-terminus of the heavy chain and the N-terminus of the light chain only.

[0266] In one embodiment, a complex is provided in which the PD-L1 binding polypeptide is attached to the C-terminus or N-terminus of the heavy or light chain of the antibody or its antigen-binding fragment.

[0267] In one particular embodiment, a complex according to any of the foregoing items is provided, wherein the antibody or its antigen-binding fragment has an affinity for an antigen, such as an antigen associated with an infectious disease or a cancer-related antigen. For example, the antigen may be PD-1 or CTLA-4.

[0268] In one embodiment, a fusion protein, conjugate, or complex as described herein is provided, wherein the second or additional moiety or moieties, or antibody or antigen-binding fragment thereof, is an inhibitor selected from the group consisting of inhibitors such as PD-1, CTLA-4, T-cell immunoglobulins, and mucin-containing protein-3. Protein-3 (TIM-3), galactagogue 9 (GAL-9), lymphocyte activation gene-3 (LAG-3), PD-L2, B7 homologue 3 (B7-H3), B7 homologue 4 (B7-H4), V domain inhibitor of T cell activation (VISTA), carcinoembryonic antigen-associated cell adhesion molecule 1 (CEACAM1), B and T lymphocyte attenuators (BTLA), colony-stimulating factor 1 receptor (CSF1R), herpesvirus invasion mediator (HVEM), killer immunoglobulin receptor (KIR), adenosine, adenosine A2a receptor (A2aR), CD200-CD200R, and T cell Ig and ITIM domains.

[0269] In one embodiment, the second portion or antibody or its antigen-binding fragment is an inhibitor of PD-1, such as an inhibitor selected from the group consisting of nivolumab, pidilizumab, BMS 936559, MPDL328OA (Roche), and pembrolizumab. In a particular embodiment, the inhibitor is pembrolizumab.

[0270] In one embodiment, the second portion or antibody or its antigen-binding fragment is an inhibitor of CTLA-4, such as an inhibitor selected from the group consisting of belacicept, abatacept, tremelimumab, and iplimumab. In a specific embodiment, the inhibitor is iplimumab.

[0271] In one embodiment, a fusion protein, conjugate, or complex as described herein is provided, wherein the second portion or antibody or its antigen-binding fragment is an agonist selected from the group consisting of agonists of CD134, CD40, 4-1BB, and glucocorticoid-induced TNFR-associated protein (GITR).

[0272] The above aspects also include peptides in which the PD-L1 binding peptide according to the first aspect, such as those contained in a fusion protein or conjugate according to the second aspect, or in a complex according to the third aspect, further comprises a marker, such as a marker selected from the group consisting of fluorescent dyes and metals, chromophore dyes, chemiluminescent compounds, bioluminescent proteins, enzymes, radionuclides, radioactive particles, and pre-targeted recognition tags. For example, such markers can be used to detect the peptide. For example, in some embodiments, such labeled peptides can be used, for example, to label or target tumors with high PD-L1 expression.

[0273] Recently, the indirect labeling of Z variant peptides using pre-targeted recognition tags has been demonstrated (Westerlund et al. (2015), Bioconjugate Chem 26:1724-1736). Similarly, this disclosure provides PD-L1 binding peptides as described herein labeled with a pre-targeted moiety, which can then be indirectly labeled with a moiety complementary to the pre-targeted moiety. When the pre-targeted moiety is included, the PD-L1 binders of this disclosure are capable of associating with the complementary pre-targeted moiety, and such complementary pre-targeted moiety can then contain or be attached to a suitable radionuclide. Those skilled in the art are aware of suitable radionuclides for therapeutic, diagnostic, and / or prognostic purposes. Such radionuclides can chelate with the complementary pre-targeted moiety via a chelating environment, as generally described below with respect to PD-L1 binders.

[0274] In one embodiment, the complementary pair of the pre-targeting portion includes stept (avidin) / biotin, oligonucleotide / complementary oligonucleotides such as DNA / complementary DNA, RNA / complementary RNA, phosphorothioate nucleic acid / complementary phosphorothioate nucleic acid, peptide nucleic acid (PNA) / complementary peptide nucleic acid (cPNA), and marpholinos / complementary marpholinos. In a particular embodiment, the pre-targeting portion is a PNA oligonucleotide, such as a 10-20-mer PNA sequence, such as a 15-mer PNA sequence.

[0275] In embodiments where peptides, fusion proteins, conjugates, or complexes are directly or indirectly (e.g., via pre-targeting as described above) labeled with an imaging agent (e.g., a radioactive agent), the amount of labeled peptide present in the tumor can be measured using an imaging device by means such as obtaining a radioactivity count or a radiation density image or a derivative thereof, such as radiation concentration. Non-limiting examples of radionuclides suitable for direct labeling of PD-L1 binders according to any aspect of this disclosure, or suitable for indirect labeling via labeling complementary pre-targeting portions, include... 68 Ga、 110m In、 18 F, 45 Ti、 44 Sc、 61 Cu、 66 Ga、 64 Cu、 55 Co、 72 As、 86 Y、 89 Zr、 124 I, 76 Br、 111 In、 99m Tc, 123 I, 131 I and 67 Ga.

[0276] In one embodiment, the imaging device used in such measurements is a positron emission tomography (PET) device, in which case a radionuclide is selected to be suitable for PET. The technician is familiar with the radionuclides suitable for use with PET. For example, the PET radionuclide is selected from… 68 Ga、 110m In、 18 F, 45 Ti、 44 Sc、 61 Cu、 66 Ga、 64 Cu、 55 Co、 72 As、 86 Y、 89 Zr、 124 I and 76 A group composed of Br.

[0277] In another embodiment, the imaging device used is a single-photon emission computed tomography (SPECT) device, in which case a radionuclide is selected to be suitable for SPECT. Those skilled in the art are familiar with radionuclides suitable for use with SPECT. For example, the SPECT radionuclide is selected from… 111 In、 99m Tc, 123I, 131 I and 67 The group consisting of Ga.

[0278] Therefore, in one embodiment, a PD-L1 binding peptide, fusion protein, or complex as described herein is provided, said PD-L1 binding peptide, fusion protein, or complex comprising a direct or indirect radionuclide label, such as selected from 68 Ga、 110m In、 18 F, 45 Ti、 44 Sc、 61 Cu、 66 Ga、 64 Cu、 55 Co、 72 As、 86 Y、 89 Zr、 124 I, 76 Br、 111 In、 99m Tc, 123 I, 131 I and 67 Groups composed of Ga, such as those composed of Ga 68 Ga、 110m In、 18 F, 45 Ti、 44 Sc、 61 Cu、 66 Ga、 64 Cu、 55 Co、 72 As、 86 Y、 89 Zr、 124 I and 76 Groups composed of Br, such as 18 F is a radioactive nuclide.

[0279] In some embodiments, the labeled PD-L1 binding peptide is present as a portion of a fusion protein, conjugate, or complex that also comprises a second portion having the desired biological activity. In some cases, the label may be coupled only to the PD-L1 binding peptide, and in others, to both the PD-L1 binding peptide and the second portion of the fusion protein or conjugate and / or an antibody or antigen-binding fragment of the complex. Furthermore, it is possible for the label to be coupled only to the second portion or the antibody or its antigen-binding fragment without being coupled to the PD-L1 binding portion. Thus, in yet another embodiment, a PD-L1 binding peptide comprising a second portion is provided, wherein the label is coupled only to the second portion. In yet another embodiment, a complex as defined herein is provided, wherein the label is coupled only to an antibody or its antigen-binding fragment.

[0280] When referring to a labeled peptide, this should be understood to refer to all aspects of the peptide as described herein, including PD-L1-binding peptides, fusion proteins containing PD-L1-binding peptides, conjugates, and complexes. Therefore, a labeled peptide may consist only of a PD-L1-binding peptide and, for example, a therapeutic radionuclide, which may be chelated or covalently coupled to the PD-L1-binding peptide, or may consist of a PD-L1-binding peptide, a therapeutic radionuclide, and a second portion such as a small molecule with desired biological activity, for example, therapeutic efficacy. A labeled peptide may consist of a PD-L1-binding peptide in heterodimeric form and, for example, a therapeutic radionuclide, which may be chelated or covalently coupled to the PD-L1-binding peptide, or may consist of a PD-L1-binding peptide in heterodimeric form, a therapeutic radionuclide, and a second portion such as a small molecule with desired biological activity, for example, therapeutic efficacy. A complex is also envisioned comprising a PD-L1 binding peptide, an antibody or an antigen-binding fragment thereof as defined herein, and, for example, a therapeutic radionuclide, which may be chelated or covalently coupled to the PD-L1 binding peptide or antibody or its antigen-binding fragment. Other possible variations are known to those skilled in the art.

[0281] In embodiments where PD-L1-binding peptides, fusion proteins, conjugates, or complexes are radiolabeled, such radiolabeled peptides may contain radionuclides. Most radionuclides are metallic, and metals typically cannot form stable covalent bonds with elements present in proteins and peptides. For this reason, labeling proteins and peptides with radioactive metals is carried out using chelating agents (i.e., polydentate ligands) that form non-covalent compounds (called chelates) with metal ions. In embodiments where PD-L1-binding peptides, fusion proteins, conjugates, or complexes are implemented, the incorporation of the radionuclide is achieved by providing a chelating environment through which the radionuclide can be coordinated, chelated, or complexed to the peptide. An example of a chelating agent is a polyaminopolycarboxylic acid type chelating agent. Such polyaminopolycarboxylic acid chelating agents can be classified into two categories: macrocyclic chelating agents and acyclic chelating agents.

[0282] In one embodiment, the PD-L1 binding peptide, fusion protein, conjugate, or complex includes a chelating environment provided by a polyamino-polycarboxylic acid chelating agent conjugated to the PD-L1 binding peptide via a thiol group of a cysteine ​​residue or an ε-amino group of a lysine residue.

[0283] The most commonly used macrocyclic chelating agents for radioisotopes of indium, gallium, yttrium, bismuth, radioactinides, and radiolanthanides are various derivatives of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). In one embodiment, the chelating environment for PD-L1 binding peptides, PD-L1 binding peptides in heterodimeric form, fusion proteins, conjugates, or complexes is provided by DOTA or its derivatives. More specifically, in one embodiment, the chelating polypeptide included in this disclosure is obtained by reacting the polypeptide with a DOTA derivative 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide (maleimidomonoamide-DOTA). In one embodiment, the chelating polypeptide included in this disclosure is obtained by reacting the DOTA derivative DOTAGA(2,2',2”-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid) with the polypeptide. Alternatively, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and its derivatives can be used as chelating agents. Therefore, in one embodiment, chelation of PD-L1 binding polypeptides, PD-L1 binding polypeptides in heterodimeric form, fusion proteins, conjugates, or complexes is achieved. The environment is provided by NOA or its derivatives. In one embodiment, the chelating polypeptide included in this disclosure is obtained by reacting the polypeptide with the NOA derivative NODAGA(2,2'-(7-(1-carboxy-4-((2,5-dioxopyrrolidone-1-yl)oxy)-4-oxobutyl)-1,4,7-triazacyclononane-1,4-diyl)diacetic acid. The most commonly used acyclic polyaminopolycarboxylic acid chelating agents are various derivatives of DTPA (diethylenetriaminepentaacetic acid). Therefore, polypeptides having a chelating environment provided by diethylenetriaminepentaacetic acid or its derivatives are also included in this disclosure.

[0284] In another aspect of this disclosure, polynucleotides encoding PD-L1 binding peptides, fusion proteins, or complexes as described herein are provided; expression vectors comprising said polynucleotides; and host cells comprising said expression vectors.

[0285] This disclosure also includes a method for producing a PD-L1 binding peptide, fusion protein, or complex as described above, the method comprising culturing the host cell under conditions that allow the peptide to be expressed by its expression vector, and isolating the peptide.

[0286] The PD-L1 binding peptides, fusion proteins, or complexes of this disclosure can optionally be prepared via non-biological peptide synthesis using amino acids and / or amino acid derivatives having protected reactive side chains, said non-biological peptide synthesis including

[0287] - Stepwise coupling of amino acids and / or amino acid derivatives to form peptides, fusion proteins, or complexes as described herein, with protected reactive side chains.

[0288] -Removal of protecting groups from reactive side chains of peptides, fusion proteins, or complexes, and

[0289] - Folding peptides in aqueous solutions.

[0290] The complexes disclosed herein can also be generated by conjugating at least one PD-L1-binding peptide or fusion protein, as described herein, to at least one antibody or its antigen-binding fragment. Those skilled in the art are familiar with conjugation methods, such as conventional chemical conjugation methods using, for example, charged succinimide esters or carbodiimides.

[0291] It should be understood that the PD-L1 binding peptides according to this disclosure can be used independently as therapeutic agents, diagnostic agents, and / or prognostic agents, or as means for targeting other therapeutic agents or diagnostic agents that have direct or indirect effects on PD-L1. Direct therapeutic effects can be achieved, for example, by inhibiting PD-L1 signaling. Indirect therapeutic effects can be achieved, for example, by pre-targeting with the PD-L1 binding peptides as described above.

[0292] Therefore, in another aspect, a composition is provided comprising a PD-L1 binding peptide, fusion protein, conjugate, or complex as described herein, and at least one pharmaceutically acceptable excipient or carrier. In one embodiment, the composition further comprises at least one additional active agent, such as at least two additional active agents, such as at least three additional active agents. Non-limiting examples of additional active agents that may prove useful in such combinations are immune response modulators and anticancer agents as described herein.

[0293] The small size and robustness of the PD-L1 binding peptides of this disclosure offer several advantages over conventional monoclonal antibody-based therapies. These advantages include superior formulation, administration methods such as alternative routes of administration, administration at higher doses than antibodies, and the absence of Fc-mediated side effects. The formulations of this disclosure are intended for oral, topical, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, sublingual, sublingual, or suppository administration, such as for topical application. Furthermore, many diseases and disorders, such as cancer and infectious diseases, are associated with more than one factor. Therefore, complexes as defined herein offer the advantage of targeting additional antigens along with PD-L1.

[0294] In another aspect of this disclosure, PD-L1 binding peptides, fusion proteins, conjugates, complexes, or compositions as described herein are provided for use as pharmaceuticals, prognostic agents, and / or diagnostic agents. In one embodiment, PD-L1 binding peptides, fusion proteins, conjugates, complexes, or compositions are provided for use in the treatment, diagnosis, or prognosis of PD-L1-related disorders.

[0295] In one embodiment, the PD-L1 binding peptide, fusion protein, conjugate, complex, or composition is provided for use as a medicine. In more specific embodiments, the PD-L1 binding peptide, fusion protein, conjugate, complex, or composition as described herein is provided for use as a medicine to modulate PD-L1 function in vivo. As used herein, the term "modulation" refers to altering activity, such as making PD-L1 a functional sub-allele that partially or completely inhibits PD-L1 function.

[0296] In one embodiment, a PD-L1 binding peptide, fusion protein, conjugate, complex, or composition is provided for use in the treatment of PD-L1-related disorders.

[0297] In one embodiment, a PD-L1 binding peptide, fusion protein, conjugate, complex, or composition is provided for use in the diagnosis of PD-L1-related disorders.

[0298] In one embodiment, a PD-L1 binding peptide, fusion protein, conjugate, complex, or composition is provided for use in the prognosis of PD-L1-related disorders.

[0299] As used herein, the term "PD-L1-related disorder" refers to any disorder, disease, or condition in which PD-L1 signaling plays a regulatory role. Examples of such PD-L1-related disorders include infectious diseases and cancer.

[0300] It should be understood that the PD-L1 binding peptide, fusion protein, conjugate, complex or composition can be used as a standalone diagnostic agent or prognostic agent or as a companion diagnostic agent and / or prognostic agent.

[0301] In one embodiment, the PD-L1-related disorder is selected from the group consisting of infectious diseases and cancer. Non-limiting examples of infectious diseases include chronic viral infections, such as those selected from the group consisting of human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV). Those skilled in the art will appreciate that cancers suitable for treatment, diagnosis, and / or prognosis using PD-L1-binding peptides, fusion proteins, conjugates, complexes, or compositions can be solid tumor cancers or non-solid tumor cancers characterized by PD-L1 overexpression. Non-limiting examples of such cancers include skin cancer; such as melanoma and non-melanoma skin cancer (NMSC); lung cancer such as small cell lung cancer and non-small cell lung cancer (NSCLC); head and neck cancer, renal cell carcinoma (RCC), bladder cancer, breast cancer, colorectal cancer, stomach cancer, ovarian cancer, pancreatic cancer, prostate cancer, glioma, glioblastoma, liver cancer, gallbladder cancer, thyroid cancer, bone cancer, cervical cancer, uterine cancer, vulvar cancer, endometrial cancer, testicular cancer, kidney cancer, esophageal cancer, brain / CNS cancer, neuronal cancer, mesothelioma, sarcoma, small intestinal adenocarcinoma, and pediatric malignancies; leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, and multiple myeloma.

[0302] In one particular implementation, the cancer is selected from the group consisting of melanoma, NSCLC, head and neck cancer, RCC, bladder cancer, breast cancer, colorectal cancer, stomach cancer, ovarian cancer, pancreatic cancer, and prostate cancer, such as cancers selected from the group consisting of melanoma, NSCLC, head and neck cancer, RCC, and bladder cancer.

[0303] In one embodiment, it may be beneficial to administer a therapeutically effective amount of a PD-L1 binding peptide, fusion protein, conjugate, complex, or composition as described herein, together with at least one second pharmaceutical substance, such as an anticancer agent or an immunomodulator.

[0304] In one embodiment, a PD-L1 binding peptide, fusion protein, conjugate, complex, or composition is provided, together with at least one cell proliferation marker, for use in prognosis and / or diagnosis. Non-limiting examples of anticipated cell proliferation markers are those selected from the group consisting of Ki-67, AgNOR, choline, claspin, cyclin A, CYR61, Cdk1, histone H3, HsMCM2, IL-2, Ki-S1, Ki-S2, LigI, MCM2, MCM6, MCM7, mitosin, p120, PCNA, PDPK, PLK, STK1, TK-1, topoisomerase IIα, and TPS.

[0305] In a related aspect, a method for treating PD-L1-related disorders is provided, the method comprising administering an effective amount of a PD-L1-binding peptide, fusion protein, conjugate, complex, or composition as described herein to a subject in need. In a more specific embodiment of the method, the PD-L1-binding peptide, fusion protein, conjugate, complex, or composition as described herein modulates PD-L1 function in vivo. Those skilled in the art will appreciate that any description of the use of the PD-L1-binding peptide, fusion protein, conjugate, complex, or composition as described herein for the treatment of diseases or disorders is equivalent to related treatment methods. For the sake of brevity, such descriptions will not be repeated herein.

[0306] In one particular implementation, the treatment method, especially the treatment method related to PD-L1-related cancers, includes the following steps:

[0307] -Contact the subject with a PD-L1 binding peptide, fusion protein, conjugate, or complex containing a pre-targeting moiety as described herein, or with a composition containing said PD-L1 binding peptide, fusion protein, conjugate, or complex containing a pre-targeting moiety, and

[0308] -The subject is brought into contact with a complementary pre-targeted portion containing a radionuclide.

[0309] In another aspect of this disclosure, a method for detecting PD-L1 is provided, the method comprising: providing a sample suspected of containing PD-L1; contacting the sample with a PD-L1-binding peptide, fusion protein, conjugate, complex, or composition as described herein; and detecting the binding of the PD-L1-binding peptide, fusion protein, conjugate, complex, or composition to indicate the presence of PD-L1 in the sample.

[0310] In one embodiment, the method further includes an intermediate washing step after contact with the sample to remove unbound peptides, fusion proteins, conjugates, complexes, or compositions.

[0311] In another embodiment, the method is a diagnostic or prognostic method for determining the presence of PD-L1 in a subject, the method comprising the following steps:

[0312] a) Contacting the subject or a sample isolated from the subject with a PD-L1 binding peptide, fusion protein, conjugate, complex, or composition as described herein, and

[0313] b) Obtain a value corresponding to the amount of PD-L1 binding peptide, fusion protein, conjugate, complex, or composition that has been bound in the subject or bound to the sample.

[0314] In one embodiment, the method further includes an intermediate washing step after contact with the subject or sample and before obtaining values ​​to remove unbound peptides, fusion proteins, conjugates, or compositions.

[0315] In one embodiment of the diagnostic or prognostic method, the PD-L1 binding peptide, fusion protein, conjugate, or complex comprises a pre-targeting moiety as described herein, and the contact step a) of the method further includes contacting the subject with a complementary pre-targeting moiety labeled with a detectable marker such as a radionuclide marker.

[0316] In one embodiment, the method further includes the step of comparing the value with a reference. The reference may be a numerical value, a threshold, or a visual indicator, such as one based on a colorimetric reaction. Those skilled in the art will appreciate that different methods of comparison with a reference are known in the art and may be suitable for use.

[0317] In one embodiment of this method, the subject is a mammalian subject, such as a human subject. In one embodiment, the method is performed in vivo. In another embodiment, the method is performed in vitro.

[0318] In one embodiment, the diagnostic or prognostic method is a method for in vivo medical imaging as discussed above. Such methods include systemic administration of a PD-L1 binding entity (i.e., the polypeptide itself, or a fusion protein, conjugate, complex, or composition containing the polypeptide) as disclosed herein to a mammalian subject. The PD-L1 binding entity is directly or indirectly labeled with a tag containing a radionuclide suitable for medical imaging (see the list of intended radionuclides above). Furthermore, the method for medical imaging includes obtaining one or more images of at least a portion of the subject's body using a medical imaging instrument, said one or more images indicating the presence of a radionuclide within the body.

[0319] Although the invention has been described with reference to various exemplary aspects and embodiments, those skilled in the art will understand that many changes can be made and equivalents can be substituted for its elements without departing from the scope of the invention. Furthermore, many modifications can be made to adapt particular situations or molecules to the teachings of the invention without departing from its essential scope. Therefore, it is intended that the invention be limited to any particular embodiment contemplated, but rather that the invention encompass all embodiments falling within the scope of the appended claims. Brief description of the attached diagram

[0321] Figure 1 Examples of PD-L1 binding peptides of this disclosure (SEQ ID NO:1-814), and PD-L1 binding monoclonal antibody Lam heavy chain (HC) Lam ; SEQ ID NO:815) and light chains (LC Lam ; SEQ ID NO:816), and the heavy chain (HC) of the CTLA-4-binding monoclonal antibody Ipi. Ipi ; SEQ ID NO:817) and light chains (LC Ipi List of amino acid sequences (SEQ ID NO: 818). In the PD-L1 binding polypeptides of this disclosure, the inferred PD-L1 binding motif (BM) extends from residue 8 to residue 36 in each sequence. The amino acid sequence of the 49-amino acid residue long polypeptide (BMod) predicted to constitute the complete triple helix bundle within each of these Z variants extends from residue 7 to residue 55.

[0322] Figure 2 illustrates the binding of two first-generation peptides to human PD-L1 as analyzed by Biacore, as described in Example 3. The Z variants (A) Z13091 (SEQ ID NO: 776) and (B) Z13156 (SEQ ID NO: 781) were injected onto a CM5 chip immobilized with hPD-L1 at concentrations of 50 nM (black), 5 nM (dark gray), and 0.5 nM (light gray).

[0323] Figure 3 The absence of SPR responses to (A)hPD-L2, (B)hB7-H3, and (C)hB7-H4 is illustrated here using PD-L1 binding peptides Z13091 (SEQ ID NO:776) and Z13156 (SEQ ID No:781) injected at concentrations of 50 nM, 5 nM, and 0.5 nM, respectively.

[0324] Figure 4Circular dichroism (CD) spectra of two first-generation PD-L1 binding peptides are shown. The CD spectra of (A) Z15168-Cys (SEQ ID NO: 809) and (B) Z15169-Cys (SEQ ID NO: 810) collected at 20 °C before (dashed line) and after (solid line) variable temperature measurement (VTM) at wavelengths ranging from 250 nm to 195 nm are shown.

[0325] Figure 5 The binding of two second-generation peptides to human PD-L1 as described in Example 7 by Biacore analysis is shown. Variants Z (A) Z17964 (SEQ ID NO:2) and (B) Z18064 (SEQ ID NO:1) were injected onto CM5 chips immobilized with hPD-L1 at concentrations of 135 nM (black), 45 nM (dark gray), and 15 nM (light gray).

[0326] Figure 6 Circular dichroism (CD) spectra of two second-generation PD-L1 binding peptides are shown. The CD spectra of (A) Z18064 (SEQ ID NO:1) and (B) Z18090 (SEQ ID NO:17) collected at 20 °C before (dashed line) and after (solid line) variable temperature measurement (VTM) at wavelengths ranging from 250 nm to 195 nm are shown.

[0327] Figure 7 This is a schematic diagram of the complex designed according to the present disclosure, as described in Example 8. “Z” represents PD-L1 targeting the Z variant Z15170 (SEQ ID NO: 814), which is fused to the N-terminus (7A and 7B) or C-terminus (7C and 7D) of the heavy chain (7A and 7D) or light chain (7B and 7C) of the anti-PD-1 monoclonal antibody Lam or the anti-CTLA-4 monoclonal antibody Ipi via a 15-residue (GGGGS) 3-linker gene.

[0328] Figure 8 illustrates the dual binding specificity of the complex analyzed in the Biacore capture assay as described in Example 8. (A)Z15170-HC Lam and (B)Z15170-LC Lam PD-L1 was injected separately over 5 minutes onto a chip surface immobilized with PD-1, followed by injection of PD-L1 at concentrations of 100 nM and / or 500 nM. (C)Z15170-HC Ipi and (D)Z15170-LC Ipi PD-L1 was injected over 5 minutes onto a chip surface immobilized with CTLA-4, followed by injection at concentrations of 100 nM and 500 nM.

[0329] Figure 9 illustrates the results of the Ipi-based complex's inhibition of PD-L1 and CTLA-4 in a mixed lymphocyte assay as described in Example 8. With the Ipi-based complex HC Ipi -Z15170, LC Ipi -Z15170, Z15170-HC Ipi and Z15170-LC Ipi An increase in the concentration of MDA-MB231 cells leads to a decrease in the number of MDA-MB231 cells and an increase in the number of CD3+ T cells.

[0330] Figure 10 shows PET maximum intensity projection (MIP) images of xenograft mice. (A) After administration of [ 18 F] MIP in mice with LOX tumors (left) and SUDHL6 tumors (right) xenografts 30–90 min after administration of AlF-NOTA-Z15168. (B) At baseline administration of [ 18 MIPs of mice with LOX tumor xenografts after pre-blocking with 400 μg of Nota-Z15168 (left side) 30–90 min and (right side) after AlF-NOTA-Z15168.

[0331] Figure 11 shows the ex vivo biodistribution results of the LOX and SUDHL6 mouse xenograft models, analyzed directly after PET data acquisition. Results are presented in (A) standard uptake values ​​(SUVs) and (B) tumor:blood ratio. Error bars represent standard deviation.

[0332] Figure 12 shows the results of a whole-body scan of rhesus monkeys. (A)[ 18 F]AlF-NOTA-Z15168 and (B)[ 18 F]MIP (sum over 90–180 min; color inverted image) of rhesus monkeys of AlF-NOTA-Z18609. (C) Mean tracer uptake in different organs, displayed in SUV units over ≈120–180 min. Error bars represent standard deviation. Example

[0333] Overview

[0334] The following examples disclose novel Z-variant molecules targeting human programmed death-ligand 1 (PD-L1) (also known as human B7 homologue 1 (B7-H1) and differentiation cluster 274 (CD274)) developed based on phage display technology. The PD-L1 binding peptides described herein were sequenced, and their amino acid sequences are enclosed in sequence identifier SEQ ID NO:1-808. Figure 1The examples are listed below. The examples also describe the characterization of the PD-L1 binding peptide and demonstrate the in vitro function of the peptide.

[0335] Example 1

[0336] PD-L1 combined with Z variant selection and screening

[0337] In this embodiment, human PD-L1 (hPD-L1) was used as the target in the phage display selection of the phage library using the Z variant. The selected clones were DNA sequenced, generated in E. coli periplasmic fractions, and measured against PD-L1 in an ELISA (enzyme-linked immunosorbent assay).

[0338] Materials and Methods

[0339] Biotinylation of target proteins hPD-L1 (human PD-L1 Fc chimera, R&D Systems, catalog 156-B7-100) was biotinylated with a 10× mol excess of No-Weigh EZ-LinkSulfo-NHS-LC-Biotin (Thermo Scentific, catalog 21327) as recommended by the manufacturer. The reaction was carried out at room temperature (RT) for 40 min. Subsequent buffer exchanges to PBS (10 mM phosphate, 137 mM NaCl, 2.68 mM KCl, pH 7.4) were performed using a Slide-a-lyzer dialysis kit (10000 MWCO, Thermo Scientific, catalog 66383) according to the manufacturer's instructions.

[0340] PD-L1-binding Z variant phage display selection Basically, as The library of random variants of protein Z displayed on a phage, constructed in phage particle pAY02592, described in J Biotechnol (2007), 128:162-183, was used to select PD-L1 binding variants of Z. In this library, the albumin-binding domain (ABD, GA3 of the G protein from Streptococcus strain G148) was used as the fusion partner for the Z variant. This library was designated Zlib006Naive.II and had a size of 1.5 × 10⁻⁶. 10Size of the Z variant members of the phage library. *E. coli* RRIΔM15 cells (Rüther et al., (1982) Nucleic Acids Res 10:5765-5772) from a glycerol stock solution containing the phage plasmid library Zlib006Naive.II were inoculated in 20 μL proline-free limited medium supplemented with 100 μg / ml ampicillin [3 g / L KH2PO4, 2 g / L K2HPO4, 0.02 g / L uracil, 6.7 g / L YNB (Difco)]. TM A yeast nitrogen source base without amino acids (Becton Dickinson), 5.5 g / L glucose monohydrate, 0.3 g / L L-alanine, 0.24 g / L L-arginine monohydrochloride, 0.11 g / L L-asparagine monohydrate, 0.1 g / L L-cysteine, 0.3 g / L L-glutamic acid, 0.1 g / L L-glutamine, 0.2 g / L glycine, 0.05 g / L L-histidine, 0.1 g / L L-isoleucine, 0.1 g / L L-leucine, 0.25 g / L L-lysine monohydrochloride, 0.1 g / L L-methionine, 0.2 g / L L-phenylalanine, 0.3 g / L L-serine, 0.2 g / L L-threonine, 0.1 g / L L-tryptophan, 0.05 g / L L-tyrosine, 0.1 g / L L-valine. The culture was grown in a fermenter (Belach Bioteknik, BR20) at 37°C. The cells reached an optical density (OD) of 0.75 at 600 nm. 600When using 10× molar excess of M13K07 helper phage (New England Biolabs, catalog number N0315S), approximately 2.6 μL of culture was infected. Cells were incubated for 30 min, at which point they were switched to a culture medium supplemented with 100 μM isopropyl-β-D-1-thiogalactopyranoside (IPTG) (for induction of expression) and 50 μg / ml ampicillin, 12.5 μg / ml carbenicillin, 25 μg / ml kanamycin, 35 ml / L 1.217 M MgSO4, and 10 ml trace element solution [129 mM FeCl3; 36.7 mM ZnSO4; 10.6 mM CuSO4; 78.1 mM MnSO4; 94.1 mM CaCl2 dissolved in 1.2 M HCl] (2.5 g / L (NH4)2SO4; 5.0 g / L yeast extract (Merck 1.03753.0500); 25 g / L peptone (Scharlau 07-119); 2 g / L The fermenter was filled to 20 liters with K₂HPO₄ (3 g / L KH₂PO₄), 1.25 g / L Na₃C₆H₅O₇·2H₂O), and 0.1 ml / L Breox FMT30 antifoaming agent. A fed-batch culture with limited glucose was initiated, in which a 600 g / L glucose solution was fed into the reactor (15 g / h initially, increasing to 40 g / h at the end of fermentation after 17 h). The pH was maintained at 7 by automatic addition of 25% NH₄OH, air was added (10 L / min), and a stirrer was set to maintain dissolved oxygen levels above 30%. Cells were removed from the culture by tangential flow filtration.

[0341] The phage particles were precipitated twice from the supernatant in PEG / NaCl (polyethylene glycol / sodium chloride), filtered, and dissolved in PBS and glycerol. As described above. Store the phage stock solution at -80°C before use.

[0342] Selection for biotinylated hPD-L1 was performed in four cycles, initially divided into two distinct tracks (tracks 1 and 2). As selection proceeded, the tracks were further subdivided based on target concentration and the number of washes and / or time, ultimately ending with nine tracks in cycle 4. More specifically, track 1 (1) was subdivided in the second through fourth cycles, resulting in a total of 2 tracks (1-1 to 1-2) in cycle 2, 4 tracks (1-1-1 to 1-2-2) in cycle 3, and 6 tracks (1-1-1-1 to 1-2-2-1) in cycle 4. Track 2 (2) was subdivided in the third through fourth cycles, resulting in a total of 2 tracks (2-1-1 to 2-1-2) in cycle 3 and 3 tracks (2-1-1-1 to 2-1-2-1) in cycle 4.

[0343] In orbital 1 with descendants, using M-280 streptavidin (SA-bead, Invitrogen, catalog number 11206D) captures the hPD-L1:Z variant complex. In orbital 2, instead of using... Protein A (SPA-bead, Invitrogen, catalog number 10002D) captures the hPD-L1:Z variant complex by binding to the Fc portion of the hPD-L1 Fc chimeric protein.

[0344] Phage stock preparation, selection procedures, and phage amplification between selection cycles are generally performed as described in WO2009 / 077175 for selection of another biotinylated target, with the following exception: the selection buffer consists of PBS supplemented with 10% fetal bovine serum (FBS, Gibco, catalog 10108-165) and 0.1% Tween20 (AcrosOrganics, catalog 233362500).

[0345] To reduce the amount of background adhesive, pre-selection is performed in each cycle. In pre-selection, the same type of beads as during selection is used: SA beads in track 1 and SPA beads in track 2. In all tracks of cycles 1-4, pre-selection is performed using SA beads or SPA beads coated with biotinylated human IgG-Fc (Jackson ImmunoResearch Lab, catalog number 009-060-008). Additionally, in cycle 1, track 1, pre-selection is performed using SA beads coated with a mixture of biotinylated hPD-L2 (human PD-L2 Fc chimera; R&D Systems, catalog number 1224-PL-100), hB7-H3 (human B7-H3 Fc chimera; R&D Systems, catalog number 1027-B3-100), and hB7-H4 (human B7-H4; R&D Systems, catalog number 6576-B7-50) as previously described with respect to hPD-L1. In cycle 1, track 2, preselection was performed using SPA-beads coated with a mixture of biotinylated PD-L2 and biotinylated B7-H3. During preselection, the phage stock solution was incubated with the coated beads at RT (end-over-end) for 30–90 min. All tubes and beads used in preselection or selection were pre-blocked with PBS supplemented with 3% bovine serum albumin (BSA, Sigma A3059-100G) and 0.1% Tween 20. Selection was performed at RT in solution for approximately 120 min, followed by washing with PBS + 0.1% Tween 20, and target-phage complexes were captured on SA-beads or SPA-beads using 1 mg beads / 1.6 or 8.5 μg biotinylated hPD-L1, respectively.

[0346] To amplify phage particles between selection cycles 1 and 2, *E. coli* strain ER2738 cells (Lucigen, Middleton, WI, USA) were used for infection and grown in medium supplemented with 20 μg / ml tetracycline. A 5× excess of M13K07 helper phages compared to bacteria was allowed to infect bacteria in logarithmic growth phase.

[0347] Table 2: Selection of biotinylated hPD-L1 Fc chimeras

[0348]

[0349] Amplification of phage particles between selection cycles 2 and 4 was performed via bacterial infection in solution as follows: After infecting logarithmically growing *E. coli* ER2738 with phage particles, TSB supplemented with 2% glucose, 10 μg / ml tetracycline, and 100 μg / ml ampicillin was added, followed by incubation at 37°C with rotation for 30 min. Subsequently, bacteria were infected with M13K07 helper phage at a 5× overdose. The infected bacteria were centrifuged, resuspended in TSB-YE medium supplemented with 100 μM IPTG, 25 μg / ml kanamycin, and 100 μg / ml ampicillin, and grown overnight at 30°C. The overnight culture was centrifuged, and the phage particles in the supernatant were precipitated twice with PEG / NaCl buffer. Finally, the phage particles were resuspended in selection buffer and then proceeded to the next selection cycle.

[0350] In the final selection cycle, logarithmic growth phase bacteria are infected with eluent, diluted, and then plated on TBAB plates (30 g / L tryptone agar, Oxoid catalog number CMO233B) supplemented with 0.2 g / L ampicillin to form single colonies to be used in ELISA screening.

[0351] Table 2 shows an overview of the selection strategy, which describes the increased stringency obtained by using a reduced target concentration and an increased number of washes in subsequent cycles. Unless otherwise stated in Table 2, wash for 1 min with 0.1% PBST (PBS supplemented with 0.1% Tween-20) and elute as described in WO2009 / 077175.

[0352] Generation of Z variants for ELISA The Z variant was generated by inoculating selected single colonies into 1 ml of TSB-YE medium supplemented with 100 μg / ml ampicillin and 1 mM IPTG in deep-well plates (Nunc, catalog number 278752). The plates were incubated at 37°C with rotation for 24 h. Cells were pelleted by centrifugation, resuspended in 200 μl of 0.05% PBST, and frozen at -80°C to release the periplasmic fraction. The frozen sample was thawed in a water bath, and the freeze-thaw procedure was repeated 8 times. 600 μl of 0.05% PBST was added to the thawed sample, and the cells were pelleted by centrifugation.

[0353] The final periplasmic extract supernatant contained the Z-variant as a fusion with ABD, denoted as AQHDEALE-[Z#####]-VDYV-[ABD]-YVPG( (Same as above). Z##### refers to a single Z variant of 58 amino acid residues.

[0354] ELISA screening for Z variantThe binding of the Z variant to hPD-L1 was analyzed in an ELISA assay. A 96-well ELISA plate (Costar, catalog number 3690) was coated overnight at 4°C with anti-ABD goat antibody (internally generated) diluted in coating buffer (50 mM sodium carbonate, pH 9.6; Sigma, catalog number C3041) in coating buffer. The antibody solution was decanted, and the wells were washed in water and blocked at RT for 1 h to 3 h with 100 μl of PBSC (PBS supplemented with 0.5% casein (Sigma, catalog number C8654)). The blocking solution was discarded, and 50 μl of periplasmic solution diluted 1:1 with 0.05% PBST was added to the wells, and the plate was incubated at RT with slow agitation for 1.5 h to 2.5 h. As a blank control, 0.05% PBST was added instead of periplasmic sample. The supernatant was decanted, and the wells were washed four times with 0.05% PBST. Next, 50 μl of biotinylated hPD-L1 at a concentration of 0.32 nM in PBSC was added to each well. The plate was incubated at RT for 1 h, followed by washing as described above. Streptomycin-conjugated HRP (Thermo Scientific, catalog number N100) diluted 1:30,000 in PBSC was added to the wells and the plate was incubated for approximately 1 h. After washing as described above, 50 μl of ImmunoPure TMB substrate (Thermo Scientific, catalog number 34021) was added to the wells, and the plate was processed according to the manufacturer's recommendations. A Victor multiwell plate reader was used. 3 (Perkin Elmer) was used to measure the absorbance at 450 nm.

[0355] sequencing In parallel with ELISA screening, all clones were sequenced. Essentially, as described in WO2009 / 077175, PCR fragments were amplified from individual colonies, sequenced, and analyzed.

[0356] EC50 analysis of the Z variantThis assay analyzed the response of selected PD-L1-binding Z variants to serial dilutions of biotinylated hPD-L1 after undergoing the procedure described above. The Z variants were diluted 1:1 in 0.05% PBST. Biotinylated hPD-L1 was added at a concentration of 40 nM and step-diluted 1:4 to 32 pM. As a background control, all Z variants were also assayed without the target protein. Periplasmic samples containing the PD-L1-binding Z variant Z13112 (SEQ ID. NO: 777) were included in each plate and analyzed as a positive control. Periplasmic samples containing only the ABD portion were used as a negative control. In the same assay, the specificity of the Z variants was tested by incubating periplasmic samples with four different biotinylated control proteins—hPD-L2, hB7-H3, hB7-H4, and IgGFc—added at a concentration of 8 nM. Data were analyzed using GraphPad Prism 5 and nonlinear regression, and EC50 values ​​(half-maximum effective concentration) were calculated.

[0357] result

[0358] PD-L1-binding Z variant phage display selection After four cycles of phage display selection targeting biotinylated hPD-L1, individual clones were obtained.

[0359] ELISA screening for Z variant Clones obtained after four cycles of selection were generated in 96-well plates and screened for hPD-L1 binding activity by ELISA. Several unique Z variants were found to produce responses of 0.3 AU or higher against hPD-L1 at a concentration of 0.32 nM (corresponding to at least 3 × blank controls). The mean response of the blank controls was 0.067 AU.

[0360] sequencing After four cycles of selection, the resulting clones were sequenced. Each variant was assigned a unique identifier #####, and the individual variant was designated Z#####. The amino acid sequence of the 58-amino acid-residue-long Z variant is shown in the figure. Figure 1 The neutralization sequence is listed as SEQ ID NO:774-808 in the sequence listing. In each sequence, the inferred PD-L1 binding motif extends from residue 8 to residue 36. The amino acid sequence of the 49-amino acid residue long polypeptide predicted to constitute the complete triple helix bundle within each of these Z variants extends from residue 7 to residue 55.

[0361] EC50 analysis of the Z variantIn the ELISA screening assay described above, the subgroup of the Z variant with the highest ELISA value was selected and subjected to target titration in ELISA format. Periplasmic samples were incubated with serially diluted biotinylated hPD-L1. Periplasmic samples containing Z13112 (SEQ ID NO: 777) that were confirmed to bind PD-L1 in the ELISA screening were selected as positive controls and used to normalize the different plates to each other. The obtained values ​​were analyzed and their respective EC50 values ​​were calculated (Table 3).

[0362] No significant binding was detected to the included B7 family (hPD-L2, hB7-H3, and hB7-H4) control proteins, nor to the control protein IgGFc (included in this paper because Fc chimeric proteins are used for selection and screening). These results indicate that the selected Z variants are specific for PD-L1.

[0363] Table 3: EC50 values ​​calculated from ELISA titration analysis

[0364] Z variant SEQ ID NO: EC50(M) Z variant SEQ ID NO: EC50(M) Z13080 774 <![CDATA[2.8×10 -10 ]]> Z13164 783 <![CDATA[2.2×10 -10 ]]> Z13088 775 <![CDATA[3.8×10 -10 ]]> Z13165 784 <![CDATA[2.4×10 -10 ]]> Z13091 776 <![CDATA[2.2×10 -10 ]]> Z13169 785 <![CDATA[1.5×10 -10 ]]> Z13104 788 <![CDATA[4.1×10 -10 ]]> Z13186 792 <![CDATA[4.7×10 -10 ]]> Z13112 777 <![CDATA[2.2×10 -10 ]]> Z13190 793 <![CDATA[2.6×10 -10 ]]> Z13115 789 <![CDATA[4.0×10 -10 ]]> Z13198 786 <![CDATA[1.6×10 -10 ]]> Z13117 790 <![CDATA[2.9×10 -10 ]]> Z13210 794 <![CDATA[3.5×10 -10 ]]> Z13134 791 <![CDATA[4.5×10 -10 ]]> Z13304 787 <![CDATA[3.2×10 -10 ]]> Z13147 779 <![CDATA[2.8×10 -10 ]]> Z13368 795 <![CDATA[4.8×10 -10 ]]> Z13154 780 <![CDATA[1.1×10 -10 ]]> Z13447 796 <![CDATA[2.9×10 -10 ]]> Z13158 782 <![CDATA[2.5×10 -10 ]]>

[0365] Example 2

[0366] Subcloning and generation of subgroups of primary PD-L1-binding Z variants

[0367] Materials and Methods

[0368] Subcloning of Z variants with His6-tagThe following 14 PD-L1 binding Z variant DNAs were amplified from the library vector pAY02592: Z13080 (SEQ ID NO:774), Z13088 (SEQ ID NO:775), Z13091 (SEQ ID NO:776), Z13112 (SEQ ID NO:777), Z13120 (SEQ ID NO:778), Z13147 (SEQ ID NO:779), Z13154 (SEQ ID NO:780), Z13156 (SEQ ID NO:781), Z13158 (SEQ ID NO:782), Z13164 (SEQ ID NO:783), Z13165 (SEQ ID NO:784), Z13169 (SEQ ID NO:785), Z13198 (SEQ ID NO:786), and Z13304 (SEQ ID NO:787). Standard molecular biology techniques (basically as described in detail in WO2009 / 077175 for Z variants binding to another target) were used to apply a subcloning strategy for constructing monomeric Z variant molecules with an N-terminal His6-tag. The Z gene fragment was subcloned into the expression vector pAY01448, resulting in the coding sequence MGSSHHHHHHLQ-[Z#####]-VD.

[0369] Subcloning of Z variants with C-terminal Cys Two Z variants, Z13091 (SEQ ID NO: 776) and Z13156 (SEQ ID NO: 781), were further subcloned using standard molecular biology techniques by mutating them to begin with the N-terminal amino acid AE instead of VD and adding the amino acid VDC to the C-terminus (incorporating a unique cysteine ​​residue into the polypeptide). The resulting coding sequences were designated Z15168-Cys (SEQ ID NO: 809) and Z15169-Cys (SEQ ID NO: 810), respectively.

[0370] nourish E. coli T7E2 cells (GeneBridges) were transformed with plasmids containing gene fragments of each of their respective PD-L1-binding Z variants and cultured at 37°C in 940 ml of TSB-YE medium supplemented with 50 μg / ml kanamycin. To induce protein expression, [the following was observed] at OD [the following temperature range]. 600 When the concentration was 2, IPTG was added to a final concentration of 0.2 mM and the culture was incubated at 37°C for another 5 hours. Cells were harvested by centrifugation.

[0371] Purification of PD-L1 binding Z variants with His6-tag Resuspend approximately 1-2g of each cell precipitate in 30ml of supplemental solution. (Merck, Catalogue No. 1.01654.0001) The protein buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4) was added to a 15 U / ml binding buffer. After cell lysis by sonication, cell debris was removed by centrifugation, and the supernatants were applied to a 1 ml His GraviTrap IMAC column (GE Healthcare, Catalogue No. 11-0033-99). Contaminants were removed by washing with wash buffer (20 mM sodium phosphate, 0.5 M NaCl, 60 mM imidazole, pH 7.4), and the PD-L1-binding Z variant was subsequently eluted with elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4). Following IMAC purification, the protein buffer was exchanged with PBS using a PD-10 column (GE Healthcare, Catalogue No. 17-0851-01).

[0372] Purification of PD-L1 binding Z variant with C-terminal Cys Resuspend the respective cell pellets in 20 mM Tris-HCl, pH 8 (10 ml buffer / g cell pellet) and lyse by heat treatment in a 90°C water bath for 10 min, followed by cooling on ice to approximately 20°C. Add (1 μl / g cell pellet) and each cell lysate was incubated at RT for 30 min, then cell debris was removed by centrifugation. To reduce the disulfide, dithiothreitol (DTT; Acros organics, catalog number 165680250) was added to a final concentration of 20 mM, followed by incubation at RT for 1 h. Purification was performed by anion exchange followed by reversed-phase chromatography (RPC). Buffer exchange was performed using a HiPrep 26 / 10 column (GE Healthcare, catalog number 17-5087-01) with 20 mM HEPES, 1 mM EDTA, pH 7.2. Finally, in Purify each Z variant on a red bar (Hyglos, catalog number 321063) to ensure low endotoxin content.

[0373] For each protein purified by any of the methods described above, the concentration is determined by using... The absorbance at 280 nm and the extinction coefficient of the protein were determined using an ND-1000 spectrophotometer. Purity was analyzed by SDS-PAGE stained with Coomassie Brilliant Blue, and the identity of each purified Z variant was confirmed by HPLC-MS analysis (HPLC-MS 1100; Agilent Technologies).

[0374] result

[0375] Cultivation and purificationPD-L1-binding Z variants with His6-tags or C-terminal Cys were expressed as soluble gene products in *E. coli*. SDS-PAGE analysis of each final protein product showed that they primarily contained PD-L1-binding Z variants. The correct identity and molecular weight of each Z variant were confirmed by HPLC-MS analysis.

[0376] Example 3

[0377] Characterization of primary PD-L1 binding Z variant

[0378] In this embodiment, subgroups of the Z variant were characterized in terms of stability and in vitro binding properties. The specificity and affinity of the Z variant for human PD-L1 were analyzed by SPR, and binding to PD-L1-expressing cells was analyzed using fluorescence activated cell sorting (FACS). Furthermore, the ability of the Z variant to block the binding of PD-L1 to its receptor PD1 was investigated using AlphaLISA.

[0379] Materials and Methods

[0380] Biacore dynamics and specificity analysis The kinetic constants (kJ) of hPD-L1 for 14 His6-tagged Z variants were determined using a Biacore 2000 instrument (GE Healthcare). a and k d ) and affinity (K D The binding of some Z variants to sequence-related proteins hPD-L2, hB7-H3, hB7-H4, and mPD-L1 (mouse PD-L1 Fc chimera, R&D Systems, catalog number 1019-B7) was also tested.

[0381] hPD-L1, hPD-L2, hB7-H3, hB7-H4, and mPD-L1 were immobilized on carboxylated dextran layers on different CM5 chip surfaces (GE Healthcare, catalog BR100012) in different flow cells. Immobilization was performed using amine coupling chemistry and HBS-EP as the running buffer (0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% v / v surfactant P20, GE Healthcare, catalog BR100188) according to the manufacturer's protocol. Ligand immobilization levels on the surfaces were 468-894 RU for hPD-L1, 537-742 RU for hPD-L2, 383 RU for hB7-H3, 538-659 RU for hB7-H4, and 482 RU for mPD-L1. One flow cell surface on each chip was activated and inactivated to serve as a blank during analyte injection. In kinetic experiments, HBS-EP was used as the run buffer at a flow rate of 50 μl / min. The analyte, the Z variant, was individually diluted in HBS-EP buffer to concentrations ranging from 1000 nM to 0.01 nM and injected for 5 min, followed by dissociation in the run buffer for 15–25 min. After dissociation, the surface was regenerated by one or two injections of 0.1% SDS. Kinetic constants were calculated from the sensor plots using the Langmuir 1:1 model in BiaEvaluation software 4.1 (GE Healthcare).

[0382] AlphaLISA blocking assay:The potential of the Z variant to inhibit PD-L1 binding to PD-1 was analyzed by AlphaLISA and recorded in an EnSpire multiplate reader 2300 (Perkin Elmer). hPD-1 (human PD-1 Fc-chimer; R&D Systems, catalog 1086-PD-050) was immobilized on AlphaLISA receptor beads (Perkin Elmer, catalog 6772002) according to the manufacturer's recommendations. His6-tagged Z variants were serially diluted 1:3 at 1:3 ratios to a final concentration of 250 nM to 12 pM in 384-well plates (Perkin Elmer, catalog G6005350) and incubated with 10 nM biotinylated hPD-L1 in AlphaLISA buffer (Perkin Elmer, catalog AL000F) for 1 h. hPD-1-coated receptor beads were added at a final concentration of 10 μg / ml and incubated for 1 h. Finally, streptavidin-coated donor beads (PerkinElmer, catalog number 6772002) were added to a final concentration of 40 μg / ml and incubated for 30 min. All incubations were performed at RT in the dark. The plates were analyzed in an EnSpire instrument, and IC50 values ​​were calculated using a GraphPad Prism 5.

[0383] Cell binding analysis via FACSThe potential of the Z variant to bind to PD-L1-expressing cells was investigated using fluorescence activated cell sorting (FACS). THP-1 cells cultured in RPMI (Lonza, catalog BE12-702F) containing 10% FBS were stimulated overnight with 10 ng / ml IFNg (R&D Systems, catalog 285-IF-100), which resulted in upregulation of PD-L1. 150,000 stimulated and unstimulated cells were pipetted into each well of a V-bottom 96-well plate (Nunc, catalog 277143), and the cells in the plate were subsequently pelleted at 400 g for 3 min on RT. The supernatant was removed, and the cells were resuspended in 100 μl PBS containing 10 μg / ml of different His-tagged Z variants plus 2.5% FBS (staining buffer). 1 μg / ml mouse anti-PD-L1 antibody (R&D Systems, catalog MAB1561) was used as a positive control. Cells incubated with a separate buffer were used as negative controls. Cells were incubated in the dark at 8°C for 1 h, washed twice with 100 μl of staining buffer, and resuspended in 100 μl of staining buffer containing goat anti-Z antibody (in-house manufactured) at a concentration of 5 μg / ml. Cells stained with the positive controls were treated with buffer only. Cells were incubated in the dark at 8°C for 1 h, washed twice with 100 μl of staining buffer, and resuspended in 100 μl of staining buffer containing either Alexa Fluor 647 chicken anti-goat IgG antibody (Life Technologies, catalog number A21469) or Alexa Fluor 647 goat anti-mouse IgG antibody (Life Technologies, catalog number A21236). Cells were again incubated in the dark at 8°C for 1 h, washed twice with 100 μl of staining buffer, and resuspended in 200 μl of staining buffer. Data from 10,000 cells were obtained using FACS Calibur (Beckman Coulter), and the data were analyzed using Flowing software 2.5.0 (Turku University). Mean fluorescence intensity (MFI) was used as a readout for binding capacity.

[0384] Circular dichroism (CD) spectral analysisTwo purified Z variants, Z15168-Cys (SEQ ID NO: 809) and Z15169-Cys (SEQ ID NO: 810), containing C-terminal cysteine, were diluted to 0.5 mg / mL in 20 mM HEPES, 1 mM EDTA, pH 7.2. For each diluted Z variant, CD spectra in the 250–195 nm range were obtained at 20 °C. Variable temperature measurement (VTM) was also performed to determine the melting temperature (Tm). In VTM, absorbance was measured at 221 nm while the temperature was increased from 20 °C to 90 °C at a rate of 5 °C / min. New CD spectra were obtained at 20 °C after the heating procedure to investigate the refolding ability of the Z variants. CD measurements were performed using cuvettes with a 1 mm optical path length on a Jasco J-810 spectropolarimeter (Jasco Scandinavia AB).

[0385] result

[0386] Biacore dynamics and specificity analysis The interaction between 14 His6-tagged PD-L1-binding Z-variants and hPD-L1 was analyzed in a Biacore instrument by injecting different concentrations of Z-variants onto surfaces containing a fixed amount of hPD-L1. All tested Z-variants showed binding to hPD-L1. The kinetic parameters (KL1) of Z-variant binding to hPD-L1 were obtained using a 1:1 interaction model. D k a and k d The summaries are given in Table 4. Typical curves obtained for the two selected Z variants Z13091 (SEQ ID NO: 776) and Z13156 (SEQ ID NO: 781), with the responses from the blank surface subtracted, are shown in Figure 2.

[0387] Table 4: Kinetic parameters of Z variant binding to hPD-L1

[0388]

[0389] Binding of Z variant subgroups to four fixed sequence-associated proteins: hPD-L2, hB7-H3, hB7-H4, and mPD-L1 was also tested. No binding to hPD-L2, hB7-H3, hB7-H4, or mPD-L1 was detected at Z variant concentrations up to 50 nM. Some responses against B7-H4 were observed in Z13156 and Z13165 when small doses of selected Z variants (Z13088, Z13091, Z13112, Z13147, Z13154, Z13156, Z13165, Z13169, Z13198) were observed. Results of the binding specificity analysis are summarized in Table 5. Typical non-interacting traces from SPR analysis of hPD-L2, hB7-H3, and hB7-H4 are shown in Table 5. Figure 3 middle.

[0390] Table 5: Binding specificity against mPD-L1, hPD-L2, hB7-H3, and hB7-H4

[0391]

[0392] Na was not measured; nd was not detected as a binding agent.

[0393] AlphaLISA blocking assay: In an AlphaLISA blocking assay, the ability of 14 His6-tagged Z variants to inhibit the binding of hPD-L1 to hPD-1 was tested. Serial dilutions of the Z variants were incubated with biotinylated hPD-L1, and the blocking ability of each variant was measured after the addition of hPD-L1-coated receptor beads and subsequently streptoacidin-coated donor beads. Inhibition was measured as a reduction in the AlphaLISA count for positive Z variants. The calculated IC50 values ​​for all 14 variants that showed blocking of PD-L1-PD-1 binding in this assay are shown in Table 6.

[0394] Table 6: IC50 values ​​of Z variants that inhibit PD-L1 binding to PD-1

[0395]

[0396]

[0397] Table 7: Normalized MFI for the binding of the Z variant to THP-1 cells

[0398] Z variant SEQ ID NO: MFI (Normalized) Z13080 774 1.06 Z13088 775 1.00 Z13091 776 1.00 Z13112 777 0.76 Z13120 778 0.71 Z13147 779 0.56 Z13154 780 1.04 Z13156 781 1.09 Z13158 782 0.91 Z13164 783 0.81 Z13165 784 1.24 Z13169 785 0.91 Z13198 786 0.89 Z13304 787 0.75 Anti-PD-L1 antibody - 0.40

[0399] Cell binding analysis via FACSThis experiment confirmed the binding of the PD-L1-specific Z variant to cells expressing PD-L1. THP-1 cells stimulated overnight with IFNγ (which increases PD-L1 expression) were stained with 10 μg / ml of each His6-tagged Z variant. Analysis was performed under two different conditions, and the MFI values ​​for Z13091 normalized in both assays are presented in Table 7.

[0400] CD Analysis CD spectra of two selected PD-L1-binding Z variants, Z15168-Cys (SEQ ID NO: 809) and Z15169-Cys (SEQ ID NO: 810), with C-terminal cysteine ​​residues, showed that both variants possess an α-helical structure at 20 °C based on typical minimums at 208 nm and 222 nm. For both Z variants, reversible folding was observed when the spectra measured before and after heating to 90 °C were overlapped. Figure 4 The noise observed in the far UV region was expected to be due to the buffer effect (HEPES, used as the analytical buffer, has strong absorption at 200 nM and below). The melting temperatures (Tm) of Z15168-Cys and Z15169-Cys were determined to be 50 °C and 58 °C, respectively (Table 8).

[0401] Table 8: Deconvolution Temperature (Tm)

[0402] Z variant SEQ ID NO: Tm (°C) Z15168-Cys 809 50 Z15169-Cys 810 58

[0403] Example 4

[0404] Design and construction of a mature library of PD-L1 combined with Z variants

[0405] In this embodiment, a mature library was constructed. The PD-L1 binding Z variant was selected using this library. Selection from a mature library can produce conjugates with increased affinity (Orlova et al., (2006) Cancer Res 66(8):4339-48). In this study, split-pool DNA synthesis was used to generate randomized single-stranded oligonucleotides, allowing defined codons to be incorporated into the desired positions during synthesis.

[0406] Materials and Methods

[0407] Document DesignThe library is based on the sequences of the PD-L1 binding Z variants described, identified, and characterized in Examples 1 and 3. In the new library, 13 variable positions in the Z molecular scaffold are biased toward certain amino acid residues according to a strategy based on the Z variant sequences defined in SEQ ID NO:774-808. Two oligonucleotides with complementary 3' ends, one positive and one negatively complementary, are generated using cleavage synthesis. Two oligonucleotides were annealed and extended by PCR using external primers to generate a gene fragment covering 147 bp, corresponding to the amino acid sequence flanking the restriction sites of XhoI and SacI: 5'-AA ATA AAT CTC GAG GTA GAT GCC AAA TAC GCC AAA GAA CGT AAC NNN GCG GCTNNN GAG ATC CTG NNN CTG CCT AAC CTC ACC NNN NNN CAA NNN TGG GCC TTC ATC TGGAAA TTA NNN GAT GAC CCA AGC CAG AGC TCA TTA TTT A-3' (SEQ ID NO: 819; randomized codons are interpreted as NNN) partially randomized helices 1 and 2. The oligonucleotides were ordered from Ella Biotech GmbH (Martinsried Germany).

[0408] Table 9: Design of Mature Document Libraries

[0409]

[0410] The theoretical distribution of amino acid residues in a new library comprising seven variable positions (11, 14, 18, 24, 25, 27, and 35) within the Z-molecule scaffold is given in Table 9. The resulting theoretical library size is 5.3 × 10⁻⁶. 7 Variations.

[0411] Library Construction The library was amplified using AmpliTaq Gold polymerase (Life Technologies, catalog 4311816) during 12 cycles of PCR, as recommended by the supplier, and the pooled products were purified using the QIAquick PCR Purification Kit (QIAGEN, catalog 28106), as recommended by the supplier. The purified pools of randomized library fragments were digested with restriction endonucleases XhoI and SacI-HF (New England Biolabs, catalog 0146L and catalog 3156M, respectively) and concentrated using the PCR Purification Kit. Subsequently, the products were subjected to preparative 2.5% agarose gel (NuSieve) as recommended by the supplier. The sample was run on an agarose gel electrophoresis (Lonza, catalog number 50080) and purified using a QIAGEN gel extraction kit (QIAGEN, catalog number 28706).

[0412] Phage vector pAY02592 (basically as follows) pAffi1, as described above, was restriction endonucleated with the same enzyme and purified using phenol / chloroform extraction and ethanol precipitation. The restriction endonuclease fragments and the restriction endonuclease vector were ligated at a 5:1 molar ratio using T4 DNA ligase (Thermo Scientific, catalog number EL0011) at RT for 2 h, followed by overnight incubation at 4°C. The ligated DNA was recovered by phenol / chloroform extraction and ethanol precipitation, and then dissolved in 10 mM Tris-HCl, pH 8.5. Thus, the resulting library in vector pAY02592 encodes Z variants, each Z variant fused to an albumin-binding domain (ABD) derived from a streptococcal G protein.

[0413] The ligation reaction (approximately 160 ng DNA / transformation) was electroporated into electrocompetent *E. coli* ER2738 cells (Lucigen, Middleton, WI, USA, 50 μl). Immediately after electroporation, approximately 1 ml of recovery medium (supplying *E. coli* ER2738 cells) was added. The transformed cells were incubated at 37°C for 60 min. Samples were collected for titration and to determine the number of transformants. Cells were then pooled and cultured overnight at 37°C in 1 L of TSB-YE medium supplemented with 2% glucose, 10 μg / ml tetracycline, and 100 μg / ml ampicillin. Cells were pelleted at 4,000 g for 15 min and resuspended in PBS / glycerol solution (approximately 40% glycerol). Cells were aliquoted and stored at -80°C. Clones from the Z variant library were sequenced to validate inclusions and to evaluate the results compared to the designed library. Sequencing was performed as described in Example 1, and the amino acid distribution was confirmed.

[0414] Preparation of phage stock solutionCells from a glycerol stock solution containing the phage plasmid library were seeded into 3.5 μL of TSB-YE supplemented with 1 g / L glucose, 100 mg / L ampicillin, and 10 mg / L tetracycline. Cells were cultured at 37°C with vortexing (100 RPM). When the cells reached an optical density (OD600) of 0.59 at 600 nm, approximately 620 μL of the culture was infected with a 5 × 10⁻⁶ M13K07 helper phage. Cells were incubated for 30 min, then centrifuged at 3,000 g to pellet the cells and resuspended in 3 μL of fresh TSB-YE supplemented with 100 mg / L ampicillin, 25 mg / L kanamycin, and 0.1 mM IMPTG. The culture was aliquoted into 6 × 5 μL shake flasks and cultured at 30°C with vortexing. After approximately 18 h, the cells were centrifuged at 4,700 g to pellet the cells. Phage particles were precipitated twice from the supernatant in PEG / NaCl as described in Example 1, filtered, and dissolved in PBS and glycerol. The phage stock solution was stored at -80°C until selected for use.

[0415] result

[0416] Library Construction A new library was designed based on a set of PD-L1 binding Z variants (Examples 1 and 3) with proven binding properties. The theoretical size of the designed library is 5.3 × 10⁻⁶. 7 The Z variant was used. The actual size of the library, determined by titration after transformation into *E. coli* ER2738 cells, was 2.8 × 10⁻⁶. 9 Transformed strains.

[0417] Library quality was tested by sequencing 116 transformants and comparing their actual sequences with the theoretical design. The contents of the actual library showed satisfactory results compared to the designed library. Therefore, a mature library of potential PD-L1 binders was successfully constructed.

[0418] Example 5

[0419] Selection, screening, and characterization of Z variants from mature libraries

[0420] Materials and Methods

[0421] PD-L1-binding Z variant phage display selectionThe target protein PD-L1 was biotinylated as described in Example 1. Phage display selection was performed in four cycles targeting hPD-L1 using a novel library of Z variant molecules constructed as described in Example 4, essentially as described in Example 1, with the following exceptions: Exception 1: SA-beads were used to capture the PD-L1:Z variant complex in all selection orbitals. Exception 2: Pre-selection was performed only for SA-beads coated with biotinylated human IgG-Fc prior to cycles 1 and 2. Additionally, in cycle 1, another pre-selection was performed for SA-beads coated with a mixture of PD-L2, B7-H3, and B7-H4, as previously described in Example 1. Exception 3: Selection for biotinylated human PD-L1 was performed in four cycles initially divided into two distinct orbitals (1 and 2). As selection proceeded, the orbitals were further divided based on target concentration and number and / or time of washes, ultimately ending with 11 orbitals in cycle 4. More precisely, the first track (1) is divided in the second through fourth cycles, resulting in a total of 2 tracks (1-1 to 1-2) in cycle 2, 4 tracks (1-1-1 to 1-2-2) in cycle 3, and 7 tracks (1-1-1-1 to 1-2-2-2) in cycle 4. The second track (2) is divided in the second through fourth cycles, resulting in a total of 2 tracks (2-1 to 2-2) in cycle 2, 4 tracks (2-1-1 to 2-2-2) in cycle 3, and 4 tracks (2-1-1-1 to 2-2-2-1) in cycle 4. Exception 4: During the 19-hour wash step of selected cycle 1-1-2-3, a 20-fold molecular excess of unbiotinylated hPD-L1 is added to the wash buffer. An overview of the selection strategy is shown in Table 10, describing the increased stringency obtained in subsequent cycles by using reduced target concentrations and increased washes.

[0422] Table 10. Selection of biotinylated hPD-L1 Fc using established libraries

[0423]

[0424] Generation of Z variants for ELISAThe Z variant was generated by inoculating selected single colonies into 1.2 ml of TSB-YE medium supplemented with 100 μg / ml ampicillin and 1 mM IPTG in a deep-well plate (Nunc, catalog number 278752). The plate was incubated at 37°C with rotation for 24 h. The cells were pelleted by centrifugation at 3300 g and resuspended in 150 μl of 0.05% PBST and frozen at -80°C to release the periplasmic fraction. The frozen sample was thawed in a water bath and the freeze-thaw procedure was repeated 8 times. The periplasmic fraction was then separated in a deep-well plate (Axygen, catalog number 391-01-101) by filtration through an EMD Millipore filter (MSNANLY50). The final periplasmic extract supernatant contained the Z-variant as a fusion with ABD, denoted as AQHDEALE-[Z#####]-VDYV-[ABD]-YVPG( (Same as above). Z##### refers to a single Z variant of 58 amino acid residues.

[0425] ELISA screening for Z variant As described in Example 1, the binding of the Z variant to human PD-L1 was analyzed in an ELISA assay with the following exceptions: Exception 1: The periplasmic fraction was diluted 1:8 with 0.05% PBST, added to the wells, and incubated for 1.7 h. Exception 2: A negative control containing the fusion protein ABD but without the Z-fusion partner was used instead of a blank control. Exception 3: A periplasmic sample containing the primary PD-L1 binding Z variant Z13091 (SEQ ID. NO: 776) was included in duplicate on each plate and analyzed as a positive control. Exception 4: 50 μl of biotinylated hPD-L1 at a concentration of 40 pM in PBSC was added to each well, and the plate was incubated at RT for 1.8 h.

[0426] sequencing As described in Example 1, all clones were sequenced in parallel with the ELISA screening.

[0427] ELISA EC50 analysis The selected PD-L1 binding Z variant was subjected to analysis of the response to a series of dilutions of biotinylated human PD-L1 as described in Example 1, with the following exceptions: Exception 1: The Z variant was diluted 1:8 in 0.05% PBST and then added to the wells. Exception 2: Biotinylated human PD-L1 was added at a concentration of 15 nM and step-diluted 1:3 to 0.25 pM. Exception 3: Periplasmic samples containing the primary PD-L1 binding Z variant Z13091 (SEQ ID. NO: 776) were included for comparison and analysis with the mature Z variant.

[0428] result

[0429] PD-L1-binding Z variant phage display selection After four cycles of phage display selection targeting biotinylated hPD-L1, individual clones were obtained.

[0430] ELISA screening for Z variant Clones obtained after four cycles of selection were generated in 96-well plates and screened for hPD-L1 binding activity by ELISA. Most unique Z variants were found to provide a higher mean response against hPD-L1 at a concentration of 40 pM than the positive control Z13091 (mean 0.264 AU). The mean response of the negative control was 0.051 AU.

[0431] sequencing After four cycles of selection, the resulting clones were sequenced. Each variant was given a unique identification number Z##### as described in Example 1. The amino acid sequence of the 58-amino acid residue-long Z variant is shown in... Figure 1 The neutralization sequence is listed as SEQ ID NO:1-773 in the sequence listing. In each sequence, the presumed PD-L1 binding motif extends from position 8 to position 36. The amino acid sequence of the 49-amino acid residue long polypeptide predicted to constitute the complete triple helix bundle within each of these Z variants extends from residue 7 to residue 55.

[0432] EC50 analysis of the Z variant In the ELISA screening assay described above, the subgroup of Z variants with the highest ELISA values ​​was selected and subjected to target titration in ELISA format. Periplasmic samples were incubated with serially diluted biotinylated hPD-L1. Periplasmic samples containing Z13091 (SEQ ID NO:776) (the isolated primary Z variant that showed the highest binding affinity to hPD-L1) were included as positive controls. Values ​​obtained using GraphPad Prism 5 analysis were used, and their respective EC50 values ​​were calculated (Table 11). All mature Z variants showed lower EC50 values ​​than the optimal primary Z variant Z13091.

[0433] Table 11: Calculated EC50 values ​​from mature Z-ABD variants

[0434]

[0435]

[0436] Example 6

[0437] Subcloning and generation of subgroups of mature PD-L1-binding Z variants

[0438] Materials and Methods

[0439] Subcloning of Z variants with His6-tag The following 24 mature PD-L1 binding Z variant DNAs were amplified from the library vector pAY02592 and subcloned using the His6-tag as described in Example 2 above: (Z17746 (SEQ ID NO:8), Z17748 (SEQ ID NO:11), Z17756 (SEQ ID NO:7), Z17825 (SEQ ID NO:5), Z17911 (SEQ ID NO:3), Z17964 (SEQ ID NO:2), Z17972 (SEQ ID NO:19), Z17978 (SEQ ID NO:13), Z18022 (SEQ ID NO:9), Z18039 (SEQ ID NO:20), Z18048 (SEQ ID NO:4), Z18052 (SEQ ID NO:14), Z18054 (SEQ ID NO:22), Z18064 (SEQ ID NO:1), Z18066 (SEQ ID NO:8), Z17748 (SEQ ID NO:1), Z17756 (SEQ ID NO:7), Z17825 (SEQ ID NO:5), Z17911 (SEQ ID NO:3), Z17964 (SEQ ID NO:2), Z17972 (SEQ ID NO:14), Z18054 (SEQ ID NO:22), Z18064 (SEQ ID NO:1), Z18066 (SEQ ID NO:1), Z18066 (SEQ ID NO:2), Z18066 (SEQ ID NO:2), Z18066 (SEQ ID NO:2), Z18066 (SEQ ID NO:2), Z18064 ...4 (SEQ ID NO:2), Z18066 (SEQ ID NO: NO:12), Z18070 (SEQ ID NO:10), Z18074 (SEQ ID NO:6), Z18090 (SEQ ID NO:17), Z18101 (SEQ ID NO:23), Z18129 (SEQ ID NO:16), Z18149 (SEQ ID NO:18), Z18233 (SEQ ID NO:21), Z18353 (SEQ ID NO:15) and Z18418 (SEQ ID NO:24)).

[0440] Subcloning of Z variants with C-terminal Cys Three Z variants, Z18064 (SEQ ID NO:1), Z17964 (SEQ ID NO:2), and Z18090 (SEQ ID NO:17), were further subcloned using standard molecular biology techniques by mutating them to begin with the N-terminal amino acid AE instead of VD and adding the amino acid VDC to the C-terminus (incorporating a unique cysteine ​​residue into the polypeptide). The resulting sequences were designated Z18608-Cys (SEQ ID NO:811), Z18609-Cys (SEQ ID NO:812), and Z18610-Cys (SEQ ID NO:813), respectively.

[0441] nourish Typically, *E. coli* T7E2 cells (GeneBridges) are transformed with plasmids containing gene fragments of each of their respective PD-L1-binding Z variants and cultured at 37°C in approximately 940 ml of TSB-YE medium supplemented with 50 μg / ml kanamycin. To induce protein expression, [the process is repeated in the original text].600 At OD2, IPTG was added to a final concentration of 0.2 mM and the culture was incubated at 37°C for another 5 h. Cells were harvested by centrifugation. Specifically, Z18608-Cys and Z18609-Cys were fed-batch cultured in approximately 700 ml of defined mineral medium supplemented with 50 μg / ml kanamycin at 37°C. To induce protein expression, at OD200... 600 At 75°C, add IPTG to a final concentration of 0.5 mM and incubate the culture for another 7 hours. Harvest the cells by centrifugation.

[0442] Purification of PD-L1 binding Z variants with His6-tag The IMAC purification, buffer exchange with PBS, and concentration determination were basically performed as described in Example 2.

[0443] Purification of PD-L1 binding Z variant with C-terminal Cys The cell pellets were resuspended in 20 mM Tris-HCl, 0.5 mM EDTA, 0.1% Tween 80, pH 7.5 (10 ml buffer / g cell pellet) and lysed by heat treatment in an 80°C water bath for 10 min, followed by cooling on ice to approximately 20°C. Add Cell lysates were incubated at RT for 30 min (1 μl / g cell pellet) and cell debris was removed by centrifugation. To reduce disulfide, dithiothreitol (DTT; Acros Organics, catalog number 165680250) was added to a final concentration of 10 mM, followed by incubation at RT for 20 min. The lysates were then filtered through a 0.45 μm syringe filter (Millipore). Purification was performed by anion exchange followed by reversed-phase chromatography (RPC). Buffer exchange with 20 mM HEPES, 1 mM EDTA, pH 7.2 was performed using Sephadex G-25 medium (GE Healthcare) packaged in XK-50 columns.

[0444] For any protein purified using any of the methods described above, the concentration is determined by using... The absorbance at 280 nm and the extinction coefficient of the protein were determined using an ND-1000 spectrophotometer. Purity was analyzed by SDS-PAGE stained with Coomassie Brilliant Blue, and the identity of each purified Z variant was confirmed by HPLC-MS analysis (HPLC-MS 1100; Agilent Technologies).

[0445] result

[0446] Cultivation and purificationThe PD-L1-binding Z variant was expressed as a soluble gene product in *E. coli*. The amount of purified protein from approximately 2.0–2.4 g of bacterial precipitate was determined by spectrophotometric measurement of absorbance at 280 nm, and ranged from approximately 18 mg to 29 mg for different His6-tagged PD-L1-binding Z variants. SDS-PAGE analysis of each final protein product showed that these products primarily contained the PD-L1-binding Z variant. The correct identity and molecular weight of each Z variant were confirmed by HPLC-MS analysis.

[0447] Example 7

[0448] Further characterization of the subgroup of primary PD-L1-binding Z variant

[0449] In this embodiment, subgroups of the Z variant were characterized in terms of stability and various binding properties. The specificity and affinity of the Z variant for PD-L1 were analyzed using Biacore, and the ability of the Z variant to block the binding of PD-L1 to its receptor PD-1 was investigated using AlphaLISA.

[0450] Materials and Methods

[0451] Biacore dynamics and specificity analysis : Determine the kinetic constants (k) of 24 mature His6-tagged Z variants (specified in Example 6) for human PD-L1 and rhesus monkey PD-L1 (RhPD-L1; rhesus monkey PD-L1 / Fc chimera, Sino Biological Inc., catalog number 90251-C02H). a and k d ) and affinity (K DThe binding of the Z variants to the sequence-related proteins hPD-L2, hB7-H3, and hB7-H4 was also tested. Biacore analysis was performed essentially as described in Example 3, however, using a flow rate of 30 μl / min. The ligand immobilization levels on the surface were 1030 RU for hPD-L1, 1060 RU for RhPD-L1, 1070 RU for hPD-L2, 1090 RU for hB7-H3, and 770 RU for hB7-H4. In the first binding kinetic analysis, 24 Z variants were injected at concentrations of 5 nM and 50 nM, respectively, onto chips immobilized with hPD-L1 and RhPD-L1. In the first experiment, 12 mature PD-L1 binding Z variants that showed the highest affinity for hPD-L1 were analyzed in more detail and injected onto immobilized hPD-L1 and RhPD-L1 at concentrations of 135 nM, 45 nM, 15 nM, 5 nM, and 1.67 nM. In the specificity assay, i.e., binding analysis against hPD-L2, hB7-H3, and hB7-H4, 24 Z variants were injected at a concentration of 500 nM.

[0452] AlphaLISA blocking assay: As described in Example 3, the potential of the Z variant to inhibit the binding of PD-L1 to its natural partner PD-1 was analyzed in an AlphaLISA assay, with the following exceptions: Exception 1: The His6-tagged Z variant was serially diluted 1:3 to a final concentration of 250 nM to 4 pM in a 384SW plate (Perkin Elmer, catalog number 6008350) and incubated with 8 nM biotinylated hPD-L1 (R&D Systems) in AlphaLISA buffer (Perkin Elmer, catalog number AL000F) for 45 min. Exception 2: hPD-1-coated receptor beads were added to a final concentration of 10 μg / ml and incubated for 50 min.

[0453] Circular dichroism (CD) spectral analysis As described in Example 3, the purified His6-tagged Z variant subgroups were analyzed by CD spectroscopy, with the following exceptions: the analysis buffer was PBS and the temperature was raised to 80°C in VTM.

[0454] result

[0455] Biacore kinetics and specificity analysisThe interactions of 24 mature His6-tagged Z variants with human PD-L1 and rhesus monkey PD-L1 were analyzed in a Biacore instrument by injecting different concentrations of Z variants onto surfaces containing fixed hPD-L1 and RhPD-L1, respectively. First kinetic analyses were performed to rank the Z variants according to their affinity for hPD-L1 and RhPD-L1, and to compare their binding kinetics with the primary PD-L1-binding Z variant Z13091. The approximate affinity constants obtained from the ranking experiments using a 1:1 interaction model are summarized in Table 12.

[0456] The 12 mature Z variants that exhibited the highest binding affinity for hPD-L1 were further analyzed, and the more precisely determined kinetic parameters for these 12 Z variants are given in Table 13. Typical curves obtained for two selected variants (with the response from the blank surface subtracted) are shown in Table 13. Figure 5 middle.

[0457] Table 12: Approximate affinity constants of Z variants for binding to hPD-L1 and RhPD-L1

[0458]

[0459]

[0460] In addition, the binding of all 24 mature His6-tagged Z variants to three sequence-related proteins, hPD-L2, hB7-H3, and hB7-H4, was tested. Consistent with the results in Example 3, no binding to any control protein was detected at a Z variant concentration of 500 nM.

[0461] Table 13: Kinetic parameters of Z variant binding to hPD-L1 and RhPD-L1

[0462]

[0463]

[0464] AlphaLISA blocking assayThe ability of 24 mature His6-tagged monomeric Z variants to inhibit the binding of hPD-L1 to hPD-1 was tested in an AlphaLISA blocking assay. The primary Z variant Z13091 was included as a reference. Serial dilutions of the Z variants were incubated with biotinylated hPD-L1, and the blocking ability of each respective variant was measured after the addition of hPD-L1-coated receptor beads and subsequently streptoacidin-coated donor beads. Inhibition was measured as a reduction in the AlphaLISA count for positive Z variants. The calculated IC50 values ​​for the 25 variants demonstrating blocking of PD-L1 to PD-1 binding in this assay are shown in Table 14.

[0465] Table 14: IC50 values ​​of Z variants blocking PD-1 / PD-L1 interaction

[0466]

[0467]

[0468] CD Analysis CD spectra of 24 mature PD-L1-binding Z variants with the His6 tag showed that each variant possessed an α-helical structure at 20 °C. The melting temperature (Tm) was determined using variable-temperature measurements (Table 15). For all PD-L1-binding Z variants, reversible folding was observed when the spectra measured at 20 °C before heating to 80 °C were overlapped with those measured after heating to 80 °C. Figure 6 The two selected Z variants are shown.

[0469] Table 15: Demothering temperature of mature PD-L1-binding Z variant

[0470]

[0471] Example 8

[0472] Characterization of anti-PD-L1 / PD-1 and anti-PD-L1 / CTLA-4 complexes

[0473] Materials and Methods

[0474] Production of complex and control antibody This study describes the construction of four different complexes targeting PD-L1 and PD-1, four different complexes targeting PD-L1 and CTLA-4, and a control antibody targeting PD-1. Heavy chain (HC) and light chain (LC) sequences were used. Lam (SEQ ID NO:815) and LC Lam(SEQ ID NO:816) Construct an antibody designated "Lam" that has the same CDR sequence and specificity as the commercially available monoclonal antibody targeting PD-1, pembrolizumab (formerly known as lambolizumab). The heavy chain (HC) sequence and the light chain (LC) sequence were used. Ipi (SEQ ID NO:817) and LC Ipi (SEQ ID NO:818) Constructed an antibody designated "Ipi" with the same CDR sequence and specificity as the commercially available monoclonal antibody targeting CTLA-4, ipilimumab. The Z variant Z15170 (SEQ ID NO:814; identical to Z13165 (SEQ ID NO:784), but starting with amino acid residue AE instead of VD), targeting PD-L1, with a C-terminal VD sequence, was coupled to HC via a flexible 15-residue (GGGGS) 3-linker. Lam LC Lam HC Ipi and LC Ipi The N-terminal genes were fused to produce the complex Z15170-HC. Lam Z15170-LC Lam Z15170-HC Ipi and Z15170-LC Ipi Alternatively, it can be fused with the C-terminal gene of the same strand to produce the complex HC. Lam -Z15170, LC Lam -Z15170, HC Ipi -Z15170 and LC Ipi -Z15170. Gene synthesis, cloning, generation by transient gene expression in CHO cells, purification by protein A chromatography, and verification of the construct by gel electrophoresis were performed using Evitria AG (Switzerland).

[0475] Biacore dynamics analysis For all eight generated complexes, the kinetic constants (ka) for hPD-L1, human PD-1 (hPD-1; R&D Systems catalog number 1086-PD-050), and human CTLA-4 (hCTLA-4; R&D Systems catalog number 325-Ct-200) were determined using a Biacore 2000 instrument (GE Healthcare). a and k d ) and affinity (K DThe binding of the control antibody Lam to PD-1 was also analyzed. A 5 μg / mL solution of each of the proteins hPD-L1, hPD-1, and hCTLA-4 was prepared in 10 mM NaAc buffer (pH 5.0 for PD-L1 and pH 4.5 for PD-1 and CTLA-4) and used for immobilization on carboxylated dextran layers on different CM5 chip surfaces (GE Healthcare, catalog number BR100012) in different flow cells. Immobilization was performed using amine coupling chemistry according to the manufacturer's protocol, and HBS-EP with 500 mM NaCl was used as the run buffer. The obtained immobilization levels were ~110–140 RU. In addition to analyzing the binding of PD-L1 to constructs having Z15170 located at the C-terminus of their respective antibodies (using solutions at concentrations of 30 nM, 90 nM, 270 nM, and 900 nM), a series of solutions of the respective complexes and Lam at concentrations of 3.33 nM, 10 nM, 30 nM, 90 nM, and 270 nM were injected and the responses were recorded.

[0476] In separate experiments, dual binding specificity was evaluated using capture assays on a Biacore 2000 instrument. The complex Z15170-HC was described above. Lam Z15170-LC Lam Z15170-HC Ipi and Z15170-LC Ipi Lam and Iprimma ( Bristol-Myers Squibb / Astra Zeneca via Apoteket AB, catalog number 065544, lot number 4A85968) was injected at a concentration of 300 nM onto a chip surface fixed with PD-1 or CTLA-4. In all cases, the injection duration was 5 min, the flow rate was 30 μl / min, with a 5 min waiting / dissociation step, followed by a second injection of 100 nM or 500 nM PD-L1 (5 min). HBS-EP with 500 mM NaCl was used as the running buffer and for protein dilution.

[0477] Cell binding analysis via FACSThe potential of the complex to bind to PD-L1-expressing cells was investigated using FACS. 150,000 cells of the breast cancer cell line MDA-MB-231, cultured in DMEM (ATCC catalog 30-2002) containing 10% FBS, were pipetted into each well of a V-bottom 96-well plate (Nunc, catalog 277143), and the cells were subsequently pelleted at 400g for 3 min at RT. The supernatant was removed, and the cells were resuspended in 100 μl PBS plus 2.5% FBS (staining buffer) containing 0.625 μg / ml of the complex Z15170-HC. Lam Z15170-LC Lam HC Lam -Z15170, LC Lam -Z15170, Z15170-HC Ipi Z15170-LC Ipi HC Ipi -Z15170 and LC Ipi -Z15170, or 0.625 μg / ml of antibody Lam or Iprimma. Mouse anti-PD-L1 antibody (RnD Systems, catalog number MAB1561) at a concentration of 1 μg / ml was used as a positive control. Cells incubated with a separate buffer were used as negative controls. Cells were incubated in the dark at 8°C for 1 h, washed twice with 100 μl of staining buffer, and resuspended in 100 μl of staining buffer containing 2.5 μg / ml goat anti-human IgG-Alexa488 (Molecular Probes, catalog number A11013), or, for cells stained with the positive control antibody, resuspended in goat anti-mouse IgG-Alexa647 antibody (Life Technologies, catalog number A21236). Cells were incubated in the dark at 8°C for 1 h, washed twice with 100 μl of staining buffer, and resuspended in 300 μl of staining buffer. Data from 10,000 cells were obtained using FACS Calibur (Beckman Coulter), and Flowing software 2.5.0 (Turku) was used. Data were analyzed by the University. Mean fluorescence intensity (MFI) was used as a readout for binding ability.

[0478] Co-culture of MDA-MB-231 and PBMCThis study used a mixed lymphocyte assay to analyze whether an Ipi-based complex could affect T cell proliferation or cytotoxicity, thereby increasing cancer cell elimination. In this study, peripheral blood mononuclear cells (PBMCs) and MDA-MB-231 cells were co-cultured for six days, and the number of T cells and cancer cells was evaluated. 20,000 MDA-MB-231 cells cultured in DMEM containing 10% FBS were pipetted into each well of a flat-bottomed 96-well plate and placed to adhere to the bottom of the well by incubation at 37°C in a humidified 5% CO2 atmosphere. On day 2 of the experiment, serial dilutions (200–0.064 nM) of the Ipi-based complex were prepared in separate plates using RPMI 1640 supplemented with 10% FCS and 1% Pen-Strep (Lonza, catalog number DE17-603E) with L-glutamate (Lonza). The DMEM medium was discarded from the MDA-MB-231 cells, and 100 μl of the diluted complex was added. PBMCs were prepared from the erythrocyte sedimentation rate (ESR) amber layer using Ficoll Paque PLUS (GE Healthcare, catalog number 17-1440-02). Briefly, the ESR amber layer was diluted 2× in PBS. 10 ml of the diluted ESR amber layer was layered on top of 5 ml of Ficoll in a 15 ml Falcon tube and centrifuged at 400 g for 30 min at RT. The lymphocyte layer was collected, and the cells were washed twice in RPMI 1640 supplemented medium as described above. Cell counts were performed, and the concentration was adjusted to 1 million cells / ml in supplemented RPMI medium. 100 μl of the cell suspension was added to a plate containing MDA-MB-231 cells. The plate was incubated at 37°C in a humidified 5% CO2 atmosphere for 6 days. On day 7 of the experiment, the number of MDA-MB-231 cells and CD3+ T cells was counted by FACS. PBMCs were transferred to V-shaped plates, washed twice with PBS containing 2% FBS (also used as staining buffer), and stained with mouse anti-CD3 antibody (EXBIO Praha, catalog number 12-631-M001) at a concentration of 2 μg / ml at 4°C for 1 h. MDA-MB231 cells were digested with trypsin (20 μl / well) and transferred to another V-shaped plate, washed twice with PBS containing 2% FBS, and stained with rabbit anti-EGFR-antibody (Abcam, catalog number ab2430-1) at a concentration of 2 μg / ml at 4°C for 1 h.Cells were washed twice with PBS containing 2% FBS and incubated at 4°C for 1 h with Alexa-fluor 488-goat-anti-rabbit antibody (Invitrogen, catalog number A11008) and Alexa-fluor 647-goat-anti-mouse antibody (Life technologies, catalog number A21236) at a concentration of 1 μg / ml.

[0479] result

[0480] Generation of complex constructs :exist Figure 7 The diagram shows a schematic of the design for each of the four types of complexes produced.

[0481] Biacore dynamics analysis The affinity of each relevant complex for the target proteins PD-L1, PD-1, and CTLA-4 was determined separately. The control antibody Lam was also analyzed against its target PD-1. The kinetic parameters of the interaction with PD-L1 are summarized in Table 16. The ability of the Z-moment of the complex to interact with PD-L1 was maintained, although the affinity decreased and was affected by the localization of the Z-moment on the antibody. For comparison, the K-value of His6-Z13165 interacting with PD-L1 was... D The K of the complex with an N-terminal localized Z moiety is 0.64 nM (as presented in Example 3), while the K of the complex with an N-terminal localized Z moiety is... D K-type complexes with a molecular weight of 1.5–2.6 nM and a C-terminal-localized Z-momentum. D The KzOz is 12-41 nM. Therefore, the N-terminal localization of the Z-part is superior to the C-terminal localization, exhibiting approximately 10-fold higher affinity. This effect is evident in both Lam and Ipi constructs. Whether the heavy or light chain of the antibody is fused is less important for the N-terminal localization of the Z-part, but has a significant impact on the C-terminal localization, particularly the KzOzOz compared to the 29-41 nM of the heavy chain fusion. D The light chain fusion has a K+ of 12-18 nM. D .

[0482] Table 16: Kinetic parameters of the binding of the specified complex to hPD-L1

[0483] Analytes <![CDATA[k a (1 / Ms)]]> <![CDATA[k d (1 / s)]]> <![CDATA[K D (M)]]> <![CDATA[HC Lam -Z15170]]> <![CDATA[2.44×10 4 ]]> <![CDATA[7.17×10 -4 ]]> <![CDATA[2.9×10 -8 ]]> <![CDATA[LC Lam -Z15170]]> <![CDATA[3.06×10 4 ]]> <![CDATA[3.58×10 -4 ]]> <![CDATA[1.2×10 -8 ]]> <![CDATA[Z15170-HC Lam ]]> <![CDATA[1.16×10 5 ]]> <![CDATA[2.84×10 -4 ]]> <![CDATA[2.4×10 -9 <!-- 49 -->]]> <![CDATA[Z15170-LC Lam ]]> <![CDATA[2.33×10 5 ]]> <![CDATA[6.01×10 -4 ]]> <![CDATA[2.6×10 -9 ]]> <![CDATA[HC Ipi -Z15170]]> <![CDATA[1.78×10 4 ]]> <![CDATA[7.27×10 -4 ]]> <![CDATA[4.1×10 -8 ]]> <![CDATA[LC Ipi -Z15170]]> <![CDATA[3.07×10 4 ]]> <![CDATA[5.63×10 -4 ]]> <![CDATA[1.8×10 -8 ]]> <![CDATA[Z15170-HC Ipi ]]> <![CDATA[2.05×10 5 ]]> <![CDATA[4.83×10 -4 ]]> <![CDATA[2.4×10 -9 ]]> <![CDATA[Z15170-LC Ipi ]]> <![CDATA[2.30×10 5 ]]> <![CDATA[3.49×10 -4 ]]> <![CDATA[1.5×10 -9 ]]>

[0484] The interactions of the complex with PD-1 and CTLA-4 follow a bivalent model, respectively. The Ki of PD-1 and CTLA-4... D1 K D2 k a1 k a2 k d1 and k d2The results are summarized in Tables 17 and 18, respectively. The affinity constant K for the interaction between PD-1 and the generated Lam control antibody is also shown. D1 The affinity was determined to be 18.6 nM. For all Lam-based complexes, stronger affinity was observed, with K0 values ​​ranging from 0.8 to 2.7 nM. D1 Range. For Z15170-HC Lam A slightly slower association rate k was observed. a1 However, the differences between complexes are usually small, meaning that the localization of the Z part on the antibody appears to have little effect on the interaction between the antibody and PD-1.

[0485] Table 17: Parameters for the binding of specified complexes and Lam to hPD-1

[0486]

[0487] Table 18: Kinetic parameters of the binding of the specified complex to hCTLA-4

[0488]

[0489] The affinity constant K of the complex interacting with CTLA-4 D1 Within the 8-10 nM range, and this is consistent with the K reported by Iprimma. D Consistent (5.25±3.62 nM; European Medicines Agency's assessment report 2011:EMA / CHMP / 557664 / 2011). For all Ipi-based constructs, the kinetic spectra were similar, but Z15170-HC... Ipi It has a slightly slower association and dissociation rate.

[0490] Biacore capture assay confirmed the presence of all the complexes included in the assay, namely Z15170-HC. Lam Z15170-LC Lam Z15170-HC Ipi and Z15170-LC Ipi The dual binding specificity is shown in Figure 8. The complex first binds to immobilized PD-1 or CTLA-4, and PD-L1 subsequently binds to its respective captured complex. In separate control experiments, PD-L1 was shown not to bind to CTLA-4 or Ipi, and no further binding of PD-L1 was observed after injection of PD-L1 into Lam captured on PD-1.

[0491] Cell binding analysis via FACSThis experiment was performed to analyze whether the complexes could bind to cells expressing PD-L1. MDA-MB-231 cells naturally expressing PD-L1 were stained with each complex at 0.625 μg / ml. MFI values ​​are presented in Table 19, showing the ability of the complexes to bind to PD-L1-expressing cells. For the Ipi and Lam-based complexes, the Z portion, located at the N-terminus of the antibody light chain, yielded the highest MFI value.

[0492] Table 19: MFI of the complex binding to cells expressing PD-L1

[0493] Analytes MFI Lam 62. <![CDATA[Z15170-HC Lam ]]> 161 <![CDATA[HC Lam -Z15170]]> 274 <![CDATA[Z15170-LC Lam ]]> 327 <![CDATA[LC Lam -Z15170]]> 145 Iprimma 64 <![CDATA[Z15170-HC Ipi ]]> 488 <![CDATA[HC Ipi -Z15170]]> 264 <![CDATA[Z15170-LC Ipi ]]> 582 <![CDATA[LC Ipi -Z15170]]> 192 negative control 69 Anti-PD-L1 antibody 610

[0494] Co-culture of MDA-MB-231 and PBMC To evaluate whether Ipi-based complexes could affect the inhibitory mechanisms induced by CTLA-4 and PD-L1, a mixed lymphocyte assay was used. Breast cancer cells MDA-MB-231 were co-cultured with PBMCs for 6 days, and the number of cancer cells and T cells was evaluated. Analysis revealed a concentration-dependent effect of the complexes, with an increase in T cell numbers and a decrease in cancer cell numbers. Figure 9A shows the decrease in MDA-MB231 cell numbers with increasing complex concentration. This decrease was significant for all complexes, with the construct with the Z portion located at the N-terminus of the antibody light chain showing the best effect. In contrast, the ipilimumab control antibody did not induce a concentration-dependent reduction in cancer cell numbers. Therefore, blocking the PD-1 / PD-L1 interaction appears to be essential for reducing cancer cell numbers. Figure 9B shows the increase in T cell numbers with increasing complex concentration. Again, this effect was most significant for the construct with the Z portion located at the N-terminus of the antibody light chain.

[0495] Example 9

[0496] PD-L1 conjugation and radiolabeling of Z variant

[0497] This embodiment describes the conjugation and radiolabeling of Z15168-Cys (SEQ ID NO:809), Z18608-Cys (SEQ ID NO:811), Z18609-Cys (SEQ ID NO:812), and Z18610-Cys (SEQ ID No:813) as described in Examples 2 and 6 and further used in in vivo imaging studies described in Examples 10 and 11.

[0498] Materials and Methods

[0499] Reduction and NOTA conjugation: 3 molar equivalents of tris(2-carboxyethyl)phosphine (TCEP) in 0.5 mL of degassed 0.2 M ammonium acetate buffer (pH 7.0) were added to 5 mg of the Z variant in [20 mM HEPES, 1 mM EDTA, pH 7.2]. The reaction mixture was kept at RT for 60 min, then transferred to an Ultracel 3K centrifuge filter and centrifuged at 4000 rpm for 90 min. The flow-through was discarded, and another 1 mL of 0.2 M ammonium acetate buffer was added, and the process was repeated. The reduced Z variant was then transferred to 2 mL of 0.2 M anoxic ammonium acetate buffer (pH 7.0) in a second reaction vessel. Then, 4 mg of NOTA-maleimide (macrocyclic) was added to 0.5 mL of 0.2 M ammonium acetate buffer (pH 7.0), and the reaction vessel was purged with argon. The mixture was then heated to 40 °C for 3 h. The reaction mixture was then transferred to an Ultracel 3K centrifuge filter and centrifuged at 4000 rpm for 90 min. The eluent was discarded, and 2 mL of milliQ water was added. The mixture was centrifuged again for 90 min, and the eluent was discarded. The purified NOTA-conjugated Z variant was collected in 1 mL of milliQ water, lyophilized, and stored at -70 °C before use. The purity of the final product was determined by LC / MS.

[0500] Radioactive labeling First, wash the contents with 1.5ml of ultrapure water. 18 F-fluoride column (cartridge), then eluted with 1.0 ml 0.4 M KHCO3 [ 18 F]-fluoride. 100 μl of eluted [ 18 F]-fluoride solution was added to a stem vial containing 10 μl acetic acid, 50 μl AlCl3 (2 mM, in 0.1 M NaOAc buffer, pH 4), and 125 μl 0.1 M NaOAc pH 4. The solution was incubated at RT for 2 min, then 1 mg of NOTA-conjugated Z variant was added to 400 μl acetonitrile and 0.1 M NaOAc pH 4 in a 1:1 solution, and the mixture was heated to 100 °C for 15 min. After heating, the sample was transferred to a vial containing 0.7 mL of 0.1% formic acid, mixed, and purified by HPLC [Waters Xselect CSH C18 column (250 × 10 mm, 130 μm)] using a gradient of 10%–30% MeCN at a flow rate of 5 mL / min over 15 min, equilibrated to 0.1% formic acid. The sample was collected corresponding to […]. 18The peak of F]AlF-NOTA-Z##### was removed under vacuum, and the sample was transferred to a sterile vial using physiological saline as a washing solution to obtain [ 18 F]AlF-NOTA-Z#####. Specific activity and radiochemical purity were determined using a Waters Acquity LC / MS system (Milford, MA, USA) and a β-RAM model 4 Radio-HPLC detector (IN / US Systems, Brandon, FL, USA).

[0501] result

[0502] PD-L1 binds to Z variants Z15168-Cys (SEQ ID NO:809), Z18608-Cys (SEQ ID NO:811), Z18609-Cys (SEQ ID NO:812), and Z18610-Cys (SEQ ID NO:813) to specifically conjugate to the NOA site at their respective unique C-terminal cysteine ​​residues. This is followed by […]. 18 Radiolabeled F]AlF typically yields 97%–100% radiochemical purity and a specific activity of 14.6 ± 6.5 GBq / mmol. The radiolabeled Z variant will be referred to as […]. 18 F]AlF-NOTA-Z[#####].

[0503] Example 10

[0504] In vivo imaging and biodistribution of tumor-bearing mice

[0505] Materials and Methods

[0506] animal models Female SCID Beige mice (6-8 weeks old, Charles River Laboratories) were kept in a temperature- and humidity-controlled indoor environment and maintained on a regular diet. LOXIMVI (human melanoma cell line; PD-L1 positive) or SUDHL-6 (PD-L1 negative) cells were cultured at 37°C with 5% CO2 in complete growth medium containing RPMI 1640 medium with 10% fetal bovine serum. The growth medium was changed 2 or 3 times per week, and cells were passaged at a ratio of 1:10 as needed. Cells were then subcutaneously injected with 1×10⁻⁶ cells in 100 μl PBS. 6 LOXIMVI cells or 10 × 10⁶ cells in 100 μl PBS 6A tumor was implanted into the right shoulder using SUDHL-6 cells plus growth factor reduced Matrigel (1:1). When the tumor reached a mass of 100-400 mg, mice were used for micro-PET and in vitro studies 5-7 days and 3 weeks after injection of LOXIMVI cells and SUDHL-6 cells, respectively.

[0507] PET data acquisition Mice were anesthetized with isoflurane (4%-5% for induction, 1%-3% for maintenance), prepared using a tail vein catheter, and placed in a dedicated small animal PET scanner (microPET Focus 220, Siemens Preclinical Solutions). Results were obtained using... 57 A 20-minute transmission scan of Co was performed to correct for photon attenuation and scattering. Then, 0.2–0.6 MBq of each [ ] was administered via a tail vein catheter. 18 F-labeled Z variant, and PET data were collected for 90 min. In a separate pre-blocking experiment, 400 μg of unlabeled NOA-conjugated Z15168-Cys was administered, followed by […]. 18 F]AlF-NOTA-Z15168.

[0508] In vitro biodistribution measurement Mice were euthanized immediately after PET scans. Tumors, heart, lungs, spleen, liver, kidneys, blood, plasma, and muscle were collected and measured using a gamma counter (PerkinElmer). For each mouse, biodistribution measurements were converted to standard uptake values ​​(SUV) units. Regions of interest (ROIs) were plotted on all tumors identifiable in the PET images, and time-activity curves (TACs) were calculated.

[0509] result

[0510] Will[ 18 Representative PET images of tumor-bearing mice after injection of the [F]-labeled Z variant show the highest uptake in the kidneys and bladder. PD-L1-positive LOX tumors are clearly visible in the images, while PD-L1-negative SUDHL6 tumors are not visible. 18 A representative PET image of [F]AlF-NOTA-Z15168 is shown in Figure 10A. The in vitro biodistribution measurements at 90 min post-injection are consistent with the PET images. 18The uptake of the F-labeled Z variants Z15168-Cys (SEQ ID NO: 809), Z18608-Cys (SEQ ID NO: 811), Z18609-Cys (SEQ ID NO: 812), and Z18610-Cys (SEQ ID NO: 813) was significantly higher in LOX tumors than in SUDHL6 tumors, and tumor uptake increased with increasing PD-L1 binding affinity of the Z variants. Figure 11A-B ). Target specificity was confirmed in pre-blocking experiments, where pre-administration of 400 μg of NOTA-Z15168 induced [ ] in LOX tumors. 18 Decreased uptake of [F]AlF-NOTA-Z15168 (Fig. 10B). Altered distribution, including faster clearance as indicated by decreased hematogenous uptake at 90 min, was also observed. Renal tracer retention (mean SUV range between 57 and 84 in LOX tumor xenografts) is likely due to tubular reuptake of the protein, where [ 18 The F]AlF marker was captured after Z variant cleavage. In summary, the results show that the Z variant ligand is effective in targeting PD-L1 positive tumors in vivo, exhibiting specific binding and rapid clearance.

[0511] Example 11

[0512] Imaging in rhesus monkeys

[0513] Materials and Methods

[0514] Fasted rhesus monkeys were sedated with ketamine (10 mg / kg, intramuscularly). Intravenous catheters were inserted into the right and left saphenous veins, and the animals were maintained under propofol anesthesia (5 mg / kg for induction and 0.45 mg / kg / min throughout the scan). After initial induction with propofol, the animals were cannulated and positioned approximately 10 cm away from the cannula. 3 Animals were given an oxygen / air mixture at a rate of 23 breaths per minute, administered at a rate of / kg / breath. A temperature probe, pulse oximeter, and end-tidal CO2 monitor were applied. Body temperature was maintained using a K-module heating pad. Routine fluid therapy was maintained throughout the scan procedure with lactated Ringer's solution (10 ml / kg / h IV). 84–138 MBq of [unspecified medication] was administered via 2-minute infusions. 18 F]AlF-NOTA-Z15168 and 147-227MBq of [ 18[F]AlF-NOTA-Z18609. A whole-body dynamic scan was initiated at the start of tracer injection using a Siemens Biograph 64 TPTV PET / CT scanner and lasted for 180 minutes. Whole-body reconstruction was performed using software provided by the PET / CT scanner vendor. PET image analysis was performed using custom Matlab-based software.

[0515] result

[0516] [ were applied separately] 18 F]AlF-NOTA-Z15168 and [ 18 Representative maximum intensity projection images of the [F]AlF-NOTA-Z18609 rhesus monkey are shown in Figures 12A-B, and the average tracer uptake (~120-180 min) is illustrated in Figure 12C. As in mice, the highest uptake was observed in the kidney (SUV≈100-112) and bladder, but lymph node and spleen targeting were also observed, consistent with PD-L1 expression.

[0517] Item-by-item listing of the implementation plan

[0518] 1. A PD-L1 binding polypeptide, wherein the PD-L1 binding polypeptide comprises a PD-L1 binding motif BM, the motif consisting of an amino acid sequence selected from the following:

[0519] i)ERNX4AAX7EIL X 11 LPNLX 16 X 17 X 18 QX 20 WAFIWX 26 LX 28 D

[0520] Among them, independently of each other,

[0521] X4 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, and Y;

[0522] X7 is selected from A, E, F, H, N, Q, S, T, V, W, and Y;

[0523] X 11 Selected from A, D, E, F, H, K, L, N, Q, R, S, T, V, W, and Y;

[0524] X 16 Selected from N and T;

[0525] X 17 Selected from A, H, K, N, Q, R, and S;

[0526] X18 Selected from A, D, E, G, H, K, L, N, Q, R, S, T, V, and Y;

[0527] X 20 Selected from H, I, K, L, N, Q, R, T, V, and Y;

[0528] X 26 Selected from K and S; and

[0529] X 28 Selected from A, D, and E;

[0530] and

[0531] ii) An amino acid sequence that has at least 96% identity with the sequence defined in i).

[0532] 2. The PD-L1 binding peptide according to Project 1, wherein, in sequence i):

[0533] X4 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, and Y;

[0534] X7 is selected from E, F, H, N, Q, S, T, V, W, and Y;

[0535] X 11 Selected from A, D, H, L, Q, R, T, V, W, and Y;

[0536] X 16 Selected from N and T;

[0537] X 17 Selected from A, H, K, N, Q, R, and S;

[0538] X 18 Selected from A, D, E, G, H, K, L, N, Q, R, S, T, V, and Y;

[0539] X 20 Selected from H, I, K, L, Q, R, T, V, and Y;

[0540] X 26 Selected from K and S; and

[0541] X 28 Choose from A, D, and E.

[0542] 3. The PD-L1 binding peptide according to Project 1, wherein, in sequence i):

[0543] X4 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, and Y;

[0544] X7 is selected from A, E, F, H, N, Q, S, T, V, W, and Y;

[0545] X 11 Selected from A, D, E, F, H, K, L, N, Q, R, S, T, V, W, and Y;

[0546] X 16 Selected from N and T;

[0547] X 17 Selected from A, H, K, N, Q, R, and S;

[0548] X 18 Selected from A, D, E, G, H, K, L, N, Q, R, S, T, V, and Y;

[0549] X 20 Selected from H, I, K, L, N, Q, R, T, V, and Y;

[0550] X 26 Selected from K and S; and

[0551] X 28 Choose from A, D, and E.

[0552] 4. The PD-L1 binding peptide according to item 2 or 3, wherein, in sequence i):

[0553] X4 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, and V;

[0554] X7 is selected from F, H, Q, and Y;

[0555] X 11 Selected from H, Q, W, and Y;

[0556] X 16 Selected from N and T;

[0557] X 17 Selected from A, H, K, N, Q, and S;

[0558] X 18 Selected from A, E, G, H, K, L, N, Q, R, S, T, V, and Y;

[0559] X 20 Selected from H, I, K, Q, R, and V;

[0560] X 26 Selected from K and S; and

[0561] X 28 Choose from A and D.

[0562] 5. A PD-L1 binding peptide based on any one of items 1-4, wherein sequence i) satisfies at least 4 of the following 7 conditions I-VII:

[0563] I.X7 is selected from F, H, Q, and Y;

[0564] II.X 11 Selected from H and Y;

[0565] III.X 16 Let T be the value of T.

[0566] IV.X 17 Selected from N, Q, and S;

[0567] VX 20 Selected from H, I, K, and R;

[0568] VI.X 26 For K; and

[0569] VII.X 28 The answer is either A or D.

[0570] 6. The PD-L1 binding polypeptide according to Project 5, wherein sequence i) satisfies at least 5 of the 7 conditions I-VII.

[0571] 7. The PD-L1 binding polypeptide according to Project 6, wherein sequence i) satisfies at least 6 of the 7 conditions I-VII.

[0572] 8. The PD-L1 binding polypeptide according to Project 7, wherein sequence i) satisfies all of the seven conditions I-VII.

[0573] 9. The PD-L1 binding peptide according to any one of items 1-8, wherein X7X 11 X 20 Selected from FYK and YYK.

[0574] 10. The PD-L1 binding peptide according to any one of items 1-9, wherein X 11 X 17 X 20 Selected from YNK and YQK.

[0575] 11. The PD-L1 binding peptide according to any one of items 1-10, wherein X 11 X 18 X 20 For YAK.

[0576] 12. The PD-L1 binding polypeptide according to any of the preceding items, wherein sequence i) corresponds to the sequence from position 8 to position 36 of the sequence selected from the group consisting of SEQ ID NO: 1-808.

[0577] 13. The PD-L1 binding polypeptide according to item 12, wherein sequence i) corresponds to the sequence from position 8 to position 36 in the sequence selected from the group consisting of SEQ ID NO: 1-93 and 774-796.

[0578] 14. The PD-L1 binding polypeptide according to item 13, wherein sequence i) corresponds to the sequence from position 8 to position 36 in the sequence selected from the group consisting of SEQ ID NO: 1-93 and 774-787.

[0579] 15. The PD-L1 binding polypeptide according to item 14, wherein sequence i) corresponds to the sequence from position 8 to position 36 in the sequence selected from the group consisting of SEQ ID NO:1-93, 775, 776, 779-781 and 784-786, such as the group consisting of SEQ ID NO:1-93, 776, 780, 781, 784 and 786, such as the group consisting of SEQ ID NO:1-93, 776 and 784, such as the group consisting of SEQ ID NO:1-93, 776 and 784, or the group consisting of SEQ ID NO:1-93, 776 and 781, for example the group consisting of SEQ ID NO:1-93 and 776 or the group consisting of SEQ ID NO:1-93 and 781 or the group consisting of SEQ ID NO:1-93 and 784.

[0580] 16. The PD-L1 binding polypeptide according to item 15, wherein sequence i) corresponds to the sequence selected from the group consisting of SEQ ID NO:1-93, 774, 775, 780-786, such as the sequence from position 8 to position 36 in the sequence consisting of the group consisting of SEQ ID NO:1-93, 775, 780, 781, 784 and 786.

[0581] 17. The PD-L1 binding polypeptide according to any one of items 14-16, wherein sequence i) corresponds to the sequence from position 8 to position 36 of the sequence selected from the group consisting of SEQ ID NO:1-93.

[0582] 18. The PD-L1 binding polypeptide according to item 17, wherein sequence i) corresponds to the sequence from position 8 to position 36 in the sequence selected from the group consisting of SEQ ID NO:1-24.

[0583] 19. The PD-L1 binding polypeptide according to item 18, wherein sequence i) corresponds to the sequence from position 8 to position 36 in the sequence selected from the group consisting of SEQ ID NO: 1-12, 14 and 17-21.

[0584] 20. The PD-L1 binding polypeptide according to item 19, wherein sequence i) corresponds to the sequence selected from the group consisting of SEQ ID NO:1-12 and 17, such as the group consisting of SEQ ID NO:1-5 and 17, such as the sequence from position 8 to position 36 in the group consisting of SEQ ID NO:1, 2 and 17.

[0585] 21. The PD-L1 binding polypeptide according to item 19, wherein sequence i) corresponds to the group selected from SEQ ID NO:1, 4, 5, 6, 9, 14 and 18-21, such as the group composed of SEQ ID NO:4, 5, 18 and 21, such as the sequence from position 8 to position 36 in the sequence of the group composed of SEQ ID NO:4, 5 and 21.

[0586] 22. The PD-L1 binding polypeptide according to item 20 or 21, wherein sequence i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:1.

[0587] 23. The PD-L1 binding polypeptide according to item 20 or 21, wherein sequence i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:4.

[0588] 24. The PD-L1 binding polypeptide according to item 20 or 21, wherein sequence i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:5.

[0589] 25. The PD-L1 binding polypeptide according to item 21, wherein sequence i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:21.

[0590] 26. The PD-L1 binding polypeptide according to any of the preceding items, wherein the PD-L1 binding motif forms part of a triple-helix bundle protein domain.

[0591] 27. The PD-L1 binding polypeptide according to item 26, wherein the PD-L1 binding motif substantially forms part of a double helix with interconnected loops within the triple-helix bundle protein domain.

[0592] 28. The PD-L1 binding polypeptide according to item 27, wherein the triple-helix bundle protein domain is selected from the bacterial receptor domain.

[0593] 29. The PD-L1 binding polypeptide according to Item 28, wherein the triple-helix bundle protein domain is selected from the domain of protein A from Staphylococcus aureus or a derivative thereof.

[0594] 30. The PD-L1 binding polypeptide according to any of the preceding items, wherein the PD-L1 binding polypeptide comprises a binding module BMod, the amino acid sequence of which is selected from the following:

[0595] iii)K-[BM]-DPSQSX a X b LLX c EAKKLX d X e X f Q;

[0596] in,

[0597] [BM] is the PD-L1 binding motif as defined in any of items 1-25;

[0598] X a Selected from A and S;

[0599] X b Selected from N and E;

[0600] X c Selected from A, S, and C;

[0601] X d Selected from E, N, and S;

[0602] X e Selected from D, E, and S; and

[0603] X f Selected from A and S; and

[0604] The amino acid sequence that has at least 93% identity with the sequence defined in iv) and iii).

[0605] 31. The PD-L1 binding polypeptide according to any of the preceding items, wherein sequence iii) corresponds to the sequence from position 7 to position 55 of the sequence selected from the group consisting of SEQ ID NO:1-808.

[0606] 32. The PD-L1 binding polypeptide according to item 31, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in the sequence selected from the group consisting of SEQ ID NO: 1-93 and 774-796.

[0607] 33. The PD-L1 binding polypeptide according to item 32, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in the sequence selected from the group consisting of SEQ ID NO: 1-93 and 774-787.

[0608] 34. The PD-L1 binding polypeptide according to item 33, wherein sequence iii) corresponds to the sequence from position 7 to position 55 of the sequence selected from the group consisting of SEQ ID NO:1-93, 775, 776, 779-781 and 784-786, such as the group consisting of SEQ ID NO:1-93, 776, 780, 781, 784 and 786, such as the group consisting of SEQ ID NO:1-93, 776 and 784, such as the group consisting of SEQ ID NO:1-93, 776 and 784, or the group consisting of SEQ ID NO:1-93, 776 and 781, for example the group consisting of SEQ ID NO:1-93 and 776 or the group consisting of SEQ ID NO:1-93 and 781 or the group consisting of SEQ ID NO:1-93 and 784.

[0609] 35. The PD-L1 binding polypeptide according to item 33, wherein sequence iii) corresponds to the group consisting of SEQ ID NO:1-93, 774, 775 and 780-786, such as the sequence from position 7 to position 55 in the sequence consisting of SEQ ID NO:1-93, 775, 780, 781, 784 and 786.

[0610] 36. The PD-L1 binding polypeptide according to any one of items 33-35, wherein sequence iii) corresponds to the sequence from position 7 to position 55 of the sequence selected from the group consisting of SEQ ID NO:1-93.

[0611] 37. The PD-L1 binding polypeptide according to item 36, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in the sequence selected from the group consisting of SEQ ID NO:1-24.

[0612] 38. The PD-L1 binding polypeptide according to item 37, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in the sequence selected from the group consisting of SEQ ID NO: 1-12, 14 and 17-21.

[0613] 39. The PD-L1 binding polypeptide according to item 38, wherein sequence iii) corresponds to the sequence selected from the group consisting of SEQ ID NO:1-12 and 17, such as the group consisting of SEQ ID NO:1-5 and 17, such as the sequence from position 7 to position 55 in the group consisting of SEQ ID NO:1, 2 and 17.

[0614] 40. The PD-L1 binding polypeptide according to item 38, wherein sequence iii) corresponds to the group consisting of SEQ ID NO:1, 4, 5, 6, 9, 14 and 18-21, such as the group consisting of SEQ ID NO:4, 5, 18 and 21, such as the sequence from position 7 to position 55 in the sequence of the group consisting of SEQ ID NO:4, 5 and 21.

[0615] 41. The PD-L1 binding polypeptide according to item 40 or 41, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:1.

[0616] 42. The PD-L1 binding polypeptide according to item 40 or 41, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:4.

[0617] 43. The PD-L1 binding polypeptide according to item 40 or 41, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:5.

[0618] 44. The PD-L1 binding polypeptide according to item 41, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:21.

[0619] 45. The PD-L1 binding polypeptide according to any of the preceding items, wherein the PD-L1 binding polypeptide comprises an amino acid sequence selected from:

[0620] v)YA-[BMod]-AP;

[0621] Where [BMod] is the PD-L1 combination module as defined in any of items 30-44; and

[0622] vi) has an amino acid sequence that is at least 90% identical to the sequence defined in v).

[0623] 46. ​​The PD-L1 binding polypeptide according to any one of items 1-44, wherein the PD-L1 binding polypeptide comprises an amino acid sequence selected from the following:

[0624] vii)FN-[BMod]-AP;

[0625] Where [BMod] is the PD-L1 combination module as defined in any of items 30-44; and

[0626] The sequences defined in viii) and vii) have at least 90% amino acid sequence identity.

[0627] 47. The PD-L1 binding polypeptide according to any of the preceding items, wherein the PD-L1 binding polypeptide comprises an amino acid sequence selected from:

[0628] ADNNFNK-[BM]-DPSQSANLLSEAKKLNESQAPK;

[0629] ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK;

[0630] ADNKFNK-[BM]-DPSVSKEILAEAKKLNDAQAPK;

[0631] ADAQQNNFNK-[BM]-DPSQSTNVLGEAKKLNESQAF

[0632] AQHDE-[BM]-DPSQSANVLGEAQKLNDSQAPK;

[0633] VDNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK;

[0634] AEAKYAK-[BM]-DPSESSELLSEAKKLNKSQAPK;

[0635] VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK;

[0636] VDAKYAK-[BM]-DPSQSSELLAEAKKLNDSQAPK;

[0637] AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;

[0638] AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAP;

[0639] AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;

[0640] AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAP;

[0641] AEAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK;

[0642] AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK;

[0643] AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAP;

[0644] AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK;

[0645] AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAP;

[0646] AEAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK;

[0647] AEAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK;

[0648] AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK;

[0649] AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAP;

[0650] AEAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK;

[0651] AEAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK;

[0652] AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK;

[0653] AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAP;

[0654] AEAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK;

[0655] AEAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK;

[0656] AEAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK;

[0657] VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;

[0658] VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK;

[0659] VDAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK;

[0660] VDAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK;

[0661] VDAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK;

[0662] VDAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK;

[0663] VDAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK;

[0664] VDAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK;

[0665] VDAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK;

[0666] VDAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK;

[0667] VDAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK;

[0668] VDAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK;

[0669] VDAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK;

[0670] AEAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK; and

[0671] ADAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;

[0672] Where [BM] is the PD-L1 binding motif as defined in any of items 1-25;

[0673] 48. The PD-L1 binding polypeptide according to any one of items 1-47, wherein the PD-L1 binding polypeptide comprises an amino acid sequence selected from the following:

[0674] xvii)VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;

[0675] Where [BM] is the PD-L1 binding motif as defined in any of items 1-25; and

[0676] The sequences defined in xviii) and xvii) have at least 89% amino acid identity.

[0677] 49. The PD-L1 binding polypeptide according to any one of items 1-47, wherein the PD-L1 binding polypeptide comprises an amino acid sequence selected from the following:

[0678] xix)AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK;

[0679] Where [BM] is the PD-L1 binding motif as defined in any of items 1-25; and

[0680] The amino acid sequence that has at least 89% identity with the sequence defined in xix) is xx).

[0681] 50. The PD-L1 binding polypeptide according to any one of items 1-47, wherein the PD-L1 binding polypeptide comprises an amino acid sequence selected from the following:

[0682] xxi)AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;

[0683] Where [BM] is the PD-L1 binding motif as defined in any of items 1-25; and

[0684] The amino acid sequences defined in xxii) and xxi) have at least 89% identity.

[0685] 51. The PD-L1 binding polypeptide according to any one of items 1-47, wherein the PD-L1 binding polypeptide comprises an amino acid sequence selected from:

[0686] xxiii)AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;

[0687] Where [BM] is the PD-L1 binding motif as defined in any of items 1-25; and

[0688] The amino acid sequences defined in xxiv) and xiii) have at least 89% identity.

[0689] 52. The PD-L1 binding polypeptide according to any of the preceding items, wherein the sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 of the sequence selected from the group consisting of SEQ ID NO:1-814.

[0690] 53. The PD-L1 binding polypeptide according to item 52, wherein the sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in the sequence selected from the group consisting of SEQ ID NO: 1-93, 774-796 and 809-814.

[0691] 54. The PD-L1 binding polypeptide according to item 53, wherein the sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in the sequence selected from the group consisting of SEQ ID NO: 1-93, 774-787 and 809-814.

[0692] 55. The PD-L1 binding polypeptide according to item 54, wherein sequence xvii) or xxi) corresponds to the group selected from SEQ ID NO: 1-93, 775, 776, 779-781, 784-786 and 809-814, such as the group selected from SEQ ID NO: 1-93, 776, 780, 781, 784, 786 and 809-814, such as the group selected from SEQ ID NO: 1-93, 776, 781, 784 and 809-814, such as the group selected from SEQ ID NO: 1-93, 776, 784, 809 and 811-814, or the group selected from SEQ ID NO: 1-93, 776, 781, 809 and 811-814, for example the group selected from SEQ ID NO: 1-93, 776, 809 and 811-814 or the group selected from SEQ ID NO: 1-93, 776, 809 and 811-814. The sequence from position 1 to position 58 in the group consisting of NO:1-93, 781, 809 and 811-814 or the sequence consisting of 1-93, 784 and 811-814.

[0693] 56. The PD-L1 binding polypeptide according to item 55, wherein the sequence xvii) or xxi) corresponds to the group consisting of SEQ ID NO:1-93, 774, 775, 780-786 and 810-814, such as the sequence from position 1 to position 58 in the sequence of the group consisting of SEQ ID NO:1-93, SEQ ID NO:775, 780, 781, 784, 786 and 810-814.

[0694] 57. The PD-L1 binding polypeptide according to any one of items 54-56, wherein the sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in the sequence selected from the group consisting of SEQ ID NO: 1-93 and 811-813.

[0695] 58. The PD-L1 binding polypeptide according to item 57, wherein the sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in the sequence selected from the group consisting of SEQ ID NO: 1-24 and 811-813.

[0696] 59. The PD-L1 binding polypeptide according to item 58, wherein the sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in the sequence selected from the group consisting of SEQ ID NO: 1-12, 14, 17-21 and SEQ ID NO: 811-812.

[0697] 60. The PD-L1 binding polypeptide according to item 59, wherein the sequence xvii) or xxi) corresponds to the sequence selected from the group consisting of SEQ ID NO:1-12, 17, 811 and 812, such as the group consisting of SEQ ID NO:1-5, 17, 811 and 812, such as the sequence from position 1 to position 58 in the sequence of the group consisting of SEQ ID NO:1, 2, 17, 811 and 812.

[0698] 61. The PD-L1 binding polypeptide according to item 58, wherein the sequence xvii) or xxi) corresponds to the sequence selected from the group consisting of SEQ ID NO: 1, 4, 5, 6, 9, 14, 18, 19, 20, 21 and 811, such as the group consisting of SEQ ID NO: 4, 5, 18 and 21, such as the sequence from position 1 to position 58 in the sequence of the group consisting of SEQ ID NO: 4, 5 and 21.

[0699] 62. The PD-L1 binding polypeptide according to item 60 or 61, wherein the sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in SEQ ID NO: 1 or 811.

[0700] 63. The PD-L1 binding polypeptide according to item 60, wherein the sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in SEQ ID NO:2 or 812.

[0701] 64. The PD-L1 binding polypeptide according to item 60 or 61, wherein the sequence xvii) corresponds to the sequence from position 1 to position 58 in SEQ ID NO:4.

[0702] 65. The PD-L1 binding polypeptide according to item 60 or 61, wherein the sequence xvii) corresponds to the sequence from position 1 to position 58 in SEQ ID NO:5.

[0703] 66. The PD-L1 binding polypeptide according to item 61, wherein the sequence xvii) corresponds to the sequence from position 1 to position 58 in SEQ ID NO:21.

[0704] 67. The PD-L1 binding peptide according to any of the preceding items, wherein the PD-L1 binding peptide is capable of blocking PD-L1-dependent signal transduction.

[0705] 68. The PD-L1 binding peptide according to item 67, wherein the half-maximum inhibitory concentration (IC50) for blocking is at most 5 × 10⁻⁶. -8 M, such as at most 1×10 -8 M, such as at most 5×10 -9 M, such as at most 3.5 × 10 -9 M, such as at most 1×10 -9 M, such as at most 5×10 -10 M, such as at most 1×10 -10 M.

[0706] 69. The PD-L1 binding polypeptide according to any of the preceding items, wherein the PD-L1 binding polypeptide is capable of blocking the interaction between PD-L1 and PD-1.

[0707] 70. The PD-L1 binding polypeptide according to any of the preceding items, wherein the PD-L1 binding polypeptide is capable of binding PD-L1 such that the interacting K D The value is at most 2×10 -8 M, such as at most 1×10 -8 M, such as at most 1×10 -9 M, such as at most 5×10 -10 M, such as at most 3×10 -10 M.

[0708] 71. The PD-L1 binding polypeptide according to any of the preceding items, wherein the PD-L1 binding polypeptide is capable of binding PD-L1 such that the interacting k d The value is at most 1×10 -3 s -1 Such as at most 6×10 -4 s -1 .

[0709] 72. The PD-L1 binding peptide according to any of the foregoing items, wherein the PD-L1 binding peptide is capable of binding PD-L1 such that the EC50 value of the interaction is at most 1 × 10⁻⁶. -9 M, such as at most 1×10 -10 M, such as at most 7×10 -11 M.

[0710] 73. The PD-L1 binding peptide according to any of the preceding items, wherein the PD-L1 is human PD-L1.

[0711] 74. The PD-L1 binding polypeptide according to any of the preceding items, wherein the PD-L1 binding polypeptide contains additional amino acids at the C-terminus and / or N-terminus.

[0712] 75. The PD-L1 binding peptide according to item 74, wherein the additional amino acids improve the generation, purification, in vivo or in vitro stability, conjugation or detection of the peptide.

[0713] 76. The PD-L1 binding polypeptide according to any of the preceding items, wherein the PD-L1 binding polypeptide is in a polymeric form, the polymeric form comprising at least two PD-L1 binding polypeptide monomer units, the amino acid sequences of the monomer units being the same or different.

[0714] 77. The PD-L1 binding polypeptide according to item 76, wherein the PD-L1 binding polypeptide monomer units are covalently coupled together.

[0715] 78. The PD-L1 binding polypeptide according to item 77, wherein the PD-L1 binding polypeptide monomer unit is expressed as a fusion protein.

[0716] 79. The PD-L1 binding polypeptide according to any one of items 76-78, wherein the PD-L1 binding polypeptide is in dimer form.

[0717] 80. A fusion protein or conjugate, said fusion protein or conjugate comprising:

[0718] - A first portion, the first portion comprising a PD-L1 binding polypeptide according to any of the preceding items; and

[0719] - The second part consists of a polypeptide having the desired biological activity.

[0720] 81. The fusion protein or conjugate according to item 80, wherein the desired biological activity is a therapeutic activity.

[0721] 82. The fusion protein or conjugate according to item 80, wherein the desired biological activity is binding activity.

[0722] 83. The fusion protein or conjugate according to item 80, wherein the desired biological activity is enzyme activity.

[0723] 84. The fusion protein or conjugate according to item 82, wherein the binding activity is albumin-binding activity, and the albumin-binding activity increases the in vivo half-life of the fusion protein or conjugate.

[0724] 85. The fusion protein or conjugate according to item 84, wherein the second portion comprises an albumin-binding domain of a streptococcal G protein or a derivative thereof.

[0725] 86. The fusion protein or conjugate according to item 82, wherein the binding activity acts to block biological activity.

[0726] 87. The fusion protein or conjugate according to item 81, wherein the second part is a therapeutically active polypeptide.

[0727] 88. The fusion protein or conjugate according to item 87, wherein the second part is an immune response modulator.

[0728] 89. The fusion protein or conjugate according to item 87, wherein the second part is an anticancer agent.

[0729] 90. The fusion protein or conjugate according to any one of items 80-83 and 86-89, wherein the second part is selected from the group consisting of human endogenous enzymes, hormones, growth factors, chemokines, cytokines and lymphokines.

[0730] 91. The fusion protein according to any one of items 80-91, wherein the second part further comprises a linker.

[0731] 92. A complex comprising at least one PD-L1 binding polypeptide according to any one of the preceding items and at least one antibody or an antigen-binding fragment thereof.

[0732] 93. The complex according to item 92, wherein the at least one antibody or its antigen-binding fragment is selected from the group consisting of full-length antibody, Fab fragment, Fab' fragment, F(ab')2 fragment, Fc fragment, Fv fragment, single-chain Fv(scFv) fragment, (scFv)2 and domain antibody.

[0733] 94. The complex according to item 93, wherein the at least one antibody or its antigen-binding fragment is selected from the group consisting of a full-length antibody, a Fab fragment, and an scFv fragment.

[0734] 95. The complex according to item 94, wherein the at least one antibody or its antigen-binding fragment is a full-length antibody.

[0735] 96. The complex according to any one of items 92-95, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

[0736] 97. The complex according to any one of items 92-96, wherein the antibody or its antigen-binding fragment is selected from the group consisting of human antibodies, humanized antibodies and chimeric antibodies, and their antigen-binding fragments.

[0737] 98. The complex according to item 97, wherein the antibody or its antigen-binding fragment is a human antibody or a humanized antibody, or its antigen-binding fragment.

[0738] 99. The complex according to any one of items 92-98, wherein the PD-L1 binding polypeptide is attached to the C-terminus or N-terminus of the heavy or light chain of the antibody or its antigen-binding fragment.

[0739] 100. The composite according to any one of items 92-99, wherein the composite further comprises a connector.

[0740] 101. The complex according to any one of items 92-100, wherein the antibody or its antigen-binding fragment has an affinity for an antigen, such as an antigen associated with an infectious disease or a cancer-related antigen.

[0741] 102. A fusion protein or conjugate according to any one of items 79-90 or a complex according to any one of items 92-101, wherein the second portion or the antibody or its antigen-binding fragment is an inhibitor selected from the group consisting of: PD-1, CTLA-4, T-cell immunoglobulins, and mucin domain protein-3 (mucin Containing protein-3 (TIM-3), galactagogue 9 (GAL-9), lymphocyte activation gene-3 (LAG-3), PD-L2, B7 homologue 3 (B7-H3), B7 homologue 4 (B7-H4), V domain inhibitor of T cell activation (VISTA), carcinoembryonic antigen-associated cell adhesion molecule 1 (CEACAM1), B and T lymphocyte attenuators (BTLA), colony-stimulating factor 1 receptor (CSF1R), herpesvirus invasion mediator (HVEM), killer immunoglobulin receptor (KIR), adenosine, adenosine A2a receptor (A2aR), CD200-CD200R, and T cell Ig and ITIM domains.

[0742] 103. The fusion protein, conjugate, or complex according to item 102, wherein the second part, antibody, or antigen-binding fragment thereof is an inhibitor of PD-1, such as an inhibitor selected from the group consisting of nivolumab, pidilizumab, BMS 936559, MPDL328OA, and pembrolizumab, such as pembrolizumab.

[0743] 104. The fusion protein, conjugate, or complex according to item 102, wherein the second part, antibody, or antigen-binding fragment thereof is an inhibitor of CTLA-4, such as an inhibitor selected from the group consisting of berazip, abatacept, and iplimumab, such as iplimumab.

[0744] 105. A fusion protein or conjugate according to any one of items 80-91 or a complex according to any one of items 92-101, wherein the second portion or the antibody or its antigen-binding fragment is an agonist selected from the group consisting of agonists of CD134, CD40, 4-1BB and glucocorticoid-induced TNFR-related protein (GITR).

[0745] 106. The PD-L1 binding peptide, fusion protein, conjugate or complex according to any one of items 1-105, wherein the PD-L1 binding peptide, fusion protein, conjugate or complex further comprises a marker.

[0746] 107. The PD-L1 binding peptide, fusion protein, conjugate or complex according to item 106, wherein the marker is selected from the group consisting of fluorescent dyes and metals, chromophore dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radionuclides, radioactive particles and pre-targeted recognition tags.

[0747] 108. The PD-L1 binding peptide, fusion protein, conjugate, or complex according to item 107, wherein the PD-L1 binding peptide, fusion protein, conjugate, or complex comprises a chelating environment provided by a polyaminopolycarboxylic acid chelating agent conjugated to the PD-L1 binding peptide via a thiol group of a cysteine ​​residue or an amino group of a lysine residue.

[0748] 109. The PD-L1 binding peptide, fusion protein, conjugate, or complex according to item 106, wherein the PD-L1 binding peptide, fusion protein, conjugate, or complex includes a pre-targeting recognition tag that forms part of a complementary pair of a pre-targeting portion, the complementary pair of the pre-targeting portion being, for example, selected from stept (avidin) / biotin, oligonucleotides / complementary oligonucleotides such as DNA / complementary DNA, RNA / complementary RNA, phosphorothioic acid / complementary phosphorothioic acid and peptide nucleic acid / complementary peptide nucleic acid, and morpholino oligomers / complementary morpholino oligomers.

[0749] 110. The PD-L1 binding peptide, fusion protein, conjugate or complex as described in Item 109, wherein the pre-targeting recognition tag is a peptide nucleic acid tag.

[0750] 111. The PD-L1 binding peptide, fusion protein, conjugate or complex according to any one of items 110, wherein the pre-targeting recognition tag is a 10-20-mer peptide nucleic acid sequence, such as a 15-mer peptide nucleic acid sequence.

[0751] 112. A polynucleotide encoding a polypeptide according to any one of items 1-105.

[0752] 113. An expression vector comprising the polynucleotides described in item 112.

[0753] 114. A host cell comprising the expression vector described in item 113.

[0754] 115. A method for preparing a polypeptide according to any one of items 1-105, said method comprising:

[0755] -Culturing host cells according to item 114 under conditions that allow the polypeptide to be expressed by the expression vector, and

[0756] - Isolate the polypeptide.

[0757] 116. A composition comprising a PD-L1 binding peptide, fusion protein, conjugate or complex according to any one of items 1-111, and at least one pharmaceutically acceptable excipient or carrier.

[0758] 117. The composition according to item 116, wherein the composition further comprises at least one additional active agent, such as an agent selected from immune response modulators and anticancer agents.

[0759] 118. The PD-L1 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-111, or the composition according to any one of items 116-117, for oral, topical, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, sublingual, sublingual or suppository administration, such as for topical application.

[0760] 119. The PD-L1 binding peptide, fusion protein, conjugate or complex according to any one of items 1-111, or the composition according to any one of items 116-117, for use as a pharmaceutical, diagnostic and / or prognostic agent.

[0761] 120. The PD-L1 binding peptide, fusion protein, conjugate, complex or composition described in item 119 for use as a medicine.

[0762] 121. The PD-L1 binding peptides, fusion proteins, conjugates, complexes or compositions described in Item 119 may be used as diagnostic agents and / or prognostic agents.

[0763] 122. A PD-L1 binding peptide, fusion protein, conjugate, complex or composition used as a medicine according to item 120, wherein the peptide, fusion protein, conjugate or composition regulates PD-L1 function in vivo.

[0764] 123. The PD-L1 binding peptide, fusion protein, conjugate, complex or composition according to any one of items 119-121 for use in the treatment, prognosis or diagnosis of PD-L1-related disorders.

[0765] 124. The PD-L1 binding peptide, fusion protein, conjugate, complex or composition for use as described in item 122, wherein the PD-L1-related disorder is selected from the group consisting of infectious diseases and cancer.

[0766] 125. The PD-L1 binding polypeptide, fusion protein, conjugate, complex or composition for use as described in item 124, wherein the PD-L1-related disorder is an infectious disease, such as a chronic viral infection, for example selected from the group consisting of human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV).

[0767] 126. The PD-L1 binding peptide, fusion protein, conjugate, complex, or composition for use as described in item 124, wherein the PD-L1-related disorder is cancer, such as cancer selected from the group consisting of:

[0768] - Cancers presenting as solid tumors, such as those selected from the following groups: skin cancer, such as melanoma and non-melanoma skin cancer (NMSC); lung cancer, such as small cell lung cancer and non-small cell lung cancer (NSCLC); head and neck cancer; renal cell carcinoma (RCC); bladder cancer; breast cancer; colorectal cancer; stomach cancer; ovarian cancer; pancreatic cancer; prostate cancer; glioma; glioblastoma; liver cancer; gallbladder cancer; thyroid cancer; bone cancer; cervical cancer; uterine cancer, vulvar cancer, endometrial cancer, testicular cancer; kidney cancer; esophageal cancer; brain / CNS cancer; neuronal cancer; mesothelioma; sarcoma; small intestinal adenocarcinoma and pediatric malignancies; and

[0769] - Cancers that present as non-solid tumors, such as leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, and multiple myeloma.

[0770] 127. The PD-L1 binding polypeptide, fusion protein, conjugate, complex or composition for use as described in item 126, wherein the cancer is selected from the group consisting of melanoma, NSCLC, head and neck cancer, RCC, bladder cancer, breast cancer, colorectal cancer, gastric cancer, ovarian cancer, pancreatic cancer and prostate cancer, such as the group consisting of melanoma, NSCLC, head and neck cancer, RCC and bladder cancer.

[0771] 128. A method for treating PD-L1-related disorders, the method comprising administering an effective amount of any one of the PD-L1-binding peptides, fusion proteins, conjugates or compositions according to any one of items 1-111, or a composition according to any one of items 116-117, to a subject in need.

[0772] 129. The method according to item 128, wherein the PD-L1-related disorder is selected from the group consisting of infectious diseases and cancer.

[0773] 130. The method according to item 129, wherein the PD-L1-related disorder is an infectious disease, such as a chronic viral infection, for example selected from the group consisting of human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV).

[0774] 131. The method according to item 129, wherein the PD-L1-related disorder is cancer, such as cancer selected from the group consisting of:

[0775] - Cancers presenting as solid tumors, such as those selected from the following groups: skin cancer, such as melanoma and non-melanoma skin cancer (NMSC); lung cancer, such as small cell lung cancer and non-small cell lung cancer (NSCLC); head and neck cancer; renal cell carcinoma (RCC); bladder cancer; breast cancer; colorectal cancer; stomach cancer; ovarian cancer; pancreatic cancer; prostate cancer; glioma; glioblastoma; liver cancer; gallbladder cancer; thyroid cancer; bone cancer; cervical cancer; uterine cancer, vulvar cancer, endometrial cancer, testicular cancer; kidney cancer; esophageal cancer; brain / CNS cancer; neuronal cancer; mesothelioma; sarcoma; small intestinal adenocarcinoma and pediatric malignancies; and

[0776] - Cancers that present as non-solid tumors, such as leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, and multiple myeloma.

[0777] 132. The method according to item 131, wherein the cancer is selected from the group consisting of melanoma, NSCLC, head and neck cancer, RCC, bladder cancer, breast cancer, colorectal cancer, gastric cancer, ovarian cancer, pancreatic cancer and prostate cancer, such as the group consisting of melanoma, NSCLC, head and neck cancer, RCC and bladder cancer.

[0778] 133. The method according to any one of items 131-132, the method comprising the following steps:

[0779] -Contact the subject with a PD-L1 binding peptide, fusion protein, conjugate, or complex according to any one of items 109-111 containing a pre-targeted recognition tag, or with a composition containing such a PD-L1 binding peptide, fusion protein, conjugate, or complex, and

[0780] -The subject is brought into contact with a complementary pre-targeted portion containing a radionuclide.

[0781] 134. A method for detecting PD-L1, the method comprising: providing a sample suspected of containing PD-L1, contacting the sample with a PD-L1-binding peptide, fusion protein, conjugate or complex according to any one of items 1-111 or a composition according to any one of items 116-117, and detecting the binding of the PD-L1-binding peptide, fusion protein, conjugate, complex or composition to indicate the presence of PD-L1 in the sample.

[0782] 135. A method for determining the presence of PD-L1 in a subject, the method comprising the steps of:

[0783] a) Contacting the subject, or a sample separated from the subject, with the PD-L1 binding peptide, fusion protein, conjugate, or complex according to any one of items 1-111, or the composition according to any one of items 116-117, and

[0784] b) Obtain a value corresponding to the amount of PD-L1 binding peptide, fusion protein, conjugate, or composition that has been bound in the subject or bound to the sample.

[0785] 136. The method of claim 135, wherein the PD-L1 binding peptide, fusion protein, conjugate, or complex is a PD-L1 binding peptide, fusion protein, conjugate, or complex according to any one of claims 109-111, or the composition comprises such a PD-L1 binding peptide, fusion protein, conjugate, or complex, and step a) further comprises contacting the subject with a complementary pre-targeted portion labeled with a detectable marker such as a radionuclide marker.

[0786] 137. The method according to item 135 or 136, the method further comprising the step of comparing the value with a reference.

[0787] 138. The method according to any one of items 134-137, wherein the subject is a mammalian subject, such as a human subject.

[0788] 139. The method according to any one of items 134-138, wherein the method is performed in vivo.

[0789] 140. The method described in item 139 is a method for medical imaging, wherein...

[0790] Step a) includes systemic administration of the PD-L1 binding peptide, fusion protein, conjugate, complex or composition to a mammalian subject;

[0791] - The PD-L1 binding peptide, fusion protein, conjugate, complex, composition, or pre-targeted portion contains a radionuclide label suitable for medical imaging; and

[0792] Step b) includes obtaining one or more images of at least a portion of the subject's body using a medical imaging instrument, the one or more images indicating the presence of radionuclides in the body. sequence list <110> Afibodi Company <120> New peptides <130> 21084269 <150> EP15192364.6 <151> 2015-10-30 <150> EP16157154.2 <151> 2016-02-24 <160> 836 <170> PatentIn version 3.5 <210> 1 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 1 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Lys Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 2 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 2 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ala Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 3 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 3 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asn Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 4 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 4 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ser Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 5 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 5 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Leu Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 6 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 6 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Gln Ala Ala Phe Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 7 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 7 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ala Ala Ala Phe Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 8 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 8 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 9 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 9 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ser Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Gln Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 10 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 10 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ala Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn His Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 11 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 11 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ala Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Ser Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 12 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 12 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Ser Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 13 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 13 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Ser Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 14 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 14 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ile Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Ser Gln Ile Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 15 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 15 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 16 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 16 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ser Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 17 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 17 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asp Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Gln Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Ala Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 18 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 18 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asn Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 19 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 19 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ile Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Asn Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 20 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 20 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Ser Gln Ile Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> twenty one <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> twenty one Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Thr Ala Ala Gln Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Gly Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> twenty two <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> twenty two Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asp Ala Ala Phe Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Ala Gln Ile Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> twenty three <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> twenty three Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ser Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Leu Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> twenty four <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> twenty four Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Ser Tyr Gln His Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 25 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 25 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Lys Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Lys Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 26 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 26 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Lys Ala Ala His Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 27 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 27 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ala Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Lys Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 28 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 28 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ala Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Lys Gln Arg Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 29 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 29 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ala Ala Ala Gln Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Ser Gln Gln Arg Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 30 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 30 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Gln Ala Ala Phe Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Gln Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 31 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 31 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn His Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Lys Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 32 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 32 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Arg Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 33 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 33 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Thr Ala Ala Gln Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Tyr Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 34 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 34 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Leu Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Lys Gln Arg Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 35 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 35 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asp Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Glu Gln Arg Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 36 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 36 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Val Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Gln Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 37 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 37 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Val Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 38 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 38 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Thr Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Ser Gln Arg Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 39 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 39 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Phe Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Ser Gln Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 40 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 40 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asn Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Ser Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 41 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 41 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Gln Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Lys Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 42 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 42 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Glu Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 43 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 43 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Thr Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 44 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 44 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ser Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Lys Gln Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 45 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 45 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Lys Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 46 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 46 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Ala Ser Gln Arg Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 47 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 47 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Thr Ala Ala Gln Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Ser Gly Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 48 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 48 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ile Ala Ala Tyr Glu Ile 1 5 10 15 Leu Trp Leu Pro Asn Leu Thr Asn Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 49 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 49 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ala Ala Ala Phe Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Ser Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 50 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 50 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Ser Ser Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 51 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 51 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala His Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Lys Gln Ile Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 52 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 52 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Phe Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Ser Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 53 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 53 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ala Ala Ala Phe Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Ser Ala Gln Val Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 54 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 54 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asp Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Lys Gln Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 55 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 55 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Lys Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Gln Gln Ile Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 56 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 56 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn His Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Gln Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 57 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 57 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Phe Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 58 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 58 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ala Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 59 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 59 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Lys Ala Ala Phe Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Glu Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 60 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 60 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ala Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Ala Gly Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 61 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 61 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Gln Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Ser Gln Ile Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 62 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 62 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asp Ala Ala Tyr Glu Ile 1 5 10 15 Leu Gln Leu Pro Asn Leu Thr Asn Gln Gln His Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 63 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 63 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ser Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Gln Gln Arg Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 64 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 64 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ser Ala Ala Gln Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Arg Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 65 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 65 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Ala Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 66 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 66 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Gln Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Lys Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 67 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 67 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Thr Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Lys Ser Gln Gln Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 68 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 68 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ser Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Ser Gln Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 69 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 69 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ile Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Asn Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 70 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 70 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ile Ala Ala Gln Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 71 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 71 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asp Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Ser Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 72 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 72 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn His Ala Ala Phe Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 73 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 73 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Lys Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 74 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 74 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Gln Ala Ala Phe Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Ser Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 75 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 75 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Val Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Asn Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 76 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 76 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ile Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Gln Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 77 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 77 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Phe Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Arg Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 78 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 78 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Arg Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr His Ser Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 79 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 79 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Thr Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Glu Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 80 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 80 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Asn Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 81 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 81 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ser Ala Ala Phe Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 82 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 82 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Gln Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Arg Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 83 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 83 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asp Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Ala Ser Gln Arg Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 84 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 84 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Leu Ala Ala Gln Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Lys Gln Arg Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 85 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 85 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Gln Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Lys Gln Ile Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 86 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 86 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Ser Lys Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 87 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 87 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Lys Ala Ala Gln Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Asn Gln Ile Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 88 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 88 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Gln Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 89 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 89 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ala Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Leu Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 90 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 90 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Phe Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Gln Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 91 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 91 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ala Ala Ala Phe Glu Ile 1 5 10 15 Leu Trp Leu Pro Asn Leu Thr Asn Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 92 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 92 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Phe Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Lys Ala Gln His Trp Ala Phe Ile Trp 20 25 30 Lys Leu Ala Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 93 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 93 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asp Ala Ala Phe Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Thr Gln Ile Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 94 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 94 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Ser Ala Gln Arg Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 95 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 95 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Thr Ala Ala His Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Ser Gln Ile Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 96 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 96 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Thr Ala Ala Tyr Glu Ile 1 5 10 15 Leu Lys Leu Pro Asn Leu Thr Lys Tyr Gln His Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 97 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 97 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asp Ala Ala Tyr Glu Ile 1 5 10 15 Leu Gln Leu Pro Asn Leu Thr Gln Ser Gln Leu Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 98 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 98 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ile Ala Ala Tyr Glu Ile 1 5 10 15 Leu Lys Leu Pro Asn Leu Thr Glu Asn Gln Arg Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 99 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 99 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ile Ala Ala Ser Glu Ile 1 5 10 15 Leu Lys Leu Pro Asn Leu Thr Lys Glu Gln His Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 100 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 100 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asp Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Tyr Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 101 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 101 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asp Ala Ala Glu Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Asn Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 102 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 102 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ala Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Trp Ser Gln Gln Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 103 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 103 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Lys Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Ala Ser Gln His Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 104 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 104 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asn Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Gln Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 105 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 105 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Thr Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Lys Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 106 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 106 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asp Ala Gln Ile Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 107 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 107 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Thr Ala Ala Ser Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Ser Gln Gln Arg Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 108 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 108 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ile Ala Ala Phe Glu Ile 1 5 10 15 Leu Trp Leu Pro Asn Leu Thr Ala His Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 109 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 109 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asp Ala Ala Phe Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Ser Glu Gln Ile Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 110 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 110 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn His Ala Ala Asp Glu Ile 1 5 10 15 Leu Lys Leu Pro Asn Leu Thr Ser Asp Gln Asn Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 111 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 111 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asp Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Glu Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 112 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 112 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ile Ala Ala Glu Glu Ile 1 5 10 15 Leu Ile Leu Pro Asn Leu Thr Gln Asn Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 113 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 113 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Gln Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Asn Gln Ile Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 114 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 114 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ser Ala Ala Gln Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Asn Gln Ile Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 115 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 115 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Phe Ala Ala Gln Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Lys Gln Ile Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 116 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 116 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asn Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Lys Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 117 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 117 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Gly Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 118 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 118 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ala Ala Ala Gln Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 119 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 119 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ala Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Lys Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 120 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 120 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Lys Ala Ala Phe Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Asn Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 121 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 121 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asn Ala Ala Gln Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Lys Gln Ile Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 122 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 122 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala His Glu Ile 1 5 10 15 Leu Thr Leu Pro Asn Leu Thr Ala Glu Gln Tyr Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 123 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 123 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Trp Ala Ala Ala Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Ala Gln Thr Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 124 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 124 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asp Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Asp Gln Ile Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 125 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 125 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala His Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 126 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 126 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ala Ala Ala Gln Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn His Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Glu Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 127 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 127 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Arg Ala Ala His Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Arg Gln Gln Gln Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 128 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 128 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Lys Gln Arg Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 129 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 129 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn His Ala Ala Phe Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Glu Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 130 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 130 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Ser Gln Gln Ile Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 131 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 131 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Thr Ala Ala Tyr Glu Ile 1 5 10 15 Leu Gln Leu Pro Asn Leu Thr Lys Ser Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Ala Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 132 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 132 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn His Ala Ala Ala Glu Ile 1 5 10 15 Leu Ser Leu Pro Asn Leu Thr Lys Lys Gln Asn Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 133 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 133 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr His Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 134 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 134 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ile Ala Ala Ser Glu Ile 1 5 10 15 Leu Thr Leu Pro Asn Leu Thr Lys Ser Gln Thr Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 135 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 135 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Lys Ala Ala Tyr Glu Ile 1 5 10 15 Leu Asn Leu Pro Asn Leu Thr Gln Ser Gln Leu Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 136 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 136 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ala Ala Ala Gln Glu Ile 1 5 10 15 Leu Glu Leu Pro Asn Leu Thr Trp Ala Gln His Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 137 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 137 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ser Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Ser Gln Ile Trp Ala Phe Ile Trp 20 25 30 Lys Leu Ala Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 138 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 138 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Tyr Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Glu Ala Gln Arg Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 139 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 139 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asn Ala Ala Tyr Glu Ile 1 5 10 15 Leu Leu Leu Pro Asn Leu Thr Gln Glu Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 140 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 140 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asn Ala Ala Gln Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Tyr Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 141 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 141 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Gln Ala Ala Tyr Glu Ile 1 5 10 15 Leu Gln Leu Pro Asn Leu Thr Asn Asp Gln Tyr Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 142 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 142 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Thr Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Asn Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 143 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 143 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Lys Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Lys Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 144 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 144 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Thr Ala Ala Glu Glu Ile 1 5 10 15 Leu Lys Leu Pro Asn Leu Thr Asn Lys Gln His Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 145 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 145 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Arg Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Ser Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Ala Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 146 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 146 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ser Ala Ala His Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Thr Ala Gln Arg Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 147 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 147 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ser Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Ala Asn Gln Arg Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 148 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 148 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Lys Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Leu Gln Arg Trp Ala Phe Ile Trp 20 25 30 Lys Leu Ala Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 149 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 149 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Gln Ala Ala Ser Glu Ile 1 5 10 15 Leu Ser Leu Pro Asn Leu Thr Lys Ser Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 150 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 150 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn His Ala Ala Trp Glu Ile 1 5 10 15 Leu Lys Leu Pro Asn Leu Thr Lys Ser Gln Glu Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 151 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 151 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Val Ala Ala Gln Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Lys Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 152 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 152 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Leu Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Arg Ala Gln Gln Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 153 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 153 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Val Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Ala Asn Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 154 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 154 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asp Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Ser Ala Gln Val Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 155 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 155 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Gln Ala Ala Glu Glu Ile 1 5 10 15 Leu Arg Leu Pro Asn Leu Thr Trp Glu Gln Asn Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 156 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 156 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Asn Ala Ala Tyr Glu Ile 1 5 10 15 Leu Gln Leu Pro Asn Leu Thr Gln Leu Gln Thr Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 157 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 157 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ser Ala Ala Tyr Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Lys Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 158 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 158 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Phe Ala Ala Gln Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Lys Ala Gln Thr Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 159 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 159 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn His Ala Ala Gln Glu Ile 1 5 10 15 Leu Glu Leu Pro Asn Leu Thr Asn Lys Gln Arg Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 160 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 160 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ile Ala Ala Gln Glu Ile 1 5 10 15 Leu Phe Leu Pro Asn Leu Thr Gln Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 161 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 161 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Phe Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Glu Lys Gln Arg Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 162 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 162 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ile Ala Ala Asn Glu Ile 1 5 10 15 Leu Lys Leu Pro Asn Leu Thr Ala Asn Gln Tyr Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 163 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 163 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Val Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Asn Ser Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 164 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 164 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala Gln Glu Ile 1 5 10 15 Leu Asn Leu Pro Asn Leu Thr Gln Ser Gln Glu Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 165 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 165 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Ile Ala Ala Tyr Glu Ile 1 5 10 15 Leu Gln Leu Pro Asn Leu Thr Gln Arg Gln Ile Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 166 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 166 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Tyr Ala Ala Tyr Glu Ile 1 5 10 15 Leu Gln Leu Pro Asn Leu Thr Ala Ala Gln Tyr Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 167 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 167 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala His Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Gln Gly Gln Ile Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 168 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 168 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn Glu Ala Ala His Glu Ile 1 5 10 15 Leu Gln Leu Pro Asn Leu Thr Ser Leu Gln Thr Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 169 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Engineered PD-L1 binding peptide <400> 169 Val Asp Ala Lys Tyr Ala Lys Glu Arg Asn His Ala Ala Phe Glu Ile 1 5 10 15 Leu Tyr Leu Pro Asn Leu Thr Ala Ala Gln Lys Trp Ala Phe Ile Trp 20 25 30 Lys Leu Asp Asp Asp Pro Ser Gln Ser Ser Glu Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 170 <211> 58 <212> PRT <213> Artificial...

Claims

1. A PD-L1 binding polypeptide, wherein the amino acid sequence of the PD-L1 binding polypeptide is shown in SEQ ID NO: 1-93, 774-796, 809-814, and wherein the amino acid sequence of the PD-L1 binding polypeptide contains: xvii) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; or xxi) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; in, [BM] is the PD-L1 binding motif.

2. The PD-L1 binding polypeptide according to claim 1, wherein the PD-L1 binding motif forms part of a triple-helix bundle protein domain.

3. The PD-L1 binding polypeptide according to claim 1, wherein the amino acid sequence of the PD-L1 binding polypeptide is shown in SEQ ID NO: 1-93, 811-813.

4. The PD-L1 binding polypeptide according to claim 1, wherein the amino acid sequence of the PD-L1 binding polypeptide is shown in SEQ ID NO: 1-24, 811-813.

5. The PD-L1 binding polypeptide according to claim 1, wherein the amino acid sequence of the PD-L1 binding polypeptide is as shown in SEQ ID NO: 1, 2, 4, 5, 21, 811, 812.

6. The PD-L1 binding polypeptide according to claim 1, wherein the amino acid sequence of the PD-L1 binding polypeptide is shown in SEQ ID NO: 1, 2, 811, 812.

7. The PD-L1 binding peptide according to claim 1, wherein the PD-L1 binding peptide is capable of blocking PD-L1-dependent signal transduction.

8. The PD-L1 binding peptide according to claim 7, wherein the half-maximum inhibitory concentration (IC50) for blocking is at most 5 × 10⁻⁶. -8 M.

9. The PD-L1 binding peptide according to claim 7, wherein the half-maximum inhibitory concentration (IC50) for blocking is at most 1 × 10⁻⁶. -8 M.

10. The PD-L1 binding peptide of claim 7, wherein the half-maximum inhibitory concentration (IC50) for blocking is at most 5 × 10⁻⁶. -9 M.

11. The PD-L1 binding peptide of claim 7, wherein the half-maximum inhibitory concentration (IC50) for blocking is at most 3.5 × 10⁻⁶. -9 M.

12. The PD-L1 binding peptide of claim 7, wherein the half-maximum inhibitory concentration (IC50) for blocking is at most 1 × 10⁻⁶. -9 M.

13. The PD-L1 binding peptide of claim 7, wherein the half-maximum inhibitory concentration (IC50) for blocking is at most 5 × 10⁻⁶. -10 M.

14. The PD-L1 binding peptide of claim 7, wherein the half-maximum inhibitory concentration (IC50) for blocking is at most 1 × 10⁻⁶. -10 M.

15. The PD-L1 binding polypeptide according to claim 1, wherein the PD-L1 binding polypeptide is capable of blocking the interaction between PD-L1 and PD-1.

16. The PD-L1 binding peptide of claim 1, wherein the PD-L1 binding peptide is capable of binding PD-L1 such that the interacting K D The value is at most 2 × 10 -8 M.

17. The PD-L1 binding peptide according to claim 1, wherein the PD-L1 binding peptide is capable of binding PD-L1 such that the interacting K D The value is at most 1 × 10 -8 M.

18. The PD-L1 binding peptide according to claim 1, wherein the PD-L1 binding peptide is capable of binding PD-L1 such that the interacting K D The value is at most 1 × 10 -9 M.

19. The PD-L1 binding polypeptide of claim 1, wherein the PD-L1 binding polypeptide is capable of binding PD-L1 such that the interacting K D The value is at most 5 × 10 -10 M.

20. The PD-L1 binding peptide of claim 1, wherein the PD-L1 binding peptide is capable of binding PD-L1 such that the interacting K D The value is at most 3 × 10 -10 M.

21. A fusion protein, said fusion protein comprising the following: - A first portion, wherein the first portion comprises a PD-L1 binding polypeptide according to any one of claims 1-20; and - The second part consists of peptides having binding or therapeutic activity.

22. The fusion protein according to claim 21, wherein, The binding activity is albumin binding activity.

23. The fusion protein according to claim 21, wherein, The second part is an antibody or its antigen-binding fragment.

24. A conjugate, said conjugate comprising: - A first portion, comprising the PD-L1 binding polypeptide according to any one of claims 1-20; and - The second part consists of a polyamino-polycarboxylic acid chelating agent conjugated to the PD-L1 binding peptide via a thiol group of a cysteine ​​residue or an ε-amino group of a lysine residue.

25. A polynucleotide encoding a PD-L1 binding polypeptide according to any one of claims 1-20, a fusion protein according to any one of claims 21-23, or a conjugate according to claim 24.

26. A composition comprising a PD-L1 binding polypeptide according to any one of claims 1-20, a fusion protein according to any one of claims 21-23, or a conjugate according to claim 24, and at least one pharmaceutically acceptable excipient or carrier.

27. Use of the PD-L1 binding peptide according to any one of claims 1-20, the fusion protein according to any one of claims 21-23, the conjugate according to claim 24, or the composition according to claim 26 in the preparation of pharmaceuticals, diagnostic agents, and / or prognostic agents, wherein, The drugs, diagnostic agents, and / or prognostic agents are used to treat, predict, or diagnose PD-L1-related disorders selected from a group consisting of infectious diseases and cancers.

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