Polypeptides targeting pd-l1 and uses thereof

Abstract

The present disclosure relates to the technical field of biological medicine, in particular to a polypeptide targeting PD-L1 and use thereof. The polypeptide provided by the present disclosure has high affinity and good stability. Based on the RDC of the polypeptide, the tumor primary lesion or metastatic lesion can be precisely targeted, and the polypeptide will not be retained in the tissues near the tumor and the tissues in non-tumor parts of the whole body, and the polypeptide can also avoid being rapidly degraded by proteases.

Description

[0001] Priority Statement This disclosure claims priority to the following patent applications: The Chinese patent application, filed on January 22, 2025, with application number 2025101074585, and entitled "Peptide Targeting PD-L1 and Its Use Thereof", is titled "Peptide Targeting PD-L1 and Its Use Thereof". Technical Field

[0002] This disclosure relates to the field of biomedical technology, specifically to a polypeptide targeting PD-L1 and its uses. Background Technology

[0003] PD-L1 (Programmed cell death 1 ligand), an immune checkpoint expressed in situ on the surface of tumor cells, can be used as a target for non-invasive imaging diagnostic techniques such as PET (Positron Emission Tomography) and SPECT (Single-Photon Emission Tomography). This allows for the monitoring of PD-L1 expression throughout the body, facilitating pre-treatment diagnosis, intra-treatment monitoring, and post-treatment evaluation of primary and metastatic tumors. Due to the ubiquity of PD-L1 throughout the entire process of tumor development and progression, using PD-L1 as a detection target effectively avoids the influence of tumor heterogeneity on the detection results.

[0004] Immune checkpoint PD-L1 can also serve as a target for cancer therapeutics. Molecules that specifically target and bind to PD-L1 with high affinity can block the binding of the immune checkpoint receptor PD-1 on the surface of immune cells to the immune checkpoint ligand PD-L1 on the surface of tumor cells, preventing tumor cells from evading the immune response. Simultaneously, these specific targeting molecules can also act as targeting elements, conjugating with active ingredients that inhibit or kill tumor cells to obtain effective cancer treatments.

[0005] Whether used for diagnosis or treatment, molecules targeting tumor cells need excellent tissue permeability to ensure successful delivery of the active ingredients for detection or treatment to the tumor cell surface. Currently, the common solution is to reduce the molecular weight (or volume) of the targeting molecule to improve its transport capacity. However, for antibody-based targeting molecules, as the molecular weight decreases, its specificity also decreases. This leads to non-specific retention of detection reagents or therapeutic drugs in healthy tissue near the tumor. Retention of detection reagents in healthy tissue can result in inaccurate test results, while retention of therapeutic drugs in healthy tissue can damage the patient's healthy cells or tissues. Furthermore, since the active ingredients for detection or treatment usually have some damaging effect on healthy tissue cells, the metabolic rate of the targeting molecule has a significant impact on diagnostic or treatment efficacy. An excessively rapid metabolic rate of the targeting molecule can lead to unstable diagnostic results.

[0006] Therefore, on the one hand, there is an urgent need to develop an active molecule that targets the immune checkpoint PD-L1 with high specificity, and on the other hand, it is also necessary to ensure that it has sufficient stability. Summary of the Invention

[0007] The purpose of this disclosure is to provide a polypeptide that targets the immune checkpoint PD-L1 with high specificity, thereby enhancing its targeting effect on tumor cells and avoiding its retention in adjacent tissues.

[0008] Another objective of this disclosure is to provide a highly stable small molecule polypeptide targeting the immune checkpoint PD-L1, which enhances its resistance to proteases in vivo, thus meeting the requirements for its use as a detection reagent or a targeted molecule for therapeutic drugs.

[0009] In a first aspect, this disclosure provides a polypeptide targeting the immune checkpoint PD-L1, said polypeptide comprising the amino acid sequence NH2-CWCWRX1PGX2...X 2+m -COOH, where m is selected from 0 to 5, and X1, X2, ..., X... 2+m Selected from natural amino acid residues, wherein X2...X 2+m It contains at least one cysteine ​​residue, and the amino acid residue on the N-terminal side of P forms at least one cyclic structure with the amino acid residue on the C-terminal side of G.

[0010] In an optional embodiment, the cyclic structure is linked by disulfide bonds.

[0011] In an optional implementation, X1 is selected from C or D.

[0012] In an optional embodiment, m=5, X6 is cysteine, and X6 forms a cyclic structure with any cysteine ​​residue on the N-terminal side of P. In one embodiment, X2...X 2+m For RSGGCK (SEQ ID NO:12). Optionally, X6 (C) is the first N-terminal cysteine ​​residue in NH2-CWCWRX1PGX2...X7-COOH ( C WCWR (SEQ ID NO:13) forms a cyclic structure.

[0013] In an optional embodiment, the amino acid sequence of the polypeptide is NH2-X. 2+m+1 ...X 2+m+ n CWCWRX1PGX2...X 2+m -COOH, where n is selected from 0 to 7, and X 2+m+1 ... X 2+m+n Selected from natural amino acid residues. Optionally, n is 7, and X... 2+m+1 ...X 2+m+7 It is SGQYASYH (SEQ ID NO:14).

[0014] In an optional embodiment, the amino acid sequence of the polypeptide is SGQYASYH. C WCWRDPGRSGG C K (SEQ ID NO:2), the structure of the polypeptide is: .

[0015] In a second aspect, this disclosure provides a polypeptide targeting the immune checkpoint PD-L1, the polypeptide comprising the amino acid sequence NH2-CWCWRDX1X2RSX3GX4K-COOH, wherein X1, X2, X3 and X4 are selected from natural amino acid residues or any chiral molecule thereof; wherein X3 and X4 are both tryptophan or neither is tryptophan.

[0016] In an optional implementation, the dipeptide fragment X1X2 is non- L P L G-dipeptide fragment.

[0017] In an optional embodiment, X1 in the dipeptide fragment X1X2 is... D P, X2 are selected from L P or L G.

[0018] In an optional implementation, the combination of X3 and X4 is selected from (a) or (b): (a) X3 is L G, X4 is LS, (b)X3 is L W, X4 is L W.

[0019] In an optional implementation, X1~X4 are selected from (a) or (b): (a) X1 is D P, X2 is L G, X3 is L G, X4 is L S, (b)X1 is D P, X2 is L P, X3 is L W, X4 is L W.

[0020] In an optional embodiment, the amino acid sequence of the polypeptide is NH2-X5...X. 5+ n CWCWRDX1X2RSX3GX4K-COOH, wherein X5...X 5+n It is an amino acid sequence of length n, where n is selected from 0 to 7, wherein X5, ..., X 5+n Selected from naturally occurring amino acid residues with chirality L. In one embodiment, n is 7, and X5...X 12 The value is SGQYASYH. In another embodiment, n is 0, and X5 is selected from K or E. Optionally, X5 forms a cyclized structure with the C-terminal amino acid K via an amide bond.

[0021] In an optional embodiment, the amino acid sequence of the polypeptide is SGQYASYHCWCWRD D PPRSWGWK (SEQ ID NO:8) or CWCWRD D PPRSWGWK (SEQ ID NO:9); Alternatively, the amino acid sequence of the polypeptide is: K CWCWRD D PPRSWGW K (SEQ ID NO:10), wherein the K at the N-terminus is connected to the K at the C-terminus via an amide bond; Alternatively, the amino acid sequence of the polypeptide is: E CWCWRD D PPRSWGW K (SEQ ID NO:11), wherein the E at the N-terminus and the K at the C-terminus are connected by an amide bond.

[0022] A third aspect of this disclosure provides the use of the polypeptide described in any embodiment of the first aspect in the preparation of tumor in situ diagnostic reagents or tumor therapeutic agents targeting the immune checkpoint PD-L1.

[0023] In optional embodiments, the tumor in situ diagnostic reagent or tumor therapeutic drug includes RDC (Radionuclide Drug Conjugates) or ADC (Antibody-Drug Conjugates).

[0024] In a fourth aspect, this disclosure provides a monomeric RDC molecule containing a polypeptide as described in any embodiment of the first aspect, a linker arm, a chelate, and a radionuclide, wherein the chelate and the radionuclide are linked to the polypeptide via the linker arm.

[0025] In an optional embodiment, the connecting arm has the structural formula shown in Formula I: .

[0026] In an optional embodiment, the chelate is a NOA derivative represented by Formula I: .

[0027] In an optional embodiment, the radionuclide is selected from... 14 C 15 N、 18 F, 32 P, 33 P, 35 S, 45 Ti、 47 Sc、 52 Fe、 55 Co、 58m Co、 59 Fe、 60 Cu、 61 Cu、 62 Cu、 63 Zn, 64 Cu、 66 Ho、 67 Cu、 67 Ga、 68 Ga、 75 Br、 76 Br、 77 Br、 82 Rb、 86 Y、 87 Y、 89 Zr、 89 Sr、 90 Y、 97 Ru、 99m Tc, 103 Pd, 103m Rh、 105 Rh、 109Pd, 111 In、 117m Sn、 119 Sb、 123 I, 124 I, 125 I, 131 I, 149 Pm, 149 Tb, 153 Sm、 153 Gd, 161 Ho、 161 Tb, 177 Lu、 186 Re、 188 Re、 191m Pt, 193m Pt, 195m Pt, 187 Pt, 198 Au、 199 Au、 201 Tl、 203 Pb, 211 At、 212 Pb, 212 Bi、 213 Bi、 223 Ra、 224 Ra、 225 Ac、 227 Th or 229 Th.

[0028] Optionally, the monomeric RDC molecule is a tumor in situ diagnostic reagent, and the radioactive factor is... 18 F or 64 Cu; preferably 64 Cu.

[0029] Optionally, the monomeric RDC molecule includes the polypeptide described in the foregoing embodiments, the linker arm shown in Formula I, the chelate shown in Formula II, and... 64 Cu, the chelate chelate and radionuclide, and linked to a polypeptide via a linker arm.

[0030] Optionally, the monomeric RDC molecule includes the polypeptide described in the foregoing embodiments, the linker arm shown in Formula I, the chelate shown in Formula II, and the radionuclide. 64 Cu, the chelate chelate and radionuclide, and linked to a polypeptide via a linker arm.

[0031] In an optional embodiment, the monomeric RDC molecule is: .

[0032] In a fifth aspect, this disclosure provides an RDC molecular polymer, the RDC molecular polymer comprising a monomeric RDC molecule as described in any embodiment of the third aspect and a multi-arm linker molecule connecting the monomeric RDC molecule, the RDC molecular polymer comprising an RDC dimer, an RDC tetramer, an RDC hexamer, or an RDC octamer; optionally, an octamer.

[0033] In an optional embodiment, the multi-arm linker molecule is multi-arm PEG (multi-arm polyethylene glycol). Optionally, the multi-arm PEG is linked to the side chain thiol group of the cysteine ​​residue of the polypeptide via a linker. Optionally, the linker is maleimide.

[0034] In an optional embodiment, the RDC polymer comprises the monomeric RDC molecule described in the foregoing embodiments and an eight-armed PEG, wherein the eight-armed PEG is linked to eight monomeric RDC molecules via maleimide.

[0035] In an optional embodiment, the average molecular weight of the eight-armed PEG is 5-40 kDa, or 5-20 kDa, for example 10 kDa.

[0036] In a sixth aspect, this disclosure provides a tumor in situ diagnostic reagent, the tumor in situ diagnostic reagent comprising a monomeric RDC molecule as described in any embodiment of the third aspect, or an RDC molecule polymer as described in any embodiment of the fourth aspect, wherein the nuclide of the RDC molecule is selected from... 18 F, 68 Ga、 89 Zr、 64 Cu、 99 mTc or 111 In, preferably 64 Cu.

[0037] In an optional embodiment, the molecular structure of the RDC molecule is as follows: .

[0038] In a seventh aspect, this disclosure provides a tumor therapeutic agent comprising a monomeric RDC molecule as described in any embodiment of the third aspect, or an RDC molecule polymer as described in any embodiment of the fourth aspect, wherein the nuclide of the RDC molecule is selected from... 225 Ac、 223 Ra、 213 Bi、 211 At、 188 Re、 177 Lu、 131 I, 90 Y、 89 Sr、 153 Sm、 125I, 77 Br or 195m Pt.

[0039] In an optional embodiment, the treatment is administered via intravenous injection; optionally, the dosage is 2-3 mCi / time / week.

[0040] The peptide disclosed herein has a high affinity for binding to the immune checkpoint PD-L1, which can prevent retention in healthy tissue adjacent to the tumor.

[0041] On the other hand, the peptides provided in this disclosure have good stability, which can prevent them from being rapidly degraded by proteases after administration into the body. At the same time, their affinity for the immune checkpoint PD-L1 is also improved.

[0042] This disclosure provides tumor in situ diagnostic reagents or tumor therapeutic drugs based on peptides, which can accurately target primary or metastatic tumor lesions without remaining in adjacent tissues, thereby improving the accuracy of diagnosis or treatment and avoiding damage to healthy tissues.

[0043] This disclosure provides tumor in situ diagnostic reagents or tumor therapeutic drugs based on polypeptides, which can stably deliver the detection active ingredient or therapeutic active ingredient to tumor cells to fully exert its efficacy, avoiding rapid degradation by proteases, thereby reducing the dosage and improving safety and subject compliance. Attached Figure Description

[0044] Figure 1 This is a comparison of the imaging effects of the RDC molecules of monomer cTPP-1 and monomer TPP-1 in mice in Example 2.

[0045] Figure 2 The results are the long-term detection results of the RDC polymer of cTPP-1 in Example 3.

[0046] Figure 3 This is a comparison of the therapeutic effects of cTPP-1 RDC multimer in Example 4 with the control group. Detailed Implementation

[0047] Based on the above content of this disclosure, and in accordance with common technical knowledge and practices in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical ideas of this disclosure.

[0048] I. Definition Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0049] The term “optional” or “optionally” means that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0050] Throughout this specification and the appended claims, unless the context otherwise requires, the words “comprising” or “including” are to be understood as implying the inclusion of the stated member, integer, or step, or a group of members, integers, or steps, but do not exclude any other member, integer, or step, or a group of members, integers, or steps. However, in some embodiments, such other members, integers, or steps, or a group of members, integers, or steps may be excluded, i.e., the subject matter is to include the stated member, integer, or step, or a group of members, integers, or steps. Unless otherwise indicated herein or clearly contradicted by the context, the terms “a” and “the” and similar references used in the context of describing this disclosure (especially in the context of the claims) shall be considered to cover both the singular and the plural. The range of values ​​enumerated herein is intended only as a shorthand for individually referring to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually enumerated herein. Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein can be performed in any suitable order. The use of any and all instances or exemplary language (e.g., “such”) provided herein is intended only to better illustrate this disclosure and does not limit the scope of this disclosure as otherwise claimed. All language in this specification should not be construed as indicating that any unclaimed element is necessary for the practice of this disclosure.

[0051] Several documents are referenced in full throughout this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that this disclosure is not prior to any prior invention disclosed herein.

[0052] As used herein, the term "amino acid" or "amino acid residue" refers to naturally occurring amino acids, non-natural amino acids that function in a similar manner to naturally occurring amino acids, amino acid analogs, and amino acid mimics, all of which are stereoisomers if their structure allows for their D and L stereoisomer forms. Amino acids are referred to herein by their names, their well-known three-letter symbols, or the single-letter symbols recommended by the IUPAC-IUB Committee on Biochemistry Nomenclature.

[0053] When used in conjunction with amino acids, the term “naturally occurring” refers to the 20 common amino acids (i.e., alanine (A), cysteine ​​(C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y)), as well as selenocysteine, pyrrolidone (PYL), and pyrrolidone-carboxylysine (PCL).

[0054] As used herein, the term "non-natural amino acid" means an amino acid that is not encoded by the genetic code of any organism or that has not been found in any organism. It can be, for example, a purely synthetic compound. Examples of non-natural amino acids include, but are not limited to, hydroxyproline, γ-carboxyglutamic acid, O-phosphoserine, azetidinecarboxylicacid, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid, desmosine, 2, 2'-Diaminopimelic acid, 2,3-Diaminopropionic acid, N-ethylglycine, N-methylglycine, N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesin, allo-isoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthylalanine, valine, leucine, ornithine, D-ornithine, D-arginine, p-aminophenylalanine, pentylglycine, pipecolic acid, and thioproline.

[0055] As used herein, the term "amino acid analogue" refers to a compound having the same basic chemical structure as a naturally occurring amino acid. Amino acid analogues include natural and non-natural amino acids that have been reversibly or irreversibly chemically blocked, or whose C-terminal carboxyl group, N-terminal amino group, and / or side-chain functional groups have been chemically modified. Such analogues include, but are not limited to, methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine ​​sulfoxide, S-(carboxymethyl)-cysteine ​​sulfone, aspartic acid-(β-methyl ester), N-ethylglycine, alanine carboxamide, homoserine, ortholeucine, and methionine methylsulfonium.

[0056] As used herein, the term "amino acid analogue" refers to a chemical compound that has a structure different from the general chemical structure of amino acids, but functions in a manner similar to that of naturally occurring amino acids.

[0057] In some embodiments, the variant includes at least one additional amino acid at its N-terminus. In one embodiment, the at least one additional amino acid is selected from naturally occurring amino acids other than proline, non-natural amino acids, amino acid analogs, and amino acid mimics. In one embodiment, the at least one additional amino acid is selected from G, A, N, and C. In a particular embodiment, the at least one additional amino acid is G.

[0058] As used in this article, the term "D-amino acid," in contrast to L-amino acid, refers to two isomers of the same amino acid with different optical rotations. According to the Fischer projection, L-amino acids have the amino group on the left and D-amino acids have the amino group on the right. Typically, naturally occurring amino acids are L-amino acids, while D-amino acids must be obtained through artificial synthesis.

[0059] As used herein, families of amino acid residues with similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with nonpolar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0060] Examples of amino acids that can be conservedly substituted for each other are shown in the table below: The term "diagnosis" means detecting a disease or condition or determining the stage or extent of a disease or condition. The term "diagnosis" also includes detecting a predisposition to a disease or condition, determining the efficacy of a drug therapy, or predicting the mode of action of a drug therapy. The diagnostic methods of this disclosure can be used alone or in combination with other diagnostic and / or grading methods known in the medical field for a particular disease or condition. Thus, in one embodiment, the diagnostic methods described herein can be used to detect PD-L1 overexpression in tissues or organs of a patient. In an exemplary embodiment, the above-described diagnostic methods were administered to a small group of patients suspected of having the disease or condition due to other clinical signs or symptoms of the disease.

[0061] The term "treatment" includes the suppression, relief, prevention, or elimination of one or more symptoms or side effects associated with the treated disease, condition, or disorder. The term "effective dose" or "therapeutic effective dose" refers to a dose sufficient to treat, suppress, or reduce one or more symptoms of the treated disease state or otherwise provide the desired pharmacological and / or physiological effect. Precise dosage will vary depending on various factors, such as subject-dependent variables (e.g., age, immune system health, etc.), the disease or disorder, and the treatment administered. The effect of an effective dose can be relative to a control. These controls are known in the art and discussed herein, and can be, for example, the condition of the subject before or without administration of the drug or combination of drugs, or, in the case of a combination of drugs, the combined effect can be compared to the effect of administration of a single drug. The peptides, monomeric RDCs, or multimeric RDCs provided in this disclosure can bind to cell surface PD-L1, fundamentally blocking the activation of the PD-1 / PD-L1 signaling pathway, potentially enhancing immunotherapeutic effects and maintaining endogenous anti-tumor effects, leading to durable tumor control.

[0062] The term "PD-L1" refers to programmed death receptor ligand-1 (PD-L1, also known as B7-H1), a typical negative costimulatory molecule. As a representative negative costimulatory molecule, PD-L1 induces phosphorylation of the tyrosine inhibitory motif of the immune receptor by binding to PD-1 on the surface of T lymphocytes. Both in vivo and in vitro, it promotes apoptosis of antigen-specific human T cell clones, inhibits T cell proliferation and differentiation, and induces the depletion of effector T cells. Tumor cells can abnormally upregulate the expression of both PD-L1 and PD-1, suppressing T cell immune activity, causing tumor immune escape, and leading to tumor development and progression. By examining the co-crystal structure of PD-L1 and PD-1 and investigating the specific characteristics of their binding interface, it can be found that the binding epitopes of both proteins are mainly composed of beta-sheeted polypeptides.

[0063] The term "ADC" refers to a compound in which an antibody / antibody functional fragment, a linker arm, and a drug moiety are chemically linked together. Its structure typically consists of three parts: an antibody or antibody-like ligand, a drug moiety, and a linker arm that couples the antibody or antibody-like ligand and the drug.

[0064] The term "RDC" typically includes antibodies, peptides or small molecules (ligands), link arms, chelators, and cytotoxic / imaging factors (radioisotopes). Its biggest difference from ADCs is the drug payload; RDCs are no longer small molecules but radionuclides. Different medical radionuclides can be used to achieve different functions, such as imaging or therapy, and some radionuclides possess both capabilities.

[0065] The term "connector arm" as used herein refers to the portion of an RDC or ADC that links a peptide to a chelate / drug, which may be cleavable or non-cleavable. A cleavable connector arm (i.e., a breakable or biodegradable connector arm) can cleave within or on target cells, thereby releasing the drug. In some embodiments, the connector arm of this disclosure is selected from cleavable connectors, such as disulfide-based connectors (which selectively cleave in tumor cells with higher thiol concentrations), peptide connectors (which are cleaved by enzymes in tumor cells), and hydrazone connectors. In other embodiments, the connector arm of this disclosure is selected from non-cleavable connectors (i.e., non-breakable connectors), such as thioether connectors. Selectable connectors in this disclosure include chain structures having the following structures: .

[0066] The chelates described herein are linked to peptides via linker arms, which can be achieved through conventional covalent bonding of reactive functional groups. N-hydroxysuccinimide (NHS), thiocyanate (SCN), and acid anhydrides are the most commonly used reactive electrophilic groups in this strategy. Under these conditions, chelating agents containing NHS or SCN can readily couple with peptides to form strong covalent bonds. After attachment, radiolabeling is achieved through a complexation process. Optional chelating agents disclosed herein include DOTA, TETA, NOA, NETA, NOTAGA, DTPA, HBED, or 5HBED.

[0067] II. Implementation Method The present disclosure is further illustrated below with reference to embodiments. The description of specific exemplary embodiments of the present disclosure is for illustrative and explanatory purposes. These descriptions are not intended to limit the present disclosure to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the teachings of this specification. The exemplary embodiments were chosen and described in order to explain the specific principles of the present disclosure and their practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the present disclosure, as well as various different choices and variations.

[0068] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0069] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0070] In a first aspect, this disclosure provides a polypeptide targeting the immune checkpoint PD-L1, said polypeptide comprising the amino acid sequence NH2-CWCWRX1PGX2...X 2+m -COOH, where m is selected from 0 to 5, and X1, X2, ..., X... 2+m Selected from natural amino acid residues, wherein X2...X 2+m The peptide contains at least one cysteine ​​residue, and at least one cyclization structure is formed between the amino acid residue at the N-terminus of P and the amino acid residue at the C-terminus of G. Constructing cyclization structures at both ends of PG in the peptide significantly improves the peptide's protease resistance, thereby enhancing its stability. Furthermore, experimental verification has shown that this cyclization method also significantly improves the affinity of the peptide for PD-L1.

[0071] In an optional embodiment, the cyclic structure is linked by disulfide bonds.

[0072] In an optional implementation, X1 is selected from C or D.

[0073] In an optional embodiment, m=5, X6 is cysteine, and X6 forms a cyclic structure with any cysteine ​​residue on the N-terminal side of P. In one embodiment, X2...X 2+m For RSGGCK (SEQ ID NO:12). Optionally, X6 (C) is the first N-terminal cysteine ​​residue in NH2-CWCWRX1PGX2...X7-COOH ( C WCWR (SEQ ID NO:13) forms a cyclic structure.

[0074] In an optional embodiment, the amino acid sequence of the polypeptide is NH2-X. 2+m+1 ...X 2+m+ n CWCWRX1PGX2...X 2+m -COOH, where n is selected from 0 to 7, and X 2+m+1 ... X 2+m+n Selected from natural amino acid residues. Optionally, n is 7, and X... 2+m+1 ...X 2+m+7 It is SGQYASYH (SEQ ID NO:14).

[0075] In an optional embodiment, the amino acid sequence of the polypeptide is SGQYASYH. C WCWRDPGRSGG C K (SEQ ID NO:2), the structure of the polypeptide is: .

[0076] In a second aspect, this disclosure provides a polypeptide targeting the immune checkpoint PD-L1, the polypeptide comprising the amino acid sequence NH2-CWCWRDX1X2RSX3GX4K-COOH, wherein X1, X2, X3 and X4 are selected from natural amino acid residues or any chiral molecule thereof; wherein X3 and X4 are both tryptophan or neither is tryptophan.

[0077] In an optional implementation, the dipeptide fragment X1X2 is non- L P L G-dipeptide fragment.

[0078] In an optional embodiment, X1 in the dipeptide fragment X1X2 is... D P, X2 are selected from L P or L G.

[0079] In an optional implementation, the combination of X3 and X4 is selected from (a) or (b): (a) X3 is L G, X4 is L S, (b)X3 is L W, X4 is L W.

[0080] In an optional implementation, X1~X4 are selected from (a) or (b): (a) X1 is D P, X2 is L G, X3 is L G, X4 is L S, (b)X1 is D P, X2 is L P, X3 is L W, X4 is L W.

[0081] In an optional embodiment, the amino acid sequence of the polypeptide is NH2-X5...X. 5+ n CWCWRDX1X2RSX3GX4K-COOH, wherein X5...X 5+n It is an amino acid sequence of length n, where n is selected from 0 to 7, wherein X5, ..., X 5+n Selected from naturally occurring amino acid residues with chirality L. In one embodiment, n is 7, and X5...X 12 The value is SGQYASYH. In another embodiment, n is 0, and X5 is selected from K or E. Optionally, X5 forms a cyclized structure with the C-terminal amino acid K via an amide bond.

[0082] In an optional embodiment, the amino acid sequence of the polypeptide is SGQYASYHCWCWRD D PPRSWGWK (SEQ ID NO:8) or CWCWRD D PPRSWGWK (SEQ ID NO:9); Alternatively, the amino acid sequence of the polypeptide is: K CWCWRD D PPRSWGW K (SEQ ID NO:10), wherein the N-terminal K and the C-terminal K are linked by an amide bond; or, the amino acid sequence of the polypeptide is as follows: E CWCWRD D PPRSWGW K (SEQ ID NO:11), where the E at the N-terminus and the K at the C-terminus are connected by an amide bond.

[0083] A third aspect of this disclosure provides the use of the polypeptide described in any embodiment of the first aspect in the preparation of tumor in situ diagnostic reagents or tumor therapeutic agents targeting the immune checkpoint PD-L1.

[0084] In optional embodiments, the tumor in situ diagnostic reagent or tumor therapeutic drug includes RDC (Radionuclide Drug Conjugates) or ADC (Antibody-Drug Conjugates).

[0085] In a fourth aspect, this disclosure provides a monomeric RDC molecule containing a polypeptide as described in any embodiment of the first aspect, a linker arm, a chelate, and a radioactive factor, wherein the chelate chelates and a radionuclide are linked to the polypeptide via the linker arm.

[0086] In an optional embodiment, the connecting arm has the structural formula shown in Formula I: .

[0087] In an optional embodiment, the chelate is a NOA derivative represented by Formula I: .

[0088] In an optional embodiment, the radionuclide is selected from... 14 C 15 N、 18 F, 32 P, 33 P, 35 S, 45 Ti、 47 Sc、52 Fe, 55 Co, 58m Co, 59 Fe, 60 Cu, 61 Cu, 62 Cu, 63 Zn, 64 Cu, 66 Ho, 67 Cu, 67 Ga, 68 Ga, 75 Br, 76 Br, 77 Br, 82 Rb, 86 Y, 87 Y, 89 Zr, 89 Sr, 90 Y, 97 Ru, 99m Tc, 103 Pd, 103m Rh, 105 Rh, 109 Pd, 111 In, 117m Sn, 119 Sb, 123 I, 124 I, 125 I, 131 I, 149 Pm, 149 Tb, 153 Sm, 153 Gd, 161 Ho, 161 Tb, 177 Lu, 186 Re, 188 Re, 191m Pt, 193m Pt, 195m Pt, 187 Pt, 198 Au, 199 Au, 201 Tl, 203 Pb, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 224 Ra, 225 Ac, 227 Th or 229 Th.

[0089] Optionally, the monomeric RDC molecule is a tumor in situ diagnostic reagent, and the radioactive factor is... 18 F or 64 Cu; preferably 64 Cu.

[0090] Optionally, the monomeric RDC molecule includes the polypeptide described in the foregoing embodiments, the linker arm shown in Formula I, the chelate shown in Formula II, and the radioactive factor. 64 Cu, the chelate chelate and radioactive factor, and linked to the polypeptide via a linker arm.

[0091] Optionally, the monomeric RDC molecule includes the polypeptide described in the foregoing embodiments, the linker arm shown in Formula I, the chelate shown in Formula II, and the radioactive factor. 64 Cu, the chelate chelate and radioactive factor, and linked to the polypeptide via a linker arm.

[0092] In an optional embodiment, the monomeric RDC molecule is: .

[0093] In a fifth aspect, this disclosure provides an RDC molecular multimer, comprising a monomeric RDC molecule as described in any embodiment of the third aspect and a multi-arm linker molecule connecting the monomeric RDC molecule. The RDC molecular multimer includes an RDC dimer, an RDC tetramer, an RDC hexamer, or an RDC octamer; optionally, it is an octamer. The multimer exhibits stronger and more stable tumor-specific recognition capabilities, further enhancing the tumor-targeting ability on the basis of peptides, while prolonging the retention time of RDC molecules within the tumor. This improves diagnostic or therapeutic efficacy while avoiding damage to normal healthy tissues.

[0094] In an optional embodiment, the multi-armed linker molecule is a multi-armed PEG (multi-armed polyethylene glycol), including but not limited to two-armed PEG, four-armed PEG, six-armed PEG, or eight-armed PEG. Optionally, the multi-armed PEG is linked to the side chain thiol group of the cysteine ​​residue of the polypeptide via a linker. Optionally, the linker is maleimide.

[0095] In an optional embodiment, the RDC polymer comprises the monomeric RDC molecule described in the foregoing embodiments and an eight-armed PEG (e.g., an eight-armed PEG with an average molecular weight of about 10k), wherein the eight-armed PEG is linked to eight monomeric RDC molecules via maleimide.

[0096] In an optional embodiment, the average molecular weight of the eight-arm PEG is 5~40 kDa, or 5~20 kDa, including but not limited to 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, 16 kDa, 17 kDa, 18 kDa, 19 kDa, or 20 kDa.

[0097] In a sixth aspect, this disclosure provides a tumor in situ diagnostic reagent, the tumor in situ diagnostic reagent comprising a monomeric RDC molecule as described in any embodiment of the third aspect, or an RDC molecule polymer as described in any embodiment of the fourth aspect, wherein the nuclide of the RDC molecule is selected from... 18 F, 68 Ga、 89 Zr、 64 Cu、 99 mTc or 111 In, preferably 64 Cu.

[0098] In an optional embodiment, the molecular structure of the RDC molecule is as follows: .

[0099] In a seventh aspect, this disclosure provides a tumor therapeutic agent comprising a monomeric RDC molecule as described in any embodiment of the third aspect, or an RDC molecule polymer as described in any embodiment of the fourth aspect, wherein the nuclide of the RDC molecule is selected from... 225 Ac、 223 Ra、 213 Bi、 211 At、 188 Re、 177 Lu、 131 I, 90 Y、 89 Sr、 153 Sm、 125 I, 77 Br or 195 mPt.

[0100] In an optional embodiment, the treatment is administered via intravenous injection; optionally, the dosage is 2-3 mCi / time / week.

[0101] TPP-1 is a polypeptide that can specifically bind to PD-L1, with the amino acid sequence SGQYASYHCWCWRDPGRSGGSK (SEQ ID NO: 1). In vivo and in vitro experiments have shown that this polypeptide can effectively inhibit the binding of PD-L1 to PD-1, reactivate the anti-tumor immune response, and inhibit tumor growth. However, the specificity and stability of this polypeptide need to be improved.

[0102] Example 1: In this embodiment, a point mutation of cysteine ​​is introduced on the right side of PG based on TPP-1, and then the sulfhydryl groups of cysteine ​​on both sides of PG are linked by disulfide bonds to form a polypeptide of TPP-1, so as to improve the stability of the short peptide TPP-1 in vivo and avoid it from being rapidly metabolized.

[0103] One feasible implementation is to mutate the serine S at position 21 to cysteine ​​C, which is then used to cyclize with the cysteine ​​preceding P.

[0104] A further feasible specific implementation is to cyclize the mutated cysteine ​​at position 21 with the cysteine ​​at position 9 to form a cyclic peptide, the amino acid sequence of which is as follows: (cTPP-1, SEQ ID NO: 2), the structure of cTPP-1 is as follows: .

[0105] The cTPP-1 peptide and subsequent labeled precursor molecules were ordered from Peptide International (Louisville, USA). Linear peptides were prepared using the Fmoc solid-phase synthesis method described on page 61 and subsequent chapters of "Fmoc Solid Phase Synthesis, WC Chan, PD White, Oxford University Press, 2000". The Cys protecting group was then removed, and the peptides were cyclized by oxidation to form disulfide bonds to obtain cyclic peptides (Reference: Peptide Macrocycles: Methods and Protocols, Chapter 2, https: / / link.springer.com / book / 10.1007 / 978-1-0716-1689-5). The purity of the cyclic peptide product was determined by HPLC (HPLC 214 nm > 95%).

[0106] 1.2 Affinity determination The affinity of cTPP-1 and TPP-1 was detected using surface plasmon resonance (SPR) technology with a Biocore T100. The chip (CM5) surface was esterified using cross-linked EDC / NHS at pH 4.5. Human recombinant PD-L1 protein was coupled to the chip at a concentration of 5 μg / ml; excess active carboxyl groups on the chip were blocked with 1 M ethanolamine hydrochloride (pH 8.5). Channels uncoated with human recombinant PD-1 protein were used as reference channels, and all SPR signals were calibrated by subtracting the corresponding values ​​from the reference channels. The cTPP-1 and TPP-1 stock solutions were diluted to a series of concentrations using running buffer (pH 7.4, 100 mM Tris, 150 nM NaCl, 0.005% Tween-20). After each round of binding and dissociation, the CM5 chip was regenerated with 10 mM glycine-HCl (pH 2.5). Finally, the affinity (K) between cTPP-1 and TPP-1 was calculated by fitting the sensor curves on the Biacore software. D value).

[0107] The results showed that TPP-1 and cTPP-1 before and after cyclization had a significant impact on the K-cell activity of PD-L1. D The nM values ​​were 94.7 nM and 38.4 nM, respectively. It can be seen that while improving stability through cyclization, the affinity of cyclized TPP-1 for PD-L1 was not only not adversely affected, but was actually greatly improved. This is related to the more complete exposure of the binding epitope CWCWR after cyclization.

[0108] Example 2: In this embodiment, cTPP-1 obtained in Example 1 and TPP-1 were used as the target molecule, respectively. A NOOTA chelating agent was linked to the N-terminus of the target peptide molecule via a linker to obtain the precursor molecule. Then, a radionuclide was prepared. 64 The method for preparing Cu monomeric RDC molecules by radionuclide labeling is as follows: 10 μg each of cTPP-1 and TPP-1 conjugated with NOA were dissolved in 100 μL of 0.05 mol / L sodium acetate solution (pH 4.5–7). Additionally, cyclotron-prepared... 64 CuCl2 (10~20 mCi) solution was dissolved in 200 μL of 0.05 mol / L sodium acetate. The two liquids were mixed and reacted at room temperature for 20 minutes. The reaction solution was then passed through a sterile filter and diluted with physiological saline to obtain […]. 64 Cu]cTPP-1 and the product solution of TPP-1. The radiochemical purity of the product was determined by Radio-HPLC under the following analytical conditions. The radiochemical purity was greater than 98%.

[0109] The detection conditions for radio-HPLC are as follows: JASCO HPLC system; YMC-Triat-C18 column (4.6 mm inner diameter × 150 mm, 5 μm); Flow rate 1 ml / min; Mobile phase A: (0.1 vol% trifluoroacetic acid [TFA] aqueous solution); Mobile phase B: (0.1 vol% TFA acetonitrile [MeCN] solution); Initially: A 90%, B 10%; After 20 minutes: A 0%, B 100%; The structures of the obtained cTPP-1 monomer RDC molecule and TPP-1 monomer RDC molecule are as follows: ; .

[0110] The imaging performance of the two obtained monomeric RDC molecules was then evaluated in tumor-bearing mice. The specific methods are as follows: MC38 tumor cells were subcutaneously inoculated into the legs of C57BL / 6 mice (6-8 weeks old), with a cell count of 5 × 10⁶ cells. 6 .

[0111] Five weeks after subcutaneous inoculation of MC38 cells into mice, the largest tumor reached 50 mm. 3 The mice were defined as tumor-bearing mice.

[0112] Will[ 64 Cu]cTPP-1 and [ 64 Cu]TPP-1 (0.2 mCi) was subcutaneously implanted into tumor-bearing mice via tail vein injection, and images were taken using PET scans to study the tumor and its distribution throughout the body.

[0113] PET scans were performed using an Inveon PET Scanner, with 1–2% (v / v) Isoflurane as the anesthetic. One hour post-injection, dynamic scanning for 30 minutes was performed to acquire MIP images of the tumor-bearing mice.

[0114] The results are as follows Figure 1 As shown, it can be seen that, with [ 64 Compared to Cu]TPP-1, [ 64 Cu]cTPP-1 significantly reduced hepatic uptake, decreased hepatotoxicity, and significantly improved image contrast. 64 Cu]cTPP-1 shows the best imaging results in vivo.

[0115] Example 3: In this embodiment, to further improve the tumor retention and internalization of RDC molecules, the monomeric RDC molecules obtained in Example 2 were polymerized using an eight-arm PEG as the backbone (BPG-1138-1 8-Arm PEG-MAL, MW 10k) to obtain multimeric RDCs. The specific method is as follows: The eight-arm PEG is linked to the cTPP-1 peptide via maleimide, specifically by the addition of the thiol group of the cysteine ​​residue at position 11 of the peptide to the double bond of the maleimide, as shown below: .

[0116] Then, the PET imaging of RDC multimers in mice was examined using the following experimental method.

[0117] MC38 tumor cells were subcutaneously inoculated into the legs of C57BL / 6 mice (6-8 weeks old), with a cell count of 5 × 10⁶ cells. 6 .

[0118] Five weeks after subcutaneous inoculation of MC38 cells into mice, the largest tumor reached 50 mm. 3 The mice were defined as tumor-bearing mice.

[0119] Will[ 64 Cu]cTPP-1 (0.2 mCi) was injected subcutaneously into tumor-bearing mice via tail vein injection, and images were taken using PET scans to study the tumor and its distribution throughout the body.

[0120] PET scans were performed using an Inveon PET Scanner, with 1–2% (v / v) Isoflurane as the anesthetic. Dynamic in-vitro pulsaring (MIP) images of the tumor-bearing mice were acquired at 28, 43, 52, and 65 hours post-injection, with each scan lasting 30 minutes.

[0121] The results are as follows Figure 2 As shown, after eight-arm PEG modification, the uptake of RDC polymers in the tumor lasts for more than three days, and the uptake in the liver and kidneys is much lower than that in the tumor. Therefore, the eight-arm PEG-modified RDC polymers provided in this embodiment can be used to prepare long-acting tumor detection products.

[0122] Example 4: This embodiment is to verify [ 64 The therapeutic effect of Cu]PEG8-cTPP1 was investigated in mice carrying MC38, using BMS (Bristol-Myers Squibb)-developed WL12 (HY-P3440, MCE China) as a positive control. The drug was administered via microintravenous injection of 2-3 mCi of […]. 64 Cu]PEG8-cTPP1 was injected twice, with a one-week interval between each injection. The specific experimental method is as follows: Dilute 2 mCi (with physiological saline) with [ 64 Cu]PEG8-cTPP1 (n=5) or WL12 was injected into experimental mice via the tail vein. The control group received an equal volume of saline (n=5). Subsequently, mice were weighed every 3 days, and tumor size and diameter were measured. Tumor volume was determined using the following formula: V=ab 2 / 2 (a: maximum major axis, b: vertical minor axis).

[0123] In addition, a second injection (2 mCi) was administered 7 days after the first injection. 64 [Cu]PEG8-cTPP1 and WL12). Two weeks later, the tumor size in the drug-injected mice was significantly smaller than that in the control mice (p<0.05). At the end of treatment (day 23), tumor tissue was collected for further evaluation.

[0124] During this process, the condition of tumor-bearing mice was observed (diet, weight, skin color, mental state, activity level, and survival status), and no drug-induced toxic side effects were observed. Results are as follows: Figure 3 As shown, it can be seen that, 64 Cu]PEG8-cTPP1 significantly inhibited tumor growth. Using the BMS-developed WL12 peptide as a control, the results showed that... 64 Cu]PEG8-cTPP1 is significantly more effective than WL12. Since WL12 is currently undergoing clinical trials, the above results fully demonstrate that […]. 64 Cu]PEG8-cTPP1 has extremely high clinical translational value.

[0125] This study proves that [ 64 [Cu] compounds, a novel radiopharmaceutical, can be used for PET cancer diagnosis and treatment.

[0126] Example 5: To further improve the affinity of the peptide, point mutants of TPP-1, namely TPP-2, TPP-3, TPP-4, TPP-5, TPP-6 and TPP-7, were constructed according to the preparation method in Example 1, as shown in Table 5-1 (from N-terminus to C-terminus, SEQ ID NOs: 3~8).

[0127] Table 5-1 Comparison of TPP-1 mutant sequences In Table 5-1, the epitopes that specifically bind to PD-L1 are CWCWR at positions 9 to 13. This disclosure did not perform any mutation optimization on these epitopes. The results show that the Pro residue at position 15 has a significant impact on the affinity of TPP for PD-L1. When position 15 is D-type Pro, the affinity is significantly improved. Furthermore, replacing glycine at position 16 with D-type Pro at position 15 also significantly affects the affinity of TPP for PD-L1. Therefore, it can be inferred that the amino acid residue combination at positions 15 and 16 has a crucial influence on the function of the binding epitope during the specific affinity of TPP for PD-L1.

[0128] Furthermore, surprisingly, when either glycine at position 19 or serine at position 21 is mutated to tryptophan, the affinity of TPP for PD-L1 decreases significantly (K). D The value increased more than threefold), and when glycine at position 19 or serine at position 21 was replaced with tryptophan, the affinity of TPP for PD-L1 was significantly increased (K). D The value dropped to single digits, which was beyond the inventor's expectations, and the reason for this phenomenon needs further research.

[0129] Based on TPP-7, this embodiment also attempted to truncate the amino acid fragment at the N-terminus of the binding epitope, resulting in TPP-8 (CWCWRD). D PP RS W G W K, SEQ ID NO:9) was detected to contain K D The value is 3.7 nM, which is comparable to TPP-7, indicating that the amino acid fragment at the N-terminus of the TPP epitope has no effect on affinity.

[0130] Example 6 Based on TPP-8 obtained in Example 6, this embodiment attempts to cyclize the peptide to improve its stability, and constructs TPP-9 and TPP-10 respectively.

[0131] TPP-9: K CWCWRD D PP RS W G W K-COOH, SEQ ID NO:10, wherein the amino group of the N-terminal lysine residue is cyclized with the carboxyl group of the C-terminal lysine residue to obtain the cyclized TPP-9 polypeptide. The affinity of TPP-9 was detected using the same detection method as in Example 1, and the result was 1.8 nM, which is comparable to that of TPP-7 and TPP-8. It can be seen that the activity was not affected after cyclization improved stability.

[0132] TPP-10: E CWCWRD D PP RS W G W K, SEQ ID NO:11, wherein the carboxyl group on the side of the N-terminal glutamic acid forms a ring with the amino group on the side of the C-terminal lysine to obtain the cyclized TPP-10 polypeptide. The affinity of TPP-10 was tested using the same detection method as in Example 1, and the result was 2.1 nM, comparable to TPP-7, TPP-8, and TPP-9. This indicates that the cyclization scheme, while improving stability, did not adversely affect TPP activity.

Claims

1. A peptide targeting the immune checkpoint PD-L1, containing the amino acid sequence NH2-CWCWRX1PGX2...X 2+m -COOH, where m is selected from 0 to 5, and X1, X2, ..., X... 2+m Selected from natural amino acid residues, wherein X2...X 2+m It contains at least one cysteine ​​residue, and the amino acid residue on the N-terminal side of P forms at least one cyclic structure with the amino acid residue on the C-terminal side of G. Optionally, the cyclic structure is linked by disulfide bonds.

2. The polypeptide according to claim 1, wherein, X1 is selected from C or D; Optionally, m=5, X6 is cysteine, and X6 forms a cyclized structure with any cysteine ​​on the N-terminal side of P; Optionally, the X2...X 2+m For RSGGCK; Optionally, X6 forms a cyclized structure with the first N-terminal cysteine ​​residue in NH2-CWCWRX1PGX2...X7-COOH.

3. The polypeptide according to claim 1 or 2, wherein, The amino acid sequence of the polypeptide is NH2-X. 2+m+1 ...X 2+m+ n CWCWRX1PGX2...X 2+m -COOH, where n is selected from 0 to 7, and X 2+m+1 ... X 2+m+n Selected from natural amino acid residues; Optionally, n is 7, and X 2+m+1 ...X 2+m+7 It is SGQYASYH.

4. A peptide targeting the immune checkpoint PD-L1, comprising the amino acid sequence NH2-CWCWRDX1X2RSX3GX4K-COOH, wherein... X1, X2, X3, and X4 are selected from natural amino acid residues or any chiral molecule thereof; wherein X3 and X4 are both tryptophan or neither is tryptophan. Optionally, the dipeptide fragment X1X2 is non- L P L G-dipeptide fragment; for example, X1 is D P, where X2 is selected from L P or L G; Optionally, the combination of X3 and X4 is selected from (a) or (b): (a) X3 is L G, X4 is L S, (b)X3 is L W, X4 is L W; Optionally, X1~X4 are selected from (a) or (b): (a) X1 is D P, X2 is L G, X3 is L G, X4 is L S, (b)X1 is D P, X2 is L P, X3 is L W, X4 is L W.

5. The polypeptide according to claim 4, wherein, The amino acid sequence of the polypeptide is NH2-X5...X 5+ n CWCWRDX1X2RSX3GX4K-COOH, wherein X5...X 5+n It is an amino acid sequence of length n, where n is selected from 0 to 7, wherein X5, ..., X 5+n Selected from natural amino acid residues with chirality L; Optionally, n is 7, and X5...X 12 For SGQYASYH; Optionally, n is 0, and X5 is selected from K or E; Optionally, X5 and the amino acid K at the C-terminus form a cyclized structure via an amide bond.

6. Use of the polypeptide according to any one of claims 1 to 5 in the preparation of tumor in situ diagnostic reagents or tumor therapeutic drugs targeting the immune checkpoint PD-L1; Optionally, the tumor in situ diagnostic reagent or tumor therapeutic drug includes RDC or ADC.

7. A monomeric RDC molecule containing the polypeptide of any one of claims 1 to 5, comprising the polypeptide of any one of claims 1 to 5, a linker arm, a chelate, and a radioactive factor, wherein the chelate and the radioactive factor are linked to the polypeptide via the linker arm; Optionally, the connecting arm has the structural formula shown in Formula I: ； Optionally, the chelate is a NOA derivative represented by Formula II: ； Optionally, the radioactive nuclide is selected from... 11 C 15 N、 18 F, 32 P, 33 P, 35 S, 38 K, 43 Sc、 44 Sc, 44 Ti、 47 Sc、 52 Fe、 55 Co、 58m Co、 55 Fe、 59 Fe、 61 Cu、 62 Cu、 63 Zn, 64 Cu、 66 Ho、 67 Cu、 67 Ga、 68 Ga、 75 Br、 76 Br、 77 Br、 82 Rb、 86 Y、 87 Y、 89 Zr、 89 Sr、 90 Y、 97 Ru、 99m Tc, 103 Pd, 103m Rh、 105 Rh、 109 Pd, 111 In、 117m Sn、 119 Sb, 123 I, 124 I, 125 I, 131 I, 149 Pm, 149 Tb, 153 Sm、 153 Gd, 161 Ho、 161 Tb, 177 Lu、 186 Re、 188 Re、 191m Pt, 193m Pt, 195m Pt, 187 Pt, 198 Au、 199 Au、 201 Tl、 203 Pb, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 224 Ra, 225 Ac, 227 Th or 229 Th; Optionally, the monomeric RDC molecule is a tumor in situ diagnostic reagent, and the radionuclide is... 18 F, 64 Cu、 68 Ga、 177 Lu; preferably 64 Cu; Optionally, the monomeric RDC molecule comprises the polypeptide of claim 3, the linker arm shown in Formula I, the chelate shown in Formula II, and the radionuclide. 64 Cu, the chelate chelate and radionuclide, and linked to a polypeptide via a linker arm; Optionally, the monomeric RDC molecule comprises the polypeptide of claim 5, the linker arm shown in Formula I, the chelate shown in Formula II, and the radionuclide. 64 Cu, the chelate chelate and radionuclide, and linked to a polypeptide via a linker arm.

8. An RDC molecular polymer, comprising the monomeric RDC molecule of claim 7 and a multi-arm linker molecule connecting the monomeric RDC molecule, wherein the RDC molecular polymer comprises an RDC dimer, an RDC tetramer, an RDC hexamer, or an RDC octamer; Optionally, the multi-armed linker molecule is a multi-armed PEG, which is linked to the side chain thiol group of the cysteine ​​residue of the polypeptide via a linker. Optionally, the linker is maleimide; Optionally, the RDC polymer comprises the monomeric RDC molecule of claim 7 and an eight-arm PEG, wherein the eight-arm PEG is linked to eight monomeric RDC molecules via maleimide; Optionally, the average molecular weight of the eight-arm PEG is 5~40 kDa, or 5~20 kDa, for example 10 kDa.

9. A tumor in situ diagnostic reagent, comprising the monomeric RDC molecule of claim 7, or the RDC molecule polymer of claim 8, wherein, The radionuclides of the RDC molecules are selected from... 18 F, 68 Ga、 89 Zr、 177 Lu、 64 Cu、 99m Tc or 111 In, preferably 64 Cu.

10. A tumor therapeutic agent comprising the monomeric RDC molecule of claim 7, or the RDC molecule polymer of claim 8, wherein, The nuclides of the RDC molecule are selected from... 225 Ac、 223 Ra、 213 Bi、 211 At、 188 Re、 177 Lu、 131 I, 90 Y、 89 Sr、 153 Sm、 125 I, 77 Br or 195 mPt; Optionally, the treatment can be administered via intravenous, intraperitoneal, or direct intratumoral injection; optionally, the dosage is 2-3 mCi / time / week.

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