Miniprotein binding to PD-l1 and use thereof

Abstract

The present invention belongs to the field of biomedicine. Provided are a miniprotein binding to PD-L1 and the use thereof. Further provided are a nucleic acid molecule encoding the miniprotein, a vector, a host cell, and a conjugate, and the use thereof for treating tumors.

Description

A microprotein that binds to PD-L1 and its applications

[0001] This invention claims priority to Chinese patent application filed on August 29, 2024 (application number: CN202411206504.9, invention title: A microprotein that binds to PD-L1 and its application), the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biomedicine and relates to microproteins that bind to PD-L1 and their applications. This invention also relates to nucleic acid molecules encoding this microprotein, vectors, and host cells, and their applications in the treatment of tumors. Technical Background

[0003] PD-1 (Programmed Death-1, PD1) and PD-L1 (Programmed Death-Ligand1, PDL1) are a pair of proteins that play important roles in the immune system. PD-1 is a surface receptor belonging to the CD28 superfamily and is mainly expressed on activated T cells, B cells, and some other immune cells. PD-L1 is the major ligand of PD-1 and is widely expressed on various cell types, including tumor cells. The binding of PD-L1 to PD-1 can transmit immunosuppressive signals, leading to a decrease in T cell function, thereby helping to maintain tissue homeostasis and self-tolerance.

[0004] In the tumor microenvironment, tumor cells express PD-L1, which binds to PD-1 on the surface of T cells, inhibiting T cell activity and evading the body's immune surveillance. Blocking the PD-1 and PD-L1 signaling pathways can effectively restore T cell activity and activate the immune system to attack tumors. Targeted PD-1 or PD-L1 therapy has become one of the most popular tumor immunotherapies in recent years, and several drugs have been approved for the treatment of melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck cancer, and other tumors. Although PD-1 / PD-L1 inhibitors have shown good therapeutic effects in various types of tumors, they also have limitations such as low clinical response rates, treatment resistance, and treatment-related adverse reactions, requiring further improvement.

[0005] Radiolabeled drugs are therapeutic agents that combine a radioactive isotope (nucleus) with a targeted molecule, and can be used for the diagnosis and treatment of cancer. These drugs conjugate a radioactive isotope to a specific targeted drug (such as an antibody), allowing the drug to specifically target diseased tissue or pathogens, while the radioactive isotope can exert its therapeutic or imaging effects. Radiolabeled drugs combine the advantages of precise targeting and potent killing. Antibodies or peptides carrying radioactive nuclides target cancer cells, concentrating energy release within a range several times the cell diameter, killing cancer cells while minimizing damage to nearby normal tissue cells, resulting in good efficacy and few adverse reactions. Summary of the Invention

[0006] Through in-depth research, the inventors of this invention discovered that antibody drugs, with their large molecular weight, long half-life, and weak permeability, tend to accumulate locally in organs such as the liver and kidneys. Furthermore, antibody conjugates carrying radionuclides may cause excessive radiation damage to the human body. Conjugates constructed using microproteins with small molecular weights, controllable half-lives, and strong permeability can effectively solve this problem. Developing microproteins that bind to PD-L1 has significant medical application value. After extensive research, the inventors of this invention have finally developed a microprotein capable of binding to PD-L1, thus completing this invention. In some embodiments, the microprotein of this invention can bind to human PD-L1 with a higher affinity than wild-type human PD-1, can block the interaction between the full-length human PD-1 polypeptide and the full-length human PD-L1 polypeptide, and / or possesses good stability (including physical and chemical stability).

[0007] In a first aspect, the present invention provides a microprotein that can specifically bind to human PD-L1, and compared with wild-type human PD-1, the microprotein can bind to human PD-L1 with a higher affinity; in some embodiments, the microprotein has a protein backbone fold of the human PD-L1 binding portion of the PD-1 polypeptide as shown in SEQ ID NO:2.

[0008] On the other hand, the present invention provides a microprotein comprising the amino acid sequence shown in SEQ ID NO:23:

[0009] X1X2X3X4X5X6X7X8X9X 10 YVX 11 GX 12 IX 13 LX 14 PX 15 IX 16 IX 17 ESX 18 RX 19 X 20 X21 X 22 X 23 X 24 AX 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 X 42 X 43 X 44 X 45 X 46 HX 47 VX 48 HX 49 EX 50 X 51 SGQLDTLX 52 AX 53 X 54 X 55 X 56 X 57 X 58 X 59 X 60 (SEQ ID NO:23)

[0010] in,

[0011] X1 is M, P, or empty;

[0012] X2 is E, A, M or blank;

[0013] X3 is I, L, P or empty;

[0014] X4 is P, A, D or blank;

[0015] X5 can be E, D, P, A, L, or be empty;

[0016] X6 can be E, D, P, I, or L;

[0017] X7 is either E or P;

[0018] X8 is G, D, P, or K;

[0019] X9 can be A, Y, P, G, or H;

[0020] X 10For L, R, or T;

[0021] X 11 For V or I;

[0022] X 12 V, A, or I;

[0023] X 13 It can be C, T, V, S, I, or A;

[0024] X 14 For E or T;

[0025] X 15 It can be E or K;

[0026] X 16 For R, K, or M;

[0027] X 17 For L, E, F, V, or I;

[0028] X 18 For E or P;

[0029] X 19 It can be R, K, H, Q, V, or A;

[0030] X 20 It can be P, A, E, or T;

[0031] X 21 For T or A;

[0032] X 22 The options are L, I, R, A, E, or blank;

[0033] X 23 It can be A or E;

[0034] X 24 It can be E or A;

[0035] X 25 For L, R, or P;

[0036] X 26 It can be E or A;

[0037] X 27 For Q, S, L, or A;

[0038] X 28 It can be E, G, L, V, or A;

[0039] X 29 It can be P, K, A, or D;

[0040] X 30 It can be A, P, E, K, or R;

[0041] X31 For L, V, or K;

[0042] X 32 For I or L;

[0043] X 33 It can be A or E;

[0044] X 34 It can be E or K;

[0045] X 35 It can be A or L;

[0046] X 36 It can be K, A, R, or I;

[0047] X 37 It can be A, K, or E;

[0048] X 38 It can be Q, V, L, K, E, or Q;

[0049] X 39 The answer is G, A, S, or blank;

[0050] X 40 G or empty;

[0051] X 41 It can be P, N, D, E, or a blank;

[0052] X 42 V, P, or vacancy;

[0053] X 43 It can be C, S, G, N, A, or Y;

[0054] X 44 It can be K, A, L, R, or S;

[0055] X 45 It can be K, V, E, or N;

[0056] X 46 It can be L, Y, F, or I;

[0057] X 47 V, L, or R;

[0058] X 48 V, I, or L;

[0059] X 49 For R, K, or L;

[0060] X 50 For S, L, K, or T;

[0061] X 51 For P or A;

[0062] X 52 The options are D, W, S, E, A, or Y;

[0063] X 53 For R, E, Q or blank;

[0064] X 54 It can be K, L, R or empty;

[0065] X 55 V, I, or blank;

[0066] X 56 V, E, L, P, or a blank;

[0067] X 57 V, I, L, E, or a blank;

[0068] X 58 P or blank;

[0069] X 59 For E, P, L, or a blank; and

[0070] X 60 It can be L or empty.

[0071] Secondly, in some embodiments, the present invention provides a fusion protein comprising the microprotein described in any of the preceding embodiments; in some embodiments, the fusion protein comprises an antibody or a fragment;

[0072] Thirdly, in some embodiments, the present invention provides a conjugate comprising the microprotein or fusion protein described in any of the preceding embodiments, and a coupling portion; in some embodiments, the conjugate wherein the coupling portion is a radionuclide;

[0073] Fourthly, in some embodiments, the present invention provides a nucleic acid molecule that encodes the microprotein or fusion protein described in any of the preceding embodiments.

[0074] Fifthly, in some embodiments, the present invention provides a recombinant vector comprising the nucleic acid molecule described in any of the preceding claims.

[0075] Sixthly, in some embodiments, the present invention provides a host cell comprising the nucleic acid molecule or recombinant vector described in any of the preceding claims.

[0076] In a seventh aspect, in some embodiments, the present invention provides a pharmaceutical composition comprising the microprotein, fusion protein, or conjugate described in any of the preceding claims, and one or more pharmaceutically acceptable carriers.

[0077] Eighthly, in some embodiments, the present invention provides the use of the microproteins, fusion proteins, conjugates, nucleic acid molecules, recombinant vectors, host cells, or pharmaceutical compositions described in any of the preceding claims in the preparation of a medicament for treating tumors;

[0078] On the other hand, in some embodiments, the present invention provides a method for treating tumors, the method comprising administering to a subject in need a therapeutically effective amount of any of the preceding microproteins, fusion proteins, conjugates, nucleic acid molecules, recombinant vectors, host cells, or pharmaceutical compositions;

[0079] On the other hand, in some embodiments, the present invention provides microproteins, fusion proteins, conjugates, nucleic acid molecules, recombinant vectors, host cells, or pharmaceutical compositions described in any of the preceding embodiments as medicaments; in some embodiments, the medicaments are used to treat tumors. Attached Figure Description

[0080] Figure 1: Schematic diagram of the Miniprotein-Fc fusion protein structure;

[0081] Figure 2: Experimental results of the binding of Miniprotein-Fc fusion protein to CHO-K1 PD-L1;

[0082] Figure 3: Experimental results of the binding of Miniprotein-Fc fusion protein to CHO-K1;

[0083] Figure 4: Experimental results of L04-His and CHO-K1 PD-L1 binding;

[0084] Figure 5: Experimental results of Miniprotein-Fc fusion protein blocking PD-1 / PD-L1 interaction.

[0085] Invention Details

[0086] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular biology, biochemistry, nucleic acid chemistry, immunology, and other operational procedures used herein are all conventional procedures widely used in their respective fields. All references cited herein, including but not limited to patents, patent applications, articles, textbooks, etc., and all references cited therein, are entirely incorporated herein.

[0087] As used herein, the terms “for example,” “such as,” “like,” “including,” “contains,” or variations thereof will be interpreted as meaning “including but not limited to.”

[0088] Unless otherwise specified herein or clearly contradicted by the context, the terms “an” and “a kind” as well as “the” and similar designations shall be interpreted to cover both the singular and the plural in the context of describing the invention (especially in the context of the following claims).

[0089] As used herein, the terms “first” and “second” are used for descriptive purposes only and should not be construed as implying relative importance or the number of technical features indicated.

[0090] The term “multiple” means at least two, such as 2, 3, 4, etc., unless it is explicitly stated in the text that this is not the case.

[0091] As used herein, the term "and / or" means that it includes both the meanings of "and" and "or". For example, the phrase "A, B and / or C" is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0092] Unless otherwise stated, when referring to nucleic acid sequences in this article, the direction is from left to right 5′ to 3′; when referring to amino acid sequences, the direction is from left (upstream) to right (downstream) amino (N) to carboxyl (C).

[0093] The twenty common amino acids referred to in this article are written in accordance with conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Goluband DRGren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. Furthermore, in this invention, amino acids are represented using single-letter and three-letter abbreviations well known in the art (amino acid three-letter codes and single-letter codes are as described in J. Biol. Chem., 243, p3558 (1968)). For example, alanine can be represented as A or Ala. The single-letter and three-letter abbreviations for other amino acids are as follows: arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0094] As used herein, the terms “peptide,” “polypeptide,” “protein molecule,” “protein,” and “protein” are used interchangeably and are defined as a biomolecule consisting of amino acid residues linked together by peptide bonds.

[0095] As used herein, the term "miniprotein" refers to a protein molecule with a small molecular weight (usually not exceeding 15 kDa) and a specific conformation and function. For example, a miniprotein can be a protein molecule composed of 10-150 (e.g., 150, 100, 90, 85, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 60, 50, 40, 30, 20, 10, or any value between them) amino acid residues and possessing protein-protein (e.g., ligand-receptor, such as PD1-PDL1) interaction function. Without limitation, a miniprotein may compete with a second protein (e.g., PD1) for binding epitopes in a third protein (e.g., PDL1), or the miniprotein may block the binding of a second protein (e.g., PD1) to a third protein (e.g., PDL1), or the binding of a miniprotein to a third protein (e.g., PDL1) may enhance the activity of the third protein. Despite their small size, many microproteins exhibit high structural and functional stability, which is crucial for maintaining their activity under various environmental conditions. Furthermore, some microproteins can bind specifically to target molecules at levels comparable to antibodies, and due to their smaller size, they offer better tissue penetration and shorter metabolic half-lives. These characteristics strongly support the application of microproteins in tissue and cell-targeted recognition and delivery, such as chemically conjugated drugs, radionuclide-conjugated drugs, or radionuclide probes.

[0096] As used herein, the term “protein backbone” refers to a protein portion having surface regions that are highly resistant to amino acid insertions, substitutions, or deletions. Numerous protein backbones are well known to those skilled in the art (Binz etl., 2005, loc.cit.; Binz etal., 2004, loc.cit.).

[0097] As used herein, the term "protein backbone folding" refers to the three-dimensional structure of a protein backbone. In some embodiments, the microprotein of the present invention has a protein backbone fold of the human PD-L1 binding portion of the PD-1 polypeptide (e.g., shown in SEQ ID NO:2), such that the microprotein has sufficient exposure of the PD-L1 binding epitope to enable it to specifically bind to human PD-L1.

[0098] As used herein, the term "specific binding" refers to a non-random binding reaction between two protein molecules. Examples include the reaction between a ligand and its targeted receptor, and the reaction between an antibody and its targeted antigen. The strength or affinity of a specific binding between two molecules can be represented by the equilibrium dissociation constant (KD) of their interaction.

[0099] As used in this article, the term "KD" refers to the dissociation equilibrium constant of a specific molecular interaction, which describes the binding affinity between different molecules. The smaller the equilibrium dissociation constant, the tighter the intermolecular binding and the higher the affinity.

[0100] The specific binding force between two protein molecules can be determined using methods known in the art. For example, the receptor-ligand binding activity can be determined by measuring the rate of formation and dissociation of the protein receptor-ligand complex. Both the "binding rate constant" (ka or kon) and the "dissociation rate constant" (kd, kdis, or koff) can be calculated from the concentration and the actual rates of association and dissociation. The ratio of kdis / kon is equal to the dissociation constant KD. KD, kon, and kdis values ​​can be measured using any effective method. The smaller the equilibrium dissociation constant KD, the tighter the receptor-ligand binding, the higher the affinity between the receptor and ligand, and the stronger the binding ability.

[0101] In some embodiments, the binding affinity between the receptor protein (e.g., a microprotein that binds to PD-L1) and its ligand (e.g., PD-L1) is determined by detecting the KD value, EC50 value, and / or Emax value (also known as the TOP value) of the receptor protein binding to its ligand. The “EC50” value refers to the half-maximum effective concentration of the substance; the “Emax” value refers to the maximum effect achievable by the substance. In some embodiments, the EC50 value is determined by measuring the half-maximum binding concentration using the fluorescence intensity of the medium, and then nonlinearly fitting an S-shaped dose response within GraphPadPrism software; the Emax value is determined by nonlinear regression analysis of a four-parameter logarithmic equation (De Lean, A., A.A. Hancock, and R.J. Lefkowitz, Mol. Pharmacol. 21:5-16 (1981)).

[0102] As used herein, the term "epitope" refers to an area (or region) on a ligand (e.g., PD-L1) of a protein that binds specifically to a receptor (e.g., PD-1). Epitopes can be formed from consecutive amino acids (linear epitopes) or non-consecutive amino acids (conformal epitopes), for example, by the folding of the ligand (i.e., by the tertiary folding of the ligand, a protein property), which allows non-consecutive amino acids to be spatially close. For example, an epitope can contain at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for proteins (e.g., ligands, antibodies) that bind to specific epitopes can be performed using methods known in the art, including but not limited to alanine scanning, Western blotting (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see “Antibodies”, Harlowand Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).

[0103] When “competition” is used to describe the competition between binding proteins (e.g., receptors or antibodies) for the same epitope, it means that the binding proteins (e.g., receptors or antibodies) competitively bind to a common ligand or antigen, which can typically be determined by measuring that the binding protein to be detected (e.g., receptor or antibody) inhibits (e.g., reduces) the specific binding of a reference binding protein (e.g., reference receptor or antibody) to the ligand or antigen. Numerous types of competitive binding assays can be used to determine whether one binding protein competes with another, including but not limited to: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, for example, Stahli et al., 1983, Methods in Enzymology 9: 242-253); solid-phase direct biotin-avidin EIA (see, for example, Kirkland et al., 1986, J. Immunol. 137: 3614-3619), solid-phase direct labeled sandwich assay (see, for example, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeled RIA of I-125 (see, for example, Morel et al., 1988, Molec. Immunol. 25: 7-15), etc. In some implementations, the binding of the reference receptor (e.g., wild-type human PD-1) to the ligand (human PD-L1) is inhibited by at least 40% (e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, 90%, 95%, 97%, or 98% or more) by a microprotein that competitively binds to it.

[0104] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, for optimal comparison purposes, a gap may be introduced in the first amino acid sequence or nucleic acid sequence to best align it with the second amino acid sequence, and then the amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. The percentage identity between two sequences is calculated as a function of the number of identity positions shared by the sequences (i.e., percentage identity = number of identical overlapping positions / total number of positions × 100%). In some embodiments, the two sequences are of the same length. The determination of percentage identity between two sequences can be achieved using mathematical algorithms; a non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as improved in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is integrated into the NBLAST and XBLAST procedures of Altschul et al., 1990, J. Mol. Biol. 215:403.

[0105] A polypeptide sequence having at least 80% identity with its parent sequence includes polypeptide variants with altered amino acid sequences (e.g., polypeptide variants obtained through conserved substitution), and polypeptide variants that have been modified (e.g., by covalently linking the molecule to the polypeptide) (e.g., through glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linking to cellular ligands or other proteins, etc.). Polypeptide variants can be generated through chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin. In some embodiments, the polypeptide variant has a function similar to, identical to, or improved upon that of the polypeptide from which it is derived.

[0106] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a polypeptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include replacing amino acid residues with amino acid residues having similar side chains, for example, substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0107] As used herein, the term "antibody" refers to a protein molecule capable of specifically binding to an antigen. Unless the context clearly indicates otherwise, the term "antibody" is used in the broadest sense and encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, full-length antibodies, or fragments thereof, provided they exhibit the desired functional activity. A "complete antibody" (or full-length antibody) typically consists of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). The antibody light chain can be divided into κ (kappa) and λ (lambda) light chains. The heavy chain can be divided into μ, δ, γ, α, or ε, corresponding to the isotypes of antibodies IgM, IgD, IgG (IgG1, IgG2, IgG3, IgG4), IgA (IgA1 and IgA2), and IgE, respectively. The full-length heavy chain consists of a variable region (VH) and a constant region (CH). A typical IgG heavy chain constant region includes three domains (CH1, CH2, and CH3). The full-length light chain consists of a variable region (VL) and a constant region (CL). The constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions include highly variable domains (called hypervariable regions or complementarity-determining regions (CDRs)). More conserved framework regions (FRs) are interspersed between the CDRs. Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (from the N-terminus to the C-terminus).The boundaries of antibody regions or domains can be defined according to various numbering systems known in the field, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883), the Abm numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268–9272), and the Contact numbering system (MacCallum, RM, Martin, ACR, & Thornton, JM (1996). Antibody-antigen Interactions: Contact Analysis and Binding Site Topography. Journal of Molecular Biology, 262(5), 732-745.) or the definition in the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody variable region, those skilled in the art can readily determine the CDR according to the definition of the numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0108] As used herein, the term "antibody fragment" refers to a polypeptide comprising a portion of a complete antibody, distinct from the complete antibody, which possesses the desired functional activity. In some embodiments, the antibody fragment is an Fc. In some embodiments, the antibody fragment is an antigen-binding fragment (i.e., a portion of a complete antibody possessing antigen-binding activity). In some embodiments, the antigen-binding fragment can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of a complete antibody. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, disulfide-linked Fv, scFv, di-scFv, diabody, and other polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0109] As used herein, the term "Fc" or "Fc region" refers to a region of the antibody heavy chain consisting of the CH2 and CH3 domains, or a region of the antibody heavy chain consisting of a hinge or portion thereof and the CH2 and CH3 domains. A typical IgG antibody's Fc is the region of the heavy chain from cysteine ​​226 (EU system number) to the C-terminus, or the region of the heavy chain from proline 230 (EU system number) to the C-terminus. The Fc can be a natural Fc or a modified Fc. A natural Fc contains an amino acid sequence identical to that of Fcs found in nature, such as the natural sequence human IgG1Fc, natural sequence human IgG2Fc, natural sequence human IgG3Fc, or natural sequence human IgG4Fc. A modified Fc contains an amino acid sequence that differs from the amino acid sequence of a natural Fc due to at least one amino acid modification. In some implementations, the variant Fc may have altered functions compared to the natural Fc (e.g., altered Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, complement function, half-life, etc.), for example, substitution of amino acid residues at positions 238, 265, 269, 270, 297, 327, and 329 (EU system number) of IgG1 Fc to reduce effector function.

[0110] Effector cell function refers to the biological activity attributable to the antibody's Fc domain and varying with antibody isotypes; examples of effector cell function include, but are not limited to, C1q binding and complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent phagocytosis (ADCP). ADCC is a mechanism that induces cell death by relying on the interaction between antibody-coated target cells and lytically active effector cells (such as natural killer (NK) cells, monocytes, macrophages, and neutrophils) via Fcγ receptors expressed on the effector cells (e.g., FcγRIIIa on NK cells, FcγRI, FcγRII, and FcγRIIIa on monocytes). ADCP is a mechanism by which antibody-coated target cells are eliminated through internalization by phagocytes (such as macrophages or dendritic cells). CDC refers to a mechanism that induces cell death in which target cells bind to the Fc effector domain of an antibody and activate the complement component C1q, which in turn activates the complement cascade, leading to target cell death. Effector cell function can be detected by methods known in the art. For example, ADCC activity can be determined by known in vitro methods, such as using cells expressing antigens as target cells and NK cells as effector cells, and detecting cell lysis rate based on markers (e.g., radioactive substrates, fluorescent dyes, or native intracellular proteins) released from lysed cells. Other methods for determining ADCC can be found in, for example, WO2006082515, WO2012130831, etc.

[0111] As used herein, the term "fusion protein" refers to a protein composed of two or more polypeptide motifs. Typically, fusion proteins are produced by linking genes encoding two or more individual proteins, and the translation of these fusion genes produces the fusion protein, which usually possesses the functional properties of each individual protein.

[0112] As used herein, the term "conjugate" refers to a polypeptide chemically or biologically linked to another pharmaceutical agent (e.g., including but not limited to: radionuclides, protein tags, detectable markers, other therapeutic agents, or diagnostic agents). Examples of conjugates include, for example, polypeptides formed by chelating microproteins of the present invention with radionuclides using a chelating agent.

[0113] As used herein, the terms "linker" or "connector" refer to a peptide composed of amino acids of a certain length (typically amino acids with low hydrophobicity and low charge effect). Those skilled in the art can select appropriate linkers as needed. Typical amino acid residues in linkers include glycine, serine, tyrosine, cysteine, lysine, glutamic acid, and aspartic acid. An exemplary example is (GmS)n, where m and n are each independently integers from 1 to 10.

[0114] As used herein, the term "radionoid" refers to an atom with an unstable nuclide, characterized by excess energy that can be transferred to newly generated radioactive particles or electrons within the atomic nucleus. Radionoids can undergo radioactive decay and emit subatomic ionizing particles during this decay process. Exemplary subatomic ionizing particles are α-particles, β-particles, and γ-particles, but are not limited thereto.

[0115] As used herein, the term "chelating agent" refers to a formulation containing a compound capable of forming at least two coordinate covalent bonds with a metal ion, thereby producing a stable, water-soluble complex. Most of these compounds are organic ligands and typically contain multiple functional groups that can coordinate with metal ions. These functional groups can form stable coordination compounds with metal ions, exerting various biological and chemical effects by altering the electronic structure, spatial configuration, and chemical properties of the metal ion.

[0116] As used herein, the terms "nucleotide," "polynucleotide," "nucleic acid," and "nucleic acid molecule" are used interchangeably and refer to a molecule composed of multiple nucleotides linked by 3'-5'-phosphodiester bonds, wherein the nucleotides include ribonucleotides and deoxyribonucleotides. The sequences of the polynucleotides of the present invention can be codon-optimized for different host cells (such as *E. coli*) to improve the expression of fusion proteins. Methods for codon optimization are known in the art.

[0117] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of the protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells.

[0118] As used in this article, the term "host cell" refers to a cell that can be used to introduce a vector.

[0119] As used in this article, the term "pharmaceutically acceptable carrier" refers to a substance that facilitates the delivery of an active substance to a subject and its absorption by that subject.

[0120] As used herein, the term "treatment" refers to the relief of at least one symptom. This term includes administering medication to a subject and / or applying one or more of the microproteins, fusion proteins, nucleic acids, host cells, pharmaceutical compositions, or conjugates described herein to provide treatment for a disease. As used herein, "treatment" of a subject with cancer means that the subject's cancer is partially or completely eliminated, or remains stable and no longer progresses after treatment. Treatment includes prevention, treatment, and / or cure. Prevention refers to preventing the occurrence of potential cancer and / or preventing the progression or worsening of cancer; preventing cancer includes mitigating or eliminating one or more risk factors that contribute to cancer development; because it is generally not possible to determine whether cancer has never occurred, prevention also includes reducing the risk of developing or having cancer. When used herein to address harmful proliferating cells (including cancer), "treatment" includes the partial or complete destruction of said harmful proliferating cells, but with minimal impact on normal cells.

[0121] As used herein, the term "patient" or "object of need" means an organism that suffers from or is susceptible to a disease or condition that can be treated by administration of the microproteins, fusion proteins, nucleic acids, host cells, pharmaceutical compositions, or conjugates provided herein. Non-limiting examples include humans, other mammals such as cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammals. In some embodiments, the patient or object is a human.

[0122] As used herein, “therapeutic effective amount” or “therapeutic effective dose” means an amount of a pharmaceutical agent, compound, or material in a preparation that is at least sufficient to produce a therapeutic effect in a subject. The exact amount depends on the therapeutic purpose and can be determined by a person skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1–3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0123] In a first aspect, the present invention provides a microprotein that can specifically bind to human PD-L1, and compared with wild-type human PD-1, the microprotein can bind to human PD-L1 with a higher affinity.

[0124] In some embodiments, the microprotein described in any of the preceding claims, wherein the wild-type human PD-1 is a full-length wild-type human PD-1 polypeptide or a PD-1 polypeptide as shown in SEQ ID NO:2; and the human PD-L1 is a full-length wild-type human PD-L1 polypeptide or a PD-L1 polypeptide as shown in SEQ ID NO:1.

[0125] In some embodiments, the microproteins described in any of the preceding embodiments, wherein the wild-type human PD-1 is the full-length wild-type human PD-1 polypeptide, and the human PD-L1 is the full-length wild-type human PD-L1 polypeptide.

[0126] In some embodiments, the microprotein described in any of the preceding embodiments has a protein backbone fold of the human PD-L1 binding portion of the PD-1 polypeptide as shown in SEQ ID NO:2.

[0127] In some embodiments, the microprotein described in any of the preceding embodiments, wherein the affinity of the microprotein for binding to human PD-L1 is more than 1 times higher than the affinity of wild-type human PD-1 for binding to human PD-L1 (e.g., greater than or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60 or more times).

[0128] In some embodiments, the microprotein described in any of the preceding embodiments, wherein the affinity of the microprotein for binding to the full-length wild-type human PD-L1 polypeptide is more than 1 times higher than the affinity of the full-length wild-type human PD-1 polypeptide for binding to the full-length wild-type human PD-L1 polypeptide (e.g., greater than or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60 or more).

[0129] In some embodiments, the microprotein described in any of the preceding embodiments has an affinity for binding to the full-length wild-type human PD-L1 polypeptide that is more than 4 times higher than the affinity between the full-length wild-type human PD-1 polypeptide and the full-length wild-type human PD-L1 polypeptide.

[0130] In some embodiments, the microprotein described in any of the preceding embodiments has an affinity for binding to the full-length wild-type human PD-L1 polypeptide that is more than 8 times higher than the affinity between the full-length wild-type human PD-1 polypeptide and the full-length wild-type human PD-L1 polypeptide.

[0131] In some embodiments, the microprotein described in any of the preceding embodiments has an affinity for binding to the full-length wild-type human PD-L1 polypeptide that is more than 30 times higher than the affinity between the full-length wild-type human PD-1 polypeptide and the full-length wild-type human PD-L1 polypeptide.

[0132] In some embodiments, the microprotein described in any of the preceding embodiments has an affinity for binding to the full-length wild-type human PD-L1 polypeptide that is more than 60 times higher than the affinity between the full-length wild-type human PD-1 polypeptide and the full-length wild-type human PD-L1 polypeptide.

[0133] In some implementations, the affinity of the microprotein described in any of the preceding embodiments is detected by surface plasmon resonance technology.

[0134] In some implementations, the affinity of the microprotein described in any of the preceding embodiments is determined by detecting the KD value using an Octet-RH16 analyzer.

[0135] In some embodiments, the microprotein described in any of the preceding embodiments is capable of blocking the interaction between human PD-1 peptide and human PD-L1 peptide.

[0136] In some embodiments, the microprotein described in any of the preceding embodiments is capable of being expressed in IC50 at a concentration of less than 100 nM (e.g., less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM or less). 50 The interaction between the full-length wild-type human PD-1 peptide and the full-length wild-type human PD-L1 peptide was blocked.

[0137] In some embodiments, the microprotein described in any of the preceding embodiments is capable of being expressed at an IC50 concentration of less than 30 nM. 50 The interaction between the full-length wild-type human PD-1 peptide and the full-length wild-type human PD-L1 peptide was blocked.

[0138] In some embodiments, the microprotein described in any of the preceding embodiments is capable of being expressed at an IC50 concentration of less than 10 nM. 50 The interaction between the full-length wild-type human PD-1 peptide and the full-length wild-type human PD-L1 peptide was blocked.

[0139] In some embodiments, the IC50 value of any of the preceding microproteins is detected by enzyme-linked immunosorbent assay (ELISA).

[0140] In some embodiments, the microprotein described in any of the preceding claims comprises an amino acid sequence having 80% or more (e.g., 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity with at least one sequence in SEQ ID NO:3-22.

[0141] In some embodiments, the microprotein described in any of the preceding claims comprises an amino acid sequence having 80% or more (e.g., 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity with SEQ ID NO:3.

[0142] In some embodiments, the microprotein described in any of the preceding claims comprises an amino acid sequence having 80% or more (e.g., 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity with SEQ ID NO:4.

[0143] In some embodiments, the microprotein described in any of the preceding claims comprises an amino acid sequence having 80% or more (e.g., 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity with SEQ ID NO:5.

[0144] In some embodiments, the microprotein described in any of the preceding embodiments comprises an amino acid sequence as shown in any of SEQ ID NO:3-22.

[0145] In some embodiments, the microprotein described in any of the preceding embodiments has an amino acid sequence as shown in any of SEQ ID NO:3-22.

[0146] In some embodiments, the microprotein described in any of the preceding embodiments has an amino acid sequence as shown in any of SEQ ID NO: 3, 4, 5, 7, 8, 11, 12, 15, 17, 19.

[0147] In some embodiments, the microprotein described in any of the preceding embodiments comprises an amino acid sequence as shown in SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

[0148] In some embodiments, the microprotein described in any of the preceding embodiments has 70-80 (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80) amino acid residues.

[0149] In some embodiments, the present invention provides a microprotein having the amino acid sequence shown in SEQ ID NO:3.

[0150] In some embodiments, the present invention provides a microprotein having the amino acid sequence shown in SEQ ID NO:4.

[0151] In some embodiments, the present invention provides a microprotein having the amino acid sequence shown in SEQ ID NO:5.

[0152] In some embodiments, the present invention provides a microprotein comprising the amino acid sequence shown in SEQ ID NO:23, the amino acid sequence of SEQ ID NO:23 being as follows:

[0153] X1X2X3X4X5X6X7X8X9X 10 YVX 11 GX 12 IX 13 LX 14 PX 15 IX 16 IX 17 ESX 18 RX 19 X 20 X 21 X 22 X 23 X 24 AX 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 X 42 X 43 X 44 X 45 X 46 HX 47 VX 48 HX 49 EX50 X 51 SGQLDTLX 52 AX 53 X 54 X 55 X 56 X 57 X 58 X 59 X 60 ,

[0154] in,

[0155] X1 is M, P, or empty;

[0156] X2 is E, A, M or blank;

[0157] X3 is I, L, P or empty;

[0158] X4 is P, A, D or blank;

[0159] X5 can be E, D, P, A, L, or be empty;

[0160] X6 can be E, D, P, I, or L;

[0161] X7 is either E or P;

[0162] X8 is G, D, P, or K;

[0163] X9 can be A, Y, P, G, or H;

[0164] X 10 For L, R, or T;

[0165] X 11 For V or I;

[0166] X 12 V, A, or I;

[0167] X 13 It can be C, T, V, S, I, or A;

[0168] X 14 For E or T;

[0169] X 15 It can be E or K;

[0170] X 16 For R, K, or M;

[0171] X 17 For L, E, F, V, or I;

[0172] X 18 For E or P;

[0173] X 19It can be R, K, H, Q, V, or A;

[0174] X 20 It can be P, A, E, or T;

[0175] X 21 For T or A;

[0176] X 22 The options are L, I, R, A, E, or blank;

[0177] X 23 It can be A or E;

[0178] X 24 It can be E or A;

[0179] X 25 For L, R, or P;

[0180] X 26 It can be E or A;

[0181] X 27 For Q, S, L, or A;

[0182] X 28 It can be E, G, L, V, or A;

[0183] X 29 It can be P, K, A, or D;

[0184] X 30 It can be A, P, E, K, or R;

[0185] X 31 For L, V, or K;

[0186] X 32 For I or L;

[0187] X 33 It can be A or E;

[0188] X 34 It can be E or K;

[0189] X 35 It can be A or L;

[0190] X 36 It can be K, A, R, or I;

[0191] X 37 It can be A, K, or E;

[0192] X 38 It can be Q, V, L, K, E, or Q;

[0193] X 39 The answer is G, A, S, or blank;

[0194] X40 G or empty;

[0195] X 41 It can be P, N, D, E, or a blank;

[0196] X 42 V, P, or vacancy;

[0197] X 43 It can be C, S, G, N, A, or Y;

[0198] X 44 It can be K, A, L, R, or S;

[0199] X 45 It can be K, V, E, or N;

[0200] X 46 It can be L, Y, F, or I;

[0201] X 47 V, L, or R;

[0202] X 48 V, I, or L;

[0203] X 49 For R, K, or L;

[0204] X 50 For S, L, K, or T;

[0205] X 51 For P or A;

[0206] X 52 The options are D, W, S, E, A, or Y;

[0207] X 53 For R, E, Q or blank;

[0208] X 54 It can be K, L, R or empty;

[0209] X 55 V, I, or blank;

[0210] X 56 V, E, L, P, or a blank;

[0211] X 57 V, I, L, E, or a blank;

[0212] X 58 P or blank;

[0213] X 59 For E, P, L, or a blank; and

[0214] X 60It can be L or empty.

[0215] In some embodiments, the present invention provides a microprotein having the amino acid sequence shown in SEQ ID NO:23.

[0216] In some embodiments, the microprotein has 70-80 (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80) amino acid residues.

[0217] In some implementations, X1 is left blank.

[0218] In some implementations, the X 40 X 41 and X 42 This is a vacancy.

[0219] In some implementations, the X 54 X 55 X 56 X 57 X 58 X 59 and X 60 This is a vacancy.

[0220] In some implementations, X7 is P, X 11 Let V, X 12 Let V, X 14 For E, X 18 For E, X 21 Let T, X 24 For E, X 25 Let L and X be the numbers. 32 For I, X 47 Let V, X 49 Let R be the value.

[0221] In some implementations, X4 is P, X7 is P, and X... 11 Let V, X 12 Let V, X 14 For E, X 18 For E, X 20 Let P, X 21 Let T, X 24 For E, X 25 Let L and X be the numbers. 26 For E, X 31 Let L and X be the numbers. 32 For I, X 44 Let K, X 47 Let V, X 49 Let R, X 50 Let it be S.

[0222] In some implementations, X1 is M or vacancy; X2 is E, A or vacancy; X3 is I, L or vacancy; X4 is P or A; X5 is E, D or P; X6 is E, D or I; X7 is P; X8 is G or D; X9 is A or Y; X 10 For L or R; X 11 For V; X 12 For V; X 13 For C, T, or V; X 14 For E; X 15 For E or K; X 16 For R or K; X 17 For L, E, or I; X 18 For E; X 19 For R, K, or V; X 20 For P or A; X 21 For T; X 22 For L, I, or E; X 23 It is A or E; X 24 For E; X 25 For L; X 26 For E or A; X 27 For Q, S, or L; X 28 For E, G, or V; X 29 For P or K; X 30 For A, P, or E; X 31 For L or V; X 32 For I; X 33 It is A or E; X 34 For E or K; X 35 For A; X 36 For K or A; X 37 For A, K, or E; X 38 For Q, V, or E; X 39 For G, S, or a blank; X 40 G or missing; X 41 E or blank; X 42 P or blank; X 43 For C, S, or N; X 44 For K or L; X 45 For K or V; X 46 For L or Y; X 47 For V; X 48 V, I, or L; X 49 R; X 50 S or L; X 51 For P or A; X 52 For D, W, or S; X 53 For R, E, or a blank; X 54 K or missing; X 55 V or blank; X 56 V or blank; X 57V or blank; X 58 P or blank; X 59 E or a blank; and X 60 It can be L or empty.

[0223] In some implementations, X1 is M or vacancy; X2 is E or A; X3 is I or L; X4 is P; X5 is E or D; X6 is E or D; X7 is P; X8 is G or D; X9 is A or Y; X 10 For L; X 11 For V; X 12 For V; X 13 For C, T; X 14 For E; X 15 For E; X 16 For R or K; X 17 For L, E; X 18 For E; X 19 For R, K; X 20 For P; X 21 For T; X 22 For L, I; X 23 It is A or E; X 24 For E; X 25 For L; X 26 For E; X 27 For Q, S; X 28 For E, G; X 29 For P or K; X 30 For A, P; X 31 For L or V; X 32 For I; X 33 It is A or E; X 34 For E or K; X 35 For A; X 36 For K or A; X 37 For A, K; X 38 For Q, V; X 39 G or missing; X 40 The blank is missing; X 41 The blank is missing; X 42 The blank is missing; X 43 For C, S; X 44 K; X 45 For K or V; X 46 For L or Y; X 47 For V; X 48 For V, I; X 49 R; X 50 S; X 51 For P or A; X 52 For D, W; X 53 For R, E; X 54 K or missing; X 55V or blank; X 56 V or blank; X 57 V or blank; X 58 P or blank; X 59 E or a blank; and X 60 It can be L or empty.

[0224] In some embodiments, the microprotein described in any of the preceding claims has an amino acid sequence identity of 80% or more (e.g., 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the sequence of SEQ ID NO:3, 4, 5, 7, 8, 11, 12, 15, 17 or 19.

[0225] In some embodiments, the microprotein described in any of the preceding claims has an amino acid sequence as shown in SEQ ID NO:3, 4, 5, 7, 8, 11, 12, 15, 17, or 19. In some embodiments, the microprotein described in any of the preceding claims has an amino acid sequence as shown in SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

[0226] In some embodiments, the microprotein described in any of the preceding embodiments comprises an amino acid sequence as shown in SEQ ID NO:3.

[0227] In some embodiments, the microprotein described in any of the preceding embodiments comprises an amino acid sequence as shown in SEQ ID NO:4.

[0228] In some embodiments, the microprotein described in any of the preceding embodiments comprises an amino acid sequence as shown in SEQ ID NO:5.

[0229] Secondly, in some embodiments, the present invention provides a fusion protein comprising the microprotein described in any of the preceding claims.

[0230] In some embodiments, the fusion protein described in any of the preceding embodiments comprises an antibody or a fragment.

[0231] In some embodiments, the fusion protein described in any of the preceding embodiments comprises an antibody Fc.

[0232] In some embodiments, the fusion protein described in any of the preceding embodiments, wherein the Fc is the Fc of human IgG1, IgG2, IgG3 or IgG4.

[0233] In some implementations, Fc is an Fc mutant that enhances effector cell function.

[0234] In some implementations, the Fc mutant exhibits enhanced ADCC / ADCP / CDC and / or affinity for the Fcγ receptor compared to wild-type Fc. For example, the Fc mutant can be obtained by amino acid substitutions at one or more of the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305. 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439, also see WO200042072 for description. Furthermore, binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII, and FcRn have been located, and variants with enhanced binding have been described (see Shieldsetal., 2001 J. Biol. Chem. 276: 6591-6604). Additionally, specific mutations at positions 256, 290, 298, 333, 334, and 339 enhance binding to FcγRIII, such as the T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A mutation combinations. In some embodiments, the Fc mutant is the DLE mutation (S239D / A330L / I332E), which enhances binding to FcγRIIIa and enhances the ADCC effect. The mutation sites of the aforementioned Fc mutants are numbered according to the EU index by Kabat et al.

[0235] In some embodiments, the fusion protein described in any of the preceding embodiments, wherein the amino acid sequence of the antibody Fc is as shown in SEQ ID NO:24.

[0236] In some embodiments, the fusion protein described in any of the preceding embodiments has the amino acid sequence shown in SEQ ID NO:25. In some embodiments, the microprotein or fusion protein described in any of the preceding embodiments has at least one of the following functions:

[0237] A) Capable of binding to human PD-L1 peptide; optionally, the microprotein or fusion protein can be expressed at a concentration of less than 15.00E-06M (e.g., less than 15.00E-06M, less than 10.00E-06M, less than 4.00E-06M, less than 1.00E-06M, less than 7.00E-07M, less than 5.00E-07M, less than 3.00E-07M, less than 1.00E-07M, ... The KD value (less than 9.00E-08M, less than 8.00E-08M, less than 7.00E-08M, less than 6.00E-08M, less than 1.00E-08M, less than 1.00E-08M, less than 5.00E-09M, less than 1.00E-09M, less than 1.00E-10M, or smaller) is combined with a human PD-L1 polypeptide (e.g., the full-length wild-type human PD-L1 polypeptide or the PD-L1 polypeptide as shown in SEQ ID NO:1); the KD value is detected by surface plasmon resonance; optionally, the KD value is detected by the method of the embodiments of the present invention;

[0238] B) capable of blocking the interaction between human PD-1 peptide and human PD-L1 peptide; optionally, the microprotein or fusion protein can be expressed at an IC50 concentration of less than 100 nM (e.g., less than 100 nM, less than 80 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 35 nM, less than 30 nM, less than 25 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 7 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM, less than 0.01 nM or less). 50 The IC blocks the interaction between human PD-1 peptides (e.g., full-length PD-1 or PD-1 peptides as shown in SEQ ID NO:2) and human PD-L1 peptides (e.g., wild-type full-length human PD-L1 peptides or PD-L1 peptides as shown in SEQ ID NO:1); 50 The KD value is detected by enzyme-linked immunosorbent assay (ELISA); optionally, the KD value is detected by the method of the embodiments of the present invention.

[0239] C) Capable of binding to cells overexpressing PD-L1; optionally, the microprotein or fusion protein is capable of binding at EC50 concentrations of less than 20 nM (e.g., less than 20 nM, less than 15 nM, less than 10 nM, less than 7 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM, less than 0.01 nM or less). 50 The value binds to CHO-K1 cells overexpressing PD-L1; the EC 50 The value was detected by flow cytometry fluorescence sorting; optionally, the EC 50 The value is detected by the method of the embodiments of the present invention;

[0240] D) Possesses good stability; optionally, the microprotein or fusion protein, after being placed in a 40°C water bath for 2 weeks, has a protein purity greater than 90% (e.g., greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or higher), and the protein purity is detected by the SEC method; in some embodiments, the microprotein or fusion protein has physical and / or chemical stability; in some embodiments, the physical and / or chemical stability of the microprotein or fusion protein is detected by PSR, BVP / OVA / DNA, HIC, SEC, AC-SINS, or DLS methods; optionally, the stability of the microprotein or fusion protein is detected by the methods of the embodiments of the present invention.

[0241] In some implementations, the microproteins or fusion proteins described in any of the preceding embodiments are capable of effectively inhibiting tumor growth.

[0242] Thirdly, in some embodiments, the present invention provides a conjugate comprising the microprotein or fusion protein described in any of the preceding claims, and a coupling portion.

[0243] In some implementations, the coupling portion is selected from protein tags, detectable markers, and therapeutic agents.

[0244] In some implementations, the conjugation portion is a cytotoxic drug, which is linked to the microprotein or fusion protein via a linker.

[0245] In some embodiments, the coupling portion is selected from protein tags. Such protein tags are well known in the art and include, but are not limited to, His, Flag, GST, MBP, HA, Myc, GFP, or biotin. Those skilled in the art know how to select appropriate protein tags (e.g., purification tags, detection tags, or tracer tags) according to the desired purpose. In some exemplary embodiments, a purification tag is attached to the C-terminus of the microprotein or fusion protein of the present invention.

[0246] In some embodiments, the coupling portion is selected from detectable markers, such as enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (e.g., chemiluminescent substances), or biotin. The detectable markers described in this invention can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Such markers are well known in the art. In some embodiments, such markers are suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescent immunoassay, etc.). In some embodiments, the detectable markers described above can be linked to the microproteins or fusion proteins of this invention via linkers of varying lengths to reduce potential steric hindrance.

[0247] In some embodiments, the coupling portion is selected from therapeutic agents, such as antitumor drugs.

[0248] In some embodiments, the coupling moiety is selected from other bioactive peptides.

[0249] In some embodiments, the conjugate comprises a microprotein or fusion protein as described above, and a coupling portion consisting of a cytotoxic drug, the coupling portion being linked to the microprotein or fusion protein via a linker.

[0250] In some implementations, the linker is a non-cleavable linker (e.g., SMCC) or a protease-cleavable linker.

[0251] In some implementations, the coupling portion is a radionuclide.

[0252] In some embodiments, the radionuclide is chelated with the microprotein or fusion protein by a chelating agent.

[0253] In some embodiments, the conjugate is obtained by chelating the radionuclide with the microprotein using a chelating agent.

[0254] In some embodiments, conjugate preparation mainly includes the steps of: a conjugation step, in which a chelating agent is conjugated with a microprotein; and a complex formation step, in which a complex of a radionuclide and a chelating agent is formed. The conjugation step can be performed before or after the complex formation step. Various chemical modification methods can be used in the conjugation step. For example, amine coupling (using a chelating agent or chelate having a carboxyl group activated with an N-hydroxysuccinimide ester group to modify the amino group of the lysine residue of the microprotein). The complex formation step only requires the ability to form a complex with the radionuclide; the order in which the radionuclide is added to the chelating agent is not limited. For example, a solution containing radionuclide ions dissolved in a water-based solvent can be used as the radionuclide. After complex formation, the resulting complex can be purified using a filter, membrane filter, column packed with various packing materials, chromatography, etc.

[0255] Fourthly, in some embodiments, the present invention provides a nucleic acid molecule that encodes the microprotein or fusion protein described in any of the preceding embodiments.

[0256] In one embodiment, the nucleic acid is contained in a nucleic acid construct, such as an expression construct. In the expression nucleic acid construct of the present invention, a sequence of polynucleotides encoding the microprotein or fusion protein is operatively linked to an expression control sequence to perform desired transcription and ultimately produce the microprotein or fusion protein in a host cell. Suitable expression control sequences include, but are not limited to, promoters, enhancers, ribosome-acting sites such as ribosome binding sites, polyadenylation sites, transcription splicing sequences, transcription termination sequences, and sequences stabilizing mRNA, etc.

[0257] Vectors used to construct the expression constructs of the present invention include those that replicate autonomously in host cells, such as plasmid vectors; and also include vectors capable of integrating into and replicating with the host cell DNA. Many commercially available vectors suitable for the present invention are readily available. In one embodiment, the plasmid is a plasmid suitable for prokaryotic or eukaryotic expression systems.

[0258] In some embodiments, the nucleic acid molecule encoding the protein according to the invention is codon-optimized for expression in mammalian cells (e.g., human cells). Methods of codon optimization are known in the art. A sequence is considered codon-optimized if at least one non-preferred codon is replaced by a more preferred codon compared to a wild-type sequence. Codon usage frequencies for a particular organism can be found in codon frequency tables, such as http: / / www.kazusa.or.jp / codon. In some embodiments, the preferred codon replacement results in higher expression.

[0259] Those skilled in the art will understand that, due to the degeneracy of the genetic code, many different polynucleotide and nucleic acid molecules can encode the same protein. Therefore, unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate to each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may or may not include introns.

[0260] Nucleic acid sequences can be generated using conventional molecular biology techniques or regenerated through DNA synthesis.

[0261] Fifthly, in some embodiments, the present invention provides a recombinant vector comprising the nucleic acid molecule described in any of the preceding claims.

[0262] In some embodiments, the vector is, but is not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain a replication initiation site. Those skilled in the art can select suitable expression vectors and functionally insert them into the nucleic acid sequences of the present invention.

[0263] Sixthly, in some embodiments, the present invention provides a host cell comprising the nucleic acid molecule or recombinant vector described in any of the preceding claims.

[0264] In some implementations, the host cell includes prokaryotes, yeast, and higher eukaryotic cells. Exemplary prokaryotic hosts include bacteria of the genera *Escherichia*, *Bacillus*, *Salmonella*, *Pseudomonas*, and *Streptomyces*. In a preferred embodiment, the host cell is an Escherichia coli cell, a mammalian cell (e.g., Chinese hamster ovary (CHO) cell, tumor cell line, BHK cell, human cell line (e.g., HEK293 cell, PER.C6 cell), or an insect cell. In a more preferred embodiment, the host cell is Escherichia coli, Bacillus subtilis, or Bacillus megaterium. In a specific embodiment of the invention, the host cell used is an Escherichia coli DH5α strain cell. The nucleic acid of the invention can be introduced into the host cell to express the encoded amino acid sequence by one of many well-known techniques, including but not limited to: heat shock conversion, electroporation, DEAE-glucan transfection, microinjection, liposome-mediated transfection, calcium phosphate precipitation, protoplasmic fusion, particle bombardment, viral transformation, and similar techniques.

[0265] In one embodiment, the nucleic acid can be integrated into the genome of a host cell, which can express or constitutively express the encoded microprotein or fusion protein under appropriate conditions.

[0266] In one implementation, the nucleic acid encoding the microprotein or fusion protein exists in the host cell in an extrachromosomal form (e.g., a plasmid or construct such as an expression vector).

[0267] In a seventh aspect, in some embodiments, the present invention provides a pharmaceutical composition comprising the microprotein, fusion protein, or conjugate described in any of the preceding claims, and one or more pharmaceutically acceptable carriers.

[0268] In some embodiments, non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, physiological saline, etc. Those skilled in the art will understand that other drug carriers can be used in this invention. If desired, the pharmaceutical composition may be contained in a kit, vial, or dispenser, which may, for example, contain one or more unit doses of the microproteins, fusion proteins, nucleic acids, or host cells or conjugates described in this invention. The kit, vial, or dispenser may be accompanied by instructions for use.

[0269] In some embodiments, the pharmaceutical composition may also contain additional therapeutic agents, such as drugs for treating tumors.

[0270] Eighthly, in some embodiments, the present invention provides the use of the microproteins, fusion proteins, conjugates, nucleic acid molecules, recombinant vectors, host cells, or pharmaceutical compositions described in any of the preceding claims in the preparation of a medicament for treating tumors;

[0271] In some implementations, the tumor is a tumor that highly expresses PD-L1;

[0272] In some implementations, the tumor is selected from melanoma, liver cancer, lung cancer, breast cancer, renal cell carcinoma, ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, colorectal cancer, nasopharyngeal carcinoma, cervical cancer, head and neck tumors, and hematologic malignancies.

[0273] On the other hand, in some embodiments, the present invention provides a method for treating tumors, the method comprising administering to a subject in need a therapeutically effective amount of any of the preceding microproteins, fusion proteins, conjugates, nucleic acid molecules, recombinant vectors, host cells, or pharmaceutical compositions;

[0274] In some implementations, the tumor is a tumor that highly expresses PD-L1;

[0275] In some implementations, the tumor is selected from melanoma, liver cancer, lung cancer, breast cancer, renal cell carcinoma, ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, colorectal cancer, nasopharyngeal carcinoma, cervical cancer, head and neck tumors, and hematologic malignancies.

[0276] In some implementations, the subject is a mammal, such as a human.

[0277] On the other hand, in some embodiments, the present invention provides microproteins, fusion proteins, conjugates, nucleic acid molecules, recombinant vectors, host cells, or pharmaceutical compositions described in any of the preceding embodiments for use as medicaments; in some embodiments, the medicaments are used to treat tumors;

[0278] In some implementations, the tumor is a tumor that highly expresses PD-L1;

[0279] In some implementations, the tumor is selected from melanoma, liver cancer, lung cancer, breast cancer, renal cell carcinoma, ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, colorectal cancer, nasopharyngeal carcinoma, cervical cancer, head and neck tumors, and hematologic malignancies.

[0280] The microproteins, fusion proteins, conjugates, nucleic acid molecules, recombinant vectors, host cells, or pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field.

[0281] In some embodiments, the dosage form is an injection. The injection may be a sterile injectable solution. For example, a sterile injectable solution may be prepared by incorporating an appropriate dose of the microprotein, fusion protein, or conjugate of the present invention into a suitable solvent, and optionally, by incorporating other desired components, followed by sterilization by filtration.

[0282] The microproteins, fusion proteins, conjugates, nucleic acid molecules, recombinant vectors, host cells, or pharmaceutical compositions of the present invention can be administered by any suitable method known in the art, including but not limited to parenteral administration. Those skilled in the art will understand that the route and / or manner of administration will vary depending on the intended purpose. In some embodiments, the microproteins, fusion proteins, conjugates, nucleic acid molecules, recombinant vectors, host cells, or pharmaceutical compositions of the present invention are administered by intravenous injection.

[0283] Furthermore, those skilled in the art will foresee that any of the preceding technical solutions can be freely combined with one or more other technical solutions of the present invention to form new technical solutions (without mutual exclusion), all of which are within the scope of protection claimed by the present invention. Detailed Implementation

[0284] The present invention is further illustrated by the following embodiments, but any embodiment or combination thereof should not be construed as limiting the scope or implementation of the invention. The scope of the invention is defined by the appended claims, and those skilled in the art can clearly understand the scope defined by the claims in conjunction with this specification and common knowledge in the art. Without departing from the spirit and scope of the invention, those skilled in the art can make any modifications or changes to the technical solutions of the invention, and such modifications and changes are also included within the scope of the invention. Unless otherwise specified, the methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used can be readily obtained from commercial companies.

[0285] Example 1. Obtaining microproteins that specifically bind to PD-L1

[0286] Based on the target sequence structure information (PDBid: 4ZQK), the protein backbone of the PD-1 peptide (SEQ ID NO:2) that binds to the human PD-L1 peptide (SEQ ID NO:1) was analyzed. Using artificial intelligence (AI) methods, several miniproteins that can specifically bind to PD-L1 were designed.

[0287] Human PD-L1 polypeptide fragment (SEQ ID NO:1):

[0288] Human PD-1 polypeptide fragment (SEQ ID NO:2):

[0289] Using yeast display technology, AI-designed PD-L1-binding sequences were cloned into the yeast display vector pYD1 to construct a miniprotein yeast display library. The obtained miniprotein library was then subjected to magnetic bead sorting. First, it was incubated with 100 nM biotinylated antigen PD-L1 (ACRO, PD1-H82F3) for 1 hour, followed by magnetic bead sorting (Invitrogen Cat: 11206D). Then, in the second round, different concentrations of biotinylated antigen were incubated at room temperature for 30 minutes (min refers to minutes) to wash away unbound antigen. Next, 100-fold non-biotinylated antigen was added for overnight competition, followed by flow cytometry enrichment and sorting. In each round of sorting, 0.1–0.5% of the clones from the library were collected for culture before the next round of sorting. After multiple rounds of sorting, several unique miniprotein sequences were finally obtained (sequences are shown in Table 1).

[0290] Table 1. Miniprotein sequences obtained from yeast display screening

[0291] Example 2. Expression and purification of a fusion protein that specifically binds to PD-L1 microprotein

[0292] The pcDNA3.4 vector was used as the expression vector. The C-terminus of the miniprotein obtained in Example 1 was directly linked to the N-terminus of human IgG1Fc (SEQ ID NO:24) to construct a Miniprotein-Fc fusion protein. Nucleotide sequences of various fusion proteins were synthesized. The vector was double-digested with HindIII and XhoI, recovered, and then seamlessly cloned using DNA homologous recombinase. The clones were then transformed into *E. coli* competent cells DH5α. Positive clones were selected, and plasmids were extracted and sequenced for verification. Plasmids were then extracted and transfected. One day before transfection, *Expi293F* cells were cultured in serum-free Expi293F expression medium (Gibco). On the day of transfection, the cell density was adjusted to 3 × 102 cells / mL using fresh Expi293F expression medium. 6The concentration of each cell was determined by mixing 1 μg of plasmid DNA and 3.2 μL of LExpi Fectamine (Gibco) with 116 μL of Opti-MEMI medium (Thermo Fisher) per mL of culture medium, and then adding this mixture to Expi293F cells. 18-22 h post-transfection (h refers to "hour"), 6 μL of Enhancer1 and 60 μL of Enhancer2 feed (Gbico) were added, and the cells were cultured for 5-7 days. The supernatant was then collected for further purification. Purification was performed on an AKTApure protein purifier. The first step of purification was performed using a MabSelect PrismA affinity purification column, and the purity of the purified protein was assessed by SEC-HPLC. The second step of purification was performed using a HiLoad 16 / 600 Superdex 200 pg molecular sieve purification column. The final purified product was used... (IMPLEN) Protein concentration was determined by absorbance at 280 nm, and protein purity was assessed by SDS-PAGE and SEC-HPLC. Endotoxin levels were detected using a recombinant factor C endotoxin assay kit (Adamaslife). SDS-PAGE and SEC-HPLC analysis confirmed the correct molecular weight of the fusion protein; endotoxin levels were confirmed to be below 1 EU mg⁻¹, meeting the standards for activity and physicochemical analysis. A schematic diagram of the Miniprotein-Fc fusion protein is shown in Figure 1, and protein purification yield and purity are shown in Table 2.

[0293] Table 2. Miniprotein-Fc protein expression yield and purity

[0294] Note: In Table 2, for example, “L04-Fc” is a fusion protein constructed by directly linking the C-terminus of the microprotein L01 that specifically binds to PD-L1 with the N-terminus of human IgG1Fc (SEQ ID NO:24), and so on.

[0295] The amino acid sequence of human IgG1Fc is as follows:

[0296] For example, the amino acid sequence of the fusion protein L04-Fc is as follows:

[0297] Example 3. Detection of the binding ability of miniprotein to PD-L1

[0298] The KD value of the protein containing the miniprotein of this invention binding to PD-L1 was detected using an Octet-RH16 molecular interaction analyzer. First, the Miniprotein-Fc fusion protein was captured using a biosensor coated with protein A. Then, its binding to PD-L1 (ACRO, PD1-H52H3) at concentrations of 50 nM and 200 nM was detected sequentially. The test solution was 0.1% PBST, with equilibration for 60 s (s represents "second"), loading for 180 s, washing for 120 s, a binding run for 120 s, and a dissociation run for 300 s.

[0299] Some experimental results are shown in Table 3. The experimental results show that the Miniprotein-Fc constructed in this invention can specifically bind to human PD-L1. In particular, L04-Fc / L08-Fc / L09-Fc have relatively high affinity, and their KD values ​​are close to the nM level.

[0300] Table 3. Detection of binding affinity between Miniprotein-Fc fusion protein and PD-L1

[0301] Example 4. Miniprotein binding experiment with PD-L1 overexpressing cells

[0302] The binding ability of the protein containing the miniprotein of the present invention to CHO-K1 cells (China National Cell Bank, 3101HAMGNHa7) and CHO-K1 cells overexpressing PD-L1 (CHO-K1 PD-L1, Nanjing Kebai Biotechnology, CBP74032) was determined using the FACS method.

[0303] Experimental procedure: After centrifugation and washing, the cells were adjusted to a density of 1×10⁻⁶. 6 Cells / mL. Transfer cells to 96-well V-type cell culture plates, adding 100 μL of cell suspension to each well and centrifuging. Add Miniprotein-Fc fusion protein diluted with FACS buffer (1% FBS, PBS) (starting concentration 200 nM, 5-fold serial dilutions), and incubate at 4°C for one hour. After washing, add 100 μL of diluted... The conjugated goat anti-human IgG-Fc antibody (Jackson Immuno Research Inc.) was incubated at 4°C for 30 minutes; after washing, it was detected by flow cytometry (IntelligentiQue3, Sartorius). The dose-response data and fluorescence signals were fitted by a four-parameter logistic model using GraphPadPrism. (The control molecule KN035-Fc is a fusion protein constructed by directly linking the C-terminus of a single variable domain to the end of Fc. The variable region sequence of KN035-Fc is shown in the variable region of hu56v1 in US11225522B2 patent. The amino acid sequence of Fc is shown in SEQ ID NO:24. The Anti-HEL IgG1 sequence is shown in J Mol Biol. 1992 Mar 20; 224(2):487-99).

[0304] Some experimental results are shown in Figures 2 and 3, and Table 4. The experimental results show that the microprotein of the present invention can specifically bind to PD-L1 overexpressing cells.

[0305] Table 4. Results of the binding experiment between Miniprotein-Fc fusion protein and CHO-K1, PD-L1 and CHO-K1 cells.

[0306] NA: Not available indicates that the software cannot fit the data.

[0307] In addition, the binding ability of L04-His (a miniprotein with eight consecutive histidine residues linked to the C-terminus of L04) to CHO-K1 cells overexpressing PD-L1 (CHO-K1 PD-L1, Nanjing Kebai Biotechnology, CBP74032) was determined using the FACS method. After centrifugation and washing, 1e5 cells per well were transferred to 96-well V-type cell culture plates, and 100 nM and 1 μM of L04-His fusion protein diluted with FACS buffer (1% FBS, PBS) were added. The plates were incubated at 4°C for one hour; after washing, 100 μL of diluted... The conjugated anti-His tag antibody (Anti-His AF647, GenScript, A01802) was incubated at 4°C for 30 minutes; after washing, it was detected using flow cytometry (IntelligentiQue3, Sartorius). The dose-response data and fluorescence signals were fitted using a four-parameter logistic model via GraphPadPrism.

[0308] Some experimental results are shown in Figure 4. The experimental results show that the microprotein of the present invention can specifically bind to PD-L1 overexpressing cells.

[0309] Example 5. Detection of the ability of miniprotein to block the interaction between PD-1 and PD-L1

[0310] The effect of proteins containing the miniprotein of this invention on blocking the interaction between PD-1 and PD-L1 was determined.

[0311] 100 μL of 1 μg / mL human PD-1 (ACRO, PD1-H5257) protein was used to coat a 96-well Costa ELISA plate (Corning, 9018) overnight. The next day, the plate was washed three times with washing buffer (PBS containing 0.05% Tween-20), and then 250 μL of blocking buffer (5% skim milk powder, 0.05% Tween-20, PBS) was added. The plate was blocked at room temperature for one hour. After washing, 100 μL of a mixture containing serially diluted Miniprotein-Fc fusion protein (control KN035-Fc as described in Example 4) and 50 nM biotinylated human PD-L1 (ACRO, PD1-H82E5) was added, and the plate was incubated at room temperature for one hour. After washing five times, 100 μL of horseradish peroxidase (H2O) was added. Streptomycin (Invitrogen, Cat#:434323) labeled with RP was incubated at room temperature for 1 hour. After washing the plate 5 times, 100 μL TMB single-component chromogenic solution (Solarbio, Cat#:PR1200) was added, and the plate was incubated in the dark for 10 minutes. Then, 100 μL LELISA stop solution (Solarbio, Cat#:C1058) was added to terminate the reaction. The absorbance (OD) value was read at 450 nm using a Multiskan FC microplate reader (Thermo Scientific). The dose-response data and absorbance data were fitted using a four-parameter logistic model using GraphPadPrism.

[0312] Some experimental results are shown in Figure 5 and Table 5. The experimental results show that the miniprotein of the present invention can effectively block the interaction between human PD-1 and human PD-L1 at low concentrations, especially the L04-Fc protein, which has the strongest activity and an IC50 of 6.8 nM.

[0313] Table 5. Experimental results of Miniprotein-Fc fusion protein blocking PD-1 / PD-L1 interaction.

[0314] Example 6. Detection of protein physicochemical properties

[0315] The physicochemical properties of proteins containing the miniprotein of this invention were determined to assess their exploitability. The assay methods are well known in the art (see JARASCHA, et al. Journal of Pharmaceutical Sciences, 2015, 104(6):1885-1898., ZURDO J. Pharmaceutical Bioprocessing, 2013, 1(1):29-50.).

[0316] DLS. Centrifuge the sample (≥6000g, 10-30 min) to remove any large aggregates or precipitates that may be present in the sample solution. Then transfer 25 μL of sample to a 384-well plate (3540; Corning) and cover with a membrane, using a scraper to ensure the membrane adheres tightly to the plate. Centrifuge (2000g, ≥5 min) to ensure no air bubbles remain in the wells. Open the sample chamber of the laser particle size analyzer (DynaproPlateReaderIII; Wyatt), place the 384-well plate correctly, and close the sample chamber. In the Dynamics software, set the parameters: select Fixed Temperature (25℃) for Experimenttype, Select No for Static Light Scattering, select the sample wells to be measured, and enter {well}-{sample}-{value:temp} in Label Measurement. Keep all other parameters at their default values ​​and collect data. Analyze the particle size (Radius), percentage of polydispersity (%PD), and the content of each component based on scattered light intensity (%Intensity).

[0317] Tagg. Sample preparation follows the same procedure described in the DLS section above. In Dynamics software, set the parameters: Click Experiment Designer > Edit Experiment, select "Continuous Temperature" for Experimenttype, select "No" for Static Light Scattering Measurement, then select the corresponding sample well. In Tag Measurement, enter {well}-{sample}-{value:temp}, change the DLS acquisition time (s) to 3, the number of DLS acquisitions to 3, the starting temperature to 35℃, and the ending temperature to 85℃. Select sealing; keep all other parameters at their default values. After data acquisition, analyze the data using Dynamics software and report the aggregation temperature value (Taggvalue).

[0318] Tm. The melting temperature of a protein was calculated by detecting changes in the fluorescent dye signal during protein heating using a quantitative real-time PCR instrument. Experimental method: 58.5 μL of sample (1.0 mg / mL) and 1.5 μL of 200× SYPROorange solution (Sigma-Aldrich / S5692, specification: 5000×, diluted to 200× with sample buffer before sample preparation) were mixed thoroughly. 25 μL of the mixture was then analyzed using the instrument, in duplicate. Experimental parameters: Real-time quantitative PCR instrument (Quant Studio6 Pro; Thermo Fisherscientific); detection temperature: 25℃ to 99℃, heating rate: 0.05℃ / s. Analysis was performed using Protein Thermal Shift software (Thermo Fisherscientific).

[0319] BVP-ELISA. This method is used to assess antibody nonspecificity. Experimental method: Add 10 μL of rod-shaped virus particle stock solution (prepared in our laboratory) diluted with 90 μL of 50 mM, pH 9.6 sodium carbonate solution (V900096-500G; Sigma-Aldrich) to each well of a 96-well plate (40303; Beave), and incubate overnight at 4°C. The next day, after blocking, add 100 μL of 0.015 mg / mL test sample PBS solution, incubate for 1 hour, and then wash six times with 250 μL of PBST. Add 100 μL of 5000-fold diluted goat anti-human IgG antibody, HRP-conjugated (A0170; Sigma-Aldrich), to each well, incubate for 1 hour, and wash six times with PBST. Add 90 μL of TMB chromogenic solution (PR1200; Solarbio) to each well, and incubate for 2–5 minutes. Finally, 40 μL of 1M hydrochloric acid was added to terminate the reaction. The absorbance at 450 nm of each well was read using a microplate reader (SpectraMax190; Moleculardevices), and the BVPScore was calculated by the ratio of the absorbance of the sample wells to that of the blank control wells.

[0320] The OVA-ELISA method is used to assess antibody nonspecificity. In brief, 100 μL of OVA (0.1 mg / mL dissolved in PBS, A5503-5G; Sigma) is coated onto a 96-well plate (40303; Beave) and incubated overnight at 4°C. The remaining steps follow the same procedure as described in the BVP-ELISA above.

[0321] The DNA-ELISA method is used to assess antibody nonspecificity. In short, DNA (10 μg / mL dissolved in PBS, D1626; Sigma-Aldrich) is coated onto 96-well plates (40303; Beave), 100 μL per well, and incubated overnight at 4°C. The remaining steps follow the same procedure as described in the BVP-ELISA section above.

[0322] AC-SINS experimental method: Goat anti-IgG antibody (109-005-098; Jackson Immuno Research) and goat non-specific antibody (005-000-003; Jackson Immuno Research) were diluted to 0.4 mg / mL using 20 mM potassium acetate (pH 4.3). The antibodies were mixed at an 8:2 ratio to prepare a coating solution. The coating solution and gold nanoparticles (15705; Ted Pella Inc.) were then thoroughly mixed at a 1:9 ratio and incubated at room temperature for 1 hour. Polyethylene glycol monomethyl ether thiol (10 μM / L dissolved in water, 729140; Sigma-Aldrich) was added according to the volume of the gold nanoparticle-antibody complex to achieve a final concentration of 0.1 μM / L, and the mixture was incubated at room temperature for 1 hour. A syringe and a needle-type 0.22 μm PVDF filter membrane (SLGVX) were used. 13 The solution obtained in the previous step was filtered using NK (Millipore). The gold nanoparticle antibody complex on the filter membrane was eluted with 1 / 10 volume of PBS and collected. 50 μL of 0.05 mg / mL sample was added to a 384-well plate (781091; GreinerBio-One), followed by 5 μL of the concentrated gold nanoparticle antibody complex. After thorough repositioning, the plate was incubated at room temperature for 2 hours. Centrifugation (3000g, 3 minutes) was performed before detection. Absorbance was collected from 510 nm to 570 nm using a microplate reader (SPARK; Tecan) (collected every 2 nm). After detection, the data were imported into Excel software for analysis, and the difference between the maximum absorption peak of the sample and the blank was calculated.

[0323] Hydrophobic interaction chromatography (HIC) method: High performance liquid chromatography (Agilent, HPLC1260) and Butyl-NPR HPLC column were used. TOSOH), mobile phase A (1.5M ammonium sulfate and 50mM dipotassium hydrogen phosphate, pH 7.0) and mobile phase B (50mM dipotassium hydrogen phosphate, pH 7.0) were used for linear gradient elution over 20 minutes at a flow rate of 0.5 mL / min (0-100% mobile phase B). The detection wavelength was 280 nm, and the column temperature was 25 °C. The protein injection volume was 20 μg. The retention time of the peak with the largest reported area percentage is recorded.

[0324] The whole-column imaging capillary isoelectric focusing electrophoresis (iCIEF) was used to separate protein variants based on their isoelectric point (pI) characteristics. The isoelectric point of each protein variant was determined and the relative percentage content was calculated.

[0325] Sample preparation for instrumentation: First, prepare a premix solution with the following composition: 0.5 μL each of pI marker 4.65 (102223; Protein Simple) and pI marker 9.91 (C19100042; Guanyu), 4.0 μL of Pharmalyte 3-10 (17-0456-01; GE Healthcare), 35 μL of 1% methylcellulose solution (101876; Protein Simple), 37.5 μL of 8M urea, and 2.5 μL of ultrapure water. Replace the sample solution with ultrapure water to achieve a final sample concentration of 1.0 mg / mL. Mix 20 μL of the replaced sample with 80 μL of the premix solution, centrifuge at 13000 rpm for 1 minute, and then transfer 95 μL of the supernatant to a 96-well plate and centrifuge at 3000 rpm for at least 5 minutes.

[0326] Capillary isoelectric focusing electrophoresis analysis parameters and data processing: Instrument information: Fully automated protein characterization system (Maurice; Protein Simple), sample tray temperature: 10℃, sample injection time: 60 seconds. Sample focusing voltage and time: First stage: 1500V for 1 minute, second stage: 3000V for 5.5 minutes. After sample injection, the spectra are imported into Empower software for analysis.

[0327] Capillary electrophoresis (CE) purity analysis is divided into reducing and non-reducing methods. Non-reducing CE-SDS uses NEM (N-ethylmaleimide) to protect possible free disulfide bonds in proteins. Reducing CE-SDS uses BME (β-mercaptoethanol) to open the disulfide bonds in proteins and then measures the purity of the reduced protein. Experimental Methods: Transfer 50 μg of protein from ultrapure water to a 1.5 mL centrifuge tube, add at least 50 μL of LDS sample buffer, add ultrapure water to a final volume of 100 μL, then add 5 μL of NEM (non-reducing) or 5 μL of NEM (reducing), mix thoroughly, and centrifuge at 10000 g for 1 minute. Prepare blank and reference samples using the same method. Incubate all samples on a constant temperature metal bath at 70 °C for 10 minutes, then remove each sample and allow it to cool to room temperature for at least 3 minutes. Centrifuge the samples at 10000 g for 1 minute at room temperature. Before analysis, transfer the samples to a 96-well plate, centrifuge at 3000 g for 10 minutes, and then load them into the centrifuge. Instrument Information and Experimental Parameters: Automated Protein Characterization System (Maurice; Protein Simple sample preparation, sample tray temperature: 15℃, sample injection time: 4600V, 20 seconds. Sample separation voltage and time: 5750V, ≥35 minutes (non-reduction) or ≥25 minutes (reduction). After sample injection, import the spectrum into Empower software for analysis.

[0328] Size-exclusion chromatography (SEC) separates components in a mixture based on the different molecular volumes of the components and the different times required for the stationary phase to separate them. Protein purity is determined based on the retention time of different molecular volumes. Experimental method: An ultra-high performance liquid chromatograph (ACQUITY™ PREMIER; Waters) and a Waters ACQUITY UPLC Protein BEHSEC column (186005225; Waters) were used. The mobile phase (50 mM phosphate, 300 mM sodium chloride, pH 6.8) was used for isocratic elution at a flow rate of 0.4 mL / min over 7 minutes. The detection wavelength was 280 nm, and the column temperature was 25 °C. The protein injection volume was 10 μg. The peak area percentages of monomers, high molecular weight components, and low molecular weight components in each sample were recorded and reported.

[0329] Some experimental results are shown in Table 6. The experimental results show that the Miniprotein-Fc constructed by the present invention has good physical / chemical properties and is suitable for drug development.

[0330] Table 6. Physicochemical evaluation of Miniprotein-Fc fusion protein

[0331] Example 7. Protein thermal stability test

[0332] The thermal stability of proteins containing the miniproteins of this invention was determined using SDS-CE-NR, iCIEF, and SEC methods.

[0333] 1 mg / mL Miniprotein-Fc fusion proteins (e.g., L04-Fc, L08-Fc, and L18-Fc) were subjected to accelerated stability testing at 40 °C. Samples were taken at Day 0, 1 week, and 2 weeks, and SDS-CE-NR, iCIEF, and SEC tests were performed to detect changes in the physicochemical properties of the fusion proteins.

[0334] Some experimental results are shown in Tables 7, 8, and 9. In the iCIEF test, samples L04-Fc, L08-Fc, and L18-Fc, after being placed at 40℃ for 2 weeks, all showed a decrease in the main peak content of <11%. In the SDS-CE-NR experiment, the main peak content decreased with increasing incubation time, but even after 2 weeks, the decrease was less than 6%. In the SEC test, after 2 weeks of placement at 40℃, the main peak content remained above 90%, and the proportion of fragment peaks remained essentially unchanged, demonstrating good stability.

[0335] Table 7. Accelerated Stability Test of Miniprotein-Fc Fusion Protein - iCIEF

[0336] Table 8. Accelerated Stability Test of Miniprotein-Fc Fusion Protein - SDS-CE-NR

[0337] Table 9. Accelerated Stability Test of Miniprotein-Fc Fusion Protein - SEC

[0338] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A microprotein that specifically binds to human PD-L1, and which binds to human PD-L1 with a higher affinity than wild-type human PD-1; Optionally, the wild-type human PD-1 is the full-length wild-type human PD-1 polypeptide or the PD-1 polypeptide as shown in SEQ ID NO:2; the human PD-L1 is the full-length wild-type human PD-L1 polypeptide or the PD-L1 polypeptide as shown in SEQ ID NO:1; Optionally, the microprotein has a protein backbone fold of the human PD-L1 binding portion of the PD-1 polypeptide as shown in SEQ ID NO:2; Optionally, the microprotein comprises an amino acid sequence having more than 80% sequence identity with at least one sequence in SEQ ID NO:3-22.

2. The microprotein according to claim 1, wherein, The affinity of the microprotein to human PD-L1 is more than twice that of wild-type human PD-1 to human PD-L1. Optionally, the affinity of the microprotein to the full-length wild-type human PD-L1 peptide is more than 4 times higher than the affinity of the full-length wild-type human PD-1 peptide to the full-length wild-type human PD-L1 peptide. Optionally, the affinity of the microprotein to the full-length wild-type human PD-L1 peptide is more than 8 times higher than the affinity of the full-length wild-type human PD-1 peptide to the full-length wild-type human PD-L1 peptide. Optionally, the affinity of the microprotein to the full-length wild-type human PD-L1 peptide is more than 30 times higher than the affinity of the full-length wild-type human PD-1 peptide to the full-length wild-type human PD-L1 peptide. Optionally, the affinity of the microprotein to the full-length wild-type human PD-L1 peptide is more than 60 times higher than the affinity of the full-length wild-type human PD-1 peptide to the full-length wild-type human PD-L1 peptide. Optionally, the affinity is detected by surface plasmon resonance technology; Optionally, the affinity is determined by detecting the KD value using an Octet-RH16 analyzer.

3. The microprotein according to claim 1 or 2, wherein, The microprotein can block the interaction between human PD-1 peptide and human PD-L1 peptide; Optionally, the microprotein can be expressed at an IC50 concentration of less than 100 nM. 50 The interaction between the full-length wild-type human PD-1 peptide and the full-length wild-type human PD-L1 peptide was blocked. Optionally, the microprotein can be expressed at an IC50 concentration of less than 30 nM. 50 The interaction between the full-length wild-type human PD-1 peptide and the full-length wild-type human PD-L1 peptide was blocked. Optionally, the microprotein can be expressed at an IC50 concentration of less than 10 nM. 50 The interaction between the full-length wild-type human PD-1 peptide and the full-length wild-type human PD-L1 peptide was blocked. Optionally, the IC50 value is detected by enzyme-linked immunosorbent assay (ELISA).

4. The microprotein according to any one of claims 1 to 3, wherein, The amino acid sequence of the microprotein has more than 90% sequence identity with at least one sequence in SEQ ID NO:3-22; Optionally, the microprotein comprises an amino acid sequence as shown in any of SEQ ID NO:3-22; Optionally, the amino acid sequence of the microprotein is as shown in any of SEQ ID NO:3-22; Optionally, the amino acid sequence of the microprotein is shown in SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:

5.

5. A microprotein comprising the amino acid sequence shown in SEQ ID NO:23: X1X2X3X4X5X6X7X8X9X 10 YVX 11 GX 12 IX 13 LX 14 PX 15 IX 16 IX 17 ESX 18 RX 19 X 20 X 21 X 22 X 23 X 24 AX 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 X 42 X 43 X 44 X 45 X 46 HX 47 VX 48 HX 49 EX 50 X 51 SGQLDTLX 52 AX 53 X 54 X 55 X 56 X 57 X 58 X 59 X 60 , in, X1 is M, P, or empty; X2 is E, A, M or blank; X3 is I, L, P or empty; X4 is P, A, D or blank; X5 can be E, D, P, A, L, or be empty; X6 can be E, D, P, I, or L; X7 is either E or P; X8 is G, D, P, or K; X9 can be A, Y, P, G, or H; X 10 For L, R, or T; X 11 For V or I; X 12 V, A, or I; X 13 It can be C, T, V, S, I, or A; X 14 For E or T; X 15 It can be E or K; X 16 For R, K, or M; X 17 For L, E, F, V, or I; X 18 For E or P; X 19 It can be R, K, H, Q, V, or A; X 20 It can be P, A, E, or T; X 21 For T or A; X 22 The options are L, I, R, A, E, or blank; X 23 It can be A or E; X 24 It can be E or A; X 25 For L, R, or P; X 26 It can be E or A; X 27 For Q, S, L, or A; X 28 It can be E, G, L, V, or A; X 29 It can be P, K, A, or D; X 30 It can be A, P, E, K, or R; X 31 For L, V, or K; X 32 For I or L; X 33 It can be A or E; X 34 It can be E or K; X 35 It can be A or L; X 36 It can be K, A, R, or I; X 37 It can be A, K, or E; X 38 It can be Q, V, L, K, E, or Q; X 39 The answer is G, A, S, or blank; X 40 G or empty; X 41 It can be P, N, D, E, or a blank; X 42 V, P, or vacancy; X 43 It can be C, S, G, N, A, or Y; X 44 It can be K, A, L, R, or S; X 45 It can be K, V, E, or N; X 46 It can be L, Y, F, or I; X 47 V, L, or R; X 48 V, I, or L; X 49 For R, K, or L; X 50 For S, L, K, or T; X 51 For P or A; X 52 The options are D, W, S, E, A, or Y; X 53 For R, E, Q or blank; X 54 It can be K, L, R or empty; X 55 V, I, or blank; X 56 V, E, L, P, or a blank; X 57 V, I, L, E, or a blank; X 58 P or blank; X 59 E, P, L, or blank; and X 60 For L or blank; Optionally, the amino acid sequence of the microprotein is shown in SEQ ID NO:23; Optionally, the microprotein has 70 to 80 amino acid residues; Optionally, the X 54 X 55 X 56 X 57 X 58 X 59 X 60 The position is vacant. Optionally, X7 is P, X 11 Let V, X 12 Let V, X 14 For E, X 18 For E, X 21 Let T, X 24 For E, X 25 Let L and X be the numbers. 32 For I, X 47 Let V, X 49 R; Optionally, X4 is P, X7 is P, and X... 11 Let V, X 12 Let V, X 14 For E, X 18 For E, X 20 Let P, X 21 Let T, X 24 For E, X 25 Let L and X be the numbers. 26 For E, X 31 Let L and X be the numbers. 32 For I, X 44 Let K, X 47 Let V, X 49 Let R, X 50 S; Optionally, X1 is M or a vacancy; X2 is E, A or a vacancy; X3 is I, L or a vacancy; X4 is P or A; X5 is E, D or P; X6 is E, D or I; X7 is P; X8 is G or D; X9 is A or Y; X 10 For L or R; X 11 For V; X 12 For V; X 13 For C, T, or V; X 14 For E; X 15 For E or K; X 16 For R or K; X 17 For L, E, or I; X 18 For E; X 19 For R, K, or V; X 20 For P or A; X 21 For T; X 22 For L, I, or E; X 23 It is A or E; X 24 For E; X 25 For L; X 26 For E or A; X 27 For Q, S, or L; X 28 For E, G, or V; X 29 For P or K; X 30 For A, P, or E; X 31 For L or V; X 32 For I; X 33 It is A or E; X 34 For E or K; X 35 For A; X 36 For K or A; X 37 For A, K, or E; X 38 For Q, V, or E; X 39 For G, S, or a blank; X 40 G or missing; X 41 E or blank; X 42 P or blank; X 43 For C, S, or N; X 44 For K or L; X 45 For K or V; X 46 For L or Y; X 47 For V; X 48 V, I, or L; X 49 R; X 50 S or L; X 51 For P or A; X 52 For D, W, or S; X 53 For R, E, or a blank; X 54 K or missing; X 55 V or blank; X 56 V or blank; X 57 V or blank; X 58 P or blank; X 59 E or a blank; and X 60 For L or blank; Optionally, X1 is M or vacancy; X2 is E or A; X3 is I or L; X4 is P; X5 is E or D; X6 is E or D; X7 is P; X8 is G or D; X9 is A or Y; X 10 For L; X 11 For V; X 12 For V; X 13 For C, T; X 14 For E; X 15 For E; X 16 For R or K; X 17 For L, E; X 18 For E; X 19 For R, K; X 20 For P; X 21 For T; X 22 For L, I; X 23 It is A or E; X 24 For E; X 25 For L; X 26 For E; X 27 For Q, S; X 28 For E, G; X 29 For P or K; X 30 For A, P; X 31 For L or V; X 32 For I; X 33 It is A or E; X 34 For E or K; X 35 For A; X 36 For K or A; X 37 For A, K; X 38 For Q, V; X 39 G or missing; X 40 The blank is left; X 41 The blank is left; X 42 The blank is left; X 43 For C, S; X 44 K; X 45 For K or V; X 46 For L or Y; X 47 For V; X 48 For V, I; X 49 R; X 50 S; X 51 For P or A; X 52 For D, W; X 53 For R, E; X 54 K or missing; X 55 V or blank; X 56 V or blank; X 57 V or blank; X 58 P or blank; X 59 E or a blank; and X 60 For L or blank; Optionally, the amino acid sequence of the microprotein has more than 80% sequence identity with SEQ ID NO:3, 4, 5, 7, 8, 11, 12, 15, 17 or 19; Optionally, the amino acid sequence of the microprotein is as shown in SEQ ID NO:3, 4, 5, 7, 8, 11, 12, 15, 17 or 19; Optionally, the amino acid sequence of the microprotein is shown in SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:

5.

6. A fusion protein comprising the microprotein of any one of claims 1 to 5; Optionally, the fusion protein comprises an antibody or an antibody fragment; Optionally, the fusion protein comprises an antibody Fc; Optionally, the Fc is the Fc of human IgG1, IgG2, IgG3 or IgG4; Optionally, the amino acid sequence of the antibody Fc is shown in SEQ ID NO:24; Optionally, the amino acid sequence of the fusion protein is shown in SEQ ID NO:

25.

7. The microprotein according to any one of claims 1 to 5 or the fusion protein according to claim 6, wherein it has at least one of the following functions: A) Capable of binding human PD-L1 peptide; Optionally, the microprotein or fusion protein is capable of binding the full-length human PD-L1 peptide with a KD value of less than 15.00E-06M; The KD value is detected by surface plasmon resonance. B) Capable of blocking the interaction between human PD-1 peptide and human PD-L1 peptide; optionally, the microprotein or fusion protein can be injected at an IC50 concentration of less than 100 nM. 50 The IC value blocks the interaction between the full-length wild-type human PD-1 peptide and the full-length human PD-L1 peptide; 50 The value was detected by enzyme-linked immunosorbent assay (ELISA). C) Cells that can bind to PD-L1 overexpression; Optionally, the microprotein or fusion protein can be expressed at an EC50 concentration of less than 20 nM. 50 The value binds to CHO-K1 cells overexpressing PD-L1, the EC 50 The values ​​were detected by flow cytometry fluorescence sorting. D) It has good stability; Optionally, the microprotein or fusion protein is placed in a 40°C water bath for 2 weeks, and the protein purity is greater than 90%, wherein the protein purity is detected by the SEC method.

8. A conjugate comprising the microprotein of any one of claims 1 to 7 or the fusion protein of claim 6 or 7, and a coupling moiety; Optionally, the coupling portion is selected from protein tags, detectable markers, or therapeutic agents; Optionally, the conjugation portion is a cytotoxic drug, and the conjugation portion is linked to the microprotein or fusion protein via a linker.

9. The conjugate according to claim 8, wherein the coupling portion is a radionuclide; Optionally, the radionuclide is chelated with the microprotein or fusion protein by a chelating agent.

10. A nucleic acid molecule encoding the microprotein of any one of claims 1 to 5, 7 or the fusion protein of claim 6 or 7.

11. A recombinant vector comprising the nucleic acid molecule of claim 10.

12. A host cell comprising the nucleic acid molecule of claim 10, or comprising the recombinant vector of claim 11.

13. A pharmaceutical composition comprising the microprotein of any one of claims 1 to 7, the fusion protein of claim 6 or 7, the conjugate of claim 8 or 9, the isolated nucleic acid molecule of claim 10, the carrier of claim 11 or the host cell of claim 12, and one or more pharmaceutically acceptable carriers.

14. Use of the microprotein of any one of claims 1 to 7, the fusion protein of claim 6 or 7, the conjugate of claim 8 or 9, the isolated nucleic acid molecule of claim 10, the carrier of claim 11, the host cell of claim 12, or the pharmaceutical composition of claim 13 in the preparation of a drug for treating tumors; Optionally, the tumor is a tumor that highly expresses PD-L1; Optionally, the tumor is selected from melanoma, liver cancer, lung cancer, breast cancer, renal cell carcinoma, ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, colorectal cancer, nasopharyngeal carcinoma, cervical cancer, head and neck tumors, and hematologic malignancies.

15. A method of treating a tumor, the method comprising administering to a subject in need a therapeutically effective amount of any one of claims 1 to 7, a fusion protein of claim 6 or 7, a conjugate of claim 8 or 9, an isolated nucleic acid molecule of claim 10, a carrier of claim 11, a host cell of claim 12, or a pharmaceutical composition of claim 13; Optionally, the tumor is a tumor that highly expresses PD-L1; Optionally, the tumor is selected from melanoma, liver cancer, lung cancer, breast cancer, renal cell carcinoma, ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, colorectal cancer, nasopharyngeal carcinoma, cervical cancer, head and neck tumors, and hematologic malignancies.

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