Protein with self-assembly polymerization performance and preparation method thereof

Abstract

The invention provides a protein with self-assembly polymerization performance and a preparation method thereof. In particular, the present invention provides individual polypeptides constituting a trimeric protein, nucleic acid sequences encoding the polypeptides, and methods of forming the trimeric protein.

Description

[0001] This application is a divisional application of the invention patent application with the application date of September 29, 2024, application number 202411374094.9, and invention name “A self-assembling trimeric protein and its preparation method”. Technical Field

[0002] The present invention relates to the field of biomedicine, and in particular to a protein with self-assembly and aggregation properties and a preparation method thereof. Background Art

[0003] Targeted antibodies / proteins offer advantages such as high specificity, minimal side effects, and a long half-life, making them a highly promising biotherapeutic approach. Targeted protein drugs have gradually become an important clinical treatment. However, due to the complexity and multifactorial nature of disease development and progression, relying solely on single-target antibodies to achieve optimal therapeutic outcomes is difficult. Currently, targeted antibodies / proteins primarily utilize a bivalent structure formed by dimers based on the CH3 domain of the antibody Fc. The vast majority of multivalent antibodies / fusion proteins are engineered based on the CH3 dimer structure. This limits the design and application of multivalent antibodies / fusion proteins with more complex structures.

[0004] Therefore, there is a need in the art to develop a self-assembling multimeric protein. Summary of the Invention

[0005] The purpose of the present invention is to provide a self-assembling multimeric protein.

[0006] In a first aspect of the present invention, a self-assembling small protein is provided, wherein the small protein comprises an amino acid sequence selected from the group consisting of:

[0007] (1) the amino acid sequence shown in SEQ ID NO: 1, 3 or 5;

[0008] (2) An amino acid sequence that has a homology of ≥90% (preferably ≥95%, more preferably ≥98%) with the amino acid sequence shown in SEQ ID NO: 1, 3 or 5, and that can spontaneously form a trimeric protein.

[0009] In the second aspect of the present invention, a fusion protein is provided, wherein the fusion protein comprises the self-assembling miniprotein as described in the first aspect of the present invention and one or more functional polypeptides.

[0010] In another preferred embodiment, the fusion protein has a structure as shown in any one of Formula I to Formula III from N-terminus to C-terminus,

[0011] M-L1-Fx (Formula I)

[0012] Fx-L1-M (Formula II)

[0013] Fx-L1-M-L2-Fx (Formula III)

[0014] in,

[0015] M is the self-assembling miniprotein as described in the first aspect of the present invention;

[0016] L1 and L2 are each independently none or a linker;

[0017] Fx is x functional polypeptides;

[0018] “-” represents a peptide bond, connecting peptide or linker connecting the above elements;

[0019] wherein x is an integer selected from 1, 2, 3 or 4.

[0020] In another preferred embodiment, the functional polypeptide is a targeting polypeptide.

[0021] In another preferred embodiment, the functional polypeptide is selected from the following group: an antibody, a ligand, a receptor, or an active fragment thereof, or a combination thereof.

[0022] In another preferred embodiment, the functional polypeptide is selected from the group consisting of an antigen-binding fragment (Fab), a single-chain antibody (scFv), a single-domain antibody (sdAb), an extracellular domain of a receptor protein, a ligand, or a combination thereof.

[0023] In another preferred embodiment, the functional polypeptide is a PD-L1 binding small protein.

[0024] In another preferred embodiment, the amino acid sequence of the functional polypeptide is shown in SEQ ID NO:11.

[0025] In another preferred embodiment, the connector is a flexible connector.

[0026] In another preferred embodiment, the amino acid sequence of the linker is as shown in (G4S)n, wherein n is an integer selected from 1-6.

[0027] In another preferred embodiment, the amino acid sequence of the linker is shown in SEQ ID NO: 12.

[0028] In another preferred embodiment, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 7 or 9.

[0029] In the third aspect of the present invention, a multimeric protein is provided, wherein the multimeric protein comprises a plurality of protein monomers, wherein the protein monomers are selected from: the self-assembling small protein as described in the first aspect of the present invention, the fusion protein as described in the second aspect of the present invention, or a combination thereof.

[0030] In another preferred embodiment, the multimeric protein is a trimeric protein, which comprises or consists of three protein monomers.

[0031] In another preferred embodiment, the multimeric protein is a homopolymer or a heteropolymer (such as a homotrimer or a heterotrimer).

[0032] In another preferred embodiment, the multimeric protein is formed by the combination of the self-assembling small proteins.

[0033] In another preferred embodiment, the plurality of protein monomers contain self-assembled small proteins with the same or different amino acid sequences.

[0034] In another preferred embodiment, the plurality of protein monomers contain self-assembling small proteins with the same amino acid sequence.

[0035] In another preferred embodiment, the plurality of protein monomers contain the same or different functional polypeptides.

[0036] In another preferred embodiment, the functional polypeptide is a targeting polypeptide, and the targeting polypeptides contained in the multiple protein monomers specifically bind to different targets respectively.

[0037] In the fourth aspect of the present invention, a polynucleotide is provided, which encodes the self-assembling miniprotein as described in the first aspect of the present invention, or the fusion protein as described in the second aspect of the present invention.

[0038] In another preferred embodiment, the sequence of the polynucleotide is shown in SEQ ID NO: 2, 4, 6, 8 or 10.

[0039] In the fifth aspect of the present invention, a vector is provided, wherein the vector contains the polynucleotide according to the fourth aspect of the present invention.

[0040] In the sixth aspect of the present invention, a host cell is provided, wherein the host cell contains the vector as described in the fifth aspect of the present invention, or the polynucleotide as described in the fourth aspect of the present invention is integrated into the genome.

[0041] In a seventh aspect of the present invention, a drug conjugate is provided, wherein the drug conjugate comprises:

[0042] (a) the self-assembling miniprotein according to the first aspect of the present invention, the fusion protein according to the second aspect of the present invention, or the multimeric protein according to the third aspect of the present invention; and

[0043] (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0044] In another preferred embodiment, the coupling moiety is a drug or a toxin.

[0045] In another preferred embodiment, the coupling moiety is a detectable label.

[0046] In another preferred embodiment, the conjugate is selected from the following group: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agents.

[0047] In an eighth aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0048] (a) the fusion protein according to the second aspect of the present invention, the multimeric protein according to the third aspect of the present invention, the polynucleotide according to the fourth aspect of the present invention, the vector according to the fifth aspect of the present invention, the host cell according to the sixth aspect of the present invention, or the drug conjugate according to the seventh aspect of the present invention; and

[0049] (b) a pharmaceutically acceptable carrier.

[0050] In another preferred embodiment, the pharmaceutical composition is used for immunotherapy.

[0051] In another preferred embodiment, the content of component (a) is 0.1-99.9 wt%, preferably 10-99.9 wt%, and more preferably 70%-99.9 wt%.

[0052] In another preferred embodiment, the pharmaceutical composition is in the form of an oral dosage form, an injection, or an external use pharmaceutical dosage form.

[0053] In another preferred embodiment, the dosage form of the pharmaceutical composition includes tablets, granules, capsules, oral solutions, or injections.

[0054] In another preferred embodiment, the pharmaceutical composition or preparation is selected from the following group: a suspension preparation, a liquid preparation or a lyophilized preparation.

[0055] In another preferred embodiment, the liquid preparation is an aqueous injection preparation.

[0056] In another preferred embodiment, the pharmaceutically acceptable carrier includes: a surfactant, a solution stabilizer, an isotonicity regulator, a buffer, or a combination thereof.

[0057] In another preferred embodiment, the subject of administration of the pharmaceutical composition or preparation includes humans or non-human animals.

[0058] In another preferred embodiment, the non-human animals include: rodents (such as rats and mice) and primates (such as monkeys).

[0059] In another preferred embodiment, in the administration of the pharmaceutical composition or preparation, the administered amount is 0.01-10 g / day, preferably 0.05-5000 mg / day, and more preferably 0.1-3000 mg / day.

[0060] In another preferred embodiment, the pharmaceutical composition or preparation is used to inhibit and / or treat tumors.

[0061] In another preferred embodiment, for the treatment of tumors, the pharmaceutical composition or preparation can be administered in combination with other anti-tumor drugs.

[0062] In a ninth aspect of the present invention, there is provided a method for preparing the multimeric protein according to the third aspect of the present invention, comprising the steps of:

[0063] A plurality of self-assembling miniproteins according to the first aspect of the present invention and / or a plurality of fusion proteins according to the second aspect of the present invention are brought into contact with each other to obtain the trimeric protein.

[0064] In the tenth aspect of the present invention, there is provided a method for preparing the self-assembling miniprotein according to the first aspect of the present invention, or the fusion protein according to the second aspect of the present invention, or the multimeric protein according to the third aspect of the present invention, comprising the steps of:

[0065] (a) culturing the host cell according to the sixth aspect of the present invention under suitable conditions to obtain a culture containing the self-assembling miniprotein, fusion protein or trimeric protein; and

[0066] (b) purifying and / or separating the culture obtained in step (a) to obtain the self-assembling small protein, fusion protein or trimeric protein.

[0067] In the eleventh aspect of the present invention, provided is the use of the fusion protein as described in the second aspect of the present invention, the multimeric protein as described in the third aspect of the present invention, the polynucleotide as described in the fourth aspect of the present invention, the vector as described in the fifth aspect of the present invention, the host cell as described in the sixth aspect of the present invention, or the drug conjugate as described in the seventh aspect of the present invention in the preparation of a drug for treating a disease.

[0068] In another preferred embodiment, the disease is a disease with high expression of PD-L1.

[0069] In another preferred embodiment, the tumor is a tumor that expresses PD-L1 protein (i.e., PD-L1 positive).

[0070] In another preferred embodiment, the tumor includes but is not limited to: acute myeloid leukemia, chronic myeloid leukemia, multiple myelopathy, non-Hodgkin's lymphoma, colorectal cancer, breast cancer, colon cancer, gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, prostate cancer, cervical cancer, lymphoma, adrenal tumor, bladder tumor, or a combination thereof.

[0071] In the twelfth aspect of the present invention, a method for treating a disease is provided, comprising the steps of administering to a subject in need thereof the fusion protein as described in the second aspect of the present invention, the multimeric protein as described in the third aspect of the present invention, or the drug conjugate as described in the seventh aspect of the present invention.

[0072] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] The following drawings are used to illustrate specific embodiments of the present invention and are not used to limit the scope of the present invention defined by the claims.

[0074] Figure 1 The schematic diagram of the spatial structure of the trimeric protein is shown, where:

[0075] A is a schematic diagram of the spatial structure of the C3-50-130-11 trimer protein;

[0076] B is a schematic diagram of the spatial structure of the C3-50-130-14 trimer protein;

[0077] C is a schematic diagram of the spatial structure of the C3-50-130-16 trimer protein;

[0078] D is a schematic diagram of the spatial structure of the C3-50-130-18 trimer protein.

[0079] Figure 2 Schematic diagrams showing several structural combinations of trimeric proteins are shown, including:

[0080] A is an antibody Fab, a single-chain antibody (scFv), a receptor protein extramembrane region or a ligand, an antibody hinge region (hinge) or linker, and a trimeric protein single chain connected in series to form a polypeptide chain, and a specific trimer combination is formed with the help of the trimeric protein single chain (or fragment) provided by the present invention;

[0081] B is a polypeptide chain formed by a trimeric protein single chain and an antibody hinge region (hinge) or linker, an antibody Fab, a single-chain antibody (scFv), a receptor protein extramembrane region, or a ligand in series, and a specific trimeric combination is formed by means of the trimeric protein single chain (or fragment) provided by the present invention;

[0082] C is an antibody Fab, a single-chain antibody (scFv), a receptor protein extramembrane region, or a ligand connected to an antibody Fab, a single-chain antibody (scFv), a receptor protein extramembrane region, or a ligand through a linker, and then connected in series with an antibody hinge region (hinge) or a linker and a trimer protein single chain to form a polypeptide chain, with the help of the trimer protein single chain (or fragment) provided by the present invention to form a specific combination;

[0083] D is a trimeric protein single chain and an antibody hinge region (hinge) or linker, as well as an antibody Fab, a single-chain antibody (scFv), a receptor protein extramembrane region or a ligand, which are connected in series with the antibody Fab, a single-chain antibody (scFv), a receptor protein extramembrane region or a ligand through a linker to form a polypeptide chain, and a specific combination is formed with the help of the trimeric protein single chain (or fragment) provided by the present invention;

[0084] E is an antibody Fab, a single-chain antibody (scFv), a receptor protein extramembrane region or a ligand, an antibody hinge region (hinge) or linker, a trimeric protein single chain, an antibody hinge region (hinge) or linker, an antibody Fab, a single-chain antibody (scFv), a receptor protein extramembrane region or a ligand connected in series to form a polypeptide chain, and a specific combination is formed with the help of the trimeric protein single chain (or fragment) provided by the present invention.

[0085] Figure 3 is the molecular screening test result of trimer protein, where:

[0086] A is the molecular weight determination result of the standard sample using the gel filtration calibration kit;

[0087] B is the molecular sieve test result of C3-50-130-11 trimer protein;

[0088] C is the molecular sieve test result of C3-50-130-14 trimer protein;

[0089] D is the molecular sieve test result of C3-50-130-16 trimer protein;

[0090] E is the molecular sieve test result of C3-50-130-18 trimer protein.

[0091] Figure 4 is the thermal stability test result of the trimeric protein structure, where:

[0092] A is the Tm test result of the trimer protein;

[0093] B is the Tagg detection result of trimeric protein.

[0094] Figure 5 is the thermal recovery test result of the trimeric protein structure, where:

[0095] A is the conformational thermal recovery test result of the trimeric protein;

[0096] B is the aggregation thermal recovery test result of the trimeric protein.

[0097] Figure 6 Binding activity assay of a self-assembling trimeric protein targeting PD-L1 is shown. DETAILED DESCRIPTION

[0098] After extensive and in-depth research, the inventors have developed, for the first time, a protein with self-assembling properties and a method for its preparation. The trimeric protein of the present invention comprises three polypeptides of identical sequence, which interact to promote the spontaneous assembly of the three polypeptides into a trimer. Furthermore, binding proteins can be linked to the N-terminus and / or C-terminus of the trimeric protein to form a multivalent targeting protein. This has led to the completion of the present invention.

[0099] the term

[0100] In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings generally understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can change. It should also be understood that the terms used herein are intended only to describe specific embodiments and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0101] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0102] As used herein, the terms "comprise," "include," and "contain" are used interchangeably to include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of," "consisting essentially of."

[0103] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reaction), ie, has a reasonable benefit / risk ratio.

[0104] As used herein, the term "therapeutically effective amount" refers to an amount that produces a function or activity in humans and / or animals and is tolerated by humans and / or animals. Those skilled in the art will appreciate that the "therapeutically effective amount" may vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and combination therapy with other drugs.

[0105] Self-assembling small protein and fusion protein of the present invention

[0106] The present invention provides a self-assembling small protein and a fusion protein or a multimer thereof comprising the small protein.

[0107] As used herein, the terms "miniprotein of the present invention" and "self-assembling miniprotein of the present invention" are used interchangeably to refer to miniproteins capable of self-assembly to form multimers (e.g., trimers) as described in the first aspect of the present invention. The miniprotein of the present invention is composed of a single peptide chain that primarily forms three α-helical secondary structures.

[0108] Preferably, the miniprotein of the present invention has an amino acid sequence as shown in SEQ ID NO: 1, 3 or 5.

[0109] As used herein, the term "fusion protein of the present invention" refers to a fusion protein formed by the self-assembling miniprotein of the present invention and another fusion element. For example, the fusion element can be a functional polypeptide, such as a specific targeting polypeptide. In some embodiments, the fusion element can be an antibody, a ligand, a receptor, or an active fragment thereof, or a combination thereof.

[0110] As used herein, the term "multimer" refers to a molecule formed by the combination of multiple small proteins or fusion proteins of the present invention. The term "homomultimer" refers to a molecule formed by the combination of multiple identical small proteins or fusion proteins. The term "heteromultimer" refers to a molecule formed by the combination of multiple different small proteins or fusion proteins. Multiple monomers in the multimer of the present invention form a multimer through self-assembly of small protein regions. In one embodiment, the multimer of the present invention is a heteromultimer, which contains multiple fusion proteins containing different functional polypeptides. For example, the multimer of the present invention can include multiple fusion proteins, each of which has a functional polypeptide targeting different targets, thereby achieving multi-target therapeutic purposes.

[0111] Typically, the fusion protein of the present invention has a structure as shown in any one of Formula I to Formula III from N-terminus to C-terminus,

[0112] M-L1-Fx (Formula I)

[0113] Fx-L1-M (Formula II)

[0114] Fx-L1-M-L2-Fx (Formula III)

[0115] in,

[0116] M is the self-assembling small protein according to claim 1;

[0117] L1 and L2 are each independently none or a linker;

[0118] Fx is x functional polypeptides;

[0119] “-” represents a peptide bond, connecting peptide or linker connecting the above elements;

[0120] wherein x is an integer selected from 1, 2, 3 or 4.

[0121] Typically, the multimer of the present invention is a trimeric protein, and its structural type can be one of the following structures:

[0122] Contains three functional polypeptide-trimeric polypeptide single chains;

[0123] Contains a trimer polypeptide single chain - three functional polypeptides;

[0124] It contains three functional polypeptide-functional polypeptide-trimeric polypeptide single chains;

[0125] It contains a trimer polypeptide single chain-three functional polypeptides-functional polypeptides;

[0126] Contains three functional polypeptides - trimer polypeptide single chain - three functional polypeptides,

[0127] Among them, the trimer polypeptide single chain is the self-assembling small protein of the present invention.

[0128] In another preferred embodiment, the functional polypeptide is selected from the following group: Fab, scFv, fusion receptor or ligand.

[0129] It should be understood that the above structural types are only examples and do not limit the present invention. Some representative structures are as follows Figure 2 The functional polypeptide connected to the self-assembling miniprotein can be single or multiple (such as 2, 3 or 4 functional polypeptides in series).

[0130] As used herein, the term "self-assembling miniprotein" or "fusion protein" also includes variant forms with self-assembly activity. These variant forms include (but are not limited to): deletion, insertion and / or substitution of 1-3 (usually 1-2, more preferably 1) amino acids, addition or deletion of one or several (usually within 3, preferably within 2, more preferably within 1) amino acids at the C-terminus and / or N-terminus, or addition of amino acid fragments with smaller amino acid side chains as linkers (such as glycine, serine, etc.) at the N-terminus or C-terminus of the miniprotein. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is generally not changed. For another example, the addition or deletion of one or several amino acids at the C-terminus and / or N-terminus generally does not change the structure and function of the protein. The term also includes linear and non-linear polypeptides (such as cyclic peptides).

[0131] The present invention also includes active fragments, derivatives and analogs of the above-mentioned self-assembling miniproteins or fusion proteins (especially fusion proteins formed with specific targeting peptides). As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the function or activity of the self-assembling miniprotein or fusion protein of the present invention.

[0132] The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a polypeptide with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (fusion proteins formed by fusion with a leader sequence, secretory sequence or tag sequence such as 6His). Based on the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0133] A preferred class of active derivatives refers to polypeptides in which no more than 5, preferably no more than 3, and more preferably no more than 1 amino acid is replaced by an amino acid with similar or similar properties compared to the amino acid sequence of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0134] Table A

[0135] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile;Leu;Met;Phe;Ala Leu

[0136] The present invention also provides analogs of the self-assembling miniproteins or fusion proteins of the present invention. These analogs may differ from the polypeptides of the present invention in terms of amino acid sequence, in terms of modifications that do not affect the sequence, or in terms of both. Analogs also include analogs having residues other than natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., β- and γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0137] In addition, the self-assembling miniproteins or fusion proteins of the present invention can also be modified. Modifications (usually without changing the primary structure) include: chemical derivatization of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during polypeptide synthesis and processing or in further processing steps. This modification can be achieved by exposing the polypeptide to a glycosylation enzyme (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, and phosphothreonine). Also included are polypeptides that have been modified to improve their resistance to proteolysis or optimize their solubility.

[0138] The term "polynucleotide of the present invention" may include a polynucleotide encoding the self-assembling miniprotein or fusion protein of the present invention, or may also include additional coding and / or non-coding sequences.

[0139] The present invention also relates to variants of the aforementioned polynucleotides, which encode fragments, analogs, and derivatives of polypeptides or fusion proteins having the same amino acid sequence as the present invention. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide, which may contain one or more nucleotide substitutions, deletions, or insertions that do not substantially alter the function of the self-assembling miniprotein or fusion protein it encodes.

[0140] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides of the present invention under stringent conditions (or stringent conditions). In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably at least 95%.

[0141] The self-assembling miniproteins or fusion proteins and polynucleotides of the present invention are preferably provided in an isolated form, and more preferably, purified to homogeneity.

[0142] The full-length sequences of the polynucleotides of the present invention can generally be obtained by PCR amplification, recombinant methods, or synthetic methods. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. For long sequences, two or more PCR amplifications are often required, followed by splicing the fragments amplified in the correct order.

[0143] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0144] In addition, the method of artificial synthesis can also be used to synthesize the relevant sequence, especially when the fragment length is shorter. Usually, by synthesizing multiple small fragments first and then connecting them, a very long fragment of sequence can be obtained.

[0145] Currently, DNA sequences encoding proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0146] Methods using PCR techniques to amplify DNA / RNA are preferably used to obtain the polynucleotides of the present invention. In particular, when full-length cDNA is difficult to obtain from a library, the RACE method (RACE - rapid amplification of cDNA ends) is preferably used. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods, such as gel electrophoresis.

[0147] The present invention also provides a method for forming a trimer between self-assembling miniproteins or fusion proteins thereof, the method comprising contacting a plurality of the self-assembling miniproteins and / or fusion proteins to obtain the trimer protein. Interacting amino acid pairs are formed on the three interacting surfaces of the self-assembling miniproteins that comprise the trimer, thereby forming a specifically interacting trimer protein.

[0148] expression vector

[0149] The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the coding sequence of the self-assembling miniprotein or fusion protein of the present invention, and a method for producing the polypeptide of the present invention by recombinant technology.

[0150] The polynucleotide sequences of the present invention can be used to express or produce recombinant fusion proteins using conventional recombinant DNA techniques. Generally, the following steps are involved:

[0151] (1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding the fusion protein of the present invention, or a recombinant expression vector containing the polynucleotide;

[0152] (2) Host cells cultured in a suitable culture medium;

[0153] (3) Isolate and purify proteins from culture medium or cells.

[0154] In the present invention, the polynucleotide sequence encoding the fusion protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. Any plasmid or vector can be used as long as it can replicate and be stable in the host. An important feature of an expression vector is that it generally contains an origin of replication, a promoter, a marker gene, and translation control elements.

[0155] In the method for preparing the self-assembling small protein or its fusion protein of the present invention, any suitable vector can be used, which can be selected from pET, pDR1, pcDNA3.1(+), pcDNA3.1 / ZEO(+), pDHFR, and the expression vector includes a fusion DNA sequence connected to a suitable transcription and translation regulatory sequence.

[0156] Both eukaryotic and prokaryotic host cells can be used to express the self-assembling miniprotein or its fusion protein of the present invention. Eukaryotic host cells are preferably mammalian or insect host cell culture systems, preferably COS, CHO, NS0, sf9 and sf21 cells; prokaryotic host cells are preferably DH5a, BL21 (DE3), or TG1.

[0157] Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence encoding the fusion protein of the present invention and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. The DNA sequence can be operatively linked to an appropriate promoter within the expression vector to direct mRNA synthesis. Representative examples of such promoters include the lac or trp promoters of Escherichia coli; the lambda phage PL promoter; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoter, retroviral LTRs, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0158] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0159] A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell to enable it to express the protein.

[0160] Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, and plant cells (such as ginseng cells).

[0161] When the polynucleotides of the present invention are expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting DNA factors, typically about 10 to 300 base pairs in length, that act on promoters to increase gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs on the late replication origin side), the polyoma enhancer on the late replication origin side, and adenovirus enhancers.

[0162] Those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers and host cells.

[0163] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0164] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0165] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0166] Affinity chromatography can be used to separate and purify the self-assembling small protein or its fusion protein disclosed in the present invention. Depending on the characteristics of the affinity column used, conventional methods such as high salt buffer, changing pH, etc. can be used to elute the self-assembling small protein or its fusion protein bound to the affinity column.

[0167] By using the above method, the self-assembling small protein or its fusion protein can be purified into a substantially uniform substance, for example, a single protein peak (OD 280 or OD 210 ).

[0168] Pharmaceutical composition

[0169] The present invention also provides a pharmaceutical composition containing the self-assembling miniprotein, fusion protein, polymer or drug conjugate of the present invention.

[0170] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the self-assembling small protein, fusion protein or polymer (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used in conjunction with other therapeutic agents. The self-assembling small protein, fusion protein, polymer or drug conjugate can be combined with pharmaceutically acceptable excipients to form a pharmaceutical preparation so as to exert therapeutic effects more stably. These preparations can ensure the structural integrity of the amino acid core sequence of the self-assembling small protein, fusion protein or polymer of the present invention, while also protecting the multifunctional groups of the protein to prevent degradation (including but not limited to aggregation, deamination or oxidation). The preparation can be in various forms. Under normal circumstances, for liquid preparations, it can usually be stably stored at 2°C-8°C for at least one year, and for freeze-dried preparations, it can remain stable at 30°C for at least six months. Here, the preparation can be a suspension, water injection, freeze-dried preparation and other preparations commonly used in the pharmaceutical field, preferably a water injection or freeze-dried preparation.

[0171] For the pharmaceutical composition of the present invention (such as an aqueous injection or a lyophilized preparation), pharmaceutically acceptable excipients include one or a combination of surfactants, solution stabilizers, isotonicity regulators, and buffers. Surfactants include non-ionic surfactants such as polyoxyethylene sorbitan fatty acid esters (Tween 20 or 80); poloxamer (such as poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; tetradecyl, linoleyl, or octadecyl sarcosine; Pluronics; MONAQUAT™, etc., and the amount of the excipients added should minimize the tendency of the protein to granulate. The solution stabilizer can be a carbohydrate, including reducing sugars and non-reducing sugars, an amino acid including monosodium glutamate or histidine, and an alcohol including one or a combination of triols, higher sugar alcohols, propylene glycol, and polyethylene glycol. The amount of the solution stabilizer added should be such that the final preparation can maintain a stable state for a stable period of time as considered by those skilled in the art. The isotonicity regulator can be one of sodium chloride and mannitol, and the buffer can be one of TRIS, histidine buffer, and phosphate buffer.

[0172] When using a pharmaceutical composition, a safe and effective amount of the miniprotein or fusion protein of the present invention or its immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 50 μg / kg body weight, and in most cases does not exceed about 100 mg / kg body weight. Preferably, the dose is about 100 μg / kg body weight to about 50 mg / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician. Typically, the total dosage does not exceed a certain range, for example, the intravenous dose is 10 to 3000 mg / day / 50 kg.

[0173] The trimeric protein of the present invention and the pharmaceutical preparation containing the same can be used as an anti-tumor drug for tumor treatment. The anti-tumor drug referred to in the present invention refers to a drug that has the function of inhibiting and / or treating tumors, which can include delaying the development of symptoms associated with tumor growth and / or reducing the severity of these symptoms. It further includes alleviating symptoms associated with existing tumor growth and preventing the occurrence of other symptoms, and also reducing or preventing metastasis.

[0174] The above-mentioned trimeric protein and its pharmaceutical preparation can also be administered in combination with other anti-tumor drugs for the treatment of tumors. These anti-tumor drugs for combined administration include but are not limited to: 1. Cytotoxic drugs (1) Drugs that act on the chemical structure of DNA: alkylating agents such as nitrogen mustards, nitrosoureas, and methanesulfonates; platinum compounds such as cisplatin, carboplatin, and oxaliplatin; mitomycin C (MMC); (2) Drugs that affect nucleic acid synthesis: dihydrofolate reductase inhibitors such as methotrexate (MTX) and Alimta; thymidine synthesis inhibitors. Enzyme inhibitors such as fluorouracil (5FU, FT-207, capecitabine), purine nucleoside synthetase inhibitors such as 6-mercaptopurine (6-MP) and 6-TG, ribonucleotide reductase inhibitors such as hydroxyurea (HU), DNA polymerase inhibitors such as cytarabine (Ara-C) and Gemzar (Gemz), etc. (3) Drugs acting on nucleic acid transcription: drugs that selectively act on DNA templates, inhibit DNA-dependent RNA polymerase, and thus inhibit RNA synthesis, such as actinomycin D, daunorubicin, and doxorubicin. , epirubicin, aclarubicin, mithramycin, etc.; (4) Drugs that mainly act on microtubule protein synthesis: paclitaxel, taxotere, vinblastine, vinorelbine, podophyllotoxin, homoharringtonine; (5) Other cytotoxic drugs: asparaginase mainly inhibits protein synthesis; 2. Hormonal antiestrogens: tamoxifen, droloxifene, exemestane, etc.; aromatase inhibitors: aminoglutethimide, lantron, letrozole, arimide, etc.; anti-androgen: flutamide RH-LH agonists / antagonists: nivolumab, enaton, etc.; 3. 1. Phosphodiesterase inhibitors: mainly inhibit tumor interferon through the body's immune function; interleukin-2; thymosin peptides; 2. Monoclonal antibodies: MabThera; Cetuximab (C225); Herceptin (Trastuzumab); Bevacizumab (Avastin); Yervoy (Ipilimumab); 3. Others include some drugs whose mechanisms are currently unknown and require further research; cell differentiation inducers such as retinoids; apoptosis inducers.

[0175] The main advantages of the present invention include:

[0176] 1) The present invention provides a small protein that self-assembles to form a trimer, and a binding protein can be connected to the N-terminus and / or C-terminus of the trimeric protein to form a multivalent targeting protein;

[0177] 2) The self-assembling small protein of the present invention has strong thermal stability and conformational thermal recovery.

[0178] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0179] Example 1: Construction, expression and purification of self-assembling trimeric protein

[0180] The trimer protein nucleotide sequence was synthesized using gene synthesis and inserted into the pET29b vector in the format of MGS-trimer protein nucleotide sequence-GSENLYFQSLEHHHHHH (ENLYFQSLE sequence is used for enzyme digestion, HHHHHH is used for protein purification). This vector was transformed into Escherichia coli and cultured in LB medium at 37°C, 270 rpm to an OD600 of 0.6. Protein expression was then induced overnight with 1 mM IPTG. The bacteria were harvested by centrifugation and resuspended, then disrupted with a low-temperature mechanical disruptor and centrifuged at high speed to obtain the supernatant. After purification using a Ni column, the protein sample was desalted and concentrated, and the concentration of the purified protein was measured using a microspectrophotometer. The purified protein was sterilized by filtration using a 0.22 μm filter membrane and stored in a 4°C refrigerator for subsequent experiments.

[0181] Example 2: Evaluation of the assembly efficiency of self-assembling trimeric proteins

[0182] The protein purified by Ni column was further purified using AKTApure protein purification system. TM 75Increase 10 / 300GL gel filtration column, first replace 20% ethanol in the column with deionized water at a low flow rate of 0.45ml / min, then use 1×PBS buffer at a flow rate of 0.75ml / min to balance the system. After the pressure and conductivity values ​​are stable, take 500ul of purified protein for sample loading and correct the UV absorption 215 and UV absorption 280 to zero. Observe the changes in the UV absorption peak and collect the sample. Calculate the molecular weight standard curve using the gel filtration calibration kit (GelFiltration Calibration KitLMW) and analyze the OD 280 The peak elution volume parameters and peak area are used to evaluate the protein molecular weight and protein purity. This method can obtain high-purity candidate proteins for subsequent experiments. Figure 3 As shown, proteins of corresponding molecular weight were collected and set aside for use.

[0183] Example 3: Thermal stability test of self-assembled trimer structure of monomeric small protein

[0184] The thermal stability of the protein structure was tested with the help of Unchained Labs' all-purpose protein stability analyzer Uncle. The sample was heated in the range of 25°C-95°C at a rate of 0.5°C / min. Static light scattering was measured at an excitation wavelength of 266nm. As the protein unfolded, the fluorescence usually decreased and shifted to a longer wavelength. The data analysis software determined the Tm value based on the center of gravity mean (BCM) of the intensity curve of the fluorescence from 300-430nm, and observed the aggregation Tagg based on the intensity of light scattered at 266nm. The changes in the secondary structure conformation of the protein at different temperatures were obtained, and the structural stability of the binding protein was evaluated.

[0185] like Figure 4 As shown, the denaturation curves showed that the samples had no obvious Tm values; the aggregation curves showed that the samples had no obvious aggregation as the temperature increased, showing super strong thermal stability.

[0186] Example 4: Thermal recovery test of self-assembled trimer structures of monomeric small proteins

[0187] The thermal recovery of protein structure was tested with the help of Unchained Labs' all-purpose protein stability analyzer Uncle. The sample was heated from 20℃ to 95℃ and then cooled to 20℃ with a step temperature of 15℃ to detect changes in protein conformation and aggregation during the heating and cooling process. Figure 5 As shown in A, the conformation of the sample opens as the temperature rises, and folds back when the temperature drops. The conformation of the sample has a good thermal recovery ability. Figure 5 As shown in B, with the change of temperature, the aggregation signal changes little and there is no obvious aggregation.

[0188] Example 5: Binding activity detection of self-assembling trimeric protein targeting PD-L1

[0189] In this example, the affinity of the high-affinity blocking protein was detected by ForteBio Octet. First, 3 μg / ml of biotin-labeled human PD-L1 protein was loaded onto the avidin-coupled detection probe (1200s), and the unbound biotin-labeled human PD-L1 protein was eluted in PBST solution. The detection probe with human PD-L1 protein was then immersed in a two-fold diluted target PD-L1 self-assembled trimeric protein solution at the same time, and the binding signal was detected (180 seconds). The probe was then immersed in PBST (300 seconds) to detect the dissociation signal of the bound protein. Finally, the affinity of the trimeric protein was calculated. The monomers that make up the trimeric protein are composed of a high-affinity small protein PD-L1-3 (SEQ ID NO: 11) targeting PD-L1 and a self-assembling small protein C3_50_130-18 of the present invention.

[0190] like Figure 6 As shown, both PD-L1-3-C3-50-130-18 (SEQ ID NO: 7) and C3-50-130-18-PD-L1-3 (SEQ ID NO: 9) exhibited super strong binding activity, with an affinity unexpectedly stronger than that of the PD-L1-3 small protein monomer, exceeding the detection limit of the BLI device and reaching the femtomolar level.

[0191] Table 2 Sequences of the present invention

[0192]

[0193]

[0194] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A self-assembling small protein, characterized in that The small protein comprises an amino acid sequence selected from the group consisting of: (1) the amino acid sequence shown in SEQ ID NO: 1; (2) An amino acid sequence that has a homology of ≥90% (preferably ≥95%, more preferably ≥98%) to the amino acid sequence shown in SEQ ID NO: 1 and is capable of spontaneously forming a trimeric protein.

2. A fusion protein, characterized in that The fusion protein comprises the self-assembling small protein according to claim 1 and one or more functional polypeptides.

3. The fusion protein according to claim 2, wherein The fusion protein has a structure as shown in any one of Formula I to Formula III from N-terminus to C-terminus, M-L1-Fx (Formula I) Fx-L1-M (Formula II) Fx-L1-M-L2-Fx (Formula III) in, M is the self-assembling small protein according to claim 1; L1 and L2 are each independently none or a linker; Fx is x functional polypeptides; "-" represents a peptide bond, connecting peptide or linker connecting the above elements; wherein x is an integer selected from 1, 2, 3 or 4.

4. The fusion protein according to claim 2, wherein The functional polypeptide is a targeting polypeptide.

5. A multimeric protein, characterized in that The multimeric protein comprises a plurality of protein monomers, and the protein monomers are selected from: The self-assembling miniprotein according to claim 1, the fusion protein according to claim 2, or a combination thereof.

6. The multimeric protein according to claim 5, characterized in that The multimeric protein is a trimeric protein, which contains or consists of three protein monomers.

7. A polynucleotide, characterized in that The polynucleotide encodes the self-assembling miniprotein according to claim 1 or the fusion protein according to claim 2.

8. A carrier, characterized in that The vector contains the polynucleotide according to claim 7.

9. A host cell, characterized in that The host cell contains the vector according to claim 8, or the polynucleotide according to claim 7 is integrated into its genome.

10. A drug conjugate, characterized in that: The drug conjugate contains: (a) the self-assembling miniprotein according to claim 1, the fusion protein according to claim 2, or the multimeric protein according to claim 5; and (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

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