Self-assembled trimeric protein and preparation method therefor

Abstract

Provided are a self-assembled trimeric protein and a preparation method therefor. Specifically, provided are individual polypeptides constituting the trimeric protein, a nucleic acid sequence encoding the polypeptides, and a method of forming the trimeric protein.

Description

Self-assembling trimeric protein and preparation method thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine. Specifically, the present application relates to a self-assembling trimeric protein and a preparation method thereof. BACKGROUND

[0002] Targeted antibodies / proteins have the advantages of high specificity, small side effects, long half-life, etc., and are a very promising method of biological treatment. Targeted protein drugs have gradually become an important means of clinical treatment. However, due to the complexity and multifactorial nature of disease occurrence and development, single-target antibodies relying on a single target are difficult to achieve better efficacy. At present, targeted antibodies / proteins are mainly based on the CH3 domain of antibody Fc to form a bivalent structure of a dimer. Most multivalent antibodies / fusion proteins are based on the modification of the dimer structure formed by antibody CH3. This limits the design and conversion application of multivalent antibodies / fusion proteins with more complex structures.

[0003] Therefore, there is a need in the art to develop a self-assembling multimeric protein. SUMMARY

[0004] The purpose of the present application is to provide a self-assembling multimeric protein.

[0005] In a first aspect of the present application, a self-assembling small protein is provided, which comprises an amino acid sequence selected from the group consisting of:

[0006] (1) an amino acid sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11;

[0007] (2) an amino acid sequence with a homology of ≥ 90% (preferably ≥ 95%, more preferably ≥ 98%) to the amino acid sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11, and capable of spontaneously forming a trimeric protein.

[0008] In a second aspect of the present application, a fusion protein is provided, which comprises a self-assembling small protein as described in the first aspect of the present application and one or more functional polypeptides.

[0009] In another preferred embodiment, the fusion protein has a structure as shown in any one of Formula I-Formula III from N-terminal to C-terminal, M-L1-Fx (Formula I) Fx-L1-M (Formula II) Fx-L1-M-L2-Fx (Formula III)

[0010] wherein,

[0011] M is a self-assembling small protein as described in the first aspect of the present application;

[0012] L1 and L2 are each independently nothing or a linker;

[0013] Fx is x functional polypeptides;

[0014] “-” represents a peptide bond, a connecting peptide or a linker connecting the above elements;

[0015] wherein x is an integer selected from 1, 2, 3 or 4.

[0016] In another preferred embodiment, the functional polypeptide is a targeting polypeptide.

[0017] In another preferred embodiment, the functional polypeptide is selected from the group consisting of an antibody, a ligand, a receptor, or an active fragment thereof, or a combination thereof.

[0018] 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 ectodomain of a receptor protein, a ligand, or a combination thereof.

[0019] In another preferred embodiment, the functional polypeptide is a PD-L1 binding mini-protein.

[0020] In another preferred embodiment, the functional polypeptide has an amino acid sequence as set forth in SEQ ID NO: 17.

[0021] In another preferred embodiment, the linker is a flexible linker.

[0022] In another preferred embodiment, the linker has an amino acid sequence as set forth in (G4S)n, wherein n is an integer selected from 1-6.

[0023] In another preferred embodiment, the linker has an amino acid sequence as set forth in SEQ ID NO: 18.

[0024] In another preferred embodiment, the fusion protein has an amino acid sequence as set forth in SEQ ID NO: 13 or 15.

[0025] In a third aspect of the present application, a multimeric protein is provided, which comprises a plurality of protein monomers selected from the group consisting of a self-assembling mini-protein according to the first aspect of the present application, a fusion protein according to the second aspect of the present application, or a combination thereof.

[0026] In another preferred embodiment, the multimeric protein is a trimeric protein comprising or consisting of 3 protein monomers.

[0027] In another preferred embodiment, the multimeric protein is a homo-multimer or a hetero-multimer (e.g., a homo-trimer or a hetero-trimer).

[0028] In another preferred embodiment, the multimeric protein is formed by the association of the self-assembling small proteins.

[0029] In another preferred embodiment, the plurality of protein monomers comprises self-assembling small proteins with identical or different amino acid sequences.

[0030] In another preferred embodiment, the plurality of protein monomers comprises self-assembling small proteins with identical amino acid sequences.

[0031] In another preferred embodiment, the plurality of protein monomers comprises functional polypeptides with identical or different amino acid sequences.

[0032] In another preferred embodiment, the functional polypeptides are targeting polypeptides, and the plurality of protein monomers comprises targeting polypeptides that specifically bind to different targets, respectively.

[0033] In a fourth aspect of the present application, a polynucleotide is provided, which encodes the self-assembling small protein of the first aspect of the present application, or the fusion protein of the second aspect of the present application.

[0034] In another preferred embodiment, the polynucleotide has a sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16.

[0035] In a fifth aspect of the present application, a vector is provided, which comprises the polynucleotide of the fourth aspect of the present application.

[0036] In a sixth aspect of the present application, a host cell is provided, which comprises the vector of the fifth aspect of the present application, or has integrated into its genome the polynucleotide of the fourth aspect of the present application.

[0037] In a seventh aspect of the present application, a drug conjugate is provided, which comprises:

[0038] (a) the self-assembling small protein of the first aspect of the present application, the fusion protein of the second aspect of the present application, or the multimeric protein of the third aspect of the present application; and

[0039] (b) a conjugating moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0040] In another preferred embodiment, the conjugating moiety is a drug or a toxin.

[0041] In another preferred embodiment, the conjugating moiety is a detectable label.

[0042] In another preferred embodiment, the conjugate is selected from the group consisting of a fluorescent or luminescent marker, a radioactive marker, an MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agent.

[0043] In an eighth aspect of the present application, a pharmaceutical composition is provided, comprising:

[0044] (a) the fusion protein according to the second aspect of the present application, the multimeric protein according to the third aspect of the present application, the polynucleotide according to the fourth aspect of the present application, the vector according to the fifth aspect of the present application, the host cell according to the sixth aspect of the present application, or the drug conjugate according to the seventh aspect of the present application; and

[0045] (b) a pharmaceutically acceptable carrier.

[0046] In another preferred embodiment, the pharmaceutical composition is used for immunotherapy.

[0047] In another preferred embodiment, the component (a) is present in an amount of 0.1-99.9 wt%, preferably 10-99.9 wt%, more preferably 70-99.9 wt%.

[0048] In another preferred embodiment, the pharmaceutical composition is in a dosage form of an oral dosage form, an injection, or a topical pharmaceutical dosage form.

[0049] In another preferred embodiment, the pharmaceutical composition is in a dosage form of a tablet, a granule, a capsule, an oral solution, or an injection.

[0050] In another preferred embodiment, the pharmaceutical composition or formulation is selected from the group consisting of a suspension formulation, a liquid formulation, or a lyophilized formulation.

[0051] In another preferred embodiment, the liquid formulation is an aqueous injection.

[0052] In another preferred embodiment, the pharmaceutically acceptable carrier comprises a surfactant, a solution stabilizer, an isotonicity adjusting agent, a buffer, or a combination thereof.

[0053] In another preferred embodiment, the subject of administration of the pharmaceutical composition or formulation is a human or a non-human animal.

[0054] In another preferred embodiment, the non-human animal comprises a rodent (e.g., a rat, a mouse), a primate (e.g., a monkey).

[0055] In another preferred embodiment, in the administration of the pharmaceutical composition or formulation, the amount of administration is 0.01-10 g / day, preferably 0.05-5000 mg / day, more preferably 0.1-3000 mg / day.

[0056] In another preferred embodiment, the pharmaceutical composition or formulation is used for inhibiting and / or treating a tumor.

[0057] In another preferred embodiment, for the treatment of a tumor, the pharmaceutical composition or formulation can be administered in combination with other anti-tumor drugs.

[0058] In a ninth aspect of the present application, a method for preparing the multimeric protein as described in the third aspect of the present application is provided, comprising the steps of:

[0059] contacting a plurality of the self-assembling small proteins as described in the first aspect of the present application and / or the fusion proteins as described in the second aspect of the present application with each other, thereby obtaining the trimeric protein.

[0060] In a tenth aspect of the present application, a method for preparing the self-assembling small protein as described in the first aspect of the present application, or the fusion protein as described in the second aspect of the present application, or the multimeric protein as described in the third aspect of the present application is provided, comprising the steps of:

[0061] (a) culturing the host cell as described in the sixth aspect of the present application under suitable conditions, thereby obtaining a culture containing the self-assembling small protein or the fusion protein or the trimeric protein; and

[0062] (b) purifying and / or isolating the culture obtained in step (a), thereby obtaining the self-assembling small protein or the fusion protein or the trimeric protein.

[0063] In an eleventh aspect of the present application, the fusion protein as described in the second aspect of the present application, the multimeric protein as described in the third aspect of the present application, the polynucleotide as described in the fourth aspect of the present application, the vector as described in the fifth aspect of the present application, the host cell as described in the sixth aspect of the present application, or the drug conjugate as described in the seventh aspect of the present application is used for preparing a medicament for treating a disease.

[0064] In another preferred embodiment, the disease is a disease with high expression of PD-L1.

[0065] In another preferred embodiment, the tumor is a tumor expressing PD-L1 protein (i.e., PD-L1 positive).

[0066] In another preferred embodiment, the tumor includes, but is not limited to, acute myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, 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 gland tumor, bladder tumor, or a combination thereof.

[0067] In a twelfth aspect of the present application, a method for treating a disease is provided, comprising the step of administering to a subject in need thereof the fusion protein according to the second aspect of the present application, the multimeric protein according to the third aspect of the present application, or the drug conjugate according to the seventh aspect of the present application.

[0068] It should be understood that, within the scope of the present application, each of the technical features described above and each of the technical features specifically described hereinafter (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0069] The following drawings are used to illustrate specific embodiments of the present application, and are not intended to limit the scope of the present application as defined by the claims.

[0070] Figure 1 shows a schematic diagram of the spatial structure of a trimeric protein, wherein:

[0071] A is a schematic diagram of the spatial structure of C3-50-70-11 trimeric protein;

[0072] B is a schematic diagram of the spatial structure of C3-50-70-12 trimeric protein;

[0073] C is a schematic diagram of the spatial structure of C3-50-70-14 trimeric protein.

[0074] D is a schematic diagram of the spatial structure of C3-50-70-15 trimeric protein;

[0075] E is a schematic diagram of the spatial structure of C3-50-70-17 trimeric protein;

[0076] F is a schematic diagram of the spatial structure of C3-50-70-19 trimeric protein.

[0077] Figure 2 shows a schematic diagram showing several structural combinations of trimeric proteins, wherein:

[0078] A is that an antibody Fab, a single-chain antibody (scFv), an extracellular region of a receptor protein, or a ligand is linked to a hinge region or a linker of an antibody and a single chain of a trimeric protein to form a polypeptide chain, and a specific trimeric combination is formed by means of the single chain (or fragment) of the trimeric protein provided by the present application;

[0079] B is that a single chain of a trimeric protein is linked to a hinge region or a linker of an antibody, an antibody Fab, a single-chain antibody (scFv), an extracellular region of a receptor protein, or a ligand to form a polypeptide chain, and a specific trimeric combination is formed by means of the single chain (or fragment) of the trimeric protein provided by the present application;

[0080] C is antibody Fab, single chain antibody (scFv), receptor protein extracellular region or ligand, which is connected with antibody Fab, single chain antibody (scFv), receptor protein extracellular region or ligand through linker, and forms polypeptide chain with antibody hinge or linker and trimeric protein single chain in series, and forms specific combination with trimeric protein single chain (or fragment) provided by the present application;

[0081] D is trimeric protein single chain, which is connected with antibody hinge or linker, and antibody Fab, single chain antibody (scFv), receptor protein extracellular region or ligand, which is connected with antibody Fab, single chain antibody (scFv), receptor protein extracellular region or ligand through linker, and forms polypeptide chain in series, and forms specific combination with trimeric protein single chain (or fragment) provided by the present application;

[0082] E is antibody Fab, single chain antibody (scFv), receptor protein extracellular region or ligand, which is connected with antibody hinge or linker, trimeric protein single chain, antibody hinge or linker, antibody Fab, single chain antibody (scFv), receptor protein extracellular region or ligand, which is connected with antibody Fab, single chain antibody (scFv), receptor protein extracellular region or ligand through linker, and forms polypeptide chain in series, and forms specific combination with trimeric protein single chain (or fragment) provided by the present application.

[0083] Figure 3 shows the results of trimeric protein molecular sieve detection, wherein:

[0084] A is the results of standard molecular weight determination by gel filtration calibration kit;

[0085] B is the results of C3-50-70-11 trimeric protein molecular sieve detection;

[0086] C is the results of C3-50-70-12 trimeric protein molecular sieve detection;

[0087] D is the results of C3-50-70-14 trimeric protein molecular sieve detection;

[0088] E is the results of C3-50-70-15 trimeric protein molecular sieve detection;

[0089] F is the results of C3-50-70-17 trimeric protein molecular sieve detection;

[0090] G is the results of C3-50-70-19 trimeric protein molecular sieve detection.

[0091] Figure 4 shows the results of trimeric protein structure thermal stability detection, wherein:

[0092] A is the results of Tm detection of trimeric protein;

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

[0094] Figure 5 shows the result of the structural thermal recovery test of the trimeric protein, wherein:

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

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

[0097] Figure 6 shows the binding activity test of the self-assembled trimeric protein targeting PD-L1.

[0098] Figure 7 shows the result of the structural thermal stability test of the trimeric protein targeting PD-L1.

[0099] Figure 8 shows the result of the structural thermal recovery test of the trimeric protein targeting PD-L1. DETAILED DESCRIPTION

[0100] The present inventors, through extensive and in-depth research, have for the first time developed a class of self-assembled trimeric proteins and a preparation method thereof. The trimeric protein of the present application comprises three polypeptides of the same sequence, which interact to promote the spontaneous assembly of the three polypeptides into a trimer. On this basis, a binding protein can be connected to the N- and / or C-terminus of the trimeric protein, thereby forming a multivalent targeting protein. On this basis, the present application is completed.

[0101] TERMINOLOGY

[0102] For easier understanding of the present application, certain technical and scientific terms are defined in detail below. Unless otherwise clearly defined in this text, all other technical and scientific terms used in this text have the meanings commonly understood by those of ordinary skill in the art to which the present application belongs. Before describing the present application, it should be understood that the present application is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terms used herein are intended to be descriptive only and are not intended to be limiting, the scope of the present application being limited only by the claims appended hereto.

[0103] As used herein, the term "about," used in reference to a particular value, means that the value can vary from the recited value by not more than 1%. 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.).

[0104] As used herein, the terms "comprising", "including", "containing", are used interchangeably and mean to include but not to the exclusion of, half-opened, and open-ended definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0105] As used herein, the term "pharmaceutically acceptable carrier" refers to a material that is suitable for use with humans and / or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio when used as described herein.

[0106] As used herein, the term "therapeutically effective amount" refers to an amount that produces functional or therapeutic effects for which it is administered, and which can be accepted by a human and / or an animal. It will be understood by one of ordinary skill in the art that the "therapeutically effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipient used, the severity of the disease, and the combination with other drugs, etc.

[0107] Self-assembling small proteins and fusion proteins of the present application

[0108] In the present application, a class of self-assembling small proteins and a fusion protein or a multimer thereof comprising the small proteins are provided.

[0109] As used herein, the terms "small protein of the present application", "self-assembling small protein of the present application" are used interchangeably, and refer to a small protein that can self-assemble to form a multimer (e.g., a trimer) as described in the first aspect of the present application. The small protein of the present application is composed of one peptide chain, and mainly forms three alpha-helix secondary structures.

[0110] Preferably, the small protein of the present application has an amino acid sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, or 11.

[0111] As used herein, the term "fusion protein of the present application" refers to a fusion protein formed by the self-assembling small protein of the present application and other fusion elements. 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.

[0112] As used herein, the term "multimer" refers to a molecule formed by the combination of multiple small proteins or fusion proteins of the present application. 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. The multiple monomers in the multimer of the present application form the multimer through the self-assembly of the small protein region. In one embodiment, the multimer of the present application is a heteromultimer, which contains multiple fusion proteins comprising different functional polypeptides. For example, the multimer of the present application can include multiple fusion proteins, each having a functional polypeptide targeting a different target, thereby achieving the purpose of multi-target therapy.

[0113] Typically, the fusion protein of the present application has a structure as shown in any one of Formula I-III from N-terminus to C-terminus, M-L1-Fx (Formula I) Fx-L1-M (Formula II) Fx-L1-M-L2-Fx (Formula III)

[0114] wherein,

[0115] M is the self-assembling small protein as described in claim 1 ;

[0116] L1 and L2 are each independently nothing or a linker;

[0117] Fx is x functional polypeptides;

[0118] “-” represents a peptide bond, a connecting peptide or a linker connecting the above elements;

[0119] wherein x is an integer selected from 1, 2, 3 or 4.

[0120] Typically, the multimer of the present application is a trimeric protein, which can have one of the following structural types:

[0121] comprising three functional polypeptides - a trimeric polypeptide single chain;

[0122] comprising a trimeric polypeptide single chain - three functional polypeptides;

[0123] comprising three functional polypeptides - a functional polypeptide - a trimeric polypeptide single chain;

[0124] comprising a trimeric polypeptide single chain - three functional polypeptides - a functional polypeptide;

[0125] comprising three functional polypeptides - a trimeric polypeptide single chain - three functional polypeptides,

[0126] wherein the trimeric polypeptide single chain is the self-assembling small protein of the present application.

[0127] In another preferred embodiment, the functional polypeptide is selected from the group consisting of Fab, scFv, fusion receptor or ligand.

[0128] It should be understood that the above structural types are only exemplary forms and do not limit the present application. Some representative structures are shown in Figure 2. Among them, the functional polypeptide connected with the self-assembling small protein can be single or multiple (such as 2, 3 or 4 functional polypeptides in tandem form).

[0129] As used herein, the term "self-assembling small protein" or "fusion protein" also includes variants having self-assembly activity. These variants include, but are not limited to, deletions, insertions and / or substitutions of one to three (usually one to two, more preferably one) amino acids, addition or deletion of one or several (usually three or fewer, more preferably two or fewer, still more preferably one) amino acids at the C-terminus and / or the N-terminus, or addition of an amino acid fragment with small side chains as a linker (e.g., glycine, serine, etc.) at the N- or C-terminus of the small protein. For example, in the art, substitutions of amino acids with similar or identical properties are usually not considered to change the function of the protein. Also, addition or deletion of one or several amino acids at the C-terminus and / or the N-terminus usually does not change the structure and function of the protein. The term also includes linear and non-linear polypeptides (e.g., cyclic peptides).

[0130] The present application also includes active fragments, derivatives and analogs of the self-assembling small proteins or fusion proteins described above, especially those formed with a specific targeting peptide. As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially maintain the function or activity of the self-assembling small proteins or fusion proteins of the present application.

[0131] The polypeptide fragments, derivatives or analogs of the present application can be (i) polypeptides having one or several conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) polypeptides having a substituent group at one or more amino acid residues, or (iii) polypeptides formed by fusing the polypeptide to another compound (such as a compound that extends the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) polypeptides formed by fusing additional amino acid sequences to the polypeptide sequence (fusion proteins formed by fusing a leader sequence, a secretion sequence or a 6His tag sequence, etc.). These fragments, derivatives and analogs are within the scope of those skilled in the art according to the teachings herein.

[0132] One preferred class of active derivatives refers to polypeptides having up to five, preferably up to three, more preferably up to one amino acid replaced by an amino acid of similar or identical properties as compared to the amino acid sequence of the present application. These conservative variant polypeptides are preferably generated by amino acid replacement according to Table A.

[0133] Table A

[0134] The present application also provides analogs of the self-assembling small proteins or fusion proteins of the present application. These analogs can differ from the polypeptides of the present application by differences in the amino acid sequence, by differences in the form of the modification that does not affect the sequence, or by both. Analog also includes analogs having residues other than the naturally occurring L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., β, γ-amino acids). It is to be understood that the polypeptides of the present application are not limited to the representative polypeptides exemplified above.

[0135] In addition, the self-assembling small proteins or fusion proteins of the present application can be modified. Modifications, which generally do not alter the primary structure, include chemical derivatization of the polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those polypeptides that are glycosylated modified during synthesis and processing or further processing steps. Such modifications can be accomplished by exposing the polypeptides to enzymes that glycosylate (e.g., mammalian glycosylation enzymes or deglycosylation enzymes). Modifications also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that are modified to increase their resistance to proteolysis or to optimize solubility.

[0136] The term "polynucleotide of the present application" can include polynucleotides that encode the self-assembling small proteins or fusion proteins of the present application, as well as polynucleotides that further include additional coding and / or non-coding sequences.

[0137] The present application also relates to variants of the above-described polynucleotides that encode fragments, analogs, and derivatives of the polypeptides or fusion proteins having the same amino acid sequence as the present application. 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 can result from a natural process such as mutation or genetic recombination. A substitution variant is one in which at least one residue has been replaced by different residue, a deletion variant is one in which at least one residue has been deleted, and an insertion variant is one in which at least one residue has been added. However, the function of the self-assembling small proteins or fusion proteins encoded by the polynucleotide will not be substantially altered.

[0138] The present application also relates to polynucleotides that hybridize to the above-described sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present application particularly relates to polynucleotides that hybridize to the polynucleotides of the present application under stringent conditions (or stringency conditions). In the present application, "stringent conditions" means: (1) hybridization and washing under low ionic strength and high temperature, such as 0.2 x SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization only when the identity between the two sequences is at least 90%, more preferably 95%.

[0139] The self-assembling miniprotein or fusion protein and polynucleotide of the present application is preferably provided in an isolated form, more preferably, purified to homogeneity.

[0140] The full-length sequence of the polynucleotide of the present application can be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequence disclosed herein, particularly the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art can be used as a template for amplification. When the sequence is long, two or more PCR amplifications can be performed, and then the amplified fragments can be spliced together in the correct order.

[0141] Once the relevant sequence is obtained, the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then into cells, and then separating the relevant sequence from the proliferated host cells by conventional methods.

[0142] In addition, the relevant sequence can also be synthesized by artificial synthesis method, especially when the length of the fragment is short. Generally, a long fragment can be obtained by synthesizing a plurality of small fragments first and then connecting them.

[0143] At present, the DNA sequence encoding the protein (or fragment thereof, or derivative thereof) of the present application can be completely obtained by chemical synthesis. Then the DNA sequence can be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0144] The method of amplifying DNA / RNA by PCR technology is preferably used to obtain the polynucleotide of the present application. In particular, when it is difficult to obtain a full-length cDNA from a library, the RACE method (RACE-cDNA rapid amplification of the end) can be preferably used. The primers used in PCR can be appropriately selected based on the sequence information of the present application disclosed herein, and can be synthesized by conventional methods. The amplified DNA / RNA fragments can be separated and purified by conventional methods such as gel electrophoresis.

[0145] The present application also provides a method for forming a trimer between self-assembling miniproteins or fusion proteins thereof, which comprises contacting a plurality of said self-assembling miniproteins and / or fusion proteins with each other, thereby obtaining said trimer protein. On the three interaction surfaces of the self-assembling miniproteins constituting said trimer, amino acid pairs with interaction are formed to form a specific interaction trimer protein.

[0146] Expression vector

[0147] The present application also relates to a vector comprising the polynucleotide of the present application, and a host cell genetically engineered with the vector of the present application or the self-assembling small protein or fusion protein coding sequence of the present application, and a method for producing the polypeptide of the present application by recombination technology.

[0148] The polynucleotide sequence of the present application can be used to express or produce a recombinant fusion protein by conventional recombination DNA technology. Generally, the following steps are involved:

[0149] (1) transforming or transducing a suitable host cell with the polynucleotide of the present application encoding the fusion protein of the present application (or a variant), or with a recombinant expression vector containing the polynucleotide;

[0150] (2) culturing the host cell in a suitable medium;

[0151] (3) isolating and purifying the protein from the medium or the cell.

[0152] In the present application, the polynucleotide sequence encoding the fusion protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vector well known in the art. Any plasmid and vector can be used as long as it can replicate and stabilize in the host. An important feature of the expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0153] In the method for preparing the self-assembling small protein or fusion protein thereof of the present application, any suitable vector can be used, which can be selected from one of pET, pDR1, pcDNA3.1(+), pcDNA3.1 / ZEO(+), pDHFR, and the expression vector includes a fusion DNA sequence linked with suitable transcription and translation regulatory sequences.

[0154] Both eukaryotic and prokaryotic host cells can be used for the expression of the self-assembling small protein or fusion protein thereof of the present application, and the eukaryotic host cell is preferably a mammalian or insect host cell culture system, preferably cells such as COS, CHO, NS0, sf9 and sf21; and the prokaryotic host cell is preferably one of DH5a, BL21(DE3) and TG1.

[0155] Methods well known to those skilled in the art can be used to construct expression vectors containing DNA sequences encoding the fusion proteins of the present application 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 sequences are operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of such promoters are the lac or trp promoter of E. coli, the PL promoter of bacteriophage lambda, eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the LTRs of retroviruses, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for initiation of translation and a transcription terminator.

[0156] 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 for eukaryotic cell culture, neomycin resistance for eukaryotic and prokaryotic cell culture, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E. coli.

[0157] Vectors containing the appropriate DNA sequences as described above, and appropriate promoters or control sequences, can be employed to transform appropriate host cells to enable them to express the proteins.

[0158] The host cells can be prokaryotic cells, such as bacterial cells, or lower eukaryotic cells, such as yeast cells, or higher eukaryotic cells, such as mammalian cells. Representative examples of useful host cells are prokaryotic cells, such as bacterial cells of E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast, plant cells (e.g., ginseng cells), and mammalian cells.

[0159] The polynucleotides of the present application, when expressed in higher eukaryotic cells, will be enhanced by the insertion of enhancer sequences into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, which act to increase the transcription of a gene. Examples of enhancers include the SV40 enhancer, which is in the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and enhancers associated with adenovirus.

[0160] The selection of appropriate vectors, promoters, enhancers, and host cells is well within the level of skill in the art.

[0161] 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 prokaryote, such as E. coli, the transformation of the host cell can be effected by the use of techniques such as calcium chloride precipitation. If necessary, the transformation can be performed by electroporation. When the host is a eukaryote, the transformation can be effected by the use of techniques such as calcium phosphate precipitation, conventional mechanical procedures such as microinjection, electroporation, and the like.

[0162] The transformants obtained can be cultured in conventional nutrient media using standard procedures known in the art. The culture conditions, such as temperature, pH, and the like, are those previously determined to be appropriate for the host cell used. When the host cells grow to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period.

[0163] The recombinant polypeptides of the above methods can be expressed intracellularly, on the cell membrane, or secreted from the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their 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 protein precipitants (salting-out method), centrifugation, osmotic lysis, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods.

[0164] The self-assembling small proteins or fusion proteins of the present application can be isolated and purified by affinity chromatography. Depending on the properties of the affinity column used, the self-assembling small proteins or fusion proteins bound to the affinity column can be eluted using conventional methods such as high salt buffer, pH change, and the like.

[0165] Using the above methods, the self-assembling small proteins or fusion proteins can be purified to a substantially homogeneous substance, such as a single protein peak (OD 280 or OD 210 ) of a similar molecular weight in a molecular sieve.

[0166] Pharmaceutical composition

[0167] In the present application, a pharmaceutical composition containing the self-assembling small proteins, fusion proteins, multimers, or drug conjugates of the present application is also provided.

[0168] The pharmaceutical composition of the present application contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the self-assembling small protein, fusion protein or multimer (or conjugate thereof) of the present application 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 be matched with the administration mode. The pharmaceutical composition of the present application can be prepared in the form of a needle, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition such as a needle, a solution is preferably manufactured under sterile conditions. The administration amount of the active ingredient is a therapeutically effective amount, for example, about 10 μg / kg body weight to about 50 mg / kg body weight per day. In addition, the polypeptide of the present application can also be used with other therapeutic agents. The self-assembling small protein, fusion protein, multimer or drug conjugate can be combined with a pharmaceutically acceptable adjuvant to form a pharmaceutical preparation to more stably exert the therapeutic effect, which can ensure the structural integrity of the amino acid core sequence of the self-assembling small protein, fusion protein or multimer of the present application, and also protect the multifunctional groups of the protein from degradation (including but not limited to condensation, deamination or oxidation). The preparation can be in various forms, and in general, for liquid preparations, it is generally stable at 2-8°C for at least one year, and for lyophilized preparations, it is stable at 30°C for at least six months. Here, the preparation can be a suspension, an aqueous needle, a lyophilized preparation commonly used in the pharmaceutical field, and a water needle or a lyophilized preparation is preferred.

[0169] For the pharmaceutical composition (e.g., aqueous needle or lyophilized preparation) of the present application, the pharmaceutically acceptable adjuvant includes one or a combination of a surfactant, a solution stabilizer, an isotonicity adjusting agent and a buffer, wherein the surfactant includes a non-ionic surfactant such as polyoxyethylene sorbitan fatty acid ester (Tween 20 or 80); poloxamer (e.g., poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; myristyl, linoleyl or stearyl sarcosine; Pluronics; MONAQUAT™, etc., which is added in an amount to minimize the tendency of protein particles, the solution stabilizer can be a sugar including a reducing sugar and a non-reducing sugar, an amino acid including monosodium glutamate or histidine, an alcohol including a trihydric alcohol, a higher sugar alcohol, propylene glycol, polyethylene glycol, or a combination thereof, the solution stabilizer is added in an amount to allow the finally formed preparation to be considered by those skilled in the art to be stable for a stable period of time, the isotonicity adjusting agent can be one of sodium chloride, mannitol, and the buffer can be one of TRIS, histidine buffer, phosphate buffer.

[0170] When the pharmaceutical composition is used, a safe and effective amount of the small protein or fusion protein of the present application or its immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 50 micrograms per kilogram of body weight, and in most cases, no more than about 100 milligrams per kilogram of body weight, preferably the dosage is about 100 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight. Of course, the specific dose will also take into account the route of administration, the health condition of the patient, etc., which are within the skill of the skilled physician. Typically, the total amount of administration usually cannot exceed a certain range, for example, the dosage for intravenous injection is 10 to 3000 mg / day / 50 kg.

[0171] The trimeric protein of the present application and the pharmaceutical preparation containing the same can be used as an antitumor drug for tumor treatment. The antitumor drug as referred to in the present application means a drug having the function of inhibiting and / or treating tumor, which can include the delay of the development of symptoms associated with tumor growth and / or the reduction of the severity of these symptoms, and further includes the reduction of the symptoms associated with the growth of already existing tumor and the prevention of the occurrence of other symptoms, and also reduces or prevents metastasis.

[0172] The 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 combination administration include but are not limited to: 1. Cytotoxic drugs (1) Drugs acting on the chemical structure of DNA: alkylating agents such as nitrogen mustard, nitrous urea, methyl sulfonate; platinum compounds such as cisplatin, carboplatin and oxaliplatin, etc.; mytomycin (MMC); (2) Drugs affecting nucleic acid synthesis: dihydrofolate reductase inhibitors such as methotrexate (MTX) and Alimta, etc.; thymidine synthetase inhibitors such as fluorouracil (5FU, FT-207, capecitabine), etc.; purine nucleoside synthetase inhibitors such as 6-mercaptopurine (6-MP) and 6-TG, etc.; nucleotide reductase inhibitors such as hydroxyurea (HU), etc.; DNA polymerase inhibitors such as cytarabine (Ara-C) and gemcitabine (Gemz), etc.; (3) Drugs acting on nucleic acid transcription: drugs selectively acting on DNA template, inhibiting DNA-dependent RNA polymerase, thereby inhibiting RNA synthesis such as: actinomycin D, daunorubicin, doxorubicin, epirubicin, aclacinomycin, and so on; (4) Drugs mainly acting on microtubulin synthesis: paclitaxel, taxotere, vinblastine, vinorelbine, podophyllotoxin, homoharringtonine; (5) Other cytotoxic drugs: asparaginase mainly inhibits protein synthesis; 2. Hormones Anti-estrogens: tamoxifen, droloxifene, exemestane, etc.; Aromatase inhibitors: aminoglutethimide, lantronex, letrozole, arimidex, etc.; Anti-androgens: flutamide, RH-LH agonists / antagonists: zoladex, enantone, etc.; 3. Biological response modifiers: mainly interferon, which interferes with tumor through body immune function; interleukin-2; thymic peptides; 4. Monoclonal antibodies: MabThera; Cetuximab (C225); Trastuzumab (Herceptin); Bevacizumab (Avastin); Yervoy (Ipilimumab); 5. Other drugs including some drugs whose mechanisms are not yet clear and need further study; cell differentiation inducers such as retinoids; apoptosis inducers.

[0173] The main advantages of the present application include:

[0174] 1) The present application provides small proteins that self-assemble to form trimers, which can be connected to binding proteins at the N- and / or C-terminus of the trimeric protein, thereby forming multivalent targeting proteins;

[0175] 2) The self-assembling small proteins of the present application have strong thermal stability and conformational thermal recovery.

[0176] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0177] Example 1: Construction, expression, and purification of self-assembled trimer proteins

[0178] Trimeric protein nucleotide sequences were synthesized using gene synthesis methods and then inserted into the pET29b vector in the sequence MGS-trimeric protein nucleotide sequence-GSENLYFQSLEHHHHHH (ENLYFQSLE sequence for enzyme digestion, HHHHHH for protein purification). After transforming *E. coli* into this vector, the culture was carried out in LB medium at 37°C and 270 rpm until OD600 = 0.6. Protein expression was then induced overnight with 1 mM IPTG. After centrifugation and resuspending, the bacteria were lysed using a cryo-mechanical lysing device, followed by high-speed centrifugation to collect the supernatant. The protein samples were purified using a Ni column, desalted, concentrated, and the concentration was determined using a micro-spectrophotometer. The purified protein was then filtered through a 0.22 μm filter for sterilization and stored at 4°C for subsequent experiments.

[0179] Example 2: Evaluation of the assembly efficiency of self-assembled trimer proteins

[0180] The protein purified using the AKTApure protein purification system was further purified from the Ni column. Superdex was selected. TM 75. Increase the flow rate of the 10 / 300GL gel filtration column. First, replace the 20% ethanol in the column with deionized water at a low flow rate of 0.45 ml / min. Then, equilibrate the system using 1×PBS buffer at a flow rate of 0.75 ml / min. After the pressure, conductivity, and other values ​​stabilize, load 500 μL of purified protein onto the column and zero the column at UV absorbance values ​​of 215 nm and 280 nm. Observe the changes in UV absorption peaks and collect the sample. Calculate the molecular weight standard curve using the Gel Filtration Calibration Kit (LMW) and analyze the OD value. 280 Elution volume parameters and peak area were used to evaluate protein molecular weight and purity. High-purity candidate proteins were obtained using this method for subsequent experiments. As shown in Figure 3, proteins of corresponding molecular weights were collected for later use.

[0181] Example 3: Detection of thermal stability of self-assembled trimeric structure of monomeric small protein

[0182] The thermal stability of the protein structure was detected by means of the Unchained Labs all-in-one protein stability analyzer Uncle. The sample was heated from 25°C to 95°C at a rate of 0.5°C / min, and static light scattering was measured at an excitation wavelength of 266 nm. As the protein unfolds, the fluorescence generally decreases and shifts to a longer wavelength. The data analysis software determines the Tm value according to the barycentric mean (BCM) of the intensity curve of the fluorescence from 300 to 430 nm, and the light intensity scattered at 266 nm is observed to determine the aggregation Tagg. The changes in the secondary structure conformation of the protein at different temperatures are obtained, and the structural stability of the binding protein is evaluated.

[0183] As shown in FIG. 4, the denaturation curve shows that none of the samples has a significant Tm value; the aggregation curve shows that the samples do not have significant aggregation as the temperature increases, indicating that they have super-strong thermal stability.

[0184] Example 4: Detection of thermal recovery of self-assembled trimeric structure of monomeric small protein

[0185] The thermal recovery of the protein structure was detected by means of the Unchained Labs all-in-one protein stability analyzer Uncle. The sample was heated from 20°C to 95°C and then cooled to 20°C, with a step temperature of 15°C. The changes in the protein conformation and aggregation during the heating and cooling of the protein were detected. As shown in FIG. 5A, as the temperature increases, the conformation also opens, and when the temperature decreases, the conformation folds back, indicating that the sample has good conformational thermal recovery ability. As shown in FIG. 5B, the aggregation signal changes little with the change in temperature, and there is no significant aggregation.

[0186] Example 5: Detection of binding activity of self-assembled trimeric protein targeting PD-L1

[0187] In this example, the affinity of the high-affinity blocking protein was detected by means of ForteBio Octet. First, 3 μg / ml of biotin-labeled human PD-L1 protein was loaded onto the detection probe coupled with avidin (1200 s), and the biotin-labeled human PD-L1 protein that had not been bound was eluted in a PBST solution. Then, the detection probe with the human PD-L1 protein was simultaneously immersed in a solution of the self-assembled trimeric protein targeting PD-L1 diluted by two times, and the binding signal was detected (180 seconds). Then, the probe was immersed in PBST (300 seconds), and the dissociation signal of the bound protein was detected. Finally, the affinity of the high-affinity blocking protein was calculated. The monomers constituting the trimeric protein are composed of the high-affinity small protein PD-L1-3 (SEQ ID NO: 17) targeting PD-L1 and the self-assembled small protein C3-50-70-11 of the present application.

[0188] As shown in Figure 6, PD-L1-3-C3-50-70-11 (SEQ ID NO: 13) and C3-50-70-11-PD-L1-3 (SEQ ID NO: 15) both exhibit super-strong binding activity, and the affinity is unexpectedly stronger than that of the PD-L1-3 small protein monomer, exceeding the upper limit of the BLI device detection, reaching the femto level.

[0189] Example 6: Detection of thermal stability of self-assembly trimeric protein structure targeting PD-L1

[0190] The thermal stability of the protein structure was detected by the Uncle protein stability analyzer of Unchained Labs. The sample temperature range was 25-95°C, the temperature rising rate was 0.5°C / min, the static light scattering was measured at an excitation wavelength of 266 nm, and the fluorescence usually decreased and shifted to a longer wavelength direction as the protein unfolded. The data analysis software determined the Tm value according to the barycenter average (BCM) of the intensity curve of 300-430 nm fluorescence, and the light intensity scattering at 266 nm was observed to observe the aggregation Tagg. The change of the protein secondary structure conformation of the protein at different temperatures was obtained, and the structural stability of the binding protein was evaluated.

[0191] As shown in Figure 7, the denaturation curves of PD-L1-3-C3-50-70-11 and C3-50-70-11-PD-L1-3 showed that the samples had no obvious Tm value; the aggregation curves of PD-L1-3-C3-50-70-11 and C3-50-70-11-PD-L1-3 showed that the samples had no obvious aggregation with the increase of temperature, and exhibited super-strong thermal stability.

[0192] Example 7: Detection of thermal recovery of self-assembly trimeric protein structure targeting PD-L1

[0193] The thermal recovery of the protein structure was detected by the Uncle protein stability analyzer of Unchained Labs. The sample was heated from 20°C to 95°C and then cooled to 20°C, with a step temperature of 15°C. The changes of protein conformation and aggregation during the heating and cooling of the protein were detected. As shown in Figure 8, with the increase of temperature, the conformation of PD-L1-3-C3-50-70-11 and C3-50-70-11-PD-L1-3 also opened, and the conformation folded back when the temperature decreased. The conformational thermal recovery ability of PD-L1-3-C3-50-70-11 and C3-50-70-11-PD-L1-3 was good. With the change of temperature, the aggregation signal of PD-L1-3-C3-50-70-11 and C3-50-70-11-PD-L1-3 changed less, and there was no obvious aggregation.

[0194] Table 2 Inventive sequences

[0195] All documents referred to in the present application are incorporated herein by reference as if each individual document were incorporated by reference. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that each disclosed embodiment can be implemented with or without the corresponding use of the other embodiments. Further, it is intended that contributions in the art by the applicant be utilized by the public and can not necessarily be limited to use herein.

Claims

1. A self-assembling miniprotein, characterized in that, The small protein comprises an amino acid sequence selected from the group consisting of: (1) an amino acid sequence as set forth in SEQ ID NO: 1, 3, 5, 7, 9, or 11; (2) an amino acid sequence with a homology of ≥ 90% (preferably ≥ 95%, more preferably ≥ 98%) to the amino acid sequence as set forth in SEQ ID NO: 1, 3, 5, 7, 9, or 11, and capable of spontaneously forming a trimeric protein.

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

3. The fusion protein of claim 2, wherein, The fusion protein has a structure as set forth in any one of Formula I-III from N-terminus to C-terminus, M-L1-Fx (Formula I) Fx-L1-M (Formula II) Fx-L1-M-L2-Fx (Formula III) wherein, M is the self-assembling small protein of claim 1; L1 and L2 are each independently nothing or a linker; Fx is x functional polypeptides; "-" represents a peptide bond, a connecting peptide, or a linker connecting the above elements; wherein x is an integer selected from 1, 2, 3, or 4.

4. The fusion protein of 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 selected from the group consisting of: The self-assembling small protein of claim 1, the fusion protein of claim 2, or a combination thereof.

6. The multimeric protein of claim 5, wherein, The multimeric protein is a trimeric protein, which comprises or consists of 3 protein monomers.

7. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of any one of claims 1-6. The polynucleotide encodes the self-assembling small protein of claim 1, or the fusion protein of claim 2.

8. A vector, characterized in that, The vector comprises the polynucleotide of claim 7.

9. A host cell, characterized in that, The host cell comprises the vector of claim 8, or the polynucleotide of claim 7 is integrated into the genome of the host cell.

10. A drug conjugate, characterized in that, The drug conjugate comprises: (a) the self-assembling small protein of claim 1, the fusion protein of claim 2, or the multimeric protein of claim 5; and (b) a conjugating 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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