Self-assembled tripolymer protein and preparation method thereof

Abstract

The invention provides a self-assembled tripolymer protein 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

Technical Field

[0001] The present invention relates to the field of biomedicine and in particular to a self-assembling trimer protein and a preparation method thereof. Background Art

[0002] Targeted antibodies / proteins have the advantages of high specificity, few side effects, and long half-life, 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 the occurrence and development of diseases, it is difficult to achieve better therapeutic effects by relying solely on single-targeted antibodies with a single target. At present, targeted antibodies / proteins are mainly based on the bivalent structure of dimers formed by the CH3 domain of antibody Fc. The vast majority of multivalent antibodies / fusion proteins are modified based on the dimer structure formed by antibody CH3. This limits the design and transformation 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 of the invention

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

[0005] In the 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:

[0006] (1) the amino acid sequence shown in SEQ ID NO: 1, 3 or 5;

[0007] (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, 3 or 5 and is capable of spontaneously forming a trimer protein.

[0008] In the second aspect of the present invention, a fusion protein is provided, wherein the fusion protein comprises the self-assembling small protein as described in the first aspect of the present invention 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 to Formula III from N-terminus to C-terminus,

[0010] M-L1-Fx (Formula I)

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

[0012] Fx-L1-M-L2-Fx (Formula III)

[0013] in,

[0014] M is the self-assembling small protein as described in the first aspect of the present invention;

[0015] L1 and L2 are each independently none or a linker;

[0016] Fx is x functional polypeptides;

[0017] “-” represents a peptide bond, connecting peptide or linker connecting the above elements;

[0018] wherein x is an integer selected from 1, 2, 3 or 4.

[0019] In another preferred embodiment, the functional polypeptide is a targeting polypeptide.

[0020] In another preferred embodiment, the functional polypeptide is selected from the following group: antibodies, ligands, receptors, or active fragments thereof, or a combination thereof.

[0021] In another preferred embodiment, the functional polypeptide is selected from the following group: antigen binding fragment (Fab), single chain antibody (scFv), single domain antibody (sdAb), receptor protein extracellular domain, ligand, or a combination thereof.

[0022] In another preferred embodiment, the functional polypeptide is a PD-L1 binding small protein.

[0023] In another preferred embodiment, the amino acid sequence of the functional polypeptide is shown in SEQ ID NO:11.

[0024] In another preferred embodiment, the connector is a flexible connector.

[0025] 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.

[0026] In another preferred embodiment, the amino acid sequence of the linker is shown in SEQ ID NO:12.

[0027] In another preferred embodiment, the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 7 or 9.

[0028] 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.

[0029] In another preferred embodiment, the multimeric protein is a trimeric protein, which comprises or consists of three of the protein monomers.

[0030] In another preferred embodiment, the multimeric protein is a homopolymer or a heteropolymer (such as a homotrimer or a heterotrimer).

[0031] In another preferred embodiment, the multimeric protein is formed by the combination of the self-assembling small proteins.

[0032] In another preferred embodiment, the plurality of protein monomers contain self-assembling small proteins with the same or different amino acid sequences.

[0033] In another preferred embodiment, the plurality of protein monomers contain self-assembling small proteins with the same amino acid sequence.

[0034] In another preferred embodiment, the multiple protein monomers contain the same or different functional polypeptides.

[0035] 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.

[0036] In the fourth aspect of the present invention, a polynucleotide is provided, wherein the polynucleotide 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.

[0037] In another preferred example, the sequence of the polynucleotide is as shown in SEQ ID NO: 2, 4, 6, 8 or 10.

[0038] In the fifth aspect of the present invention, a vector is provided, wherein the vector contains the polynucleotide as described in the fourth aspect of the present invention.

[0039] 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.

[0040] In the seventh aspect of the present invention, a drug conjugate is provided, wherein the drug conjugate comprises:

[0041] (a) the self-assembling miniprotein as described in the first aspect of the present invention, the fusion protein as described in the second aspect of the present invention, or the multimeric protein as described in the third aspect of the present invention; and

[0042] (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0043] In another preferred embodiment, the coupling moiety is a drug or a toxin.

[0044] In another preferred embodiment, the coupling moiety is a detectable label.

[0045] In another preferred embodiment, the conjugate is selected from the following group: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents.

[0046] In an eighth aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0047] (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

[0048] (b) a pharmaceutically acceptable carrier.

[0049] In another preferred embodiment, the pharmaceutical composition is used for immunotherapy.

[0050] In another preferred embodiment, the content of component (a) is 0.1-99.9wt%, preferably 10-99.9wt%, and more preferably 70%-99.9wt%.

[0051] In another preferred embodiment, the pharmaceutical composition is in the form of an oral dosage form, an injection, or an external pharmaceutical dosage form.

[0052] In another preferred embodiment, the dosage form of the pharmaceutical composition includes tablets, granules, capsules, oral solutions, or injections.

[0053] 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.

[0054] In another preferred embodiment, the liquid preparation is an aqueous injection preparation.

[0055] In another preferred embodiment, the pharmaceutically acceptable carrier includes: a surfactant, a solution stabilizer, an isotonicity regulator, a buffer, or a combination thereof.

[0056] In another preferred embodiment, the subject of administration of the pharmaceutical composition or preparation includes humans or non-human animals.

[0057] In another preferred embodiment, the non-human animals include: rodents (such as rats, mice), primates (such as monkeys).

[0058] In another preferred embodiment, in the administration of the pharmaceutical composition or preparation, the amount administered is 0.01-10 g / day, preferably 0.05-5000 mg / day, and more preferably 0.1-3000 mg / day.

[0059] In another preferred embodiment, the pharmaceutical composition or preparation is used to inhibit and / or treat tumors.

[0060] In another preferred embodiment, for the treatment of tumors, the pharmaceutical composition or preparation can be administered in combination with other anti-tumor drugs.

[0061] In the ninth aspect of the present invention, there is provided a method for preparing the multimeric protein as described in the third aspect of the present invention, comprising the steps of:

[0062] A plurality of self-assembling miniproteins as described in the first aspect of the present invention and / or a plurality of fusion proteins as described in the second aspect of the present invention are contacted with each other to obtain the trimeric protein.

[0063] In the tenth aspect of the present invention, there is provided a method for preparing 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, or the multimeric protein as described in the third aspect of the present invention, comprising the steps of:

[0064] (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 trimer protein; and

[0065] (b) purifying and / or separating the culture obtained in step (a) to obtain the self-assembling small protein or fusion protein or trimer protein.

[0066] 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.

[0067] In another preferred embodiment, the disease is a disease with high expression of PD-L1.

[0068] In another preferred embodiment, the tumor is a tumor that expresses PD-L1 protein (ie, PD-L1 positive).

[0069] 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.

[0070] 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.

[0071] 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 specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] 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.

[0073] Figure 1 The schematic diagram of the spatial structure of the trimer protein is shown, where:

[0074] A is a schematic diagram of the spatial structure of the C3-50-130-11 trimer protein;

[0075] B is a schematic diagram of the spatial structure of the C3-50-130-14 trimer protein;

[0076] C is a schematic diagram of the spatial structure of the C3-50-130-16 trimer protein;

[0077] D is a schematic diagram of the spatial structure of the C3-50-130-18 trimer protein.

[0078] Figure 2 Schematic diagrams showing several structural combinations of the trimeric protein are shown, including:

[0079] A is an antibody Fab, a single-chain antibody (scFv), a receptor protein extracellular region or a ligand, an antibody hinge region (hinge) or linker, and a trimer protein single chain connected in series to form a polypeptide chain, and a specific trimer combination is formed by means of the trimer protein single chain (or fragment) provided by the present invention;

[0080] 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 extracellular 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;

[0081] C is an antibody Fab, a single-chain antibody (scFv), a receptor protein extracellular region or a ligand connected to an antibody Fab, a single-chain antibody (scFv), a receptor protein extracellular 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, and a specific combination is formed with the help of the trimer protein single chain (or fragment) provided by the present invention;

[0082] D is a trimeric protein single chain and an antibody hinge region (hinge) or linker, and an antibody Fab, a single-chain antibody (scFv), a receptor protein extracellular region or a ligand are connected in series through a linker to form a polypeptide chain, and a specific combination is formed by means of the trimeric protein single chain (or fragment) provided by the present invention;

[0083] E is a polypeptide chain formed by connecting antibody Fab, single-chain antibody (scFv), receptor protein extracellular region or ligand with antibody hinge region (hinge) or linker, trimeric protein single chain, antibody hinge region (hinge) or linker, antibody Fab, single-chain antibody (scFv), receptor protein extracellular region or ligand in series, and a specific combination is formed by means of the trimeric protein single chain (or fragment) provided by the present invention.

[0084] Figure 3 It is the molecular screening test result of trimer protein, where:

[0085] A is the molecular weight determination result of the standard product by gel filtration calibration kit;

[0086] B is the molecular sieve test result of C3-50-130-11 trimer protein;

[0087] C is the test result of molecular sieve of C3-50-130-14 trimer protein;

[0088] D is the molecular sieve test result of C3-50-130-16 trimer protein;

[0089] E is the molecular sieve test result of C3-50-130-18 trimer protein.

[0090] Figure 4 is the thermal stability test result of the trimer protein structure, where:

[0091] A is the Tm test result of the trimer protein;

[0092] B is the Tagg detection result of the trimer protein.

[0093] Figure 5 is the thermal recovery test result of the trimer protein structure, where:

[0094] A is the conformational thermal recovery test result of the trimer protein;

[0095] B is the aggregation thermal recovery test result of the trimer protein.

[0096] Figure 6 Binding activity assay of a self-assembled trimer protein targeting PD-L1 is shown. DETAILED DESCRIPTION

[0097] After extensive and in-depth research, the inventors have developed a self-assembling trimeric protein and a preparation method thereof for the first time. The trimeric protein of the present invention comprises three polypeptides of the same sequence, which interact with each other to cause the three polypeptides to spontaneously assemble to form a trimer. On this basis, a binding protein can be connected to the N-terminus and / or C-terminus of the trimeric protein to form a multivalent targeting protein. On this basis, the present invention is completed.

[0098] the term

[0099] In order to more easily understand the present invention, some 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 commonly 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 be changed. 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.

[0100] As used herein, when used in reference to a specific recited numerical 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.).

[0101] As used herein, the terms "comprise", "include", and "contain" are used interchangeably and include not only closed definitions, but also semi-closed and open definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0102] 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 response), ie, has a reasonable benefit / risk ratio.

[0103] 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 acceptable to humans and / or animals. It should be understood by those skilled in the art that the "therapeutically effective amount" may vary depending on the form of the pharmaceutical composition, the route of administration, the adjuvants of the drug used, the severity of the disease, and the combination with other drugs.

[0104] Self-assembling small protein and fusion protein of the present invention

[0105] The present invention provides a self-assembling small protein and a fusion protein or a multimer thereof comprising the small protein.

[0106] As used herein, the terms "small protein of the present invention" and "self-assembling small protein of the present invention" are used interchangeably, and both refer to small proteins that can self-assemble to form polymers (e.g., trimers) as described in the first aspect of the present invention. The small protein of the present invention is composed of a peptide chain, which mainly forms three α-helical secondary structures.

[0107] Preferably, the small protein described in the present invention has an amino acid sequence as shown in SEQ ID NO: 1, 3 or 5.

[0108] 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 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.

[0109] As used herein, the term "polymer" refers to a molecule formed by the combination of multiple small proteins or fusion proteins of the present invention. The term "homopolymer" refers to a molecule formed by the combination of multiple identical small proteins or fusion proteins. The term "heteropolymer" 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 polymer through self-assembly of small protein regions. In one embodiment, the multimer of the present invention is a heteropolymer, 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 a multi-target therapeutic purpose.

[0110] 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,

[0111] M-L1-Fx (Formula I)

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

[0113] Fx-L1-M-L2-Fx (Formula III)

[0114] in,

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

[0116] L1 and L2 are each independently none or a linker;

[0117] Fx is x functional polypeptides;

[0118] “-” represents a peptide bond, connecting peptide or 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 invention is a trimeric protein, and its structural type can be one of the following structures:

[0121] It contains three functional polypeptides - trimer polypeptide single chains;

[0122] Contains a trimer polypeptide single chain - three functional polypeptides;

[0123] It contains three functional polypeptide-functional polypeptide-trimeric polypeptide single chains;

[0124] It comprises a trimer polypeptide single chain-three functional polypeptides-functional polypeptide;

[0125] Contains three functional polypeptides - trimer polypeptide single chain - three functional polypeptides,

[0126] Among them, the trimer polypeptide single chain is the self-assembling small protein of the present invention.

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

[0128] 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 As shown, the functional polypeptide connected to the self-assembling miniprotein can be single or multiple (such as 2, 3 or 4 functional polypeptides in series).

[0129] As used herein, the term "self-assembling small protein" 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, 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 small protein. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding or deleting one or several amino acids at the C-terminus and / or N-terminus usually does not change the structure and function of the protein. The term also includes linear and non-linear polypeptides (such as cyclic peptides).

[0130] The present invention also includes active fragments, derivatives and analogs of the above-mentioned self-assembling small protein or fusion protein (especially the fusion protein formed with the specific targeting peptide). As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the function or activity of the self-assembling small protein or fusion protein of the present invention.

[0131] 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 substitution groups in one or more amino acid residues, or (iii) polypeptides formed by fusion of a polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to this polypeptide sequence (fusion proteins formed by fusion with a leader sequence, a secretory sequence or a tag sequence such as 6His). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0132] A preferred class of active derivatives refers to polypeptides formed by replacing at most 5, preferably at most 3, and more preferably at most 1 amino acid with similar or similar properties compared to the amino acid sequence of the present invention. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.

[0133] Table A

[0134] 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

[0135] The present invention also provides analogs of the self-assembling miniprotein or fusion protein of the present invention. The difference between these analogs and the polypeptide of the present invention may be a difference in amino acid sequence, or a difference in modification form that does not affect the sequence, or both. Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.

[0136] In addition, the self-assembling small protein or fusion protein of the present invention can also be modified. Modifications (usually without changing the primary structure) include: chemical derivatization forms of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing of polypeptides or in further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylation enzyme). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes polypeptides that have been modified to improve their anti-proteolytic properties or optimize their solubility properties.

[0137] 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 include additional coding and / or non-coding sequences.

[0138] The present invention also relates to variants of the above-mentioned 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 known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the self-assembling miniprotein or fusion protein encoded by it.

[0139] 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 can 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 lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably 95%.

[0140] The self-assembling miniprotein or fusion protein and polynucleotide of the present invention are preferably provided in an isolated form, and more preferably, purified to homogeneity.

[0141] The full-length sequence of the polynucleotide of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art are used as templates to amplify and obtain the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each time together in the correct order.

[0142] 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.

[0143] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0144] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. Then the DNA sequence can be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0145] The method of amplifying DNA / RNA using PCR technology is preferably used to obtain the polynucleotides of the present invention. In particular, when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-cDNA terminal rapid amplification method) can be preferably used. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. The amplified DNA / RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

[0146] The present invention also provides a method for forming a trimer between self-assembling small proteins or their fusion proteins, the method comprising contacting a plurality of the self-assembling small proteins and / or fusion proteins to obtain the trimer protein. On the three interaction surfaces of the self-assembling small proteins constituting the trimer, amino acid pairs with interactions are formed to form a trimer protein with specific interactions.

[0147] Expression vector

[0148] 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.

[0149] The polynucleotide sequence of the present invention can be used to express or produce a recombinant fusion protein by conventional recombinant DNA technology. Generally speaking, there are the following steps:

[0150] (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;

[0151] (2) Host cells cultured in a suitable culture medium;

[0152] (3) Isolate and purify proteins from culture medium or cells.

[0153] 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. As long as they can replicate and be stable in the host, any plasmid and vector can be used. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0154] 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.

[0155] Both eukaryotic and prokaryotic host cells can be used for the expression of the self-assembling small protein or its fusion protein of the present invention. The eukaryotic host cells are preferably mammalian or insect host cell culture systems, preferably COS, CHO, NS0, sf9 and sf21 cells; the prokaryotic host cells are preferably one of DH5a, BL21 (DE3) and TG1.

[0156] 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 transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include: lac or trp promoters of Escherichia coli; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, retroviral LTRs and other known promoters that can 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.

[0157] 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.

[0158] The vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins.

[0159] 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 include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, plant cells (such as ginseng cells).

[0160] When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, which act on the promoter to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0161] Those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers and host cells.

[0162] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0163] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture 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 by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0164] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using 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, ultra-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.

[0165] Affinity chromatography can be used to separate and purify a self-assembled small protein or a fusion protein thereof disclosed in the present invention. According to the characteristics of the affinity column used, conventional methods such as high salt buffer, changing pH, etc. can be used to elute the self-assembled small protein or a fusion protein thereof bound to the affinity column.

[0166] 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 ).

[0167] Pharmaceutical composition

[0168] The present invention also provides a pharmaceutical composition containing the self-assembling miniprotein, fusion protein, polymer or drug conjugate of the present invention.

[0169] 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, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. 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 milligrams / kg body weight per day. In addition, the polypeptide of the present invention can also be used 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 the therapeutic effect 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 protecting the multifunctional groups of the protein to prevent its degradation (including but not limited to coagulation, deamination or oxidation). The preparation can be in various forms. Under normal circumstances, for liquid preparations, it can usually be stably stored for at least one year at 2°C-8°C, and for freeze-dried preparations, it can be stable at 30°C for at least six months. Here, the preparation can be a suspension, water injection, freeze-dried preparation commonly used in the pharmaceutical field, preferably a water injection or freeze-dried preparation.

[0170] For the pharmaceutical composition of the present invention (such as aqueous injection or lyophilized preparation), the pharmaceutically acceptable excipients include one or a combination of surfactants, solution stabilizers, isotonicity regulators and buffers, wherein the 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; MONAQUATTM, etc., and the amount of addition should minimize the granulation tendency of the protein. The solution stabilizer can be a carbohydrate, including reducing sugars and non-reducing sugars, amino acids include monosodium glutamate or histidine, and alcohols include one or a combination of triols, higher sugar alcohols, propylene glycol, polyethylene glycol, and the amount of solution stabilizer added should be such that the final preparation formed can maintain a stable state within a stable period of time as considered by technicians in the field. The isotonicity regulator can be one of sodium chloride and mannitol, and the buffer can be one of TRIS, histidine buffer, and phosphate buffer.

[0171] 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 usually at least about 50 micrograms / kg body weight, and in most cases does not exceed about 100 milligrams / kg body weight, preferably the dose is about 100 micrograms / kg body weight-about 50 milligrams / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians. Typically, usually, the total dosage cannot exceed a certain range, for example, the dose for intravenous injection is 10 to 3000 mg / day / 50kg.

[0172] The trimeric protein of the present invention and the pharmaceutical preparation containing the same can be used as anti-tumor drugs for tumor treatment. The anti-tumor drugs referred to in the present invention refer to drugs that have the function of inhibiting and / or treating tumors, which may 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.

[0173] The above-mentioned trimer protein and its pharmaceutical preparation can also be co-administered with other anti-tumor drugs for the treatment of tumors. These anti-tumor drugs for co-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 methyl sulfonates; platinum compounds such as cisplatin, carboplatin, and oxaloplatin; 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 gemcitabine (Gemz), (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: serotonin, enantone, etc.; 3. 1. Physiological response modifiers: mainly inhibit tumor interferon through the body's immune function; interleukin-2; thymosin peptides; 4. Monoclonal antibodies: MabThera; Cetuximab (C225); Herceptin (Trastuzumab); Bevacizumab (Avastin); Yervoy (Ipilimumab); 5. Others include some drugs whose mechanisms are currently unknown and require further research; cell differentiation inducers such as retinoids; apoptosis inducers.

[0174] The main advantages of the present invention include:

[0175] 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 trimer protein to form a multivalent targeting protein;

[0176] 2) The self-assembling small protein of the present invention has strong thermal stability and conformational thermal recovery.

[0177] 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 present invention. The experimental methods in the following examples where specific conditions are not specified are usually 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 under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0178] Example 1: Construction, expression and purification of self-assembling trimer protein

[0179] The trimer protein nucleotide sequence was synthesized by gene synthesis, and the sequence was loaded into the pET29b vector in the form of MGS-trimer protein nucleotide sequence-GSENLYFQSLEHHHHHH (ENLYFQSLE sequence was used for enzyme digestion, HHHHHH was used for protein purification). After the vector was transformed into Escherichia coli, it was cultured in LB medium at 37°C and 270rpm until OD600=0.6. Then 1mM IPTG was used to induce bacterial protein expression overnight. After centrifugation and resuspending, the bacteria were broken by a low-temperature mechanical crusher and then centrifuged at high speed to obtain the supernatant. After purification by Ni column, the protein sample was desalted and concentrated, and the concentration of the purified protein was measured by a micro-spectrophotometer. The purified protein was filtered and sterilized with a 0.22um filter membrane and placed in a 4°C refrigerator for subsequent experiments.

[0180] Example 2: Evaluation of the assembly efficiency of self-assembling trimer proteins

[0181] 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 conductivity and other values ​​are stable, take 500ul of purified protein for sample loading, and correct and zero the UV absorption 215 and UV absorption 280. Observe the changes in the UV absorption peak and collect the samples. Calculate the molecular weight standard curve using the Gel Filtration Calibration Kit (Gel Filtration Calibration KitLMW) and use OD 280 The peak elution volume parameters and peak area are used to evaluate the protein molecular weight and protein purity. This method can be used to obtain high-purity candidate proteins for subsequent experiments. Figure 3 As shown, proteins with corresponding molecular weights were collected and set aside for later use.

[0182] Example 3: Thermal stability test of self-assembled trimer structure of small monomeric proteins

[0183] The thermal stability of the protein structure was tested with the help of Unchained Labs' all-purpose protein stability analyzer Uncle. The sample heating range was 25℃-95℃, the heating rate was 0.5℃ / min, and static light scattering was measured at an excitation wavelength of 266nm. As the protein unfolds, the fluorescence usually decreases and shifts to a longer wavelength. The data analysis software determines the Tm value based on the center of gravity mean (BCM) of the intensity curve of the fluorescence at 300-+430nm, and observes 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 are obtained, and the structural stability of the binding protein is evaluated.

[0184] like Figure 4 As shown, the denaturation curves show that the samples have no obvious Tm values; the aggregation curves show that the samples have no obvious aggregation as the temperature increases, showing super strong thermal stability.

[0185] Example 4: Thermal recovery test of self-assembled trimer structure of small monomeric proteins

[0186] The thermal recovery of protein structure was tested with the help of Uncle, an all-purpose protein stability analyzer from 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, to detect changes in protein conformation and aggregation during the heating and cooling process. Figure 5 As shown in A, as the temperature of the sample increases, the conformation opens up, and when the temperature decreases, the conformation folds back. The thermal recovery ability of the sample conformation is relatively good. Figure 5 As shown in B, with the change of temperature, the aggregation signal changes little and there is no obvious aggregation.

[0187] Example 5: Binding activity detection of self-assembled trimer protein targeting PD-L1

[0188] In this embodiment, the affinity detection of high-affinity blocking protein was performed with the aid of ForteBio Octet. First, 3 μg / ml of biotin-labeled human PD-L1 protein was loaded onto the detection probe coupled with avidin (1200s), and the unbound biotin-labeled human PD-L1 protein was eluted in a PBST solution. Then the detection probe with human PD-L1 protein was simultaneously immersed in a two-fold diluted targeted PD-L1 self-assembled trimer protein solution to detect the binding signal (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 trimer protein was calculated. The monomers constituting the trimer 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.

[0189] 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, and their affinity was 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.

[0190] Table 2 Sequences of the present invention

[0191]

[0192]

[0193]

[0194] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

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, 3 or 5; (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, 3 or 5 and is capable of spontaneously forming a trimer protein.

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

3. The fusion protein according to claim 2, characterized in that 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 as described in 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, characterized in that 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 trimer 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 as claimed in claim 8, or the polynucleotide as claimed in 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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