Self-assembled trimeric protein and preparation method therefor

Abstract

Provided are a self-assembled trimeric protein and a preparation method therefor. Specifically, provided are an individual polypeptide for forming the trimeric protein, a nucleic acid sequence encoding the polypeptide, and the method for forming the trimeric protein.

Description

A self-assembling trimeric protein and a 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 dimeric 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, the small protein comprising an amino acid sequence selected from the group consisting of:

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

[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 or 5, and capable of spontaneously forming a trimeric protein.

[0008] In a second aspect of the present application, a fusion protein is provided, the fusion protein comprising 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] L1and L2are 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: 11.

[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: 12.

[0024] In another preferred embodiment, the fusion protein has an amino acid sequence as set forth in SEQ ID NO: 7 or 9.

[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 as described in the first aspect of the present application, a fusion protein as described in the second aspect of the present application, or a combination thereof.

[0026] In another preferred embodiment, the multimeric protein is a trimeric protein, which comprises or consists 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 identical or different functional polypeptides.

[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, or 10.

[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 label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed 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 oral dosage form, injection, or external pharmaceutical dosage form.

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

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

[0051] In another preferred embodiment, the liquid preparation 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 preparation is a human or a non-human animal.

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

[0055] In another preferred embodiment, in the administration of the pharmaceutical composition or preparation, 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 preparation is used for inhibiting and / or treating a tumor.

[0057] In another preferred embodiment, for the treatment of tumor, the pharmaceutical composition or preparation 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 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, so as to obtain 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 steps of:

[0061] (a) culturing the host cell as described in the sixth aspect of the present application under suitable conditions, so as to obtain 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), so as to obtain 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 drug 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, 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 steps of: administering 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, or the drug conjugate as described in the seventh aspect of the present application to a subject in need.

[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 described in detail below (e.g., in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0069] The following drawings are used to illustrate the specific embodiments of the present application, and are not used to limit the scope of the present application 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 C3-50-130-11 trimeric protein;

[0072] B is a schematic diagram of the spatial structure of C3-50-130-14 trimeric protein;

[0073] C is a schematic diagram of the spatial structure of C3-50-130-16 trimeric protein;

[0074] D is a schematic diagram of the spatial structure of C3-50-130-18 trimeric protein.

[0075] Figure 2 shows a schematic diagram of several structural combinations of trimeric proteins, wherein:

[0076] A is that an antibody Fab, a single-chain antibody (scFv), an extracellular region of a receptor protein, or a ligand is connected to an antibody hinge region or a linker and a trimeric protein single chain in series to form a polypeptide chain, and a specific trimeric combination is formed by means of the trimeric protein single chain (or fragment) provided by the present application;

[0077] B is that a trimeric protein single chain is connected to an antibody hinge region or a linker, an antibody Fab, a single-chain antibody (scFv), an extracellular region of a receptor protein, or a ligand in series to form a polypeptide chain, and a specific trimeric combination is formed by means of the trimeric protein single chain (or fragment) provided by the present application;

[0078] C is that an antibody Fab, a single-chain antibody (scFv), an extracellular region of a receptor protein, or a ligand is connected to an antibody Fab, a single-chain antibody (scFv), an extracellular region of a receptor protein, or a ligand through a linker, and then connected to an antibody hinge region or a linker and a trimeric protein single chain in series 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 application;

[0079] D is a trimeric protein single chain and antibody hinge or linker, and antibody Fab, single chain antibody (scFv), receptor protein extracellular region or ligand in series to form a polypeptide chain, and the trimeric protein single chain (or fragment) provided by the application forms a specific combination;

[0080] E is antibody Fab, single chain antibody (scFv), receptor protein extracellular region or ligand and antibody hinge or linker, trimeric protein single chain, antibody hinge or linker, antibody Fab, single chain antibody (scFv), receptor protein extracellular region or ligand in series to form a polypeptide chain, and the trimeric protein single chain (or fragment) provided by the application forms a specific combination.

[0081] Figure 3 is the detection result of trimeric protein molecular sieve, wherein:

[0082] A is the standard molecular weight determination result of gel filtration calibration kit;

[0083] B is the detection result of C3-50-130-11 trimeric protein molecular sieve;

[0084] C is the detection result of C3-50-130-14 trimeric protein molecular sieve;

[0085] D is the detection result of C3-50-130-16 trimeric protein molecular sieve;

[0086] E is the detection result of C3-50-130-18 trimeric protein molecular sieve.

[0087] Figure 4 is the detection result of trimeric protein structure thermal stability, wherein:

[0088] A is the Tm detection result of trimeric protein;

[0089] B is the Tagg detection result of trimeric protein.

[0090] Figure 5 is the detection result of trimeric protein structure thermal recovery, wherein:

[0091] A is the conformational thermal recovery detection result of trimeric protein;

[0092] B is the aggregation thermal recovery detection result of trimeric protein.

[0093] Figure 6 shows the binding activity detection of self-assembly trimeric protein targeting PD-L1. DETAILED DESCRIPTION

[0094] The present inventors have made extensive and in-depth research and first developed a self-assembling trimeric protein and a preparation method thereof. The trimeric protein of the present application comprises three polypeptides with 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-terminus and / or C-terminus of the trimeric protein, thereby forming a multivalent targeting protein. On this basis, the present application is completed.

[0095] The terms

[0096] For the purpose of facilitating the understanding of the present application, certain technical and scientific terms are defined below in detail. Unless otherwise defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one 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 particular methodology and experimental conditions described, as such methodology and conditions can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, the scope of the present application being limited only by the appended claims.

[0097] As used herein, the term "about," when used in reference to a numerically recited 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.).

[0098] As used herein, the terms "comprising", "including", "containing", are interchangeable and are meant to encompass both the open- and the semi-closed-ended definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0099] As used herein, the term "pharmaceutically acceptable carrier" means a material that is not biologically or otherwise undesirable, i.e., with respect to the patient and / or the administration of a drug, has a reasonable benefit / risk ratio in connection with its administration.

[0100] As used herein, the term "therapeutically effective amount" means an amount that produces a functional or a biological activity and is acceptable to the person and / or the animal. It should be understood by those skilled in the art that the "therapeutically effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and the combination with other drugs, etc.

[0101] Self-assembling small protein and fusion protein of the present application

[0102] In the present application, a self-assembling small protein and a fusion protein comprising the small protein or a multimer thereof are provided.

[0103] As used herein, the terms "small protein of the invention", "self-assembling small protein of the invention" are used interchangeably and refer to a small protein that is capable of self-assembling to form a multimer (e.g. a trimer) as described in the first aspect of the invention. The small protein of the invention is composed of one peptide chain and mainly forms three alpha-helix secondary structures.

[0104] Preferably, the small protein of the invention has an amino acid sequence as set forth in SEQ ID NO: 1, 3 or 5.

[0105] As used herein, the term "fusion protein of the invention" refers to a fusion protein of a self-assembling small protein of the invention with 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.

[0106] As used herein, the term "multimer" refers to a molecule formed by the association of multiple small proteins or fusion proteins of the invention. The term "homomultimer" refers to a molecule formed by the association of multiple identical small proteins or fusion proteins. The term "heteromultimer" refers to a molecule formed by the association of multiple different small proteins or fusion proteins. The multiple monomers in a multimer of the invention form the multimer through the self-assembly of the small protein region. In one embodiment, the multimer of the invention is a heteromultimer containing multiple fusion proteins comprising different functional polypeptides. For example, the multimer of the invention can comprise multiple fusion proteins each having a functional polypeptide targeting a different target, thereby achieving the purpose of multi-target therapy.

[0107] Typically, the fusion protein of the invention 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)

[0108] wherein,

[0109] M is a self-assembling small protein as described in claim 1 ;

[0110] L1and L2are each independently absent or a linker;

[0111] Fx is x functional polypeptides;

[0112] "-" represents a peptide bond, a connecting peptide or a linker connecting the above elements;

[0113] wherein x is an integer selected from 1, 2, 3 or 4.

[0114] Typically, the multimer of the invention is a trimeric protein, which can be of one of the following structural types:

[0115] comprising three functional polypeptides - a trimeric polypeptide single chain;

[0116] comprising a trimeric polypeptide single chain - three functional polypeptides;

[0117] comprising three functional polypeptides - a functional polypeptide - a trimeric polypeptide single chain;

[0118] comprising a trimeric polypeptide single chain - three functional polypeptides - a functional polypeptide;

[0119] comprising three functional polypeptides - a trimeric polypeptide single chain - three functional polypeptides,

[0120] wherein the trimeric polypeptide single chain is the self-assembling small protein of the present application.

[0121] In another preferred embodiment, the functional polypeptide is selected from the group consisting of Fab, scFv, fusion receptor or ligand.

[0122] It should be understood that the above structural types are merely exemplary forms and do not limit the present application. Some representative structures are shown in Figure 2. In these structures, the functional polypeptides linked to the self-assembling small protein can be single or multiple (e.g., 2, 3 or 4 functional polypeptides in tandem).

[0123] 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, deletion, insertion and / or substitution of 1-3 (usually 1-2, more preferably 1) amino acids, addition or deletion of one or several (usually 3 or less, preferably 2 or less, more preferably 1 or less) amino acids at the C-terminus and / or N-terminus, or addition of an amino acid fragment with small side chain as linker (e.g., glycine, serine, etc.) at the N- or C-terminus of the small protein. For example, in the art, substitution with an amino acid having similar or similar properties usually does not change the function of the protein. For another example, addition or deletion of 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 (e.g., cyclic peptides).

[0124] The present application also includes active fragments, derivatives and analogs of the above self-assembling small proteins or fusion proteins (especially fusion proteins formed with specific targeting peptides). As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially maintain the function or activity of the self-assembling small proteins or fusion proteins of the present application.

[0125] The polypeptide fragments, derivatives or analogs of the present application can be (i) a polypeptide having one or more conservative or non-conservative amino acid residue substitutions (preferably conservative amino acid residue substitutions), (ii) a polypeptide having a substituent group at one or more amino acid residues, (iii) a polypeptide fused to another compound (such as a compound that extends the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) a polypeptide having an additional amino acid sequence fused to the polypeptide sequence (such as a fusion protein having a leader sequence, a secretion sequence or a 6His tag sequence). These fragments, derivatives and analogs are within the scope of those skilled in the art in light of the teachings herein.

[0126] One preferred class of active derivatives are polypeptides having up to 5, preferably up to 3, more preferably up to 1 amino acid replaced by a similar or related amino acid compared to the amino acid sequence of the present application. These conservative variant polypeptides are preferably generated by making amino acid substitutions in accordance with Table A.

[0127] Table A

[0128] 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 in terms of the amino acid sequence, in terms of modifications that do not affect the sequence, or both. Analogues also include those 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 understood that the polypeptides of the present application are not limited to the representative polypeptides exemplified above.

[0129] 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 during synthesis and processing or further processing steps. Such modifications can be accomplished by exposing the polypeptide to an enzyme that glycosylates (e.g., a mammalian glycosylating enzyme or deglycosylating enzyme). Modifications also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

[0130] The term "polynucleotide of the present application" can be a polynucleotide that encodes a self-assembling small protein or fusion protein of the present application, or a polynucleotide that further includes additional coding and / or non-coding sequences.

[0131] The present application also relates to variants of the above polynucleotides, which 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 known in the art, an allelic variant is an alternative form of a polynucleotide which can be one or more nucleotides different from the corresponding sequence, but which do not change the functional properties of the encoded self-assembling small protein or fusion protein substantially.

[0132] The present application also relates to polynucleotides which hybridize to the above sequences and which 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 which 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, etc.; or (3) hybridization only when the identity between the two sequences is at least 90%, more preferably 95% or more.

[0133] The self-assembling small proteins or fusion proteins and polynucleotides of the present application are preferably provided in isolated form, more preferably, purified to homogeneity.

[0134] The full-length sequences of the polynucleotides of the present application can be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed according to the nucleotide sequences disclosed herein, especially the open reading frame sequences, and a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art is used as a template for amplification. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

[0135] Once the relevant sequence is obtained, recombination can be used to obtain the relevant sequence in large quantities. This is usually done by cloning into a vector, then transforming into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods.

[0136] In addition, artificial synthesis can also be used to synthesize the relevant sequence, especially when the length of the fragment is relatively short. Usually, a long fragment of the sequence can be obtained by first synthesizing a plurality of small fragments and then ligating them together.

[0137] At present, it is possible to obtain the DNA sequence encoding the protein (or fragment thereof, or derivative thereof) of the present application entirely by chemical synthesis. Then the DNA sequence can be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art.

[0138] The method of amplifying DNA / RNA by PCR 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- rapid amplification of cDNA ends) can be preferably used, and primers for PCR can be appropriately selected based on the sequence information of the present application disclosed herein and synthesized by a conventional method. The amplified DNA / RNA fragment can be separated and purified by a conventional method such as gel electrophoresis.

[0139] The present application also provides a method of forming a trimer between self- assembling small proteins or fusion proteins thereof, the method comprising contacting a plurality of said self-assembling small proteins and / or fusion proteins with each other, thereby obtaining said trimeric protein. On three interaction surfaces of the self-assembling small proteins constituting said trimer, interaction-forming amino acid pairs are formed to form a specific interaction trimeric protein.

[0140] Expression vector

[0141] 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 polynucleotide sequence encoding the self-assembling small protein or fusion protein of the present application, and a method of producing the polypeptide of the present application by a recombination technique.

[0142] The polynucleotide sequence of the present application can be used to express or produce a recombinant fusion protein by a conventional recombinant DNA technique. In general, the following steps are involved:

[0143] (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;

[0144] (2) culturing the host cell in a suitable medium;

[0145] (3) isolating and purifying the protein from the medium or the cell.

[0146] 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 vectors well known in the art. Any plasmid and vector can be used as long as it can replicate and be stable in the host. An important feature of the expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and a translation control element.

[0147] In the method for preparing the self-assembling small protein or fusion protein thereof according to the present application, any suitable vector can be used, which can be selected from one of pET, pDRl, pcDNA3.1(+), pcDNA3.1 / ZEO(+), pDHFR, and the expression vector includes the fusion DNA sequence linked with suitable transcription and translation regulatory sequences.

[0148] Both eukaryotic and prokaryotic host cells can be used for the expression of the self-assembling small protein or fusion protein thereof according to the present application, and the eukaryotic host cell is preferably a mammalian or insect host cell culture system, preferably cells such as COS, CHO, NSO, sf9 and sf21; and the prokaryotic host cell is preferably one of DH5a, BL21(DE3), and TG1.

[0149] Methods well known to those skilled in the art can be used to construct the expression vector containing the DNA sequence encoding the fusion protein according to the present application and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be operably linked to a suitable promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: the lac or trp promoters of E. coli; the PL promoter of lambda phage; 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 includes a ribosome binding site for translation initiation and a transcription terminator.

[0150] 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 in mammalian cells, and green fluorescent protein (GFP) in bacteria, or tetracycline or ampicillin resistance in E. coli.

[0151] The vector containing the appropriate DNA sequence as described above and a suitable promoter or control sequence can be used to transform an appropriate host cell to enable it to express the protein.

[0152] The host cell can be a prokaryotic cell such as a bacterial cell; or a lower eukaryotic cell such as a yeast cell; or a higher eukaryotic cell such as a mammalian cell. Representative examples are: bacterial cells of E. coli, Streptomyces; Salmonella typhimurium; fungal cells such as yeast, plant cells (such as ginseng cells).

[0153] The polynucleotide of the present application, when expressed in higher eukaryotic cells, will be transcribed more efficiently if an enhancer sequence is inserted in the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, which act on a promoter to increase the transcription of a gene. Examples of enhancers include the SV40 enhancer, which is about 250 base pairs in length and is located near the late side of the replication origin; the polyoma enhancer, which is about 100 base pairs in length and is located near the replication origin; the adenovirus enhancer, which is about 200 base pairs in length and contains two 55 base pair repeat motifs; and the enhancer of the long terminal repeat (LTR) of the Rous Sarcoma Virus.

[0154] The selection of the appropriate vector, promoter, enhancer, and host cell is a matter of choice depending on the requirements of the particular application and the polynucleotide of the present application.

[0155] The transformation of host cells with recombinant DNA can be performed using conventional techniques known to those skilled in the art. When the host is a prokaryote, such as E. coli, competent host cells can be made by the method of Competent Cell Preparation, which is well known in the art. Another method is to use MgCl2. If desired, the transformation can be performed by electroporation. When the host is a eukaryote, DNA can be introduced into the host cell by methods such as calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, and liposome packaging.

[0156] The resulting transformants can be cultured in conventional media using conventional techniques, the medium being selected so as to provide an appropriate growth medium for the host cell. The culture conditions, such as temperature, pH and the like, are those under which the host cell grows optimally. When the host cell has grown to an appropriate cell density, the selected promoter is induced by the appropriate method (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period.

[0157] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If desired, the recombinant protein can be isolated 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 lysis, ultra-treatment, ultra-centrifugation, 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.

[0158] The self-assembling small protein or its fusion protein disclosed in the present application can be isolated and purified using affinity chromatography. Depending on the properties of the affinity column used, the self-assembling small protein or its fusion protein bound to the affinity column can be eluted using conventional methods such as high-salt buffer, changing the pH, etc.

[0159] By the above method, the self-assembling small protein or fusion protein thereof can be purified into a substantially uniform substance, such as a single protein peak of similar molecular weight in a molecular sieve (OD 280 or OD 210 ).

[0160] Pharmaceutical composition

[0161] In the present application, a pharmaceutical composition containing the self-assembling small protein, fusion protein, multimer or drug conjugate of the present application is also provided.

[0162] 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 and a solution should be 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 together 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 generally, for liquid preparations, it can be stably stored at 2-8°C for at least one year, and for freeze-dried preparations, it can be stably stored at 30°C for at least six months. Here, the preparation can be a suspension, water needle, freeze-dried preparation, etc. commonly used in the pharmaceutical field, and the water needle or freeze-dried preparation is preferred.

[0163] For the pharmaceutical composition (such as water injection or lyophilized preparation) of the present application, the pharmaceutically acceptable adjuvant includes one or a combination of a surface active agent, a solution stabilizer, an isotonicity adjusting agent and a buffer, wherein the surface active agent includes a non-ionic surface active agent such as polyoxyethylene sorbitan fatty acid ester (Tween 20 or 80), poloxamer (such as 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 the protein to be granulated, 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 one or a combination of a trihydric alcohol, a higher sugar alcohol, propylene glycol, polyethylene glycol, 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.

[0164] The pharmaceutical composition is used by administering a safe and effective amount of the small protein or fusion protein or immunoconjugate thereof of the present application to a mammal, wherein the safe and effective amount is generally at least about 50 μg / kg body weight and in most cases not more than about 100 mg / kg body weight, preferably the dose is about 100 μg / kg body weight to about 50 mg / kg body weight. Of course, the specific dose will also take into account a route of administration, the condition of the patient, etc., which are all within the skill of a skilled medical practitioner. Typically, the total amount administered will generally not exceed a certain range, for example, the dose for intravenous injection is 10 to 3000 mg / day / 50 kg.

[0165] 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 an inhibitory and / or therapeutic effect on a tumor, which can include a delay in the development of symptoms associated with tumor growth and / or a decrease in the severity of these symptoms, and further includes a reduction in symptoms associated with the growth of an already existing tumor and prevention of the occurrence of other symptoms, and also reduces or prevents metastasis.

[0166] The trimeric protein and its pharmaceutical preparation can also be combined with other anti-tumor drugs for the treatment of tumors. These anti-tumor drugs for combined administration include but are not limited to: 1. Cytotoxic drugs (1) Drugs 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; mytomycin (MMC); (2) Drugs affecting nucleic acid synthesis: dihydrofolate reductase inhibitors such as methotrexate (MTX) and Alimta; thymidine synthetase inhibitors such as fluorouracil (5FU, FT-207, capecitabine) and the like; purine nucleoside synthetase inhibitors such as 6-mercaptopurine (6-MP) and 6-TG and the like; nucleotide reductase inhibitors such as hydroxyurea (HU) and the like; DNA polymerase inhibitors such as cytarabine (Ara-C) and gemcitabine (Gemz) and the like; (3) Drugs acting on nucleic acid transcription: drugs selectively acting on DNA templates, inhibiting DNA-dependent RNA polymerase, thereby inhibiting RNA synthesis such as: actinomycin D, daunorubicin, doxorubicin, epirubicin, aclacinomycin, and the like; (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 and the like; aromatase inhibitors: aminoglutethimide, lantronex, letrozole, arimidex and the like; anti-androgens: flutamide, RH-LH agonists / antagonists: zoladex, enantone and the like; 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.

[0167] The main advantages of the present application include:

[0168] 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;

[0169] 2) The self-assembling small proteins of the present application have strong thermal stability and conformational thermal recovery.

[0170] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate but not to limit the scope of the application. The experimental methods in the following examples, if not otherwise specified, are generally carried out according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions suggested by the manufacturer. Unless otherwise specified, the percentages and parts are weight percentages and weight parts.

[0171] Example 1: Construction, expression and purification of self-assembling trimeric protein

[0172] The trimeric protein nucleotide sequence was synthesized by gene synthesis method, and the sequence was loaded into pET29b vector according to the sequence form of MGS-trimeric protein nucleotide sequence-GSENLYFQSLEHHHHHH (ENLYFQSLE sequence for enzyme cutting, HHHHHH for protein purification). After transforming the vector into E. coli, the bacteria were cultured in LB medium at 37°C and 270 rpm until OD600=0.6. Then the bacteria were induced to express proteins overnight using 1 mM IPTG. After centrifugation to collect the bacteria and resuspension, the bacteria were broken by low-temperature mechanical disrupter 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 determined by microspectrophotometer. The purified protein was filtered by 0.22 um filter membrane to remove bacteria and stored in 4°C refrigerator for subsequent experiments.

[0173] Example 2: Evaluation of assembly efficiency of self-assembling trimeric protein

[0174] The protein purified by Ni column was further purified using AKTA pure protein purification system. Superdex TM 75 Increase 10 / 300 GL gel filtration column was used to replace 20% ethanol in the column with low flow rate of 0.45 ml / min, and then 1x PBS buffer was used to equilibrate the system at a flow rate of 0.75 ml / min. After the pressure conductivity values were stable, 500 ul of purified protein was loaded, and the ultraviolet absorption 215 and ultraviolet absorption 280 were corrected and zeroed. The change of ultraviolet absorption peak was observed to collect the sample. The molecular weight standard curve was calculated by gel filtration calibration kit (Gel Filtration Calibration Kit LMW), and the protein molecular weight and protein purity were evaluated by the peak elution volume parameter and the peak area. High purity candidate protein was obtained by this method for subsequent experiments. As shown in Figure 3, the protein corresponding to the molecular weight was collected and used. 280 Peak elution volume parameter and peak area were used to evaluate the protein molecular weight and protein purity. High purity candidate protein was obtained by this method for subsequent experiments. As shown in Figure 3, the protein corresponding to the molecular weight was collected and used.

[0175] Example 3: Thermal stability detection of self-assembled trimeric structure of monomeric small protein

[0176] The thermal stability of the protein structure was detected by means of the Unchained Labs all-purpose 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. The fluorescence generally decreases and shifts to a longer wavelength as the protein unfolds. The data analysis software determines the Tm value according to the barycentric average (BCM) of the intensity curve of the fluorescence at 300-430 nm, and the light intensity scattering 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.

[0177] As shown in FIG. 4, the denaturation curve shows that none of the samples has a significant Tm value; the aggregation curve shows that there is no significant aggregation of the samples as the temperature increases, indicating that the samples have super-strong thermal stability.

[0178] Example 4: Thermal recovery detection of self-assembled trimeric structure of monomeric small protein

[0179] The thermal recovery of the protein structure was detected by means of the Unchained Labs all-purpose 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.

[0180] Example 5: Binding activity detection of self-assembled trimeric protein targeting PD-L1

[0181] 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 PBST solution. Then the detection probe with human PD-L1 protein was simultaneously immersed in a solution of twice-diluted target PD-L1 self-assembled trimeric protein, 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 trimeric protein was calculated. The monomers constituting the trimeric protein are composed of the high-affinity small protein PD-L1-3 (SEQ ID NO: 11) targeting PD-L1 and the self-assembled small protein C3_50_130-18 of the present application.

[0182] As shown in Figure 6, PD-L1-3-C3-50-130-18 (SEQ ID NO: 7) and C3-50-130-18-PD-L1-3 (SEQ ID NO: 9) both showed super strong binding activity, and the affinity was unexpectedly stronger than that of the PD-L1-3 small protein monomer, beyond the detection upper limit of the BLI device, reaching the level of femto.

[0183] Table 2 Sequences of the present application

[0184] All documents mentioned in the present application are incorporated herein 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 combined with one another in the application.

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 or 5; (2) an amino acid sequence with homology ≥ 90% (preferably ≥ 95%, more preferably ≥ 98%) to the amino acid sequence as set forth in SEQ ID NO: 1, 3 or 5, 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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