CN117886945A - Ferritin fusion, nanoparticle thereof and application thereof - Google Patents

Abstract

The present disclosure relates to ferritin fusion, nanoparticles formed from the fusion, and their use in the preparation of a medicament, in particular, ferritin fusion carrier platforms, self-assembled nanoparticles, which are capable of supporting exogenous bioactive moieties of different functions and maintaining their original functions, have multiple uses as carrier platforms.

Description

Ferritin fusion, nanoparticle thereof and application thereof

Technical Field

The present disclosure is in the field of recombinant proteins. In particular, the present disclosure relates to ferritin fusion and nanoparticles formed by self-assembly of the fusion and uses thereof, and to molecules with different bioactive functions carried by ferritin to achieve different biological functions such as treatment, targeting, labeling, tracing, modification, and the like.

Background

Natural ferritin is composed of two different ferritin subunits (H subunit and L subunit) with molecular masses of 21kDa and 19kDa, respectively. Typical ferritin structures are spherical shell structures formed by self-assembly of 24 light chain/heavy chain subunits, with an outer diameter of 12nm and an inner cavity structure with a diameter of 8nm formed inside. Ferritin fusion proteins have been reported to be prepared by recombinant means, i.e. fusion of peptides, proteins (e.g. antibodies, antigen fragments, ligands capable of binding to receptors, targeting peptides, therapeutic peptides, labelling peptides, receptor binding peptides, cleavage peptides, signal peptides, pro-apoptotic peptides, fluorescent proteins, etc.) with the N-or C-terminus of human ferritin HFn in order to confer targeting, tracer, therapeutic and prophylactic properties to HFn (see documents for fusion means construction: WO2017039382A1, WO2016122259A1, KR-2018008349, WO 2013055055508A 2, WO 2018010252A 1, CNC104017088A, "Ferritin nanocages with biologically orthogonal conjugation for vascular targeting and imaging, bioconjugate Chem,2018,29,4,1209-1218", etc.) with biological functions, as well as prolonged half-life of exogenous bioactive moieties, enhanced immunogenicity, increased single molecule titers, etc. At present, ferritin fusion proteins with specific biological activities can be obtained stably, rapidly and economically through a recombinant expression mode, and the ferritin fusion platform is considered to have good drug carrier development potential and drug property.

However, in the construction of fusion proteins, it is conventional to attach the target protein sequence to be fused to the N-or C-terminus of the ferritin coding sequence, which is believed to be likely to allow at least one end of the target protein to be active and facilitate folding into a spatially active conformation. In fact, it has been found in practice that a large amount of the desired fragment cannot be expressed smoothly, or that it is difficult to maintain the original biological function of the desired protein after expression. Therefore, development of a ferritin carrier platform suitable for protein expression of a greater variety of categories is a need to expand the use of ferritin in a greater number of fields.

Disclosure of Invention

In the process of developing ferritin carrier systems with different bioactive functions by using a genetic engineering recombination method, according to the protein high-level structural characteristics of a ferritin self-assembled caged protein structure, the results of different exogenous fragment insertion regions and different exogenous connection positions are compared, and according to unexpected findings, compared with the traditional method of sequentially connecting target proteins at the N end or the C end of ferritin monomer subunits, the method has the advantages that the target proteins are easier to be expressed and the biological activity of the exogenous target proteins is improved. The discovery of the insertion site expands the variety of exogenous proteins which can use ferritin as a carrier, so that the ferritin carrier platform has wider application fields and wider application.

The present disclosure contemplates the use of exogenous biologically active fragments of different sequences, linked or inserted into different loop regions of ferritin, AB loop, BC loop, CD loop and DE loop, while comparing the effect of N-terminal and C-terminal sequential linkages, thereby creatively finding several modes of construction, selecting the insertion of exogenous fragments in DE loop regions, more conducive to protein expression, and formation of the active conformation of the inserted fragments.

The ferritin monomer subunit sequences used for constructing the fusion protein can be selected from wild type or variants of ferritin monomer subunits subjected to point mutation modification, and the variants have the advantages of reduced aggregate proportion, increased hydrophilicity and increased bioavailability after self-assembly to form ferritin.

Specifically, the present disclosure proposes the following technical solutions:

in one aspect, the present disclosure provides a ferritin fusion.

In one aspect, the present disclosure provides a nanoparticle comprising the foregoing ferritin fusion.

In one aspect, the present disclosure provides a method for producing the foregoing ferritin fusion or nanoparticle.

In one aspect, the present disclosure provides a composition comprising the foregoing ferritin fusion or nanoparticle.

In one aspect, the present disclosure provides the use of the foregoing ferritin fusion, nanoparticle, and composition in the preparation of a targeting molecule, therapeutic molecule, detection molecule, tracer molecule, regulatory molecule, or modification molecule.

In one aspect, the present disclosure provides the use of the foregoing ferritin fusion, nanoparticle or composition in the preparation of a targeted anti-cancer drug.

In one aspect, the present disclosure provides the foregoing ferritin fusions, nanoparticles, and compositions for preparing targeting, therapeutic, detection, tracer, regulatory, or modifying molecules. In one aspect, the present disclosure provides the foregoing ferritin fusions, nanoparticles, and compositions for the preparation of targeted anticancer drugs.

In one aspect, the present disclosure provides a method of treating a tumor comprising providing to a patient in need thereof a therapeutically effective amount of the foregoing ferritin fusion, nanoparticle, and composition.

Compared with the prior art, the method has the following beneficial effects:

(1) The ferritin fusion constructed by the method can self-assemble to form cage-shaped nano-particles, and the inserted exogenous target fragment is loaded on the outer surface of the ferritin nano-particles and can form a space conformation with bioactivity, so that a ferritin carrier system with specific functions is formed.

(2) Compared with the exogenous target fragment, the half-life period of the nanoparticle formed by self-assembly of the constructed ferritin fusion is prolonged, the single-molecule titer is up to 24, and the biological titer and activity of the single molecule are improved.

(3) The human heavy chain ferritin has active targeting combined with transferrin receptor TFR1, and the target is expressed in various tumor cells, so when the human heavy chain ferritin is selected as the inserting carrier of the exogenous target fragment, the exogenous target protein can be brought to the tumor site by utilizing the active targeting, and the tracing and treating functions are exerted. Meanwhile, the ferritin nano-particles have EPR effect (high permeability and retention effect of solid tumor) of nano-particles, and the effect enables the nano-particles to enter the tumor more easily, so that the tracing and therapeutic effects are exerted.

(4) The ferritin fusion is obtained by using the escherichia coli expression system, the nanoparticle with binding activity can be formed simply and in high yield through self-assembly, the preparation method is simple, the cost is greatly reduced, the operation is easy, and the high-patent-medicine value and the industrialization value are realized.

Drawings

FIG. 1 shows a plasmid map of pET-30a (+) for fusion protein expression.

FIG. 2 shows SDS-PAGE results of Mut-HFn-Fc-257 and Mut-HFn-Fc-258 expression.

FIG. 3 shows SDS-PAGE results of Mut-HFn-PD1-422 expression.

FIG. 4 shows SDS-PAGE results of Mut-HFn-PD1-430 expression.

FIG. 5 shows SDS-PAGE results of Mut-HFn-PD1-436 expression.

FIG. 6 shows SDS-PAGE results of Mut-HFn-PD1-431 expression.

FIG. 7 shows SDS-PAGE results of HFn-CD206-4 expression.

FIG. 8 shows SDS-PAGE results of HFn-CD206-2 expression.

FIG. 9 shows the affinity profile of Mut-HFn-258 binding to FcRn as determined by ELISA.

Detailed Description

Terminology

In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. Meanwhile, in order to better understand the present disclosure, definitions and explanations of related terms are provided below.

"ferritin" refers to an iron storage structure consisting of two parts, a protein outer shell and an iron inner core. Naturally, the protein shell of ferritin is a cage-like protein structure (12 nm outside diameter, 8nm inside diameter) formed by self-assembly of 24 subunits, while the main component of the iron core is ferrihydrite. The protein coat of ferritin, which does not contain an iron core, is also known as "deferiprone". The term "ferritin" as used herein includes eukaryotic ferritin and prokaryotic ferritin, preferably eukaryotic ferritin, more preferably mammalian ferritin, e.g. human ferritin. Eukaryotic ferritin typically includes a heavy chain H subunit and a light chain L subunit. The proportion of H and L subunits contained in ferritin molecules varies in different tissues and organs of the body. However, "H ferritin (HFn)" assembled from only H subunits or "L ferritin (LFn)" assembled from only L subunits can also be obtained by recombinant means.

The term "ferritin monomeric subunit" refers to a wild-type ferritin monomeric subunit, including but not limited to mammalian ferritin monomeric subunits, such as a human ferritin monomeric subunit or a horse ferritin monomeric subunit, preferably a human ferritin monomeric subunit. The human ferritin monomer subunit includes a human heavy chain ferritin monomer subunit (H subunit) and also includes a human light chain ferritin monomer subunit (L subunit), and an exemplary wild-type human ferritin H subunit comprises the amino acid sequence shown in SEQ ID No. 1.

The term "variant" refers to a fragment obtained after substitution, partitioning or deletion of the amino-terminal, carboxy-terminal or internal amino acid sequence of a ferritin monomeric subunit of the present disclosure, as compared to the amino acid sequence, which variant may result in new biological activity and function due to substitution, partitioning or deletion, but still retains all or part of the biological activity and function of the wild-type ferritin monomeric subunit. Variants of the monomeric subunits of ferritin described in the present disclosure also include fragments obtained by conservative substitution of an amino acid residue at a particular amino acid residue position (conservatively substituted).

A "conservative substitution" is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined and are well known in the art to which the present disclosure pertains. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), beta-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

It is contemplated that variants of the monomeric subunits of ferritin of the present disclosure may retain activity despite conservative amino acid substitutions.

"nanoparticle", also referred to as "nanocage", refers to a three-dimensional protein structure, i.e., a cage-like structure, having an internal central cavity formed by a plurality of polypeptides (subunits) capable of self-assembly. The number of polypeptides (subunits) assembled into the nanoparticle is not particularly limited as long as it is capable of forming the nanoparticle. The nanoparticles may have a symmetrical structure or an asymmetrical structure depending on their subunit composition. Typical nanoparticles comprise ferritin/deferiprone.

The term "self-assembled" protein refers to a protein capable of forming nanoparticles by regularly arranging to form multimers while being expressed without the aid of a specific inducer.

The terms "peptide segment", "peptide" and "protein" are used interchangeably in this disclosure to refer to a polymer of amino acids. The term applies to amino acid polymers in which one or more amino acids are artificial chemical analogues of the corresponding natural amino acid, as well as to polymers of natural amino acids. In the present disclosure, amino acids may be mentioned by their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission (IUPAC-IUB biochemical nomenclature committee).

"bioactive moiety" refers to a functional molecule having a biological activity, which in the present disclosure comprises at least one amino acid residue component, including but not limited to a polypeptide, peptide or protein consisting of only amino acid components; including but not limited to polypeptides, peptides or proteins modified, conjugated, encapsulated by non-amino acid components; including but not limited to non-amino acid functional molecules modified with amino acids, conjugated, and encapsulated.

The modification, conjugation, encapsulation may be accomplished physically, chemically, or a combination of both, wherein the "modification" includes, but is not limited to, glycosylation, lipid linkage, sulfation, gamma carboxylation of glutamic acid, hydroxylation, and ADP-ribosylation; the term "conjugation" includes, but is not limited to, covalent bond formation, hydrogen bonding, non-covalent bonds (e.g., van der Waals forces), ionic bonds, disulfide bonds, intramolecular forces, coordination bonds, intermolecular forces, and the like; the term "encapsulate" refers to a polypeptide, peptide, protein, or polypeptide that spatially forms or is surrounded by non-amino acid components.

"ferritin fusion" refers to a natural or synthetic molecule formed by fusion of a ferritin monomeric subunit or variant thereof with a biologically active moiety, said fusion being between the ferritin monomeric subunit and the biologically active moiety by chemical means, biological means or a combination of both, optionally via a chemical or amino acid based linker molecule. The ligation may be achieved by C-N fusion or N-C fusion (in the 5 '. Fwdarw.3' direction).

The term "comprising" is used herein to describe a sequence of a protein or nucleic acid, which may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present disclosure. Furthermore, it will be clear to those skilled in the art that the methionine encoded by the start codon at the N-terminus of a polypeptide may be retained in some practical situations (e.g., when expressed in a particular expression system) without substantially affecting the function of the polypeptide. Thus, in describing a particular polypeptide amino acid sequence in the present disclosure and claims, a sequence comprising methionine is also contemplated at this time, although it may not comprise a methionine encoded at the N-terminus by the start codon. Accordingly, the coding nucleotide sequence may also comprise an initiation codon.

The term "expression construct" refers to a vector, such as a recombinant vector, suitable for expression of a nucleotide sequence of interest in an organism. "expression" refers to the production of a functional product. For example, expression of a nucleotide sequence may refer to transcription of the nucleotide sequence (e.g., transcription into mRNA or functional RNA) and/or translation of RNA into a precursor or mature protein. The "expression construct" of the present disclosure may be a linear nucleic acid fragment, a circular plasmid, a viral vector, or may be an RNA (e.g., mRNA) capable of translation. Typically, in an expression construct, the nucleotide sequence of interest is operably linked to regulatory sequences.

The term "self-assembled" protein refers to a protein capable of forming nanoparticles by regularly arranging to form multimers while being expressed without the aid of a specific inducer.

The term "linker peptide" refers to a peptide segment that links two active molecules, which can be part of a fusion protein, to provide a spatial separation between the two proteins to be fused so as to achieve a tendency to fold properly without interfering with each other to link the two protein active molecules, without affecting the function and stability of the active molecules.

The "tumor" as described in the present disclosure includes, but is not limited to, pancreatic cancer, liver cancer, colon cancer, rectal cancer, stomach cancer, lymphoma, basal cell carcinoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal carcinoma, breast cancer, endometrial cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, biliary tract cancer, esophageal cancer, renal cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. Wherein the leukemia is selected from acute lymphoblastic (lymphoblastic) leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia; the lymphoma is selected from hodgkin's lymphoma and non-hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and waldenstrom's macroglobulinemia; the sarcoma is selected from osteosarcoma, ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. Preferably, the neoplasm is selected from pancreatic cancer, liver cancer, oral squamous carcinoma, colon cancer, ovarian cancer and gastric cancer.

The term "diagnosis" as used in this disclosure refers to ascertaining whether a patient has a disease or condition in the past, at the time of diagnosis, or in the future, or ascertaining the progression or likely progression of a disease in the future, or assessing a patient's response to treatment.

The term "treatment" as used in this disclosure means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily refer to the complete elimination of all disease-related signs, symptoms, conditions, or disorders, and refers to a therapeutic intervention that ameliorates the signs, symptoms, etc. of a disease or pathological state after the disease has begun to develop.

"homology" as used in the present disclosure means that a person skilled in the art can adjust the sequence according to actual working needs without changing the main structure or function of the original sequence, using sequences having (including but not limited to) 1%,2%,3%,4%,5%,6%,7%,8%,9%,10%,11%,12%,13%,14%,15%,16%,17%,18%,19%,20%,21%,22%,23%,24%,25%,26%,27%,28%,29%,30%,31%,32%,33%,34%,35%,36%,37%,38%,39%,40%,41%,42%,43%,44%,45%,46%,47%,48%,49%,50%,51%,52%,53%,54%,55%,56%,57%,58%,59%,60%,70%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%, 96%,97%, 99% or 100% homology to specific sequences as described in the present disclosure. For example, the "amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID No.1, 2, 12, 3 or 13" in the present disclosure is that the amino acid sequence of SEQ ID No.1, 2, 12, 3 or 13 may be modified according to actual working requirements, including one or more changes such as substitution, deletion and/or insertion of one or more amino acids, etc., truncation, or lengthening of one or both ends, so long as it retains 80% or more homology, and the changes may be substitution, addition or deletion. May be a substitution between amino acids of the same polarity, a substitution between amino acids of the same charge, a substitution between uncharged amino acids, a substitution between aliphatic amino acids, a substitution between aromatic amino acids, a substitution between nonpolar amino acids or a substitution between amino acids having side chain moieties of a relevant nature. Wherein the basic side chains include, but are not limited to, lysine, arginine or histidine. Acidic side chains include, but are not limited to, aspartic acid or glutamic acid. Uncharged amino acids include, but are not limited to aspartyl, glutamine, serine, threonine or tyrosine. Nonpolar side chains include, but are not limited to, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, or cysteine. Wherein said at least 80% includes, but is not limited to 80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or 100%.

Ferritin fusion

In a first aspect, the present disclosure provides a ferritin fusion, wherein the ferritin fusion comprises:

(a) A ferritin monomer subunit or variant thereof, and (b) a biologically active moiety; the biologically active portion comprises an amino acid component and is inserted into the sequence of (a).

In some embodiments, the ferritin monomer subunit or variant thereof is a human ferritin monomer subunit or variant thereof, human ferritin is a protein that regulates iron metabolic balance in humans, 24 human ferritin monomer subunits constitute a hollow caged protein that is a nanoparticle with an inner diameter of 8nm and an outer diameter of 12nm, and is divided into a human heavy chain ferritin monomer subunit (human H subunit) and a human light chain ferritin subunit (human L subunit) according to different subunit types.

In one embodiment, the ferritin monomer subunit or variant thereof is a human heavy chain ferritin monomer subunit or variant thereof. The human heavy chain ferritin monomer subunit consists of 182 amino acids, the molecular weight of 21kDa, consists of 5 alpha helices and 4 loop regions between the helices, wherein the 5 alpha helices are ABCDE helices respectively corresponding to amino acids 13-42, 48-77, 95-125, 126-159 and 163-174, and the 4 flexible loop regions are AB/BC/CD/DE respectively corresponding to amino acids 43-47, 78-94, 125-126 and 160-162. The 24 human heavy chain ferritin monomer subunits self-assemble into cage-shaped nano particles, the human heavy chain ferritin monomer subunits can be natural extracts, can be prepared by artificial synthesis or by genetic engineering technology, and can self-assemble to form the ferritin fusion.

In some of these embodiments, the human heavy chain ferritin monomer subunit is a wild-type human heavy chain ferritin monomer subunit having an amino acid sequence as shown in SEQ ID No. 1.

In some of these embodiments, the variant of the human heavy chain ferritin monomer subunit is a variant sequence that has been mutated by an amino acid; preferably, the mutant amino acid is cysteine (Cys); more preferably, the cysteines are each independently mutated to glutamic acid (Glu), serine (Ser), or alanine (Ala).

In particular embodiments, the heavy chain ferritin monomeric subunit variants may also be conservatively substituted variants.

In particular embodiments, the heavy chain ferritin monomer subunit variant may also be a truncation mutant; the truncation mutant is an alpha-helical truncation mutant at the C-terminus of the monomeric subunit of heavy chain ferritin (H).

In one embodiment, the human heavy chain ferritin monomer subunit variant comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in any one of SEQ ID nos. 2-6, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity; preferably, the amino acid sequence of the human heavy chain ferritin monomer subunit or variant thereof is shown in any one of SEQ ID NO. 2-6.

In some of these embodiments, the biologically active moiety is inserted into the loop region of a monomeric subunit of ferritin or a variant thereof, such as a human H subunit or a variant thereof. The inventor finds that the loop area is a flexible area and is positioned on the surface of the nano-particle after the ferritin monomer subunits are self-assembled into the nano-particle composed of 24 polymers, so that the peptide fragments can be displayed on the surface of the ferritin nano-particle by inserting the bioactive part into the area, meanwhile, the assembly of the ferritin monomer subunits is not influenced, a flexible space is provided to the greatest extent, and the biological activity and the function of the inserted part are ensured.

The loop region of the human heavy chain ferritin monomer subunit or variant thereof is divided into AB loop region, BC loop region, CD loop region or DE loop region, and the insertable site comprises between 44D/45D of AB loop, between 126D/127P of CD loop, between 161P/162E or 162E/163S of DE loop, preferably between DE loop regions, such as between 160A/161P, or between 161P/162E, or between 162E/163S, more preferably between 161P/162E or between 162E/163S. The amino acid sequence of the human heavy chain ferritin monomer subunit or variant thereof is as described above.

The bioactive moiety is a functional molecule comprising an amino acid component, including but not limited to a polypeptide, peptide or protein that is modified, conjugated, or encapsulated by a non-amino acid component, or a functional molecule that is modified, conjugated, or encapsulated by an amino acid, in addition to a polypeptide, peptide or protein that is composed of only amino acid components. The peptides include polypeptides, oligopeptides, and the peptides and proteins include, but are not limited to, antibodies, antigenic proteins, targeting peptides, therapeutic peptides, labeling peptides, receptor binding peptides, cleavage peptides, signal peptides, the peptides or proteins being 3-40 amino acids in length, preferably 3-30 amino acids.

In some embodiments, the biologically active moiety is a peptide, which may be selected from the group consisting of: peptides synthesized from the gastrointestinal tract, including gastrin (34 peptide), secretin (27 peptide), cholecystokinin (8 peptide), motilin (22 peptide), vasoactive intestinal peptide (28 peptide), neurotensin (13 peptide) and the like; tumor targeting peptides, such as RDG peptide, NGR peptide, RVG29; cell penetrating peptide CPPs including natural protein penetrating peptide RQIKIWFQNRRMKWKK (SEQ ID NO. 29), chimeric peptides such as MPG (GALFLGFLGAAGSTMGA (SEQ ID NO. 30)), pep-1 (KETWWETWWTEWSQPKKRKV (SEQ ID NO. 31)), transporter (AGYLLGKINLKALAALAKKIL (SEQ ID NO. 32)), M918 (MVTVLFRRLRIRRACGPPRVRV (SEQ ID NO. 33)), YTA2 (YTAIAWVKAFIRKLRK-NH 2 (SEQ ID NO. 34)), YTA4 (IAWVKAFIRKLRKGPLG (SEQ ID NO. 35)), synthetic peptide penetrating peptides such as bipolar polypeptide MAP (KLALKLALKALKAALKLA (SEQ ID NO. 36)), PH-sensitive penetrating peptide or PH-selective penetrating peptide AGYLLGHINLHHLAHL (Aib) HHIL-NH2 (SEQ ID NO. 37), positive charge sequences (octameric or nonarginine RRRRRR (SEQ ID NO. 38), RRRRRRRRR (SEQ ID NO. 39)), CADY (GLWRALWRLLRSLWRLLWRA (SEQ ID NO. 40)) and also ocular transporter peptide POD (GGGARKKAAKAARKKAAKAARKKAAKAARKKAAKA (SEQ ID NO. 41)).

In some embodiments, the biologically active moiety is a protein, including antibodies and/or antigens, ligand/receptor binding fragments, including but not limited to Fab fragments, fab' fragments, fc fragments, nanobodies, single domain antibodies, VHHs, and the like. Such ligand/receptor binding fragments include, but are not limited to, viral surface antigen binding fragments, viral binding target fragments, tumor antigen binding fragments such as peptide fragments capable of binding CD20, CD206, PD-1, PDL1, or peptide fragments capable of binding neonatal Fc receptor (FcRn), and the like.

In some embodiments, the peptide fragment capable of binding human FcRn comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID No.7, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity; preferably, the amino acid sequence of the peptide fragment capable of binding to human FcRn is shown in SEQ ID NO. 7.

In some embodiments, the peptide fragment capable of binding to human programmed death receptor 1 (PD-1) comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID No.8, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity; preferably, the amino acid sequence of the peptide fragment capable of binding to human PD-1 is shown in SEQ ID NO. 8.

In some embodiments, the peptide fragment capable of binding human CD206 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID No.9, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity; preferably, the amino acid sequence of the peptide fragment capable of binding to human CD206 is shown in SEQ ID NO. 9.

The amino acid of the bioactive part can be directly connected with the amino acid of the ferritin monomer subunit or the variant thereof through peptide bonds, or can be connected with the amino acid of the ferritin monomer subunit or the variant thereof through adding connecting peptides at two ends; the connecting peptide is a short peptide which is commonly used in the field for connecting different functional proteins when fusion proteins are expressed, and comprises a GS structure, such as SGGC (SEQ ID NO. 23), GGSS (SEQ ID NO. 24) and GGGGS _n N=1-5 (SEQ ID No. 25), GSTSGSGKSSEGKG (SEQ ID No. 26), GSGGGSG (SEQ ID No. 27), GGGSGGSG (SEQ ID No. 28), and the like.

In some of these embodiments, the ferritin fusion comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in any one of SEQ ID nos. 10-18, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity; preferably, the amino acid sequence of the ferritin fusion is as shown in any one of SEQ ID NO. 10-18.

Nanoparticles

In a second aspect, the present disclosure provides a nanoparticle formed from self-assembly of at least one ferritin fusion and/or ferritin monomeric subunit or variant thereof according to the first aspect, and wherein the nanoparticle exhibits on its surface said bioactive moiety which retains at least 50% of its bioactive function in its native state, preferably retains at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98% or 99% of its bioactive function in its native state.

In one embodiment, the ferritin monomer subunit or variant thereof is selected from the group consisting of a heavy chain ferritin monomer subunit or variant thereof, and a light chain ferritin monomer subunit or variant thereof.

In one embodiment, the heavy chain ferritin monomer subunit or variant thereof is a human heavy chain ferritin monomer subunit or variant thereof.

In a preferred embodiment, the light chain ferritin monomer subunit or variant thereof is a human light chain ferritin monomer subunit or variant thereof.

In particular embodiments, the source of ferritin comprises any one or a combination of at least two of natural extraction products, artificial synthesis products, or genetic engineering products.

In specific embodiments, the ferritin monomeric subunit variant is a variant sequence after amino acid mutation; preferably, the mutant amino acid is cysteine (Cys); more preferably, the cysteine is mutated to glutamic acid (Glu), serine (Ser), or alanine (Ala).

In particular embodiments, the ferritin monomeric subunit variants may also be variants obtained by conservative substitutions.

In specific embodiments, the ferritin monomer subunit variant is a truncation mutant; in one embodiment, the truncation mutant is an alpha-helical truncation mutant at the C-terminus of the monomeric subunit of heavy chain ferritin (H); in one embodiment, the truncation mutant is an epsilon helix truncation mutant at the C-terminus of the monomeric subunit of light chain ferritin (L).

In one embodiment, the heavy chain ferritin monomer subunit or variant thereof comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in any one of SEQ ID nos. 1-6, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity; more preferably, the amino acid sequence of the heavy chain ferritin monomer subunit or variant thereof is shown in any one of SEQ ID NO. 1-6.

In one embodiment, the light chain ferritin monomer subunit or variant thereof comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in any one of SEQ ID nos. 19-22, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity; more preferably, the amino acid sequence of the monomeric subunit of light chain ferritin or a variant thereof is shown in any one of SEQ ID NO. 19-22.

In one embodiment, the heavy chain ferritin monomer subunit or variant thereof and/or the light chain ferritin monomer subunit or variant thereof forms a nanoparticle with the ferritin fusion.

In one embodiment, the nanoparticle is a 24-mer formed by self-assembly of the ferritin fusion and optionally a ferritin monomer subunit or variant thereof. The nanoparticle displays on its surface a biologically active moiety comprised by the ferritin fusion, which biologically active moiety can perform biological functions in vivo including, but not limited to, treatment, diagnosis, targeting, modulation of signal pathways, tracking, etc. In the nanoparticle, the amount of ferritin fusion may be 1-24, i.e. the ratio of ferritin fusion to ferritin monomer subunits or variants thereof may be 24:0-1:23. The amount of ferritin fusion in the nanoparticle determines the amount of active molecules in the nanoparticle, depending on the amount of ferritin fusion, a nanoparticle having a specific biological activity of 1-24 valency can be formed.

In one embodiment, the nanoparticle is a 24-mer formed by self-assembly of 24 ferritin fusions, and does not comprise ferritin monomer subunits or variants thereof.

In a third aspect, the present disclosure provides a method for producing a ferritin fusion according to the first aspect, or a nanoparticle according to the second aspect, the method comprising introducing one or more nucleic acid molecules encoding the ferritin fusion and optionally encoding the ferritin monomeric subunit or variant thereof into a cell and culturing the cell under conditions suitable for expression of the encoded ferritin fusion and/or encoding the ferritin monomeric subunit or variant thereof, or for nanoparticle formation.

In a fourth aspect, the present disclosure provides a composition comprising at least one of a ferritin fusion according to the first aspect, a nanoparticle according to the second aspect or a ferritin fusion or nanoparticle produced by the method according to the third aspect;

in one embodiment, the composition is a pharmaceutical composition, which comprises a bioactive fraction in ferritin fusion with therapeutic or prophylactic effect, and the pharmaceutical composition may be an antitumor drug, an antiallergic drug, an antiinfective drug, an antiviral drug, an anti-aging drug, an antimetabolite drug, etc., according to the therapeutic effect of the bioactive fraction.

In a specific embodiment, the composition is an anti-tumor pharmaceutical composition.

In one embodiment, the composition is a diagnostic composition comprising a ferritin fusion with a tracer, diagnostic marker, which depending on the biologically active moiety, may be used for in vivo as well as ex vivo diagnostics, including but not limited to in vivo tracers, such as tumour diagnostic tracers, lymphatic tracers; in vitro diagnostics include, but are not limited to, detection of various target biomarkers, detection of various metabolic index parameters, and the like.

In one embodiment, the composition is a composition for modulating a biological function, comprising a ferritin fusion having a physiological modulating function, which may be used to modulate the level of protein expression, the intensity of nucleic acid expression, the hormone level, the blood oxygen level, etc. in vivo, depending on the biologically active moiety, by activating, inhibiting the activity of a target, or by modulating the expression of genes, mRNAs.

In a fifth aspect, the present disclosure provides the use of a ferritin fusion according to the first aspect, a nanoparticle according to the second aspect or a ferritin fusion or nanoparticle produced by a method according to the third aspect for the preparation of a targeting molecule, a therapeutic molecule, a tracer molecule, a regulatory molecule and a modifying molecule.

In a sixth aspect, the present disclosure provides the use of a ferritin fusion according to the first aspect, a nanoparticle according to the second aspect, a ferritin fusion or nanoparticle produced by a method according to the third aspect, and a composition according to the fourth aspect in the manufacture of an anti-tumour medicament. In some embodiments, the tumor may be a solid tumor or a hematological tumor, wherein hematological tumors include, but are not limited to, acute lymphoblastic leukemia, acute non-lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, hodgkin's lymphoma, non-hodgkin's lymphoma, multiple myeloma, and the like; the solid tumors include, but are not limited to, breast cancer, gastric cancer, liver cancer, non-small cell lung cancer, melanoma, esophageal cancer, glioma, bone cancer, skin cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer and ovarian cancer.

Examples

A further understanding of the present disclosure may be obtained by reference to the specific examples presented herein which are intended to be illustrative of the present disclosure and are not intended to limit the scope of the present disclosure in any way. It will be apparent that various modifications and variations can be made to the present disclosure without departing from the spirit of the disclosure, and therefore, such modifications and variations are also within the scope of the claimed subject matter.

The experimental materials and experimental methods used in the following examples were as follows:

1. candidate mutant HFn monomeric protein subunits

To increase the solubility and patentability of HFn, the amino acid sequence of the H subunit mutant was designed based on the wild-type amino acid sequence of the H subunit of human ferritin (SEQ ID NO.1; see PDB: 3AJO_A), and the specific design is shown in Table 1. The resulting subunit mutants were designated as Mut-HFn-212 (SEQ ID NO. 2), mut-HFn-243 (SEQ ID NO. 3), mut-HFn-104 (SEQ ID NO. 4), mut-HFn-212-S (SEQ ID NO. 5) and Mut-HFn-243-S (SEQ ID NO. 6), respectively.

TABLE 1 design of HFn mutants

2. Candidate exogenous fragments of interest

Exogenous fragments of different functions and sequences used in the present disclosure are inserted or sequentially linked to different regions of ferritin to compare the effect of the different regions on expression of the exogenous protein, the amino acid sequence of the selected exogenous fragment of interest being as follows:

(1) Human IgG Fc portion sequence 1 (F423-L441, C425S):

FSSSVMHEALHNHYTQKSL(SEQ ID NO.7)

(2) PD-1-Vaxx polypeptide fragments:

GAISLAPKAQIKESLRAEL(SEQ ID NO.8)

(3) Binding to CD206 peptide fragment:

SSPGAK(SEQ ID NO.9)

3. fusion of heavy chain ferritin monomer subunit and exogenous target fragment

3.1 human IgG Fc portion sequence 1 insertion or ligation of different regions of ferritin

(1) Mut-HFn-Fc-254 fusion proteins

Using Mut-HFn-243-S as a template, human IgG Fc portion sequence 1 (F423-L441, C425S) was then passed through a linker: GSGGGSG is linked to the C-terminus of Mut-HFn-243-S to form a Mut-HFn-Fc-254 fusion protein having the amino acid sequence (SEQ ID NO: 10):

TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEKREGAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLDAMEAALHLEENVNQSLLELHKLATDKNDPHLADFIETHYLNEQVKAIKELGDHVTNLHKMGSGGGSGFSSSVMHEALHNHYTQKSL

(2) Mut-HFn-Fc-257 fusion proteins

Human IgG Fc portion sequence 1 (F423-L441, C425S) was then passed through a linker using Mut-HFn-212-S as template: GSGGGSG is linked to the C-terminus of Mut-HFn-212-S to form a Mut-HFn-Fc-257 fusion protein, the amino acid sequence of which is (SEQ ID NO. 11):

TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMESALHLEKNVNQSLLELHKLATDKNDPHLSDFIETHYLNEQVKAIKELGDHVTNLRKMGSGGGSGFSSSVMHEALHNHYTQKSL

(3) Mut-HFn-Fc-258 fusion proteins

Human IgG Fc portion sequence 1 (F423-L441, C425S) was inserted between DE loops (P161/E162) of Mut-HFn-212 using Mut-HFn-212 as a template to form a Mut-HFn-Fc-258 fusion protein having the amino acid sequence (SEQ ID NO. 12):

TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMESALHLEKNVNQSLLELHKLATDKNDPHLSDFIETHYLNEQVKAIKELGDHVTNLRKMGAPFSSSVMHEALHNHYTQKSLESGLAEYLFDKHTLGDSDNES

3.2 insertion of PD-1-Vaxx polypeptide fragments into or ligation of different regions of ferritin

(1) Mut-HFn-PD1-422 fusion proteins

The PD-1-Vaxx polypeptide sequence is inserted between A/B loops (44D/45D) of Mut-HFn-104 to form a Mut-HFn-PD1-422 fusion protein, the amino acid sequence of which is (SEQ ID NO. 13):

TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDGAISLAPKAQIKESLRAELDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDEDDWESGLNAMEAALHLEKNVNQSLLELHKLATDKNDPHLADFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES

(2) Mut-HFn-PD1-430 fusion proteins

The PD-1-Vaxx polypeptide sequence was inserted between the C/D loops of Mut-HFn-104 (126D/127P) to form a Mut-HFn-PD1-430 fusion protein having the amino acid sequence (SEQ ID NO. 14):

TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDEDDWESGLNAMEAALHLEKNVNQSLLELHKLATDKNDGAISLAPKAQIKESLRAELPHLADFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES

(3) Mut-HFn-PD1-431 fusion proteins

The PD-1-Vaxx polypeptide sequence was inserted between D/E loop (162E/163S) of Mut-HFn-104 to form a Mut-HFn-PD1-431 fusion protein having the amino acid sequence (SEQ ID NO. 15):

TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDEDDWESGLNAMEAALHLEKNVNQSLLELHKLATDKNDPHLADFIETHYLNEQVKAIKELGDHVTNLRKMGAPEGAISLAPKAQIKESLRAELSGLAEYLFDKHTLGDSDNES

(4) Mut-HFn-PD1-436 fusion proteins

Passing the PD-1-Vaxx polypeptide sequence through a linker: GSGGGSG is linked to the C-terminal of Mut-HFn-104 to form Mut-HFn-PD1-431 fusion protein, the amino acid sequence of which is (SEQ ID NO. 16):

TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDEDDWESGLNAMEAALHLEKNVNQSLLELHKLATDKNDPHLADFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNESGSGGGSGGAISLAPKAQIKESLRAEL

3.3 insertion or ligation of CD 206-binding peptide fragments into different regions of ferritin

(1) The peptide segment combined with CD206 is connected with the N end of Mut-HFn-212, and the middle is connected with connecting peptide GGGGS, so that the Mut-HFn-CD206-2 fusion protein is constructed and obtained, and the amino acid sequence of the fusion protein is (SEQ ID NO. 17):

SSPGAKGGGGSTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMESALHLEKNVNQSLLELHKLATDKNDPHLSDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES

(2) The CD 206-binding peptide was inserted between DE loop (P161/E162) of Mut-HFn-212. During insertion, a connecting peptide GGGGS is added to two sides of the inserted fragment to construct and obtain the Mut-HFn-CD206-4 fusion protein, and the amino acid sequence of the fusion protein is as follows (SEQ ID NO. 18):

TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMESALHLEKNVNQSLLELHKLATDKNDPHLSDFIETHYLNEQVKAIKELGDHVTNLRKMGAPGGGGSSSPGAKGGGGSESGLAEYLFDKHTLGDSDNES

EXAMPLE 1 expression and purification of ferritin fusions

1. Gene synthesis

The peptide fragment, the protein or the fusion protein are all obtained by expression in prokaryotic expression host bacteria E.coli, the Shanghai JieRui bioengineering Limited company is entrusted according to the amino acid sequences of the peptide fragment, the protein and the fusion protein, the gene synthesis and the plasmid vector construction work are completed according to codons preferred by a host, and the sequence of the constructed recombinant plasmid is correct after confirmation.

2. Construction of expression strains

A common vector pET-30a (+) for expressing foreign proteins of escherichia coli is selected, the resistance is kanamycin resistance (Kan+), nde I and Hind III enzyme cutting sites are selected to be embedded into the coding genes of the human IgG Fc part sequence 1, PD-1-Vaxx and peptide fragments, ferritin or ferritin fusion combined with CD206, so as to obtain the recombinant pET-30a (+) carrying the target coding genes. The successful construction of the expression vector is confirmed by enzyme digestion map and gene sequencing. Wherein, the plasmid map of pET-30a (+) is shown in FIG. 1. After double enzyme digestion, 1% agarose gel electrophoresis is carried out, and the position of the band after enzyme digestion is correct in reference to the molecular weight marker molecular weight, which indicates that the target gene is constructed into the expression plasmid.

E.coli BL21 (DE 3) was selected as host strain, and recombinant plasmids containing ferritin-human IgG Fc part sequence 1, ferritin-PD-1-Vaxx and ferritin-CD 206 fusion genes were transformed into competent cells of host strain, and positive clones were selected by a resistance plate containing 50. Mu.g/ml kanamycin, and recombinant strains were determined as follows.

3. Recombinant strain construction

i. Recombinant plasmid resuspension

Taking 10 mug of two recombinant plasmid freeze-dried powder, respectively using 200 mug TE buffer solution to re-suspend uniformly, split charging 10 mug/tube, respectively leaving 1 tube for standby, and freezing the rest in a refrigerator at-80 ℃ for standby.

Transformation of

(1) Taking out competent cells from a refrigerator at-80 ℃, placing the competent cells on ice for melting (about 5 min), taking a certain amount of plasmid heavy suspension (different carrier and host bacteria dosage) in an ice bath, adding the plasmid heavy suspension into 20 mu l of competent cells, fully mixing the competent cells uniformly, and standing and incubating the competent cells on ice for 30min.

(2) The sample was heat-shocked in a water bath at 42℃for 90s, immediately placed on ice and allowed to stand for 2min.

(3) 280. Mu.L of sterile LB liquid medium was added to the heat-shocked sample and activated at 37℃for 1h at 220 rpm.

(4) The transformed bacterial solutions were respectively spread on LB resistant plates, cultured overnight in an incubator at 37℃and observed for colony growth.

4. Protein expression

i. Shake flask culture

Respectively taking 3 large and full clones on a resistance plate, respectively inoculating the clones into 40mL LB culture medium, culturing at 37 ℃ until the OD600 is 1.0-2.0, adding 0.5mM IPTG, and carrying out induced expression at 25 ℃ for 4-6h, and respectively detecting the expression condition of target proteins by SDS-PAGE.

Preparation of test samples

Cell lysis: 30mL of the bacterial liquid is taken, centrifuged for 15min at 5000r/min, the supernatant is discarded, 25mL of 20mM Tris-HCl buffer solution is added for uniform resuspension, and the bacterial liquid is crushed for 3 times by a high-pressure homogenizer at 1000 bar.

SDS-PAGE sample preparation: taking 1000 mu L of the thallus lysate, centrifuging at 10000rpm for 10min, taking 20 mu L of supernatant into another centrifuge tube, adding 5 mu L of 5×loading buffer, mixing well, and incubating at 95 ℃ for 5min to obtain lysate supernatant; the remainder of the pellet was resuspended in 1000. Mu.L of 20mM Tris-HCl, pH8.0 buffer, and 5. Mu.L of 5 Xloading buffer was added to the 20. Mu.L of the suspension and mixed well, and incubated at 95℃for 5min, which was a pellet sample of lysate.

The expression of the different ferritin fusions is shown in FIGS. 2-8.

FIG. 2 shows the expression of ferritin-human IgG Fc portion sequence 1 fusion. In the figure, arabidopsis numbers represent sample numbers (i.e., products of different recombinant strains of the same fusion), S represents supernatant, C represents precipitated Mut-HFn-Fc-257 and Mut-HFn-Fc-258, which are expressed in cell supernatants, and HFn-Fc-258 is expressed in a larger amount. Subsequent activity experiments (see example 3 for specific methods and results) showed that only Mut-HFn-Fc-258 had binding activity and Mut-HFn-Fc-257 had no activity, indicating that the selection of different positions for insertion of the exogenous fragment of interest had different effects on its biological activity, and that insertion in the D/E loop region better maintained the biological function of the inserted fragment.

FIGS. 3-6 show the expression of the ferritin PD-1-Vaxx fusions Mut-HFn-PD1-422, mut-HFn-PD1-430, mut-HFn-PD1-436, mut-HFn-PD1-431, respectively. In the figure, arabic numerals represent sample numbers, S represents supernatant, and C represents precipitate. The results showed that the Mut-HFn-PD1-422 fusion protein (inserted in the A/B loop region) was not expressed in the supernatant, but in the form of inclusion bodies in the pellet (FIG. 3); mut-HFn-PD1-430 (inserted C/D loop region) (FIG. 4) and Mut-HFn-PD1-436 (linked to the N-terminus) were not expressed in the host strain (FIG. 5), whereas Mut-HFn-PD1-431 (inserted D/E loop region) was observed to express the target protein in the supernatant (FIG. 6), thus demonstrating that the selection of the D/E loop region for insertion of the foreign target protein enabled the fusion protein to be expressed in large amounts from soluble forms.

FIGS. 7-8 show the expression of the ferritin-CD 206 fusions Mut-HFn-CD206-4 and Mut-HFn-CD206-2, respectively. As can be seen from the results, HFn-CD206-4 was stable in all the parallel samples and expressed more (FIG. 8), while HFn-CD206-2 was expressed unstably (FIG. 7). The selection of the D/E loop region for insertion of the foreign protein of interest allows for a large and stable expression of the fusion in soluble form for different types of foreign proteins.

Example 2 fusion protein purification and purity detection

The ferritin-human IgG Fc portion sequence 1, ferritin-PD-1-Vaxx and ferritin-binding CD206 fusions prepared above were purified by the following steps:

(1) Example 1 after shake flask induced expression, a certain amount of bacterial sludge is weighed, bacterial cells are resuspended in 50mM Tris-HCl, pH8.0 buffer solution with a solid-to-liquid ratio of about 1:20 (m/v), and fully mixed;

(2) Crushing 3 times at 2-10 ℃ and 800-1000 bar high pressure by using a high pressure homogenizer to obtain a bacterial lysate;

(3) Heating thallus lysate in 75 deg.c water bath (magnetic stirring), timing when the sample solution is raised to 70 deg.c for 5min, and cooling the sample in ice water bath to below 25 deg.c;

(4) Centrifuging at 12000rpm and 2-8deg.C for 20min, collecting supernatant, and filtering the supernatant with 0.45 μm;

(5) Purified ferritin fusion was obtained by treatment with hydrophobic chromatography (column: phenyl HP), ion exchange chromatography (column: EMD TMAE) and desalting (G25).

The purified ferritin fusion was measured for purity and monomer/polymer by Size Exclusion Chromatography (SEC) as follows:

(1) Taking 1ml of protein samples with the protein concentration of 1mg/ml respectively, and placing the protein samples in a clean 1.5ml EP tube;

(2) A10. Mu.l sample was taken and analyzed for ferritin monomer and polymer peaks by SEC in a high performance liquid chromatography system (Agilent Technologies 1260 Infinicity II) through a gel filtration column (Agilent Advance Bio SEC 300A 2.7um 7.8*300nm, column number: ARD-007 flow rate: 0.5 ml/min), mobile phase: 50mM Tris buffer,pH7.2. Detection wavelength: column temperature 280 nm: 25 ℃.

The particle size of the ferritin fusion is in the range of 15-21nm through SEC detection, the ferritin fusion is self-assembled into 24-mer complex, and SEC detection results show that the ferritin nanoparticle is formed by the fusion proteins in the form of 24-mer, and no obvious aggregate is found.

Meanwhile, the endotoxin detection is carried out on the protein obtained by purification, and the result shows that the endotoxin is 10-100Eu/ml and reaches the standard.

Example 3 detection of fusion protein Activity

The binding activity of the purified proteins to FcRn and CD206 proteins was examined using an indirect ELISA method, thereby confirming the presence or absence of the protein of interest having binding activity.

The experimental operation flow is as follows:

(1) Coating a sample: the prepared mutant fusion protein samples of each group are diluted to 6 mug/mL, diluted by 3 times of gradient, added into an ELISA plate after being uniformly mixed, 100 mug/hole, placed into a wet box and placed into a refrigerator at 4 ℃ for overnight.

(2) And (5) taking out the ELISA plate washing plate: wash 3 times with 1 XPBST (300. Mu.l Tween-20 was added to PBS 100ml for mixing).

(3) Closing: taking 5% skimmed milk powder, adding 300 μl/well respectively, covering with sealing plate membrane, and incubating in incubator at 37deg.C for 2 hr.

(4) Washing the plate: wash 3 times with 1 XPBST.

(5) FcRn protein (Beijing Yiqiao China Biotechnology Co., ltd.) was diluted to 0.4. Mu.g/mL with 1% BSA, or CD206 antigen (Beijing Baips Biotechnology Co., ltd.) was diluted to 6. Mu.g/mL, 100. Mu.l per well was mixed at room temperature and incubated for 2 hours.

(6) Washing the plate: wash 3 times with 1 XPBST.

(7) Incubating the antibody: a primary Antibody (Anti-His Tag Antibody, boster) was diluted 1:1000 with 1% BSA, 100. Mu.L/Kong Jiazhi ELISA plates were designed as well as plates, covered with plate sealing membranes and incubated for 2h at ambient temperature.

(8) Washing the plate: 1 XPBST was washed 3 times.

(9) Incubating a secondary antibody: the secondary antibody (Anti-mouse HRP, boster) was diluted 1:5000 with 1% BSA, mixed well, 100. Mu.L/Kong Jiazhi ELISA plate, covered with plate membrane and incubated at 37℃for 1.5h.

(10) Washing the plate: 1 XPBST was washed 3 times.

(11) Color development: TMB 100. Mu.L/well was added to each well, covered with a membrane and incubated in an incubator at 37℃for 15min.

(12) And (3) terminating: after the completion of the color development, 50. Mu.L of a stop solution was added to each well in sequence, and the mixture was read after the stop (wavelength: 450 nm).

The results were repeated several times and found: the ferritin-human IgG Fc portion sequence 1 fusion Mut-HFn-Fc-258, which showed distinct results (fig. 9) with excellent FcRn binding activity, showed an EC50 value of 2.067 μg/ml, i.e. 3.6nM, with affinity comparable to that of mab, was not yet detected at coating concentrations as high as 0.8 mg/ml. It can be seen that selection of the D/E loop region for insertion of an exogenous fragment of interest maintains the biological activity of the insert.

The binding activity of ferritin-binding CD206 fusion protein and CD206 protein is shown in table 2 below: mut-HFn-CD206-4 > Mut-HFn-CD206-2 (Table 2). It can be seen that the selection of the D/E loop region for insertion of the exogenous fragment of interest allows the exogenous fragment to retain higher biological activity and function.

TABLE 2 affinity of caged proteins for CD206 antigen

Sample of	EC50(μg/mL)
		HFn-CD206-2	6.03
HFn-CD206-4	4.21

Claims (10)

1. A ferritin fusion comprising: (a) A ferritin monomer subunit or variant thereof, and (b) a biologically active moiety; the biologically active portion comprises an amino acid component and is inserted into the sequence of (a).

2. The ferritin fusion protein according to claim 1, wherein the ferritin monomeric subunit or variant thereof is a human ferritin monomeric subunit or variant thereof, preferably a human heavy chain ferritin monomeric subunit or variant thereof;

the biologically active moiety is inserted into the DE loop region of the ferritin monomer subunit or variant thereof, preferably between proline (P) at position 161 and glutamic acid (E) at position 162 of the DE loop region of the ferritin monomer subunit or variant thereof; or between glutamic acid (E) at position 162 and serine (S) at position 163;

preferably, a linker peptide is also added at both ends of the inserted biologically active moiety; preferably, the connecting peptide comprises G, S, C amino acids, preferably SGGC (SEQ ID NO. 23), GGSS (SEQ ID NO. 24), (GGGGS) _n N=1-5 (SEQ ID No. 25), GSTSGSGKSSEGKG (SEQ ID No. 26), GSGGGSG (SEQ ID No. 27) or GGGSGGSG (SEQ ID No. 28).

3. Ferritin fusion according to claim 1 or 2, wherein the amino acid sequence of the ferritin monomer subunit or variant thereof comprises an amino acid sequence having 80% or more identity to the amino acid sequence shown in any one of SEQ ID nos. 1 to 6, preferably an amino acid sequence having more than 85%, 90%, 95%, 96%, 97%, 98%, 99% identity, more preferably an amino acid sequence having more than 98% or 99% identity; more preferably, the amino acid sequence of the heavy chain ferritin monomer subunit or variant thereof is shown in any one of SEQ ID NO. 1-6.

4. A ferritin fusion according to any one of claims 1 to 3, wherein the biologically active moiety is a peptide fragment or protein, preferably wherein the peptide fragment or protein is selected from the group consisting of antibodies, antigens, targeting peptides, therapeutic peptides, labelling peptides, receptor binding peptides, cleavage peptides or signal peptides, and wherein the peptide fragment or protein is 3 to 40 amino acids in length, preferably 3 to 30 amino acids;

preferably, the biologically active moiety is a peptide that binds to human FcRn, a peptide that binds to human PD-1, or a peptide that binds to human CD 206;

preferably, the peptide fragment is a peptide fragment which binds human FcRn and the amino acid sequence comprises an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID No.7, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity to the amino acid sequence shown in SEQ ID No. 7; more preferably, the amino acid sequence of the peptide fragment binding to human FcRn is shown in SEQ ID No. 7;

preferably, the peptide fragment is a peptide fragment binding to human PD-1, the amino acid sequence of which comprises an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO.8, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity; more preferably, the amino acid sequence of the peptide fragment binding to human PD-1 is shown in SEQ ID NO. 8;

Preferably, the peptide fragment is a peptide fragment which binds to human CD206 and the amino acid sequence thereof comprises an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID No.9, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity to the amino acid sequence shown in SEQ ID No. 9; more preferably, the amino acid sequence of the peptide fragment binding to human CD206 is shown in SEQ ID NO. 9.

5. The ferritin fusion according to any one of claims 1 to 4, wherein said ferritin fusion comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in any one of SEQ ID nos. 12 to 15 and 18, preferably an amino acid sequence having more than 85%, 90%, 95%, 96%, 97%, 98%, 99% identity, more preferably an amino acid sequence having more than 98% or 99% identity; more preferably, the amino acid sequence of the ferritin fusion is as shown in any one of SEQ ID NO.12-15 and 18.

6. A nanoparticle formed from self-assembly of at least one ferritin fusion according to any one of claims 1 to 5 and optionally a ferritin monomeric subunit or variant thereof, and wherein the nanoparticle exhibits on its surface said bioactive moiety which retains at least 50% of its bioactive function in its native state;

Preferably, the ferritin monomer subunit or variant thereof is selected from the group consisting of a heavy chain ferritin monomer subunit or variant thereof, or a light chain ferritin monomer subunit or variant thereof;

preferably, the heavy chain ferritin monomer subunit or variant thereof is a human heavy chain ferritin monomer subunit or variant thereof; preferably, the light chain ferritin monomer subunit or variant thereof is a human light chain ferritin monomer subunit or variant thereof;

preferably, the heavy chain ferritin monomer subunit or variant thereof comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in any one of SEQ ID nos. 1 to 6, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity; more preferably, the amino acid sequence of the heavy chain ferritin monomer subunit or variant thereof is shown in any one of SEQ ID NO. 1-6;

preferably, the light chain ferritin monomer subunit or variant thereof comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in any one of SEQ ID nos. 19-22, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity; more preferably, the amino acid sequence of the monomeric subunit of light chain ferritin or a variant thereof is shown in any one of SEQ ID NO. 19-22;

Preferably, the nanoparticle is a 24-mer formed by self-assembly of the ferritin fusion, or by self-assembly of the ferritin fusion with the ferritin monomer subunit or variant thereof; more preferably, the nanoparticle is a 24-mer formed by self-assembly of 24 ferritin fusions.

7. A method for producing the ferritin fusion of any one of claims 1-5, or the nanoparticle of claim 6, the method comprising introducing a nucleic acid molecule encoding the ferritin fusion and optionally encoding the ferritin monomer subunit or variant thereof into a cell and culturing the cell under conditions suitable for expression of the encoded ferritin fusion and/or encoded ferritin monomer subunit or variant thereof, or nanoparticle formation; preferably, the cell is an E.coli cell.

8. A composition comprising at least one of the ferritin fusion of any one of claims 1-5, the nanoparticle of claim 6, or the ferritin fusion or nanoparticle produced according to the method of claim 7.

9. Use of a ferritin fusion according to any one of claims 1 to 5, a nanoparticle according to claim 6, a ferritin fusion or nanoparticle produced by a method according to claim 7 or a composition according to claim 8 for the preparation of a targeting molecule, a therapeutic molecule, a tracer molecule, a regulatory molecule or a modifying molecule.

10. Use of a ferritin fusion according to any one of claims 1 to 5, a nanoparticle according to claim 6, a ferritin fusion or nanoparticle produced by a method according to claim 7 or a composition according to claim 8 for the manufacture of an anti-tumour medicament, characterised in that the anti-tumour medicament is a targeted anti-cancer medicament.