Human ferritin light chain variants and glycosylated modified forms thereof

Abstract

The invention relates to the technical field of protein drugs, in particular to a human ferritin light chain variant and a glycosylation modified human ferritin light chain variant. According to the invention, an N-glycan modification sequence is introduced at a proper position, and the obtained human ferritin light chain variant can be used for constructing a glycosylation modified human ferritin light chain. The glycosylation-modified human ferritin light chain has good stability and protease degradation resistance, the immunogenicity is remarkably reduced, and meanwhile, a natural cavity structure is reserved, so that the glycosylation-modified human ferritin light chain can be used as a drug carrier to realize good embedding of drugs.

Description

Technical Field

[0001] This invention relates to the field of protein drug technology, and more particularly to human ferritin light chain variants and their glycosylated modifications. Background Technology

[0002] Human ferritin is an iron-containing protein synthesized by the human body, primarily found in tissues such as the liver, spleen, and bone marrow. Its core function is to store iron ions, maintain iron homeostasis, and prevent oxidative damage from iron excess or anemia from iron deficiency. Human ferritin is assembled from 24 subunits of a heavy chain (H chain) and a light chain (L chain). The heavy chain contains ferrooxidase activity, which catalyzes the oxidation of iron... 2+ Oxidized to Fe 3+ It facilitates the binding and storage of iron ions; the light chain has no enzymatic activity and its main function is to stabilize the ferritin shell structure, regulate iron storage capacity, and also regulate the release and transport of iron by binding to cell receptors.

[0003] Ferritin light chain (FTL), an endogenous protein, naturally forms a 24-mer spherical cage-like structure. Its internal cavity can efficiently load small molecule drugs, nucleic acids, nanoparticles, and other active ingredients, while its external surface can be genetically engineered or chemically modified to couple with targeting ligands, achieving precise drug delivery. FTL exhibits good biocompatibility, being derived from the human body's own protein and unlikely to induce acute toxic reactions. Furthermore, FTL can release drugs at acidic lesion sites (such as the tumor microenvironment) through a pH-sensitive mechanism, increasing local drug concentration. Its moderate molecular weight avoids rapid clearance by small molecule carriers and allows for accumulation through the high permeability and retention effect (EPR) of tumor tissue, demonstrating broad application prospects in cancer treatment and gene therapy.

[0004] Using wild-type free-float polymeric substances (FTLs) directly as drug carriers presents several key challenges. Regarding stability, their cage-like structure is susceptible to depolymerization under physiological conditions due to pH and temperature fluctuations, leading to premature drug leakage and reduced delivery efficiency. Protease degradation is also a significant issue; wild-type FTLs are easily hydrolyzed by proteases in the blood or cells, damaging the carrier structure and shortening its circulating half-life. Immunogenicity risks are substantial; although FTLs originate from the human body, exogenous administration or structural modification may trigger an immune response, producing antibodies that clear the carrier, affecting therapeutic efficacy and potentially causing allergic reactions. Furthermore, wild-type FTLs have limited drug loading capacity and lack active targeting capabilities, making it difficult to accurately identify specific diseased cells, thus limiting their clinical application. Therefore, optimizing the FTL structure through genetic engineering and chemical modification is necessary to address these shortcomings. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide human ferritin light chain variants and their glycosylation modified forms.

[0006] The present invention provides a variant of the human ferritin light chain, wherein at least one N-glycosylation site is mutated at at least one site at positions 1-10, 80-90, or 150-160 in the amino acid sequence of the wild-type human ferritin light chain.

[0007] The N-glycosylation site is either NXS or NXT; where X is any amino acid that is not P.

[0008] This invention uses human ferritin light chain (FTL, uniprot ID: P02792) as a model protein and designs and screens glycosylation sites. Based on the three-dimensional structure and functional region analysis of FTL, engineeringable regions with hydrophilic surfaces and minimal impact on protein folding, assembly, and function are screened, away from multimer interfaces, hydrophobic cores, and key interaction surfaces. The resulting variants are used to prepare glycosylated human ferritin light chain variants, which exhibit significantly improved thermal stability (Tm increases by 15-25℃), enhanced resistance to protease degradation, reduced immunogenicity by 6-18 times, and reversible acid-base stability, possessing drug loading capabilities (such as highly efficient encapsulation of paclitaxel).

[0009] In this invention, the mutation includes the substitution, deletion, and / or addition of amino acids. The mutation can be the insertion of the N-glycosylation site between two amino acid residues, or it can utilize amino acid residues in the sequence to form the N-glycosylation site by substituting or adding amino acids. This invention does not limit the formation method of the N-glycosylation site. This invention also does not limit the number and location of N-glycosylation sites. The N-glycosylation site can be a single site, multiple identical sites repeatedly linked together, or multiple different sites randomly repeated. There may or may not be spacer sequences between the multiple sites. The N-glycosylation site can also be formed by the overlap of two or more glycosylation sequences, such as NNST, NNTS, NNSS, or NNTT.

[0010] In this invention, the sequences of N-glycosylation sites are optimized to better suit their locations, ensuring that the introduced N-glycosylation sites are correctly exposed on the protein surface during protein folding. This facilitates efficient glycan addition and subsequent glycosylation modification. Through screening and optimization, the three site sequences used in the examples are more effective at modifying the target locations compared to other N-glycosylation site sequences. In specific embodiments, the N-glycosylation sites are NSS, NNST, or NKTLNGTS.

[0011] In the variants described in this invention, the number of N-glycosylation sites, as mentioned above, can be one or more. When multiple N-glycosylation sites are present, their sequences can be identical or different. In this invention, the N-glycosylation site can be located at the N-terminus, the middle, or the C-terminus. As a feasible example, the N-terminal N-glycosylation site is located between any two amino acid sites from position 1 to 10; the middle N-glycosylation site is located between any two amino acid sites from position 80 to 90; and the C-terminal N-glycosylation site is located between any two amino acid sites from position 150 to 160.

[0012] In some embodiments, the mutation includes at least one of the following:

[0013] Insert at least one amino acid residue at any position between 1 and 10 to make it contain NSS;

[0014] Insert at least one amino acid residue at any position between positions 80 and 90 to make it contain NNST;

[0015] Insert at least one amino acid residue between positions 150 and 160 to make it contain NKTLNGTS;

[0016] In some specific embodiments, the mutation is:

[0017] Insert N between 2 and 3 positions;

[0018] Or it could be inserting NNST between bits 86 and 87;

[0019] Or it could be that NKTLNGTS is inserted between bits 157 and 158;

[0020] Alternatively, N can be inserted between positions 2 and 3, and NNST can be inserted between positions 86 and 87.

[0021] Alternatively, N can be inserted between bits 2 and 3, and NKTLNGTS can be inserted between bits 157 and 158.

[0022] Alternatively, NNST could be inserted between bits 86 and 87, and NKTLNGTS could be inserted between bits 157 and 158.

[0023] Alternatively, N can be inserted between bits 2 and 3, NNST between bits 86 and 87, and NKTLNGTS between bits 157 and 158.

[0024] This invention validated the effects of the seven variants described above. The results showed that N-glycan modification significantly enhances the protein's resistance to heat denaturation, resistance to protease degradation, and reduction of immunogenicity. Variants with N inserted between positions 2 and 3, NNST inserted between positions 86 and 87, and NKTLNGTS inserted between positions 157 and 158 of the wild-type human ferritin light chain exhibited the best anti-degradation ability.

[0025] This invention does not limit the sequence of the wild-type human ferritin light chain. In the embodiments of this invention, the amino acid sequence of the wild-type human ferritin light chain is shown in SEQ ID NO:1. If the amino acid sequence of the wild-type human ferritin light chain is different from that shown in SEQ ID NO:1, then the N-glycosylation site is located at the equivalent position of the site described above.

[0026] In this invention, a variant of the human ferritin light chain is glycosylated to form a glycosylated human ferritin light chain. The glycosylation method includes introducing the variant into the ER / Golgi secretory pathway, enabling it to participate in endogenous N-glycosylation processing, thereby completing co- / post-translational modification of the N-glycan. Alternatively, the FTL can be obtained through biosynthesis (e.g., by directly expressing the FTL glycosylated sequence in E. coli and purifying it to obtain an FTL containing glycosylation sites but without N-glycosylation modification), followed by chemical synthesis to add a controllable N-glycan to the asparagine residue of the FTL glycosylated sequence.

[0027] Based on this, for better expression, especially for introducing variants of the human ferritin light chain into the ER / Golgi secretion pathway, the N-terminus of the variants described in this invention is also linked to a signal peptide. As a feasible example, the signal peptide is at least one of the following: Mouse IgG kappa signal peptide, IL-2 signal peptide, INS signal peptide, SPN signal peptide, and CTRB2 signal peptide.

[0028] To enable the isolation and purification of variants of the expressed human ferritin light chain, a purification tag is also included at its N-terminus. As a feasible example, the purification tag is at least one of the following: 6×His tag, 8×His tag, 10×His tag, Strep tag, HA tag, GST tag, MBP tag, GFP tag, Flag tag, and AlFA tag.

[0029] Furthermore, the present invention also provides biomaterials, including at least one of the following:

[0030] a) Nucleic acid, which encodes the variants described above;

[0031] b) An expression unit containing a promoter and the nucleic acid described in a);

[0032] c) An expression vector containing the nucleic acid described in a) and / or the expression unit described in b);

[0033] d) Transformation bodies containing the plasmid vector described in c), or whose genomes are integrated with the nucleic acid described in a) and / or the expression unit described in b).

[0034] The nucleic acid encoding variants described in this invention can be wild-type sequences or codon-optimized sequences; this invention does not limit the specific type of sequence.

[0035] As a feasible example, the promoter can be a eukaryotic promoter or a prokaryotic promoter; this invention does not limit this. For example, the promoter can be a CMV promoter, CAG promoter, EF1a promoter, PGK promoter, U6 and H1 promoters, EFS promoter, CBh promoter, SFFV promoter, MSCV promoter, SV40 promoter, UBC promoter, or TRE promoter. The expression unit of this invention may also selectively include a terminator, for example, the terminator is selected from T7 phage terminator, T0 phage terminator, λ phage terminator, SV40 terminator, CMV terminator, rrnB terminator, bGH terminator, hGH terminator, or rbGlob terminator. The expression unit of this invention may also selectively include a terminator. For example, the enhancer is selected from SV40 enhancer, CMV enhancer, SV-1 enhancer, ROSA26 enhancer, EF1α enhancer, HARE5 enhancer, UBC enhancer, EF1A enhancer, PGK enhancer, CAGG enhancer, COPIA enhancer, or ACT5C enhancer.

[0036] In this invention, the expression vector is used for the storage, amplification, or preparation of nucleic acids and / or expression units as described above. The expression vector is a cloning vector, expression vector, or viral vector. In this invention, the expression vector is used for the storage, amplification, or expression of the nucleic acid or expression unit, or for the expression of the variant. In some embodiments, the vector is a prokaryotic expression vector (e.g., pET-28a, pGEX-4T-1), a yeast expression vector (e.g., pPIC9K, pYES2), an insect cell expression vector (e.g., pFastBac1, pVL1393), a plant expression vector (e.g., pCAMBIA1301, pBI121), a mammalian expression vector (e.g., pcDNA3.1 (+), pCMV-Myc), an adenovirus vector (e.g., pAdEasy-1, pShuttle-CMV), a lentiviral vector (e.g., pLVX-IRES-ZsGreen1, pLKO.1), or a retroviral vector (e.g., pMXs, pBABE-puro).

[0037] In this invention, the transformant is used for the storage, amplification, or preparation of nucleic acids and / or expression units as described above. The transformant is a prokaryotic cell or a eukaryotic cell. As feasible examples, the prokaryotic cell includes, but is not limited to, *Escherichia coli*, and the eukaryotic cell includes, but is not limited to, yeast cells (e.g., *Pichia pastoris* GS115, *Saccharomyces cerevisiae* BY4741), insect cells (e.g., Sf9 cells, Sf21 cells), mammalian cells (e.g., HEK293 cells, CHO cells, HeLa cells), or plant cells (e.g., *Arabidopsis thaliana* protoplasts, tobacco BY-2 cells, rice callus cells). In a specific embodiment of this invention, mammalian cells are used to express the variant. As feasible examples, the mammalian cells include, but are not limited to, HEK293F / 293T cells and CHO cells.

[0038] Furthermore, the present invention also provides a glycosylated human ferritin light chain variant, wherein the N residue at the N-glycosylation site is glycosylated in the variant as described above.

[0039] In this invention, the glycosylation includes the reducing end of the sugar chain being linked to the amide nitrogen atom of an N residue via an N-glycosidic bond;

[0040] In this invention, the sugar chain includes a core pentasaccharide, which comprises two N-acetylglucosamine residues and three mannose residues. The sugar chain also includes at least one mannose residue, or includes mannose and / or hybrid branches containing N-acetylglucosamine, galactose, sialic acid, etc., or includes multiple complex branches containing N-acetylglucosamine, galactose, sialic acid, fucose, etc.

[0041] Furthermore, the present invention also provides a method for preparing glycosylated modified human ferritin light chain variants.

[0042] In some embodiments, the glycosylated human ferritin light chain variant comprises culturing the transformant as described above to obtain the variant as described above, and chemically modifying it to link the N-glycan chain to the amide nitrogen atom of the N residue.

[0043] As a feasible example, the chemical modification includes, but is not limited to, reductive amination coupling, N-hydroxysuccinimide (NHS) ester-mediated coupling, click chemical coupling, and enzyme-assisted chemical coupling.

[0044] In this embodiment, the host of the transformant does not undergo glycosylation modification at the N-glycosylation site. For example, the transformant is an *E. coli* bacterium. When the host is *E. coli*, the tag in the fusion protein is GST.

[0045] In other embodiments, the method for preparing the glycosylated human ferritin light chain variant includes culturing the transformant as described above to obtain a culture product containing the glycosylated human ferritin light chain variant.

[0046] In this embodiment, the host of the transformant includes, but is not limited to, HEK293F, 293S, 293T cells, CHO cells, insect sf9 cells, or yeast cells. HEK293F cells are preferred.

[0047] Furthermore, the present invention also provides the application of the aforementioned variants, the glycosylated modified human ferritin light chain variants, or the culture products obtained by the aforementioned preparation method in the preparation of packaging carriers.

[0048] In this invention, the packaging carrier is used to package active substances. The ferritin variant of this invention retains the natural cavity structure of ferritin, possessing excellent drug loading capacity and capable of efficiently encapsulating various small molecule drugs, nucleic acid drugs, and other active substances. Its surface glycans also provide sites for further functionalization modifications. Therefore, the glycosylated human ferritin light chain variant of this invention has significant application prospects in the preparation of efficient, safe, and targeted drug packaging carriers.

[0049] Furthermore, the present invention also provides a formulation whose raw materials include an active ingredient and a glycosylated human ferritin light chain variant as described above or a culture product prepared by the method described above.

[0050] As a feasibility example, the active ingredient includes at least one of the following: paclitaxel, doxorubicin, epirubicin, camptothecin, cisplatin, carboplatin, gefitinib, everolimus, temozolomide, curcumin, zinc phthalocyanine photosensitizers (such as zinc hexadecafluorophthalocyanine), ATP, and nucleic acid drugs.

[0051] Furthermore, the present invention also provides a method for preparing the formulation as described above, which includes mixing the active ingredient with the glycosylated modified human ferritin light chain variant and assembling it to obtain the formulation.

[0052] In this invention, the assembly conditions include: treating the glycosylated human ferritin light chain variant at pH 2.0 for 20 minutes, then mixing it with the active ingredient, and then adjusting the pH to 7.0.

[0053] Furthermore, the present invention also provides a medicament comprising the formulation as described above and pharmaceutically acceptable excipients.

[0054] In this invention, the pharmaceutically acceptable excipients include, but are not limited to, diluents, excipients, fillers, binders, humectants, disintegrants, lubricants, flow aids, colorants, flavoring agents, preservatives, antioxidants, pH adjusters, osmotic pressure adjusters, surfactants, solubilizers, suspending agents, emulsifiers, chelating agents, or adsorbents.

[0055] In this invention, the dosage form of the drug includes, but is not limited to, injections, microspheres, liposomes, suspensions, solutions, sterile powders for injection, concentrated solutions for injection, emulsions for injection, suspensions for injection, or infusions.

[0056] Furthermore, the present invention also provides a method of administration, which includes oral or injectable administration of the drug as described above.

[0057] In this invention, the routes of administration include, but are not limited to, intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intrathecal injection, intra-articular injection, intralesional injection, or inhalation.

[0058] In this invention, the drug is administered to animals including, but not limited to, mammals, birds, reptiles, amphibians, or fish. Mammals include humans, mice, rats, rabbits, monkeys, dogs, cats, pigs, cattle, sheep, or horses. The dosage of the drug can be adjusted according to factors such as the species, age, weight, health status, disease type, and severity of the animal.

[0059] In this invention, by introducing an N-glycan receptor sequence at an appropriate location, the obtained human ferritin light chain variant can be used to construct glycosylated human ferritin light chain variants. These glycosylated human ferritin light chain variants exhibit good stability, resistance to protease degradation, and significantly reduced immunogenicity, while retaining their natural cavity structure. Therefore, they can be used as drug carriers to achieve effective drug encapsulation. Attached Figure Description

[0060] Figure 1 The image shows an SDS-PAGE of the FTL-glycosylated mutant;

[0061] Figure 2 The image shown is a diagram of the FTL-glycosylated mutant PNGaseF restriction enzyme.

[0062] Figure 3 The diagram shows the structural verification of the FTL-glycosylation mutant;

[0063] Figure 4 The figure shows that N-glycosylation significantly improves the thermal stability of FTLs;

[0064] Figure 5 The image shows how N-glycosylation enhances the resistance of FTL to Protease K degradation.

[0065] Figure 6 The figure shows that N-glycosylation significantly reduces the immunogenicity of FTLs in mice;

[0066] Figure 7 The image shows how N-glycosylation enhances the acid-base stability of FTLs and imparts them with drug loading capacity.

[0067] Figure 8 The image shows the release effect of paclitaxel after encapsulation with a carrier. Detailed Implementation

[0068] This invention provides human ferritin light chain variants and their glycosylation modifications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0069] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0070] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0071] The term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist simultaneously, or B exists alone. A and B can be singular or plural.

[0072] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0073] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0074] In this application, the terms "variant" or "mutant" are used interchangeably in this invention, referring to a protein molecule obtained by introducing one or more amino acid substitutions, insertions, or deletions based on the amino acid sequence of wild-type human ferritin light chain (FTL), wherein at least one newly introduced N-glycosylation modification site is included. This site is typically composed of a conserved sequence of "Asn-X-Ser / Thr" (where X is any amino acid other than proline), which enables the variant to be recognized by glycosyltransferases and undergo N-glycosylation modification.

[0075] In this application, "nucleic acid" refers to a polymer composed of nucleotides linked by phosphodiester bonds, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). In this invention, the nucleic acid can be a DNA molecule, such as genomic DNA, cDNA, or synthetic DNA, or an RNA molecule, such as mRNA, tRNA, rRNA, or synthetic RNA. It encodes a variant of the human ferritin light chain as described above, i.e., a nucleotide sequence containing a signal peptide, a purified tag, and a mature FTL peptide sequence with N-glycosylation sites. This nucleic acid sequence can be obtained by modifying the wild-type FTL gene through site-directed mutagenesis or other methods, or it can be a synthetic sequence optimized according to codon preferences to facilitate efficient expression in specific host cells. For example, to improve expression efficiency in mammalian cells such as HEK293F, the codons in the coding region can be humanized.

[0076] In this application, the "signal peptide" is a short peptide located at the N-terminus of a protein. Its main function is to guide the variant from the ribosome to specific subcellular structures such as the endoplasmic reticulum for subsequent glycosylation modification. In this invention, the addition of a specific signal peptide enables the correct folding, glycosylation modification, and extracellular secretion of the human ferritin light chain variant in eukaryotic host cells (such as HEK293F cells). In this invention, the signal peptide is derived from mouse immunoglobulin, human interleukin-2, human insulin, human CD43 precursor protein, or human chymotrypsinogen B2. Its specific name may vary depending on the source or literature, but it is acceptable as long as it can guide the human ferritin light chain variant into the endoplasmic reticulum for glycosylation modification. For example, the signal peptide of Mouse IgG κappa involved in the examples is derived from the mouse immunoglobulin G kappa (κ) light chain signal peptide, and its amino acid sequence is METDTLLLWVLLLWVPGSTGDTPT.

[0077] In this application, the term "expression unit" refers to a functional DNA fragment capable of driving the transcription and translation of a target gene in a host cell. Its core components include a promoter and a coding sequence (such as the nucleic acid sequence encoding a human ferritin light chain variant in this invention). It may also selectively include terminators, enhancers, ribosome binding sites, Kozak sequences, and other regulatory elements as needed. These elements work synergistically to ensure that the exogenous gene is transcribed from DNA into mRNA in a specific host, and further translated into a biologically active protein. For example, when the expression unit contains a CMV promoter, it can efficiently initiate the transcription of downstream coding sequences in mammalian cells; while the polyA terminator helps ensure the stability of the mRNA and promotes its transport from the nucleus to the cytoplasm, thereby improving protein expression efficiency.

[0078] In this application, the term "expression vector" refers to a DNA molecule capable of carrying exogenous nucleic acids or expression units and autonomously replicating and expressing them within host cells. It typically includes an origin of replication, selection marker genes for screening transformants (such as antibiotic resistance genes, e.g., ampicillin resistance genes, kanamycin resistance genes, puromycin resistance genes, etc.), and a multiple cloning site consisting of multiple restriction endonuclease recognition sites to facilitate the insertion and cloning of exogenous nucleic acid fragments. For example, when the mammalian expression vector pcDNA3.1(+) is used, its CMV promoter can drive high-level transcription of the inserted human ferritin light chain variant coding sequence in mammalian cells such as HEK293F, while the neo resistance gene on the vector can be used to screen cell lines that have successfully transformed into and stably express the recombinant vector.

[0079] In this application, the term "transformer" refers to a cell obtained by introducing a foreign nucleic acid (such as a nucleic acid encoding a human ferritin light chain variant) or expression unit into a host cell. This cell is capable of carrying and stably maintaining the foreign nucleic acid or expression unit, thereby enabling its storage, amplification, or expression of the target protein (i.e., the human ferritin light chain variant) within the host. The transformed cell line can proliferate rapidly under suitable culture conditions, thereby amplifying the foreign nucleic acid it carries, or efficiently synthesizing and secreting the target variant protein under inducible conditions (if the expression unit contains an inducible promoter), providing sufficient protein samples for subsequent glycosylation modification, purification, and functional studies.

[0080] In this application, "N-glycosylation modification" refers to a biochemical reaction in which the reducing end of a glycan chain covalently links to the amide nitrogen atom of a specific asparagine (N) residue via an N-glycosidic bond. Specifically, it involves the transfer and linkage of glycan chains catalyzed by glycosyltransferases at asparagine residues in the conserved sequence "Asn-X-Ser / Thr" (where X is any amino acid except proline) appearing in the primary structure of a protein, thereby forming a glycoprotein. The "glycan chain" refers to an oligosaccharide or polysaccharide chain composed of multiple monosaccharide molecules linked by glycosidic bonds, and its monosaccharide composition includes, but is not limited to, glucose, mannose, galactose, N-acetylglucosamine, N-acetylglucosamine, fucose, etc.

[0081] In this application, the term "packaging carrier" refers to a biomaterial or carrier system capable of encapsulating or loading active substances (such as small molecule drugs, nucleic acid drugs, protein drugs, etc.) within its internal cavity or surface using its own structural characteristics. In this invention, the glycosylated human ferritin light chain retains the natural hollow spherical cage structure of ferritin, possessing a large internal cavity volume. This allows for the encapsulation of active substances through physical embedding, hydrophobic interactions, electrostatic adsorption, etc., thereby achieving stable loading of the active substances. Furthermore, the glycan structure on its surface not only improves the biocompatibility and stability of the carrier but also serves as a site for targeted modification, enabling targeted delivery to specific tissues or cells through conjugation with targeting ligands (such as antibodies, peptides, aptamers, etc.).

[0082] In this application, the term "formulation" refers to a stable complex formed by an active ingredient and a glycosylated human ferritin light chain variant under specific assembly conditions. This complex can effectively encapsulate the active ingredient, thereby improving its stability, bioavailability, or targeting. For example, when the active ingredient is paclitaxel, a paclitaxel-glycosylated ferritin light chain formulation can be formed by mixing paclitaxel with a glycosylated human ferritin light chain treated at pH 2.0 for 20 minutes, and then adjusting the pH to 7.0 for assembly. This formulation can protect paclitaxel from external environmental influences and may achieve specific in vivo distribution through the properties of ferritin.

[0083] In this application, the term "drug" refers to a substance composed of the aforementioned formulation and one or more pharmaceutically acceptable excipients, used for the prevention, diagnosis, treatment, or alleviation of human or animal diseases. Its core lies in the glycosylated human ferritin light chain variant in the formulation serving as a drug carrier, efficiently encapsulating and delivering the active ingredient to the target lesion or tissue.

[0084] In this application, "equivalent position" refers to the corresponding amino acid position within the corresponding domain or functional region determined by sequence homology analysis (such as using bioinformatics tools like BLAST or ClustalW) when compared with the amino acid sequence shown in SEQ ID NO:1. Its spatial structure and role in the overall protein function are similar to positions 1-10, 80-90, or 150-160 in SEQ ID NO:1. For example, if a wild-type FTL has a few amino acid insertions or deletions at the N-terminus compared to SEQ ID NO:1, but the overall folding structure is consistent, then the equivalent position of its N-glycosylation site should be within the N-terminal flexible region corresponding to SEQ ID NO:1. Similarly, for the equivalent positions in the middle and C-terminus, it is also necessary to combine secondary structure prediction (such as the distribution of α-helices, β-sheets, and random coils) and tertiary structure simulation to ensure that the selected equivalent sites are also located in surface-exposed regions with minimal impact on the stability of the protein core structure. This ensures that the introduced N-glycosylation sites can effectively perform glycosylation modification and exert the expected functional improvement effect.

[0085] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0086] The amino acid sequences of the seven variants involved in the examples (shown in parentheses) are His tags. The glycosylation sites shown are:

[0087] FTL-N1a: METDTLLLWVLLLWVPGSTGDTPT(HHHHHH)M NSS QIRQ NYS TDVEAAVNSLVNLYLQASYTYLSLGFYFDDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLKHD* (SEQID NO: 3)

[0088] FTL-N1b:METDTLLLWVLLLWVPGSTGDTPT(HHHHHH)MSSQIRQ NYS TDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPA NNST EDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLKHD*(SEQ ID NO:4)

[0089] FTL-N1c:METDTLLLWVLLLWVPGSTGDTPT(HHHHHH)MSSQIRQ NYS TDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEWGKTPDAMKAAMALEKKNLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGG NKTLNGTS PEAGLGEYLFERLTLKHD*(SEQ ID NO:5)

[0090] FTL-N2ab:METDTLLLWVLLLWVPGSTGDTPT(HHHHHH)M NSS FORTY NYS TDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPA NNST EDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLKHD*(SEQ ID NO:6)

[0091] FTL-N2ac:METDTLLLWVLLLWVPGSTGDTPT(HHHHHH)M NSS FORTY NYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGG NKTLNGTS PEAGLGEYLFERLTLKHD*(SEQ ID NO:7)

[0092] FTL-N2bc:METDTLLLWVLLLWVPGSTGDTPT(HHHHHH)MSSQIRQ NYS TDVEAAVNSLVNLYLQASYTYLSLGFYFDDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPA NNST EDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGG NKTLNGTS PEAGLGEYLFERLTLKHD*(SEQ ID NO:8)

[0093] FTL-N3abc:METDTLLLWVLLLWVPGSTGDTPT(HHHHHH)M NSS QIRQ NYS TDVEAAVNSLVNLYLQASYTYLSLGFYFDDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPA NNST EDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGG NKTLNGTS PEAGLGEYLFERLTLKHD*(SEQ ID NO:9)

[0094] It should be understood that in the various embodiments of this application, the sequence numbers of the above processes do not imply the order of execution. Some or all steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application. The present invention is further illustrated below with reference to embodiments:

[0095] Example

[0096] 1. Construction of FTL expression vector containing signal peptide

[0097] (1) Template and carrier selection

[0098] Vector: pCDNA3.1(+)

[0099] Gene coding for human FTL: MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGPEAGLGEYLFERLTLKHD (SEQ IDNO: 1)

[0100] (2) Insertion of signal peptide and purification tag

[0101] Signal peptide sequence: METDTLLLWVLLLWVPGSTGDTPT (SEQ ID NO:2) (Alternatively, the IL-2 signal peptide, sequence MYRMQLLSCIALSLALVTNS; the INS signal peptide, sequence MALWMRLLPLLALLALWGPDPAAA; the SPN signal peptide, sequence MATLLLLLGVLVVSPDALGST; and the CTRB2 signal peptide, sequence MAFLWLLSCWALLGTTFG can be used. The effects of these signal peptides are comparable to those shown in SEQ ID NO:2.)

[0102] Purification tag: HHHHHH (6× His tag)

[0103] By synthesizing oligonucleotides, the signal peptide coding sequence and protein purification tag were designed before the FTL start codon, and a "signal peptide-His-FTL" fusion expression cassette was constructed at the vector multiple cloning site.

[0104] Signal peptide-His-FTL fusion expression cassette sequence: METDTLLLWVLLLWVPGSTGDTPTHHHHHHMSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLKHD* (* is the stop codon)

[0105] The ligation method can be either restriction endonuclease Hind III / Xba I digestion followed by T7 ligase ligation or homologous recombination splicing. After plasmid construction, sequencing is used to confirm that the inserted sequence and reading frame are correct.

[0106] 4.2.2 Design of N-glycosylation modification sites and construction of site-directed mutagenesis

[0107] (1) Structural analysis and site selection

[0108] The overall conformation of FTL was analyzed using its protein structure (PDB: 6WX6); key residues at the multimer interface and in the core hydrophobic region were excluded; and amino acid residues located on the surface, hydrophilic, in the flexible loop region, or in non-critical secondary structures were selected as potential engineering sites.

[0109] (2) Introduction of N-glycosylation sequence

[0110] Based on the above screening results, three engineerable regions were selected in FTL. The N-glycosylation sequence Asn-X-Ser / Thr (i.e., NXS or NXT, where X is any amino acid other than proline) was inserted into suitable sites, or one or more amino acids were deleted and / or replaced in the original sequence to form the N-glycosylation sequence as described above. FTL-N was constructed respectively. 1 a、FTL-N 1 b, FTL-N 1 c mutant. Building on the success of the single-point mutation, we continued to construct conjugates containing 2-3 N-glycosites to obtain a higher density of glycocalyx. The final modified glycosylated FTL contained the three newly introduced glycosylation sites and was named FTL-N. 3 abc. Site-directed mutagenesis can be performed using PCR. Primers containing the target mutated codon are designed for amplification. The amplified product is inserted into the vector pCDNA3.1(+) via homologous recombination. The homologous recombination vector is then transformed into E. coli, and single clones are selected for sequencing verification.

[0111] PCR system (50 μl system)

[0112]

[0113] PCR procedure:

[0114]

[0115] Vector digestion system:

[0116]

[0117] Vector / fragment homologous recombination system:

[0118]

[0119] After homologous recombination of the fragment and vector, a frozen DH5α chemicompetent cell was completely thawed on ice. 10 μL of the recombinant product was added to the cells, and the mixture was gently pipetted to ensure adequate DNA contact with the cell membrane. The mixture was incubated on ice for approximately 15 minutes to promote DNA binding. Subsequently, the centrifuge tube was rapidly transferred to a 42°C water bath for heat shock for 60 seconds, and immediately returned to ice for 2 minutes to allow for cell membrane recovery. The bacterial culture was evenly spread onto LB agar plates containing ampicillin and incubated at 37°C for 12–16 hours, observing colony growth. Morphologically normal and well-dispersed single clones were selected, inoculated into liquid LB agar, and plasmids were extracted for sequencing to verify the correctness of the recombinant fragment and mutation sites.

[0120] The final modified FTL-N 3 abc sequence:

[0121] METDTLLLWVLLLWVPGSTGDTPT(HHHHHH)M N SSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPA NNST EDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGG NKTLNGTS PEAGLGEYLFERLTLKHD*(( ) marks the purification tag. The marked sites are the three sites where glycosylation sequences were inserted in the modified sequence, named site 1, site 2, and site 3, respectively.

[0122] 3. Transient expression and secretion in HEK293F cells

[0123] (1) Cell preparation

[0124] HEK293F suspension cells (Human Embryonic Kidney 293F) were used and cultured in serum-free suspension medium specifically formulated for HEK293F by SinoBiological. Culture conditions were: 37℃, 5% CO2, shake-flask culture at 120 rpm. Cell status was monitored beforehand to ensure a cell density of approximately 2.0 × 10⁻⁶ cells before transfection. 6 The expression rate must be at least 95% and the number of cells / mL must be greater than 95% to ensure optimal transient expression efficiency.

[0125] (2) Instantaneous transfection

[0126] The constructed N-glycosylated FTL expression plasmid was prepared at a dosage of 2 μg DNA / mL cell culture volume. Linear polyethyleneimine (PEI) was used as the transfection reagent, and a DNA-PEI complex was prepared at a DNA:PEI ratio of 1:2 (mass ratio). The complex was incubated in serum-free medium at room temperature for 15 minutes to allow for the formation of the nanoparticle complex. Subsequently, the complex was slowly added to the HEK293F cell suspension and gently shaken to ensure uniform distribution.

[0127] (3) Protein expression and secretion

[0128] After transfection, the culture was continued at 37 °C and 120 rpm for 72 hours. During this process, the signal peptide mediates the entry of the target protein into the endoplasmic reticulum and Golgi apparatus secretion pathway, completes N-glycosylation modification, and is finally released into the culture supernatant in a secretory form.

[0129] (4) Collection of supernatant

[0130] 72 hours after transfection, the cell pellet was separated from the culture supernatant by centrifugation (3000×g, 15 min, 4 ℃) for subsequent Ni-NTA affinity chromatography purification of N-glycosylated FTL glycoprotein.

[0131] Two-step purification of 4 N-glycosylated FTLs

[0132] (1) Ni-NTA affinity chromatography (His-tag affinity purification)

[0133] First, Ni-NTA affinity chromatography was used to initially enrich the His-tagged glycosylated FTLs. The procedure is as follows:

[0134] ① Pre-equilibrate the Ni-NTA agarose packing material with PBS (pH 7.4) buffer to reduce weak nonspecific binding in the sample;

[0135] Load the HEK293F supernatant into a Ni-NTA column. After the protein is completely bound, wash thoroughly with 15 column volume (CV) of PBS buffer to remove non-specifically bound proteins.

[0136] ② Use a washing buffer containing 20 mM imidazole to further remove weakly bound impurities;

[0137] ③ Finally, the bound glycosylated FTL protein was eluted with 250 mM imidazole elution buffer, and the eluent was collected.

[0138] (2) 100 kDa ultrafiltration concentration (preliminary desalination and volume concentration)

[0139] Ni-NTA eluents are typically large in volume and contain high concentrations of imidazole, requiring further processing before entering a gel filtration system. Therefore, a centrifugal ultrafiltration tube with a molecular weight cutoff (MWCO) of 100 kDa was used to concentrate and desalinate the FTL.

[0140] ① Transfer the Ni-NTA eluent to a 100 kDa ultrafiltration centrifuge tube;

[0141] ② Centrifuge at 4 ℃ and 1500g until the sample volume reaches the required range (0.5-1 mL). The concentrated sample can then be directly used for SR6 chromatography separation.

[0142] (3) Gel chromatography

[0143] To further remove contaminating proteins and obtain high-purity glycosylated proteins, this invention employs an SR6 separation column (Superose 6 Increase) on an AKTA chromatography system for gel chromatography. The specific procedures are as follows:

[0144] ① Equilibrate the SR6 column thoroughly with PBS (pH 7.4) buffer at a flow rate of 0.3 ml / min;

[0145] ② The FTL sample concentrated by 100 kDa ultrafiltration was injected into the sample loop of the AKTA system; elution was performed at a flow rate of 0.3 ml / min, and the elution peak at UV 280 nm was monitored in real time.

[0146] ③ Collect the main peak portion (generally corresponding to the 24-mer nanocage structure of FTL), and further verify its purity and aggregation state by SDS-PAGE; finally, obtain high-purity N-glycosylated modified FTL protein.

[0147] 5. Identification of N-glycosylated FTL proteins

[0148] After obtaining high-purity N-glycosylated FTL protein, we further verified the multidimensional structure of the modified protein by SDS-PAGE, PNGaseF digestion, and cryo-electron microscopy, confirming that all three sites of the modified FTL were successfully N-glycosylated.

[0149] result Figures 1-3 :

[0150] Figure 1 In this dataset, FTL-WT is a wild-type FTL without a signal peptide; FTL-spWT is a wild-type FTL with an inserted signal peptide; FTL-N1a is an FTL with an inserted signal peptide and a glycosylation sequence at site 1; FTL-N1b is an FTL with an inserted signal peptide and a glycosylation sequence at site 2; FTL-N1c is an FTL with an inserted signal peptide and a glycosylation sequence at site 3; FTL-N2ab is an FTL with an inserted signal peptide and glycosylation sequences at sites 1 and 2; FTL-N2ac is an FTL with an inserted signal peptide and glycosylation sequences at sites 1 and 3; FTL-N2bc is an FTL with an inserted signal peptide and glycosylation sequences at sites 2 and 3; and FTL-N3abc is an FTL with an inserted signal peptide and glycosylation sequences at sites 1, 2, and 3. Figure 1 As can be seen, the molecular weight of glycoprotein gradually increases with the increase of glycosylation sites, and all three sites representing FTL can be successfully modified by N-glycosylation.

[0151] Figure 2 PNGase F is an endoglucosidase that cleaves N-glycans from asparagine (Asn) residues in glycoproteins, and is the gold standard for detecting the presence or absence of N-glycans. The results in the figure show that after PNGase F digestion, the N-glycosylated mutants of FTL all exhibited band sizes consistent with the wild-type FTL without glycosylation, indicating that the protein indeed acquired site-specific N-glycosylation during HEK293F expression.

[0152] Figure 3The Cryo-EM structure of the FTL N-glycosylation mutant is shown. The top left image is a schematic diagram of the three-dimensional structure of the wild-type FTL (FTL-spWT) carrying the signal peptide; the magnified view on the right shows an N-glycosylation site inherent in FTL-spWT after secretion, where the structure of the N-glycan core pentasaccharide connected to this site can be well aligned with the electron density. The middle and bottom rows show multiple site-specific N-glycosylation mutants constructed in this invention, with each corresponding N-glycan site marked with a colored area on the surface. Significant additional electron density was observed at each designed N-glycan site, located above the Asn side chain groove and extending to the outer surface of the protein, consistent with typical oligosaccharide structures, proving that all three N-glycosylation sites in the mutant have successfully undergone glycan modification. The magnified view in the lower right corner further shows the density characteristics around some key mutation sites, indicating a clear and distinguishable spatial relationship between the glycan and the surrounding amino acid structure.

[0153] In summary, this invention introduces three novel N-glycosylation sequences, Asn-X-Ser / Thr, into the FTL. Furthermore, the FTL protein itself possesses a natural N-glycosylation sequence, NFS, but this natural site is not glycosylated during soluble protein expression. After adding a signal peptide to the N-terminus of the FTL using this invention, the natural NFS sequence was successfully introduced into the ER / Golgi secretion pathway, thus achieving genuine N-glycan modification at the NFS site. These results provide direct, precise, and structurally resolution support for the proposed "signal peptide introduction + multi-site N-glycosylation design" strategy.

[0154] The effect of 6 N-glycosylation on FTL proteins

[0155] 6.1 FTL N-glycosylation significantly improves protein thermal stability

[0156] This invention addresses the problem of insufficient thermal stability of natural soluble proteins by introducing multiple N-glycosylation sites onto the surface of the fibroblast-like protein (FTL), thereby enabling programmable glycan modification. The introduction of glycans is expected to enhance local structural rigidity, increase the hydration shell, and reduce some unfolded intermediate states, thus improving the high-temperature stability of the protein.

[0157] To systematically evaluate the effect of glycosylation on FTL stability, this study used differential scanning calorimetry (DSC) to determine the thermal denaturation temperature (Tm) of each mutant. Experimental conditions included: protein concentration of 0.5 mg / mL, PBS (pH 7.4) as the buffer, heating rate of 2 °C / min, and a temperature range of 30–110 °C. The curves showing the change in heat capacity with temperature were recorded, with the peak value corresponding to Tm.

[0158] like Figure 4As shown, FTL-spWT and several site-directed N-glycosylation mutants (FTL-N) 1 b, FTL-N 2 ab、FTL-N 3 Differences in thermal stability (abc). Differential scanning calorimetry (DSC) was used to determine the thermal denaturation curves of the proteins during heating, and the melting temperature (Tm) was calculated based on the temperature corresponding to the peak heat capacity. The Tm of wild-type FTL was 84.9℃, while that of FTL-N with introduced N-glycans was... 1 b, FTL-N 2 ab and FTL-N 3 The Tm values ​​of abc were significantly increased to 103.9℃, 103.7℃, and 102.1℃, respectively.

[0159] DSC results showed that multi-site N-glycosylation significantly improved the thermal stability of FTLs. The Tm of FTL-spWT was 84.9℃, while that of FTL-N... 1 b, FTL-N 2 ab、FTL-N 3 The heat maturities (Tm) of abc reached 103.9℃, 103.7℃, and 102.1℃, respectively, which are 15-25℃ higher than the Tm of wild-type FTL (approximately 80℃). These data demonstrate that N-glycan modification can significantly enhance the protein's resistance to heat denaturation, providing core evidence that this invention can solve the key technical problem of poor protein stability.

[0160] 6.2 FTL N-glycans enhance the protein's resistance to protease degradation

[0161] Proteins are susceptible to degradation by proteases in the in vivo environment, leading to functional decline and accelerated clearance. To enhance the protein's resistance to degradation, this invention inserts N-glycans into structurally exposed regions, enabling the glycans to form steric barriers against potential protease action sites on the protein surface, thereby improving anti-protease capabilities.

[0162] Experimentally, we used protease K (PK) enzymatic digestion to verify the degradation resistance of glycosylated FTLs. The expressed and purified FTL-spWT and FTL-N... 1 b, FTL-N 2 ab、FTL-N 3 abc was mixed with 150 ug / ml PK and reacted at 58 ℃ for 0, 5, 10, 15 and 20 minutes. 5 mM PMSF was added to the reaction system and the reaction was immediately terminated. The amount of remaining protein was quantitatively detected by Western blot using anti-His antibody.

[0163] Figure 5The figure shows the resistance to degradation of FTL-spWT and several site-directed N-glycosylation mutants under the action of proteinase K (PK). Each protein sample was digested with PK at 58℃, and the reaction was terminated at 0, 5, 10, 15, and 20 minutes. The amount of remaining intact FTL protein was detected by Western blot. The gray-black bands represent FTLs that still maintain their intact structure, and the colored dots indicate the relative degradation trend from intact to complete degradation.

[0164] Experimental results showed that FTL-spWT, containing only a small amount of sugar chains, was almost completely degraded within 5 minutes, while FTL-N, with multiple glycosylation sites, was significantly degraded. 1 b, FTL-N 2 ab and FTL-N 3 abc still retains a large number of intact bands within 20 minutes. Especially FTL-N 3 The bands abc showed almost no significant attenuation, demonstrating that the glycans form an effective barrier on the outer surface of the structure, significantly enhancing the protein's resistance to proteases. This result directly illustrates that the present invention can improve the technical problem of protein degradation and lays the foundation for subsequent half-life extension.

[0165] 6.3 Modification of FTL with human complex N-glycans significantly reduced protein immunogenicity.

[0166] Protein drugs often induce immune responses due to their "non-self" characteristics, aggregates, or surface epitope exposure. This invention introduces multiple N-glycan chains to form a barrier on the protein surface similar to the host's glycocalyx, and utilizes naturally occurring human complex, highly sialylated N-glycans produced by HEK293F cells to reduce recognition by the immune system, thereby fundamentally reducing immunogenicity.

[0167] To assess the in vivo immunogenicity of glycosylated FTLs, we compared FTL-spWT and FTL-N with different degrees of glycosylation. 3 abc and PBS controls were injected into the tail vein of mice on days 1 and 14, respectively. Peripheral blood was subsequently collected on days 7, 14, 21, and 28, and serum levels of FTL-spWT and FTL-N were detected using ELISA. 3 The IgG antibody titers of abc were calculated, and the ED was calculated based on the curve. 50 The value serves as a quantitative indicator of immunogenicity.

[0168] Figure 6 This demonstrates the difference between FTL-spWT and the multi-site N-glycosylation mutant FTL-N. 3The difference in the ability of abc to induce an immune response in mice. The experimental procedure is illustrated above: mice were given two tail vein injections (PBS, FTL-spWT, or FTL-N) on Day 0 and Day 14. 3 (abc) and peripheral blood was collected on Day 7, Day 14, Day 21, and Day 28, respectively. The titer of anti-FTL IgG in serum was then detected using enzyme-linked immunosorbent assay (ELISA), and its ED was calculated. 50 (half-maximal effective dilution) serves as a quantitative indicator of immunogenicity.

[0169] Experimental results showed that no immune response was detected in the PBS control group; low-glycosylated FTL-spWT showed an effective response at ED in weeks 2–4. 50 The values ​​reached the range of 1600-1800, while the multi-site glycosylated FTL-N 4abc The ED50 value is only about 100–300, which is more than 6–18 times lower than that of FTL-spWT, and the difference is significant. This fully demonstrates that N-glycans can effectively mask immunosensitive epitopes, and the "autologous" hypersialylated glycans derived from HEK293 cells further reduce immune recognition, clearly solving the problem of high immunogenicity of existing protein drugs.

[0170] 6.4 FTL N-glycosylation significantly improves the acid-base reversible stability of FTLs, enabling them to acquire drug loading capacity.

[0171] Natural ferritin heavy chains (FTH) possess strong subunit interactions and a more stable cavity, enabling them to dissociate into monomers under acidic conditions (e.g., pH 2–3) and reassemble under neutral conditions, thus facilitating the encapsulation of hydrophobic small molecule drugs. However, ferritin light chains (FTL) exhibit poor acid-base cycling stability, particularly at pH 2, where they are prone to partial irreversible conformational damage or aggregation, making it difficult for them to reform into complete nanocage structures during refolding. Therefore, wild-type FTLs are almost incapable of achieving effective drug loading under conventional acid-base triggering modes.

[0172] To verify whether N-glycans improve the acid-base cycling stability of FTLs and enable them to encapsulate drugs, this invention treated low-glycosylated FTL-spWT and multi-site glycosylated FTL-N3abc at pH 2.0 for 20 minutes to dissociate their protein structures, while simultaneously adding a fixed dose of paclitaxel (PTX). The pH was then adjusted back to 7.0 to induce the reassembly of the 24-mer. After renaturation, reversed-phase high-performance liquid chromatography (HPLC) was used to detect the loading of PTX onto the FTLs, thereby determining whether the drug was encapsulated within the protein nanocages.

[0173] Figure 7 Demonstrates multi-site N-glycosylation of FTLs (FTL-N) 3 The differences between FTL-spWT (abc) and low-glycosylation sites in acid-base induced drug loading experiments. The schematic diagram on the left illustrates the experimental procedure: First, FTL was treated at pH 2.0 for 20 minutes to induce its dissociation, temporarily opening the nanocage structure, while the hydrophobic small molecule paclitaxel (PTX) was added simultaneously; then, the FTL was allowed to reassemble at pH 7.0 to evaluate the protein's ability to encapsulate drugs during refolding.

[0174] Experimental results showed that low-glycosylated FTL-spWT was difficult to refold correctly at pH 7.0 after treatment at pH 2.0, and no PTX absorption peak appeared in HPLC, indicating loading failure; while multi-site N-glycosylated FTL-N 3 abc exhibits a clear PTX absorption peak with a significant peak area, indicating that glycosylation significantly improves the refolding ability of FTL after acidic dissociation, enabling it to complete the entire "dissociation-loading-refolding" process like FTH, thus achieving efficient PTX packaging.

[0175] and Figure 8 The display shows the release effect after encapsulation. SUM159 is a tumor cell, and FTL(-PTX) is a blank FTL without PTX encapsulation, which does not cause tumor cell death. However, after adding FTL(+PTX), PTX release leads to its killing effect on tumor cells; at this point, the survival rate of SUM159 cells is significantly reduced. This indicates that PTX is successfully encapsulated in FTL and can also be successfully released to exert a tumor-killing effect.

[0176] Based on the above, it is shown that N-glycans not only endow FTLs with advantages in structural stability and immunological dimensions, but also enhance their acid-base response adaptability and functional plasticity, enabling light chain ferritin to acquire drug carrier capabilities that natural FTLs do not possess.

[0177] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A variant of the human ferritin light chain, wherein at least one N-glycosylation site is formed at position 1-10, 80-90 or 150-160 in the amino acid sequence of the wild-type human ferritin light chain. The N-glycosylation site is NXS or NXT; wherein... X is any amino acid other than proline.

2. The variant according to claim 1, characterized in that, The mutations include the substitution, deletion, and / or addition of amino acids, and the N-glycosylation site is NSS, NNST, or NKTLNGTS.

3. The variant according to claim 2, characterized in that, The mutation includes at least one of the following: Insert at least one amino acid residue at any position between 1 and 10 to make it contain NSS; Insert at least one amino acid residue at any position between positions 80 and 90 to make it contain NNST; Insert at least one amino acid residue at any position between 150 and 160 to make it contain NKTLNGTS.

4. The variant according to claim 3, characterized in that, The mutation is: Insert N between 2 and 3 positions; Or it could be inserting NNST between bits 86 and 87; Or it could be that NKTLNGTS is inserted between bits 157 and 158; Alternatively, N can be inserted between positions 2 and 3, and NNST can be inserted between positions 86 and 87. Alternatively, N can be inserted between bits 2 and 3, and NKTLNGTS can be inserted between bits 157 and 158. Alternatively, NNST could be inserted between bits 86 and 87, and NKTLNGTS could be inserted between bits 157 and 158. Alternatively, N can be inserted between bits 2 and 3, NNST between bits 86 and 87, and NKTLNGTS between bits 157 and 158.

5. The variant according to any one of claims 1 to 4, characterized in that, The amino acid sequence of the wild-type human ferritin light chain is shown in SEQ ID NO:

1.

6. The variant according to any one of claims 1 to 5, characterized in that, Its N-terminus also includes a signal peptide and / or a purified tag. The signal peptide is at least one of Mouse IgG kappa signal peptide, IL-2 signal peptide, INS signal peptide, SPN signal peptide, and CTRB2 signal peptide; The purification tag is at least one of the following: 6×His tag, 8×His tag, 10×His tag, Strep tag, HA tag, GST tag, MBP tag, GFP tag, Flag tag, and AlFA tag.

7. Biomaterials, including at least one of the following: a) A nucleic acid encoding a variant as described in any one of claims 1 to 6; b) An expression unit containing a promoter and the nucleic acid described in a); c) An expression vector containing the nucleic acid described in a) and / or the expression unit described in b); d) Transformation bodies containing the plasmid vector described in c), or whose genomes are integrated with the nucleic acid described in a) and / or the expression unit described in b).

8. A glycosylated human ferritin light chain variant, wherein in any one of claims 1 to 6, the N residue at the N-glycosylation site is glycosylated.

9. The glycosylated human ferritin light chain variant according to claim 8, characterized in that, The glycosylation involves the reducing end of the sugar chain being linked to the amide nitrogen atom of an N residue via an N-glycosidic bond.

10. A method for preparing the glycosylated human ferritin light chain variant of claim 8 or 9, comprising culturing the transformant of claim 7 to obtain the variant of any one of claims 1 to 6, and chemically modifying it by linking the N-glycan chain to the amide nitrogen atom of the N residue; Alternatively, the transformant as described in claim 7 can be cultured to obtain a culture product containing a glycosylated human ferritin light chain variant.

11. The use of the variants of any one of claims 1 to 6, the glycosylated human ferritin light chain variants of claim 8 or 9, or the culture product obtained according to claim 10, in the preparation of packaging carriers.

12. A formulation, the raw materials for which are prepared include the active ingredient and the glycosylated modified human ferritin light chain variant as described in claim 8 or 9, or the culture product obtained in claim 10.

13. The formulation according to claim 12, characterized in that, The active ingredients include at least one of the following: paclitaxel, doxorubicin, epirubicin, camptothecin, cisplatin, carboplatin, gefitinib, everolimus, temozolomide, curcumin, zinc phthalocyanine photosensitizers, ATP, and nucleic acid drugs.

14. A method for preparing the formulation of claim 12 or 13, comprising mixing the active ingredient with the glycosylated modified human ferritin light chain variant of claim 8 or 9, and assembling the mixture to obtain the formulation.

15. A pharmaceutical product comprising the formulation of claim 12 or 13 and pharmaceutically acceptable excipients.

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