CN116804049A - Ferritin tracer polypeptide conjugates and uses thereof - Google Patents

Ferritin tracer polypeptide conjugates and uses thereof Download PDF

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Publication number
CN116804049A
CN116804049A CN202310278157.XA CN202310278157A CN116804049A CN 116804049 A CN116804049 A CN 116804049A CN 202310278157 A CN202310278157 A CN 202310278157A CN 116804049 A CN116804049 A CN 116804049A
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ferritin
polypeptide
subunit
mutant
seq
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柯天一
丁会
姚德惠
劳芳
刘岩
赵梦蕊
张泽译
闫西冲
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Kunshan Xinyunda Biotech Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/644—Transferrin, e.g. a lactoferrin or ovotransferrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Abstract

The present disclosure relates to a ferritin tracer polypeptide conjugate and uses thereof. The ferritin tracer polypeptide conjugate comprises a ferritin H subunit (HFn) mutant polypeptide comprising an amino acid sequence selected from one of SEQ ID NOs 5-8. The disclosure also relates to a polypeptide conjugate, a caged protein formed from the polypeptide or the polypeptide conjugate, and uses thereof. The polypeptide or the polypeptide conjugate and the caged protein formed by the polypeptide or the polypeptide conjugate have better hydrophilicity and coupling characteristics, and better resolution, accuracy and durability on tumor sentinel lymph tracking.

Description

Ferritin tracer polypeptide conjugates and uses thereof
Technical Field
The present disclosure belongs to the biomedical field, relates to a polypeptide conjugate containing ferritin heavy chain subunit mutant and application thereof in tracking, in particular to a ferritin nano contrast agent and a preparation method thereof, and application thereof in lymph node staining and imaging diagnosis in tumor surgery.
Background
Sentinel Lymph Node (SLN) refers to the first lymph node at which a primary tumor occurs at a specific site in an organ, and the tumor cells invade earliest. Sentinel lymph node biopsies are a reliable method of assessing early breast cancer axillary lymph node metastasis status. The selection of a proper sentinel lymph node tracing technology is a key for accurately searching sentinel lymph nodes in operation, so that the detection rate of the sentinel lymph nodes is improved, the false negative rate is reduced, and the axilla injury of patients can be reduced. The sentinel lymph node tracing method mainly comprises a dye method (including blue dye, fluorescent dye and the like), a nano-carbon method, a radionuclide method, a superparamagnetic iron oxide method and a combined tracing method of the dyes. The blue dye method has low cost and wide application, but is easy to transition. The nano-carbon method has high biocompatibility, but the dyeing time is required to be strictly controlled. The detection rate and the false negative rate of the superparamagnetic iron oxide are similar to those of the standard combination method, but the requirements on the materials of surgical instruments are high. The fluorescent dye method can image in real time, has high sensitivity, but has complicated operation in operation. The fluorescent dye most commonly used at present is indocyanine green (Indocyanine Green, ICG), a highly sensitive near infrared fluorescein that has been approved for clinical use by the us FDA. However, the imaging of lymph nodes also has obvious limitation, namely ICG lacks specificity, and sentinel lymph nodes with tumor metastasis and secondary lymph nodes with inflammatory hyperplasia cannot be distinguished; secondly, the problem of ICG quenching during surgery also affects its accuracy. The nuclide method has high detection rate, but cannot be directly identified by naked eyes.
There is still a lack of effective methods for tracing sentinel nodes in oncology surgery that are rapid, safe, low-damaging and accurate. Ferritin (Ferritin) is a large protein of about 450kDa self-assembled from 24 subunits into a spherical cage-like structure with internal and external dimensions of about 8nm and about 12nm, respectively. Since ferritin has activity of binding to transferrin receptor (TfR 1), tfR1 is found to be highly expressed in a variety of tumor cells with active targeting. Meanwhile, the ferritin has the characteristic of small size and can reach the deep part of tumor tissues, so that the ferritin is tried to be used as a carrier for conveying various living active molecules in the tumor field in the field of diagnosis, image tracing and treatment. The delivery modes of ferritin as a transport carrier are divided into two modes, namely, bioactive molecules such as small molecular medicine doxorubicin are loaded in a cage-shaped cavity; the other delivery mode is to couple bioactive molecules on the outer surface of the ferritin, and compared with the inner wrapping mode of the ferritin, the delivery mode has more types of the bioactive molecules which can be carried, is not limited by the inner space of the ferritin, does not need to consider the problems of releasing the molecules in the cage after reaching the target position, and the like, so that the delivery mode has wider application prospect.
The methods reported in the literature for coupling bioactive molecules to the outer surface of ferritin can be performed biologically or chemically. Compared with the complex biological construction mode, the chemical method for coupling the bioactive molecules to the outer surface of the ferritin is more convenient and flexible, can couple various bioactive molecules to the surface of the ferritin rapidly through various chemical reactions with high flux, and can couple various functional molecules simultaneously. The key of the chemical coupling method is to provide optimized ferritin mutant suitable for the chemical coupling scene. Because wild-type ferritin has already had good pH and temperature stability compared to other carrier proteins and is easy to prepare on a large scale by means of prokaryotic expression, it is unavoidable that it is easy to polymerize, produce impurities, have low coupling efficiency (low bonding rate), and the coupled product is unstable in chemical reaction conditions. There is currently little research and development on ferritin mutants suitable for use in chemical coupling scenarios, and there is an urgent need in the art. Coupling of ferritin mutants to different bioactive molecules can play a positive role in tumor treatment, diagnosis or tracking.
Summary of The Invention
The ferritin mutant polypeptide with better drug formation, stability and coupling effect is constructed, and is conjugated with sentinel lymph node imaging molecules in a chemical coupling mode, so that the obtained mutant polypeptide conjugate and caged protein can solve the problems of high effective dose, low resolution and easiness in quenching existing in the current sentinel lymph node tracing technology, and provide a more sensitive, accurate, safe and small-damage imaging agent for locating a sentinel lymph region in tumor diagnosis and operation.
In a first aspect, the present disclosure provides a mutant ferritin H subunit polypeptide comprising an amino acid substitution at a position corresponding to position 98, 108, and/or 156 of SEQ ID No. 1 relative to a wild-type ferritin H subunit.
In certain embodiments, the mutant polypeptide has amino acids at positions corresponding to positions 98, 108, and/or 156 of SEQ ID NO. 1 substituted with more hydrophilic amino acids, such as amino acids with a carboxyl side chain, relative to the wild type ferritin H subunit.
In certain embodiments, the mutant polypeptide wherein the amino acid with a carboxyl side chain is selected from glutamic acid (E), aspartic acid (D), or histidine (H).
In certain embodiments, the mutant polypeptide has an amino acid such as asparagine (N) substituted with aspartic acid (D) at a position corresponding to position 98 of SEQ ID NO. 1.
In certain embodiments, the mutant polypeptide has an amino acid such as lysine (K) substituted with glutamic acid (E) at a position corresponding to position 108 of SEQ ID NO. 1.
In certain embodiments, the mutant polypeptide has an amino acid, e.g., arginine (R), substituted with histidine (H) at a position corresponding to position 156 of SEQ ID NO. 1.
In certain embodiments, the mutant polypeptide has an amino acid such as asparagine (N) substituted with aspartic acid (D) at a position corresponding to position 98 of SEQ ID NO. 1, and an amino acid such as lysine (K) substituted with glutamic acid (E) at a position corresponding to position 108 of SEQ ID NO. 1.
In certain embodiments, the mutant polypeptide has an amino acid such as asparagine (N) substituted with aspartic acid (D) at a position corresponding to position 98 of SEQ ID NO. 1, an amino acid such as lysine (K) substituted with glutamic acid (E) at a position corresponding to position 108 of SEQ ID NO. 1, and an amino acid such as arginine (R) substituted with histidine (H) at a position corresponding to position 156 of SEQ ID NO. 1.
In certain embodiments, the mutant polypeptide comprises an amino acid substitution at a position corresponding to position 62 and/or 65 of SEQ ID NO. 1 relative to the wild-type ferritin H subunit.
In certain embodiments, the mutant polypeptide has an amino acid, such as glutamic acid (E), substituted with lysine (K) at a position corresponding to position 62 of SEQ ID NO. 1.
In certain embodiments, the mutant polypeptide has an amino acid such as histidine (H) substituted with glycine (G) at a position corresponding to position 65 of SEQ ID NO. 1.
In certain embodiments, the mutant polypeptide has an amino acid, e.g., glutamic acid (E), substituted with lysine (K) at a position corresponding to position 62 of SEQ ID NO. 1, and an amino acid, e.g., histidine (H), substituted with glycine (G) at a position corresponding to position 65 of SEQ ID NO. 1.
In certain embodiments, wherein the mutant polypeptide comprises reduced cysteine relative to a wild type ferritin H subunit.
In certain embodiments, the mutant polypeptide has at least one substitution of a cysteine at a position corresponding to positions 90, 102, and 130 of SEQ ID NO. 1. Wherein the cysteine is substituted with an amino acid selected from the group consisting of: serine, threonine, asparagine, glutamine, glutamic acid, aspartic acid, lysine, arginine, histidine, alanine, glycine, preferably by serine or by an amino acid at the corresponding position of the wild-type ferritin light chain (L) subunit polypeptide.
In certain embodiments, the mutant polypeptide comprises a cysteine at a position corresponding to position 90 of SEQ ID NO. 1, and a cysteine at a position corresponding to position 102 of SEQ ID NO. 1, preferably a serine or an alanine, relative to the wild type ferritin H subunit, optionally a cysteine at a position corresponding to position 130 of SEQ ID NO. 1, preferably a serine or an alanine.
In certain embodiments, wherein the mutant polypeptide comprises a cysteine at a position corresponding to position 102 of SEQ ID NO. 1, and a cysteine at a position corresponding to position 90 of SEQ ID NO. 1, preferably serine or glutamic acid, relative to the wild type ferritin H subunit, optionally a cysteine at a position corresponding to position 130 of SEQ ID NO. 1, preferably serine or alanine.
In certain embodiments, wherein the mutant polypeptide comprises one cysteine in the loop region relative to the wild-type ferritin H subunit, the cysteine at the position corresponding to position 102 of SEQ ID No. 1 is substituted, and optionally the cysteine at the position corresponding to position 130 of SEQ ID No. 1 is substituted.
In certain embodiments, wherein the mutant polypeptide does not comprise additional cysteines except for one cysteine in the loop region and optionally a cysteine at a position corresponding to position 130 of SEQ ID NO. 1.
In certain embodiments, wherein the mutant polypeptide does not comprise a cysteine outside the loop region.
In certain embodiments, wherein the mutant polypeptide has a substitution of a cysteine at positions corresponding to positions 90 and 102 of SEQ ID No. 1 relative to the wild-type ferritin H subunit, optionally a substitution of a cysteine at position 130 of SEQ ID No. 1; and the mutant polypeptide has a cysteine substituted for the amino acid at a position corresponding to one of positions 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and 91 of SEQ ID NO. 1.
In certain embodiments, wherein the mutant polypeptide has a substitution of a cysteine at a position corresponding to positions 90, 102 and 130 of SEQ ID NO. 1 relative to the wild type ferritin H subunit; and the mutant polypeptide has a cysteine substituted for the amino acid at a position corresponding to one of positions 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and 91 of SEQ ID NO. 1.
In certain embodiments, wherein the mutant polypeptide comprises an amino acid sequence selected from one of SEQ ID NOs 5-8.
In a second aspect, the present disclosure provides a polypeptide conjugate comprising a ferritin H subunit mutant polypeptide according to the first aspect and a biologically active moiety conjugated thereto via a thiol group of the ferritin H subunit mutant polypeptide.
In a third aspect, the present disclosure provides a method of preparing a polypeptide conjugate according to the second aspect.
In a fourth aspect, the present disclosure provides a caged protein comprising at least one ferritin H subunit mutant polypeptide according to the first aspect, and/or at least one polypeptide conjugate according to the second aspect.
In a fifth aspect, the present disclosure provides a pharmaceutical composition comprising the ferritin H subunit mutant polypeptide of the first aspect, or the polypeptide conjugate of the second aspect, or the caged protein of the third aspect.
In a sixth aspect, the present disclosure provides uses comprising the polypeptides, polypeptide conjugates, caged proteins, and pharmaceutical compositions described above.
Drawings
FIG. 1 shows the results of protein expression of the ferritin H subunit mutants.
FIG. 2 shows the transmission electron microscopy results of samples of the ferritin H subunit mutants.
FIG. 3 shows SEC spectra for Mut-HFn-212, mut-HFn-241, mut-HFn-242, and Mut-HFn-243.
FIG. 4 shows the RP-HPLC profile of the coupling molecule Mut-HFn-243-ICG.
FIG. 5 shows SEC spectra of the coupling molecules HFn-ICG and Mut-HFn-243-ICG.
FIG. 6 shows the results of fluorescence imaging of ICG, HFn-ICG and Mut-HFn-243-ICG conjugated molecules on sentinel and secondary lymph nodes of rats.
Detailed Description
1. Definition of the definition
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.
As used herein, the term "and/or" encompasses all combinations of items connected by the term, and should be viewed as having been individually listed herein. For example, "a and/or B" encompasses "a", "a and B", and "B". For example, "A, B and/or C" encompasses "a", "B", "C", "a and B", "a and C", "B and C" and "a and B and C".
"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 ferritin H subunit of the present disclosure includes, but is not limited to, a mammalian ferritin H subunit, such as a human ferritin H subunit or a horse ferritin H subunit, preferably a human ferritin H subunit. An exemplary wild-type human ferritin H subunit comprises the amino acid sequence shown in SEQ ID NO. 1.
"caged proteins", also known as "nanocages", refer to three-dimensional protein structures, i.e., cage structures, formed from a plurality of polypeptides (subunits) capable of self-assembly, having an internal central cavity. The number of polypeptides (subunits) assembled into a cage protein is not particularly limited as long as it is capable of forming the cage structure. Cage proteins may have a symmetrical structure or may have an asymmetrical structure depending on their subunit composition. Typical caged proteins comprise ferritin/deferiprone.
"polypeptide," "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. The terms "polypeptide," "peptide," "amino acid sequence," and "protein" may also include modified forms including, but not limited to, glycosylation, lipid attachment, sulfation, gamma carboxylation of glutamic acid, hydroxylation, and ADP-ribosylation.
As used herein, "polynucleotide" refers to a macromolecule in which multiple nucleotides are linked by phosphodiester linkages, wherein the nucleotides include ribonucleotides and deoxyribonucleotides. The sequences of the polynucleotides of the present disclosure may be codon optimized for different host cells (e.g., E.coli) to improve expression of the polypeptide. Methods for performing codon optimization are known in the art.
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 specification 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 initiation codon. Accordingly, the coding nucleotide sequence may also comprise an initiation codon.
As used herein, the term "bioactive moiety" refers to a molecule capable of performing a biological function such as therapeutic, diagnostic, labeling, or prophylactic functions in an organism, and may be an organic macromolecule such as a protein, polypeptide, hormone, oligopeptide, nucleic acid, or an inorganic molecule such as a dye molecule, fluorescent molecule, nanoparticle, metal complex, or radioisotope. Generally, the "bioactive moiety" does not comprise a pharmaceutical carrier or excipient.
As used herein, a "detectable molecule" refers to a molecule that is capable of performing an indicated, detected, diagnosed, tagged, predicted biological function in an organism, as it can help an operator distinguish between lesions and normal tissue.
As used herein, "linker", "linker structure" or "linker" or "linking unit" refers to a fragment or bond of chemical structure that is linked at one end to a mutant ferritin H subunit polypeptide and at the other end to a drug (pharmaceutical compound), or to a drug compound after linking other linkers. The linker structures of the invention may be synthesized by methods known in the art, or by methods described herein.
As used herein, "pharmaceutically acceptable excipient" refers to any ingredient used in formulating pharmaceutical products that is not pharmacologically active and non-toxic, including but not limited to disintegrants, binders, fillers, buffers, tonicity agents, stabilizers, antioxidants, surfactants or lubricants.
2. Mutant polypeptides of ferritin heavy chain (H) subunit
In order to improve the properties of ferritin heavy chain subunits as drug carriers, the inventors have previously obtained improved mutants of mutations in the ferrite core and/or in the cysteine site (PCT/CN 2020/101312).
As a drug carrier, ferritin can be chemically coupled to a functional molecule (antibody, tracer molecule, small molecule peptide) through thiol groups (SH) on its surface, thereby obtaining a ferritin-functional molecule conjugate. Based on the previous work, the inventors have further engineered the ferritin H subunit to provide more flexible coupling sites, reduce side reactions and improve uniformity of the coupled product, and reduce ferritin aggregation and improve ferritin solubility. Unexpectedly, the ferritin H subunit mutants of the present disclosure also have significantly improved chemical coupling reaction efficiency (binding rate).
Accordingly, in one aspect, the present disclosure provides a ferritin heavy chain (H) subunit mutant polypeptide comprising an amino acid substitution at a position corresponding to position 98, 108, and/or 156 of SEQ ID No. 1 relative to a wild-type ferritin H subunit.
Without being bound by any theory, it is believed that substitution of the amino acid at positions corresponding to positions 98, 108, and/or 156 of SEQ ID NO. 1 with a more hydrophilic amino acid will increase the hydrophilicity of ferritin and reduce its aggregation.
In some embodiments, the amino acid at positions corresponding to positions 98, 108, and/or 156 of SEQ ID NO. 1 is substituted with a more hydrophilic amino acid, such as an amino acid with a carboxyl side chain. In some embodiments, the amino acid with a carboxyl side chain comprises glutamic acid (E), aspartic acid (D), or histidine (H).
In some embodiments, the mutant polypeptide is substituted with aspartic acid (D), also known as N98D, at a position corresponding to position 98 of SEQ ID NO. 1, for example asparagine (N). In some embodiments, the mutant polypeptide has an amino acid such as lysine (K) substituted with glutamic acid (E), also referred to as K108E, at a position corresponding to position 108 of SEQ ID NO. 1. In some embodiments, the mutant polypeptide has an amino acid such as arginine (R) substituted with histidine (H), also known as R156H, at a position corresponding to position 156 of SEQ ID NO. 1.
In some embodiments, the mutant polypeptide has an amino acid, e.g., asparagine (N), substituted with aspartic acid (D) at a position corresponding to position 98 of SEQ ID NO. 1, and an amino acid, e.g., lysine (K), substituted with glutamic acid (E) at a position corresponding to position 108 of SEQ ID NO. 1.
In some embodiments, the mutant polypeptide has an amino acid such as asparagine (N) substituted with aspartic acid (D) at a position corresponding to position 98 of SEQ ID NO. 1, an amino acid such as lysine (K) substituted with glutamic acid (E) at a position corresponding to position 108 of SEQ ID NO. 1, and an amino acid such as arginine (R) substituted with histidine (H) at a position corresponding to position 156 of SEQ ID NO. 1.
In some embodiments, the mutant polypeptide comprises an amino acid substitution at a position corresponding to position 27, 61, 62 and/or 65 of SEQ ID No. 1 relative to the wild-type ferritin H subunit. Without being bound by any theory, it is believed that amino acid substitutions at positions corresponding to positions 27, 61, 62 and/or 65 of SEQ ID NO. 1 may reduce the iron storage capacity of the ferritin formed, thereby rendering the ferritin more safe when used as a coupling carrier.
In some embodiments, the mutant polypeptide has glutamic acid (E) substituted with phenylalanine (F) at a position corresponding to position 27 of SEQ ID NO. 1. In some embodiments, the mutant polypeptide has glutamic acid (E) substituted with tryptophan (W) at a position corresponding to position 61 of SEQ ID NO. 1. In some embodiments, the mutant polypeptide has an amino acid, such as glutamic acid (E), substituted with lysine (K) at a position corresponding to position 62 of SEQ ID NO. 1. In some embodiments, the mutant polypeptide is substituted with glycine (G) at a position corresponding to position 65 of SEQ ID NO. 1, for example histidine (H). In some embodiments, the mutant polypeptide is substituted with lysine (K) at a position corresponding to position 62 of SEQ ID No. 1, e.g., glutamic acid (E), and is substituted with glycine (G) at a position corresponding to position 65 of SEQ ID No. 1, e.g., histidine (H).
The wild type human ferritin H subunit has 3 sulfhydryl groups, which are respectively positioned in the Loop region between the 2 nd and 3 rd alpha helices (the sulfhydryl group of the 90 th cysteine of the wild type human ferritin H subunit), the 3 rd alpha helices (the sulfhydryl group of the 102 th cysteine of the wild type human ferritin H subunit) and the 4 th alpha helices are near the triple symmetry axis region (the sulfhydryl group of the 130 th cysteine of the wild type human ferritin H subunit). However, during conjugation, if multiple reactive sites are present, the specific location of conjugation, and the ratio of functional molecules to HFn reaction, cannot be controlled.
Thus, in some embodiments, the mutant polypeptide comprises reduced cysteines relative to a wild-type ferritin H subunit.
In some embodiments, the mutant polypeptide is substituted with at least one of the cysteines at positions corresponding to positions 90, 102, and 130 of SEQ ID NO. 1. In some embodiments, the cysteine is substituted with an amino acid selected from the group consisting of: serine, threonine, asparagine, glutamine, glutamic acid, aspartic acid, lysine, arginine, histidine, alanine, glycine, preferably by serine or by an amino acid at the corresponding position of the wild-type ferritin light chain (L) subunit polypeptide. An exemplary wild-type ferritin light chain (L) subunit polypeptide has the amino acid sequence shown in SEQ ID NO. 17.
In some embodiments, the mutant polypeptide comprises a cysteine at a position corresponding to position 90 of SEQ ID NO. 1, and a cysteine at a position corresponding to position 102 of SEQ ID NO. 1, preferably a serine or an alanine, relative to the wild type ferritin H subunit, optionally a cysteine at a position corresponding to position 130 of SEQ ID NO. 1, preferably a serine or an alanine.
In some embodiments, the mutant polypeptide comprises a cysteine at a position corresponding to position 102 of SEQ ID NO. 1, and a cysteine at a position corresponding to position 90 of SEQ ID NO. 1, preferably serine or glutamic acid, relative to the wild type ferritin H subunit, optionally a cysteine at a position corresponding to position 130 of SEQ ID NO. 1, preferably serine or alanine.
Furthermore, it is also possible to leave only one site for chemical conjugation per ferritin subunit by only including one cysteine in the loop region of the ferritin H subunit (corresponding to amino acids 79 to 91 of SEQ ID NO: 1) while removing other surface sulfhydryl groups.
In some embodiments, the mutant polypeptide comprises one cysteine in the loop region relative to the wild-type ferritin H subunit, the cysteine at the position corresponding to position 102 of SEQ ID No. 1 is substituted, and optionally the cysteine at the position corresponding to position 130 of SEQ ID No. 1 is substituted. In some embodiments, the mutant polypeptide does not comprise additional cysteines except for one cysteine in the loop region and optionally a cysteine at a position corresponding to position 130 of SEQ ID NO. 1. In some preferred embodiments, the mutant polypeptide does not comprise a cysteine outside the loop region.
In some embodiments, the mutant polypeptide has a substitution of a cysteine at positions corresponding to positions 90 and 102 of SEQ ID No. 1, and optionally a substitution of a cysteine at position 130 of SEQ ID No. 1, relative to the wild-type ferritin H subunit; and the mutant polypeptide has a cysteine substituted for the amino acid at a position corresponding to one of positions 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and 91 of SEQ ID NO. 1. In some embodiments, the mutant polypeptide has a substitution of a cysteine at a position corresponding to positions 90, 102 and 130 of SEQ ID NO. 1 relative to the wild type ferritin H subunit; and the mutant polypeptide has a cysteine substituted for the amino acid at a position corresponding to one of positions 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and 91 of SEQ ID NO. 1. In some embodiments, the mutant polypeptide has an amino acid such as arginine (R) substituted with cysteine (C) at a position corresponding to position 79 of SEQ ID NO. 1. In some embodiments, the mutant polypeptide has an amino acid such as isoleucine (I) substituted with a cysteine at a position corresponding to position 80 of SEQ ID NO. 1. In some embodiments, the mutant polypeptide has an amino acid, such as phenylalanine (F), substituted with a cysteine at a position corresponding to position 81 of SEQ ID NO. 1. In some embodiments, the mutant polypeptide has an amino acid, such as leucine (L), substituted with a cysteine at a position corresponding to position 82 of SEQ ID NO. 1. In some embodiments, the mutant polypeptide has an amino acid such as glutamine (Q) substituted with a cysteine at a position corresponding to position 83 of SEQ ID NO. 1. In some embodiments, the mutant polypeptide has an amino acid, such as aspartic acid (D), substituted with a cysteine at a position corresponding to position 84 of SEQ ID NO. 1. In some embodiments, the mutant polypeptide has an amino acid such as isoleucine (I) substituted with a cysteine at a position corresponding to position 85 of SEQ ID NO. 1. In some embodiments, the mutant polypeptide has an amino acid, such as lysine (K), substituted with cysteine at a position corresponding to position 86 of SEQ ID NO. 1. In some embodiments, the mutant polypeptide has an amino acid, such as lysine (K), substituted with cysteine at a position corresponding to position 87 of SEQ ID NO. 1. In some embodiments, the mutant polypeptide has an amino acid such as proline (P) substituted with a cysteine at a position corresponding to position 88 of SEQ ID NO. 1. In some embodiments, the mutant polypeptide has an amino acid, such as aspartic acid (D), substituted with a cysteine at a position corresponding to position 89 of SEQ ID NO. 1. In some embodiments, the mutant polypeptide has an amino acid, such as aspartic acid (D), substituted with a cysteine at a position corresponding to position 91 of SEQ ID NO. 1.
In some embodiments, the mutant polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs 5-8.
In some embodiments, the mutant polypeptide is capable of assembling into a caged protein and/or is capable of conferring the caged protein the ability to bind, treat, diagnose, track, etc., upon assembly into a caged protein.
3. Polynucleotide, expression construct, host cell and method for preparing ferritin H subunit mutant polypeptide
In another aspect, the present disclosure provides an isolated polynucleotide comprising a nucleotide sequence encoding a recombinant ferritin H subunit polypeptide of the present disclosure.
In some embodiments, the polynucleotides of the present disclosure comprise a nucleotide sequence selected from one of SEQ ID NOs 13-16, for example.
In another aspect, the present disclosure provides an expression construct comprising a polynucleotide of the present disclosure operably linked to an expression control sequence.
Vectors for use in the expression constructs of the present disclosure include those that autonomously replicate in the host cell, such as plasmid vectors; also included are vectors that are capable of integrating into and replicating with host cell DNA. Many vectors suitable for the present disclosure are commercially available. In a specific embodiment, the expression construct of the present disclosure is derived from pET22b from Novagen.
In another aspect, the present disclosure provides a host cell comprising a polynucleotide of the present disclosure or transformed with an expression construct of the present disclosure, wherein the host cell is capable of expressing a ferritin H subunit mutant polypeptide of the present disclosure.
Host cells useful for expressing the ferritin H subunit mutant polypeptides of the present disclosure include prokaryotes, yeast, and higher eukaryotic cells. Exemplary prokaryotic hosts include bacteria of the genera Escherichia (Escherichia), bacillus (Bacillus), salmonella (Salmonella) and Pseudomonas (Pseudomonas) and Streptomyces (Streptomyces). In a preferred embodiment, the host cell is an Escherichia cell, preferably E.coli. In a specific embodiment of the present disclosure, the host cell used is a cell of the E.coli BL21 (DE 3) strain.
The recombinant expression constructs of the present disclosure may be introduced into a host cell by one of many well known techniques including, but not limited to: heat shock transformation, electroporation, DEAE-dextran transfection, microinjection, liposome-mediated transfection, calcium phosphate precipitation, protoplast fusion, microprojectile bombardment, viral transformation and the like.
In another aspect, the present disclosure provides a method of producing a ferritin H subunit mutant polypeptide of the present disclosure, comprising:
a) Culturing a host cell of the disclosure under conditions that allow expression of the polypeptide;
b) Obtaining from the culture obtained from step a) a polypeptide expressed by the host cell; a kind of electronic device with high-pressure air-conditioning system
c) Optionally further purifying the polypeptide from step b).
However, the ferritin H subunit mutant polypeptides of the present disclosure may also be obtained by chemical synthesis methods.
4. Polypeptide conjugates
In another aspect, the present disclosure provides a polypeptide conjugate comprising a ferritin H subunit mutant polypeptide of the present disclosure and a functional moiety conjugated thereto by a thiol group of the ferritin H subunit mutant polypeptide.
In some embodiments, the functional moiety is selected from a therapeutic molecule, a detectable molecule, or a targeting molecule.
Such therapeutic molecules include, but are not limited to, small molecule drugs, therapeutic polypeptides, therapeutic antibodies, and the like. Exemplary therapeutic small molecules include, but are not limited to, toxins, immunomodulators, antagonists, apoptosis inducers, hormones, radiopharmaceuticals, anti-angiogenic agents, monoclonal antibodies, siRNA, cytokines, chemokines, prodrugs, chemotherapeutic drugs, and the like.
Such detectable molecules include, but are not limited to, dye molecules, fluorescent molecules, luminescent chemicals, enzymes, isotopes, tags.
In some embodiments, the dye molecule is selected from the group consisting of merocyanine, evans blue, isothiocyanic blue, methylene blue, and patent blue.
In some embodiments, the fluorescent molecule is selected from the group consisting of near infrared fluorescent dyes, including but not limited to cyanine dyes, rhodamine dyes, aromatic acids, porphyrins, exemplary fluorescent dyes being indocyanine green, cy3, cy3.5, cy5, cy5.5, cy7, IR-782, IR820, and IR-783.
In one embodiment, the fluorescent dye molecule is indocyanine green.
In some embodiments, the isotopes include, but are not limited to 99m Tc。
Such targeting molecules include, but are not limited to, targeting antibodies, specific receptor ligands, and the like. For example, the targeting molecule may be an antibody that specifically targets a tumor antigen.
In some embodiments, the biologically active moiety is conjugated to the ferritin H subunit mutant polypeptide through a linker.
In some embodiments, the polypeptide conjugate is capable of assembling into a caged protein and/or is capable of conferring the caged protein the ability to bind, treat, diagnose, track, or otherwise, upon assembly into a caged protein.
In some embodiments, the polypeptide conjugate is an isolated polypeptide conjugate, e.g., that does not assemble into a caged protein. In some embodiments, the polypeptide conjugates are assembled into caged proteins.
5. Cage-like proteins
Because of the retained self-assembly and/or receptor binding capacity of the wild-type ferritin H subunit, the ferritin H subunit mutant polypeptides and/or polypeptide conjugates of the present disclosure may assemble alone into a caged protein (i.e., H ferritin/deferiprotein) in a suitable medium, may also form a caged protein with ferritin L subunits or other ferritin H subunits or other self-assembled polypeptides, and are capable of conferring biological activity and function to the caged protein, such as the ability to specifically target ferritin receptor (Tfr 1).
Thus, in another aspect, the present disclosure provides a caged protein comprising at least one ferritin H subunit mutant polypeptide of the present disclosure and/or a polypeptide conjugate of the present disclosure.
Exemplary such caged proteins may comprise, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 ferritin H subunit mutant polypeptides of the present disclosure and/or polypeptide conjugates of the present disclosure. In some preferred embodiments, the caged protein comprises 24 ferritin H subunit mutant polypeptides of the disclosure and/or polypeptide conjugates of the disclosure.
In some embodiments, the caged proteins comprise only ferritin H subunit mutant polypeptides of the disclosure and/or polypeptide conjugates of the disclosure, e.g., only polypeptide conjugates of the disclosure. For example, in some preferred embodiments, the caged proteins are assembled from 24 polypeptide conjugates of the present disclosure.
In some embodiments, the caged protein comprises a plurality of polypeptide conjugates of the present disclosure comprising the same or different biologically active moiety.
In some embodiments, the caged protein comprises a biologically active moiety that is indocyanine green or 99m Tc。
In some embodiments, the caged protein further comprises a ferritin L subunit. In some embodiments, the caged protein comprises at least one ferritin H subunit mutant polypeptide of the disclosure and at least one ferritin L subunit, preferably the ratio of ferritin H subunit mutant polypeptide to ferritin L subunit can range, for example, from 1:23 to 23:1.
In some embodiments, the caged protein does not comprise a ferritin L subunit.
6. Conjugation methods
Various methods of conjugating functional molecules to proteins via sulfhydryl groups are known in the art and may be employed in the present disclosure. One skilled in the art can determine the appropriate conjugation method based on the particular functional molecule and the linker selected. Exemplary methods can be found in Moon, S.J., et al, antibody conjugates of, 7-methyl-10-hydro-amphetamine (SN-38) for targeted cancer chemicotherapy.J Med Chem,2008.51 (21): p.6916-26.
In one aspect, the present disclosure provides a method of preparing a polypeptide conjugate of the present disclosure, the method comprising:
(1) Preparing a third portion of the ferritin H subunit mutant polypeptide;
(2) Coupling the ferritin H subunit mutant polypeptide obtained in the step (1) with a bioactive part under the condition suitable for coupling reaction to obtain the polypeptide conjugate of the fourth part.
In some embodiments, the biologically active moiety is conjugated to a depolymerized ferritin H subunit mutant polypeptide of the present disclosure through a linker.
In some embodiments, the bioactive moiety is indocyanine green (ICG). ICG can be prepared by condensing ICG-mal of formula (I) with a maleimide molecule (linker) and then coupling with thiol groups on a mutant polypeptide of ferritin H subunit.
In some embodiments, wherein step (2) comprises contacting a compound of formula (I) (ICG with linker) with a depolymerized ferritin H subunit mutant polypeptide of the present disclosure of step (1).
In one aspect, the present disclosure provides a method of making a caged protein of the present disclosure comprising at least one polypeptide conjugate of the present disclosure, the method comprising:
a) Providing a caged protein comprising at least one ferritin H subunit mutant polypeptide of the disclosure, and
b) Conjugation of a functional molecule to a ferritin H subunit mutant polypeptide of the disclosure in the caged protein.
In some embodiments, the functional molecule is conjugated to a ferritin H subunit mutant polypeptide of the disclosure in the caged protein through a linker.
In some embodiments, the functional molecule is ICG. In some embodiments, wherein step b) comprises contacting a compound of formula (I) (ICG with linker) with the caged protein.
7. Caged protein-API complexes
In another aspect, the present invention provides a caged protein-API complex, wherein the caged protein-API complex comprises a caged protein of the present invention, and a pharmaceutically active ingredient (API) loaded inside the caged protein.
In some embodiments, the caged proteins in the complex comprise a polypeptide conjugate of the invention comprising a ferritin H subunit mutant polypeptide of the invention and a detectable molecule. The caged proteins of the present invention conjugated with a detectable molecule can be used to display the target to be detected, monitor (e.g., monitor in real time) the delivery of a drug.
The pharmaceutically active ingredient (API) loaded inside the cage protein is not particularly limited as long as it is suitable for loading in the cage protein of the present invention, for example, the API does not disrupt the cage structure of the cage protein and/or its size is suitable for being accommodated by the cage structure. Examples of such APIs include, but are not limited to, alkylating agents, platins, antimetabolites, tumor antibiotics, natural extracts, hormones, radiopharmaceuticals, neurotransmitters, dopamine receptor agonists, nerve center anticholinergic agents, choline receptor agonists, gamma secretase inhibitors, antioxidants, anesthetics.
8. Pharmaceutical composition and application thereof
In another aspect, the present disclosure provides a pharmaceutical composition comprising a ferritin H subunit mutant polypeptide of the disclosure, a polypeptide conjugate of the disclosure, a caged protein of the disclosure, and/or a caged protein-API complex of the disclosure, and a pharmaceutically acceptable excipient.
In some embodiments, the pharmaceutical composition comprises a ferritin H subunit mutant polypeptide of the present disclosure or a polypeptide conjugate of the present disclosure, and a caged protein of the present disclosure, and/or a caged protein-API complex of the present disclosure, wherein the ferritin H subunit mutant polypeptide, polypeptide conjugate of the present disclosure is provided in a form that is not assembled into a caged protein. The ferritin H subunit mutant polypeptide or polypeptide conjugate may self-assemble into a caged protein under suitable conditions, either in vitro or after delivery to the body.
In some embodiments, where the polypeptide conjugates of the present disclosure comprise a detectable molecule, the pharmaceutical composition comprising the polypeptide conjugates of the present disclosure may further comprise an additional API.
The ferritin H subunit mutant polypeptides, polypeptide conjugates, caged proteins, caged protein-API complexes and/or pharmaceutical compositions of the present disclosure may be used to diagnose and track a disease depending on the therapeutic molecule or API that it comprises.
In another aspect, the present disclosure provides the use of a ferritin H subunit mutant polypeptide, polypeptide conjugate, caged protein-API complex and/or pharmaceutical composition of the disclosure in the preparation of a medicament, diagnostic reagent, tracer reagent.
In some embodiments, the drug, diagnostic reagent, tracer reagent is a tumor drug, tumor diagnostic reagent, tumor tracer reagent.
In some embodiments, the tumor tracer reagent is a tumor sentinel lymph node tracer reagent.
In another aspect, the present disclosure provides a method of diagnosing a disease in a subject, the method comprising administering to the subject an effective amount of a ferritin H subunit mutant polypeptide, polypeptide conjugate, caged protein-API complex and/or pharmaceutical composition of the disclosure. The disease is as defined above, preferably a tumour.
The polypeptides, ferritin H subunit mutant polypeptides, polypeptide conjugates, caged proteins, caged protein-API complexes and/or pharmaceutical compositions of the present disclosure may be administered by any suitable method known to one of ordinary skill in the art (see, e.g., remington: the Science and Practice of Pharmacy, 21 st edition, 2005). The pharmaceutical compositions may be administered, for example, by intravenous, intramuscular, intraperitoneal, intracerebroventricular, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical or inhalation routes.
9. Method for preparing caged protein-API complex
In another aspect, the present disclosure provides a method of making a caged protein-API complex of the present disclosure, the method comprising contacting a ferritin H subunit mutant polypeptide of the present disclosure, a polypeptide conjugate of the present disclosure, and/or a caged protein of the present disclosure with an API, thereby obtaining the caged protein-API complex.
In some embodiments, the method comprises:
a) Contacting a depolymerized caged protein of the present disclosure with an API; and
b) Reassembling the caged protein, thereby obtaining a caged protein-API complex.
As used in this disclosure, "deagglomeration" refers to the process by which the tightly closed globular structure of a caged protein is opened to separate all or part of its subunits from one another under conditions such as protein denaturation conditions, e.g., buffer solutions containing high concentrations of urea.
As used in this disclosure, "reassembly" refers to the process of reassembling a deaggregated caged protein, i.e., an isolated subunit, into a caged protein by changing conditions, e.g., changing to physiologically compatible conditions. During the recombinant assembly of the caged proteins, the API will be encapsulated within the caged proteins, thereby forming a caged protein-API complex. The physiologically compatible condition is, for example, a physiological buffer solution.
In some embodiments, the method further comprises a step of depolymerizing the caged proteins of the present disclosure prior to step a). In some embodiments, the caged proteins of the present disclosure are depolymerized therein by the presence of high concentrations (e.g., at least 6M, preferably 8M) of urea. In some embodiments, wherein the caged protein is reassembled by stepwise decreasing urea concentration (e.g., gradient dialysis).
In some embodiments, the method comprises:
a) Contacting a caged protein of the present disclosure with an API under non-deagglomerating conditions, thereby allowing the API to bind to the caged protein and/or to load into the internal central cavity of the caged protein,
b) The caged protein-API complex is obtained.
In some embodiments, the non-deagglomerating conditions comprise placing the caged protein and API in a physiologically acceptable buffer. Suitable physiologically acceptable buffers include, but are not limited to, PBS solution, physiological saline, pure water, HEPES buffer, and the like.
In some embodiments, the API shuttles to the internal central cavity of the caged protein by passive diffusion. By placing the caged protein and the API in a physiologically acceptable buffer, the API can diffuse into the internal lumen of the caged protein without depolymerizing the caged protein.
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 application.
Example 1 construction of human H-ferritin with improved binding Rate and Water solubility
1.1 design of mutations in the H subunit of ferritin
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), with mutations in the H subunit at sites likely to be involved in iron loading, at the ferrite core sites of glutamic acid (E62) at position 62 and histidine (H65) at position 65. Simultaneously, in order to increase the hydrophilicity of ferritin and reduce aggregation, lysine (K108) with an amino side chain at the 108 th position is replaced by amino acid with a carboxyl side chain; substitution of asparagine (N98) at position 98 with an amino acid having a side chain with a carboxyl group; and/or the arginine with amino side chain at position 156 (R156) is substituted with an amino acid with carboxyl side chain. All amino acid positions are referred to SEQ ID NO. 1. Meanwhile, in order to increase the uniformity of ferritin coupling drugs, two of the three active sulfhydryl sites (positions 90 and 130) of the H subunit are mutated into other amino acids, and only the 102 th cysteine is reserved as the coupling site.
The specific design of the mutants is shown in Table 1. The resulting subunit mutants were designated as Mut-HFn-240 (SEQ ID NO: 5), mut-HFn-241 (SEQ ID NO: 6), mut-HFn-242 (SEQ ID NO: 7) and Mut-HFn-243 (SEQ ID NO: 8), respectively.
In addition, the mutants Mut-HFn-212 (SEQ ID NO: 2), mut-HFn-233 (SEQ ID NO: 3) and Mut-HFn-203 (SEQ ID NO: 4) were used as controls, the mutants were not modified for the hydrophilic site and the iron loading-related site, only 1 Cys was retained as the chemical coupling site, and the other two Cys on the outer surface were mutated (C102S and C130S).
TABLE 1 design of HFn mutants
1.2 preparation and purification of H-ferritin
After obtaining the mutated amino acid sequence, the coding sequence was codon optimized for E.coli. The codon-optimized nucleotide sequences of the wild-type ferritin and 5 mutants are shown in SEQ ID NOs: 9-16. The construction methods of wild HFn, mut-HFn-212, mut-HFn-203, mut-HFn-233 and Mut-HFn-242 are as follows: the common vector pET-30a (+) for expressing foreign proteins in E.coli was selected, kanamycin resistance (Kan+), and NdeI and HindIII cleavage sites were selected for insertion of the target gene. The successful construction of the expression vector is confirmed by enzyme digestion map and gene sequencing.
The construction methods of the Mut-HFn-240, the Mut-HFn-241 and the Mut-HFn-243 are as follows: the common vector pET-28a (+) for expressing exogenous proteins of escherichia coli, kanamycin resistance (Kan+), and the restriction sites of Nco I and Xho I are selected to be embedded into the target genes. The successful construction of the expression vector is confirmed by enzyme digestion map and gene sequencing.
After the expression vector is successfully constructed, E.coli BL21 (DE 3) is selected as host bacteria, recombinant plasmids containing target genes are transformed into competent cells of the host bacteria, positive clones are screened through a kanamycin-containing resistance plate, and recombinant strains are determined.
The recombinant strain is inoculated in 1L LB culture medium/2L shake flask, cultured at a low speed of 37 ℃ until the OD600 is about 1.0, added with 0.5mM IPTG, induced to express for 3-4 h, and centrifuged to collect bacterial sludge. 25mL of 20mM Tris-HCl buffer solution is added into every 30ml of bacterial liquid for centrifugation and collection, the bacterial mud is resuspended uniformly, the bacterial mud is crushed for 0.5 to 2min by a high-pressure homogenizer at 1000bar, the lysate is centrifuged for 30min at 5000r/min, and the supernatant is taken for SDS-PAGE, and the Loading amount is 10 μl (sample: loading buffer=1:1). The results of the expression of each protein are shown in FIG. 1. All proteins were expressed in the supernatant.
The protein purification method comprises the following steps: removing coliform fragments from the high-pressure homogenized and crushed thallus lysate by centrifugation (1500 rpm,10 min); heating the supernatant at 72 ℃ for 15 minutes; precipitating the impurity protein, and centrifuging to remove the precipitate; separating and purifying the supernatant on a chromatography-exclusion Superdex 200pg column; purity was identified by SDS-PAGE electrophoresis; BCA assay protein concentration. All samples reach over 96% purity.
EXAMPLE 2 characterization of ferritin H subunit mutants
2.1. Mutant HFn TEM results
The sample preparation method comprises the following steps: ferritin samples (20 μl,0.1 mg/mL) were applied to plain carbon support membranes for 1min, then washed twice with 2% uranyl acetate, and finally stained with 2% uranyl acetate for one minute. FEI Tecnai Spirit (120 kV) electron microscope observation conditions: observation conditions were observed using a transmission electron microscope (FEI Tecnai Spirit (120 kV): HT100Kv. The transmission electron microscopy results (FIG. 2) show that both the mutant H subunit polypeptide and the wild-type H subunit polypeptide can form a uniform and regular cage-like protein structure with a diameter of between about 12-19 nm.
2.2 SEC results for mutant HFn
1ml protein samples with a protein concentration of 1mg/ml were taken separately, placed in clean 1.5ml EP tubes, 10. Mu.l samples were taken and analyzed for ferritin monomer and polymer peaks in a high performance liquid chromatography system (Agilent Technologies 1260 InfiniI II) by SEC on 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 DEG C
The SEC results are shown in Table 2, and FIG. 3 shows in detail the SEC spectra of Mut-HFn-212, mut-HFn-241, mut-HFn-242 and Mut-HFn-243. Under the same preparation conditions, the particle size of Mut-HFn-212 is slightly larger, and aggregation easily occurs.
Table 2 SEC results for mutant HFn
Sample of Particle size (nm)
Mut-HFn-212 19.23
Mut-HFn-233 20.31
Mut-HFn-203 20.98
Mut-HFn-240 14.73
Mut-HFn-241 16.97
Mut-HFn-242 16.89
Mut-HFn-243 17.076
Example 3 coupling of HFn to a tracer molecule
3.1 coupling of wild-type HFn, mut-HFn-243 with ICG
Reagent (table 3):
table 3: coupling reagent
Reagent name Manufacturing factories Lot number
ICG-mal New Chaozhimei R00210890
EPPS Tertbe chemistry 21082903
EDTA disodium salt Microphone forest C12297614
DMSO Alatine F2110074
NaH2PO4·2H2O Chinese medicine 20190903
Na2HPO4·12H2O Alatine D1825031
L-cys Beijing enokie KYEGV07
3.1.1 coupling methods
The wild-type HFn, mut-HFn-243 purified protein prepared in example 1 was changed to a solution of 50mM EPPS+5mM EDTA (pH 8.0) using a 100KD ultrafiltration tube and concentrated to a concentration of greater than 6mg/ml. ICG-mal was dissolved in DMSO. The HFn solutions of each group are placed in an ice-water bath, when the temperature of the solution reaches 4 ℃, the HFn 28 molar equivalent of ICG-mal DMSO solution (the final DMSO concentration is 15%) is added while mixing, and after fully mixing, the mixed solution is placed in a refrigerator at 4 ℃ for standing reaction for 40min. HFn concentration in the system should be 5mg/ml. After the reaction was completed, L-Cys (L-cysteine) was added in an amount of 28 molar equivalents to HFn, and the mixture was stirred at 25℃in a shaker at 30rpm for 10 minutes. Finally, the reacted solution was desalted and changed to 20mM PB (pH 7.5) using Sephadex G-25 packing and concentrated to a concentration of 0.2mg/ml ICG. Sterile filtered and stored at 4 ℃.
3.2 detection of coupled products
3.2.1RP-HPLC detection
3.1.1 using RP-HPLC (Agilent, 1260 information II) liquid chromatography columns for HFn-ICG and Mut-HFn-243-ICG prepared BEH C18.7 μm 2.1X100 mm), detection of 280nm, 397nm wavelength, gradient elution, mobile phase: phase A: 0.1% tfa/water; and B phase: acetonitrile, column temperature 20 ℃,the flow rate was 0.4ml/min, and the sample injection amount was 2. Mu.l.
The gradient elution conditions were:
time (min) Flow rate (ml/min) M.P.A(％) M.P.B(％)
0 0.4 62.0 38.0
7.0 0.4 55.0 45.0
According to RP-HPLC result measurement, the bonding rate of each wild type HFn-ICG is 8.8, the bonding rate of Mut-HFn-243-ICG coupling is 12.3, namely the bonding rate of HFn mutant and small molecules is improved.
FIG. 4 shows a RP-HPLC specific pattern of Mut-HFn-243-ICG. The upper line in the figure shows the detection of the sample at 280nm, wherein the left peak is unconjugated Mut-HFn-243 and the right peak is Mut-HFn-243-ICG after ICG conjugation. The lower line shows the detection at 397nm, and the wavelength is the absorption peak of ICG-mal. As can be seen, mut-HFn-243 showed overlapping peak times at ICG-mal small molecule (397 nm) and HFn (280 nm), indicating that ICG-mal was coupled to the ferritin mutant.
3.2.2SEC detection
The wild-type HFn-ICG, mut-HFn-243-ICG conjugate products prepared in section 3.1.1 were subjected to SEC (TSK gel G4000SWxl 7.8X300 mm,8 μm) detection at 280nm, 363nm, column temperature 30℃and isocratic elution with mobile phases: 0.1M PB&0.2M NaCl&5%IPA,pH7.0. The flow rate was 0.4ml/min and the sample injection amount was 30. Mu.l.
SEC results are shown in figure 5. Under the same preparation conditions, the HFn-ICG had a slightly larger particle size and was liable to aggregation. The Mut-HFn-243-ICG had smaller particle sizes and no aggregates were detected, indicating improved uniformity and stability of HFn mutant conjugated small molecules.
Example 4, HFn-ICG, mut-HFn-243-ICG conjugate tracer assay
4.1 animal tracking methods
(1) The experimental animals were divided into 5 groups of 8 male sterile grade SD rats (this experiment was completed in Peking Violet laboratory animal technologies Co., ltd.), and the detailed grouping and tracer usage dosages (dosages calculated as ICG concentration) are shown in Table 4;
(2) After the SD rats are anesthetized, 5 different kinds or doses of lymph node tracers are respectively injected under the hind limb foot pad;
(3) After adequate drainage, sentinel lymph nodes of hind limb lymphatic drainage pathways of each rat were located using fluorescence (excitation 780nm, emission 800 nm). The fluorescence method is to detect the 1 st fluorescence imaging lymph node by the fluorescence imaging lymph tube. Dissecting the drainage paths of the hind limb and the abdomen lymph of the rat at 1h, and separating the sentinel lymph node of the popliteal fossa and the secondary lymph node of the abdominal cavity of the rat;
(4) The fluorescent imaging of the different sets of isolated sentinel and secondary lymph nodes was compared in the fluorescent imaging mode to determine the imaging of the different tracers at the different lymphatic sites.
Table 4: tracer animal grouping and dosage for use
4.2 Trace results
The trace images of each group of section 4.1 are shown in FIG. 6. The results showed that Mut-HFn-243-ICG clearly developed sentinel lymph nodes at low doses (0.2 mg/ml) and 1.3 times stronger fluorescence intensity (fluorescence intensity calculated by software imageJ assay, EXCEL) than ICG at standard doses of 2.5mg/ml, demonstrating that mutant-ICG obtained according to the present disclosure still clearly developed sentinel lymph nodes at least one order of magnitude lower. In addition, ICG at a standard dose of 2.5mg/ml also developed secondary lymph nodes, while Mut-HFn-243-ICG did not substantially enter the secondary lymph nodes and did not stain the secondary lymph nodes, which demonstrates that the mutant-ICG obtained by the present disclosure can better distinguish between sentinel and secondary lymph nodes, which gives a surgeon in tumor surgery good imaging guidance, does not cause the secondary lymph nodes to be cleared due to development, and can greatly reduce the injury to tumor patients caused by mistakenly sweeping the secondary lymph.

Claims (10)

1. A mutant ferritin H subunit (HFn) polypeptide comprising an amino acid sequence selected from one of SEQ ID NOs 5 to 8.
2. A polypeptide conjugate comprising a mutant ferritin H subunit polypeptide, and a biologically active moiety conjugated thereto by a thiol group of said mutant ferritin H subunit polypeptide, said mutant ferritin H subunit polypeptide sequence comprising an amino acid sequence selected from one of SEQ ID NOs 5 to 8.
3. The polypeptide conjugate of claim 2, wherein the biologically active moiety is selected from a therapeutic molecule, a detectable molecule, or a targeting molecule;
preferably, the detectable molecule is selected from dye molecules, fluorescent molecules, luminescent chemicals, enzymes, isotopes, tags;
alternatively, the biologically active moiety is conjugated to the thiol group of the ferritin H subunit mutant polypeptide via a linker.
4. The polypeptide conjugate of claim 3 whereinThe dye molecule is selected from the group consisting of merocyanine, evans blue, isothioblue, methylene blue and patent blue; the fluorescent molecule is near infrared fluorescent dye, such as cyanine dye, rhodamine, aromatic acid and porphyrin, preferably indocyanine green, cy3, cy3.5, cy5, cy5.5, cy7, IR-782, IR820 and IR-783; the isotope is 99m Tc。
5. The polypeptide conjugate of claim 3 or 4, wherein the biologically active moiety is indocyanine green; the connector is maleimide.
6. A method of preparing the polypeptide conjugate of any one of claims 2-5 comprising the steps of:
(1) Preparing the ferritin H subunit mutant polypeptide of claim 1;
(2) And (3) performing coupling reaction on the ferritin H subunit mutant polypeptide obtained in the step (1) and a bioactive part under the condition suitable for the coupling reaction to obtain a polypeptide conjugate.
7. A caged protein comprising at least one ferritin H subunit mutant polypeptide according to claim 1, and/or at least one polypeptide conjugate according to any one of claims 2 to 5;
preferably, said caged protein comprises 24 of said ferritin H subunit mutant polypeptides and/or said polypeptide conjugates.
8. A caged protein-API complex, wherein the caged protein-API complex comprises the caged protein of claim 7, and a pharmaceutically active ingredient (API) loaded inside the caged protein.
9. A pharmaceutical composition comprising the ferritin H subunit mutant polypeptide of claim 1, the polypeptide conjugate of any one of claims 2-5, the caged protein of claim 7, and/or the caged protein-API complex of claim 8, and a pharmaceutically acceptable excipient.
10. Use of the ferritin H subunit mutant polypeptide according to claim 1, the polypeptide conjugate according to any one of claims 2 to 5, the caged protein according to claim 7, the caged protein-API complex according to claim 8, and/or the pharmaceutical composition according to claim 9 in the preparation of a medicament, a diagnostic reagent, a tracer reagent; preferably, the drug, diagnostic reagent, tracer reagent is a tumor drug, tumor diagnostic reagent, tumor tracer reagent; more preferably, the tumor tracer reagent is a tumor sentinel lymph node tracer reagent.
CN202310278157.XA 2022-03-23 2023-03-21 Ferritin tracer polypeptide conjugates and uses thereof Pending CN116804049A (en)

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CN2022102958005 2022-03-23

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