Polypeptide specifically binding to transferrin receptor and application thereof

Abstract

The invention is applicable to the technical field of molecular biology, and provides a polypeptide specifically bound with a transferrin receptor, the polypeptide comprises a general formula of H1-H2-E1-E2-H3-H4-E3, and H1, H2, H3 and H4 independently comprise an alpha helical domain; the E1, the E2 and the E3 respectively and independently comprise a beta folding structural domain; when the polypeptide is combined with the hTfR1, transferrin is allowed to be combined to the hTfR1; the polypeptides are capable of binding and delivering oligonucleotides. The invention also provides a recombinant nucleic acid encoding the polypeptide, an expression vector comprising the recombinant nucleic acid, a recombinant host cell comprising the polypeptide, the nucleic acid and / or the expression vector, a conjugate of the polypeptide and oligonucleotide, and a corresponding pharmaceutical composition. The polypeptide disclosed by the invention can be highly specifically combined with hTfR1, so that accurate delivery of oligonucleotide is realized, and a practical basis is provided for treating diseases related to gene mutation.

Description

Technical Field

[0001] The present application belongs to the field of molecular biology technology, and specifically relates to a polypeptide that specifically binds to human transferrin receptor 1 (hTfR1) and its application, and also relates to a conjugate of the polypeptide coupled with an oligonucleotide, which can be used for targeted regulation of gene expression. Background Art

[0002] The blood-brain barrier (BBB) ​​refers to the barrier between plasma and brain cells formed by the walls of brain capillaries and glial cells, and the barrier between plasma and cerebrospinal fluid formed by the choroid plexus. These barriers can strictly control specific nutrients and prevent harmful substances from entering the brain tissue from the blood. Although they ensure the relative safety of the brain, they also bring great difficulties and challenges to the treatment and drug development of some central nervous system diseases.

[0003] Human transferrin receptor 1 (hTfR1) is a transmembrane glycoprotein that mediates iron absorption and transport across the blood-brain barrier (BBB) ​​by interacting with transferrin (Tf). Therefore, this process can be exploited to deliver therapeutic drugs that would otherwise be blocked by the BBB to the brain parenchyma, and peptide ligands that specifically bind to hTfR1 can serve as delivery vehicles for therapeutic payloads that cross the BBB.

[0004] Chinese patent application CN 116075524 A provides a polypeptide that specifically binds to hTfR1. However, the C-terminal domain of this polypeptide is not optimally arranged, resulting in suboptimal binding affinity to hTfR1. Therefore, it is urgent to obtain transferrin receptor-binding polypeptides with improved thermal stability and higher affinity by rearranging the polypeptide structure. Summary of the Invention

[0005] To solve the above problems, the present invention provides a polypeptide that specifically binds to human transferrin receptor 1 (hTfR1), wherein the polypeptide has a structure represented by the general formula H1-H2-E1-E2-H3-H4-E3, wherein each of H1, H2, H3 and H4 independently comprises an α-helical domain with a length of between 11 and 20 amino acids;

[0006] Each of the E1, E2 and E3 independently comprises a β-pleated domain with a length of 4-6 amino acids;

[0007] There are one or more amino acid linkers between any two adjacent α-helical domains and / or β-pleated domains;

[0008] The polypeptide binds to hTfR1 and simultaneously allows transferrin to bind to hTfR1.

[0009] Preferably, the polypeptide binds to hTfR1 comprising the amino acid sequence shown in SEQ ID NO: 95 within the range of C89 to F760.

[0010] Preferably, at least three amino acids in each of E1, E2 and E3 are hydrophobic.

[0011] Preferably, the polypeptide comprises the amino acid sequence of DVVVVT (SEQ ID NO: 133), wherein the amino acid V at positions 2 and 4 can be replaced by I, the first D residue and the last T residue can be deleted, and the sequence can be located at any position of E1, E2 or E3.

[0012] Preferably, the polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 46, 47, 48, 49, SEQ ID NO: 55, SEQ ID NO: 84 or SEQ ID NO: 85, and the sequence may be located at any position of E1, E2 or E3.

[0013] Preferably, the E1 domain comprises an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 98% or 100% identical to any one of SEQ ID NOs: 46-55, wherein the amino acid substitutions in the E1 domain are conservative amino acid substitutions; or wherein the E2 domain comprises an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 98% or 100% identical to any one of SEQ ID NOs: 56-69, wherein the amino acid substitutions in the E2 domain are conservative amino acid substitutions; or wherein the E3 domain comprises an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 98% or 100% identical to any one of SEQ ID NOs: 70-85, wherein the amino acid substitutions in the E3 domain are conservative amino acid substitutions.

[0014] Preferably, the polypeptide has an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 2, 5, 6, 8, 9 and 12.

[0015] Preferably, the polypeptide binds hTfR1 with an affinity of at least 3 micromolar, more preferably at least 1 micromolar, more preferably at least 500 nanomolar, more preferably at least 250 nanomolar, more preferably at least 100 nanomolar, more preferably at least 50 nanomolar, more preferably at least 5 nanomolar, more preferably at least 1 nanomolar, and most preferably at least 0.1 nanomolar.

[0016] Preferably, the polypeptide comprises one or more additional functional domains, wherein the additional functional domains include the following amino acid fragments: MHHHHHH (SEQ ID NO: 109), MHHHHHHC (SEQ ID NO: 110), (GS)n, (GS)nC, (SG)n, (SG)nC, (GGGGS)n (SEQ ID NO: 111), (GGGGS)nC (SEQ ID NO: 112), FLAG (DYKDDDDK)n (SEQ ID NO: 113), HA (YPYDVPDYA) (SEQ ID NO: 114), HSV (QPELAPEDPED) (SEQ ID NO: 115), Myc (EQKLISEEDL) (SEQ ID NO: 116), constant region of immunoglobulin heavy chain, glutathione S-transferase (GST), green fluorescent protein (GFP), maltose binding protein (MBP), ubiquitination tag (SUMO), Trx tag, V5 (GKPIPNPLLGLDST) (SEQ ID NO: 117). NO:117), NO:123)), VSVG (YTDIEMNRLGK) (SEQ ID NO:124), SV40 NLS (PKKKRKV (SEQ ID NO:125) or PKKKRKVG (SEQ ID NO:126)), Protein C (EDQVDPRLIDGK) (SEQ ID NO:127), S tag (KETAAAKFERQHMDS) (SEQ ID NO:128), OneStrap(SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK)(SEQ ID NO: 129), SB1 (PRPSNKRLQQ) (SEQ ID NO: 130), wherein n = any integer between 1 and 15. In all of these embodiments, the comparison of polypeptide homology does not involve any additional functional domains that may be incorporated into the polypeptide.

[0017] Preferably, the functional domain is present at the N-terminus and / or C-terminus of the polypeptide and is linked to the polypeptide via a peptide bond or cysteine.

[0018] Preferably, the functional domain includes a detection domain, a stabilizing domain, a therapeutic moiety, a diagnostic moiety and a drug delivery vehicle.

[0019] Preferably, the stabilizing domain comprises polyethylene glycol (PEG), albumin, lipids, hydroxyethyl starch (HES), a conformationally disordered polypeptide sequence consisting of amino acids Pro, Ala and / or Ser ("AS acylation"), and / or a mucin diffusible polypeptide consisting of amino acids Lys and Ala with or without Glu.

[0020] Furthermore, the polypeptide does not bind to human transferrin receptor 2 (hTfR2).

[0021] In some embodiments, the polypeptides described herein are produced using any method known in the art for polypeptide (protein) synthesis, particularly by chemical synthesis or by recombinant expression, and preferably by recombinant expression technology.

[0022] In some embodiments, the polypeptide (protein) or its binding fragment is produced by recombinant expression, wherein a nucleic acid encoding the polypeptide or its binding fragment is assembled using chemically synthesized oligonucleotides, which includes synthesizing overlapping oligonucleotides comprising a sequence portion encoding the polypeptide, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0023] The present invention also provides a conjugate obtained by coupling the above-mentioned human anti-transferrin receptor polypeptide (protein) with an oligonucleic acid molecule. In some cases, the ratio of the oligonucleic acid molecule to the anti-transferrin receptor polypeptide (protein) (drug to polypeptide ratio or DAR ratio) is about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1 or 16:1.

[0024] Preferably, the oligonucleic acid molecule is a small interfering RNA, an antisense oligonucleotide, or a phosphoramidite morpholino oligomer (PMO).

[0025] Another object of the embodiments of the present application is to provide a recombinant nucleic acid encoding the above-mentioned polypeptide that specifically binds to transferrin receptor.

[0026] Another object of an embodiment of the present application is to provide an expression vector comprising the above-mentioned recombinant nucleic acid operably linked to a promoter.

[0027] Another object of the embodiments of the present application is to provide a recombinant host cell comprising the above-mentioned polypeptide, recombinant nucleic acid and / or expression vector.

[0028] Another object of the embodiments of the present application is to provide uses of the polypeptide, recombinant nucleic acid, expression vector and / or recombinant host cell, which uses include preparing drugs for treating or limiting arenavirus infection, preparing drugs for gene mutation-related diseases, preparing drugs for treating tumors, and / or for fusion with biological products to increase their therapeutic serum half-life.

[0029] Another object of an embodiment of the present application is to provide a pharmaceutical composition comprising the above-mentioned polypeptide, recombinant nucleic acid, expression vector and / or recombinant host cell, wherein, preferably, the polypeptide is covalently linked to a therapeutic agent, and the therapeutic agent includes a protein, nucleic acid, lipid, PEG or a drug compound; preferably, the therapeutic agent is an oligonucleotide, and the oligonucleotide is a small interfering RNA, an antisense oligonucleotide, or a phosphoramidite morpholino oligomer (PMO), and the oligonucleotide is linked to the polypeptide by chemical covalent coupling and is used to regulate gene expression; preferably, its target tissues include skeletal muscle, myocardium, diaphragm and central nervous system; preferably, its delivery to the central nervous system includes crossing the blood-brain barrier through receptor-mediated endocytosis.

[0030] Compared with the prior art, this application has the following outstanding advantages:

[0031] 1. The arrangement of the C-terminal domains E2-H3-H4-E3 of the polypeptide provided in this application is different from that of similar polypeptides in the prior art. The polypeptides screened out that specifically bind to the transferrin receptor have better thermal stability and higher affinity than the polypeptides in the prior art.

[0032] 2. The polypeptides provided herein have precise targeting properties and can accurately act on the target area. They can bind highly specifically to human transferrin receptor 1 (hTfR1) and not to human transferrin receptor 2 (hTfR2) (hTfR2 does not participate in the iron transport process), enabling the precise delivery of oligonucleotides and providing a practical basis for the treatment of diseases related to gene mutations.

[0033] 3. The polypeptide provided in this application does not occupy the site on hTfR1 where hTfR1 binds to transferrin (Tf), and thus does not affect the normal iron transport process in the human body. This means that the polypeptide provided in this application has good blood safety and can function without interfering with normal physiological functions.

[0034] 4. After the polypeptide provided in the present application is combined with the oligonucleotide, its binding ability to the human transferrin receptor will not be weakened, which indicates that the binding of the polypeptide to the oligonucleotide will not interfere with its binding to the human transferrin receptor, and can ensure the stable delivery of the oligonucleotide and the targeting of the polypeptide. Therefore, the polypeptide can play an important role in drug delivery and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a flowchart of computer-assisted peptide design provided in the examples of this application;

[0036] Figure 2 This is a design diagram of a polypeptide (protein) that specifically binds to transferrin receptor provided in the examples of the present application;

[0037] Figure 3 Flow cytometry sorting results of W30029-PC and W30029-PC-Long linker respectively binding to Ag1 provided in the examples of the present application;

[0038] Figure 4 This is a flow chart of the flow cytometry screening of the polypeptide library provided in the examples of this application;

[0039] Figure 5 : This is a graph showing the results of flow cytometry screening of a polypeptide library provided in an example of the present application, wherein APC-A represents affinity and PE-A represents expression level;

[0040] Figure 6 This is a diagram showing the binding effect of the candidate polypeptide provided in the examples of this application on mTfR1;

[0041] Figure 7 This is a flow cytometry sorting result diagram of the binding of the candidate polypeptides provided in the examples of the present application to hTfR2;

[0042] Figure 8 1 is a graph showing the ELISA results of competition between the candidate polypeptide provided in the examples of the present application and human transferrin (Tf);

[0043] Figure 9 This is a graph showing the quality control results of the synthesis of the polypeptide oligonucleotide conjugate CGBC-1018 provided in the examples of this application;

[0044] Figure 10 is the SAXHPLC spectrum of the polypeptide oligonucleotide conjugate CGBC-1022 provided in the examples of the present application;

[0045] Figure 11 : is a diagram showing the knockdown results of the target gene by the polypeptide oligonucleotide conjugate provided in the examples of the present application in human rhabdomyoma cells at 72 hours and 96 hours respectively; and

[0046] Figure 12 This is a graph showing the detection results of the effect of the polypeptide provided in the examples of the present application on delivering oligonucleotides across the blood-brain barrier in hTfR1 transgenic mice. DETAILED DESCRIPTION

[0047] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings, examples and implementation schemes. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0048] In all embodiments of the polypeptides disclosed herein, any N-terminal methionine residue is optional (i.e., the N-terminal methionine residue may be present or absent and may be included or excluded when determining percent amino acid sequence identity compared to another polypeptide).

[0049] One aspect of the present application provides a polypeptide that specifically binds to the transferrin receptor with the general formula H1-H2-E1-E2-H3-H4-E3, which is used to bind to the human transferrin receptor hTfR1 but not to hTfR2, wherein H1, H2, H3 and H4 each independently contain an α-helical domain with a length between 11-20 amino acids; E1, E2 and E3 each independently contain a β-pleated domain with a length of 4-6 amino acids.

[0050] Furthermore, the polypeptide further comprises a linker of 1 to 5 optional amino acids connecting adjacent domains (α-helical domain and β-sheet domain).

[0051] Specifically, the polypeptide of the present application is bound to the hTfR1 apical domain, which is also the site where New World Arenaviruses enter cells. Some New World Arenaviruses, such as Machupo virus, Junín virus, Guanarito virus, and Sabiá virus, can cause hemorrhagic fever with a high mortality rate. Therefore, the polypeptide of the present application can be used to block these viruses from entering cells. In addition, hTfR1 is overexpressed in many tumors, so the polypeptide of the present application can be used for targeted therapy of tumors expressing hTfR1. Similarly, since hTfR1 is expressed throughout the body, the polypeptide of the present application can be used as a general delivery platform, particularly, to cross the blood-brain barrier (BBB). In addition, hTfR1 continuously circulates between the cell surface and endocytic vesicles, and this circulation is part of the natural function of hTfR1 to transport serum Tf into cells. Therefore, the fusion of biological preparations with the polypeptide of the present application can be used to increase the in vivo life of biological preparations.

[0052] The length of the various helical domains (H1, H2, H3 and H4) of the present application is between 11-20 amino acids and can be any amino acid composition, as long as these domains have an alpha helical structure. In various embodiments, the length of the helical domain can be 12-20, 13-20, 14-20, 15-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-19, 13-18, 13-17, 13-16, 13-15 or 13-14 amino acids.

[0053] The length of the various β-pleated domains (E1, E2 and E3) of the present application is between 4 and 6 amino acids, and contains at least 3, 4 or 5 hydrophobic amino acids, including but not limited to norleucine, Met, Ala, Val, Leu, Ile, and may also include unnatural amino acids.

[0054] In one embodiment, the human anti-transferrin receptor binding polypeptide comprises an amino acid sequence D(V / I)V(V / I)VT sequence, which may appear at any position of E1, E2 or E3, wherein the residues in brackets are alternative residues at the given positions, wherein the first D residue and the last T residue may be deleted.

[0055] In another embodiment, the E1 domain comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% homologous to any one of SEQ ID NOs: 46-55 of Table 1, wherein the amino acid substitutions are conservative amino acid substitutions.

[0056] In another embodiment, the E2 domain comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% homologous to any one of SEQ ID NOs: 56-69 of Table 1, wherein the amino acid substitutions are conservative amino acid substitutions.

[0057] In another embodiment, the E2 domain comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% homologous to any one of SEQ ID NOs: 70-85 of Table 1, wherein the amino acid substitutions are conservative amino acid substitutions.

[0058] Table 1 Sequence information of the β-folded domain contained in the polypeptide that specifically binds to transferrin receptor

[0059] E1 E2 E3 DIVVV (SEQ ID NO: 46) AIVT (SEQ ID NO: 56) EVILVE (SEQ ID NO: 70) DIVVVT (SEQ ID NO: 47) AVVVVK (SEQ ID NO: 57) EVVL (SEQ ID NO: 71) DVVVV (SEQ ID NO: 48) DIVIVK (SEQ ID NO: 58) KVIL (SEQ ID NO: 72) IVVVT (SEQ ID NO: 49) DIVVVK (SEQ ID NO: 59) KVILVP (SEQ ID NO: 73) VIAH (SEQ ID NO: 50) DIVVVR (SEQ ID NO: 60) KVVLVP (SEQ ID NO: 74) VIAY (SEQ ID NO: 51) GIVA (SEQ ID NO: 61) RVIL (SEQ ID NO: 75) VILH (SEQ ID NO: 52) GIVV (SEQ ID NO: 62) RVILV (SEQ ID NO: 76) VILY (SEQ ID NO: 53) GIVVVK (SEQ ID NO: 63) RVILVE (SEQ ID NO: 77) VIYY (SEQ ID NO: 54) GIVVVQ (SEQ ID NO: 64) RVILY (SEQ ID NO: 78) VVVVT (SEQ ID NO: 55) GIVVVS (SEQ ID NO: 65) RVVL (SEQ ID NO: 79) GVVVVK (SEQ ID NO: 66) RVVLV (SEQ ID NO: 80) HIVIVK (SEQ ID NO:67) RVVLVP (SEQ ID NO: 81) IVIVK (SEQ ID NO: 68) TVILV (SEQ ID NO: 82) LVVC (SEQ ID NO: 69) TVILVP (SEQ ID NO: 83) VVIV (SEQ ID NO: 84) VVVV (SEQ ID NO: 85)

[0060] In one embodiment, the amino acid replacement is positioned at the surface residue place that is not in the interface or near the interface relative to above-mentioned polypeptide.Table 2 has been listed the surface residue position that is not in the interface or near the interface.As those skilled in the art will appreciate, these residues are not present in the binding interface place or near (as described in detail in the embodiment) of polypeptide of the present application and transferrin receptor, are therefore more easily variable and do not affect the binding activity of transfer receptor.

[0061] Table 2 All surface residues in each sequence that are not within or near the interface:

[0062]

[0063] In one embodiment, the transferrin receptor binding polypeptide comprises the following additional functional domain amino acid fragments at the N-terminus or C-terminus: MHHHHHH (SEQ ID NO: 109), MHHHHHHC (SEQ ID NO: 110), (GS)n, (GS)nC, (SG)n, (SG)nC, (GGGGS)n (SEQ ID NO: 111), (GGGGS)nC (SEQ ID NO: 112), FLAG (DYKDDDDK)n (SEQ ID NO: 113), HA (YPYDVPDYA) (SEQ ID NO: 114), HSV (QPELAPEDPED) (SEQ ID NO: 115), Myc (EQKLISEEDL) (SEQ ID NO: 116), the constant region of the immunoglobulin heavy chain, glutathione S-transferase (GST), green fluorescent protein (GFP), maltose binding protein (MBP), ubiquitination tag (SUMO), Trx tag, V5 (GKPIPNPLLGLDST) (SEQ ID NO: 117), SEQ ID NO: 118. NO:117), NO:123)), VSVG (YTDIEMNRLGK) (SEQ ID NO:124), SV40 NLS (PKKKRKV (SEQ ID NO:125) or PKKKRKVG (SEQ ID NO:126)), Protein C (EDQVDPRLIDGK) (SEQ ID NO:127), S tag (KETAAAKFERQHMDS) (SEQ ID NO:128), OneStrap(SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK)(SEQ ID NO:129), SB1 (PRPSNKRLQQ) (SEQ ID NO:130), wherein n=any integer between 1 and 15.Common tags refer to Pina AS, et al. (2014) Affinity tags in protein purification and peptide enrichment: An overview. Methods in molecular biology (Clifton, NJ) 1129: 147-168; Kimple ME, et al. (2013) Overview of affinity tags for protein purification. Curr Protoc Protein Sci 73: 9.9.1-9.9.23; Amarasinghe C, et al. (2015) The use of affinity tags to overcome obstacles in recombinant protein expression and purification. Protein Pept Lett 22 (10): 885-892. In all of these embodiments, the comparison of polypeptide homology does not involve any additional functional domains that may be incorporated into the polypeptide.

[0064] The polypeptide of the specific binding transferrin receptor of the present application can comprise one or more amino acid linkers between adjacent domains.This amino acid linker can only be present between two adjacent domains (for example, only having an amino acid linker between H1 and H2 domain, not having a linker between other domains), can also be present between multiple adjacent domains, or be present between all adjacent domains.This amino acid linker can have any appropriate length and be made up of any amino acid.In a preferred embodiment, this amino acid linker length is generally 2 to 4 amino acid.

[0065] In another embodiment, the polypeptide that specifically binds to transferrin receptor comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 2, 5, 6, 8, 9 and 12.

[0066] The polypeptide that specifically binds to transferrin receptor of the present application may include additional amino acid residues. In certain embodiments, the polypeptide may include additional amino acid residues at the N-terminus and / or C-terminus. Depending on the intended purpose, any appropriate additional residues may be added. In various non-limiting embodiments, the polypeptide may further include a functional domain, and the additional functional domains include: a detection domain, a stabilizing domain, a therapeutic group, a diagnostic group, and a drug delivery vehicle. The functional domains may be added as a transcriptional fusion with the polypeptide or may be chemically coupled to the polypeptide. Any appropriate chemical coupling method may be used, such as covalent bonding with cysteine ​​residues. Any surface amino acid residue outside the binding interface or its vicinity in the polypeptide of the present application may be mutated to cysteine. In one embodiment, one or more additional functional domains are present at the N-terminus and / or C-terminus of the polypeptide as a transcriptional fusion. In one embodiment, the one or more functional domains include a stabilizing domain, such as polyethylene glycol (PEG), albumin, hydroxyethyl starch (HES), a conformationally disordered polypeptide sequence (PASylation) composed of Pro, Ala and / or Ser, and a mucin diffusible polypeptide composed of Lys and Ala (with or without Glu).

[0067] In some embodiments, a given amino acid can be replaced by a residue with similar physicochemical properties, for example, by replacing one aliphatic residue with another (e.g., replacing Ile, Val, Leu, or Ala with each other), or by replacing one polar residue with another (e.g., replacing between Lys and Arg; between Glu and Asp; or between Gln and Asn). Other such conservative substitutions can also be included, such as replacement of entire regions with similar hydrophobicity characteristics. Amino acids can be grouped according to the similarity of their side chain properties (see ALLehninger, in Biochemistry, second ed., pp.73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into several groups based on common side chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain direction: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. "Non-conservative substitutions" in this application refer to exchanging a member of one class for another. Specific conservative substitutions include, for example: Ala for Gly or Ser; Arg for Lys; Asn for Gln or His; Asp for Glu; Cys for Ser; Gln for Asn; Glu for Asp; Gly for Ala or Pro; His for Asn or Gln; Ile for Leu or Val; Leu for Ile or Val; Lys for Arg, Gln, or Glu; Met for Leu, Tyr, or Ile; Phe for Met, Leu, or Tyr; Ser for Thr; Thr for Ser; Trp for Tyr; Tyr for Trp; and / or Phe for Val, Ile, or Leu.

[0068] In another embodiment, the polypeptide specifically binding to transferrin receptor of the present application can bind to transferrin receptor with a binding affinity of at least 3 μM, 1 μM, 500 nM, 250 nM, 100 nM, 50 nM, 5 nM, 1 nM, or 0.1 nM.

[0069] Another aspect of the present application provides recombinant nucleic acids encoding all the polypeptides in the embodiments of the present application. The nucleic acid sequence may comprise single-stranded or double-stranded RNA or DNA in the form of a genome or cDNA, or a DNA-RNA hybrid, wherein each sequence may comprise structurally modified, non-natural or derived nucleotide bases. Such a nucleic acid sequence may comprise additional sequences for promoting expression and / or purification of the encoded polypeptide, including polyadenylic acid (polyA) sequences, modified Kozak sequences, and sequences encoding epitope tags, exocytic signals, secretory signals, nuclear localization signals, and plasma membrane localization signals. Based on the above description, those skilled in the art can clearly understand all the nucleic acid sequences corresponding to the polypeptides disclosed in the present application.

[0070] Another aspect of the present invention provides an expression vector comprising the above-mentioned recombinant nucleic acid operably linked to a promoter. An "expression vector" includes a vector that effectively links a nucleic acid coding region or gene to any control sequence that can affect the expression of a gene product. A "control sequence" that is effectively linked to the nucleic acid sequence of the present invention is a nucleic acid sequence that can affect the expression of a nucleic acid molecule. The control sequence does not need to be adjacent to the nucleic acid sequence; it only needs to play a role in directing its expression. Therefore, an untranslated sequence that is used for transcription can be inserted between the promoter sequence and the nucleic acid sequence, and the promoter sequence can still be considered to be "operably linked" to the coding sequence. Other such control sequences include polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type, including plasmids and viral-based expression vectors. The control sequences used to drive the expression of the disclosed nucleic acid sequences in mammalian systems can be constitutive (driven by any of a variety of promoters, including CMV, SV40, RSV, actin, EF) or inducible (driven by any of a variety of inducible promoters, including tetracycline promoter, ecdysone promoter, and steroid response promoter). The expression vector must be replicable in the host organism either as an episome or by integration into the host chromosomal DNA.In various embodiments, the expression vector may comprise a plasmid, a viral-based vector, or any other suitable expression vector.

[0071] Another aspect of the present application also provides a recombinant host cell comprising the above polypeptide, nucleic acid and / or expression vector (exogenous plasmid or chromosome integration), and the host cell can be a prokaryotic organism or a eukaryotic organism.

[0072] Another aspect of the present application also provides uses of the polypeptide, recombinant nucleic acid, expression vector, and recombinant host cell, including uses for preparing drugs for treating or limiting arenavirus infection, for preparing drugs for gene mutation-related diseases, for preparing drugs for treating tumors, and for fusion with biological products (including proteins, nucleic acids and antibodies) to increase their therapeutic serum half-life.

[0073] Another aspect of the present application also provides a pharmaceutical composition comprising the polypeptide, recombinant nucleic acid, expression vector, or recombinant host cell and a pharmaceutically acceptable carrier. The polypeptide is covalently linked to a therapeutic agent to increase the serum half-life and tissue targeting of the therapeutic agent, and the therapeutic agent includes a protein, nucleic acid, lipid, PEG or drug compound. Preferably, the therapeutic agent is an oligonucleotide, wherein the oligonucleotide can be a small interfering RNA (siRNA), an antisense oligonucleotide (antisenseoligonucleotides), or a phosphoramide morpholino oligomer (PMO); wherein the oligonucleotide is used to regulate gene expression and is linked to the polypeptide by chemical covalent coupling. The therapeutic target tissues of the pharmaceutical composition include skeletal muscle, myocardium, diaphragm and central nervous system, and the mode of delivery of the pharmaceutical composition to the central nervous system includes crossing the blood-brain barrier by receptor-mediated endocytosis.

[0074] The present application is further described below through specific examples.

[0075] Example 1. Design of human transferrin receptor TfR1 binding polypeptide

[0076] The purpose of this example is to design the sequence of human TfR1 binding polypeptides by computer-aided design for subsequent screening of yeast display libraries. The experimental method is described as follows.

[0077] (1) Computational design of proteins

[0078] Using the complex structure of human transferrin receptor hTfR1 and its binding protein (PDB ID: 6wrw) as a template, hTfR1-targeted binding protein was designed from scratch while keeping the binding site unchanged to obtain a binding protein with high binding affinity and specificity for hTfR1 ( Figure 1 ). Retain binding protein ( Figure 2 A β-sheet structure interacting with TfR ( Figure 2 B), exhaustively enumerate and reverse all other secondary structural connection modes of the binding protein ( Figure 2 C), and then connect these secondary structures in sequence on the computer software, complete the main chain residues in the loop region, and obtain a new hTfR1 binding protein main chain skeleton structure ( Figure 2D) The most suitable structure was selected through visualization and 31 skeleton structures were obtained using the computer software pdbtool TM Complete the side chain O, N, and Cb atoms.

[0079] (2) Fixed backbone sequence design

[0080] For the binding protein backbone generated in the previous step, the neural network model ProteinMPNN was used with a sampling temperature of 0.01 (the lower the sampling temperature, the higher the sequence recovery rate and the relatively higher the sequence activity specificity; the higher the sampling temperature, the higher the sequence diversity and the larger the sequence search space). The sequence design of the binding protein was carried out under the condition of retaining and fixing the hTfR1 interface structure. 4,000 sequences were designed for each backbone, and 100,000 sequences were removed and screened out after deduplication.

[0081] (3) Sequence screening

[0082] Using the protein structure prediction Transformer model ESMFold TM High-throughput sequence folding validation was performed on the 100,000 sequences mentioned above. Using a specific prediction algorithm combined with metrics such as protein radius, relative solvent accessible surface area (SASA), and secondary structure reduction, poor sequences were filtered out. Further screening using Esmfold software identified optimal sequences.

[0083] Example 2. Flow cytometry sorting method

[0084] The purpose of this example is to construct a yeast display library of Yangshen polypeptides, determine the design of molecular clones, and use it to screen other unknown polypeptides. Secondly, a flow cytometry sorting method was established using the yeast display library of Yangshen polypeptides for screening unknown polypeptides. The experimental method is described below.

[0085] Yang Shen (positive control) polypeptide sequence: named W30029-PC, from the prior art Sahtoe et al., PNAS 2021 Vol.118 No.17 e2021569 and prior art CN 116075524 A, its sequence is DEEEIQKAIEELLRKGVSEEEAAIIIVQRFNVAVVVVVQDERQAKHISEYIRRYIPEADVILFANIVVIKVETHELRKRVWEAAQKAY (SEQ ID NO: 1).

[0086] The W30029-PC plasmid was synthesized by Azenta based on the pCTcon2 yeast display vector. The Yangshen plasmid was used as a template for the W30029-PC-Long linker plasmid. PCR conditions were: 98°C, 3 minutes; (98°C, 30 seconds; 52°C, 30 seconds; 72°C, 3 minutes), 25 cycles; 72°C, 10 minutes. The PCR product was purified using a gel and PCR Clean-up Kit, and then PCR amplified using PrimeSTAR Max DNA Polymerase. Two linkers were designed in this experiment: a standard linker and a long linker, each used to construct plasmids for subsequent flow cytometry sorting. The designs are as follows:

[0087] Common linker: GGGGS (SEQ ID NO: 131);

[0088] Long linker: GGGGSGGGGS (SEQ ID NO: 132);

[0089] The W30029-PC and W30029-PC-Long linker plasmids were introduced into yeast using a stock electrocompetent Saccharomyces cerevisiae strain EBY100. For each electroporation, 4 μg of vector was mixed with 400 μL of electrocompetent EBY100 (optical density 160 OD) and transferred to a pre-chilled cuvette with a 2 mm electrode gap. The mixture was placed on ice for 5 minutes and then electroporated using an electroporator with a voltage of 2.5 kV, a pulse width of 200 ms, and a capacitance of 25 μF. Immediately after electroporation, the cells were added to a 1:1 mixture of 1 mL of 1 M sorbitol and 1X YPD medium (containing 10 g yeast extract, 20 g peptone, and 20 g glucose) and placed on a platform shaker at 225 rpm and 30°C for 2 hours. Plate the culture onto SDCAA agar plates and incubate at 30°C for 3 days to select yeast strains harboring the TRP marker carried by the modified plasmid pCTcon2. Cultivate the selected yeast strains in one liter of SDCAA medium (containing 5.4 g NaHPO4, 7.44 g NaHPO4, 5 g Bacto Casamino Acids, 20 g glucose, and 6.7 g Yeast Nitrogen Base). Filter-sterilize the medium using a 0.22 μm vacuum filter before use.

[0090] Single colonies were picked, inoculated into SDCAA medium and grown overnight. The next day, W30029-PC and W30029-PC-Long linker yeast cells were centrifuged at 4000 RPM for two minutes and resuspended in inducing SDCAA medium to 0.5x10 7cells / mL. One liter of SGRCAA induction medium contains 5.4 g Na2HPO4, 7.44 g NaH2PO4, 20 g raffinose, 5 g Bacto Casamino Acids, 20 g galactose, and 6.7 mg yeast nitrogen base and is sterilized by filtration through a 0.22 μm membrane before use. Cultures are grown overnight in induction medium at 20°C to induce expression of the yeast cell surface peptide library.

[0091] After induction of W30029-PC and W30029-PC-Long linker yeast cells overnight, Ag1 (antigen 1, i.e., hTfR1, starting from 2.5 μM, diluted 1:3) was incubated with W30029-PC and W30029-PC-Long linker yeast cells, respectively. After incubation, the mixture was washed three times with 1× PBS, and streptavidin fluorescent dye conjugate SA-647 and fluorescent antibody reagent Anti-cMyc-RPE (200 μL, diluted 1:200) (commercially available) were added and incubated for 0.5 h. SA-647 was used to detect the binding of Ag1 to cells, and Anti-cMyc-RPE was used to detect the expression level of peptides on yeast cells. Unstained cells were used as negative controls. The incubated cells were detected by flow cytometry using BD-C6 and BD-Aria3 (BD ​​Biosciences).

[0092] like Figure 3 As shown, both W30029-PC yeast cells and W30029-PC-Long linker yeast cells can bind to Ag1 well. Subsequent flow cytometry sorting experiments use common length linkers to screen the peptide library.

[0093] Example 3. Establishment of a polypeptide library

[0094] The purpose of this example is to establish a yeast display library of unknown polypeptides based on the design of Example 1 and to control the quality of the library. The experimental method is described as follows.

[0095] The peptide oligonucleotide library DNA product was synthesized by Twist Biosciences. The synthesized DNA product was used as a template to obtain more DNA products for library construction. The PCR conditions were: 98°C, 3 minutes; (98°C, 20 seconds; 59°C, 15 seconds; 72°C, 15 seconds), 19 cycles; 72°C, 1 minute. The PCR products were obtained by The DNA fragment was then PCR amplified using PrimeSTAR Max DNA polymerase and the primers listed in Table 3. The backbone vector pCTcon2 was digested with Nhe1 and Sal1 (commercially available) to obtain a linearized vector.

[0096] Table 3 Primer information

[0097] Primer name Sequence SEQ ID NO: Oligo pool-F TAGCGGAGGCGGAGGGTCGGCTAGC 101 Oligo pool-R CTTTTGTTCGGATCCGCCCCCGTCGAC 102

[0098] The DNA product and linearized vector were then electroporated into EBY100, plated onto SDCAA plates, and incubated at 30°C. For each electroporation, 4 μg of DNA product and 12 μg of linearized vector were mixed with 400 μL of electrocompetent EBY100 (160 OD) and transferred to a pre-cooled cuvette of a BioRad GenePulser electroporator with a 2 mm electrode gap. The established W30029 peptide yeast library was submitted for high-throughput sequencing. The results (Table 4) showed that the effective library size was 1000-fold greater than the designed library diversity. Expression rate and sequence analysis indicated that the library quality was sufficient for subsequent sorting.

[0099] Table 4 Peptide yeast library detection results

[0100] name Diversity Library size Expression ratio Sequencing accuracy W30029 peptide yeast library <![CDATA[2.0E 5 ]]> <![CDATA[4E 8 ]]> 95.5％(84 / 88) 89.1％(41 / 46)

[0101] Example 4. Flow cytometric sorting of polypeptide libraries

[0102] The purpose of this example is to screen the polypeptide library by flow cytometry to obtain polypeptide sequences with higher affinity than Yang Shen. The specific steps are as follows.

[0103] 2x10 8 Cells were thawed from a single library stock, suspended in SDCAA medium, and cultured overnight. The next day, 2x10 8 The cells were centrifuged at 2000 RPM for two minutes and resuspended in SGRCAA induction medium to a concentration of 0.5 x 10 7 The culture was grown overnight in induction medium at 20°C to induce the expression of the yeast cell surface peptide library.

[0104] Induce cells from the oligonucleotide library for subsequent fluorescence activated cell sorting (FACS) protocols (see Figure 4 ), which included one competitive selection and three rounds of positive selection. Each round involved enriching for high-affinity clones by decreasing the concentration of Ag1 (antigen 1, hTfR1) and varying the sorting gate. In competitive selection, Ag2 was human transferrin (Tf), and competition with transferrin was used to select peptide clones with less competitive ability with transferrin. In positive selection, high-affinity clones were enriched by decreasing the concentration of Ag1 (human transferrin receptor hTfR1) and varying the sorting gate each round.

[0105] In the first round of the draft, 2x10 8Freshly grown yeast cells were incubated with 830 nM Ag1. After incubation, the mixture was washed three times with 1× PBS and 8300 nM Ag2, 500 μL, was added and incubated for 1 h.

[0106] In the second round of the draft, 2x10 8 Freshly grown yeast cells were incubated with 100 nM Ag1.

[0107] In the third round of selection, 2x10 8 Freshly grown yeast cells were incubated with 40 nM or 20 nM Ag1.

[0108] In the fourth round of the draft, 2x10 8 Freshly grown yeast cells were incubated with 20 nM Ag1.

[0109] After incubation, the mixture was washed three times with 1× PBS. SA-647 and Anti-cMyc-RPE (200 μL, 1:200 dilution) were added to the mixture and incubated for 0.5 hours. SA-647 was used to detect the binding of Ag1 to the cells. Anti-cMyc-RPE was used to detect the expression level of the peptide on the yeast cells. Unstained cells were used as negative controls. The incubated cells were detected and sorted by flow cytometry BD-Aria3 (BD ​​Biosciences).

[0110] After each round of sorting, cells were collected at the P4 gate for the next round of sorting. Therefore, clones with higher affinity can be obtained by narrowing the P4 gate and reducing the antigen concentration. The results showed that a certain proportion of positive cells can be obtained in each round ( Figure 5 ), and clones obtained in the third or fourth round were selected for identification.

[0111] Example 5. Screening and identification of candidate polypeptides

[0112] The purpose of this example is to identify all candidate polypeptides with higher affinity than Yangshen by flow cytometry and confirm their sequences. The specific experimental steps are as follows.

[0113] Individual colonies were picked from the plates from rounds 3 and 4 in Example 4 and inoculated into SDCAA medium and grown overnight. The next day, the yeast cells were centrifuged at 4000 RPM for two minutes and resuspended in induction SGRCA medium to 0.5 x 107 cells / mL. The cultures were grown overnight in induction medium at 20°C to induce expression of the yeast cell surface peptide library. These yeast colonies were lysed by incubation with NaOH at 100°C for 8 minutes. The mixture was then used as a template for colony PCR, and the PCR products were sequenced.

[0114] After the yeast cells were induced overnight, 40MAg1 was incubated with these yeast cells. SA-647 was used to detect the binding of Ag1 to the cells. After incubation, the mixture was washed 3 times with 1×PBS. SA-647 and Anti-cMyc-RPE (200μL, 1:200 dilution) were added to the mixture and incubated for 0.5 hours. Anti-cMyc-RPE was used to detect the expression level of peptides on yeast cells. Unstained cells were used as negative controls. The incubated cells were detected by flow cytometry BD-C6 (BD Biosciences). Positive clones with sample Ag1 binding signal intensity greater than W30029-PC were collected (Table 5) and sent for sequencing to obtain 44 polypeptide sequences (SEQ ID NO:2-45) (Table 6).

[0115] Table 5 Enrichment of positive clones and binding signal intensity with Ag1

[0116]

[0117]

[0118] Table 6 Amino acid sequences of positive clones

[0119]

[0120]

[0121]

[0122] Example 6. Comparison of affinity between candidate polypeptides and human and mouse transferrin receptors (hTfR1 and mTfR1)

[0123] The purpose of this example is to accurately determine the affinity of the preferred polypeptide to hTfR1 and mouse transferrin receptor 1 (mTfR1) after expression and purification. The experimental method is described as follows.

[0124] Based on the affinity and sequence diversity of hTfR1, six candidate polypeptides (SEQ ID NOs: 86-91) were selected from the 44 polypeptides in Example 5 and attached to the N-terminus with an MHHHHHH sequence to support subsequent purification, detection, and conjugation. The Yangshen sequence (SEQ ID NO: 92) was also expressed with a His tag, which was then removed by enzyme digestion to leave a glycine (G) at the N-terminus. Full sequence information is shown in Table 7. Binding assays were performed using a streptavidin-coated biosensor on an OctetRED384 BLI system (Sartorius, FB-20493). Cytiva PBS (pH 7.2) containing 0.03% Tween 20 was used as the baseline buffer for the streptavidin (SA) sensor (GenScript, Lot. No. 20230130P0004). The biotinylated TfR1 (hTfR1 and mTfR1) external domain was fixed to the biosensor by immersing the biosensor in a solution with 2.5ug / ml TfR1 for 120-300s. It was then immersed in fresh octet buffer (GenScript, Lot. No. 20230130P0004) to establish a 200s baseline in the buffer. The experiment was performed at 30°C while rotating at 1000rpm. It was determined that 6 polypeptides bound to hTfR1 and the affinity index KD was in the nanomolar order (Table 8), the highest of which was more than 3 orders of magnitude higher than that of Yangshen, and did not bind to mTfR1 ( Figure 8 ).

[0125] Table 7 Names and sequence information of candidate polypeptides and Yangshen polypeptides

[0126]

[0127] Table 8 Affinity of candidate peptides to human transferrin receptor (hTfR1)

[0128]

[0129]

[0130] KD: Equilibrium dissociation constant, representing the strength of affinity. The smaller the KD value, the greater the affinity.

[0131] Kdis: dissociation rate constant, representing the degradation rate of the product per unit time;

[0132] Ka: Binding rate constant, representing the rate of product formation per unit time;

[0133] Rmax: maximum analyte concentration that can be achieved;

[0134] DissocX^2: Chi-square value of dissociation, used to assess the goodness of fit between experimental data and predicted values.

[0135] DissocR^2: correlation coefficient of dissociation. The closer it is to 1, the better the regression fitting effect.

[0136] Example 7. Affinity of candidate polypeptides for human transferrin receptor 2 (TfR2)

[0137] The purpose of this example was to screen for peptides that have affinity for hTfR1 but do not bind to hTfR2. hTfR1 and hTfR2 are members of the same family of proteins and have similar functions. Under physiological conditions, hTfR1 is primarily distributed in human muscle tissue and the blood-brain barrier, while hTfR2 is primarily distributed in the liver. The specific experimental steps are described below.

[0138] CHO-K1 / human TfR2(+) stably transfected cells with high expression of human TfR2 were harvested by centrifugation and the cell density was adjusted with FACS buffer (99% Dulbecco's phosphate buffer + 1% fetal bovine serum), and then the cell suspension was transferred to the assay plate. The working solution of the test substance was prepared with FACS buffer. The test sample was added to the assay plate and incubated at 4°C for 1 hour. The assay plate was removed, centrifuged, and the supernatant was removed. The cells with FACS buffer were resuspended in the assay plate and the detection secondary antibody working solution was added, in which Var2iii was added with Alexa 647AffiniPure Goat Anti-Human IgG, Fcγfragment specific (min X Bov, Hrs, Ms Sr Prot) (Jackson, 109-605-098) was used as the secondary antibody, and OTI1B1 was supplemented with Alexa 647AffiniPure Goat Anti-Mouse IgG, Fcγfragment specific (min X Hu, Bov, Hrs Sr Prot) (Jackson, 115-605-071) was used as the secondary antibody, and 6 peptide samples (SEQ ID NO: 86-91) were added to the TMHis Tag Antibody [iFluor 647], mAb, Mouse (Genscript, A01802) was used as a secondary antibody, followed by incubation at 4°C in the dark for 20 minutes. The assay plate was removed, centrifuged, and the supernatant removed. The cell pellet was resuspended in FACS buffer in the assay plate. Data were acquired using a BD FACS Celesta. Raw FACS data were exported from BD FACSDiva software v8.0.1.1 and analyzed using FlowJo v10.8.0.

[0139] The humanized anti-hTfR1 antibody Var2iii used in the experiment is a reference antibody that binds to the apical domain of human TfR1. Its Fab end comes from 13E4-Variant 2iii of the prior art US10913800B2, and its Fc end comes from a human IgG1 subclass monoclonal antibody. The sequence is shown in Table 9 and is used as a negative control in the experiment. The mouse anti-hTfR2 binding antibody OTI1B1 used in the experiment is from Invitrogen, catalog number MA5-25932, and is used as a positive control in the experiment. The results showed that none of the 6 peptides bound to TfR2 ( Figure 7 ).

[0140] Table 9 Var2iii antibody sequence

[0141]

[0142] Example 8. Ability of candidate polypeptides to compete with human transferrin (Tf) for hTfR1

[0143] The purpose of this example is to screen for polypeptides with suitable hTfR1-binding epitopes. Under physiological conditions, the binding of hTfR1 and Tf directly regulates the transport of iron ions. Therefore, if antibodies block the hTfR1-Tf interaction, it is possible to interfere with iron homeostasis and lead to side effects such as anemia during in vivo experiments and clinical administration. The ideal hTfR1-binding polypeptide should bind to a suitable epitope of hTfR1 (e.g., the apical domain) to minimize interference with the hTfR1-Tf interaction. The specific experimental steps are as follows.

[0144] 1 μg / ml Tf was plated at a density of 100 μl / well and incubated at 4°C overnight. The cells were washed twice with 0.05% PBST, blocked with 3% MPBS, and incubated at 37°C for 1 hour. The cells were washed once with 0.05% PBST. Six peptides (SEQ ID NOs: 86-91) were diluted fourfold in PBST, starting at a maximum concentration of 3200 nM. 50 μL / well were mixed with biotinylated hTfR1 (0.06 μg / ml) in the same volume and incubated at 37°C for 1 hour. The cells were washed three times with 0.05% PBST, and SA-HRP (0.05% PBST, 1:10,000, 100 μL / well) was added and incubated at 37°C for 30 minutes. The cells were washed six times with 0.05% PBST, and TMB was added for a 10-minute reaction. Among them, AF2474 is a polyclonal antibody known to compete with human transferrin for human TfR1 (R&D System, Cat#AF2474). The results showed that none of the six peptides competed with Tf for the same epitope on TfR1, that is, the six peptides did not bind to the same position on Tf when binding to TfR1; while AF2474 at above 1 nM and Var2iii at above 100 nM all had the ability to compete with human transferrin for the same epitope on TfR1 ( Figure 8 ).

[0145] Example 9. Binding epitopes of candidate polypeptides to hTfR1

[0146] The purpose of this example is to study the binding mode between candidate polypeptides (the above 6 candidate polypeptides) and hTfR1, including the polypeptide binding epitope (epitope) of hTfR1 and the surface residues (paratope) of the polypeptide directly involved in hTfR1 binding. The experimental method is described as follows.

[0147] Identification of antigen-antibody two-dimensional peptide spectra: The two-dimensional peptide spectra of antigen and antibody were identified by using the hydrogen-deuterium exchange mass spectrometry platform LEAP PAL 3.0 using secondary mass spectrometry (MS / MS) on a mass spectrometer (Orbitrap Fusion TM Tribrid TM Protein enzymatic peptide fragments were identified using a Mass Spectrometer (Thermo Fisher). MS / MS data files were processed using Proteome Discover software for peptide identification.

[0148] Preparation of samples for hydrogen-deuterium exchange mass spectrometry: 5-10 μM of antigen, antibody, or antigen-antibody complex (1:1 molar ratio) was incubated in 50 mM HEPES, pH 7.4, 150 mM NaCl, and 4 mM TCEP at 4°C for 1 hour to allow the complex to reach a stable state. Five microliters of sample was then diluted into 20 μL of deuterated DO and subjected to HDX mass spectrometry analysis at various time points (e.g., 0, 10, 60, 300, and 900 seconds). Following the hydrogen-deuterium exchange period, the reaction was terminated by mixing with 25 μL of ice-cold 4 M guanidine hydrochloride and 1% trifluoroacetic acid. Immediately after terminating the reaction, the sample tube was placed on dry ice until injection into the HDX LEAP PAL 3.0 platform.

[0149] After injection onto the fully automated hydrogen-deuterium exchange platform, the sample passed through the fixed pepsin column at a flow rate of 120 μL / min, and the enzymatically digested peptides were captured on the C18 capture column and desalted. Within 8 minutes, the desalted peptides were separated using a 2.1 mm × 5 cm C18 column (1.9 μm Hypersil Gold, Thermo Fisher) with a linear gradient of 4-40% acetonitrile and 0.3% formic acid. During sample processing, protein enzymatic digestion and peptide separation were performed at 4°C. An Orbitrap mass spectrometer (Orbitrap Fusion TM Tribrid TM Mass spectrometer (Thermo Fisher) was used to obtain hydrogen-deuterium exchange mass spectrometry data with a resolution of 65,000 (m / z 400). Each sample was subjected to three HDX determinations (triplicates) at each time point. The average m / z centroid value of the mass spectrometry peak intensity of each enzymatic peptide was calculated by HDXWorkbench software (10 ppm accuracy) and then converted into the percentage of deuterium incorporation. The key amino acid sequences involved in the spatial epitope were calculated, and the difference in Delta%D was determined by calculating the difference between the two samples (comparing the change in the percentage of deuterium incorporation on the same peptide). Differences in Delta%D outside of -5 to 5% were considered significant. In addition, HDX Workbench detected statistically significant (p < 0.05) differences between samples at each time point using student's t test.

[0150] The binding epitope was determined to be I202, V203, D204, K205, N206, G207, R208, L209, V210, Y211, L212, and V213 on the extracellular region of hTfR1 (C89-F760, SEQ ID NO: 95). Based on the distance between the peptide and the human TfR1 binding interface, the surface residues of the candidate peptides that are speculated to be involved in hTfR1 binding are shown in Table 10.

[0151] (SEQ ID NO:95)

[0152] Table 10 Surface residues of polypeptides involved in human TfR1 binding

[0153]

[0154] Example 10. Conjugation of polypeptides and oligonucleotides

[0155] The purpose of this example is to conjugate a polypeptide to an oligonucleotide to obtain a quality-controlled polypeptide-oligonucleotide conjugate. The inventors have developed two conjugation schemes for connecting polypeptides to oligonucleotides. The candidate polypeptide sequence is derived from W30029-P1R4-1F2 (SEQ ID NO: 6) in Example 5. To meet the conjugation requirements, the candidate polypeptides were modified at the N-terminus and renamed CGBB-0002 and CGBB-0005. The detailed sequences are shown in Table 11, and the conjugation process is described below.

[0156] Table 11 Peptide sequence information

[0157]

[0158] Conjugation protocol 1:

[0159] The detailed technical route is based on Chun Guo et al. Bioconjugate Chemistry 202233 (10), 1885-1891. 6-Azidomethyl-4-methoxypicolinaldehyde (30 nmol dissolved in 1 μL dimethyl sulfoxide (DMSO)) and 3-maleimidopropionic acid (15 nmol dissolved in 1 μL DMSO) were added to 30 μL of CGBB-0002 (3 nmol) dissolved in pH 7.4 phosphate buffer. The resulting mixture was incubated at 37°C overnight, and then the resulting solution was concentrated and buffer was added; a 0.5 mL centrifugal filter was used for liquid exchange. Buffer exchange was first performed by diluting each sample to 500 μL using 50 mM pH 7.4 phosphate buffer. Each sample was then concentrated to 30 μL, and the process was repeated 3 times to obtain a polypeptide intermediate. Then, a dialkynyl glycidyl (DBCO)-modified oligonucleotide (4.5 nmol, dissolved in 4.5 μL pH 7.4 phosphate buffer, the siRNA sequence is shown in Table 12, and the target is the mouse DMPK gene) was added to 30 μL of protein solution (3 nmol). The resulting mixture was incubated overnight at room temperature, purified by SEC FPLC, and concentrated to pH 7.4 phosphate buffer to obtain the corresponding polypeptide-oligonucleotide conjugate, named CGBC-1018. The efficacy test results of the purified product are shown in Figure 9 .

[0160] Table 12siRNA sequence design 1

[0161]

[0162] Description of modifications: m = 2'-O-methyl; i2F = 2'-fluoro; * / = phosphorothioate linkage; / = phosphodiester linkage.

[0163] Conjugation protocol 2:

[0164] CGBB-0005 (0.42 mg) was dissolved in pH 7.4 phosphate buffer to a final concentration of 0.21 mg / mL. TCEP (7.64 μL of a 10 mM aqueous solution) and EDTA (20 μL of a 200 mM aqueous solution) were then added to a final concentration of 2 mM. The mixture was incubated at 37°C for 2 h. The reaction was cooled to room temperature, and then 213.94 μL of a 0.357 mM succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC)-modified oligonucleotide (siRNA sequence shown in Table 13, targeting the human DMPK gene) was added to the reaction mixture. The resulting mixture was incubated at room temperature for 1 h. The corresponding polypeptide-oligonucleotide conjugate was obtained by SAX FPLC purification and concentration and exchanged to pH 7.4 phosphate buffer, named CGBC-1022. The degree of conjugation was confirmed to be DAR1 by SAX HPLC method (Table 14), and the purity was above 90% (see test results). Figure 10 ).

[0165] Table 13 siRNA sequence design 2

[0166]

[0167] Description of modifications: m = 2'-O-methyl; i2F = 2'-fluoro; * / = phosphorothioate linkage; / = phosphodiester linkage.

[0168] Table 14 Strong anion exchange chromatography (SAX) method parameters

[0169]

[0170] Example 11. Affinity of the polypeptide-oligonucleotide conjugate for hTfR1

[0171] The purpose of this example is to evaluate whether the polypeptide component in the conjugate of polypeptide and oligonucleotide still retains affinity for the target hTfR1 in the conjugated product.

[0172] Using the affinity determination method adopted in Example 6, the affinity of the original polypeptide to hTfR1 was measured to be 3.7 nM. The affinities of the polypeptide-oligonucleotide conjugates CGBC-1018 and CGBC-1022 synthesized in Example 10 to hTfR1 were 4.2 nM and 2.9 nM, respectively (Table 15). The results indicate that the conjugated nucleic acid has no significant effect on the binding of the polypeptide to the human transferrin receptor (TfR1).

[0173] Table 15 Binding of polypeptide oligonucleotide conjugates to human TfR1

[0174]

[0175]

[0176] Example 12. In vitro activity of conjugates of polypeptides and oligonucleotides

[0177] The purpose of this example was to evaluate whether a peptide-oligonucleotide conjugate could be internalized by hTfR1-overexpressing muscle cells and exert biological activity in vitro without the aid of lipofectamine. The peptide-oligonucleotide conjugate used in the experiment was the same as that used in Example 11, and the specific experimental steps were as follows.

[0178] RD human rhabdomyosarcoma cell line (4201HUM-CCTCC00295) was cultured in DMEM (Gibco) containing 10% fetal bovine serum (Gibco). Antibody-siRNA conjugates (CGBC-1018 and CGBC-1022) were diluted to a maximum dose of 10 mM. The conjugates were transfected at final siRNA concentrations of 200, 100, 10, 1, and 0.1 nM. Cells were seeded onto 48-well culture plates 24 h before administration, with 10,000 cells per well. The conjugates were added to the wells of the 48-well plates. PBS was added to some wells as an additional negative control. The cells were placed at 37°C and 5% CO2 for 72 or 96 hours. The culture medium was removed from the wells and 150 mL of Trizol (Life) was added. The plates were frozen at -80°C overnight or longer before analysis. RNA was isolated using the Direct-zol-96 RNA kit (Zymo Research) according to the manufacturer's instructions. RNA was reverse transcribed into cDNA using the ReverTraAce™ qPCR RT MasterMix (TOYOBO) according to the manufacturer's instructions, and cDNA samples were evaluated by qPCR using TaqMan human gene expression probes (ThermoFisher). % mRNA was calculated using the standard 2-ΔΔCT method, with PBS-treated cells set to 100% expression. All experiments were performed in triplicate. Primer information is shown in Table 16.

[0179] Table 16 Human DMPK primer information

[0180] Primers Sequence (5'-3') SEQ ID NO: Human DMPK-F1 CACTGTCGGACATTCGGGAAGGTGC 103 Human DMPK-R1 GCTTGCACGTGTGGCTCAAGCAGCTG 104 human DMPK-F2 CCTATCGTTGGTTCGCAAAGT 105 Human DMPK-R2 CAAAAGCAAATTTCCCGAGTAA 106 Human GADPH-F ATGGGGAAGGTGAAGGTCG 107 human GADPH-R GGGGTCATTGATGGCAACAATA 108

[0181] The results of in vitro activity studies showed that CGBC-1018 can enter the RD cell line under in vitro co-incubation conditions without the assistance of lipofectamine and exert a dose-dependent target gene knockdown effect. Figure 11 A) or 96 hours ( Figure 11B) had no significant effect on the experimental results. In terms of dosage, the peptide-siRNA conjugate CGBC-1018 needed to be at a concentration greater than 100 nM to exert a significant knockdown effect. The maximum knockdown efficiency of the peptide-siRNA conjugate was approximately 30% (results shown in Figure 11 A and Figure 11 B).

[0182] The same detection operation was performed on CGBC-1022, and the results were similar to those above.

[0183] Example 13. In vivo studies of conjugates of polypeptides and oligonucleotides

[0184] The purpose of this example is to evaluate the ability of a conjugate of a polypeptide and an oligonucleotide to deliver the oligonucleotide across the blood-brain barrier in hTfR1 transgenic mice. The experimental method is described as follows.

[0185] The polypeptide sequence, nucleic acid sequence, and coupling process used in the experiment are exactly the same as those of CGBC-1018 in Example 10. Due to different batches and production volumes, it was renamed CGBC-1021. CGBN-0066 is the siRNA with the same sequence in the conjugate. Its sequence is identical to that of SEQ ID NO: 97-98 except for the linker modification. For the first group of mice, the polypeptide and oligonucleotide conjugate was administered; for the second group of mice, the oligonucleotide was administered. The nucleotide sequences of the two groups were the same, and hTfR1 transgenic mice were used. The dosing schedule is shown in Table 17. The dosing interval was 12 hours, and all mice in Groups 1 and 2 were injected via the tail vein. Blood was collected from the mice in the conjugate group 15 minutes, 1 hour, 4 hours, 8 hours, and 24 hours (after the third dose) after the first dose, with two mice collected at each time point; and all mice were finally blood-collected at the end of the experiment. Blood samples were collected in EDTA-K2 coated anticoagulant tubes and centrifuged at 7,000g, 4°C for 10 minutes to prepare approximately 20 μL of plasma samples for SL-RT-PCR (detection of siRNA antisense strands) to quantitatively detect the RNA concentration in plasma. The samples were temporarily stored in a -80°C refrigerator, transported on dry ice, and sent to the in vitro laboratory of the Department of Biology for testing. After the mice were euthanized, tissue samples were collected and transferred to the in vitro laboratory of the Department of Biology on dry ice. SL-PCR was used to detect the siRNA antisense strand and quantitatively detect the RNA concentration in the brain, cervical spinal cord, and plasma. The test results are shown in Figure 12 A and 12B. Figure 12A shows the concentration test results of CGBC-1021 and CGBN-0066 in tissues. The results showed that 51 hours after injection, the concentration of CGBC-1021 in brain tissue was 97.23 pmol / g, the concentration in spinal cord tissue was 71.43 pmol / g, and the concentration of CGBN-1066 in brain tissue was 53.57 pmol / g, and the concentration in spinal cord tissue was 57.83 pmol / g. Figure 12 B shows the concentration test results of CGBC-1021 in plasma. The plasma concentration reaches the maximum 15 minutes after injection, and then gradually decreases. After 24 hours, the concentration returns to the maximum, and then gradually decreases again.

[0186] Table 17 In vivo studies of conjugates of polypeptides and oligonucleotides

[0187]

[0188] The experimental results show that the polypeptide of the present application can achieve delivery of oligonucleotides across the blood-brain barrier in hTfR1 transgenic mice.

[0189] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements and improvements made within the principles of the present application should be included in the scope of protection of the present application.

Claims

1. A polypeptide that specifically binds to transferrin receptor, comprising a structure represented by the general formula H1-H2-E1-E2-H3-H4-E3, wherein: Said H1, H2, H3 and H4 each independently comprise an α-helical domain with a length between 11 and 20 amino acids; Each of the E1, E2 and E3 independently comprises a β-pleated domain with a length of 4-6 amino acids; There are one or more amino acid linkers between any two adjacent α-helical domains and / or β-pleated domains; The polypeptide binds to hTfR1 and simultaneously allows transferrin to bind to hTfR1.

2. The polypeptide according to claim 1, wherein The polypeptide binds to the amino acid sequence shown in SEQ ID NO:

95. Preferably, the polypeptide binds to the amino acid sequence at positions 1-90.

3. The polypeptide according to claim 1, wherein At least three amino acids in each of E1, E2 and E3 are hydrophobic.

4. The polypeptide according to claim 1, wherein The polypeptide comprises the amino acid sequence SEQ ID NO: 133, which is located at any position of E1, E2 or E3, wherein the amino acid V at positions 2 and 4 can be replaced by I, and the first D residue and the last T residue can be deleted.

5. The polypeptide according to claim 1, wherein It comprises an amino acid sequence as shown in SEQ ID NO: 46, 47, 48, 49, 55, 84 or 85, which is located at any position of E1, E2 or E3.

6. The polypeptide according to claim 1, wherein The E1 domain comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NOs: 46-55, wherein the amino acid substitutions are conservative amino acid substitutions; and / or the E2 domain comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NOs: 56-69, wherein the amino acid substitutions are conservative amino acid substitutions; and / or the E3 domain comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence of SEQ ID NOs: 70-85, wherein the amino acid substitutions are conservative amino acid substitutions.

7. The polypeptide according to claim 1, wherein The polypeptide has an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2-45.

8. The polypeptide according to claim 1, wherein The polypeptide has an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2, 5, 6, 8, 9 and 12.

9. The polypeptide according to claim 1, wherein The polypeptide further comprises one or more additional functional domains, wherein the additional functional domains include the following amino acid fragments: the sequence of SEQ ID NO: 109, the sequence of SEQ ID NO: 110, (GS)n, (GS)nC, (SG)n, (SG)nC, the sequence of SEQ ID NO: 111, the sequence of SEQ ID NO: 112, the sequence of SEQ ID NO: 113, the sequence of SEQ ID NO: 114, the sequence of SEQ ID NO: 115, the sequence of SEQ ID NO: 116, the constant region of an immunoglobulin heavy chain, glutathione S-transferase, green fluorescent protein, maltose binding protein, a ubiquitination tag, a Trx tag, the sequence of SEQ ID NO: 117, the sequence of SEQ ID NO: 118, the sequence of SEQ ID NO: 119, the sequence of SEQ ID NO: 120, the sequence of SEQ ID NO: 121, the sequence of SEQ ID NO: 122, the sequence of SEQ ID NO: 123, the sequence of SEQ ID NO: 124, the sequence of SEQ ID NO: 125, the sequence of SEQ ID NO: 126 The sequence shown in SEQ ID NO: 125, the sequence shown in SEQ ID NO: 126, the sequence shown in SEQ ID NO: 127, the sequence shown in SEQ ID NO: 128, the sequence shown in SEQ ID NO: 129, and the sequence shown in SEQ ID NO: 130, wherein n is any integer between 1 and 15.

10. The polypeptide according to claim 1, wherein The polypeptide binds to human TfR1 with an affinity of at least 3 micromolar, more preferably at least 1 micromolar, more preferably at least 500 nanomolar, more preferably at least 250 nanomolar, more preferably at least 100 nanomolar, more preferably at least 50 nanomolar, more preferably at least 5 nanomolar, more preferably at least 1 nanomolar, and most preferably at least 0.1 nanomolar.

11. The polypeptide according to claim 9, wherein The functional domain is present at the N-terminus and / or C-terminus of the polypeptide and is linked to the polypeptide via a peptide bond or cysteine.

12. The polypeptide according to claim 9, wherein The functional domains also include detection domains, stabilization domains, therapeutic moieties, diagnostic moieties, and drug delivery vehicles.

13. The polypeptide according to claim 12, wherein The stabilizing domain includes polyethylene glycol, albumin, lipids, hydroxyethyl starch, a conformationally disordered polypeptide sequence consisting of amino acids Pro, Ala and / or Ser, and / or a mucin diffusible polypeptide consisting of amino acids Lys and Ala with or without Glu.

14. The polypeptide according to claim 1, wherein The polypeptide does not bind to human transferrin receptor 2.

15. A recombinant nucleic acid encoding the polypeptide of any one of claims 1 to 14.

16. An expression vector comprising the recombinant nucleic acid of claim 15 operably linked to a promoter.

17. A recombinant host cell comprising the polypeptide according to any one of claims 1 to 14, and / or the recombinant nucleic acid according to claim 15, and / or the expression vector according to claim 16.

18. A conjugate of a polypeptide and an oligonucleotide, comprising an oligonucleotide and the polypeptide according to any one of claims 1 to 14; the oligonucleotide is a small interfering RNA, an antisense oligonucleotide, or a phosphoramidite morpholino oligomer, the oligonucleotide is linked to the polypeptide by chemical covalent conjugation, and is used to regulate gene expression.

19. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 14 or the conjugate of the polypeptide and oligonucleotide according to claim 18, the recombinant nucleic acid according to claim 15, the expression vector according to claim 16 and / or the recombinant host cell according to claim 17.

20. The pharmaceutical composition according to claim 19, wherein The polypeptide is covalently linked to a therapeutic agent, which includes a protein, nucleic acid, lipid, PEG, or chemical compound.

21. The pharmaceutical composition according to claim 19, wherein Its target tissues include skeletal muscle, myocardium, diaphragm, and central nervous system.

22. The pharmaceutical composition according to claim 19, wherein Its delivery to the central nervous system involves crossing the blood-brain barrier via receptor-mediated endocytosis.

23. The pharmaceutical composition according to claim 19, wherein The pharmaceutical composition is used to treat or limit arenavirus infection, treat diseases related to gene mutation, treat central nervous system related diseases, treat tumors, or improve the serum half-life of biological products during treatment.

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