Tfr1 binding protein and use thereof

Abstract

A TfR1 binding protein and a use thereof. The TfR1 binding protein comprises an immunoglobulin single variable domain, and the immunoglobulin single variable domain comprises HCDR1, HCDR2, and HCDR3 contained in the amino acid sequence as shown in any one of SEQ ID NOs: 1-74.

Description

TfR1 binding protein and its application Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a TfR1 binding protein and its applications. Background Technology

[0002] The blood-brain barrier (BBB) ​​is a special protective barrier that exists between capillaries and brain tissue. It selectively prevents certain substances from entering the brain from the blood, protecting brain tissue from harmful substances and pathogens in the blood, while ensuring that brain tissue receives the necessary nutrients and oxygen.

[0003] Neurological diseases are a class of illnesses affecting the function of the nervous system, including diseases of the central and peripheral nervous systems. They can be classified into various types, including neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and multiple sclerosis), cerebrovascular diseases (such as stroke and cerebral hemorrhage), infectious diseases (such as meningitis and encephalitis), and immune-mediated diseases (such as multiple sclerosis). Currently, the number of people suffering from neurological diseases globally is enormous. For example, according to WHO statistics, as of 2022, approximately 50 to 55 million people worldwide had Alzheimer's disease (AD), and as of 2019, approximately 8.5 million people worldwide had Parkinson's disease. However, the prospects for drugs to treat neurological diseases are not optimistic. Although many drugs with therapeutic activity have been discovered, they are difficult to translate into effective clinical outcomes because they cannot cross the blood-brain barrier.

[0004] Transferrin receptor 1 (TfR1), also known as differentiation cluster 71 (CD71), is a transmembrane glycoprotein primarily responsible for intracellular iron uptake. TfR1 binds to iron-binding transferrin (Tf) to form an iron-Tf-TfR1 complex, which then transports iron into the cell via endocytosis. As an essential receptor for cellular iron uptake, TfR1 is highly expressed in brain microvascular endothelial cells, and it can transport circulating iron across the blood-brain barrier to the brain via endocytosis, maintaining normal brain function. Therefore, TfR1 can serve as a potential target for drugs to cross the blood-brain barrier. However, the structural form (e.g., different binding valence states / conformations) and affinity of TfR1-binding proteins should facilitate receptor-mediated transendothelial transport: they should be able to efficiently bind to TfR1 on the surface of brain capillary endothelial cells on the blood side and trigger endocytosis, while avoiding excessive retention or lysosomal degradation during intracellular sorting and transport, and subsequently be released from the brain side into the brain parenchyma via exocytosis, thereby achieving the crossing of the blood-brain barrier. Therefore, developing TfR1-binding proteins with both suitable structural form and appropriate affinity is of great significance. Summary of the Invention

[0005] This disclosure provides a TfR1 binding protein comprising an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises HCDR1, HCDR2 and HCDR3 contained in any one of the amino acid sequences shown in SEQ ID NO:1 to 74.

[0006] In some embodiments, the single variable domain of the immunoglobulin is a VHH domain.

[0007] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:75, SEQ ID NO:76 and SEQ ID NO:77 or are composed of the amino acid sequences shown in SEQ ID NO:75, SEQ ID NO:76 and SEQ ID NO:77 respectively;

[0008] The HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80, or are composed of the amino acid sequences shown in SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80, respectively;

[0009] The HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:81, SEQ ID NO:82, and SEQ ID NO:83, or are composed of the amino acid sequences shown in SEQ ID NO:81, SEQ ID NO:82, and SEQ ID NO:83, respectively;

[0010] The HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:84, SEQ ID NO:85, and SEQ ID NO:86, or are composed of the amino acid sequences shown in SEQ ID NO:84, SEQ ID NO:85, and SEQ ID NO:86, respectively;

[0011] The HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89, or are composed of the amino acid sequences shown in SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89, respectively;

[0012] The HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:90, SEQ ID NO:91, and SEQ ID NO:92, or are composed of the amino acid sequences shown in SEQ ID NO:90, SEQ ID NO:91, and SEQ ID NO:92, respectively;

[0013] The HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:93, SEQ ID NO:94, and SEQ ID NO:95, or are composed of the amino acid sequences shown in SEQ ID NO:93, SEQ ID NO:94, and SEQ ID NO:95, respectively;

[0014] The HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98, or are composed of the amino acid sequences shown in SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98, respectively;

[0015] The HCDR1, HCDR2 and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:99, SEQ ID NO:100 and SEQ ID NO:101 or are composed of the amino acid sequences shown in SEQ ID NO:99, SEQ ID NO:100 and SEQ ID NO:101 respectively;

[0016] The HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:102, SEQ ID NO:103, and SEQ ID NO:104 or are composed of the amino acid sequences shown in SEQ ID NO:102, SEQ ID NO:103, and SEQ ID NO:104 respectively;

[0017] The HCDR1, HCDR2 and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:105, SEQ ID NO:106 and SEQ ID NO:107 or are composed of the amino acid sequences shown in SEQ ID NO:105, SEQ ID NO:106 and SEQ ID NO:107 respectively;

[0018] The HCDR1, HCDR2 and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:108, SEQ ID NO:109 and SEQ ID NO:110 or are composed of the amino acid sequences shown in SEQ ID NO:108, SEQ ID NO:109 and SEQ ID NO:110 respectively;

[0019] The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:111, SEQ ID NO:112, and SEQ ID NO:113 or are composed of the amino acid sequences shown in SEQ ID NO:111, SEQ ID NO:112, and SEQ ID NO:113 respectively;

[0020] The HCDR1, HCDR2, and HCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO:114, SEQ ID NO:115, and SEQ ID NO:116;

[0021] The HCDR1, HCDR2 and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:117, SEQ ID NO:118 and SEQ ID NO:119 or are composed of the amino acid sequences shown in SEQ ID NO:117, SEQ ID NO:118 and SEQ ID NO:119 respectively;

[0022] The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:120, SEQ ID NO:121, and SEQ ID NO:122 or are composed of the amino acid sequences shown in SEQ ID NO:120, SEQ ID NO:121, and SEQ ID NO:122 respectively;

[0023] The HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:123, SEQ ID NO:124, and SEQ ID NO:125 or are composed of the amino acid sequences shown in SEQ ID NO:123, SEQ ID NO:124, and SEQ ID NO:125 respectively;

[0024] The HCDR1, HCDR2 and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:126, SEQ ID NO:127 and SEQ ID NO:128 or are composed of the amino acid sequences shown in SEQ ID NO:126, SEQ ID NO:127 and SEQ ID NO:128 respectively;

[0025] The HCDR1, HCDR2 and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:129, SEQ ID NO:130 and SEQ ID NO:131 or are composed of the amino acid sequences shown in SEQ ID NO:129, SEQ ID NO:130 and SEQ ID NO:131 respectively;

[0026] The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:132, SEQ ID NO:133, and SEQ ID NO:134 or are composed of the amino acid sequences shown in SEQ ID NO:132, SEQ ID NO:133, and SEQ ID NO:134 respectively;

[0027] The HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:135, SEQ ID NO:136, and SEQ ID NO:137 or are composed of the amino acid sequences shown in SEQ ID NO:135, SEQ ID NO:136, and SEQ ID NO:137 respectively;

[0028] The HCDR1, HCDR2 and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:138, SEQ ID NO:139 and SEQ ID NO:140 or are composed of the amino acid sequences shown in SEQ ID NO:138, SEQ ID NO:139 and SEQ ID NO:140 respectively;

[0029] The HCDR1 contains the amino acid sequence shown in SEQ ID NO:87, the HCDR2 contains the amino acid sequence shown in SEQ ID NO:88, and the HCDR3 contains the amino acid sequence shown in one of SEQ ID NO:141 to 159.

[0030] The HCDR1 is composed of the amino acid sequence shown in SEQ ID NO:87, the HCDR2 is composed of the amino acid sequence shown in SEQ ID NO:88, and the HCDR3 is composed of the amino acid sequence shown in one of SEQ ID NO:141 to 159.

[0031] The HCDR1 contains the amino acid sequence shown in SEQ ID NO:99, the HCDR2 contains the amino acid sequence shown in SEQ ID NO:100, and the HCDR3 contains one of the amino acid sequences shown in SEQ ID NO:160-178; or

[0032] The HCDR1 consists of the amino acid sequence shown in SEQ ID NO:99, the HCDR2 consists of the amino acid sequence shown in SEQ ID NO:100, and the HCDR3 consists of the amino acid sequence shown in one of SEQ ID NO:160 to 178.

[0033] In some embodiments, the immunoglobulin single variable domain comprises:

[0034] (1) An amino acid sequence as shown in any one of SEQ ID NO:1 to 74; or

[0035] (2) An amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with any of the amino acid sequences shown in SEQ ID NO:1 to 74.

[0036] In some implementations, the immunoglobulin's single variable domain is humanized.

[0037] In some implementations, the TfR1 binding protein also comprises human Ig Fc.

[0038] In some embodiments, the TfR1 binding protein further comprises human Ig Fc and human Ig hinge region.

[0039] In some implementations, the human Ig is human IgG1, human IgG2, human IgG3, or human IgG4.

[0040] In some embodiments, the amino acid sequence of the human Ig G4 hinge region is shown in SEQ ID NO:179.

[0041] In some embodiments, the human IgG4 Fc comprises a Ks chain and an Hs chain, the amino acid sequences of which are shown in SEQ ID NO:180 and 181, respectively.

[0042] In some embodiments, the amino acid sequence of the human Ig G1 hinge region is shown in SEQ ID NO:182.

[0043] In some embodiments, the amino acid sequence of the human IgG1 Fc is shown in SEQ ID NO:183.

[0044] In some embodiments, the human Ig Fc contains a mutation that prolongs the half-life of the TfR1 binding protein.

[0045] In some implementations, the mutations are M252Y, S254T, and T256E, or the mutations are M428L and N434S.

[0046] In some embodiments, the TfR1 binding protein does not inhibit or weakly inhibits the binding of TfR1 to transferrin.

[0047] In some embodiments, the K of the TfR1-binding protein D No more than 1×10 -6 mol / L;

[0048] In some embodiments, the EC50 of the TfR1 binding protein for TfR1 is less than 500 nmol / L.

[0049] In some embodiments, an mRNA comprises a polynucleotide encoding a fusion protein, the fusion protein containing an active molecule and a transport molecule capable of binding to a blood-brain barrier receptor to transport the active molecule across the blood-brain barrier.

[0050] In some embodiments, the blood-brain barrier receptor is selected from one or more of the following: transferrin receptor, insulin receptor, insulin-like growth factor receptor, low-density lipoprotein receptor-associated protein 8, low-density lipoprotein receptor-associated protein 1, glucose transporter 1, and heparin-binding epidermal growth factor-like growth factor.

[0051] In some embodiments, the transferrin receptor is human TfR1.

[0052] In some embodiments, the transport molecule is an antibody or its antigen-binding fragment.

[0053] In some embodiments, the transport molecule is an antibody that binds to human TfR1 or an antigen-binding fragment thereof.

[0054] In some embodiments, the transport molecule comprises the TfR1 binding protein described above.

[0055] In some embodiments, the active molecule is a contrast agent or is used to prevent or treat neurological diseases.

[0056] In some embodiments, the neurological disease is one or more of the following: Alzheimer's disease, stroke, dementia, muscular dystrophy, multiple sclerosis, amyotrophic lateral sclerosis, cystic fibrosis, Angelman syndrome, Liddell syndrome, Parkinson's disease, Pick's disease, Paget's disease, neurological cancers, and traumatic brain injury.

[0057] In some embodiments, the active molecule is capable of binding to a brain antigen selected from the group consisting of: β-secretase 1, Aβ, epidermal growth factor receptor, human epidermal growth factor receptor 2, tau, aliphatic apolipoprotein E4, α-synuclein, CD20, huntingtin, prion protein, leucine-rich repeat kinase 2, perkinin, progerin 1, progerin 2, γ-secretase, death receptor 6, amyloid precursor protein, p75 neurotrophic protein receptor, and caspase 6.

[0058] In some embodiments, the active molecule is an antibody that specifically binds to Aβ or its antigen-binding fragment.

[0059] In some embodiments, the active molecule is iduxose-2-sulfatase (IDS).

[0060] In some embodiments, the fusion protein does not inhibit or weakly inhibits the binding of the blood-brain barrier receptor to one or more of its natural ligands.

[0061] In some embodiments, the mRNA further comprises at least one of a 5'-cap structure, a 5'-UTR, a 3'-UTR, and a poly(A) tail.

[0062] In some implementations, the mRNA contains a modified nucleoside;

[0063] Preferably, the modified nucleoside includes at least one of modified uridine, modified cytidine, modified adenosine, and modified guanosine.

[0064] This disclosure also provides a fusion protein derived from the translation of the above-described mRNA.

[0065] This disclosure also provides a multispecific antibody comprising the aforementioned TfR1 binding protein.

[0066] This disclosure also provides a conjugate comprising the above-described TfR1 binding protein and an active molecule conjugated thereto, or the above-described multispecific antibody and an active molecule conjugated thereto.

[0067] This disclosure also provides a chimeric antigen receptor (CAR) comprising the TfR1 binding protein described above.

[0068] This disclosure also provides a nucleic acid that encodes the aforementioned TfR1 binding protein, the aforementioned multispecific antibody, or the aforementioned chimeric antigen receptor.

[0069] In some implementations, the nucleic acid is mRNA.

[0070] In some implementations, the nucleic acid is DNA.

[0071] This disclosure also provides a DNA that can be transcribed into the above-described mRNA.

[0072] This disclosure also provides a genetic engineering vector containing the above-described nucleic acid or the above-described DNA.

[0073] This disclosure also provides a host cell comprising the above-described nucleic acid, the above-described DNA, or the above-described genetic engineering vector.

[0074] This disclosure also provides an immune cell that expresses the chimeric antigen receptor described above.

[0075] This disclosure also provides a pharmaceutical composition comprising the above-described TfR1 binding protein, the above-described mRNA, the above-described fusion protein, the above-described multispecific antibody, the above-described conjugate, the above-described chimeric antigen receptor, the above-described nucleic acid, the above-described DNA, the above-described genetically engineered vector, or the above-described immune cell.

[0076] In some implementations, the mRNA is formulated in lipid nanoparticles.

[0077] In some embodiments, the lipid nanoparticles comprise one or more of the following: ionizable lipids, auxiliary lipids, structural lipids, and polymer-lipids.

[0078] In some embodiments, ionizable lipids account for 20 mol% to 75 mol% of the total lipids present in LNPs, accessory lipids account for 0 mol% to 45 mol% of the total lipids present in LNPs, structural lipids account for 0 mol% to 60 mol% of the total lipids present in LNPs, and polymer-lipids account for 0.5 mol% to 15 mol% of the total lipids present in LNPs.

[0079] This disclosure also provides the use of the above-mentioned TfR1 binding protein, the above-mentioned mRNA, the above-mentioned fusion protein, the above-mentioned multispecific antibody, the above-mentioned conjugate, the above-mentioned chimeric antigen receptor, the above-mentioned nucleic acid, the above-mentioned DNA, the above-mentioned genetic engineering vector, or the above-mentioned immune cell, the above-mentioned host cell, or the above-mentioned pharmaceutical composition in the preparation of a medicament for the prevention or treatment of TfR1-related diseases.

[0080] In some implementations, the drug is used to treat tumors or cancers with high TfR1 expression.

[0081] This disclosure also provides the use of the above-mentioned TfR1 binding protein, the above-mentioned mRNA, the above-mentioned fusion protein, the above-mentioned multispecific antibody, the above-mentioned conjugate, the above-mentioned chimeric antigen receptor, the above-mentioned nucleic acid, the above-mentioned DNA, the above-mentioned genetic engineering vector, or the above-mentioned immune cell, the above-mentioned host cell, or the above-mentioned pharmaceutical composition in the preparation of a drug capable of crossing the blood-brain barrier.

[0082] In some implementations, the drug is used to treat neurological disorders. Attached Figure Description

[0083] Figures 1A to 1I show the competitive ELISA detection of the VHH-Fc fusion protein and holo-Tf competing for hTfR1 binding. In Figure 1A, "AT9-1-36 & holo-Tf" means that holo-Tf and AT9-1-36 are added simultaneously during the competitive ELISA detection, and "AT9-1-36" means that only AT9-1-36 is added during the competitive ELISA detection. The results of other competitive ELISAs are similar.

[0084] Figures 2A to 6D show the results of FACS detection of the antigen-binding activity of the VHH-Fc fusion protein;

[0085] Figures 7A to 7C show the results of FACS detection of antigen-binding activity of fusion proteins fused with IDS;

[0086] Figure 8A shows the results of the metabolic kinetics study of the VHH-Fc fusion protein;

[0087] Figures 8B and 8C show the content of VHH-Fc fusion protein in the whole brain and brain parenchyma 24 hours after injection.

[0088] Figures 9A to 9C show the antigen-binding activity results of the humanized modified hTfR1 antibody detected by FACS.

[0089] Figures 10A-10C show the competitive ELISA results of the humanized hTfR1-binding antibody and holo-Tf competing for hTfR1 binding.

[0090] Figure 11A shows the results of a metabolic kinetic study of the humanized modified hTfR1-binding antibody;

[0091] Figures 11B-11C show the content of the humanized modified hTfR1-binding antibody in the whole brain and brain parenchyma 24 hours after injection.

[0092] Figure 12 shows the results of the metabolic kinetics study of the antibody with extended half-life;

[0093] Figure 13A shows the metabolic kinetics of the humanized modified hTfR1-binding antibody and its corresponding mRNA-LNP.

[0094] Figures 13B-13C show the content of the humanized modified hTfR1-binding antibody and its corresponding mRNA-LNP in the whole brain and brain parenchyma.

[0095] Figure 14A shows the results of a metabolic kinetic study of the fusion protein of the therapeutic antibody and the antibody that binds to hTfR1;

[0096] Figures 14B and 14C show the content of the fusion protein of the therapeutic antibody and the antibody binding to hTfR1 in the whole brain and brain parenchyma 48 hours after injection.

[0097] Figure 15A shows the results of the metabolic kinetics study of Lec*hu12 mRNA-LNP;

[0098] Figures 15B to 15C show the content of the fusion protein of Lecanemab and VHH bound to hTfR1 in the whole brain and brain parenchyma after injection of Lec*hu12 mRNA-LNP.

[0099] Invention Details

[0100] I. Definition

[0101] All patents, patent applications, scientific publications, manufacturers' specifications and guidelines, etc., cited herein, are incorporated herein in their entirety, whether mentioned above or below. Nothing herein should be construed as an admission that this disclosure is not entitled to precede such disclosure.

[0102] Unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, and microbiology used herein are all widely used terms in their respective fields (see, for example, *Molecular Cloning: A Laboratory Manual, 2nd Edition*, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). To better understand this application, definitions and explanations of relevant terms are provided below.

[0103] As used herein, the expressions “comprising,” “including,” “containing,” and “having” are open-ended, meaning they include the listed elements, steps, or components but do not exclude other unlisted elements, steps, or components. The expression “composed of” excludes any unspecified elements, steps, or components. The expression “substantially composed of” means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the essential and novel nature of the claimed subject matter. It should be understood that the expressions “substantially composed of” and “composed of” are encompassed within the meaning of the expression “including.”

[0104] As used herein, unless the context otherwise indicates, the singular forms of “a,” “an,” and “the,” and similar references used in the context of describing this application (particularly in the context of the claims) should be interpreted as encompassing both the singular and plural. The terms “one or more” or “at least one” cover 1, 2, 3, 4, 5, 6, 7, 8, 9, or more.

[0105] The numerical ranges described herein should be understood to encompass any and all subranges contained therein. For example, the range “1 to 10” should be understood to include not only the explicitly stated values ​​of 1 and 10, but also any single value within the range of 1 to 10 (e.g., 2, 3, 4, 5, 6, 7, 8, and 9) and subranges (e.g., 1 to 2, 1.5 to 2.5, 1 to 3, 1.5 to 3.5, 2.5 to 4, 3 to 4.5, etc.). This principle also applies to ranges that use only one numerical value as their minimum or maximum value.

[0106] As used herein, the terms “and / or,” “any combination thereof,” and their grammatical equivalents are used interchangeably. These terms can express any combination specifically. For example, the phrases “A, B, and / or C” or “A, B, C, or any combination thereof” can refer to “A alone; B alone; C alone; A and B; B and C; A and C; and A, B, and C.”

[0107] Unless otherwise stated, all methods described herein may be performed in any suitable order.

[0108] As used herein, the term "wildtype" means that the sequence is naturally occurring and has not been artificially modified, including naturally occurring mutants.

[0109] As used herein, the term "% identity" or "% similarity" refers to the percentage of identical nucleotides or amino acids in the best alignment between sequences to be compared. Differences between the two sequences can be distributed across local regions (segments) or the entire length of the sequences being compared. Identity between two sequences is typically determined after the best alignment of a segment or "comparison window." The best alignment can be performed manually or using algorithms known in the art. Algorithms known in the art include, but are not limited to, the local homology algorithms described in Smith and Waterman, 1981, Ads App. Math. 2, 482 and Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443; the similarity search methods described in Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444; or the use of computer programs such as GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. For example, the percentage similarity between two sequences can be determined using the BLASTN or BLASTP algorithms publicly available on the National Center for Biotechnology Information (NCBI) website.

[0110] "% identity" or "% similarity" can be obtained by determining the number of identical positions corresponding to the sequences to be compared, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying the result by 100. In some embodiments, a degree of similarity is given for at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the region. In some embodiments, a degree of similarity is given for the entire length of the reference sequence. Alignment for determining sequence similarity can be performed using tools known in the art, preferably using optimal sequence alignment, such as Align, using standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, or Gap Extend 0.5.

[0111] As used herein, "nucleotide" includes deoxyribonucleotides, deoxyribonucleotides, deoxyribonucleotide derivatives, and ribonucleotide derivatives. As used herein, "ribonucleotide" is a constituent of ribonucleic acid (RNA), consisting of one base, one pentose sugar, and one phosphate molecule; it refers to a nucleotide with a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. "Deoxyribonucleotide" is a constituent of deoxyribonucleic acid (DNA), also consisting of one base, one pentose sugar, and one phosphate molecule; it refers to a nucleotide where the hydroxyl group at the 2' position of the β-D-ribofuranosyl group is replaced by hydrogen, and is a major chemical component of chromosomes.

[0112] Nucleotides are usually identified by a single letter representing the bases in them. "A" or "A nucleotide" refers to adenine deoxyribonucleotide or adenine ribonucleotide containing adenine; "C" or "C nucleotide" refers to cytosine deoxyribonucleotide or cytosine ribonucleotide containing cytosine; "G" or "G nucleotide" refers to guanine deoxyribonucleotide or guanine ribonucleotide containing guanine; "U" or "U nucleotide" refers to uracil ribonucleotide containing uracil; and "T" or "T nucleotide" refers to thymine deoxyribonucleotide containing thymine.

[0113] As used herein, the term "nucleic acid" generally refers to any compound comprising a polymer of deoxyribonucleotides (deoxyribonucleic acid, or DNA) or a polymer of ribonucleotides (ribonucleic acid, or RNA), or a combination thereof. Additionally, "nucleic acid" as used herein also includes derivatives of nucleic acids. The term "derivatives of nucleic acids" includes chemical derivatization of nucleic acids at the bases, sugars, or phosphates of nucleotides, as well as nucleic acids containing non-natural nucleotides and nucleotide analogs. Furthermore, in this document, nucleic acids can be in the form of single-stranded or double-stranded linear or covalently closed circular molecules.

[0114] The terms "polynucleotide sequence," "nucleic acid sequence," and "nucleotide sequence" are used interchangeably to refer to the sequence of nucleotides in a polynucleotide. Those skilled in the art should understand that the DNA coding strand (sense strand) and its encoded RNA can be considered to have the same nucleotide sequence, and the deoxythymidine nucleotide in the DNA coding strand sequence corresponds to the uridine nucleotide in its encoded RNA sequence. The RNA corresponding to DNA refers to a polynucleotide in DNA where all T atoms are replaced with U atoms.

[0115] A polynucleotide may contain one or more segments (nucleic acid fragments) (e.g., segments 1, 2, 3, 4, 5, 6, 7, and 8). For example, a polynucleotide may contain a segment encoding a polypeptide of interest. In a particular embodiment, a polynucleotide may contain a segment encoding a polypeptide of interest as well as a regulatory segment (including, but not limited to, segments for transcriptional and translational regulation). In one embodiment, the regulatory segment comprises a polynucleotide corresponding to one or more of the following regulatory elements: a promoter, a 5' untranslated region (5'-UTR), a 3' untranslated region (3'-UTR), and a poly(A) tail.

[0116] As used herein, the term "promoter" refers to a polynucleotide located upstream of the 5' end of the coding region of a gene. It contains a conserved sequence required for the specific binding of RNA polymerase and transcription initiation, activates RNA polymerase, and enables precise binding of RNA polymerase to the template DNA, thus achieving transcription initiation specificity. Promoters can originate from viruses, bacteria, fungi, plants, insects, and animals. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operon-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter, or SV40 late promoter and CMV IE promoter.

[0117] As used herein, the term "5' untranslated region" or "5'-UTR" can refer to an RNA sequence in mRNA that is upstream of the coding sequence and is not translated into protein. A 5'-UTR in a gene typically begins at the transcription start site and ends with a nucleotide upstream of the translation start codon in the coding sequence. The 5'-UTR can contain elements that control gene expression, such as ribosome binding sites, 5'-terminal oligopyrimidine bundles, and translation initiation signals such as the Kozak sequence. mRNA can undergo post-transcriptional modification by adding a 5' cap. Therefore, the 5'-UTR in mature mRNA can also refer to the RNA sequence between the 5' cap and the start codon.

[0118] As used herein, the term "3' untranslated region" or "3'-UTR" can refer to an RNA sequence in mRNA that is downstream of the coding sequence and is not translated into a protein. The 3'-UTR in mRNA is located between a stop codon and a poly(A) sequence of the coding sequence, for example, starting from a nucleotide downstream of the stop codon and ending at a nucleotide upstream of the poly(A) sequence.

[0119] As used herein, the terms “poly(A) nucleotide,” “poly(A) sequence,” and “poly(A) tail” are used interchangeably. Naturally occurring poly(A) sequences typically consist of adenine ribonucleotides. According to this application, the term “modified poly(A) sequence” refers to a poly(A) sequence containing nucleotides or nucleotide segments other than adenine ribonucleotides. Poly(A) sequences are typically located at the 3' end of mRNA, such as the 3' end (downstream) of the 3'-UTR.

[0120] As used herein, the term "5'-cap structure" refers to a structure that is typically located at the 5' end of mature mRNA. In some embodiments, the 5'-cap structure is linked to the 5' end of the mRNA via a 5'-5'-triphosphate bond. 5'-cap structures are typically formed from modified (e.g., methylated) ribonucleotides, particularly guanine nucleotide derivatives. For example, m7GpppN (cap0, or "cap0") is a cap structure formed by the interaction of the 5' phosphate group of hnRNA with the 5' phosphate group of m7GTP via guanylate transferase to form a 5',5'-phosphodiester bond, where N is the terminal 5' nucleotide of the nucleic acid carrying the 5'-cap structure. In some embodiments, the 5'-cap structure includes, but is not limited to, cap 0, cap 1 (a cap structure formed by further methylation of the 2'-OH of the first nucleotide glycosyl group of hnRNA on the basis of cap 0, or "cap1"), cap 2 (a cap structure formed by further methylation of the 2'-OH of the second nucleotide glycosyl group of hnRNA on the basis of cap 1, or "cap2"), cap 4, cap 0 analogue, cap 1 analogue, cap 2 analogue, or cap 4 analogue.

[0121] As used herein, the term “expression” includes the transcription and / or translation of a nucleotide sequence. Therefore, expression can involve the production of transcripts and / or peptides. The term “transcription” refers to the process of transcribing the genetic code in a DNA sequence into RNA (transcription). The term “in vitro transcription” refers to the in vitro synthesis of RNA, particularly mRNA, in a cell-free system (e.g., in a suitable cell extract) (see, e.g., Pardi N., Muramatsu H., Weissman D., Karikó K. (2013). In: Rabinovich P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation. Methods in Molecular Biology (Methods and Protocols), vol 969. Humana Press, Totowa, NJ.). Vectors that can be used to produce transcripts are also called “transcription vectors,” which contain the regulatory sequences required for transcription. The term “transcription” encompasses “in vitro transcription.”

[0122] The term "multispecific antibody" refers to an antibody that has binding specificity to at least two different sites (i.e., different epitopes on different antigens or different epitopes on the same antigen).

[0123] "Immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which can be a heavy chain or light chain domain, including VH, VHH, or VL domains) that can form a functional antigen-binding site without interacting with other variable domains (e.g., without the VH / VL interaction required between the VH and VL domains of a conventional four-chain monoclonal antibody). Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (e.g., VHH domains) are generally preferred. A specific example of an immunoglobulin single variable domain is the "VHH domain" (or simply "VHH") as defined below.

[0124] The “VHH domain,” also known as a heavy chain single-domain antibody, VHH, VHH domain, VHH antibody fragment, VHH antibody, or nanobody, is a variable domain of an antigen-binding immunoglobulin called a “heavy chain antibody” (i.e., “antibody lacking a light chain”) (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature 363, 446-448 (1993)). The term “VHH domain” is used to distinguish the variable domain from the heavy chain variable domain (referred to herein as the “VH domain”) and the light chain variable domain (referred to herein as the “VL domain”) present in conventional tetrapeptide chain antibody structures. The VHH domain specifically binds to epitopes without the need for other antigen-binding domains (unlike the VH or VL domains in conventional tetrapeptide chain antibodies, where the epitope is recognized by both the VL and VH domains). The VHH domain is a small, stable, and highly efficient antigen-recognition unit formed by a single immunoglobulin domain. Terms include "heavy chain single-domain antibody," "VHH domain," "VHH," and "V..." H The terms “VHH domain”, “VHH antibody fragment”, “VHH antibody”, and “nanobody” are used interchangeably. The “VHH domain” includes, but is not limited to, naturally occurring antibodies produced by camelids, antibodies produced by camelids that have been humanized, or antibodies obtained through phage display technology. The total number of amino acid residues in the VHH domain will typically be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described in this disclosure.

[0125] The term "coupling" refers to the association between atoms or molecules. This association can be direct (e.g., through covalent bonds) or indirect (e.g., through non-covalent bonds). Non-covalent bonds include, but are not limited to, electrostatic interactions (e.g., ionic bonds, hydrogen bonds, halogen bonds), van der Waals forces, ring stacking (π effect), and hydrophobic interactions.

[0126] As used herein, the term "host cell" refers to a cell used to receive, maintain, replicate, and express polynucleotides or vectors. The term "host cell" includes prokaryotic cells (e.g., *Escherichia coli*) or eukaryotic cells (e.g., yeast cells and insect cells). Examples include cells derived from humans, mice, hamsters, pigs, goats, and primates. Cells can be derived from a variety of tissue types and include primary cells and cell lines. Some specific examples include keratinocytes, peripheral blood leukocytes, bone marrow stem cells, and embryonic stem cells. In other embodiments, the host cell is an antigen-presenting cell, particularly dendritic cells, monocytes, or macrophages. Nucleic acids may be present in the host cell in single or multiple copies. In some embodiments, the host cell may be a cell in which the polypeptide of this application is expressed.

[0127] In the context of this application, the term "plasmid" generally refers to a circular DNA molecule, but the term can also encompass linearized DNA molecules. Specifically, the term "plasmid" also encompasses molecules obtained by linearizing a circular plasmid, for example, by digesting the circular plasmid with a restriction enzyme, thereby converting the circular plasmid molecule into a linear molecule. Plasmids can replicate, i.e., amplify genetic information in a cell independently of chromosomal DNA, and can be used for cloning, i.e., for amplifying genetic information in bacterial cells. In one optional specific example, the DNA plasmid is a medium-copy or high-copy plasmid. In another optional specific example, the DNA plasmid is a high-copy plasmid. Examples of such high-copy plasmids include, for example, pUC and pTZ plasmids or any other plasmid (e.g., pMB1, pCoIE1) containing a replication origin that supports high copy numbers.

[0128] The term "vaccine" is typically understood as a preventive or therapeutic material that provides at least one antigen or has antigenic function, which can stimulate the body's adaptive immune system to provide an adaptive immune response.

[0129] The term "treatment" and similar terms are used herein to generally mean achieving a desired pharmacological and / or physiological effect. Therefore, the treatment of this application may involve the treatment of a disease state, but may also involve preventative treatment with regard to the complete or partial prevention of the disease or its symptoms. In some embodiments, the term "treatment" should be understood as being therapeutic in terms of partially or completely curing the disease and / or the adverse effects and / or symptoms attributable to the disease. Treatment can also be prophylactic or preventive, i.e., measures taken to prevent disease, such as to prevent infection and / or the onset of disease.

[0130] As used herein, the terms "subject" and "patient" are used interchangeably. In some embodiments, the subject is a mammal, such as a human, a non-human primate (e.g., apes, chimpanzees, monkeys, and orangutans), a domesticated animal (including dogs and cats, and livestock (e.g., horses, cattle, pigs, sheep, and goats)), or other mammals. Other mammals include, but are not limited to, mice, rats, guinea pigs, rabbits, hamsters, etc. In a particular embodiment, the subject is a human. In one embodiment, the subject is a mammal (e.g., a human) suffering from an infectious disease or a neoplastic disease. In another embodiment, the subject is a mammal (e.g., a human) at risk of developing an infectious disease or a neoplastic disease.

[0131] As used herein, the term "administration" means the provision or administration of a drug to a subject by any effective route. Exemplary routes of administration include, but are not limited to, one or more of the following: injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intravenous, intraventricular, or intravenous), oral, intraluminal bile duct, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation. When used to treat a disease, condition, symptom, or symptom, the substance is usually administered after the onset of the disease, condition, symptom, or symptom. When used to prevent a disease, condition, symptom, or symptom, the substance is usually administered before the onset of the disease, condition, symptom, or symptom.

[0132] This document describes some elements of the present application. These elements are listed together with specific embodiments; however, it should be understood that they can be combined in any manner and in any number to produce other embodiments. The examples and preferred embodiments described differently should not be construed as limiting the present application to the explicitly described embodiments. This specification should be understood to support and include embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, unless the context otherwise indicates, any permutation and combination of all descriptive elements in this application should be considered as disclosed in the specification of this application.

[0133] II. TfR1 binding protein

[0134] Transferrin receptor 1 (TfR1), also known as differentiation cluster 71 (CD71), is a transmembrane glycoprotein primarily responsible for intracellular iron uptake. TfR1 binds to iron-binding transferrin (Tf) to form an iron-Tf-TfR1 complex, which then transports iron into the cell via endocytosis. As an essential receptor for cellular iron uptake, TfR1 is highly expressed in brain microvascular endothelial cells and can transport circulating iron across the blood-brain barrier to the brain via endocytosis, maintaining normal brain function. Therefore, TfR1 can serve as a potential target for drugs to cross the blood-brain barrier. Furthermore, TfR1 is widely overexpressed in many malignant tumor cells; therefore, TfR1 is considered a potential tumor marker, and TfR1 antibodies hold great potential for the diagnosis and treatment of tumors with high TfR1 expression.

[0135] Based on the above, this disclosure provides a TfR1 binding protein comprising an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises complementarity-determining region 1 (CDR1), complementarity-determining region 2 (CDR2), and complementarity-determining region 3 (CDR3) contained in any one of the amino acid sequences shown in SEQ ID NO:1 to 74.

[0136] In some implementations, CDR1 is heavy chain complementarity determination region 1 (HCDR1), CDR2 is heavy chain complementarity determination region 2 (HCDR2), and CDR3 is heavy chain complementarity determination region 3 (HCDR3).

[0137] Based on the above, this disclosure provides a TfR1 binding protein comprising an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises heavy chain complementarity-determining region 1 (HCDR1), heavy chain complementarity-determining region 2 (HCDR2), and heavy chain complementarity-determining region 3 (HCDR3) contained in any one of the amino acid sequences shown in SEQ ID NO:1 to 74.

[0138] The aforementioned TfR1 binding protein binds to TfR1 and can act as a carrier to transport active molecules (such as drugs) across the blood-brain barrier, transferring active molecules from the blood into brain tissue. This is beneficial for the treatment of diseases that require drugs to cross the blood-brain barrier, and can also target tumor cells or cancer cells with high TfR1 expression, facilitating targeted therapy for tumors or cancers.

[0139] In some embodiments, the TfR1 binding protein comprises HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable domain as shown in any of SEQ ID NO:1–74. It is understood that HCDR1, HCDR2, and HCDR3 may be defined based on encoding methods known to those skilled in the art (e.g., IMGT, Kabat, Chothia, Martin, or Aho).

[0140] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 or are composed of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively.

[0141] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO:75, SEQ ID NO:76 and SEQ ID NO:77.

[0142] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively contain or consist of the amino acid sequences shown in SEQ ID NO:78, SEQ ID NO:79 and SEQ ID NO:80.

[0143] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:81, SEQ ID NO:82 and SEQ ID NO:83 or are composed of the amino acid sequences shown in SEQ ID NO:81, SEQ ID NO:82 and SEQ ID NO:83 respectively.

[0144] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO:84, SEQ ID NO:85 and SEQ ID NO:86.

[0145] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:87, SEQ ID NO:88 and SEQ ID NO:89 or are composed of the amino acid sequences shown in SEQ ID NO:87, SEQ ID NO:88 and SEQ ID NO:89.

[0146] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92 or are composed of the amino acid sequences shown in SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92.

[0147] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:93, SEQ ID NO:94 and SEQ ID NO:95 or are composed of the amino acid sequences shown in SEQ ID NO:93, SEQ ID NO:94 and SEQ ID NO:95.

[0148] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:96, SEQ ID NO:97 and SEQ ID NO:98 or are composed of the amino acid sequences shown in SEQ ID NO:96, SEQ ID NO:97 and SEQ ID NO:98.

[0149] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:99, SEQ ID NO:100 and SEQ ID NO:101 or are composed of the amino acid sequences shown in SEQ ID NO:99, SEQ ID NO:100 and SEQ ID NO:101.

[0150] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:102, SEQ ID NO:103 and SEQ ID NO:104 or are composed of the amino acid sequences shown in SEQ ID NO:102, SEQ ID NO:103 and SEQ ID NO:104.

[0151] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO:105, SEQ ID NO:106 and SEQ ID NO:107.

[0152] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO:108, SEQ ID NO:109 and SEQ ID NO:110.

[0153] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:111, SEQ ID NO:112 and SEQ ID NO:113 or are composed of the amino acid sequences shown in SEQ ID NO:111, SEQ ID NO:112 and SEQ ID NO:113.

[0154] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO:114, SEQ ID NO:115 and SEQ ID NO:116.

[0155] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:117, SEQ ID NO:118 and SEQ ID NO:119 or are composed of the amino acid sequences shown in SEQ ID NO:117, SEQ ID NO:118 and SEQ ID NO:119.

[0156] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:120, SEQ ID NO:121 and SEQ ID NO:122 or are composed of the amino acid sequences shown in SEQ ID NO:120, SEQ ID NO:121 and SEQ ID NO:122.

[0157] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:123, SEQ ID NO:124 and SEQ ID NO:125 or are composed of the amino acid sequences shown in SEQ ID NO:123, SEQ ID NO:124 and SEQ ID NO:125.

[0158] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:126, SEQ ID NO:127 and SEQ ID NO:128 or are composed of the amino acid sequences shown in SEQ ID NO:126, SEQ ID NO:127 and SEQ ID NO:128.

[0159] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:129, SEQ ID NO:130 and SEQ ID NO:131 or are composed of the amino acid sequences shown in SEQ ID NO:129, SEQ ID NO:130 and SEQ ID NO:131.

[0160] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:132, SEQ ID NO:133 and SEQ ID NO:134 or are composed of the amino acid sequences shown in SEQ ID NO:132, SEQ ID NO:133 and SEQ ID NO:134.

[0161] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:135, SEQ ID NO:136 and SEQ ID NO:137 or are composed of the amino acid sequences shown in SEQ ID NO:135, SEQ ID NO:136 and SEQ ID NO:137.

[0162] In some embodiments, HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:138, SEQ ID NO:139 and SEQ ID NO:140 or are composed of the amino acid sequences shown in SEQ ID NO:138, SEQ ID NO:139 and SEQ ID NO:140.

[0163] In some embodiments, HCDR1 contains the amino acid sequence shown in SEQ ID NO:87, HCDR2 contains the amino acid sequence shown in SEQ ID NO:88, and HCDR3 contains the amino acid sequence shown in one of SEQ ID NO:141 to 159.

[0164] In some embodiments, HCDR1 consists of the amino acid sequence shown in SEQ ID NO:87, HCDR2 consists of the amino acid sequence shown in SEQ ID NO:88, and HCDR3 consists of the amino acid sequence shown in one of SEQ ID NO:141 to 159.

[0165] In some embodiments, HCDR1 contains the amino acid sequence shown in SEQ ID NO:99, HCDR2 contains the amino acid sequence shown in SEQ ID NO:100, and HCDR3 contains the amino acid sequence shown in one of SEQ ID NO:160 to 178.

[0166] In some embodiments, HCDR1 consists of the amino acid sequence shown in SEQ ID NO:99, HCDR2 consists of the amino acid sequence shown in SEQ ID NO:100, and HCDR3 consists of the amino acid sequence shown in one of SEQ ID NO:160 to 178.

[0167] In some embodiments, the immunoglobulin single variable domain comprises HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable domain as shown in any one of SEQ ID NO:1–74, and the VHH domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the polypeptide shown in any one of SEQ ID NO:1–74. For example, in an optional specific example, the immunoglobulin single variable domain comprises HCDR1 (SEQ ID NO:75), HCDR2 (SEQ ID NO:76), and HCDR3 (SEQ ID NO:77) of the heavy chain variable domain as shown in SEQ ID NO:1, wherein the immunoglobulin single variable domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:1.

[0168] In some embodiments, the immunoglobulin single variable domain comprises an amino acid sequence in which 1 to 15 amino acids are substituted relative to any one of SEQ ID NO:1 to 74 in its framework region (FR).

[0169] In some implementations, the single variable domain of the immunoglobulin is humanized.

[0170] In some implementations, the four backbone regions of the immunoglobulin single variable domain are derived from the human heavy chain variable domain.

[0171] In some implementations, the immunoglobulin single variable domain comprises an amino acid sequence as shown in any one of SEQ ID NO:1 to 74.

[0172] In some implementations, the immunoglobulin single variable domain comprises, as shown in SEQ ID NO:5, 9, 11, 25, 65, 70 or 73.

[0173] In some implementations, the immunoglobulin single variable domain comprises, as shown in SEQ ID NO:65, 70 or 73.

[0174] In some implementations, the immunoglobulin single variable domain comprises, as shown in SEQ ID NO:65.

[0175] In some implementations, the single variable domain of the immunoglobulin is the VHH domain.

[0176] In some implementations, the amino acid sequence of the single variable domain of the immunoglobulin is shown in any one of SEQ ID NO:1 to 74.

[0177] In some embodiments, the amino acid sequence of a single variable domain of an immunoglobulin is as shown in SEQ ID NO:5, 9, 11, 25, 65, 70 or 73.

[0178] In some implementations, the amino acid sequence of a single variable domain of the immunoglobulin is shown in SEQ ID NO:65, 70 or 73.

[0179] In some implementations, the amino acid sequence of a single variable domain of the immunoglobulin is shown in SEQ ID NO:65.

[0180] In some implementations, the TfR1 binding protein further includes human Ig Fc.

[0181] In some implementations, human Ig is human IgG.

[0182] In some implementations, human IgG is human IgG1, human IgG2, human IgG3, or human IgG4.

[0183] In some implementations, the TfR1 binding protein also includes human IgG4 Fc.

[0184] In some implementations, the TfR1 binding protein also includes human IgG1 Fc.

[0185] In some implementations, the TfR1 binding protein also includes human Ig Fc and human Ig hinge region.

[0186] In some implementations, the TfR1 binding protein also includes human IgG4 Fc and human IgG4 hinge region.

[0187] In some implementations, the TfR1 binding protein also includes human IgG1 Fc and human IgG1 hinge region.

[0188] In some implementations, the TfR1 binding protein comprises a single variable immunoglobulin domain, a human Ig hinge region, and a human Ig Fc linked to the human Ig hinge region.

[0189] In some implementations, the amino acid sequence of the human Ig G4 hinge region is shown in SEQ ID NO:179.

[0190] In some embodiments, human IgG4 Fc includes a Ks chain and an Hs chain, the amino acid sequences of which are shown in SEQ ID NO:180 and 181, respectively.

[0191] In some implementations, the amino acid sequence of the human Ig G1 hinge region is shown in SEQ ID NO:182.

[0192] In some implementations, the amino acid sequence of human IgG1 Fc is shown in SEQ ID NO:183.

[0193] In some implementations, human Ig Fc contains mutations that extend the half-life of the TfR1 binding protein.

[0194] In some implementations, mutations that extend the half-life of TfR1 binding proteins are M252Y, S254T, and T256E.

[0195] In some implementations, the mutations that extend the half-life of the TfR1 binding protein are M428L and N434S.

[0196] In some embodiments, human Ig Fc is human IgG4 Fc, and the amino acid sequences of the Ks and Hs chains of human IgG 4 Fc containing a mutation that prolongs the half-life of TfR1 binding protein are shown in SEQ ID NO:188 and 189, respectively.

[0197] In some embodiments, human Ig Fc is human IgG4 Fc, and the amino acid sequences of the Ks and Hs chains of human IgG 4 Fc containing a mutation that prolongs the half-life of TfR1 binding protein are shown in SEQ ID NO:190 and 191, respectively.

[0198] It is understood that in some other implementations, the mutations that extend the half-life of the TfR1 binding protein are not limited to those mentioned above, and may also be other mutations.

[0199] In some implementations, human Ig Fc also contains mutations that promote heterodimerization.

[0200] In some implementations, the TfR1 binding protein is VHH.

[0201] In some implementations, the TfR1 binding protein is a bivalent VHH.

[0202] In some implementations, the TfR1 binding protein is a monovalent VHH.

[0203] In some implementations, the TfR1 binding protein is a heavy chain antibody.

[0204] In some implementations, the TfR1 binding protein is a monovalent heavy chain antibody.

[0205] In some implementations, the TfR1 binding protein is a bivalent heavy chain antibody.

[0206] III. Multispecific antibodies

[0207] This disclosure also provides a multispecific antibody comprising the TfR1 binding protein of any of the above embodiments.

[0208] In some implementations, the multispecific antibody is a bispecific antibody.

[0209] In some implementations, the multispecific antibody is a trispecific antibody or more antibodies that bind to specific antibodies.

[0210] In some implementations, multispecific antibodies also include an active molecule or a binding site that binds to the active molecule.

[0211] IV. mRNA

[0212] In addition, this disclosure provides an mRNA comprising a polynucleotide encoding a fusion protein, the fusion protein comprising an active molecule and a transport molecule.

[0213] active molecules

[0214] In some implementations, the active molecule is a contrast agent or an active molecule used to prevent or treat diseases.

[0215] In some embodiments, the active molecule is a developer. A developer is a compound having one or more properties that allow its presence and / or location to be detected directly or indirectly. Examples of developers include proteins and small molecule compounds incorporating a labeling motif that enables detection.

[0216] In some implementations, the active molecule is used to prevent or treat diseases.

[0217] In some embodiments, the active molecule is used to treat anemia. In some embodiments, the active molecule is used to treat β-thalassemia.

[0218] In some embodiments, the active molecule is used to treat tumors. In some embodiments, the tumor is one or both of the following: acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL).

[0219] In some embodiments, the active molecule is used to treat neurological disorders. In some embodiments, the neurological disorder is one or more of the following: Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman's syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, neurological cancers, and traumatic brain injury.

[0220] In some embodiments, the active molecule is capable of binding to a brain antigen. In some embodiments, the brain antigen is selected from one or more of the following: β-secretase 1 (BACE1), Aβ, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), perkinin, presenilin 1, presenilin 2, γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic protein receptor (p75NTR), and caspase 6.

[0221] In some implementations, the active molecule is a protein or polypeptide.

[0222] In some implementations, the active molecule is an antibody or its antigen-binding fragment.

[0223] In some embodiments, the active molecule is one or two of the following: an antibody that binds to Aβ or its antigen-binding fragment, or idose-2-sulfatase (IDS).

[0224] In some implementations, the active molecule is a small molecule compound. Examples include the anticancer drug monomethyl arysuccinamide E (MMAE) and azathioprine, used to treat multiple sclerosis.

[0225] In some implementations, the active molecule is a nucleic acid.

[0226] In some embodiments, the active molecule is a nucleic acid based on RNA interference technology or gene therapy. In some embodiments, the active molecule is one or more of the following: small interfering RNA (siRNA), microRNA (miRNA), small ribonucleic acid (shRNA), sgRNA, or mRNA encoding a gene editing-related protein (e.g., Cas9).

[0227] In some implementations, the active molecule is mRNA.

[0228] In some embodiments, the active molecule is directly linked to the TfR1 binding protein or its antigen-binding fragment via non-covalent bonds (e.g., electrostatic interactions, hydrogen bonds, hydrophobic interactions, van der Waals forces, etc.) to enable the TfR1 binding protein or its antigen-binding fragment to transport the active molecule. For example, in some embodiments, the active molecule is an antibody binding to Aβ, and the TfR1 binding protein or its antigen-binding fragment has a binding site for the Aβ-binding antibody. In this case, the active molecule and the TfR1 binding protein or its antigen-binding fragment are linked through antigen-antibody interactions.

[0229] In some embodiments, the active molecule is indirectly linked to a TfR1 binding protein or its antigen-binding fragment via a non-covalent bond after being coupled to a linker, thereby enabling the TfR1 binding protein or its antigen-binding fragment to transport the active molecule. For example, in some embodiments, the active molecule is a nucleic acid (e.g., mRNA, siRNA, etc.) or a small molecule compound, and the TfR1 binding protein or its antigen-binding fragment has an antigen-binding site for the linker. In this case, the active molecule and the TfR1 binding protein or its antigen-binding fragment are linked using antigen-antibody interactions. In some embodiments, the linker may be cleavable or non-cleavable.

[0230] In some embodiments, the active molecule is indirectly linked to a TfR1-binding protein or its antigen-binding fragment via a covalent bond to enable the TfR1-binding protein or its antigen-binding fragment to transport the active molecule. For example, in some embodiments, the active molecule is a protein (e.g., an IDS), and the active molecule is covalently linked to the TfR1-binding protein or its antigen-binding fragment via a linker peptide (e.g., a polynucleotide encoding the active molecule and a polynucleotide encoding the TfR1-binding protein or its antigen-binding fragment are fused and expressed as a single protein). In some embodiments, the linker peptide may be cleavable so that the active molecule can be released from the TfR1-binding protein or its antigen-binding fragment after being transported to the target region by the TfR1-binding protein or its antigen-binding fragment. Of course, the linker peptide may also be non-cleavable.

[0231] In some implementations, the active molecule is one or more.

[0232] In some implementations, the active molecule is a single, binding molecule to a brain antigen.

[0233] In some implementations, there are multiple active molecules, and these multiple active molecules bind to the same brain antigen.

[0234] In some implementations, there are multiple active molecules, and these multiple active molecules bind to different brain antigens.

[0235] It is understood that, in some implementation schemes, the active molecules used to prevent or treat diseases are not limited to proteins or peptides, nucleic acids and small molecule compounds, but may also include other substances that can be used to prevent or treat diseases, such as viruses, oncolytic bacteria, etc.

[0236] Transport molecules

[0237] Transport molecules are used to transport active molecules to a target area (such as brain tissue, tumor tissue, etc.).

[0238] In some implementations, the transport molecule is a molecule capable of binding to blood-brain barrier receptors to transport the active molecule across the blood-brain barrier (BBB).

[0239] In some embodiments, the blood-brain barrier is in mammals. In some embodiments, the blood-brain barrier is in mammals with neurological diseases. In some embodiments, the mammal is a human being with a neurological disease.

[0240] In some embodiments, the blood-brain barrier receptor is selected from one or more of the following: transferrin receptor, insulin receptor, insulin-like growth factor receptor, low-density lipoprotein receptor-associated protein 8, low-density lipoprotein receptor-associated protein 1, glucose transporter 1, and heparin-binding epidermal growth factor-like growth factor. In some embodiments, the blood-brain barrier receptor is transferrin receptor 1 (TfR1).

[0241] In some implementations, the transferrin receptor is human TfR1.

[0242] In some implementations, the transport molecule comprises or is an antibody or its antigen-binding fragment.

[0243] In some implementations, the transport molecule comprises or is an antibody or antigen-binding fragment thereof that binds to human TfR1.

[0244] In some embodiments, the transport molecule comprises or is a TfR1 binding protein of any of the above embodiments.

[0245] TfR1 is a class of receptors with normal physiological function, and the binding site of TfR1 binding proteins is located as far away as possible from the binding sites of TfR1 and its natural ligands. In some embodiments, TfR1 binding proteins do not inhibit or weakly inhibit the binding of TfR1 to one or more natural ligands. Weak inhibition means that the binding of TfR1 binding proteins to one or more natural ligands is inhibited, but this inhibition does not affect the normal physiological function of its natural ligands. In some embodiments, TfR1 binding proteins do not inhibit or weakly inhibit TfR1 activity. In some embodiments, TfR1 binding proteins do not inhibit or weakly inhibit the binding of TfR1 to transferrin (TF).

[0246] In addition to structural form (such as different binding valence states / configurations), the affinity of TfR1-binding proteins for TfR1 should facilitate the smooth completion of receptor-mediated transendothelial transport: they should be able to efficiently bind TfR1 on the surface of brain capillary endothelial cells on the blood side and trigger endocytosis, and avoid excessive retention or being directed to lysosomal degradation during intracellular sorting and transport, and then be released from the brain side into the brain parenchyma via exocytosis, thereby achieving the crossing of the blood-brain barrier.

[0247] In some implementations, TfR1 binds to the K of the protein. D No more than 1×10 -6 mol / L.

[0248] In some implementations, TfR1 binds to the K of the protein. D 1×10 -10 mol / L~1×10 -6 mol / L.

[0249] In some implementations, TfR1 binds to the K of the protein. D 1×10 -9 mol / L~9×10 -7 mol / L.

[0250] In some implementations, TfR1 binds to the K of the protein. D 1×10 -8 mol / L~9.5×10 -8 mol / L.

[0251] In some implementation schemes, K D The determination was performed using biomembrane interferometry (BLI).

[0252] In some implementations, the EC50 of the TfR1 binding protein for TfR1 is no greater than 500 nmol / L.

[0253] In some implementations, the EC50 of the TfR1 binding protein for TfR1 is 0.000001 nmol / L to 300 nmol / L.

[0254] In some implementations, the EC50 of the TfR1 binding protein for TfR1 is 0.00001 nmol / L to 100 nmol / L.

[0255] In some implementations, the EC50 of the TfR1 binding protein for TfR1 is 0.0001 nmol / L to 100 nmol / L.

[0256] In some implementations, the EC50 of the TfR1 binding protein for TfR1 is 0.001 nmol / L to 80 nmol / L.

[0257] In some implementations, the EC50 of the TfR1 binding protein for TfR1 is 0.01 nmol / L to 50 nmol / L.

[0258] In some implementations, the EC50 of the TfR1 binding protein for TfR1 is 0.01 nmol / L to 30 nmol / L.

[0259] In some implementations, EC50 is measured using flow cytometry fluorescence sorting (FACS).

[0260] In some implementations, the transport molecule comprises or is a multispecific antibody of any of the above implementations.

[0261] In some embodiments, the transport molecule comprises or is the TfR1 binding protein of any of the above embodiments, and the active molecule is an antibody that binds to Aβ or its antigen-binding fragment.

[0262] In some embodiments, the transport molecule comprises or is the TfR1 binding protein of any of the above embodiments, and the active molecule is iduxose-2-sulfatase (IDS).

[0263] In some embodiments, the fusion protein is a multispecific antibody of any of the above embodiments, the transport molecule is a TfR1 binding protein of any of the above embodiments, and the active molecule is an antibody that binds to Aβ or its antigen-binding fragment.

[0264] In some embodiments, the fusion protein is a multispecific antibody of any of the above embodiments, the transport molecule is a TfR1 binding protein of any of the above embodiments, and the active molecule is IDS.

[0265] In some implementations, the fusion protein does not inhibit or weakly inhibits the binding of the blood-brain barrier receptor to one or more of its natural ligands.

[0266] In some embodiments, the fusion protein comprises the TfR1 binding protein of any of the above embodiments and the active molecule of any of the above embodiments, and the fusion protein does not inhibit or weakly inhibits TfR1 activity.

[0267] In some embodiments, the fusion protein comprises the TfR1 binding protein of any of the above embodiments and the active molecule of any of the above embodiments, and the fusion protein does not inhibit or weakly inhibits the binding of TfR1 to transferrin.

[0268] It is understandable that in the aforementioned mRNA containing polynucleotides encoding fusion proteins, whether the polynucleotide encoding TfR1 binding protein or the polynucleotide encoding the active molecule, there can be multiple (e.g., two or three) copies on the same strand. For example, if the polynucleotide encoding the TfR1 binding protein is simply referred to as "fragment A" and the polynucleotide encoding the active molecule is simply referred to as "fragment B," then the aforementioned mRNA containing fusion proteins can simultaneously contain multiple fragments A and multiple fragments B, contain one fragment A and multiple fragments B, or contain multiple fragments A and one fragment B.

[0269] In some embodiments, the polynucleotide encoding the TfR1-binding protein and the polynucleotide encoding the active molecule are each independently one or more copies. In some embodiments, the polynucleotide encoding the TfR1-binding protein and the polynucleotide encoding the active molecule are both one copy.

[0270] In some embodiments, the mRNA of any of the above embodiments further comprises at least one of a 5'-cap structure, a 5'-UTR, a 3'-UTR, and a poly(A) tail.

[0271] In some implementations, the mRNA of any of the above implementations also includes a 5'-cap structure, a 5'-UTR, a 3'-UTR, and a poly(A) tail.

[0272] In some implementations, the 5'-UTR sequence is shown in SEQ ID NO:198.

[0273] In some implementations, the 3'-UTR sequence is shown in SEQ ID NO:199.

[0274] In some implementations, the poly(A) tail sequence is as shown in SEQ ID NO:200.

[0275] In some implementations, the mRNA of any of the above implementations does not contain modified nucleotides.

[0276] In some implementations, the mRNA of any of the above implementations contains modified nucleotides.

[0277] In some implementations, the mRNA of any of the above implementations also contains a modified nucleoside.

[0278] In some embodiments, the modified nucleoside includes at least one of modified uridine, modified cytidine, modified adenosine, and modified guanosine.

[0279] In some embodiments, the modified nucleoside is modified uridine. In some embodiments, 0.1% to 100% of the uridine in the above-mentioned mRNA is modified. For example, at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% of the uridine is modified. In some embodiments, 80% to 100% of the uridine is modified. In some embodiments, 100% of the uridine is modified. Exemplary modified uridines include, but are not limited to, one or more of the following: pseudouridine (ψ), N1-methylpseudouridine, and 5-methoxy-uridine (mo5U).

[0280] In some embodiments, the modified nucleoside is modified cytidine. In some embodiments, 0.1% to 100% of the cytidine in the above-mentioned mRNA is modified. For example, at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% of the cytidine is modified. In some embodiments, 80% to 100% of the cytidine is modified. In some embodiments, 100% of the cytidine is modified. Exemplary modified cytidines include one or more of the following: 3-methyl-cytidine (m3C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), and 5-methyl-cytidine (m5C).

[0281] In some embodiments, the modified nucleoside is modified adenosine. In some embodiments, 0.1% to 100% of the adenosine in the nuclear mRNA is modified. For example, at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% of the adenosine is modified. In some embodiments, 80% to 100% of the adenosine is modified. In some embodiments, 100% of the adenosine is modified. Exemplary modified adenosines include, but are not limited to, one or more of the following: 1-methyl-adenosine (m1A) and N6-methyl-adenosine (m6A).

[0282] In some embodiments, the modified nucleoside is modified guanosine. In some embodiments, 0.1% to 100% of the guanosine in the above-mentioned mRNA is modified. For example, at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% of the guanosine is modified. In some embodiments, 80% to 100% of the guanosine is modified. In some embodiments, 100% of the guanosine is modified. Exemplary modified guanosines include, but are not limited to, one or more of the following: 7-methyl-guanosine (m7G) and 2'-O-methyl-guanosine (Gm).

[0283] In some implementations, the modified nucleotides in the mRNA of any of the above implementations include isotopic nucleotides.

[0284] In some embodiments, the mRNA of any of the above embodiments comprises a nucleotide containing a hydrogen isotope. The hydrogen isotope is not limited to deuterium or tritium. Additionally, in some embodiments, the mRNA of any of the above embodiments also comprises or contains nucleotides containing isotopes of elements other than hydrogen, wherein these other elements include, but are not limited to, carbon, oxygen, nitrogen, and phosphorus.

[0285] In some embodiments, the mRNA of any of the above embodiments further includes one or more microRNA binding sites. The microRNA binding sites are used to regulate the expression of the aforementioned nucleic acids. For example, they are used to increase or decrease the expression of the nucleic acid in which it resides, preferably to decrease the expression of the aforementioned nucleic acid in undesirable cells and / or tissues.

[0286] In some implementations, the microRNA binding site is located in the 5'-UTR, in the 3'-UTR, after the 3'-UTR and before the poly(A) tail, or in the poly(A) tail.

[0287] In some implementations, the microRNA binding site is located in the poly(A) tail.

[0288] In some implementations, the microRNA binding site is located after the 3'-UTR and before the poly(A) tail.

[0289] In some embodiments, there is one microRNA binding site. In some embodiments, there are multiple microRNA binding sites. In some embodiments, there are two, three, four, five, six, seven, eight, nine, or ten microRNA binding sites. In some embodiments, there are three microRNA binding sites.

[0290] In some implementations, multiple microRNA binding sites are linked by a linker. In some implementations, the linker sequence is GCUG.

[0291] In some implementations, the microRNA binding site is the miR142 binding site.

[0292] In some implementations, the mRNA of any of the above implementations contains three miR142 binding sites.

[0293] In some implementations, the sequence of the miR142 binding site is shown in SEQ ID NO: 210.

[0294] In some implementations, the three miR142 binding sites are linked via GCUG.

[0295] In some implementations, the sequences of the three miR142 binding sites are shown in SEQ ID NO: 197.

[0296] V. Fusion proteins and conjugates, chimeric antigen receptors

[0297] This disclosure also provides a fusion protein obtained by translating mRNA from any of the above embodiments.

[0298] This disclosure also provides a fusion protein comprising the TfR1 binding protein and an active molecule of any of the above embodiments.

[0299] In some implementations, the active molecule is as described above.

[0300] In some embodiments, the TfR1-binding protein can bind to blood-brain barrier receptors to transport the active molecule across the blood-brain barrier. This disclosure also provides a conjugate comprising the TfR1-binding protein of any of the above embodiments and the active molecule conjugated thereto.

[0301] This disclosure also provides another conjugate comprising a multispecific antibody of any of the above embodiments and an active molecule conjugated thereto.

[0302] This disclosure also provides another conjugate comprising the fusion protein of any of the above embodiments and the active molecule conjugated thereto.

[0303] In some implementations, the TfR1 binding protein, multispecific antibody, or fusion protein of the conjugate is directly conjugated to the active molecule via covalent bonds (e.g., peptide bonds, ester bonds, thioether bonds, imine bonds, disulfide bonds, etc.).

[0304] In some implementations, the TfR1-binding protein, multispecific antibody, or fusion protein of the conjugate is indirectly conjugated to the active molecule via covalent bonds (e.g., using a linker for indirect covalent conjugation).

[0305] In some implementations, the TfR1-binding protein, multispecific antibody, or fusion protein of the conjugate is conjugated to the active molecule via non-covalent bonds (e.g., electrostatic interactions, hydrogen bonds, hydrophobic interactions, van der Waals forces, etc.).

[0306] In some embodiments, the conjugate comprises the fusion protein of any of the above embodiments and another active molecule conjugated thereto, which is different from the active molecule in the fusion protein.

[0307] In some implementations, the active molecule is as described above.

[0308] In some implementations, the active molecule is a protein or peptide, such as an antibody.

[0309] In some implementations, the active molecule is a small molecule compound, such as a small molecule chemical drug.

[0310] In some implementations, the active molecule is a nucleic acid, such as mRNA or siRNA.

[0311] This disclosure also provides a chimeric antigen receptor (CAR) comprising the TfR1 binding protein of any of the above embodiments.

[0312] VI. DNA, nucleic acids and their preparation methods, gene engineering vectors, host cells, and immune cells

[0313] This disclosure also provides a nucleic acid comprising a TfR1 binding protein encoding any of the above embodiments, a multispecific antibody encoding any of the above embodiments, or a polynucleotide encoding a CAR of any of the above embodiments.

[0314] In some implementations, the nucleic acid encodes the TfR1 binding protein of any of the above implementations, the multispecific antibody of any of the above implementations, or the CAR of any of the above implementations.

[0315] In some implementations, the nucleic acid is mRNA.

[0316] In some implementations, the nucleic acid is DNA.

[0317] This disclosure also provides a DNA for preparing mRNA according to any of the above embodiments.

[0318] This disclosure also provides a DNA that can be transcribed into mRNA according to any of the above embodiments.

[0319] This disclosure also provides a DNA that can be transcribed and post-transcribed (e.g., capped) into mRNA according to any of the above embodiments.

[0320] In some embodiments, the DNA described above contains at least one of a 5'-UTR, a 3'-UTR, and a polynucleotide encoding a poly(A) tail, and a polynucleotide encoding a TfR1 binding protein of any of the embodiments described above.

[0321] In some embodiments, the DNA described above contains a 5'-UTR, a 3'-UTR, and a polynucleotide encoding a poly(A) tail, as well as a polynucleotide encoding a TfR1 binding protein of any of the embodiments described above.

[0322] In addition, this disclosure also provides a gene engineering vector comprising DNA or nucleic acid of any of the above embodiments, or the gene engineering vector comprising a polynucleotide capable of being transcribed into mRNA of any of the above embodiments, or the gene engineering vector capable of being transcribed or transcribed and post-transcribed into mRNA of any of the above embodiments.

[0323] In some embodiments, the genetic engineering vector is an expression vector. In some embodiments, the genetic engineering vector is a plasmid, a granule, a virus (e.g., adenovirus, adeno-associated virus), a bacteriophage, or another vector conventionally used in genetic engineering. In one optional specific example, the genetic engineering vector is a plasmid. In one optional specific example, the genetic engineering vector is adeno-associated virus (AAV). In some embodiments, the genetic engineering vector further comprises at least one or more of the following: origin of replication (ORI), a marker gene or a fragment thereof, a reporter gene or a fragment thereof, and a restriction site allowing the insertion of a DNA element. In one optional specific example, the restriction site allowing the insertion of a DNA element is a multiple cloning site (MCS).

[0324] In some embodiments, the above-described genetic engineering vector comprises a promoter, a 5'-UTR, a polynucleotide encoding a TfR1 binding protein of any of the above embodiments, a 3'-UTR, and a polynucleotide encoding a poly(A) tail, wherein the polynucleotide encoding the poly(A) tail, the promoter, the 5'-UTR, the polynucleotide encoding the TfR1 binding protein, and the 3'-UTR are operatively linked to each other.

[0325] In other implementations, the aforementioned genetic engineering vector is a cloning vector.

[0326] This disclosure also provides a method for preparing DNA or nucleic acid according to any of the above embodiments, the method comprising the step of introducing (e.g., in plasmid form) a gene engineering vector according to any of the above embodiments into a host cell (e.g., Escherichia coli) and then culturing the host cell containing the gene engineering vector.

[0327] In addition, this disclosure also provides another method for preparing the above-described DNA, which includes the step of preparing the DNA using a chemical synthesis method based on the nucleotide sequence of the DNA according to any of the above embodiments. It is understood that the specific method of chemical synthesis can be a method known in the art, such as the solid-phase phosphorus amide method.

[0328] It is understood that the method for preparing DNA or nucleic acid in any of the above embodiments is not limited to the above, and may also be other methods.

[0329] In addition, this disclosure also provides a host cell comprising nucleic acid of any of the above embodiments, DNA of any of the above embodiments, or genetic engineering vector of any of the above embodiments.

[0330] In some implementations, the host cell is a separated cell.

[0331] In some implementations, the host cell is used to store and / or amplify the DNA of any of the above implementations.

[0332] In some implementations, the host cell is a bacterial cell. Bacterial host cells include *Escherichia coli* (E. coli) cells, which are well-known to those skilled in the art.

[0333] The host cells of this disclosure can be prepared by transforming competent host cells with a genetically engineered vector according to any of the above embodiments. Competent host cells are cells capable of taking up free extracellular genetic material (e.g., DNA plasmids) in a sequence-independent manner. Many bacterial cells known to those skilled in the art are naturally capable of taking up exogenous DNA from the environment and can therefore serve as bacterial host cells according to this disclosure. Furthermore, those skilled in the art know that competent bacterial host cells can be obtained from naturally non-competent bacterial cells using methods such as electroporation or chemicals (e.g., treatment with calcium ions accompanied by high-temperature exposure). After take-up, the exogenous DNA preferably neither degrades nor integrates into the genome of the bacterial host cell.

[0334] In addition, this disclosure also provides a TfR1 binding protein derived from the mRNA of any of the above embodiments.

[0335] In addition, this disclosure also provides a method for preparing a TfR1 binding protein, the method comprising: transcribing a polynucleotide (e.g., DNA or a genetic engineering vector) encoding a TfR1 binding protein into RNA; and translating the transcribed RNA into a TfR1 binding protein.

[0336] In addition, this disclosure also provides another method for preparing a TfR1 binding protein, the method comprising: translating an mRNA containing a polynucleotide encoding a TfR1 binding protein according to any of the above embodiments into a TfR1 binding protein.

[0337] In some embodiments, the preparation of the TfR1 binding protein in any of the above embodiments is carried out entirely or partially in vitro.

[0338] This disclosure also provides an immune cell that expresses a chimeric antigen receptor according to any of the above embodiments.

[0339] In some implementations, the immune cells are isolated immune cells.

[0340] VII. Pharmaceutical Compositions

[0341] This disclosure also provides a pharmaceutical composition comprising the TfR1 binding protein of any of the above embodiments, the mRNA of any of the above embodiments, the fusion protein of any of the above embodiments, the multispecific antibody of any of the above embodiments, the conjugate of any of the above embodiments, the chimeric antigen receptor of any of the above embodiments, the nucleic acid of any of the above embodiments, the DNA of any of the above embodiments, the genetic engineering vector of any of the above embodiments, and the isolated immune cells of any of the above embodiments.

[0342] In some embodiments, the above-described pharmaceutical compositions further comprise a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" means approved for use in animals and / or humans by a regulatory authority (e.g., the China Food and Drug Administration, the U.S. Food and Drug Administration (FDA)) or a recognized pharmacopoeia (e.g., the Chinese Pharmacopoeia, the European Pharmacopoeia). The term "pharmaceuticalally acceptable carrier" refers to a substance that can be administered with the TfR1 binding protein, mRNA, fusion protein, chimeric antigen receptor, multispecific antibody, nucleic acid, conjugate, DNA, genetically engineered vector, host cell, or isolated immune cell of this disclosure, including but not limited to delivery carriers, diluents, sweeteners, flavoring agents, wetting agents, adjuvants, flow aids, preservatives, dyes / coloring agents, surfactants, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers.

[0343] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, and a TfR1 binding protein of any of the above embodiments, an mRNA of any of the above embodiments, a fusion protein of any of the above embodiments, a multispecific antibody of any of the above embodiments, a conjugate of any of the above embodiments, a chimeric antigen receptor of any of the above embodiments, a nucleic acid of any of the above embodiments, DNA of any of the above embodiments, a genetically engineered vector of any of the above embodiments, or an isolated immune cell of any of the above embodiments.

[0344] In some implementations, the pharmaceutically acceptable carrier is a delivery carrier.

[0345] In some embodiments, the pharmaceutically acceptable vector is a delivery vector in which the mRNA, nucleic acid, DNA, or genetically engineered vector of any of the above embodiments is formulated (e.g., encapsulated).

[0346] In some embodiments, the delivery carrier is selected from a combination or one of the following: lipid nanoparticles (LNPs), liposomes, cationic proteins, vesicles, microparticles, polymers, and micelles. In some embodiments, the delivery carrier is selected from one of the following: LNPs, liposomes, cationic proteins, vesicles, microparticles, polymers, and micelles.

[0347] In some implementations, the delivery carrier is LNPs.

[0348] In some implementations, LNPs refer to particles having a nanoscale (e.g., 1 nm to 1000 nm) size, which include one or more lipids.

[0349] In some implementations, the average diameter of LNPs is 20nm–800nm, 20nm–500nm, 20nm–400nm, 20nm–300nm, 20nm–200nm, 20nm–100nm, 30nm–700nm, 30nm–500nm, 30nm–300nm, 30nm–200nm, 30nm–100nm, 40nm–800nm, 40nm–600nm, 40nm–500nm, 40nm–300nm, etc. 0nm, 40nm~200nm, 40nm~100nm, 50nm~800nm, 50nm~600nm, 50nm~500nm, 50nm~400nm, 50nm~300nm, 50nm~200nm, 50nm~100nm, 60nm~800nm, 60nm~600nm, 60nm~500nm, 60nm~400nm, 60nm~300nm, 60nm~200nm or 60nm~100nm. In some optional specific examples, the average diameter of LNPs is 26nm, 31nm, 36nm, 41nm, 46nm, 51nm, 56nm, 61nm, 66nm, 71nm, 76nm, 81nm, 86nm, 91nm, 96nm, 101nm, 106nm, 111nm, 116nm, 121nm, 126nm, 131nm, 136nm, 141nm, 146nm, 151nm, 156nm, 161nm, 166nm, 171nm, 176nm, 181nm, 186nm, 191nm, 196nm, 201nm, 206nm, 211nm, 216nm, 221nm, 226nm, 231nm, 236nm, 241nm, 246nm, or 249nm. In this paper, the average diameter of LNPs can be represented by the z-average value determined by dynamic light scattering.

[0350] In some embodiments, LNPs include one of the following: cationic lipid nanoparticles, solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), and nonlamellar lipid nanoparticles. In one optional specific example, LNPs are cationic lipid nanoparticles.

[0351] In some embodiments, LNPs contain one or more of the following substances: ionizable lipids, auxiliary lipids, structural lipids, and polymer-lipids.

[0352] In some implementations, LNPs contain ionizable lipids.

[0353] In some implementations, LNPs contain ionizable lipids, auxiliary lipids, structural lipids, and polymer-lipids.

[0354] The term "ionizable lipid" refers to a lipid that becomes positively charged when the pH drops below the pKa of its ionizable groups, but gradually becomes neutral at higher pH values. Below the pKa, the positively charged lipid can bind to negatively charged nucleic acids. In some embodiments, ionizable lipids include zwitterionic lipids.

[0355] In some embodiments, ionizable lipids include the following compound (III), its N-oxide, its salt, or isomers thereof:

[0356] Where Y1 and Y2 are independently O or S;

[0357] Z3 represents H, halogen, and -R. b -N(R) b )2、-CN、-N3、-C(=O)OR b -OC(=O)R b -OR b -SR b -S(-O)R b -S(=O)OR b -S(=O)2OR b -N(R) b )S(=O)2R b -NHR a N(R b )2、-NHR a OR a N(Rb )2、-NHR a OR b or -N(R) a OR b )2;

[0358] Each R a Independently for C1-C 12 Alkylene, C2-C 12 alkenyl group, C2-C 12 Alynyl group, C3-C8 cyclic hydrocarbon group, C3-C8 heterocyclic group, C1-C containing O, N or S 12 Heteroalkyl groups, C2-C containing O, N, or S, having 1, 2, 3, or more double bonds. 12 Imaginal group, or C2-C containing O, N, or S with 1, 2, 3, or more triple bonds. 12 Hypo-heyne group;

[0359] Each R b Independently H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkyne group, C3-C8 cyclic hydrocarbon group, C3-C8 heterocyclic group, C1-C containing O, N or S 12 Heteroalkyl groups, C2-C containing O, N, or S, having 1, 2, 3, or more double bonds. 12 Heterene group, or C2-C containing O, N, or S with 1, 2, 3, or more triple bonds. 12 Zeyne group;

[0360] Each Z4 group is independently H, C1-C6 hydrocarbon group, or C1-C6 heterohydrocarbon group;

[0361] Z5 is H, C1-C6 hydrocarbon group, or C1-C6 heterohydrocarbon group;

[0362] L3 can be a C1-C6 alkylene group, a C3-C8 carbocyclic group, a heterocyclic group, a C1-C6 heteroene group, or a bond;

[0363] L7, L8, L 11 and L 12 Independently for C1-C 18 Hydroxyl group, C1-C 18 heteroalkyl groups or bonds;

[0364] A9 and A 10Independently -N(Z4)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -O-, -C(=O)-, -S-, -C(=O)S-, -SC(=O)-, -C(=O)N(Z4)-, -N(Z4)C(=O)-, -OC(=O )S-, -SC(=O)O-, -SC(=O)S-, -OC(=O)N(Z4)-, -N(Z4)C(=O)O-, -N(Z4)C(=O)N(Z4)-, -SC(=O)N(Z4)-, -N(Z4)C(=O)S-, -N(C(=O)L 13 OZ4)-、-N(C(=O)L 13 SZ4)-、-N(C(=O)Z4),-N(L 13 OZ4)-、-N(L 13 SZ4)-, -OS(=O)O-, -OS(=O)2O-, -OP(=O)O-, -OP(=O)(OH)O-, -OP(=O)(H)O-, -OS(=O)2NH-, -NHS(= O)2O-, -OC(=O)C(=O)O-, -N(Z4)C(=O)C(=O)O-, -OC(=O)C(=O)N(Z4)-, -N(Z4)C(=O)C(=O)N(Z4)-, OR key;

[0365] Each L 13 Independently, it is a C1-C8 alkylene group, or a C1-C8 heteroalkylene group, or a bond;

[0366] Z1 and Z2 are independently C1-C 24 hydrocarbon group, -C(Z6)(OL 14 A 12 Z7)2、-C(Z6)(SL 14 A 12 Z7)2、-C(Z6)(SL 14 A 12 Z7)(OL 14 A 12 Z7), -C(Z6)(C(=O)OL 14 A 12 Z7)2、-C(Z6)(OC(=O)L 14 A 12 Z7)2、-C(Z6)(C(=O)OL 14 A 12 Z7)Z7 or -C(Z6)(OC(=O)L 14 A 12 Z7)Z7;

[0367] Each A 12Independently -C(=O)O-, -OC(=O)-, -OC(=O)O-, -O-, -C(=O)-, -S-, -C(=O)S-, -SC(=O)-, -C(=O)N(Z4)-, -N(Z4)C(=O)-, -OC(=O)N(Z 4)-, -N(Z4)C(=O)O-, -N(Z4)C(=O)N(Z4)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -SC(=O)N(Z4)-, -N(Z4)C(=O)S-, -N(C(=O)L 13 OZ4)-、-N(C(=O)L 13 SZ4)-、-N(C(=O)Z4),-N(L 13 OZ4)-、-N(L 13 SZ4)-, -N(Z4)-, -OS(=O)O-, -OS(=O)2O-, -OP(=O)O-, -OP(=O)(OH)O-, -OP(=O)(H)O-, -OS(=O)2NH-, -NHS(=O)2O-, benzene ring or bond;

[0368] Each Z6 group is independently H, C1-C6 hydrocarbon group, or C1-C6 heterohydrocarbon group;

[0369] Each L 14 Independently for C1-C 18 Hydroxyl group, C1-C 18 heteroalkyl groups or bonds;

[0370] Each Z7 is independently C1-C 24 Hydrocarbon group or C1-C 24 heterohydrocarbon group.

[0371] In some embodiments, ionizable lipids include the following compound (III-1), its N-oxide, its salt, or isomers thereof:

[0372] Among them, Y1, Y2, Z1, Z2, Z3, Z4, L3, L7, L8, L 11 L 12 , A9 and A 10 As defined in formula (III) above.

[0373] In some embodiments, in the compounds represented by formula (III) or (III-1), Y1 and Y2 are O, Z3 is -N(H)CH3, and A9 and A 10 For -C(=O)O-, Z1 and Z2 are independently -C(H)(OZ7)2, and each Z7 is independently C1-C 12 Hydrocarbon group.

[0374] In some embodiments, ionizable lipids include compound (III) or (III-1), or its N-oxide, salt or isomer, and, where applicable, include one or more of the following features.

[0375] In some implementations, Y1 and Y2 are 0.

[0376] In some implementations, Z3 is -N(R) b )2.

[0377] In some implementations, Z3 is -N(H)CH3.

[0378] In some implementations, A9 and A 10 It can be independently -C(=O)O- or -OC(=O)-.

[0379] In some implementations, A9 and A 10 It is -C(=O)O-.

[0380] In some implementations, Z1 and Z2 are independently -C(H)(OZ7)2, and each Z7 is independently C1-C 12 Hydrocarbon group.

[0381] In some implementations, L3 is a C1-C6 alkylene group.

[0382] In some implementations, each Z4 is independently H or a C1-C6 alkyl group.

[0383] In some implementations, L7 and L8 are independently C1-C 10 Alkylene.

[0384] In some implementations, L 11 and L 12 It is independently a C1-C5 alkylene group.

[0385] In some embodiments, the ionizable lipid is compound 1-1, its salt, or an isomer thereof:

[0386] In some embodiments, the ionizable lipid is compound 1-2, its salt, or an isomer thereof:

[0387] In some embodiments, ionizable lipids include the following compounds (IV), their N-oxides, their salts, or isomers thereof:

[0388] Wherein, A1 is H, C1-C5 hydrocarbon group or C1-C5 heterohydrocarbon group, A2 is H, C1-C5 hydrocarbon group or C1-C5 heterohydrocarbon group, and A3 is C1-C5 alkylene group or bond;

[0389] A4 is a C1-C5 hydrocarbon group or bond;

[0390] A5, A6, A7, and A8 are independently C1-C 18 Hydroxyl group, C1-C 18 heteroalkyl groups or bonds;

[0391] Q1 and Q2 are independently -C(=O)O- and -OC(=O)-;

[0392] Each Z9 is independently C1-C 24 Hydrocarbon group or C1-C containing O or S 24 heterohydrocarbon group;

[0393] Each Z 11 Independently for C1-C 24 Hydrocarbon group or C1-C containing O or S 24 heterohydrocarbon group.

[0394] In some embodiments, each hydrocarbon group in formula (IV) is independently an alkyl, alkenyl, or alkynyl group.

[0395] In some embodiments, each of the alkylene groups in formula (IV) is independently an alkylene group, an alkenylene group, or an alkynylene group.

[0396] In some embodiments, each heteroalkyl group in formula (IV) is independently a heteroalkyl, heteroalkenyl, or heteroynyl group.

[0397] In some embodiments, each heteroalkyl group in formula (IV) is independently a heteroalkyl, heteroalkenyl, or heteroyne group.

[0398] In some embodiments, in the compound represented by formula (IV), Q1 and Q2 are -C(=O)O-.

[0399] In some embodiments, in the compound represented by formula (IV), Q1 and Q2 are -C(=O)O-, A4 is a bond, and A5 and A6 are independently C3-C 10 Alkylenes, A7 and A8 are independently C2-C4 alkylenes, each Z9 is independently C3-C9 alkyl, each Z 11 A1 and A2 are independently C3-C9 alkyl groups, A3 is a C2-C4 alkylene group.

[0400] Ionizable lipids include compounds (IV), or their N-oxides, salts, or isomers thereof, and, where applicable, include one or more of the following characteristics.

[0401] In some implementations, A4 is the bond, and Q1 and Q2 are C(=O)O-.

[0402] In some implementations, A5 and A6 are independently C4-C9 alkylene groups.

[0403] In some implementations, A7 and A8 are independently C2-C4 alkylene groups.

[0404] In some embodiments, each Z9 is independently a C5-C8 alkyl group, such as C6, C8, C9 ... 7、 C8 alkyl.

[0405] In some implementation schemes, each Z 11 Independently C5-C8 alkyl, such as C6, C 7、 C8 alkyl.

[0406] In some embodiments, A1 and A2 are independently C1-C3 alkyl groups.

[0407] In some embodiments, A3 is a C2-C4 alkylene, such as a C3 alkylene.

[0408] In some embodiments, the ionizable lipid is compound 2-1, its salt, or an isomer thereof:

[0409] In some embodiments, the ionizable lipid is compound 2-2, its salt, or an isomer thereof:

[0410] In some implementations, the ionizable lipid is compound 2-3, its salt, or an isomer thereof:

[0411] In some embodiments, the cofactor lipid comprises a phospholipid (phospholipid). Phospholipids are typically semi-synthetic, but may also be of natural origin or chemically modified. In one optional specific example, the cofactor lipid is a phospholipid. In some embodiments, the phospholipid comprises one or more of the following: DSPC (distearylphosphatidylcholine), DOPE (dioleoylphosphatidylethanolamine), DOPC (dioleoyllecithin), DOPS (dioleoylphosphatidylserine), DSPG (1,2-octacosanoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol)), DPPG (dispalmitoylphosphatidylglycerol), DPPC (dispalmitoylphosphatidylcholine), DGTS (1,2-dispalmitoyl-sn-glycerol-3-O-4'-(N,N,N-trimethyl)homoserine), and lysophospholipids. In some embodiments, the cofactor lipid is selected from one or more of the following: DSPC, DOPE, DOPC, and DOPS. In some implementations, the assisting lipids are DSPC and / or DOPE.

[0412] In some embodiments, the structural lipid comprises sterols. In one optional specific example, the structural lipid is a sterol. In some embodiments, the sterol comprises one or more of the following: 20α-hydroxycholesterol, cholesterol, cholesterol esters, sterol hormones, sterol vitamins, bile acids, ergosterol, β-sitosterol, and oxidized cholesterol derivatives. In some embodiments, the structural lipid comprises at least one of cholesterol, cholesterol esters, sterol hormones, sterol vitamins, and bile acids. In some embodiments, the structural lipid is cholesterol. In one optional specific example, the structural lipid is high-purity cholesterol, particularly injectable high-purity cholesterol, such as CHO-HP (produced by AVT). In other embodiments, the structural lipid is 20α-hydroxycholesterol.

[0413] Polymer-lipid conjugates are complexes comprising a polymer and a lipid coupled to that polymer. Polymer-lipid conjugates (e.g., polyethylene glycol-lipid conjugates) in LNPs can improve the stability of LNPs in vivo.

[0414] In some embodiments, the lipids used to form the polymer-lipids of LNPs include one or more of the following: 1,2-dimyristoyl-sn-glycerol (DMG), distearoyl-phosphatidyl-ethanolamine (DSPE), diacylglycerol (DAG), dialkyloxypropyl (DAA), phospholipids, ceramide (Cer), 1,2-distearoyl-rac-glycerol (DSG), and 1,2-dipalmitoyl-rac-glycero (DPG).

[0415] In some embodiments, the polymer-lipid polymers used to form LNPs include one or two of the following: hydrophilic polymers and amphoteric polymers.

[0416] In some embodiments, the polymer-lipid polymer used to form LNPs is a hydrophilic polymer. In other embodiments, the polymer-lipid polymer used to form LNPs is a zwitterionic polymer.

[0417] In some embodiments, the hydrophilic polymer includes one or more of the following: polyethylene glycol (PEG), poly(oxazolines) (POX), poly(glycerols) (PGs), poly(hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), polyvinylpyrrolidone (PVP), poly(N,N-dimethyl acrylamide) (PDMA), poly(N-acryloyl morpholine) (PAcM), polyamino acids, glycosaminoglycans (GAGs), heparin, and hyaluronic acid. The following are listed: HA (acid), polysialic acid (PSA), elastin-like polypeptides (ELPs), serum albumin, and CD47.

[0418] Correspondingly, polymer-lipids include one or more of the following: polyethylene glycol-lipids (PEG-lipids), polyoxazoline-lipids, polyglycerol-lipids, polyhydroxypropyl methacrylate-lipids, poly-2-hydroxyethyl methacrylate-lipids, poly-N-(2-hydroxypropyl)methacrylamide-lipids, polyvinylpyrrolidone-lipids, poly-N,N-dimethylacrylamide-lipids, poly-N-acryloylmorpholine-lipids, glycosaminoglycan-lipids, heparin-lipids, hyaluronic acid-lipids, polysialic acid-lipids, elastin-like lipids, serum albumin-lipids, and CD47-lipids. It should be noted that "PEG-lipids" are conjugates of polyethylene glycol and lipids, "polyoxazoline-lipids" refer to conjugates formed by coupling polyoxazoline and lipids, and "polyglycerol-lipids" refer to conjugates formed by coupling polyglycerol and lipids; the same applies to other polymer-lipids. In an optional specific example, the hydrophilic polymer includes polyethylene glycol.

[0419] In some embodiments, the polymer-lipid comprises a PEG-lipid. In one optional specific example, the polymer-lipid is a PEG-lipid. In some embodiments, the PEG-lipid comprises one or more of the following: myristoyl glycerol-PEG (DMG-PEG), distearate phosphatidylethanolamine-PEG (DSPE-PEG), diacylglycerol-PEG (DAG-PEG), dialkyloxypropyl-PEG (DAA-PEG), phospholipid-PEG, ceramide-PEG (Cer-PEG), 1,2-distearate-rac-glycerol-PEG (DSG-PEG), and 1,2-dispalmitoyl-rac-glycerol-PEG (DPG-PEG). The PEG-lipid is preferably DMG-PEG, DSG-PEG, or DPG-PEG. DMG-PEG is a polyethylene glycol derivative of 1,2-dimyristoyl glycerol. In some embodiments, the average molecular weight of the PEG in the PEG-lipid is about 2000 to 5000. In one optional specific example, the average molecular weight of PEG in the PEG-lipid is about 2000. In some embodiments, the amphoteric polymer includes one or more of the following: poly(carboxybetaine) (pCB), poly(sulfobetaine) (pSB), phosphobetaine-based polymers, and phosphorylcholine polymers.In some embodiments, the amphoteric polymer includes one or more of the following: poly(carboxybetaine acrylamide, pCBAA), poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), poly(methacryloyloxyethyl phosphorylcholine), poly(vinyl-pyridinio propanesulfonate), poly(carboxybetaine) based on vinylimidazole, poly(sulfobetaine) based on vinylimidazole, and poly(sulfobetaine) based on vinylpyridine.

[0420] Correspondingly, the polymer-lipid includes one or more of the following: polyhydroxybetaine-lipid, polysulfobetaine-lipid, phosphate betaine-based polymer-lipid, and phosphate choline polymer-lipid. In some embodiments, the polymer-lipid includes one or more of the following: poly(carboxybetaine acrylamide)-lipid, poly(carboxybetaine methacrylate)-lipid, poly(sulfobetaine methacrylate)-lipid, poly(methacryloyloxyethyl phosphorylcholine)-lipid, poly(vinylpyridinylpropanesulfonate)-lipid, polyvinylimidazolyl betaine-lipid, polyvinylimidazolyl sulfobetaine-lipid, and polyvinylpyridinyl sulfobetaine-lipid.

[0421] In some implementations, PEG-lipids can alleviate or prevent accelerated blood clearance (ABC).

[0422] In some implementations, LNPs contain ionizable lipids, phospholipids, cholesterol, and PEG-lipids.

[0423] In some implementations, LNPs contain compounds 2-3, phospholipids, structural lipids, and PEG-lipids.

[0424] In some implementations, LNPs contain compounds 2-3, phospholipids, cholesterol, and PEG-lipids.

[0425] In some implementations, LNPs contain compounds 2-3, DSPC, cholesterol, and DMG-PEG.

[0426] In some embodiments, LNPs comprise ionizable lipids, accessory lipids, structural lipids, and polymer-lipids, wherein ionizable lipids comprise 25 mol% to 75 mol% of the total lipids present in the LNPs, accessory lipids comprise 0 mol% to 45 mol% of the total lipids present in the LNPs, structural lipids comprise 0 mol% to 60 mol% of the total lipids present in the LNPs, and polymer-lipids comprise 0.5 mol% to 5 mol% of the total lipids present in the LNPs.

[0427] In some embodiments, ionizable lipids account for 30 mol% to 65 mol%, 30 mol% to 60 mol%, 35 mol% to 60 mol%, 40 mol% to 60 mol%, 45 mol% to 55 mol% or 50 mol% to 55 mol% of the total lipids present in LNPs.

[0428] In some embodiments, the accessory lipids (e.g., DSPC) account for 1 mol% to 40 mol%, 5 mol% to 40 mol%, 5 mol% to 35 mol%, 5 mol% to 30 mol%, 5 mol% to 25 mol%, or 5 mol% to 15 mol% of the total lipids present in the LNPs.

[0429] In some embodiments, structural lipids (e.g., cholesterol) account for 1 mol% to 60 mol%, 1 mol% to 55 mol%, 5 mol% to 55 mol%, 10 mol% to 50 mol%, 15 mol% to 50 mol%, 15 mol% to 45 mol%, 20 mol% to 45 mol%, or 25 mol% to 40 mol% of the total lipids present in LNPs.

[0430] In some embodiments, the polymer-lipid (e.g., PEG-lipid) accounts for 0.5 mol% to 4.5 mol%, 1 mol% to 4.5 mol%, 1 mol% to 4 mol%, 1.5 mol% to 4 mol%, 1.5 mol% to 3.5 mol%, or 1.5 mol% to 3 mol% of the total lipids present in the LNPs.

[0431] In some embodiments, ionizable lipids account for 45 mol% to 55 mol% of the total lipids present in LNPs, phospholipids account for 5 mol% to 25 mol% of the total lipids present in LNPs, structural lipids account for 25 mol% to 45 mol% of the total lipids present in LNPs, and PEG-lipids account for 1 mol% to 4.5 mol% of the total lipids present in LNPs.

[0432] In some implementations, the total lipids present in LNPs consist of ionizable lipids, accessory lipids, structural lipids, and polymer-lipids.

[0433] In some embodiments, the pharmaceutical composition comprises an mRNA of any of the above embodiments, the mRNA being formulated within lipid nanoparticles.

[0434] In some embodiments, the pharmaceutical composition comprises multiple mRNAs of any of the above embodiments, with the multiple mRNAs co-formulated within lipid nanoparticles (i.e., a single lipid nanoparticle contains (e.g., encapsulates) multiple mRNAs).

[0435] In some embodiments, the pharmaceutical composition comprises multiple mRNAs of any of the above embodiments, with each mRNA individually formulated within a lipid nanoparticle (i.e., a single lipid nanoparticle contains (e.g., encapsulates) a nucleic acid).

[0436] VIII. Application

[0437] In addition, this disclosure also provides the use of any of the above-described embodiments of a TfR1 binding protein, any of the above-described embodiments of mRNA, any of the above-described embodiments of a fusion protein, any of the above-described embodiments of a multispecific antibody, any of the above-described embodiments of a conjugate, any of the above-described embodiments of a chimeric antigen receptor, any of the above-described embodiments of DNA, any of the above-described embodiments of nucleic acid, any of the above-described embodiments of a genetic engineering vector, any of the above-described embodiments of a host cell, any of the above-described embodiments of an isolated immune cell, or any of the above-described embodiments of a pharmaceutical composition in the preparation of a medicament for the prevention or treatment of a disease.

[0438] In some implementations, the disease is associated with TfR1. For example, tumors or cancers with high TfR1 expression.

[0439] In some implementations, the disease is a neurological disease. In some implementations, the neurological disease is as described above.

[0440] In some implementations, the neurological disease is one or more of the following: Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman's syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, neurological cancers, and traumatic brain injury.

[0441] 1. Methods for transporting active molecules across the blood-brain barrier

[0442] This disclosure also provides a method for transporting an active molecule across the blood-brain barrier, the method comprising administering to a subject any of the following embodiments: a TfR1-binding protein, mRNA, fusion protein, multispecific antibody, conjugate, DNA, nucleic acid, genetically engineered vector, or pharmaceutical composition thereof. It is understood that the active molecule in this method is capable of or has been conjugated to a TfR1-binding protein to enable the active molecule to be transported across the blood-brain barrier.

[0443] In some embodiments, the method of transporting an active molecule across the blood-brain barrier includes administering to a subject a pharmaceutical composition comprising the mRNA of any of the embodiments described above. The mRNA comprises a polynucleotide encoding a TfR1-binding protein and a polynucleotide encoding the active molecule. Upon translation, the mRNA forms a fusion protein in which the TfR1-binding protein or its antigenic fragment is conjugated to the active molecule. Under the action of the TfR1-binding protein or its antigenic fragment, the active molecule is transported from the bloodstream to brain tissue, thereby achieving crossing of the blood-brain barrier.

[0444] In some embodiments, the method of transporting the active molecule across the blood-brain barrier includes administering to a subject any of the above-described embodiments of mRNA formulated in lipid nanoparticles. The mRNA contains a polynucleotide encoding a TfR1-binding protein and a polynucleotide encoding the active molecule. Upon in vivo translation, the mRNA forms a fusion protein in which the TfR1-binding protein or its antigenic fragment is conjugated to the active molecule, thereby achieving passage across the blood-brain barrier. It is understood that in some embodiments, the polynucleotide encoding the TfR1-binding protein and the polynucleotide encoding the active molecule can be on different mRNAs, as long as the translated TfR1-binding protein can conjugate to the active molecule. Furthermore, the mRNA containing the polynucleotide encoding the TfR1-binding protein and the mRNA containing the polynucleotide encoding the active molecule may not be in the same lipid nanoparticle, as long as the TfR1-binding protein or its antigenic fragment can conjugate to the active molecule.

[0445] In some embodiments, the method of transporting the active molecule across the blood-brain barrier includes administering to a subject a DNA or genetically engineered vector capable of being transcribed into mRNA as described in any of the above embodiments. Upon transcription and translation, this DNA or genetically engineered vector can produce a fusion protein in which a TfR1-binding protein or its antigenic fragment is conjugated to the active molecule, thus also enabling crossing of the blood-brain barrier.

[0446] In some embodiments, the method of transporting the active molecule across the blood-brain barrier includes administering to a subject in need a conjugate containing the active molecule conjugated in any of the above embodiments, or a fusion protein containing the active molecule conjugated in any of the above embodiments. The conjugate or fusion protein containing the active molecule and a TfR1-binding protein or a multispecific antibody can bind to TfR1, thereby utilizing the TfR1 iron transport mechanism to transport the active molecule from the blood to brain tissue, achieving cross-brain barrier transport.

[0447] In some embodiments, the method of transporting the active molecule across the blood-brain barrier includes administering the active molecule and administering a multispecific antibody from any of the above embodiments to a subject in need, wherein the active molecule is capable of conjugating to the multispecific antibody. Following administration, the active molecule is able to bind to the multispecific antibody, thereby enabling it to cross the blood-brain barrier after the multispecific antibody has bound to TfR1.

[0448] 2. Methods for preventing or treating diseases

[0449] This disclosure provides a method for preventing or treating a disease, the method comprising administering to a subject in need an effective dose of mRNA of any of the above embodiments, fusion protein of any of the above embodiments, multispecific antibody of any of the above embodiments, conjugate of any of the above embodiments, chimeric antigen receptor of any of the above embodiments, DNA of any of the above embodiments, nucleic acid of any of the above embodiments, genetically engineered vector of any of the above embodiments, or pharmaceutical composition of any of the above embodiments.

[0450] This method involves administering an active molecule for treating a disease, the active molecule being capable of or already conjugated to a TfR1-binding protein that transports it, thereby enabling the active molecule for treating the disease to be transported to a target region (e.g., brain tissue, tumor tissue) to achieve the therapeutic purpose. For example, when administering a fusion protein or conjugate of any of the above embodiments, the active molecule is already conjugated to a TfR1-binding protein. After administration, the fusion protein or conjugate carries the active molecule to the target region, thereby allowing the active molecule to exert its effect. As another example, when administering a TfR1-binding protein or multispecific antibody of any of the above embodiments, it must be capable of conjugating to the active molecule for treating the disease. Of course, when the active molecule is not already conjugated to a TfR1-binding protein, the active molecule and the TfR1-binding protein or mRNA that can be translated into a TfR1-binding protein can be administered separately (e.g., the TfR1-binding protein or mRNA that can be translated into a TfR1-binding protein precedes the active molecule).

[0451] In some implementations, the disease is a neurological disease, and the active molecule is a substance used to treat the neurological disease.

[0452] In some embodiments, the neurological disease is as described above. In some embodiments, the active molecule is as described above.

[0453] In some implementations, the disease is a tumor or cancer, and the active molecule is a substance used to treat the tumor or cancer.

[0454] This disclosure also provides a method for preventing or treating a disease, the method comprising administering an effective dose of the TfR1 binding protein of any of the above embodiments to a subject in need.

[0455] This method uses a TfR1-binding protein as a therapeutic protein to treat TfR1-related diseases, such as tumors or cancer.

[0456] It is understood that the frequency and dosage of administration of the above-mentioned TfR1 binding protein, mRNA, fusion protein, multispecific antibody, conjugate, DNA, nucleic acid, genetically engineered vector, or pharmaceutical composition should take into account factors including the specific disease to be treated, the specific mammal to be treated, the individual patient's clinical condition, the cause of the disease, the delivery site of the reagent, the method of administration, the timing of administration, and other factors known to the medical practitioner. The effective amount administered depends on the amount of TfR1 binding protein present in or after translation of the formulation, the type of disease or treatment, and other factors discussed above, and can be used at any dose and via any route determined empirically / clinically.

[0457] 3. Increase the content of active molecules in brain tissue

[0458] This disclosure also provides a method for increasing the content of active molecules in brain tissue, the method comprising administering a TfR1 binding protein of any of the above embodiments, mRNA of any of the above embodiments, a fusion protein of any of the above embodiments, a multispecific antibody of any of the above embodiments, a conjugate of any of the above embodiments, DNA of any of the above embodiments, nucleic acid of any of the above embodiments, a genetically engineered vector of any of the above embodiments, or a pharmaceutical composition of any of the above embodiments. The active molecule is capable of or has been conjugated to a TfR1 binding protein that transports it across the blood-brain barrier, thereby enabling the active molecule to be transported to brain tissue, thereby increasing the content of the active molecule in the brain tissue.

[0459] 4. Increase the content of active molecules in tumor or cancerous tissue.

[0460] This disclosure also provides a method for increasing the content of active molecules in tumor tissue or cancerous tissue, the method comprising administering a TfR1 binding protein of any of the above embodiments, mRNA of any of the above embodiments, a fusion protein of any of the above embodiments, a multispecific antibody of any of the above embodiments, a conjugate of any of the above embodiments, DNA of any of the above embodiments, nucleic acid of any of the above embodiments, a genetic engineering vector of any of the above embodiments, or a pharmaceutical composition of any of the above embodiments. The active molecule can be or has been conjugated to the TfR1 binding protein, and the tumor tissue or cancerous tissue highly expresses TfR1, thus the active molecule can be transported to the tumor tissue or cancerous tissue, thereby increasing the content of the active molecule in the tumor tissue or cancerous tissue and improving the efficiency of tumor or cancer treatment.

[0461] IX. Test Reagents

[0462] 1. TfR1 detection

[0463] As described above, the TfR1-binding protein of any of the above embodiments is capable of binding TfR1. Therefore, it can be used for the detection of TfR1 levels, for example, using ELISA for TfR1 level detection. Therefore, this disclosure also provides a detection reagent comprising the TfR1-binding protein of any of the above embodiments.

[0464] In addition, a method for detecting the content of TfR1 is provided, which uses the detection reagents of any of the above embodiments. In some embodiments, the method for detecting the content of TfR1 is based on ELISA.

[0465] 2. Development

[0466] As described above, the TfR1 binding protein has the function of transporting active molecules across the blood-brain barrier, and the active molecules can be contrast agents. Therefore, this disclosure also provides a detection reagent for contrast agent formation, which comprises the TfR1 binding protein of any of the above embodiments, and a contrast agent that has been or can be conjugated to the TfR1 binding protein.

[0467] Furthermore, this disclosure also provides a method for development, which includes using a detection reagent for development of an object according to any of the above embodiments on an object requiring development. Example

[0468] To make the objectives and technical solutions of this disclosure clearer, the following detailed description is provided in conjunction with specific embodiments. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the reagents and instruments used in the embodiments are conventionally selected in the art. Experimental methods not specifying specific conditions in the embodiments are implemented according to conventional conditions, such as those described in literature, books, or methods recommended by the manufacturer.

[0469] In the following examples, recombinant human transferrin receptor 1 (hTfR1) was purchased from Sinocare Pharmaceutical Co., Ltd. (catalog number 11020-H07H), recombinant human transferrin (hTF) was purchased from Sinocare Pharmaceutical Co., Ltd. (catalog number 11019-H08H), recombinant cynomolgus monkey transferrin receptor 1 (cynoTfR1 for short) was purchased from Sinocare Pharmaceutical Co., Ltd. (catalog number 90253-C07H), Anti-HEL IgG1 was purchased from Baiying Biotechnology Co., Ltd. (catalog number B117901), and hTfR-KI mice (8-10 weeks old) were purchased from Nanmo Biotechnology Co., Ltd. (NM-HU-215059).

[0470] Example 1: Screening of single-domain antibodies (VHH) combining TfR1

[0471] 1. Alpaca Immunity

[0472] One 1.5-year-old unvaccinated male alpaca (Vicugna pacos) was selected and injected with recombinant human transferrin receptor 1 (hTfR1) immunogen every two weeks. For the first immunization, 300 μg of immunogen was mixed with an equal volume of complete Freund's adjuvant to prepare an emulsion. For the second to fifth immunizations, 300 μg of immunogen was mixed with an equal volume of incomplete Freund's adjuvant to prepare an emulsion.

[0473] 2. Construction of an immune phage library

[0474] Before each animal immunization, 5 mL of blood was collected, allowed to coagulate at room temperature for 2 hours, and then centrifuged at 3000g for 5 minutes. The supernatant was collected as serum and stored at -20℃ for antibody titer determination. Seven days after the last immunization, 50 mL of blood was collected into an anticoagulant tube and analyzed using Ficoll-Paque. TM Peripheral blood mononuclear cells (PBMCs) were extracted using PLUS Media (purchased from GE). Total RNA was extracted from PBMCs and reverse transcribed into cDNA using the total RNA as a template. The VHH sequence was amplified by PCR and ligated into a phage display vector, which was then transformed into TG1 cells to obtain a phage library.

[0475] 3. Screening of immune phage libraries

[0476] The phage library was screened using ELISA. First, phages were added to negative selection wells pre-coated with hTF, incubated, and then transferred to positive selection wells. The positive selection wells were also pre-coated with hTF, blocked, and then excess hTfR1 or cynoTfR1 was added before being added to the negative selection wells to obtain phages. Phages binding to hTfR1 or cynoTfR1 were eluted and amplified. Finally, 96 clones were randomly selected from each of the third and fourth rounds of screening for further identification.

[0477] 4. Identification of candidate VHHs

[0478] Periplasmic proteins (without phage-soluble forms) from the 192 clones obtained in step 3 were identified by ELISA, yielding 120 positive clones. These 120 positive clones were then sequenced, resulting in 22 VHHs that bind to TfR1. The amino acid sequences of these 22 TfR1-binding VHHs are shown in Table 1-1, and the HCDR sequence numbers of the VHHs in Table 1-1 are shown in Table 1-2.

[0479] Table 1-1

[0480] The underscore indicates a CDR defined in Kabat.

[0481] Table 1-2

[0482] Example 2 Preparation of VHH-Fc fusion protein

[0483] 1. Preparation of bivalent VHH-Fc fusion protein

[0484] (1) Construction of bivalent VHH-Fc fusion protein expression plasmid: The gene sequence encoding VHH was synthesized based on the amino acid sequence of VHH. The 3' end of the VHH gene sequence was ligated to the 5' end of the gene sequence encoding human IgG1 Fc with the IgG1 hinge region by PCR amplification. Then, it was constructed into the eukaryotic expression vector pcDNA3.1 by homologous recombination. After being transformed into DH5α competent cells, it was plated and cultured. Single clones were selected for sequencing. The single clones with correct sequencing were cultured overnight at 37°C. Then, the plasmid was extracted using an endotoxin-free plasmid extraction kit (purchased from Qiagen) to prepare the bivalent VHH-Fc fusion protein expression plasmid.

[0485] The amino acid sequence of the IgG1 Fc segment connecting the human IgG1 hinge region (underlined is the human IgG1 hinge region):

[0486] (2) Expression and purification of fusion protein

[0487] The bivalent VHH-Fc fusion protein expression plasmid extracted in (1) was transfected into the human embryonic kidney cell 293 cell line (Expi293F, purchased from Thermo Fisher Scientific). The antibody protein was then purified from the cell culture supernatant using a Protein A affinity chromatography column. After testing, the bivalent VHH-Fc fusion protein was obtained. The bivalent VHH-Fc fusion protein was named using the VHH designation, for example, AT9-1-36.

[0488] It refers to the bivalent AT9-1-36VHH-Fc fusion protein.

[0489] 2. Preparation of monovalent VHH-scFc fusion protein

[0490] (1) Construction of monovalent VHH-scFc fusion protein expression plasmid: The 3' end of the VHH gene sequence was amplified by PCR and ligated to the 5' end of the gene sequence encoding scFc (two human IgG1 Fc cells with hinge regions connected by a linker). Then, the recombinant protein expression vector pcDNA3.1 was constructed by homologous recombination. The constructed recombinant protein expression vector was transformed into DH5α competent cells for plate culture. Single clones were selected for sequencing. The single clones with correct sequencing were cultured overnight at 37°C. Then, the plasmid was extracted using an endotoxin-free plasmid extraction kit (purchased from Qiagen) to prepare the monovalent VHH-scFc fusion protein expression plasmid.

[0491] The amino acid sequence of scFc connected to the hinge region (underlined region, wavy line connector):

[0492] (2) The monovalent VHH-scFc fusion protein expression plasmid extracted in (1) was transfected into the human embryonic kidney cell 293 cell line (Expi293F, purchased from Thermo Fisher Scientific) and cultured. The antibody protein was then purified from the cell culture supernatant using a protein A affinity chromatography column. After detection, the monovalent VHH-scFc fusion protein was obtained.

[0493] 3. Monovalent VHH-IgG4 Fc fusion protein

[0494] (1) Construction of monovalent VHH-IgG4 Fc fusion protein expression vector: The corresponding gene sequences were synthesized based on the amino acid sequences of VHH, IgG4 Fc Ks with hinge region, and IgG4 Fc Hs with hinge region. The VHH gene sequence was ligated to the 5' or 3' end of the gene sequence encoding IgG4 Fc Ks with hinge region by PCR amplification, and then constructed into the eukaryotic expression vector pcDNA3.1 by homologous recombination; the gene sequence encoding IgG4 Fc Hs with hinge region was constructed into the eukaryotic expression vector pcDNA3.1 by homologous recombination. The constructed recombinant protein expression vector was transformed into DH5α competent cells and plated for culture. Single clones were selected for sequencing. The single clones with correct sequencing were cultured overnight at 37°C. Then, plasmids were extracted using an endotoxin-free plasmid extraction kit (purchased from Qiagen) to obtain expression plasmids containing gene sequences encoding IgG4 Fc Hs with hinge regions and gene sequences containing VHH and IgG4 Fc Ks with hinge regions.

[0495] The amino acid sequence of IgG4 Fc Ks with hinge regions (underlined regions):

[0496] The amino acid sequence of IgG4 Fc Hs with hinge regions (underlined regions):

[0497] (2) Expression and purification of VHH-Fc fusion protein

[0498] The expression plasmid containing the gene sequence encoding IgG4 Fc Hs with a hinge region obtained in (1) and the expression plasmid containing the gene sequence encoding VHH and IgG4 Fc Ks with a hinge region were co-transfected into the human embryonic kidney cell 293 cell line (Expi293F, purchased from Thermo Fisher Scientific). After culture, the antibody protein was purified from the cell culture supernatant using a protein A affinity chromatography column. After detection, the VHH-Fc fusion protein was obtained. The fusion protein with VHH at the N-terminus of IgG4 Fc Ks connected to the hinge region is named VHH Fc(KIH), for example, 1-42Fc(KIH) indicates that AT9-1-42 VHH is at the N-terminus of IgG4 Fc Ks connected to the hinge region; the protein with VHH at the C-terminus of IgG4 Fc Ks connected to the hinge region is named Fc(KIH)VHH, for example, Fc(KIH)1-42 indicates that AT9-1-42 VHH is at the C-terminus of IgG4 Fc Ks connected to the hinge region.

[0499] Example 3: Antigen Binding Activity Assay

[0500] 1. Competitive ELISA detection of VHH-Fc fusion protein competitively binding to hTfR1 with holo-Tf

[0501] The competitive ELISA was used to detect the competitive binding of the VHH-Fc fusion protein of Example 2 to hTfR1 with human holo-Tf (human transferrin, abbreviated as holo-Tf, purchased from R&D, catalog number 2914-HT).

[0502] The results are shown in Figures 1A to 1I.

[0503] As shown in Figures 1A to 1I, AT9-1-44, AT9-1-89, and 1-69Fc(KIH) compete with human holo-Tf for binding to hTfR1.

[0504] 2. FACS detection of antigen-binding activity of VHH-Fc fusion protein

[0505] The CHO-K1 human TFRC cell line was constructed using the CHO-K1 cell line. This CHO-K1 human TFRC cell line expresses hTfR1 and was used to detect the binding of the VHH-Fc fusion protein from Example 2 to hTfR1 on the cell surface. Similarly, the CHO-K1 cyno TFRC cell line was constructed using the CHO-K1 cell line. This CHO-K1 cyno TFRC cell line expresses cynoTfR1 and was used to detect whether the antibody bound to cynoTfR1 on the cell surface. Furthermore, the binding of the VHH-Fc fusion protein from Example 2 to CHO-K1 cells, HepG2 cells, and 293T cells was also detected. CHO-K1 cells themselves do not express hTfR1 or cynoTfR1, while the HepG2 and 293T cells used expressed hTfR1. The steps for detecting the antigen-binding activity of the VHH-Fc fusion protein using FACS included:

[0506] (1) Resuspend the cells and adjust the cell density to 2.0 × 10⁻⁶. 6 Add 100 μL / well to a 96-well plate.

[0507] (2) Centrifuge at 300g and discard the supernatant. Resuspend the cells in 2.5% FBS-PBS and wash twice.

[0508] (3) Add serially diluted candidate antibody, resuspend cells at 100 μL / well, and incubate at room temperature in the dark for 30 min. Centrifuge at 300 g for 5 min, discard the supernatant, resuspend cells in 2.5% FBS-PBS, and wash twice.

[0509] (4) Add APC-labeled anti-human IgG Fc flow cytometry antibody (Biolegend, 410712), resuspend cells at 100 μL / well, and incubate at room temperature in the dark for 30 min. Centrifuge at 300 g for 5 min, discard the supernatant, and wash twice.

[0510] (5) Resuspend cells in 2.5% FBS-PBS and detect fluorescence intensity using flow cytometer.

[0511] The test results are shown in Figures 2A-2B, 3A-3C, 4A-4D, 5A-5D, and 6A-6D.

[0512] As shown in Figures 2A and 2B, except for AT9-2-13, other bivalent VHH-Fc fusion proteins can bind to 293T cells and HepG2 cells expressing hTfR1.

[0513] As shown in Figures 3A-3C, except for AT9-1-34 which binds nonspecifically to CHO-K1 cells, the other bivalent VHH-Fc fusion proteins can specifically bind to both CHO-K1 human TFRC and CHO-K1 cyno TFRC cells, exhibiting cross-species binding activity between humans and monkeys. Among them, AT9-2-13 binds relatively weakly to CHO-K1 human TFRC cells, while AT9-2-13, AT9-2-64, and AT9-2-92 bind relatively weakly to CHO-K1 cyno TFRC cells.

[0514] As shown in Figures 4A-4D, 5A-5D, and 6A-6D, the monovalent VHH-IgG4 Fc fusion protein exhibited binding activity in both 293T cells and CHO-K1 human TFRC cells. For CHO-K1 cyno TFRC cells, except for the monovalent VHH-IgG4 Fc fusion proteins constructed using AT9-1-7VHH, AT9-1-24VHH, AT9-1-39VHH, AT9-1-76VHH, AT9-1-84VHH, AT9-1-90VHH, AT9-2-64VHH, and AT9-2-92 VHH, which showed extremely weak binding ability, most fusion proteins still exhibited significant binding activity.

[0515] 3. Detection of antigen-binding activity of VHH-Fc fusion protein based on BLI technology

[0516] The binding activity of the VHH-Fc fusion protein of Example 2 to hTfR1 and cynoTfR1 was determined using OctetRED96 (Sartorius). Specific steps included:

[0517] (1) Dilute the proteins to be tested serially with SD buffer (1×PBS with 2% BSA and 0.02% Tween 20, pH 7.4). Dilute hTfR1 and cynoTfR1 with SD buffer to 10 μg / mL.

[0518] (2) Add the diluted sample solution to a 96-well black ELISA plate. Add SD buffer to the baseline, dissociation, and neutralization wells. Add 10 mM glycine-hydrochloric acid (pH 1.7) to the regeneration wells. The biosensor used is HIS1K (Sartorius, catalog number 18-5120).

[0519] (3) The OctetRED96 was used to test under the conditions specified in Table 2. After obtaining the data, the OctetRED96 analysis software was used to fit the binding and dissociation curves according to the 1:1 binding model to obtain the binding rate constant (Kon), dissociation rate constant (Koff), and dissociation constant (KD) of the antibody with hTfR1 and cynoTfR1.

[0520] The results are shown in Tables 3 and 4. Table 3 shows the binding activity of the VHH-Fc fusion protein with hTfR1, and Table 4 shows the binding activity of the VHH-Fc fusion protein with cynoTfR1. In this paper, among the parameters used to represent the antibody-antigen binding activity, KD is the dissociation constant, Kon is the binding rate constant, and Koff is the dissociation rate constant.

[0521] As shown in Tables 3 and 4, most bivalent VHH-Fc fusion proteins exhibit cross-binding activity to hTfR1 and cynoTfR1, but no binding was detected between AT9-2-13 and either hTfR1 or cynoTfR1, and no binding was detected between AT9-2-92 and cynoTfR1.

[0522] Most monovalent VHH-IgG4 Fc fusion proteins exhibit cross-binding activity to hTfR1 and cynoTfR1 proteins, but some monovalent VHH-IgG4 Fc fusion proteins do not bind to cynoTfR1.

[0523] Table 2

[0524] Table 3

[0525] ND indicates not detected; the same applies below.

[0526] Table 4

[0527] Example 4 Affinity Modification

[0528] The amino acid sequences of CDR3 in Fc(KIH)1-36 and Fc(KIH)1-61 are as follows:

[0529] The CDR3 amino acid sequence of AT9-1-36VHH is: DPRIEAAWGTTWYRASTYEYDY (SEQ ID NO:89).

[0530] The CDR3 amino acid sequence of AT9-1-61VHH is: TKTYYHGTYNSPPAENHFYY (SEQ ID NO: 101). The amino acids of the CDR3 in the VHH domain of Fc(KIH)1-36 and Fc(KIH)1-61 were sequentially mutated to alanine to obtain multiple VHH domains with different affinities due to alanine mutation in CDR3 (SEQ ID NO: 23-60). Referring to Example 2, based on the VHH domains after the above-mentioned alanine mutation in CDR3, Fc(KIH)1-36 and Fc(KIH)1-61 with alanine mutation in CDR3 were prepared to obtain alanine mutants of Fc(KIH)1-36 and Fc(KIH)1-61. The HCDR sequence numbers of the alanine mutants of Fc(KIH)1-36 and Fc(KIH)1-61 are shown in Table 5.

[0531] Table 5

[0532] Referring to Example 3, the binding activities of the mutated Fc(KIH)1-36 and Fc(KIH)1-61 antibodies with hTfR1 and cynoTfR1 were determined. The results are shown in Tables 6-1 and 6-2. Table 6-1 shows the binding activities of the mutated Fc(KIH)1-36 and Fc(KIH)1-61 antibodies with hTfR1, and Table 6-2 shows the binding activities of the mutated Fc(KIH)1-36 and Fc(KIH)1-61 antibodies with cynoTfR1.

[0533] As shown in Tables 6-1 and 6-2, most alanine mutants of Fc(KIH)1-36 exhibit reduced binding affinity to hTfR1 and cynoTfR1 compared to their parent lines. Among them, alanine mutants with affinity close to hTfR1 and cynoTfR1 include Fc(KIH)1-36 R101A, Fc(KIH)1-36 W110A, Fc(KIH)1-36 R112A, Fc(KIH)1-36 S114A, and Fc(KIH)1-36 D119A. Alanine mutants of Fc(KIH)1-61 show reduced binding affinity to hTfR1 and cynoTfR1 compared to their parent lines, and no alanine mutants exhibited affinity comparable to hTfR1 and cynoTfR1.

[0534] Table 6-1

[0535] Table 6-2

[0536] Example 5: Fusion protein of IDS and hTfR1 protein

[0537] 1. Preparation of a fusion protein of IDS and an antibody binding to hTfR1

[0538] Referring to Example 2, gene sequences were synthesized based on the amino acid sequences of VHH of Fc(KIH)1-36 R101A and iduronic acid 2-sulfatase (IDS). The VHH gene sequence of Fc(KIH)1-36 R101A was ligated to the 5' or 3' end of the gene sequence encoding IgG4 Fc Ks with a hinge region. The IDS gene sequence was then ligated to the 5' or 3' end of the above gene sequences (or the IDS gene sequence was not introduced). These sequences were then constructed into the eukaryotic expression vector pcDNA3.1 via homologous recombination. Similarly, the IDS gene sequence was ligated to the 5' or 3' end of the IgG4 Fc Hs gene sequence (or the IDS gene sequence was not introduced), and these sequences were constructed into the eukaryotic expression vector pcDNA3.1 via homologous recombination. The constructed recombinant protein expression vectors were transformed into *E. coli* DH5α and cultured overnight at 37°C. Plasmids were then extracted using an endotoxin-free plasmid extraction kit (Qiagen), and expressed in human embryonic kidney cell line 293F. After purification and detection, a fusion protein containing IgG4 Fc, IDS, and an antibody binding to hTfR1 was obtained. The fusion protein is named as follows:

[0539] Fc(KIH)1-36 R101A IDS CO1: The C-terminus of Fc(KIH)1-36 R101A IgG4 Fc Hs has one IDS fused to it;

[0540] Fc(KIH)1-36 R101A IDS N01: The N-terminus of IgG4 Fc Hs of Fc(KIH)1-36 R101A is fused with one IDS;

[0541] Fc(KIH)1-36 R101A IDS N10: The N-terminus of Fc(KIH)1-36 R101A IgG4 Fc Ks is fused with one IDS;

[0542] Fc(KIH)1-36 R101A IDS N11: Fc(KIH)1-36 R101A IgG4 Fc Ks and Hs each have one IDS fused to their N-terminus.

[0543] The amino acid sequence of the IDS is as follows:

[0544] 2. Assay for antigen binding activity

[0545] (1) Referring to Example 3, the binding activity of the fusion protein to hTfR1 was detected by OctetRED96 (Sartorius Corporation, working conditions are shown in Table 2) using BLI technology, and the results are shown in Table 7.

[0546] As shown in Table 7, the affinity of Fc(KIH)1-36 R101A fused with IDS for hTfR1 was not significantly affected.

[0547] Table 7

[0548] (2) Referring to Example 3, the binding of the fusion protein to CHO-K1 cells, CHO-K1 human TFRC cells and CHO-K1 cyno TFRC cells was detected by FACS. The results are shown in Figures 7A to 7C.

[0549] As shown in Figures 7A-7C, the Fc(KIH)1-36 R101A fused with IDS still maintains its binding with CHO-K1 human TFRC cells and CHO-K1cyno TFRC cells.

[0550] Example 6: Brain Penetration Performance and Pharmacokinetics Study

[0551] Mice whose extracellular region gene encoding the mouse transferrin receptor was replaced with the extracellular region gene of the human transferrin receptor gene were called hTfR-KI mice (purchased from Nanmo Biotechnology). These mice were used to evaluate antibody translocation into the brain by crossing the blood-brain barrier (BBB).

[0552] Multiple antibodies binding to hTfR1 (the fusion protein prepared in Example 2 and the mutant prepared in Example 4) were administered intravenously to hTfR-KI mice (8-10 weeks old) at a dose of 77 nmol / kg. Whole blood was collected from the mice at 0.25 h, 1 h, 4 h, and 24 h post-injection in anticoagulant tubes for analysis. Approximately 24 h after intravenous injection, the mice were perfused with PBS, and brain tissue was collected. After determining the wet weight of brain tissue, PBS containing twice the wet weight of the brain was added and homogenized. The homogenate was divided into two portions. One portion was mixed with an equal volume of T-per (Thermo) containing a mixture of protease inhibitors (Sigma), centrifuged, and the supernatant was collected and labeled as whole brain lysis buffer. The other portion was mixed with an equal volume of 30% dextran (Sigma), vortexed thoroughly, centrifuged at 5400g for 15 minutes, and the upper brain parenchyma was collected. T-per (Thermo) containing a mixture of protease inhibitors (Sigma) was added, mixed, centrifuged, and the supernatant was collected and labeled as brain parenchyma lysis buffer.

[0553] The levels of antibodies binding to hTfR1 in the blood samples, whole brain lysates, and brain parenchyma lysates were determined using an enzyme-linked immunosorbent assay kit for human immunoglobulin G Fc fragment (Fcγ) (Elabscience). The results are shown in Figures 8A to 8C.

[0554] As shown in Figures 8A-8C, the monovalent Fc(KIH)1-75 has a higher exposure in brain parenchyma than the bivalent AT9-1-75, indicating that the monovalent hTfR1 antibody may be more likely to penetrate the blood-brain barrier. The Fc(KIH)1-36 R101A and Fc(KIH)1-36 R101A have a higher exposure in brain parenchyma than Fc(KIH)1-36, indicating that relatively low affinity molecules may be more likely to penetrate the blood-brain barrier.

[0555] Example 7: Humanization Modification

[0556] 1. Human-centered design

[0557] Humanization of AT9-1-36VHH R101A and AT9-1-75VHH was performed using CDR substitution and reverse mutation to obtain the following VHHs, with the underlined CDRs defined in Kabat:

[0558] hu 1-36 R101A 01 VHH:

[0559] hu 1-36 R101A 02 VHH:

[0560] hu 1-36 R101A 03 VHH:

[0561] hu 1-36 R101A 11 VHH:

[0562] hu 1-36 R101A 12 VHH:

[0563] hu 1-36 R101A 13 VHH:

[0564] hu 1-36 R101A 14 VHH:

[0565] hu 1-36 R101A 15 VHH:

[0566] hu 1-36 R101A 16 VHH

[0567] hu 1-36 R101A 17 VHH:

[0568] hu 1-36 R101A 18 VHH:

[0569] hu 1-75 01 VHH:

[0570] hu 1-75 02 VHH:

[0571] hu 1-75 03 VHH:

[0572] 2. Preparation of humanized antibodies binding to hTfR1

[0573] Referring to Example 2 (3. Monovalent VHH-IgG4 Fc Fusion Protein), a humanized antibody fused with IgG4 Fc and monovalently binding to hTfR1 was prepared based on the humanized VHH (VHH is located at the C-terminus of IgG4 Fc). The humanized antibody fused with IgG4 Fc and monovalently binding to hTfR1 was named Hu VHH, for example, Hu 1-36 R101A 01 represents the hu 1-36 R101A01VHH fused with IgG4 Fc and monovalently binding to hTfR1.

[0574] 3. Assay for antigen binding activity

[0575] (1) Referring to Example 3, the binding activity of the humanized modified antibody fused with IgG4 Fc to hTfR1 was detected using the OctetRED96 (Sartorius, operating conditions as shown in Table 2) with BLI technology. The results are shown in Table 8.

[0576] As shown in Table 8, most of the humanized antibodies of Fc(KIH)1-36 R101A and Fc(KIH)1-75 can still bind to hTfR1. Among them, the affinity of some humanized antibodies is comparable to or even slightly stronger than that of the parent antibodies, while the affinity of some humanized antibodies decreases to varying degrees.

[0577] Table 8

[0578] (2) Referring to Example 3, FACS was used to detect the binding of the humanized modified hTfR1-binding antibody to CHO-K1 cells, CHO-K1 human TFRC cells, and CHO-K1 cyno TFRC cells. The results are shown in Figures 9A to 9C.

[0579] As shown in Figures 9A to 9C, the humanized antibodies of Fc(KIH)1-36 R101A still retain their ability to bind to CHO-K1 human TFRC cells and CHO-K1 cyno TFRC cells, although the binding ability of some antibodies is weakened, such as Hu 1-36 R101A 12.

[0580] (3) Referring to Example 3, a competitive ELISA method was used to detect whether the humanized modified antibody binding to hTfR1 competed with holo-Tf for binding to hTfR1. The results are shown in Figures 10A to 10C.

[0581] As shown in Figures 10A to 10C, Fc(KIH)1-36 R101A, even after humanization, still does not compete with human holo-Tf for binding to hTfR1.

[0582] 4. Evaluation of brain penetration performance and pharmacokinetic studies

[0583] Referring to Example 6, the efficacy of humanized hTfR1-binding antibodies in penetrating the blood-brain barrier and transferring to brain tissue was evaluated and pharmacokinetic studies were conducted using hTfR-KI mice. The results are shown in Figures 11A to 11C.

[0584] As shown in Figures 11A to 11C, Fc(KIH)1-36 R101A retains its ability to penetrate the blood-brain barrier after humanization, and some antibodies have improved penetration ability, which may be related to changes in antibody affinity.

[0585] Example 8: Antibody with Extended Half-Life

[0586] 1. Preparation of half-life extended antibodies Fc(KIH)1-36 R101A YTE and Fc(KIH)1-36 R101A LS

[0587] Referring to Example 2, based on the amino acid sequences after introducing YTE or LS mutations into the IgG4 Fc Ks and Hs chains that connect the hinge regions, a monovalent VHH-IgG4 Fc fusion protein (half-life extended antibody, Fc(KIH)1-36 R101A YTE) with YTE mutations introduced into the IgG4 Fc Ks and Hs chains and a monovalent VHH-IgG4 Fc fusion protein (half-life extended antibody, Fc(KIH)1-36 R101A LS) with LS mutations introduced into the IgG4 Fc Ks and Hs chains were prepared.

[0588] The amino acid sequence of the IgG4 Fc Ks chain with the hinge region after the introduction of the YTE mutation (underlined region):

[0589] The amino acid sequence of the IgG4 Fc Hs chain with the hinge region after the introduction of the YTE mutation (underlined region):

[0590] The amino acid sequence of the IgG4 Fc Ks chain with the hinge region after the introduction of the LS mutation (underlined region):

[0591] The amino acid sequence of the IgG4 Fc Hs chain with the hinge region after the introduction of the LS mutation (underlined region):

[0592] 2. Assay for antigen binding activity

[0593] Referring to Example 3, the binding activity of the extended-life antibody to hFcRn (human neonatal Fc receptor, Sinocare, catalog number CT009-H08H) at different pH values ​​(6.0 and 7.4) was detected using the BLI technique with OctetRED96 (Sartorius, operating conditions as shown in Table 9). The results are shown in Table 10.

[0594] Table 9

[0595] Table 10

[0596] Therefore, it can be seen that the antibody binding to hTfR1 after mutation has enhanced affinity for hFcRn under pH 6.0 and pH 7.4 conditions.

[0597] 3. Pharmacokinetic Studies

[0598] FcRn humanized mice were purchased from Biocytogen, and the construction strategy involved inserting the full-length human FcRn gene into the mouse FcRn gene.

[0599] The half-life extension antibody was administered intravenously to FcRn humanized mice (8-10 weeks old) at a dose of 77 nmol / kg. Whole blood samples were collected from mice at 0.5h, 1h, 4h, 8h, 24h, 72h, 168h, and 336h post-injection. The half-life extension antibody content in the blood samples was determined using an enzyme-linked immunosorbent assay kit for human immunoglobulin G Fc fragment (Fcγ) (Elabscience). The results are shown in Figure 12.

[0600] This indicates that the mutated antibody binding to hTfR1 has a prolonged half-life in hFcRn humanized mice.

[0601] Example 9: Metabolic kinetics of the antibody binding to hTfR1 and the LNP encapsulating the mRNA encoding the antibody binding to hTfR1.

[0602] 1. Prepare a humanized antibody fused with IgG4 Fc and monovalently binding to hTfR1 according to Example 7.

[0603] 2. Preparation of mRNA-LNP

[0604] (1) mRNA preparation: A plasmid containing the gene sequence of an antibody (Hu 1-36 R101A12) binding to hTfR1 was prepared using subcloning techniques (e.g., subcloning based on PCR and restriction endonuclease digestion or in-fusion techniques). The plasmid was then linearized, transcribed in vitro, and capped to obtain the mRNA encoding the antibody binding to hTfR1. This mRNA contains Cap1, the following 5'-UTR, 3'-UTR, poly(A) tail, and a 3* microRNA binding site located between the 3'-UTR and the poly(A) tail. All uridine was replaced by N1-methylpseudouridine.

[0605] 5'-UTR:

[0606] 3'-UTR:

[0607] poly(A) tail:

[0608] 3* microRNA binding sites:

[0609] (2) Using a microsyringe and a microfluidic chip (SN.000038), the mRNA encoding the antibody that binds to hTfR1 was encapsulated at a rate of 9 mL / min in the aqueous phase and 3 mL / min in the alcohol phase to prepare lipid nanoparticles (mRNA-LNP) containing the mRNA encoding the antibody that binds to hTfR1. The aqueous phase was an acetate-sodium acetate solution (pH 5.0) containing the mRNA encoding the antibody that binds to hTfR1. The alcohol phase contained compound 2-3:DSPC:cholesterol, DMG-PEG2000 and ethanol. The molar ratio of compound 2-3, DSPC, cholesterol and DMG-PEG2000 was 49:5.2:43.3:2.5.

[0610] 3. Evaluation of brain penetration performance and pharmacokinetic studies

[0611] Referring to Example 6, hTfR-KI mice (8-10 weeks old) were intravenously injected with an antibody binding to hTfR1 at a dose of 77 nmol / kg and mRNA-LNP at a dose of 0.3 mpk. Whole blood samples were collected from the mice at 0.5 h, 8 h, 24 h, 48 h, 120 h, and 168 h after intravenous injection in anticoagulant tubes for analysis. Whole brain lysate and brain parenchyma lysate were prepared as described in Example 6 at 48 h and 168 h after intravenous injection. The content of the hTfR1-binding antibody in the blood samples, whole brain lysate, and brain parenchyma lysate was determined using an Enzyme-Linked Immunosorbent Assay Kit for Human Immunoglobulin G Fc Fragment (Fcγ) (Elabscience).

[0612] The results are shown in Figures 13A-13C. In Figures 13A-13C, "Hu 1-36 R101A 12" represents the group injected with the Hu 1-36R101A 12 antibody, and "Hu 1-36 R101A 12 mRNA-LNP" represents the group injected with lipid nanoparticles encapsulating the mRNA encoding Hu 1-36 R101A 12. As shown in Figures 13A-13C, mRNA-LNP can enable the expression of the hTfR1-binding antibody in vivo, and after 48 hours of administration, the antibody exposure of 0.3 mpk mRNA-LNP in the brain parenchyma is very high.

[0613] Example 10: Evaluation of brain penetration performance of therapeutic antibodies (Lecanemab or Donanemab) and VHH fusion proteins binding to hTfR1.

[0614] 1. Preparation of fusion proteins of Lecanemab, Donanemab, therapeutic antibodies (Lecanemab or Donanemab) and VHH bound to hTfR1:

[0615] (1) Construction of expression plasmids: Gene sequences were synthesized based on the amino acid sequences of the light and heavy chains of Lecanemab (see below), and the gene sequences corresponding to the light and heavy chains were constructed into the expression vector of pcDNA3.1 to obtain expression plasmids containing the gene sequences of the light or heavy chains of Lecanemab. In order to prepare a fusion protein of Lecanemab and hTfR1-binding VHH, and a fusion protein in which the hTfR1-binding VHH is monovalently fused to the C-terminus of the heavy chain of Lecanemab, a knock-in-holes structure is required to achieve heterodimerization of the two heavy chains of Lecanemab (one heavy chain C-terminus fused with the hTfR1-binding VHH, and the other heavy chain C-terminus without any fused protein). Specifically, an S354C / T366W chain, designated Ks, was introduced into one of the heavy chains of Lecanemab via a point mutation. Then, a VHH (hu 1-36 R101A 12 VHH, hu 1-36 R101A 17 VHH, or hu 1-75 02 VHH, amino acid sequence see Example 7) binding to hTfR1 was fused to the C-terminus of the Ks chain. The Ks chain and the hTfR1-binding VHH were linked by a (G4S)4 linker. Another heavy chain, designated Hs, was introduced with Y349C / T366S / L368A / Y407V. Finally, gene sequences were synthesized based on the amino acid sequences of each chain (see below), and the corresponding gene sequences were constructed into expression plasmids for pcDNA3.1. Similarly, a fusion protein of Donanemab and the hTfR1-binding VHH was designed, and expression plasmids were prepared.

[0616] The amino acid sequence of the heavy chain of Lecanemab:

[0617] The amino acid sequence of the light chain of Lecanemab:

[0618] The amino acid sequence of the Lecanemab Ks chain:

[0619] The amino acid sequence of the Lecanemab Hs chain:

[0620] The amino acid sequence of the heavy chain of Donanemab:

[0621] The amino acid sequence of the light chain of Donanemab:

[0622] The amino acid sequence of the Donanemab Ks chain:

[0623] The amino acid sequence of the Donanemab Hs chain:

[0624] (2) Plasmid extraction: The above expression plasmids were transformed into DH5α competent cells and plated for culture. Single clones with correct sequencing were selected and cultured overnight at 37°C. Then, plasmids were extracted using an endotoxin-free plasmid extraction kit.

[0625] (3) Protein expression and purification: Expression plasmids containing the gene sequence of the light chain of Lecanemab and the gene sequence of the heavy chain of Lecanemab were co-transfected into 293 cell lines and cultured. Lecanemab was purified from the culture supernatant using a protein A affinity chromatography column. Similarly, expression plasmids containing the gene sequence of the light chain of Donanemab and the gene sequence of the heavy chain of Donanemab were co-transfected into 293 cell lines and purified using a protein A affinity chromatography column to obtain Donanemab. Similarly, co-transfection with an expression plasmid containing the gene sequence of a fusion protein consisting of a Lecanemab Hs chain, a Lecanemab light chain, and a Lecanemab Ks chain and a VHH binding hTfR1 yielded a fusion protein of Lecanemab and an antibody binding hTfR1; co-transfection with an expression plasmid containing the gene sequence of a Donanemab Hs chain, a Donanemab light chain, and a Donanemab Ks chain and a VHH binding hTfR1 yielded a fusion protein of Donanemab and an antibody binding hTfR1.

[0626] 2. Evaluation of brain penetration performance and pharmacokinetic studies

[0627] Lecanemab and Donanemab prepared in step 1 were intravenously injected into hTfR-KI mice at a dose of 333 nmol / kg, and the fusion protein prepared in step 1 was intravenously injected into hTfR-KI mice at a dose of 67 nmol / kg. Whole blood was collected from mice at 0.5 h, 2 h, 8 h, 24 h, and 48 h after intravenous injection in anticoagulant tubes for analysis. Approximately 48 h after intravenous injection, the mice were perfused with PBS, and brain tissue was collected. Whole brain lysate and brain parenchyma lysate were prepared according to Example 6. The levels of antibodies binding to hTfR1 in the blood samples, whole brain lysate, and brain parenchyma lysate were measured using a human immunoglobulin G Fc fragment (Fcγ) enzyme-linked immunosorbent assay kit (Elabscience).

[0628] The results are shown in Figures 14A to 14C. In Figures 14A to 14C, "Lecanemab" represents the group injected with Lecanemab, "Lec*hu1-36 R101A 12" represents the fusion protein group injected with Lecanemab and hu 1-36 R101A 12 VHH, and "Don*hu 1-36 R101A12" represents the fusion protein group injected with Donanemab and hu 1-36 R101A 12 VHH, and so on.

[0629] As shown in Figures 14A-14C, the fusion proteins of Lecanemab with hu 1-36 R101A 12 VHH, hu 1-36 R101A 17 VHH, or hu 1-75 02 VHH achieved the same or even higher brain parenchyma exposure as Lecanemab at only 1 / 5 the dosage of Lecanemab, indicating that VHHs such as hu 1-36 R101A 12 VHH that bind to hTfR1 can enhance the brain penetration ability of Lecanemab. Similarly, the fusion protein of Donanemab with hu 1-36 R101A 12 VHH also achieved the same brain parenchyma exposure as Donanemab monoclonal antibody at a lower dosage, indicating that hu 1-36 R101A12 VHH can enhance the ability of Donanemab to penetrate the blood-brain barrier.

[0630] Example 11: Evaluation of LNP brain penetration performance of the encapsulated mRNA encoding the fusion protein of Lecanemab and hTfR1-binding VHH.

[0631] 1. Preparation of mRNA encoding the fusion protein of Lecanemab and an antibody binding to hTfR1, and mRNA of Lecanemab:

[0632] (1) Referring to Example 9, mRNA encoding the light chain of Lecanemab, mRNA encoding the heavy chain of Lecanemab, mRNA encoding the Ks chain of Lecanemab, and mRNA encoding a fusion protein containing the hu 1-36 R101A 12 VHH and the Lecanemab Hs LALA chain were prepared.

[0633] The amino acid sequences of the light chain, heavy chain, and Ks chain of Lecanemab are shown in Example 10. The fusion protein containing hu 1-36 R101A 12 VHH and the Lecanemab Hs LALA chain is formed by fusing hu 1-36 R101A 12 VHH (amino acid sequence shown in Example 7) to the C-terminus of the Lecanemab Hs LALA chain (amino acid sequence shown below). The Lecanemab Hs LALA chain and hu 1-36 R101A 12 VHH are linked by a (G4S)4 linker.

[0634] The amino acid sequence of the Lecanemab Hs LALA chain:

[0635] 2. Preparation of mRNA-LNP

[0636] Referring to Example 9, lipid nanoparticles were prepared by encapsulating mRNA encoding the light chain of Lecanemab and mRNA encoding the heavy chain of Lecanemab, denoted as "Lecanemab mRNA-LNP"; lipid nanoparticles were prepared by encapsulating mRNA encoding the light chain of Lecanemab, mRNA encoding the Lecanemab Ks chain, and mRNA encoding a fusion protein containing the hu1-36 R101A12 VHH and Lecanemab Hs LALA chains, denoted as "Lec*hu12 mRNA-LNP".

[0637] 3. Evaluation of brain penetration performance and pharmacokinetic studies

[0638] Lecanemab mRNA-LNP was administered intravenously to hTfR-KI mice at a dose of 1.5 mg / kg, and Lec*hu12 mRNA-LNP was administered intravenously at a dose of 0.6 mg / kg. Whole blood samples were collected from mice at 2 h, 8 h, 24 h, and 48 h post-injection in anticoagulant tubes for analysis. Approximately 48 h after injection, the mice underwent systemic perfusion with PBS, and brain tissue was collected.

[0639] Whole-brain lysate and brain parenchyma lysate were prepared according to Example 6. The levels of antibodies binding to hTfR1 in the blood samples, whole-brain lysate, and brain parenchyma lysate were measured using a human immunoglobulin G Fc fragment (Fcγ) enzyme-linked immunosorbent assay kit (Elabscience). The results are shown in Figures 15A-15C. In Figures 15A-15C, "Lecanemab" represents the Lecanemab mRNA-LNP injection group, and "Lec*hu12" represents the Lec*hu12 mRNA-LNP injection group.

[0640] As shown in Figures 15A-15C, compared with the Lecanemab mRNA-LNP group, the Lec*hu12 mRNA-LNP group had a higher brain parenchyma exposure, indicating that the fusion of hu 1-36 R101A 12 VHH enhanced the ability of Lecanemab to penetrate the blood-brain barrier.

[0641] Sequence (excluding the aforementioned embodiments)

[0642] SEQ ID NO:23

[0643] SEQ ID NO:24

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Claims

1. A TfR1 binding protein comprising an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises HCDR1, HCDR2 and HCDR3 contained in any one of the amino acid sequences shown in SEQ ID NO:1 to 74.

2. The TfR1 binding protein according to claim 1, wherein the single variable domain of the immunoglobulin is a VHH domain.

3. The TfR1 binding protein according to claim 1 or 2, The HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77 or are composed of the amino acid sequences shown in SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77 respectively; The HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80, or are composed of the amino acid sequences shown in SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80, respectively; The HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:81, SEQ ID NO:82, and SEQ ID NO:83, or are composed of the amino acid sequences shown in SEQ ID NO:81, SEQ ID NO:82, and SEQ ID NO:83, respectively; The HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:84, SEQ ID NO:85, and SEQ ID NO:86, or are composed of the amino acid sequences shown in SEQ ID NO:84, SEQ ID NO:85, and SEQ ID NO:86, respectively; The HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89, or are composed of the amino acid sequences shown in SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89, respectively; The HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:90, SEQ ID NO:91, and SEQ ID NO:92, or are composed of the amino acid sequences shown in SEQ ID NO:90, SEQ ID NO:91, and SEQ ID NO:92, respectively; The HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:93, SEQ ID NO:94, and SEQ ID NO:95, or are composed of the amino acid sequences shown in SEQ ID NO:93, SEQ ID NO:94, and SEQ ID NO:95, respectively; The HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98, or are composed of the amino acid sequences shown in SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98, respectively; The HCDR1, HCDR2 and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:99, SEQ ID NO:100 and SEQ ID NO:101 or are composed of the amino acid sequences shown in SEQ ID NO:99, SEQ ID NO:100 and SEQ ID NO:101 respectively; The HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:102, SEQ ID NO:103, and SEQ ID NO:104 or are composed of the amino acid sequences shown in SEQ ID NO:102, SEQ ID NO:103, and SEQ ID NO:104 respectively; The HCDR1, HCDR2 and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:105, SEQ ID NO:106 and SEQ ID NO:107 or are composed of the amino acid sequences shown in SEQ ID NO:105, SEQ ID NO:106 and SEQ ID NO:107 respectively; The HCDR1, HCDR2 and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:108, SEQ ID NO:109 and SEQ ID NO:110 or are composed of the amino acid sequences shown in SEQ ID NO:108, SEQ ID NO:109 and SEQ ID NO:110 respectively; The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:111, SEQ ID NO:112, and SEQ ID NO:113 or are composed of the amino acid sequences shown in SEQ ID NO:111, SEQ ID NO:112, and SEQ ID NO:113 respectively; The HCDR1, HCDR2, and HCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO:114, SEQ ID NO:115, and SEQ ID NO:116; The HCDR1, HCDR2 and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:117, SEQ ID NO:118 and SEQ ID NO:119 or are composed of the amino acid sequences shown in SEQ ID NO:117, SEQ ID NO:118 and SEQ ID NO:119 respectively; The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:120, SEQ ID NO:121, and SEQ ID NO:122 or are composed of the amino acid sequences shown in SEQ ID NO:120, SEQ ID NO:121, and SEQ ID NO:122 respectively; The HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:123, SEQ ID NO:124, and SEQ ID NO:125 or are composed of the amino acid sequences shown in SEQ ID NO:123, SEQ ID NO:124, and SEQ ID NO:125 respectively; The HCDR1, HCDR2 and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:126, SEQ ID NO:127 and SEQ ID NO:128 or are composed of the amino acid sequences shown in SEQ ID NO:126, SEQ ID NO:127 and SEQ ID NO:128 respectively; The HCDR1, HCDR2 and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:129, SEQ ID NO:130 and SEQ ID NO:131 or are composed of the amino acid sequences shown in SEQ ID NO:129, SEQ ID NO:130 and SEQ ID NO:131 respectively; The HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:132, SEQ ID NO:133, and SEQ ID NO:134 or are composed of the amino acid sequences shown in SEQ ID NO:132, SEQ ID NO:133, and SEQ ID NO:134 respectively; The HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:135, SEQ ID NO:136, and SEQ ID NO:137 or are composed of the amino acid sequences shown in SEQ ID NO:135, SEQ ID NO:136, and SEQ ID NO:137 respectively; The HCDR1, HCDR2 and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:138, SEQ ID NO:139 and SEQ ID NO:140 or are composed of the amino acid sequences shown in SEQ ID NO:138, SEQ ID NO:139 and SEQ ID NO:140 respectively; The HCDR1 contains the amino acid sequence shown in SEQ ID NO:87, the HCDR2 contains the amino acid sequence shown in SEQ ID NO:88, and the HCDR3 contains the amino acid sequence shown in one of SEQ ID NO:141 to 159. The HCDR1 is composed of the amino acid sequence shown in SEQ ID NO:87, the HCDR2 is composed of the amino acid sequence shown in SEQ ID NO:88, and the HCDR3 is composed of the amino acid sequence shown in one of SEQ ID NO:141 to 159. The HCDR1 contains the amino acid sequence shown in SEQ ID NO:99, the HCDR2 contains the amino acid sequence shown in SEQ ID NO:100, and the HCDR3 contains one of the amino acid sequences shown in SEQ ID NO:160-178; or The HCDR1 is composed of the amino acid sequence shown in SEQ ID NO:99, the HCDR2 is composed of the amino acid sequence shown in SEQ ID NO:100, and the HCDR3 is composed of the amino acid sequence shown in one of SEQ ID NO:160 to 178. Preferably, HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:87, SEQ ID NO:88 and SEQ ID NO:143 or are composed of the amino acid sequences shown in SEQ ID NO:87, SEQ ID NO:88 and SEQ ID NO:

143.

4. The TfR1 binding protein according to any one of claims 1 to 3, wherein the immunoglobulin single variable domain comprises: (1) An amino acid sequence as shown in any one of SEQ ID NO:1 to 74; or (2) An amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the amino acid sequences shown in SEQ ID NO:1 to 74 and less than 100%; Preferably, the amino acid sequence of the single variable domain of the immunoglobulin is as shown in SEQ ID NO:65, 70 or 73. Preferably, the TfR1 binding protein is VHH.

5. The TfR1 binding protein according to any one of claims 1 to 4, wherein the immunoglobulin single variable domain is humanized.

6. The TfR1 binding protein according to any one of claims 1 to 5, wherein the TfR1 binding protein is a heavy chain antibody; Preferably, the TfR1 binding protein further comprises human Ig Fc; Preferably, the TfR1 binding protein further comprises human Ig Fc and human Ig hinge region; Preferably, the human Ig is human IgG1, human IgG2, human IgG3, or human IgG4; Preferably, the TfR1 binding protein further comprises human IgG4 Fc.

7. The TfR1 binding protein according to claim 6, wherein the amino acid sequence of the human Ig G4 hinge region is shown in SEQ ID NO:179; Preferably, the human IgG4 Fc comprises a Ks chain and an Hs chain, the amino acid sequences of which are shown in SEQ ID NO:180 and 181, respectively; Preferably, the amino acid sequence of the human Ig G1 hinge region is shown in SEQ ID NO:182; Preferably, the amino acid sequence of the human IgG1 Fc is shown in SEQ ID NO:

183.

8. The TfR1 binding protein according to any one of claims 6 to 7, wherein the human Ig Fc comprises a mutation that prolongs the half-life of the TfR1 binding protein.

9. The TfR1 binding protein according to claim 8, wherein the mutation is M252Y, S254T, and T256E; or the mutation is M428L and N434S.

10. The TfR1 binding protein according to any one of claims 1 to 9, wherein the TfR1 binding protein does not inhibit or weakly inhibits the binding of TfR1 to transferrin.

11. The TfR1-binding protein according to any one of claims 1 to 10, wherein the K of the TfR1-binding protein is... D No more than 1×10 - 6 mol / L; Alternatively, the EC50 of the TfR1 binding protein for TfR1 is less than 500 nmol / L.

12. An mRNA comprising a polynucleotide encoding a fusion protein, the fusion protein comprising an active molecule and a transport molecule capable of binding to a blood-brain barrier receptor to transport the active molecule across the blood-brain barrier.

13. The mRNA according to claim 12, wherein the blood-brain barrier receptor is selected from one or more of the following: transferrin receptor, insulin receptor, insulin-like growth factor receptor, low-density lipoprotein receptor-associated protein 8, low-density lipoprotein receptor-associated protein 1, glucose transporter 1, and heparin-binding epidermal growth factor-like growth factor.

14. The mRNA according to claim 13, wherein the transferrin receptor is human TfR1.

15. The mRNA according to any one of claims 12 to 14, wherein the transport molecule is an antibody or an antigen-binding fragment thereof.

16. The mRNA according to any one of claims 11 to 15, wherein the transport molecule is an antibody that binds to human TfR1 or an antigen-binding fragment thereof.

17. The mRNA according to any one of claims 11 to 15, wherein the transporter comprises the TfR1 binding protein according to any one of claims 1 to 10.

18. The mRNA according to any one of claims 12 to 17, wherein the active molecule is a contrast agent or the active molecule is used for the prevention or treatment of neurological diseases.

19. The mRNA according to claim 18, wherein the neurological disease is one or more of the following: Alzheimer's disease, stroke, dementia, muscular dystrophy, multiple sclerosis, amyotrophic lateral sclerosis, cystic fibrosis, Angelman syndrome, Liddell syndrome, Parkinson's disease, Pick's disease, Paget's disease, neurological cancers, and traumatic brain injury.

20. The mRNA according to any one of claims 12 to 19, wherein the active molecule is capable of binding to a brain antigen selected from the group consisting of: β-secretase 1, Aβ, epidermal growth factor receptor, human epidermal growth factor receptor 2, tau, aliphatic apolipoprotein E4, α-synuclein, CD20, huntingtin, prion protein, leucine-rich repeat kinase 2, perkinin, progerin 1, progerin 2, γ-secretase, death receptor 6, amyloid precursor protein, p75 neurotrophic protein receptor, and caspase 6.

21. The mRNA according to any one of claims 12 to 20, wherein the active molecule is an antibody that specifically binds to Aβ or an antigen-binding fragment thereof.

22. The mRNA according to any one of claims 12 to 18, wherein the active molecule is iduxose-2-sulfatase (IDS).

23. The mRNA according to any one of claims 12 to 22, wherein the fusion protein does not inhibit or weakly inhibits the binding of the blood-brain barrier receptor to one or more of its natural ligands.

24. The mRNA according to any one of claims 12 to 23, wherein the mRNA further comprises at least one of a 5'-cap structure, a 5'-UTR, a 3'-UTR, and a poly(A) tail.

25. The mRNA according to any one of claims 12 to 24, wherein the mRNA contains a modified nucleoside; Preferably, the modified nucleoside includes at least one of modified uridine, modified cytidine, modified adenosine, and modified guanosine.

26. A fusion protein derived from the translation of the mRNA according to any one of claims 12 to 25.

27. A multispecific antibody comprising the TfR1 binding protein as described in any one of claims 1 to 11.

28. A conjugate comprising the TfR1 binding protein and its conjugated active molecule as described in any one of claims 1 to 11, or the multispecific antibody and its conjugated active molecule as described in claim 27.

29. A chimeric antigen receptor (CAR) comprising the TfR1 binding protein according to any one of claims 1 to 11.

30. A nucleic acid comprising a polynucleotide encoding the TfR1 binding protein of any one of claims 1 to 11, the multispecific antibody of claim 27, or the chimeric antigen receptor of claim 29.

31. The nucleic acid according to claim 30, wherein the nucleic acid is mRNA.

32. The nucleic acid according to claim 30, wherein the nucleic acid is DNA.

33. A DNA that can be transcribed into the mRNA according to any one of claims 12 to 25.

34. A gene engineering vector comprising the nucleic acid of claim 30 or the DNA of claim 32.

35. A host cell comprising the nucleic acid of any one of claims 30 to 32, the DNA of claim 33, or the genetic engineering vector of claim 34.

36. An immune cell that expresses the chimeric antigen receptor of claim 29.

37. A pharmaceutical composition comprising the TfR1 binding protein of any one of claims 1 to 11, the mRNA of any one of claims 12 to 25, the fusion protein of claim 26, the multispecific antibody of claim 27, the conjugate of claim 28, the chimeric antigen receptor of claim 29, the nucleic acid of any one of claims 30 to 32, the DNA of claim 33, the genetic engineering vector of claim 34, or the immune cell of claim 36.

38. The pharmaceutical composition of claim 37, wherein the mRNA is formulated in lipid nanoparticles.

39. The pharmaceutical composition of claim 38, wherein the lipid nanoparticles comprise one or more of the following: ionizable lipids, auxiliary lipids, structural lipids, and polymer-lipids; Preferably, the lipid nanoparticles comprise ionizable lipids, auxiliary lipids, structural lipids, and polymer-lipids; Preferably, the ionizable lipid is: The auxiliary lipid is a phospholipid; preferably, the phospholipid is DSPC. The structural lipid is cholesterol; The polymer-lipid is PEG-lipid; preferably, the PEG-lipid is DMG-PEG.

40. The pharmaceutical composition according to claim 39, wherein ionizable lipids comprise 20 mol% to 75 mol% of the total lipids present in the LNPs, accessory lipids comprise 0 mol% to 45 mol% of the total lipids present in the LNPs, structural lipids comprise 0 mol% to 60 mol% of the total lipids present in the LNPs, and polymer-lipids comprise 0.5 mol% to 15 mol% of the total lipids present in the LNPs.

41. The use of the TfR1 binding protein according to any one of claims 1 to 11, the mRNA according to any one of claims 12 to 25, the fusion protein according to claim 26, the multispecific antibody according to claim 27, the conjugate according to claim 28, the chimeric antigen receptor according to claim 29, the nucleic acid according to any one of claims 30 to 32, the DNA according to claim 33, the genetic engineering vector according to claim 34, the host cell according to claim 35, the immune cell according to claim 36, or the pharmaceutical composition according to any one of claims 37 to 40 in the preparation of a medicament for the prevention or treatment of TfR1-related diseases; Preferably, the drug is used to treat tumors or cancers with high TfR1 expression.

42. The use of the TfR1 binding protein according to any one of claims 1 to 11, the mRNA according to any one of claims 12 to 25, the fusion protein according to claim 26, the multispecific antibody according to claim 27, the conjugate according to claim 28, the chimeric antigen receptor according to claim 29, the nucleic acid according to any one of claims 30 to 32, the DNA according to claim 33, the genetic engineering vector according to claim 34, the host cell according to claim 35, the immune cell according to claim 36, or the pharmaceutical composition according to any one of claims 37 to 40 in the preparation of a drug capable of crossing the blood-brain barrier; Preferably, the drug is used to treat neurological diseases.

Images