Anti-TfR1 single-domain antibody and application thereof

Abstract

The invention provides an anti-TfR1 single domain antibody and application thereof. The single-domain antibody comprises a heavy chain variable region, and the heavy chain variable region comprises a heavy chain CDR1 as shown in any one of SEQ ID NO: 53 to SEQ ID NO: 60, a heavy chain CDR2 as shown in any one of SEQ ID NO: 61 to SEQ ID NO: 72 and a heavy chain CDR3 as shown in any one of SEQ ID NO: 73 to SEQ ID NO: 82.

Description

Single domain antibodies against TfR1 and their uses Technical Field

[0001] The present disclosure relates to the field of biomedicine. Specifically, the present disclosure relates to transferrin receptor 1 (TfR1) epitope peptides, TfR1 antigens, anti-TfR1 single domain antibodies, recombinant proteins, antibody complexes, pharmaceutical compositions, kits, and their uses in preventing or treating diseases. Background Art

[0002] Transferrin receptor (TfR) is a type II homodimer transmembrane glycoprotein, is composed of two identical 90kDa subunits connected by two disulfide bridges (Jing and Trowbridge 1987, McClelland et al., 1984). Each TfR molecule includes a 61 amino acid whose short cytoplasmic N-terminal domain containing YTRF (tyrosine-threonine-arginine-phenylalanine) internalization motif, a 27 amino acid whose single hydrophobic transmembrane segment and a 670 amino acid whose large C-terminal extracellular domain containing trypsin cleavage site and transferrin binding site (Aisen, 2004). Each subunit of TfR molecule can bind to transferrin molecule. The extracellular domain of TfR has an O-glycosylation site and three N-glycosylation sites, which are important for the correct folding of TfR and for transport to cell membranes (Hayes et al., 1997). There is also a palmitoylation site in the endodomain of TfR. It is speculated that this palmitoylation site allows TfR to be anchored and internalized (Alvarez et al., 1990, Omary and Trowbridge, 1981). Many studies have shown that the expression level of TfR is higher in cancer cells compared to healthy cells and is correlated with tumor grade and disease stage or prognosis.

[0003] There are two types of transferrin receptors: TfR1 receptor and the homologous receptor TfR2, which is mainly expressed in the liver. It has been reported that the expression level of TfR1 receptor in malignant proliferating cells is significantly higher than its expression level in normal cells. TfR1 is considered to be an important target for tumor treatment. In recent years, TfR1 receptor is also considered to be an important target for drug transport across the blood-brain barrier.

[0004] However, there is still room for improvement in antibodies that specifically recognize transferrin receptor TfR1.

[0005] Summary of the Invention

[0006] A first aspect of the present disclosure provides a human TfR1 antigen epitope peptide, wherein the amino acid sequence of the TfR1 antigen epitope peptide is selected from the group consisting of: (1) SEQ ID NO: 85; (2) SEQ ID NO: 97; (3) SEQ ID NO: 98; (4) SEQ ID NO: 100; and (5) SEQ ID NO: 101. The TfR1 antigen epitope peptide can be used to prepare an anti-TfR1 antibody that specifically binds to the TfR1 protein with high affinity.

[0007] The second aspect of the present invention provides a TfR1 antigen, which is a conjugate of the human TfR1 antigen epitope peptide and a carrier protein as described in the first aspect. The TfR1 antigen can be used to prepare anti-TfR1 antibodies that specifically bind to the TfR1 protein with high affinity.

[0008] The third aspect of the present invention provides a human TfR1 antibody, which is an anti-TfR1 antibody produced using the TfR1 antigen described in the second aspect as an immunogen.

[0009] A fourth aspect of the present invention provides an anti-TfR1 single-domain antibody, comprising a heavy chain variable region comprising a heavy chain CDR1 as shown in any one of SEQ ID NO: 53 to SEQ ID NO: 60, a heavy chain CDR2 as shown in any one of SEQ ID NO: 61 to SEQ ID NO: 72, and a heavy chain CDR3 as shown in any one of SEQ ID NO: 73 to SEQ ID NO: 82. The anti-TfR1 single-domain antibody can specifically bind to the TfR1 protein with high affinity.

[0010] In some embodiments, the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 are selected from the group consisting of (1) to (12):

[0011] (1) CDR1 shown in SEQ ID NO: 53, CDR2 shown in SEQ ID NO: 62, and CDR3 shown in SEQ ID NO: 76;

[0012] (2) CDR1 shown in SEQ ID NO:54, CDR2 shown in SEQ ID NO:72, and CDR3 shown in SEQ ID NO:80;

[0013] (3) CDR1 shown in SEQ ID NO: 55, CDR2 shown in SEQ ID NO: 64, and CDR3 shown in SEQ ID NO: 73;

[0014] (4) CDR1 shown in SEQ ID NO: 56, CDR2 shown in SEQ ID NO: 61, and CDR3 shown in SEQ ID NO: 75;

[0015] (5) CDR1 shown in SEQ ID NO: 57, CDR2 shown in SEQ ID NO: 66, and CDR3 shown in SEQ ID NO: 74;

[0016] (6) CDR1 shown in SEQ ID NO: 58, CDR2 shown in SEQ ID NO: 68, and CDR3 shown in SEQ ID NO: 78;

[0017] (7) CDR1 shown in SEQ ID NO: 59, CDR2 shown in SEQ ID NO: 67, and CDR3 shown in SEQ ID NO: 82;

[0018] (8) CDR1 shown in SEQ ID NO: 59, CDR2 shown in SEQ ID NO: 63, and CDR3 shown in SEQ ID NO: 81;

[0019] (9) CDR1 shown in SEQ ID NO: 59, CDR2 shown in SEQ ID NO: 71, and CDR3 shown in SEQ ID NO: 77;

[0020] (10) CDR1 shown in SEQ ID NO: 59, CDR2 shown in SEQ ID NO: 69, and CDR3 shown in SEQ ID NO: 79;

[0021] (11) CDR1 shown in SEQ ID NO: 59, CDR2 shown in SEQ ID NO: 70, and CDR3 shown in SEQ ID NO: 77; and

[0022] (12) CDR1 shown in SEQ ID NO:60, CDR2 shown in SEQ ID NO:65, and CDR3 shown in SEQ ID NO:73.

[0023] In some embodiments, the heavy chain variable region further comprises a framework region FR. The framework region FR comprises FR1, FR2, FR3 and FR4.

[0024] In some embodiments, the amino acid sequence of the FR1 region is as shown in any one of SEQ ID NOs: 25-33 or has at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% sequence homology to any one of SEQ ID NOs: 25-33. Optionally, the amino acid sequence of the FR1 region comprises up to 5 amino acid substitutions relative to any one of SEQ ID NOs: 25-33.

[0025] In some embodiments, the amino acid sequence of the FR2 region is as set forth in any one of SEQ ID NOs: 34-40 or has at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% sequence homology to any one of SEQ ID NOs: 34-40. Optionally, the amino acid sequence of the FR2 region comprises up to 5 amino acid substitutions relative to any one of SEQ ID NOs: 34-40.

[0026] In some embodiments, the amino acid sequence of the FR3 region is as set forth in any one of SEQ ID NOs: 41-51 or has at least about 60%, about 70%, about 72%, about 74%, about 76%, about 78%, about 80%, about 82%, about 85%, about 88%, about 90%, about 92%, about 94%, about 96% sequence homology to any one of SEQ ID NOs: 41-51. Optionally, the amino acid sequence of the FR3 region comprises up to 10 amino acid substitutions relative to any one of SEQ ID NOs: 41-51.

[0027] In some embodiments, the amino acid sequence of the FR4 region is as set forth in SEQ ID NO: 52 or has at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% sequence homology to SEQ ID NO: 52. Optionally, the amino acid sequence of the FR4 region comprises up to 5 amino acid substitutions relative to SEQ ID NO: 52.

[0028] The present inventors have obtained a variety of anti-TfR1 single-domain antibodies through extensive research. These anti-TfR1 single-domain antibodies can have a wide range of affinities for TfR1 protein and thus have a wide range of applications.

[0029] In some embodiments, the amino acid sequence of the anti-TfR1 single domain antibody is as shown in any one of SEQ ID NOs: 1 to 12, or has at least about 80% sequence homology with any one of SEQ ID NOs: 1 to 12. The anti-TfR1 single domain antibody can specifically bind to the TfR1 protein with high affinity.

[0030] In some embodiments, the amino acid sequence of the anti-TfR1 single domain antibody is as shown in any one of SEQ ID NOs: 1 to 12. Alternatively, the amino acid sequence of the anti-TfR1 single domain antibody has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence homology to any one of SEQ ID NOs: 1 to 12, and is capable of specifically binding to the TfR1 protein.

[0031] In some embodiments, the anti-TfR1 single-domain antibody of the present disclosure may be an Fc fusion antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, or a polyclonal antibody.

[0032] The fifth aspect of the present disclosure provides a recombinant protein comprising the amino acid sequence of the anti-TfR1 single-domain antibody according to the fourth aspect.

[0033] In some embodiments, the recombinant protein can be a multi-epitope antibody, a multivalent antibody, or a multispecific antibody.

[0034] The sixth aspect of the present disclosure provides an antibody complex comprising the anti-TfR1 single-domain antibody according to the fourth aspect or the recombinant protein according to the fifth aspect.

[0035] In some embodiments, the antibody complex can be a conjugate formed by coupling the anti-TfR1 single domain antibody described in the fourth aspect or the recombinant protein described in the fifth aspect with an active ingredient selected from a therapeutic agent, a diagnostic agent, a carrier protein and a liposome.

[0036] The seventh aspect of the present disclosure provides a nucleic acid encoding the anti-TfR1 single-domain antibody of the fourth aspect or the recombinant protein of the fifth aspect.

[0037] In some embodiments, the nucleotide sequence of the nucleic acid is as shown in SEQ ID NO: 13-24. Alternatively, the nucleotide sequence of the nucleic acid has at least about 80% sequence homology with any one of SEQ ID NO: 13-24, such as at least about 85% homology, at least about 90% homology, at least about 95% homology, at least about 98% homology or at least about 99% homology, and the anti-TfR1 single domain antibody encoded by the nucleic acid can specifically bind to the TfR1 protein.

[0038] The eighth aspect of the present disclosure provides an expression vector comprising the nucleic acid of the seventh aspect, wherein the expression vector expresses the anti-TfR1 single domain antibody of the fourth aspect or the recombinant protein of the fifth aspect.

[0039] The ninth aspect of the present disclosure provides a host cell comprising the expression vector according to the eighth aspect, wherein the host cell expresses the anti-TfR1 single domain antibody according to the fourth aspect or the recombinant protein according to the fifth aspect.

[0040] The tenth aspect of the present disclosure provides a pharmaceutical composition comprising the anti-TfR1 single-domain antibody of the fourth aspect, the recombinant protein of the fifth aspect, the antibody complex of the sixth aspect, the nucleic acid of the seventh aspect, the expression vector of the eighth aspect, or the host cell of the ninth aspect, and a pharmaceutically acceptable carrier. The pharmaceutical composition is used to prevent or treat a disease.

[0041] The eleventh aspect of the present disclosure provides the use of the anti-TfR1 single-domain antibody described in the fourth aspect, the recombinant protein described in the fifth aspect, the antibody complex described in the sixth aspect, the nucleic acid described in the seventh aspect, the expression vector described in the eighth aspect, the host cell described in the ninth aspect, or the pharmaceutical composition described in the tenth aspect in the preparation of a medicament for preventing or treating diseases associated with abnormal TfR1 receptor expression.

[0042] The twelfth aspect of the present disclosure provides the anti-TfR1 single-domain antibody of the fourth aspect, the recombinant protein of the fifth aspect, the antibody complex of the sixth aspect, the nucleic acid of the seventh aspect, the expression vector of the eighth aspect, the host cell of the ninth aspect, or the pharmaceutical composition of the tenth aspect for use as a medicine.

[0043] In this aspect, the present disclosure further provides the anti-TfR1 single-domain antibody described in the fourth aspect, the recombinant protein described in the fifth aspect, the antibody complex described in the sixth aspect, the nucleic acid described in the seventh aspect, the expression vector described in the eighth aspect, the host cell described in the ninth aspect, or the pharmaceutical composition described in the tenth aspect for use in preventing or treating diseases associated with abnormal TfR1 receptor expression.

[0044] The thirteenth aspect of the present disclosure provides a method for preventing or treating a disease associated with abnormal TfR1 receptor expression, comprising administering to a subject in need thereof a therapeutically effective amount of the anti-TfR1 single-domain antibody described in the fourth aspect, the recombinant protein described in the fifth aspect, the antibody complex described in the sixth aspect, the nucleic acid described in the seventh aspect, the expression vector described in the eighth aspect, the host cell described in the ninth aspect, or the pharmaceutical composition described in the tenth aspect.

[0045] In some embodiments, the disease associated with abnormal TfR1 receptor expression is a tumor that abnormally expresses the TfR1 receptor.

[0046] In some embodiments, the tumor includes but is not limited to breast cancer, glioma, lung adenocarcinoma, leukemia, lymphoma, non-Hodgkin's lymphoma, ovarian cancer, etc.

[0047] The fourteenth aspect of the present disclosure provides a kit for detecting the TfR1 receptor, which includes the anti-TfR1 single-domain antibody described in the fourth aspect, the recombinant protein described in the fifth aspect, the antibody complex of the sixth aspect, the nucleic acid described in the seventh aspect, the expression vector described in the eighth aspect, the host cell described in the ninth aspect, or the pharmaceutical composition described in the tenth aspect.

[0048] In some embodiments, the kit is used to diagnose a disease associated with abnormal TfR1 receptor expression.

[0049] A fifteenth aspect of the present disclosure provides the anti-TfR1 single-domain antibody of the fourth aspect, the recombinant protein of the fifth aspect, or the antibody complex of the sixth aspect for use as a drug for crossing the blood-brain barrier.

[0050] It should be understood that within the scope of the present disclosure, the above-mentioned technical features of the present disclosure and the technical features described below (such as in the embodiments) can be combined with each other to form new technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the technical solution of the present disclosure, the following briefly introduces the drawings required for use in the embodiments. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0052] FIG1 is an SDS-PAGE electrophoresis pattern of the purified humanized TfR1 protein in Example 1 of the present disclosure.

[0053] FIG2 is a diagram showing library enrichment for screening antibodies targeting humanized TfR1 protein in Example 3 of the present disclosure.

[0054] FIG3 is a dose-effect curve and measurement results of the antibody-antigen binding in Example 12 of the present disclosure.

[0055] FIG4 is a dose-effect curve and measurement results of the antibody-antigen binding in Example 12 of the present disclosure.

[0056] FIG5 is a dose-effect curve and measurement results of the antibody-antigen binding in Example 12 of the present disclosure.

[0057] FIG6 is a dose-effect curve and measurement results of the antibody-antigen binding in Example 12 of the present disclosure.

[0058] FIG7 is a dose-effect curve and measurement results of the antibody-antigen binding in Example 12 of the present disclosure.

[0059] FIG8 is a dose-effect curve and measurement results of the antibody-antigen binding in Example 12 of the present disclosure. DETAILED DESCRIPTION

[0060] The present disclosure will now provide a clear and complete description of the technical solutions in the embodiments of the present disclosure in conjunction with the accompanying drawings. Obviously, the described embodiments are only a portion of the embodiments of the present disclosure, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present disclosure without creative effort are within the scope of protection of the present disclosure.

[0061] definition

[0062] As used herein, "single-domain antibody" (sdAb, also known as nanobody or VHH) refers to an antibody that includes only the heavy chain variable region of a heavy chain antibody, which is the smallest functional antigen-binding fragment. Single-domain antibodies can be natural antibodies with only the heavy chain variable domain, single-domain antibodies derived from conventional antibodies, and engineered single-domain antibodies.

[0063] As used herein, the term "Fc fusion antibody" refers to a novel protein produced by fusing the Fc segment of an antibody of interest with a functional protein molecule having biological activity using genetic engineering technology.

[0064] The term "humanized antibody" refers to an antibody obtained by fusing the heavy chain variable region of a target antibody (such as an animal antibody) with the constant region of a human antibody, or by transplanting the complementarity determining regions (CDR1, CDR2, and CDR3 sequences) of a target antibody into the variable region of a human antibody, or by subjecting the target antibody to individual amino acid mutations based on the structure of the human antibody framework region (FR1, FR2, FR3, and FR4). Humanized antibodies can be produced by synthesis or point mutagenesis.

[0065] The term "polyepitopic antibody" refers to an antibody that has the ability to specifically bind to two or more different epitopes on the same or different targets.

[0066] The term "multivalent antibody" refers to an antibody that can bind to multiple antigenic epitopes. The number of antigenic epitopes an antibody binds to is called its antigen-binding valency.

[0067] The term "multispecific antibody" refers to an artificial antibody containing two or more specific antigen-binding sites, which can stimulate a targeted immune response in a host (eg, human).

[0068] Herein, the terms “TfR1”, “TfR1 protein”, “TfR1 receptor” or “TfR1 antigen” are used interchangeably to refer to transferrin receptor 1, which can specifically bind to the anti-TfR1 single domain antibody of the present disclosure.

[0069] The term "tumor" refers to a group of diseases that can be defined as any abnormal benign or malignant growth of tissue that has no physiological function and that arises from uncontrolled, often rapid, cell proliferation and has the potential to invade or spread to other parts of the body.

[0070] The terms "subject" and "patient" are used interchangeably and generally refer to humans, but may also refer to non-human species such as mice, camels, goats, rabbits, or cows. A "subject" can be of any age, sex, and physical condition.

[0071] "Administering" a drug to a subject refers to direct administration (which can be by a healthcare professional), self-administration, and / or indirect administration (which can be the act of prescribing a drug). For example, a physician administering a drug to a subject by instructing the subject to self-administer a drug or providing a prescription for the drug to the subject is administering the drug to the subject.

[0072] As used herein, the term "effective amount" of an agent (e.g., a single domain antibody against TfR1) refers to an amount sufficient to produce a beneficial or desired result (e.g., a clinical result). Therefore, the "effective amount" depends on the actual application. The term "effective amount" can be used interchangeably with "effective dose" or "therapeutically effective amount."

[0073] Complementarity determining regions (CDRs) refer to the hypervariable regions within the variable regions of antibody molecules. Each heavy or light chain variable region consists of three CDRs and four framework regions (FWs). These three CDR regions (CDR1, CDR2, and CDR3) collectively form the antigen-binding site of an antibody.

[0074] TfR1 antigen epitope peptide

[0075] According to one aspect of the present disclosure, a human TfR1 antigen epitope peptide is provided, wherein the amino acid sequence of the TfR1 antigen epitope peptide is selected from the group consisting of: (1) SEQ ID NO: 85; (2) SEQ ID NO: 97; (3) SEQ ID NO: 98; (4) SEQ ID NO: 100; and (5) SEQ ID NO: 101. By using the human TfR1 antigen epitope peptide, an anti-TfR1 antibody that specifically binds to the TfR1 protein with high affinity can be prepared.

[0076] In some embodiments, a TfR1 antigen is provided, which is a conjugate of the above-mentioned human TfR1 antigen epitope peptide and a carrier protein.

[0077] In the present disclosure, the carrier protein includes, but is not limited to, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or OVA protein (chicken egg albumin). In some embodiments, when used as an immunogen to immunize animals to produce antibodies, the carrier protein is keyhole limpet hemocyanin (KLH). In addition, when used as a coating source to screen monoclonal cell lines, the carrier protein is bovine serum albumin (BSA).

[0078] The use of the human TfR1 antigen epitope peptide or the TfR1 antigen as an immunogen to prepare anti-TfR1 antibodies also falls within the scope of protection of the present disclosure, wherein the application can be non-disease diagnosis or treatment applications, or disease diagnosis or treatment applications.

[0079] Therefore, the present disclosure also provides human TfR1 antibodies, which are produced using the above-mentioned TfR1 antigen as an immunogen.

[0080] Single domain antibody against TfR1

[0081] According to one aspect of the present disclosure, a single-domain antibody against TfR1 is provided, wherein the single-domain antibody against TfR1 includes a heavy chain variable region, the heavy chain variable region includes a heavy chain CDR1 shown in any one of SEQ ID NO:53 to SEQ ID NO:60, a heavy chain CDR2 shown in any one of SEQ ID NO:61 to SEQ ID NO:72, and a heavy chain CDR3 shown in any one of SEQ ID NO:73 to SEQ ID NO:82.

[0082] In some embodiments, sequences having at least about 60% homology, at least about 70% homology, at least about 75% homology, at least about 80% homology, at least about 85% homology, at least about 90% homology, at least about 95% homology, at least about 98% homology, or at least about 99% homology to the heavy chain CDR1 shown in SEQ ID NO:53 to SEQ ID NO:60, the heavy chain CDR2 shown in SEQ ID NO:61 to SEQ ID NO:72, and the heavy chain CDR3 shown in SEQ ID NO:73 to SEQ ID NO:82 can also achieve the purposes of the present disclosure.

[0083] In some embodiments, the heavy chain variable region further comprises a framework region FR. The framework region FR comprises FR1 as shown in any one of SEQ ID NOs: 25-33, FR2 as shown in any one of SEQ ID NOs: 34-40, FR3 as shown in any one of SEQ ID NOs: 41-51, and FR4 as shown in SEQ ID NO: 52.

[0084] In some embodiments, the framework region sequences FR1, FR2, FR3, and FR4 may also have conservative amino acid substitutions. In some embodiments, a single domain antibody against TfR1 may include a FR1 having at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, sequence homology to any one of SEQ ID NOs: 25-33; a FR2 having at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% sequence homology to any one of SEQ ID NOs: 34-40; a FR3 having at least about 60%, about 70%, about 72%, about 74%, about 76%, about 78%, about 80%, about 82%, about 85%, about 88%, about 90%, about 92%, about 94%, about 96% sequence homology to any one of SEQ ID NOs: 41-51; and a FR4 having at least about 60%, about 70%, about 72%, about 74%, about 76%, about 78%, about 80%, about 82%, about 85%, about 88%, about 90%, about 92%, about 94%, about 96% sequence homology to any one of SEQ ID NOs: 41-51; and a FR5 having at least about 60%, about 70%, about 72%, about 74%, about 76%, about 78%, about 80%, about 82%, about 85%, about 88%, about 90%, about 92%, about 94%, about 96% sequence homology to any one of SEQ ID NOs: 41-51; and a FR6 having at least about 60%, about 70%, about 72%, about 74%, about 76%, about 78%, about 80%, about 82%, about 85%, about 88%, about 90%, about 92%, about NO:52 has a FR4 with at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% sequence homology.

[0085] In some embodiments, the amino acid sequences of FR1, FR2, FR3, and FR4 have up to 10 amino acid substitutions relative to any one of SEQ ID NOs: 25-33, any one of SEQ ID NOs: 34-40, any one of SEQ ID NOs: 41-51, and SEQ ID NO: 52, respectively, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions.

[0086] In some embodiments, the anti-TfR1 single-domain antibody of the present disclosure may be an Fc fusion antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, or a polyclonal antibody.

[0087] In some embodiments, the Fc fragment of the Fc fusion antibody is derived from IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, or IgE.

[0088] According to another aspect of the present disclosure, an anti-TfR1 single-domain antibody is provided, the amino acid sequence of the anti-TfR1 single-domain antibody being shown in any one of SEQ ID NOs: 1-12.

[0089] In some embodiments, sequences having at least about 80% homology, at least about 85% homology, at least about 90% homology, at least about 95% homology, at least about 98% homology, or at least about 99% homology to any one of SEQ ID NOs: 1-12 can also achieve the purposes of the present disclosure.

[0090] In some embodiments, sequences that replace only one or a few amino acids compared to any one of SEQ ID NOs: 1-12, for example, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions, can also achieve the purposes of the present disclosure.

[0091] As used herein, the term "sequence homology" refers to the extent to which two (nucleotide or amino acid) sequences have identical residues at identical positions in an alignment, and is usually expressed as a percentage. Homology is determined over the entire length of the sequences being compared. Thus, two copies of the exact same sequence would have 100% homology.

[0092] In fact, when determining the degree of sequence homology between two amino acid sequences or when determining the combination of CDR1, CDR2 and CDR3 in a single domain antibody, a skilled person may consider a "conservative" amino acid substitution. In the case of a substitution, the substitution is a conservative amino acid substitution. The conservative amino acid can generally be described as an amino acid substitution in which an amino acid residue is replaced by another amino acid residue of similar chemical structure, and the substitution has little or substantially no effect on the function, activity or other biological properties of the polypeptide. The conservative amino acid substitution is common in the art.

[0093] For example, a conservative amino acid substitution is one or a few amino acids in the following groups (a)-(d) replaced by another or a few amino acids in the same group: (a) polar negatively charged residues and their uncharged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. In some embodiments, conservative amino acid substitutions are as follows: Asp is replaced by Glu; Asn is replaced by Gln or His; Glu is replaced by Asp; Gln is replaced by Asn; His is replaced by Asn or Gln; Arg is replaced by Lys; Lys is replaced by Arg, Gln; Phe is replaced by Met, Leu, Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe, Trp; Ala is replaced by Gly or Ser; Ser is replaced by Thr; Thr is replaced by Ser; Gly is replaced by Ala or Pro; Met is replaced by Leu, Tyr or Ile; Leu is replaced by Ile or Val; Ile is replaced by Leu or Val; Val is replaced by Ile or Leu; Cys is replaced by Ser.

[0094] In the present disclosure, “single-domain antibodies against TfR1” include not only complete single-domain antibodies, but also fragments, derivatives and analogs of single-domain antibodies against TfR1. As used herein, the terms “fragment”, “derivative” and “analog” have the same meaning, and all refer to polypeptides that substantially maintain the same biological function or activity as the antibodies of the present disclosure. The polypeptide fragments, derivatives or analogs disclosed herein may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (especially conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substituent group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence, a secretory sequence, or a sequence for purifying the polypeptide, or a fusion protein formed with an Fc tag). According to the teachings herein, these fragments, derivatives and analogs all fall within the scope of protection of the present disclosure.

[0095] The inventors of the present disclosure unexpectedly discovered that the single-domain antibody against TfR1 has a high affinity for the TfR1 protein. Furthermore, TfR1 is highly expressed on the cell surface of some disease tissues, especially on the cell surface of some cancer tissues. The inventors have verified that the use of the single-domain antibody against TfR1 disclosed in the present disclosure can induce an immune response in vivo, and thus the above-mentioned single-domain antibody against TfR1 can be used to effectively prevent or treat diseases associated with abnormal expression of the TfR1 receptor. For example, the disease is a tumor that abnormally expresses the TfR1 receptor. For example, the tumor is breast cancer, glioma, lung adenocarcinoma, leukemia, lymphoma, non-Hodgkin's lymphoma or ovarian cancer. In view of the above, the single-domain antibody against TfR1 disclosed in the present disclosure can also be used to diagnose diseases associated with abnormal expression of the TfR1 receptor. For example, the disease is a tumor that abnormally expresses the TfR1 receptor. For example, the tumor is breast cancer, glioma, lung adenocarcinoma, leukemia, lymphoma, non-Hodgkin's lymphoma or ovarian cancer.

[0096] In addition, the blood-brain barrier is composed of a network of endothelial cells, capillary basement membranes, astrocyte end feet and pericytes embedded in the capillary basement membrane. Many compounds in the blood (for example, all macromolecular drugs with a molecular weight greater than 1 kD and more than 98% of small molecule drugs) are almost difficult to cross the blood-brain barrier and enter the brain parenchyma network. The inventors also unexpectedly found that the expression level of TfR1 receptors in the blood-brain barrier is higher than that in normal tissues. Transferrin receptor TfR1 can mediate transcellular transport mechanisms. By adopting the single-domain antibodies against TfR1 provided by the present disclosure, specific drugs can be effectively transported across the blood-brain barrier to the brain parenchyma, thereby significantly improving the treatment of diseases, especially the treatment of neurological diseases. For example, the neurological diseases include, but are not limited to: Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), Parkinson's disease, traumatic brain injury, etc.

[0097] The anti-TfR1 single-domain antibody disclosed herein specifically targets the TfR1 protein but has low immunogenicity, thus not producing adverse side effects in the subject. It has good compatibility with the subjects, especially humans, and can be advantageously used as a carrier for delivering pharmaceutical agents or directly as a drug.

[0098] In addition, the anti-TfR1 antibody disclosed herein is a single-domain antibody, which has a small molecular size and a simple structure, and is therefore easy to express in a variety of expression systems. For example, it can be expressed in both prokaryotic systems and eukaryotic systems such as yeast cells or mammalian cells. Therefore, it can be prepared at a lower production cost and is suitable for industrial large-scale production.

[0099] recombinant protein

[0100] According to one aspect of the present disclosure, a recombinant protein is provided, which comprises the amino acid sequence of the above-mentioned single-domain antibody against TfR1.

[0101] In some embodiments, the recombinant protein may contain, in addition to the amino acid sequence of the anti-TfR1 single domain antibody described above, additional amino acid sequences, such as a leader sequence, a linker sequence, a tag sequence, a green fluorescent protein sequence, and the like.

[0102] In some embodiments, the recombinant protein can be a multi-epitope antibody, a multivalent antibody, or a multispecific antibody.

[0103] In some embodiments, the recombinant protein is a multi-epitope antibody. For example, the multi-epitope antibody comprises a plurality of sequences selected from SEQ ID NOs: 1-12. In one embodiment, any one sequence in the multi-epitope antibody is directly linked to another adjacent sequence. In another embodiment, any two adjacent sequences in the multi-epitope antibody can be linked via a linker sequence. In another embodiment, the multi-epitope antibody consists of a plurality of sequences selected from SEQ ID NOs: 1-12. In other embodiments, the multi-epitope antibody has 2 to 10 different epitopes, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 different epitopes.

[0104] In some embodiments, the recombinant protein is a multivalent antibody. In some embodiments, the multivalent antibody comprises several repeats of any one of SEQ ID NOs: 1-12. In some embodiments, the repeats are linked together directly or via a linker sequence. In other embodiments, the multivalent antibody consists of several repeats of any one of SEQ ID NOs: 1-12. In some embodiments, the multivalent antibody has 2 to 20 repeats, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 repeats.

[0105] In some embodiments, the recombinant protein is a multispecific antibody. In some embodiments, the multispecific antibody includes but is not limited to a bispecific antibody, a trispecific antibody, a tetraspecific antibody, or a pentaspecific antibody. Typically, the multispecific antibody is a bispecific antibody. For example, in addition to targeting the TfR1 antigen, the multispecific antibody can also target other antigens, such as PD-1, PD-L1, CD19, CD22, EGFR, etc.

[0106] In the present disclosure, the anti-TfR1 single-domain antibody has a small molecular size and a simple structure, and is therefore easy to be modified to obtain multi-epitope antibodies, multivalent antibodies or multispecific antibodies, thereby expanding the application scope of the anti-TfR1 single-domain antibody.

[0107] Antibody complex

[0108] According to another aspect of the present disclosure, an antibody complex is provided, which comprises the above-mentioned single-domain antibody against TfR1 or the above-mentioned recombinant protein.

[0109] In some embodiments, the antibody complex can be a conjugate formed by coupling the above-mentioned single-domain antibody against TfR1 or the above-mentioned recombinant protein with an active ingredient selected from therapeutic agents, diagnostic agents, carrier proteins, and liposomes.

[0110] In some embodiments, the above-mentioned anti-TfR1 single domain antibody or the above-mentioned recombinant protein can be coupled to a therapeutic agent, a diagnostic agent or a carrier protein via a linker. The linker has 5 to 30 amino acid sequences, such as 5 to 25 amino acid sequences, such as 5 to 20 amino acid sequences, such as 5 to 15 amino acid sequences, such as 5 to 10 amino acid sequences.

[0111] In some embodiments, the above-mentioned single-domain antibody against TfR1 or the above-mentioned recombinant protein can be coupled to the liposome via a covalent bond or a non-covalent bond. In some embodiments, the covalent bond coupling includes coupling to the liposome via a disulfide bond on the single-domain antibody against TfR1 or the recombinant protein, or coupling to the single-domain antibody against TfR1 or the recombinant protein via a hydrophobic group on the liposome.

[0112] In some embodiments, the therapeutic agent may include, but is not limited to, cisplatin, paclitaxel, cyclophosphamide, doxorubicin, daunorubicin, idarubicin, gemcitabine, interferons (e.g., interferon-α, interferon-β, interferon-γ), interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), tumor necrosis factor (TNF) (e.g., TNFα), granulocyte colony-stimulating factor (G-CSF), and granulocyte macrophage colony-stimulating factor (GM-CSF)), and the like.

[0113] In some embodiments, the carrier protein can include, but is not limited to, keyhole limpet hemocyanin (KLH), serum albumin (eg, bovine serum albumin, BSA) or ovalbumin, immunoglobulin Fc domain, tetanus toxoid, diphtheria toxoid, or a combination thereof.

[0114] In some embodiments, the diagnostic agent may include a developer or label. Useful labels include magnetic beads, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, green fluorescent protein, etc.), radiolabels, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), colorimetric labels (such as colloidal gold or colored glass or plastic beads or nanoparticles (e.g., polystyrene, polypropylene, latex, etc.)), etc.

[0115] The present disclosure can provide a conjugate of an anti-TfR1 single domain antibody or a recombinant protein thereof with a therapeutic agent. Thus, based on the transcellular transport mechanism mediated by transferrin receptor TfR1, the anti-TfR1 single domain antibody described herein can be used to effectively deliver the therapeutic agent to the target site of interest.

[0116] In some embodiments, the antibody complexes can cross the blood-brain barrier and deliver therapeutic agents to the brain parenchyma, thereby significantly improving the treatment of brain diseases, particularly neurological diseases. For example, the neurological diseases include, but are not limited to, Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), Parkinson's disease, traumatic brain injury, etc.

[0117] In some embodiments, the above-mentioned antibody complex can also deliver therapeutic agents to tumor tissues based on the transcellular transport mechanism mediated by transferrin receptor TfR1, thereby significantly enhancing the ability to kill tumors.

[0118] Similarly, the present disclosure may also provide a conjugate of an anti-TfR1 single domain antibody or a recombinant protein thereof with a diagnostic agent, thereby enabling the anti-TfR1 single domain antibody conjugated with a diagnostic agent to cross the blood-brain barrier and be delivered to the target site, thereby being used to diagnose diseases.

[0119] Therefore, in another aspect, the present disclosure provides a method for transporting a drug across the blood-brain barrier, comprising exposing the anti-TfR1 single-domain antibody conjugated to a drug of the present disclosure to the blood-brain barrier, so that the anti-TfR1 single-domain antibody transports the conjugated drug across the blood-brain barrier to achieve specific therapeutic or diagnostic purposes as well as non-therapeutic or diagnostic purposes. The above-mentioned drug can be a therapeutic agent or a diagnostic agent.

[0120] Nucleic Acids

[0121] According to another aspect of the present disclosure, a nucleic acid is provided, which encodes the anti-TfR1 single domain antibody or recombinant protein described in the present disclosure.

[0122] In some embodiments, the nucleotide sequence of the nucleic acid is shown in SEQ ID NOs: 13-24.

[0123] In some embodiments, a nucleotide sequence having at least about 80% sequence homology, such as at least about 85% homology, at least about 90% homology, at least about 95% homology, at least about 98% homology, or at least about 99% homology to any one of SEQ ID NOs: 13-24 can also achieve the purposes of the present disclosure. The single-domain antibody against TfR1 encoded by the nucleic acid can specifically bind to the TfR1 protein.

[0124] In some embodiments, the nucleic acid can be used as a drug. The nucleic acid can accurately express the anti-TfR1 single domain antibody in a subject, and thus can be used to prevent or treat diseases associated with abnormal TfR1 receptor expression.

[0125] expression vector

[0126] According to another aspect of the present disclosure, an expression vector is provided, comprising the nucleic acid described in the present disclosure, wherein the expression vector expresses the anti-TfR1 single domain antibody or a recombinant protein thereof described in the present disclosure.

[0127] In one embodiment, the expression vector is obtained by integrating a nucleic acid encoding an anti-TfR1 single domain antibody or a recombinant protein thereof into the vector RJK-V4-hFC1 by genetic engineering means. The RJK-V4-hFC1 is prepared according to Example 10 of the present disclosure.

[0128] By using the expression vector disclosed herein, the above-mentioned anti-TfR1 single-domain antibody or its recombinant protein can be accurately and stably expressed.

[0129] host cells

[0130] According to another aspect of the present disclosure, a host cell is provided, comprising the expression vector described in the present disclosure, wherein the host cell expresses the anti-TfR1 single domain antibody or recombinant protein described in the present disclosure.

[0131] In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0132] In some embodiments, the host cell comprises a prokaryotic cell or a eukaryotic cell, including bacteria or fungi.

[0133] In some embodiments, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophage, or a combination thereof.

[0134] In some embodiments, the prokaryotic cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, Lactobacillus, Streptomyces, Proteus mirabilis, or a combination thereof.

[0135] In some embodiments, the eukaryotic cell is selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, or a combination thereof.

[0136] In some embodiments, the eukaryotic cells are selected from the group consisting of insect cells such as fall armyworm, plant cells such as tobacco, Syrian baby hamster kidney cells (BHK cells), Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (COS cells), myeloma cells, or a combination thereof.

[0137] In other embodiments, the host cell is a suspension ExpiCHO-S cell.

[0138] In other embodiments, the host cell is a suspension human embryonic kidney epithelial 293F cell.

[0139] The anti-TfR1 antibody disclosed herein is a single-domain antibody, which has a small molecular size and a simple structure, and is therefore easy to express in a variety of expression systems. For example, it can be expressed in both prokaryotic systems and eukaryotic systems such as yeast cells or mammalian cells, and can therefore be prepared at a lower production cost.

[0140] Pharmaceutical composition

[0141] According to another aspect of the present disclosure, a pharmaceutical composition is provided, which comprises the anti-TfR1 single-domain antibody, its recombinant protein, its antibody complex, the nucleic acid encoding it, the expression vector expressing it, or the host cell expressing it in the present disclosure as an active ingredient, and a pharmaceutically acceptable carrier.

[0142] In some embodiments, the pharmaceutical composition comprises the anti-TfR1 single domain antibody, its recombinant protein or its antibody complex as an active ingredient, and a pharmaceutically acceptable carrier.

[0143] In another embodiment, the pharmaceutical composition comprises the anti-TfR1 single-domain antibody as an active ingredient, and a pharmaceutically acceptable carrier.

[0144] Typically, the anti-TfR1 single-domain antibody can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier. The pH is typically determined based on the isoelectric point of the anti-TfR1 single-domain antibody. The pH of the aqueous carrier should deviate from the isoelectric point of the anti-TfR1 single-domain antibody and should differ from the isoelectric point of the anti-TfR1 single-domain antibody by approximately 2.

[0145] The pharmaceutical composition of the present disclosure contains a safe and effective amount, for example, 0.001 wt% to 99 wt%, 0.01 wt% to 90 wt%, or 0.1 wt% to 80 wt% of an anti-TfR1 single domain antibody, a recombinant protein thereof, or an antibody complex thereof, and a pharmaceutically acceptable carrier.

[0146] In some embodiments, the carrier includes, but is not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.

[0147] The pharmaceutical formulation should be compatible with the route of administration. The pharmaceutical compositions disclosed herein can be prepared as injections, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical composition dosage forms such as injections and solutions are preferably manufactured under sterile conditions.

[0148] As described herein, the anti-TfR1 single domain antibody, its recombinant protein, its antibody complex, or a pharmaceutical composition comprising the same can be administered parenterally, topically, intravenously, orally, intragastricly, subcutaneously, intraarterially, intracranially, intraperitoneally, intranasally, or intramuscularly for therapeutic and / or prophylactic treatment. In some embodiments, the anti-TfR1 single domain antibody, its recombinant protein, its antibody complex, or a pharmaceutical composition comprising the same is administered to a subject by intramuscular injection or intravenous injection.

[0149] According to embodiments of the present disclosure, the pharmaceutical composition can be used to effectively prevent or treat a disease associated with abnormal expression of the TfR1 receptor. For example, the disease is a tumor that abnormally expresses the TfR1 receptor. For example, the tumor is breast cancer, glioma, lung adenocarcinoma, leukemia, lymphoma, non-Hodgkin's lymphoma, or ovarian cancer.

[0150] In addition, according to embodiments of the present disclosure, the transferrin receptor TfR1 can mediate a transcellular transport mechanism, and by using the pharmaceutical compositions provided by the present disclosure, specific drugs can be effectively transported across the blood-brain barrier to the brain parenchyma, thereby significantly improving the treatment of diseases, especially the treatment of neurological diseases. For example, the neurological diseases include, but are not limited to: Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), Parkinson's disease, traumatic brain injury, etc.

[0151] Reagent test kit

[0152] In another aspect of the present disclosure, a kit is provided, which includes the anti-TfR1 single-domain antibody, recombinant protein, antibody complex, nucleic acid, expression vector, host cell or pharmaceutical composition described in the present disclosure.

[0153] In some embodiments, the kit further comprises one or more containers, or instructions for use. The anti-TfR1 single-domain antibody, the recombinant protein, the antibody complex, the nucleic acid, the expression vector, the host cell or the pharmaceutical composition may be contained in one or more containers to facilitate administration to a subject. The one or more containers may also be used to hold additional agents for preventing or treating a disease. The kit may further comprise a device for delivering the active ingredients in the kit, such as a needle or a syringe. The instructions for use indicate information such as the method of administration, dosage, side effects, etc. for preventing or treating a disease that expresses the TfR1 receptor.

[0154] According to an embodiment of the present disclosure, the kit can be used to prevent or treat diseases associated with abnormal expression of TfR1 receptors, such as tumors that abnormally express TfR1 receptors. For example, the tumor is breast cancer, glioma, lung adenocarcinoma, leukemia, lymphoma, non-Hodgkin's lymphoma or ovarian cancer. The kit can also be used to prevent or treat brain diseases, such as nervous system diseases. The single-domain antibody against TfR1 is used to transport the therapeutic agent across the blood-brain barrier to achieve the therapeutic purpose.

[0155] In some embodiments, the kit is used to detect TfR1 protein in vitro. The kit can be an in vitro detection device for detecting the level of TfR1 protein.

[0156] In some embodiments, the kit includes an anti-TfR1 single domain antibody, a lysis medium for dissolving the sample, and general reagents for detection (e.g., a detection label such as an enzyme or a fluorescent or radioactive label, a detection substrate, a buffer, etc.). In one embodiment, the kit may further include an anti-TfR1 single domain antibody (i.e., an anti-antibody) as a second antibody.

[0157] According to an embodiment of the present disclosure, the kit can be used to diagnose diseases associated with abnormal expression of TfR1 receptors, with high sensitivity and specificity.

[0158] Application of single-domain antibodies against TfR1

[0159] In another aspect of the present disclosure, provided is the use of the anti-TfR1 single-domain antibody, the recombinant protein, the antibody complex, the nucleic acid, the expression vector, the host cell, or the pharmaceutical composition in the preparation of a medicament for preventing or treating a disease associated with abnormal expression of the TfR1 receptor.

[0160] In yet another aspect of the present disclosure, provided is the anti-TfR1 single-domain antibody, the recombinant protein, the antibody complex, the nucleic acid, the expression vector, the host cell, or the pharmaceutical composition for use as a medicament.

[0161] In another aspect of the present disclosure, provided is the anti-TfR1 single-domain antibody, the recombinant protein, the antibody complex, the nucleic acid, the expression vector, the host cell, or the pharmaceutical composition for preventing or treating a disease associated with abnormal expression of the TfR1 receptor.

[0162] In some embodiments, the disease associated with abnormal expression of the TfR1 receptor is a tumor associated with abnormal expression of the TfR1 receptor. In some embodiments, the tumor includes but is not limited to breast cancer, glioma, lung adenocarcinoma, leukemia, lymphoma, non-Hodgkin's lymphoma, ovarian cancer, etc. The single-domain antibody to TfR1 disclosed herein, its recombinant protein, antibody complex, or pharmaceutical composition can effectively prevent or treat the disease.

[0163] In yet another aspect of the present disclosure, provided is the anti-TfR1 single-domain antibody, the recombinant protein, the antibody complex, or the pharmaceutical composition for use as a drug for crossing the blood-brain barrier.

[0164] The inventors also unexpectedly discovered that the expression level of TfR1 receptors in the blood-brain barrier is higher than that in normal tissues. The transferrin receptor TfR1 can mediate a transcellular transport mechanism. By using the anti-TfR1 single-domain antibodies provided by the present disclosure, specific drugs can be effectively transported across the blood-brain barrier to the brain parenchyma, thereby significantly improving the treatment of diseases, especially the treatment of neurological diseases. For example, the neurological diseases include, but are not limited to: Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), Parkinson's disease, traumatic brain injury, etc.

[0165] Methods of preventing or treating disease

[0166] In another aspect of the present disclosure, a method for preventing or treating a disease associated with abnormal expression of the TfR1 receptor is provided, comprising administering a therapeutically effective amount of the anti-TfR1 single-domain antibody, the recombinant protein, the antibody complex, the nucleic acid, the expression vector, the host cell or the pharmaceutical composition to a subject in need thereof.

[0167] In some embodiments, the disease associated with abnormal expression of the TfR1 receptor is a tumor associated with abnormal expression of the TfR1 receptor. In some embodiments, the tumor includes but is not limited to breast cancer, glioma, lung adenocarcinoma, leukemia, lymphoma, non-Hodgkin's lymphoma, ovarian cancer, etc. The anti-TfR1 single domain antibody provided by the present disclosure can effectively prevent or treat the disease.

[0168] In another aspect of the present disclosure, a method for transporting a drug across the blood-brain barrier is provided, comprising exposing a drug-conjugated anti-TfR1 single-domain antibody of the present disclosure (i.e., an antibody complex of the present disclosure) to the blood-brain barrier, so that the anti-TfR1 single-domain antibody transports the drug conjugated thereto across the blood-brain barrier to achieve a specific preventive or therapeutic purpose. The above method is used to prevent or treat brain diseases, particularly neurological diseases. For example, the neurological diseases include, but are not limited to: Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), Parkinson's disease, traumatic brain injury, etc.

[0169] Method and system for diagnosing disease

[0170] In another aspect of the present disclosure, a method for diagnosing a disease associated with abnormal TfR1 receptor expression is provided. According to an embodiment of the present disclosure, the diagnostic method comprises: contacting the above-mentioned anti-TfR1 single-domain antibody with a biological sample from a subject to detect the expression level of TfR1 protein in the biological sample; comparing the detected expression level value of the TfR1 protein with the normal expression level value of the TfR1 protein in the same biological sample from a normal subject, and the detected expression level value being higher than the normal expression level value indicates that the subject is at risk of suffering from a disease associated with abnormal TfR1 receptor expression.

[0171] In some embodiments, the disease is a tumor that aberrantly expresses the TfR1 receptor. In some embodiments, the tumor is breast cancer, glioma, lung adenocarcinoma, leukemia, lymphoma, non-Hodgkin's lymphoma, or ovarian cancer.

[0172] In another aspect of the present disclosure, a system for diagnosis is provided. The diagnostic system includes: a detection device; and a diagnostic result determination device. The detection device is configured to contact the above-mentioned anti-TfR1 single-domain antibody with a biological sample from a subject to detect the expression level of the TfR1 protein in the biological sample. The diagnostic result determination device is connected to the detection device and is configured to compare the detected expression level value of the TfR1 protein with the normal expression level value of the TfR1 protein from the same biological sample of a normal subject, wherein the detected expression level value is higher than the normal expression level value, indicating that the subject has a risk of suffering from a disease associated with abnormal TfR1 receptor expression.

[0173] Preparation method

[0174] In one embodiment of the present disclosure, a method for producing an anti-TfR1 single-domain antibody is provided, comprising: (a) culturing the above-mentioned host cell under conditions suitable for producing the single-domain antibody to obtain a culture containing the anti-TfR1 single-domain antibody; and (b) isolating the anti-TfR1 single-domain antibody from the culture; (c) optionally, purifying and / or modifying the anti-TfR1 single-domain antibody obtained in (b).

[0175] In another embodiment of the present disclosure, a method for preparing an anti-TfR1 single-domain antibody includes: using TfR1 protein or a truncated form thereof as an immunogen to immunize camels to produce anti-TfR1 single-domain antibodies; harvesting gene fragments of the anti-TfR1 single-domain antibodies from immunized peripheral blood lymphocytes or spleen cells; recombining the above gene fragments into a vector for phage display; screening out anti-TfR1 single-domain antibodies against TfR1 protein; and isolating and purifying the above anti-TfR1 single-domain antibodies.

[0176] The following examples of the present disclosure are described in detail. It should be noted that the following examples are illustrative and are intended only to explain the present invention and are not to be construed as limiting the present invention. In addition, unless otherwise expressly stated, all reagents used in the following examples are commercially available or can be synthesized according to the literature or known methods. Reaction conditions not listed are also readily available to those skilled in the art.

[0177] Example 1: Preparation of humanized TfR1 protein

[0178] The humanized recombinant TfR1 protein used in this example was obtained by expression and purification by Nanjing Rongjiekang Biotechnology Co., Ltd. The expression vector of the humanized recombinant TfR1 protein was designed according to the following scheme.

[0179] (1) The coding sequence of TfR1 protein was retrieved from NCBI (Accession No.: NM_001128148.2). The coding sequence generated the amino acid sequence with accession No. NP_001121620.1.

[0180] (2) The coding sequence of amino acids 89 to 760 aa of the TfR1 protein was cloned into the vector pcDNA3.4 (from Invitrogen) by gene synthesis. The constructed vector was Sanger sequenced by Jinweizhi Company, and the sequence obtained was compared with the original sequence to confirm that the correct recombinant plasmid was obtained. The recombinant plasmid was then subjected to a large amount of extraction, endotoxin removal, and transfected into suspended 293F cells for expression and purification of the TfR1 protein. The purity of the obtained humanized TfR1 protein reached more than 90%, meeting the requirements of animal immunization. The SDS-PAGE electrophoresis pattern of the purified humanized TfR1 protein is shown in Figure 1.

[0181] Example 2: Construction of a single domain antibody library against humanized TfR1 protein

[0182] 1 mg of the humanized recombinant TfR1 protein obtained in Example 1 was mixed with an equal volume of Freund's complete adjuvant (purchased from Sigma-Aldrich, catalog number F5881-6X10ML) and used to immunize Bactrian camels in Alxa, Inner Mongolia, once a week for a total of 7 consecutive immunizations. Except for the first immunization, the remaining six immunizations were all performed by mixing 1 mg of TfR1 protein with an equal volume of Freund's incomplete adjuvant (purchased from Sigma-Aldrich) for animal immunization. This immunization process is used to stimulate Bactrian camels to produce antibodies against the TfR1 protein.

[0183] After the animal immunization is completed, 150 mL of peripheral blood lymphocytes of Bactrian camels are extracted to extract RNA from peripheral blood lymphocytes. The extracted total RNA is used to synthesize cDNA, and the VHH fragment (i.e., the heavy chain variable region of the antibody) is amplified using cDNA as a template through a nested PCR reaction. The pMECS vector and VHH fragment are then digested using restriction endonucleases (PstⅠ (Product No. R3140L), NotⅠ (Product No. R3189L), and BstEⅡ (Product No. R3162S), all purchased from NEB (New England BioLabs)). The digested fragments and the vector are then connected, and the vector connected to the fragment is electroporated into competent cells TG1 (purchased from Agilent). A phage display library of TfR1 protein was constructed and the library capacity was determined. The library capacity was approximately 1×10 9 At the same time, colony PCR was used to detect the correct insertion rate of the target fragment in the library.

[0184] The results showed that after PCR amplification of 30 clones randomly selected from the library, 28 clones could amplify bands of the predicted size, and the amplified bands of 2 clones were incorrect, so the correct insertion rate was about 93%.

[0185] Example 3: Screening of single domain antibodies against humanized TfR1 protein

[0186] 200 μL of the recombinant TG1 cells obtained in Example 2 were cultured in 2×TY medium (purchased from Thermo Fisher). 40 μL of helper phage VCSM13 (purchased from Agilent) was added to infect the TG1 cells and cultured overnight to amplify the phage. The next day, the phage was precipitated using PEG / NaCl, and the amplified phage was collected by centrifugation.

[0187] 500 ng of TfR1 protein was diluted in 100 mM, 100 μL of NaHCO3 at pH 8.3. The mixture of TfR1 protein was added to the wells of the ELISA plate and placed at 4°C overnight to coat the TfR1 protein on the well surface. At the same time, a negative control well (i.e., culture medium control) was set up. The next day, 200 μL of 3% skim milk was added and blocked at room temperature for 2 hours. After blocking, 100 μL of amplified phage library (approximately 2×10 11 phage particles) and reacted at room temperature for 1 hour. Afterwards, each well was washed 15 times with a mixture of PBS + 0.05% Tween-20 to wash away unbound phage particles.

[0188] Phage that specifically bound to the TfR1 protein were dissociated using trypsin at a final concentration of 25 mg / mL. These phage were then used to infect Escherichia coli TG1 cells in the logarithmic growth phase and cultured at 37°C for 1 hour to produce and collect phage for the next round of screening. The same screening process was repeated for one more round, gradually achieving enrichment. Two rounds of screening were performed for single-domain antibodies against humanized TfR1 protein using the above screening method. Screening was concluded when the enrichment factor for phage against humanized TfR1 protein reached 10 or greater. The enrichment effect is shown in Figure 2.

[0189] In Figure 2, P / N = the number of monoclonal bacteria grown after phage eluted from the positive wells in biopanning infected with TG1 bacteria / the number of monoclonal bacteria grown after phage eluted from the positive wells infected with TG1 bacteria. This P / N parameter gradually increases after enrichment.

[0190] I / E = total amount of phage added to positive wells in each round of biopanning / total amount of phage eluted from positive wells in each round of biopanning. This I / E parameter gradually approaches 1 after enrichment.

[0191] This example shows that the single domain antibody against humanized TfR1 protein was enriched after two rounds of solid phase panning. The P / N of the second round was 103.57 and I / E was 68.96, indicating a good enrichment effect.

[0192] Example 4: Screening of specific positive clones against humanized TfR1 protein using phage enzyme-linked immunosorbent assay (ELISA)

[0193] 384 single colonies were selected from the positive clones obtained by the screening method of Example 3 and inoculated into 96-well plates in 2×TY medium containing 100 μg / mL ampicillin. A blank control was also set up and cultured at 37°C until the logarithmic phase. Subsequently, isopropylthiogalactoside (IPTG) (purchased from Sangon Biotech, Catalog No. A600168-0100) was added to a final concentration of 1 mM and cultured overnight at 28°C.

[0194] 100 ng of humanized TfR1 protein was diluted in 100 mM, 100 μL, pH 8.3 NaHCO 3 , and the mixture containing TfR1 protein was added to the wells of an ELISA plate and placed at 4° C. overnight to coat the TfR1 protein on the well surface.

[0195] The crude antibody was obtained by osmotic swelling method. 100 μL of the obtained crude antibody extract was transferred to an ELISA plate coated with TfR1 protein and incubated at room temperature for 1 hour. Unbound antibodies were washed away with PBST and Tween-20 buffer (PBS was purchased from Sangon Biotech, item number B548117-0500; Tween-20 was purchased from Beyotime Biotechnology (Shanghai Biyuntian Biotechnology Co., Ltd.), item number ST825). 100 μL of mouse anti-HA horseradish peroxidase-labeled antibody (Thermo Fisher) diluted 1:2000 was added and incubated at room temperature for 1 hour. Unbound antibodies were washed away with PBST buffer, horseradish peroxidase colorimetric solution was added, and after reacting at 37°C for 15 minutes, the stop solution was added and the absorbance value was read at a wavelength of 450 nm on a microplate reader (purchased from TECAN).

[0196] When the OD value of the sample well is more than 5 times greater than the OD value of the control well, it is determined to be a positive clone well. The bacterial solution of the positive clone well is transferred to LB medium containing 100 μg / mL ampicillin and cultured with shaking. The plasmid is then extracted and sequenced.

[0197] The gene sequences of each clone were analyzed using the sequence alignment software VectorNTI. Strains containing identical CDR1, CDR2, and CDR3 sequences were considered the same clone, while strains containing different sequences were considered different clones. Finally, single-domain antibodies specific for humanized TfR1 protein were obtained (as well as single-domain antibody clones whose sequences are not shown).

[0198] The single-domain antibody against humanized TfR1 protein has a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The harvested recombinant plasmid can be expressed in a prokaryotic system to produce the single-domain antibody against humanized TfR1 protein.

[0199] Twelve single-domain antibodies against humanized TfR1 protein were obtained (i.e., 2H7, 4C5, 4D11, 5A3, 5D1, 5D3, 7A3, 7D10, 7G10, 7G5, 8B8 and 8E10), whose amino acid sequences are shown in SEQ ID NOs: 1-12, and whose nucleotide sequences are shown in SEQ ID NOs: 13-24.

[0200] The CDR1, CDR2, and CDR3 sequences of the 12 anti-TfR1 single-domain antibodies are shown in Tables 1-3. These CDR sequences were numbered according to the IMGT numbering convention. For modifications involving the Fc segment, the "EU numbering" convention was used.

[0201] Table 1 CDR1 sequences of 12 single-domain antibodies

[0202] Table 2 CDR2 sequences of 12 single-domain antibodies

[0203] Table 3 CDR3 sequences of 12 single-domain antibodies

[0204] The FR1 sequences, FR2 sequences, FR3 sequences, and FR4 sequences of the 12 anti-TfR1 single domain antibodies are shown in Tables 4 to 7, respectively. These FR sequences are numbered according to the "IMGT coding rules."

[0205] Table 4 FR1 sequences of 12 single-domain antibodies

[0206] Table 5 FR2 sequences of 12 single domain antibodies

[0207] Table 6 FR3 sequences of 12 single domain antibodies

[0208] Table 7 FR4 sequences of 12 single domain antibodies

[0209] The 12 anti-TfR1 single-domain antibodies have amino acid sequences represented by SEQ ID NOs: 1-12, respectively, as shown in Table 8.

[0210] Table 8 Amino acid sequences of single domain antibodies

[0211] The nucleic acid molecules encoding the 12 anti-TfR1 single-domain antibodies have the nucleotide sequences shown in SEQ ID NOs: 13-24, respectively, as shown in Table 9.

[0212] Table 9 Nucleotide sequences of nucleic acids of single domain antibodies

[0213] Example 5: Purification and expression of single domain antibodies against humanized TfR1 protein in Escherichia coli

[0214] Plasmids of the different clones obtained in Example 4 (i.e., pMECS vectors containing VHHs) were electroporated into Escherichia coli HB2151 (purchased from Agilent), plated onto LB medium containing ampicillin and glucose, and cultured overnight at 37°C. A single colony was selected and inoculated into 5 mL of LB medium containing ampicillin (purchased from Sangon Biotech, Catalog No. A507002-0250), and cultured overnight at 37°C in a shaker.

[0215] Inoculate 1 mL of overnight cultured bacterial solution into 330 mL of TB culture medium (TB culture medium is a mixture of TB(A) and TB(B). TB(A) includes tryptone (purchased from OXOID, product number 73049-73-7); yeast extract (purchased from OXOID, product number 8013-01-2) and glycerol (purchased from Sangon Biotech, product number A501745). TB(B) includes potassium dihydrogen phosphate (purchased from Sangon Biotech, product number A600445) and dipotassium hydrogen phosphate (purchased from Sangon Biotech, product number A501212)). Culture at 37°C in a shaker until the OD 600nm value reaches 0.6-0.9. Then add 1 M IPTG and culture overnight in a shaker at 28°C. Centrifuge and collect the E. coli. Obtain a crude antibody extract using the osmotic burst method. Purify the antibody by nickel column affinity chromatography.

[0216] Example 6: Construction of a recombinant eukaryotic expression vector for Fc fusion antibody of a single domain antibody against humanized TfR1

[0217] The nucleotide sequences of the 12 single-domain antibodies obtained in Example 4 (as shown in SEQ ID NO: 13-24) and the nucleotide sequences of other single-domain antibodies whose sequences are not shown are respectively merged into the eukaryotic expression vector RJK-V4-hFC1 designed and modified by our company to obtain a recombinant eukaryotic expression vector. The modification method of the eukaryotic expression vector RJK-V4-hFC is as described in Example 10. The obtained recombinant eukaryotic expression vector is transformed into DH5α Escherichia coli, cultured and plasmid extracted to remove endotoxins. The extracted plasmid is sequenced to confirm that the correct recombinant eukaryotic expression vector is obtained. The determined recombinant eukaryotic expression vector is subsequently used to transfect eukaryotic cells. The fusion antibody of VHH and Fc is expressed by the method in Example 7 or 8, and the fusion antibody of VHH and Fc is purified by the method of Example 9.

[0218] Example 7: Expression of single-domain antibodies against humanized TfR1 protein in ExpiCHO-S cells

[0219] 3 days before transfection, 2.5×10 5 The cell concentration of ExpiCHO-S cells / mL TM Cells (from Thermo Fisher) were passaged and expanded. The required volume of cell culture medium was transferred to a fresh, pre-warmed 120 mL (final volume) of ExpiCHO TM The cells were cultured in a 500 mL shake flask containing expression medium until the cell concentration reached approximately 4 × 10 6 -6×10 6 Live cells / mL. One day before transfection, TM Cells were diluted to 3.5 × 10 6 The concentration of viable cells / mL was set and cultured overnight.

[0220] On the day of transfection, the cell density and percentage of viable cells were determined. The cell density was raised to approximately 7 × 10 6 -10×10 6 Viable cells / mLc.

[0221] Use fresh ExpiCHO preheated to 37°C TM The cells were diluted to 6×10 6 Then, transfer the required volume of cells to a 100 mL (final volume) of fresh pre-warmed ExpiCHO TM Gently invert and mix the ExpiFectamine TM CHO reagent (from Thermo Fisher). Use 3.7 mL OptiPRO TMExpiFectamine was diluted in culture medium (from Thermo Fisher) TM CHO reagent, vortex or mix. Use 4mL OptiPRO TM The recombinant eukaryotic expression vector expressing the VHH and Fc fusion antibody prepared in Example 6 was diluted with culture medium and mixed by vortexing.

[0222] Incubate the diluted ExpiFectamine CHO reagent with the diluted recombinant eukaryotic expression vector at room temperature for 1-5 minutes, then gently add the prepared ExpiCHO-S TM The cell suspension was gently shaken during the incubation period. The cell suspension was cultured in a cell incubator at 37°C and 8% CO2.

[0223] On the first day after transfection (about 18-22 hours later), add 600 μl of ExpiFectamine TM CHO Enhancer and 24 mL ExpiCHO feed. Approximately 8 days after transfection, cell viability was less than 70%, and the cell culture supernatant was collected.

[0224] Example 8: Expression of single-domain antibodies against humanized TfR1 protein in 293F cells

[0225] 3 days before transfection, 2.5×10 5 The cell concentration of 293F cells / mL was used for passage and expansion of 293F cells. The required volume of cell culture medium was transferred to a 500 mL shake flask filled with fresh pre-warmed 120 mL (final volume) of OPM-293CD05 Medium (purchased from OPM Biotech (Suzhou) Co., Ltd., catalog number 81075-001) to make the cell concentration reach about 2×10 6 -3×10 6 Live cells / mL. On the day of transfection, measure the cell density and percentage of live cells. 6 -3×10 6 The viable cells / mL of cells were used for transfection.

[0226] The cells were diluted to 1×10 6 The cells were counted at 100 viable cells / mL and the required volume of cells was transferred to a 500 mL shake flask containing 100 mL (final volume) of fresh pre-warmed OPM-293CD05 Medium.

[0227] Dilute polyethyleneimine reagent (PEI, 1 mg / mL, purchased from PolyScience Inc., Catalog No. A795153) with 4 mL of Opti-MEM medium and mix thoroughly by vortexing or pipetting. Dilute the recombinant eukaryotic expression vector expressing the VHH and Fc fusion antibody prepared in Example 6 with 4 mL of Opti-MEM medium, mix thoroughly by vortexing, filter through a 0.22 μm filter, and incubate at room temperature for 5 min.

[0228] Incubate the diluted PEI reagent and the diluted recombinant eukaryotic expression vector at room temperature for 15-20 minutes, then gently add the prepared 293F cell suspension while gently shaking the flask. Incubate the cell suspension at 120 rpm in a 37°C, 5% CO2 incubator. Add 5 mL of OPM-CHO PFF05 feed 24 and 72 hours after transfection. Collect the cell culture supernatant approximately 7 days after transfection, when cell viability falls below 70%.

[0229] Example 9: Purification of single domain antibodies against humanized TfR1 protein

[0230] The supernatant obtained in Example 7 or the supernatant obtained in Example 8 was filtered using a 0.45 μm disposable filter to remove insoluble impurities.

[0231] The filtrate was purified by affinity chromatography using a protein purifier. The antibody was purified using a Protein A-coupled agarose filler (purchased from Suzhou Nanovitamin Technology Co., Ltd., catalog number 17010-250100-2100) utilizing the ability of the humanized Fc to bind to Protein A.

[0232] The filtrate was passed through a Protein A prepacked column at a flow rate of 1 mL / min to allow the antibody in the filtrate to bind to Protein A. Low-salt buffer and high-salt buffer were used to wash away impure proteins.

[0233] The antibody was eluted with a low pH buffer.

[0234] The eluate containing the antibody was quickly added to a Tris-HCl solution at pH 9.0 for neutralization.

[0235] The neutralized antibody solution was dialyzed and then analyzed by SDS-PAGE. The resulting antibody had a purity of over 95%. The antibody solution with a concentration greater than 0.5 mg / mL was stored at low temperature for later use.

[0236] Example 10: Modification of eukaryotic expression vector for single domain antibodies

[0237] The eukaryotic expression vector RJK-V4-hFC1 for single-domain antibodies was prepared by fusing the coding sequence of the human IgG1 Fc region to the vector pCDNA3.4 (obtained from Invitrogen). The vector RJK-V4-hFC1 contains the IgG1 heavy chain hinge region (Hinge), CH2 region, and CH3 region.

[0238] The transformation plan is as follows:

[0239] The restriction enzyme sites XbaI and AgeI on the vector pcDNA3.4 were selected;

[0240] A multiple cloning site and a 6×His tag were introduced into the 5′ and 3′ ends of the coding sequence of the Fc region of human IgG1, respectively, by overlapping PCR;

[0241] The above sequence was amplified by PCR using primer pairs with XbaI and AgeI restriction sites, respectively;

[0242] pcDNA3.4 and the amplified sequence obtained above were digested with restriction endonucleases XbaI and AgeI, respectively;

[0243] The digested vector and the digested amplified sequence were ligated using T4 ligase, and the ligated product was then transformed into Escherichia coli, amplified, and sequenced for verification, thereby obtaining an expression vector containing the coding sequence of the Fc region.

[0244] Example 11: Expression and purification of tool antibody (Tab) targeting humanized TfR1 protein

[0245] Herein, Tab1 is the antibody AB405 targeting humanized TfR1, and its sequence is from WO2014089209A2. Tab2 is the antibody RG6102 targeting humanized TfR1, and its sequence is from WO2017055540A1. Tab4 is pabinafusp alfa targeting humanized TfR1 (see, US11111308B2). Tab1, Tab2 and Tab4 are used as positive control antibodies for the single-domain antibodies against TfR1 disclosed herein. The searched sequence was entrusted to Universal Biosystems (Anhui) Co., Ltd. for mammalian cell expression system codon optimization, cloned into pcDNA3.1 vector, and transformed into Escherichia coli. After resistance screening, positive clones were selected for amplification, and then the plasmid was extracted using a plasmid extraction kit (Macherey Nagel, Cat#740412.50). For transient expression, a solution of polyethylenimine (PEI) diluted with plasmids expressing Tab1, Tab2, or Tab4 was added to 293F cell cultures (FreeStyle 293 Expression medium, Thermo Fisher, Cat#12338026, and F-68, Thermo Fisher, Cat#24040032) at a volume of 100 μg plasmid (40 μg heavy chain + 60 μg light chain) per 100 mL of cells. Six to 24 hours after transfection, 5% volume of 10% Peptone (Sigma, Cat#P0521-100G) was added and cultured at 130 rpm under 8% CO2 for approximately 7 to 8 days. The supernatant was collected when cell viability dropped to 50%. Antibodies were purified using a Protein A (GE, Cat#17-5438-02) gravity column. The purified antibody was dialyzed against PBS, and the concentration was determined using Nanodrop, the purity was identified using SEC, and the binding capacity was verified using indirect ELISA.

[0246] The concentration of Tab1, Tab2, Tab3 and Tab4 obtained by the method is not less than 2 mg / ml and the purity is greater than 95%.

[0247] Example 12: Determination of the dose-effect curve of antibody-antigen binding

[0248] This example uses standard enzyme-linked immunosorbent assay (ELISA) for determination.

[0249] (1) Coat the wells of an ELISA plate with 50 μL of 1 μg / mL TfR1 protein at 4°C overnight.

[0250] (2) Rinse the wells of the ELISA plate several times with PBST buffer. Add 200 μL of 5% milk and block at 37°C for 2 h.

[0251] (3) The purified single-domain antibody Fc fusion antibody against humanized TfR1 prepared in Example 9 (i.e., VHH-hFc fusion antibody expressed in 293F cells) was diluted to 2 μg / mL and then diluted in a 5-fold gradient to obtain a total of 8 concentration gradients. In addition, control wells for hIgG, Tab1, Tab2, and Tab4 were set up respectively. Tab1, Tab2, and Tab4 were prepared by Example 11. hIgG refers to the isotype control, which does not bind to any TfR1 protein and is purchased commercially.

[0252] (4) Rinse the wells of the ELISA plate in step (2) several times with PBST buffer. Add 50 μL of the diluted single domain antibody, hIgG, Tab1, Tab2, or Tab4 in step (3) to each well, duplicate wells, and incubate at 37°C for 1 hour.

[0253] (5) Rinse the wells of the ELISA plate in step (4) several times with PBST buffer. Add 50 μL of HRP-Goat anti-hIgG secondary antibody to each well and incubate at 37°C for 30 min.

[0254] (6) Rinse the wells of the ELISA plate in step (5) several times with PBST buffer. Add 50 μL of TMB colorimetric solution to each well and react at room temperature in the dark for 15 minutes.

[0255] (7) Add 50 μL of stop solution (1 mol / L HCl) to each well to terminate the reaction. Measure the OD450 using a microplate reader and save the reading.

[0256] (8) Draw the curve and calculate EC50.

[0257] The experimental results are shown in Figures 3 to 8. As shown in Figures 3 to 8, the EC50 of antibodies 5D3, 7G10, 8E10, 5A3, 4C5, 2H7, 4D11, 5D1, 7A3, 7D10, 8D8, and 7G5 of the present invention are lower or comparable to that of Tab, demonstrating that they all have excellent antigen-binding activity.

[0258] Example 13: Internalization of human erythroid leukemia cells (TF-1 cells)

[0259] 1. Preparation of PHrodo conjugates

[0260] 1) Using pHrodo TMiFL Green STP ester (Thermo Fisher, product number P36013) was used to label 100 μg of the purified Fc fusion antibody of the anti-humanized TfR1 single-domain antibody prepared in Example 9 (i.e., the VHH-hFc fusion antibody expressed in 293F cells), 100 μg of Tabs prepared in Example 11, and 100 μg of hIgGTfR1 to obtain pHrodo-VHH-hFcs, pHrodo-Tabs, or pHrodo-hIgG solutions.

[0261] 2) Desalting the pHrodo-RS40-VHH-hFcs, pHrodo-Tabs, or pHrodo-hIgG solution.

[0262] 3) Using Pierce TM The protein concentration in the solution was detected using the BCA protein assay kit (from Thermo Fisher, cat. no. 23227).

[0263] 2. Internalization Experiment

[0264] 1) TF-1 cells (Procell CL-0232, from Wuhan Procell Life Science Co., Ltd.) were harvested by trypsinization and centrifugation.

[0265] 2) 1×10 6 The TF-1 cells were washed once with cold PBS and 10 μg / ml pHrodo-Tabs, 10 μg / ml pHrodo-hIgG, or 10 μg / ml pHrodo-VHH-hFcs were added to cold PBS.

[0266] 3) The cell solution in 2) was divided into two parts. One part of the cell solution was incubated on ice, and the other part of the cell solution was incubated at 37°C for 4 hours.

[0267] 4) Wash the cells after incubation in step 3) once with cold PBS and resuspend the cells in 200 μl of cold PBS.

[0268] 5) CytoFlex flow cytometer (from Beckman Coulter) was used to detect FITC signals.

[0269] The internalization results of 12 Fc-fusion antibodies of anti-humanized TfR1 single-domain antibodies, Tab4 as a positive control, and hIgG as a negative control are shown in Table 10 below. In this table, "Fold" represents the multiple of the MFI at 37°C divided by the MFI at 4°C. The MFI at 4°C represents the background value. This example demonstrates that anti-humanized TfR1 single-domain antibodies can be internalized by target cells to exert their pharmacological effects in target cells.

[0270] Table 10 TF-1 cell internalization assay results

[0271] Example 14: Internalization of CHO-K1-TfR1 cells

[0272] 1. Preparation of PHrodo conjugates

[0273] 1) Use pHrodo TM iFL Green STP ester (Thermo Fisher, product number P36013) was used to label 100 μg of the purified Fc fusion antibody of the anti-humanized TfR1 single-domain antibody prepared in Example 9 (i.e., the VHH-hFc fusion antibody expressed in 293F cells), 100 μg of Tabs prepared in Example 11, and 100 μg of hIgGTfR1 to obtain pHrodo-VHH-hFcs, pHrodo-Tabs or pHrodo-hIgG solutions.

[0274] 2) Desalting the pHrodo-VHH-hFcs, pHrodo-Tabs, or pHrodo-hIgG solution.

[0275] 3) Using Pierce TM The protein concentration in the solution was detected using the BCA protein assay kit (from Thermo Fisher, cat. no. 23227).

[0276] 2. Internalization Experiment

[0277] 1) CHO-K1-TfR1 cells were harvested by trypsin digestion and centrifugation. CHO-K1-TfR1 cells are derived from the CHO-K1-TfR1 cell line. The steps for the CHO-K1-TfR1 cell line are as follows: the CDS region sequence information of TfR1 (accession number: NM_003234.4) was obtained from the NCBI database, the DNA sequence of the CDS region was synthesized by gene synthesis, and the DNA sequence was cloned into the PHB-Puro expression vector to construct the PHB-TfR1-Puro plasmid vector. The lentivirus was then packaged and infected with the lentivirus to introduce the CMV-TfR1-EF1a-Puromycin gene into the CHO-K1 genome. Puromycin resistance screening and monoclonal screening were performed to obtain a CHO-K1-TfR1 stable cell line that stably expressed the TfR1 protein.

[0278] 2) 1×10 6The CHO-K1-TfR1 cells were washed once with cold PBS. 10ug / ml pHrodo-RS40-Tabs, 10ug / ml pHrodo-hIgG, or 10ug / ml pHrodo-RS40-VHH-hFcs were added to cold PBS.

[0279] 3) The cell solution in 2) was divided into two parts. One part of the cell solution was incubated on ice, and the other part of the cell solution was incubated at 37°C for 4 hours.

[0280] 4) Wash the cells after incubation in step 3) once with cold PBS and resuspend the cells in 200 μl of cold PBS.

[0281] 5) CytoFlex flow cytometer (from Beckman Coulter) was used to detect FITC signals.

[0282] The internalization results of 12 Fc-fusion antibodies of single-domain antibodies against humanized TfR1, Tab4 as a positive control, and hIgG as a negative control are shown in Table 11 below. In this table, "Fold" represents the multiple of the MFI at 37°C divided by the MFI at 4°C. The MFI at 4°C represents the background value. This example demonstrates that single-domain antibodies against humanized TfR1 can be internalized by target cells and exert their pharmacological effects in target cells.

[0283] Table 11 CHO-K1-TfR1 internalization experimental results

[0284] Example 15 Antigen epitope experiment

[0285] 1. Epitope peptides of TfR1 protein

[0286] After extensive theoretical research and experimental exploration, the inventors of the present invention performed protein three-dimensional structure preprocessing through computer-assisted protein structure analysis, and docked the processed antigen and antibody (2H7, 5D1) structures with the 1,000 output conformations. The docking structure with the highest score was selected as the final result, and the binding region of the antigen and antibody in these results was determined to be the epitope peptide region.

[0287] Table 12 Peptide names and amino acid sequences

[0288] Based on the analysis results, Nanjing Jiepeptide Biotechnology Co., Ltd. was commissioned to synthesize a peptide based on the above amino acid sequence. The binding between the peptide and the antibody was identified by ELISA. A positive control was also set up to determine the final epitope of the antigen and antibody binding.

[0289] 1.3 Identification of Peptide-Antibody Binding

[0290] Standard enzyme-linked immunosorbent assay (ELISA) was used to identify the binding of peptides to antibodies. The steps are as follows:

[0291] (1) Coat the wells of an ELISA plate with 100 μL of 2 μg / mL SA protein (streptavidin) at 4°C overnight.

[0292] (2) Rinse the wells of the ELISA plate with PBST buffer, add 200 μL of 2% BSA, and block at 37°C for 1 hour.

[0293] (3) Dilute the peptide protein to 2 μg / mL and set up a negative control and a positive control (Tfr1 protein containing an AVI tag). Set up a set of duplicate controls for each experimental group. In the results, the result of the positive control is indicated as N.

[0294] (4) Rinse the wells of the ELISA plate several times with PBST buffer. Add 100 μL of the peptide protein solution diluted in step (3) in duplicate, and set up a negative control well. Incubate at 37°C for 1 hour.

[0295] (5) Rinse the wells of the ELISA plate several times with PBST buffer. Add 100 μL of 2 μg / ml TfR1 antibody 2H7-hFc solution and incubate at 37°C for 1 hour. 2H7-hFc here refers to an Fc fusion antibody of a single-domain antibody against TfR1 expressed and purified in 293F cells.

[0296] (6) Rinse the wells of the ELISA plate several times with PBST buffer. Add 100 μL of 2× anti-hFC HRP secondary antibody and incubate at 37°C for 1 h.

[0297] (7) Rinse the wells of the ELISA plate several times with PBST buffer. Add 100 μL of TMB colorimetric solution and react at room temperature for 30 minutes in the dark.

[0298] (8) Add 100 μL of stop solution (1 mol / L HCl) to terminate the reaction, measure OD450 using a microplate reader, and save the reading.

[0299] Table 13 Peptide names and their determination results

[0300] Where N represents the result of the positive control, and a P / N value greater than 2 indicates binding between the antigen and the antibody. The above results indicate that the antigen epitope peptides include D1-helix4, D2-loop1-loop2, D1-helix3, D1-helix1-helix2, and D1-helix3-helix4 peptides, and their amino acid sequences are numbered as SEQ ID NO: 85, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 100, and SEQ ID NO: 101.

[0301] Table 14 Amino acid sequence of epitope peptide

[0302] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0303] The above-described embodiments merely represent several implementation methods of the present disclosure, and although the descriptions are relatively detailed, they should not be construed as limiting the scope of the patent application. It should be noted that a person of ordinary skill in the art may make various modifications and improvements without departing from the spirit of the present disclosure, and these modifications and improvements fall within the scope of protection of the present disclosure. Therefore, the scope of protection of the patent application of the present disclosure shall be determined by the appended claims.

Claims

1. A human TfR1 antigen epitope peptide, wherein the amino acid sequence of the TfR1 antigen epitope peptide is selected from: (1) SEQ ID NO: 85; (2) SEQ ID NO: 97; (3) SEQ ID NO: 98; (4) SEQ ID NO: 100; and (5) SEQ ID NO:

101. 2 . A TfR1 antigen, which is a conjugate of the human TfR1 antigen epitope peptide according to claim 1 and a carrier protein. 3 . A human TfR1 antibody, which is an anti-TfR1 antibody produced using the TfR1 antigen according to claim 2 as an immunogen.

4. A single-domain antibody against TfR1, wherein the single-domain antibody against TfR1 comprises a heavy chain variable region, and the heavy chain variable region comprises a heavy chain CDR1 shown in any one of SEQ ID NO:53 to SEQ ID NO:60, a heavy chain CDR2 shown in any one of SEQ ID NO:61 to SEQ ID NO:72, and a heavy chain CDR3 shown in any one of SEQ ID NO:73 to SEQ ID NO:

82.

5. The anti-TfR1 single-domain antibody according to claim 4, wherein the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are selected from the group consisting of (1) to (12): (1) CDR1 shown in SEQ ID NO:53, CDR2 shown in SEQ ID NO:62, and CDR3 shown in SEQ ID NO:76; (2) CDR1 shown in SEQ ID NO:54, CDR2 shown in SEQ ID NO:72, and CDR3 shown in SEQ ID NO:80; (3) CDR1 shown in SEQ ID NO:55, CDR2 shown in SEQ ID NO:64, and CDR3 shown in SEQ ID NO:73; (4) CDR1 shown in SEQ ID NO:56, CDR2 shown in SEQ ID NO:61, and CDR3 shown in SEQ ID NO:75; (5) CDR1 shown in SEQ ID NO:57, CDR2 shown in SEQ ID NO:66, and CDR3 shown in SEQ ID NO:74; (6) CDR1 shown in SEQ ID NO:58, CDR2 shown in SEQ ID NO:68, and CDR3 shown in SEQ ID NO:78; (7) CDR1 shown in SEQ ID NO:59, CDR2 shown in SEQ ID NO:67, and CDR3 shown in SEQ ID NO:82; (8) CDR1 shown in SEQ ID NO:59, CDR2 shown in SEQ ID NO:63, and CDR3 shown in SEQ ID NO:81; (9) CDR1 shown in SEQ ID NO:59, CDR2 shown in SEQ ID NO:71, and CDR3 shown in SEQ ID NO:77; (10) CDR1 shown in SEQ ID NO:59, CDR2 shown in SEQ ID NO:69, and CDR3 shown in SEQ ID NO:79; (11) CDR1 shown in SEQ ID NO:59, CDR2 shown in SEQ ID NO:70, and CDR3 shown in SEQ ID NO:77; and (12) CDR1 shown in SEQ ID NO:60, CDR2 shown in SEQ ID NO:65, and CDR3 shown in SEQ ID NO:73 CDR3.

6. The anti-TfR1 single-domain antibody according to claim 4 or 5, wherein the heavy chain further comprises a framework region FR; the framework region FR comprises FR1, FR2, FR3 and FR4, wherein: The amino acid sequence of the FR1 region is as shown in any one of SEQ ID NOs: 25-33 or has at least about 60% sequence homology with any one of SEQ ID NOs: 25-33; The amino acid sequence of the FR2 region is as shown in any one of SEQ ID NOs: 34-40 or has at least about 60% sequence homology with any one of SEQ ID NOs: 34-40; The amino acid sequence of the FR3 region is as shown in any one of SEQ ID NOs: 41-51 or has at least about 60% sequence homology with any one of SEQ ID NOs: 41-51; and The amino acid sequence of the FR4 region is as shown in SEQ ID NO:52 or has at least about 60% sequence homology to SEQ ID NO:

52.

7. An anti-TfR1 single-domain antibody, wherein the amino acid sequence of the anti-TfR1 single-domain antibody is as shown in any one of SEQ ID NOs: 1-12 or has at least about 80% sequence homology with any one of SEQ ID NOs: 1-12. 8 . The anti-TfR1 single-domain antibody according to any one of claims 4 to 7 , wherein the anti-TfR1 single-domain antibody is an Fc fusion antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody or a polyclonal antibody. 9 . A recombinant protein comprising the amino acid sequence of the anti-TfR1 single domain antibody according to any one of claims 4 to 8 . 10 . The recombinant protein according to claim 9 , wherein the recombinant protein is a multi-epitope antibody, a multivalent antibody or a multispecific antibody. 11 . An antibody complex comprising the anti-TfR1 single domain antibody according to any one of claims 4 to 8 or the recombinant protein according to claim 9 or 10.

12. The antibody complex according to claim 11, wherein the antibody complex is a conjugate formed by coupling the anti-TfR1 single domain antibody according to any one of claims 4 to 8 or the recombinant protein according to claim 9 or 10 with an active ingredient selected from a therapeutic agent, a diagnostic agent, a carrier protein and a liposome. 13 . A nucleic acid encoding the anti-TfR1 single domain antibody according to any one of claims 4 to 8 , or the recombinant protein according to claim 9 or 10 .

14. The nucleic acid according to claim 10, wherein the nucleotide sequence of the nucleic acid is as shown in any one of SEQ ID NOs: 13-24 or has at least about 80% sequence homology with any one of SEQ ID NOs: 13-24.

15. An expression vector comprising the nucleic acid according to claim 13 or 14. A host cell comprising the expression vector according to claim 15 .

17. A pharmaceutical composition comprising the anti-TfR1 single domain antibody according to any one of claims 4 to 8, the recombinant protein according to claim 9 or 10, the antibody complex according to claim 11 or 12, the nucleic acid according to claim 13 or 14, the expression vector according to claim 15, or the host cell according to claim 16, and a pharmaceutically acceptable carrier.

18. Use of the anti-TfR1 single domain antibody according to any one of claims 4 to 8, the recombinant protein according to claim 9 or 10, the antibody complex according to claim 11 or 12, the nucleic acid according to claim 13 or 14, the expression vector according to claim 15, or the host cell according to claim 16, or the pharmaceutical composition according to claim 17 in the preparation of a medicament for preventing or treating a disease associated with abnormal TfR1 receptor expression.

19. An anti-TfR1 single domain antibody according to any one of claims 4 to 8, a recombinant protein according to claim 9 or 10, an antibody complex according to claim 11 or 12, a nucleic acid according to claim 13 or 14, an expression vector according to claim 15, or a host cell according to claim 16, or a pharmaceutical composition according to claim 17 for use in preventing or treating a disease associated with abnormal TfR1 receptor expression.

20. A method for preventing or treating a disease associated with abnormal TfR1 receptor expression, comprising administering to a subject in need thereof a therapeutically effective amount of the anti-TfR1 single domain antibody of any one of claims 4 to 8, the recombinant protein of claim 9 or 10, the antibody complex of claim 11 or 12, the nucleic acid of claim 13 or 14, the expression vector of claim 15, or the host cell of claim 16, or the pharmaceutical composition of claim 17.

21. The use according to claim 18, the anti-TfR1 single domain antibody, the recombinant protein, the antibody complex, the nucleic acid, the expression vector, the host cell or the pharmaceutical composition used according to claim 19, or the method according to claim 20, wherein the disease is a tumor that abnormally expresses the TfR1 receptor.

22. The use according to claim 21, the anti-TfR1 single domain antibody, the recombinant protein, the antibody complex, the nucleic acid, the expression vector, the host cell or the pharmaceutical composition, or the method, wherein the tumor is breast cancer, glioma, lung adenocarcinoma, leukemia, lymphoma, non-Hodgkin's lymphoma or ovarian cancer.

23. A kit for detecting the TfR1 receptor, wherein the kit comprises the anti-TfR1 single domain antibody described in any one of claims 4 to 8, the recombinant protein described in claim 9 or 10, the antibody complex described in claim 11 or 12, or the nucleic acid described in claim 13 or 14, and optionally the kit is used to diagnose diseases associated with abnormal TfR1 receptor expression. 24 . The anti-TfR1 single domain antibody according to claim 4 , the recombinant protein according to claim 9 or 10 , or the antibody complex according to claim 11 or 12 for use as a drug that crosses the blood-brain barrier.

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