Anti-TFR1 antibody and application thereof

Abstract

The present disclosure relates to anti-TFR1 (transferrin receptor 1) antibodies, antigen binding fragments and uses thereof.

Description

[0001] Priority Claim

[0002] This application claims the priority of PCT / CN2022 / 136246 filed on December 2, 2022. All of the above are incorporated herein by reference. Technical Field

[0003] The present invention relates to anti-TFR1 (transferrin receptor 1) antibodies, antigen-binding fragments, and their uses. Background Art

[0004] Therapeutic antibodies are the fastest-growing class of therapeutic compounds, growing faster than small molecule drugs. For example, monoclonal antibodies have revolutionized cancer treatment. However, the large size of conventional antibodies hinders their delivery to tumor cells in vivo. The minimal target recognition module of conventional antibodies consists of two non-covalently bound variable domains (VH and VL). The intrinsic hydrophobic interactions of the VH and VL domains limit the stability and solubility of engineered antibodies, often causing aggregation and / or mispairing of the V-domains.

[0005] The discovery of heavy chain antibodies has brought unprecedented opportunities for cancer treatment. These unique antibody forms derived from camelids lack a complete light chain and CH1 domain and consist of only a single variable domain called VHH. Recombinant VHH molecules are small (15-20 kDa) and strictly monomeric; they bind targets with nanomolar affinity and are stable over a wide range of pH and temperature. Molecular manipulation of VHH is also easier; compared to traditional recombinant antibodies and their fragments, this helps to prepare multivalent forms of monoclonal antibodies because traditional recombinant antibodies and their fragments have problems due to aggregation and reduced affinity. In addition, the epitopes that VHH usually binds to are less immunogenic for traditional antibodies.

[0006] Generally, therapeutic antibodies are human or humanized antibodies. Human or humanized antibodies can be obtained by humanizing rodent antibodies (such as mouse antibodies) or by using phage libraries. However, these animal or phage libraries usually cannot produce heavy chain antibodies. Instead, heavy chain antibodies are usually obtained from the heavy chain antibodies of camelids. These camelid heavy chain antibodies need to be humanized. The humanization process may have an adverse effect on binding affinity and introduce immunogenic epitopes into the antibody. Repeated and time-consuming experiments are usually required to improve the properties of these antibodies. In some cases, these antibodies may also be immunogenic in patients, resulting in a decrease in their efficacy over time. Therefore, it is necessary to develop more types of antibodies to treat or prevent human diseases. Summary of the Invention

[0007] The present disclosure relates to an antibody or antigen-binding fragment thereof that binds to transferrin receptor 1 (TFR1), comprising: a heavy-chain single variable domain (VHH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein in some embodiments, the VHH CDR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a selected VHH CDR1 amino acid sequence, the VHH CDR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identity to a selected VHH CDR2 amino acid sequence, and the VHH CDR3 region comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identity to a selected VHH CDR3 amino acid sequence; in some embodiments, the selected VHH CDRs 1, 2, and 3 amino acid sequences are one of the following:

[0008] (1) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 1, 2, and 3, respectively;

[0009] (2) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 4, 5, and 6, respectively;

[0010] (3) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 7, 8, and 9, respectively;

[0011] (4) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 10, 11, and 12, respectively;

[0012] (5) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 13, 14, and 15, respectively;

[0013] (6) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 16, 17, and 18, respectively;

[0014] (7) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 19, 20, and 21, respectively; and

[0015] (8) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 22, 23, and 24, respectively.

[0016] In some embodiments, the VHH comprises CDR 1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 1, 2, and 3, respectively. In some embodiments, the VHH comprises CDR 1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 4, 5, and 6, respectively. In some embodiments, the VHH comprises CDR 1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 7, 8, and 9, respectively. In some embodiments, the VHH comprises CDR 1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 10, 11, and 12, respectively.

[0017] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to TFR1 and comprises a heavy-chain single variable domain (VHH) that comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a selected VHH sequence. In some embodiments, the selected VHH sequences are selected from SEQ ID NO: 25, 26, 27, and 28. In some embodiments, the VHH comprises the sequence of SEQ ID NO: 25. In some embodiments, the VHH comprises the sequence of SEQ ID NO: 26. In some embodiments, the VHH comprises the sequence of SEQ ID NO: 27. In some embodiments, the VHH comprises the sequence of SEQ ID NO: 28. In some embodiments, the antibody or antigen-binding fragment specifically binds to human TFR1, simian TFR1, murine TFR1, or chimeric TFR1. In some embodiments, the antibody or antigen-binding fragment is a human or humanized antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a multispecific antibody (e.g., a bispecific antibody).

[0018] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof that comprises the VHH CDR 1, 2, and 3 of the antibody or antigen-binding fragment described herein.

[0019] In some embodiments, the antibody or antigen-binding fragment comprises a human IgG Fc (e.g., a human IgG1 Fc). In some embodiments, the human IgG Fc comprises a non-asparagine residue (e.g., alanine) at position 297 according to EU numbering. In some embodiments, the antibody or antigen-binding fragment comprises two or more heavy-chain single variable domains.

[0020] In one aspect, the present disclosure relates to a nucleic acid that comprises a polynucleotide encoding the antibody or antigen-binding fragment thereof described herein. In some embodiments, the nucleic acid is cDNA.

[0021] On the one hand, the present disclosure relates to a vector comprising one or more nucleic acids as described herein.

[0022] On the one hand, the present disclosure relates to a cell comprising the vector as described herein. In some embodiments, the cell is a CHO cell.

[0023] On the one hand, the present disclosure relates to a cell comprising one or more nucleic acids as described herein.

[0024] On the one hand, the present disclosure relates to a method for producing an antibody or an antigen-binding fragment thereof, the method comprising: (a) culturing the cell as described herein under conditions sufficient for the cell to produce the antibody or the antigen-binding fragment thereof; and (b) collecting the antibody or the antigen-binding fragment thereof produced by the cell.

[0025] On the one hand, the present disclosure relates to an antibody-drug conjugate comprising the antibody or an antigen-binding fragment thereof as described herein covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or a cell growth inhibitor.

[0026] On the one hand, the present disclosure relates to a method for treating a subject suffering from a brain disease (e.g., brain cancer), the method comprising administering to the subject a therapeutically effective amount of a composition comprising the antibody or an antigen-binding fragment thereof or an antibody-drug conjugate as described herein. In some embodiments, the antibody or an antigen-binding fragment thereof or an antibody-drug conjugate can cross the blood-brain barrier (BBB) of the subject.

[0027] On the one hand, the present disclosure relates to a method for treating a subject suffering from cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising the antibody or an antigen-binding fragment thereof or an antibody-drug conjugate as described herein. In some embodiments, the cancer is brain cancer, lung cancer, gastric cancer, colorectal cancer, liver cancer, ovarian cancer, prostate cancer, leukemia, or breast cancer.

[0028] On the one hand, the present disclosure relates to a method for identifying a subject suffering from a brain disease (e.g., brain cancer), the method comprising detecting a sample collected from the subject with the antibody or an antigen-binding fragment thereof as described herein, thereby identifying that the subject suffers from a brain disease. In some embodiments, the sample is a brain parenchyma sample from the subject. In some embodiments, the subject as described herein is a human subject.

[0029] On the one hand, the present disclosure relates to a method for delivering an agent across the blood-brain barrier, the method comprising administering to the subject an agent covalently linked to the antibody or an antigen-binding fragment thereof as described herein. In some embodiments, the agent is an antibody or an antibody-drug conjugate. In some embodiments, the agent is an anti-amyloid antibody.

[0030] In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described herein and a pharmaceutically acceptable carrier. In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody-drug conjugate as described herein and a pharmaceutically acceptable carrier.

[0031] In one aspect, the present disclosure relates to an antibody or an antigen-binding fragment thereof that cross-competes with an antibody or an antigen-binding fragment thereof as described herein.

[0032] In one aspect, the present disclosure provides a method for preparing an antibody that specifically binds an antigen. The method includes exposing an animal as described herein to the antigen; obtaining (e.g., by sequencing) a nucleic acid sequence encoding a human heavy-chain immunoglobulin variable region in a cell that expresses a chimeric heavy-chain antibody that specifically binds the antigen; and operably linking the nucleic acid encoding the human heavy-chain immunoglobulin variable region to a nucleic acid encoding a human heavy-chain immunoglobulin constant region in the cell.

[0033] The present disclosure also relates to the offspring of a non-human mammal. In some embodiments, the non-human mammal is a rodent. In some embodiments, the non-human mammal is a mouse.

[0034] The present disclosure also provides a cell comprising a targeting vector as described herein. The present disclosure also relates to a cell (e.g., a stem cell, an embryonic stem cell, an immune cell, a B cell, a T cell, or a hybridoma) or cell line, or a primary cell culture derived from the non-human mammal or its offspring. The present invention also relates to a tissue, an organ, or a culture thereof from the non-human mammal or its offspring.

[0035] The present invention also relates to the use of the non-human mammal or its offspring, an animal model produced by the method described herein, in the development of products related to the immune process, the production of human antibodies, or in a model system for pharmacological, immunological, microbiological, and medical research.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference. In case of conflict, the present specification (including definitions) will prevail.

[0037] Other features and advantages of the present invention will be apparent from the following detailed description, the drawings, and the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 List the CDR sequences of the anti-TFR1 antibody heavy chain variable region according to Kabat numbering.

[0039] Figure 2 List the CDR sequences of the anti-TFR1 antibody heavy chain variable region according to IMGT numbering.

[0040] Figure 3 List the amino acid sequences discussed in this disclosure.

[0041] Figure 4A Show the antibody concentrations in the total brain protein of hTFR1 mice within 72 hours after intravenous injection (i.v.) of hIgG1 (G1), JR141-N (G2), 23B8-N (G3), 24A1-N (G4), 24G5-N (G5), or 24C9-N (G6).

[0042] Figure 4B Show the ratio of the antibody concentration in the total brain protein of hTFR1 mice to the antibody concentration in the serum within 72 hours after intravenous injection (i.v.) of hIgG1 (G1), JR141-N (G2), 23B8-N (G3), 24A1-N (G4), 24G5-N (G5), or 24C9-N (G6).

[0043] Figure 4C Show the antibody concentrations in the brain parenchyma of hTFR1 mice within 72 hours after intravenous injection (i.v.) of hIgG1 (G1), JR141-N (G2), 23B8-N (G3), 24A1-N (G4), 24G5-N (G5), or 24C9-N (G6).

[0044] Figure 4D Show the ratio of the antibody concentration in the brain parenchyma of hTFR1 mice to the serum antibody concentration within 72 hours after intravenous injection (i.v.) of hIgG1 (G1), JR141-N (G2), 23B8-N (G3), 24A1-N (G4), 24G5-N (G5), or 24C9-N (G6).

[0045] Figure 5A Show the detection results of the antibody concentrations in the brain parenchyma 24 hours after intravenous injection (i.v.) of hIgG1 (G1), JR141-N (G2), 23B8-N (G3), 24A1-N (G4), or 24G5-N (G5).

[0046] Figure 5B Show the detection results of the antibody concentrations in the total brain protein (whole brain) 24 hours after intravenous injection (i.v.) of hIgG1 (G1), JR141-N (G2), 23B8-N (G3), 24A1-N (G4), or 24G5-N (G5).

[0047] Figure 6 Show the results of antibody concentration after intravenous (i.v.) injection of JR141-N (G2 - G4) or 24G5-N (G5 - G7) for 6 hours or 24 hours. hIgG1 was used as a negative control.

[0048] Figure 7 Show the results of ADC concentration detection in the brain parenchyma after intravenous (i.v.) injection of 24G5-ADC or 24G5-mono-ADC for 72 hours.

[0049] Detailed description

[0050] Heavy chain antibodies (or antibodies consisting only of heavy chains) are antibodies that contain only heavy chains (usually two heavy chains) and lack the two light chains commonly found in antibodies. Naturally occurring heavy chain antibodies have been found in cartilaginous fish (such as sharks) and camelids (such as llamas). For example, in cartilaginous fish, the immunoglobulin new antigen receptor (IgNAR) is a heavy chain antibody. IgNAR has significant structural differences from other antibodies. It has five constant regions (CH) per chain instead of the usual three, has multiple disulfide bonds in uncommon positions, and the complementarity-determining region 3 (CDR3) forms an extended loop that covers the site where the light chain binds in other antibodies. These differences, combined with the phylogenetic age of cartilaginous fish, lead to the hypothesis that IgNAR may be more closely related to the primordial antigen-binding proteins than mammalian immunoglobulins.

[0051] The only mammals that possess heavy chain (IgG-like) antibodies are camelids such as dromedaries, camels, llamas, and alpacas. Like all mammals, camelids (such as llamas) can produce conventional antibodies (such as IgG1) consisting of two heavy chains and two light chains linked by disulfide bonds into a Y shape. However, they also produce two unique IgG subclasses: IgG2 and IgG3, also known as heavy chain IgG. These antibodies consist of only two heavy chains, lack the CH1 region, but still have an antigen-binding domain (such as VHH) at their N-terminus. Conventional Igs require the variable regions of the heavy and light chains to bind to each other to achieve a high degree of diversity in antigen-antibody interactions. Although isolated heavy and light chains still exhibit this ability, they show very low affinity compared to paired heavy and light chains. The unique feature of heavy chain IgG is that its monomeric antigen-binding region can specifically and highly affinity bind to antigens and can rival conventional antibodies in terms of diversity without the need to pair with other regions. This feature is mainly due to some major variations within the amino acid sequences of the variable regions of the two heavy chains, which lead to significant conformational changes compared to conventional Igs. The major substitutions in the variable regions prevent light chain binding to the heavy chain and also prevent unbound heavy chains from being recycled by immunoglobulin-binding proteins.

[0052] These single variable domains of heavy-chain antibodies (named VHH, sdAb, or nanobody) are the smallest antigen-binding domains produced by the adaptive immune system. Complementary determining region 3 (CDR3) of these antibody variable regions is twice as long as that of conventional antibodies. This results in an increased interaction surface with antigens and an increased diversity of antigen-antibody interactions, thus compensating for the absence of the light chain. With the longer complementary determining region 3 (CDR3), VHH can extend into protein clefts inaccessible to conventional antibodies, including some functionally significant sites such as the active site of an enzyme or the receptor-binding canyon on the surface of a virus. In addition, the additional cysteine residues make the structure more stable, thus increasing the strength of the interaction.

[0053] Compared with conventional antibodies carrying variable domains of conventional antibodies (VH and VL), VHH has many other advantages, including higher stability, solubility, expression yield, and refolding ability, as well as better in vivo tissue penetration. In addition, different from the VH domain of conventional antibodies, VHH does not show an inherent tendency to bind to the light chain. This facilitates the induction of heavy-chain antibodies in the presence of a functional light-chain locus. Moreover, since VHH does not bind to the VL domain, it is much easier to engineer VHH into bispecific antibody constructs than constructs containing a conventional VH-VL pair or VH-domain-based single-domain constructs.

[0054] The significant differences between camelid VHH and human VH domains are the length and orientation of the CDR3 loop. CDR3 corresponds to the unique region in the antibody molecule encoded by newly generated DNA elements during B-cell development. Gene recombination results in the fusion of D elements with flanking V and J elements. During the recombination process, further genetic diversity is generated by adding and / or deleting nucleotides at the junctions. Therefore, the CDR3 loop plays a major role in antibody diversity and specificity. In some early transgenic heavy-chain antibody animals, a limited number of variable region genes (IGHV, IGHD, and IGHJ) caused these animals to be unable to recognize certain antigens, although wild-type animals could mount a strong immune response to these antigens (Janssens, Rick et al., "Generation of heavy-chain-only antibodies in mice." Proceedings of the National Academy of Sciences 103.41 (2006): 15130-15135). The present disclosure provides fully humanized heavy-chain antibodies produced by genetically engineered animals that possess a complete human heavy-chain antibody repertoire.

[0055] As used herein, the term "antibody" refers to an antigen-binding molecule that comprises at least one (e.g., one, two, three, four, five, or six) complementarity-determining region (CDR) (e.g., any one of the three CDRs of an immunoglobulin light chain or any one of the three CDRs of an immunoglobulin heavy chain) and is capable of specifically binding an epitope. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, heavy-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody may comprise the Fc region of a human antibody. The term "antibody" also includes derivatives such as bispecific antibodies, single-chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0056] As used herein, the term "antigen-binding fragment" refers to a portion of a full-length antibody, wherein the portion is capable of specifically binding an antigen. In some embodiments, an antigen-binding fragment comprises at least one variable domain (e.g., the variable domain of a heavy chain or the variable domain of a light chain). Non-limiting examples of antibody fragments include, for example, Fab, Fab’, F(ab’)2, and Fv fragments.

[0057] As used herein, the term "human antibody" refers to an antibody encoded by nucleic acids present in humans (e.g., rearranged human immunoglobulin heavy or light chain loci). In some embodiments, human antibodies are collected from humans or produced in human cell cultures (e.g., human hybridoma cells). In some embodiments, human antibodies are produced in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, human antibodies are produced in bacterial or yeast cells. In some embodiments, human antibodies are produced in transgenic non-human animals (e.g., mice) containing unrearranged or rearranged human immunoglobulin loci (e.g., human immunoglobulin heavy or light chain loci).

[0058] As used herein, the term "chimeric antibody" refers to an antibody that comprises sequences present in at least two different antibodies (e.g., antibodies from two different mammalian species, such as human and mouse antibodies). A non-limiting example of a chimeric antibody is an antibody that comprises human antibody variable domain sequences (e.g., all or part of the light chain and / or heavy chain variable domain sequences) and non-human antibody constant domains. Other examples of chimeric antibodies are described herein and are known in the art.

[0059] As used herein, the term "humanized antibody" refers to a non-human antibody that comprises sequences derived from a non-human (e.g., mouse) immunoglobulin and sequences derived from a human immunoglobulin.

[0060] As used herein, the term "single-chain antibody" refers to a single polypeptide comprising at least two immunoglobulin variable domains (e.g., variable domains of mammalian immunoglobulin heavy or light chains) and capable of specifically binding an antigen.

[0061] As used herein, the term "heavy-chain antibody" refers to an antibody molecule consisting only of heavy chains (usually two) and lacking any light chains.

[0062] As used herein, the term "VHH" refers to the variable domain derived from a heavy-chain antibody. A VHH can specifically recognize an antigen without pairing with a VL. In some embodiments, the VHHs (also referred to as sdAbs or nanobodies) described herein are derived from any of the humanized heavy-chain antibodies described herein. In some embodiments, the VHHs, sdAbs or nanobodies described herein are derived from heavy-chain antibodies produced by any genetically modified non-human animal described herein.

[0063] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification to describe an animal, including a human or non-human. The present disclosure encompasses veterinary and non-veterinary applications. A human patient can be an adult or an adolescent (e.g., a person under 18 years old). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. For example, it includes non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., pigs, mini-pigs), horses, dogs, cats, cows, and other domestic, farm, and zoo animals.

[0064] As used herein, when referring to an antibody, the phrases "specifically binding" and "specifically binds" mean that the interaction of the antibody with its target molecule is preferred over its interaction with other molecules, because this interaction depends on the presence of a specific structure (i.e., antigenic determinant or epitope) on the target molecule; in other words, the reagent recognizes and binds to a molecule comprising the specific structure, rather than all molecules in general. An antibody that specifically binds a target molecule can be referred to as a target-specific antibody.

[0065] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably and refer to an amino acid polymer of any length composed of at least two amino acids.

[0066] As used herein, the terms "polynucleotide", "nucleic acid molecule" and "nucleic acid sequence" are used interchangeably and refer to a nucleotide polymer of any length composed of at least two nucleotides, including but not limited to DNA, RNA, DNA / RNA hybrids and their modified forms.

[0067] Antibodies and antigen-binding fragments

[0068] The present disclosure provides antibodies and antigen-binding fragments thereof (e.g., heavy-chain antibodies, humanized heavy-chain antibodies, or multispecific antibodies) produced by the methods described herein.

[0069] Generally, conventional antibodies are composed of two types of polypeptide chains, namely light chains and heavy chains. The non-limiting antibodies of the present disclosure can be intact four-chain immunoglobulin antibodies comprising two heavy chains and two light chains. The heavy chain of the antibody can be any isotype (including IgM, IgG, IgE, IgA, or IgD) or subclass (including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc.). The light chain can be a κ light chain or a λ light chain. The antibody can comprise two identical copies of the light chain and two identical copies of the heavy chain. Each heavy chain comprises a variable domain (or variable region, VH) and multiple constant domains (or constant regions), which are bound to each other by disulfide bonds within the constant region to form the "backbone" of the antibody. Each light chain comprises a variable domain (or variable region, VL) and a constant domain (or constant region), which are bound to one heavy chain each by disulfide bonds. The variable region of each light chain aligns with the variable region of the heavy chain to which it binds. The variable regions of the light and heavy chains each contain three hypervariable regions, which are sandwiched between more conserved framework regions (FRs).

[0070] These hypervariable regions, i.e., complementarity-determining regions (CDRs), form loop structures that constitute the major antigen-binding surface of the antibody. The four framework regions mostly adopt a β-sheet conformation, while the CDRs form loop structures that connect the β-sheet structures and, in some cases, also form part of the β-sheet structure. The CDRs in each chain are brought close together by the framework regions and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding region.

[0071] Methods for determining the CDR regions of an antibody by analyzing its amino acid sequence are well known, and there are multiple commonly used CDR definitions. The Kabat definition is based on sequence variability, while the Chothia definition is based on the positions of structural loop regions. These methods and definitions are described, for example, in Martin's "Protein sequence and structure analysis of antibody variable domains," Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439;

[0072] "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains," by Abhinandan et al., Molecular immunology 45.14 (2008): 3832-3839; Wu, T.T. and Kabat, E.A. (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203: 121-53 (1991); Morea et al., Biophys Chem. 68(1-3): 9-16 (Oct. 1997); Morea et al., J Mol Biol. 275(2): 269-94 (Jan. 1998); Chothia et al., Nature 342(6252): 877-83 (Dec. 1989); Ponomarenko and Bourne, BMC Structural Biology 7: 64 (2007); each of which is incorporated herein by reference.

[0073] CDRs are very important for recognizing epitopes of antigens. As used herein, an "epitope" is the smallest portion of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope may be about three, four, five, six, or seven amino acids, but these amino acids need not be in a continuous linear sequence in the primary structure of the antigen, since the epitope may depend on the three-dimensional conformation of the antigen based on its secondary and tertiary structures.

[0074] In some embodiments, the antibody is a full-length immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgG4, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved and differ in their constant regions, particularly in the hinge region and the upper CH2 domain. The sequences and differences of the IgG subclasses are known in the art, such as "IgG subclasses and allotypes: from structure to effector functions." by Vidarsson et al., Frontiers in immunology 5 (2014); "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases." by Irani et al., Molecular immunology 67.2 (2015): 171-182; The human IgG subclasses: molecular analysis of structure, function and regulation. by Shakib, Farouk et al., Elsevier, 2016; each of which is incorporated herein by reference. The heavy chain constant region in a heavy chain antibody can be derived from any of the immunoglobulin molecules described herein (e.g., IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4, IgM, IgD, IgE, IgA).

[0075] Antibodies can also be immunoglobulin molecules derived from any species (e.g., human, rodent, mouse, rat, camel). Antibodies disclosed herein also include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, and chimeric antibodies comprising an immunoglobulin binding domain fused to another polypeptide. The term "antigen-binding domain" or "antigen-binding fragment" refers to the portion of an antibody that retains the specific binding activity of the intact antibody, i.e., any portion of the antibody that is capable of specifically binding to an epitope on the target molecule of the intact antibody. It includes, for example, Fab, Fab’, F(ab’)2, and variants of these fragments. Thus, in some embodiments, the antibody or its antigen-binding fragment can be, for example, scFv, Fv, Fd, dAb, bispecific antibody, bispecific scFv, diabody, linear antibody, single-chain antibody molecule, multispecific antibody formed from antibody fragments, and any polypeptide comprising a binding domain that is identical or homologous to an antibody binding domain. Non-limiting examples of antigen-binding domains include, for example, the heavy-chain and / or light-chain CDRs of an intact antibody, the heavy-chain and / or light-chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or a single CDR from the heavy or light chain of an intact antibody.

[0076] In some embodiments, the antigen-binding fragment can form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of a VHH as described herein with a CD3-ζ transmembrane and intracellular domain.

[0077] Antibodies and their antigen-binding fragments (e.g., humanized antibodies or chimeric antibodies) produced by the methods described herein have a variety of advantages. In some embodiments, the desired properties (e.g., binding affinity, thermal stability, and / or limited aggregation) can be obtained without further optimization.

[0078] In some embodiments, the antibody (or its antigen-binding fragment) specifically binds to the target with a dissociation rate (koff) of less than 0.1 s -1 , less than 0.01 s -1 , less than 0.001 s -1 , less than 0.0001 s -1 or less than 0.00001 s -1 . In some embodiments, the dissociation rate (koff) is greater than 0.01 s -1 , greater than 0.001 s -1 , greater than 0.0001 s -1 , greater than 0.00001 s -1 or greater than 0.000001 s -1 .

[0079] In some embodiments, the kinetic binding rate (kon) is greater than 1x10 2 / Ms, greater than 1x10 3 / Ms, greater than 1x10 4 / Ms, greater than 1x10 5 / Ms or greater than 1x10 6 / Ms. In some embodiments, the kinetic binding rate (kon) is less than 1x10 5 / Ms, less than 1x10 6 / Ms or less than 1x10 7 / Ms.

[0080] The affinity can be derived from the quotient of the kinetic rate constants (KD = koff / kon). In some embodiments, KD is less than 1x10 -6 M, less than 1x10 -7 M, less than 1x10 -8 M, less than 1x10 -9 M or less than 1x10 -10 M. In some embodiments, KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM or 1 nM. In some embodiments, KD is greater than 1x10 -7 M, greater than 1x10 -8 M, greater than 1x10 -9 M, greater than 1x10 -10 M, greater than 1x10 -11 M or greater than 1x10 -12 M. In some embodiments, the antibody binds the target with a KD of less than or equal to about 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nM.

[0081] In some embodiments, the thermal stability is determined. The Tm of the antibody or antigen-binding fragment described herein can be higher than 60 °C, 61 °C, 62 °C, 63 °C, 64 °C, 65 °C, 66 °C, 67 °C, 68 °C, 69 °C, 70 °C, 71 °C, 72 °C, 73 °C, 74 °C, 75 °C, 76 °C, 77 °C, 78 °C, 79 °C, 80 °C, 81 °C, 82 °C, 83 °C, 84 °C, 85 °C, 86 °C, 87 °C, 88 °C, 89 °C, 90 °C, 91 °C, 92 °C, 93 °C, 94 °C or 95 °C.

[0082] In various embodiments, the parental heavy-chain antibody sequence is replaced to prepare a variant heavy-chain antibody. Generally, the heavy-chain antibody variant of the parental heavy-chain antibody has a binding affinity for a specific antigen that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% (e.g., at least 150%, at least 200%, at least 500%, at least 1000% or up to at least 10,000%) of the binding affinity of the parental heavy-chain antibody. In some embodiments, the variant heavy-chain antibody will contain a single amino acid replacement compared to the parental heavy-chain antibody. However, in other embodiments, several amino acids (e.g., up to about 5 or 10 or more) are replaced compared to the parental heavy-chain antibody sequence derived from other human heavy-chain sequences having the same amino acid at a given position. In various embodiments, the resulting variant heavy-chain antibody is tested to confirm that the desired binding affinity and / or specificity has not been significantly reduced. In some embodiments, improved variant heavy-chain antibodies are prepared by replacing amino acids of different human heavy-chain sequences. In various embodiments, the VHH has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the parental VHH.

[0083] The VHHs described herein can be used to prepare multispecific (e.g., bispecific) antibodies. In one aspect, the invention provides a multispecific antibody comprising: a first antigen-binding portion and a second antigen-binding portion. In some embodiments, the first antigen-binding portion comprises a heavy-chain variable domain (VH) and a light-chain variable domain (VL), wherein the VH and VL together form an antigen-binding site that specifically binds a first epitope. In some embodiments, the first antigen-binding portion comprises a VHH that specifically binds a first epitope. In some embodiments, the second antigen-binding portion comprises a VHH that specifically binds a second epitope. In some embodiments, the first epitope and the second epitope are from the same antigen.

[0084] In some embodiments, the first epitope and the second epitope are from different antigens.

[0085] In some embodiments, the first antigen-binding portion is a full-length antibody consisting of two heavy chains and two light chains. In some embodiments, the first antigen-binding portion is an antibody fragment comprising a heavy chain and a light chain, the heavy chain comprising VH and the light chain comprising VL. In some embodiments, the second antigen-binding portion comprises a single polypeptide chain. In some embodiments, the C-terminus of the second antigen-binding portion is fused to the N-terminus of at least one heavy chain of the first antigen-binding portion. In some embodiments, the C-terminus of the second antigen-binding portion is fused to the N-terminus of at least one light chain of the first antigen-binding portion. In some embodiments, the N-terminus of the second antigen-binding portion is fused to the C-terminus of at least one heavy chain of the first antigen-binding portion. In some embodiments, the N-terminus of the second antigen-binding portion is fused to the C-terminus of at least one light chain of the first antigen-binding portion. In some embodiments, the second antigen-binding portion is a Fab-like domain comprising a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising a first VHH fused to a CH1 domain and the second polypeptide chain comprising a second VHH fused to a CL domain.

[0086] In some embodiments, the antibody or its antigen-binding fragment is a trispecific antibody. In some embodiments, the trispecific antibody is a trispecific VHH-Fc. In some embodiments, the trispecific antibody comprises the same VHH. In some embodiments, the trispecific antibody comprises different VHHs. In some embodiments, the VHHs bind to the same epitope. In some embodiments, the VHHs bind to different epitopes.

[0087] In some embodiments, the antibody or its antigen-binding fragment has four or more VHHs. In some embodiments, for increased developability, at least four VHHs are combined without adding an IgG Fc domain to construct a tetravalent VHH. Compared to bispecific and trispecific VHH-Fc, these molecules have the additional advantage of increased affinity and avidity for antigens despite the lack of Fc effector functions.

[0088] In some embodiments, these antibodies or their antigen-binding fragments (e.g., comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10) have a functional Fc.

[0089] In some embodiments, the heavy chain antibodies produced by the genetically modified non-human animals described herein have a VHH domain comprising CDR1, CDR2, and CDR3. In some embodiments, the CDR3 length is between 6 and 23, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the CDR3 length is at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23.

[0090] Transferrin receptor 1 (TFR1)

[0091] Transferrin receptor 1 (TFR1), also known as cluster of differentiation 71 (CD71), is widely expressed and can bind transferrin (Tf) with high affinity. Human TFR1 is a 90 kDa type II transmembrane glycoprotein composed of 760 amino acids and exists on the cell surface as a dimer (180 kDa) linked by disulfide bonds. The TFR1 monomer consists of a large extracellular C-terminal domain of 671 amino acids containing the Tf binding site, a transmembrane region (28 amino acids), and an intracellular N-terminal domain (61 amino acids). The C-terminal extracellular domain contains three N-linked glycosylation sites at asparagine residues 251, 317, and 727 and one O-linked glycosylation site at threonine 104, all of which are necessary for the receptor to function fully.

[0092] Transferrin (Tf) is an 80 kDa glycoprotein composed of two 40 kDa subunits called the N-lobe and the C-lobe, which are separated by a short linker sequence. Each subunit can bind one free ferric ion (Fe 3+ ), so Tf can bind up to two iron atoms at most. The iron-free form of Tf, i.e., apo-transferrin (apo-Tf), can bind Fe 3+ efficiently in the blood and transport it to the cell surface for endocytosis by interacting with TFR1. As a membrane protein that regulates iron uptake, TFR1 is a member of the TFR family and exhibits nanomolar affinity for transferrin (Tf) bound to Fe 3+ . The Tf-TFR1 complex is endocytosed by clathrin-mediated endocytosis, and when the pH drops to 5.5, Fe 3+ dissociates from Tf. Under this pH condition, apo-Tf and TFR1 remain bound and are recycled back to the cell surface under physiological pH conditions, and then apo-Tf is released.

[0093] Iron uptake through the transferrin receptor is an important pathway for tumor cells to absorb iron. Increasing evidence indicates that TFR1 is involved in the occurrence and development of tumors, and its expression is significantly dysregulated in many tumors. The relationship between TFR1 and cancer has been revealed, making TFR1 a valuable drug target for cancer intervention.

[0094] In preclinical studies, TFR1 expressed on the endothelial cells of the blood-brain barrier has also been used to deliver macromolecules, including antibodies, to the brain. Antibodies targeting TFR1 can cross the blood-brain barrier without interfering with iron uptake.

[0095] For a detailed description of TFR1, Tf, and their functions, see, for example, Candelaria, P.V. et al., "Antibodies targeting the transferrin receptor 1 (TFR1) as direct anti-cancer agents", Frontiers in Immunology 12 (2021): 607692; and Shen, Y. et al., "Transferrin receptor 1 in cancer: a new sight for cancer therapy", American Journal of Cancer Research 8.6 (2018): 916; both of which are incorporated herein by reference in their entirety.

[0096] Heavy-chain single variable domain (VHH) antibody

[0097] Monoclonal antibodies and recombinant antibodies are important tools in the fields of medicine and biotechnology. Like all mammals, camelids (such as llamas) can produce conventional antibodies (such as IgG1) with a Y-shaped structure formed by two heavy chains and two light chains linked together by disulfide bonds. However, they can also produce two unique IgG subclasses: IgG2 and IgG3, also known as heavy-chain IgG. These antibodies consist of only two heavy chains, lack the CH1 region, but still have an antigen-binding domain called VHH (or nanobody) at their N-terminus. Conventional Igs require the combination of heavy-chain and light-chain variable regions to achieve a high degree of diversity in antigen-antibody interactions. Although isolated heavy and light chains still possess this ability, their affinity is very low compared to paired heavy and light chains. The unique feature of heavy-chain IgG is that its monomeric antigen-binding region can specifically and highly affinity bind to antigens and is comparable in diversity to conventional antibodies without the need to pair with other regions. This property is mainly attributed to some major variations in the amino acid sequences of the two heavy-chain variable regions, which trigger deep conformational changes compared to conventional Igs. The major substitutions in the variable region prevent the binding of the light chain to the heavy chain and also prevent the unbound heavy chain from being recycled by immunoglobulin-binding proteins.

[0098] The single variable domain of these antibodies (called VHH, sdAb, or nanobody) is the smallest antigen-binding domain produced by the adaptive immune system. It has been found that the length of the third complementarity-determining region (CDR3) of the variable region of these antibodies is twice that of conventional antibodies. This results in an increased interaction surface with antigens and an increased diversity of antigen-antibody interactions, thus compensating for the absence of the light chain. With the longer complementarity-determining region 3 (CDR3), VHH can penetrate into protein clefts that are inaccessible to conventional antibodies, including functionally significant sites such as the active site of an enzyme or the receptor-binding canyon on the surface of a virus.

[0099] Compared with conventional antibodies carrying the variable domains (VH and VL) of conventional antibodies, VHH has many other advantages, including higher stability, solubility, expression yield, and refolding ability, as well as better in vivo tissue penetration and internalization ability. In addition, different from the VH domain of conventional antibodies, VHH has no intrinsic tendency to bind to the light chain. Since VHH does not bind to the VL domain, it is much easier to engineer VHH into multi-specific (such as bispecific antibody) constructs than constructs containing conventional VH-VL pairs or VH-domain-based single-domain constructs.

[0100] The present invention provides, for example, anti-TFR1 antibodies, modified antibodies thereof, chimeric antibodies thereof, and humanized antibodies thereof.

[0101] According to the Kabat definition, the CDR sequences of 23B8 and its derivative antibodies (such as humanized antibodies) respectively include the VHH CDRs shown in SEQ ID NO: 1, 2, and 3. The CDRs can also be defined according to the IMGT system. According to the IMGT definition, the CDRs of the VHH domain are respectively shown in SEQ ID NO: 13, 14, and 15.

[0102] According to the Kabat definition, the CDR sequences of 24A1 and its derivative antibodies (such as humanized antibodies) respectively include the VHH CDRs shown in SEQ ID NO: 4, 5, and 6. The CDRs can also be defined according to the IMGT system. According to the IMGT definition, the CDRs of the VHH domain are respectively shown in SEQ ID NO: 17 and 18.

[0103] According to the Kabat definition, the CDR sequences of 24C9 and its derivative antibodies (such as humanized antibodies) respectively include the VHH CDRs shown in SEQ ID NO: 7, 8, and 9. The CDRs can also be defined according to the IMGT system. According to the IMGT definition, the CDRs of the VHH domain are respectively shown in SEQ ID NO: 19, 20, and 21.

[0104] According to the Kabat definition, the CDR sequences of 24G5 and its derivative antibodies (such as humanized antibodies) respectively include the VHH CDRs shown in SEQ ID NO: 10, 11, and 12. The CDRs can also be defined according to the IMGT system. According to the IMGT definition, the CDRs of the VHH domain are respectively shown in SEQ ID NO: 22, 23, and 24.

[0105] The amino acid sequence of the VHH domain of the 23B8 antibody is shown in SEQ ID NO: 25. The amino acid sequence of the VHH domain of the 24A1 antibody is shown in SEQ ID NO: 26. The amino acid sequence of the VHH domain of the 24C9 antibody is shown in SEQ ID NO: 27. The amino acid sequence of the VHH domain of the 24G5 antibody is shown in SEQ ID NO: 28.

[0106] The amino acid sequences of various modified or humanized VHHs are also provided. Since there are multiple ways to modify or humanize heavy-chain antibodies (for example, the sequence can undergo different amino acid substitutions), the VHH domain of the heavy-chain antibody can have more than one version of the humanized sequence. In some embodiments, the humanized VHH domain has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences in SEQ ID NO: 25 - 28.

[0107] In addition, in some embodiments, the antibodies or antigen-binding fragments thereof described herein may further comprise one, two, or three VHH CDRs selected from the group consisting of SEQ ID NOs: 1-3, SEQ ID NOs: 4-6, SEQ ID NOs: 7-9, SEQ ID NOs: 10-12, SEQ ID NOs: 13-15, SEQ ID NOs: 16-18, SEQ ID NOs: 19-21, and SEQ ID NOs: 22-24.

[0108] In some embodiments, an antibody may have a heavy-chain single variable domain (VHH) comprising complementarity-determining regions (CDR) 1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity to a selected VHH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity to a selected VHH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity to a selected VHH CDR3 amino acid sequence. The selected VHH CDR 1, 2, and 3 amino acid sequences are as Figure 1 and Figure 2 shown.

[0109] In some embodiments, the antibodies or antigen-binding fragments thereof described herein may comprise a heavy-chain single variable domain (VHH) comprising one, two, or three VHH CDR1s having 0, 1, or 2 amino acid insertions, deletions, or substitutions; VHH CDR2s having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and VHH CDR3s having 0, 1, or 2 amino acid insertions, deletions, or substitutions, wherein the VHH CDR1, VHH CDR2, and VHH CDR3 are selected from the CDRs Figure 3 as set forth herein.

[0110] In some embodiments, the antibodies or antigen-binding fragments thereof described herein may comprise a heavy-chain single variable domain (VHH) comprising one, two, or three of the following CDRs: SEQ ID NO: 1 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 3 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0111] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a heavy-chain single variable domain (VHH) comprising one, two, or three of the following CDRs: SEQ ID NO: 4 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 5 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 6 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0112] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a heavy-chain single variable domain (VHH) comprising one, two, or three of the following CDRs: SEQ ID NO: 7 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 9 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0113] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a heavy-chain single variable domain (VHH) comprising one, two, or three of the following CDRs: SEQ ID NO: 10 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 12 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0114] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a heavy-chain single variable domain (VHH) comprising one, two, or three of the following CDRs: SEQ ID NO: 13 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 14 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 15 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0115] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a heavy-chain single variable domain (VHH) comprising one, two, or three of the following CDRs: SEQ ID NO: 16 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 17 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 18 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0116] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a heavy-chain single variable domain (VHH) that comprises one, two, or three of the following CDRs: SEQ ID NO: 19 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 21 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0117] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a heavy-chain single variable domain (VHH) that comprises one, two, or three of the following CDRs: SEQ ID NO: 22 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 23 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 24 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0118] Insertions, deletions, and substitutions may occur within the CDR sequences or at one or both termini of the CDR sequences. In some embodiments, the CDRs are defined according to Kabat numbering. In some embodiments, the CDRs are defined according to Chothia numbering. In some embodiments, the CDRs are defined according to combined numbering. In some embodiments, the CDRs are defined according to IMGT numbering.

[0119] The present disclosure also provides an antibody or an antigen-binding fragment thereof that binds to TFR1 (human TFR1). The antibody or antigen-binding fragment thereof comprises a heavy-chain single variable region (VHH) that comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity to a selected VHH sequence. In some embodiments, the selected VHH sequence is SEQ ID NO: 25. In some embodiments, the selected VHH sequence is SEQ ID NO: 26. In some embodiments, the selected VHH sequence is SEQ ID NO: 27. In some embodiments, the selected VHH sequence is SEQ ID NO: 28.

[0120] To determine the percent identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). Then the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the two molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. For illustration, the comparison of two sequences and the determination of percent identity can be accomplished, for example, using the Blossum62 scoring matrix (gap penalty 12, gap extension penalty 4, frameshift gap penalty 5).

[0121] The present disclosure also provides a nucleic acid comprising a polynucleotide encoding a polypeptide comprising a single variable domain of an immunoglobulin heavy chain (VHH). The VHH comprises CDRs as shown in Figure 1 and Figure 2 or has a sequence as shown in Figure 3

[0122] Antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of an antibody or an antibody fragment, and multispecific (e.g., bispecific) antibodies or antibody fragments. Other antibodies provided herein are polyclonal antibodies, monoclonal antibodies, multispecific (multimeric, e.g., bispecific) antibodies, human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, intracellularly produced antibodies (i.e., intrabodies), and antigen-binding fragments thereof.

[0123] In some embodiments, the antibody or its antigen-binding fragment comprises an Fc domain, which can be derived from various types (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), classes (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclasses. In some embodiments, the Fc domain is derived from an IgG antibody or its antigen-binding fragment. In some embodiments, the Fc domain comprises one, two, three, four, or more heavy chain constant regions.

[0124] ​The present disclosure also provides an antibody or an antigen-binding fragment thereof that cross-competes with any antibody or antigen-binding fragment described herein. Cross-competition assays are known in the art and are described, for example, in Moore et al., “Antibody Cross-Competition Analysis of the Human Immunodeficiency Virus type 1 gp120 exterior envelope glycoprotein.” Journal of Virology 70.3 (1996): 1863-1872, which is incorporated herein by reference in its entirety.

[0125] In one aspect, the present disclosure also provides an antibody or an antigen-binding fragment thereof that binds to the same epitope or region as any antibody or antigen-binding fragment described herein. Cross-competition assays are known in the art and are described, for example, in Estep et al., “High throughput solution-based measurement of antibody-antigen affinity and epitope binning”. MAbs. Vol. 5. No. 2. Taylor & Francis, 2013, which is incorporated herein by reference in its entirety.

[0126] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain single variable domain (VHH) CDR1 selected from SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, and 22.

[0127] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain single variable domain (VHH) CDR2 selected from SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, and 23.

[0128] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain single variable domain (VHH) CDR3 selected from SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, and 24.

[0129] Antibody Characteristics

[0130] TFR1 plays a key role in cellular iron uptake by interacting with transferrin (TF) that binds iron. Iron is essential for multiple cellular processes and is crucial for DNA synthesis and cell proliferation. Since TFR1 plays a central role in the pathological processes of cancer cells, malignant cells usually overexpress TFR1, and this increased expression may be associated with poor prognosis in various cancers. The elevated expression level of TFR1 on malignant cells, together with its extracellular accessibility, endocytic ability, and central role in the pathological processes of cancer cells, makes this receptor an ideal target for antibody-mediated therapy.

[0131] In some embodiments, the antibodies or antigen-binding fragments thereof described herein do not block the binding between TFR1 and TF. In some embodiments, the antibodies or antigen-binding fragments thereof described herein are capable of blocking the binding between TFR1 and TF. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be conjugated to an anti-cancer agent and enter cells through receptor-mediated endocytosis. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can disrupt receptor function. In some embodiments, the antibodies or antigen-binding fragments thereof described herein do not induce Fc effector function, thereby avoiding or reducing their negative impact on normal cells.

[0132] The present disclosure provides antibodies or antigen-binding fragments thereof comprising a human Fc domain that have an ability to induce Fc-dependent effector functions that is at least or about 1-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, or at least or about 100-fold compared to when the antibodies or antigen-binding fragments thereof described herein are not present.

[0133] The present disclosure provides antibodies or antigen-binding fragments thereof comprising a human Fc domain that have an ability to induce a host immune response that is at least or about 1-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, or at least or about 100-fold compared to when the antibodies or antigen-binding fragments thereof of the present invention are not present.

[0134] The antibodies or antigen-binding fragments thereof provided by the present disclosure can be internalized into human brain cells (e.g., cortical microvascular endothelial cells), and the internalization rate is at least 50%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. In some embodiments, compared with the isotype control antibody, the internalization rate of the antibody or antigen-binding fragment thereof described herein is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 500-fold or 1000-fold.

[0135] In some embodiments, the antibody or antigen-binding fragment thereof provided herein comprises one heavy chain. In some embodiments, the antibody or antigen-binding fragment thereof provided herein comprises a pair of heavy chains. In some embodiments, the pair of heavy chains is linked by a disulfide bond. In some embodiments, the pair of heavy chains includes a knob-in-hole modification. In some embodiments, the heavy chain comprises the Fc domain of human IgG. In some embodiments, each heavy chain of the antibody or antigen-binding fragment thereof comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 VHH domains. In some embodiments, the VHH domains in each heavy chain specifically bind to the same epitope. In some embodiments, the VHH domains in each heavy chain specifically bind to different epitopes. In some embodiments, the VHH domains of each heavy chain bind to at least 1, 2, 3, 4 or 5 different epitopes.

[0136] In some embodiments, the antibody or antigen-binding fragment thereof is a bispecific antibody or a trispecific antibody. In some embodiments, the antibody or antigen-binding fragment thereof can specifically bind to at least 4, 5 or 6 antigens.

[0137] In some embodiments, the antibody (or antigen-binding fragment thereof) specifically binds to TFR1 with a dissociation rate (koff) of less than 0.1 s -1 , less than 0.01 s -1 , less than 0.001 s -1 , less than 0.0001 s -1 or less than 0.00001 s -1 . In some embodiments, the dissociation rate (koff) is greater than 0.01 s -1 , greater than 0.001 s -1 , greater than 0.0001 s -1 , greater than 0.00001 s -1 or greater than 0.000001 s -1 .

[0138] In some embodiments, the kinetic association rate (kon) is greater than 1×10 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×10 5 / Ms or greater than 1×10 6 / Ms. In some embodiments, the kinetic association rate (kon) is less than 1×10 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 / Ms.

[0139] The affinity can be derived from the quotient of the kinetic rate constants (KD = koff / kon). In some embodiments, KD is less than 1×10 -6 M, less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M or less than 1×10 -10 M. In some embodiments, KD is less than 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM or 1 nM. In some embodiments, KD is greater than 1×10 -7 M, greater than 1×10 -8 M, greater than 1×10 -9 M, greater than 1×10 -10 M, greater than 1×10 -11 M or greater than 1×10 -12 M.

[0140] Common techniques for measuring the affinity of an antibody for an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR). In some embodiments, the antibody binds to human TFR1, monkey TFR1, mouse TFR1, or chimeric TFR1.

[0141] In some embodiments, the antibody does not bind to human TFR1, monkey TFR1, mouse TFR1, or chimeric TFR1.

[0142] In some embodiments, the thermal stability was determined. The antibodies or antigen-binding fragments described herein can have a Tm greater than 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C. In some embodiments, the Tm is less than 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C. The Tagg (aggregation temperature, e.g., Tagg at 266 nm (Tagg266) or Tagg at 473 nm (Tagg473)) of the antibodies or antigen-binding fragments as described herein is greater than 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C. In some embodiments, the Tagg is less than 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.

[0143] In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.

[0144] In some embodiments, the antibody or its antigen-binding fragment has a functional Fc region. In some embodiments, the antibody or its antigen-binding fragment comprises a human IgG1 Fc region. In some embodiments, the human IgG1 Fc region comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 34.

[0145] In some embodiments, the antibody or antigen-binding fragment does not have an Fc region. For example, the antibody (or its antigen-binding fragment) is a polypeptide comprising one or more VHH domains interconnected by a linker peptide. In some embodiments, the antibody comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 VHH domains. In some embodiments, the VHH domains specifically bind the same epitope. In some embodiments, the VHH domains bind different epitopes. In some embodiments, the VHH domains bind at least 1, 2, 3, 4, or 5 different epitopes.

[0146] In some embodiments, the antibody or its antigen-binding fragment does not have a functional Fc region. In some embodiments, the Fc region has an LALA mutation (EU numbering: L234A and L235A), or an LALA-PG mutation (EU numbering:

[0147] L234A, L235A, P329G). In some embodiments, according to the EU numbering, the Fc region has a mutation at position 297 (e.g., N297A). In some embodiments, the mutated human IgG1 Fc region comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 35.

[0148] In some embodiments, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, or 72 hours after administering the drug to a subject, the concentration of the antibody or its antigen-binding fragment described herein in the brain (e.g., whole brain or brain parenchyma) can be greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the concentration immediately after administration (e.g., 0.5 hours). In some embodiments, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, or 72 hours after administering the drug to a subject, the concentration of the antibody or its antigen-binding fragment described herein in the brain (e.g., whole brain or brain parenchyma) can be at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold,

[0149] 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, 2000-fold, 5000-fold, or 10000-fold of the concentration of a control antibody (e.g., hIgG1 or JR141-N) or the concentration in the subject's serum.

[0150] Method for preparing an anti-TFR1 antibody

[0151] Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing suitable nucleotide variants into the DNA encoding a human, humanized, or chimeric antibody or its antigen-binding fragment, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues in the amino acid sequence that constitutes the antibody antigen-binding site or antigen-binding domain. Among such variant populations, some antibodies or antigen-binding fragments have increased affinity for a target protein (such as TFR1). Any combination of deletions, insertions, and / or combinations can be made to obtain an antibody or its antigen-binding fragment with higher binding affinity for the target. Amino acid changes introduced into an antibody or antigen-binding fragment can also alter or introduce new post-translational modifications, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence so that intracellular enzymes attach different sugars), or introducing new glycosylation sites. In some embodiments, the heavy-chain antibodies or their antigen-binding fragments described herein are obtained by immunizing any genetically modified animal (such as a mouse in which the human heavy-chain variable region is completely replaced in situ and combined with a modified constant region), as described in PCT / CN2022 / 119188.

[0152] Humanized antibodies include antibodies having variable and constant regions with human germline immunoglobulin sequences (or having the same amino acid sequence as that derived from human germline immunoglobulin sequences) derived from human immunoglobulin framework sequences. A humanized antibody can contain amino acid residues encoded by non-human germline immunoglobulin sequences (such as mutations introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutations). Thus, a "humanized" antibody is a chimeric antibody in which the sequences of non-human species are replaced by the corresponding human sequences.

[0153] Typically, amino acid sequence variants of human, humanized, or chimeric anti-TFR1 antibodies have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequence in the original antibody VHH domain.

[0154] Identity or homology with the original sequence generally refers to the percentage of amino acid residues in the candidate sequence that are the same as the sequence in the human, humanized, or chimeric anti-TFR1 antibody or fragment after aligning the sequences and introducing gaps as necessary to obtain the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity.

[0155] Additional modifications can be made to the antibody or antigen-binding fragment. For example, one or more cysteine residues can be introduced into the Fc region, allowing for the formation of interchain disulfide bonds in this region. The resulting homodimeric antibody may have an extended half-life in vitro and / or in vivo. Homodimeric antibodies with an extended half-life in vitro and / or in vivo can also be prepared using, for example, heterobifunctional crosslinking linkers as described by Wolff et al. ("Monoclonal antibody homodimers: enhanced antitumor activity in nude mice." Cancer research 53.11 (1993): 2560-2565). Alternatively, an antibody with two Fc regions can be engineered.

[0156] In some embodiments, the anti-TFR1 antibody or its antigen-binding fragment can be covalently modified. These covalent modifications can be achieved by chemical synthesis or enzymatic synthesis, or by enzymatic cleavage or chemical cleavage. Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting the target amino acid residue of the antibody or fragment with an organic derivatizing reagent capable of reacting with the selected side chain or N-terminal or C-terminal residue.

[0157] In some embodiments, antibody variants are provided that have a carbohydrate structure lacking (directly or indirectly) fucose linked to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all sugar structures linked to Asn297 (e.g., complex, hybrid, and high-mannose structures), as by MALDI-TOF mass spectrometry, as described in WO2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering); however, due to minor sequence variations in the antibody, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such defucosylated variants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody can be further engineered to replace the asparagine at position 297 with alanine (N297A).

[0158] The present disclosure also provides recombinant vectors (such as expression vectors) comprising the isolated polynucleotides disclosed herein (such as polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors have been introduced (i.e., such that the host cells contain the polynucleotide and / or the vector containing the polynucleotide), and recombinant antibody polypeptides or fragments thereof produced by recombinant techniques.

[0159] As used herein, a "vector" refers to any construct that, when introduced into a host cell, is capable of delivering one or more polynucleotides of interest to the host cell. An "expression vector" is capable of delivering one or more polynucleotides of interest into a host cell into which the expression vector has been introduced and expressing them as encoded polypeptides. Thus, in an expression vector, the polynucleotide of interest is positioned in the vector for expression by being operably linked to regulatory elements (such as promoters, enhancers, and / or poly-A tails), which may be located within the vector or at, near, or flanking the site of integration of the polynucleotide of interest in the host cell genome, such that the polynucleotide of interest can be translated in the host cell into which the expression vector has been introduced.

[0160] Vectors can be introduced into host cells by methods known in the art, such as electroporation, chemical transfection (such as the DEAE-dextran method), transformation, transfection, and infection and / or transduction (such as using recombinant viruses). Thus, non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0161] In some embodiments, viral expression systems such as vaccinia or other poxviruses, retroviruses, or adenoviruses are used to introduce the polynucleotides disclosed herein (such as polynucleotides encoding the polypeptides disclosed herein), which may involve the use of non-pathogenic (defective), replication-competent viruses, or the use of replication-defective viruses. In the latter case, viral replication typically occurs only in complementary virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. N.Y. Acad Sci. 569:86-103; Flexner et al., 1990, Vaccine, 8:17-21; U.S. Patent Nos. 4,603,112, 4,769,330, and 5,017,487; WO89 / 01973; U.S. Patent No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91 / 02805; Berchner-Biotechniques, 6:616-627, 1988; Rosenfeld et al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisle et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for integrating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA can also be "naked", as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749, and Cohen, 1993, Science, 259:1691-1692. Uptake of naked DNA can be increased by coating the DNA on biodegradable microbeads that are efficiently transported into cells.

[0162] For expression, a DNA insert comprising a polynucleotide encoding an antibody or polypeptide disclosed herein can be operably linked to a suitable promoter, such as a heterologous promoter, for example, the bacteriophage λPL promoter, the E. coli lac, trp, and tac promoters, the SV40 early and late promoters, and the promoters of retroviral LTRs, etc. Other suitable promoters are known to those skilled in the art. In some embodiments, the promoter is the cytomegalovirus (CMV) promoter. The expression construct can further comprise transcription start and stop sites and, within the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct can include a translation initiation codon at the start and appropriate stop codons (UAA, UGA, or UAG) positioned at the end of the polypeptide to be translated.

[0163] As described above, the expression vector can comprise at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance markers for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culture in E. coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells such as yeast cells; insect cells such as Drosophila S2 and Spodoptera frugiperda Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Suitable media and culture conditions for the host cells described herein are known in the art.

[0164] Non-limiting vectors for bacteria include pQE70, pQE60, and pQE-9 from Qiagen; PBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A from Stratagene; ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 from Pharmacia. Non-limiting vectors for eukaryotes include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG from Stratagene; pSVK3, pBPV, pMSG, and pSVL from Pharmacia. Other suitable vectors will be apparent to those skilled in the art.

[0165] Suitable non-limiting bacterial promoters include the E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoter, λPR and PL promoters, and trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, promoters of retroviral LTRs such as the promoter of Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0166] In Saccharomyces cerevisiae, many vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used.

[0167] The construct can be introduced into the host cell by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. These methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986), the full text of which is incorporated herein by reference.

[0168] By inserting enhancer sequences into the vector, the transcription level of the DNA encoding the antibodies of the present disclosure in higher eukaryotes can be increased. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp, which function to increase the transcriptional activity of promoters in a given host cell type. Examples of enhancers include the SV40 enhancer located 100 to 270 bp downstream of the replication origin, the cytomegalovirus early promoter enhancer, the polyomavirus enhancer located downstream of the replication origin, and the adenovirus enhancer.

[0169] To secrete the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space, or the extracellular environment, a suitable secretion signal can be added to the expressed polypeptide. These signals can be endogenous to the polypeptide or heterologous signals.

[0170] Polypeptides (such as antibodies) can be expressed in modified forms, such as fusion proteins (such as GST fusion proteins) or with a histidine tag, and can include not only secretion signals but also additional heterologous functional regions. For example, an additional segment of amino acids, especially charged amino acids, can be added to the N-terminus of the polypeptide to improve its stability and persistence in the host cell, as well as its stability during purification or subsequent processing and storage. In addition, peptide segments can be added to the polypeptide to facilitate purification. Such regions can be removed before the final preparation of the polypeptide. Before the final preparation of the polypeptide, these regions can be removed. Adding peptide segments to the polypeptide to achieve secretion or excretion, improve stability, and facilitate purification are well-known conventional techniques in the art.

[0171] The present disclosure also provides nucleotide sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any nucleotide sequence described herein, and amino acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any amino acid sequence described herein. In some embodiments, the present disclosure relates to nucleotide sequences encoding any peptide described herein, or any amino acid sequence encoded by any nucleotide sequence described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acid residues.

[0172] In some embodiments, the amino acid sequence (i) comprises the amino acid sequence; or (ii) consists of the amino acid sequence, wherein the amino acid sequence is any one of the sequences described herein.

[0173] In some embodiments, the nucleic acid sequence (i) comprises the nucleic acid sequence; or (ii) consists of the nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences described herein.

[0174] In some embodiments, the antibody or its antigen-binding fragment is expressed in yeast, insect cells, or mammalian cells (e.g., CHO cells).

[0175] Therapeutic and diagnostic methods

[0176] The anti-TFR1 antibody or antigen-binding fragment thereof of the present invention can be used for various therapeutic purposes. On the one hand, the present disclosure provides methods for treating brain diseases (such as brain cancer, dementia, or Alzheimer's disease) in a subject, methods for identifying a subject with a brain disease (such as brain cancer, dementia, or Alzheimer's disease), methods for reducing the risk of developing a brain disease, or methods for reducing the risk of a subject developing additional symptoms. In some embodiments, the treatment can stop, slow down, delay, or inhibit the progression of a brain disease (such as brain cancer, dementia, or Alzheimer's disease). In some embodiments, the treatment can reduce the number, severity, and / or duration of one or more symptoms of a brain disease (such as brain cancer, dementia, or Alzheimer's disease) in a subject.

[0177] On the one hand, the present disclosure features a method that includes administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof disclosed herein to a subject in need (such as a subject having, identified, or diagnosed with a brain disease).

[0178] On the one hand, the present disclosure features a method for delivering a therapeutic agent across the blood-brain barrier. In some embodiments, the antibody or antigen-binding fragment thereof described herein is linked to a therapeutic agent. In some embodiments, the therapeutic agent is an antibody, its antigen-binding fragment, a small molecule, or an antibody-drug conjugate.

[0179] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk of having a brain disease (such as brain cancer, dementia, or Alzheimer's disease). Patients having a brain disease (such as brain cancer, dementia, or Alzheimer's disease) can be identified by various methods known in the art.

[0180] In some embodiments, the brain disease is brain cancer.

[0181] On the one hand, the present disclosure relates to a method for reducing the tumor growth rate, including contacting tumor cells with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof or an antibody-drug conjugate described herein. On the one hand, the present disclosure relates to a method for killing tumor cells, including contacting tumor cells with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof or an antibody-drug conjugate described herein.

[0182] As used herein, "effective amount" refers to an amount or dose sufficient to achieve a beneficial or desired result, and these results include terminating, slowing down, delaying, or inhibiting the progression of a disease (such as cancer). The effective amount will vary depending on, for example, the age and weight of the subject to whom the antibody, antigen-binding fragment, antibody-drug conjugate, antibody-encoding polynucleotide, vector comprising the polynucleotide, and / or its composition are to be administered, the severity of the symptoms, and the route of administration, and thus can be determined based on the individual for administration.

[0183] An effective amount can be administered in one or more administrations. For example, an effective amount of an antibody, antigen-binding fragment, or antibody-drug conjugate is an amount sufficient to ameliorate, terminate, stabilize, reverse, inhibit, slow, and / or delay the progression of an autoimmune disease or cancer in a patient, or an amount sufficient to ameliorate, terminate, stabilize, reverse, slow, and / or delay the proliferation of cells (e.g., biopsy cells, any of the cancer cells described herein, or a cell line (e.g., a cancer cell line)) in vitro. As understood in the art, the effective amount of an antibody, antigen-binding fragment, or antibody-drug conjugate can vary depending on, inter alia, the patient's medical history and other factors such as the type (and / or dose) of the antibody used.

[0184] The effective amount and dosing regimen of the antibodies, polynucleotides encoding the antibodies, antibody-drug conjugates, and / or compositions disclosed herein can be determined empirically and making such determinations is within the skill of the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal to which the antibodies, polynucleotides encoding the antibodies, antibody-drug conjugates, and / or compositions disclosed herein will be administered, the route of administration, the specific type of the antibodies, polynucleotides encoding the antibodies, antigen-binding fragments, antibody-drug conjugates, and / or compositions disclosed herein used, and other drugs being administered to the mammal. Guidance for selecting a suitable dose of an antibody or antigen-binding fragment can be found in the literature regarding the therapeutic use of antibodies and antigen-binding fragments, such as Handbook of Monoclonal Antibodies, Ferrone et al., Noges Publications, Park Ridge, N.J., 1985, ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., Raven Press, New York, 1977, pp. 365-389.

[0185] The typical daily dose of an effective amount of the antibody is from 0.01 mg / kg to 100 mg / kg. In some embodiments, the dose can be less than 100 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg or 0.1 mg / kg. In some embodiments, the dose can be greater than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg or 0.01 mg / kg. In some embodiments, the dose is about 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg or 0.1 mg / kg.

[0186] In any of the methods described herein, at least one antibody, an antigen-binding fragment thereof or a pharmaceutical composition (e.g., any antibody, antigen-binding fragment or pharmaceutical composition described herein) and optionally at least one additional therapeutic agent can be administered at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day or three times a day). In some embodiments, at least two different antibodies and / or antigen-binding fragments are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition). In some embodiments, the at least one antibody or antigen-binding fragment and the at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, at least one additional therapeutic agent is administered as a pill, tablet or capsule. In some embodiments, the at least one additional therapeutic agent is administered in the form of a sustained-release oral formulation.

[0187] In some embodiments, one or more additional therapeutic agents may be administered to a subject before or after administration of at least one antibody, antigen-binding antibody fragment, antibody-drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein). In some embodiments, one or more additional therapeutic agents and at least one antibody, antigen-binding antibody fragment, antibody-drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) are administered to a subject such that there is an overlap in the bioactive periods of one or more additional therapeutic agents and at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) in the subject.

[0188] In some embodiments, at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) may be administered to a subject over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or tracking treatment efficacy (e.g., observing at least one symptom of the disease). As described herein, a skilled medical professional may also vary the type and amount (e.g., increase or decrease) of the antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to the subject and may also adjust the dose or dosing frequency of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to the subject based on an assessment of treatment efficacy (e.g., increase or decrease).

[0189] In some embodiments, one or more additional therapeutic agents may be administered to a subject. The additional therapeutic agents may include one or more inhibitors selected from B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, PI3K / mTOR dual inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors.

[0190] In some embodiments, the additional therapeutic agent may include one or more inhibitors selected from the group consisting of: HER3 inhibitors, LSD1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, inhibitors of the activated hedgehog signaling pathway, and agents that selectively degrade estrogen receptor.

[0191] In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from the group consisting of: trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, zykadia, sutent, temsirolimus, axitinib, sorafenib, Votrient, IMA-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

[0192] In some embodiments, the additional therapeutic agent may comprise one or more therapeutic agents selected from the group consisting of: adjuvants, TLR agonists, tumor necrosis factor (TNF)α, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, IL-17 antagonists, HVEM antagonists, ICOS agonists, therapies targeting CX3CL1, therapies targeting CXCL9, therapies targeting CXCL10, therapies targeting CCL5, LFA-1 agonists, ICAM1 agonists, and selectin agonists.

[0193] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX or FOLFIRI is administered to a subject.

[0194] In some embodiments, the additional therapeutic agent is an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody or an anti-GITR antibody.

[0195] Drug Compositions and Routes of Administration

[0196] The present invention also provides a pharmaceutical composition comprising at least one (e.g., one, two, three or four) antibody or antigen-binding fragment described herein. Two or more (e.g., two, three or four) of any of the antibodies or antigen-binding fragments described herein may be present in the pharmaceutical composition in any combination. The pharmaceutical composition can be formulated in any manner known in the art.

[0197] The pharmaceutical composition is formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous or intraperitoneal). The composition may include a sterile diluent (e.g., sterile water or saline), a fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents, antibacterial or antifungal agents (such as benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (such as ascorbic acid or sodium bisulfite), chelating agents (such as ethylenediaminetetraacetic acid), buffers (such as acetate, citrate or phosphate) and isotonic agents (such as sugars (e.g., glucose), polyols (e.g., mannitol or sorbitol) or salts (e.g., sodium chloride)), or any combination thereof. Liposome suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811). The formulation of the composition can be prepared and encapsulated in ampoules, disposable syringes or multi-dose vials. When required (such as in injectable formulations), appropriate fluidity can be maintained, for example, by using coatings (such as lecithin) or surfactants. The absorption of the antibody or its antigen-binding fragment can be prolonged by including agents that delay absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by implants and microencapsulation delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene-vinyl acetate copolymer, polyanhydride, polyglycolic acid, collagen, polyorthoester and polylactic acid; Alza Corporation and Nova Corporation).

[0198] A composition comprising one or more of any of the antibodies or antigen-binding fragments described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in unit dosage form (i.e., physically discrete units containing a predetermined quantity of the active compound, to facilitate administration and ensure dose uniformity).

[0199] The toxicity and efficacy of the composition can be determined in cell cultures or experimental animals (e.g., monkeys) by standard pharmaceutical methods. For example, the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) can be determined: the therapeutic index is the ratio of LD50:ED50. Agents with a high therapeutic index are preferably shown. When an agent exhibits adverse side effects, care should be taken to minimize potential damage (i.e., reduce adverse side effects). The toxicity and efficacy can be determined by other standard pharmaceutical methods.

[0200] Data obtained from cell culture assays and animal studies can be used to formulate an appropriate dose of any given agent for a subject (e.g., a human). A therapeutically effective amount of one or more (e.g., one, two, three, or four) antibodies or their antigen-binding fragments (e.g., any of the antibodies or antibody fragments described herein) will be an amount that treats a disease (e.g., kills cancer cells), reduces the severity, frequency, and / or duration of one or more symptoms of the disease in a subject (e.g., a human subject identified as having cancer) or a subject identified as being at risk of developing the disease (e.g., a subject who had cancer previously but is now cured). The effectiveness and dose of any of the antibodies or antigen-binding fragments described herein can be determined by healthcare or veterinary professionals using methods known in the art and by observing one or more symptoms of the disease in a subject (e.g., a human). Certain factors can affect the dose and schedule required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health status and / or age of the subject, and the presence of other diseases).

[0201] Exemplary doses include milligram or microgram amounts of any of the antibodies or antigen-binding fragments per kilogram of subject body weight (e.g., about 1 μg / kg to about 500 mg / kg; about 100 μg / kg to about 500 mg / kg; about 100 μg / kg to about 50 mg / kg; about 10 μg / kg to about 5 mg / kg; about 10 μg / kg to about 0.5 mg / kg; or about 1 μg / kg to about 50 μg / kg). Although these doses cover a wide range, one of ordinary skill in the art will understand that the potencies of therapeutic agents, including antibodies and their antigen-binding fragments, vary and that an effective amount can be determined by methods known in the art. Generally, a relatively low dose is administered first, and then the attending health care professional or veterinary professional (in the case of therapeutic applications) or researcher (when working in the development stage) can subsequently increase the dose gradually until an appropriate response is obtained. Additionally, it should be understood that the specific dose level for any particular subject will depend on a variety of factors, including the activity of the specific compound used, the age, weight, general health, sex, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the in vivo half-life of the antibody or antibody fragment.

[0202] The pharmaceutical composition may be contained in a container, package, or dispenser together with a dosing instruction. The present invention also provides methods for preparing antibodies or their antigen-binding fragments for the various uses described herein. Examples

[0203] The present invention is further described in the following examples, which do not limit the scope of the present invention as claimed.

[0204] Example 1. Generation of Human Anti-TFR1 Antibodies

[0205] Immunize RenNano

[0206] mice (Beijing Biocytogen, in situ replacement of the human heavy chain variable domain, combined with a modified constant region, the description of this mouse can be found in PCT / CN2022 / 119188, which is hereby incorporated by reference in its entirety) with His-tagged human TFR1 (transferrin receptor 1) protein (hTFR1-His, ACROBiosystems, Cat#: TM CD1-H5243) to obtain anti-TFR1 antibodies. Before immunization, orbital blood was collected as a negative control. Complete Freund's adjuvant (CFA) was used for the first immunization, and incomplete Freund's adjuvant (IFA) was used for the second and third immunizations. A total of three immunizations were performed (once every two weeks). One week after the 3rd immunization, orbital blood was collected, and the serum antibody titer was detected by flow cytometry.

[0207] At least 14 days after the above immunization, a booster immunization was also carried out. Through intraperitoneal injection of TFR1 protein and tail vein injection of CHO-S cells expressing human TFR1 antigen.

[0208] Antigen-specific immune cells were isolated from the immunized mice to further obtain anti-TFR1 antibodies or the heavy chain variable region sequences of anti-TFR1 antibodies. For example, single-cell techniques (e.g., using Optofluidic System, Berkeley Lights Inc.) were used to screen and discover plasma cells secreting antigen-specific monoclonal antibodies. Antibody variable region sequences were obtained by reverse transcription and PCR sequencing. The obtained variable region sequences were used for antibody expression, and the binding affinity of the antibody to TFR1 was verified by FACS. Because of the lack of the CH1 domain, the heavy chain variable region (VH) of the obtained antibody is also called the heavy chain single variable domain (VHH).

[0209] Specifically, the obtained VHH sequences were respectively ligated to the human IgG1 constant region (e.g., hinge region, CH2 domain, and CH3 domain). Exemplary antibodies obtained by this method include: 23B8, 24A1, 24C9, and 24G5. Their heavy chain CDR1-3 are as Figure 1 and Figure 2 shown. The VHHs of 23B8, 24A1, 24C9, and 24G5 are as Figure 3 shown.

[0210] The constant region of the antibody can be further modified by replacing asparagine at position 297 with alanine (N297A). For example, when the N297A mutation was introduced into the constant region of 24G5, the resulting antibody was named 24G5-N.

[0211] In addition, single-arm antibodies 23B8-mono, 24A1-mono, 24C9-mono, and 24G5-mono were also constructed, which have an anti-TFR1 arm containing the VHH region and a heavy chain fragment containing the CH2 and CH3 domains of IgG1 with the N297A mutation.

[0212] Example 2. Cross-species binding of anti-TFR1 antibodies

[0213] CHO-S-hTFR1 cells or CHO-S-fasTFR1 cells were respectively transferred to a 96-well plate at a density of 10 5 cells / well. Serial dilutions of anti-TFR1 antibody samples were added to the 96-well plate and incubated at 4 °C for 30 minutes. PBS was used as a negative control (NC). Then, the cells were incubated with the secondary antibody anti-hIgG-Fc-Alex Flour TM647 (Jackson ImmunoResearch Laboratories, Cat#: 109 - 606 - 170)) was incubated in the dark at 4 °C for 15 minutes and then subjected to flow cytometry analysis.

[0214] CHO - S - hTFR1 cells or CHO - S - fasTFR1 cells were obtained by transfecting CHO - S cells with vectors expressing human TFR1 (hTFR1, SEQ ID NO: 29) or the amino acid sequence of Macaca fascicularis TFR1 (fasTFR1, SEQ ID NO: 30), respectively. The detection results are shown in the following table.

[0215] JR141 is a humanized IgG1 antibody targeting human TFR1 and conjugated with human iduronate - 2 - sulfatase. It was first approved in Japan in March 2021 for intravenous injection treatment of mucopolysaccharidosis type II. The VH and VL sequences of JR141 are shown in SEQ ID NO: 31 and SEQ ID NO: 32, respectively. Connecting the VH and VL of JR141 to the human IgG1 constant region with N297A mutation was used as a positive control (JR141 - N).

[0216] Table 1

[0217]

[0218] Example 3. Binding Affinity of Anti - TFR1 Antibodies

[0219] On a Biacore TM (Biacore, Inc., Piscataway N.J.) 8K biosensor equipped with a pre - immobilized protein A sensor chip, surface plasmon resonance (SPR) was used to determine the binding affinity of anti - TFR1 antibodies to His - tagged human TFR1 (hTFR1 - His, ACROBiosystems, Cat#: CD1 - H5243) or monkey TFR1 (fasTFR1 - His, ACROBiosystems, Cat#: TFR - C524a).

[0220] The purified anti-TFR1 antibody was captured using a Protein A chip (Series S Sensor Chip Protein A) for detection. The purified anti-TFR1 antibody at 1 μg / mL was loaded at a rate of 10 μL / min to bind hTFR1-His and fasTFR1-His (200 nM). The flow rate was 30 μL / min. The binding and dissociation times were set at 180 seconds and 600 seconds, respectively. After the last injection in each round of titration, the chip was regenerated with glycine solution (pH 2.0) at a rate of 30 μL / min for 30 seconds.

[0221] Data analysis was performed in Biacore TM 8K evaluation software 3.0 with global fitting to a 1:1 Langmuir binding model (Karlsson, R., Roos, H., Fagerstam, L., Petersson, B., 1994. Methods Enzymology 6.99 - 110) to obtain the kinetic binding rate (kon) and dissociation rate (koff). The affinity value was derived from the quotient of the kinetic rate constants (KD = koff / kon).

[0222] As understood by those of ordinary skill in the art, the same method was used for each tested anti-TFR1 antibody, and appropriate adjustments were made to parameters such as antibody concentration. The results of testing the antibodies are summarized in the table below. The results show that all 4 anti-TFR1 antibodies can bind to human and monkey TFR1 with high affinity.

[0223] Table 2

[0224]

[0225] Example 4. Epitope analysis of anti-TFR1 antibodies

[0226] The relative positions of target protein epitopes between pairs of purified anti-TFR1 antibodies were analyzed by Biolayer Interference (BLI) using the ForteBio Octet system at 30 °C. 1× HBS-EP+ buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA), and 0.05% P20, pH 7.4) diluted from HBS-EP+ buffer (10×) was used as the running buffer for the entire experiment. Approximately 10 μg / mL of hTFR1-His protein was captured by HIS1K (Anti-Penta-HIS) for 200 seconds, and 200 nM of antibody (Analyte 1) was injected at a flow rate of 30 μL / min to bind the ligand. Another antibody (Analyte 2) was injected under the same conditions to determine whether the binding of different antibodies interfered with each other. The binding time for each antibody was 300 seconds.

[0227] Binding values for each antibody were obtained using Data Analysis HT 12.0. To quantify the interference of one antibody's binding with another, binding ratios were calculated to compare each pair of antibodies. The binding ratio was defined as the binding value of the second antibody (Analyte 2) divided by the binding ratio of the first antibody (Analyte 1). The binding ratios for each antibody pair are summarized in the table below. Specifically, if Analyte 1 showed a blocking effect on Analyte 2, the binding ratio was between 0.0 and 0.5; if Analyte 1 did not show a blocking effect on Analyte 2, the binding ratio was between 0.5 - 1.1. Generally, antibody pairs that interfere with each other have the same or overlapping epitopes.

[0228] The epitope binding experiment showed that 24A1 and 24G5 recognize the same epitope, and 23B8, 24C9, and JR141-N recognize different epitopes.

[0229] Table 3

[0230]

[0231] Example 5. Endocytic Activity of Anti-TFR1 Antibodies

[0232] Anti-TFR1 antibodies were added to human cerebral microvascular endothelial cells (hCMEC / D cells) together with pHAb-goat anti-human IgG secondary antibody and incubated for 3 hours. After incubation, the cells were centrifuged and washed with FACS buffer. The mean fluorescence intensity (MFI) was measured using a flow cytometer. The endocytic rate of the antibody was calculated. Human IgG1 protein (CrownBio, Cat#: C0001) was used as an isotype control (ISO). The results are shown in the table below, indicating that all four antibodies showed good endocytic activity in human cerebral microvascular endothelial cells.

[0233] Table 4

[0234] Antibody MFI Positive population ISO 4288 0.9％ 23B8 11392 67.0％ 24A1 26972 96.6％ 24C9 37086 96.0％ 24G5 26406 96.0％

[0235] Example 6. Developability Analysis of Anti-TFR1 Antibodies

[0236] The developability of anti-TFR1 antibodies 23B8, 24A1, 24C9, and 24G5 was evaluated. The antibodies were diluted with water to 1 mg / mL. The following tests were specifically performed: (1) observing the appearance of the solution and the presence of visible insoluble matter; (2) detecting the purity change of the antibody by size exclusion ultra-high performance liquid chromatography (SEC-UPLC) (expressed as the percentage of the main peak area in the total area of all peaks (purity, %)); (3) detecting the change in the apparent hydrophobicity of the antibody using hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) (expressed as the retention time of the main peak (HIC, min)); (4) detecting the charge variation in the antibody by capillary isoelectric focusing (cIEF) (expressed as the percentages of the main component, acidic component, and basic component); and (5) detecting the thermal stability of the antibody by the UNcle system (expressed as the melting temperature (Tm) and the aggregation temperature (Tagg)).

[0237] In the SEC-UPLC experiment, an Agilent 1290 chromatographic system (connected to an XBridge TM Protein BEH SEC column ( Waters Corporation) was used. The antibody sample was diluted to 1 mg / mL with purified water. The following parameters were used: mobile phase: 25 mM phosphate buffer (PB) (pH 6.8) + 0.3 M NaCl; flow rate: 1.8 mL / min; column temperature: 25 °C; detection wavelength: 280 nm; injection volume: 10 μL; sample tray temperature: 6 °C; run time: 7 minutes.

[0238] In the HIC-HPLC experiment, an Agilent 1260 chromatographic system (connected to a ProPac TM HIC-10 column (4.6 × 100 mm, Thermo Scientific)) was used, and the sample was diluted to 0.5 mg / mL with mobile phase A. The following parameters were used: mobile phase A: 0.9 M ammonium sulfate, 0.1 M PB, 10% acetonitrile pH 6.5; mobile phase B: 0.1 M PB, 10% acetonitrile pH 6.5; flow rate: 0.8 mL / min; gradient: 0 minutes 100% A, 2 minutes 100% A, 32 minutes 100% B, 34 minutes 100% B, 35 minutes 100% A, and 45 minutes 100% A; column temperature: 30 °C; detection wavelength: 280 nm; injection volume: 10 μg; sample tray temperature: approximately 6 °C; run time: 45 minutes.

[0239] In the cIEF experiment, a kit (Protein Simple, Cat#: PS-MDK01-C) was developed using the Maurice cIEF method for sample preparation. Specifically, 40 μg of protein sample was mixed with the following reagents in the kit: 1 μL of Maurice cIEF pI Marker-4.05, 1 μL of Maurice cIEF pI Marker-9.99, 35 μL of 1% MethylCellulose Solution, 2 μL of Maurice cIEF, 500 mM arginine, 4 μL of Ampholytes (Pharmalyte pH range 3 - 10), and water (added to a final volume of 100 μL). On a Maurice analyzer (Protein Simple, Santa Clara, CA), imaging capillary isoelectric focusing spectra were generated using Maurice cIEF Cartridges (PS-MC02-C). The sample was focused for a total of 10 minutes. The analysis software installed on the instrument was used to analyze the absorbance of the focused protein at 280 nm.

[0240] In the thermal stability experiment, a 60 mg / mL antibody solution was heated from 25 °C to 95 °C in 1 °C increments, with a 1-minute equilibration time before each measurement.

[0241] Furthermore, the antibody was diluted to 1 mg / mL using PBS buffer and the following tests were performed: (1) The specificity of the antibody was detected using the cross-interaction chromatography (CIC) method (expressed as retention time (CIC, min)); (2) The colloidal stability of the antibody was detected by the standing monolayer chromatography (SMAC) method (expressed as retention time (SMAC, % / min)).

[0242] In the CIC analysis, a CIC column was prepared by conjugating human polyclonal IgG (Sigma, Cat#: I4506) to HiTrap NHS-activated resin (GE Healthcare, Cat#: 17-0716-01), and then passivating it with ethanolamine according to the published procedure. The column was then connected to an Agilent 1260 chromatographic system and run at a flow rate of 0.1 mL / min using 1×PBS as the mobile phase until a flat baseline was achieved. Then, 10 μg of a 1 mg / mL antibody PBS solution was injected. The peak retention time on the column was monitored at 280 nm; the running time: 50 minutes.

[0243] In the SMAC analysis, a Zenix chromatographic column (4.6 mm × 30 cm, Sepax, Cat#: 213300-4630) was connected to the column oven, and appropriate tubing was placed in the mobile phase. The chromatographic column was equilibrated with the mobile phase buffer at a flow rate of 0.350 mL / min for 60 min. The antibody was loaded into the injection sequence. Mobile phase A: 150 mM sodium phosphate, pH 7.0; flow rate: 0.35 mL / min; run time: 25 minutes; column temperature: 30 °C; detection wavelengths: 280 nm, 220 nm.

[0244] The detailed results are shown in the following table.

[0245] Table 5

[0246]

[0247] For the Tagg of 24C9, a slight fluctuation was observed at 33.63 °C on the Tagg curve (data not shown), while large-scale aggregation occurred at 50 - 60 °C. To confirm the thermal stability, the SEC-UPLC of 24C9 was tested after treatment at 40 °C for 14 days. After heating, the purity of 24C9 remained above 97%. These results indicate that all 4 antibodies have good developability.

[0248] Example 7. Pharmacokinetic (PK) analysis

[0249] The humanized TFR1 mouse model (hTFR1 mouse) was engineered to express a chimeric TFR1 protein (SEQ ID NO: 33), in which the extracellular domain of the mouse TFR1 protein was replaced by the corresponding human TFR1 extracellular domain. A detailed description of the humanized TFR1 mouse model can be found in PCT application PCT / CN2022 / 105924, which is incorporated herein by reference in its entirety.

[0250] The concentration of the anti-TFR1 antibody was determined in hTFR1 mice. Specifically, the mice were divided into different groups (8 mice per group), and approximately equimolar doses of JR141-N (G2), 23B8-N (G3), 24A1-N (G4), 24G5-N (G5), or 24C9-N (G6) were administered by intravenous injection (i.v.). The control group (G1) mice were administered human IgG1 (hIgG1). The detailed dosing regimen is shown in the following table.

[0251] Table 6

[0252] Group Number of mice Antibody Dose Administration method Frequency G1 8 hIgG1 18.4 mg / kg i.v. Single dose G2 8 JR141-N 18.4 mg / kg i.v. Single dose G3 8 23B8-N 10.0 mg / kg i.v. Single dose G4 8 24A1-N 10.0 mg / kg i.v. Single dose G5 8 24G5-N 10.0 mg / kg i.v. Single dose G6 8 24C9-N 10.0 mg / kg i.v. Single dose

[0253] Blood samples and brain samples were collected at 0.5, 6, 24, and 72 hours after drug administration. Two mice were sampled at each time point, and the mice were anesthetized after orbital blood collection. To avoid interference from residual blood in the brain, the mice were perfused with saline at room temperature for 10 minutes. Specifically, saline was perfused through the systemic circulation from the left ventricle to the right ventricle. The brain samples were excised and divided into two hemispheres by the sagittal plane. The left hemisphere was used for quantification of the injected antibody, while the right hemisphere was fixed with formalin and embedded in paraffin for serial sectioning. The brain samples were minced and homogenized in DPBS (Dulbecco’s phosphate-buffered saline) containing 1× protease inhibitor cocktail. The brain homogenates were aliquoted for protein extraction, and then antibody quantification was performed by electrochemiluminescence. For the remaining homogenates, capillaries were removed by gradient density centrifugation at 5400 g for 15 minutes using 15% dextran. After centrifugation, the upper part of the centrifuge tube was saved as the brain parenchyma, and protein extraction and antibody quantification were performed. Figures 4A - 4D showed the antibody concentration in total brain protein ( Figure 4A ), the ratio of the antibody concentration in total brain protein to the serum antibody concentration ( Figure 4B ), the antibody concentration in the brain parenchyma ( Figure 4C ), and the ratio of the antibody concentration in the brain parenchyma to the serum antibody concentration at each time point ( Figure 4D ). The results showed that 24G5-N (Group G5) was the most abundant both in the brain parenchyma and in the whole brain.

[0254] In a similar experiment, hTFR1 mice were divided into 5 groups (3 mice per group) and were administered 18.4 mg / kg JR141-N (G2), 10 mg / kg 23B8-N (G3), 10 mg / kg 24A1-N (G4), or 10 mg / kg 24G5-N (G5) (administered once in total) by intravenous injection. Control group (G1) mice were administered hIgG1. At 24 hours after administration, brain samples were collected to measure the concentration of anti-TFR1 antibody. Figures 5A - 5B The results of antibody concentration detection in the brain parenchyma and total brain protein are shown respectively. The concentrations of all tested antibodies in the brain were higher than those of hIgG1 (G1), and compared with the positive control JR141-N (G2), 23B8-N (G3) and 24G5-N (G5) could better cross the blood-brain barrier and enter the brain parenchyma.

[0255] In another similar experiment, hTFR1 mice were divided into 7 groups (6 mice per group) and were administered JR141-N (G2-G4) or 24G5-N (G5-G7) by intravenous injection (i.v.). Control group (G1) mice were administered hIgG1. The detailed dosing schedule is shown in the following table.

[0256] Table 7

[0257] Group Number of mice Antibody Dose (mg / kg) Administration method Frequency G1 6 hIgG1 5.52 mg / kg i.v. Single dose G2 6 JR141-N 1.84 mg / kg i.v. Single dose G3 6 JR141-N 5.52 mg / kg i.v. Single dose G4 6 JR141-N 18.4 mg / kg i.v. Single dose G5 6 24G5-N 1 mg / kg i.v. Single dose G6 6 24G5-N 3 mg / kg i.v. Single dose G7 6 24G5-N 10 mg / kg i.v. Single dose

[0258] At 6 hours and 24 hours after administration, blood and brain samples were collected using the above method. Three mice were sampled at each time point. The processing of tissues and the quantification of antibodies were also carried out as described above. The results of the determination of the concentration of anti-TFR1 antibody in the brain parenchyma are as Figure 6 shown. The results showed that at each dose condition, the antibody concentration of 24G5-N accumulated in the brain parenchyma was significantly higher than that of hIgG1. In addition, the concentrations of JR141-N and 24G5-N in the brain parenchyma both showed a dose-dependent trend.

[0259] To detect the distribution of anti-TFR1 antibody 24G5-N in the mouse brain, immunofluorescence assay was performed by staining hIgG, hTFR1, and mCD31 on the right half-brain sections of the mice in the above experiment. The results showed that mCD31 was well-labeled in the microvessels. hTFR1 was also detected on the microvessels, which co-localized with mCD31. In addition, the expression of hTFR1 was also detected on some neurons in the brain parenchyma. In particular, the anti-TFR1 antibody 24G5-N was stained with a secondary antibody IgG antibody conjugated with 488. Similar to hTFR1, 24G5-N was detected in the microvessels and parenchyma, and its signal overlapped with the hTFR1 signal. Therefore, for the quantification of 24G5-N in the whole brain or brain parenchyma, or the visual evidence of the immunofluorescence of 24G5-N in the brain parenchyma, it was shown that the anti-TFR1 antibody 24G5-N could effectively cross the blood-brain barrier (BBB).

[0260] Example 8. Blocking assay

[0261] By Biolayer Interferometry (BLI) using ForteBio System, the blocking effects of anti-TFR1 antibodies 23B8, 24A1, 24C9, and 24G5 on the binding of TFR1 to TF (transferrin) were detected at 30 °C. Specifically, 1× HBS-EP+ buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20, pH 7.4) diluted from HBS-EP+ buffer (10×) was used as the running buffer for the entire experiment. Antibodies at approximately 10 μg / mL were captured with AHC (Anti-Human IgG Fc Capture) for 200 seconds, and 800 nM of hTFR1-His (ACROBiosystems, Cat#: CD1-H5243) and hTF-His (human transferrin, Kactus Biosystems, Cat#: TFN-HM101) were injected to bind the ligands. The binding time for each antibody was 300 seconds. The binding values of each antibody were obtained using Data Analysis HT 12.0. The results showed that none of these 4 antibodies blocked the binding of TFR1 to TF. Therefore, such non-blocking antibodies are unlikely to interfere with the TFR1-TF interaction in normal cells.

[0262] Example 9. Antibody-Drug Conjugate (ADC)

[0263] Each purified antibody (23B8-N, 24A1-N, 24C9-N, 24G5-N, 23B8-mono, 24A1-mono, 24C9-mono, and 24G5-mono) was conjugated to Dxd (Deruxtecan) via a GGFG linker.

[0264] For the name of the antibody-drug conjugate, "ADC" was directly added after the antibody name. For example, if 24G5-N was coupled to GGFG-Dxd, it was named 24G5-ADC. Another example, if 24G5-mono was coupled to GGFG-Dxd, it was named 24G5-mono-ADC. HIC-HPLC (Reversed Phase High Performance Liquid Chromatography) was used to detect the conjugation of the antibody and the drug molecule. The results showed that the drug-antibody ratio (DAR) of the ADC was approximately 3.

[0265] hTFR1 mice were divided into different groups (6 mice per group) and administered 10 mg / kg of bivalent anti-TFR1 ADCs (24G5-ADC, 23B8-ADC, 24A1-ADC, 24C9-ADC) or the same molar dose of 8.34 mg / kg of single-arm monovalent anti-TFR1 ADCs (23B8-mono-ADC, 24A1-mono-ADC, 24C9-mono-ADC, or 24G5-mono-ADC) by intravenous (i.v.) injection (administered once). At 0.5 hour, 18 hours, and 72 hours after administration, blood and brain samples were collected using the method described in Example 7. As described above, tissue processing and quantitative analysis of antibodies and ADCs were also performed. The results showed that all tested ADCs could effectively carry Dxd across the BBB. Exemplary results are as Figure 7 shown, 24G5-ADC and 24G5-mono-ADC carried Dxd across the BBB, and the monovalent 24G5-mono-ADC had a better penetration effect than the bivalent form 24G5-ADC.

[0266] Other embodiments

[0267] It should be understood that although the present invention has been described in connection with specific embodiments of the present invention, the foregoing description is intended to illustrate rather than limit the scope of the present invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to transferrin receptor 1 (TFR1), said antibody or antigen-binding fragment comprising a heavy-chain single variable domain (VHH) comprising complementary determining regions (CDRs) 1, 2, and 3, wherein said VHH CDR1 comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a selected VHH CDR1 amino acid sequence, said VHH CDR2 comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identity to a selected VHH CDR2 amino acid sequence, and said VHH CDR3 comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identity to a selected VHH CDR3 amino acid sequence, wherein the selected VHH CDR1, 2, and 3 amino acid sequences are one of the following: (1) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 1, 2, and 3, respectively; (2) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 4, 5, and 6, respectively; (3) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 7, 8, and 9, respectively; (4) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 10, 11, and 12, respectively; (5) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 13, 14, and 15, respectively; (6) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 16, 17, and 18, respectively; (7) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 19, 20, and 21, respectively; and (8) The selected VHH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 22, 23, and 24, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein said VHH comprises CDRs 1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 1, 2, and 3, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein said VHH comprises CDRs 1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 4, 5, and 6, respectively.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein said VHH comprises CDRs 1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 7, 8, and 9, respectively.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein said VHH comprises CDRs 1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 10, 11, and 12, respectively.

6. An antibody or antigen-binding fragment thereof that binds to TFR1, said antibody or antigen-binding fragment comprising a heavy chain single variable domain (VHH), said VHH comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a selected VHH sequence, wherein said selected VHH sequence is selected from SEQ ID NO: 25, 26, 27 and 28.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein said VHH comprises SEQ ID NO:

25.

8. The antibody or antigen-binding fragment thereof according to claim 6, wherein said VHH comprises SEQ ID NO:

26.

9. The antibody or antigen-binding fragment thereof according to claim 6, wherein said VHH comprises SEQ ID NO:

27.

10. The antibody or antigen-binding fragment thereof according to claim 6, wherein said VHH comprises SEQ ID NO:

28.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1-10, wherein said antibody or antigen-binding fragment specifically binds to human TFR1, monkey TFR1, mouse TFR1, or chimeric TFR1.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1-11, wherein said antibody or antigen-binding fragment is a human or humanized antibody or antigen-binding fragment thereof.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1-12, wherein said antibody or antigen-binding fragment is a multispecific antibody (e.g., bispecific antibody).

14. An antibody or antigen-binding fragment thereof that comprises the VHH CDR1, 2, 3 of the antibody or antigen-binding fragment thereof according to any one of claims 1-13.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1-14, wherein said antibody or antigen-binding fragment comprises a human IgG Fc (e.g., human IgG1 Fc).

16. The antibody or antigen-binding fragment thereof according to claim 15, wherein said human IgG Fc comprises a non-asparagine residue (e.g., alanine) at position 297, according to EU numbering.

17. The antibody or antigen-binding fragment thereof according to any one of claims 1-16, wherein said antibody or antigen-binding fragment comprises two or more heavy chain single variable domains.

18. A nucleic acid comprising a polynucleotide, wherein said polynucleotide encodes an antibody or antigen-binding fragment thereof according to any one of claims 1-17.

19. The nucleic acid according to claim 18, wherein said nucleic acid is cDNA.

20. A vector that comprises one or more nucleic acids according to claim 18 or 19.

21. A cell that comprises the vector according to claim 20.

22. The cell according to claim 21, wherein said cell is a CHO cell.

23. A cell, said cell comprising one or more nucleic acids as claimed in claim 18 or 19.

24. A method for producing an antibody or an antigen-binding fragment thereof, said method comprising: (a) culturing the cell as claimed in any one of claims 21-23 under conditions sufficient for the cell to produce an antibody or an antigen-binding fragment; and (b) collecting the antibody or antigen-binding fragment produced by the cell.

25. An antibody-drug conjugate, said antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-17 covalently bound to a therapeutic agent.

26. The antibody-drug conjugate according to claim 25, wherein the therapeutic agent is a cytotoxic agent or a cell growth inhibitor.

27. A method of treating a subject having a brain disease (such as brain cancer), the method comprising: Administering to the subject a therapeutically effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-17, or an antibody-drug conjugate as claimed in claim 25 or 26.

28. The method according to claim 27, wherein the antibody or its antigen-binding fragment or antibody-drug conjugate can cross the blood-brain barrier (BBB) of the subject.

29. A method of treating a subject having cancer, the method comprising: Administering to the subject a therapeutically effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-17, or an antibody-drug conjugate as claimed in claim 25 or 26.

30. The method according to claim 29, wherein the cancer is brain cancer, lung cancer, gastric cancer, colorectal cancer, liver cancer, ovarian cancer, prostate cancer, leukemia or breast cancer.

31. A method for identifying a subject having a brain disease (such as brain cancer), the method comprising: By the antibody or its antigen-binding fragment as claimed in any one of claims 1- 17 detecting a sample collected from a subject, thereby identifying that the subject has a brain disease.

32. The method according to claim 31, wherein the sample is a brain parenchymal sample from the subject.

33. The method according to any one of claims 27-32, wherein the subject is a human subject.

34. A method for delivering an agent across the blood-brain barrier, said method comprising administering to a subject an agent covalently linked to an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-17.

35. The method according to claim 34, wherein the agent is an antibody or an antibody-drug conjugate.

36. The method according to claim 34 or 35, wherein the agent is an anti-amyloid antibody.

37. A pharmaceutical composition, said pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-17 and a pharmaceutically acceptable carrier.

38. A pharmaceutical composition, said pharmaceutical composition comprising an antibody-drug conjugate as claimed in claim 25 or 26 and a pharmaceutically acceptable carrier.

39. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof cross-competing with an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-17.

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