Anti-TFR1 antibodies and uses thereof

Abstract

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

Description

[Technical Field]

[0001] Priority claims This application claims priority to PCT Application No. PCT / CN2022 / 136246, filed December 2, 2022. The entire contents of the foregoing are incorporated herein by reference.

[0002] Technical Field The present disclosure relates to anti-TFR1 (transferrin receptor 1) antibodies, antigen-binding fragments, and uses thereof. [Background technology]

[0003] Therapeutic antibodies are one of the fastest-growing classes of therapeutic compounds, rapidly outpacing the growth of small molecule drugs. For example, monoclonal antibodies have revolutionized cancer treatment. However, the large size of traditional antibodies hinders their delivery to tumor cells in vivo. The minimal target recognition module of a traditional antibody consists of two non-covalently linked variable domains (VH and VL). The inherent hydrophobic interactions between the VH and VL domains limit the stability and solubility of engineered antibodies and often result in V domain aggregation and / or mispairing.

[0004] The discovery of heavy-chain antibodies has provided unprecedented opportunities to impact cancer therapy. These unique forms of camelid-derived antibodies lack the entire light chain and CH1 domain and consist only of a single variable domain, termed VHH. Recombinant VHHs are small (15–20 kDa) and strictly monomeric. They bind to their targets with nM affinity and are stable over a wide pH and temperature range. Molecular engineering is also easier with VHHs, facilitating the production of multivalent monoclonal antibodies compared to traditional recombinant antibodies and their fragments, which are subject to aggregation and reduced affinity. Furthermore, VHHs often bind epitopes that are less immunogenic than traditional antibodies.

[0005] Typically, therapeutic antibodies are human or humanized. Human or humanized antibodies can be generated by humanizing rodent antibodies (e.g., murine antibodies) or by using phage libraries. However, these animals and phage libraries typically cannot produce heavy chain antibodies. Instead, heavy chain antibodies are often derived from camelid heavy chain antibodies. These camelid heavy chain antibodies must be humanized. The humanization process can adversely affect binding affinity and introduce immunogenic epitopes into the antibody. Repetitive and time-consuming experiments are often required to improve the properties of these antibodies. In some cases, these antibodies can also be immunogenic in patients, resulting in a decline in their effectiveness over time. Therefore, there is a need to develop more forms of antibodies to treat or prevent human diseases. Summary of the Invention

[0006] 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 that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of a selected VHH CDR1; the VHH CDR2 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of a selected VHH CDR2; and the VHH CDR3 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of a selected VHH CDR3. In some embodiments, the amino acid sequences of the selected VHH CDR1, 2, and 3 are one of the following: (1) The amino acid sequences of the selected VHH CDR1, 2, and 3 are shown in SEQ ID NOs: 1, 2, and 3, respectively; (2) The amino acid sequences of the selected VHH CDR1, 2, and 3 are shown in SEQ ID NOs: 4, 5, and 6, respectively; (3) The amino acid sequences of the selected VHH CDR1, 2, and 3 are shown in SEQ ID NOs: 7, 8, and 9, respectively; (4) The amino acid sequences of the selected VHH CDR1, 2, and 3 are shown in SEQ ID NOs: 10, 11, and 12, respectively; (5) The amino acid sequences of the selected VHH CDR1, 2, and 3 are shown in SEQ ID NOs: 13, 14, and 15, respectively; (6) The amino acid sequences of the selected VHH CDR1, 2, and 3 are shown in SEQ ID NOs: 16, 17, and 18, respectively; (7) The amino acid sequences of the selected VHH CDR1, 2, and 3 are shown in SEQ ID NOs: 19, 20, and 21, respectively; and (8) The amino acid sequences of the selected VHH CDR1, 2, and 3 are shown in SEQ ID NOs: 22, 23, and 24, respectively.

[0007] In some embodiments, a VHH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively. In some embodiments, a VHH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, a VHH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively. In some embodiments, a VHH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.

[0008] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof that binds to TFR1, comprising a heavy chain single variable region (VHH) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a selected VHH sequence; in some embodiments, the selected VHH sequence is selected from the group consisting of SEQ ID NOs: 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, monkey TFR1, mouse TFR1, or chimeric TFR1. In some embodiments, the antibody or antigen-binding fragment is a human antibody 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).

[0009] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof comprising VHH CDR1, 2, 3 of an antibody or antigen-binding fragment thereof described herein.

[0010] 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., an 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.

[0011] In one aspect, the disclosure relates to a nucleic acid comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof described herein. In some embodiments, the nucleic acid is a cDNA.

[0012] In one aspect, the disclosure relates to a vector comprising one or more of the nucleic acids described herein.

[0013] In one aspect, the present disclosure relates to a cell comprising the vector described herein. In some embodiments, the cell is a CHO cell. In one aspect, the present disclosure relates to a cell comprising one or more of the nucleic acids described herein.

[0014] In one aspect, the disclosure relates to a method for producing an antibody or antigen-binding fragment thereof, the method comprising: (a) culturing a cell described herein under conditions sufficient for the cell to produce the antibody or antigen-binding fragment thereof; and (b) recovering the antibody or antigen-binding fragment thereof produced by the cell.

[0015] In one aspect, the present disclosure relates to an antibody drug conjugate comprising an antibody or antigen-binding fragment thereof described herein covalently attached to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.

[0016] In one aspect, the present disclosure relates to a method of treating a subject having a brain disease (e.g., brain cancer), the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof, or an antibody-drug conjugate described herein. In some embodiments, the antibody or antigen-binding fragment thereof, or antibody-drug conjugate is capable of crossing the blood-brain barrier (BBB) ​​of the subject.

[0017] In one aspect, the present disclosure relates to 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 antigen-binding fragment thereof, or an antibody-drug conjugate, 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. In one aspect, the present disclosure relates to a method of identifying a subject as having a brain disease (e.g., brain cancer), the method comprising detecting a sample collected from the subject as having the brain disease with an antibody or antigen-binding fragment thereof described herein, thereby identifying the subject as having the brain disease. In some embodiments, the sample is a brain parenchyma sample from the subject. In some embodiments, the subject described herein is a human subject.

[0018] In one aspect, the present disclosure relates to a method of delivering an agent across the blood-brain barrier, the method comprising administering to a subject an agent covalently linked to an antibody or antigen-binding fragment thereof described herein. In some embodiments, the agent is an antibody or antibody-drug conjugate. In some embodiments, the agent is an anti-amyloid antibody.

[0019] In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier. In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody drug conjugate described herein and a pharmaceutically acceptable carrier.

[0020] In one aspect, the present disclosure relates to antibodies, or antigen-binding fragments thereof, that cross-compete with the antibodies, or antigen-binding fragments thereof, described herein.

[0021] In one aspect, the disclosure provides a method of making an antibody that specifically binds to an antigen, the method comprising exposing an animal described herein to the antigen, obtaining (e.g., by sequencing) the sequence of a nucleic acid encoding a human heavy chain immunoglobulin variable region in a cell that expresses a chimeric heavy chain antibody that specifically binds to 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.

[0022] The present disclosure also relates to progeny of the non-human mammal. In some embodiments, the non-human mammal is a rodent. In some embodiments, the non-human mammal is a mouse.

[0023] The present disclosure also provides cells comprising the targeting vectors described herein. The present disclosure also relates to cells (e.g., stem cells, embryonic stem cells, immune cells, B cells, T cells, or hybridomas) or cell lines derived from non-human mammals or their descendants, or primary cell cultures thereof. The present disclosure further relates to tissues, organs, or cultures thereof derived from non-human mammals or their descendants.

[0024] The present disclosure further relates to the use of non-human mammals or their progeny, animal models produced by the methods described herein, in the development of products related to the immunization process, the production of human antibodies, or in model systems for the study of pharmacology, immunology, microbiology, and medicine.

[0025] 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 use in the present invention are described herein; other suitable methods and materials well known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0026] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. [Brief explanation of the drawings]

[0027] [Figure 1] The CDR sequences of the heavy chain variable regions of anti-TFR1 antibodies are listed according to Kabat numbering. [Figure 2] 1 lists the CDR sequences of the heavy chain variable regions of anti-TFR1 antibodies according to IMGT numbering. [Figure 3] Listed below are the amino acid sequences discussed in this disclosure. [Figure 4A] 1 shows antibody concentrations in total brain protein of hTFR1 mice within 72 hours after intravenous (iv) administration of hIgG1 (G1), JR141-N (G2), 23B8-N (G3), 24A1-N (G4), 24G5-N (G5), or 24C9-N (G6). [Figure 4B] This shows the ratio of antibody concentration in total brain protein to serum antibody concentration in hTFR1 mice within 72 hours after intravenous (iv) administration of hIgG1 (G1), JR141-N (G2), 23B8-N (G3), 24A1-N (G4), 24G5-N (G5), or 24C9-N (G6). [Figure 4C]1 shows antibody concentrations in the brain parenchyma of hTFR1 mice within 72 hours after intravenous (iv) administration of hIgG1 (G1), JR141-N (G2), 23B8-N (G3), 24A1-N (G4), 24G5-N (G5), or 24C9-N (G6). [Figure 4D] 1 shows the ratio of antibody concentration in the brain parenchyma to serum antibody concentration in hTFR1 mice within 72 hours after intravenous (iv) administration of hIgG1 (G1), JR141-N (G2), 23B8-N (G3), 24A1-N (G4), 24G5-N (G5), or 24C9-N (G6). [Figure 5A] 1 shows the results of antibody concentration tests in the brain parenchyma 24 hours after intravenous (iv) administration of hIgG1 (G1), JR141-N (G2), 23B8-N (G3), 24A1-N (G4), or 24G5-N (G5). [Figure 5B] FIG. 1 shows the results of an antibody concentration test in total brain protein (whole brain) 24 hours after intravenous (iv) administration of hIgG1 (G1), JR141-N (G2), 23B8-N (G3), 24A1-N (G4), or 24G5-N (G5). [Figure 6] The results of antibody concentrations 6 hours or 24 hours after intravenous (iv) administration of JR141-N (G2 to G4) or 24G5-N (G5 to G7) are shown. hIgG1 was used as a negative control. [Figure 7] 1 shows the results of testing ADC concentrations in brain parenchyma 72 hours after intravenous (iv) administration of 24G5-ADC or 24G5-mono-ADC. DETAILED DESCRIPTION OF THE INVENTION

[0028] Heavy-chain antibodies (or heavy-chain-only antibodies) are antibodies that have only heavy chains (typically two heavy chains) and lack the two light chains typically found in antibodies. Naturally occurring heavy-chain antibodies have been found in cartilaginous fish (e.g., sharks) and camelids (e.g., llamas). For example, in cartilaginous fish, the immunoglobulin new antigen receptor (IgNAR) is a heavy-chain antibody. IgNARs have significant structural differences from other antibodies: they have five constant domains (CH) per chain instead of the usual three, several disulfide bonds in unusual positions, and the complementarity-determining region 3 (CDR3) forms an extended loop that covers the binding site for the light chain of other antibodies. These differences, combined with the phylogenetic age of cartilaginous fish, have led to the hypothesis that IgNARs may be more closely related to primitive antigen-binding proteins than mammalian immunoglobulins.

[0029] The only mammals with 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 (e.g., IgG1) consisting of two heavy chains and two light chains linked in a Y-shape by disulfide bonds. However, they also produce two unique subclasses of IgG (also called heavy-chain IgGs): IgG2 and IgG3. These antibodies lack the CH1 region but still consist of only two heavy chains with antigen-binding domains (e.g., VHH) at their N-termini. Conventional IgGs require the combination of both heavy and light chain variable regions to enable a high diversity of antigen-antibody interactions. While single heavy and light chains still exhibit this ability, they exhibit significantly lower affinity compared to paired heavy and light chains. A unique feature of heavy-chain IgGs is the ability of their monomeric antigen-binding regions to bind antigens with specificity, affinity, and especially diversity comparable to conventional antibodies, without the need for pairing. This feature is mainly due to several key mutations in the amino acid sequences of the variable regions of the two heavy chains, which cause major structural changes compared to conventional Igs. The key substitutions in the variable regions not only prevent the light chains from binding to the heavy chains, but also prevent the unbound heavy chains from being recycled by immunoglobulin-binding proteins.

[0030] These heavy-chain antibody single variable domains (designated VHHs, sdAbs, or nanobodies) are the smallest antigen-binding domains generated by the adaptive immune system. It is well known that the complementarity-determining region 3 (CDR3) of the variable region of these antibodies is twice as long as that of conventional antibodies. This results in an expanded interaction surface with antigens, increasing the diversity of antigen-antibody interactions and compensating for the lack of light chains. The long CDR3 allows VHHs to reach crevices on proteins inaccessible to conventional antibodies, including functionally interesting sites such as the active site of an enzyme or the receptor-binding canyon on the surface of a virus. Furthermore, the addition of cysteine ​​residues makes the structure more stable, thus enhancing the strength of the interaction.

[0031] Compared to conventional antibodies with conventional antibody variable domains (VH and VL), VHHs offer numerous other advantages, including higher stability, solubility, expression yield, and refolding ability, as well as better in vivo tissue penetration. Furthermore, in contrast to the VH domain of conventional antibodies, VHHs exhibit no intrinsic tendency to bind to light chains. This facilitates the induction of heavy-chain antibodies in the presence of a functional light chain locus. Furthermore, because VHHs do not bind to VL domains, it is much easier to reformat VHHs into bispecific antibody constructs than constructs containing conventional VH-VL pairs or single domains based on VH domains.

[0032] A notable difference between camelid VHH and human VH domains is the length and orientation of the CDR3 loop. CDR3 corresponds to a unique region of the antibody molecule encoded by newly generated DNA elements during B cell development. Genetic recombination results in the fusion of the D element with adjacent V and J elements. During recombination, additional genetic diversity is generated by the addition and / or deletion of nucleotides at the junctions. Thus, the CDR3 loop provides a major contribution to antibody diversity and specificity. Some early transgenic heavy chain antibody animals have a limited number of variable region genes (IGHV, IGHD, and IGHJ), resulting in the inability to recognize some antigens despite the strong antigen response of wild-type animals (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 engineered into transgenic animals that have a fully human heavy chain antibody repertoire.

[0033] As used herein, the term "antibody" refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementarity-determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to 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 can contain the Fc region of a human antibody. The term antibody also includes derivatives, e.g., bispecific antibodies, single-chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0034] As used herein, the term "antigen-binding fragment" refers to a portion of a full-length antibody, which portion is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a heavy chain variable domain or a light chain variable domain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0035] As used herein, the term "human antibody" means an antibody encoded by nucleic acid present in a human (e.g., a rearranged human immunoglobulin heavy or light chain locus). In some embodiments, a human antibody is recovered from a human or produced in human cell culture (e.g., in human hybridoma cells). In some embodiments, a human antibody is produced in a non-human cell (e.g., a mouse or hamster cell line). In some embodiments, a human antibody is produced in a bacterial cell or yeast cell. In some embodiments, a human antibody is produced in a transgenic non-human animal (e.g., a mouse) containing unrearranged or rearranged human immunoglobulin loci (e.g., a heavy or light chain human immunoglobulin locus).

[0036] As used herein, the term "chimeric antibody" refers to an antibody that contains sequences present in at least two different antibodies (e.g., antibodies from two different mammalian species, such as human and murine antibodies). Non-limiting examples of chimeric antibodies include antibodies that contain variable domain sequences (e.g., all or part of the light and / or heavy chain variable domain sequences) of a human antibody and the constant domain of a non-human antibody. Further examples of chimeric antibodies are described herein and are well known in the art.

[0037] As used herein, the term "humanized antibody" means a non-human antibody that contains sequences derived from non-human (e.g., murine) immunoglobulin and that contains sequences derived from human immunoglobulin.

[0038] As used herein, the term "single-chain antibody" means a single polypeptide containing at least two immunoglobulin variable domains (e.g., the variable domains of a mammalian immunoglobulin heavy or light chain) that is capable of specifically binding to an antigen.

[0039] As used herein, the term "heavy chain antibody" refers to an antibody molecule that is composed only of heavy chains (usually two) and does not have any light chains.

[0040] As used herein, the term "VHH" refers to a variable domain derived from a heavy chain antibody. A VHH can specifically recognize an antigen without necessarily being paired with a VL. In some embodiments, a VHH (also referred to as an sdAb or nanobody) described herein is derived from any of the humanized heavy chain antibodies described herein. In some embodiments, a VHH, sdAb, or nanobody described herein is derived from a heavy chain antibody produced by any of the genetically modified non-human animals described herein.

[0041] As used herein, the terms "subject" and "patient" are used interchangeably throughout and refer to an animal, human, or non-human. Veterinary and non-veterinary applications are contemplated by the present disclosure. A human patient can be an adult human or a juvenile human (e.g., a human under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. For example, patients include non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pigs, minipigs), equines, canines, felines, bovines, and other domestic, livestock, and zoo animals.

[0042] As used herein, the phrases "specifically bind" and "specifically binds" when referring to an antibody mean that the antibody preferably interacts with the target molecule in preference to other molecules because the interaction is dependent on the presence of a particular structure (i.e., an antigenic determinant or epitope) on the target molecule; in other words, the reagent generally recognizes and binds to molecules containing a particular structure rather than whole molecules. An antibody that specifically binds to a target molecule can also be referred to as a target-specific antibody.

[0043] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to polymers of amino acids of any length, of at least two amino acids.

[0044] As used herein, the terms "polynucleotide," "nucleic acid molecule," and "nucleic acid sequence" are used interchangeably herein and refer to polymers of nucleotides of any length, of at least two nucleotides, including, but not limited to, DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0045] Antibodies and antigen-binding fragments 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.

[0046] Generally, traditional antibodies are composed of two types of polypeptide chains, light chains and heavy chains. Non-limiting antibodies of the present disclosure can be intact four immunoglobulin chain antibodies, including two heavy chains and two light chains. The heavy chain of the antibody can be of 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 kappa light chain or a lambda light chain. An antibody can include two identical copies of the light chain and two identical copies of the heavy chain, each containing one variable domain (or variable region, V H ), and heavy chains containing multiple constant domains (or constant regions) linked together via disulfide bonds within their constant domains to form the "stem" of the antibody. L Each light chain, containing a constant domain (or constant region) and one light chain, binds to one heavy chain via disulfide bonds. The variable region of each light chain aligns with the variable region of the heavy chain to which it is bound. The variable regions of both the light and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FRs).

[0047] The hypervariable regions, known as complementarity-determining regions (CDRs), form the loops that comprise the principal antigen-binding surface of an antibody. The four framework regions largely conform to a β-sheet structure, and the CDRs form connecting loops that, in some cases, form part of the β-sheet structure. The CDRs of each chain are held in close proximity by the framework regions and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding region.

[0048] Methods for identifying CDR regions of antibodies by analyzing their amino acid sequences are well known, and several definitions of CDRs are commonly used: the Kabat definition is based on sequence variability, and the Chothia definition is based on the location of structural loop regions. These methods and definitions are described, for example, by Martin, “Protein sequence and structure analysis of antibody variable domains,” Antibody engineering, Springer Berlin Heidelberg, 2001.422-439; Abhinandan, et al. Kabat,EA(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 by reference herein in its entirety.

[0049] CDRs are important for recognizing the epitope of an antigen. As used herein, "epitope" refers to 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 can be approximately 3, 4, 5, 6, or 7 amino acids, but these amino acids do not need to be in a contiguous linear sequence in the primary structure of the antigen, as the epitope may depend on the three-dimensional structure of the antigen based on the secondary and tertiary structure of the antigen.

[0050] In some embodiments, antibodies are intact immunoglobulin molecules (e.g., IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved and differ in their constant regions, particularly the hinge and upper CH2 domains. The sequences and differences between IgG subclasses are well known in the art and are described, for example, in Vidarsson, et al., "IgG subclasses and allotypes: from structure to effector functions," Frontiers in Immunology 5 (2014); Irani, et al., "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases," Molecular Immunology 67.2 (2015): 171-182; Shakib, Farouk, ed., The human IgG subclasses: molecular analysis of structure, function, and regulation. Elsevier, 2016, each of which is incorporated by reference in its entirety herein. The heavy chain constant region of the 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).

[0051] An antibody can also be an immunoglobulin molecule from any species (e.g., human, rodent, mouse, rat, camelid). Antibodies disclosed herein include, but are not limited to, polyclonal, monoclonal, monospecific, 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 any portion of an antibody that retains the specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specifically binding to an epitope on the intact antibody's target molecule. This includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, an antibody or antigen-binding fragment thereof can be, for example, an scFv, Fv, Fd, dAb, diabody, bispecific scFv, diabody, linear antibody, single-chain antibody molecule, multispecific antibody formed from antibody fragments, and any polypeptide comprising a binding domain that is, or is homologous to, an antibody-binding domain. Non-limiting examples of antigen-binding domains include, for example, the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or individual CDRs derived from either the heavy or light chain of an intact antibody.

[0052] In some embodiments, the antigen-binding fragment can form part of a chimeric antigen receptor (CAR), which in some embodiments is a fusion of a VHH described herein fused to a CD3-ζ transmembrane domain and endodomain.

[0053] The antibodies and antigen-binding fragments thereof (e.g., humanized or chimeric antibodies) produced by the methods described herein have various advantages. In some embodiments, no further optimization is required to obtain the desired properties (e.g., binding affinity, thermal stability, and / or limited aggregation).

[0054] In some embodiments, the antibody (or antigen-binding fragment thereof) is -1 Less than 0.01s -1 Less than 0.001s -1 Less than 0.0001s -1 Less than or 0.00001s -1 Specifically binds to a target with a k of less than 0.01 s. In some embodiments, the k is less than 0.01 s. -1 Super, 0.001s -1 Super, 0.0001s -1 Super, 0.00001s -1 Over or 0.000001s -1 It's super.

[0055] In some embodiments, the kinetic association rate (k) is 1×10 2 / Ms super, 1×10 3 / Ms super, 1×10 4 / Ms super, 1×10 5 / Ms or more than 1 × 10 6 In some embodiments, the kinetic association rate (k) is greater than 1×10 5 / Ms less than 1 × 10 6 / Ms or less than 1 x 10 7 / Ms is less than.

[0056] Affinity can be estimated from the quotient of the kinetic rate constants (K = k / k). In some embodiments, K is greater than or equal to 1 x 10 -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M or 1 x 10 -10 In some embodiments, the 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, the KD is less than 1 x 10 -7 Super M, 1x10 -8 Super M, 1x10 -9 Super M, 1x10 -10 Super M, 1x10-11 Over M or 1x10 -12 In some embodiments, the antibody binds to the target with a KD of 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 or less.

[0057] In some embodiments, thermal stability is measured. The antibodies or antigen-binding fragments described herein may have a Tm of greater than 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.

[0058] In various embodiments, substitutions are made to the parent heavy chain antibody sequence to generate a mutant heavy chain antibody. Generally, the heavy chain antibody variant of the parent heavy chain antibody has an antigen-binding affinity of 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 parent heavy chain antibody's binding affinity for a particular antigen. In some embodiments, the mutant heavy chain antibody contains a single substitution compared to the parent heavy chain antibody. However, in other embodiments, several amino acids, e.g., up to about 5 or 10 or more, are substituted compared to the parent heavy chain antibody sequence derived from another human heavy chain sequence that shares identity at a particular position. In various embodiments, the resulting mutant heavy chain antibody is tested to ensure that the substituted residues do not significantly reduce the desired binding affinity and / or specificity. In some embodiments, improved mutant heavy chain antibodies are produced by substitution of amino acids from different human heavy chain sequences. In various embodiments, the VHH is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the parent VHH.

[0059] The VHHs described herein can be used to generate multispecific antibodies (e.g., bispecific antibodies). In one aspect, the present disclosure provides multispecific antibodies 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 to a first epitope. In some embodiments, the first antigen-binding portion comprises a VHH that specifically binds to a first epitope. In some embodiments, the second antigen-binding portion comprises a VHH that specifically binds to a second epitope. In some embodiments, the first epitope and the second epitope are from the same antigen. In some embodiments, the first epitope and the second epitope are from different antigens.

[0060] In some embodiments, the first antigen-binding moiety is a full-length antibody consisting of two heavy chains and two light chains. In some embodiments, the first antigen-binding moiety is an antibody fragment comprising a heavy chain comprising a VH and a light chain comprising a VL. In some embodiments, the second antigen-binding moiety comprises a single polypeptide chain. In some embodiments, the C-terminus of the second antigen-binding moiety is fused to the N-terminus of at least one heavy chain of the first antigen-binding moiety. In some embodiments, the C-terminus of the second antigen-binding moiety is fused to the N-terminus of at least one light chain of the first antigen-binding moiety. In some embodiments, the N-terminus of the second antigen-binding moiety is fused to the C-terminus of at least one heavy chain of the first antigen-binding moiety. In some embodiments, the N-terminus of the second antigen-binding moiety is fused to the C-terminus of at least one light chain of the first antigen-binding moiety. In some embodiments, the second antigen-binding portion is a Fab-like domain comprising a first polypeptide chain comprising a first VHH fused to a CH1 domain and a second polypeptide chain comprising a second VHH fused to a CL domain.

[0061] In some embodiments, the antibody or antigen-binding fragment thereof 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 VHH. In some embodiments, the VHHs bind to the same epitope. In some embodiments, the VHHs bind to different epitopes.

[0062] In some embodiments, an antibody or antigen-binding fragment thereof comprises four or more VHHs. In some embodiments, to enhance developability, at least four VHHs are combined to construct a tetraspecific VHH without the addition of an IgG Fc domain. These molecules lack Fc effector functions but have the added advantage of improved affinity and avidity for antigen compared to bispecific and trispecific VHH-Fc.

[0063] In some embodiments, these antibodies or antigen-binding fragments thereof (e.g., comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) have a functional Fc.

[0064] In some embodiments, heavy chain antibodies produced by the transgenic non-human animals described herein have a VHH domain comprising CDR1, CDR2, and CDR3. In some embodiments, the length of CDR3 is 6 to 23, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the length of CDR3 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.

[0065] Transferrin receptor 1 (TFR1) TFR1, also known as cluster of differentiation 71 (CD71), is ubiquitously expressed and can bind transferrin (Tf) with high affinity. Human TFR1 is a 90-kDa type II transmembrane glycoprotein consisting of 760 amino acids found as a dimer (180 kDa) held together by disulfide bonds on the cell surface. The TFR1 monomer consists of a large extracellular C-terminal domain of 671 amino acids, which contains the Tf-binding site, a transmembrane domain (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 required for full receptor function.

[0066] Transferrin (Tf) is an 80 kDa glycoprotein composed of two 40 kDa subunits, known as the N- and C-lobes, separated by a short linker sequence. Each subunit contains one free ferric ion (Fe 3+ Tf can have up to two bound iron atoms because it can bind to iron-containing ATP. Tf in its iron-free form, apoTf, is highly efficient at transporting Fe in the blood. 3+ bound to Fe 3+ Upon interaction with TFR1, TFR1 transports Fe to the cell surface for internalization. As a membrane protein that regulates iron import, TFR1 transports Fe 3+ It is a member of the TFR family that exhibits nanomolar affinity for transferrin (Tf) bound to TFR1. The Tf-TFR1 complex is internalized by clathrin-mediated endocytosis and releases Fe upon a decrease in pH to 5.5. 3+ At this pH, apoTf and TFR1 still associate and are recycled to the cell surface at physiological pH, releasing apoTf.

[0067] Because iron uptake via the transferrin receptor is an important method for cancer cells to absorb iron, accumulating evidence demonstrates that TFR1 is involved in tumor initiation and progression, and its expression is significantly dysregulated in many cancers. The relationship between TFR1 and cancer has become clear, making TFR1 a valuable pharmaceutical target for cancer intervention.

[0068] TFR1, which is expressed on the endothelial cells of the blood-brain barrier, can also be used in preclinical studies to deliver macromolecules, including antibodies, to the brain. Antibodies targeting TFR1 can cross the blood-brain barrier without interfering with iron uptake.

[0069] A detailed description of TFR1, Tf, and their functions can be found, for example, in Candelaria, PV, 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, each of which is incorporated by reference in its entirety.

[0070] Heavy chain single variable domain (VHH) antibodies Monoclonal and recombinant antibodies are important tools in medicine and biotechnology. Like all mammals, camelids (e.g., llamas) can produce conventional antibodies (e.g., IgG1) consisting of two heavy chains and two light chains linked in a Y-shape by disulfide bonds. However, they also produce two unique subclasses of IgG (also called heavy-chain IgGs): IgG2 and IgG3. These antibodies lack the CH1 region but still consist of only two heavy chains with antigen-binding domains at their N-termini, called VHHs (or nanobodies). Conventional IgGs require the combination of both heavy and light chain variable regions to enable high diversity in antigen-antibody interactions. While single heavy and light chains still exhibit this ability, they exhibit significantly lower affinity compared to paired heavy and light chains. A unique feature of heavy-chain IgGs is the ability of their monomeric antigen-binding regions to bind antigens with specificity, affinity, and especially diversity comparable to conventional antibodies, without the need for pairing. This feature is mainly due to several key mutations in the amino acid sequences of the variable regions of the two heavy chains, which cause major structural changes compared to conventional Igs. The key substitutions in the variable regions prevent the light chains from binding to the heavy chains, but also prevent the unbound heavy chains from being recycled by immunoglobulin-binding proteins.

[0071] The single variable domains of these antibodies (designated VHHs, sdAbs, or nanobodies) are the smallest antigen-binding domains generated by the adaptive immune system. It is well known that the third complementarity-determining region (CDR3) of the variable regions of these antibodies is twice as long as that of conventional antibodies. This results in an expanded interaction surface with the antigen, increasing the diversity of antigen-antibody interactions and compensating for the lack of a light chain. The long complementarity-determining region 3 (CDR3) allows VHHs to reach niches on proteins inaccessible to conventional antibodies, including functionally interesting sites such as the active site of an enzyme or the receptor-binding canyon on the surface of a virus.

[0072] Compared to conventional antibodies with conventional antibody variable domains (VH and VL), VHHs offer numerous other advantages, including higher stability, solubility, expression yield, and refolding ability, as well as better in vivo tissue penetration and internalization. Furthermore, in contrast to the VH domain of conventional antibodies, VHHs do not exhibit an intrinsic tendency to bind to light chains. Because VHHs do not bind to VL domains, it is much easier to reformat VHHs into multispecific (e.g., bispecific) constructs than constructs containing conventional VH-VL pairs or single domains based on VH domains.

[0073] The present disclosure provides, for example, anti-TFR1 antibodies, modified antibodies thereof, chimeric antibodies thereof, and humanized antibodies thereof.

[0074] The CDR sequences of 23B8 and 23B8-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as defined by Kabat numbering and set forth in SEQ ID NOS: 1, 2, and 3, respectively. CDRs can also be defined by the IMGT system, in which the CDRs of the VHH domain are set forth in SEQ ID NOS: 13, 14, and 15, respectively.

[0075] The CDR sequences of 24A1 and 24A1-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domains set forth in SEQ ID NOS: 4, 5, and 6, respectively, as defined by Kabat numbering. CDRs can also be defined by the IMGT system, in which the CDRs of the VHH domains are set forth in SEQ ID NOS: 16, 17, and 18, respectively.

[0076] The CDR sequences of 24C9 and 24C9-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as defined by Kabat numbering and set forth in SEQ ID NOs: 7, 8, and 9, respectively. CDRs can also be defined by the IMGT system, in which the CDRs of the VHH domain are set forth in SEQ ID NOs: 19, 20, and 21, respectively.

[0077] The CDR sequences of 24G5 and 24G5-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as defined by Kabat numbering and set forth in SEQ ID NOS: 10, 11, and 12, respectively. CDRs can also be defined by the IMGT system, in which the CDRs of the VHH domain are set forth in SEQ ID NOS: 22, 23, and 24, respectively.

[0078] 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.

[0079] The amino acid sequences of various modified or humanized VHHs are also provided. Because there are different methods for modifying or humanizing heavy chain antibodies (e.g., the sequence can be altered with different amino acid substitutions), multiple versions of the humanized sequence of the VHH domain of a heavy chain antibody may exist. In some embodiments, the humanized VHH domain is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the sequences set forth in SEQ ID NOs: 25-28.

[0080] Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein may also contain one, two, or three VHH domain CDRs selected from the group consisting of SEQ ID NOs: 1-3, 4-6, 7-9, 10-12, 13-15, 16-18, 19-21, and 22-24.

[0081] In some embodiments, the antibody may have a heavy chain single variable domain (VHH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of a selected VHH CDR1, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of a selected VHH CDR2, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of a selected VHH CDR3. The amino acid sequences of selected VHH CDR1, 2, and 3 are shown in Figures 1 and 2.

[0082] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing one, two, or three of: a VHH CDR1 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; a VHH CDR2 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; and a VHH CDR3 with 0, 1, or 2 amino acid insertions, deletions, or substitutions, wherein VHH CDR1, VHH CDR2, and VHH CDR3 are selected from the CDRs in Figure 3.

[0083] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing one, two, or three of the CDRs of SEQ ID NO: 1 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 3 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0084] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing one, two, or three of the CDRs of 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; or SEQ ID NO: 6 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0085] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing one, two, or three of the CDRs of 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; or SEQ ID NO: 9 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0086] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing one, two, or three of the CDRs of 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; or SEQ ID NO: 12 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0087] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing one, two, or three of the CDRs of 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; or SEQ ID NO: 15 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0088] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing one, two, or three of the CDRs of 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; or SEQ ID NO: 18 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0089] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing one, two, or three of the CDRs of 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; or SEQ ID NO: 21 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0090] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing one, two, or three of the CDRs of 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; or SEQ ID NO: 24 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0091] Insertions, deletions, and substitutions can occur within the CDR sequences or at either or both ends of the CDR sequences. In some embodiments, the CDRs are determined according to the Kabat numbering scheme. In some embodiments, the CDRs are determined according to the Chothia numbering scheme. In some embodiments, the CDRs are determined according to a combined numbering scheme. In some embodiments, the CDRs are determined according to the IMGT numbering scheme.

[0092] The present disclosure also provides an antibody or antigen-binding fragment thereof that binds to TFR1 (human TFR1). The antibody or antigen-binding fragment thereof includes a heavy chain single variable region (VHH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical 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.

[0093] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then 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, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to optimally align the two sequences. By way of example, sequence comparison and percent identity determination between two sequences can be performed using, for example, the Blossum62 scoring matrix, with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0094] The present disclosure also provides nucleic acids comprising polynucleotides encoding polypeptides comprising immunoglobulin heavy chain single variable domains (VHHs), the VHHs comprising CDRs as shown in Figures 1 and 2, or having a sequence as shown in Figure 3.

[0095] The antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments, as well as multispecific (e.g., bispecific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal, monoclonal, multispecific (multimeric, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, intracellularly produced antibodies (i.e., intrabodies), and antigen-binding fragments thereof.

[0096] In some embodiments, the antibody or antigen-binding fragment thereof 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 antigen-binding fragment thereof. In some embodiments, the Fc domain comprises one, two, three, four, or more heavy chain constant regions.

[0097] The present disclosure also provides antibodies or antigen-binding fragments thereof that cross-compete with any of the antibodies or antigen-binding fragments described herein. Cross-competition assays are well 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, the entire contents of which are incorporated herein by reference. In one aspect, the present disclosure also provides antibodies or antigen-binding fragments thereof that bind to the same epitope or region as any of the antibodies or antigen-binding fragments described herein. Epitope binding assays are well 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, the entire contents of which are incorporated herein by reference.

[0098] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain single variable domain (VHH) CDR1 selected from SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, and 22.

[0099] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain single variable domain (VHH) CDR2 selected from SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, and 23.

[0100] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain single variable domain (VHH) CDR3 selected from SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, and 24.

[0101] Antibody characteristics TFR1 plays a key role in cellular iron uptake through its interaction with iron-binding TF. Iron is required for multiple cellular processes and is essential for DNA synthesis and, therefore, cell proliferation. Due to its central role in cancer cytopathology, malignant cells often overexpress TFR1, and this increased expression may be associated with poor prognosis in different types of cancer. The elevated expression levels of TfR1 in malignant cells, its extracellular accessibility, internalization ability, and its central role in cancer cytopathology make this receptor an attractive target for antibody-mediated therapy.

[0102] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are unable to 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 anti-cancer drugs that are internalized by receptor-mediated endocytosis. In some embodiments, the antibodies or antigen-binding fragments thereof described herein are capable of disrupting receptor function. In some embodiments, the antibodies or antigen-binding fragments thereof described herein are unable to induce Fc effector function, thus preventing or ameliorating their adverse effects on normal cells.

[0103] The present disclosure provides antibodies or antigen-binding fragments thereof comprising a human Fc domain that induce Fc-dependent effector function by at least or about 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 the absence of an antibody or antigen-binding fragment thereof described herein.

[0104] The present disclosure provides antibodies or antigen-binding fragments thereof comprising a human Fc domain that induce a host immune response at least or about 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 the absence of an antibody or antigen-binding fragment thereof described herein.

[0105] The present disclosure provides antibodies or antigen-binding fragments thereof capable of internalizing into human brain cells (e.g., cortical microvascular endothelial cells) with an endocytosis rate of 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, the endocytosis rate of the antibodies or antigen-binding fragments 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 higher than that of an isotype control antibody.

[0106] In some embodiments, provided herein are antibodies or antigen-binding fragments thereof comprising a single heavy chain. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof comprising a pair of heavy chains. In some embodiments, the heavy chain pair is linked by a disulfide bond. In some embodiments, the heavy chain pair comprises knobs-in-holes modifications. In some embodiments, the heavy chain comprises a human IgG Fc domain. In some embodiments, the antibody or antigen-binding fragment thereof comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 VHH domains in each heavy chain. In some embodiments, the VHH domains in each heavy chain specifically bind the same epitope. In some embodiments, the VHH domains in each heavy chain specifically bind different epitopes. In some embodiments, the VHH domains in each heavy chain bind at least 1, 2, 3, 4, or 5 different epitopes.

[0107] 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 four, five, or six antigens.

[0108] In some embodiments, the antibody (or antigen-binding fragment thereof) is -1 Less than 0.01s -1 Less than 0.001s -1 Less than 0.0001s -1 Less than or 0.00001s -1 Specifically binds to TFR1 with a koff of less than 0.01 s. In some embodiments, the koff is less than 0.01 s. -1 Super, 0.001s -1 Super, 0.0001s -1 Super, 0.00001s -1 Over or 0.000001s -1 It's super.

[0109] In some embodiments, the kinetic association rate (k) is 1×10 2 / Ms super, 1×103 / Ms super, 1×10 4 / Ms super, 1×10 5 / Ms or more than 1 × 10 6 In some embodiments, the kinetic association rate (k) is greater than 1×10 5 / Ms less than 1 × 10 6 / Ms or less than 1 x 10 7 / Ms is less than.

[0110] Affinity can be estimated from the quotient of the kinetic rate constants (K = k / k). In some embodiments, K is greater than or equal to 1 x 10 -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M or 1 x 10 -10 In some embodiments, the 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, the KD is less than 1 x 10 -7 Super M, 1×10 -8 Super M, 1×10 -9 Super M, 1×10 -10 Super M, 1×10 -11 More than M or 1 x 10 -12 It's over M.

[0111] Common techniques for measuring the affinity of an antibody to 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. In some embodiments, the antibody does not bind to human TFR1, monkey TFR1, mouse TFR1, or chimeric TFR1.

[0112] In some embodiments, thermal stability is measured. The antibodies or antigen-binding fragments described herein may have a Tm (melting temperature) of 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 antibodies or antigen-binding fragments described herein can have a Tag (aggregation temperature, e.g., Tag at 266 nm (Tagg266) or Tag at 473 nm (Tagg473)) 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, 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.

[0113] In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.

[0114] In some embodiments, the antibody or antigen-binding fragment thereof has a functional Fc region. In some embodiments, the antibody or antigen-binding fragment thereof comprises a human IgG1 Fc region. In some embodiments, the human IgG1 Fc region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34.

[0115] In some embodiments, the antibody or antigen-binding fragment does not have an Fc region. For example, an antibody (or antigen-binding fragment thereof) is a polypeptide comprising one or more VHH domains interconnected by linker peptides. 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 to the same epitope. In some embodiments, the VHH domains bind to different epitopes. In some embodiments, the VHH domains bind to at least 1, 2, 3, 4, or 5 different epitopes.

[0116] In some embodiments, the antibody or antigen-binding fragment thereof does not have a functional Fc region. In some embodiments, the Fc region has a LALA mutation (L234A and L235A mutations in EU numbering) or a LALA-PG mutation (L234A, L235A, P329G mutations in EU numbering). In some embodiments, the Fc region has a mutation at position 297 according to EU numbering (e.g., N297A). In some embodiments, the mutated human IgG1 Fc region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 35.

[0117] In some embodiments, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or 72 hours after administration to a subject, the concentration of an antibody or antigen-binding fragment thereof described herein in the brain (e.g., whole brain or parenchyma) may be greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of its 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 administration to a subject, the concentration of an antibody or antigen-binding fragment thereof described herein in the brain (e.g., whole brain or 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, 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 the concentration of a control antibody (e.g., hIgG1 or JR141-N) or the concentration in the serum of the subject.

[0118] Method for producing anti-TFR1 antibodies Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into DNA encoding a human, humanized, or chimeric antibody, or an antibody or antigen-binding fragment thereof described herein, or by peptide synthesis. Such variants include, for example, deletion, insertion, or substitution of residues within the amino acid sequence that makes up the antigen-binding site or domain of the antibody. In a population of such variants, some antibodies or antigen-binding fragments have increased affinity for a target protein, e.g., TFR1. Any combination of deletion, insertion, and / or a combination can be achieved in an antibody or antigen-binding fragment thereof with increased binding affinity for the target. Antibodies or antigen-binding fragments can be altered by introducing amino acid changes into the antibody or antigen-binding fragment, such as changing the number (e.g., increasing or decreasing) of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence so that different sugars are attached by enzymes present in cells), or introducing new glycosylation sites, or by introducing new post-translational modifications into the antibody or antigen-binding fragment. In some embodiments, the heavy chain antibodies or antigen-binding fragments thereof described herein are obtained by immunizing any of the transgenic animals described, for example, in PCT / CN2022 / 119188 (e.g., mice with in situ fully human heavy chain variable domain replacements combined with modified constant regions).

[0119] Humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequences as) human germline immunoglobulin sequences of a human immunoglobulin scaffold sequence. Humanized antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). Thus, "humanized" antibodies are chimeric antibodies in which sequences from a non-human species are replaced by corresponding human sequences.

[0120] Typically, an amino acid sequence variant of a human, humanized, or chimeric anti-TFR1 antibody contains an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% percent identity with the sequence present in the VHH domain of the original antibody.

[0121] Identity or homology to the original sequence is typically the percentage of amino acid residues present in the candidate sequence that are identical to sequences present in a human, humanized, or chimeric anti-TFR1 antibody or fragment, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity.

[0122] Further modifications can be made to the anti-TFR1 antibody or antigen-binding fragment. For example, cysteine ​​residues can be introduced into the Fc region to allow interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have some increased in vitro and / or in vivo half-life. Homodimeric antibodies with increased in vitro and / or in vivo half-life can also be prepared using heterobifunctional cross-linkers, for example, as described by Wolff et al. ("Monoclonal antibody homodimers: enhanced antitumor activity in nude mice," Cancer research 53.11 (1993):2560-2565). Alternatively, antibodies having dual Fc regions can be engineered.

[0123] In some embodiments, covalent modifications can be made to anti-TFR1 antibodies or antigen-binding fragments thereof. These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of antibodies or antibody fragments are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with organic derivatizing agents capable of reacting with selected side chains or with the N- or C-terminal residues.

[0124] In some embodiments, antibody variants are provided that have carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibody compositions may 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 in the glycan at Asn297 relative to the sum of all glycans (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at position 297 in the Fc region (position 314 in the EU numbering of Fc region residues or Kabat numbering). However, Asn297 may also be located approximately ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to minor sequence variation in antibodies. Such fucosylation variants may have improved ADCC function. In some embodiments, the Fc region of the antibody can be further modified to replace the asparagine at position 297 with alanine (N297A) to reduce glycan heterogeneity.

[0125] The present disclosure also provides recombinant vectors (e.g., expression vectors) comprising the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors have been introduced (i.e., such that the host cell contains the polynucleotides and / or vectors comprising the polynucleotides), and the production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.

[0126] As used herein, a "vector" is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An "expression vector" is capable of delivering and expressing one or more polynucleotides of interest as encoded polypeptides in a host cell into which the expression vector is introduced. Thus, in an expression vector, a polynucleotide of interest is positioned for expression in the vector by being operably linked to control elements, such as a promoter, enhancer, and / or polyA tail, at, near, or adjacent to the integration site of the polynucleotide of interest, either within the vector or in the genome of the host cell, such that the polynucleotide of interest is translated in a host cell into which the expression vector is introduced.

[0127] Vectors can be introduced into host cells by methods well known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., by recombinant viruses). Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0128] In some embodiments, a polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein) is introduced using a viral expression system (e.g., variola or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication-competent virus, or may employ a replication-defective virus, in which case viral propagation generally occurs only in complementary viral packaging cells. For example, Fisher-Hoch et al.,1989,Proc.Natl.Acad.Sci.USA 86:317-321;Flexner et al.,1989,Ann.NYAcad Sci.569:86-103;Flexner et al. al., 1990, Vaccine, 8:17-21; U.S. Patent Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89 / 01973; 91 / 02805;Berkner-Biotechniques,6:616-627,1988;Rosenfeld et al. Suitable systems are disclosed in Kolls et al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler 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 incorporating DNA into such expression systems are well known to those skilled 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 onto biodegradable beads that are efficiently transported into cells.

[0129] For expression, a DNA insert containing an antibody-encoding or polypeptide-encoding polynucleotide disclosed herein can be operably linked to a suitable promoter (e.g., a heterologous promoter), such as the phage lambda PL promoter, the E. coli lac, trp, and tac promoters, the SV40 early and late promoters, and promoters of retroviral long terminal repeats, to name a few. Other suitable promoters are known to those of skill in the art. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. The expression construct can further contain sites for transcription initiation and termination, and, within the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the construct can include a translation initiation codon at the beginning and a termination codon (UAA, UGA, or UAG) positioned approximately at the end of the polypeptide to be translated.

[0130] As indicated, the expression vector can include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance genes 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 melanogaster S2 and Spodoptera litura Sf9 cells, animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells, and plant cells. Appropriate culture media and conditions for the host cells described herein are well known in the art.

[0131] Non-limiting vectors for use in bacteria include pQE70, pQE60, and pQE-9 available from Qiagen, pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A available from Stratagene, and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG available from Stratagene, and pSVK3, pBPV, pMSG, and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to those of skill in the art.

[0132] Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters, and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, promoters of retroviral LTRs such as those of Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0133] In the yeast Saccharomyces cerevisiae, several vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, can be used.

[0134] Introduction of the construct into the host cell can be accomplished by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0135] Transcription of DNA encoding the antibodies of the present disclosure in more eukaryotic organisms can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp, that serve to increase transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0136] For secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment, appropriate secretion signals can be incorporated into the expressed polypeptide. The signals can be endogenous to the polypeptide or they can be heterologous signals.

[0137] Polypeptides (e.g., antibodies) can be expressed in modified forms, such as fusion proteins (e.g., GST fusions) or with histidine tags, and can contain not only secretion signals but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of a polypeptide to improve stability and durability in host cells during purification or during subsequent handling and storage. Peptide moieties can also be added to polypeptides to facilitate purification. Such regions can be removed before final preparation of the polypeptide. The addition of peptide moieties to polypeptides to cause secretion or excretion, improve stability, and facilitate purification are, inter alia, well-known and routine techniques in the art.

[0138] The present disclosure also provides nucleic acid sequences that are 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%, 99% identical to any of the nucleotide sequences described herein, and Also provided are amino acid sequences that are 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%, 99% identical to any amino acid sequence described herein. In some embodiments, the present disclosure relates to a nucleotide sequence encoding any of the peptides described herein or any amino acid sequence encoded by any of the nucleotide sequences 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.

[0139] In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences described herein.

[0140] In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences described herein.

[0141] In some embodiments, the antibody or antigen-binding fragment thereof is expressed in yeast, insect cells, or mammalian cells (eg, CHO cells).

[0142] Treatment and diagnostic methods The anti-TFR1 antibodies of the present disclosure, or antibodies or antigen-binding fragments thereof, can be used for various therapeutic purposes. In one aspect, the present disclosure provides methods for treating a brain disease (e.g., brain cancer, dementia, or Alzheimer's disease) in a subject, methods for identifying a subject with a brain disease (e.g., brain cancer, dementia, or Alzheimer's disease), methods for reducing the risk of developing a brain disease, or methods for reducing the risk of developing additional symptoms in a subject. In some embodiments, treatment can halt, slow, delay, or inhibit the progression of the brain disease (e.g., brain cancer, dementia, or Alzheimer's disease). In some embodiments, treatment can reduce the number, severity, and / or duration of one or more symptoms of a brain disease (e.g., brain cancer, dementia, or Alzheimer's disease) in a subject.

[0143] In one aspect, the disclosure features a method that includes administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, a brain disorder).

[0144] In one aspect, the disclosure features a method of delivering a therapeutic agent across the blood-brain barrier. In some embodiments, an antibody or antigen-binding fragment thereof described herein is linked to a therapeutic agent. In some embodiments, the therapeutic agent is an antibody, an antigen-binding fragment thereof, a small molecule, or an antibody-drug conjugate.

[0145] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk for brain disorders (e.g., brain cancer, dementia, or Alzheimer's disease). Patients with brain disorders (e.g., brain cancer, dementia, or Alzheimer's disease) can be identified by a variety of methods known in the art.

[0146] In some embodiments, the brain disease is brain cancer.

[0147] In one aspect, the present disclosure relates to a method of reducing tumor growth rate, comprising contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof, or an antibody drug conjugate described herein. In one aspect, the present disclosure relates to a method of killing tumor cells, comprising contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof, or an antibody drug conjugate described herein.

[0148] As used herein, "effective amount" means an amount or dosage sufficient to bring about a beneficial or desired result, including halting, slowing, preventing, or inhibiting the progression of a disease, e.g., cancer. The effective amount will vary depending on, for example, the age and weight of the subject to whom the antibody, antigen-binding fragment, polynucleotide encoding the antibody, vector comprising the polynucleotide, and / or composition is administered, the severity of the symptoms, and the route of administration, and thus, dosing can be determined on an individual basis.

[0149] An effective amount can be administered in one or more administrations. By way of example, an effective amount of an antibody or antigen-binding fragment thereof is an amount sufficient to ameliorate, halt, stabilize, reverse, inhibit, slow, and / or delay the progression of a patient's disease. As understood in the art, an effective amount of an antibody or antigen-binding fragment may vary depending on other factors, such as, inter alia, the patient's medical history, as well as the type (and / or dosage) of antibody used.

[0150] Effective amounts and schedules for administering the antibodies, antibody-encoding polynucleotides, and / or compositions disclosed herein can be determined empirically, and making such determinations is within the skill of one in the art. One of skill in the art will understand that the dosage required to be administered will vary depending, for example, on the mammal receiving the antibodies, antibody-encoding polynucleotides, and / or compositions disclosed herein, the route of administration, the particular type of antibody, antibody-encoding polynucleotide, antigen-binding fragment, and / or composition disclosed herein used, and other agents administered to the mammal. Guidance for selecting appropriate doses for antibodies or antigen-binding fragments can be found in literature on the therapeutic use of antibodies and antigen-binding fragments, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, 1985, ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York, 1977, pp. 365-389.

[0151] A typical daily dose of an effective amount of antibody is 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.

[0152] In any of the methods described herein, at least one antibody, antigen-binding fragment thereof, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, or pharmaceutical compositions described herein), and optionally at least one additional therapeutic agent, can be administered to a subject 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, at least one antibody or antigen-binding fragment and 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 a sustained release oral formulation.

[0153] In some embodiments, one or more additional therapeutic agents can be administered to a subject before or after administration of 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). In some embodiments, the one or more additional therapeutic agents and 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) are administered to a subject such that there is overlap in the period of biological activity of the one or more additional therapeutic agents with the period of biological activity of the at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) in the subject.

[0154] In some embodiments, a subject can be administered 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) for an extended period of time (e.g., for 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, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional can determine the length of treatment period using any of the methods described herein to diagnose or track the effectiveness of the treatment (e.g., by observing at least one symptom of the disease). As described herein, a skilled medical professional can also vary (e.g., increase or decrease) the identity and number of antibodies or antigen-binding antibody fragments (and / or one or more additional therapeutic agents) administered to a subject, and can adjust (e.g., increase or decrease) the dosage or frequency of administration of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) to a subject based on an evaluation of the effectiveness of the treatment (e.g., using any of the methods described herein and well known in the art).

[0155] In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agents can include one or more inhibitors selected from the group consisting of inhibitors of B-Raf, EGFR inhibitors, inhibitors of MEK, inhibitors of ERK, inhibitors of K-Ras, inhibitors of c-Met, inhibitors of anaplastic lymphoma kinase (ALK), inhibitors of phosphatidylinositol 3-kinase (PI3K), inhibitors of Akt, inhibitors of mTOR, dual PI3K / mTOR inhibitors, inhibitors of Bruton's tyrosine kinase (BTK), and inhibitors of isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2).

[0156] In some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of inhibitors of HER3, inhibitors of LSD1, inhibitors of MDM2, inhibitors of BCL2, inhibitors of CHK1, inhibitors of the activated hedgehog signaling pathway, and agents that selectively degrade the estrogen receptor.

[0157] In some embodiments, the additional therapeutic agent is trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, leolysin, Alimta, Dicaida, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, pazopanib, IMA-901, AGS-00 The present invention may include one or more therapeutic agents selected from the group consisting of 3, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, lenalidomide, bortezomib, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0158] In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a CX3CL1 targeted therapy, a CXCL9 targeted therapy, a CXCL10 targeted therapy, a CCL5 targeted therapy, an LFA-1 agonist, an ICAM1 agonist, and a selectin agonist.

[0159] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI is administered to the subject.

[0160] In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-4-1BB antibody, an anti-CTLA-4 antibody, an anti-CD40 antibody, an anti-OX40 antibody, or an anti-GITR antibody.

[0161] Pharmaceutical Compositions and Routes of Administration Also provided herein are pharmaceutical compositions containing at least one (e.g., 1, 2, 3, or 4) of the antibodies or antigen-binding fragments described herein. Two or more (e.g., 2, 3, or 4) of any of the antibodies or antigen-binding fragments described herein can be present in the pharmaceutical composition, in any combination. Pharmaceutical compositions can be formulated in any manner known in the art.

[0162] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). Compositions may contain a sterile diluent (e.g., sterile water or saline), fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antibacterial or antifungal agents (e.g., benzyl alcohol, methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffers (e.g., acetate, citrate, or phosphate), and isotonic agents (e.g., sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride)), or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Pat. No. 4,522,811). The composition preparation can be formulated and enclosed in ampoules, disposable syringes, or multiple-dose vials. Where necessary (e.g., in injectable formulations), proper fluidity can be maintained, for example, by the use of a coating such as lecithin or a surfactant. Absorption of the antibody or antigen-binding fragment thereof can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin). Alternatively, sustained release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

[0163] Compositions containing 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 amount of active compound(s) for ease of administration and uniformity of dosage).

[0164] The toxicity and therapeutic efficacy of a composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). 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, and the therapeutic index is the ratio of LD50:ED50. Drugs that exhibit a high therapeutic index are preferred. If a drug exhibits undesirable side effects, care should be taken to minimize the potential for harm (i.e., reduce the undesirable side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0165] Data obtained from cell culture assays and animal studies can be used in formulating an appropriate dose of any given agent for use in a subject (e.g., a human). A therapeutically effective amount of one or more (e.g., one, two, three, or four) antibodies or antigen-binding fragments thereof (e.g., any of the antibodies or antibody fragments described herein) is an amount that treats a disease in a subject, a subject, or a subject identified as being at risk for developing the disease, and reduces the severity, frequency, and / or duration of one or more symptoms of the disease in a subject (e.g., a human). The efficacy and administration of any of the antibodies or antigen-binding fragments described herein can be determined by a health care professional or veterinary professional using methods well known in the art, as well as by observing one or more symptoms of the disease in a subject (e.g., a human). Certain factors can affect the dose and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the subject's overall health and / or age, and the presence of other diseases).

[0166] Exemplary doses include amounts (milligrams or micrograms) of any of the antibodies or antigen-binding fragments described herein 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). While these doses cover a wide range, those of skill in the art will appreciate that the efficacy and effective amounts of therapeutic agents comprising antibodies and antigen-binding fragments thereof can be determined by methods well known in the art. Typically, a relatively low dose is administered initially, and the dose can be subsequently and gradually increased by the attending health care professional or veterinary professional (for therapeutic uses) or by a researcher (if still working in the development phase) until an appropriate response is obtained. In addition, it is 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 subject's age, weight, general health, sex, and diet, time of administration, route of administration, rate of excretion, and half-life of the antibody or antibody fragment in vivo.

[0167] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.The present disclosure also provides methods for producing antibodies or antigen-binding fragments thereof for the various uses described herein. [Example]

[0168] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0169] Example 1. Generation of human anti-TFR1 antibodies RenNano TMMice (Biocytogen, in situ fully human heavy chain variable domain replacement combined with modified constant region). For example, mice described in PCT / CN2022 / 119188, the entire contents of which are incorporated herein by reference, were immunized with His-tagged human TFR1 (transferrin receptor 1) protein (hTFR1-His, ACROBiosystems, catalog number: CD1-H5243) to obtain anti-TFR1 antibodies. Prior to immunization, retro-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 (every two weeks) were performed. One week after the third immunization, retro-orbital blood was collected, and serum antibody titers were detected by FACS.

[0170] A booster immunization procedure was also performed at least 14 days after the previous immunization: TFR1 protein was injected intraperitoneally, and CHO-S cells expressing human TFR1 antigen were injected via the tail vein.

[0171] Antigen-specific immune cells were isolated from immunized mice, and anti-TFR1 antibodies were obtained, or heavy chain variable region sequences of anti-TFR1 antibodies were obtained. For example, plasma cells secreting antigen-specific monoclonal antibodies were screened using single-cell technology (e.g., using the Beacon® Optofluidic System, Berkeley Lights Inc.). Antibody variable region sequences were obtained using reverse transcription and PCR sequencing. The obtained variable region sequences were used for antibody expression, and their binding activity to TFR1 was verified using FACS. Because the CH1 domain is missing, the heavy chain variable region (VH) of the obtained antibody is also called a heavy chain single variable domain (VHH).

[0172] Specifically, the resulting VHH sequences were each connected to a 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. The heavy chain CDR1-3 sequences are shown in Figures 1 and 2. The VHH region sequences of 23B8, 24A1, 24C9, and 24G5 are shown in Figure 3.

[0173] The constant region of the antibody can be further modified to replace the asparagine at position 297 with alanine (N297A). For example, if the N297A mutation is introduced into the constant region of 24G5, the resulting antibody is designated 24G5-N.

[0174] Furthermore, single-arm antibodies 23B8-mono, 24A1-mono, 24C9-mono, and 24G5-mono were constructed, which have an anti-TFR1 arm containing a VHH region and a heavy chain fragment containing the CH2 and CH3 domains of IgG1 with the N297A mutation.

[0175] Example 2. Cross-species binding of anti-TFR1 antibodies CHO-S-hTFR1 cells or CHO-S-fasTFR1 cells were cultured at 10 5 The cells were transferred to a 96-well plate at a density of 100 cells / well. Serially diluted sample anti-TFR1 antibodies were added to the 96-well plate and incubated at 4°C for 30 minutes. PBS was used as a negative control (NC). The cells were then incubated with a secondary antibody, anti-hIgG-Fc-Alex Flour, prior to flow cytometry analysis. TM 647 (Jackson ImmunoResearch Laboratories, Catalog No.: 109-606-170) in the dark at 4°C for 15 minutes.

[0176] 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 Macaca fascicularis (cynomolgus monkey) TFR1 amino acid sequence (fasTFR1, SEQ ID NO: 30), respectively. The test results are shown in the table below.

[0177] JR141, a humanized IgG1 antibody targeting human TFR1 conjugated to human iduronate-2-sulfatase, was the first approved for the intravenous treatment of mucopolysaccharidosis type II in Japan in March 2021. The VH and VL sequences of JR141 are shown in SEQ ID NO: 31 and SEQ ID NO: 32, respectively. In the positive control (JR141-N), the VH and VL of JR141 were ligated to a human IgG1 constant region with the N297A mutation.

[0178] [Table 1]

[0179] Example 3. Binding affinity of anti-TFR1 antibodies The binding affinity of anti-TFR1 antibodies to human (hTFR1-His, ACROBiosystems, Cat. No.: CD1-H5243) or monkey (fasTFR1-His, ACROBiosystems, Cat. No.: TFR-C524a) His-tagged TFR1 proteins was measured using a Biacore™ system equipped with a pre-immobilized Protein A sensor chip. TM (Biacore, Inc., Piscataway, NJ) Validation was performed using surface plasmon resonance (SPR) on an 8K biosensor.

[0180] Purified anti-TFR1 antibodies were captured on a Protein A chip (Series S SensorChip Protein A) for detection. Purified anti-TFR1 antibodies (1 μg / mL) were loaded at 10 μL / min to bind to hTFR1-His and fasTFR1-His (200 nM). The flow rate was 30 μL / min. The binding and dissociation times were set to 180 and 600 seconds, respectively. After the last injection of each titration solution, the chip was regenerated with glycine solution (pH 2.0) at 30 μL / min for 30 seconds.

[0181] Biacore TM The kinetic association rate (k) and dissociation rate (k) were obtained simultaneously by fitting the entire data set to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110) using 8K Evaluation software 3.0. The affinity was estimated from the quotient of the kinetic rate constants (K = k / k).

[0182] As will be understood by those skilled in the art, the same method was performed for each test antibody, adjusting parameters (e.g., antibody concentration) appropriately. The results for the test antibodies are summarized in the table below. The results show that all four anti-TFR1 antibodies can bind to human and monkey TFR1 with high affinity.

[0183] [Table 2]

[0184] Example 4. Epitope analysis of anti-TFR1 antibodies The relative location of target protein epitopes between pairs of purified anti-TFR1 antibodies was analyzed by biolayer interferometry (BLI) using a ForteBio Octet system at 30°C. 1x 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 (10x), was used as the running buffer throughout the experiment. Approximately 10 μg / mL hTFR1-His protein was captured with HIS1K (anti-PentaHIS) for 200 s, and 200 nM antibody (analyte 1) was injected at a flow rate of 30 μL / min to bind to the ligand. To determine whether the binding of different antibodies interfered with each other, another antibody (analyte 2) was injected under the same conditions. The binding time was 300 seconds for each antibody.

[0185] Binding values ​​for each antibody were obtained using Data Analysis HT 12.0. To quantify the interference of one antibody with another, binding ratios were calculated and compared for each pair of antibodies. The binding ratio was defined by dividing the binding value of the second antibody (sample 2) by the binding value of the first antibody (sample 1). The binding ratios for each antibody pair are summarized in the matrix table below. Specifically, if sample 1 exhibited an inhibitory effect on sample 2, the binding ratio was 0.0 to 0.5; if sample 1 did not exhibit a blocking effect on sample 2, the binding ratio was 0.5 to 1.1. Generally, antibody pairs that interfere with each other have identical or overlapping epitopes.

[0186] The results of the epitope binding assay indicate that 24A1 and 24G5 can recognize the same epitope, while 23B8, 24C9 and JR141-N can recognize different epitopes.

[0187] [Table 3]

[0188] Example 5. Internalization of anti-TFR1 antibodies Anti-TFR1 antibody and pHAb-goat anti-human IgG secondary antibody were added to human cortical microvascular endothelial cells (hCMEC / D3 cells) 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 endocytosis rate of the antibodies was calculated. Human IgG1 protein (CrownBio, catalog number: C0001) was used as an isotype control (ISO). The results are shown in the table below, which indicates that all four antibodies exhibited good endocytosis activity in human cortical microvascular endothelial cells.

[0189] [Table 4]

[0190] Example 6. Analysis of the development feasibility of anti-TFR1 antibodies The anti-TFR1 antibodies 23B8, 24A1, 24C9, and 24G5 were evaluated for development potential. The antibodies were diluted to 1 mg / mL in water. Specifically, the following tests were performed: (1) observing the appearance of the solution and the presence of visible insoluble material; (2) detecting changes in antibody purity by size-exclusion ultra-performance liquid chromatography (SEC-UPLC) (expressed as the percentage of the main peak area relative to the sum of all peak areas (purity, %)); (3) detecting changes in 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, minutes)); (4) detecting antibody charge variants by capillary isoelectric focusing (cIEF) (expressed as the percentage of the main, acidic, and alkaline components); and (5) detecting the thermal stability of the antibody using the UNcle system (expressed as the melting temperature (Tm) and aggregation temperature (Tag)).

[0191] For SEC-UPLC experiments, an Agilent 1290 chromatographic system (XBridge TMA Protein BEH SEC column (200 Å, 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; and run time: 7 min.

[0192] For HIC-HPLC experiments, an Agilent 1260 chromatographic system (ProPac TM A 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 min 100% A, 2 min 100% A, 32 min 100% B, 34 min 100% B, 35 min 100% A, and 45 min 100% A; column temperature: 30 °C; detection wavelength: 280 nm; injection volume: 10 μg; sample tray temperature: approximately 6 °C; and run time: 45 min.

[0193] For cIEF experiments, a Maurice cIEF Method Development Kit (Protein Simple, catalog number: PS-MDK01-C) was used 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). Imaging capillary isoelectric focusing spectra were generated using a Maurice cIEF cartridge (PS-MC02-C) on a Maurice analyzer (Protein Simple, Santa Clara, CA). The sample was focused for a total of 10 min. The absorbance of the proteins, focused at 280 nm, was analyzed using the analytical software installed on the instrument.

[0194] For thermal stability experiments, 60 mg / mL antibody solutions were heated from 25°C to 95°C in 1°C increments, with a 1 minute equilibration period before each measurement.

[0195] Furthermore, the antibody was diluted to 1 mg / mL using PBS buffer and the following tests were performed: (1) detection of antibody specificity using cross-interaction chromatography (CIC) method (shown as retention time (CIC, min)); (2) detection of antibody colloidal stability using stand-up single layer chromatography (SMAC) method (shown as retention time (SMAC, % / min)).

[0196] For the CIC assay, a CIC column was prepared by coupling human polyclonal IgG (Sigma, catalog number I4506) to HiTrap NHS-activated resin (GE Healthcare, catalog number 17-0716-01) and then passivating with ethanolamine according to published procedures. The column was then connected to an Agilent 1260 chromatographic system and run at 0.1 mL / min using 1x PBS as the mobile phase until a flat baseline was reached. 10 μg of antibody at 1 mg / mL in PBS was then injected. Peak retention times on the column were monitored at 280 nm with a run time of 50 min.

[0197] For the SMAC assay, connect a Zenix column (4.6 mm x 30 cm, Sepax, catalog number: 213300-4630) to the column compartment and place the appropriate lines in the mobile phase. Equilibrate the column with mobile phase buffer for 60 minutes at a flow rate of 0.350 mL / min. Load the antibody 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. Detailed results are summarized in the table below.

[0198] [Table 5]

[0199] For the Tagg of 24C9, slight fluctuations were observed at 33.63°C from the Tagg curve (data not shown), while significant aggregation occurred at 50-60°C. To confirm thermal stability, SEC-UPLC of 24C9 was examined after treatment at 40°C for 14 days. After heating, the purity of 24C9 remained above 97%. These results indicate that all four antibodies have good development potential.

[0200] Example 7. Pharmacokinetic (PK) Analysis A 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 with the corresponding human TFR1 extracellular domain. A detailed description of the humanized TFR1 mouse model can be found in PCT Application No. PCT / CN2022 / 105924, which is incorporated herein by reference in its entirety.

[0201] The concentration of anti-TFR1 antibodies was determined in hTFR1 mice. Specifically, mice were placed in different groups (8 mice per group) and administered approximately equimolar amounts of JR141-N (G2), 23B8-N (G3), 24A1-N (G4), 24G5-N (G5), or 24C9-N (G6) intravenously (iv). Control group (G1) mice were administered human IgG1 (hIgG1). The details of the administration scheme are shown in the table below.

[0202] [Table 6]

[0203] Blood and brain samples were collected 0.5, 6, 24, and 72 hours after administration. Two mice were sampled at each time point. After retro-orbital blood collection, the mice were anesthetized. To avoid interference from residual blood in the brain, the mice were perfused with saline for 10 minutes at room temperature. Specifically, saline was perfused from the left ventricle to the right ventricle via the systemic circulation. Brain samples were excised and divided into two hemibrains along the sagittal plane. The left hemibrain was subjected to quantification of the injected antibodies, while the right hemibrain was fixed in formalin and embedded in paraffin for serial sectioning. Brain samples were minced and homogenized in Dulbecco's phosphate-buffered saline (DPBS) containing 1x mixed protease inhibitors. Aliquots of the brain homogenate were used for protein extraction, followed by antibody quantification using electrochemiluminescence. The remaining homogenate was subjected to gradient density centrifugation at 5400 g for 15 minutes using 15% dextran to remove capillaries. After centrifugation, the upper fraction of the centrifuge tube was saved as the parenchyma and subjected to protein extraction and antibody quantification. Figures 4A-4D show the antibody concentration in total brain protein (Figure 4A), the ratio of antibody concentration in total brain protein to serum antibody concentration (Figure 4B), the antibody concentration in the brain parenchyma (Figure 4C), and the ratio of antibody concentration in the brain parenchyma to serum antibody concentration at each time point (Figure 4D). These results demonstrated that 24G5-N (group G5) was most abundant in either the parenchyma or the whole brain.

[0204] In a similar experiment, hTFR1 mice were divided into five groups (three mice per group) and 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) intravenously (one total dose). Control group (G1) mice were administered hIgG1 (G1). Brain samples were collected 24 h after administration, and the concentration of anti-TFR1 antibodies was determined. Figures 5A-5B show the results of antibody concentration tests in the brain parenchyma and total brain protein, respectively. All tested antibodies showed higher concentrations in the brain than hIgG1 (G1), and compared to the positive control JR141-N (G2), 23B8-N (G3) and 24G5-N (G5) showed better ability to cross the blood-brain barrier and enter the brain parenchyma.

[0205] In another similar experiment, hTFR1 mice were divided into seven groups (six mice per group) and administered JR141-N (G2-G4) or 24G5-N (G5-G7) intravenously (iv). Control group (G1) mice were administered hIgG1. The details of the administration scheme are shown in the table below.

[0206] [Table 7]

[0207] Six and 24 hours after administration, blood and brain samples were collected using the methods described above. Three mice were sampled at each time point. Tissue processing and antibody quantification were also performed as described above. Measurement results of humanized anti-TFR1 antibody concentrations in the brain parenchyma are shown in Figure 6. The results show that under each administration condition, the antibody concentrations accumulated in the brain parenchyma of 24G5-N were significantly higher than those of hIgG1. Furthermore, the concentrations of both JR141-N and 24G5-N showed a dose-dependent trend in the brain parenchyma.

[0208] To detect the distribution of the humanized anti-TFR1 antibody 24G5-N in the mouse brain, we performed immunofluorescence assays by staining for hIgG, hTFR1, and mCD31, respectively, on right hemibrain sections from the mice used in the above experiments. The results showed that mCD31 was well labeled in microvessels. hTFR1 was also detected in microvessels and colocalized with mCD31. Furthermore, hTFR1 expression was also detected in some neurons within the parenchyma. Notably, the anti-TFR1 antibody 24G5-N was stained with a secondary anti-IgG antibody conjugated with DyLight® 488. Similar to hTFR1, 24G5-N was detected in microvessels and the parenchyma, and its signal overlapped with the hTFR1 signal. Therefore, whether quantifying 24G5-N in the whole brain or parenchyma, or visually demonstrating immunofluorescence of 24G5-N in the parenchyma, the results indicate that the anti-TFR1 antibody 24G5-N can efficiently cross the blood-brain barrier (BBB).

[0209] Example 8. Blocking assay Blocking of TFR1 binding to TF (transferrin) by anti-TFR1 antibodies 23B8, 24A1, 24C9, and 24G5 was tested by biolayer interferometry (BLI) using a ForteBio Octet® system 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 throughout the experiment. Approximately 10 μg / mL of antibody was captured with AHC (anti-human IgG Fc capture) for 200 seconds, and 800 nM of hTFR1-His (ACROBiosystems, catalog number: CD1-H5243) and hTF-His (human transferrin protein, Kactus Biosystems, catalog number: TFN-HM101) were injected to allow binding to the ligands. The binding time was 300 seconds for each antibody. Binding values ​​for each antibody were obtained using Data Analysis HT 12.0. The results showed that these four antibodies did not block TFR1 binding to TF. Therefore, such non-blocking antibodies are unlikely to interfere with TFR1-TF interaction in normal cells.

[0210] Example 9. Antibody Drug Conjugates (ADCs) Each purified antibody (23B8-N, 24A1-N, 24C9-N, 24G5-N, 23B8-mono, 24A1-mono, 24C9-mono, and 24G5-mono) was coupled to Dxd (deruxtecan) via a GGFG linker.

[0211] The antibody-drug conjugate is named by adding "ADC" immediately after the antibody name. For example, when 24G5-N is coupled to GGFG-Dxd, it is named 24G5-ADC. As another example, when 24G5-mono is coupled to GGFG-Dxd, it is named 24G5-mono-ADC. The binding of the antibody to the drug molecule was detected using HIC-HPLC (reverse-phase high-performance liquid chromatography). The HIC-HPLC detection results showed that the drug-antibody ratio (DAR) of the ADC was approximately 3.

[0212] hTFR1 mice were placed in 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 an equimolar dose of 8.34 mg / kg of one-arm monovalent anti-TFR1 ADCs (23B8-mono-ADC, 24A1-mono-ADC, 24C9-mono-ADC, or 24G5-mono-ADC) via intravenous (iv) injection (total of one dose). Blood and brain samples were collected 0.5, 18, and 72 hours after administration using the methods described in Example 7. Tissue processing and antibody and ADC quantification were also performed as described above. The results indicate that all tested ADCs can cross the BBB and efficiently induce Dxd. Exemplary results are shown in Figure 7, where 24G5-ADC and 24G5-mono-ADC cross the BBB to provide Dxd, and among these, the monovalent 24G5-mono-ADC has better penetration effect than the bivalent format 24G5-ADC.

[0213] Other embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the above description is intended to be illustrative and not limiting of the scope of the invention, which is defined by 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), a heavy chain single variable domain (VHH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VHH CDR1 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of a selected VHH CDR1; the VHH CDR2 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of a selected VHH CDR2; and the VHH CDR3 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of a selected VHH CDR3; The amino acid sequences of the selected VHH CDR1, 2, and 3 are one of the following: (1) The amino acid sequences of the selected VHH CDR1, 2, and 3 are shown in SEQ ID NOs: 1, 2, and 3, respectively; (2) The amino acid sequences of the selected VHH CDR1, 2, and 3 are shown in SEQ ID NOs: 4, 5, and 6, respectively; (3) The amino acid sequences of the selected VHH CDR1, 2, and 3 are shown in SEQ ID NOs: 7, 8, and 9, respectively; (4) The amino acid sequences of the selected VHH CDR1, 2, and 3 are shown in SEQ ID NOs: 10, 11, and 12, respectively; (5) The amino acid sequences of the selected VHH CDR1, 2, and 3 are set forth in SEQ ID NOs: 13, 14, and 15, respectively; (6) The amino acid sequences of the selected VHH CDR1, 2, and 3 are set forth in SEQ ID NOs: 16, 17, and 18, respectively; (7) The amino acid sequences of the selected VHH CDR1, 2, and 3 are set forth in SEQ ID NOs: 19, 20, and 21, respectively; and (8) The amino acid sequences of the selected VHH CDR1, 2, and 3 are shown in SEQ ID NOs: 22, 23, and 24, respectively.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the VHH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the VHH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the VHH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9, respectively.

5. The antibody or antigen-binding fragment thereof of claim 1, wherein the VHH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 10, 11, and 12, respectively.

6. An antibody or antigen-binding fragment thereof that binds to TFR1, comprising a heavy chain single variable region (VHH) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a selected VHH sequence, wherein the selected VHH sequence is selected from the group consisting of SEQ ID NOs: 25, 26, 27, and 28.

7. The antibody or antigen-binding fragment thereof of claim 6, wherein the VHH comprises the sequence of SEQ ID NO:

25.

8. The antibody or antigen-binding fragment thereof of claim 6, wherein the VHH comprises the sequence of SEQ ID NO:

26.

9. The antibody or antigen-binding fragment thereof of claim 6, wherein the VHH comprises the sequence of SEQ ID NO:

27.

10. The antibody or antigen-binding fragment thereof of claim 6, wherein the VHH comprises the sequence of SEQ ID NO:

28.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the 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 to 11, wherein the antibody or antigen-binding fragment is a human antibody or a humanized antibody or an antigen-binding fragment thereof.

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

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

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

16. The antibody or antigen-binding fragment thereof of claim 15, wherein the 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 of any one of claims 1 to 16, wherein the antibody or antigen-binding fragment comprises two or more heavy chain single variable domains.

18. A nucleic acid comprising a polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 17.

19. 19. The nucleic acid of claim 18, wherein the nucleic acid is a cDNA.

20. 20. A vector comprising one or more of the nucleic acids of claim 18 or 19.

21. A cell comprising the vector of claim 20.

22. 22. The cell of claim 21, wherein the cell is a CHO cell.

23. 20. A cell comprising one or more of the nucleic acids of claim 18 or 19.

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

25. 18. An antibody drug conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 17 covalently attached to a therapeutic agent.

26. 26. The antibody drug conjugate of claim 25, wherein the therapeutic agent is a cytotoxic or cytostatic agent.

27. 27. A method of treating a subject having 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 antigen-binding fragment thereof of any one of claims 1 to 17, or the antibody-drug conjugate of claim 25 or 26.

28. 28. The method of claim 27, wherein the antibody or antigen-binding fragment thereof, or the antibody-drug conjugate is capable of crossing the blood-brain barrier (BBB) ​​of the subject.

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

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

31. 1. A method for identifying a subject as having a brain disease (e.g., brain cancer), the method comprising: detecting a sample collected from the subject as having the brain disease using the antibody or antigen-binding fragment thereof of any one of claims 1 to 17; thereby identifying the subject as having the brain disorder; Including, method.

32. 32. The method of claim 31 , wherein the sample is a brain parenchyma sample from the subject.

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

34. 1. A method of delivering a drug across the blood-brain barrier, said method comprising: Administering to a subject the agent covalently bound to the antibody or antigen-binding fragment thereof of any one of claims 1 to 17; Including, method.

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

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

37. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 and a pharmaceutically acceptable carrier.

38. 27. A pharmaceutical composition comprising the antibody-drug conjugate of claim 25 or 26 and a pharmaceutically acceptable carrier.

39. An antibody or an antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 17.

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