ANTI-TfR1 ANTIBODIES, ANTIGEN-BINDING FRAGMENTS THEREOF OR HUMANIZED

Abstract

The invention discloses an anti-transferrin receptor (TfR1) antibody or an antigen binding fragment thereof. The preparation method comprises the following steps: carrying out phage immunization library-building screening by using a rabbit anti-technology to obtain an anti-TfR1 monoclonal antibody with high affinity, high specificity and high blood safety, and carrying out humanization and affinity maturation treatment on the anti-TfR1 monoclonal antibody to obtain the humanized anti-TfR1 monoclonal antibody which has high affinity and does not cause immunogenicity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to an anti-TfR1 antibody, an antigen binding fragment thereof or a humanized antibody thereof, and uses thereof. BACKGROUND

[0002] Transferrin receptor (TfR) is a type II transmembrane glycoprotein and a glycoprotein expressed on the cell surface, which is cross-linked by disulfide bond by two homodimeric subunits of about 90 kDa in size. TfR is composed of 760 amino acids and participates in the transport of iron ions in the form of a dimer (180 kDa). Each subunit contains an extracellular C-terminal region, a single transmembrane region and a short N-terminal region. The C-terminal region consists of 671 amino acids, which serves as an external functional region and contains a site for binding to transferrin (Tf). The transmembrane domain consists of 28 amino acids, and the intracellular N-terminal domain consists of 61 amino acids. The extracellular domain of TfR1 is composed of three subdomains: apical region (A), helical region (H) and protease-like region (P). Two types of transferrin receptors are known in humans: transferrin receptor 1 (TfR1, also known as CD71) and transferrin receptor 2 (TfR2). The former is mainly expressed on cancer cells, muscle cells and endothelial cells of the blood-brain barrier, and the latter is mainly expressed on hepatocytes.

[0003] The most important physiological function of TfR1 is to bind to transferrin (Tf) and mediate cellular uptake of iron through endocytosis. Tf is composed of a polypeptide chain of 679 amino acids and two sugar chains. It is a 80 kDa glycoprotein composed of two 40 kDa subunits (called N leaf and C leaf) separated by a short linker sequence. Each subunit can bind one free trivalent iron (Fe 3+ ), so Tf can bind up to two iron atoms. The apo-Tf form of Tf (apo-Tf) binds Fe 3+ efficiently in the blood and is transported to the cell surface for internalization by interacting with TfR1. Therefore, the Tf-TfR1 system is considered an important pathway for the body to obtain iron ions. Specifically, Tf and iron ions (Fe 3+ or Fe 2+After binding, the spatial structure of TfR1 changes accordingly to form holo-Tf. Secondly, TfR1 binds to holo-Tf at physiological pH to form a Tf-TfR1 complex, which is endocytosed by a clathrin-dependent pathway. In the cell, the Tf-TfR1 complex is acidified in the endosome, causing a conformational change that facilitates the release of Fe 3+ The apo-Tf is recycled to the cell surface by Golgi complex exocytosis to complete the iron ion transport. At the cell surface, Tf and TfR1 are separated, and apo-Tf recombines with extracellular Fe 3+ to become holo-Tf for the next iron transport cycle.

[0004] Iron ions are essential for cell growth and metabolism, and TfR1 is highly expressed in various healthy tissues such as muscle and vascular endothelium, as well as in tumors. Therefore, ADCs targeting TfR1 can be designed to achieve enrichment of anticancer drugs in tumors and inhibit tumor growth. For example, CytomX's TfR1-targeting Probody-ADC (CX-2029) (Clin Cancer Res (2021) 27(16): 4521-4530). CX-2029 is hydrolyzed by proteases in the tumor microenvironment, exposing the TfR1 binding site, internalized by tumor cells, and finally releasing MMAE to exert cytotoxicity. On the other hand, designing TfR1 antibodies targeting exclusive epitopes as delivery systems can achieve drug delivery to TfR1 highly expressed healthy tissues without affecting iron ion absorption, treating related diseases. For example, Avidity's product AOC 1001 targets type 1 myotonic dystrophy (DM1), which consists of three parts: a full-length monoclonal antibody targeting TfR1, a chemical linker, and an siRNA targeting DMPK mRNA. AOC 1001 uses TfR1 antibodies to deliver therapeutic siRNA to muscle, which is a major breakthrough in the extracorporeal application of siRNA drugs. SUMMARY

[0005] To solve the above problems, the present application uses rabbit anti-technology to screen for anti-TfR1 monoclonal antibodies with high affinity, high specificity, and high blood safety by phage immunization library screening, and to humanize and affinity mature the anti-TfR1 monoclonal antibodies to obtain a humanized anti-TfR1 monoclonal antibody with high affinity and no immunogenicity.

[0006] The present application provides in a first aspect an anti-transferrin receptor (TfRl) antibody or antigen binding fragment thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises: a VH CDR1 comprising SEQ ID NO: 001 or a sequence at least 80% homologous to SEQ ID NO: 001; a VH CDR2 comprising SEQ ID NO: 004 or a sequence at least 80% homologous to SEQ ID NO: 004; a VH CDR3 comprising SEQ ID NO: 007 or a sequence at least 80% homologous to SEQ ID NO: 007; and the VL region comprises: a VL CDR1 comprising SEQ ID NO: 010 or a sequence at least 80% homologous to SEQ ID NO: 010; a VL CDR2 comprising SEQ ID NO: 013 or a sequence at least 80% homologous to SEQ ID NO: 013; a VL CDR3 comprising SEQ ID NO: 016 or a sequence at least 80% homologous to SEQ ID NO: 016;

[0007] or the VH region comprises: a VH CDR1 comprising SEQ ID NO: 002 or a sequence at least 80% homologous to SEQ ID NO: 002; a VH CDR2 comprising SEQ ID NO: 005 or a sequence at least 80% homologous to SEQ ID NO: 005; a VH CDR3 comprising SEQ ID NO: 008 or a sequence at least 80% homologous to SEQ ID NO: 008; and the VL region comprises: a VL CDR1 comprising SEQ ID NO: 011 or a sequence at least 80% homologous to SEQ ID NO: 011; a VL CDR2 comprising SEQ ID NO: 014 or a sequence at least 80% homologous to SEQ ID NO: 014; a VL CDR3 comprising SEQ ID NO: 017 or a sequence at least 80% homologous to SEQ ID NO: 017;

[0008] or, the VH region comprises: a VH CDR1 comprising SEQ ID NO: 003 or a sequence at least 80% homologous to SEQ ID NO: 003; a VH CDR2 comprising SEQ ID NO: 006 or a sequence at least 80% homologous to SEQ ID NO: 006; a VH CDR3 comprising SEQ ID NO: 009 or a sequence at least 80% homologous to SEQ ID NO: 009; and the VL region comprises: a VL CDR1 comprising SEQ ID NO: 012 or a sequence at least 80% homologous to SEQ ID NO: 012; a VL CDR2 comprising SEQ ID NO: 015 or a sequence at least 80% homologous to SEQ ID NO: 015; a VL CDR3 comprising SEQ ID NO: 018 or a sequence at least 80% homologous to SEQ ID NO: 018.

[0009] Further, the anti-TfRl antibody or antigen binding fragment thereof specifically binds to TfRl but not to TfR2, preferably, specifically binds to human TfRl, cynomolgus TfRl, but not to mouse TfRl.

[0010] Further, the anti-TfRl antibody or antigen binding fragment thereof does not inhibit the binding of transferrin (Tf) to TfRl. (The anti-TfRl antibody or antigen binding fragment thereof has no significant effect on the binding of transferrin (Tf) and TfRl at a given drug concentration.)

[0011] Further, the anti-TfRl antibody or antigen binding fragment thereof is a Fab, Fab', F(ab'), chemically linked F(ab'), monospecific Fab, bispecific Fab, trispecific Fab, monovalent IgG, scFv (single chain variable fragment), di-scFv (bivalent scFv), bispecific diabody, trispecific triabody, scFv-Fc, minibody, or sdAb (single domain antibody).

[0012] Further, the anti-TfRl antibody or antigen binding fragment thereof binds to TfRl with a KD in the sub-nanomolar range, preferably, the anti-TfRl antibody or antigen binding fragment thereof binds to TfRl with a KD lower than 1 nM.

[0013] Further, the anti-TfRl antibody or antigen binding fragment thereof further comprises a heavy chain constant region (CH) or a portion thereof, preferably, the CH is from human IgGl, preferably, the CH is a mutated human IgGl, preferably, the mutation is a LALA mutation and a LR mutation, the amino acid sequence of the CH is as set forth in SEQ ID NO: 170.

[0014] Further, the anti-TfRl antibody or antigen-binding fragment thereof further comprises a light chain constant region (CL), preferably, the CL is a kappa light chain, lambda light chain or kappa light chain, preferably, the amino acid sequence of the light chain constant region is as set forth in SEQ ID NO: 171.

[0015] The present application provides a humanized anti-transferrin receptor (TfRl) antibody or antigen-binding fragment thereof, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VL region independently has 1 or more amino acid substitutions or mutations in at least one CDR relative to SEQ ID NO: 197;

[0016] and / or, the VH region independently has 1 or more amino acid substitutions or mutations in at least one CDR relative to SEQ ID NO: 179;

[0017] The humanized anti-TfRl antibody or antigen-binding fragment thereof has at least the same affinity for the receptor TfRl as the anti-TfRl antibody or antigen-binding fragment thereof described above.

[0018] Further, the amino acid substitutions or mutations in the CDRs of the VL are at positions 32, 33, 50, 53 and 91 of SEQ ID NO: 197, and the corresponding amino acids are substituted as follows: 32Y, 32F, 32W, 33R, 50R, 53R, 91V, 91I.

[0019] Further, the amino acid substitutions or mutations in the CDRs of the VH are at positions 32, 54 and 101 of SEQ ID NO: 179, and the corresponding amino acids are substituted as follows: 32W, 54F, 101Y, 101W.

[0020] Further, the VH comprises a VH CDR1 comprising SEQ ID NO: 001 or a sequence having at least 80% homology to SEQ ID NO: 001; a VH CDR2 comprising SEQ ID NO: 004 or a sequence having at least 80% homology to SEQ ID NO: 004; and a VH CDR3 comprising SEQ ID NO: 007 or a sequence having at least 80% homology to SEQ ID NO: 007; and the VL region comprises a VL CDR1 comprising SEQ ID NO: 072 or a sequence having at least 80% homology to SEQ ID NO: 072; a VL CDR2 comprising SEQ ID NO: 080 or a sequence having at least 80% homology to SEQ ID NO: 080; and a VL CDR3 comprising SEQ ID NO: 081 or a sequence having at least 80% homology to SEQ ID NO: 081.

[0021] or, the VH comprises: a VH CDR1 comprising SEQ ID NO: 001 or a sequence at least 80% homologous to SEQ ID NO: 001; a VH CDR2 comprising SEQ ID NO: 004 or a sequence at least 80% homologous to SEQ ID NO: 004; a VH CDR3 comprising SEQ ID NO: 070 or a sequence at least 80% homologous to SEQ ID NO: 070; and the VL region comprises: a VL CDR1 comprising SEQ ID NO: 073 or a sequence at least 80% homologous to SEQ ID NO: 073; a VL CDR2 sequence comprising SEQ ID NO: 080 or a sequence at least 80% homologous to SEQ ID NO: 080; a VL CDR3 sequence comprising SEQ ID NO: 081 or a sequence at least 80% homologous to SEQ ID NO: 081;

[0022] or, the VH comprises: a VH CDR1 comprising SEQ ID NO: 001 or a sequence at least 80% homologous to SEQ ID NO: 001; a VH CDR2 comprising SEQ ID NO: 004 or a sequence at least 80% homologous to SEQ ID NO: 004; a VH CDR3 comprising SEQ ID NO: 007 or a sequence at least 80% homologous to SEQ ID NO: 007; and the VL region comprises: a VL CDR1 comprising SEQ ID NO: 071 or a sequence at least 80% homologous to SEQ ID NO: 071; a VL CDR2 sequence comprising SEQ ID NO: 078 or a sequence at least 80% homologous to SEQ ID NO: 078; a VL CDR3 sequence comprising SEQ ID NO: 081 or a sequence at least 80% homologous to SEQ ID NO: 081.

[0023] Further, the VH region comprises a sequence comprising SEQ ID NO: 179 and the VL region comprises a sequence comprising SEQ ID NO: 212;

[0024] or, the VH region comprises a sequence comprising SEQ ID NO: 183 and the VL region comprises a sequence comprising SEQ ID NO: 200;

[0025] or, the VH region comprises a sequence comprising SEQ ID NO: 179 and the VL region comprises a sequence comprising SEQ ID NO: 209.

[0026] Further, the humanized anti-TfRl antibody or antigen binding fragment thereof has mutations in the VL CDR1, VL CDR2, VH CDR1 and VH CDR3 regions.

[0027] Further, the amino acid sequence of the VL CDR1 is selected from any one of SEQ ID NOs: 010, 095-127 and 132-147;

[0028] and / or the amino acid sequence of the VL CDR2 is selected from any one of SEQ ID NOs: 013, 128-131 and 148-150;

[0029] and / or the amino acid sequence of the VH CDR1 is selected from any one of SEQ ID NOs: 001, 083-091 and 152-159;

[0030] and / or the amino acid sequence of the VH CDR3 is selected from any one of SEQ ID NOs: 007, 092-094, 160 and 161.

[0031] Further, the amino acid sequences of the VL CDR1, VL CDR2, VH CDR1 and VH CDR3 regions are shown in Table 1 below:

[0032] Table 1

[0033]

[0034] The sequence of the VL CDR1 of the antibody of Sample ID NO. 30001-30012 is SEQ ID NO: 010, the sequence of the VL CDR2 is SEQ ID NO: 013, and the sequence of the VL CDR3 is SEQ ID NO: 016;

[0035] and / or as shown in Table 2:

[0036] Table 2

[0037]

[0038] The sequence of the VH CDR1 of the antibody of Sample ID NO. 30013-30049 is SEQ ID NO: 001, the sequence of the VH CDR2 is SEQ ID NO: 004, and the sequence of the VH CDR3 is SEQ ID NO: 007; and / or as shown in Table 3:

[0039] Table 3

[0040]

[0041]

[0042] The sequence of the VH CDR2 of the antibody with the sample ID NO. 30050-30097 is SEQ ID NO: 004, and the sequence of the VL CDR3 is SEQ ID NO: 016.

[0043] The third aspect of the present application provides a method for preparing the humanized anti-TfR1 antibody or the antigen binding fragment thereof as described above, comprising the following steps:

[0044] S1. Searching the sequence against the IgBLAST database to determine the best template for the Fv fragment. Selecting a best template for VH / VL matching the human framework sequence;

[0045] S2. Designing a humanized antibody using CDR grafting, grafting the CDR of the parent antibody into the human framework sequence to obtain a chimeric antibody, while comparing the sequence of the chimeric antibody and the parent antibody in the CDR, residues, stem loop structure, and hydrophobic core;

[0046] S3. Designing to gradually incorporate one or more reverse mutations in the grafted antibody sequence, and selecting antibodies with corresponding mutation combinations for affinity maturation according to the affinity detection results and the number of accumulated mutations;

[0047] S4. Based on the selected antibodies with corresponding mutation combinations, obtaining antibodies with comparable affinity to the positive control antibody by single-point saturation mutation of the CDR region;

[0048] S5. Based on the results of single-point mutation, further improving the affinity by mutation combination;

[0049] S6. Based on the selected antibodies with corresponding mutation combinations, improving the affinity of the monoclonal antibody by yeast (Sc) display platform technology and block mutation method, so as to obtain antibodies with comparable affinity to the positive control antibody.

[0050] Further, the step S5 comprises the following steps:

[0051] 1) Designing a special primer, and using PCR method to mutate several amino acids (1-5) in the VH and VL CDR regions of the selected antibodies with corresponding mutation combinations, which can introduce all 20 amino acid combinations at each amino acid position;

[0052] 2) Enriching high-affinity clones in the library by 2-3 rounds of target protein FACS sorting, and selecting 48 clones for sequencing each round to monitor the enrichment of wild type.

[0053] 3) After sorting, the yeast clones are selected and single concentration of target protein FACS experiment is carried out for verification, and the clones with improved FACS signal value are sequenced;

[0054] 4) 10 polypeptide clones with different sequences are selected, and gradient dilution of target protein FACS experiment is carried out for sorting.

[0055] The fourth aspect of the present application provides a nucleic acid molecule for encoding the anti-TfR1 antibody or the antigen binding fragment thereof or the humanized anti-TfR1 antibody or the antigen binding fragment thereof as described above.

[0056] The fifth aspect of the present application provides an expression vector comprising the nucleic acid molecule as described above.

[0057] The sixth aspect of the present application provides a host cell comprising the nucleic acid molecule as described above or the expression vector as described above.

[0058] The seventh aspect of the present application provides a conjugate comprising a humanized TfR1 antibody or an antigen binding fragment thereof as described above covalently linked to an active component for targeting the active component to muscle cells or mediating the transport of the active component across the blood-brain barrier, preferably, the active component is an oligonucleotide, preferably, the active component is siRNA.

[0059] The eighth aspect of the present application provides a pharmaceutical composition comprising the conjugate as described above and optionally a pharmaceutically acceptable adjuvant.

[0060] The ninth aspect of the present application provides the use of the humanized anti-TfR1 antibody or the antigen binding fragment thereof as described above or the conjugate as described above or the pharmaceutical composition as described above in the preparation of a medicament for treating tumor, muscle and central nervous system related diseases.

[0061] The tenth aspect of the present application provides the use of the anti-TfR1 antibody or the antigen binding fragment thereof as described above or the humanized anti-TfR1 antibody or the antigen binding fragment thereof as described above in the preparation of a kit for detecting the presence or level of TfR1 in a sample.

[0062] Compared with the prior art, the present application has the following beneficial effects:

[0063] The application utilizes rabbit anti-technology, and the anti-TfR1 monoclonal antibody with high affinity, high specificity and high blood safety is obtained by phage immunization library screening, and the anti-TfR1 monoclonal antibody is humanized and affinity matured (single point mutation, combination of single point mutation, yeast display technology and block mutation method), so that a humanized anti-TfR1 monoclonal antibody with high affinity and without immunogenicity is obtained. BRIEF DESCRIPTION OF DRAWINGS

[0064] In order to more clearly illustrate the technical solutions of the embodiments of the application, the following will briefly introduce the drawings needed to be used in the description of the embodiments of the application. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0065] Figure 1 Flow chart for screening of anti-human TfR1 antibody;

[0066] Figure 2 IC50 results of competitive experiment of three preferred antibodies;

[0067] Figure 3 First round library construction - first round FACS panning results;

[0068] Figure 4 First round library construction - second round FACS panning results;

[0069] Figure 5 First round library construction - third round FACS panning results;

[0070] Figure 6 Second round library construction - first round FACS panning results;

[0071] Figure 7 Second round library construction - second round FACS panning results;

[0072] Figure 8 KD-FACS results;

[0073] Figure 9 Effect diagram of knockdown of candidate antibody siRNA conjugate in RD cells. DETAILED DESCRIPTION

[0074] In order to make the technical problems, technical solutions and beneficial effects to be solved by the application more clear and obvious, the application will be further described in detail in combination with embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the application, and are not used to limit the application.

[0075] TERMS DEFINITION

[0076] Other aspects of the disclosure are provided below, including a description defining terms. Unless otherwise indicated or defined, all terms used have the ordinary meaning that would be understood by one of skill in the art.

[0077] The term "antibody" also known as immunoglobulin, refers to a polypeptide comprising at least one immunoglobulin variable domain or at least one antigenic determinant. In some embodiments, the immunoglobulin has a symmetrical structure of four polypeptide chains, two heavy (H) chains and two light (L) chains, linked by disulfide bonds and noncovalent bonds to form a monomeric molecule of four polypeptide chains. There are two types of light chains, kappa and lambda, and five types of heavy chains, mu, delta, gamma, epsilon, and alpha. In some embodiments, the immunoglobulin has no light chains and is composed of two heavy chains. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody includes a humanized antibody or antigen-binding fragment thereof or a chimeric antibody or antigen-binding fragment thereof. In some embodiments, the antibody includes a multispecific antibody or antigen-binding fragment thereof. In some embodiments, the antibody includes a bispecific antibody or antigen-binding fragment thereof. In some embodiments, the antibody includes full-length antibodies, individual chains thereof, and all portions, domains, or fragments thereof, including but not limited to antigen-binding domains or fragments, e.g., VHH domains or VH / VL domains, respectively.

[0078] The term "sequence" (e.g., in the terms "immunoglobulin sequence," "antibody sequence," "single variable domain sequence," "FR sequence," "CDR sequence," "VH sequence," "VL sequence," or "protein sequence," etc.), unless otherwise specifically limited, refers herein to both the relevant amino acid sequence as well as the nucleic acid sequence or nucleotide sequence encoding the sequence.

[0079] The term "chimeric antibody" refers to an antibody comprising heavy and light chain variable region sequences from one species and constant region sequences from another species, such as an antibody having rabbit heavy and light chain variable regions linked to human constant regions.

[0080] The term "humanized antibody" refers to an antibody comprising heavy and light chain variable region sequences from a non-human species (e.g., mouse, rabbit) but in which at least a portion of the VH and / or VL sequences are replaced with human sequences by genetic engineering. In some embodiments, it refers to the constant region portion of the antibody (i.e., CH and CL regions) or all of the antibody is encoded by human antibody genes. In some embodiments, humanized anti-TfR antibodies and antigen-binding portions are provided.

[0081] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes) of the antigen, each monoclonal antibody is directed against a single determinant on the antigen.

[0082] The term "CDR" refers to the antibody complementarity determining region or complementarity determining site, located in the variable region of immunoglobulins, which is complementary to the structure of an antigenic determinant. A typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which generally participate in antigen binding. In some embodiments, "immunoglobulin variable domain" refers to an immunoglobulin domain composed of four "framework regions" (FRs), FR1, FR2, FR3, and FR4, which are relatively conserved in sequence, and three "complementarity determining regions" (CDRs), CDR1, CDR2, and CDR3, which are relatively hypervariable in sequence, interspersed with the FRs. That is, each VH and VL is typically composed of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxy-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The location of the framework and complementarity determining regions can be identified using methods known in the art, such as by the definitions of Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)), IMGT definition, Chothia definition, etc. In some embodiments, complementarity determining region can refer to the CDR region defined by any of the definition methods.

[0083] The term "framework region" or "framework sequence" refers to the relatively conserved regions of an immunoglobulin variable region that are flanking the complementarity determining regions. There are four framework regions in each of the heavy and light chains, designated FR1, FR2, FR3, and FR4. Each VH and VL is typically composed of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxy-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Human heavy and light chain acceptor sequences are known in the art. In one embodiment, the acceptor sequences known in the art can be used for the antibodies disclosed herein.

[0084] The term "conservative amino acid substitution" also known as conservative mutation or conservative substitution, is a substitution in a protein in which a particular amino acid is changed to another amino acid with similar biochemical properties (charge, hydrophobicity, size, etc.). A substitution or group substitution is an amino acid substitution of a first amino acid with a final amino acid having different physicochemical properties. Variants can be prepared according to methods known to those of ordinary skill in the art for altering polypeptide sequences (Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2012). Conservative substitutions of amino acids include substitutions among amino acids within the following groups: (1) M, I, L, V; (2) F, Y, W; (3) K, R, H; (4) A, G; (5) S, T; (6) E, D.

[0085] The term "covalently linked" refers to two or more atoms interacting with one another using the form of a covalent bond. In some embodiments, two molecules can be covalently linked together through a single bond, e.g., a disulfide bond. However, in some embodiments, two or more molecules can be covalently linked together through multiple covalent bonds. In some embodiments, the linker can be a cleavable linker. However, in some embodiments, the linker can be a non-cleavable linker.

[0086] The term "epitope" or the term "antigenic determinant" used interchangeably, refers to any antigenic determinant on an antigen to which the paratope of an antibody binds, refers to a chemical group of an antigen on the surface of that antigen which determines its specificity to an antibody, a B cell, or a T cell of the immune system. Epitopes of a given antigen can be identified using a number of epitope mapping techniques well known in the art, for example, epitope identification using surface plasmon resonance (SPR) can be used for early antibody drug screening. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996). Antibodies can be screened for competitive binding to the same epitope using routine techniques known to those skilled in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete or cross-compete for binding to an antigen with one another.

[0087] The term“cross-reactivity” refers to two or more antigens of different origin (e.g., antigens of multiple homologs, paralogs, or orthologs) that can have identical antigenic determinants from each other, whereby the antibodies stimulated by the determinants in the body can bind to the corresponding epitope of the respective antigen on its own surface, but also to the same epitope of another antigen with similar affinity. For example, in some embodiments, human and non-human primate antigens have similarities (e.g., human transferrin receptor and non-human primate transferrin receptor), whereby an antibody having cross-reactivity is able to bind to the human antigen and the non-human primate antigen with similar affinity. In some embodiments, an antibody has cross-reactivity to a human antigen or a similar rodent antigen. In some embodiments, an antibody has cross-reactivity to a human antigen or a similar rodent or a similar non-human primate antigen.

[0088] The term“transferrin receptor 1” (also known as TFRC, CD71, p90, TfRl, or TFR1) is a type II transmembrane glycoprotein with a molecular weight of about 180 kDa, composed of two 90 kD subunits by disulfide bonds, which controls the supply of iron through the binding and endocytosis of the transporter protein (main iron carrier protein), playing a key role in cell proliferation. In some embodiments, the transferrin receptor can be of human (NCBI Gene ID 7037), non-human primate (NCBI Gene ID 711568 or NCBI Gene ID 102136007), or rodent (NCBI Gene ID 22042) origin. In addition, multiple human transcript variants have been characterized as encoding different isoforms of the receptor (e.g., as annotated under GenBank RefSeq Accession Nos. NP_001121620.1, NP_003225.2, NP_001300894.1, and NP_001300895.1).

[0089] An example of a human transferrin receptor amino acid sequence, corresponding to >NP_003225.2 transferrin receptor protein 1 isoform 1 [Homo sapiens], is as follows:

[0090] SEQ ID NO: 175

[0091] Target gene: human TfRl

[0092] >NP_003225.2 transferrin receptor protein 1 isoform 1 [Homo sapiens]

[0093] MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDEEENADNNTKANVTKPKRCSGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTESPVREEPGEDFPAARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTD STCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPELNKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGLSLQWLYSARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHVFWGSGSHTLPALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF

[0094] Non-human primate transferrin receptor amino acid sequence examples, corresponding sequences are as follows:

[0095] Generation of stable CHO-K1 / cTfR1 cell line

[0096] Target gene: cyno TfR1

[0097] SEQ ID NO: 174

[0098] >XP_045243212.1 transferrin receptor protein 1 [Macaca fascicularis]

[0099] MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLGVDEEENTDNNTKANGTKPKRCGGNICYGTIAVIIFFLIGFMIGYLGYCKGVEPKTECERLAGTESPAREEPEEDFPAAPRLYWDDLKRKLSEKLDTTDFTSTIKLLNENLYVPREAGSQKDENLALYIENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGGLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLDSPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVKADLSFFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCKMVTSENKSVKLTVSNVLKETKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSSVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQDVKHPVTGRSLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELVERIPELNKVARAAAEVAGQFVIKLTHDTELNLDYERYNSQLLLFLRDLNQYRADVKEMGLSLQWLYSARGDFFRATSRLTTDFRNAEKRDKFVMKKLNDRVMRVEYYFLSPYVSPKESPFRHVFWGSGSHTLSALLESLKLRRQNNSAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF

[0100] The term“affinity” is the degree or strength of binding of an antibody to an epitope or antigen. The dissociation constant K d and the affinity constant K a are quantitative measures of affinity. KD is the equilibrium dissociation constant between an antibody and its antigen, and is the ratio of the dissociation rate K dThe ratio of the rate at which an antibody dissociates from an antigen (its off-rate) to the antibody association rate K a The ratio of the rate at which an antibody dissociates from an antigen (its off-rate) to the antibody association rate K d The smaller the value, the greater the affinity of the antibody for its target. The K d values of most antibodies are in the low micromolar (10 -6 ) to nanomolar (10 -7 to 10 -9 ) range. High affinity antibodies are generally considered to be in the low nanomolar range (10 -9 ), while very high affinity antibodies are in the picomolar (10 -12 ) range.

[0101] The term "antibody affinity maturation" refers to a normal state of immune function that exists in the body. In humoral immunity, the average affinity of antibodies produced in secondary responses is higher than that in primary immune responses, and this phenomenon is called antibody affinity maturation. "Affinity maturation" described herein refers to an in vitro antibody affinity maturation technique, mainly including antibody library construction and screening techniques.

[0102] The present application uses rabbit anti-technology to obtain anti-TfR1 monoclonal antibodies with high affinity, high specificity and high blood safety through phage immunization library screening.

[0103] In certain embodiments, the present application provides an anti-transferrin receptor (TfRl) antibody or antigen-binding fragment thereof, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises: a VH CDR1 comprising SEQ ID NO: 001 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous thereto; a VH CDR2 comprising SEQ ID NO: 004 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous thereto; a VH CDR3 comprising SEQ ID NO: 007 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous thereto; and the VL region comprises: a VL CDR1 comprising SEQ ID NO: 010 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous thereto; a VL CDR2 comprising SEQ ID NO: 013 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous thereto; a VL CDR3 comprising SEQ ID NO: 016 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous thereto.

[0104] In certain embodiments, the VH region comprises: a VH CDR1 comprising SEQ ID NO: 002 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 002; a VH CDR2 comprising SEQ ID NO: 005 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 005; a VH CDR3 comprising SEQ ID NO: 008 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 008; and the VL region comprises: a VL CDR1 comprising SEQ ID NO: 011 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 011; a VL CDR2 comprising SEQ ID NO: 014 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 014; a VL CDR3 comprising SEQ ID NO: 017 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 017.

[0105] In certain embodiments, the VH region comprises: a VH CDR1 comprising SEQ ID NO: 003 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 003; a VH CDR2 comprising SEQ ID NO: 006 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 006; a VH CDR3 comprising SEQ ID NO: 009 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 009; and the VL region comprises: a VL CDR1 comprising SEQ ID NO: 012 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 012; a VL CDR2 comprising SEQ ID NO: 015 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 015; a VL CDR3 comprising SEQ ID NO: 018 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 018.

[0106] In certain embodiments, the VH region comprises: a VH CDR1 comprising SEQ ID NO: 157 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 157; a VH CDR2 comprising SEQ ID NO: 004 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 004; a VH CDR3 comprising SEQ ID NO: 007 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 007; and the VL region comprises: a VL CDR1 comprising SEQ ID NO: 134 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 134; a VL CDR2 comprising SEQ ID NO: 149 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 149; a VL CDR3 comprising SEQ ID NO: 016 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 016.

[0107] In certain embodiments, the VH region comprises: a VH CDR1 comprising SEQ ID NO: 156 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 156; a VH CDR2 comprising SEQ ID NO: 004 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 004; a VH CDR3 comprising SEQ ID NO: 007 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 007; and the VL region comprises: a VL CDR1 comprising SEQ ID NO: 133 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 133; a VL CDR2 comprising SEQ ID NO: 150 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 150; a VL CDR3 comprising SEQ ID NO: 016 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 016.

[0108] In certain embodiments, the VH region comprises: a VH CDR1 comprising SEQ ID NO: 155 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 155; a VH CDR2 comprising SEQ ID NO: 004 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 004; a VH CDR3 comprising SEQ ID NO: 007 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 007; and the VL region comprises: a VL CDR1 comprising SEQ ID NO: 132 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 132; a VL CDR2 comprising SEQ ID NO: 149 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 149; a VL CDR3 comprising SEQ ID NO: 016 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 016.

[0109] In certain embodiments, the anti-TfRl antibody or antigen-binding fragment thereof comprises at least one immunoglobulin single variable domain capable of specifically binding TfRl. In certain embodiments, the anti-TfRl antibody or antigen-binding fragment thereof specifically binds TfRl but does not bind TfR2. In certain embodiments, the anti-TfRl antibody or antigen-binding fragment thereof specifically binds transferrin receptor from human, non-human primates, mice, rats, rabbits, and the like. In certain embodiments, the anti-TfRl antibody or antigen-binding fragment thereof specifically binds human TfRl, cynomolgus TfRl, but shows no or substantially no binding to mouse TfRl or binds less to mouse TfRl. In certain embodiments, the anti-TfRl antibody or antigen-binding fragment thereof is capable of specifically binding both human TfRl and cynomolgus TfRl.

[0110] In certain embodiments, the anti-TfRl antibody or antigen-binding fragment thereof selectively specifically binds transferrin receptor 1 (TfRl) or a homodimer thereof but does not bind TfR2. In certain embodiments, the antibody specifically binds to the apical domain of TfRl.

[0111] In certain embodiments, the binding of the anti-TfRl antibody or antigen-binding fragment thereof to TfRl does not inhibit the binding of transferrin (Tf) to TfRl. In certain embodiments, the anti-TfRl antibody or antigen-binding fragment thereof has no significant effect on the binding of transferrin (Tf) and TfRl at a given drug concentration. In certain embodiments, the binding of the anti-TfRl antibody or antigen-binding fragment thereof to TfRl does not inhibit the binding of HFE-beta2-microglobulin to TfRl.

[0112] In certain embodiments, the anti-TfRl antibody or antigen-binding fragment thereof binds to an amino acid segment corresponding to E169-V169, I190-I201, N372-E383 of human transferrin receptor, which is not in the human transferrin receptor and Tf binding domain. In certain embodiments, the TfRl antibody binds to an amino acid segment corresponding to V200-Y211 of human transferrin receptor, which is not in the human transferrin receptor and Tf binding domain. In certain embodiments, the TfRl antibody binds to an amino acid segment corresponding to L274-Y282, L427-M432 of human transferrin receptor, which is not in the human transferrin receptor and Tf binding domain.

[0113] In certain embodiments, the antigen binding fragment includes, but is not limited to, Fab, Fab', F(ab'), chemically linked F(ab'), monospecific Fab, bispecific Fab, trispecific Fab, monovalent IgG, scFv (single chain variable fragment), di-scFv (bivalent scFv), bispecific diabody, trispecific triabody, scFv-Fc, minibody, or sdAb (single domain antibody).

[0114] In certain embodiments, the anti-TfRl antibody or antigen binding fragment thereof specifically binds to TfRl with an affinity. In certain embodiments, the anti-TfRl antibody or antigen binding fragment thereof binds to TfRl with a KD in the sub-nanomolar range, preferably, the anti-TfRl antibody or antigen binding fragment thereof binds to TfRl with a KD lower than 1 nM.

[0115] In certain embodiments, the anti-TfRl antibody or antigen binding fragment thereof further comprises a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). In certain embodiments, the heavy chain constant region can be from any suitable source, such as human, mouse, rat, or rabbit. In certain embodiments, the heavy chain constant region can be from human IgG (gamma heavy chain), such as IgGl, IgG2, or IgG4. Preferably, the CH is from human IgGl. An example of human IgGl constant region is given below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWD VS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK

[0116] (SEQ ID NO: 173)

[0117] In certain embodiments, the heavy chain constant region comprises a mutated human IgGl constant region. In certain embodiments, introduction of LALA mutations (substitution of residues Leu234 Leu235 with Ala234 and Ala235) and LR mutations (substitution of residue Leu327 with Arg327) in the CH2 domain of the human IgGl constant region reduces Fc receptor binding. In certain embodiments, the amino acid sequence of the CH is SEQ ID NO: 170.

[0118] In certain embodiments, the anti-TfRl antibody or antigen-binding fragment thereof further comprises a light chain constant region (CL). In certain embodiments, the CL is a kappa light chain, a lambda light chain, or a kappa light chain. In certain embodiments, the CL is a kappa light chain, the sequence of which is provided below: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0119] (SEQ ID NO: 171)

[0120] In certain embodiments, other antibody heavy and light chain constant regions are well known in the art, for example, those provided in the IMGT database (www.imgt.org).

[0121] In certain embodiments, the present application provides a humanized anti-transferrin receptor (TfRl) antibody or antigen-binding fragment thereof, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein at least one amino acid substitution or mutation (e.g., single amino acid substitution, deletion, or insertion) is made in the VL region and / or the VH region.

[0122] In certain embodiments, the heavy chain variable region and / or the light chain variable region further comprises a humanized framework region, which is grafted between the CDR sequences. In certain embodiments, the sequence of the heavy chain variable region is selected from any one of SEQ ID NOs: 177-181. In certain embodiments, the sequence of the light chain variable region is selected from any one of SEQ ID NOs: 196-197.

[0123] In certain embodiments, it comprises at least one mutation in at least one CDR of the light chain variable region. In certain embodiments, it comprises at least one mutation in at least one CDR of the heavy chain variable region. In certain embodiments, when there is a single mutation, it is in CDR1 or CDR2 or CDR3. In certain embodiments, when there is more than one mutation, the second mutation and further mutations can be in CDR1 and / or CDR2 and / or CDR3 and / or any other region of the antibody.

[0124] In the present invention, the inventors found that when at least one mutation is made in CDR1 or CDR2 or CDR3 of a humanized anti-TfRl antibody, not only the activity of the humanized antibody can be retained, but also a mutant humanized antibody is obtained which has at least equal or better affinity to the non-mutant chimeric antibody and does not cause or causes a smaller immune reaction.

[0125] In certain embodiments, the VL region independently has 1 or more amino acid substitutions or mutations in at least one CDR relative to SEQ ID NO: 196. In certain embodiments, the amino acid substitutions or mutations in CDRs in the VL are at positions 32, 33, 50, 53 and 91 of SEQ ID NO: 196. In certain embodiments, the substitutions of the corresponding amino acids are: 32Y, 32F, 32W, 33R, 50R, 53R, 91V, 91I.

[0126] In certain embodiments, the VH region independently has 1 or more amino acid substitutions or mutations in at least one CDR relative to SEQ ID NO: 179. In certain embodiments, the amino acid substitutions or mutations in CDRs in the VH region are at positions 32, 54 and 101 of SEQ ID NO: 179. In certain embodiments, the substitutions of the corresponding amino acids are: 32W, 54F, 101Y, 101W.

[0127] In certain embodiments, the humanized anti-TfRl antibody or antigen binding fragment thereof has at least equal affinity to the receptor TfRl as the chimeric antibody.

[0128] In certain embodiments, the VH comprises: a VH CDR1 comprising SEQ ID NO: 001 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 001; a VH CDR2 comprising SEQ ID NO: 004 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 004; a VH CDR3 comprising SEQ ID NO: 007 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 007; and the VL region comprises: a VL CDR1 comprising SEQ ID NO: 072 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 072; a VL CDR2 sequence comprising SEQ ID NO: 080 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 080; a VL CDR3 sequence comprising SEQ ID NO: 081 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 081.

[0129] In certain embodiments, the VH comprises: a VH CDR1 comprising SEQ ID NO: 001 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous thereto; a VH CDR2 comprising SEQ ID NO: 004 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous thereto; a VH CDR3 comprising SEQ ID NO: 070 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous thereto; and the VL region comprises: a VL CDR1 comprising SEQ ID NO: 073 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous thereto; a VL CDR2 sequence comprising SEQ ID NO: 080 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous thereto; a VL CDR3 sequence comprising SEQ ID NO: 081 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous thereto;

[0130] In certain embodiments, the VH comprises: a VH CDR1 comprising SEQ ID NO: 001 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 001; a VH CDR2 comprising SEQ ID NO: 004 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 004; a VH CDR3 comprising SEQ ID NO: 007 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 007; and the VL region comprises: a VL CDR1 comprising SEQ ID NO: 071 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 071; a VL CDR2 sequence comprising SEQ ID NO: 078 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 078; a VL CDR3 sequence comprising SEQ ID NO: 081 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 081.

[0131] In certain embodiments, the VH region comprises a sequence comprising SEQ ID NO: 179 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 179, and the VL region comprises a sequence comprising SEQ ID NO: 212 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 212. In certain embodiments, the VH region comprises a sequence comprising SEQ ID NO: 183 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 183, and the VL region comprises a sequence comprising SEQ ID NO: 200 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 200. In certain embodiments, the VH region comprises a sequence comprising SEQ ID NO: 179 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 179, and the VL region comprises a sequence comprising SEQ ID NO: 209 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous to SEQ ID NO: 209.

[0132] In certain embodiments, the humanized anti-TfRl antibody or antigen-binding fragment thereof has mutations in the VL CDR1, VL CDR2, VH CDR1, and VH CDR3 regions. In certain embodiments, the amino acid sequence of the VL CDR1 is any one sequence selected from the group consisting of SEQ ID NOs: 010, 095-127, and 132-147; in certain embodiments, the amino acid sequence of the VL CDR2 is any one sequence selected from the group consisting of SEQ ID NOs: 013, 128-131, and 148-150; in certain embodiments, the amino acid sequence of the VH CDR1 is any one sequence selected from the group consisting of SEQ ID NOs: 001, 083-091, and 152-159; and the amino acid sequence of the VH CDR3 is any one sequence selected from the group consisting of SEQ ID NOs: 007, 092-094, 160, and 161.

[0133] In certain embodiments, the amino acid sequences of the VL CDR1, VL CDR2, VH CDR1, and VH CDR3 regions are as shown in Table 1 below:

[0134] Table 1

[0135]

[0136] The sequence of VL CDR1 of the antibody with Sample ID NO. 30001-30012 is SEQ ID NO: 010, the sequence of VL CDR2 is SEQ ID NO: 013, and the sequence of VL CDR3 is SEQ ID NO: 016;

[0137] and / or:

[0138] As shown in Table 2:

[0139] Table 2

[0140]

[0141]

[0142] The sequence of VH CDR1 of the antibody with Sample Name 30013-30049 is SEQ ID NO: 001, the sequence of VH CDR2 is SEQ ID NO: 004, and the sequence of VH CDR3 is SEQ ID NO: 007;

[0143] and / or:

[0144] As shown in Table 3:

[0145] Table 3

[0146]

[0147]

[0148] The sequence of VH CDR2 of the antibody with Sample ID NO. 30050-30097 is SEQ ID NO: 004, and the sequence of VL CDR3 is SEQ ID NO: 016.

[0149] In certain embodiments, the present application provides a method for preparing a humanized anti-TfRl antibody or antigen-binding fragment thereof as described above, comprising the following steps:

[0150] S1. Search the sequence against the IgBLAST database to determine the best template for the Fv fragment. Select a best template for VH / VL matching the human framework sequence;

[0151] S2. Design a humanized antibody using CDR grafting, graft the CDRs of the parent antibody into a human framework sequence to obtain a chimeric antibody, while comparing the chimeric antibody and the parent antibody in CDR, residue, stem loop structure, sequence near the hydrophobic core;

[0152] S3. Design stepwise incorporation of one or more reverse mutations in the transplanted antibody sequence, select antibodies with corresponding mutation combinations for affinity maturation according to the affinity detection results and the number of accumulated mutations;

[0153] S4. Based on the selected antibodies with corresponding mutation combinations, obtain antibodies with comparable affinity to the Gypsophila oldhamiana antibody through single-point saturation mutation of CDR regions;

[0154] S5. Based on the results of single-point mutation, further improve the affinity through mutation combination;

[0155] S6. Based on the selected antibodies with corresponding mutation combinations, improve the affinity of the monoclonal antibody through the yeast (Sc) display platform technology and block mutation method, so as to obtain antibodies with comparable affinity to the Gypsophila oldhamiana antibody.

[0156] In certain embodiments, the step S5 comprises the following steps:

[0157] 1) Design a special primer, and use PCR method to mutate several amino acids (1-5) positions in the VH and VL CDR regions of the selected antibodies with corresponding mutation combinations, which can introduce all 20 amino acids combinations at each amino acid position;

[0158] 2) Enrich high-affinity clones in the library through 2-3 rounds of target protein FACS sorting, and select 24 clones for sequencing each round to monitor the enrichment of wild type;

[0159] 3) After sorting, select yeast clones and perform single-concentration target protein FACS experiment verification, and sequence the clones with improved FACS signal value;

[0160] 4) Select 10 polypeptide clones with different sequences, and perform sorting through gradient dilution target protein FACS experiment.

[0161] In certain embodiments, the present application provides a nucleic acid molecule for encoding an anti-TfR1 antibody or an antigen-binding fragment thereof as described above or a humanized anti-TfR1 antibody or an antigen-binding fragment thereof as described above. In certain embodiments, the nucleic acid molecule can include DNA and RNA sequences.

[0162] In certain embodiments, the present application provides an expression vector comprising the nucleic acid molecule as described above. In certain embodiments, the expression vector is a lentiviral expression vector, a retroviral expression vector, an adenoviral expression vector, a DNA vector, or an RNA vector. In certain embodiments, the expression vector is well known to those skilled in the art, including but not limited to: a plasmid, a bacteriophage (such as lambda phage or M13 filamentous phage, etc.), a cosmid (i.e., a cos plasmid), a viral vector (such as a baculovirus vector, a retrovirus (including lentivirus), an adenovirus, an adeno-associated virus, or a herpes virus (such as a herpes simplex virus), etc.). In one embodiment of the present application, the vector can be specifically pUC57 or pCGS3.

[0163] In certain embodiments, the present application provides a host cell comprising the nucleic acid molecule as described above or the expression vector as described above. In certain embodiments, the host cell includes but is not limited to: a eukaryotic cell (such as a yeast cell, an Aspergillus), an animal cell (such as a mammalian cell, an insect cell), or a prokaryotic cell. In certain embodiments, the host cell can be specifically ExpiCHO-S cell.

[0164] The anti-TfRl antibodies of the present application can be produced by recombinant expression. The above-described nucleic acids encoding the light and heavy chain variable regions, optionally linked to constant regions, can be inserted into expression vectors. Vectors comprising nucleic acids encoding the antibodies described herein are aspects of the present application per se. The light and heavy chains can be cloned into the same or different expression vectors. The nucleic acids encoding the antibody chains described herein can be operably linked to one or more control sequences in the expression vector to ensure expression of the antibody chains. Expression control sequences include, but are not limited to, promoters (e.g., naturally-associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Preferably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells (such as COS, CHO, or Expi293 cells). Such vectors can be incorporated into the appropriate host, which is then maintained in a suitable condition to allow for high-level expression of the nucleotide sequences and collection and purification of the antibodies.

[0165] In certain embodiments, the present application provides a conjugate product comprising a covalently linked humanized TfRl antibody or antigen-binding fragment thereof as described above and an active payload for targeting the active payload to muscle cells or mediating transport of the active payload across the blood-brain barrier. Preferably, the active payload is an oligonucleotide, preferably, the active payload is an siRNA.

[0166] In certain embodiments, the present application provides a pharmaceutical composition comprising the conjugate as described above and optionally a pharmaceutically acceptable excipient. The pharmaceutically acceptable carrier can be, but is not limited to, a diluent, an excipient, a filler, a binder, a humectant, a disintegrant, an absorption enhancer, an adsorption carrier, a surfactant or a lubricant.

[0167] In certain embodiments, the present application provides use of a humanized anti-TfRl antibody or an antigen-binding fragment thereof as described above or a conjugate as described above or a pharmaceutical composition as described above in the manufacture of a medicament for treating tumor, muscle and central nervous system related diseases.

[0168] In certain embodiments, the pharmaceutical composition can be administered by any suitable means, such as intranasally, subcutaneously, intramuscularly, intravenously, intra-arterially, intra-articularly, intra-lesionally, orally or topically. According to some embodiments, the pharmaceutical composition is administered parenterally. In certain embodiments, the pharmaceutical composition is administered intravenously (i.v).

[0169] In certain embodiments, the present application provides use of an anti-TfRl antibody or an antigen-binding fragment thereof as described above or a humanized anti-TfRl antibody or an antigen-binding fragment thereof as described above in the manufacture of a kit for detecting the presence or level of TfRl in a sample.

[0170] The following is illustrated in conjunction with specific examples:

[0171] Unless otherwise specified, the experimental reagents used in the following examples are all conventional reagents in the art, which can be prepared according to conventional methods in the art or purchased. Unless otherwise specified, the experimental methods used in the following examples are all conventional methods in the art, which can be referred to relevant experimental manuals, such as Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: A Laboratory Manual, 2001), or manufacturer's instructions. 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 application belongs.

[0172] Example 1 Preparation of humanized anti-human TfRl monoclonal antibody from rabbit-derived mAb

[0173] Human TfRl was used as an immunogen to establish a rabbit anti-human TfRl library by immunizing two New Zealand white rabbits. Rabbit-derived anti-human TfRl monoclonal antibodies were prepared using phage display screening technology. Specifically, total RNA was extracted from spleen cells, VHand VLgenes were amplified by RT-PCR, and were constructed into M13 phage vectors to become chimeric Fab phage libraries with a library capacity of 2 x 1011. 8After the phage display library was constructed, 4 rounds of panning were performed using human monkey TfR1. The enrichment strategy included: ① the ability to bind to the apical region of TfR1, ② cross-immunogenicity with monkey TfR1, ③ minimal potential deamidation, isomerization or oxidation sites, ④ no ADCC or CDC effect, ⑤ no binding to TfR2, ⑥ no binding to Tf and HFE. The enrichment process included first immobilizing TfR1, adding the positive control Var2iii (an antibody that can bind to TfR1 but does not affect the binding of Tf and HFE to TfR1), then adding the supernatant, and selecting and collecting the supernatant remaining in the stationary phase. Antibody clones that were simultaneously reactive with human and cynomolgus monkey TfR1 were obtained by phage ELISA, and single clones with good antibody expression were selected for sequencing. A total of 262 positive monoclonal antibodies were obtained.

[0174] The 262 rabbit-derived antibodies enriched by the phage display library were analyzed for human and cynomolgus monkey TfR1 binding activity and sequencing, and finally 40 antibodies were selected based on the sequencing results of the 262 antibody positive monoclonals. Based on the requirement for sequence stability, the last amino acid of VH CDR3 of 30775 sequence was mutated from P→A to obtain 30775A sequence, and a total of 41 antibody sequences were subjected to CHO cell transient transfection and expression purification.

[0175] Human-rabbit chimeric anti-human TfR1 monoclonal antibodies were constructed. In terms of sequence design, the rabbit anti-heavy chain variable region was grafted into the human IgG1 antibody heavy chain constant region, and the rabbit anti-light chain variable region was grafted into the human IgG1 antibody light chain constant region for antibody transient expression. The heavy chain constant region of this embodiment contains some characteristic mutations, such as LALA mutation (substituting hinge residues Leu234 and Leu235 with Ala234 and Ala235, respectively) and LR mutation (substituting Leu327 with Arg327), thereby reducing the effector function of Fc.

[0176] The following is the sequence of the human IgG1 antibody heavy chain constant region for grafting:

[0177] STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKARPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLLSLPGK

[0178] The sequence of the human IgGl antibody light chain constant region for grafting is as follows:

[0179] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0180] (SEQ ID NO: 171)

[0181] The chimeric antibodies were further evaluated according to the detection results of human and cynomolgus TfRl affinity kinetics, the blocking activity of the chimeric antibodies on Tf and TfR binding, and the binding activity of the chimeric antibodies and human TfR2. Among them, the third amino acid of VL CDR3 of 28158 was mutated from C to S to meet the requirement of antibody stability.

[0182] The light and heavy chain variable regions containing CDRs of the three antibodies were chimerized into human IgG constant regions, and the transient expression, purity, affinity, competition experiment, and sequence (Table 4) were further compared. At the same time, the comparison was made with positive control Var2iii (an antibody capable of binding to human TfRl but not blocking Tf binding) and negative control AF2474 (a polyclonal antibody capable of binding to human TfRl and blocking Tf binding) (Table 4).

[0183] Table 4 Comparison of the three preferred rabbit-derived antibodies

[0184]

[0185] wherein VH_CDR1, VH_CDR2, VH_CDR3 are the three complementarity determining regions (CDRs) in the variable region of the heavy chain; VL_CDR1, VL_CDR2, VL_CDR3 are the three complementarity determining regions (CDRs) in the variable region of the light chain.

[0186] According to the expression, purity, affinity, competition experiment and sequence analysis results of the preferred antibodies, the antibody 28158 was finally selected for further humanization modification. The amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) of the rabbit-derived monoclonal antibody and the contained complementarity determining regions (CDRs) are shown in Table 5.

[0187] Table 5 Statistics of CDR sequences of heavy and light chains of rabbit-derived monoclonal antibody

[0188]

[0189] By analyzing the framework region sequences of existing humanized antibodies, the amino acids of the light and heavy chain framework regions were first determined, and then the CDR region amino acids of the light and heavy chains of the antibody 28158 were spliced into the determined framework region, i.e. to form the precursor light and heavy chains of the humanized rabbit monoclonal antibody. According to the amino acid sequences of the light and heavy chains, the nucleotide sequences encoding the same were optimized (for reference, https: / / sg.idtdna.com / pages / tools / codon-optimization-tool), such as avoiding commonly used enzyme digestion sites, avoiding consecutive high GC sites, avoiding codon sites preferred by mammalian cells, optimizing potential intron splicing sequences, optimizing potential stem-loop structures of mRNA, etc. The specific scheme is as follows:

[0190] Selection of human antibody framework

[0191] The selection method includes: by analyzing the framework region sequences of existing humanized antibodies, evaluating the similarity of the existing human germline and the framework region of the antibody 28158, and evaluating the amino acid at each position by means of 3D structure model. For the antibody 28158, the human germline IGHV3-53*01, IGKV1-12*01 was selected as the starting human receptor framework.

[0192] Design of humanized VH and VL highly close to

[0193] Based on the key points of classical residues, interaction loop region, core region, mutation hot spots, etc., suitable sites were selected for combination design to obtain the amino acid sequences of the variable region of the humanized antibody. Five humanized amino acid sequences were designed for the heavy chain 28158_VH gene, and two humanized amino acid sequences were designed for the light chain 28158_VL gene. The combination of VL and VH produced a total of 10 mutants, which were the candidate clones of humanized rabbit monoclonal antibodies, for subsequent recombinant antibody expression plasmid construction.

[0194] The five humanized amino acid sequences designed for the heavy chain 28158_VH gene are as follows:

[0195] > 28158_VH1 (SEQ ID NO: 177)

[0196] EVLVESGGGLIQPGGSLRLSCAASGFSLSSYYMIWVRQAPGKGLEWIGFMHN

[0197] DGTTYFASWAKGRFTISKSKTTLDLQMNSLRAEDTAVYFCARYGGSNSGAD

[0198] LWGQGTLVTVSS

[0199] > 28158_VH2 (SEQ ID NO: 178)

[0200] EVQLVESGGGLIQPGGSLRLSCAASGFSLSSYYMIWVRQAPGKGLEYIGFMH

[0201] NDGTTYFASWAKGRFTISKDNSKTTLYLQMNSLRAEDTAVYYCARYGGSNS

[0202] GADLWGQGTLVTVSS

[0203] > 28158_VH3 (SEQ ID NO: 179)

[0204] EVQLVESGGGLIQPGGSLRLSCAASGFSLSSYYMIWVRQAPGKGLEYIGFMH

[0205] NDGTTYFASWAKGRFTISKDNSKNTVDLQMNSLRAEDTAVYFCARYGGSNS

[0206] GADLWGQGTLVTVSS

[0207] >28158_VH4 (SEQ ID NO: 180)

[0208] EVQLVESGGGLIQPGGSLRLSCAASGFSLSSYYMIWVRQAPGKGLEYIGFMH

[0209] NDGTTYFASWAKGRFTISKSSTTVDLQMNSLRAEDTAVYFCARYGGSNSGA

[0210] DLWGQGTLVTVSS

[0211] >28158_VH5 (SEQ ID NO: 181)

[0212] EVQLVESGGGLIQPGGSLRLSCAASGFSLSSYYMIWVRQAPGKGLEYIGFMH

[0213] NDGTTYFASWAKGRFTISKDNSKTTVDLQMNSLRAEDTAVYFCARYGGSNS

[0214] GADLWGQGTLVTVSS

[0215] Two humanized amino acid sequences designed for the light chain 28158_VL gene:

[0216] >28158_VL1 (SEQ ID NO: 196)

[0217] DIQMTQSPSSVSASVGDRVTITCQASQNIGSNLAWYQQKPGKAPKLLIYSAST

[0218] LASGVSSRFSGSGSGTDFTLTISSLQPEDFATYYCQSSGAIANYGGTFGGGTK

[0219] LEIK

[0220] >28158_VL2 (SEQ ID NO: 197)

[0221] DIQMTQSPSSVSASVGDRVTITCQASQNIGSNLAWYQQKPGKAPKLLIYSAST

[0222] LASGVSSRFKGSGSGTDFTLTISSLQPEDFATYYCQSSGAIANYGGTFGGGTK

[0223] LEIK

[0224] Predicted structure analysis of mutation list

[0225] Analysis of sequence to detect PTM, and homology modeling of antibody by MOE software, analysis of the risk of mutation of amino acid at different sites on stability, the risk of increasing immunogenicity, and the risk of maintaining the affinity of target binding. For example, in the initial design, by mutating C in the FR3 region to P, the redundant rabbit disulfide bond was removed. In addition, the unpaired cysteine in the VL CDR3 region was also mutated from C to S. These attempts were found to weaken the binding affinity in the test construct, so a subsequent round of design was carried out. The sequence analysis results are shown in Table 6.

[0226] Table 6 Sequence analysis results of preferred antibodies

[0227]

[0228] Based on the sequence analysis results, the antibody 28158_VH was modified, for example, the glycosylation site introduced in VH2 and VH5 was removed. Homology modeling was performed on the selected Fv fragment. The sequence was searched against the IgBLAST database to determine the best template for the Fv fragment. A best template matching the human framework sequence was selected for VH / VL, for example, the heavy chain corresponds to the human framework sequence: IGHV3-53*01 (similarity 58.5%), and the light chain corresponds to the human framework sequence: IGKV1-12*01 (similarity 67.7%). The CDR grafting was used to design a humanized antibody, and the CDR of the parent antibody was grafted into the human framework sequence to obtain a chimeric antibody. At the same time, the sequence of the chimeric antibody and the parent antibody in the CDR, residues, stem loop structure, and hydrophobic core was compared. One or more reverse mutations were gradually incorporated into the grafted antibody sequence. The humanization ratio (Humanness) was calculated according to the number of accumulated mutated amino acids and the comparison of human framework sequences. The affinity of the humanized antibody was detected by SPR. According to the affinity detection results (Table 7) and the number of accumulated mutations (Table 8), the combination of 28158_VH3 and 28158_VL2 was selected for affinity maturation.

[0229] Table 7 Affinity determination results of selected antibodies

[0230] Antibody ID Chi 2 (RU 2 )]]> ka (1 / Ms) kd (1 / s) KD (M) Rmax (RU) 28158_VH1+VL1 2.07 6.75E+04 2.51E-03 3.72E-08 49.9 28158_VH1+VL2 2.11 7.02E+04 2.49E-03 3.55E-08 48.0 28158_VH2+VL1 1.46 9.45E+04 3.00E-03 3.18E-08 30.3 28158_VH2+VL2 1.19 8.68E+04 3.17E-03 3.66E-08 25.4 28158_VH3+VL1 1.59 6.31E+04 1.81E-03 2.86E-08 97.0 28158_VH3+VL2 1.84 6.55E+04 1.55E-03 2.37E-08 119.0 28158_VH4+VL1 1.96 6.85E+04 1.38E-03 2.01E-08 84.4 28158_VH4+VL2 1.55 6.81E+04 1.38E-03 2.03E-08 77.7 28158_VH5+VL1 2.30 7.46E+04 1.35E-03 1.81E-08 140.8 28158_VH5+VL2 2.28 7.02E+04 1.32E-03 1.89E-08 152.8 28158_VH+VL 0.0449 6.20E+04 6.12E-04 9.86E-09 63.0 28158 3.56E-02 1.43E+05 1.31E-04 9.17E-10 123.2

[0231] Table 8 Analysis of humanization mutations of selected antibody variable region sequences

[0232]

[0233] Example 2 Antibody affinity maturation-single point mutation

[0234] The experimental purpose is to obtain antibodies with comparable affinity to the Gynura japonica antibody by single-point saturation mutation of the CDR region based on antibody 28158_VH3+VL2. The sequence of antibody 28158_VH3+VL2 is shown in Table 9 as follows:

[0235] Table 9 Sequence of antibody 28158_VH3+VL2

[0236]

[0237] Based on the sequence of antibody 28158_VH3+VL2, the amino acids at each site of the CDR region were subjected to saturation mutation, and expression was cultured in CHO-S cells. For the well-expressed saturation mutation sequences, ELISA experiment was used to determine the affinity of Human TfR1 antigen, and the steps of ELISA experiment are described as follows: Biotin-labeled Human TfR1 antigen (0.3 μg / ml, CBS, 100 μl / well) and BSA (10 μg / ml, CBS, 100 μL / well) were incubated overnight in the enzyme-labeled plate. 50 μl FASEBA supernatant (100 μl / well) was diluted with 50 μl 0.1% PBST and added to the wells coated with antigen as the first antibody. After incubation for 1 h, 0.1 μg / ml Peroxidase AffiniPure Goat Anti-Human IgG (F(ab')2 fragment specific, 0.05% PBST, 100 ul / well) was used as the second antibody, and the reaction was performed for 30-60 min. 3,3',5,5'-tetramethylbenzidine (TMB) was used as a rapid color developing indicator. The color development reaction was terminated by 1M hydrochloric acid, and the absorbance OD value was measured by an enzyme-labeled instrument at a wavelength of OD 450 nm (Table 6). According to the absorbance OD value, and according to the formula Ratio=OD(Mutation) / OD(WT), the ratio was calculated, and the ratio≥1.75 and top 5 of each plate were selected, obtaining 69 antibodies, and sequencing found that some of them were repeated sequences, and the results are shown in Table 10.

[0238] Table 10 Affinity maturation of antibody single-point mutation results

[0239]

[0240]

[0241] After sequencing 69 antibodies, remove repeated sequences to obtain 47 antibody CDR region single point mutation designs. Among them, antibody names 23472-23480 only mutate VH CDR1, other CDR and framework region sequences are consistent with 28158_VH3+VL2, and the mutation sites of VH CDR1 are shown in Table 11; among them, antibody names 23481-23493 only mutate VH CDR2, other CDR and framework region sequences are consistent with 28158_VH3+VL2, and the mutation sites of VH CDR2 are shown in Table 12; among them, antibody names 23494-23496 only mutate VH CDR3, other CDR and framework region sequences are consistent with 28158_VH3+VL2, and the mutation sites of VH CDR3 are shown in Table 13.

[0242] Table 11 Mutation design of VH CDR1

[0243]

[0244]

[0245] Table 12 Mutation design of VH CDR2

[0246] Antibody Name Mutational Design of VH CDR2 SEQ ID NO. 28158_VH3+VL2 FMHNDGTTYFASWAKG 004 23481 FFHNDGTTYFASWAKG 026 23482 FMHNRGTTYFASWAKG 027 23483 FMHNFGTTYFASWAKG 028 23487 FMHNDGKTYFASWAKG 029 23488 FMHNDGTTSFASWAKG 030 23489 FMHNDGTTYFQSWAKG 031 23490 FMHNDGTTYFVSWAKG 032 23491 FMHNDGTTYFASWAKS 033 23492 FMHNDGTTYFASWAKH 034 23493 FMHNDGTTYFASWAKK 035

[0247] Table 13 Mutation design of VH CDR3

[0248] Antibody Name Mutational Design of VH CDR3 SEQ ID NO. 28158_VH3+VL2 YGGSNSGADL 007 23494 YGGYNSGADL 036 23496 YGGWNSGADL 037

[0249] Antibody names 23500-23513 only mutate VL CDR1, other CDR and framework region sequences are consistent with 28158_VH3+VL2, and the mutation sites of VL CDR1 are shown in Table 14; among them, antibody names 23514-23534 only mutate VL CDR2, other CDR and framework region sequences are consistent with 28158_VH3+VL2, and the mutation sites of VL CDR2 are shown in Table 15; among them, antibody names 23537-23541 only mutate VL CDR3, other CDR and framework region sequences are consistent with 28158_VH3+VL2, and the mutation sites of VL CDR3 are shown in Table 16.

[0250] Table 14 Mutation design of VL CDR1

[0251]

[0252]

[0253] Table 15 Mutation design of VL CDR2

[0254] Antibody Name Mutational Design of VL CDR1 SEQ ID NO. 28158_VH3+VL2 SASTLAS 013 23514 RASTLAS 050 23516 SGSTLAS 051 23517 SRSTLAS 052 23520 SKSTLAS 053 23521 SAKTLAS 054 23524 SAHTLAS 055 23525 SARTLAS 056 23526 SASKLAS 057 23527 SASLLAS 058 23528 SASRLAS 059 23531 SASTLKS 060 23533 SASTLAR 061 23534 SASTLAK 062

[0255] Table 16 Mutation design of VL CDR3

[0256] Antibody Name Mutational Design of VL CDR1 SEQ ID NO. 28158_VH3+VL2 QSSGAIANYGGT 016 23537 QSVGAIANYGGT 063 23540 QSAGAIANYGGT 064 23541 QSIGAIANYGGT 065

[0257] The affinity of the above antibodies and human TfRl was detected by surface plasmon resonance (SPR), and the results are shown in Table 17.

[0258] Table 17 Affinity detection results of single point mutation

[0259]

[0260]

[0261] According to the affinity results, the mutation sites that have greater impact on the affinity of the antibody 28158_VH3+VL2 were screened, for example, the 32nd, 54th and 101st sites of the heavy chain variable region, and the 32nd, 33rd, 50th, 53rd and 91st sites of the light chain variable region, as shown in Table 18.

[0262] Table 18 Design of mutation hot spot region

[0263] Mutated Position WT Mutational Design VH CDR1 32Y 32W VH CDR2 54D 54F VH CDR3 101S 101Y VH CDR3 101S 101W VL CDR1 32N 32Y VL CDR1 32N 32F VL CDR1 32N 32W VL CDR1 33L 33R VL CDR2 50S 50R VL CDR2 53T 53R VL CDR3 91S 91V VL CDR3 91S 91I

[0264] Example 3 Affinity maturation of antibodies - combination of single point mutations

[0265] The purpose of the experiment is to further improve the affinity by combining mutations based on the results of single point mutations. The 32nd, 54th and 101st sites of the heavy chain variable region, and the 32nd, 33rd, 50th, 53rd and 91st sites of the light chain variable region were selected for combination by previous single point mutations. Table 19 shows the design of combination mutation of antibody heavy chain variable region, and Table 20 shows the design of combination mutation of antibody light chain variable region. According to the sequence design of Table 15 and Table 16, CHO cell transient expression purification was used, and the affinity with human TfRl was detected by surface plasmon resonance (SPR), and the results are shown in Table 21.

[0266] Table 19 Design of combination mutation of antibody heavy chain CDR region

[0267]

[0268]

[0269] Table 20 Design of combination mutation of antibody light chain CDR region

[0270]

[0271]

[0272] Table 21 Antibody combination mutation affinity results

[0273]

[0274]

[0275] According to the determination results of KDand Rmax, 23669, 23673, 23699 were selected as preferred 3 antibodies, and the specific sequences are referred to Table 22.

[0276] Table 22 Preferred 3 antibody sequences

[0277]

[0278]

[0279] Example 4 Antibody affinity maturation - yeast display technology and block mutation method

[0280] The purpose of the experiment is to improve the affinity of monoclonal antibodies based on antibody 28158_VH3+VL2(WT) by using EBY100 yeast through yeast (Sc) display platform technology and block mutation method, so as to obtain an antibody with comparable affinity to the variegated antibody (Var2iii, PC). According to the template antibody 28158_VH3+VL2(WT) of affinity maturation, the antibody Fab fragment (scFab) antibody library is constructed.

[0281] High-affinity protein variants are isolated from the yeast library by FACS. First round of library construction: first incubate with 111 nM biotinylated antigen for 1 hour, use cMyc-RPE and SA647 staining to sort, use FACS special tube for instrument detection and analysis, isolate high-affinity protein variants from the yeast library by FACS, refer to Figure 3 . Then incubate with different concentrations of biotinylated antigen at room temperature for 30 min, wash away the unbound antigen, adjust the incubation temperature and time according to the previous sorting results, select the yeast cells at the appropriate time point for flow sorting, collect the library clones for culture in each round of sorting, and then perform the next round of sorting, and finally after a total of 3 rounds of sorting Figures 3-5 ). The yeast strains are selected from each antibody library for sequencing analysis, and finally several unique mutant clones are obtained from the heavy chain antibody library and the light chain antibody library (the sequences are shown in Table 23).

[0282] Analysis of the results of the three rounds of FACS screening sequences, a total of 49 clones were obtained. Antibody names 30001-30012 only exist in VH CDR1 and VH CDR3 region mutation, other CDR and skeleton region sequence and 28158_VH3+VL2 consistent, as shown in Table 23. Antibody names 30013-30049 only exist in VL CDR1 and VL CDR2 region mutation, other CDR and skeleton region sequence and 28158_VH3+VL2 consistent, as shown in Table 24.

[0283] Table 23 First round of library sorting monoclonal_VH sequence

[0284]

[0285] Table 24 First round of library sorting monoclonal_VL sequence

[0286]

[0287]

[0288] Second round of library construction: using the obtained VH CDR1, VH CDR3, VL CDR1 and VL CDR2 sequence and the same method as the first round of library construction, finally after a total of 2 rounds of sorting Figures 6-7 ) Analysis of the results of the two rounds of FACS screening sequences, a total of 48 clones were obtained. Antibody names 30050-30097 exist in VL CDR1, VL CDR2, VH CDR1 and VH CDR3 region mutation, other CDR and skeleton region sequence and 28158_VH3+VL2 consistent, as shown in Table 25.

[0289] Table 25 Second round of library sorting monoclonal sequence

[0290]

[0291]

[0292]

[0293] According to the MFI results of 48 clones (Table 26), 14 clones better than that of the Patrinia Var2iii were screened for KD-FACS detection, and the detection results are referred to Figure 8 and Table 27.

[0294] Table 26 MFI results of 48 clones

[0295]

[0296] Table 27 KD-FACS detection results

[0297]

[0298]

[0299] According to the KD-FACS results, 11 clones (Table 28) were selected for recombinant expression verification compared with the Ligilactobacillus brevis antibody. Among them, the 11 clones have mutations in VL CDR1, VL CDR2, VH CDR1 and VH CDR3 regions, and their VL CDR3 and VH CDR2 as well as FR region, C region are the same as the WT (28158_VH3+VL2) sequence.

[0300] Table 28 11 clones for recombinant expression verification

[0301]

[0302] The 11 clones for recombinant expression were cultured and expressed in CHO-S cells using the antibody transient expression method. After antibody purification, the supernatant concentration was determined using A280, the antibody purity was analyzed by SDS-PAGE, and the affinity for Human / Cyno TfR1 and the binding ability to TfR2 were determined, and the results are shown in Table 29. The results show that the preferred 11 clones can all bind to Human / Cyno TfR1 and do not bind to TfR2. Among them, clones 30068, 30082, 30052 and 30054 have relatively high expression levels.

[0303] Table 2911 clones expression quality control results

[0304]

[0305] Then, using the polyclonal antibody AF2474 of TfR1 as the Ligilactobacillus brevis antibody, and using the clinically tested monoclonal antibody Var2iii as an internal reference, the competition ability of the 11 antibodies and 23699 with transferrin was evaluated. The results are shown in Table 30. The results show that the 11 antibodies do not affect the binding of transferrin and TfR.

[0306] Table 30 Competition ability of 11 antibodies with transferrin

[0307]

[0308]

[0309] According to the above screening results, the sequences of 30068, 30082 and 30054 were finally selected, as shown in Table 31.

[0310] Table 31 Sequences of the three antibodies finally selected

[0311]

[0312]

[0313] Example 5. Binding epitope of candidate antibodies to hTfRl

[0314] The purpose of this example is to investigate the binding mode of the candidate antibodies (3 candidate antibodies described above) to hTfRl, the experimental method is described as follows:

[0315] Identification of antigen-antibody two-dimensional polypeptide spectrum: The enzymatic peptides of the protein were identified by hydrogen-deuterium exchange mass spectrometry platform LEAP PAL 3.0 using secondary mass spectrometry (MS / MS) on a mass spectrometer (Orbitrap Fusion Tribrid Mass Spectrometer, Thermo Fisher). The MS / MS data file was processed by Proteome Discover software for peptide identification. TM Tribrid TM Mass Spectrometer, Thermo Fisher). The MS / MS data file was processed by Proteome Discover software for peptide identification.

[0316] Preparation of hydrogen-deuterium exchange mass spectrometry sample: 5-10 μM antigen or antibody or antigen-antibody complex (1:1 molar ratio) (50 mM HEPES, pH 7.4, 150 mM NaCl, 4 mM TCEP) was placed at 4°C for 1 hour to form a stable state of complex. Then 5 microliters of sample was diluted into 20 microliters of D2O (deuterium) and placed into different HDX time points for mass spectrometry analysis (e.g. 0, 10, 60, 300, 900 seconds). After the reaction, the reaction was terminated by mixing 4M guanidine hydrochloride and 1% trifluoroacetic acid. Immediately after stopping the reaction, the sample tube was placed on dry ice until the sample was injected into the HDX LEAP PAL 3.0 platform. After injection into the fully automated hydrogen-deuterium exchange platform, the sample was passed through a fixed pepsin column at a flow rate of 120 μL / min, and the enzymatic peptides were captured on a C18 capture column and desalted. The desalted peptides were separated by a 2.1 mm x 5 cm C18 column (1.9 μm Hypersil Gold, Thermo Fisher) with a linear gradient of 4-40% acetonitrile and 0.3% formic acid in 8 minutes. During sample processing, protein enzymolysis and peptide separation were carried out at 4°C. Orbitrap mass spectrometer (Orbitrap Fusion Tribrid Mass Spectrometer, Thermo Fisher) was used to analyze the captured peptides. TM Tribrid TMMass spectrometer, Thermo Fisher) to obtain hydrogen-deuterium exchange mass spectrometry data with a resolving power of 65,000 (m / z 400). Each sample had triplicates at each time point. The mass spectrometry peak intensity average m / z centroid values (10 ppm precision) for each enzymatic peptide were calculated by the HDX Workbench software, followed by conversion to percentage deuterium incorporation. The key amino acid sequences involved in the spatial epitope were calculated, and the difference in Delta %D was determined by calculating the difference between two samples (comparing the change in percentage deuterium incorporation on the same peptide). Differences in Delta %D outside of -5 to 5% were considered to be significantly different. In addition, the HDX Workbench detected statistically significant (p < 0.05) differences between samples at each time point by student’s t test.

[0317] In certain embodiments, the anti-TfRl antibody or antigen-binding fragment thereof binds to an amino acid segment corresponding to E169-V169, I190-I201, N372-E383 of human transferrin receptor, outside of the human transferrin receptor and Tf binding domain. In certain embodiments, the TfRl antibody binds to an amino acid segment corresponding to V200-Y211 of human transferrin receptor, outside of the human transferrin receptor and Tf binding domain. In certain embodiments, the TfRl antibody binds to an amino acid segment corresponding to I274-Y282, L429-M432 of human transferrin receptor, outside of the human transferrin receptor and Tf binding domain.

[0318] Example 6. In vitro application of candidate antibodies

[0319] Using the antibody, the candidate antibody siRNA conjugate was constructed by linking the candidate antibody with a nucleic acid chain assembly structure (Linker-SiRNA). The materials used in the coupling method are described as follows: the candidate anti-human TfRl antibody; the siRNA target is human DMPK mRNA, wherein the Linker is designed as SMCC-5’NH-C6. The candidate antibody was conjugated with siRNA using the SMCC-5’NH-C6 linker. The siRNA passenger strand contains a C6-NH2 conjugate at the 5’ end. Through the conventional coupling steps of antibody reduction by DTT, oxidation, coupling, purification, etc., the candidate antibody siRNA conjugate was obtained.

[0320] In vitro cell transfection experiments were performed using candidate antibody conjugated nucleic acid drug complexes. The AOC potency assay procedure is described below: RD human rhabdomyosarcoma cell line (4201HUM-CCTCC00295) was cultured in DMEM (Gibco) containing 10% fetal bovine serum (Gibco). Antibody siRNA conjugates were diluted to a maximum dose of 20 mM. Antibody siRNA conjugates were added directly to culture media at a final concentration of 200, 100, 10, 1 and 0.1 nM siRNA either incubated directly or transfected with Invitrogen TM Lipofectamine TM 3000 (Lipo3000). Cells were seeded onto 96 well plates at 6000 cells per well prior to dosing. Conjugates were added to the wells of the 96 well plate. PBS and vehicle groups were set up as negative controls. Cells were left for 72 h at 37 °C and 5% C02 post dosing. First strand cDNA was synthesized directly from cell lysate using Superscript TM IV CellsDirect TM cDNA synthesis kit (Thermo Fisher) and cDNA samples were evaluated by qPCR using TaqMan Human Gene Expression probes (Thermo Fisher). %mRNA was calculated using the standard 2-ΔΔCT method with PBS treated cells set at 100% expression. All experiments were performed in duplicate.

[0321] The results of the experiments show that the candidate antibody siRNA conjugate is able to knock down the target gene DMPK in RD cells under Lipo3000 transfection conditions. Figure 9 The results also show that the candidate antibody is able to act as a molecular vehicle for siRNA, delivering it to the cell to exert its effect.

[0322] The above description is only the preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the principle of the present application should be included in the protection scope of the present application.

Claims

1. An anti-transferrin receptor (TfRl) antibody or antigen-binding fragment thereof, characterized in that, comprises a heavy chain variable (VH) region and a light chain variable light (VL) region, wherein the VH region comprises: a VH CDR1 comprising SEQ ID NO: 001 or a sequence with at least 80% homology to SEQ ID NO: 001; a VH CDR2 comprising SEQ ID NO: 004 or a sequence with at least 80% homology to SEQ ID NO: 004; a VH CDR3 comprising SEQ ID NO: 007 or a sequence with at least 80% homology to SEQ ID NO: 007; the VL region comprises: a VL CDR1 comprising SEQ ID NO: 010 or a sequence with at least 80% homology to SEQ ID NO: 010; a VL CDR2 comprising SEQ ID NO: 013 or a sequence with at least 80% homology to SEQ ID NO: 013; a VL CDR3 comprising SEQ ID NO: 016 or a sequence with at least 80% homology to SEQ ID NO: 016; or, the VH region comprises: a VH CDR1 comprising SEQ ID NO: 002 or a sequence with at least 80% homology to SEQ ID NO: 002; a VH CDR2 comprising SEQ ID NO: 005 or a sequence with at least 80% homology to SEQ ID NO: 005; a VH CDR3 comprising SEQ ID NO: 008 or a sequence with at least 80% homology to SEQ ID NO: 008; the VL region comprises: a VL CDR1 comprising SEQ ID NO: 011 or a sequence with at least 80% homology to SEQ ID NO: 011; a VL CDR2 comprising SEQ ID NO: 014 or a sequence with at least 80% homology to SEQ ID NO: 014; a VL CDR3 comprising SEQ ID NO: 017 or a sequence with at least 80% homology to SEQ ID NO: 017; or, the VH region comprises: a VH CDR1 comprising SEQ ID NO: 003 or a sequence with at least 80% homology to SEQ ID NO: 003; a VH CDR2 comprising SEQ ID NO: 006 or a sequence with at least 80% homology to SEQ ID NO: 006; a VH CDR3 comprising SEQ ID NO: 009 or a sequence with at least 80% homology to SEQ ID NO: 009; the VL region comprises: a VL CDR1 comprising SEQ ID NO: 012 or a sequence with at least 80% homology to SEQ ID NO: 012; a VL CDR2 comprising SEQ ID NO: 015 or a sequence with at least 80% homology to SEQ ID NO: 015; a VL CDR3 comprising SEQ ID NO: 018 or a sequence at least 80% homologous to SEQ ID NO: 018; or, the VH region comprises: a VH CDR1 comprising SEQ ID NO: 156 or a sequence at least 80% homologous to SEQ ID NO: 156; a VH CDR2 comprising SEQ ID NO: 004 or a sequence at least 80% homologous to SEQ ID NO: 004; a VH CDR3 comprising SEQ ID NO: 007 or a sequence at least 80% homologous to SEQ ID NO: 007; the VL region comprises: a VL CDR1 comprising SEQ ID NO: 133 or a sequence at least 80% homologous to SEQ ID NO: 133; a VL CDR2 comprising SEQ ID NO: 150 or a sequence at least 80% homologous to SEQ ID NO: 150; a VL CDR3 comprising SEQ ID NO: 016 or a sequence at least 80% homologous to SEQ ID NO: 016; or, the VH region comprises: a VH CDR1 comprising SEQ ID NO: 155 or a sequence at least 80% homologous to SEQ ID NO: 155; a VH CDR2 comprising SEQ ID NO: 004 or a sequence at least 80% homologous to SEQ ID NO: 004; a VH CDR3 comprising SEQ ID NO: 007 or a sequence at least 80% homologous to SEQ ID NO: 007; the VL region comprises: a VL CDR1 comprising SEQ ID NO: 132 or a sequence at least 80% homologous to SEQ ID NO: 132; a VL CDR2 comprising SEQ ID NO: 149 or a sequence at least 80% homologous to SEQ ID NO: 149; ​ ​ ​ ​ ​ ​ ​ ​ ​ a VL CDR3 comprising SEQ ID NO: 016 or a sequence having at least 80% homology to SEQ ID NO:

016.

2. The anti-TfRl antibody or antigen-binding fragment thereof of claim 1, wherein, The anti-TfRl antibody or antigen-binding fragment thereof specifically binds to transferrin receptor 1 (TfRl) but not to transferrin receptor 2 (TfR2), preferably, specifically binds to human TfRl and cynomolgus TfRl but not to mouse TfRl.

3. The anti-TfRl antibody or antigen-binding fragment thereof of claim 1, wherein, The anti-TfRl antibody or antigen-binding fragment thereof does not inhibit the binding of transferrin (Tf) to TfRl.

4. The anti-TfRl antibody or antigen-binding fragment thereof of claim 1, wherein, The anti-TfRl antibody or antigen-binding fragment thereof is a Fab, Fab', F(ab'), chemically linked F(ab'), monospecific Fab, bispecific Fab, trispecific Fab, monovalent IgG, scFv, di-scFv, bispecific di-chain antibody, trispecific tri-chain antibody, scFv-Fc, minibody, or sdAb.

5. The anti-TfRl antibody or antigen-binding fragment thereof of claim 1, wherein, The anti-TfRl antibody or antigen-binding fragment thereof binds to TfRl with a KD in the sub-nanomolar range, preferably, the anti-TfRl antibody or antigen-binding fragment thereof binds to TfRl with a KD lower than 1 nM.

6. The anti-TfRl antibody or antigen-binding fragment thereof of claim 1, wherein, The anti-TfRl antibody or antigen-binding fragment thereof further comprises a heavy chain constant region (CH), preferably, the CH is from human IgGl or mutated human IgGl, preferably, the mutation is LALA mutation and LR mutation, the amino acid sequence of the CH is set forth in SEQ ID NO:

170.

7. The anti-TfRl antibody or antigen-binding fragment thereof of claim 1, wherein, The anti-TfRl antibody or antigen-binding fragment thereof further comprises a light chain constant region (CL), preferably, the CL is kappa light chain, lambda light chain or kappa light chain, preferably, the amino acid sequence of the light chain constant region is set forth in SEQ ID NO:

171.

8. A humanized anti-transferrin receptor (TfRl) antibody or antigen binding fragment thereof, characterized in that, comprises a heavy chain variable (VH) region and a light chain variable (VL) region, the VL region independently has 1 or more amino acid substitutions or mutations in at least one CDR relative to SEQ ID NO: 197; and / or, the VH region independently has 1 or more amino acid substitutions or mutations in at least one CDR relative to SEQ ID NO:

179.

9. The humanized anti-TfRl antibody or antigen-binding fragment thereof of claim 8, wherein, The amino acid substitutions or mutations in the CDRs of the VL are at positions 32, 33, 50, 53 and 91 of SEQ ID NO: 197, and the corresponding amino acids are substituted as follows: 32Y, 32F, 32W, 33R, 50R, 53R, 91V, 91I.

10. The humanized anti-TfRl antibody or antigen-binding fragment thereof of claim 8, wherein, The amino acid substitutions or mutations in the CDRs of the VH are at positions 32, 54 and 101 of SEQ ID NO: 179, and the corresponding amino acids are substituted as follows: 32W, 54F, 101Y, 101W.

11. The humanized anti-TfRl antibody or antigen-binding fragment thereof of claim 8, wherein, The humanized anti-TfRl antibody or antigen-binding fragment thereof has at least the same affinity to the receptor TfRl as the anti-TfRl antibody or antigen-binding fragment thereof of any one of claims 1-7.

12. The humanized anti-TfRl antibody or antigen-binding fragment thereof of claim 8, wherein, The VH comprises: a VH CDR1 comprising SEQ ID NO: 001 or a sequence at least 80% homologous to SEQ ID NO: 001; a VH CDR2 comprising SEQ ID NO: 004 or a sequence at least 80% homologous to SEQ ID NO: 004; a VH CDR3 comprising SEQ ID NO: 007 or a sequence at least 80% homologous to SEQ ID NO: 007; the VL region comprises a VL CDR1 comprising SEQ ID NO: 072 or a sequence at least 80% homologous to SEQ ID NO: 072; a VL CDR2 sequence comprising SEQ ID NO: 080 or a sequence at least 80% homologous to SEQ ID NO: 080; a VL CDR3 sequence comprising SEQ ID NO: 081 or a sequence at least 80% homologous to SEQ ID NO: 081; or, the VH comprises a VH CDR1 comprising SEQ ID NO: 001 or a sequence at least 80% homologous to SEQ ID NO: 001; a VH CDR2 comprising SEQ ID NO: 004 or a sequence at least 80% homologous to SEQ ID NO: 004; a VH CDR3 comprising SEQ ID NO: 070 or a sequence at least 80% homologous to SEQ ID NO: 070; the VL region comprises a VL CDR1 comprising SEQ ID NO: 073 or a sequence at least 80% homologous to SEQ ID NO: 073; a VL CDR2 sequence comprising SEQ ID NO: 080 or a sequence at least 80% homologous to SEQ ID NO: 080; a VL CDR3 sequence comprising SEQ ID NO: 081 or a sequence at least 80% homologous to SEQ ID NO: 081; or, the VH comprises a VH CDR1 comprising SEQ ID NO: 001 or a sequence at least 80% homologous to SEQ ID NO: 001; a VH CDR2 comprising SEQ ID NO: 004 or a sequence at least 80% homologous to SEQ ID NO: 004; a VH CDR3 comprising SEQ ID NO: 007 or a sequence at least 80% homologous to SEQ ID NO: 007; the VL region comprises a VL CDR1 comprising SEQ ID NO: 071 or a sequence at least 80% homologous to SEQ ID NO: 071; a VL CDR2 sequence comprising SEQ ID NO: 078 or a sequence at least 80% homologous to SEQ ID NO: 078; a VL CDR3 sequence comprising SEQ ID NO: 081 or a sequence at least 80% homologous to SEQ ID NO:

081.

13. The humanized anti-TfRl antibody or antigen-binding fragment thereof of claim 12, wherein, the VH region comprises a sequence comprising SEQ ID NO: 179 and the VL region comprises a sequence comprising SEQ ID NO: 212; or, the VH region comprises a sequence comprising SEQ ID NO: 183 and the VL region comprises a sequence comprising SEQ ID NO: 200; or, the VH region comprises a sequence comprising SEQ ID NO: 179 and the VL region comprises a sequence comprising SEQ ID NO:

209.

14. The humanized anti-TfRl antibody or antigen-binding fragment thereof of claim 8, wherein, the humanized anti-TfRl antibody or antigen binding fragment thereof has mutations in the VL CDR1, VL CDR2, VH CDR1 and VH CDR3 regions.

15. The humanized anti-TfRl antibody or antigen-binding fragment thereof of claim 14, wherein, the amino acid sequence of the VL CDR1 is any one sequence selected from the group consisting of SEQ ID NO: 010, 095-127 and 132-147; and / or, the amino acid sequence of the VL CDR2 is any one sequence selected from the group consisting of SEQ ID NO: 013, 128-131 and 148-150; and / or, the amino acid sequence of the VH CDR1 is any one sequence selected from the group consisting of SEQ ID NO: 001, 083-091 and 152-159; and / or, the amino acid sequence of the VH CDR3 is any one sequence selected from the group consisting of SEQ ID NO: 007, 092-094, 160 and 161.

16. The humanized anti-TfRl antibody or antigen-binding fragment thereof of claim 14, wherein, the amino acid sequences of the VL CDR1, VL CDR2, VH CDR1 and VH CDR3 regions are as follows: the sequence of the VL CDR1 of the antibody with Sample ID NO. 30001-30012 is SEQ ID NO: 010, the sequence of the VL CDR2 is SEQ ID NO: 013, and the sequence of the VL CDR3 is SEQ ID NO: 016; and / or: the sequence of the VH CDR1 of the antibody with Sample ID NO. 30013-30049 is SEQ ID NO: 001, the sequence of the VH CDR2 is SEQ ID NO: 004, and the sequence of the VH CDR3 is SEQ ID NO: 007; and / or: the sequence of the VH CDR2 of the antibody with Sample ID NO. 30050-30097 is SEQ ID NO: 004, and the sequence of the VL CDR3 is SEQ ID NO:

016.

17. The humanized anti-TfRl antibody or antigen-binding fragment thereof of claim 16, wherein, the VH comprises: a VH CDR1 comprising a sequence comprising SEQ ID NO: 157 or a sequence having at least 80% homology with SEQ ID NO: 157; a VH CDR2 comprising a sequence comprising SEQ ID NO: 004 or a sequence having at least 80% homology with SEQ ID NO: 004; a VH CDR3 comprising a sequence comprising SEQ ID NO: 007 or a sequence having at least 80% homology with SEQ ID NO: 007; the VL region comprises a VL CDR1 comprising SEQ ID NO: 134 or a sequence at least 80% homologous to SEQ ID NO: 134; a VL CDR2 sequence comprising SEQ ID NO: 149 or a sequence at least 80% homologous to SEQ ID NO: 149; a VL CDR3 sequence comprising SEQ ID NO: 016 or a sequence at least 80% homologous to SEQ ID NO: 016; or, the VH comprises: a VH CDR1 comprising SEQ ID NO: 156 or a sequence at least 80% homologous to SEQ ID NO: 156; a VH CDR2 comprising SEQ ID NO: 004 or a sequence at least 80% homologous to SEQ ID NO: 004; a VH CDR3 comprising SEQ ID NO: 007 or a sequence at least 80% homologous to SEQ ID NO: 007; the VL region comprises a VL CDR1 comprising SEQ ID NO: 133 or a sequence at least 80% homologous to SEQ ID NO: 133; a VL CDR2 sequence comprising SEQ ID NO: 150 or a sequence at least 80% homologous to SEQ ID NO: 150; a VL CDR3 sequence comprising SEQ ID NO: 016 or a sequence at least 80% homologous to SEQ ID NO: 016; or, the VH comprises: a VH CDR1 comprising SEQ ID NO: 155 or a sequence at least 80% homologous to SEQ ID NO: 155; a VH CDR2 comprising SEQ ID NO: 004 or a sequence at least 80% homologous to SEQ ID NO: 004; a VH CDR3 comprising SEQ ID NO: 007 or a sequence at least 80% homologous to SEQ ID NO: 007; the VL region comprises a VL CDR1 comprising SEQ ID NO: 132 or a sequence at least 80% homologous to SEQ ID NO: 132; a VL CDR2 sequence comprising SEQ ID NO: 149 or a sequence at least 80% homologous to SEQ ID NO: 149; a VL CDR3 sequence comprising SEQ ID NO: 016 or a sequence at least 80% homologous to SEQ ID NO:

016.

18. The humanized anti-TfRl antibody or antigen-binding fragment thereof of claim 17, wherein, the VH region comprises a sequence comprising SEQ ID NO: 192 and the VL region comprises a sequence comprising SEQ ID NO: 228; or, the VH region comprises a sequence comprising SEQ ID NO: 193 and the VL region comprises a sequence comprising SEQ ID NO: 229; or, the VH region comprises a sequence comprising SEQ ID NO: 194 and the VL region comprises a sequence comprising SEQ ID NO:

230.

19. The humanized anti-TfRl antibody or antigen-binding fragment thereof of claim 17, wherein, the anti-TfRl antibody or antigen-binding fragment thereof binds to the amino acid segment of E169-V169, I190-I201, and N372-E383 of human transferrin receptor, or, to the amino acid segment of V200-Y211 of human transferrin receptor, or, to the amino acid segment of I274-Y282, and L429-M432 of human transferrin receptor.

20. A nucleic acid molecule, wherein, the nucleic acid molecule is for encoding the anti-TfRl antibody or antigen-binding fragment thereof of any one of claims 1-7 or the humanized anti-TfRl antibody or antigen-binding fragment thereof of any one of claims 8-19.

21. An expression vector comprising the nucleic acid of claim 20. the expression vector comprises the nucleic acid molecule of claim 20.

22. A host cell, characterized in that, the host cell comprises the nucleic acid molecule of claim 20 or the expression vector of claim 21.

23. A conjugate, characterized in that, the conjugate comprises a covalently linked humanized anti-TfRl antibody or antigen-binding fragment thereof of any one of claims 8-16 and an active component for targeting the active component to muscle cells or for mediating transport of the active component across the blood-brain barrier, preferably the active component is an oligonucleotide, preferably the active component is an siRNA.

24. A pharmaceutical composition comprising, the pharmaceutical composition comprises the conjugate of claim 23 and optionally a pharmaceutically acceptable excipient.

25. Use of the humanized anti-TfRl antibody or antigen-binding fragment thereof of any one of claims 8-19 or the conjugate of claim 23 or the pharmaceutical composition of claim 24 for the preparation of a medicament for treating tumor, muscle and central nervous system related diseases.

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