Anti-tfr1 antibody, and antigen-binding fragment thereof or humanized antibody thereof, and use thereof

Abstract

Using rabbit antibody technology, an anti-TfR1 monoclonal antibody having high affinity, high specificity and high blood safety is obtained by means of phage immune library construction and screening, and the anti-TfR1 monoclonal antibody is subjected to humanization and affinity maturation treatment to obtain a humanized anti-TfR1 monoclonal antibody having high affinity without inducing immunogenicity.

Description

Anti-TfR1 antibodies, antigen-binding fragments thereof or humanized antibodies thereof, and uses thereof 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 an 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 shown in SEQ ID NO: 171.

[0015] The second aspect of 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 to 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 replaced by: 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 replaced by: 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 with SEQ ID NO: 001; a VH CDR2 comprising SEQ ID NO: 004 or a sequence having at least 80% homology with SEQ ID NO: 004; a VH CDR3 comprising SEQ ID NO: 007 or a sequence having at least 80% homology with 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 with SEQ ID NO: 072; a VL CDR2 sequence comprising SEQ ID NO: 080 or a sequence having at least 80% homology with SEQ ID NO: 080; and a VL CDR3 sequence comprising SEQ ID NO: 081 or a sequence having at least 80% homology with 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] 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;

[0034] and / or as shown in Table 2:

[0035] Table 2

[0036] 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;

[0037] and / or as shown in Table 3:

[0038] Table 3

[0039] 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.

[0040] 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:

[0041] 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;

[0042] S2. Designing a humanized antibody using CDR grafting, grafting the CDR of the parent antibody into a human framework sequence to obtain a chimeric antibody, while comparing the sequence of the chimeric antibody and the parent antibody in CDR, residues, stem loop structure, and the vicinity of the hydrophobic core;

[0043] 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;

[0044] S4. Based on the selected antibodies with corresponding mutation combinations, obtaining antibodies with comparable affinity to the positive control antibody through single-point saturation mutation of the CDR region;

[0045] S5. Based on the results of single-point mutation, further improving the affinity through mutation combination;

[0046] S6. Based on the selected antibodies with corresponding mutation combinations, improving the affinity of the monoclonal antibody through the yeast (Sc) display platform technology and block mutation method, thereby obtaining antibodies with comparable affinity to the positive control antibody.

[0047] Further, the step S5 comprises the following steps:

[0048] 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, wherein the mutation can introduce all 20 combinations of amino acids at each amino acid position;

[0049] 2) Enriching high-affinity clones in the library through 2-3 rounds of target protein FACS sorting, and selecting 48 clones for sequencing each round to monitor the enrichment of wild type;

[0050] 3) After sorting, selecting yeast clones and performing single-concentration target protein FACS experiment verification, and sequencing the clones with improved FACS signal value.

[0051] 4) Select 10 polypeptide clones with different sequences and sort them using the target protein FACS experiment with serial dilution.

[0052] A fourth aspect of the present invention 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.

[0053] A fifth aspect of the present invention provides an expression vector comprising nucleic acid molecules as described above.

[0054] A sixth aspect of the present invention provides a host cell comprising a nucleic acid molecule as described above or an expression vector as described above.

[0055] A seventh aspect of the present invention provides a conjugate comprising a covalently linked humanized TfR1 antibody or its antigen-binding fragment as described above and an active component, wherein the humanized TfR1 antibody or its antigen-binding fragment is used to target the active component to muscle cells or to transport proteins across the blood-brain barrier. Preferably, the active component is an oligonucleotide, and more preferably, the active component is siRNA.

[0056] An eighth aspect of the present invention provides a pharmaceutical composition comprising, as described above, a conjugate and optionally pharmaceutically acceptable excipients.

[0057] The ninth aspect of the present invention provides the use of the humanized anti-TfR1 antibody as described above or its antigen-binding fragment, or the conjugate as described above, or the pharmaceutical composition as described above in the preparation of a medicament for treating tumors, muscle and central nervous system-related diseases.

[0058] The tenth aspect of the present invention provides the use of the anti-TfR1 antibody as described above or its antigen-binding fragment thereof, or the humanized anti-TfR1 antibody as described above or its antigen-binding fragment thereof, in the preparation of a kit for detecting the presence or level of TfR1 in a sample.

[0059] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0060] This invention utilizes rabbit anti-antibody technology to obtain anti-TfR1 monoclonal antibodies with high affinity, high specificity, and high blood safety through phage immunotherapy library screening. The anti-TfR1 monoclonal antibodies are then humanized and their affinity matured (using single-point mutation, combinations of single-point mutations, yeast display technology, and block mutation methods) to obtain a humanized anti-TfR1 monoclonal antibody with high affinity that does not induce immunogenicity. Attached Figure Description

[0061] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the description of the embodiments of the present application. Obviously, the drawings in the following description only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor.

[0062] Figure 1 is a flow chart of screening of anti-human TfR1 antibody;

[0063] Figure 2 is the IC50 result of competition experiment of the preferred three strains of antibodies;

[0064] Figure 3 is the result of the first round of library construction - the first round of FACS screening;

[0065] Figure 4 is the result of the first round of library construction - the second round of FACS screening;

[0066] Figure 5 is the result of the first round of library construction - the third round of FACS screening;

[0067] Figure 6 is the result of the second round of library construction - the first round of FACS screening;

[0068] Figure 7 is the result of the second round of library construction - the second round of FACS screening;

[0069] Figure 8 is the result of KD-FACS;

[0070] Figure 9 is the effect diagram of knockdown of the candidate antibody siRNA conjugate in RD cells. DETAILED DESCRIPTION

[0071] In order to make the technical problems to be solved by the present application, the technical solutions and the beneficial effects more clear and obvious, the following will be further described in detail in combination with the embodiments and the drawings. It should be understood that the specific embodiments described here are only used to explain the present application, and are not used to limit the present application.

[0072] Definition of terms

[0073] The following provides other aspects of the present disclosure, including the description of the definition of terms. Unless otherwise indicated or defined, all terms used have the usual meaning in the art, which will be understood by those skilled in the art.

[0074] 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 into a monomeric molecule of four polypeptide chains. There are two types of light chain, kappa and lambda, and five types of heavy chain, mu, delta, gamma, epsilon, and alpha. In some embodiments, the immunoglobulin has no light chain 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.

[0075] 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 to both the relevant amino acid sequence as well as the nucleic acid sequence or nucleotide sequence encoding the sequence.

[0076] 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.

[0077] 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. In some embodiments, it refers to the constant region portion of an antibody (i.e., the CH and CL regions) or to an antibody that is entirely encoded by human antibody genes. In some embodiments, humanized anti-TfR antibodies and antigen-binding portions are provided.

[0078] 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), each monoclonal antibody is directed against a single determinant on the antigen.

[0079] The term "CDR" refers to antibody complementarity determining regions or complementarity determining segments, located in the variable region of immunoglobulins, which complement 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, an "immunoglobulin variable domain" refers to an immunoglobulin domain composed of four "framework regions" (FRs), FR1, FR2, FR3, and FR4, which are of relatively conserved sequence, and three "complementarity determining regions" (CDRs), CDR1, CDR2, and CDR3, which are of relatively hypervariable sequence, interspersed therebetween. That is, each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: 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 Kabat definition (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)), the IMGT definition, the Chothia definition, and the like. In some embodiments, a complementarity determining region can refer to the CDR region defined by any of the definition methods.

[0080] The term "framework region" or "framework sequence" refers to the relatively conserved regions of an immunoglobulin variable region that are outside the complementarity determining regions. There are four framework regions in each of the heavy and light chains, denoted FR1, FR2, FR3, and FR4. Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: 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.

[0081] 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 for 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.

[0082] 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, for example, 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.

[0083] 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.

[0084] 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.

[0085] 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).

[0086] 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:

[0087] SEQ ID NO: 175

[0088] Target gene: human TfRl

[0089] >NP_003225.2 transferrin receptor protein 1 isoform 1 [Homo sapiens]

[0090] Example of the amino acid sequence of the non-human primate transferrin receptor, the corresponding sequence is as follows:

[0091] Generation of stable CHO-K1 / cTfR1 cell line

[0092] Target gene: cyno TfR1

[0093] SEQ ID NO:174

[0094] >XP_045243212.1 transferrin receptor protein 1[Macaca fascicularis]

[0095] The term "affinity" refers to the degree or strength of antibody binding to an epitope or antigen. The dissociation constant K... d Affinity constant K a KD is a quantitative measure of affinity. KD is the equilibrium dissociation constant between the antibody and its antigen, and is the antibody dissociation rate K. d (The rate at which it dissociates from the antigen) is related to the antibody association rate K. a The ratio of the rate at which an antibody binds to its antigen to the rate at which it binds to the antigen. The binding of an antibody to its antigen is a reversible process, and the rate of the binding reaction is directly proportional to the concentration of the reactant. K d The smaller the K value, the greater the affinity of the antibody for its target. Most antibodies have a K value of [missing value]. d The values ​​are all in the low micromolar range (10 -6 ) to nanomoles (10 -7 Up to 10 -9 The range of ) is generally considered to be high affinity antibodies in the low nanomolar range (10). -9 Within ), while very high affinity antibodies are present in picomoles (10). -12 Within the range of ).

[0096] The term "antibody affinity maturation" refers to a normal immune function state in the body. In humoral immunity, the average affinity of antibodies produced in a secondary immune response is higher than that of the primary immune response; this phenomenon is called antibody affinity maturation. The "affinity maturation" discussed in this article refers to in vitro antibody affinity maturation techniques, primarily including antibody library construction and screening techniques.

[0097] This invention utilizes rabbit anti-antibody technology to obtain anti-TfR1 monoclonal antibodies with high affinity, high specificity, and high blood safety through phage immunotherapy library screening.

[0098] 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 light chain (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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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 not TfR2. In certain embodiments, the anti-TfRl antibody or antigen-binding fragment thereof specifically binds transferrin receptor from human, non-human primates, mouse, rat, rabbit, etc. 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.

[0105] In certain embodiments, the anti-TfRl antibody or antigen-binding fragment thereof selectively specifically binds transferrin receptor 1 (TfRl) or a homodimer thereof but not TfR2. In certain embodiments, the antibody specifically binds to the apical domain of TfRl.

[0106] In certain embodiments, the anti-TfRl antibody or antigen-binding fragment thereof does not inhibit the binding of transferrin (Tf) to TfRl upon binding to TfRl. In certain embodiments, the anti-TfRl antibody or antigen-binding fragment thereof does not significantly affect the binding of transferrin (Tf) and TfRl at a given drug concentration. In certain embodiments, the anti-TfRl antibody or antigen-binding fragment thereof does not inhibit the binding of HFE-beta2-microglobulin to TfRl upon binding to TfRl.

[0107] 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.

[0108] In certain embodiments, the antigen binding fragment includes, but is not limited to, a Fab, a Fab', a F(ab'), a chemically linked F(ab'), a monospecific Fab, a bispecific Fab, a trispecific Fab, a monovalent IgG, a scFv (single chain variable fragment), a di-scFv (bivalent scFv), a bispecific diabody, a trispecific triabody, a scFv-Fc, a minibody, or a sdAb (single domain antibody).

[0109] 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 of less than 1 nM.

[0110] 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 a human IgGl constant region is given below:

[0111] In certain embodiments, the heavy chain constant region comprises a mutated human IgGl constant region. In certain embodiments, introducing LALA mutations (replacing residues Leu234 Leu235 with Ala234 and Ala235) and LR mutations (replacing 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.

[0112] 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:

[0113] In certain embodiments, other antibody heavy and light chain constant regions are well known in the art, such as those provided in the IMGT database (www.imgt.org).

[0114] 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.

[0115] In certain embodiments, the heavy chain variable region and / or the light chain variable region further comprises a humanized framework region that 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.

[0116] 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.

[0117] In the present application, 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 that has at least equal or better affinity than the non-mutant chimeric antibody and does not cause or causes a smaller immune response.

[0118] 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 the CDRs of 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.

[0119] 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.

[0120] In certain embodiments, the humanized anti-TfRl antibody or antigen-binding fragment thereof has at least equal affinity for the receptor TfRl as the chimeric antibody.

[0121] 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.

[0122] 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;

[0123] 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: 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: 071 or a sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologous thereto; a VL CDR2 sequence comprising SEQ ID NO: 078 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.

[0124] 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.

[0125] 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.

[0126] 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:

[0127] Table 1

[0128] 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;

[0129] and / or:

[0130] As shown in Table 2:

[0131] Table 2

[0132] 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;

[0133] and / or:

[0134] As shown in Table 3:

[0135] Table 3

[0136] 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.

[0137] In some 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:

[0138] 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;

[0139] 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 sequences of the chimeric antibody and the parent antibody in CDR, residues, stem loop structure, near the hydrophobic core;

[0140] S3. Design to gradually incorporate one or more reverse mutations in the grafted antibody sequence, select antibodies with corresponding mutation combinations for affinity maturation according to the affinity detection results and the number of accumulated mutations;

[0141] S4. Based on the selected antibodies with corresponding mutation combinations, obtain antibodies with comparable affinity to the Xiangshen antibody by single-point saturation mutation of CDR regions;

[0142] S5. Based on the results of single-point mutation, further improve the affinity by mutation combination;

[0143] S6. Based on the selected antibodies with corresponding mutation combinations, improve the affinity of monoclonal antibodies by yeast (Sc) display platform technology and block mutation method, so as to obtain antibodies with comparable affinity to the Xiangshen antibody.

[0144] In some embodiments, the step S5 comprises the following steps:

[0145] 1) Design a special primer, and use 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 acids combinations at each amino acid position;

[0146] 2) Enrich high-affinity clones in the library by 2-3 rounds of target protein FACS sorting, and select 24 clones for sequencing each round to monitor the enrichment of wild type;

[0147] 3) After sorting, select yeast clones and perform single-concentration target protein FACS experiment verification, and sequence the clones with improved FACS signal value;

[0148] 4) Select 10 polypeptide clones with different sequences, and perform sorting by gradient dilution target protein FACS experiment.

[0149] In some 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 some embodiments, the nucleic acid molecule can include DNA and RNA sequences.

[0150] In some embodiments, the present application provides an expression vector comprising a nucleic acid molecule as described above. In some 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 some embodiments, the expression vector is well known to those skilled in the art, including but not limited to: plasmids, bacteriophages (such as lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), viral vectors (such as baculovirus vectors, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, or herpes viruses (such as herpes simplex virus), etc.). In an embodiment of the present application, the vector can be specifically pUC57 or pCGS3.

[0151] 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 (e.g., a yeast cell, an Aspergillus), an animal cell (e.g., a mammalian cell, an insect cell) or a prokaryotic cell. In certain embodiments, the host cell can be specifically ExpiCHO-S cells.

[0152] 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 (e.g., 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.

[0153] In certain embodiments, the present application provides a conjugate product comprising a humanized TfRl antibody or antigen-binding fragment thereof as described above covalently linked to an active payload for targeting the active payload to muscle cells or transport of the active payload across the blood-brain barrier mediated protein. Preferably, the active payload is an oligonucleotide, preferably, the active payload is an siRNA.

[0154] 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.

[0155] In certain embodiments, the present application provides use of a humanized anti-TfRl antibody or 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.

[0156] 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).

[0157] In certain embodiments, the present application provides use of an anti-TfRl antibody or antigen binding fragment thereof as described above or a humanized anti-TfRl antibody or antigen binding fragment thereof as described above in the manufacture of a kit for detecting the presence or level of TfRl in a sample.

[0158] The following is illustrated in conjunction with specific examples:

[0159] 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 commercially. 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.

[0160] Example 1 Preparation of rabbit-derived monoclonal antibody against human TfRl

[0161] Two New Zealand white rabbits were immunized with human TfRl as an immunogen to establish a rabbit anti-immune library. Rabbit-derived monoclonal antibody against human TfRl was prepared by 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 vector to become chimeric Fab phage library with a library capacity of 2 x 10 8After the construction of the phage display library, the antibodies were enriched by 4 rounds of panning against human 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 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 immobilized phase. Antibody clones that were simultaneously reactive with human and cynomolgus monkey TfR1 were obtained by phage ELISA, and antibody-expressing monoclonal antibodies were selected and sequenced. A total of 262 positive monoclonal antibodies were obtained.

[0162] The 262 rabbit-derived antibodies enriched by the phage display library were analyzed for human and cynomolgus monkey TfR1 binding activity and sequencing, and based on the sequencing results of the 262 antibody positive monoclonals, 40 antibodies were selected, and based on the sequence stability requirement, the last amino acid of VH CDR3 of 30775 sequence was mutated from P→A to obtain 30775A sequence, a total of 41 antibody sequences were subjected to CHO cell transient transfection and expression purification.

[0163] 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.

[0164] The following is the sequence of the human IgG1 antibody heavy chain constant region for grafting:

[0165] The following is the sequence of the human IgG1 antibody light chain constant region for grafting:

[0166] The human and cynomolgus monkey TfR1 affinity kinetic parameters of the chimeric antibodies were detected, the blocking activity of the chimeric antibodies on the binding of Tf to TfR was detected, and the binding activity of the chimeric antibodies to human TfR2 was detected, and based on the detection results, the chimeric antibodies 28158, 28162 and 28169 with high human and cynomolgus monkey TfR1 affinity, no effect on the binding activity of Tf to TfR, and no binding to human TfR2 were selected for further evaluation. The third amino acid of VL CDR3 of 28158 was mutated from C→S to meet the stability requirement of the antibody.

[0167] The light and heavy chain variable regions of the three antibodies containing CDRs were chimerized into human IgG constant regions, and further compared in terms of transient expression, purity, affinity, competition experiments, and sequences (Table 4). Comparison was made with positive control Var2iii (an antibody capable of binding to human TfR1 but not blocking Tf binding) and negative control AF2474 (a polyclonal antibody capable of binding to human TfR1 and blocking Tf binding) (Table 4).

[0168] Table 4 Comparison of the three preferred rabbit-derived antibodies

[0169] wherein VH_CDR1, VH_CDR2, VH_CDR3 are three complementarity determining regions (CDRs) in the heavy chain variable region; VL_CDR1, VL_CDR2, VL_CDR3 are three complementarity determining regions (CDRs) in the light chain variable region.

[0170] According to the results of expression, purity, affinity, competition experiment and sequence analysis 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.

[0171] Table 5 Statistics of CDR sequences of heavy and light chains of rabbit-derived monoclonal antibodies

[0172] 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 antibody 28158 were spliced into the determined framework regions, i.e. to form the precursor light and heavy chains of humanized rabbit monoclonal antibodies. According to the amino acid sequences of the light and heavy chains, the nucleotide sequences encoding them were optimized (reference https: / / sg.idtdna.com / pages / tools / codon-optimization-tool), such as avoiding commonly used restriction 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:

[0173] Selection of human antibody framework

[0174] The selection method includes: by analyzing the framework region sequence of the existing humanized antibody, evaluating the similarity of the existing human germline and the framework region of antibody 28158, and evaluating the amino acid of each position by means of 3D structure model. For antibody 28158, human germline IGHV3-53*01, IGKV1-12*01 is selected as the starting human receptor framework.

[0175] Design of humanized highly close VH and VL

[0176] Based on the key points such as classic residues, interaction loop region, core region, mutation hot spot, etc., suitable sites are selected for combination design to obtain the variable region amino acid sequence of humanized antibody. Five humanized amino acid sequences are designed for the heavy chain 28158_VH gene, and two humanized amino acid sequences are designed for the light chain 28158_VL gene. The combination of VL and VH generates 10 mutants, which are humanized rabbit monoclonal candidate clones, used for subsequent recombinant antibody expression plasmid construction.

[0177] The five humanized amino acid sequences designed for the heavy chain 28158_VH gene are as follows:

[0178] >28158_VH1 (SEQ ID NO: 177)

[0179] >28158_VH2 (SEQ ID NO: 178)

[0180] >28158_VH3 (SEQ ID NO: 179)

[0181] >28158_VH4 (SEQ ID NO: 180)

[0182] >28158_VH5 (SEQ ID NO: 181)

[0183] The two humanized amino acid sequences designed for the light chain 28158_VL gene are as follows:

[0184] >28158_VL1 (SEQ ID NO: 196)

[0185] >28158_VL2 (SEQ ID NO: 197)

[0186] Research prediction structure analysis mutation list

[0187] The sequences were analyzed for PTM and homology modeling of the antibodies was performed using MOE software to analyze the risk of stability that might be caused by mutations in different amino acids, the risk of maintaining the binding affinity to the target, and the risk of increasing immunogenicity. For example, in the initial design, the C in the FR3 region was mutated from C to P to remove the extra rabbit disulfide bond. In addition, the C in the unpaired cysteine VL CDR3 region was also mutated from C to S. These attempts were found to weaken the binding affinity in the test constructs, so a subsequent round of design was performed. The results of the sequence analysis are shown in Table 6.

[0188] Table 6 Sequence analysis results of preferred antibodies

[0189] Based on the results of the sequence analysis, the antibody 28158_VH was modified, for example, to remove the glycosylation sites introduced in VH2 and VH5. Homology modeling was performed on the selected Fv fragments. The sequences were searched against the IgBLAST database to determine the best templates for the Fv fragments. A best template matching the human framework sequence was selected for VH / VL, for example, the heavy chain corresponding to the human framework sequence: IGHV3-53*01 (similarity 58.5%), and the light chain corresponding to the human framework sequence: IGKV1-12*01 (similarity 67.7%). A humanized antibody was designed using CDR grafting, in which the CDRs of the parent antibody were grafted into a human framework sequence to obtain a chimeric antibody. At the same time, the sequences of the chimeric antibody and the parent antibody were compared in terms of CDRs, residues, stem loop structures, and the vicinity of the hydrophobic core. One or more reverse mutations were gradually incorporated into the grafted antibody sequence. The humanization ratio (Humanness) was calculated based on the number of accumulated mutated amino acids and the human framework sequence. The affinity of the humanized antibody was detected by SPR. Based on the results of the affinity detection (Table 7) and the number of accumulated mutations (Table 8), the combination of 28158_VH3 and 28158_VL2 was selected for affinity maturation.

[0190] Table 7 Affinity determination results of selected antibodies

[0191] Table 8 Analysis of humanization mutations in the variable region sequences of selected antibodies

[0192] Example 2 Antibody affinity maturation - single point mutation

[0193] The purpose of the experiment was to obtain an antibody with comparable affinity to the Xanthium antibody based on antibody 28158_VH3+VL2 through single point saturation mutation in the CDR region. The sequence of antibody 28158_VH3+VL2 is shown in Table 9 below:

[0194] Table 9 Antibody 28158_VH3+VL2 sequence

[0195] Based on the antibody 28158_VH3+VL2 sequence, the amino acids at each site of the CDR region were saturated mutated, and the expression was cultured in CHO-S cells. The results of the affinity for Human TfRl antigen of the well-expressed saturated mutation sequence were determined using ELISA experiments, and the steps of the ELISA experiments are described as follows: Biotin-labeled Human TfRl antigen (0.3 μg / ml, CBS, 100 μl / well) and BSA (10 μg / ml, CBS, 100 μL / well) were incubated in an enzyme-labeled plate overnight. After 50 μl FASEBA supernatant (100 μl / well) was diluted with 50 μl 0.1% PBST, it was added to the antigen-coated wells 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 with 1M hydrochloric acid, and the absorbance OD value was measured at a wavelength of OD 450 nm using an enzyme-labeled instrument (Table 6). According to the absorbance OD value, and according to the formula Ratio = OD (Mutation) / OD (WT), the ratio was calculated, and according to the ratio ≥ 1.75 and the top 5 of each plate, 69 antibodies were selected, and sequencing found that some of them were repeated sequences, and the results are shown in Table 10.

[0196] Table 10 Antibody affinity maturation single point mutation results

[0197] After sequencing the 69 antibodies, repeated sequences were removed, and 47 antibodies CDR region single point mutation designs were obtained. Among them, the antibody names 23472-23480 only performed single point mutation on VH CDR1, and the other CDR and framework region sequences were consistent with 28158_VH3+VL2, and the mutation sites of VH CDR1 are shown in Table 11; among them, the antibody names 23481-23493 only performed single point mutation on VH CDR2, and the other CDR and framework region sequences were consistent with 28158_VH3+VL2, and the mutation sites of VH CDR2 are shown in Table 12; among them, the antibody names 23494-23496 only performed single point mutation on VH CDR3, and the other CDR and framework region sequences were consistent with 28158_VH3+VL2, and the mutation sites of VH CDR3 are shown in Table 13.

[0198] Table 11 Mutation design of VH CDR1

[0199] Table 12 Mutation design of VH CDR2

[0200] Table 13 Mutation design of VH CDR3

[0201] The antibody names 23500-23513 only carry out single-point mutation on VL CDR1, other CDR and skeleton region sequences are consistent with 28158_VH3+VL2, and the mutation sites of VL CDR1 are shown in Table 14; wherein the antibody names 23514-23534 only carry out single-point mutation on VL CDR2, other CDR and skeleton region sequences are consistent with 28158_VH3+VL2, and the mutation sites of VL CDR2 are shown in Table 15; wherein the antibody names 23537-23541 only carry out single-point mutation on VL CDR3, other CDR and skeleton region sequences are consistent with 28158_VH3+VL2, and the mutation sites of VL CDR3 are shown in Table 16.

[0202] Table 14 Mutation design of VL CDR1

[0203] Table 15 Mutation design of VL CDR2

[0204] Table 16 Mutation design of VL CDR3

[0205] The affinity of the above-mentioned antibodies and human TfR1 was detected by surface plasmon resonance (SPR), and the results are shown in Table 17.

[0206] Table 17 Affinity detection results of single-point mutation

[0207] According to the affinity results, the mutation sites that have greater influence 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.

[0208] Table 18 Mutation hot spot region design

[0209] Example 3 Antibody affinity maturation-single-point mutation combination

[0210] The experimental purpose is to further improve the affinity by combining mutations based on the results of single-point mutations. The heavy chain variable region 32nd, 54th and 101st sites and the light chain variable region 32nd, 33rd, 50th, 53rd and 91st sites were selected for combination according to the previous single-point mutation. Table 19 shows the design of the combination mutation of the antibody heavy chain variable region, and Table 20 shows the design of the combination mutation of the antibody light chain variable region. According to the sequence design of Table 15 and Table 16, the CHO cell transient expression purification was used, and the human TfR1 affinity was detected by surface plasmon resonance (SPR), and the results are shown in Table 21.

[0211] Table 19 Combination mutation design of antibody heavy chain CDR region

[0212] Table 20 Combination mutation design of antibody light chain CDR region

[0213] Table 21 Antibody combination mutation affinity results

[0214] According to the determination results of KD and Rmax, 23669, 23673 and 23699 were selected as the preferred 3 antibodies, and the specific sequences are referred to Table 22.

[0215] Table 22 Sequence of the preferred 3 antibodies

[0216] Example 4 Antibody affinity maturation-yeast display technology and block mutation method

[0217] The experimental purpose is to improve the affinity of the monoclonal antibody by using EBY100 yeast based on the antibody 28158_VH3+VL2(WT) through the yeast (Sc) display platform technology and the 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) for affinity maturation, the antibody Fab fragment (scFab) antibody library was constructed.

[0218] High affinity protein variants were isolated from the yeast library by FACS. First round library construction: high affinity protein variants were isolated from the yeast library by FACS, using 111 nM biotinylated antigen for 1 hour, sorting with cMyc-RPE and SA647 staining, instrument detection and analysis using FACS specific tubes, referring to FIG. 3. Then different concentrations of biotinylated antigen were used for 30 min at room temperature, unbound antigen was washed away, the temperature and time of incubation were adjusted according to the results of the previous sorting, and yeast cells at the appropriate time point were selected for flow sorting, library clones were collected for culture after each round of sorting, and the next round of sorting was performed, and finally a total of 3 rounds of sorting were performed (FIGS. 3-5). Yeast strains were selected from each antibody library for sequencing analysis, and finally several unique mutant clones were obtained from the heavy chain antibody library and the light chain antibody library (sequences are shown in Table 23).

[0219] Analysis of three rounds of FACS screening sequence results, a total of 49 clones were obtained. Antibody names 30001-30012 only have VH CDR1 and VH CDR3 region mutations, other CDR and framework region sequences are consistent with 28158_VH3+VL2, as shown in Table 23. Antibody names 30013-30049 only have VL CDR1 and VL CDR2 region mutations, other CDR and framework region sequences are consistent with 28158_VH3+VL2, as shown in Table 24.

[0220] Table 23 First round library sorting single clone_VH sequence

[0221] Table 24 First round library sorting single clone_VL sequence

[0222] Second round library construction: using the obtained VH CDR1, VH CDR3, VL CDR1 and VL CDR2 sequences and the same method as the first round library, the second round of combined library construction was carried out, and finally a total of 2 rounds of sorting were performed (FIGS. 6-7). Analysis of two rounds of FACS screening sequence results, a total of 48 clones were obtained. Antibodies of antibody names 30050-30097 have VL CDR1, VL CDR2, VH CDR1 and VH CDR3 region mutations, other CDR and framework region sequences are consistent with 28158_VH3+VL2, as shown in Table 25.

[0223] Table 25 Second round library sorting single clone sequence

[0224] According to the MFI results of 48 clones (Table 26), 14 clones better than the Anemone Var2iii were screened for KD-FACS detection. The detection results are shown in Figure 8 and Table 27.

[0225] Table 26 MFI results of 48 clones

[0226] Table 27 KD-FACS detection results

[0227] According to the KD-FACS results, 11 clones (Table 28) were selected for recombinant expression verification compared with the Anemone 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.

[0228] Table 28 11 clones for recombinant expression verification

[0229] The 11 clones were 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 bind to Human / Cyno TfR1, and none of them binds to TfR2. Among them, clones 30068, 30082, 30052 and 30054 have relatively high expression levels.

[0230] Table 29 Quality control results of 11 clones

[0231] Then, using the polyclonal antibody AF2474 of TfR1 as Anemone, and using the clinically tested monoclonal antibody Var2iii as an internal reference, the competition ability of 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.

[0232] Table 30 Competition ability of 11 antibodies with transferrin

[0233] According to the above screening results, the sequences of 30068, 30082 and 30054 were finally selected, as shown in Table 31.

[0234] Table 31 Sequences of the three antibodies finally selected

[0235] Example 5. Epitope of candidate antibodies binding to hTfRl

[0236] 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:

[0237] Identification of antigen antibody two-dimensional polypeptide profile: enzymatic peptides of proteins 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). MS / MS data files were processed by Proteome Discover software for peptide identification. TM Tribrid TM Mass Spectrometer, Thermo Fisher). MS / MS data files were processed by Proteome Discover software for peptide identification.

[0238] Preparation of hydrogen-deuterium exchange mass spectrometry samples: 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) were placed at 4°C for 1 hour, respectively, to form a stable state of complex. Then 5 microliters of sample were 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 both carried out at 4°C. Orbitrap mass spectrometer (Orbitrap Fusion Tribrid Mass Spectrometer, Thermo Fisher) was used to analyze the captured peptides. TM Tribrid TM Mass 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 proteolytic 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.

[0239] 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.

[0240] Example 6. In vitro application of candidate antibodies

[0241] 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.

[0242] In vitro cell transfection experiments were performed using candidate antibody conjugated nucleic acid drug complexes. The AOC potency assay procedure is described as follows: 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% CO2 after 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 to 100% expression. All experiments were performed in duplicate.

[0243] 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). This also shows that the candidate antibody is able to act as a molecular carrier for siRNA, delivering it to the cell to exert its effect.

[0244] 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 NOs: 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 NOs: 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 NOs: 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 NOs: 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 a sequence comprising SEQ ID NO: 134 or a sequence having at least 80% homology with 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 segments E169-V169, I190-I201, and N372-E383 of human transferrin receptor, the anti-TfRl antibody or antigen binding fragment thereof binds to the amino acid segments E169-V169, I190-I201, and N372-E383 of human transferrin receptor, or an amino acid segment of V200-Y211 bound to human transferrin receptor, or I274-Y282 and L429-M432 amino acid segments bound to human transferrin receptor.

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

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

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

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

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

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