Humanized antibody targeting tfr1 and use thereof

Abstract

Provided are a humanized antibody targeting TfR1 and the use thereof. A new humanized anti-TfR1 antibody, which can bind to a TfR1 antigen with high specificity, and has high affinity and biological activity, good endocytic activity, low immunogenicity and structural stability. The humanized antibody can be used in the preparation of a drug for preventing or treating TfR1-associated diseases.

Description

Humanized antibodies targeting TfR1 and uses thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biomedicine, and in particular relates to humanized antibodies targeting TfR1 and uses thereof. BACKGROUND

[0002] High expression of transferrin receptor 1 (TfR1) is closely related to the occurrence and development of a variety of tumors, including AML, ALL, lymphoma, multiple myeloma, breast cancer, gastric cancer, glioblastoma, prostate cancer, uroepithelial bladder cancer, pancreatic cancer, esophageal cancer, colorectal cancer, ovarian cancer, liver cancer and other malignant tumors. These tumor cells need a large amount of iron to maintain their high proliferation rate, and TfR1, as a key receptor for iron uptake, plays an important role in the uptake and transport of iron in tumor cells.

[0003] Therefore, based on the high expression of TfR1, researchers have developed various targeted delivery systems to deliver chemotherapeutic drugs, toxins, nucleic acids and the like directly into tumor cells, thereby improving the therapeutic effect and reducing side effects. In addition, directly inhibiting the function of TfR1 is also considered a potential tumor treatment strategy.

[0004] However, current research still needs to be improved. For example, although TfR1 has potential as a target for tumor treatment, the efficiency of current TfR1-based targeted delivery systems in practical applications still needs to be improved.

[0005] Moreover, antibodies or antibody conjugate drugs based on TfR1 still need to be improved in terms of stability and immunogenicity.

[0006] Therefore, there is an urgent need in the art to develop an antibody targeting TfR1 with good affinity, good endocytosis activity and low immunogenicity. SUMMARY

[0007] The present application provides an antibody targeting TfR1 with good affinity, good endocytosis activity and low immunogenicity.

[0008] In a first aspect of the present application, a humanized antibody targeting TfR1 or an antigen binding fragment thereof is provided, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region being selected from the group consisting of:

[0009] (a1) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 9;

[0010] (a2) a heavy chain variable region derived from the sequence set forth in SEQ ID NO: 9 having the function of the heavy chain variable region of (a1) formed by substitution, deletion, modification, and / or addition of at least one (e.g., 1 to 20, preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 8, more preferably 1 to 3, most preferably 1 or 2) amino acid residues of the sequence set forth in SEQ ID NO: 9;

[0011] (a3) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 11;

[0012] The light chain variable region is selected from the group consisting of:

[0013] (b1) a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 15;

[0014] (b2) a light chain variable region derived from the sequence set forth in SEQ ID NO: 15 having the function of the light chain variable region of (b1) formed by substitution, deletion, modification, and / or addition of at least one (e.g., 1 to 20, preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 8, more preferably 1 to 3, most preferably 1 or 2) amino acid residues of the sequence set forth in SEQ ID NO: 15;

[0015] (b3) a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 17.

[0016] In another preferred embodiment, the substitution, deletion, modification, and / or addition of amino acid residues occurs in the CDR region and / or in the framework region.

[0017] In another preferred embodiment, the substitution, deletion, modification, and / or addition of amino acid residues occurs in the CDR region.

[0018] In another preferred embodiment, the mutation of the heavy chain variable region is selected from the group consisting of G56A, H59L, T28F, T28L, T28D, T28S, G56H, G56T, G56R, H59L, H59W, D101P, D101H, or a combination thereof.

[0019] In another preferred embodiment, the mutation of the light chain variable region is selected from the group consisting of N31M, G33W, S32E, S32L, Q95M, N31E, S32T, G33D, G33H, Q95L, Q95F, or a combination thereof.

[0020] In another preferred aspect of the present application, a humanized antibody or antigen binding fragment thereof targeting TfR1 is provided, comprising a heavy chain variable region and a light chain variable region selected from the group consisting of:

[0021] (1) a heavy chain variable region of an amino acid sequence as set forth in SEQ ID NO: 9, and a light chain variable region of an amino acid sequence as set forth in SEQ ID NO: 15;

[0022] (2) a heavy chain variable region of an amino acid sequence as set forth in SEQ ID NO: 9, and a light chain variable region of an amino acid sequence as set forth in SEQ ID NO: 17;

[0023] (3) a heavy chain variable region of an amino acid sequence as set forth in SEQ ID NO: 11, and a light chain variable region of an amino acid sequence as set forth in SEQ ID NO: 15;

[0024] (4) a heavy chain variable region of an amino acid sequence as set forth in SEQ ID NO: 9 and having the following mutations on SEQ ID NO: 9: G56A, and a light chain variable region of an amino acid sequence as set forth in SEQ ID NO: 15 and having the following mutations on SEQ ID NO: 15: N31M; or

[0025] a heavy chain variable region of an amino acid sequence as set forth in SEQ ID NO: 19, and a light chain variable region of an amino acid sequence as set forth in SEQ ID NO: 21;

[0026] (5) a heavy chain variable region of an amino acid sequence as set forth in SEQ ID NO: 9 and having the following mutations on SEQ ID NO: 9: G56A, and a light chain variable region of an amino acid sequence as set forth in SEQ ID NO: 15 and having the following mutations on SEQ ID NO: 15: G33W; or

[0027] a heavy chain variable region of an amino acid sequence as set forth in SEQ ID NO: 19, and a light chain variable region of an amino acid sequence as set forth in SEQ ID NO: 23. In another preferred embodiment, the antibody is a humanized antibody.

[0028] In another preferred embodiment, the antibody is specific for binding to TfR1.

[0029] In another preferred embodiment, the antibody has a KD value (M) of 2.0E-10 to 2.0E-11 for the affinity to human TfR1.

[0030] In another preferred embodiment, the antibody is a diabody, or a single chain antibody.

[0031] In another preferred embodiment, the antibody is a single chain antibody.

[0032] In another preferred embodiment, the antibody is a monoclonal antibody.

[0033] In another preferred embodiment, the antibody is a bispecific antibody.

[0034] In another preferred embodiment, the antibody is in the form of a drug conjugate.

[0035] In another preferred embodiment, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region, the amino acid sequences of which are set forth in SEQ ID NO: 28 and SEQ ID NO: 30, respectively.

[0036] In a second aspect of the present application, there is provided a recombinant protein, the recombinant protein having:

[0037] (i) an antibody or antigen-binding fragment thereof as described in the first aspect of the present application; and

[0038] (ii) an optional tag sequence for facilitating expression and / or purification.

[0039] In another preferred embodiment, the tag sequence comprises a 6His tag.

[0040] In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.

[0041] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

[0042] In a third aspect of the present application, there is provided an antibody preparation, the antibody preparation comprising:

[0043] (a) an antibody or antigen-binding fragment thereof as described in the first aspect of the present application; and

[0044] (b) a carrier, the carrier comprising: a buffer, sterile water, and optionally a surfactant.

[0045] In a fourth aspect of the present application, there is provided a kit, the kit comprising the antibody preparation of the third aspect of the present application, and a container holding the antibody preparation.

[0046] In a fifth aspect of the present application, there is provided a CAR construct, the scFv segment of the antigen-binding region of the CAR construct being a binding region that specifically binds to TfRl, and the scFv having a heavy chain variable region and a light chain variable region as in the antibody or antigen-binding fragment thereof of the first aspect of the present application.

[0047] In a sixth aspect of the present application, there is provided an antibody drug conjugate, the antibody drug conjugate comprising:

[0048] (a) an antibody moiety selected from the group consisting of an antibody or antigen-binding fragment thereof as described in the first aspect of the present application; and

[0049] (b) a conjugate moiety conjugated to the antibody moiety, the conjugate moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0050] In another preferred embodiment, the antibody moiety is conjugated to the conjugate moiety via a chemical bond or linker.

[0051] In a seventh aspect of the present application, there is provided a recombinant immune cell expressing an exogenous CAR construct as described in the fifth aspect of the present application.

[0052] In another preferred embodiment, the immune cell is selected from the group consisting of an NK cell, a T cell.

[0053] In another preferred embodiment, the immune cell is from a human or a non-human mammal (e.g. murine).

[0054] In an eighth aspect of the present application, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:

[0055] (1) an antibody or antigen-binding fragment thereof as described in the first aspect of the present application;

[0056] (2) a recombinant protein as described in the second aspect of the present application; and / or

[0057] (3) a CAR construct as described in the fifth aspect of the present application.

[0058] In another preferred embodiment, the nucleotide sequence encoding the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof as described in the first aspect of the present application is selected from the group consisting of:

[0059] (1) a nucleotide sequence encoding a heavy chain variable region as set forth in SEQ ID NO: 10, and a nucleotide sequence encoding a light chain variable region as set forth in SEQ ID NO: 16;

[0060] (2) a nucleotide sequence encoding a heavy chain variable region as set forth in SEQ ID NO: 10, and a nucleotide sequence encoding a light chain variable region as set forth in SEQ ID NO: 18;

[0061] (3) a nucleotide sequence encoding a heavy chain variable region as set forth in SEQ ID NO: 12, and a nucleotide sequence encoding a light chain variable region as set forth in SEQ ID NO: 16;

[0062] (4) a nucleotide sequence encoding a heavy chain variable region as set forth in SEQ ID NO: 20, and a nucleotide sequence encoding a light chain variable region as set forth in SEQ ID NO: 22;

[0063] (5) a nucleotide sequence encoding a heavy chain variable region as set forth in SEQ ID NO: 20, and a nucleotide sequence encoding a light chain variable region as set forth in SEQ ID NO: 24.

[0064] In a ninth aspect of the present application, a vector is provided, wherein the vector comprises the polynucleotide of the eighth aspect of the present application.

[0065] In a tenth aspect of the present application, a genetically engineered host cell is provided, wherein the host cell comprises the vector of the ninth aspect of the present application or the polynucleotide of the eighth aspect of the present application is integrated into the genome of the host cell.

[0066] In an eleventh aspect of the present application, a use of an active ingredient is provided, wherein the active ingredient is selected from the group consisting of the antibody or antigen binding fragment thereof of the first aspect of the present application, the recombinant protein of the second aspect of the present application, the antibody drug conjugate of the seventh aspect of the present application, or the immune cell of the sixth aspect of the present application, or a combination thereof, for:

[0067] (a) preparing a detection reagent or a kit; and / or

[0068] (b) preparing a medicament or a preparation for preventing and / or treating a TfRl -related disease.

[0069] In another preferred embodiment, the TfRl -related disease comprises a TfRl -positive cancer or tumor.

[0070] In another preferred embodiment, the TfRl -positive cancer or tumor is a TfRl -high cancer or tumor.

[0071] In another preferred embodiment, the cancer or tumor is selected from the group consisting of a hematological tumor, a solid tumor, or a combination thereof.

[0072] In another preferred embodiment, the TfRl -positive cancer or tumor is selected from the group consisting of AML, ALL, lymphoma, multiple myeloma, breast cancer, gastric cancer, glioblastoma, prostate cancer, urothelial bladder cancer, pancreatic cancer, esophageal cancer, colorectal cancer, ovarian cancer, liver cancer, or a combination thereof.

[0073] In another preferred embodiment, the medicament or preparation is for preparing a medicament or a preparation for preventing and / or treating a disease associated with TfRl (expression positive).

[0074] In another preferred embodiment, the antibody is in the form of an antibody drug conjugate (ADC).

[0075] In another preferred embodiment, the detection reagent or kit is used for diagnosing a TfRl -related disease.

[0076] In another preferred embodiment, the detection reagent or kit is used for detecting TfRl protein in a sample.

[0077] In another preferred embodiment, the detection reagent is a test strip.

[0078] In a twelfth aspect of the present application, a pharmaceutical composition is provided, which comprises:

[0079] (i) an active ingredient selected from the group consisting of an antibody or antigen binding fragment thereof according to the first aspect of the present application, a recombinant protein according to the second aspect of the present application, an antibody drug conjugate according to the sixth aspect of the present application, an immune cell according to the seventh aspect of the present application, or a combination thereof; and

[0080] (ii) a pharmaceutically acceptable carrier.

[0081] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0082] In another preferred embodiment, the pharmaceutical composition is an injection.

[0083] In another preferred embodiment, the pharmaceutical composition is used for treating cancer or tumor.

[0084] In another preferred embodiment, the tumor is a cancer or tumor that highly expresses TfRl.

[0085] In a thirteenth aspect of the present application, a detection plate is provided, which comprises: a substrate (support plate) and a test strip, wherein the test strip comprises an antibody or antigen binding fragment thereof according to the first aspect of the present application or an antibody drug conjugate according to the sixth aspect of the present application.

[0086] In a fourteenth aspect of the present application, a kit is provided, which comprises:

[0087] (1) a first container comprising an antibody or antigen binding fragment thereof according to the first aspect of the present application; and / or

[0088] (2) a second container comprising a secondary antibody against an antibody or antigen binding fragment thereof according to the first aspect of the present application.

[0089] Alternatively, the kit comprises a detection plate according to the thirteenth aspect of the present application.

[0090] In a fifteenth aspect of the present application, there is provided a method of producing a recombinant polypeptide, the method comprising:

[0091] (a) culturing the host cell of the tenth aspect of the present application under conditions suitable for expression;

[0092] (b) isolating the recombinant polypeptide from the culture, the recombinant polypeptide being the antibody or antigen-binding fragment thereof of the first aspect of the present application or the recombinant protein of the second aspect of the present application.

[0093] In a sixteenth aspect of the present application, there is provided a method of detecting (including diagnosing or non-diagnosing) TfR1 protein in a sample in vitro, the method comprising the steps of:

[0094] (1) contacting, in vitro, the sample with the antibody of the first aspect of the present application;

[0095] (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of TfR1 protein in the sample.

[0096] In a seventeenth aspect of the present application, there is provided a method of treating a TfR1 -related disease, the method comprising: administering to a subject in need thereof the antibody or antigen-binding fragment thereof of the first aspect of the present application, the antibody-drug conjugate of the antibody, or the CAR-T cell expressing the antibody, or a combination thereof.

[0097] In another preferred embodiment, the TfR1 -related disease comprises a TfR1 -positive cancer or tumor.

[0098] In another preferred embodiment, the TfR1 -positive cancer or tumor is a cancer or tumor that overexpresses TfR1.

[0099] In another preferred embodiment, the cancer or tumor is selected from the group consisting of a hematological tumor, a solid tumor, or a combination thereof.

[0100] In another preferred embodiment, the TfR1 -positive cancer or tumor is selected from the group consisting of AML, ALL, lymphoma, multiple myeloma, breast cancer, gastric cancer, glioblastoma, prostate cancer, urothelial bladder cancer, pancreatic cancer, esophageal cancer, colorectal cancer, ovarian cancer, liver cancer, or a combination thereof.

[0101] It should be understood that, within the scope of the present application, all combinations between the above technical features of the present application and the technical features specifically described hereinafter (e.g. in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0102] Figure 1 shows the results of SDS-PAGE of antibodies.

[0103] Figure 2 shows the reduced and non-reduced SDS-PAGE of the parental antibody.

[0104] Figure 3 shows the NGS sequencing map.

[0105] Figure 4 shows the light and heavy chain sequencing results.

[0106] Figures 5A and 5B show the purified antibody SDS-PAGE map.

[0107] Figure 6 shows the antibody endocytosis experiment results. DETAILED DESCRIPTION

[0108] The present inventors, through extensive and in-depth research, obtained humanized antibodies targeting TfRl (including: VH4+VL1, VH4+VL2 and VH5+VL1) through a large number of screenings, the humanized antibodies of the present application retain the binding affinity of the protein antigen, the binding epitope is consistent with the parent antibody (i.e. the murine antibody MAb 11-22.1 targeting TfRl), and the endocytosis activity is basically consistent with the endocytosis activity of the chimeric antibody. And on this basis, PTM risk removal was carried out, and antibodies with good affinity and endocytosis function were obtained (for example, VH4-DA+VL1-MSG). On this basis, the present application is completed.

[0109] TERMINOLOGY

[0110] In order that the disclosure can be more readily understood, certain terms are first defined. As used in this application, unless specifically stated otherwise, each of the following terms has the meaning given below. Additional definitions are set forth throughout the application.

[0111] The term“about” can refer to a value or a composition that is within an acceptable error range for the particular value or composition determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression“about 100” includes all values between 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0112] As used herein, the term“containing” or“including” can be open, semi-closed and closed. In other words, the term also includes“consisting essentially of’ or“consisting of’.

[0113] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range is to be understood to include the values expressly indicated within the range, as well as implicitly suggested by the language of the range (e.g., tenths and hundredths of an integer).

[0114] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0115] As used herein, the terms "administering" and "treatment" refer to the application of an exogenous drug, therapeutic agent, diagnostic agent, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid. "Administering" and "treatment" can refer to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contact of the reagent with the cell, and contact of the reagent with a fluid, and contact of the fluid with the cell. "Administering" and "treatment" also mean in vitro and ex vivo treatment by a reagent, diagnostic, binding composition, or by another cell. "Treatment" when applied to a human, animal, or research subject, refers to therapeutic treatment, prevention, or prophylactic measures, research, and diagnosis; including contact of an anti-TfRl antibody with a human or animal, subject, cell, tissue, physiological compartment, or physiological fluid.

[0116] As used herein, the term "treatment" refers to the administration of a therapeutic agent to a patient, including any of the anti-TfRl antibodies of the present application and compositions thereof, who has one or more symptoms of a disease, where the therapeutic agent is known to have a therapeutic effect on the symptoms. Typically, the patient is administered the therapeutic agent in an amount effective to alleviate one or more symptoms of the disease (therapeutically effective amount).

[0117] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance can, but need not, occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of the particular sequence can, but need not, have one, two, or three.

[0118] "Sequence identity" as described herein means the extent to which two nucleic acid or two amino acid sequences are identical, when optimally aligned and compared with appropriate mutations, such as substitutions, insertions or deletions. The sequence identity between a sequence described herein and a sequence identical thereto can be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

[0119] As used herein, the terms "PTM", "Post-translational modification" and "post- translational modification" have the same meaning, are used interchangeably, and all refer to chemical modifications of a protein after translation. Compared with small molecule drugs, the synthesis and secretion process of monoclonal antibodies is complex, and various post-translational modification variants can be produced during production, storage and clinical use. Common PTMs include glycosylation modification, deamidation modification, oxidation, isomerization, C-terminal lysine deletion, N-terminal pyroglutamic acid cyclization, etc. These PTMs can cause changes in the charge and structure of antibody drugs, affect the affinity with target antigens and Fc receptors, and thus change the activity and half-life of antibody drugs, etc.; in addition, PTMs can even reduce the stability of antibodies, produce immunogenicity, etc., ultimately affecting the effectiveness, safety and stability of monoclonal antibody drugs.

[0120] TfR1 and antibody MAb 11-22.1 targeting TfR1

[0121] TfR1: Transferrrin R (human transferrrin R, TfR1) is also known as Cluster of Differentiation 71 (CD71) or TFRC (Transferrin Receptor Protein 1), all of which refer to the same type II transmembrane glycoprotein. TfR1 plays a key role in iron uptake and regulation of cell growth by being linked by two disulfide bonds to form a 180-kDa homodimer.

[0122] Antibody MAb 11-22.1 targeting TfR1: is a monoclonal antibody (IgG1, K) produced by one of the hybridomas generated after immunizing mice with multiple acute myeloid leukemia (AML) live cell lines, which is identified as a specific monoclonal antibody against human TfR1 expressed on AML and other cancer cells (WO2023103922A1).

[0123] The humanized antibody targeting TfR1 of the present application is based on the humanization of MAb 11-22.1 antibody.

[0124] Antibody

[0125] As used herein, the term "antibody" refers to an immunoglobulin, which is a four polypeptide chain structure linked by interchain disulfide bonds, consisting of two identical heavy chains and two identical light chains. The antigenicity of immunoglobulin heavy chain constant region differs according to the amino acid composition and the arrangement order. Accordingly, the immunoglobulin can be classified into five types, or called as isotypes, i.e. IgM, IgD, IgG, IgA and IgE, with the corresponding heavy chains of μ chain, δ chain, γ chain, α chain and ε chain, respectively. The same type of Ig can be further classified into different subtypes according to the difference of amino acid composition of the heavy chain region and the number and position of disulfide bonds, such as IgG can be classified into IgG1, IgG2, IgG3 and IgG4. The light chain can be classified into κ chain or λ chain according to the constant region. Each type of Ig can have κ chain or λ chain. The subunit structure and three-dimensional configuration of different types of immunoglobulin are well known to those skilled in the art.

[0126] The antibody light chain of the present application can further comprise a light chain constant region, which comprises human or murine κ, λ chain or variant thereof.

[0127] In the present application, the antibody heavy chain of the present application can further comprise a heavy chain constant region, which comprises human or murine IgG1, IgG2, IgG3, IgG4 or variant thereof. The sequence of about 110 amino acids near the N-terminus of antibody heavy chain and light chain varies greatly, which is the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable, which is the constant region. The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from the amino terminal to the carboxyl terminal. The three CDR regions of light chain are LCDR1, LCDR2 and LCDR3; the three CDR regions of heavy chain are HCDR1, HCDR2 and HCDR3.

[0128] The term "murine antibody" in the present application refers to a monoclonal antibody against TfR1 prepared according to the knowledge and skills in the art. In preparation, the test subject is injected with TfR1 antigen, and then the hybridoma expressing antibody with the desired sequence or functional characteristics is isolated. In a preferred embodiment of the present application, the murine TfR1 antibody or antigen binding fragment thereof can further comprise a light chain constant region of murine κ, λ chain or variant thereof, or further comprise a heavy chain constant region of murine IgG1, IgG2, IgG3 or variant thereof.

[0129] The term "chimeric antibody" is an antibody in which the variable region of a murine antibody is fused with the constant region of a human antibody, and can reduce the immune response reaction induced by the murine antibody.

[0130] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody in which the CDR sequences of a mouse are grafted into a human antibody variable region framework, i.e., an antibody produced in a different type of human germline antibody framework sequence. The humanized antibody can overcome the heterogeneity reaction induced by the chimeric antibody due to the presence of a large amount of murine protein components. Such framework sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. To avoid the decrease in immunogenicity while causing a decrease in activity, the human antibody variable region framework sequence can be subjected to a minimum of reverse mutation or back mutation to maintain the activity.

[0131] The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., TfR1). It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment of an antibody" include

[0132] (i) an Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains;

[0133] (ii) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region;

[0134] (iii) an Fd fragment consisting of the VH and CH1 domains;

[0135] (iv) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody.

[0136] An Fv antibody contains the variable region of the heavy chain, the variable region of the light chain, but no constant region, and has the smallest antibody fragment of all antigen-binding sites. Generally, the Fv antibody further comprises a polypeptide linker between the VHand VLdomains, and is capable of forming a structure required for antigen binding.

[0137] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that mainly contributes to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat E.A et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242.

[0138] The term "epitope" or "antigenic determinant" refers to a site (e.g., a specific site on a TfRl molecule) on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes are generally formed both by contiguous amino acids and non-contiguous amino acids juxtaposed in three dimensional space. Epitopes formed from two or more non-contiguous amino acids often occur where the individual amino acids are spaced apart from one another by 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in the primary amino acid sequence.

[0139] The terms "specifically binds," "selectively binds," "selectively binds to," and "binds specifically to" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, an antibody will bind to its antigen with an affinity of about less than 10 -7 M, for example, less than about 10 -8 M, 10 -9 M, or 10 -10 M or less.

[0140] The term "competes for binding" refers to an antibody that recognizes and binds to the same epitope (also referred to as an antigenic determinant) or a portion of the same epitope on the extracellular region of TfRl as a monoclonal antibody of the present application, and binds to the antigen. An antibody that binds to the same epitope as a monoclonal antibody of the present application refers to an antibody that recognizes and binds to the same amino acid sequence of TfRl recognized by the monoclonal antibody of the present application.

[0141] The term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, the antibodies of the present application bind to TfRl with a dissociation equilibrium constant (KD) of less than about 10 -7 M, for example, less than about 10 -8 M, 10 -9 M, or 10 -10 M or less.

[0142] The term "antigenic determinant" as used herein refers to a discontinuous, three dimensional site on an antigen that is recognized by an antibody or antigen binding fragment of the present application.

[0143] The present application includes not only intact antibodies, but also fragments of an antibody that are immunologically active or a fusion protein of an antibody with other sequences. Thus, the present application also includes fragments, derivatives, and analogs of the antibodies.

[0144] In the present application, antibodies include murine, chimeric, humanized, or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, can be prepared using DNA recombination techniques well known in the art.

[0145] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a clonal secretion of a single cell source. Monoclonal antibodies are highly specific, directed to a single epitope of an antigen. The cell can be a clonal cell strain of eukaryotic, prokaryotic or phage.

[0146] In the present application, the antibody can be mono-specific, bi-specific, tri-specific, or more multi-specific.

[0147] In the present application, the antibody of the present application also includes conservative variants thereof, which means that a polypeptide is formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or similar properties, compared to the amino acid sequence of the antibody of the present application. These conservative variant polypeptides are preferably generated by amino acid replacement according to Table A.

[0148] Table A

[0149] Anti-TfRl humanized antibody

[0150] The present application provides an anti-TfRl humanized antibody (hereinafter referred to as TfRl antibody). Specifically, the present application provides a humanized antibody against TfRl with high specificity and high affinity, which includes a heavy chain containing a heavy chain variable region (VH) amino acid sequence and a light chain containing a light chain variable region (VL) amino acid sequence.

[0151] Jones et al. first transplanted the heavy chain CDR of a murine monoclonal antibody to the human antibody heavy chain framework region in 1986, and then assembled it with the light chain of a murine monoclonal antibody to form a complete antibody and maintained similar affinity to the original murine monoclonal antibody, providing an idea for the development of antibody humanization technology. In 1989, Queen et al. successfully constructed an anti-CD25 humanized antibody by CDR grafting method, which used human antibody Eu framework region for humanization, and retained some amino acids of murine antibody in the framework region to maintain the affinity. In 1992, Presta et al. reported a method of constructing humanized antibodies by CDR grafting using human antibody consensus sequence as a template. In 1994, Pedersen et al. reported a method of humanizing antibodies by surface resurfacing. In 1994, Hsiao et al. reported a method of humanizing antibodies by CDR grafting using human antibody Germline sequence framework region. In 1994, Jespers et al. successfully constructed a humanization method by using phage library (shuffling library).

[0152] There are two choices for the human framework region in antibody humanization, one is the known mature antibody, and the other is the human germline sequence. The known mature antibody framework region usually contains somatic mutation sites, which may bring potential immunogenicity. Compared with the mature antibody, the human germline sequence framework region is theoretically less immunogenic, and the structure is more flexible, with strong plasticity and easy to accept different CDR regions. The frequency of use of human antibody germline genes in the human body has a certain bias, and the antibody humanized by selecting a high-frequency germline framework region has the advantages of low immunogenicity, high expression, stable structure and the like. Therefore, the present application does not select the germline sequence with the highest similarity to the murine antibody during humanization, but considers the similarity and the frequency of use in the human body, and selects the germline corresponding to VH4 and VL1 through a large number of experiments. The present application selects the human antibody germline framework region for CDR grafting and back mutation, so that the structure of the humanized antibody constructed in this way is more stable, has high expression, low immunogenicity and higher drugability.

[0153] In particular, as described in the first aspect of the present application.

[0154] In the first aspect of the present application, a humanized antibody or antigen binding fragment thereof targeting TfR1 is provided, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region being selected from the group consisting of:

[0155] (a1) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 9;

[0156] (a2) a heavy chain variable region derived from the sequence set forth in SEQ ID NO: 9 by substitution, deletion, modification and / or addition of at least one (such as 1-20, preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-3, most preferably 1 or 2) amino acid residues, having the function of the heavy chain variable region of (a1);

[0157] (a3) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 11;

[0158] The light chain variable region is selected from the group consisting of:

[0159] (b1) a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 15;

[0160] (b2) a light chain variable region derived from the sequence set forth in SEQ ID NO: 15 having the function of the light chain variable region of (b1) formed by substitution, deletion, modification and / or addition of at least one (e.g. 1-20, preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-3, most preferably 1 or 2) amino acid residues of the sequence set forth in SEQ ID NO: 15;

[0161] (b3) a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 17.

[0162] In another preferred embodiment, the constant region of the humanized antibody is different from the constant region of anti-TfRl murine monoclonal antibody MAb 11-22.1 (application number PCT / CN2022 / 136411).

[0163] In another preferred embodiment, the sequence formed by addition, deletion, modification and / or substitution of at least one amino acid sequence is preferably an amino acid sequence having a homology of at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95%.

[0164] The antibody of the present application can be a double-chain or single-chain antibody.

[0165] The antibody derivative of the present application can be a single-chain antibody, and / or an antibody fragment such as Fab, Fab', (Fab')2, or other known antibody derivatives in the art, and any one or several of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0166] The antibody of the present application can be a humanized antibody, a CDR grafted and / or modified antibody targeting TfRl.

[0167] In the above description of the present application, the number of amino acids added, deleted, modified and / or substituted is preferably not more than 40% of the total number of amino acids in the original amino acid sequence, more preferably not more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.

[0168] The present application successfully humanizes the TfRl murine monoclonal antibody, and the affinity of the humanized antibody reaches that of the chimeric antibody, which has the potential to be further developed into a humanized monoclonal antibody drug for targeted therapy in the future.

[0169] Preparation of antibodies

[0170] Any method suitable for producing monoclonal antibodies can be used to produce the TfRl antibodies of the application. For example, an animal can be immunized with a linked or naturally occurring TfRl protein or fragment thereof. Suitable immunization protocols can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes of administration can be used.

[0171] Any suitable form of TfRl can be used as an immunogen (antigen) for the production of non-human antibodies specific for TfRl, and for screening the antibodies for biological activity. The immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art. The immunogen can be purified from a natural source or produced in a genetically modified cell. The DNA encoding the immunogen can be genomic or non-genomic (e.g., cDNA) in origin. The DNA encoding the immunogen can be expressed using a suitable genetic vector, including but not limited to adenoviral vectors, baculoviral vectors, plasmids, and non-viral vectors.

[0172] Humanized antibodies can be selected from any species of immunoglobulin, including IgM, IgD, IgG, IgA, and IgE. Likewise, any class of light chain can be used in the compounds and methods herein. In particular, kappa, lambda chains, or variants thereof, are useful in the compounds and methods of the application.

[0173] An exemplary method for humanizing the TfRl antibodies of the application is described in Example 1.

[0174] The sequence of the DNA molecule of the antibody or fragment thereof of the application can be obtained using conventional techniques, such as by PCR amplification or screening of genomic libraries. In addition, the coding sequences for the light and heavy chains can be fused together to form a single chain antibody.

[0175] Once the relevant sequences have been obtained, they can be obtained in large quantities using recombinant methods. This is typically done by cloning them into vectors, which are then introduced into cells, and then isolating the relevant sequences from the propagated host cells using conventional methods.

[0176] In addition, the relevant sequences can be synthesized using artificial synthesis methods, particularly for shorter fragments. Typically, longer fragments are obtained by first synthesizing a number of smaller fragments, which are then ligated together. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art.

[0177] The term "nucleic acid molecule" refers to DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA. Nucleic acids are "operably linked" when they are functionally connected. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0178] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.

[0179] The present application also relates to vectors comprising the appropriate DNA sequences described above and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0180] The term "host cell" refers to a cell into which an expression vector has been introduced. The host cell can be a prokaryotic cell, such as a bacterial cell, or a eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a plant or animal cell (e.g., a mammalian cell).

[0181] The steps of transforming host cells with recombinant DNA described in the present application can be performed using techniques well known in the art. The transformants obtained can be cultured using conventional methods, and the transformants express the polypeptides encoded by the genes of the present application. The culture is performed under suitable conditions using conventional media, depending on the host cell used.

[0182] Generally, the resulting host cells are cultured under conditions suitable for expression of the antibodies of the present application. The antibodies of the present application are then purified using conventional immunoglobulin purification methods, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, size exclusion chromatography, or affinity chromatography, as well as other conventional separation and purification methods known to those skilled in the art.

[0183] The resulting monoclonal antibodies can be identified using conventional means. For example, the binding specificity of the monoclonal antibodies can be determined using immunoprecipitation or an in vitro binding assay, such as a radioimmunoassay (RIA) or an enzyme-linked immunosorbent assay (ELISA).

[0184] Antibody formulation

[0185] Antibodies have different stability in different formulation buffers, which is manifested as changes in charge heterogeneity, degradation of antibody molecules, aggregation, etc. These changes in quality properties are related to the physicochemical properties of the antibody itself, and therefore, in the process of developing antibody drugs, the formulation buffer suitable for the physicochemical properties of the antibody itself needs to be screened, as well as an appropriate amount of surfactant to maintain the stability of the antibody.

[0186] The antibody formulation of the present application is as described in the third aspect of the present application.

[0187] Pharmaceutical composition

[0188] The present application also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the antibody or active fragment thereof or fusion protein thereof or ADC thereof or corresponding CAR-T cell described above, and a pharmaceutically acceptable carrier. The prepared pharmaceutical composition can be administered by conventional routes, including but not limited to intratumoral, intraperitoneal, intravenous, or local administration.

[0189] The antibody of the present application can also be expressed in cells by nucleotide sequences for cell therapy, such as the antibody for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

[0190] The pharmaceutical composition of the present application can be directly used to bind TfR1 protein molecules, and thus can be used for preventing and treating TfR1 related diseases. In addition, other therapeutic agents can also be used simultaneously.

[0191] Detection purposes and kits

[0192] The antibody of the present application can be used for detection purposes, for example for detecting a sample to provide diagnostic information.

[0193] In the present application, the sample (sample) used includes cells, tissue samples and biopsy specimens. The term "biopsy" used in the present application should include all kinds of biopsies known to those skilled in the art. Therefore, the biopsy used in the present application can include, for example, a tissue sample prepared by endoscopic methods or puncture or needle biopsy of an organ.

[0194] The sample used in the present application includes fixed or preserved cells or tissue samples.

[0195] The present application also provides a kit comprising the antibody (or fragment thereof) of the present application. In a preferred embodiment of the present application, the kit further comprises a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present application can be immobilized on a detection plate.

[0196] The main advantages of the present application include

[0197] (a) The human VH, VL skeleton region template structure of the humanized antibody of the present application is stable, and well adapted with the CDR region of the murine monoclonal antibody, and the light and heavy chain variable regions can be well paired together, with high affinity and stable structure.

[0198] (b) Compared with the chimeric antibody, the humanized antibody of the present application has excellent biological activity, specificity, endocytosis activity, while retaining the affinity of TfR1, it has lower immunogenicity and higher expression.

[0199] (c) The humanized antibody of the present application only recognizes TfR1 expressed on malignant tumor cells, and has very low risk of causing bone marrow suppression.

[0200] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. The experimental methods in the following examples, if not specified, are generally carried out according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0201] Materials and Methods

[0202] I. Instruments, Materials and Reagents

[0203] (1) The instrument information is shown in Table 1 below.

[0204] Table 1

[0205] (2) The consumable information is shown in Table 2 below.

[0206] Table 2

[0207] (3) The reagent information is shown in Table 3 below.

[0208] Table 3

[0209] (4) The sample information is shown in Table 4 below.

[0210] Table 4

[0211] II. Antibody Humanization Experimental Methods

[0212] (1) Antibody humanization based on CDR grafting and back mutation technology

[0213] The structure of the parent antibody (i.e., the mouse-derived antibody Mab 11-22.1 targeting TfR1) was modeled by the MOE homology modeling program. Humanized antibodies were designed using CDR grafting. Briefly, the CDRs of the parent antibody were grafted into a human antibody scaffold to obtain a humanized light chain and a heavy chain for each parent antibody. The 5 heavy chains and 2 light chains were paired for affinity ranking experiments. The specific sequences are shown in Table B.

[0214] (2) Construction and production of antibodies

[0215] DNA sequences encoding the heavy and light chains of the chimeric antibody were synthesized, inserted into pcDNA3.4 expression vector, and full-length IgG expression plasmid was constructed. The expression of the antibody was performed in ExpiCHO-S cell culture, and the supernatant was purified by protein A affinity column. The purified antibody buffer was replaced with PBS using a PD-10 desalting column. The concentration and purity of the purified protein were determined by OD280 and SDS-PAGE, respectively.

[0216] (3) Affinity determination of humanized antibody

[0217] The affinity of the antibody to the antigen was determined by surface plasmon resonance (SPR). The antibody was captured onto a protein A sensor chip, and the antigen flowed through the sensor chip surface as an analyte. The dissociation rate (kd) and the association rate (ka) were obtained using analysis software. The equilibrium dissociation constant (KD) was calculated from the ratio of kd to ka.

[0218] (4) ELISA IC of antibody 50 determination

[0219] To evaluate the epitope competition of humanized antibody with murine antibody, ELISA IC 50 determination was performed. Briefly, 96-well ELISA microtiter plates were coated with 1 μg / ml antigen (TfR1 / CD71) in CBS at 4°C overnight, respectively. The plates were washed twice and blocked with 4% MPBS at 37°C for 1 hour. After washing the plates once, the humanized antibody was added to the blocked wells after gradient dilution, with the first well at a concentration of 25 μg / ml, using PBS 3-fold dilution, and a non-cognate antibody as a negative control, 100 μl / well. Incubate at 37°C for 1 hour, wash the plates once, and add 0.1 μg / ml of murine antibody, incubate at 37°C for 1 hour. After washing the plates three times with washing buffer, Anti-mouse IgG Fc [HRP] was added to the plates and incubated at room temperature for 45 minutes. After washing 6 more times, color developing solution was added to the wells to react. The absorbance was measured at 450 nm using a spectrometer.

[0220] (5) Antibody endocytosis experiment-warm difference method

[0221] After the antibody binds to the target cells (human TfR1 target CHO-K1 overexpression cell line), no endocytosis occurs at 4°C, endocytosis occurs when incubated at 37°C, and after the antibody is endocytosed into the cells, the antibody bound to the surface of the cells decreases, the secondary antibody indicator is added to bind to the surface antibody, and the difference in fluorescence signal on the surface of the cells incubated at 4°C and 37°C is detected by flow cytometry as an indicator of the efficiency of antibody endocytosis.

[0222] III. Affinity maturation method

[0223] (1) Production and detection of parent antibody

[0224] The parent antibody is the preferred antibody after humanization as the parent antibody. DNA sequences encoding the heavy and light chains of the parent antibody were synthesized and inserted into pcDNA3.4 expression vector to construct full-length IgG expression plasmid. The expression of antibody was performed in ExpiCHO-S cell culture and the supernatant was purified by Protein A affinity column. The purified antibody buffer was replaced to PBS using PD-10 desalting column. The concentration and purity of purified protein were determined by OD280 and SDS-PAGE, respectively.

[0225] The affinity of the parent antibody to the antigen TfR1 / CD71 was determined by the method of surface plasmon resonance (SPR). The antibody was captured onto a Protein A sensor chip and the antigen was flowed over the sensor chip surface as the analyte. The dissociation rate (kd) and the association rate (ka) were obtained using the analysis software and the equilibrium dissociation constant (KD) was calculated from the ratio of kd to ka.

[0226] (2) Production and detection of parent Fab FASEBA samples

[0227] 1) Production of parent Fab FASEBA samples

[0228] DNA sequences encoding the heavy and light chains of the parent Fab were synthesized and inserted into FASEBA vector to construct the expression plasmid of the parent Fab. The expression plasmid was transduced in TG1 and positive clones were selected for culture and IPTG induction of the expression of the Fab parent antibody.

[0229] 2) Affinity determination and ELISA verification of parent Fab FASEBA samples

[0230] Affinity determination: The affinity between the supernatant of the parent FASEBA prokaryotic expression and the antigen TfR1 / CD71 was determined by the method of surface plasmon resonance (SPR). The antibody was captured onto a CM5 sensor chip coupled with BSA and the antigen was flowed over the sensor chip surface as the analyte. The dissociation rate (kd) and the association rate (ka) were obtained using the analysis software and the equilibrium dissociation constant (KD) was calculated from the ratio of kd to ka.

[0231] ELISA validation: ELISA plates were coated with 10 pg / ml BSA (expresser plate) and antigen TfR1 / CD71 (binder plate), antigens were diluted with CBS, initial concentration 2 pg / ml, 2-fold dilution gradient, 100 mΐ / well plated, incubated at 4°C overnight. Blocking solution of 4% MPBS was added, incubated at 37°C for 1 hour. After washing the plate, 50 mΐ 0.05% PBST and 50 mΐ FASEBA supernatant were added to each well, incubated at room temperature for 2 hours. After washing the plate, 0.1 pg / ml Anti-Human IgG, F(ab')2 secondary antibody was added, incubated for 45 minutes. After washing the plate, 100 mΐ TMB substrate was added to react at room temperature for 10 minutes, and the reaction was stopped by adding 50 mΐ 1 M HC1. The absorbance at 450 nm was read using a microplate reader.

[0232] (3) PML library construction

[0233] 20 residues in CDR regions were mutated to other amino acids. NGS was used to guarantee the quality of the library and to guarantee a minimum coverage of 90%. 48 clones were randomly selected from each PML library for expression in E. coli.

[0234] (4) FASEBA screening and affinity ranking of PML libraries

[0235] Each library clone was transferred and induced for expression in 96-deep well plates. Expression and binding affinity were evaluated by ELISA analysis of secreted proteins in the culture medium against BSA and TfR1 / CD71, compared to parental FASEBA supernatant, NC (non-relevant FASEBA supernatant), and blank (2YT medium), respectively.

[0236] Clones with improved value were selected for sequencing. Affinity ranking confirmed the sites that affect affinity. Antibody affinity to antigen was determined by surface plasmon resonance (SPR). Antibodies were captured onto a sensor chip coupled with BSA, and antigens were flowed over the sensor chip surface as analytes. Dissociation rate (kd) and association rate (ka) were obtained using analysis software. Equilibrium dissociation constant (KD) was calculated from the ratio of kd to ka. The surface was regenerated before injecting other selected clones.

[0237] The process was repeated until all samples were analyzed. The dissociation rate of Fab-SASA clones was obtained by locally fitting the experimental data to a 1 : 1 interaction model using Biacore T200 evaluation software. Selected mutants were ranked according to their dissociation rate constant (dissociation rate, kd).

[0238] (5) Combination library construction and screening

[0239] A combinatorial library was constructed by randomly combining the identified mutations by PCR. Analysis was performed by ELISA, and a subset of clones were selected for DNA sequencing, affinity ranking. The final combination of affinity improved antibodies without affecting expression was selected by affinity ranking.

[0240] (6) TOP cloning gene synthesis and eukaryotic expression

[0241] Synthetic heavy chain (VH) and light chain (VL) variable domains encoding affinity matured antibodies were inserted into pCDNA3.4 vector to construct full length IgG expression vectors, respectively. Transfection was performed using heavy chain and light chain expression plasmids. Supernatants were purified by Protein A affinity column. Finally, the concentration and purity of the protein were evaluated by OD280 and SDS-PAGE, respectively.

[0242] (7) TOP cloning eukaryotic antibody affinity detection

[0243] The affinity of antibodies to antigens was determined by surface plasmon resonance (SPR). Antibodies were captured to a sensor chip either on Protein A or coupled to anti-mouse IgG-Fc secondary antibody, and antigens were flowed over the sensor chip surface as analytes. Dissociation rate (kd) and association rate (ka) were obtained using analysis software.

[0244] Equilibrium dissociation constant (KD) was calculated from the ratio of kd to ka.

[0245] (8) TOP cloning epitope identification and endocytosis verification

[0246] ELISA IC 50 The experiment verified whether the epitope bound by the TOP clone and the mouse-derived antibody was consistent; the secondary antibody method endocytosis experiment was used to detect the endocytosis activity of the test antibody and purified antibody.

[0247] Example 1 Production of chimeric antibodies and humanized antibodies

[0248] After expression of the chimeric antibodies, SDS-PAGE was used for purification. Under non-reducing conditions, the purified IgG migrated as a ~ 150 kDa band in SDS-PAGE, and under reducing conditions, as a ~ 50 kDa and ~ 25 kDa band. The purity of IgG was evaluated by SDS-PAGE results to be ≥ 95% (Figure 1). The concentration, yield and endotoxin level of all antibodies are shown in Table 5.

[0249] Table 5 Production results of chimeric antibodies and humanized antibodies

[0250] Note: VH1, VH2, VH3, VH4, VH5 in the table are the heavy chain variable regions after humanization, VL1, VL2 are the light chain variable regions after humanization; VH+VL indicates the antibody containing the heavy chain variable region and the light chain variable region.

[0251] Example 2 Affinity ranking of chimeric antibody and humanized antibody

[0252] The data processing adopts the Biacore T200 analysis software version 3.1. The injection of channel 1 and running buffer is deducted as double background signal. The affinity detection data of the experiment is shown in Table 6.

[0253] Table 6 Detection data of antigen and antibody ranking

[0254] Note: VH1, VH2, VH3, VH4, VH5 in the table are the heavy chain variable regions after humanization, VL1, VL2 are the light chain variable regions after humanization; VH+VL indicates the antibody containing the heavy chain variable region and the light chain variable region.

[0255] Example 3 Affinity determination of humanized antibody

[0256] The data processing adopts the Biacore T200 analysis software version 3.1. The injection of channel 1 and running buffer is deducted as double background signal. The affinity detection data of the experiment is shown in Table 7.

[0257] Table 7 Affinity detection data of antigen and antibody

[0258] As can be seen from Table 7, the humanized antibodies containing the following heavy chain variable regions and light chain variable regions retain the binding affinity (or the affinity is equivalent or superior to the mouse-derived antibody) to the protein antigen, and the binding position is consistent with the parent antibody (i.e. the mouse-derived antibody Mab11-22.1 targeting TfR1): VH4+VL1; VH4+VL2; or VH5+VL1.

[0259] Example 4 ELISA IC of antibody 50 Determination

[0260] Through ELISA epitope identification, the IC of the chimeric antibody (VH+VL-chimeric), VH4+VL1, VH4+VL2, VH5+VL1 and the mouse-derived antibody competing for the antigen epitope is respectively: 0.5288 μg / ml, 0.8474 μg / ml, 0.4362 μg / ml, and 0.4706 μg / ml. 50

[0261] Therefore, the humanized antibody of the application has the same antigen binding epitope as the mouse-derived antibody.

[0262] ​Considering the affinity, the number of back mutations, the antibody yield, etc., the VH4+VL1 antibody was finally selected for subsequent experiments.

[0263] Example 5 Antibody endocytosis experiment

[0264] The results of the antibody endocytosis experiment are shown in Table 8.

[0265] Table 8

[0266] As can be seen from Table 8, the in vivo endocytosis activity of the humanized antibody is basically the same as that of the chimeric antibody.

[0267] The following example is based on the strategy of PML unsaturated mutation and FASEBA screening to mature the affinity, so as to ensure that the affinity of the parent antibody (i.e. VH4+VL1 obtained by humanization) screened in the above example to the human transferrin receptor 1 / CD71 protein antigen is not lower than that of the mouse antibody, and the PTM risk site is removed.

[0268] The specific steps for removing the PTM risk site include: determining the high-risk amino acid combination through amino acid sequence analysis, then selecting the mutant that maintains or improves the affinity by screening the CDR region saturation point mutation library, and then sequencing the mutant to select the heavy chain and light chain combination that removes the PTM.

[0269] Example 6 Production and detection of parent antibody (VH4+VL1)

[0270] 6.1 Production of parent antibody

[0271] SDS-PAGE band of purified antibody: non-reduced band size 150 kDa, reduced band size 50 kDa and 25 kDa. The purity of the parent antibody (VH4+VL1) by SDS-PAGE is >98% (Figure 2). The concentration, yield and endotoxin content of the parent antibody are shown in Table 9.

[0272] Table 9

[0273] 6.2 Binding verification of parent antibody

[0274] All data were processed using Biacore T200 analysis software 4.0. The detection results are shown in Table 10.

[0275] Table 10 Antigen and antibody affinity detection data

[0276] 6.3 ELISA verification of parent Fab FASEBA sample

[0277] The results of ELISA detection of parent FASEBA supernatant are shown in Table 11.

[0278] Table 11 Indirect ELISA detection results

[0279] Note: NC is non-related FASEBA supernatant, Blank is 2YT medium.

[0280] The affinity of the parental Fab FASEBA supernatant to antigen was determined by Biacore 8K. The results are shown in Table 12.

[0281] Table 12

[0282] Note: NC is non-related FASEBA supernatant, Blank is 2YT medium.

[0283] Example 7 PML library construction

[0284] For the parental antibody in Example 6, a point mutation library was synthesized and cloned into C4738175P0-WT-Fab-pFASEBA vector as sub-libraries. Based on the FASEBA platform, each individual PML library was generated for each residue with a theoretical diversity of 16-18. The libraries are shown in Table 13. Library QC was ensured by NGS, and the results are shown in Figure 3.

[0285] Table 13 PML library sites

[0286] Example 8 PML library ELISA screening and top clone DNA sequencing

[0287] From each PML library in Example 7, 920 individual clones were cultured and tested for their binding activity by ELISA, compared with parental FASEBA supernatant, NC (non-related FASEBA supernatant), and blank (2YT medium).

[0288] And the 32 clones with improved affinity were sequenced, and the DNA sequence alignment results are shown in Table 14 and Figure 4.

[0289] Table 14

[0290] Example 9 PML screening top clone analysis

[0291] According to the ELISA results, a total of 23 unique clones were subjected to affinity ranking further analysis.

[0292] The affinity of TfR1 / CD71 to the supernatant of the selected clones is summarized in Table 15.

[0293] Table 15 Affinity ranking of unique clones to antigen

[0294] As can be seen from Table 15, the following mutations in the heavy chain variable region and / or the light chain variable region of the wild type antibody or the parent antibody (i.e. VH4+VL1) in this embodiment have an improved affinity or a comparable affinity relative to the wild type antibody: VH4-G56A, VH4-H59L, VL1-G33W, VL1-S32E, VL1-S32L, VL1-Q95M, VL1-N31M.

[0295] Example 10 TOP clone eukaryotic expression and affinity detection

[0296] According to sequence analysis and Probio’s SOP, 6 antibodies were expressed and purified. Antibody expression information is shown in Table 16, and SDS-PAGE is shown in Figure 5A and Figure 5B. The purified antibodies migrated as ~150 kDa bands under non-reducing conditions and as ~50 kDa and ~25 kDa bands under reducing conditions in SDS-PAGE.

[0297] Table 16 TOP antibody expression information

[0298] The affinity detection data of the antibodies to the antigen TfR1 / CD71 are shown in Table 17.

[0299] Table 17 antigen antibody binding detection data

[0300] VH4-DA in Table 16-17, i.e. VH4 chain and has the following mutations on the VH4 chain: G56A; VL1-MSG and VL1-NSW in Table 16-17, i.e. VL1 chain and has the following mutations on the VL1 chain respectively: N31M, G33W.

[0301] As can be seen from the results in Table 17, the affinity of VH4-DA+VL1-MSG and VH4-DA+VL1-NSW after removing the medium-high risk PTM sites is comparable to or superior to the parent antibody (i.e. VH4+VL1).

[0302] Example 11 TOP clone epitope identification

[0303] Through ELISA epitope identification, the IC50 of the chimeric antibody (VH+VL-chimeric), VH4+VL1, VH4+VL1-NSW, VH4-DA+VL1, VH4-DA+VL1-MSG, and VH4-DA+VL1-NSW were obtained. 50The values are: 0.8214 μg / ml, 1.118 μg / ml, 0.6191 μg / ml, 0.9562 μg / ml, 1.015 μg / ml, and 0.6965 μg / ml, respectively.

[0304] It can be seen that the humanized antibody after removal of the PTM risk site has the same antigen binding epitope as the chimeric antibody.

[0305] Example 12 Endocytosis experiment

[0306] The results of the antibody endocytosis experiment are shown in Table 18 and Figure 6.

[0307] Table 18

[0308] The results show that the EC50values of the humanized antibody (VH4+VL1) and the antibody after removal of the PTM are similar to those of the chimeric antibody. 50 The endocytosis function is comparable. The EC50value of the antibody VH4-DA+VL1-MSG after removal of the PTM is lower than that of the humanized antibody (VH4+VL1) and the chimeric antibody. 50 The EC50value of the antibody VH4-DA+VL1-NSW is comparable to that of the humanized antibody (VH4+VL1) and the chimeric antibody. 50 The EC50value of the antibody VH4-DA+VL1-NSW is comparable to that of the humanized antibody (VH4+VL1) and the chimeric antibody.

[0309] The sequence information of the present application is shown in Table B below.

[0310] Table B

[0311] All documents mentioned in the present application are incorporated herein by reference as if each document were individually incorporated by reference. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that embodiments can be practiced without materials details, quantifications, and other specific items described herein. These equivalents are also intended to be within the scope of the application. Accordingly, many modifications and variations will be apparent to those skilled in the art upon reading this above specification, the embodiments described herein being given by way of example only.

Claims

1. A humanized antibody or antigen-binding fragment thereof targeting TfRl, comprising a heavy chain variable region and a light chain variable region, characterized in that, the heavy chain variable region is selected from the group consisting of: (a1) a heavy chain variable region having an amino acid sequence of SEQ ID NO: 9; (a2) a heavy chain variable region derived from the sequence of SEQ ID NO: 9 by substitution, deletion, modification, and / or addition of at least one (e.g., 1 to 20, preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 8, more preferably 1 to 3, most preferably 1 or 2) amino acid residues, said heavy chain variable region having the function of the heavy chain variable region of (a1); (a3) a heavy chain variable region having an amino acid sequence of SEQ ID NO: 11; the light chain variable region is selected from the group consisting of: (b1) a light chain variable region having an amino acid sequence of SEQ ID NO: 15; (b2) a light chain variable region derived from the sequence of SEQ ID NO: 15 by substitution, deletion, modification, and / or addition of at least one (e.g., 1 to 20, preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 8, more preferably 1 to 3, most preferably 1 or 2) amino acid residues, said light chain variable region having the function of the light chain variable region of (b1); (b3) a light chain variable region having an amino acid sequence of SEQ ID NO:

17.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, the heavy chain variable region and the light chain variable region are selected from the group consisting of: (1) a heavy chain variable region having an amino acid sequence of SEQ ID NO: 9, and a light chain variable region having an amino acid sequence of SEQ ID NO: 15; (2) a heavy chain variable region having an amino acid sequence of SEQ ID NO: 9, and a light chain variable region having an amino acid sequence of SEQ ID NO: 17; (3) a heavy chain variable region having an amino acid sequence of SEQ ID NO: 11, and a light chain variable region having an amino acid sequence of SEQ ID NO: 15; (4) a heavy chain variable region having an amino acid sequence of SEQ ID NO: 9 and having the following mutations on SEQ ID NO: 9: G56A, and a light chain variable region having an amino acid sequence of SEQ ID NO: 15 and having the following mutations on SEQ ID NO: 15: N31M; or a heavy chain variable region having an amino acid sequence of SEQ ID NO: 19, and a light chain variable region having an amino acid sequence of SEQ ID NO: 21; (5) a heavy chain variable region having an amino acid sequence of SEQ ID NO: 9 and having the following mutations on SEQ ID NO: 9: G56A, and a light chain variable region having an amino acid sequence of SEQ ID NO: 15 and having the following mutations on SEQ ID NO: 15: G33W; or a heavy chain variable region having an amino acid sequence of SEQ ID NO: 19, and a light chain variable region having an amino acid sequence of SEQ ID NO:

23.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein The antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region, the amino acid sequences of which are set forth in SEQ ID NO: 28 and SEQ ID NO: 30, respectively.

4. A recombinant protein, characterized in that, The recombinant protein has: (i) the antibody or antigen-binding fragment thereof of claim 1; and (ii) an optional tag sequence that facilitates expression and / or purification.

5. A CAR construct, wherein the scFv segment of the antigen-binding region of the CAR construct is a binding region that specifically binds to TfRl, and the scFv has the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof of claim 1.

6. An antibody drug conjugate, characterized in that, The antibody drug conjugate comprises: (a) an antibody moiety selected from the group consisting of: the antibody or antigen-binding fragment thereof of claim 1; and (b) a conjugating moiety conjugated to the antibody moiety, the conjugating moiety being selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

7. The antibody drug conjugate of claim 6, wherein, The antibody moiety is conjugated to the conjugating moiety via a chemical bond or a linker.

8. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of any one of claims 1-7. The polynucleotide encodes a polypeptide selected from the group consisting of: (1) the antibody or antigen-binding fragment thereof of claim 1; (2) the recombinant protein of claim 4; and / or (3) the CAR construct of claim 5.

9. The polynucleotide of claim 8, wherein, The nucleotide sequence encoding the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof of claim 1 is selected from the group consisting of: (1) a nucleotide sequence encoding the heavy chain variable region set forth in SEQ ID NO: 10, and a nucleotide sequence encoding the light chain variable region set forth in SEQ ID NO: 16; (2) a nucleotide sequence encoding the heavy chain variable region set forth in SEQ ID NO: 10, and a nucleotide sequence encoding the light chain variable region set forth in SEQ ID NO: 18; (3) a nucleotide sequence encoding the heavy chain variable region set forth in SEQ ID NO: 12, and a nucleotide sequence encoding the light chain variable region set forth in SEQ ID NO: 16; (4) a nucleotide sequence encoding the heavy chain variable region set forth in SEQ ID NO: 20, and a nucleotide sequence encoding the light chain variable region set forth in SEQ ID NO: 22; (5) a nucleotide sequence encoding the heavy chain variable region set forth in SEQ ID NO: 20, and a nucleotide sequence encoding the light chain variable region set forth in SEQ ID NO:

24.

10. A vector, characterized in that, The vector comprises the polynucleotide of claim 8.

11. A genetically engineered host cell, characterized in that, The host cell comprises the vector of claim 10 or the polynucleotide of claim 8 integrated into the genome of the host cell.

12. Use of an active ingredient, characterized in that The active ingredient is selected from the group consisting of: the antibody or antigen-binding fragment thereof of claim 1, the recombinant protein of claim 4, the antibody drug conjugate of claim 6, or a combination thereof, for use in: (a) preparing a detection reagent or a kit; and / or (b) preparing a medicament or a preparation for preventing and / or treating a cancer or a tumor that is positive for TfRl.

13. The use according to claim 12, characterized in that, The TfR1-positive cancer or tumor is selected from the group consisting of AML, ALL, lymphoma, multiple myeloma, breast cancer, gastric cancer, glioblastoma, prostate cancer, urothelial bladder cancer, pancreatic cancer, esophageal cancer, colorectal cancer, ovarian cancer, liver cancer, or a combination thereof.

14. A pharmaceutical composition, characterized by, The pharmaceutical composition contains: (i) an active ingredient selected from the group consisting of: The antibody or antigen-binding fragment thereof of claim 1, the recombinant protein of claim 4, the antibody drug conjugate of claim 6, or a combination thereof; and (ii) a pharmaceutically acceptable carrier.

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