CN115594762A

CN115594762A - Ferritin heavy chain antibody and application thereof

Info

Publication number: CN115594762A
Application number: CN202110777534.5A
Authority: CN
Inventors: 柯天一; 郁倩文; 劳芳; 孙雯倩; 姚德惠; 张鹏飞; 刘岩; 阎西冲
Original assignee: Kunshan Xinyunda Biotech Co ltd
Current assignee: Kunshan Xinyunda Biotech Co ltd
Priority date: 2021-07-09
Filing date: 2021-07-09
Publication date: 2023-01-13

Abstract

The present disclosure relates to a ferritin heavy chain antibody and uses thereof, the antibody comprising a heavy chain variable region and/or a light chain variable region, wherein: the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5 respectively; or HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences therefrom, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10 respectively; or LCDR1, LCDR2 and LCDR3 with 1, 2 or 3 amino acid differences therefrom. The antibody has higher affinity with the heavy chain of ferritin through competitive screening.

Description

Ferritin heavy chain antibody and application thereof

Technical Field

The invention relates to a monoclonal antibody of a Ferritin heavy chain (Ferritin heavy chain), a hybridoma cell strain secreting the monoclonal antibody and application of the antibody.

Background

Iron is one of trace elements necessary for cell survival, and iron deficiency causes iron-deficiency anemia and child development retardation of a human body; however, excessive iron in cells induces the increase of free radicals, which may cause damage to biological macromolecules such as lipid, protein, carbohydrate, and nucleic acid, and finally result in cell death. Ferritin is essential for maintaining the iron balance in the cell. Ferritin (ferricin) is a large protein of approximately 450kDa self-assembled from 24 subunits into a spherical cage-like structure with internal and external dimensions of approximately 8 and approximately 12nm, respectively, which contains an iron core containing up to 4500 iron atoms. Eukaryotic ferritin comprises a heavy chain (H; 21 kDa) and a light chain (L; 19 kDa). The H chain is responsible for the oxidation of Fe (II) to Fe (III) and includes a catalytic iron oxidase site, while the L chain plays a role in iron nucleation. The H and L chains together assemble a 24-mer heteromeric ferritin.

The level of ferritin is one of the indicators of a healthy level of the body. Decreased ferritin levels are indicative of iron-deficiency anemia, and increased levels reflect the effects of many factors including malignancy, inflammation, liver disease, and repeated blood transfusions. Typically, serum ferritin levels are maintained within a relatively stable range, whereas WHO specifies normal concentrations of ferritin in the range of 30-300ng/mL and 15-200ng/mL for men and women, respectively. Abnormal elevation of serum ferritin may reflect a variety of diseases such as hepatitis, cirrhosis, liver cancer, lung cancer, leukemia, and the like. In addition, a decrease in serum ferritin can lead to anemia and malnutrition. Therefore, the detection of ferritin in serum has clinical guidance value.

Meanwhile, since ferritin has a cage structure capable of encapsulating a drug, remarkable stability, small size, and uniform size, the art has attempted to use ferritin as a drug carrier to deliver a drug. The mode of drug delivery of ferritin may be to load a drug, such as the small molecule drug doxorubicin, into the cage cavity. In addition, because the H-ferritin has the activity of binding with a receptor TfR1, the H-ferritin can target a plurality of tumor tissues with high expression of TfR1 and can cross a blood brain barrier. Therefore, the H-ferritin is tried to be used as a tumor drug carrier and a nano drug carrier capable of potentially crossing a blood brain barrier in the field, and the coupling of the H-ferritin and different therapeutic agents, tracers and the like can also achieve the effects of treatment and diagnosis.

The above studies have also raised the need for the measurement of ferritin itself, although several methods for measuring ferritin have been established, such as electrochemical immunosensors, radioimmunoassays and Inductively Coupled Plasma Mass Spectrometry (ICPMS). Although these methods are relatively sensitive and have excellent selectivity, these instruments are expensive, complicated to operate, require special expertise and highly trained operators. Therefore, the fast and convenient immunoassay method is still the most potential ferritin assay method, but the existing immunoassay kit becomes the bottleneck of immunoassay accuracy and reliability due to the problems of antibody sensitivity and specificity, and is also one of the reasons that the common sensitivity of the ferritin immunoassay used in hospitals at present is not high. At present, there are several reports related to ferritin antibodies, for example, CN110468108B and CN105821004A disclose monoclonal antibodies of light chain ferritin and applications thereof, but effective antibodies still lack for detection of H ferritin with huge drug loading potential, so there is still a need in the art to develop ferritin monoclonal antibodies with high sensitivity and good specificity.

Disclosure of Invention

In order to solve the above problems, the disclosure provides, in a first aspect, a hybridoma cell strain 3A71B6, which is deposited in the common microorganism center of the china committee for culture collection and management of microorganisms (CGMCC), wherein the hybridoma cell strain 3A71B6 has a collection number of CGMCC NO:21906, and is capable of secreting a monoclonal antibody 3A that specifically recognizes a heavy chain of human ferritin.

In a second aspect, the present disclosure provides a monoclonal antibody 3A secreted by a hybridoma cell line 3A71B6 that specifically recognizes the heavy chain of human ferritin, having a light chain and a heavy chain.

And wherein the heavy chain variable region of said heavy chain comprises the following three heavy chain CDRs:

SEQ ID NO.:3VH-CDR1：SYWMH

4VH-CDR2: EINPSNARTNYNEKFKS and

5VH-CDR3: SSYGKNAMDY. The light chain variable region of the light chain comprises the following three light chain CDRs:

SEQ ID NO.:8VL-CDR1：RTSQDISNYLN

9VL-CDR2: YTSRLHS and

SEQ ID NO.:10VL-CDR3：QEGNTLPRT

in another preferred embodiment, any one of the above amino acid sequences further comprises a derivative sequence optionally added, deleted, modified and/or substituted with at least one (e.g., 1-3, preferably 1-2, and more preferably 1) amino acid and capable of retaining heavy chain ferritin binding affinity.

In another preferred embodiment, the light chain of said antibody comprises said three light chain CDRs and a light chain framework region for linking the light chain CDRs; and the heavy chain of said antibody comprises said three heavy chain CDRs and a heavy chain framework region for linking the heavy chain CDRs.

In another preferred embodiment, the heavy chain variable region further comprises a human FR region or a murine FR region.

In another preferred embodiment, the heavy chain variable region has the sequence shown in SEQ ID No. 1.

In another preferred embodiment, the heavy chain of the antibody further comprises a heavy chain constant region.

In another preferred embodiment, the heavy chain constant region is of human, murine or rabbit origin, preferably of human origin.

In another preferred embodiment, the light chain variable region further comprises an FR region of human or murine origin.

In another preferred embodiment, the light chain variable region is as shown in SEQ ID NO. 6.

In another preferred embodiment, the light chain of the antibody further comprises a light chain constant region.

In another preferred embodiment, the light chain constant region is of human, murine or rabbit origin, preferably human.

In another preferred embodiment, the antibody is a humanized antibody.

In another preferred embodiment, the antibody is selected from the group consisting of: an antibody of animal origin, a chimeric antibody and/or a humanized antibody.

In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

In another preferred embodiment, the antibody is a monoclonal antibody.

In another preferred embodiment, the antibody comprises a monospecific, bispecific or trispecific antibody.

In another preferred embodiment, the antigen binding fragment is selected from the group consisting of Fab, fab '-SH, fv, scFv, and F (ab') 2.

In a third aspect of the present disclosure, there is provided a recombinant protein having the following elements:

(i) Heavy and/or light chains or antigen-binding fragments of said antibodies, or antibodies against heavy chain ferritin formed by said heavy and said light chains,

in another preferred embodiment, the heavy chain variable region of the heavy chain comprises 3 heavy chain CDRs as set forth in SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5, and the light chain variable region of the light chain comprises 3 light chain CDRs as set forth in SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10; and

(ii) Optionally a tag sequence to facilitate expression and/or purification.

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

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

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

In a fourth aspect of the present disclosure, there is provided an antibody preparation comprising:

(a) An antibody or antigen-binding fragment thereof according to the second aspect of the disclosure; and

(b) A pharmaceutically acceptable carrier, said carrier comprising: a buffer, sterile water, and optionally a surfactant.

In a fifth aspect of the present disclosure, there is provided a kit comprising an antibody or antigen-binding fragment thereof according to the second aspect of the present disclosure, a recombinant protein according to the third aspect of the present disclosure and/or an antibody preparation according to the fourth aspect of the present disclosure, and a container holding the antibody preparation.

In a sixth aspect of the present disclosure, there is provided an antibody drug conjugate comprising:

(a) An antibody portion selected from the group consisting of: an antibody or antigen-binding fragment thereof according to the second aspect of the present disclosure and/or a recombinant protein according to the third aspect of the present disclosure; and

(b) A coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide and/or enzyme.

In a seventh aspect of the present disclosure, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:

(1) An antibody or antigen-binding fragment thereof according to the second aspect of the disclosure; or

(2) A recombinant protein according to the third aspect of the disclosure.

In an eighth aspect of the present disclosure, there is provided a vector comprising a polynucleotide according to the seventh aspect of the present disclosure.

In another preferred embodiment, the carrier comprises: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retroviruses, or other vectors.

In a ninth aspect of the present disclosure, there is provided a genetically engineered host cell comprising a vector or genome according to the eighth aspect of the present disclosure integrated with a polynucleotide according to the seventh aspect of the present disclosure.

In a tenth aspect of the present disclosure, there is provided a use of an active ingredient selected from the group consisting of: an antibody or antigen-binding fragment thereof according to the second aspect of the present disclosure, a recombinant protein according to the third aspect of the present disclosure, an antibody preparation according to the fourth aspect of the present disclosure, an antibody drug conjugate according to the sixth aspect of the present disclosure, a polynucleotide according to the seventh aspect of the present disclosure, a vector according to the eighth aspect of the present disclosure, and/or a host cell according to the ninth aspect of the present disclosure, wherein the active ingredient is for:

(a) Preparing a detection reagent or kit;

(b) Preparing a medicament or preparation for preventing and/or treating ferritin related diseases; and/or

(c) Preparing a medicament or preparation for preventing and/or treating cancer.

In another preferred embodiment, the cancer or tumor is selected from the group consisting of: lung cancer, melanoma, colon cancer, pancreatic cancer, bladder cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, liver cancer, lymphoma, myeloma, leukemia.

In an eleventh aspect of the present disclosure, there is provided a method for detecting heavy chain ferritin in a sample in vitro for non-diagnostic purposes, the method comprising the steps of:

(1) Contacting the sample in vitro with an antibody or antigen-binding fragment thereof according to the second aspect of the present disclosure, a recombinant protein according to the third aspect of the present disclosure, an antibody preparation according to the fourth aspect of the present disclosure, and/or an antibody drug conjugate according to the sixth aspect of the present disclosure;

(2) Detecting the formation of an antigen-antibody complex, wherein the formation of a complex is indicative of the presence of heavy chain ferritin in the sample.

In a twelfth aspect of the disclosure, there is provided an in vitro method of detecting (including diagnostic or non-diagnostic purpose) heavy chain ferritin in a sample, the method comprising the steps of:

In a thirteenth aspect of the present disclosure, there is provided a method of treating a ferritin related disease, the method comprising:

administering to a subject in need thereof an antibody or antigen-binding fragment thereof according to the second aspect of the present disclosure, a recombinant protein according to the third aspect of the present disclosure, an antibody preparation according to the fourth aspect of the present disclosure and/or a drug conjugate of an antibody according to the sixth aspect.

In the fourteenth aspect of the disclosure, a hybridoma cell strain secreting a monoclonal antibody specifically recognizing a heavy chain of human ferritin is provided, and the preservation number is CGMCC NO. 21906.

It is to be understood that within the scope of the present disclosure, each of the above-described technical features of the present disclosure and each of the technical features specifically described below (e.g., examples) may be combined with each other to constitute a new or preferred technical solution. Not to be repeated herein, depending on the space.

Drawings

FIG. 1 shows the results of SDS-PAGE detection of the monoclonal antibodies of the disclosure.

Figure 2 shows an antibody concentration detection curve.

FIG. 3 shows the results of the WESTERN-BLOT assay for the monoclonal antibodies of the disclosure.

Detailed Description

The inventor obtains a hybridoma capable of secreting the heavy chain ferritin resisting monoclonal antibody with excellent affinity through extensive and intensive research and massive screening, and obtains the heavy chain ferritin monoclonal antibody through secretion of the hybridoma. Experiments show that the antibodies of the present disclosure are capable of high affinity binding to heavy chain ferritin.

I. Term(s) for

In the present disclosure, unless otherwise indicated, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms, and laboratory procedures used herein are all terms and routine procedures used extensively in the corresponding arts. Meanwhile, for better understanding of the present disclosure, definitions and explanations of related terms are provided below.

As used herein and unless otherwise specified, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where integers are required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur.

As used herein, the term "antibody" refers to an immunoglobulin, a tetrapeptide chain structure made up of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus, their antigenicity. Accordingly, immunoglobulins can be classified into five classes, or different types called immunoglobulins, namely IgM, igD, igG, igA and IgE, and the heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, d δ, ε, γ, and μ, respectively. IgG represents the most important class of immunoglobulins, which can be divided into 4 subclasses due to chemical structure and biological function differences: igG1, igG2, igG3, and IgG4. Light chains are classified as kappa or lambda chains by differences in the constant regions. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, as are the variable regions (V-regions); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable regions include 3 hypervariable regions (HVRs) and 4 FR Regions (FRs) which are relatively conserved in sequence. The amino acid sequences of the 4 FRs are relatively conserved and do not directly participate in the binding reaction. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) consists of 3 CDR regions and 4 FR regions, and the sequence from amino terminus to carboxy terminus is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain, the light chain hypervariable region (LCDR), refer to LCDR1, LCDR2 and LCDR3; the 3 CDR regions of the heavy chain, the heavy chain hypervariable region (HCDR), refer to HCDR1, HCDR2 and HCDR3. The CDR amino acid residues in the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the invention are in number and position in accordance with known Kabat numbering convention (LCDR 1-3, HCDR2-3), or in accordance with Kabat and chothia numbering convention (HCDR 1). The four FR regions in the native heavy and light chain variable regions are in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held together tightly by the FR regions and form the antigen binding site of the antibody with the CDRs of the other chain. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type. The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.

The term "antigen-binding fragment," as used herein, refers to a Fab fragment, fab 'fragment, F (ab') 2 fragment, or single Fv fragment having antigen-binding activity. Fv antibodies contain the variable regions of the antibody heavy chain, the variable regions of the light chain, but no constant regions, and have the smallest antibody fragment of the entire antigen binding site. Generally, fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding.

The present disclosure includes not only intact antibodies, but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the disclosure also includes fragments, derivatives and analogs of the antibodies.

In the present disclosure, antibodies include murine, chimeric, humanized antibodies comprising CDR regions of the present disclosure prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using DNA recombination techniques well known in the art.

As used herein, the term "monoclonal antibody" refers to an antibody secreted by a clone obtained from a single cell source. Monoclonal antibodies are highly specific, being directed against a single epitope. The cell may be a eukaryotic, prokaryotic, or phage clonal cell line.

As used herein, the term "chimeric antibody" is an antibody molecule expressed by a host cell transfected with a vector after splicing a V region gene of a murine antibody to a C region gene of a human antibody into a chimeric gene. Not only retains the high specificity and affinity of the parent mouse antibody, but also ensures that the humanized Fc segment can effectively mediate the biological effect function.

As used herein, the term "humanized antibody", is a variable region engineered version of a murine antibody of the present disclosure having CDR regions derived from (or substantially derived from) a non-human antibody (preferably a mouse monoclonal antibody), and FR regions and constant regions substantially derived from human antibody sequences; that is, the CDR sequence of mouse antibody is grafted to the framework sequence of different types of human germline antibody. Because the CDR sequences are responsible for most of the antibody-antigen interactions, recombinant antibodies that mimic the properties of a particular naturally occurring antibody can be expressed by constructing an expression vector.

As used herein, the term "monospecific antibody" refers to an antibody (including but not limited to a bivalent antibody) capable of binding to one particular epitope.

As used herein, the term "Bispecific antibody" (BsAb) refers to an antibody capable of binding to two different antigens or two different epitopes on the same antigen, typically a Bispecific antibody comprising two antigen binding sites, each of which is specific for a different epitope. In certain embodiments, the bispecific antibody is capable of binding two epitopes simultaneously, in particular two epitopes expressed on two different cells.

As used herein, the term "trispecific antibody" refers to an antibody capable of binding to three different antigens or three different epitopes on the same antigen or three different epitopes present on two different antigens.

As used herein, the term "multispecific antibody" refers to an antibody capable of binding to two or more different antigens or two or more different epitopes on the same antigen. Thus, multispecific antibodies include bispecific and trispecific antibodies.

In the present disclosure, the antibody may be monospecific, bispecific, trispecific, or more multispecific.

In the present disclosure, the antibody of the present disclosure also includes conservative variants thereof, which means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to the amino acid sequence of the antibody of the present disclosure to form a polypeptide. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table 1.

TABLE 1

Initial residue(s)	Representative substitutions	Preferred substitutions
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

As used herein, the term "nucleic acid molecule" refers to DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

The term "vector" as used herein 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.

As used herein, the term "expression" refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. The expression level of the polypeptide can be assessed using any method known in the art, including, for example, methods that determine the amount of polypeptide produced from the host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, coomassie blue staining after gel electrophoresis, lowry protein assay, and Bradford protein assay.

The term "host cell" as used herein refers to a cell into which an expression vector has been introduced. The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or a higher eukaryotic cell, such as a plant or animal cell (e.g., a mammalian cell).

As used herein, the term "pharmaceutical formulation" refers to such forms of formulation: the formulation allows the biological activity of the active ingredients contained therein to be effective and is free of additional ingredients having unacceptable toxicity to the subject to which the formulation is to be administered.

"pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation that is not an active ingredient and that is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

As used herein, the term "treatment" refers to the administration of a therapeutic agent, either internally or externally, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect, including any of the heavy chain ferritin antibodies of the present disclosure and compositions thereof. Typically, the therapeutic agent is administered to the patient in an amount (therapeutically effective amount) effective to alleviate one or more symptoms of the disease. By "treating" an individual having a disease or condition is meant that the individual's symptoms are partially or fully alleviated, or remain unchanged after treatment. Thus, treatment includes prophylaxis, treatment and/or cure. Prevention refers to prevention of the underlying disease and/or prevention of worsening of symptoms or progression of the disease. Treatment also includes any antibody or antigen-binding fragment thereof provided as well as any pharmaceutical use of the compositions provided herein.

As used herein, "therapeutic effect" means an effect resulting from treatment of an individual that alters, typically ameliorates or improves a symptom of a disease or disease condition, or cures the disease or disease condition.

As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect upon administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, block, or partially block the symptoms of the disease or disorder.

As used herein, a "prophylactically effective amount" or a "prophylactically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset or recurrence of a disease or symptom, reducing the likelihood of onset or recurrence of a disease or symptom. A complete prophylactically effective dose need not occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

As used herein, the terms "administration" 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. "administration" and "treatment" may refer to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells, and contacting the reagent with a fluid, and contacting the fluid with the cells. "administering" and "treating" also mean treating by a reagent, diagnostic, binding composition, or by another cell in vitro and ex vivo. "treatment" when applied to a human, animal or subject under study refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnosis; including contact of ferritin antibodies with humans or animals, subjects, cells, tissues, physiological compartments or physiological fluids.

As used herein, the term "patient" or "individual" refers to a mammal, such as a human.

Anti-heavy chain ferritin antibodies

As used herein, the term "heavy chain ferritin" generally refers to native or recombinant human heavy chain ferritin (HFn), as well as non-human homologues of human heavy chain ferritin.

The present disclosure provides a highly specific and high affinity antibody to human heavy chain ferritin comprising a heavy chain comprising heavy chain variable region (VH) amino acid sequences and a light chain comprising light chain variable region (VL) amino acid sequences.

In the present disclosure, a monoclonal antibody specifically binding to human heavy chain ferritin is obtained by selecting a high quality human heavy chain ferritin antigen to immunize a mouse, and then forming hybridoma cells by fusing mouse spleen cells and B cells.

Preferably, the CDRs of the heavy chain variable region (VH) are selected from the group consisting of:

VH-CDR1 shown in SEQ ID NO. 3,

VH-CDR2 shown in SEQ ID No. 4, and

VH-CDR3 shown in SEQ ID NO. 5; and/or

The CDRs of the light chain variable region (VL) are selected from the group consisting of:

VL-CDR1 shown in SEQ ID NO. 8,

VL-CDR2 of SEQ ID No. 9, and

VL-CDR3 shown in SEQ ID NO. 10.

Wherein, any one of the above amino acid sequences further comprises a derivative sequence with ferritin binding affinity, which is added, deleted, modified and/or substituted with at least one (such as 1-5, 1-3, preferably 1-2, more preferably 1) amino acid.

In another preferred embodiment, the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence is preferably an amino acid sequence with homology of at least 80%,85%,90%,95%,96%,97%,98% or 99%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

The antibody of the present disclosure may be a double-chain or single-chain antibody, and may be selected from an animal-derived antibody, a chimeric antibody, a humanized antibody, more preferably a humanized antibody, a human-animal chimeric antibody.

The antibody derivatives of the present disclosure may be single chain antibodies, and/or antibody fragments, such as: fab, fab ', (Fab') 2 or other antibody derivatives known in the art, and the like, as well as any one or more of IgA, igD, igE, igG, and IgM antibodies or antibodies of other subtypes.

Among them, the animal is preferably a mammal such as a mouse.

The antibodies of the present disclosure may be murine, chimeric, humanized, CDR-grafted and/or modified antibodies that target human ferritin.

In a preferred embodiment of the present disclosure, any one or more of the above-mentioned SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5, or sequences thereof having heavy chain ferritin binding affinity, which have been subjected to addition, deletion, modification and/or substitution of at least one amino acid, are located in the CDR regions of the heavy chain variable region (VH).

In a preferred embodiment of the present disclosure, any one or more of the above SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10, or sequences thereof having ferritin binding affinity with at least one amino acid added, deleted, modified and/or substituted, are located in the CDR regions of the light chain variable region (VL).

In a more preferred embodiment of the present disclosure, VH CDR1, CDR2, CDR3 are each independently selected from any one or more of SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5, or sequences thereof with ferritin binding affinity with at least one amino acid added, deleted, modified and/or substituted; VL CDR1, CDR2 and CDR3 are respectively and independently selected from any one or more sequences of SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10, or sequences with heavy chain ferritin binding affinity, wherein at least one amino acid is added, deleted, modified and/or substituted.

In the above-mentioned context of the present disclosure, the number of amino acids to be added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, more preferably 15 to 20% of the total number of amino acids in the original amino acid sequence.

In the present disclosure, the number of the amino acids to be added, deleted, modified and/or substituted is usually 1, 2, 3, 4 or 5, preferably 1 to 3, more preferably 1 to 2, and most preferably 1.

In a preferred embodiment of the present disclosure, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5, respectively; or HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5, respectively.

In a preferred embodiment of the present disclosure, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences as set forth in SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10, respectively.

In a preferred embodiment of the present disclosure, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in amino acid sequences SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10.

In a preferred embodiment of the present disclosure, the heavy chain variable region further comprises a human FR region or a murine FR region.

In a preferred embodiment of the present disclosure, the light chain variable region further comprises an FR region of human or murine origin.

In a preferred embodiment of the present disclosure, the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID No. 1, or an amino acid sequence having at least 80%,85%,90%,95%,96%,97%,98% or 99% sequence identity to the amino acid sequence shown in SEQ ID No. 1, preferably at least 85%, more preferably at least 90%, most preferably at least 95%.

In a preferred embodiment of the present disclosure, the light chain variable region comprises the amino acid sequence as shown in SEQ ID No. 6, or an amino acid sequence having at least 80%,85%,90%,95%,96%,97%,98% or 99% sequence identity to the amino acid sequence shown in SEQ ID No. 6, preferably at least 85%, more preferably at least 90%, most preferably at least 95%.

In a preferred embodiment of the present disclosure, the antibody further comprises a heavy chain constant region and/or a light chain constant region.

In a preferred embodiment of the present disclosure, the heavy chain constant region is of human, murine or rabbit origin, preferably human.

In a preferred embodiment of the present disclosure, the light chain constant region is of human, murine or rabbit origin, preferably of human origin.

In a preferred embodiment of the present disclosure, the antibody is a humanized antibody.

In a preferred embodiment of the present disclosure, the antibody is selected from the group consisting of: an antibody of animal origin, a chimeric antibody and/or a humanized antibody.

In a preferred embodiment of the present disclosure, the antibody is a double-chain antibody, or a single-chain antibody.

In a preferred embodiment of the present disclosure, the antibody is a monoclonal antibody.

In a preferred embodiment of the present disclosure, the antibody comprises a monospecific, bispecific, or trispecific antibody.

In a preferred embodiment of the present disclosure, the antigen binding fragment is selected from the group consisting of Fab, fab '-SH, fv, scFv, and F (ab') 2.

In a preferred embodiment of the present disclosure, the heavy chain of the antibody comprises the amino acid sequence of the heavy chain of the hybridoma-producing antibody with the collection number of CGMCC No. 21906, or an amino acid sequence having at least 80%,85%,90%,95%,96%,97%,98% or 99% sequence identity with the amino acid sequence of the heavy chain of the hybridoma-producing antibody, preferably at least 85%, more preferably at least 90%, most preferably at least 95%.

In a preferred embodiment of the present disclosure, the light chain of the antibody comprises the amino acid sequence of the light chain of the hybridoma-producing antibody with the accession number of CGMCC No. 21906, or an amino acid sequence having at least 80%,85%,90%,95%,96%,97%,98% or 99% sequence identity with the amino acid sequence of the light chain of the hybridoma-producing antibody, preferably at least 85%, more preferably at least 90%, most preferably at least 95%.

In a preferred embodiment of the present disclosure, the antibody comprises the amino acid sequence of the heavy chain and the amino acid sequence of the light chain of the hybridoma-producing antibody with the preservation number of CGMCC NO. 21906.

In a preferred embodiment of the present disclosure, there is provided an antibody against human heavy chain ferritin 3A:

3A heavy chain variable region amino acid sequence (SEQ ID NO: 1):

EVQLQQPGAELVKPGTSVKLSCKASGYIFTSYWMHWVKQRPGQGLEWIGEINPSNARTNYNEKFKSKATLT VDKSSSTTYMQLSSLTSEDSAVYYCARSSYGKNAMDYWGQGTSVTVSS

3A light chain variable region amino acid sequence (SEQ ID No.: 6):

DIQMTQTPSSLSASLGDRVTISCRTSQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTAYSLTI SNLEQEDIATYLCQEGNTLPRTFGGGTKLEIKR

the disclosure also provides nucleotide sequences encoding the above amino acids:

3A VH nucleotide sequence (SEQ ID No.: 2):

gaggtccagctgcagcagcctggggctgaactggtgaagcctgggacttcagtgaagctgtcctgcaaggcttctggctacatcttcaccagctactggatgcactgggtga agcagaggcctggacaaggccttgagtggattggagagattaatcctagcaacgctcgtactaactacaatgagaagttcaagagcaaggccacactgactgtagacaaatcctcc agcacaacctacatgcaactcagcagcctgacatctgaggactctgcggtctattactgtgcaagatcctcttatggtaaaaatgctatggactactggggtcaaggaacctcagtcac cgtctcctca

3A VL nucleotide sequence (SEQ ID No.: 7):

gatatccagatgactcagaccccatcctccctgtctgcctctctgggagacagagtcaccatcagttgcaggacaagtcaggacattagtaattatttaaactggtatcagcaga aaccagatggaactgttaaactcctgatctactacacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagcttattctctgaccattagcaacctgg agcaagaagatattgccacttacctttgccaagagggtaatacgcttccgaggacgttcgggggaggcaccaagctggaaataaaacgg

preparation of antibodies

Any method suitable for producing monoclonal antibodies can be used to produce the heavy chain ferritin antibodies of the disclosure. For example, an animal can be immunized with a linked or naturally occurring heavy chain ferritin protein or fragment thereof. Suitable immunization methods, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes may be used.

Monoclonal antibodies can also be produced by hybridomas that include B cells obtained from a transgenic non-human animal, such as a transgenic mouse, having a genome comprising all the components of human immunoglobulin heavy and light chain gene segments fused to immortalized cells. For example, monoclonal antibodies that specifically bind human heavy chain ferritin are obtained by selecting a high quality human heavy chain ferritin antigen to immunize mice, and then fusing mouse spleen cells and B cells to form hybridoma cells. The inventor obtains a hybridoma capable of secreting the heavy chain ferritin monoclonal antibody with excellent affinity through extensive and intensive research and large-scale screening, and the preservation number of the hybridoma is CGMCC NO:21906.

Ferritin in any suitable form may be used as an immunogen (antigen) for the production of non-human antibodies specific for ferritin, which antibodies are screened for biological activity. The immunogen may be used alone or in combination with one or more immunogenicity enhancing agents known in the art. Immunogens can be purified from natural sources or produced in genetically modified cells. The DNA encoding the immunogen may be genomic or non-genomic in origin (e.g., cDNA). DNA encoding the immunogen may be expressed using suitable genetic vectors, including but not limited to adenoviral vectors, baculovirus vectors, plasmids, and non-viral vectors.

Humanized antibodies may be selected from any class of immunoglobulins, including IgM, igD, igG, igA, and IgE. Likewise, any type 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 present disclosure.

An exemplary method for making ferritin antibodies of the disclosure is described in example 1.

The sequence of the DNA molecule of the antibodies or fragments thereof of the present disclosure can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Alternatively, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. The DNA sequence can then be introduced into various existing DNA molecules (or e.g., vectors) and cells known in the art.

The disclosure also relates to vectors comprising suitable DNA sequences as described above and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell so that it can express the protein.

The steps described in this disclosure for transforming a host cell with a recombinant DNA can be performed using techniques well known in the art. The resulting transformants, which express the polypeptides encoded by the genes of the present disclosure, can be cultured by conventional methods. Depending on the host cell used, it is cultured in a conventional medium under suitable conditions.

Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the disclosure. The antibodies of the present disclosure are then purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, using conventional separation and purification means well known to those skilled in the art.

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

Antibody-drug conjugates (ADC)

The present disclosure also provides antibody-conjugated drugs (ADCs) based on the antibodies of the present disclosure.

Typically, the antibody-conjugated drug comprises the antibody, and an effector molecule to which the antibody is conjugated, and preferably chemically conjugated. Wherein the effector molecule is preferably a therapeutically active drug. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

The antibody of the present disclosure may be conjugated to the effector molecule by a coupling agent. Examples of the coupling agent may be any one or more of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond. The non-selective coupling agent is a compound which enables effector molecules and antibodies to form covalent bonds, such as glutaraldehyde and the like. The coupling agent using carboxyl can be any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (coupling site is acylhydrazone).

Certain residues on the antibody (e.g., cys or Lys, etc.) are used to attach to a variety of functional groups, including imaging agents (e.g., chromophores and fluorophores), diagnostic agents (e.g., MRI contrast agents and radioisotopes), stabilizing agents (e.g., ethylene glycol polymers) and therapeutic agents. The antibody may be conjugated to a functional agent to form an antibody-functional agent conjugate. Functional agents (e.g., drugs, detection reagents, stabilizers) are coupled (covalently linked) to the antibody. The functional agent may be directly linked to the antibody, or indirectly linked through a linker.

Antibodies may be conjugated to drugs to form Antibody Drug Conjugates (ADCs). Typically, the ADC comprises a linker between the drug and the antibody. The linker may be degradable or non-degradable. Degradable linkers are typically susceptible to degradation in the intracellular environment, e.g., degradation of the linker at the site of interest, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, e.g., glucuronide-containing linkers that can be degraded by glucuronidase. Peptidyl linkers may include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH sensitive linkers (e.g., linkers that hydrolyze at a pH of less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions in which the antibody is hydrolyzed by a protease.

Prior to attachment to the antibody, the linker has reactive groups capable of reacting with certain amino acid residues, and attachment is achieved by the reactive groups. Thiol-specific reactive groups are preferred and include: such as maleimide compounds, haloamides (e.g., iodine, bromine or chlorine); halogenated esters (e.g., iodo, bromo, or chloro); halomethyl ketones (e.g., iodo, bromo, or chloro), benzyl halides (e.g., iodo, bromo, or chloro); vinyl sulfone, pyridyl disulfide; mercury derivatives such as 3, 6-bis- (mercuric methyl) dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may comprise, for example, a maleimide linked to the antibody via a thiosuccinimide.

The drug may be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, the linker links the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or keto group that may form a bond with the linker. In the case of a drug attached directly to a linker, the drug has a reactive active group prior to attachment to the antibody.

Useful classes of drugs include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. In the present disclosure, a drug-linker can be used to form an ADC in one simple step. In other embodiments, bifunctional linker compounds may be used to form ADCs in a two-step or multi-step process. For example, a cysteine residue is reacted with a reactive moiety of a linker in a first step and in a subsequent step a functional group on the linker is reacted with a drug, thereby forming an ADC.

Generally, the functional group on the linker is selected to facilitate specific reaction with a suitable reactive group on the drug moiety. As a non-limiting example, azide-based moieties may be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker by a 1, 3-dipolar cycloaddition between the azide and the alkynyl group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other ligation strategies, such as those described in bioconjugation technology, second edition (Elsevier), are well known to those skilled in the art. It will be appreciated by those skilled in the art that for selective reaction of the drug moiety and linker, each member of a complementary pair may be used for both the linker and the drug when the reactive functional group of the complementary pair is selected.

Antibody formulations

The disclosure also provides pharmaceutical formulations comprising the antibodies. The pharmaceutical formulations generally include one or more optional pharmaceutically acceptable carriers or excipients.

The selection of the carrier is determined in part by the particular cell and/or method of administration. Thus, there are a number of suitable formulations. For example, the pharmaceutical formulation may comprise a preservative. Suitable preservatives can include, for example, methyl paraben, propyl paraben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is employed. Preservatives or mixtures thereof are typically present in an amount of from about 0.0001% to about 2% (by total weight of the formulation). Carriers are described, for example, in Remington's Pharmaceutical Sciences, 16 th edition, osol, a. Eds (1980). At the dosages and concentrations employed, pharmaceutically acceptable carriers are generally non-toxic to the recipient and include, but are not limited to: buffers such as phosphate, citrate and other organic acid buffers; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride, quaternary ammonium salts of hexa-hydrocarbonic acid, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, such as methyl or propyl parabens, catechol, m-benzenediol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other sugars including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zn-protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG).

In some embodiments, the pharmaceutical formulation comprises an amount, such as a therapeutically or prophylactically effective amount of cells, effective to treat or prevent a disease or disorder. In some embodiments, therapeutic or prophylactic efficacy is monitored by periodically evaluating the treated subject. The desired dose may be delivered by single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.

The antibody has different stability in different preparation buffers, and is represented by the change of charge heterogeneity, degradation, polymerization and the like of antibody molecules, and the change of the quality properties is related to the physicochemical properties of the antibody, so that the preparation buffers suitable for the antibody need to be screened according to the physicochemical properties of different antibodies in the development process of antibody drugs. The currently commonly used antibody preparation buffer systems include phosphate buffer, citric acid buffer, histidine buffer, and the like, and according to the antibody properties, saline ions with different concentrations or excipients such as sorbitol, trehalose, sucrose, and the like, and a proper amount of surfactants such as tween 20 or tween 80 and the like are added to maintain the stability of the antibody.

The antibody drug combination preparation disclosed by the invention can effectively inhibit side reactions such as aggregation precipitation, hydrolysis, oxidation and deamidation of the humanized antibody, and can effectively improve the stability of the product under pressurization (high temperature, strong light irradiation, freeze thawing and the like), acceleration and long-term refrigeration conditions.

Methods of treatment and compositions

Any of the antibodies or antigen-binding fragments thereof or antibody-drug conjugates of the present disclosure may be used in a method of treatment.

In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof or antibody-drug conjugate of the present disclosure for use as a medicament. In other aspects, an antibody or antigen-binding fragment thereof or antibody-drug conjugate of the disclosure for use in treating a disease is provided.

In some embodiments, an antibody or antigen-binding fragment thereof or antibody-drug conjugate of the present disclosure as used in a method of treatment is provided. In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof or antibody-drug conjugate of the present disclosure for use in a method of treating an individual, the method comprising administering to the individual an effective amount of the antibody or antigen-binding fragment thereof or antibody-drug conjugate of the present disclosure. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. An "individual" according to any of the above embodiments may be a human.

In yet another aspect, the present disclosure provides the use of an antibody or antigen-binding fragment thereof or antibody-drug conjugate of the present disclosure in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treating a disease. In yet another embodiment, the medicament is for use in a method of treating a disease, the method comprising administering to an individual having a disease an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. An "individual" according to any of the above embodiments may be a human.

In yet another aspect, the present disclosure provides a method for treating a disease. In one embodiment, the method comprises administering to an individual having such a disease an effective amount of an antibody or antigen-binding fragment thereof or an antibody-drug conjugate as described in the present disclosure. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent as described below. An "individual" according to any of the above embodiments may be a human.

In yet another aspect, the disclosure provides a pharmaceutical formulation comprising an antibody or antigen-binding fragment thereof or antibody-drug conjugate of the disclosure, e.g., for use in any of the methods of treatment described above. In one embodiment, a pharmaceutical formulation comprises an antibody or antigen-binding fragment thereof or an antibody-drug conjugate described in the present disclosure, and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation comprises an antibody or antigen-binding fragment thereof or antibody-drug conjugate described in the present disclosure and at least one additional therapeutic agent, e.g., as described below.

The antibodies or antigen-binding fragments thereof or antibody-drug conjugates described in the present disclosure may be used alone or in combination with other active agents in therapy. For example, an antibody or antigen-binding fragment thereof or antibody-drug conjugate described in the present disclosure can be co-administered with at least one additional therapeutic agent.

Such combination therapies indicated above encompass both combined administration (where two or more therapeutic agents are contained in the same or separate formulations), and separate administration, in which case administration of the antibodies of the disclosure can occur prior to, concurrently with, and/or subsequent to administration of additional therapeutic agents and/or adjuvants. The antibodies or antigen-binding fragments thereof or antibody-drug conjugates described in the present disclosure may also be used in combination with radiotherapy.

The antibodies or antigen-binding fragments thereof or antibody-drug conjugates of the present disclosure (and any additional therapeutic agent) may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is transient or chronic. Various dosing regimens are contemplated herein, including but not limited to multiple administrations, single or at multiple time points, bolus administrations, and pulsed infusions.

The antibody or antigen-binding fragment thereof or antibody-drug conjugate will be formulated, administered and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the plan of administration and other factors known to the practitioner. The antibody or antigen-binding fragment thereof or antibody-drug conjugate need not be formulated with, but is optionally formulated with, one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody or antigen-binding fragment thereof or antibody-drug conjugate present in the formulation, the type of disease or therapy, and other factors discussed above. These are typically used at the same dosages and using the routes of administration as described herein, or at about 1% to 99% of the dosages described herein, or at any dosage and any route empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of the antibody or antigen-binding fragment thereof or antibody-drug conjugate (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody or antigen-binding fragment thereof or antibody-drug conjugate, the severity and course of the disease, whether the antibody or antigen-binding fragment thereof or antibody-drug conjugate is administered for prophylactic or therapeutic purposes, previous therapy, the clinical history of the patient and response to the antibody or antigen-binding fragment thereof or antibody-drug conjugate, and the judgment of the attending physician. The antibody or antigen-binding fragment thereof or antibody-drug conjugate is administered to the patient, suitably at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μ g/kg to 15mg/kg (e.g., 0.5mg/kg-10 mg/kg) of the antibody or antigen-binding fragment thereof or antibody-drug conjugate may be an initial candidate dose for administration to a patient, whether or not, e.g., by one or more separate administrations or by continuous infusion. A typical daily dose may be from about 1. Mu.g/kg to 100mg/kg or more, depending on the factors mentioned above. For repeated administration over a range of days or longer, depending on the condition, treatment will generally continue until the desired suppression of disease symptoms occurs. An exemplary dose of the antibody or antigen-binding fragment thereof or antibody-drug conjugate will range from about 0.05mg/kg to about 10mg/kg. Thus, a patient may be administered a dose of one or more of about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg, or 10mg/kg (or any combination thereof). Such doses may be administered intermittently, e.g., weekly or every 3 weeks (e.g., such that the patient receives from about 2 to about 20 doses of the antibody or antigen-binding fragment thereof or antibody-drug conjugate, or, e.g., about 6 doses of the antibody or antigen-binding fragment thereof or antibody-drug conjugate). A higher initial loading dose may be administered followed by one or more lower doses. However, other dosage regimens may be used. The progress of this therapy is readily monitored by conventional techniques and assays.

It will be appreciated that any of the above-described formulations or methods of treatment may be practiced using the immune antibodies or antigen-binding fragments thereof or antibody-drug conjugates of the present disclosure instead of or in addition to antibodies or antigen-binding fragments thereof or antibody-drug conjugates as reported herein.

Detection use and kit

The antibodies of the present disclosure can be used in detection applications, for example, to detect a sample, thereby providing diagnostic information.

In the present disclosure, the samples (specimens) employed include cells, tissue samples, and biopsy specimens. The term "biopsy" as used in this disclosure shall include all kinds of biopsies known to the person skilled in the art. A biopsy as used in the present disclosure may thus comprise a tissue sample prepared, for example, by endoscopic methods or a needle or needle biopsy of an organ.

Samples used in the present disclosure include fixed or preserved cell or tissue samples.

The present disclosure also provides a kit containing the antibody (or fragment thereof) of the present disclosure, and in a preferred embodiment of the present disclosure, the kit further comprises a container, instructions for use, a buffer, and the like. In a preferred embodiment, the antibodies of the present disclosure may be immobilized to a detection plate.

The main advantages of the present disclosure include:

(1) The antibodies of the present disclosure have excellent biological activity and specificity;

(2) The antibody disclosed by the invention has higher affinity through competitive screening;

the disclosure is further set forth below in connection with the specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure. Experimental procedures without specifying the detailed conditions in the following examples, generally followed by conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: cold Spring Harbor laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

TABLE 2 Experimental Main reagents

Name of reagent	Manufacturer(s) of
		Protein marker	Thermo
Coomassie brilliant blue G250	Chemical test of national medicine groupAgents Ltd
		RPMI1640	Thermo
HAT	SIGMA
		HT	SIGMA
Enzyme-labeled secondary antibody	Jackson
		PEG	MERCK
Fetal bovine serum	All-season green tea
		Superdex 200pg	GE Healthcare
Nano drop	Thermo

Example 1:

1. preparation of antigens

The coding nucleotide sequence (SEQ ID NO: 12) was obtained from the wild-type amino acid sequence (SEQ ID NO:11; see PDB:3AJO _A) of the human ferritin H subunit, and Nde1 cleavage site was added before the above nucleotide sequence, and after the sequence, a stop codon and Hind III cleavage site sequence were added, followed by full-gene synthesis and sequencing, and ligated into pET-30a (+) expression plasmid vector. After the expression vector is successfully constructed, E.coli BL21 (DE 3) is selected as a host bacterium, and the target gene-containing strainThe recombinant plasmid is transformed into host bacterium competent cells, and positive clones are screened by a kanamycin-containing resistant plate to determine a recombinant strain. The recombinant strain is inoculated in an LB culture medium for shake culture and is subjected to shake culture at 37 ℃ at a low speed until OD is reached ₆₀₀ About 1.0, adding 0.5mM IPTG, inducing expression for 3-4 h, and centrifuging to obtain bacterial sludge. And adding 25mL of 20mM Tris-HCl buffer solution into bacterial sludge centrifugally collected by every 30ml of bacterial liquid, uniformly suspending, and crushing for 0.5-2 min at 1000bar in a high-pressure homogenizer. Homogenizing the crushed thallus lysate under high pressure, and centrifuging (1500rpm, 10min) to remove the thallus fragments of the Escherichia coli; heating the supernatant at 72 deg.C for 15 min; precipitating the foreign protein, and centrifuging to remove the precipitate; separating and purifying the supernatant on an exclusion chromatography Superdex 200pg column; performing SDS-PAGE electrophoresis to identify purity; nano drop determines protein concentration. The purity of all samples reaches over 96 percent.

2. Preparation and purification of monoclonal antibodies

(1) Immunizing animals

Female BALB/c mice, 6-8 weeks old, were used for 4 immunizations according to the pre-established immunization protocol.

The first immunization was performed at a dose of 50. Mu.g/mouse, and the antigen was diluted to 100. Mu.l with PBS, mixed with an equal volume of complete Freund's adjuvant, and completely emulsified, and injected subcutaneously into the groin of each mouse with a 2ML syringe. The second immunization was separated by two weeks, and the third and second immunizations were separated by two weeks, with the same procedures and amounts as the first immunization except that Freund's complete adjuvant was changed to Freund's incomplete adjuvant. And 7 days after the third immunization, taking blood, and detecting the antibody titer by an ELISA method. Two weeks after the third immunization, a fourth booster immunization was performed, and the mice from which the spleen was to be removed were boosted, injected intraperitoneally, and contained 50. Mu.g of antigen (100. Mu.l diluted in PBS), and an equal volume of incomplete Freund's adjuvant. Splenocytes with titers above 100 ten thousand were selected for cell fusion. Before fusion, 50. Mu.g/mouse of heavy chain ferritin antigen was used for intraperitoneal challenge.

(2) Cell fusion

Mice were sacrificed, spleens were removed, ground in a 200 mesh screen, washed with RPMI1640 medium, and splenocytes suspensions were prepared. The prepared myeloma cells SP2/0 of the same line were washed 4 times with RPMI1640 medium and centrifuged at 15000rpm for 5min each. Mixing the prepared mouse spleen cells and myeloma cells according to a ratio of 1.

Detection of hybridoma cell culture supernatants by ELISA: the plates were coated with 2. Mu.g/ml of human heavy chain ferritin (HFn) protein, 100. Mu.l per well, washed three times with PBST (phosphate buffered saline, pH 7.4) after overnight at 4 ℃ and blocked with 5% nonfat dry milk, 300. Mu.l per well, incubated for 2 hours at 37 ℃. The blocking solution was discarded and the plates were washed 2 times with PBST. Hybridoma cell culture supernatants were added to 100. Mu.l/well of microplate, while PBST (phosphate buffered saline) was added as a negative control, and incubated at 37 ℃ for 2 hours. The plate was washed with PBS buffer and incubated for 2 hours at 37 deg.C with goat anti-mouse IgG antibody conjugated to horseradish peroxidase (HRP). After washing the plate, adding TMB substrate 100. Mu.l per well in the microplate, incubating at 37 ℃ for 15min, terminating the reaction with 2M sulfuric acid 50. Mu.l per well, and analyzing the value with a microplate reader under OD450 conditions. 30 hybridoma cell strains are selected from positive clones, culture supernatant is collected, 5 hybridoma cell strains are selected for subcloning after testing, subtype identification results show that the hybridoma cell strains are all single subtypes, namely the hybridoma cell strains with the anti-heavy chain ferritin with the best potency are named as 3A, and the subtype is identified as IgG2b.

The selected positive hybridoma cell lines are subjected to monoclonal screening (limiting dilution method) to obtain hybridoma cell clones capable of producing monoclonal antibodies with high titer (indirect ELISA dilution ratio >1, 5,000). And (4) expanding and culturing the hybridoma cell strain, and freezing and preserving the strain. The positive hybridoma cell is an anti-human heavy chain ferritin hybridoma cell line 3A71B6 which is stored in a preservation organization-China general microbiological culture Collection center, wherein the preservation organization is CGMCC for short, the address is No. 3 of Beijing Western No. 1 Hospital on North Chen of the open area, the preservation date is 2021 year, 3 months and 12 days, the suggested classification is named as Balb/c mouse hybridoma cell line, and the preservation number is CGMCC NO:21906.

(3) Preparation and purification of monoclonal antibodies

Preparation: inoculating the hybridoma cell strain 3A71B6 obtained in the step (2) to the abdominal cavity of a BALB/c mouse, preparing ascites, and then purifying the antibody from the ascites.

And (3) purification: proteinG affinity purification was used. The ProteinG packing well balanced with PB solution was loaded into the purification column, and after the ascites was diluted with 0.02M PB solution, it was filtered through 0.22 μ M filter membrane and applied to the column at a flow rate of 0.6 ml/min. After the loading was complete, the column was washed with 0.02M PB solution until Coomassie Brilliant blue G250 did not turn blue. Then, the mixture was eluted with 0.1M glycine-hydrochloric acid solution (pH3.0), and the eluate was collected until Coomassie brilliant blue G250 became blue. The pH of the eluted product was adjusted to neutral with saturated sodium carbonate. The 10kDa ultrafiltration tube is ultrafiltered and concentrated to about 1-5ml, dialyzed and concentrated and replaced by PBS pH7.4 solution. The SDS-PAGE results showed that the purity of the purified antibody was above 95% (see FIG. 1).

3. Sandwich ELISA detection and identification of monoclonal antibody

To assess the binding activity of the hybridoma clones, purified monoclonal antibodies from the clones were tested in an ELISA assay.

The method comprises the following steps:

1) Antigen coating: the antigen was diluted to 1. Mu.g/ml with the coating solution, and 100. Mu.L/well was added to a polystyrene 96-well reaction plate and left overnight at 4 ℃.

2) Washing: the next day the coating liquid was discarded and the wash was washed 1 time at 200. Mu.L/well.

3) And (3) sealing: add 200. Mu.L/well blocking solution and stand at 37 ℃ for 2h.

4) Washing: the next day the blocking solution was discarded and the wash solution 200. Mu.L/well was washed 3 times.

5) Adding the sample to be tested (i.e. the prepared monoclonal antibody): the sample to be tested (the sample needs to be diluted to the set concentration with PBS) is added at 100. Mu.L per well and left at 37 ℃ for 1h.

6) Washing: the sample to be tested is discarded, and the washing solution is washed 3 times at 200 mu L/hole.

7) Enzyme-labeled secondary antibody addition: HRP-labeled goat anti-mouse IgG (1: 10000 diluted with enzyme) was added thereto, and the mixture was stored at 37 ℃ for 40min per well.

8) Washing: the enzyme-labeled secondary antibody was discarded, and the washing solution was washed 5 times at 200. Mu.L/well.

9) Color development: adding a substrate solution prepared freshly 90 mu L/hole, and standing at 37 ℃ in a dark place for 15-20 min

10 Termination reaction, color comparison: after adding 50 mu L/well stop solution, the absorbance of each well at 450nm was measured with a microplate reader

The OD values of different antibody concentration detections are drawn into a concentration detection curve, and as shown in figure 2, the sensitivity of the monoclonal antibody for detecting ferritin antigen can reach 0.5ng/ml.

From the results in FIG. 2, the EC50 value of 3A mab prepared according to the present disclosure is 3.678ng/ml, and has good affinity binding ability with ferritin antigen.

4. WESTERN-BLOT detection and identification of monoclonal antibody

1) Preparation of Polyacrylamide gels

(1) Prepare 12% strength separation gel solution (10 ml): 3.3ml of deionized water, 4ml of 30% acrylamide, 2.5ml of Tris at pH8.8, 100. Mu.L of 10% ammonium persulfate, 100. Mu.L of 10% SDS, and 6. Mu.L of TEMED were added in this order (the gel immediately started to polymerize after the TEMED was added, and therefore, the gel was immediately and rapidly mixed).

(2) The separating glue solution is added into the gap between the two glass plates quickly to leave the space for filling the concentrated glue.

(3) After the separation gel polymerization was complete, a 5% concentrated gel solution (4 ml) was prepared: 2.7ml of deionized water, 670. Mu.L of 30% acrylamide, 500. Mu.L of Tris at pH6.8, 40. Mu.L of 10% ammonium persulfate, 40. Mu.L of 10% SDS, 6. Mu.L of TEMED were added in this order, the concentrated gel was poured directly onto the polymerized gel, and a clean comb was immediately inserted into the concentrated gel solution.

2) Preparation of samples

Then the recombinant heavy chain ferritin was diluted to 10 ng/. Mu.l with 8M urea, 2.5. Mu.l to 0.5ml EP tube was added with 7.5. Mu.l 8M urea (total volume was about 10. Mu.l), 10. Mu.l 2 Xloading buffer was added and mixed well, centrifuged for 2min in boiling water bath 5-10min at 2500rpm, and the tube wall solution was centrifuged.

3) Electrophoresis

(1) The sample mixture was slowly added to the sample well with a pipette, pre-stained with Marker, and loaded at 10 μ L.

(2) And (4) carrying out 80V electrophoresis for 30min, changing the voltage to 120V, and carrying out electrophoresis until the bromophenol blue loading buffer solution migrates to the bottom of the gel in the gel.

4) Rotary film

(1) Proteins from the gel were transferred to PVDF membrane and blocked.

5) Binding antibodies

(1) The membrane is taken out and soaked in monoclonal antibody diluent at 25 ℃ for 2h or 4 ℃ overnight. And diluting the monoclonal antibody to 5-10 mu g/ml. The dilution was 500ml of 1 × TBS +2.5g casein +250 μ l Tween.

(2) The membranes were removed and washed 5 times with TBST (5 min each time with shaking on a horizontal shaker).

(3) The membrane was removed and soaked in a secondary antibody (goat anti mouse-HRP) diluted in TBS diluent at 25 ℃ for 1h. Wherein, the goat anti-mouse-HRP and TBS are diluted according to the proportion of 1.

6) Exposure method

The results are shown in fig. 3, and it can be seen from the results that the monoclonal antibody prepared by the present disclosure can specifically recognize heavy chain ferritin at 20 KD.

Example 2: identification of 3A antibody variable region sequences

The 3A antibody protein was subjected to sequencing (Wuhan Kingkurui bioengineering, inc.) to obtain Anti-HFn candidate sequences.

3A VH amino acid sequence (SEQ ID No.: 1)

EVQLQQPGAELVKPGTSVKLSCKASGYIFTSYWMHWVKQRPGQGLEWIGEINPSNARTNYNEKFKSKATLTV DKSSSTTYMQLSSLTSEDSAVYYCARSSYGKNAMDYWGQGTSVTVSS

The corresponding nucleotide sequence is (SEQ ID NO: 2):

3A VL amino acid sequence (SEQ ID No.: 6)

DIQMTQTPSSLSASLGDRVTISCRTSQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTAYSLTIS NLEQEDIATYLCQEGNTLPRTFGGGTKLEIKR

The corresponding nucleotide sequence (SEQ ID No.: 7):

wherein the underlining region is CDRs (defined by IMGT, as follows):

TABLE 3 murine anti-HFn antibody light chain variable regions and CDR sequences

TABLE 4 murine anti-HFn antibody heavy chain variable regions and CDR sequences

Sequence listing

<110> Kunshan New Nentada Biotech Co., ltd

<120> ferritin heavy chain antibody and application thereof

<130> MTI21191

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 119

<212> PRT

<213> 3A heavy chain variable region (Mouse)

<400> 1

Glu Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn Pro Ser Asn Ala Arg Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Thr Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Ser Tyr Gly Lys Asn Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser

115

<210> 5

<211> 357

<212> DNA

<213> 3A heavy chain variable region nucleotide sequence (Mouse)

<400> 5

gaggtccagc tgcagcagcc tggggctgaa ctggtgaagc ctgggacttc agtgaagctg 60

tcctgcaagg cttctggcta catcttcacc agctactgga tgcactgggt gaagcagagg 120

cctggacaag gccttgagtg gattggagag attaatccta gcaacgctcg tactaactac 180

aatgagaagt tcaagagcaa ggccacactg actgtagaca aatcctccag cacaacctac 240

atgcaactca gcagcctgac atctgaggac tctgcggtct attactgtgc aagatcctct 300

tatggtaaaa atgctatgga ctactggggt caaggaacct cagtcaccgt ctcctca 357

<210> 2

<211> 5

<212> PRT

<213> 3A heavy chain variable region CDR1 (Mouse)

<400> 2

Ser Tyr Trp Met His

1 5

<210> 3

<211> 17

<212> PRT

<213> 3A heavy chain variable region CDR2 (Mouse)

<400> 3

Glu Ile Asn Pro Ser Asn Ala Arg Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Ser

<210> 4

<211> 10

<212> PRT

<213> 3A heavy chain variable region CDR3 (Mouse)

<400> 4

Ser Ser Tyr Gly Lys Asn Ala Met Asp Tyr

1 5 10

<210> 6

<211> 108

<212> PRT

<213> 3A light chain variable region (Mouse)

<400> 6

Asp Ile Gln Met Thr Gln Thr Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Thr Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Ala Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Leu Cys Gln Glu Gly Asn Thr Leu Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 7

<211> 324

<212> DNA

<213> 3A light chain variable region nucleotide sequence (Mouse)

<400> 7

gatatccaga tgactcagac cccatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca ggacaagtca ggacattagt aattatttaa actggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctactac acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagct tattctctga ccattagcaa cctggagcaa 240

gaagatattg ccacttacct ttgccaagag ggtaatacgc ttccgaggac gttcggggga 300

ggcaccaagc tggaaataaa acgg 324

<210> 8

<211> 11

<212> PRT

<213> 3A light chain variable region CDR1 (Mouse)

<400> 8

Arg Thr Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 9

<211> 7

<212> PRT

<213> 3A light chain variable region CDR2 (Mouse)

<400> 9

Tyr Thr Ser Arg Leu His Ser

1 5

<210> 10

<211> 9

<212> PRT

<213> 3A light chain variable region CDR3 (Mouse)

<400> 10

Gln Glu Gly Asn Thr Leu Pro Arg Thr

1 5

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds a ferritin heavy chain comprising a heavy chain variable region and/or a light chain variable region wherein:

the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID No.:3, SEQ ID No.:4 and SEQ ID No.:5, respectively; or HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5, respectively;

the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10 respectively; or LCDR1, LCDR2 and LCDR3 which have 1, 2 or 3 amino acid differences with the amino acid sequences shown in SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10 respectively.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10 respectively;

preferably, the heavy chain variable region further comprises an FR region of human or murine origin;

preferably, the light chain variable region further comprises an FR region of human or murine origin;

preferably, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID No. 1, or an amino acid sequence having at least 80%,85%,90%,95%,96%,97%,98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 1, preferably at least 85%, more preferably at least 90%, most preferably at least 95%;

preferably, the light chain variable region comprises the amino acid sequence as set forth in SEQ ID No. 6, or an amino acid sequence having at least 80%,85%,90%,95%,96%,97%,98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 6, preferably at least 85%, more preferably at least 90%, most preferably at least 95%.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody further comprises a heavy chain constant region and/or a light chain constant region;

preferably, the heavy chain constant region is of human, murine or rabbit origin, preferably of human origin;

preferably, the light chain constant region is of human, murine or rabbit origin, preferably human;

preferably, the antibody is a humanized antibody;

preferably, the antibody is selected from the group consisting of: an antibody of animal origin, chimeric antibody and/or humanized antibody;

preferably, the antibody is a double-chain antibody or a single-chain antibody;

preferably, the antibody is a monoclonal antibody;

preferably, the antibody comprises a monospecific, bispecific, or trispecific antibody;

preferably, the antigen binding fragment is selected from the group consisting of Fab, fab '-SH, fv, scFv, and F (ab') 2;

preferably, the heavy chain of the antibody comprises the amino acid sequence of the heavy chain of the hybridoma-producing antibody with the collection number of CGMCC NO. 21906, or an amino acid sequence having at least 80%,85%,90%,95%,96%,97%,98% or 99% sequence identity with the amino acid sequence of the heavy chain of the hybridoma-producing antibody, preferably at least 85%, more preferably at least 90%, most preferably at least 95%;

preferably, the light chain of the antibody comprises the amino acid sequence of the light chain of the hybridoma-producing antibody with the preservation number of CGMCC NO. 21906, or an amino acid sequence with at least 80%,85%,90%,95%,96%,97%,98% or 99% sequence identity with the amino acid sequence of the light chain of the hybridoma-producing antibody, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%;

preferably, the antibody comprises the amino acid sequence of the heavy chain and the amino acid sequence of the light chain of the hybridoma-producing antibody with the preservation number of CGMCC NO:21906.

4. A recombinant protein having the following elements:

(i) The heavy and/or light chain of the antibody of any one of claims 1-3 or an antigen-binding fragment of said antibody, or an anti-heavy chain ferritin antibody formed by said heavy and light chains, and

(ii) Optionally a tag sequence to facilitate expression and/or purification;

preferably, the tag sequence comprises a 6His tag;

preferably, the recombinant protein (or polypeptide) comprises a fusion protein;

preferably, the recombinant protein is a monomer, dimer, or multimer.

5. A polynucleotide encoding a polypeptide selected from the group consisting of:

(1) The antibody or antigen-binding fragment thereof of any one of claims 1-3; or

(2) The recombinant protein of claim 4.

6. A vector comprising the polynucleotide of claim 5;

preferably, the vector is selected from the group consisting of bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

7. A genetically engineered host cell comprising a genome into which has been integrated the polynucleotide of claim 5, or comprising the vector of claim 6.

8. An antibody preparation, said antibody preparation comprising:

(a) The antibody or antigen-binding fragment thereof of any one of claims 1-3; and

9. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 3, the recombinant protein of claim 4 and/or the antibody preparation of claim 5, and a container holding the antibody preparation.

10. An antibody drug conjugate, said antibody drug conjugate comprising:

(a) An antibody moiety selected from the group consisting of: the antibody or antigen-binding fragment thereof of any one of claims 1-3 and/or the recombinant protein of claim 4; and

(b) A coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, and/or an enzyme.

11. Use of an active ingredient selected from the group consisting of: the antibody or antigen-binding fragment thereof of any one of claims 1 to 3, the recombinant protein of claim 4, the polynucleotide of claim 5, the vector of claim 6, the host cell of claim 7, the antibody preparation of claim 8, and/or the antibody drug conjugate of claim 10, wherein the active ingredient is for:

(a) Preparing a detection reagent or kit;

(c) Preparing a medicament or preparation for preventing and/or treating cancer or tumor;

preferably, the cancer or tumor is selected from the group consisting of: lung cancer, melanoma, colon cancer, pancreatic cancer, bladder cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, liver cancer, lymphoma, myeloma, leukemia.

12. An in vitro method for detecting (including diagnostic or non-diagnostic) heavy chain ferritin in a sample, comprising the steps of:

(1) Contacting said sample in vitro with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, a recombinant protein according to claim 4 and/or an antibody preparation according to claim 8 and/or an antibody drug conjugate according to claim 10;

(2) Detecting the formation of an antigen-antibody complex, wherein the formation of the complex is indicative of the presence of heavy chain ferritin in the sample.

13. A method of treating a ferritin related disease comprising:

administering to a subject in need thereof an antibody or antigen-binding fragment thereof according to any one of claims 1-3, a recombinant protein according to claim 4, an antibody preparation according to claim 8 and/or an antibody drug conjugate according to claim 10.

14. A hybridoma cell strain secreting a monoclonal antibody specifically recognizing a human ferritin heavy chain has a preservation number of CGMCC NO. 21906.