CN111303289B

CN111303289B - Anti-human Tn-type glycosylated MUC1 antibody and application thereof

Info

Publication number: CN111303289B
Application number: CN202010146410.2A
Authority: CN
Inventors: 张君; 劳滔滔; 林潮喜; 张雁
Original assignee: Shenzhen Beinuobo Biotechnology Co ltd
Current assignee: Shenzhen Beinuobo Biotechnology Co ltd
Priority date: 2020-03-05
Filing date: 2020-03-05
Publication date: 2022-10-14
Anticipated expiration: 2040-03-05
Also published as: CN111303289A

Abstract

The application provides an antibody or an antigen-binding portion thereof binding to human Tn-type glycosylated MUC1 and a use thereof, a detection kit comprising the antibody or the antigen-binding portion thereof and a use thereof, and a method for detecting the level of human Tn-type glycosylated MUC1 in a sample using the antibody or the antigen-binding portion thereof.

Description

Anti-human Tn-type glycosylated MUC1 antibody and application thereof

Technical Field

The present application relates generally to the field of biological detection, and in particular, provides antibodies or antigen-binding portions thereof that bind human Tn-glycosylated MUC1 and uses thereof. The present application provides novel antibodies or antigen-binding portions thereof that bind to human Tn-glycosylated MUC1, and provides kits comprising the antibodies or antigen-binding portions thereof, and methods of detecting the level of human Tn-glycosylated MUC1 in a sample using the antibodies or antigen-binding portions thereof.

Background

The human tumor-associated mucin MUC1, also known as cancer antigen 15-3 (CA 15-3) or epitopic protein, is a highly glycosylated, high molecular weight protein with >50% glycosylation and a molecular refraction of >200kD, and its coding sequence is 1821bp long and contains 7 exons, of which the 2 nd exon contains tandem repeats (VNTR), and the VNTR levels of different people vary from 20 to 125, and constitutes a polymorphism of the MUC1 gene, belonging to a transmembrane molecule. Each VNTR contains 60 base pairs and contains 5 potential O-glycosylation sites. When abnormal O-glycosylation is accompanied by cellular malignant transformation, the original O sugar chain structure becomes a tumor-specific sugar chain structure such as Tn antigen, sialylated Tn antigen, or T antigen. Due to autoimmune tolerance, the MUC1 peptide chain alone cannot stimulate the body to produce a strong enough immune response. Sorensen et al, which use recombinant glycosyltransferase to add O sugar chain structure on MUC1, synthesize Tn antigen, sialylated Tn antigen and O-glycosylated complete glycopeptide antigen, and the complete glycopeptide antigen induced immunization method with complete O-glycosylation at 5 sites should be the most strong. Tn antigen is a precursor of T antigen, mucin is expressed in a concealed way under normal conditions, and Tn antigen is exposed and generates stronger immune response when cells are degenerated with incomplete glycosylation, so that the Tn antigen can be used as a marker of potential tumor threat.

MUC1 is a specific molecular marker on the surface of about 90 percent of tumor cells of breast cancer, lung cancer, colon cancer, cervical cancer and the like. The National Cancer Institute (NCI) considers MUC1 as the second most promising tumor-specific target of the 75 priority tumor-associated antigens now discovered. MUC1 is not only a specific marker on the surface of tumor cells, but also the quantification of the MUC1 level in blood is an important index for clinicians to follow up cancer patients and monitor the recurrence and metastasis of tumors. However, due to the diversity of glycosylation and instability of MUC1 in serum, MUC1 is not a homogeneous polypeptide. Distinguishing healthy and tumor cell-derived MUC1 requires accurate measurement of tumor-associated, specifically glycosylated MUC1 in clinical samples, and is therefore very challenging.

At present, the clinical detection of MUC1 in China seriously depends on diagnostic antibody products developed by foreign enterprises. The existing diagnostic antibody can recognize MUC1 at different sites, and the sensitivity and accuracy of each diagnostic product are different due to the limitation of recognizing the tumor marker in the initial establishment of the detection. Recent studies on MUC1 by the professor Carl June at the university of Pennsylvania were published in 2016, and have shown that Tn-type glycosylation is critical in determining MUC1 as a tumor cell-specific antigen.

Therefore, the Tn-type glycosylated MUC1 peptide fragment is specially subjected to immune screening to develop an antibody product for clinical blood immune diagnosis and tumor tissue immunochemistry diagnosis, which not only has prospective and innovative properties, but also can fill the blank of high-titer Tn-type glycosylated MUC1 specific antibody diagnostic reagent products in China, and can be extended to other diagnostic projects which are particularly sensitive to titer and even develop MUC1 specific tumor targeted drugs.

Summary of The Invention

In a first aspect, the present application provides an antibody that binds human Tn-type glycosylated MUC1, comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 amino acid sequences and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 amino acid sequences, wherein

The amino acid sequence of the HCDR1 is shown as SEQ ID NO. 2, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 3, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 4, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 6, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 7; or alternatively

The amino acid sequence of the HCDR1 is shown as SEQ ID NO. 8, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 9, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 10, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 11, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 12, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 13;

wherein the amino acid sequences of the HCDR and LCDR are defined according to Kabat or Chothia.

In some embodiments of the first aspect, the antibody is a whole antibody, a Fab fragment, a F (ab') ₂ Fragment or single chain Fv fragment (scFv).

In some embodiments of the first aspect, the antibody is a monoclonal antibody.

In some embodiments of the first aspect, the antibody binds to the C-terminus of human Tn-glycosylated MUC1, said C-terminus comprising the amino acid sequence set forth in SEQ ID No. 1.

In some embodiments of the first aspect, the C-terminus comprises an epitope defined by the amino acids:

the amino acid sequence shown in the 1 st to 12 th positions of SEQ ID NO.1, the amino acid sequence shown in the 1 st to 16 th positions of SEQ ID NO.1, the amino acid sequence shown in the 17 th to 26 th positions of SEQ ID NO.1, the amino acid sequence shown in the 35 th to 45 th positions of SEQ ID NO.1, the amino acid sequence shown in the 49 th to 68 th positions of SEQ ID NO.1 or the amino acid sequence shown in the 69 th to 82 th positions of SEQ ID NO. 1.

In a second aspect, the present application provides a kit for detecting the level of human Tn-glycosylated MUC1 in a sample, comprising a first antibody and/or a second antibody;

wherein the first antibody comprises a heavy chain variable region comprising HCDR1 shown in SEQ ID NO. 2, HCDR2 shown in SEQ ID NO. 3 and HCDR3 shown in SEQ ID NO. 4, and a light chain variable region comprising LCDR1 shown in SEQ ID NO. 5, LCDR2 shown in SEQ ID NO. 6 and LCDR3 shown in SEQ ID NO. 7; and/or

The second antibody comprises a heavy chain variable region comprising HCDR1 shown in SEQ ID NO. 8, HCDR2 shown in SEQ ID NO. 9 and HCDR3 shown in SEQ ID NO. 10, and a light chain variable region comprising LCDR1 shown in SEQ ID NO. 11, LCDR2 shown in SEQ ID NO. 12 and LCDR3 shown in SEQ ID NO. 13.

In some embodiments of the second aspect, the kit further comprises a standard, a quality control, and/or a buffer.

In some embodiments of the second aspect, the kit is used to aid in the detection or assessment of a tumor, such as pancreatic cancer.

In some embodiments of the second aspect, the level of Tn-type glycosylated MUC1 is associated with increased tumor risk, diagnosis of the presence of a tumor, or severity of a tumor.

In some embodiments of the second aspect, the level of Tn-type glycosylated MUC1 is an important indicator for monitoring the severity of postoperative recovery, tumor recurrence or metastasis in a tumor patient.

In a third aspect, the present application provides a method for detecting the level of human Tn-glycosylated MUC1 in a sample, the method comprising contacting a first antibody and a second antibody with the sample;

The second antibody comprises a heavy chain variable region comprising HCDR1 shown in SEQ ID NO. 8, HCDR2 shown in SEQ ID NO. 9 and HCDR3 shown in SEQ ID NO. 10 and a light chain variable region comprising LCDR1 shown in SEQ ID NO. 11, LCDR2 shown in SEQ ID NO. 12 and LCDR3 shown in SEQ ID NO. 13.

In some embodiments of the third aspect, the contacting is simultaneous or sequential.

In some embodiments of the third aspect, the level of human Tn-glycosylated MUC1 in the sample is detected by an immunological method.

In some embodiments of the third aspect, the immunological method is selected from the group consisting of: enzyme-linked immunosorbent assay, immunofluorescence, immunoturbidimetry, immunochemiluminescence, immunoprecipitation and combinations thereof.

In some embodiments of the second or third aspect, the first or second antibody is labeled with a detectable label.

In some embodiments of the second or third aspect, the detectable label is selected from the group consisting of: enzymes, fluorescent molecules, radioisotopes, electrochemiluminescent molecules, latex particles, gold particles, detectable ligands, and combinations thereof.

In some embodiments of the second or third aspect, the first or second antibody is attached to an immobilization surface.

In some embodiments of the second or third aspect, the fixed surface is a plastic or glass container, or a surface of a slide.

In a fourth aspect, the present application provides the use of an antibody according to the first aspect, or a kit according to the second aspect, for detecting the level of human Tn-glycosylated MUC1 in a sample.

In some embodiments of the fourth aspect, the level of Tn-type glycosylated MUC1 is associated with increased tumor risk, diagnosis of the presence of a tumor, or severity of a tumor.

In some embodiments of the fourth aspect, the level of Tn-type glycosylated MUC1 is an important indicator for monitoring the severity of postoperative recovery, tumor recurrence or metastasis in a tumor patient.

In a fifth aspect, the present application provides use of an antibody according to the first aspect in the preparation of a kit for detecting the level of human Tn-glycosylated MUC1 in a sample.

In some embodiments of the fifth aspect, the level of Tn-type glycosylated MUC1 is associated with increased tumor risk, diagnosis of the presence of a tumor, or severity of a tumor.

In some embodiments of the fifth aspect, the level of Tn-type glycosylated MUC1 is an important indicator for monitoring the severity of postoperative recovery, tumor recurrence or metastasis in a tumor patient.

In some embodiments of any of the above aspects, the sample is selected from the group consisting of: blood, serum, plasma, lymph, urine, gastric juice, bile, saliva, sweat, spinal fluid, feces, muscle biopsy, and combinations thereof.

Drawings

FIG. 1 is a schematic representation of the B cell in vitro cloning technique to obtain immunoglobulin antibodies of the present application that specifically recognize Tn-type glycosylated MUC1.

Figure 2 is a standard curve plotted for an ELISA assay using the antibodies of the present application.

FIG. 3 is an analysis of Tn-type glycosylated MUC1 levels in clinical samples.

FIG. 4 shows the comparison of the binding capacity of the antibody of the present application to Abbotium antibody.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO.1 shows the amino acid sequence of the C-terminus of human (homo sapiens) Tn-type glycosylated MUC1.

SEQ ID NO. 2 shows the amino acid sequence of HCDR1 of the first antibody.

SEQ ID NO 3 shows the amino acid sequence of HCDR2 of the first antibody.

SEQ ID NO. 4 shows the amino acid sequence of HCDR3 of the first antibody.

SEQ ID NO 5 shows the amino acid sequence of LCDR1 of the first antibody.

SEQ ID NO. 6 shows the amino acid sequence of LCDR2 of the first antibody.

SEQ ID NO. 7 shows the amino acid sequence of LCDR3 of the first antibody.

SEQ ID NO 8 shows the amino acid sequence of HCDR1 of the second antibody.

SEQ ID NO 9 shows the amino acid sequence of HCDR2 of the second antibody.

SEQ ID NO. 10 shows the amino acid sequence of HCDR3 of the second antibody.

SEQ ID NO 11 shows the amino acid sequence of LCDR1 of the second antibody.

SEQ ID NO. 12 shows the amino acid sequence of LCDR2 of the second antibody.

SEQ ID NO. 13 shows the amino acid sequence of LCDR3 of the second antibody.

Detailed Description

In order to achieve the measurement of the level of human Tn-type glycosylated MUC1, the inventors of the present application obtained a novel antibody or an antigen-binding portion thereof that binds to the C-terminal of human Tn-type glycosylated MUC1 by an antibody engineering technique. In various aspects of the present application, there are provided a detection kit comprising the antibody or an antigen-binding portion thereof, and uses thereof, a method for detecting the level of human Tn-glycosylated MUC1 using the antibody or the antigen-binding portion thereof, and uses of the antibody or the antigen-binding portion thereof in detecting the level of human Tn-glycosylated MUC1 and in preparing a kit for detecting the level of human Tn-glycosylated MUC1 in a sample.

The practice of the present invention will employ, unless otherwise indicated, molecular biology, microbiology, cell biology, biochemistry and immunology techniques which are conventional in the art.

Unless otherwise indicated, terms used in the present application have meanings commonly understood by those skilled in the art.

Definition of

The term "breast cancer" as used herein refers to a malignant tumor that occurs in mammary gland epithelial tissue, which may metastasize in organs such as lung, liver, bone, brain, etc., and destroy its normal tissue. The cause of breast cancer is unknown, and the survival rate of breast cancer is greatly related to the discovery of the early and late stages. According to the American cancer society, the five-year survival rate of patients with type I breast cancer reaches 88%, the higher the grade is, the lower the survival rate is, and the 5-year survival rate of type IV breast cancer is only 15%.

The term "subject" as used herein refers to a living human or non-human organism. Preferred subjects herein are human subjects.

The term "antibody" as used herein, refers to a peptide capable of passing through at least one positionThe antigen recognition site in the variable region of the immunoglobulin molecule specifically binds to the immunoglobulin molecule of the target. Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, polypeptides, and the like. As used herein, "antibody" includes not only intact (i.e., full-length) antibodies, but also antigen-binding fragments thereof (e.g., fab ', F (ab') ₂ Fv), variants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site of a desired specificity, including glycosylated variants of an antibody, amino acid sequence variants of an antibody, and covalently modified antibodies.

Typically, a complete or full-length antibody comprises two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and first, second and third constant regions (CH 1, CH2 and CH 3). Each light chain contains a light chain variable region (VL) and a constant region (CL). The full-length antibody may be any type of antibody, such as IgD, igE, igG, igA, or IgM (or subclasses thereof), but the antibody need not belong to any particular class. Depending on the antibody amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to different classes. Generally, there are five main classes of immunoglobulins: igA, igD, igE, igG and IgM, and several of these classes can be further classified into subclasses (isotypes), such as IgG1, igG2, igG3, igG4, igA1 and IgA2. The heavy chain constant domains corresponding to different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.

The term "antigen-binding portion" or "antigen-binding site" or "target-binding site" as used herein means one or more fragments of a binding protein (e.g., an antibody or receptor), such as an immunoglobulin variable domain (e.g., VH or VL), that retain the ability to specifically bind to an antigen or target. The antigen-binding portion of an antibody can be a fragment of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" include, but are not limited to: (i) Fab sheetA monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) F (ab') ₂ A fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) (ii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment comprising a single variable domain; and (vi) an isolated Complementarity Determining Region (CDR). Furthermore, the VL and VH of the Fv encoded by separate genes can be joined by a synthetic linker using recombinant methods into a single protein chain in which the VH and VL regions pair to form monovalent molecules (known as single chain Fv (scFv)). Such scfvs are also encompassed within the term "antigen-binding portion", as are other forms of single chain antibodies, such as diabodies and "linear antibodies" that comprise a pair of tandem Fv fragments (VH-CH 1-VH-CH 1) that, together with a complementary light chain polypeptide, form a pair of antigen-binding sites. Not every amino acid of the antigen-binding portion can bind to an antigen. For example, the variable domain of an antibody comprises Complementarity Determining Regions (CDRs) and Framework Regions (FRs).

The term "CDR" as used herein means the complementarity determining region within an immunoglobulin variable region sequence. There are three CDRs in the variable regions of the heavy and light chains, respectively, which are designated CDR1, CDR2, and CDR3 for the heavy and light chain variable regions, respectively. The term "set of CDRs" refers to a set of three CDRs present in a single variable region capable of binding antigen. The exact boundaries of these CDRs have been defined according to different systems. The system described by Kabat (Kabat et al (1971) Ann. NY Acad. Sci.190: 382-391. These CDRs may be referred to as Kabat CDRs. The amino acid residues of the CDR regions are more variable (e.g., hypervariable) than other amino acid residues in the variable regions of the heavy and light chains of the antibody. Chothia and colleagues (Chothia and Lesk (1987) J.mol.biol.196:901-917, chothia et al (1989) Nature 342. These sub-portions are designated as L1, L2 and L3 or H1, H2 and H3, where "L" and "H" designate the light and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs that overlap with Kabat CDRs have been described by Padlan (1995) FASEB J.9:133-139 and MacCallum (1996) J.mol.biol.262 (5): 732-45. Still other CDR boundary definitions may not strictly follow one of the systems herein, but still overlap with the Kabat CDRs. The antibodies herein, or antigen binding portions thereof, can utilize CDRs defined according to any of these systems.

The middle CDR sequence of a variable region sequence can be analyzed in a variety of ways for the variable region sequence of a given antibody, such as can be determined using the online software Abysis (http:// www. Abysis. Org. /).

The term "specific binding" as used herein refers to a non-random binding reaction between two molecules, e.g. binding of an antibody to an epitope of an antigen.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for the possible presence of naturally occurring mutations in a small number of individuals. The monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and also include fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al, proc. Natl. Acad. Sci. USA 81 6851-6855 (1984).

MUC1 (GenBank accession numbers: NC-000001.11 (gene) and NP-001018017.1 (protein)) mainly exists in certain epithelial tissues and organs, has tissue specificity in expression, is mainly expressed in epithelial tissues such as mammary gland, particularly in all normal and cancerous mammary glands, and is not expressed in lymph nodes derived from mesenchymal tissues, so that the MUC1 can be used as an effective marker for lymph node metastasis of certain epithelial tumors. After canceration, MUC1 expression is enhanced, the structure is changed in glycosylation, and particularly, the Tn-type glycosylated MUC1 is highly abnormally expressed in breast cancer, and has higher clinical diagnosis and application value for diagnosis of the breast cancer because the specificity of the Tn-type glycosylated MUC1 is higher than that of a tissue polypeptide antigen and the sensitivity of the Tn-type glycosylated MUC is higher than that of a carcinoembryonic antigen (CEA).

Initiation of protein glycosylation begins with covalent attachment of a glycan to an asparagine residue (N-link), or to a serine or threonine residue (O-link). Wherein the O-linked glycosylation is formed by the attachment of N-acetylgalactosamine (GalNac) to a serine or threonine residue catalyzed by about 20 human polypeptides galactosamine transferase (galactosamine-TS). In normal cells, the GalNAc residues attached to the protein backbone are further extended by T synthase, forming a core 1 structure (galactose-GalNAc-a-serine/threonine). On the surface of tumor cells, the most common forms of aberrant glycosylation are Tn (GalNAca 1-O-Ser/Thr) and sialylation Tn (STn) (NeuAca 2-6-GalNAca 1-O-Ser/Thr). The accumulation of Tn is mainly caused by the fact that the chaperone protein Cosmc necessary for sugar chain synthesis of normal cells is mutated in tumor cells or gene silencing is caused by epigenetics, and the loss of T synthetase activity is finally influenced. Aberrant glycosylation patterns of Tn are very common on MUC1 proteins on the surface of tumor cells. However, in healthy individuals, there is no expression of Tn antigen, and the human body has natural IgM antibodies against Tn.

The use of glycoprotein glycomodifications for cancer diagnosis and therapy has recently become a focus. Due to the diversity of glycosylation, antibodies obtained by immunization with glycoproteins have their corresponding epitopes that are difficult to identify without an antibody-antigen complex structure. MUC1 is not only a glycoprotein, but also has a relatively large molecular weight, and is expected to show a heterogeneous state in serum as various glycosylation and degradation intermediates, different configurations, and the like. The establishment of MUC1 glycosylation sites and forms which are directly and closely related to tumor antigenicity and are used as diagnostic indexes to prepare corresponding monoclonal antibodies is undoubtedly the key for improving the diagnostic sensitivity and specificity of MUC1.

Tn-type glycosylated MUC1 has been shown to increase dramatically in secretion during cellular carcinogenesis and to elicit severe immune responses. The test of the probe containing VNTR by Wanglishun et al shows that MUC1mRNA is not expressed or is low-expressed in normal tissues, but is obviously enhanced in adenocarcinoma, so that the probe can be used as a target for diagnosing and treating breast cancer. Studies on MUC1 immunoradiometric assay (IRMA) by Zhang Xin et al show that MUC1 serum level is in positive correlation with tumor malignancy degree, and has important reference value for breast cancer diagnosis. The Mensdorf et al study suggested that immunization with the MUC1 core peptide can produce second generation antibodies. MUC1 is involved in diseases such as breast cancer, tumor and adenocarcinoma, has important auxiliary diagnosis effect on the breast cancer, but has limited specificity, so that the recognition of the MUC1 by the existing diagnostic antibody acts on different sites, and simultaneously, the sensitivity and the accuracy of each diagnostic product are different due to the limitation of the recognition of the tumor marker in the initial establishment of the detection. A recent study of MUC1 by the professor Carl June at the university of Pennsylvania was published in 2016 and suggests that Tn-type glycosylation is critical in determining MUC1 as a tumor cell-specific antigen. This finding lays the theoretical foundation of the present invention.

Human Tn-type glycosylated MUC1 comprises a specific C-terminal domain comprising the following amino acid sequence:

MTPGTQSPFFLLLLLTVLTVVTGSGHASSTPGGEKETSATQRSSVPSSTEKNAFNSSLEDPSTDYYQELQRDISEMFLQIYK(SEQ ID NO:1)。

the inventors of the present application found that by using the antibodies disclosed herein in combination with immunoassays (e.g., ELISA) well known in the art, the levels of Tn-type glycosylated MUC1 in a sample can be reliably and reproducibly detected and correlated with, for example, the presence, severity and stage of breast cancer in a subject.

Exemplary C-terminal epitopes include, but are not limited to:

MTPGTQSPFLL (amino acid sequence shown in positions 1-12 of SEQ ID NO: 1);

MTPGTQSPFLLLLLLT (amino acid sequence shown in 1 st-16 th position of SEQ ID NO: 1);

VLTVVTGSGH (amino acid sequence shown at positions 17-26 of SEQ ID NO: 1);

KETSATQRSSV (amino acid sequence shown at positions 35-45 of SEQ ID NO: 1);

EKNANSSLEDDPDYYQE (amino acid sequence shown in 49-68 th position of SEQ ID NO: 1);

LQRDDISEMFLQIYK (amino acid sequence shown in positions 69-82 of SEQ ID NO: 1).

Wherein the letters represent standard amino acid codes. While these amino acid sequences describe linear epitopes, epitopes resulting from the secondary and tertiary structure of the N-terminus and comprising amino acids from discrete stretches of the sequence can also be used.

In a first aspect, the present application provides an antibody that binds human Tn-glycosylated MUC1 comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 amino acid sequences and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 amino acid sequences, wherein

In some embodiments of the first aspect, the antibody is a monoclonal antibody.

In some embodiments of the first aspect, the antibody is a rabbit monoclonal antibody.

In some embodiments of the first aspect, the antibody binds to the C-terminus of human Tn-type glycosylated MUC1, said C-terminus comprising the amino acid sequence set forth in SEQ ID No. 1.

In some embodiments of the first aspect, the C-terminus comprises an epitope defined by the amino acids: the amino acid sequence shown in the 1 st to 12 th positions of SEQ ID NO.1, the amino acid sequence shown in the 1 st to 16 th positions of SEQ ID NO.1, the amino acid sequence shown in the 17 th to 26 th positions of SEQ ID NO.1, the amino acid sequence shown in the 35 th to 45 th positions of SEQ ID NO.1, the amino acid sequence shown in the 49 th to 68 th positions of SEQ ID NO.1 or the amino acid sequence shown in the 69 th to 82 th positions of SEQ ID NO. 1.

In some embodiments of the second aspect, the standard is native cancer antigen 15-3 from human serum.

In some embodiments of the second aspect, the quality control is a negative and a positive quality control of native cancer antigen 15-3 from human serum.

In some embodiments of the second aspect, the buffer is a PBS buffer at pH 7.2 to which is added 0.1% Triton X-100.

In some embodiments of the second aspect, the kit further comprises a container for mixing the sample with the antibody. Such containers may be suitable for use in a detection instrument capable of detecting a signal generated by a detection monoclonal antibody.

In some embodiments of the second aspect, the kit may additionally comprise one or more of: (1) Instructions for using the kit to determine the level of Tn-glycosylated Muc 1; (2) A monoclonal antibody or antigen-binding portion thereof labeled with a detectable label; and (3) a solid phase on which either antibody or an antigen-binding portion thereof is immobilized. If no labeled antibody is provided, the antibody itself may be labeled with a detectable label, such as a chemiluminescent moiety, an enzymatic moiety, a fluorescent moiety, or a radioactive moiety.

In some embodiments of the third aspect, the level of Tn-type glycosylated MUC1 in the sample can be quantified using an antibody, or antigen-binding portion thereof, that binds to the C-terminal portion of human Tn-type glycosylated MUC1. According to the present application, the level of Tn-type glycosylated MUC1 can be detected immunologically using the antibodies or antigen-binding portions thereof described above. For example, tn-type glycosylated MUC1 can be detected and quantified using a "sandwich" assay. In this method, typically, an antibody is immobilized on a solid surface in order to bind and capture Tn-type glycosylated MUC1, which antibody is thus referred to herein as a capture antibody. The other antibody is detectably labeled, for example, with a fluorophore, an enzyme, or a colored particle, so that its binding to the Tn-type glycosylated MUC 1-complex indicates that the Tn-type glycosylated MUC1 has been captured. The intensity of the signal is proportional to the concentration of Tn-glycosylated MUC1 in the sample. The other antibody is therefore also referred to herein as a detection antibody or a labeled antibody.

Such assay methods may be referred to as a two-site immunoassay, "sandwich" method or (when the antibody is a binder) "sandwich immunoassay". The capture and detection antibodies can be contacted with the test sample simultaneously or sequentially as is known in the art. Sequential methods, sometimes referred to as "forward" methods, can be accomplished by incubating the capture antibody with the sample and thereafter adding a labeled detection antibody at a predetermined time. Alternatively, the labeled detection antibody can be first incubated with the sample, and the sample can then be contacted with the capture antibody (sometimes referred to as the "reverse" method). Such assays may be performed in many specific formats known to those skilled in the art, including through the use of different high throughput clinical laboratory analyzers or with point of care (care) or home test equipment.

The most commonly used enzyme immunoassay is the "enzyme-linked immunosorbent assay (ELISA)". ELISA is a technique that uses a labeled (e.g., enzyme-linked) form of an antibody to detect and measure the concentration of an antigen. There are different ELISA formats known to the person skilled in the art. Standard techniques for ELISA known in the art are described in "Methods in immunology", 2 nd edition, rose and bigzi, eds. John Wiley & Sons,1980; campbell et al, "Methods and Immunology", w.a. benjamin, inc.,1964; and Oelleric, m. (1984, j. Clin. Chem. Clin. Biochem.22.

In a "sandwich ELISA," an antibody (e.g., anti-Tn-type glycosylated MUC 1) is attached to a solid phase (i.e., a microtiter plate) and contacted with a biological sample containing an antigen (e.g., tn-type glycosylated MUC 1). The solid phase is then washed to remove unbound antigen. The labeled antibody (e.g., enzyme-linked) is then bound to the bound antigen, thereby forming an antibody-antigen-antibody sandwich. Examples of enzymes that can be linked to an antibody are alkaline phosphatase, horseradish peroxidase, luciferase, urease and β -galactosidase. The enzyme-linked antibody reacts with the substrate to produce a colored reaction product that can be measured. This measurement can be used to deduce the concentration of Tn-type glycosylated MUC1 present in the sample, for example, by comparing the measurements to a Tn-type glycosylated MUC1 standard curve.

The immunofluorescence method is based on the principle of antigen-antibody reaction, firstly, the known antigen or antibody is marked with fluorescein to prepare a fluorescent marker, and then the fluorescent antibody (or antigen) is used as a molecular probe to examine the corresponding antigen (or antibody) in cells or tissues. The antigen-antibody complex formed in the cell or tissue contains fluorescein, the specimen is observed by a fluorescence microscope, the fluorescein emits bright fluorescence when being irradiated by exciting light, and the cell or tissue where the fluorescence is located can be seen, so that the property and the location of the antigen or the antibody can be determined, and the content can be measured by a quantitative technology.

Chemiluminescence immunoassay (CLIA) is a detection and analysis technique for various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, drugs and the like by combining a chemiluminescence assay technique with high sensitivity and a high specificity immunoreaction. Is a latest immunoassay technology developed after radioimmunoassay, enzyme immunoassay, fluoroimmunoassay and time-resolved fluoroimmunoassay. Chemiluminescent immunoassays are carried out by directly labeling an antigen or antibody with a chemiluminescent agent. Among the chemiluminescent materials commonly used for labeling are Acridinium Esters (AE), which are efficient luminescent labels that emit light by initiating the action of a luminescent reagent, where strong direct luminescence is accomplished within one second and is a rapid flash luminescence. The acridinium ester is used as a marker for immunoassay, the chemical reaction is simple and rapid, no catalyst is needed, a competition method is adopted for detecting small molecular antigens, a sandwich method is adopted in a macromolecule antibody principle, the nonspecific binding is less, and the background is low; binding to macromolecules does not reduce the amount of light produced, thereby increasing sensitivity.

Immunoprecipitation is a method for purifying and enriching a target protein by using an antibody-specific reaction. After the antibody is combined with corresponding protein in a sample, the sample is incubated with agarose or agarose beads coupled with protein A/G (protein A/G) or a secondary antibody, a bead-protein A/G or a secondary antibody-target protein compound is obtained by centrifugation, the precipitate is re-suspended in an electrophoresis loading buffer solution after being washed, boiled, under the action of high temperature and a reducing agent, the antigen and the antibody are dissociated, and supernatant is collected by centrifugation, wherein the supernatant comprises the antibody, the target protein and a small amount of hybrid protein.

In some embodiments of the second or third aspects, the first or second antibody is labeled with a detectable label.

In some embodiments of the second or third aspect, the label used in the kits and detection methods of the present application may be selected from any label generally known in the art. Preferably the label is one which allows for more accurate quantitation. Examples of labels include, but are not limited to, fluorescent moieties, enzymes, electrochemically active species, radioisotopes, chemiluminescent molecules, latex particles or gold particles, detectable ligands, and the like.

In some embodiments of the second or third aspect, the label is an enzyme or a fluorescent molecule. Methods for attaching labels to antibodies are well known in the art and include covalent and non-covalent attachment.

In some embodiments of the second or third aspect, the antibody may be labeled with a fluorescent compound. When a fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence can then be detected by the emitted fluorescence. Among the most commonly used fluorescent labeling compounds are Cy3 and Cy5 (water-soluble fluorescent dyes of the cyanine dye family- "Cy" dyes), fluorescein isothiocyanate, rhodamine, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine.

In some embodiments of the second or third aspect, the detection antibody is detectably labeled by linking the antibody to an enzyme. Whereby the enzyme will react with its substrate upon contact with its substrate, which reaction can be detected, for example, by spectrophotometric, fluorimetric or visual measurements. Enzymes that can be used to detectably label the antibodies of the invention include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

Detection can also be achieved using radiolabeled antibodies. The antibodies can then be detected by using radioimmunoassay. Radioisotopes can be detected using such methods as the use of gamma counters or scintillation counters or using autoradiography. Isotopes particularly useful for the purposes of the present application are ³ H、 ¹³¹ I、 ³⁵ S、 ¹⁴ C, and preferably ¹²⁵ I。

The antibody may also be detectably labeled by coupling it to a chemiluminescent moiety. The presence of chemiluminescent antibody is then determined by detecting the presence of luminescence generated during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, luciferin, isoluminol, imidazole, acridinium salts and oxalate esters.

In some embodiments of the second or third aspect, examples of the enzyme that can be linked to an antibody include, but are not limited to, alkaline phosphatase, horseradish peroxidase, luciferase, urease, and β -galactosidase.

In some specific embodiments of the second or third aspect, the enzyme is horseradish peroxidase.

In some embodiments of the second or third aspect, one of the first and second antibodies is used as a capture antibody and is immobilized on a solid surface for capturing Tn-type glycosylated MUC1. The other of the first and second antibodies is used as a detection antibody and is linked to a detectable label.

In a fifth aspect, the present application provides the use of an antibody according to the first aspect in the preparation of a kit for detecting the level of human Tn-glycosylated MUC1 in a sample.

In some embodiments of the fifth aspect, the level of Tn-type glycosylated MUC1 is associated with an increased risk of a tumor, a diagnosis of the presence of a tumor, or a severity of a tumor.

In some embodiments of any of the above aspects, the test sample for detection of Tn-type glycosylated MUC1 may be any body fluid or tissue sample, including but not limited to blood, serum, plasma or lymph and less preferably urine, gastric juice, bile, saliva, sweat and spinal fluid, stool or muscle biopsy.

In some embodiments of any of the aspects above, the sample is a blood sample.

In some embodiments of any of the aspects above, the sample is a plasma sample.

In some embodiments of any of the aspects above, the sample is a serum sample.

In some embodiments of any of the above aspects, the bodily fluid may be treated (e.g., serum) or untreated. Methods of obtaining a bodily fluid from a subject are known to those skilled in the art.

Examples

The following examples are provided merely to illustrate some embodiments of the present application and are not intended to be limiting in any way. In addition, the methods in the examples will be performed according to conventional protocols in the art, unless otherwise specified.

Example 1: obtaining of Rabbit monoclonal antibodies

A B lymphocyte for expressing an antibody is obtained from a rabbit immunized by using human Tn-type glycosylated MUC1, and an immunoglobulin antibody for specifically identifying the Tn-type glycosylated MUC1 is obtained by adopting a B cell in-vitro cloning technology, wherein the specific method is shown in figure 1.

A cDNA encoding the C-terminal 82 amino acids of human MUC1 (SEQ ID NO: 1) was cloned into pET28 vector and the vector was transformed into BL21 bacteria. Then, separation and purification are carried out to obtain 6mg of human recombinant MUC1 protein, after glycosyltransferase treatment, tn-type glycosylation is completed, the Tn-type glycosylation is mixed with complete Freund's immunologic adjuvant according to a volume ratio of 1. Each immunization injection was separated by 3 weeks, and the rabbit blood was analyzed for its immune titer by ELISA after the last subcutaneous injection. Briefly, 96-well ELISA plates were coated with the above-described human recombinant galectins, and gradient-diluted rabbit sera (1.

Blood from rabbits with a titer of 1. The remaining leukocytes were flow cytometrically labeled with anti-CD 19, anti-IgM and anti-IgA antibodies (purchased from Biolegend) and rabbit B memory cells positive for CD19, negative for IgM and negative for IgA were selected. Each B cell was cultured in one well of a 384-well microtiter plate and cultured in MEM/F12 medium (purchased from Cellgro). 384-well microtiter plates were plated beforehand with CD 40L-expressing mouse fibroblasts. Mouse fibroblasts were extracted from 14-day gestational-age embryos and transfected with a plasmid expressing CD40L (purchased from origin) after 4-day in-vitro culture in DMEM (containing 10% FBS). Mice fibroblasts were plated at 2000/well after 48 hours. After 12 days of culture, the B memory cells proliferate and secrete monoclonal antibodies. Positive B cell clones were screened by ELISA as described above.

B cell RNA in positive wells was isolated using RNA miniprep kit (purchased from Qiagen) and reverse transcribed to cDNA using reverse transcriptase (purchased from Biosystems). PCR was then performed using the rabbit antibody specific primers described below. The amplification products of the heavy and light chains of the rabbit antibody Ig were inserted into pCDNA3.1 (purchased from LIFE TECH) expression vectors, respectively. HEK293 cells (purchased from LIFE TECH) were transfected with the vector via liposomes (Lipofectamine 2000, purchased from LIFE TECH), specific antibodies were overexpressed, and the specific rabbit antibodies were isolated and purified using a protein A column (purchased from LIFE TECH), to obtain 64 rabbit monoclonal antibodies in total.

Example 2: screening of monoclonal antibody pairs specific for Tn-type glycosylated MUC1 from the monoclonal antibodies identified in example 1

The 64 rabbit monoclonal antibodies obtained were further characterized. The capture antibody and detection antibody pairs were identified using a checkerboard method. Briefly, all 64 antibodies were plated at a concentration of 2ng/mL (antibody concentration adjusted with PBS) in an amount of 100. Mu.L per well as capture antibodies in 384-well microtiter plates, 47 wells per capture antibody, and after 18 hours, 100. Mu.L of 4-BSA-containing PBS buffer was added per well for blocking, and then the standard CA15-3 (purchased from ARCHITECT) was added to the 384-well microtiter plates at an amount of 100. Mu.L per well, and after washing 5 times with PBST buffer, biotin-labeled antibodies other than itself were used as detection antibodies (concentration of 2ng/mL, 100. Mu.L per well) to detect the corresponding wells.

The 64 antibodies obtained were detected by ELISA, respectively, and the antibody with a weak positive signal was deleted. Obtaining a rabbit monoclonal antibody with specificity to human Tn-type glycosylated MUC1.

After sequencing, the first antibody comprises a heavy chain variable region containing HCDR1 shown in SEQ ID NO. 2, HCDR2 shown in SEQ ID NO. 3 and HCDR3 shown in SEQ ID NO. 4, and a light chain variable region containing LCDR1 shown in SEQ ID NO. 5, LCDR2 shown in SEQ ID NO. 6 and LCDR3 shown in SEQ ID NO. 7; the second antibody comprises a heavy chain variable region comprising HCDR1 shown in SEQ ID NO. 8, HCDR2 shown in SEQ ID NO. 9 and HCDR3 shown in SEQ ID NO. 10, and a light chain variable region comprising LCDR1 shown in SEQ ID NO. 11, LCDR2 shown in SEQ ID NO. 12 and LCDR3 shown in SEQ ID NO. 13.

Example 3: detection of the level of cancer antigen 15-3 in standards and samples Using the antibodies obtained in example 2

The capture antibodies (two antibodies each as capture antibody and detection antibody) were diluted in 2mM sodium carbonate (pH9.0) coating to a concentration of 1. Mu.g/mL, 50. Mu.L of each well of a 384-well microtiter plate was taken, incubated at 4 ℃ for 18h, washed twice with PBS, blocked for 2h with PBST (PBS +0.1 Triton-100) containing 4% BSA, washed three times with PBST (PBS +0.1 Triton-100) and then added with 100. Mu.L of standard (purchased from ARCHITECT) at concentrations of 0, 20, 80, 160, 400 and 800U/mL, respectively, quality control (purchased from Fujirebio Diagnostics Inc, wherein the quality control was negative and positive quality control of native Tn-type glycosylated MUC1 from human serum) and clinical patient samples (whose content of cancer antigen was known, some of which were diagnosed as breast cancer, some of which were healthy human), incubated at room temperature for 30 min. During which Tn-type glycosylated MUC1 in the standards and samples bound to the capture antibody, followed by three washes with PBST (PBS +0.1% Triton-100) to wash away all unbound material, including unbound Tn-type glycosylated MUC1, from the wells. Then, 100. Mu.L of 1. Mu.g/mL HRP-labeled detection antibody was added to each well and incubated for 30 minutes. Upon incubation, antibody-antigen-antibody complexes are formed in the titer plate. The plate was washed to remove any unbound detection antibody and incubated for 10 minutes with the addition of 100. Mu.L of the reaction substrate Tetramethylbenzidine (TMB) and the HRP-containing wells were blue in color. The reaction was then stopped by adding 100. Mu.L of sulfuric acid to each well, the color turned yellow, and the absorbance was read at 450nm using a SpectraMax iD5 microplate reader. The intensity of the absorbance was positively correlated with the level of Tn-glycosylated MUC1 in the sample.

The results measured using this example were plotted in the form of a standard curve as shown in FIG. 2. The level of human Tn-type glycosylated MUC1 (CA 15-3) in breast cancer patients was significantly increased (FIG. 3), demonstrating that the pair of antibodies can be effectively used to detect the level of human Tn-type glycosylated MUC1 (CA 15-3) in blood. To compare whether the obtained TnMUC1 antibody is comparable to the same products in the market. anti-MUC 1 (CA 15-3) antibody of Yapei and its standard substance are selected for pull-down determination. Experiments showed that co-incubated CA15-3 standards were successfully pulled down by primary and secondary antibodies with plating with the same concentration of antibody. The first and second antibodies had higher ability to bind to the standard than yapei antibody (fig. 4).

Sequence listing

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Claims

1. An antibody that binds human Tn-type glycosylated MUC1 comprising a heavy chain variable region comprising the amino acid sequences of HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising the amino acid sequences of LCDR1, LCDR2 and LCDR3, wherein

2. The antibody of claim 1, wherein

The antibody is a whole antibody, a Fab fragment, F (ab') ₂ Fragment or single chain Fv fragment (scFv).

3. The antibody of claim 1, wherein the antibody is a monoclonal antibody.

4. The antibody of claim 3, wherein the antibody is a rabbit monoclonal antibody.

5. The antibody of any one of claims 1-4, wherein the antibody binds to the C-terminus of human Tn-glycosylated MUC1, said C-terminus comprising the amino acid sequence set forth in SEQ ID NO 1.

6. The antibody of claim 5, wherein the C-terminus comprises an epitope defined by the amino acids:

7. A kit for detecting the level of human Tn-glycosylated MUC1 in a sample, comprising a first antibody and/or a second antibody;

8. The kit of claim 7, wherein the first or second antibody is labeled with a detectable label selected from the group consisting of: enzymes, fluorescent molecules, radioisotopes, electrochemiluminescent molecules, latex particles, gold particles, detectable ligands, and combinations thereof.

9. The kit of claim 7, wherein the first antibody or the second antibody is attached to an immobilization surface.

10. The kit of claim 9, wherein the fixed surface is a plastic or glass container, or a surface of a slide.

11. The kit of claim 7, wherein the kit further comprises a standard, a quality control, and/or a buffer.

12. The kit of claim 11, wherein the buffer is a pH 7.2 PBS buffer supplemented with 0.1% Triton X-100.

13. The kit of claim 7, wherein the kit is used to aid in the detection or assessment of a tumor.

14. The kit of claim 13, wherein the tumor is breast cancer.

15. The kit of any one of claims 7-14, wherein the level of Tn-type glycosylated MUC1 is associated with increased tumor risk, diagnosis of the presence of a tumor, or severity of a tumor; and/or

The level of Tn-type glycosylated MUC1 is an important index for monitoring the postoperative recovery of tumor patients, the severity of tumor recurrence or metastasis.

16. Use of a first antibody and a second antibody in the preparation of a kit for detecting the level of human Tn-glycosylated MUC1 in a sample;

17. The use of claim 16, wherein the first or second antibody is labeled with a detectable label selected from the group consisting of: enzymes, fluorescent molecules, radioisotopes, electrochemiluminescent molecules, latex particles, gold particles, detectable ligands, and combinations thereof.

18. The use of claim 16, wherein the first or second antibody is attached to an immobilization surface.

19. The use of claim 18, wherein the fixed surface is a plastic or glass container, or a surface of a slide.

20. The use of claim 16, wherein the level of human Tn-glycosylated MUC1 in the sample is detected by an immunological method.

21. The use of claim 20, wherein the immunological method is selected from the group consisting of: enzyme-linked immunosorbent assay, immunofluorescence, immunoturbidimetry, immunochemiluminescence, immunoprecipitation and combinations thereof.

22. The use of claim 20, wherein the immunological method is an enzyme-linked immunosorbent assay.

23. Use of the antibody of any one of claims 1-6 in the preparation of a kit for detecting the level of human Tn-glycosylated MUC1 in a sample.

24. The kit of any one of claims 7-14, wherein the sample is selected from the group consisting of: blood, serum, plasma, lymph fluid, urine, gastric fluid, bile, saliva, sweat, spinal fluid, stool, muscle biopsy, and combinations thereof.

25. The kit of claim 24, wherein the sample is blood.

26. The kit of claim 15, wherein the sample is selected from the group consisting of: blood, serum, plasma, lymph fluid, urine, gastric fluid, bile, saliva, sweat, spinal fluid, stool, muscle biopsy, and combinations thereof.

27. The kit of claim 26, wherein the sample is blood.

28. The use of any one of claims 16-23, wherein the sample is selected from the group consisting of: blood, serum, plasma, lymph fluid, urine, gastric fluid, bile, saliva, sweat, spinal fluid, stool, muscle biopsy, and combinations thereof.

29. The use of claim 28, wherein the sample is blood.