CN111303289A

CN111303289A - Anti-human Tn-type glycosylated MUC1 antibody and application thereof

Info

Publication number: CN111303289A
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: 2020-06-19
Anticipated expiration: 2040-03-05
Also published as: CN111303289B

Abstract

The application provides an antibody or an antigen-binding part thereof for binding human Tn-type glycosylated MUC1 and application thereof, a detection kit comprising the antibody or the antigen-binding part thereof and application thereof, and a method for detecting the level of human Tn-type glycosylated MUC1 in a sample by using the antibody or the antigen-binding part 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, kits comprising the antibodies or antigen-binding portions thereof, and methods of using the antibodies or antigen-binding portions thereof to detect the level of human Tn-glycosylated MUC1 in a sample.

Background

Human tumor associated mucin MUC1, also known as cancer antigen 15-3(CA15-3) or epitopic protein, is a highly glycosylated, high molecular weight protein with glycosylation > 50%, molecular refraction >200kD, coding sequence 1821bp long, containing 7 exons, of which the 2 nd exon contains tandem repeats (VNTR), and VNTR levels vary from 20 to 125 in different people, constituting polymorphisms of the MUC1 gene, belonging to transmembrane molecules. 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, 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% 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 that have been discovered so far. 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, MUC1 is not a homogeneous polypeptide due to the diversity of glycosylation and instability of MUC1 in serum. 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 of 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 fills the blank of high-titer Tn-type glycosylated MUC1 specific antibody diagnostic reagent products in China, and can be extended to other various diagnostic projects particularly sensitive to titer and even developed 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 the amino acid sequences HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising the amino acid sequences LCDR1, LCDR2 and LCDR3, 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

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')₂Fragment 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 post-operative 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;

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 post-operative 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 post-operative 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 fluid, urine, gastric fluid, bile, saliva, sweat, spinal fluid, stool, muscle biopsy, and combinations thereof.

Drawings

Fig. 1 is a schematic diagram of the B cell in vitro cloning technology to obtain the immunoglobulin antibody of the present application specifically recognizing Tn-type glycosylated MUC 1.

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

Figure 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 MUC 1.

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 determination 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-terminus of human Tn-type glycosylated MUC1 by antibody engineering techniques. In various aspects of the application, a detection kit comprising the antibody or an antigen-binding portion thereof and application thereof, a method for detecting the level of human Tn-type glycosylated MUC1 by using the antibody or the antigen-binding portion thereof, and application of the antibody or the antigen-binding portion thereof in detection of the level of human Tn-type glycosylated MUC1 and preparation of the kit for detecting the level of human Tn-type glycosylated MUC1 in a sample are provided.

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 an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. 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')₂Fv), 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 full-length antibody comprises two heavy chains, each heavy chain comprising a heavy chain variable region (VH) and first, second and third constant regions (CH1, CH2 and CH3) and two light chains, each light chain comprising a light chain variable region (VL) and a constant region (CL). the full-length antibody can 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.

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 fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains; (ii) f (ab')₂A 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, VL and VH of 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" which comprise a pair of tandem Fv fragments (VH-CH1-VH-CH1) 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. NYAcad. Sci.190: 382-391; Kabat et al (1987) Sequences of Proteins of Immunological Interest, fourth edition US Govt. printing off.No. 165-492; Kabat et al (1991) Sequences of Proteins of Immunological Interest, fifth edition NIH publication No. 91-3242) provides not only a clear residue numbering system applicable to any antibody variable region, but also precise residue boundaries defining the three CDRs. 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 342:877-883) found that certain sub-parts within the Kabat CDRs adopt almost identical peptide backbone conformations despite large diversity at the amino acid sequence level. These subsections were 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 the Kabat CDRs have been described by Padlan (1995) FASEBJ.9: 133-. 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.

For the variable region sequence of a given antibody, the middle CDR sequence of the variable region sequence can be analyzed in a variety of ways, 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)) is mainly present 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 high abnormal expression in breast cancer is realized to form Tn type glycosylated MUC1, and as the specificity is higher than that of a tissue polypeptide antigen and the sensitivity is higher than that of carcinoembryonic antigen (CEA), the Tn type glycosylated MUC1 has higher clinical diagnosis and application value for breast cancer diagnosis.

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, GalNAc residues attached to the protein backbone are further extended by the action of 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 (GalNAca1-O-Ser/Thr) and Tn (STn) (NeuAca2-6-GalNAca1-O-Ser/Thr) sialylation. 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 protein on the surface of tumor cells. However, in healthy individuals, there is no expression of Tn antigen, and the human body has natural anti-Tn IgM antibodies.

The use of glycoprotein glycomodifications for cancer diagnosis and therapy has recently become a hotspot. 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 heterogeneous states in serum, with various glycosylation and various degradation intermediates, different configurations, and the like. The establishment of MUC1 glycosylation sites and forms directly and closely related to tumor antigenicity as diagnostic indexes for preparing corresponding monoclonal antibodies is undoubtedly the key for improving the diagnostic sensitivity and specificity of MUC 1.

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 probes containing VNTR by the Wanlichen et al shows that MUC1mRNA is not expressed or is expressed at a low degree in normal tissues, but is obviously enhanced in adenocarcinoma, so that the MUC1mRNA can be used as a target point for diagnosing and treating breast cancer. Studies of Zhangxin et al on MUC1 immunoradiometric assay (IRMA) show that MUC1 serum level is in positive correlation with tumor malignancy, 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 breast cancer, but has limited specificity, so that the recognition of MUC1 by the existing diagnostic antibody acts on different sites, and the sensitivity and 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. Recent studies of MUC1 by the professor Carl June at the university of Pennsylvania were published in 2016 and suggest 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 amino acid sequence:

MTPGTQSPFFLLLLLTVLTVVTGSGHASSTPGGEKETSATQRSSVPSSTEKNAFNSSLEDPSTDYYQELQRDISEMFLQIYK(SEQ ID NO:1)。

the inventors of the present application have found that by using the antibodies disclosed herein in combination with immunoassays (e.g., ELISA) well known in the art, the level of Tn-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:

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

MTPGTQSPFFLLLLLT (amino acid sequence shown at positions 1-16 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);

EKNAFNSSLEDPSTDYYQE (amino acid sequence shown at positions 49-68 of SEQ ID NO: 1);

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

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

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 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 pH 7.2 PBS buffer supplemented with 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 the signal generated by the detection monoclonal antibody.

In some embodiments of the second aspect, the kit may additionally comprise one or more of the following: (1) instructions for using the kit to determine the level of Tn-type 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 MUC 1. 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, thus referred to herein as a capture antibody, is immobilized on a solid surface in order to bind and capture Tn-type glycosylated MUC 1. The other antibody is detectably labeled, e.g., with a fluorophore, an enzyme, or a colored particle, such that its binding to the Tn-type glycosylated MUC 1-complex indicates that 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 immunodiagnosis", 2 nd edition, Rose and bigzi, eds. john Wiley & Sons, 1980; campbell et al, "Methods and Immunology", w.a. benjamin, inc., 1964; and Oellic, M. (1984, J.Clin.chem.Clin.biochem.22: 895-904).

In a "sandwich ELISA," an antibody (e.g., anti-Tn-glycosylated MUC1) is attached to a solid phase (i.e., a microtiter plate) and contacted with a biological sample containing an antigen (e.g., Tn-glycosylated MUC 1). the solid phase is then washed to remove unbound antigen.

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 the action of an initiating luminescent reagent, where strong direct luminescence is accomplished in one second, a rapid flash. 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 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.

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, α -glycerol phosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β -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 this application are³H、¹³¹I、³⁵S、¹⁴C, and preferably¹²⁵I。

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 MUC 1. The other of the first and second antibodies is used as a detection antibody and is linked to a detectable label.

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 rabbit immunized by human Tn-type glycosylated MUC1 is used for obtaining B lymphocytes expressing the antibody, and a B cell in-vitro cloning technology is adopted to obtain an immunoglobulin antibody specifically recognizing the Tn-type glycosylated MUC1, 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 separating and purifying to obtain 6mg of human recombinant MUC1 protein, treating by glycosyltransferase to complete Tn type glycosylation, mixing the Tn type glycosylation with complete Freund's immunologic adjuvant according to the volume ratio of 1:1, and then carrying out 4 times of subcutaneous immune injection and one time of abdominal cavity boosting immunity on the New Zealand white rabbits of about 1.5 kg at the age of 2 months. 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, gradient dilutions of rabbit sera (1:500, 1:2000, 1:8000, 1:32000 and 1:128000) were detected by coating 96-well ELISA plates with the above-described human recombinant galectins.

Blood from rabbits with a titer of 1:32000 was collected, centrifuged at 1200rpm for 5 minutes and then red blood cells were removed with red blood cell lysate (from Biolegend). 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 mouse fibroblasts expressing CD 40L. Mouse fibroblasts were extracted from 14-day gestational-age embryos and transfected with a plasmid expressing CD40L (purchased from Origene) after 4 days of in vitro culture in DMEM (10% FBS-containing) medium. After 48 hours, mouse fibroblasts were plated at 2000/well. After 12 days of culture, the B memory cells proliferated and secreted 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 rabbit antibody specific primers as described below. The amplification products of the rabbit antibody Ig heavy and light chains 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 antibodies identified in example 1 for monoclonal antibody pairs specific for Tn-type glycosylated MUC1

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 a volume 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 at a volume of 100. mu.L per well to 384-well microtiter plates, and the corresponding wells were detected with biotin-labeled antibodies other than themselves after washing 5 times with PBST buffer as detection antibodies (concentration of 2ng/mL, 100. mu.L per well).

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

By sequencing, 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; 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

Diluting capture antibodies (two antibodies are each capture antibody and detection antibody) in 2mM sodium carbonate (pH9.0) coating solution to a concentration of 1. mu.g/mL, taking 50. mu.L of each well of a 384-well microtiter plate, incubating at 4 ℃ for 18h, washing twice with PBS, blocking 2h with PBST (PBS + 0.1% Triton-100) containing 4% BSA, washing three times with PBST (PBS + 0.1% Triton-100), and sequentially adding 100. mu.L of standard (purchased from ARCHITECT) at concentrations of 0, 20, 80, 160, 400 and 800U/mL), quality control (purchased from Fujirieio Diagnostics Inc, wherein the quality control is negative and positive quality control of natural Tn-type glycosylated MUC1 from human serum) and clinical patient samples (with known content of cancer antigen 15-3, some of which are diagnosed as breast cancer, and some of which are healthy population), incubate for 30 minutes at room temperature. During this period 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 out all unbound material in the wells, including unbound Tn-type glycosylated MUC 1. 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 of the measurement using this example were plotted in a standard curve as shown in FIG. 2. The human Tn-type glycosylated MUC1(CA15-3) level of the breast cancer patient is obviously increased (figure 3), and the pair of antibodies can be proved to be effectively used for detecting the level of the human Tn-type glycosylated MUC1(CA15-3) in blood. To compare whether the TnMUC1 antibody obtained is comparable to the commercial analog. anti-MUC 1(CA15-3) antibody of Yapeh and its standard substance were selected for pull-down measurement. Experiments showed that co-incubated CA15-3 standard was successfully pulled down by the primary and secondary antibodies with plating with the same concentration of antibody. The first and second antibodies had a 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')₂A fragment or single chain Fv fragment (scFv); and/or

The antibody is a monoclonal antibody, preferably a rabbit monoclonal antibody.

3. The antibody of claim 1 or 2, 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.

4. The antibody of claim 3, wherein the C-terminus comprises an epitope defined by the amino acids:

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

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;

optionally, 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;

optionally, the first or second antibody is attached to a fixed surface, preferably the fixed surface is a plastic or glass container, or the surface of a slide;

optionally, the kit further comprises a standard, a quality control and/or a buffer; preferably, the buffer is PBS buffer with pH 7.2 added with 0.1% Triton X-100;

optionally, the kit is used to aid in the detection or assessment of a tumor, such as pancreatic cancer.

6. The kit of claim 5, 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 the Tn-type glycosylated MUC1 is an important indicator for monitoring the severity of postoperative recovery, tumor recurrence or metastasis in tumor patients.

7. 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, optionally the contacting is simultaneous or sequential;

optionally, the level of human Tn-glycosylated MUC1 in the sample is detected by an immunological method, preferably selected from the group consisting of: enzyme-linked immunosorbent assay, immunofluorescence, immunoturbidimetry, immunochemiluminescence, immunoprecipitation and combinations thereof, more preferably enzyme-linked immunosorbent assay.

8. Use of the antibody of any one of claims 1-4, or the kit of claim 5 or 6, for detecting the level of human Tn-glycosylated MUC1 in a sample.

9. Use of the antibody of any one of claims 1-4 in the preparation of a kit for detecting the level of human Tn-glycosylated MUC1 in a sample.

10. The kit of claim 5 or 6, the method of claim 7, or the use of claim 8 or 9, 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, preferably blood.