Antibodies specifically binding to CREPT and uses thereof

Abstract

The present invention discloses an antibody that specifically binds to CREPT and its use. The present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to CREPT, which comprises an antibody heavy chain variable region and an antibody light chain variable region, wherein the antibody heavy chain variable region comprises at least one HCDR selected from the following sequences: SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; and the antibody light chain variable region comprises at least one LCDR selected from the following sequences: SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. The antibody or the antigen-binding fragment thereof that specifically binds to CREPT provided by the present invention targets different epitopes of CREPT protein than the existing CREPT antibody, and can effectively detect antibodies of CREPT protein in tumor tissues and tumor cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibodies, and relates to antibodies specifically binding to CREPT and uses thereof, and more specifically, to an antibody for identifying CREPT in tumor cells or tumor tissues. Background Art

[0002] Cancer is a class of diseases involving alterations in the state and expression of multiple genes that confer survival advantages and sustained proliferative potential to somatic or germinal cells. Alterations in three broad categories of genes, namely (proto)oncogenes, tumor suppressor genes, and DNA repair genes, collectively contribute to the development of cancer genotypes and phenotypes that are resistant to the natural and intrinsic death mechanisms embedded in the cell (processes such as apoptosis) and are accompanied by dysregulation of cell proliferation events.

[0003] Cancer has a profound impact on human health. It is a complex and diverse disease that has a significant impact on the patient's life, quality of life and family. Therefore, the detection and diagnosis of cancer is of great clinical significance. Early diagnosis of cancer can provide better treatment opportunities because at the early stage of the disease, cancer cells have not yet spread to other parts, increasing the patient's chance of survival. In addition, it also helps in risk assessment and the formulation of a reasonable treatment plan.

[0004] CREPT, also known as RPRD1B, is a novel oncogene. In immunohistochemical staining analysis of clinical tumor samples, CREPT is highly expressed in a variety of tumors, and patients have a poor prognosis (Lu D, Wu Y, Wang Y, et al. CREPT accelerates tumorigenesis by regulating the transcription of cell-cycle-related genes. Cancer Cell. 2012; 21(1): 92-104. doi: 10.1016 / j.ccr.2011.12.016). Previously, monoclonal antibodies 3E10 (Ren F, Wang R, Zhang Y, et al. Characterization of a monoclonal antibody against CREPT, a novel protein highly expressed intumors. Monoclon Antib Immunodiagn Immunother. 2014; 33(6): 401-408. doi: 10.1089 / mab.2014.0043) and 4H1 (CN102559601A) have been identified as being able to specifically recognize CREPT protein, but the sequences of 3E10 and 4H1 antibodies are identical and their application scenarios are similar. Therefore, it is necessary to develop antibodies with different epitopes to further optimize the application scope of CREPT. For example, the low efficiency of 3E10 and 4H1 antibodies for ChIP-seq experiments may be due to the epitope recognition of the antibodies. Developing antibodies that recognize different CREPT protein epitopes from existing antibodies against CREPT will help expand different detections of CREPT proteins, such as improving the efficiency of experiments such as ChIP-seq, and realizing the combined use of antibodies targeting different epitopes in immunological experiments. It is also of great significance for studying CREPT signal transduction and revealing the relationship between CREPT and tumors.

[0005] Therefore, there is an urgent need to develop an antibody that targets a different epitope of the CREPT protein than the existing CREPT antibodies and can effectively detect the CREPT protein in tumor tissues and tumor cells. Summary of the invention

[0006] Problem that the invention aims to solve

[0007] In order to solve the above problems existing in the prior art, the object of the present invention is to provide a new antibody that specifically binds to CREPT.

[0008] Solutions for solving problems

[0009] In the first aspect of the present invention, an antibody or an antigen-binding fragment thereof that specifically binds to CREPT is provided, which comprises an antibody heavy chain variable region and an antibody light chain variable region, wherein the antibody heavy chain variable region comprises at least one HCDR selected from the following sequences: SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; and the antibody light chain variable region comprises at least one LCDR selected from the following sequences: SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

[0010] In some embodiments, the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:4, and HCDR3 shown in SEQ ID NO:5; and / or, the light chain variable region comprises LCDR1 shown in SEQ ID NO:6, LCDR2 shown in SEQ ID NO:7, and LCDR3 shown in SEQ ID NO:8.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CREPT comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; the light chain variable region comprises LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CREPT comprises a heavy chain variable region set forth in SEQ ID NO:1, or a heavy chain variable region that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical thereto; and / or, the antibody or antigen-binding fragment thereof that specifically binds to CREPT comprises a light chain variable region set forth in SEQ ID NO:2, or a light chain variable region that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical thereto.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CREPT comprises a heavy chain variable region set forth in SEQ ID NO:1, or a heavy chain variable region that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical thereto, and a light chain variable region set forth in SEQ ID NO:2, or a light chain variable region that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical thereto; preferably, the antibody or antigen-binding fragment thereof that specifically binds to CREPT comprises a heavy chain variable region set forth in SEQ ID NO:1, and a light chain variable region set forth in SEQ ID NO:2.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CREPT further comprises a heavy chain constant region derived from human or mouse IgG1, IgG2, IgG3 or IgG4 or a variant thereof; and / or, the antibody or antigen-binding fragment thereof that specifically binds to CREPT further comprises a light chain constant region derived from human or mouse κ chain, λ chain or a variant thereof.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CREPT comprises a murine antibody or fragment thereof, a chimeric antibody or fragment thereof, a humanized antibody or fragment thereof; and / or, the antibody or antigen-binding fragment thereof that specifically binds to CREPT is selected from at least one of a full-length antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a single-chain antibody, and a multispecific antibody.

[0016] In the second aspect of the present invention, a hybridoma cell strain is provided, which is classified and named as mouse hybridoma cell, and is deposited in the China General Microbiological Culture Collection Center (CGMCC), the storage address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the storage date is: December 20, 2023, and the storage number is: CGMCC No.45759.

[0017] In the third aspect of the present invention, a monoclonal antibody is provided, which is secreted and produced by the hybridoma cell line described in the second aspect of the present invention.

[0018] In a fourth aspect of the present invention, a biomaterial is provided, wherein the biomaterial comprises at least one of the following B1) to B8):

[0019] B1) a polynucleotide encoding the antibody or antigen-binding fragment thereof that specifically binds to CREPT as described in the first aspect of the present invention;

[0020] B2) an expression cassette containing the polynucleotide described in B1);

[0021] B3) a vector containing the polynucleotide described in B1);

[0022] B4) a vector containing the expression cassette described in B2);

[0023] B5) a host cell containing the polynucleotide described in B1);

[0024] B6) a host cell containing the expression cassette described in B2);

[0025] B7) a host cell containing the recombinant vector described in B3);

[0026] B8) A host cell containing the recombinant vector described in B4).

[0027] In some embodiments, the host cell is a microbial or mammalian cell.

[0028] In the fifth aspect of the present invention, a detection or diagnostic kit is provided, which contains the antibody or antigen-binding fragment thereof that specifically binds to CREPT as described in the first aspect of the present invention, the monoclonal antibody as described in the third aspect of the present invention, or the biological material as described in the fourth aspect of the present invention.

[0029] In the sixth aspect of the present invention, any of the following uses of the antibody or antigen-binding fragment thereof that specifically binds to CREPT described in the first aspect of the present invention, the monoclonal antibody described in the third aspect of the present invention, or the biomaterial described in the fourth aspect of the present invention is provided:

[0030] (i) Use in preparing a reagent or kit for detecting CREPT protein in a sample;

[0031] (ii) Use in the preparation of a reagent or kit for diagnosing or assisting in the diagnosis of whether a subject suffers from a disease associated with CREPT.

[0032] In the seventh aspect of the present invention, a method for detecting the presence or absence of CREPT protein or its content in a sample is provided, which comprises the step of contacting the sample with the antibody or antigen-binding fragment thereof that specifically binds to CREPT as described in the first aspect of the present invention, the monoclonal antibody as described in the third aspect of the present invention, or the biological material as described in the fourth aspect of the present invention.

[0033] Effects of the Invention

[0034] The antibody or antigen-binding fragment thereof and monoclonal antibody specifically binding to CREPT provided by the present invention targets different epitopes of CREPT protein from existing CREPT antibodies and can effectively detect CREPT protein in tumor tissues and tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the monoclonal antibody titer determination results.

[0036] Figure 2 Schematic diagram of the results of 6H2 antibody detection of CREPT-Myc and p15RS-Flag.

[0037] Figure 3 Schematic diagram of the results of 6H2 antibody detecting CREPT CID-Myc and CREPT CCT-Myc.

[0038] Figure 4 This is a schematic diagram of the PPC results of 6H2 antibody detection.

[0039] Figure 5 Schematic diagram of the results of 6H2 antibody detection CREPT (△160-168).

[0040] Figure 6 Schematic diagram of the comparison of the heavy chain and light chain variable region sequences of 6H2 antibody, 3E10 and 4H1 antibody.

[0041] Figure 7 Schematic diagram of ChIP-seq results of 6H2 antibody.

[0042] Figure 8 Schematic diagram of the results of 6H2 antibody detecting endogenous expression of CREPT in various cancer cells

[0043] Fig. 9 Schematic diagram of the differential expression of the CREPT gene in tumor tissues and adjacent tissues. DETAILED DESCRIPTION

[0044] Various exemplary embodiments, features and aspects of the present invention will be described in detail below. The word "exemplary" used here means "used as an example, embodiment or illustrative". Any embodiment described here as "exemplary" is not necessarily interpreted as being superior or better than other embodiments.

[0045] In addition, in order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

[0046] Unless otherwise stated, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0047] In this specification, the word "may" means both performing a certain process and not performing a certain process.

[0048] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0049] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints numerical values ​​A and B.

[0050] In the present specification, the use of “substantially” or “essentially” means that the standard deviation from a theoretical model or theoretical data is within a range of 5%, preferably 3%, and more preferably 1%.

[0051] In the present specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes cases where the event occurs and cases where it does not occur.

[0052] In this specification, when the term "and / or" is used to connect two or more options, it should be understood to mean any one of the options or any two or more of the options.

[0053] According to the present invention, the terms "polypeptide", "protein" and "peptide" are used interchangeably herein to refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with similar peptide backbones.

[0054] According to the present invention, the terms "nucleic acid molecule", "polynucleotide", "polynucleic acid", "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes and primers. Nucleic acid molecules can be linear or circular.

[0055] According to the present invention, the three-letter and one-letter codes for amino acids are as described in J. biol. chem, 243, p3558 (1968).

[0056] The term "antibody" as used in the present invention refers to immunoglobulin, which is a tetrapeptide chain structure formed by two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so its antigenicity is also different. Based on this, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain respectively. The same class of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into κ chain or λ chain by the difference in the constant region. Each of the five types of Ig can have κ chain or λ chain.

[0057] In the present invention, the antibody light chain may further comprise a light chain constant region, wherein the light chain constant region comprises a human κ, λ chain or a variant thereof.

[0058] In the present invention, the antibody heavy chain may further comprise a heavy chain constant region, wherein the heavy chain constant region comprises human IgG1, IgG2, IgG3, IgG4 or variants thereof.

[0059] The sequences of about 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region (C region). The variable region includes three hypervariable regions (HVRs) and four framework regions (FRs) with relatively conservative sequences. The three hypervariable regions determine the specificity of the antibody, also known as the complementary determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The number and position of the CDR amino acid residues in the VL and VH regions of the antibody or antigen-binding fragment conform to the known Kabat numbering convention and the Kabat or AbM or IMGT definition convention (http: / / bioinf.org.uk / abs / ).

[0060] The antibodies disclosed herein include murine antibodies, chimeric antibodies or humanized antibodies. In addition to full-length antibodies, the antibodies disclosed herein also include antigen-binding fragments that can bind to antigens.

[0061] The term "murine antibody" in the present disclosure refers to a monoclonal antibody derived from mice prepared according to the knowledge and skills in the art. When prepared, the test subject is injected with an antigen, and then a hybridoma expressing an antibody with the desired sequence or functional properties is isolated. When the injected test subject is a mouse, the antibody produced is a murine antibody.

[0062] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody. To establish a chimeric antibody, it is necessary to first establish a hybridoma that secretes mouse-specific monoclonal antibodies, then clone the variable region genes from the mouse hybridoma cells, and then clone the constant region genes of human antibodies as needed, connect the mouse variable region genes with the human constant region genes into a chimeric gene, insert it into an expression vector, and finally express the chimeric antibody molecule in a eukaryotic system or a prokaryotic system. The antibody light chain of the chimeric antibody further comprises a light chain constant region of a human κ, λ chain or a variant thereof. The antibody heavy chain of the chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, preferably comprises a human IgG1, IgG2 or IgG4 heavy chain constant region, or an IgG1, IgG2 or IgG4 heavy chain constant region variant using amino acid mutations (such as YTE mutations or back mutations, L234A and / or L235A mutations, or S228P mutations).

[0063] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting the mouse CDR sequence into the human antibody variable region framework, i.e., different types of human germline antibody framework sequences. The heterologous reaction induced by chimeric antibodies due to carrying a large amount of mouse protein components can be overcome. Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references. For example, the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk / vbase), and in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid the decrease in activity caused by the decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity. The humanized antibodies disclosed in the present invention also include humanized antibodies after further affinity maturation mutation of CDR by yeast display.

[0064] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and an antibody analog, which generally includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of a parental antibody. Antibody fragments retain at least some of the binding specificity of the parental antibody. Typically, when activity is expressed on a molar basis, antibody fragments retain at least 10% of the parental binding activity. Preferably, antibody fragments retain at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parental antibody to the target. Examples of antigen-binding fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, linear antibodies, single-chain antibodies (single chain antibody fragment, scFv), domain antibodies, nanobodies, and multispecific antibodies (e.g., diabodies). Engineered antibody variants are reviewed in Holliger and Hudson, 2005, Nat.Biotechnol.23:1126-1136.

[0065] The term "Fab fragment" comprises a light chain and the CH1 and variable region of a heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0066] The term "Fab' fragment" contains one light chain and a portion or fragment of one heavy chain, said portion or fragment containing the VH domain and the CH1 domain and the region between the CH1 and CH2 domains.

[0067] The term "F(ab')2 fragment" contains two light chains and two heavy chains, wherein the heavy chain contains a portion of the constant region between the CH1 and CH2 domains, so that an interchain disulfide bond is formed between the two heavy chains. The F(ab')2 fragment is thus composed of two Fab' fragments, which are linked together by a disulfide bond between the two heavy chains.

[0068] The term "Fv fragment" comprises the variable regions from the heavy and light chains, but lacks the constant regions.

[0069] The term "single-chain antibody", "single-chain Fv" or "scFv" refers to an antibody fragment comprising the VH domain and the VL domain of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker between the VH domain and the VL domain that enables the scFv to form a desired structure for antigen binding.

[0070] The term "multispecific antibody" is used in its broadest sense to cover antibodies with multiple epitope specificities. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH-VL unit has multiple epitope specificities; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, antibody fragments covalently or non-covalently linked together, etc.

[0071] The term "diabody" or "diabody" refers to a small antibody fragment with two antigen-binding sites, which comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, each domain is forced to pair with the complementary domain of another chain, thereby generating two antigen-binding sites.

[0072] The term "domain antibody" is an immunoglobulin fragment with immunological function that contains only the variable region of the heavy chain or the variable region of the light chain. In some cases, two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody fragment. The two VH regions of the bivalent domain antibody fragment can target the same or different antigens.

[0073] The term "fusion antibody", "antibody fusion protein" or "Ig fusion protein" refers to a recombinant protein that is expressed in a eukaryotic or prokaryotic expression system by linking the target gene with the immunoglobulin partial fragment gene at the genetic level. Antibody fusion protein has the characteristics of antibodies and the activity of fusion functional proteins, and can be widely used in immunodiagnosis, immunotherapy, antibody purification, and quantitative analysis of antibodies and antigens, and can be particularly used in the preparation of immune-guided drugs. Depending on the different Ig fragments bound, antibody fusion proteins can be divided into Fab fusion proteins, Fc fusion proteins and single-chain antibody (scFv) fusion proteins.

[0074] The term "antigen binding site" of the present invention refers to the three-dimensional site recognized by the antibody or antigen-binding fragment of the present invention.

[0075] The term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. An epitope can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by tertiary folding of a protein. Epitopes formed by adjacent amino acids are usually retained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with a denaturing solvent. An epitope usually includes at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitope is bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation detection analysis, etc. Methods for determining the spatial conformation of an epitope include techniques in the art, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, etc.

[0076] The terms "specific binding" and "selective binding" used in the present invention refer to the binding of an antibody to an epitope on a predetermined antigen. Generally, when measured in an instrument by surface plasmon resonance (SPR) technology, the antibody is detected at a specific binding site of less than 10 -7 M or even smaller equilibrium dissociation constant (K D ) binds to a predetermined antigen, and its affinity for binding to the predetermined antigen is at least twice its affinity for binding to a non-specific antigen (such as BSA, etc.) other than the predetermined antigen or a closely related antigen. The term "antibody that recognizes an antigen" can be used interchangeably with the term "specific binding antibody" herein.

[0077] According to the present invention, amino acid "addition" refers to the addition of amino acids at the C-terminus or N-terminus of an amino acid sequence. According to the present invention, amino acid "deletion" refers to the deletion of 1, 2 or 3 or more amino acids from an amino acid sequence. According to the present invention, amino acid "insertion" refers to the insertion of amino acid residues at appropriate positions in an amino acid sequence, and the inserted amino acid residues may be all or partly adjacent to each other, or none of the inserted amino acids may be adjacent to each other.

[0078] According to the present invention, amino acid "substitution" refers to the replacement of a certain amino acid residue at a certain position in the amino acid sequence by another amino acid residue; wherein the "substitution" may be a conservative amino acid substitution.

[0079] According to the present invention, "conservative modification", "conservative substitution" or "conservative replacement" refers to the replacement of an amino acid in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), so that changes can be made frequently without changing the biological activity of the protein. It is known to those skilled in the art that, in general, single amino acid replacements in non-essential regions of a polypeptide do not substantially change the biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). In addition, replacements of amino acids with similar structures or functions are unlikely to destroy biological activity. Exemplary conservative replacements are set forth in the following "Exemplary amino acid conservative replacements".

[0080] Exemplary conservative amino acid substitutions

[0081] Original residue Conservative substitution Ala(A) Gly; Ser Arg(R) Lys; His Asn(N) Gln;His;Asp Asp(D) Glu; Asn Cys(C) Ser; Ala; Val Gln(Q) Asn; Glu Glu(E) Asp; Gln Gly(G) Ala His(H) Asn; Gln Ile(I) Leu; Val Leu(L) Ile; Val Lys(K) Arg; His Met(M) Leu; Ile; Tyr Phe(F) Tyr;Met;Leu Pro(P) Ala Ser(S) Thr Thr(T) Ser Trp(W) Tyr; Phe Tyr(Y) Trp; Phe Val(V) Ile; Leu

[0082] "Identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if every position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences are matched or homologous, then the two sequences are 60% homologous. In general, comparison is made when the two sequences are aligned to obtain the maximum percent identity. According to some specific embodiments of the present invention, "identity" refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence or a nucleotide sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values), respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be obtained.

[0083] According to the present invention, the term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector can express a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to plasmids, bacteriophages, cosmids, etc.

[0084] According to the present invention, the term "expression cassette" is a recombinant expression element comprising a target gene and a regulatory element for regulating the expression of the target gene. In some embodiments, the target gene is a coding gene for a target polypeptide. In some embodiments, the regulatory element is a promoter element for initiating transcription of the coding gene. In some embodiments, the regulatory element may also include enhancers, silencers, insulators and other elements.

[0085] According to the present invention, the terms "cell", "cell line" and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the terms "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It should also be understood that all progeny may not be precisely identical in DNA content, due to deliberate or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included.

[0086] According to the present invention, the term "host cell" refers to a cell into which an expression vector has been introduced. The host cell may include a bacterial, microbial, plant or animal cell. Easily transformed bacteria include members of the family Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary Cell Line) and NS0 cells.

[0087] As used herein, the term "diagnosis" includes the detection or identification of a disease state or condition in a subject, determining the likelihood that a subject will develop a given disease or condition, determining the likelihood that a subject with a disease or condition will respond to treatment, determining the prognosis of a subject with a disease or condition (or its likely progression or regression), and determining the effectiveness of a treatment on a subject with a disease or condition.

[0088] In this specification, the term "subject" refers to a human (i.e., a male or female of any age, for example, a pediatric subject (e.g., an infant, child, or adolescent) or an adult subject (e.g., a young, middle-aged, or elderly person)) or a non-human animal. In certain embodiments, the non-human animal is a mammal (e.g., a primate (e.g., a cynomolgus monkey or a rhesus monkey), a commercially relevant mammal (e.g., a cow, pig, horse, sheep, goat, cat, or dog), or a bird. The non-human animal can be male or female at any stage of development. The non-human animal can be a transgenic animal or a genetically engineered animal.

[0089] In this specification, the term "sample" refers to any substance that may contain a target molecule that needs to be analyzed, including biological samples. As used herein, "biological sample" refers to any sample obtained from a live or viral (or prion) source or other macromolecule and biomolecule source, and includes any cell type or tissue of a subject from which nucleic acids, proteins and / or other macromolecules can be obtained. A biological sample can be a sample obtained directly from a biological source or a processed sample. For example, amplified isolated nucleic acids constitute a biological sample. Biological samples include, but are not limited to, body fluids (e.g., blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, sweat, semen, feces, sputum, tears, mucus, amniotic fluid, etc.), exudates, bone marrow samples, ascites, pelvic washings, pleural fluid, spinal fluid, lymph, eye fluid, extracts of nasal, throat or genital swabs, cell suspensions of digested tissues, or extracts of fecal matter, and tissue and organ samples from humans, animals (e.g., non-human mammals) and plants, and processed samples derived therefrom.

[0090] The technical solution of the present invention is described in detail below:

[0091] <Antibody or Antigen-binding Fragment thereof that Specificy Binds to CREPT>

[0092] According to some embodiments of the present invention, an antibody or an antigen-binding fragment thereof that specifically binds to CREPT is provided, which comprises an antibody heavy chain variable region and an antibody light chain variable region, wherein the antibody heavy chain variable region comprises at least one HCDR selected from the following sequences: SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; and the antibody light chain variable region comprises at least one LCDR selected from the following sequences: SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

[0093] The antibody or antigen-binding fragment thereof specifically binding to CREPT provided by the present invention has the effect of specifically binding to CREPT and can be used to detect CREPT in tumor cells or tumor tissues, and further used for diagnosis or auxiliary diagnosis of cancer.

[0094] In some embodiments of the present disclosure, CREPT is the CREPT disclosed in D Lu et al., CREPT accelerates tumorigenesis by regulating the transcription of cell-cycle related genes. Cancer Cell. 21:92-104, 2012.

[0095] In some embodiments, the sequence of the CREPT gene is recorded in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), and the identification number is: Gene ID: 58490. In some embodiments, the sequence of the expression product of the CREPT gene (e.g., mature CREPT protein) is recorded in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), and the identification number is: NP_067038.1.

[0096] In some embodiments, the amino acid sequence of the mature CREPT protein is as shown in SEQ ID NO:9.

[0097] In some embodiments, the heavy chain variable region of an antibody or antigen-binding fragment thereof that specifically binds to CREPT according to the present invention comprises:

[0098] HCDR1 shown in SEQ ID NO: 3,

[0099] HCDR2 shown in SEQ ID NO: 4 and

[0100] HCDR3 shown in SEQ ID NO:5.

[0101] In some embodiments, the light chain variable region of an antibody or antigen-binding fragment thereof that specifically binds to CREPT according to the present invention comprises:

[0102] LCDR1 shown in SEQ ID NO: 6,

[0103] LCDR2 shown in SEQ ID NO: 7 and

[0104] LCDR3 shown in SEQ ID NO:8.

[0105] In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to CREPT according to the present invention comprises:

[0106] HCDR1 shown in SEQ ID NO: 3,

[0107] HCDR2 shown in SEQ ID NO: 4 and

[0108] HCDR3 shown in SEQ ID NO: 5; and

[0109] LCDR1 shown in SEQ ID NO: 6,

[0110] LCDR2 shown in SEQ ID NO: 7 and

[0111] LCDR3 shown in SEQ ID NO:8.

[0112] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CREPT according to the present invention comprises a heavy chain variable region shown in SEQ ID NO: 1, or a heavy chain variable region that has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto.

[0113] Illustratively, at least 90% identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0114] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CREPT according to the present invention comprises a light chain variable region shown in SEQ ID NO: 2, or a light chain variable region that has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto.

[0115] In some specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CREPT according to the present invention comprises a heavy chain variable region shown in SEQ ID NO: 1, or a heavy chain variable region that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical thereto, and a light chain variable region shown in SEQ ID NO: 2, or a light chain variable region that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical thereto.

[0116] In a specific embodiment, the antibody or antigen-binding fragment thereof that specifically binds to CREPT according to the present invention comprises a heavy chain variable region as shown in SEQ ID NO:1, and a light chain variable region as shown in SEQ ID NO:2.

[0117] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CREPT according to the present invention may further comprise a light chain constant region of a human or mouse κ, λ chain or a variant thereof, or further comprise a heavy chain constant region of a human or mouse IgG1, IgG2, IgG3, IgG4 or a variant thereof.

[0118] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CREPT according to the present invention includes a murine antibody or a fragment thereof, a chimeric antibody or a fragment thereof, or a humanized antibody or a fragment thereof.

[0119] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CREPT according to the present invention is selected from at least one of a full-length antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a single-chain antibody, and a multispecific antibody.

[0120] According to some embodiments of the present invention, a fusion antibody is provided, which comprises an antibody or an antigen-binding fragment thereof that specifically binds to CREPT according to the present invention.

[0121] <Hybridoma cell lines and antibodies secreted therefrom>

[0122] According to some embodiments of the present invention, a hybridoma cell line CREPT-Ab-6H2 is provided, and the classification and naming of the hybridoma cell line CREPT-Ab-6H2 is mouse hybridoma cell, which is deposited in the China General Microbiological Culture Collection Center (CGMCC), the deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the deposit date is: December 20, 2023, and the deposit number is: CGMCC No.45759.

[0123] According to some embodiments of the present invention, a monoclonal antibody is provided, which is secreted and produced by the above-mentioned hybridoma cell line.

[0124] <Biological Materials>

[0125] According to some embodiments of the present invention, there is provided a biomaterial, the biomaterial comprising at least one of the following B1) to B8):

[0126] B1) a polynucleotide encoding an antibody or an antigen-binding fragment thereof that specifically binds to CREPT according to the present invention;

[0127] B2) an expression cassette containing the polynucleotide described in B1);

[0128] B3) a vector containing the polynucleotide described in B1);

[0129] B4) a vector containing the expression cassette described in B2);

[0130] B5) a host cell containing the polynucleotide described in B1);

[0131] B6) a host cell containing the expression cassette described in B2);

[0132] B7) a host cell containing the recombinant vector described in B3);

[0133] B8) A host cell containing the recombinant vector described in B4).

[0134] The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0135] The polynucleotide encoding the antibody or antigen-binding fragment thereof that specifically binds to CREPT of the present invention includes: a coding sequence that only encodes an antibody or antigen-binding fragment thereof that specifically binds to CREPT; a coding sequence of an antibody or antigen-binding fragment thereof that specifically binds to CREPT and various additional coding sequences; a coding sequence of an antibody or antigen-binding fragment thereof that specifically binds to CREPT (and optional additional coding sequences) and non-coding sequences.

[0136] The term "polynucleotide encoding an antibody or an antigen-binding fragment thereof that specifically binds to CREPT" may include a polynucleotide encoding the antibody or an antigen-binding fragment thereof that specifically binds to CREPT, and may also include additional coding and / or non-coding sequences.

[0137] The present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the antibody or antigen-binding fragment thereof that specifically binds to CREPT.

[0138] In some embodiments, the host cell may be a microorganism.

[0139] In some specific embodiments, the host cell is a bacterium; illustratively, the host cell is Escherichia coli, but is not limited thereto.

[0140] In another specific embodiment, the host cell is a yeast, and illustratively, the host cell is Pichia pastoris, but not limited thereto.

[0141] In other embodiments, the host cell is a mammalian cell. Exemplarily, the host cell is a mammalian cell line, such as HEK293T cells, HEK293F cells, HEK293 cells, CHO cells, but not limited thereto.

[0142] <Test or diagnostic kit>

[0143] According to some embodiments of the present invention, a detection or diagnosis kit is provided, which contains an antibody or an antigen-binding fragment thereof that specifically binds to CREPT according to the present invention, or a biological material according to the present invention.

[0144] In some embodiments, the detection or diagnosis kit can be used to detect CREPT protein in a sample (whether CREPT protein exists or the amount of CREPT protein), or can be used to diagnose or assist in diagnosing whether a subject has a disease related to CREPT.

[0145] In some specific embodiments, the sample may be a cell or a tissue. Preferably, the sample is a tumor cell or a tumor tissue, such as a cancer cell or a cancer tissue.

[0146] In some specific embodiments, diseases associated with CREPT include cancer, for example: lung cancer, liver cancer, cervical cancer, intestinal cancer, prostate cancer, breast cancer, gastric cancer, endometrial cancer, pancreatic cancer, etc. (Lu D, Wu Y, Wang Y, et al. CREPT accelerates tumorigenesis by regulating the transcription of cell-cycle-related genes. Cancer Cell. 2012; 21(1): 92-104. doi: 10.1016 / j.ccr.2011.12.016; Ma D, Zou Y, Chu Y, et al. A cell-permeable peptide-based PROTAC against the oncoprotein CREPT proficiently inhibits pancreatic cancer. Theranostics. 2020; 10(8): 3708-3721. Published 2020Feb 19. doi: 10.7150 / thno.41677).

[0147] <For testing or diagnostic purposes>

[0148] According to some embodiments of the present invention, there is provided any of the following uses of an antibody or antigen-binding fragment thereof that specifically binds to CREPT according to the present invention, or a biomaterial according to the present invention:

[0149] (i) Use in the preparation of a reagent or kit for detecting CREPT protein in a sample (whether CREPT protein is present or the amount of CREPT protein);

[0150] (ii) Use in the preparation of a reagent or kit for diagnosing or assisting in the diagnosis of whether a subject suffers from a disease associated with CREPT.

[0151] In some specific embodiments, the sample may be a cell or a tissue. Preferably, the sample is a tumor cell or a tumor tissue, such as a cancer cell or a cancer tissue.

[0152] In some specific embodiments, diseases associated with CREPT include cancer, such as lung cancer, liver cancer, cervical cancer, intestinal cancer, prostate cancer, breast cancer, gastric cancer, endometrial cancer, pancreatic cancer, etc.

[0153] <Detection or Diagnosis Method>

[0154] According to some embodiments of the present invention, an antibody or an antigen-binding fragment thereof that specifically binds to CREPT according to the present invention or a biological material according to the present invention is provided, which is used to detect CREPT protein in a sample (whether CREPT protein is present or the content of CREPT protein), or is used to diagnose or assist in diagnosing whether a subject suffers from a disease related to CREPT.

[0155] According to some embodiments of the present invention, a method is provided for detecting CREPT protein in a sample (whether CREPT protein exists or the amount of CREPT protein), or for diagnosing or assisting in diagnosing whether a subject suffers from a disease associated with CREPT, which comprises the step of contacting the sample to be tested with an antibody or an antigen-binding fragment thereof that specifically binds to CREPT according to the present invention or a biomaterial according to the present invention.

[0156] In some specific embodiments, the present invention provides a method for detecting the presence or absence of CREPT protein or its content in a sample, comprising the step of contacting the sample with the antibody or antigen-binding fragment thereof that specifically binds to CREPT described in the present invention or the biomaterial described in the present invention. In some specific embodiments, the method further comprises the step of detecting whether an antigen-antibody complex is formed or the amount of the antigen-antibody complex formed. In some embodiments, the method is a non-disease treatment and diagnosis method.

[0157] In some specific embodiments, the sample may be a cell or a tissue. Preferably, the sample is a tumor cell or a tumor tissue, such as a cancer cell or a cancer tissue.

[0158] In some specific embodiments, diseases associated with CREPT include cancer, such as lung cancer, liver cancer, cervical cancer, intestinal cancer, prostate cancer, breast cancer, gastric cancer, endometrial cancer, pancreatic cancer, etc.

[0159] Example

[0160] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged. The homologous recombination kit was from Novozymes Version 22.1

[0161] Example 1: Antigen cloning and expression

[0162] The antigen used in the present invention is constructed by a prokaryotic protein expression method known in the art. Briefly, the prokaryotic expression vector pGEX-5X-2 / CREPT is transformed into Escherichia coli (E. coli) BL21 (purchased from clontech) to obtain a recombinant bacterium. The recombinant bacterium is cultured in LB medium at 37°C and 200rpm for 12 hours, and then a new LB medium is inoculated with an inoculum of 1% (volume ratio). When the OD600 is about 0.4, IPTG is added (to make its initial concentration 0.2mM), and the timing starts from the addition of IPTG, and the culture is cultured at 16°C for 12 hours. The culture system is centrifuged at 4°C and 6000rpm for 10 minutes, and the bacterial precipitate is collected, and then resuspended and ultrasonically broken with 10 times the volume of the bacterial cell in PBS buffer (pH7.5, 0.1M) (ultrasonication for 3s and 3s, for a total of 10 minutes; the ultrasonic power is 10 watts), and then centrifuged at 4°C and 10000rpm for 10 minutes, and the supernatant is collected. The supernatant was mixed with GST beads (purchased from GE), combined and rotated at 4°C for 3 hours, and then eluted with elution buffer (the solvent was pH 8.8, 50mM Tris-cl buffer, and the solute was reduced GST, the concentration of which was 10mM).

[0163] Amino acid sequence of human CREPT antigen (SEQ ID NO: 9):

[0164] MSSFSESALEKKLSELSNSQQSVQTLSLWLIHHRKHAGPIVSVWHRELRKAKSNRKLTF

[0165] LYLANDVIQNSKRKGPEFTREFESVLVDAFSHVAREADEGCKKPLERLLNIWQERSVY

[0166] GGEFIQQLKLSMEDSKSPPPKATEEKKSLKRTFQQIQEEEDDDYPGSYSPQDPSAGPLLT

[0167] EELIKALQDLENAASGDATVRQKIASLPQEVQDVSLLEKITDKEAAERLSKTVDEACLL

[0168] LAEYNGRLAAELEDRRQLARMLVEYTQNQKDVLSEKEKKLEEYKQKLARVTQVRKE

[0169] LKSHIQSLPDLSLLPNVTGGLAPLPSAGDLFSTD

[0170] Nucleotide sequence encoding CREPT antigen (SEQ ID NO: 10):

[0171] ATGTCCTCCTTCTCTGAGTCGGCGCTGGAGAAGAAGCTCTCGGAGCTGAGCAACTC

[0172] TCAGCAGAGCGTGCAGACCCTGTCCCTTTGGCTCATCCACCACCGCAAGCACGCGG

[0173] GACCCATCGTCTCCGTGTGGCACCGCGAGCTCCGCAAAGCCAAATCAAATAGAAAG

[0174] CTTACTTTTCTGTATTTAGCGAATGATGTCATCCAAAACAGTAAAAGGAAAGGACCT

[0175] GAATTCACTAGAGAATTTGAATCTGTCCTTGTGGATGCTTTTTCTCATGTTGCCAGAG

[0176] AGGCAGATGAAGGCTGTAAAAAACCTTTAGAAAGATTGCTGAACATCTGGCAAGA

[0177] ACGAAGTGTGTATGGCGGCGAGTTCATACAGCAGCTGAAGCTGTCTATGGAGGACT

[0178] CCAAGAGCCCTCCCCCCAAAGCAACAGAAGAGAAGAAATCTCTGAAACGAACTTT

[0179] TCAGCAAATTCAGGAGGAGGAGGATGACGACTACCCTGGCAGCTACTCTCCTCAGG

[0180] ATCCTTCTGCAGGACCCCTCTTGACTGAGGAACTAATCAAAGCTTTGCAGGATCTGG

[0181] AAAATGCCGCATCAGGGGATGCTACTGTCCGACAGAAAATTGCTTCTCTGCCCCAG

[0182] GAAGTGCAAGATGTTTCTCTATTGGAAAAAATAACAGACAAAGAGGCAGCTGAAC

[0183] GTCTTTCAAAAACAGTAGATGAAGCATGTCTGTTACTAGCAGAATATAACGGGCGCC

[0184] TGGCAGCAGAACTGGAGGACCGTCGCCAGCTGGCTCGGATGTTGGTGGAGTATACC

[0185] CAGAATCAGAAAGATGTTTTGTCGGAGAAGGAGAAAAAACTAGAGGAATACAAAC

[0186] AGAAGCTTGCACGAGTAACCCAGGTCCGCAAGGAACTGAAATCCCATATTCAGAGC

[0187] TTGCCAGACCTCTCACTGCTGCCCAACGTCACAGGGGGCTTAGCCCCCCTGCCCTC

[0188] TGCTGGGGACCTGTTTTCAACTGACTAG

[0189] Amino acid sequence of GST-CREPT fusion protein (SEQ ID NO: 11): MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKMSSFSESALEKKLSELSNSQQSVQTLSLWLIHHRKHAGPIVSVWHRELRKAKSNRKLTFLYLANDVIQNSKRKGPEFTREFESVLVDAFSHVAREADEGCKKPLERLLNIWQERSVYGGEFIQQLKLSMEDSKSPPPKATEEKKSLKRTFQQIQEEEDDDYPGSYSPQDPSAGPLLTEELIKALQDLENAASGDATVRQKIASLPQEVQDVSLLEKITDKEAAERLSKTVDEACLLLAEYNGRLAAELEDRRQLARMLVEYTQNQKDVLSEKEKKLEEYKQKLARVTQVRKELKSHIQSLPDLSLLPNVTGGLAPLPSAGDLFSTD

[0190]

[0191] Example 2. Production of mouse monoclonal antibody cell lines and antibodies against human CREPT

[0192] The present invention obtains a mouse-derived anti-human CREPT monoclonal cell line by immunizing mice, fusion of spleen cells, and hybridoma screening. This method is well known in the art. For example, reference can be made to the preparation method described in Zhang C. Hybridoma technology for the generation of monoclonal antibodies. Methods Mol Biol. 2012; 901: 117-135. doi: 10.1007 / 978-1-61779-931-0_7.

[0193] Specifically, 5-week-old female BALB / C mice (Vitamin Liva, license number: SCXK (Beijing) 2002-0003) were immunized with GST-CREPT fusion protein as an antigen. Each mouse was immunized with 5 μg of antigen, and multiple subcutaneous injections were made near the lymph nodes of the limbs. The injection volume was 0.3 ml / mouse. Immunization was performed once every 7 days. The first immunization was mixed with complete Freund's adjuvant, and the second and third immunizations were mixed with incomplete Freund's adjuvant. After the third immunization, blood was collected from the eye sockets and serum was separated. The antibody titer was determined by indirect ELISA. Determination of antibody titer (method is the same as in Example 3). Serum antibody titer of 1×10 5 The mice were boosted once 3 days before fusion.

[0194] Mouse myeloma Sp2 / 0 cells in the logarithmic growth phase were fused with spleen cells from immunized mice using the conventional PEG method. The cells were cloned using the limiting dilution method. Specific antibodies were screened using indirect ELISA. The cloned cell lines were confirmed to be stable cell lines after passage and stored in liquid nitrogen. One of the cell lines was named CREPT-Ab-6H2.

[0195] Balb / c mice (Vital River Company) were intraperitoneally injected with sterile paraffin oil (0.4 mL / mouse), and hybridoma cell lines (5×105 / mouse) were intraperitoneally injected 3 days later. Ascites was collected 7 days later, which was the monoclonal antibody and stored at -80°C for future use. Monoclonal antibodies can also be prepared by incremental culture method: hybridoma cell lines are placed in cell culture medium and cultured at 37°C for 3-4 days. The obtained culture fluid is the monoclonal antibody solution (stored at -80°C).

[0196] Example 3: Antibody titer determination

[0197] The antibody titer of CREPT-Ab-6H2 hybridoma cells was tested by ELISA. The specific steps of antibody titer determination are as follows (pH 9.5, 0.1M PBS buffer was used):

[0198] (1) The GST-CREPT fusion protein solution (the concentration was adjusted with PBS buffer) was used for coating, 100 μL / well, the coating concentration was 1 μg / mL, incubated at 4°C for 16 hours, blocked and washed.

[0199] (2) Add 100 μL of serum or its dilution (gradient dilution using PBS buffer) to each well, set up wells with only PBS buffer as negative controls, incubate at room temperature for 2 h, and wash the plate.

[0200] (3) Add 50 μL of enzyme-labeled secondary antibody (horseradish peroxidase-labeled goat anti-mouse IgG) to each well, incubate at room temperature in the dark for 2 h, and wash the plate.

[0201] (4) Add 50 μL TMB (Thermofisher, WP20004) colorimetric solution to each well and color for 5 min in the dark.

[0202] (5) Add 100 μL 2M sulfuric acid to each well to terminate the reaction; read the OD 450 A positive result was considered when the ratio of the OD value of the test well to the negative control well was greater than 2.1.

[0203] The results showed that the antibody titer of CREPT-Ab-6H2 hybridoma cells could reach 1:25600 ( Figure 1 The anti-CREPT antibody provided by the present invention is a monoclonal antibody secreted by CREPT-Ab-6H2 hybridoma cells (abbreviated as 6H2), which is a CREPT antibody that can be used to identify tumor cells or tumor tissues, that is, an antibody that specifically binds to CREPT, referred to as anti-CREPT antibody or 6H2 antibody.

[0204] The hybridoma cell line CREPT-Ab-6H2 is classified and named as mouse hybridoma cell; it is deposited in the China General Microbiological Culture Collection Center (CGMCC), the deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the deposit date is: December 20, 2023, and the deposit number is: CGMCC No.45759.

[0205] Example 4: Anti-CREPT antibody sequence cloning

[0206] The single cell strain CREPT-Ab-6H2 hybridoma cells with good activity obtained in Example 3 were cDNA cloned, and then the monoclonal antibodies were recombinantly expressed and various activities were tested.

[0207] Determination of the anti-CREPT antibody sequence includes the following steps:

[0208] (1) Take 1×10 6 For 6H2 hybridoma cells, add 1 mL of Trizol to directly lyse the cells and lyse them at room temperature for 10 minutes.

[0209] (2) Add 200 μL of chloroform, mix by inverting, and let stand at room temperature for 15 minutes. Centrifuge at 4°C, 12,000 rpm for 10 minutes until stratification is visible.

[0210] (3) Transfer the upper aqueous phase to an RNase-free centrifuge tube, add 500 μL of isopropanol, mix thoroughly, and place on ice for 10 minutes.

[0211] (4) After 10 minutes, centrifuge at 4°C, 12,000 rpm for 10 minutes. The RNA will settle to the bottom of the tube. Discard the supernatant, wash twice with 75% ethanol, dry, and dissolve in RNAase-free water to test the RNA concentration and quality.

[0212] (5) Reverse transcribe the RNA using a reverse transcription kit (Novozyme HiScript IIQ RT SuperMix for GPCR, catalog number R222-01) to obtain cDNA.

[0213] (6) Using the cDNA from step (5) as a template, PCR amplification was performed on the heavy chain variable region and light chain variable region of 6H2 using primer pairs consisting of F1 and R1, and F2 and R2 as shown below, respectively, to obtain PCR amplification products.

[0214] F1:GAGGTGCAGCTGGTGGAGTC (SEQ ID NO: 13)

[0215] R1:CTGCAGAGACAGTGACCA (SEQ ID NO: 14)

[0216] F2:GACATTGTGATGTCACAG (SEQ ID NO: 15)

[0217] R2:GTTTGATTTCCAGCTTGG (SEQ ID NO: 16)

[0218] PCR reaction conditions: denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, for 30 cycles; extension at 72°C for 10 min.

[0219] (7) The PCR product is purified and sequenced.

[0220] Through the above sequencing, the sequence of the mouse hybridoma monoclonal antibody 6H2 antibody of the present invention is as follows:

[0221] Amino acid sequence of the heavy chain variable region (SEQ ID NO: 1):

[0222] EVQLVESGGGLVQPGGSRKLSCAASGFTFS SFGMH WVRQAPEKGLEWVA YISSGRSTIYYADTVKG RFTISRDNPKNTLFLQMTSLRSEDTAMYYCAR GNYGPPFAY WGQGTLVTVSA

[0223] The underlined part represents the CDR region.

[0224] Amino acid sequence of the light chain variable region (SEQ ID NO: 2):

[0225] DIVMSQSPASLAVSLGQRATISY RASKSVSTSGYSYMH WNQQKPGQPPRLLIY LVSNLES GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHIRELTRS EGGPSWKSNKIY

[0226] The underlined part represents the CDR region.

[0227] The amino acid residues of the VH / VL CDRs of the anti-human CREPT antibody were determined and annotated using the Kabat numbering system.

[0228] The CDR sequences of the anti-human CREPT antibodies of the present invention are as follows:

[0229] Heavy chain CDR1 (HCDR1) SFGMH SEQ ID NO: 3 Heavy chain CDR1 (HCDR2) YISSGRSTIYYADTVKG SEQ ID NO: 4 Heavy chain CDR1 (HCDR3) GNYGPPFAY SEQ ID NO: 5 Light chain CDR1 (LCDR1) RASKSVSTSGYSYMH SEQ ID NO: 6 Light chain CDR2 (LCDR2) LVSNLES SEQ ID NO: 7 Light chain CDR3 (LCDR3) QHIRELTRS SEQ ID NO: 8

[0230] Example 5. Determination of anti-CREPT antibody specificity

[0231] 1. Construction of eukaryotic expression vector

[0232] 1. Construction of eukaryotic expression vector pcDNA3.1-CREPT-Myc

[0233] (1) The double-stranded DNA shown in SEQ ID NO: 10 was synthesized.

[0234] (2) Using the double-stranded DNA of step (1) as a template, PCR amplification is performed using the primer pair consisting of F1 and R1 as shown below to obtain a PCR amplification product.

[0235] F1:CTAGAGCTTGGTACCATGTCCTCCTTCTCT (SEQ ID NO: 17)

[0236] R1:GTGCTGGATGATATCCGTCAGTTGAAAACAGG (SEQ ID NO: 18)

[0237] PCR reaction conditions: denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 60 s, 30 cycles; extension at 72°C for 10 min

[0238] (3) Recover the PCR product using Tiangenjiao recovery kit.

[0239] (4) The plasmid pcDNA3.1-Myc was digested with restriction endonucleases KpnI and EcoRV, and the vector backbone (about 5.5 kb) was recovered.

[0240] (5) The recovered product of step (3) and the vector backbone of step (4) are connected by homologous recombination to obtain a eukaryotic expression vector pcDNA3.1-CREPT-Myc. According to the sequencing results, the structure of the eukaryotic expression vector pcDNA3.1-CREPT-Myc is described as follows: a DNA fragment of SEQ ID NO: 10 is inserted between the KpnI and EcoRV restriction sites of the plasmid pcDNA3.1-Myc, and SEQ ID NO: 10 and the Myc tag coding sequence on the vector backbone form a fusion gene to express the CREPT-Myc fusion protein.

[0241] Amino acid sequence of CREPT-Myc fusion protein (SEQ ID NO: 27):

[0242] MSSFSESALEKKLSELSNSQQSVQTLSLWLIHHRKHAGPIVSVWHRELRKAKSNRKLTF

[0243] LYLANDVIQNSKRKGPEFTREFESVLVDAFSHVAREADEGCKKPLERLLNIWQERSVY

[0244] GGEFIQQLKLSMEDSKSPPPKATEEKKSLKRTFQQIQEEEDDDYPGSYSPQDPSAGPLLT

[0245] EELIKALQDLENAASGDATVRQKIASLPQEVQDVSLLEKITDKEAAERLSKTVDEACLL

[0246] LAEYNGRLAAELEDRRQLARMLVEYTQNQKDVLSEKEKKLEEYKQKLARVTQVRKE

[0247] LKSHIQSLPDLSLLPNVTGGLAPLPSAGDLFSTDEQKLISEEDL

[0248] 2. Construction of eukaryotic expression vector pcDNA3.1-p15RS-Flag

[0249] (1) The double-stranded DNA represented by SEQ ID NO: 28 was synthesized.

[0250] (2) Using the double-stranded DNA of step (1) as a template, PCR amplification is performed using the primer pair consisting of F1 and R1 as shown below to obtain a PCR amplification product.

[0251] F1:TATAGATATATGTCAGCCTTTCTCTG (SEQ ID NO: 19)

[0252] R1:TATACTCGAGTCAATCTTCACTGTAG (SEQ ID NO: 20)

[0253] PCR reaction conditions: denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 60 s, for 30 cycles; extension at 72°C for 10 min.

[0254] (3) The PCR amplification product of step (2) is digested with restriction endonucleases EcoRV and Xho I, and the digestion product is recovered.

[0255] (4) The plasmid pcDNA3.1-Flag was digested with restriction endonucleases EcoRV and Xho I, and the vector backbone (about 5.5 kb) was recovered.

[0256] (5) Connecting the recovered product of step (3) and the vector backbone of step (4) to obtain the eukaryotic expression vector pcDNA3.1-p15RS-Flag. According to the sequencing results, the structure of the eukaryotic expression vector pcDNA3.1-p15RS-Flag is described as follows: the DNA fragment shown in SEQ ID NO: 28 is inserted between the EcoRV and Xho I restriction sites of the plasmid pcDNA3.1-Flag, and the DNA shown in SEQ ID NO: 28 and the Flag tag coding sequence on the vector backbone form a fusion gene to express the p15RS-Flag fusion protein.

[0257] Nucleotide sequence encoding p15RS antigen (SEQ ID NO: 28):

[0258] ATGTCAGCCTTCTCTGAGGCGGCGCTGGAGAAGAAGCTGTCGGAGTTGAGCAACT

[0259] CGCAGCAGAGCGTGCAGACCTTGTCCCTGTGGCTCATTCACCACCGTAAACACTCG

[0260] CGTCCCATCGTCACCGTGTGGGAGCGGGAGCTGCGGAAAGCCAAACCAAACAGGA

[0261] AGCTTACTTTTCCTACCTAGCCAATGATGTCATACAGAACAGCAAGAGGAAGGGG

[0262] CCAGAGTTTACAAAAGATTTTGCACCAGTTATAGTGGAGGCTTTTAAGCATGTTTCA

[0263] AGTGAAACTGATGAAAGTTGTAAGAAGCACCTTGGAAGAGTGTTATCTATTTGGGA

[0264] AGAAAGGTCTGTTTATGAAAATGATGTATTAGAACAACTTAAACAAGTCCTGTATGG

[0265] TGATAAGAAGCCTAGGAAGCGAACTTATGAACAGATAAAGGTGGATGAAAATGAAA

[0266] ACTGTTCCTCTCTGGGATCTCCAAGTGAACCACCACAGACTCTAGATCTCGTTAGAG

[0267] CATTACAAGATCTGGAAAATGCAGCCTCAGGTGATGCAGCAGTTCATCAGAGGATA

[0268] GCTTCTTTACCTGTTGAAGTCCAAGAAGTATCTCTATTAGATAAAATAACAGATAAA

[0269] GAATCTGGAGAAAGGCTTTCCAAAATGGTAGAGGATGCGTGTATGTTGCTGGCAGA

[0270] TTACAATGGCAGATTGGCGGCAGAAATAGATGATAGAAAGCAACTCACTCGAATGTT

[0271] AGCAGATTTTCTTCGTTGTCAAAAGGAAGCCCTTGCAGAGAAAGAGCATAAATTGG

[0272] AAGAGTACAAGCGCAAGCTAGCCAGAGTTTCCCTGGTGCGCAAAGAACTCAGGTC

[0273] CCGGATCCAGAGCCTGCCAGACTTATCTCGATTGCCCAATGTCACTGGCAGCCACAT

[0274] GCACCTGCCCTTTGCGGGAGACATCTACAGTGAAGATTGA

[0275] Amino acid sequence of p15RS-Flag fusion protein (SEQ ID NO: 29):

[0276] MSAFSEAALEKKLSELSNSQQSVQTLSLWLIHHRKHSRPIVTVWERELRKAKPNRKLTF

[0277] LYLANDVIQNSKRKGPEFTKDFAPVIVEAFKHVSSETDESCKKHLGRVLSIWEERSVYE

[0278] NDVLEQLKQALYGDKKPRKRTYEQIKVDENENCSSLGSPSEPPQTLDLVRALQDLENA

[0279] ASGDAAVHQRIASLPVEVQEVSLLDKITDKESGERLSKMVEDACMLLADYNGRLAAEI

[0280] DDRKQLTRMLADFLRCQKEALAEKEHKLEEYKRKLARVSLVRKELRSRIQSLPDLSRL

[0281] PNVTGSHMHLPFAGDIYSEDDYKDHDGDYKDHDIDYKDDDDK

[0282] 3. Construction of the eukaryotic expression vector pcDNA3.1-CREPT CID-Myc

[0283] (1) Using the double-stranded DNA synthesized by SEQ ID NO: 10 as a template, PCR amplification was performed using the primer pair consisting of F1 and R1 shown below to obtain a PCR amplification product.

[0284] F1:CTAGAGCTTGGTACCATGTCCTCCTTCTCT (SEQ ID NO: 17)

[0285] R1:GTGCTGGATGATATCAGGGCTCTTGGAGTCC (SEQ ID NO: 21)

[0286] PCR reaction conditions: denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 60 s, for 30 cycles; extension at 72°C for 10 min.

[0287] (2) Recover the PCR product using Tiangenjiao recovery kit.

[0288] (3) The plasmid pcDNA3.1-Myc was digested with restriction endonucleases KpnI and EcoRV, and the vector backbone (about 5.5 kb) was recovered.

[0289] (4) The recovered product of step (2) and the vector backbone of step (3) are connected by homologous recombination to obtain a eukaryotic expression vector pcDNA3.1-CREPT CID-Myc. According to the sequencing results, the structure of the eukaryotic expression vector pcDNA3.1-CREPT CID-Myc is described as follows: a DNA fragment of CREPTCID is inserted between the KpnI and EcoRV restriction sites of the plasmid pcDNA3.1-Myc, and the DNA fragment of CREPT CID and the Myc tag coding sequence on the vector backbone form a fusion gene to express the CREPT CID-Myc fusion protein.

[0290] Amino acid sequence of CREPT CID-Myc fusion protein (SEQ ID NO: 30):

[0291] MSSFSESALEKKLSELSNSQQSVQTLSLWLIHHRKHAGPIVSVWHRELRKAKSNRKLTF

[0292] LYLANDVIQNSKRKGPEFTREFESVLVDAFSHVAREADEGCKKPLERLLNIWQERSVY

[0293] GGEFIQQLKLSMEDSKSPEQKLISEEDL

[0294] 4. Construction of the eukaryotic expression vector pcDNA3.1-CREPT CCT-Myc

[0295] (1) Using the double-stranded DNA synthesized by SEQ ID NO: 10 as a template, PCR amplification was performed using the primer pair consisting of F1 and R1 shown below to obtain a PCR amplification product.

[0296] F1:CTAGAGCTTGGTACCCCCCCCAAAGCAACA (SEQ ID NO: 22)

[0297] R1:GTGCTGGATGATATCCGTCAGTTGAAAACAGG (SEQ ID NO: 18)

[0298] PCR reaction conditions: denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 60 s, for 30 cycles; extension at 72°C for 10 min.

[0299] (2) Recover the PCR product using Tiangenjiao recovery kit.

[0300] (3) The plasmid pcDNA3.1-Myc was digested with restriction endonucleases KpnI and EcoRV, and the vector backbone (about 5.5 kb) was recovered.

[0301] (4) The recovered product of step (2) and the vector backbone of step (3) are connected by homologous recombination to obtain a eukaryotic expression vector pcDNA3.1-CREPT CCT-Myc. According to the sequencing results, the structure of the eukaryotic expression vector pcDNA3.1-CREPT CCT-Myc is described as follows: a DNA fragment of CREPTCCT is inserted between the KpnI and EcoRV restriction sites of the plasmid pcDNA3.1-Myc, and the DNA fragment of CREPT CCT and the Myc tag coding sequence on the vector backbone form a fusion gene to express the CREPT CCT-Myc fusion protein.

[0302] Amino acid sequence of CREPT CCT-Myc fusion protein (SEQ ID NO: 31):

[0303] PPKATEEKKSLKRTFQQIQEEEDDDYPGSYSPQDPSAGPLLTEELIKALQDLENAASGDA

[0304] TVRQKIASLPQEVQDVSLLEKITDKEAAERLSKTVDEACLLLAEYNGRLAAELEDRRQ

[0305] LARMLVEYTQNQKDVLSEKEKKLEEYKQKLARVTQVRKELKSHIQSLPDLSLLPNVTG

[0306] GLAPLPSAGDLFSTDEQKLISEEDL

[0307] 5. Construction of eukaryotic expression vector pcDNA3.1-PPC-HA

[0308] (1) The double-stranded DNA shown in SEQ ID NO: 32 was synthesized.

[0309] (2) Using the double-stranded DNA of step (1) as a template, PCR amplification is performed using the primer pair consisting of F1 and R1 as shown below to obtain a PCR amplification product.

[0310] F1:CTAGAGCTTGGTACCATGTCAGCCTTCTCTG (SEQ ID NO: 23)

[0311] R1:GTGCTGGATGATATCCGTCAGTTGAAAACAGG (SEQ ID NO: 18)

[0312] PCR reaction conditions: denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 60 s, for 30 cycles; extension at 72°C for 10 min.

[0313] (3) Recover the PCR product using Tiangenjiao recovery kit.

[0314] (4) The plasmid pcDNA3.1-HA was digested with restriction endonucleases KpnI and EcoRV to recover the vector backbone (about 5.5 kb).

[0315] (5) The recovered product of step (3) and the vector backbone of step (4) are connected by homologous recombination to obtain a eukaryotic expression vector pcDNA3.1-PPC-HA. According to the sequencing results, the structure of the eukaryotic expression vector pcDNA3.1-PPC-HA is described as follows: DNA fragments of the p15RS CID and linker regions and the CREPT CCT region are inserted between the KpnI and EcoRV restriction sites of the plasmid pcDNA3.1-HA, and the DNA fragment of PPC and the Myc tag coding sequence on the vector backbone form a fusion gene to express the PPC-HA fusion protein.

[0316] Nucleotide sequence encoding PPC (SEQ ID NO: 32):

[0317] ATGTCAGCCTTCTCTGAGGCGGCGCTGGAGAAGAAGCTGTCGGAGTTGAGCAACT

[0318] CGCAGCAGAGCGTGCAGACCTTGTCCCTGTGGCTCATTCACCACCGTAAACACTCG

[0319] CGTCCCATCGTCACCGTGTGGGAGCGGGAGCTGCGGAAAGCCAAACCAAACAGGA

[0320] AGCTTACTTTTCCTACCTAGCCAATGATGTCATACAGAACAGCAAGAGGAAGGGG

[0321] CCAGAGTTTACAAAAGATTTTGCACCAGTTATAGTGGAGGCTTTTAAGCATGTTTCA

[0322] AGTGAAACTGATGAAAGTTGTAAGAAGCACCTTGGAAGAGTGTTATCTATTTGGGA

[0323] AGAAAGGTCTGTTTATGAAAATGATGTATTAGAACAACTTAAACAAGCTCTGTATGG

[0324] TGATAAGAAGCCTAGGAAGCGAACTTATGAACAGATAAAGGTGGATGAAAATGAAA

[0325] ACTGTTCCTCTCTGGGATCTCCAAGTGAACCACCACAGACTGAGGAACTAATCAAA

[0326] GCTTTGCAGGATCTGGAAAATGCCGCATCAGGGGATGCTACTGTCCGACAGAAAAT

[0327] TGCTTCTCTGCCCCAGGAAGTGCAAGATGTTTCTCTATTGGAAAAAATAACAGACA

[0328] AAGAGGCAGCTGAACGTCTTTCAAAAACAGTAGATGAAGCATGTCTGTTACTAGCA

[0329] GAATATAACGGGCGCCTGGCAGCAGAACTGGAGGACCGTCGCCAGCTGGCTCGGAT

[0330] GTTGGTGGAGTATACCCAGAATCAGAAAGATGTTTTGTCGGAGAAGGAGAAAAAA

[0331] CTAGAGGAATACAAACAGAAGCTTGCACGAGTAACCCAGGTCCGCAAGGAACTGA

[0332] AATCCCATATTCAGAGCTTGCCAGACCTCTCACTGCTGCCCAACGTCACAGGGGGC

[0333] TTAGCCCCCCTGCCCTCTGCTGGGGACCTGTTTTCAACTGACTAG

[0334] Amino acid sequence of the PPC-HA fusion protein (SEQ ID NO: 33):

[0335] MSAFSEAALEKKLSELSNSQQSVQTLSLWLIHHRKHSRPIVTVWERELRKAKPNRKLTF

[0336] LYLANDVIQNSKRKGPEFTKDFAPVIVEAFKHVSSETDESCKKHLGRVLSIWEERSVYE

[0337] NDVLEQLKQALYGDKKPRKRTYEQIKVDENENCSSLGSPSEPPQTEELIKALQDLENA

[0338] ASGDATVRQKIASLPQEVQDVSLLEKITDKEAAERLSKTVDEACLLLAEYNGRLAAEL

[0339] EDRRQLARMLVEYTQNQKDVLSEKEKKLEEYKQKLARVTQVRKELKSHIQSLPDLSL

[0340] LPNVTGGLAPLPSAGDLFSTDYPYDVPDYA

[0341] 6. Construction of the eukaryotic expression vector pcDNA3.1-CREPT(△160-168)-Myc

[0342] (1) Using the double-stranded DNA synthesized by SEQ ID NO: 10 as a template, PCR amplification was first performed using the primer pairs consisting of F1 and R1, and F2 and R2 as shown below, respectively. Then, using the obtained PCR amplification product as a template, PCR amplification was performed using the primer pair consisting of F1 and R2 as shown below.

[0343] F1:CTAGAGCTTGGTACCATGTCCTCCTTCTCTG (SEQ ID NO: 24)

[0344] R1:GTCCTGCAGAAGGATCGTCATCCTCCTCCTC (SEQ ID NO: 25)

[0345] F2:GAGGAGGAGGATGACGATCCTTCTGCAGGAC (SEQ ID NO: 26)

[0346] R2:GTGCTGGATGATATCCGTCAGTTGAAAACAGG (SEQ ID NO: 18)

[0347] PCR reaction conditions: denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 60 s, for 30 cycles; extension at 72°C for 10 min.

[0348] (2) Recover the PCR product using Tiangenjiao recovery kit.

[0349] (3) The plasmid pcDNA3.1-Myc was digested with restriction endonucleases KpnI and EcoRV, and the vector backbone (about 5.5 kb) was recovered.

[0350] (4) The recovered product of step (2) and the vector backbone of step (3) are connected by homologous recombination to obtain a eukaryotic expression vector pcDNA3.1-CREPT(△160-168)-Myc. According to the sequencing results, the structure of the eukaryotic expression vector pcDNA3.1-CREPT(△160-168)-Myc is described as follows: a DNA fragment of CREPT(△160-168) is inserted between the KpnI and EcoRV restriction sites of the plasmid pcDNA3.1-Myc, and the DNA fragment of CREPT(△160-168) and the Myc tag coding sequence on the vector backbone form a fusion gene to express the CREPT(△160-168)-Myc fusion protein.

[0351] Amino acid sequence of CREPT(△160-168)-Myc fusion protein (SEQ ID NO: 34):MSFSESALEKKLSELSNSQQSVQTLSLWLIHHRKHAGPIVSVWHRELRKAKSNRKLTFLYLANDVIQNSKRKGPEFTREFESVLVDAFSHVAREADEGCKKPLERLLNIWQERSVYGGEFIQQLKLSMEDSKSPPPKATEEKKSLKRTFQQIQEEEDDD PSAGPLLTEELIKALQDLENAASGDATVRQKIASLPQEVQDVSLLEKITDKEAAERLSKTVDEACLLLAEYNGRLAAELEDRRQLARMLVEYTQNQKDVLSEKEKKLEEYKQKLARVTQVRKELKSHIQSLPDLSLLPNVTGGLAPLPSAGDLFSTDEQKLISEEDL

[0352] 2. Determination of Antibody Specificity

[0353] Group 1: The eukaryotic expression vector pcDNA3.1-CREPT-Myc was transfected into 293T cells, referred to as CREPT-Myc;

[0354] Group 2: The eukaryotic expression vector pcDNA3.1-p15RS-Flag was transfected into 293T cells, referred to as p15RS-Flag;

[0355] Group 3: The eukaryotic expression vector pcDNA3.1-CREPT CID-Myc was transfected into 293T cells, referred to as CREPTCID-Myc;

[0356] Group 4: 293T cells were transfected with the eukaryotic expression vector pcDNA3.1-CREPT CCT-Myc, referred to as CREPTCCT-Myc;

[0357] Group 5: The eukaryotic expression vector pcDNA3.1-PPC-HA was transfected into 293T cells, referred to as PPC-HA;

[0358] Group 6: The eukaryotic expression vector pcDNA3.1-CREPT(△160-168)-Myc was transfected into 293T cells, referred to as CREPT(△160-168)-Myc.

[0359] 24 hours after transfection, cells were lysed with cell lysis buffer and then Western blot was performed, using Myc antibody (Sigma, product number SAB2702192), Flag antibody (Sigma, product catalog number F4042), HA antibody (Thermo, product number 26183), 3E10 (from Ren F, Wang R, Zhang Y, et al. Characterization of a monoclonal antibody against CREPT, a novel protein highly expressed intumors. Monoclonal Antib Immunodiagn Immunother. 2014; 33(6): 401-408. doi: 10.1089 / mab.2014.0043), 4H1 (from (CN102559601A), 6H2 monoclonal antibody was purified from hybridoma cell supernatant and ascites, and the specific method was the same as in Example 2.

[0360] The results using Myc antibody are shown in Figure 2 to Figure 5 CREPT-Myc fusion protein, CREPT CID-Myc, CREPT CCT-Myc, and CREPT(△160-168)-Myc were detected in the first, third, fourth, and sixth groups, respectively, while there were no corresponding bands in the control group.

[0361] The results using the Flag antibody are shown in Figure 2, the second group could detect p15RS-Flag.

[0362] The results using HA antibody are shown in Figure 4 , the fifth group can detect PPC-HA

[0363] The results using 6H2 antibody are shown in Figure 2 , Figure 3 , Figure 4 , Figure 5 , 6H2 antibody can recognize the full-length CREPT protein, CREPTCCT region and CREPT (△160-168) protein.

[0364] The results using 3E10 antibody are shown in Figure 2 to Figure 5 , the 3E10 antibody can recognize the full-length CREPT protein as well as the CREPTCCT region.

[0365] The results using 4H1 antibody are shown in Figure 2 to Figure 5 , the 4H1 antibody can recognize the full-length CREPT protein as well as the CREPT CCT region.

[0366] Results and Analysis

[0367] Since the amino acid sequences of CREPT protein and p15RS are highly homologous, in order to detect the specificity of monoclonal antibody 6H2, HEK293T cells were transiently transfected with plasmids carrying CREPT-Myc and p15RS-Flag, which has high homology with CREPT-Myc. Western blot results showed that 6H2 antibody can recognize both overexpressed CREPT protein and endogenous CREPT protein (as will be confirmed in Example 7), but not p15RS protein, indicating that monoclonal antibody 6H2 has good specificity ( Figure 2 ). Furthermore, the amino-terminal CREPTCID and carboxyl-terminal CREPT CCT deletions of CREPT protein were overexpressed in 293T cells. Western blot analysis revealed that Myc antibody could detect both deletions simultaneously, while monoclonal antibody 6H2 only recognized the CREPT carboxyl terminus but not the amino terminus ( Figure 3 ). Next, the p15RS CID-p15RS linker-CREPT CCT clone (i.e., pcDNA3.1-PPC-HA, abbreviated as PPC-HA / PPC) was constructed. The full-length CREPT protein and PPC were overexpressed in 293T cells, and Western blot analysis revealed that the 6H2 antibody only recognized the full-length CREPT protein, but not PPC ( Figure 4), indicating that the recognition epitope of 6H2 is located in the linker region of CREPT. The 3E10 antibody in the prior art is a monoclonal antibody secreted by the hybridoma cell CREPT-Ab-3E10, which can specifically recognize the CREPT protein (Ren F, Wang R, Zhang Y, et al. Characterization of a monoclonal antibody against CREPT, a novel protein highly expressed in tumors. Monoclon Antib ImmunodiagnImmunother. 2014; 33(6): 401-408. doi: 10.1089 / mab.2014.0043). The 4H1 in the prior art is a monoclonal antibody secreted by the hybridoma cell CREPT-Ab-4H1, which can also specifically recognize the CREPT protein ((CN102559601A)). Previous studies have shown that the recognition epitope of 3E10 and 4H1 antibodies is amino acids 160-168 of the CREPT protein (Ren F, Wang R, Zhang Y, et al. Characterization of a monoclonal antibody against CREPT, a novel protein highly expressed in tumors. Monoclon Antib ImmunodiagnImmunother. 2014; 33(6): 401-408. doi: 10.1089 / mab.2014.0043). Therefore, after deleting amino acids 160-168 of the CREPT protein, the cells were overexpressed in 293T cells. It was found that 3E10 and 4H1 did not recognize the CREPT protein with amino acids 160-168 missing, while 6H2 could still recognize the CREPT protein with amino acids 160-168 missing and the full-length CREPT protein ( Figure 5 ), which indicates that the CREPT protein epitope recognized by 6H2 is different from that of 3E10 and 4H1. Further, as in Example 4, the 6H2 antibody of the hybridoma cell was sequenced, and the heavy chain and light chain variable regions of the 3E10 antibody, 4H1 antibody and 6H2 antibody were sequenced. The results showed that the antibody sequences of 3E10, 4H1 and 6H2 were inconsistent ( Figure 6 ).

[0368] Example 6: Application of Antibodies in ChIP-seq Experiments

[0369] (1) Place a 10 cm culture dish (approximately 10 7LN229 cells, ATCC, CRL-2611) were added with 5 mL of 1% formaldehyde (which can be prepared directly in the culture supernatant) and cross-linked at 37°C on a shaker for 10 min.

[0370] (2) Prepare 2.5 M glycine in advance, add 250 μL of 2.5 M glycine directly to the culture dish, and terminate cross-linking at 37°C for 10 min on a shaker.

[0371] (3) Scrape the cells with a cell brush and transfer them to an EP tube. Wash twice with PBS, add 500 μL of cell lysis buffer (50 mM Tris-HCl, 10 mM EDTA, 0.5% SDS, 1 mM PMSF, 1 mM DTT) to resuspend the cells, and place on ice for 30 min to lyse.

[0372] (4) Ultrasonic disruption: 40% power, 8 s pulse, 10 s interval, 40 times of ultrasonication in total. Centrifuge after the end of ultrasonication (15 min, 13000 rpm, 4°C), and transfer the supernatant to a new EP tube.

[0373] (5) Take 100 μL of the supernatant and add 9 times the volume of diluent (16.7 mM Tris-HCl, 167 mM NaCl, 1.1 mM EDTA, 1 mM PMSF, 1.1% Triton X-100, 1 mM DTT) and 20 μL of Protein G Beads (Thermofisher, 10003D) washed with cell lysis buffer, and 2 μg of CREPT antibody (3E10, 6H2 or 4H1), and then place in a 4°C refrigerator for hybridization overnight.

[0374] (6) The beads were washed once with low salt, high salt, and LiCl wash buffer, and then washed twice with TE buffer (Invitrogen, 12090015). After washing, the beads were resuspended in 500 μL of elution buffer and incubated for 30 min.

[0375] (7) Centrifuge for 3 min and transfer the supernatant to a new EP tube.

[0376] (8) Add 20 μL of 5 M NaCl and incubate at 65°C overnight to decrosslink.

[0377] (9) Add 20 μL Tris-HCl (pH=6.8), 10 μL 0.5 M EDTA, and 1 μL proteinase K (concentration: 20 μg / mL) to the test tube and incubate at 45°C for 1 h.

[0378] (10) Phenol-chloroform extraction of DNA: Add an equal volume of phenol-chloroform and vortex until a white flocculent precipitate is visible. Centrifuge at 13,000 rpm and 4°C for 15 min.

[0379] (11) Take the top aqueous phase and transfer it to a new EP tube. Add 1 / 10 volume of 3 M NaAc (PH = 5.2), shake and mix. Then add double volume of anhydrous ethanol, mix thoroughly, and place at -20°C overnight.

[0380] (12) Centrifuge for 15 min at 13,000 rpm and 4°C, and discard the supernatant.

[0381] (13) Add 70% ethanol, mix well, and centrifuge at 13,000 rpm for 15 min.

[0382] (14) Discard the supernatant, dry at room temperature, and add 20 μL of ultrapure water to dissolve.

[0383] (15) Take 2 μL and measure the concentration of the sample using a Qubit Fluorometer.

[0384] (16) Take 5 μL of the sample and perform 2% agarose gel electrophoresis to detect the integrity and purity of the sample. The voltage is set to 80 V and the electrophoresis time is 40 min. Then, the sample is exposed and photographed on a colorimeter.

[0385] ChIP-seq experiments can achieve specific matching of proteins and genomic regions. After isolating chromatin, antibodies targeting the target antigen are used to determine whether the target is bound to a specific DNA sequence. Suitable antibodies are a key factor in ChIP experiments. This experiment has high requirements for the specificity and sensitivity of antibodies, and the different recognition epitopes of antibodies will also affect the binding of proteins to DNA. In this experiment, the same amount of three monoclonal antibodies 3E10, 4H1, and 6H2 were added to the same number of lysed cells to obtain the target protein-DNA complex. Afterwards, the DNA is purified to obtain a chromatin immunoprecipitated DNA sample. The DNA precipitated by the three antibodies is further quantified. The results are as follows. Figure 7 As shown, the total amount of DNA bound by each microgram of antibody of 3E10, 4H1, and 6H2 is shown, among which 6H2 binds the largest amount of DNA and is more suitable for ChIP-seq experiments than 3E10 and 4H1.

[0386] Example 7. Expression of CREPT gene in cells and tissues

[0387] 1. Expression of CREPT gene in cells

[0388] The experiments were conducted on MCF7 (human breast cancer cell line, ATCC, HTB-22), LN229 (human glioblastoma cell line, ATCC, CRL-2611), PANC-1 (human pancreatic cancer cell line, ATCC, CRL-1469), HCT116 (human colon cancer cell line, ATCC, CCL-247), H441 (human lung adenocarcinoma cell line, ATCC, HTB-174), and Hela (human cervical cancer cell line, ATCC, CCL-2). The cells were lysed with each cell lysate and then subjected to Western blot detection using 6H2 antibody. The results are shown in Figure 8 The results showed that the 6H2 antibody could detect endogenous CREPT expression in a variety of tumor cell lines.

[0389] 2. Differential expression of CREPT gene in different tissues

[0390] Based on the informed consent of the patients, tumor tissues (including tumor tissues, adjacent cancer tissues or normal tissues) were obtained from multiple hospitals and sliced ​​for immunohistochemistry (6H2 monoclonal antibody was used as the primary antibody, HPR-goat anti-mouse antibody was used as the secondary antibody, DAB was used for color development, and hematoxylin was used for counter-staining. The secondary antibody and color development reagents were purchased from Zhongshan Company). The results are shown in Figure 8 Among different types of tumor tissues, the expression of CREPT protein in lung cancer tissue, breast cancer tissue, gastric cancer tissue, kidney cancer tissue, prostate cancer tissue and colon cancer tissue was significantly higher than that in the corresponding adjacent cancer or normal tissues.

[0391] The above results show that 6H2 monoclonal antibody can recognize the expression of endogenous CREPT protein. Western blot method was used to detect the expression of CREPT in several commonly used cell lines, including MCF7 (human breast cancer cell line), LN229 (human glioblastoma cell line), PANC-1 (human pancreatic cancer cell line), HCT116 (human colon cancer cell line), H441 (human lung adenocarcinoma cell line), and Hela (human cervical cancer cell line). The results showed that CREPT protein exists in most cell lines ( Figure 7 ).

[0392] In order to test whether the prepared CREPT monoclonal antibody recognizes tissue proteins, immunohistochemical experiments were performed using monoclonal antibodies on clinically obtained lung cancer, liver cancer, cervical cancer, intestinal cancer, prostate cancer, breast cancer, gastric cancer, endometrial cancer and adjacent tissues. The experimental results showed that the CREPT protein is located in the cell nucleus and is highly expressed in tumor tissues, while the expression in normal tissues is very low or even undetectable ( Fig. 9 ).

[0393] The above results indicate that the antibody or antigen-binding fragment thereof that specifically binds to CREPT provided by the present invention can specifically bind to CREPT protein, including CREPT protein endogenously expressed in tumor tissues or tumor cells.

[0394] It should be noted that, although the technical solution of the present invention is introduced with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto.

[0395] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or technical improvements in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to CREPT, comprising an antibody heavy chain variable region and an antibody light chain variable region, wherein: The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4 and HCDR3 shown in SEQ ID NO: 5; the light chain variable region comprises LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7 and LCDR3 shown in SEQ ID NO:

8.

2. The antibody or antigen-binding fragment thereof that specifically binds to CREPT according to claim 1, wherein: The antibody or antigen-binding fragment thereof that specifically binds to CREPT comprises a heavy chain variable region as set forth in SEQ ID NO: 1, or a heavy chain variable region that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical thereto; and / or, The antibody or antigen-binding fragment thereof that specifically binds to CREPT comprises a light chain variable region shown in SEQ ID NO: 2, or a light chain variable region that has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto.

3. The antibody or antigen-binding fragment thereof that specifically binds to CREPT according to claim 1 or 2, wherein: The antibody or antigen-binding fragment thereof that specifically binds to CREPT comprises a heavy chain variable region as shown in SEQ ID NO: 1, or a heavy chain variable region that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical thereto, and a light chain variable region as shown in SEQ ID NO: 2, or a light chain variable region that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical thereto.

4. The antibody or antigen-binding fragment thereof that specifically binds to CREPT according to claim 1 or 2, wherein: The antibody or antigen-binding fragment thereof that specifically binds to CREPT comprises a heavy chain variable region as shown in SEQ ID NO:1, and a light chain variable region as shown in SEQ ID NO:

2.

5. The antibody or antigen-binding fragment thereof that specifically binds to CREPT according to claim 1 or 2, wherein: The antibody or antigen-binding fragment thereof that specifically binds to CREPT further comprises a heavy chain constant region derived from human or mouse IgG1, IgG2, IgG3 or IgG4 or a variant thereof; and / or, The antibody or antigen-binding fragment thereof that specifically binds to CREPT further comprises a light chain constant region derived from a human or mouse κ chain, λ chain or a variant thereof.

6. The antibody or antigen-binding fragment thereof that specifically binds to CREPT according to claim 1 or 2, wherein: The antibody or antigen-binding fragment thereof that specifically binds to CREPT includes a murine antibody or a fragment thereof, a chimeric antibody or a fragment thereof, a humanized antibody or a fragment thereof; and / or, The antibody or antigen-binding fragment thereof that specifically binds to CREPT is selected from at least one of a full-length antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, and a single-chain antibody.

7. A hybridoma cell strain, which is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration with the deposit number of CGMCC No.45759.

8. A monoclonal antibody secreted and produced by the hybridoma cell line according to claim 7.

9. A biomaterial, wherein: The biological material comprises at least one of the following B1) to B8): B1) a polynucleotide encoding an antibody or an antigen-binding fragment thereof that specifically binds to CREPT according to any one of claims 1 to 6; B2) an expression cassette containing the polynucleotide described in B1); B3) a vector containing the polynucleotide described in B1); B4) a vector containing the expression cassette described in B2); B5) a host cell containing the polynucleotide described in B1); B6) a host cell containing the expression cassette described in B2); B7) a host cell containing the vector described in B3); B8) A host cell containing the vector described in B4).

10. The biomaterial according to claim 9, wherein The host cell is a microorganism or a mammalian cell. 11 . A detection or diagnosis kit comprising the antibody or antigen-binding fragment thereof that specifically binds to CREPT according to any one of claims 1 to 6, the monoclonal antibody according to claim 8, or the biomaterial according to claim 9 or 10.

12. Any of the following uses of the antibody or antigen-binding fragment thereof that specifically binds to CREPT according to any one of claims 1 to 6, the monoclonal antibody according to claim 8, or the biomaterial according to claim 9 or 10: (i) Use in preparing a reagent or kit for detecting CREPT protein in a sample; (ii) Use in the preparation of a reagent or kit for diagnosing or assisting in the diagnosis of whether a subject suffers from a disease expressing CREPT.

13. A method for detecting the presence or absence of CREPT protein or its content in a sample for non-diagnostic purposes, comprising the step of contacting the sample with an antibody or antigen-binding fragment thereof that specifically binds to CREPT according to any one of claims 1 to 6, a monoclonal antibody according to claim 8, or a biomaterial according to claim 9 or 10.

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